•REVIEW• https://doi.org/10.1007/s11427-020-1683-x. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Plant abiotic stress response and nutrient use efficiencyZhizhong Gong1†, Liming Xiong2†, Huazhong Shi3†, Shuhua Yang1†,

Luis R. Herrera-Estrella4,5,6†, Guohua Xu6†, Dai-Yin Chao7†, Jingrui Li1, Peng-Yun Wang8,Feng Qin1, Jijang Li1, Yanglin Ding1, Yiting Shi1, Yu Wang1, Yongqing Yang1, Yan Guo1* &

Jian-Kang Zhu9*

1State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193,China;

2Department of Biology, Hong Kong Baptist University, Kowlong Tong, Hong Kong, China;3Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409, USA;

4Plant and Soil Science Department (IGCAST), Texas Tech University, Lubbock, TX 79409, USA;5Unidad de Genómica Avanzada (Langebio), Centro de Investigación y de Estudios Avanzados, Irapuato 36610, México;

6College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing 210095, China;7National Key laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant

Physiology and Ecology, Chinese Academy of Sciences, Shanghai 200032, China;8School of Life Science, Henan University, Kaifeng 457000, China;

9Shanghai Center for Plant Stress Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy ofSciences, Shanghai 200032, China

Received February 3, 2020; accepted March 17, 2020; published online March 31, 2020

Abiotic stresses and soil nutrient limitations are major environmental conditions that reduce plant growth, productivity and quality.Plants have evolved mechanisms to perceive these environmental challenges, transmit the stress signals within cells as well asbetween cells and tissues, and make appropriate adjustments in their growth and development in order to survive and reproduce. Inrecent years, significant progress has been made on many fronts of the stress signaling research, particularly in understanding thedownstream signaling events that culminate at the activation of stress- and nutrient limitation-responsive genes, cellular ionhomeostasis, and growth adjustment. However, the revelation of the early events of stress signaling, particularly the identification ofprimary stress sensors, still lags behind. In this review, we summarize recent work on the genetic and molecular mechanisms ofplant abiotic stress and nutrient limitation sensing and signaling and discuss new directions for future studies.

abiotic stress, sensing, nutrient use efficiency, heavy metal, Ca2+ signaling, ROS, signal transduction, phosphorylation,transcription factor, transporter

Citation: Gong, Z., Xiong, L., Shi, H., Yang, S., Herrera-Estrella, L.R., Xu, G., Chao, D.Y., Li, J., Wang, P.Y., Qin, F., et al. (2020). Plant abiotic stress responseand nutrient use efficiency. Sci China Life Sci 63, https://doi.org/10.1007/s11427-020-1683-x

Introduction

With a continuously growing population in the world, food

security becomes a major issue, which is further complicatedby the potential impact of climate change on crop pro-ductivity. Extreme temperatures, drought and soil salinity aremajor adverse environmental conditions that plants oftenencounter.Drought is one of the most detrimental abiotic stresses for

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†Contributed equally to this manuscript*Corresponding authors (Jian-Kang Zhu, email: jkzhu@sibs.ac.cn; Yan Guo, email:guoyan@cau.edu.cn)

plant agriculture. Currently, agriculture uses more than 70%of the fresh water in the world (86% in developing coun-tries), and this consumption is estimated to increase as globalweather becomes generally drier and warmer. Therefore,understanding the molecular mechanisms underlying plantdrought stress response and developing crops with enhanceddrought resistance is of vital importance. In the past twodecades, researchers working on plant molecular biologyhave unraveled how plants cope with drought stress byproducing the phytohormone abscisic acid (ABA), repro-gramming gene expression, closing stomata, and makingosmotic adjustment, finally leading to adaptive growth anddevelopment. Identification of key genetic determinants andimproving the drought resistance of crops will be importantto meet global food demands with sustainable water supply.Soil salinity is another global problem limiting land usage

and crop production. It was estimated that approximatelyone-fifth of the irrigated lands in the world are affected bysalinity (Morton et al., 2019). In addition, there are vast areasof marginal lands such as coastal regions that are unsuitablefor crop cultivation. Utilization of these saline soils for cropproduction could mitigate the issues of food demands by theever increasing human population. However, most crops areglycophytes sensitive to salinity, and thus cultivation of thecurrently used cultivars in these saline soils is not feasible.There have been two strategies towards cropping in salinesoils; one is to reduce soil salinity through improving agri-cultural practice and/or phytoremediation and the other is tobreed or genetically engineer new varieties suitable for salinesoils. The combination of these two strategies would be idealto maximize cultivatable lands and increase crop production.Climate change is increasing the magnitude and frequency

of temperature extremes (i.e., low and high temperatures)worldwide. Crops exposed to adverse temperatures haveimpaired growth and development, restricting where theycan be grown and reducing crop yield. Therefore, to maintainagricultural production in the face of climate change, it isimportant to understand the mechanisms underlying planttemperature stress responses. Over the past few decades,these mechanisms have been extensively studied, whichshould benefit future engineering of cold and heat stress-tolerant crops.To be able to design the new generation of crop varieties

that make optimal use of nutrients naturally present in thesoil or applied as fertilizers, we must better understand themechanisms that regulate nutrient uptake and use efficiencyunder variable field conditions. Most studies that aimed atdetermining the effect of mineral nutrition on plant devel-opment and productivity have been performed under la-boratory conditions, delimiting the effect of single elementand over relatively short periods of time. Therefore, there is aneed for experimental approaches that consider the interac-tions of multiple elements and variations in weather and soil

conditions. Ion transporters are often not specific for a singleelement. In addition, changes in atmospheric CO2 will affectphotorespiration and nitrogen assimilation, and the relativeactivity of some elements, such as Mg and Mn, can haveprofound effects on photosynthetic carbon assimilation.The toxicity of heavy metals is well documented for its

impairment of plant growth and development, damage tophotosynthesis, alteration of enzymatic activities, and oxi-dative injury (Eleftheriou et al., 2015; Farooq et al., 2016;Karmous et al., 2017). Once heavy metals are accumulated inplants and enriched in the food chain, they further threatenanimals and human health (Jafari et al., 2018). To deal withthe toxicity of heavy metals, plants have evolved delicatemechanisms for minimizing heavy metal uptake or trans-portation, and for detoxification through modification orcompartmentalization. These processes involve numerousproteins, especially the heavy metal transporters such asHeavy Metal ATPases (HMAs), Natural Resistance-Asso-ciated Macrophage Proteins (NRAMPs), ATP Binding Cas-sette transporters (ABCs) and Yellow Strip-Like familytransporters (YSLs).Here, we summarize recent progress in abiotic stress

sensing and signal transduction. Readers are referred toprevious reviews for an overview of the field.

Sensing abiotic stresses

Common themes of abiotic stress sensing: The Ca2+

connections

Different stresses such as cold, drought, and high salinityshare some common features with regards to their impacts onplants and the ways by which plants perceive them. For in-stance, all these abiotic stresses generally cause osmoticstress to plant cells. They also quickly elicit a transient in-crease in cytosolic Ca2+ concentration ([Ca2+]). Ca2+ is thusconsidered as a universal second messenger for the primarystress signals. Several characteristics of Ca2+ make it suitableas a ubiquitous signaling molecule. Its low concentration inthe cytosol relative to other internal or external spaces makesthe concentration readily changeable. There are numerousproteins that can recognize and interrupt the changes in[Ca2+]. There are many Ca2+-permeable channels or trans-porters that can precisely manage these concentration chan-ges.Among the various Ca2+ permeable channels, many are

activated or gated by posttranslational modifications or othersecond messengers that result from the initial perception ofstress. These molecules include, for example, cyclic nu-cleotides (cAMP and cGMP), amino acids (glutamate andmethionine), various reactive oxygen species (ROS), andCa2+ itself. Here we focus on the initial signal perception andwill not elaborate on these second messengers or signal

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propagation processes. These and other Ca2+ channels havebeen discussed in detail in several recent reviews (De-midchik et al., 2018; Hamilton et al., 2015; McAinsh andPittman, 2009; Verret et al., 2010; Ward et al., 2009).

Challenges to identifying abiotic stress sensors

Due to the complexity of abiotic stress as well as the com-plexity of plant responses, the identification of primary stresssensors has been proven very challenging. Indeed, variousapproaches have been used to attempt to identify the initialsensors for abiotic stress, yet few components identified sofar can be confidently considered as primary stress sensors.Genetic approaches are often powerful for plant studies butsuffer from likely genetic redundancies and/or lethalitieswhen they are applied to discover abiotic stress sensors, gi-ven the importance of these sensors to plants. To identifythese sensors, choosing an early readout in the signalingpathway would avoid the complications from downstreamsignaling interactions and integration. For instance, changesin cytosolic [Ca2+] are an early readout of abiotic stresssignaling. Tracing the source of these Ca2+ transients wouldlikely lead to the discovery of primary sensors for thestresses. In fact, as discussed below, some Ca2+ channelshave been identified as candidate sensors using this ap-proach.

Subcellular locations of stress perception and the natureof stress sensors

Unlike chemical signals, abiotic stresses are mostly physicalsignals that simultaneously impact all parts of cells. As aresult, abiotic stress signals could be independently per-ceived at various locations of the cells and by various cellularmacromolecules and structures. In a recent review (Zhu,2016), abiotic stress sensing and signaling in nuclei, chlor-oplasts, mitochondria, peroxisomes, plasma membranes,endoplasmic reticulum (ER), and cell walls were discussed.In addition, there are membraneless organelles such as stressgranules, speckles, liquid-liquid phase separations and ri-bonucleoprotein condensates that may also play importantroles in stress signaling, as has been realized recently foranimal cells (Boeynaems et al., 2018). Many stress-re-sponsive genes in plants encode intrinsically disorderedproteins and a high proportion of their pre-mRNAs is notadequately processed under abiotic stress (Cui and Xiong,2015). Drought and salt stress also lower water potentialsand increase molecular crowding in cells. These factors areall conducive to the formation of various ribonucleoproteincondensates. While current studies are still limited, it is ex-pected that the involvement and importance of ribonucleo-protein condensates in stress signaling and stress tolerancewill be increasingly appreciated in the coming years.

Although abiotic stress may affect all the molecules withinthe cells, not all of the affected molecules are sensors. Asensor must transmit its molecular changes caused by stressto a downstream component, leading to cellular response(s)important for stress adaptation. Given the many outputs for aparticular stress, there could be as many branches of sig-naling pathways and multiple sensors. Thus, a sensor ismeaningful only in the context of its signaling readout. Forinstance, a low temperature sensor for the CBF-class generegulation may or may not be relevant to cold stress reg-ulation of photosynthesis.

Recent advances in the study of stress perception in plants

Temperature stressesCold and heat can decrease and increase, respectively, thefluidity of cellular membranes (Falcone et al., 2004; Zhu,2016). This change of membrane fluidity may be sensed bymembrane-anchored proteins such as calcium (Ca2+) chan-nels and receptor-like kinases (RLKs) (Gong et al., 1998;Knight et al., 1996; Zhu, 2016). Transient receptor potentialvanilloid channels (TRPVs) have been reported to sensetemperature signals in mammalian cells (Venkatachalam andMontell, 2007), but no homologs of such receptors have beenidentified in land plants. Instead, other types of Ca2+ chan-nels seem to function in temperature stress signaling inplants.The Arabidopsis genome encodes more than 40 putative

Ca2+ channels, some of which may function in temperaturesensing (Ward et al., 2009). Arabidopsis cyclic nucleotide-gated Ca2+ channel 6 (CNGC6) mediates heat-induced Ca2+

influx, which promotes the expression of heat shock protein(HSP) genes and increases thermotolerance (Gao et al.,2012). Consistent with the critical role of Ca2+ signals, cal-cium signaling components such as calmodulin 3 (CaM3)and CaM-binding protein kinase 3 (CBK3) are importantregulators of plant heat stress tolerance (Liu et al., 2008; Liuet al., 2010). Recently, the rice gene Chilling tolerance di-vergence 1 (COLD1) was identified as a potential cold sen-sor. COLD1 works together with Rice G-protein α subunit 1(RGA1) to trigger a cold-induced increase in cytosolic [Ca2+](Ma et al., 2015). Given that the generation of calcium sig-nals seems to be involved in temperature sensing, it is im-portant to identify the Ca2+ channel(s) involved.As RLKs are often involved in perceiving external signals

in plants (Liang and Zhou, 2018), it is not surprising thatseveral RLKs have been reported to play critical roles inregulating cold and heat responses. Two calcium/calmodu-lin-regulated receptor-like cytoplasmic kinases (RLCKs),CRLK1 and CRLK2, positively regulate freezing tolerance(Yang et al., 2010a; Yang et al., 2010b). Recently, a plasmamembrane-localized RLCK, Cold-responsive protein kinase1 (CRPK1), was found to play a negative role in regulating

3Gong, Z., et al. Sci China Life Sci

freezing tolerance (Liu et al., 2017b). Overexpression of theRLK ERECTA (ER) improves thermotolerance in rice andtomato (Shen et al., 2015). 25L1 and 25L2, which encodetwo leucine-rich repeat receptor-like kinases, mediate hightemperature-dependent interspecific hybrid weakness in rice(Chen et al., 2014). Thermo-sensitive genic male sterile 10(TMS10) and TMS10-like protein (TMS10L) regulate theeffects of temperature on male fertility in rice (Yu et al.,2017). Whether these proteins are responsible for sensingtemperature signals merits further investigation.Non-membrane proteins located in the nucleus and cyto-

sol, including the histone variant H2A.Z and the photo-receptor phytochrome B (phyB), may also be involved intemperature sensing (Jung et al., 2016; Kumar and Wigge,2010; Legris et al., 2016). Previous results showed that DNAis wrapped more tightly by nucleosomes containing H2A.Zthan by those containing H2A, suggesting H2A.Z canmodulate gene transcription in a temperature-dependentmanner (Kumar and Wigge, 2010). Recently, two studieshave demonstrated that phyB responds to changes in ambienttemperature by changing its form (from active Pfr to inactivePr). Moreover, phyB regulates target gene expression in atemperature-dependent manner, and phyB null mutants dis-play a constitutive warm-temperature response (Jung et al.,2016; Legris et al., 2016). Recently, phototropin, a type ofblue light receptor, was found to be involved in cold per-ception in the liverwortMarchantia polymorpha (Fujii et al.,2017). Although phyB and phototropin are implicated astemperature sensors, it is not known whether other photo-receptors may have a similar function and may be importantfor sensing temperature extremes.

Osmotic stressLike other abiotic stresses, osmotic stress can rapidly andtransiently increase cytosolic [Ca2+] probably by activatingcertain Ca2+-permeable channels. The activation of thesechannels has also been speculated to be caused by mechan-ical forces generated by the osmotic stress on the cellmembrane or cell wall. By screening for osmotic stress(caused by high concentrations of sorbitol)-induced Ca2+

transients, Yuan et al. identified the OSCA1 protein as re-quired for this [Ca2+] increase (Yuan et al., 2014). TheOSCA1 group of proteins form a new family of mechan-osensitive Ca2+-permeable ion channels conserved in yeast,plants and humans (Hou et al., 2014; Yuan et al., 2014).During the past year, the structure of members in this proteinfamily was independently solved by five groups (Jojoa-Cruzet al., 2018; Liu et al., 2018c; Maity et al., 2019; Murthy etal., 2018; Zhang et al., 2018c). These studies show thatOSCA1 family proteins (OSCA1.1, OSCA1.2, andOSCA3.1/ERD4) are pore-forming ion channels structurallysimilar to the mammalian TMEM16 family of proteins.Based on their structural features, it is believed that osmotic

stress generates tensions in the lipid bilayer that could causea conformation change and activate the channels to allow theentry of Ca2+ (Zhang et al., 2018c). The osca1 mutant plantsshowed increased sensitivity to osmotic stress (but not toABA), although the signaling events immediately down-stream of OSCA1 are currently unclear. The identification ofOSCA1 opens a door to learning the ways plant cells senseosmotic stress. Future identification of the downstreamCa2+-binding proteins and interacting proteins should pro-vide important information on how plants respond to osmoticstress at the molecular level.

Salt stressUsing the same method as that for identifying OSCA1, agenetic screen identified the moca1 (monocation inducedCa2+ increases 1) mutant that showed reduced cytosolic [Ca2+]in response to salt treatments (Jiang et al., 2019). AlthoughCa2+ levels in the mutant are also reduced when seedlingswere treated with K+ or Li+, increased sensitivity in terms ofgrowth inhibition to the ions was only seen with Na+ treat-ments. Furthermore, the growth response of the moca1 mu-tant seedlings to ABA or osmotic stress also did not change,indicating that the wild type MOCA1 functions specificallyin mediating Na+ signaling. The MOCA1 gene encodes aninositol phosphorylceramide glucuronosyltransferase(IPUT1), an enzyme catalyzing the biosynthesis of thesphingolipid glycosyl inositol phosphorylceramide (GIPC)and residing on plasma membranes and ER membranes.Indeed, the moca1 mutant plants had low contents of GIPCbut accumulated the precursor inositol phosphorylceramide(IPC). While GIPC appeared to bind Na+, the significance ofthis binding is currently not very clear. The lack of GIPC (orincreased IPC levels) may be responsible for diminishedNa+-induced Ca2+ transients in the mutant. These lipids areenriched in membrane nanodomains where many signalingproteins reside, and they may be necessary for maintaining alipid environment conducive to the activity or gating of theNa+-responsive Ca2+ channels. Alternatively, these lipidsmay be required for the production of certain lipid signalsthat in turn regulate these channels.While membrane lipids may have important roles in

regulating membrane protein activities, they may alsophysically affect the mobility of these proteins. For in-stance, lipid nanodomains in mammalian cells could sepa-rate phospholipase D2 (PLD2) from its substrates and thuskeep the signaling pathway in an ‘off’ state. Disruption ofthe lipid rafts could activate PLD2 by allowing the enzymeto mix with its substrate, thus generating phosphatidic acidto trigger downstream signaling (Petersen et al., 2016). It isknown that membrane intrinsic proteins such as ion chan-nels may move within the bilayers to some extent. Inter-estingly, the mobility of these proteins is more affected bycell walls (Martinière et al., 2012). Furthermore, cell walls

4 Gong, Z., et al. Sci China Life Sci

may also directly interact with membrane proteins duringgrowth and stress responses.As mentioned earlier, salt stress and osmotic stress have

long been suggested to cause mechanical strains on cell wallsthat might activate stress signaling, yet the potential signal-ing role of cell walls only becomes more evident in recentyears thanks to the identification of a group of related smallpeptides and their receptors that are involved in cell growth,disease resistance and stress responses. Among these areseveral rapid alkalization factors (RALFs) peptides and themalectin receptor-like kinase FERONIA (FER). The specificligand-receptor binding results in FER phosphorylation andinhibition of the plasma membrane H+-ATPase 2 (AHA2),leading to cell wall alkalization and growth inhibition(Haruta et al., 2014). A transient increase in cytosolic [Ca2+]was also observed upon ligand activation of the receptorkinase (Haruta et al., 2014). In addition to these earlierproposed events, more components are identified recentlyand the functions of these ligand-receptor complexes arerapidly being elucidated, including their roles in salt stresssignaling and salt tolerance.Salt stress weakens cell walls and this damage to the

walls appears to be sensed by the FER signaling pathwaywhose activation prevents cell burst (Feng et al., 2018).The pathway also triggers transient [Ca2+] increases al-though the downstream events are unclear. An early stepof the salt-overly-sensitive (SOS) pathway has a Ca2+-binding component SOS3 yet the SOS pathway appears tobe independent of the FER pathways (Feng et al., 2018).Several candidates have been proposed to be able to detectcell wall integrity for the FER pathway. These include theFER extracellular domain, pectins, the co-receptor LOR-ELEI-LIKE GPI-anchored protein 1 (LLG1), and theleucine-rich extension (LRX) proteins LRX3/4/5 (Feng etal., 2018; Zhao et al., 2018a). Since salt stress could in-crease the mature RALF22 ligand via Site 1 Protease(S1P) cleavage. It would be interesting to investigate howS1P is activated by salt stress. Furthermore, salt (andRALF22) can cause the internalization of FER (Zhao etal., 2018a). This may suggest multi-stage functions of theFER pathway in response to salt stress: a quick responsefrom the membrane-localized FER resulting in transientincreases in cytosolic [Ca2+] and a subsequent signaling torestore growth that involves the slower internalization ofFER, a process that may also depend on the early Ca2+

transients. Thus, FER internalization may not necessarilyrepresent the end of the signaling but it could mediatesustained signaling to allow cell growth during the re-covery stage, as reported for internalized receptors inmammalian cells (Thomsen et al., 2016). Besides osmoticstress, FER is also involved in ABA and drought stresssignaling by interacting with ABI2 and G proteins (Chenet al., 2016; Yu and Assmann, 2018).

Drought stressDetecting soil water availability and initiating appropriatestress responses are important for plants to survive drought.The soil matrix is heterogeneous in water availability androots can detect water potential gradients in the soil and growtoward areas with high water potential, a process referred toas hydrotropism. Recently, it was found that water potentialgradients at the root tip could generate cytosolic Ca2+ signalsalong the phloem that peaks and distributes asymmetricallyat the elongation zone of the root. This water gradient-eli-cited Ca2+ pattern presumably facilitates root to bend awayfrom the low water potential direction (Shkolnik et al.,2018). Interestingly, the previously isolated miz1 mutant thatlacks hydrotropism (Kobayashi et al., 2007) does not exhibitsuch Ca2+ patterns along the phloem. The source of this Ca2+

signal is related to the ER-localized type 2A Ca2+-ATPase(ECA1) since the eca1mutant showed higher Ca2+ levels andalso had enhanced root bending. Furthermore, MIZ1 inter-acts with and inhibits ECA1 (Shkolnik et al., 2018). There-fore, ECA1 appears to play an important role in determiningroot hydrotropism. Future studies will determine how waterpotential gradients are perceived and how they cause ER-originated Ca2+ to propagate along the roots, and how theCa2+ signals could be converted to root growth changes.Upon sensing water deficit in the soil, roots can transmit

the signal to the shoot. Although the nature of this sensor isstill unknown, many molecules are suggested as the long-distance messengers to trigger stress responses in the shoot.These include, for example, abscisic acid (ABA), H+ (pH),Ca2+, ROS, NO, SO4

2–, lipids, small peptides, RNA, andphysical signals such as hydraulic and electrical signals. Onesignal recently discovered is the small peptide Clavata3/en-dosperm surrounding region-related 25 (CLE25), whoseexpression in root vascular tissues is enhanced by dehydra-tion. Once arrived at the leaves, it binds to the Barely AnyMeristem (BAM1 and BAM3) receptors to activate NCED3gene expression, thus promoting ABA biosynthesis anddrought stress tolerance (Takahashi et al., 2018). One canexpect that BAM1/3 receptors may phosphorylate tran-scription factors to activate NCED3 gene expression. Thesensor and signaling pathway for the activation of CLE25gene is still unknown, but the turgor pressure changes in thevascular tissues are among likely causes and this can beexamined in future studies.

Plant response to drought stress

ABA biosynthesis and transport

Under drought stress, the concentrations of ABA can in-crease up to 50-fold (Zeevaart, 1980), which is one of themost drastic changes observed thus far in the concentrationof a plant hormone responding to an environmental stimulus.

5Gong, Z., et al. Sci China Life Sci

Not surprisingly, the functions and the signaling pathways ofABA in plants’ responses to various stresses have been ex-tensively studied, and it is now well accepted that ABA playsimportant roles in plant adaptation to environmental stressesincluding drought, cold, or high salinity (Zhu, 2016). Inaddition, ABA also regulates many developmental processesthroughout the plant life cycle, including seed maturationand dormancy, seed germination, vegetative development,nodule development, and senescence (Cutler et al., 2010;Finkelstein et al., 2002; Liu et al., 2018a; Zhu, 2016).The early steps of ABA biosynthesis occur in plastids,

beginning with the isopentenyl diphosphate (IPP). The firstidentified rate-limiting enzyme in ABA biosynthesis ismaize VP14, a 9-cis-epoxycarotenoid dioxygenase(Schwartz et al., 1997; Tan et al., 1997), and its homolog inArabidopsis is NCED3 (Iuchi et al., 2001; Ruggiero et al.,2004) (Figure 1). After several enzymatic steps catalyzed bytwo important enzymes ABA deficient 1 (ABA1) and ABA4in plastids (Dall’Osto et al., 2007; Koornneef et al., 1982;Niyogi et al., 1998; Schwartz et al., 1997; Tan et al., 1997;Xiong et al., 2001), xanthoxin is finally produced by NCEDsand then moves to the cytoplasm where it is converted toactive ABA by two oxidative steps carried out by a short-chain dehydrogenase/reductase (ABA2) and ABA aldehydeoxidase (AAO3) (González-Guzmán et al., 2002; Seo et al.,2000; Xiong et al., 2001). AAO3 requires a molybdenumcofactor (MoCo) that is produced by the MoCo sulfurase(ABA3) (Xiong et al., 2001). ABA catabolism involves ABAhydroxylation catalyzed by CYP707A, resulting in 8′-hy-droxy-ABA, which is spontaneously converted to phaseicacid (PA) and further catabolized to dihydrophaseic acid(Krochko et al., 1998; Kushiro et al., 2004). PA can alsoactivate a subset of PYL ABA receptors (Weng et al., 2016).The expression of CYP707A is induced by high humidity inArabidopsis (Okamoto et al., 2009). ABA can also be con-jugated with glucosyl ester (GE) by the UDP-glucosyl-transferases, UGT71B6 (Priest et al., 2006) and UGT71C5(Liu et al., 2015), producing physiologically inactive ABA-GE that is probably a storage form in vacuoles or a trans-location form of ABA. ABA-GE can be quickly hydrolyzedto become free active ABA by β-glucosidases; two β-glu-cosidase homologs have been identified in Arabidopsis, i.e.,AtBG1, localized on the endoplasmic reticulum (Lee et al.,2006b), and AtBG2, localized to the vacuole (Xu et al.,2012). The accumulation of AtBG1 and AtBG2 is low undernormal conditions, but significantly increases under dehy-dration stress. Both atbg1 and atbg2 mutants are more sen-sitive to drought stress than the wild type (Lee et al., 2006b;Xu et al., 2012).Based on the expression patterns of the key enzymes in-

volved in ABA biosynthesis, ABA is believed to be syn-thesized in all plant tissues including vascular tissues andguard cells. However, under water-deficit conditions which

are initially detected by roots, ABA accumulates first inshoot vascular tissues, and later appears in roots and guardcells (Christmann et al., 2005), suggesting long-distancetransport of ABA for proper ABA signaling in the wholeplant (Li et al., 2018b). Consistent with this notion, a smallpeptide, CLE25, is induced by dehydration stress in roots,which moves to leaves to stimulate ABA biosynthesis andpromote stomatal closing (Takahashi et al., 2018). CLE25 isalso involved in phloem initiation in Arabidopsis (Ren et al.,2019). Arabidopsis ATP-BINDING CASSETTE G25/26(AtABCG25/26) (Kuromori et al., 2010; Park et al., 2016),AtABCG40 (Kang et al., 2010) and ABA-IMPORTINGTRANSPORTER 1 (AIT1)/NITRATE TRANSPORTER 1:2(NRT1.2)/PEPTIDE TRANSPORTER FAMILY (NPF)member (AtNPF4.6) (Ge et al., 2017), the DetoxificationEfflux Carriers 50/Multidrug and Toxic Compound Extru-sion (MATE) transporters (AtDTX50) (Zhang et al., 2014),AtABCC1/2 (Burla et al., 2013), AtABCG22 (Kuromori etal., 2011) were characterized as ABA transporters (Figure 1).AtABCG40/22 and AIT1 were shown to function as influxtransporters of ABA (Ge et al., 2017; Kang et al., 2010;Kuromori et al., 2011), whereas AtDTX50 and AtABCG25

Figure 1 ABA metabolism and transporting in plant cells. The ABAprecursor xanthoxin is synthesized in chloroplasts and transported to cy-toplasm, where it is catalyzed by two oxidative steps carried out by ABADEFICIENT 2 (ABA2) and Aldehyde oxidase 3 (ABA3/AAO3) to becomeactive ABA. ABA can be hydroxylated into 8′-hydroxy-ABA byCYP707A, which is spontaneously converted to phaseic acid (PA). TheUDP-glucosyltransferases UGT71B6 and UGT71C5 can add glucose toABA to become inactive form ABA-GE. ABA-GE can be quickly hydro-lyzed by β-glucosidase AtBG1 on ER, or transported into vacuoles, whereit can be hydrolyzed by AtBG2, to become free ABA. ABA in cytoplasmcan be transported out of cells by ATP-binding cassette transportersAtABCG25/26 or Detoxification Efflux Carriers 50 (AtDTX50); whileAtABCG22/40, ABA-IMPORTING TRANSPORTER 1 (AtAIT1)/NI-TRATE TRANSPORTER (NRT1.2) and rice OsPM1 are influx transportersof ABA.

6 Gong, Z., et al. Sci China Life Sci

act as the efflux transporters of ABA. AtABCC1/2 transportABA-GE into vacuoles (Burla et al., 2013). PLASMAMEMBRANE PROTEIN1 (OsPM1) on the plasma mem-brane is found to be involved in ABA influx in rice, and itsgene expression is regulated by the AREB/ABF familytranscription factor OsbZIP46 (Yao et al., 2018). ABAtransporters are mostly localized on the plasma membraneexcept for AtABCC1/2 that is localized on the vacuolarmembrane.

ABA signaling and transcriptional regulation for droughtstress adaptation

The first Arabidopsis mutants showing defects in ABA re-sponse, including ABA insensitive 1 (abi1), abi2 and abi3,were identified by Koornneef et al. in 1984 (Koornneef et al.,1984). After the ABI1 and ABI2 loci were cloned, they wereshown to encode group A protein phosphatase 2Cs (PP2Cs)(Leung et al., 1994; Leung et al., 1997; Meyer et al., 1994).The breakthrough of the field was made in 2009, when twoindependent studies reported that a group of PYRABACTINRESISTANCE (PYR)/PYR1-LIKE (PYL)/regulatory com-ponents of ABA receptor (RCAR) proteins, members of afamily of 14 START-domain-containing proteins in Arabi-dopsis, function as the long-sought ABA receptors (Ma et al.,2009; Park et al., 2009). The crystal structures of the ABA-bound PYR/PYL/RCAR proteins and of the PYR/PYL/RCAR-ABA-PP2C complexes were then reported soon after(Melcher et al., 2009; Miyazono et al., 2009; Nishimura etal., 2009; Santiago et al., 2009; Yin et al., 2009). The bindingaffinity of PYR/PYL/RCAR proteins to ABA was dramati-cally increased in the presence of PP2Cs (Melcher et al.,2009; Miyazono et al., 2009; Nishimura et al., 2009; San-tiago et al., 2009; Yin et al., 2009); therefore, the PP2Cs arealso regarded as ABA co-receptors. The core ABA signalingpathway has been reconstituted in vitro and in yeast cells(Fujii et al., 2009; Ruschhaupt et al., 2019).The protein kinases in the SNF1-related protein kinase 2

(SnRK2) family, particularly SnRK2.2, SnRK2.3 andSnRK2.6/Open Stomata 1 (OST1), have been shown tofunction as pivotal positive regulators of ABA signaling(Fujii et al., 2007; Fujii and Zhu, 2009; Mustilli et al., 2002;Nakashima et al., 2009) (Figure 2). It is now well acceptedthat PYR/PYL/RCAR proteins, PP2Cs and SnRK2 kinasesconstitute the core ABA signaling module that is responsiblefor the earliest events of ABA signaling. In the absence ofABA, PP2Cs are active and repress the kinase activity ofSnRK2s as well as the downstream ABA signaling events;by contrast, ABA induces the formation of PYR/PYL/RCAR-ABA-PP2C complexes, which inactivate PP2Cs, al-lowing the activation of SnRK2s and the downstream eventsof ABA signaling (Cutler et al., 2010; Qi et al., 2018; Ra-ghavendra et al., 2010; Zhu, 2016). However, SnRK2s need

to be phosphorylated by certain protein kinases to becomeactive during this process, as OST1 does not exhibit activityeven in the ABA hypersensitive quadruple mutant ear1-1abi1-2 abi2-2 hab1-1 harboring a very low phosphatase ac-tivity (Wang et al., 2018c). EAR1 binds the N-terminal re-gions of six clade A PP2Cs and increases the phosphataseactivities through releasing the inhibition of the catalyticdomains of PP2Cs by their N-terminal domains (Wang et al.,2018c). BRASSINOSTEROID INSENSITIVE2 (BIN2), oneof GSK/SHAGGY-related kinases in the brassinosteroidpathway, is inhibited by ABI1 and ABI2, while it canphosphorylate and activate SnRK2.2/2.3 in the ABA sig-naling pathway (Wang et al., 2018b). In moss, the ABA andabiotic stress-responsive Raf-like kinase (ARK), a B3 Raf-like MAP kinase kinase kinase, plays a key role in activatingSnRK2 (Saruhashi et al., 2015). In Arabidopsis, differentRaf-like kinases (RAFs) are required for ABA or osmoticstress activation of SnRK2s (Lin et al., 2020; Takahashi etal., 2020). Clade E Growth-Regulating (EGR) Type 2Cprotein phosphatase 2 (EGR2) negatively regulates plantgrowth and osmotic stress response (Bhaskara et al., 2017),perhaps partially through inhibiting OST1 activity as it doesin cold tolerance (Ding et al., 2019). RECEPTOR DEADKINASE1 (RDK1), a receptor-like kinases (RLKs) localizedon the plasma membrane, can interact with ABI1 and is apositive regulator in plant drought stress response withoutrequirement of its kinase activity (Kumar et al., 2017). Nitricoxide is found to negatively regulate OST1 and PYLs by S-nitrosylation (Castillo et al., 2015; Feng et al., 2019; Wang etal., 2015). Under unstressed conditions, the growth-pro-moting Target of Rapamycin (TOR) kinase plays a crucialrole in preventing the stress response through phosphor-ylating PYLs, which blocks the interaction of PYLs withPP2Cs (Wang et al., 2018d). Under stress, ABA-activatedSnRK2s phosphorylate Raptor, one component in TORcomplex, leading to the dissociation of TOR complex andgrowth inhibition (Wang et al., 2018d). In contrast, CARK1kinase can phosphorylate PYLs/RCARs and enhance theirinhibition of PP2Cs (Zhang et al., 2018b). The receptor-likekinase FERONIA (FER), a positive growth regulator inauxin signaling, likely activates the Rho-like small GTPaseROP11 through interacting with guanine exchange factorsGEF1, GEF4, and GEF10 (Yu et al., 2012). ROP11/10 candirectly interact with ABI1 or ABI2, which protects the in-hibition of ABI1/2 by PYLs, and increase ABI1/2 activitiesthrough a mechanism different from EAR1 (Li et al., 2012;Yu et al., 2012). Therefore, FER plays a negative role inABA signaling (Yu et al., 2012). Thus, the cross talks amongdifferent signaling pathways coordinately balance the trade-offs between plant growth and stress responses.The ABA signaling core components are highly conserved

in plants. One example supporting this notion is that themaize OST1 could complement Arabidopsis ost1 mutant

7Gong, Z., et al. Sci China Life Sci

(Wu et al., 2019), and GHR1 from rice can complement theArabidopsis ghr1 mutant (Hua et al., 2012). These corecomponents are regulated by protein post-modifications anddegradation (Figure 2). For example, ABI1 degradatioin ispromoted by PUB12/13 upon its interaction with PYLs inboth ABA-dependent and -independent manners (Kong etal., 2015); RING DOMAIN LIGASE1 and 5 (RGLG1/5)target PP2CA/ABA-hypersensitive germination3 (AHG3)for degradation (Wu et al., 2016); DET1-, DDB1-ASSO-CIATED1 (DDA1) as a substrate receptor for CULLIN4-RING E3 ubiquitin ligases (CRL4)-COP10-DET1-DDB1(CDD) complexes facilitates the proteasomal degradation ofPYL8 (Irigoyen et al., 2014). PYR1 and PYL4 degradation ismediated by the single subunit RING-type E3 ubiquitin li-gase RSL1 localized at the plasma membrane (Bueso et al.,2014); the casein kinase EL-like (AEL) phosphorylatesPYLs and promotes their ubiquitin-mediated degradation(Chen et al., 2018). The ubiquitin E2-like protein VPS23A isinvolved in vacuole-mediated degradation of PYR1/PYL4(Yu et al., 2016). The degradation of SnRK2.3 and OST1 ismediated by the F-box protein phloem protein 2-B11(AtPP2-B11) (Cheng et al., 2017) and E3-ubiquitin-ligaseHIGH EXPRESSION OF OSMOTICALLY RESPONSIVEGENES 15 (HOS15) (Ali et al., 2019a), respectively. Theseresults suggest that ABA signaling is fine-tuned at the pro-

tein level to suit the environment. Interestingly, C2-domainABA-related (CAR) proteins in Arabidopsis exhibitCa2+-dependent phospholipid binding activity, which canrecruit PYLs to membranes and positively regulate ABAsignaling (Diaz et al., 2016; Rodriguez et al., 2014). FREE1,a component of the endosomal sorting complex required fortransport machinery (ESCRT), interacts with RSL1-receptorcomplexes and recruit PYL4 to endosomal compartments fordegradation in vacuoles (Belda-Palazon et al., 2016).LOWER TEMPERATURE 1 (LOT1) physically interactswith CAR9 and increases the membrane localization andstability of CAR9 to enhance drought tolerance (Qin et al.,2019). Thus, lot1mutant plants lose more water than the wildtype under drought stress (Qin et al., 2019). In addition to theubiquitin/26S proteasome degradation pathway, the SU-MOylation pathway (including the SUMO protease ASP1and SUMO E3 ligase SIZ1) has been found to be involved inABA signaling (Wang et al., 2018e; Zhang et al., 2017a).ABI3, ABI4 and ABI5 are transcription factors (TFs) of

basic B3, AP2/ERF and bZIP families, respectively, andwere identified by genetic screens for ABA-insensitiveArabidopsis mutants (Finkelstein, 1994; Finkelstein andLynch, 2000; Finkelstein et al., 1998; Giraudat et al., 1992;Koornneef et al., 1984) (Figure 2). In response to drought,the expression of about 30% of the total genes could be

Figure 2 ABA signaling in transcription and stomatal movement. ABA signaling is initiated by the ABA receptors binding to ABA, resulting in theinteraction of ABA receptors with PP2Cs, and inhibiting their activities, which leads to activation of SnRKs or other kinases. These activated protein kinaseseither regulate the activities of transcriptional factors for controlling the expression of stress responsive genes or modulate the plasma membrane proteins inguard cells or other cells to control cell turgor. The PYL-PP2Cs-SnRK2s module is regulated by different proteins. The blue arrows indicate activating orpromoting; the red bars indicate inhibiting; P in pink circle means phosphorylation. RDK1 can recruit more ABI1 to plasma membrane, but whether RDK1can inhibit ABI1 is not known. For detailed explanation, please see text.

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altered, which is mainly achieved by the transcriptionalregulatory roles of TFs (Maruyama et al., 2014). The droughtresponsive genes can be classified into ABA-dependent andABA-independent categories. PYLs can antagonize ABA-independent activation of SnRK2s (Zhao et al., 2018b). Inthe plant kingdom, there are at least 60 different TF genefamilies. Several of them have been determined to be in-volved in ABA signaling, among which, a clade of bZIP TFs,also known as ABA-responsive element (ABRE) bindingfactors (ABFs or AREBs or DPBFs), plays central roles bybinding to the cis-element PyACGTGG/TC in the promoterof many ABA-responsive genes, such as RD29B, RD20 andRAB18 (Fujita et al., 2005; Kim et al., 2004b). Comparisonof the transcriptomic profiles of drought-responsive genes inArabidopsis, rice and soybean demonstrated that the mostconserved hexamer sequence among all drought-responsivegene promoters was the core sequence of ABRE (ACGTGG)(Maruyama et al., 2012). Protein phosphorylation is requiredfor this group of TFs to achieve their full activity. It wasshown that SnRK2s are the direct upstream activators ofABF/AREB and related to ABI3/VP1 (RAV1) proteins inABA signaling (Feng et al., 2014; Fujita et al., 2009). RAV1can directly bind to the ABI3, ABI4 and ABI5 promoters todown-regulate their expression (Feng et al., 2014). Calcium-dependent protein kinases, CPK4 and CPK11 or CPK6, wereidentified as positive regulators of ABA signaling by med-iating ABF1 and ABF4/AREB2, and ABF3 and ABI5phosphorylation, respectively (Zhang et al., 2020a; Zhu etal., 2007b). In addition, ABFs can directly bind to the pro-moters of ABA co-receptor genes, including ABI1 and ABI2,and mediate rapid induction of their expression in responseto ABA, reflecting a feedback response to avoid over-reac-tion to the ABA signal (Wang et al., 2018e). Interestingly,ABA-activated SnRK2.2/2.3 can phosphorylate membranelocalized FREE1, which then enters nuclei and interacts withABF4 and ABI5 and represses their transcriptional activity(Li et al., 2019b).Another important TF family involved in drought response

is Apetala2/Ethylene Response Factors (AP2/ERFs), in-cluding several dehydration responsive element-bindingproteins (DREBs) and ABI4 (Finkelstein et al., 1998; Liu etal., 1998; Stockinger et al., 1997). DREBs were first isolatedfrom yeast one-hybrid screens for regulators of the stress-inducible marker gene RD29A, and later studies found thatDREB1s (also known as CBFs) are more specifically in-volved in cold response (Guo et al., 2018; Liu et al., 2018b),whereas DREB2s function in drought response (Sakuma etal., 2006a). Arabidopsis DREB2A protein is regulated by26S proteasome-mediated proteolysis, and removal of itsnegative regulatory domain can transform DREB2A into amore stable form (Morimoto et al., 2013; Qin et al., 2008;Sakuma et al., 2006b). Overexpression of the stabilizedDREB2A protein uncovered several genes whose expression

was specifically activated by DREB2A, and notably, a heatshock TF (AtHsfA3) was identified as one of its target genewhen plants are challenged by heat stress (Sakuma et al.,2006b). Similarly, the maize homologous gene ZmDREB2Awas also found to regulate both drought- and heat-responsivegene expression (Qin et al., 2007).A clade of the NAM, ATAF, and CUC (NAC) family TF

genes (which is also known as SNACs), such as RD26/NAC019, NAC055 and NAC072, are highly inducible bydrought and can specifically bind the CACG core sequenceto activate the expression of many drought-responsive genes(Tran et al., 2004). In crops, OsNAC5, OsNAC6 andZmNAC111 were also found to play positive roles in plantdrought tolerance (Hu et al., 2006; Mao et al., 2015; Naka-shima et al., 2007; Takasaki et al., 2010). Several WRKYTFs, such as WRKY40, WRKY18 and WRKY60, act torepress ABI5 expression in the absence of ABA. Upon ABAor stress treatments, they move out of the nucleus whichrelieves their repression of ABI5 expression (Shang et al.,2010). ABO3/WRKY63 can directly bind the ABF2 pro-moter and control the expression of ABF2 and the down-stream genes RD29A and COR47 (Ren et al., 2010).The homeodomain TF genes, HB7 and HB12, are strongly

induced by ABA and water-deficit stress. HB7 and HB12 candirectly activate the expression of several PP2C genes andrepress the expression of ABA receptor genes PYL5 andPYL8, thus mediating a negative feedback effect on ABAsignaling in response to water-deficit (Valdés et al., 2012). Inaddition, HB33 was identified as a positive regulator of ABAresponse, and its expression was repressed by auxin responsefactor 2 (ARF2). The arf2 mutant showed enhanced ABAsensitivity in terms of seed germination and primary rootgrowth (Wang et al., 2011). Therefore, ARF2-HB33 maymodulate the crosstalk between the ABA and auxin signalingpathways (Wang et al., 2011). Additionally, Nuclear FactorY (NF-Y) transcription factors, composed of three subunits,i.e., NF-YA, NF-YB and NF-YC, can mediate drought-regulated gene expression, possibly by coordinating withDREB2A or with other unidentified components (Nelson etal., 2007; Sato et al., 2014; Sato et al., 2019; Su et al., 2018).Importantly, discovery and characterization of TFs func-tioning in transcriptional regulation in response to droughthave provided promising genetic strategies for improving thestress tolerance of crops.

ABA-mediated stomatal movement under drought stressconditions

Stomata, formed by a pair of guard cells on the leaf surfaces,are major gateways that are opened by light under normalconditions, enabling CO2 to enter into leaves for photo-synthesis, water evaporation and plant growth (Kim et al.,2010; Qi et al., 2018; Sussmilch et al., 2017). It is well

9Gong, Z., et al. Sci China Life Sci

known that light-induced stomatal opening starts with plas-ma membrane hyperpolarization by light-activated H+-AT-Pases to create a proton gradient, which stimulates a group ofinward-rectifying K+ channels localized on the plasmamembrane in guard cells, such as KAT1 and KAT2 (Kim etal., 2010; Qi et al., 2018). Stomatal closure is regulated bydifferent stresses that activate anion channels as well asoutward K+ channels, and inhibit inward-rectifying K+

channels (Kim et al., 2010; Qi et al., 2018). Guard cellsrespond quickly to drought stress through a complex mem-brane transporter system to close stomata in order to savewater for survival and increase water-use efficiency, which isdifferent from transcriptional regulation for the long-timedrought adaptation (Raghavendra et al., 2010). During thisprocess, ABA signaling regulates the plasma membranetransporters through phosphorylation by different down-stream kinases such as the SnRK2.6/OST1 kinase andCDPKs (Qi et al., 2018).One of the key plasma membrane transporters is the S-type

anion channel slow anion channel-associated1 (SLAC1),which is required for the efflux of Cl– and NO3

– from guardcells (Negi et al., 2008; Vahisalu et al., 2008). SLAC1 wasidentified through genetic screens for mutants hypersensitiveto ozone and less responsive to CO2 than the wild type bytwo research groups independently (Negi et al., 2008; Va-hisalu et al., 2008). In the slac1 mutant, the stomata are lessresponsive to environmental signals, including ABA, CO2

and H2O2 (Negi et al., 2008; Vahisalu et al., 2008). SLAC1can be activated by either phosphorylation or interactionwith other proteins likely through its conformational change.Some kinases, including OST1 (Geiger et al., 2009), CPKs/CDPKs (CPK3, 6, 21, 23) (Brandt et al., 2012; Geiger et al.,2010; Maierhofer et al., 2014; Mori et al., 2006; Saito et al.,2018; Scherzer et al., 2012), and calcineurin-B like protein 5(CBL5)–CBL-interacting protein kinase 11 (CIPK11) (Saitoet al., 2018) and CBL1/9-CIPK23 (Maierhofer et al., 2014)modules, can directly phosphorylate and activate SLAC1.Drought stress or ABA treatment increases the con-

centration of cytosolic Ca2+ which is released from in-tracellular stores (such as ER) or flows into cells throughsome plasma membrane Ca2+ transporters (Grabov and Blatt,1998; Hamilton et al., 2001; Hamilton et al., 2000; Pei et al.,2000; Schroeder and Hagiwara, 1990). The increased Ca2+

activates CPKs and CBLs (Kim et al., 2010). The SLAC1homolog, SLAC1 Homolog3 (SLAH3), is also regulated bymost of the kinases that could activate SLAC1, such asCPK3, CPK6, CPK21, CPK23, and CBL1/CBL9-CIPK23,but not by OST1 (Demir et al., 2013; Geiger et al., 2011;Maierhofer et al., 2014). GUARD CELL HYDROGENPEROXIDE-RESISTANT1 (GHR1) can directly phosphor-ylate and activate SLAC1 in an oocyte analysis system (Huaet al., 2012). However, it was recently argued that the GHR1kinase activity is not required for SLAC1 activation based on

the observation that the mutated GHR1 leading to a kinasedead form can still complement the defect in ABA-promotedstomatal closure of the ghr1 mutant and can activate SLAC1in oocytes (Sierla et al., 2018). These results suggest that theinteraction of GHR1 with SLAC1 may change the con-formation of SLAC1 and thus activate it in guard cells (Huaet al., 2012; Sierla et al., 2018). Combining the data of ge-netic and biochemical analyses, it seems that ABI1 specifi-cally inhibits OST1 while ABI2 specifically inhibits GHR1in guard cells (Hua et al., 2012; Murata et al., 2001), al-though abi1-1 and abi2-1 display similar phenotypes interms of seed germination and seedling growth (Leung et al.,1994; Leung et al., 1997; Meyer et al., 1994; Rodriguez et al.,1998). Consistently, the extracellular H2O2 produced byOST1-phosphorylated and -activated plasma membraneNADPH oxidases acts downstream of OST1, but upstream ofCa2+ and GHR1 (Han et al., 2019; Hua et al., 2012; Sir-ichandra et al., 2009). OST1 can also phosphorylate the Ser-121 of the Plasma membrane Intrinsic Protein 2;1 (PIP2;1)aquaporin, enhancing its water and possibly also H2O2

transport activity (Grondin et al., 2015). H2O2 in the cytosolaffects the activities of different enzymes (Qi et al., 2018). Inghr1 mutant, H2O2-activated Ca

2+ channel activity could notbe detected, suggesting that GHR1 regulates plasma mem-brane Ca2+ channels in guard cells (Hua et al., 2012). Simi-larly, another LRR receptor kinase HYDROGEN-PEROXIDE-INDUCED Ca2+ INCREASES1 (HPCA1) wasfound to act as a hydrogen peroxide sensor to activate Ca2+

channels in guard cells (Wu et al., 2020). Two MAP kinasesMPK9 and MPK12 are found to positively regulate stomatalclosure in ABA and H2O2 pathways (Jammes et al., 2009).The R-type anion channel, quick anion channel 1/aluminum-activated anion channel 12 (QUAC1/ALMT12), was iden-tified to facilitate the efflux of malate, chloride and nitratefrom guard cells, but are usually not required for kinaseactivation (Meyer et al., 2010; Sasaki et al., 2010). However,OST1 can further increase the activity of QUAC1 in Xenopusoocytes (Imes et al., 2013). Similar to slac1, the slah3 mu-tants are insensitive to ABA, H2O2 and CO2 in their pro-motion of stomatal closure and lose more water than the wildtype (Meyer et al., 2010; Sasaki et al., 2010).During ABA-promoted stomatal closure, ABA-activated

OST1 can phosphorylate the inward K+ channel (KAT1) andinhibit K+ influx into guard cells (Sato et al., 2009). CPKs(such as CPK33) are found to increase the activity of theoutward potassium channel GORK to negatively regulatestomatal closure (Corratgé-Faillie et al., 2017). CPK9 ne-gatively (Chen et al., 2019a), whereas CPK10 positivelyregulates ABA- and Ca2+-mediated stomatal movement un-der drought stress (Zou et al., 2010), but their direct targetsare not known yet. The activation of SLAC1 (Geiger et al.,2009), SLAH3 (Geiger et al., 2011) and QUAC1 leads toplasma membrane depolarization, which inhibits plasma

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membrane proton H+-ATPases to prevent the plasma mem-brane hyperpolarization (Qi et al., 2018). It is also reportedthat both SLAC1 and SLAH3 are able to physically interactwith KAT1 and inhibit its inward K+ channel activity (Zhanget al., 2016a), suggesting that the plasma membrane trans-porters can influence each other during stomatal movement.These results suggest that during stomatal closure induced bydrought stress, the influx channels (such as KAT1) are in-hibited, but at the same time, the efflux channels (such asSLAC1) are activated, resulting in net exit of both cationsand anions from the guard cells, which then decreases theosmotic potential and cell turgor to close stamata.Given that the vacuoles occupy the most space in a guard

cell, the change of vacuolar volume in guard cells is one ofthe most remarkable features in the regulation of stomatalmovement (Gao et al., 2005). When stomata are open, thesmall vacuoles are fused to become big ones; in contrast,when stomata are closed, the big vacuoles become smallones (Gao et al., 2005). This dynamic change of vacuolarsize is involved in the exchange of cations and anionsthrough the tonoplast membrane (Barragán et al., 2012).Maintaining the osmolyte level in vacuoles is critical forswelling and shrinkage of guard cells. In Arabidopsis, themalate concentration of guard cells plays crucial roles inosmo-regulation of guard cell turgor, which increases 2- to 3-fold during stomatal opening. A recent study indicates thatthe BI ZUI1 (BZU1), encoding an acetyl-coenzyme A syn-thetase and converting acetate to malate in peroxisomes, isimportant for maintaining the turgor of guard cells in Ara-bidopsis (Dong et al., 2018). The bzu1 mutant contained lessmalate in guard cells and displayed a reduced stomatalopening (Dong et al., 2018). In addition, ALMT9 acts asmalate-activated Cl− channel for Cl− uptake into vacuoles toregulate stomatal opening (De Angeli et al., 2013). TheArabidopsis atalmt9 mutant lost water more slowly than thewild type and displayed significantly increased drought re-sistance due to impaired stomatal opening (De Angeli et al.,2013). In contrast to ALMT9, ALMT4 is likely activated bymitogen-activated protein kinases and mediates malate ef-flux from the vacuoles during drought stress and ABAtreatment (Eisenach et al., 2017). Consistently, the atalmt4mutants lost more water and were more sensitive to droughtstress than the wild type due to impaired stomatal closure(Eisenach et al., 2017). Stomatal movement also criticallydepends on K+ homeostasis in the vacuoles. NHX-typetransporters are found to mediate K+ uptake into the vacuolesusing the proton gradient (Andrés et al., 2014; Barragán etal., 2012). Both dark- and ABA-induced stomatal closureand light-induced stomatal opening processes were impairedin the nhx1 nhx2 double mutant (Andrés et al., 2014). On thecontrary, TPK1, another tonoplast membrane K+ channel,transports K+ out of the vacuoles (Isner et al., 2018). TPK1 isphosphorylated and activated by the receptor-like kinase

KIN7, which is involved in ABA- and CO2-mediated sto-matal closure (Isner et al., 2018). These results indicate thatthe osmolyte homeostasis in the vacuoles of guard cells isclosely related to stomatal movement.

Key scientific questions that remain to be addressed fordrought stress resistance

(1) Most of the drought and ABA signaling informationhas been obtained in the laboratory, and how to apply thisknolwledge to improve the drought resistances of variouscrops is an important challenge for both crop breeders andresearch scientists.(2) To precisely evaluate the drought resistance traits of

different germplasms of major crops in the field is anotherbig challenge in this post-genomics era.(3) Thus far, most studies have been focused on drought

stress regulation of gene expression and ABA signal trans-duction. How root growth is regulated under drought stresswarrants more attention in the future, especially for cropsunder field conditions.

Salt stress tolerance

Na+ transport

When plants are exposed to high salinity in the soil, theysequentially experience two types of stresses, one is thechange in osmotic potential resulting in reduced water up-take and the other is ion toxicity due to the accumulation oftoxic ions such as Na+. The osmotic stress is a commonconsequence of water deficit and is not specific to salt stress,and the molecular mechanisms of plant osmotic stress re-sponse have been extensively reviewed above and in a paper(Yoshida et al., 2014). This part of the review is thus con-centrated on Na+ transport and accumulation in plants. Theaccumulation of Na+ is executed by the membrane trans-porters responsible for Na+ uptake, export and compart-mentation. The molecular identities of some of thesetransporters have been revealed from the research mostly inArabidopsis and recently in crops.

The SOS signaling pathway and Na+ exclusion

An important progress in the understanding of plant salttolerance in the last two decades is the identification of theSalt Overly Sensitive (SOS) genes. Through a mutant screenin Arabidopsis, several salt-hypersensitive mutants, namedsos mutants, were isolated and the molecular identities ofthese SOS genes were identified. Among these SOS genes,SOS1, SOS2 and SOS3 encode proteins that constitute theSOS signaling pathway (Zhu, 2001). SOS1 encodes a plasmamembrane Na+/H+ antiporter, which uses the H+ gradient to

11Gong, Z., et al. Sci China Life Sci

drive Na+ efflux and thus reducing cytosolic Na+ con-centration (Shi et al., 2000; Shi et al., 2003; Shi et al., 2002).SOS2 is a protein kinase and can phosphorylate SOS1 (Liu etal., 2000; Quintero et al., 2002). SOS2 is an autoinhibitorykinase under normal conditions but its autoinhibition is re-leased by the binding of SOS3 with the autoinhibitory do-main of SOS2 under salt stress conditions (Guo et al., 2001).SOS3 is a Ca2+-binding protein with three EF-hands (Liu andZhu, 1998). All three SOS genes are critical for salt tolerancein Arabidopsis because the mutants of these genes are hy-persensitive to NaCl. In the SOS signaling pathway, SOS3senses the elevated Ca2+ elicited by salt stress, and theCa2+-bound SOS3 interacts with and activates SOS2. The N-terminus of SOS3 is myristoylated and associated with theplasma membrane, and thus the SOS3-SOS2 complex isrecruited to the plasma membrane, where SOS2 phosphor-ylates SOS1. Upon phosphorylation, the Na+/H+ antiporteractivity of SOS1 is enhanced. As a result, the intracellularNa+ concentration is reduced by Na+ efflux transport throughSOS1 (Figure 3).SOS1 ubiquitously exists in all sequenced genomes as a

signal copy gene in the diploid genome. Its unique feature ofa long cytosolic C-terminal region, which contains more than700 amino acid residues in Arabidopsis (Shi et al., 2000),distinguishes it from other Na+/H+ antiporters such as NHX-type antiporters identified from different plant species. TheSOS1 C-terminal region possesses an autoinhibitory domainat the very end, which interacts with the upstream sequencescontaining a putative cNMP-binding motif to inhibit thetransport activity of SOS1 under normal conditions. Theserine residues in the autoinhibitory domain of SOS1 arephosphorylated by SOS2, which activates SOS1 upon saltstress (Quintero et al., 2011). The putative cNMP-bindingmotif in the SOS1 C-terminal region is essential for SOS1function because mutations in this motif result in dysfunc-tional SOS1 (Shi et al., 2000). This suggests that SOS1 mightbe regulated by cyclic nucleotides, which are signaling mo-lecules mediating environmental adaptation in plants(Świeżawska et al., 2018). Cyclic nucleotides have beenproposed as signaling molecules in plant response to salt andosmotic stress because their cellular levels are rapidly in-creased in response to these stresses (Shabala et al., 2015).The large C-terminal region of SOS1 resembles its mam-malian ortholog NHE1. The C-terminal part of NHE1 in-teracts with many regulatory proteins and is subjected tophosphorylation, which regulates its antiport activity (Flie-gel, 2019). The SOS1 C-terminal part also interacts withRCD1 (Katiyar-Agarwal et al., 2006), a signaling proteininteracting with many transcription factors and playingcentral roles in plant stress responses (Kragelund et al.,2012). The cellular function of SOS1 is to exclude Na+ fromthe cytosol by transporting Na+ into the extracellular space(Shi et al., 2003; Shi et al., 2002). However, in a whole plant,

many unanswered questions remain, such as the mechanismof SOS1-mediated Na+ uptake in roots, Na+ translocationfrom root to shoot, Na+ exclusion from leaf cells, and itsrelationship with other transporters like HKT-type transpor-ters in controlling these processes. It was proposed that,under mild salt stress, SOS1 might transport Na+ to the rootxylem and promote the root to shoot translocation of Na+,allowing Na+ accumulation and storage in the vacuoles ofleaf cells, which facilitates osmotic adjustment and plantgrowth under this condition (Zhu, 2016). A recent study onrice SOS1 further supports the role of SOS1 in exporting Na+

from the xylem parenchyma cells into the xylem vessels andthus promoting Na+ accumulation in the shoot for osmoticadjustment (El Mahi et al., 2019).SOS2 belongs to a family of SNF1-related kinase 3 pro-

teins (SnRK3s, also named as PKS or CIPKs) consisting of25 members, and SOS3 is one of the 10 members of the so-called SOS3-like calcium binding proteins (SCaBPs, alsoknown as CBLs) (Yang and Guo, 2018; Zhu, 2016). TheCBL-CIPK signaling network has been implicated in plantresponses to various abiotic stresses (Kudla et al., 2018;Weinl and Kudla, 2009). In addition to its interaction withSOS3, SOS2 also interacts with and phosphorylates SCaBP8under salt stress conditions, and phosphorylation of SCaBP8enhances the association of SOS2-SCaBP8 and promotes therecruitment of SOS2 to the plasma membrane, where SOS2phosphorylates SOS1 and thus enhancing Na+ efflux trans-port (Lin et al., 2009; Quan et al., 2007). The SOS2-SOS3complex functions in the root, while the SOS2-SCaBP8functions in the shoot, to confer salt tolerance by enhancingSOS1 transport activity (Lin et al., 2009; Quan et al., 2007;Yang and Guo, 2018). The SOS2-SCaBP8 complex alsofunctions in the phosphorylation and repression of the pu-tative Ca2+-permeable transporter AtANN4, which forms anegative feedback loop to fine-tune the Ca2+ signature of saltstress response (Ma et al., 2019). Moreover, SOS2 interactswith ABI2 (Ohta et al., 2003), GIGANTEA (GI) (Kim et al.,2013), and 14-3-3 (Yang et al., 2019; Zhou et al., 2014a), andthese interactions inhibit SOS2 kinase activity. The interac-tion between SOS2 and ABI2 indicates a possible interplaybetween ABA signaling and salt stress response, but howABA regulates such an interaction and thus modulatesSOS2-mediated salt stress response is not known. Under saltstress, the flowering regulator GI is degraded to releaseSOS2 from the GI-SOS2 inhibitory complex so that SOS2 isactivated to function in the salt tolerance pathway (Kim etal., 2013). This finding reveals an important molecular linkbetween salt stress response and flowering control. SOS2bound to 14-3-3 is inactive, and this interaction is enhancedby PKS5-mediated phosphorylation of SOS2. Under saltstress, elevated cytosolic Ca2+ binds the 14-3-3 protein,which promotes the binding of 14-3-3 with PKS5 to inhibitPKS5 kinase activity but decreases the interaction between

12 Gong, Z., et al. Sci China Life Sci

14-3-3 with SOS2, thereby activating SOS2 for salt tolerance(Yang et al., 2019).

The HKT-type transporters and Na+ uptake

The important role of HKT-type transporters in plant salttolerance was first evidenced in Arabidopsis by a geneticscreen for sos3 suppressors and loss-of-function mutations inthe AtHKT1 gene were found to suppress the salt hy-persensitivity of sos1, sos2, and sos3 mutants (Rus et al.,2004; Rus et al., 2001). These studies suggested that AtHKT1mediates Na+ influx in roots. Another genetic screen for Na+

over accumulation in shoots also identified mutations in theAtHKT1 gene causing hyper-accumulation of Na+ in shootsand hypersensitivity to sodium (Berthomieu et al., 2003).This study suggested that AtHKT1 is involved in recircula-tion of Na+ from shoot to root via phloem sap. Further studieson AtHKT1 have established a consensus that AtHKT1functions in root parenchyma cells to retrieve Na+ from xy-lem and thus reduce Na+ accumulation in the shoot. This issupported by the finding that salt tolerance is conferred bytissue-specific overexpression of AtHKT1 in root stele(Møller et al., 2009). Notably, AtHKT1 is also expressed inleaf vasculature (Mäser et al., 2002). However, the molecularfunctions of leaf vasculature expressed AtHKT1 need to bedetermined.Unlike Arabidopsis that encodes only one HKT gene

(HKT1), the monocot species, such as rice, wheat and barleyencode multiple copies of HKT-type transporters (Hama-moto et al., 2015). Rice was found to have seven functionalHKT-type transporters, which can be classified into twoclasses (class I and II) according to their transport activity.The ‘class I’ HTK transporters usually mediate relativelyNa+ selective transport. However, the ‘class II’ HKT trans-porters are mostly Na+/K+ symporters that co-transport Na+

and K+ (Ali et al., 2019b; Hamamoto et al., 2015). For in-stance, one wheat ‘class II’ transporter, named TaHKT2;1,can transport both Na+ and K+. Interestingly, TaHKT2;1shows a Na+-activated K+ transport activity (Rubio et al.,1995; Schachtman and Schroeder, 1994). The riceOsHKT2;1 mediates Na+ transport under K+ starvation andthus supplements the plant with Na+ as a nutritional re-placement of K+ (Horie et al., 2007). Notably, the ‘class I’transporters are evolutionarily important salt tolerance de-terminants, which has been identified as salt tolerance QTLsand natural variations in them are related to salt tolerance(Zhang and Shi, 2013). The OsHKT1;5 is located in a salt-tolerance QTL and functions in removing Na+ from the rootxylem, thus confering salt tolerance (Kobayashi et al., 2017;Ren et al., 2005). The natural variations in OsHKT1;1 affectroot Na+ accumulation and salt tolerance in rice (Campbell etal., 2017). In wheat, Nxa1 (TmHKT1;4-A2) and Nax2(TmHKT1;5-A and TaHKT1;5-D1) are two major loci con-ferring salt-tolerance. TmHKT1;4-A2 mediates Na+ se-

Figure 3 Na+ transporters and their regulators for Na+ uptake, exclusion and compartmentation. The Na+ sensing mechanism via Na+-GIPC activation of theCa2+-permeable channel MOCA1 is also shown.

13Gong, Z., et al. Sci China Life Sci

questration into the sheath, thus reducing Na+ transport toand accumulation in leaf blades (Huang et al., 2006; James etal., 2006). TmHKT1;5-A/TaHKT1;5-D1 has a similarfunction as its Arabidopsis homolog AtHKT1 to unload Na+

from the xylem of roots (Byrt et al., 2007). Importantly,integration of TmHKT1;5-A into durum wheat cultivars in-creased grain yield by 25% when cultured in saline soils(Munns et al., 2012), which highlights the potential utility ofthis gene for improving the salt-tolerance of economicallyimportant crops.The evolutionary importance of HKT-type transporters in

salt tolerance is also supported by studies in Arabidopsis.The coastal accessions with elevated shoot Na+ contentpossess an AtHKT1 allele with ~700-nucleotide deletion ofone of the tandem repeat in the AtHKT1 promoter, whichresults in decreased expression of AtHKT1 gene in roots andincreased accumulation of Na+ in shoots (Rus et al., 2006).The tandem repeat in the promoter acts as an enhancer topromote AtHKT1 expression (Baek et al., 2011). Interest-ingly, although athkt1 knockout mutants show hypersensi-tivity to salt stress, the costal allele of AtHKT1 causing lowerexpression of AtHKT1 in roots is actually a natural variationleading to salt tolerance (Balzergue et al., 2017; Rus et al.,2006). Grafting experiments suggested that the costal alleleof AtHKT1 determines salt tolerance in shoots, while theAtHKT1 allele from Col-0 drives salt tolerance in roots. Thecoastal allele of AtHKT1 is expressed at a higher level instems to retrieve Na+ from the xylem and thus limit Na+

accumulation in flowers (Balzergue et al., 2017). This studyrevealed a critical role of AtHKT1 in salt stress adaptation forplant reproduction, a most pivotal process for all living or-ganisms. A recent study using large-scale genomic analysisfurther consolidates the role of AtHKT1 in plant adaptation tosaline conditions (Busoms et al., 2018).

The NHX-type transporters and Na+ compartmentation

It has long been known that the large central vacuoles ofplant cells accumulate Na+ through a Na+/H+ exchanger ac-tivity (Blumwald and Poole, 1985). In principle, the centralvacuole is an ideal location for Na+ sequestration to reduceNa+ accumulation in the cytoplasm. Na+ accumulated in thevacuole can also serve as an osmolyte to lower the cellularwater potential and thus promote water uptake under salineconditions. In roots, several cell layers including cortex cellsand parenchyma cells with large vacuoles can accumulateNa+, which reduces the rate of Na+ entering into the rootxylem. Na+ sequestration into the vacuoles of root cells de-creases Na+ accumulation in leaves where essential processesfor plant growth such as photosynthesis occur. The Na+

translocated from root to shoot can also be stored in thevacuoles of stem cells and leaf cells, which prevents ex-cessive accumulation of Na+ in their cytoplasm. Although

the size of vacuoles limits the amount of Na+ that can beaccumulated, Na+ sequestration into the vacuoles is an ef-fective cellular mechanism to reduce cytosolic toxicity ofNa+ under salt stress especially when plants are growingsince more vacuolar space is created during plant growth.The molecular identity of the vacuolar Na+/H+ exchanger

AtNHX1 was first reported by Gaxiola and colleagues(Gaxiola et al., 1999) (Figure 3). Overexpression of AtNHX1confers salt tolerance in Arabidopsis, tomato and canola(Apse et al., 1999; Zhang and Blumwald, 2001; Zhang et al.,2001). After these initial reports, overexpression of AtNHX1in many other plants including crops has been reported toimprove salt tolerance (Zhang and Shi, 2013). AtNHX1belongs to a subfamily of cation/H+ exchangers and has fiveadditional homologs named AtNHX2–6. Topological andactivity studies of AtNHX1 revealed that the C-terminal partof AtNHX1 interacts with a calmodulin (CaM) inside thevacuole in a Ca2+-dependent manner and this interactioninhibits AtNHX1 transport activity (Yamaguchi et al., 2005;Yamaguchi et al., 2003). These studies suggest that Na+

compartmentation into vacuoles via the NHX-type Na+/H+

antiporters is somehow controlled by Ca2+ signaling throughthe Ca2+ binding protein CaM. The AtNHX1 and AtNHX2also play important roles in K+ accumulation in vacuoles andin vacuolar pH regulation. The double mutant nhx1nhx2 inArabidopsis show defects in cell expansion, plant growth andflowering, and stomatal movement (Barragán et al., 2012;Bassil et al., 2011b). These suggest that AtNHX1 andAtNHX2 have a redundant function in K+ uptake and theregulation of cellular turgor. AtNHX5 and AtNHX6 are lo-calized in the Golgi, trans-Golgi vesicles and prevacuolarcompartments, play important roles in the regulation of pH inthese compartments, and are required for protein traffickingto the vacuoles (Bassil et al., 2011a; Reguera et al., 2015).The double mutant nhx5nhx6 also displays hypersensitivityto salt stress, which is likely due to mis-trafficking of thetransporters such as AtNHX1 and the vacuolar H+ pump,AtVP1, to the tonoplast and/or inability to remove deleter-ious Na+ from the endomembrane compartments whereAtNHX5 and AtNHX6 are localized (Bassil et al., 2011a).The NHX-type Na+/H+ antiporters are clearly important inplant growth and development. Nevertheless, evidence forthe role of these transporters, especially AtNHX1, in plantsalt tolerance is also strong since AtNHX1 overexpressionenhances salt tolerance (Zhang and Shi, 2013) and dominantgain-of-function mutations in AtNHX1 can suppress the salthypersensitivity of sos1 mutants (Shi lab, unpublished data).

Other transporters

In addition to the HKT-type transporters, cyclic nucleotide-gated channels (CNGCs) have been proposed to mediate Na+

influx transport (Isayenkov and Maathuis, 2019). CNGCs

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possess the cyclic nucleotide (cNMP) binding domain and aCaM binding domain that often overlaps with the cNMPbinding domain. Binding of cNMP to CNGCs activates thechannels, while CaM senses the intracellular Ca2+ andCa2+-CaM binding to CNGCs blocks the cNMP binding sitein CNGCs and thus inhibits the transport activity of CNGCs(Duszyn et al., 2019). The finding that Ca2+ inhibits Na+

uptake in roots (Essah et al., 2003) supports that CNGCs arelikely among the major Na+ influx transporters in plants. TheArabidopsis genome contains 20 CNGCs, but the role ofthese CNGCs in Na+ influx uptake has not been well char-acterized. Interestingly, in addition to its permeability towater molecules, the Arabidopsis aquaporin AtPIP2;1 ex-hibits a non-selective cation channel activity with a pre-ference for Na+, and this transport activity is regulated byCa2+ and pH (Byrt et al., 2017). The study suggests thattransporters like this aquaporin could also function asCa2+-regulated Na+ uptake transporters in plants.Recent work on a magnesium transporter, named

OsMGT1, indicates that Mg2+ uptake and transport affectNa+ translocation and salt tolerance in rice (Chen et al.,2017b). The rice mutant osmgt1 shows high accumulation ofNa+ in shoots and displays salt hypersensitivity, which phe-notypically resembles the mutant oshkt1;5. The double mu-tants osmgt1oshkt1;5 displays a salt hypersensitivity similarto that of the single mutants, and OsHKT1;5 transport ac-tivity is enhanced by Mg2+. This study indicates thatOsHKT1;5 activity requires Mg2+, which is modulated by theMg2+ transporter OsMGT1.

Key scientific questions that remain to be addressed forsalt stress tolerance

(1) Analytical tools need to be developed to accuratelydetermine cellular and subcellular Na+ and K+ ion contents insitu. Such tools will be critical for elucidating the cell- andtissue-specific functions of the transporters important for salttolerance.(2) Core collections of germplasms of major crops with

diversity in salt tolerance are needed for identification ofnatural variations conferring salt tolerance in crops.(3) Understanding the molecular mechanism of salt blad-

der/gland formation in halophytes will help to engineer thesehighly specialized salt tolerance structures in crops in thefuture.(4) How plants sense salt stress remains a mystery.

Temperature stress

The major effects of temperature extremes on plants

Based on the temperature thresholds at which they occur, thestresses caused by temperature extremes can be divided into

cold stress, which includes freezing stress (below 0°C) andchilling stress (0–15°C), and heat stress (10–15°C aboveambient) (Wahid et al., 2007; Zhu et al., 2007a). Cold andheat stresses can have similar adverse effects on plants, in-cluding inhibition of seed germination, reduction of plantgrowth and reproduction, and a decrease in crop yield andquality. The biochemical and molecular impacts of cold andheat stresses are also similar in some aspects. First, both coldand heat can change the fluidity of cell membranes, whichmay affect the function of membrane-localized proteins totrigger downstream responses (Zhu, 2016). Second, tem-perature extremes drastically affect the activities of enzymes;for example, reactive oxygen species (ROS)-scavengingenzymes can be affected, leading to oxidative stress (Ruel-land et al., 2009; Siddiqui and Cavicchioli, 2006; Wahid etal., 2007). Third, cold or heat temperatures cause a con-siderable impact on cell physiology, such as destabilizationof protein complexes and RNA secondary structure. As aconsequence, temperature stress can have damaging effectsincluding photoinhibition and metabolic imbalances(McClung and Davis, 2010; Ruelland et al., 2009). Plantsexposed to prolonged stressful temperatures will eventuallyperish.

Cold stress responses in plants

Chilling and freezing impact plants in different ways, and asa result, chilling and freezing tolerance involves very dif-ferent mechanisms. Freezing causes ice formation, initiallyin the cell wall. As ice crystals grow in the wall, water isdrawn from inside the cell, thus causing cellular dehydrationstress. In contrast, chilling stress mainly causes injuries as-sociated with metabolic imbalance because different en-zymes in a metabolic pathway and various cellular processesare differentially inhibited by chilling temperatures. Plantsadapted to tropical regions are generally chilling sensitivebecause they may have accumulated mutations in criticalproteins that render the proteins inactive only at chillingtemperatures, so the plants can still function normally in thetropical environment. These mutations are known as tem-perature-sensitive alleles. Similarly, plants native to coldregions may accumulate heat-sensitive alleles of genes cri-tical for cellular function. These temperature-sensitive al-leles make it more challenging to decipher the molecularmechanisms of chilling and heat sensitivities of plants innature, and to improve their chilling and heat tolerance.The C-repeat binding factor/dehydration-responsive ele-

ment binding factor 1 (CBF/DREB1) pathway is a well-studied cold regulatory pathway that plays an important rolein cold acclimation in Arabidopsis, an adaptive responsewhere plants increase freezing tolerance after exposure tolow non-freezing temperatures (Jaglo-Ottosen et al., 1998;Liu et al., 1998). CBF/DREB1 comprises a small family of

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AP2/ERF transcriptional activators, which bind to CRT/DREsequences in the promoters of cold-responsive (COR) genes(Stockinger et al., 1997). Recent studies revealed that CBF-dependent cold response involves transcriptional, post-tran-scriptional and post-translational changes, expanding ourknowledge of cold stress regulatory pathways.

Transcriptional regulation

At low temperatures, plants rapidly induce the expression ofCBFs by more than a hundred-fold (Gilmour et al., 1998).Inducer of CBF expression 1 (ICE1) activates transcriptionof CBF3 by binding to MYC recognition elements in thepromoter (Chinnusamy et al., 2003) (Figure 4). The ricehomolog of ICE1, bHLH002, positively regulates cold tol-erance by promoting the expression of OsTPP1, a gene en-coding a key enzyme for trehalose biosynthesis (Zhang et al.,2017c). This finding indicates that the function of ICE1 incold response regulation may be conserved between dicotsand monocots. The expression of CBFs is repressed byMYB15, a MYB family transcriptional repressor (Agarwal etal., 2006). MYB15 interacts with ICE1 and binds to Mybrecognition sequences in the promoters of CBF genes(Agarwal et al., 2006). Recent studies indicated that calmo-dulin binding transcription activators (CAMTAs) function aspositive regulators of CBFs (Doherty et al., 2009). CAMTA3and CAMTA5 regulate CBF1 and CBF2 expression in re-sponse to a rapid temperature decrease (Kidokoro et al.,2017).There is increasing evidence that phytohormones interact

with the CBF pathway to regulate cold responses (Shi et al.,2015). CBF1 reduces bioactive gibberellin levels, whichrestrains growth and enhances freezing tolerance (Achard etal., 2008). Brassinosteoid (BR)-regulated transcription fac-tors, Brassinazole-resistant 1 (BZR1)/BRI1-EMS suppressor1 (BES1) and CESTA, activate the expression of CBFs anddownstream COR genes (Eremina et al., 2016; Li et al.,2017b). CBF gene expression is also repressed by Ethyleneinsensitive 3 (EIN3), a transcription factor that positivelyregulates ethylene-dependent gene expression (Shi et al.,2012). Jasmonate zim-domain protein 1/4 (JAZ1/4) proteins,which are repressors of the jasmonic acid (JA) signalingpathway, interact with ICE1/2 to regulate CBF expression(Hu et al., 2013). Auxin plays an important role in a “sa-crifice-for-survival” mechanism in which cold-damagedcolumella stem cell daughters are sacrificed in order for theroot as a whole to withstand chilling stress and recovergrowth (Hong et al., 2017). These findings suggest thatplants integrate hormone and cold signaling pathways forbetter adaptation to cold stress.Under normal temperatures, the expression of CBFs is

regulated by circadian clock and light. Two core componentsof circadian clock, Circadian clock-associated 1 (CCA1) and

Late elongated hypocotyl (LHY) directly bind to CBF pro-moters to positively regulate CBF expression in the earlymorning (Dong et al., 2011). During the daytime, a decreasein the red/far-red light ratio leads to increased CBF expres-sion at 16°C (Franklin and Whitelam, 2007). Phytochromeinteracting factor 3 (PIF3), PIF4 and PIF7 directly bind toCBF promoters and negatively regulate CBF expression(Jiang et al., 2017; Kidokoro et al., 2009; Lee and Thoma-show, 2012). Blue light-regulated COR27 and COR28 ne-gatively regulate CBF expression through crosstalk withCCA1 and Pseudo response regulator 5 (PRR5) (Li et al.,2016; Wang et al., 2017). Zeitlupe (ZTL), a circadian pho-toreceptor of blue light (Kim et al., 2007), interacts withheat-shock chaperone protein 90 (HSP90) to positivelyregulate CBF expression (Norén et al., 2016). Thus, thequality of light and the circadian clock are also modulators ofCBF-dependent cold response.As only 10% to 15% of COR genes are regulated by CBFs

(Jia et al., 2016; Park et al., 2015; Zhao et al., 2016), CBF-independent pathways for regulating these genes must alsoexist. Other first-wave transcription factors, such as Heatshock transcription factor C1 (HSFC1), Responsive to highlight 41 (ZAT12/RHL41), and Salt-inducible zinc finger 2(CZF1/SZF2), also modulate COR gene expression (Jia etal., 2016; Park et al., 2015; Zhao et al., 2016). Thus, thetranscription of COR genes is orchestrated by a network ofmaster transcription factors, leading to sweeping changes inthe transcriptome.

Protein modification

Cold response also involves regulation of the stability of keyproteins by post-translational modifications, includingphosphorylation, ubiquitination, sumoylation, and myr-istoylation. For example, ICE1 can be ubiquitinated by highexpression of osmotically responsive gene 1 (HOS1), aRING-finger ubiquitin E3 ligase that mediates the degrada-tion of ICE1 through the 26S proteasome pathway under coldstress (Dong et al., 2006). ICE1 is also sumoylated (andthereby stabilized) by a SUMO E3 ligase, SIZ1 (SAP/Miz)(Miura et al., 2007) in plant response to low temperature.Phosphorylation of ICE1 determines its stability and trans-activation activity under cold stress (Ding et al., 2015; Miuraet al., 2011). SnRK2.6/OST1, a member of the SNF1-relatedprotein kinase family (Mustilli et al., 2002), was the firstkinase discovered to phosphorylate ICE1. This phosphor-ylation increases ICE1 stability by antagonizing HOS1 (Dinget al., 2015). Under prolonged cold stress, the protein kinaseBIN2 interacts with and phosphorylates ICE1, facilitating theinteraction of ICE1 with HOS1 and thereby promoting ICE1degradation (Ye et al., 2019). Previously, two groups in-dependently showed that mitogen-activated protein kinase 3/6 (MPK3/6) phosphorylate and destabilize ICE1 in response

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to cold stress (Li et al., 2017a; Zhao et al., 2017). Interest-ingly, MPK3/6 and BIN2 all phosphorylate Ser94 of ICE1(Li et al., 2017a; Ye et al., 2019; Zhao et al., 2017), sug-gesting that the two types of kinases regulate ICE1 in acoordinated manner.The combined activities of HOS1, SnRK2.6/OST1,

MPK3/6, and BIN2 finely and dynamically tune the stabilityof ICE1. Under cold stress, OST1 is activated and inhibitsHOS1-mediated ICE1 degradation, rapidly inducing CBFexpression. MPK3/6 is also activated in response to cold,promoting the degradation of overaccumulated ICE1. Theactivity of BIN2 is gradually increased by cold, which pro-motes HOS1-mediated ICE1 degradation and thus attenuatesthe induction of CBF genes. This antagonistic regulation ofICE1 protein stability is important for the transient expres-

sion of CBFs; in this way, freezing tolerance is increasedwithout causing excessive suppression of plant growth.In addition to ICE1, two other substrates of OST1 were

recently identified: basic transcription factor 3 (BTF3) andBTF3L (BTF3-like). These proteins are β-subunits of anascent polypeptide-associated complex and positively reg-ulate freezing tolerance. OST1 phosphorylates BTF3 andBTF3L, facilitating their interaction with CBFs and therebystabilizing CBF proteins under cold stress (Ding et al., 2018).It is worthy to note that the kinase activity of OST1 is acti-vated by both cold and ABA. However, its activation ismodulated by two different mechanisms. In response toABA, accumulated ABA is perceived by ABA receptors,leading to releasing the inhibitiory of type 2C (clade A)protein phosphatases such as ABI1 on OST1, thereby acti-

Figure 4 Regulatory mechanisms of plant cold response. Cold signaling involves transcriptional, post-transcriptional, and post-translational events. Coldstress activates Ca2+ channels and protein kinases that are localized on the plasma membrane. Protein kinases (OST1, MPK3/6, and BIN2), E3 ligases (HOS1and PUB25/26), and SUMO E3 ligase (SIZ1) are involved in modulating the stability of the pivotal transcription factors ICE1 and MYB15 at the post-translational level. The expression of CBF genes is regulated by both positive (red) and negative (blue) regulators at the transcriptional level, and CBF proteinstability is modulated by BTFs and 14-3-3 proteins. The major components involved in CBF-independent signaling pathways, as well as epigeneticregulators, are also illustrated.

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vating OST1 (Ma et al., 2009; Park et al., 2009). Under coldstress, the activation of OST1 is regulated by clade E growth-regulating 2 (EGR2), a member of type 2C (Clade E) proteinphosphatase, which is independent of ABA and ABA re-ceptors (Ding et al., 2015; Ding et al., 2019). Therefore, it ispossible that different sensors upstream of OST1 may or-chestrate OST1 activation under different environmentalstimuli. Some central components of cold signaling can betargeted by ubiquitin ligases (E3), which is responsible fortheir polyubiquitination and degradation via 26S proteasomepathway. As mentioned above, HOS1 can ubiquitinate ICE1,thus promoting ICE1 degradation (Dong et al., 2006). Arecent study showed that CBF proteins are also subject todegradation by the 26S-proteasome pathway (Liu et al.,2017b). Under cold stress, CRPK1 phosphorylates 14-3-3proteins and triggers their translocation from the cytoplasmto the nucleus. There, the 14-3-3 proteins interact with anddestabilize CBF1/3 proteins, thus attenuating the CBFpathway and preventing an excessive cold response (Liu etal., 2017b). Two F-box proteins, EIN3-binding F-box 1/2(EBF1/2), positively regulate CBF expression by mediatingthe degradation of EIN3 and PIF3 via the 26S proteasomepathway (Jiang et al., 2017; Shi et al., 2012). Two U-box typeE3 ubiquitin ligases, PUB25 and PUB26, trigger cold-in-duced degradation of MYB15 to positively regulate freezingtolerance. Furthermore, cold-activated OST1 phosphorylatesPUB25 and PUB26, which enhances their ligase activity(Wang et al., 2019).

Post-transcriptional regulation

The components involved in post-transcriptional regulation,including alternative splicing, RNA export and pre-mRNAprocessing play important roles in cold response. For example,cold represses the alternative transcript ofCCA1, resulting in ahigh level of CCA1α (the full-length transcript of CCA1) thatis necessary for CBF induction by cold. Also, cold-repressedCCA1β (alternative form of CCA1) interacts with CCA1α toinhibit the DNA binding activity of CCA1α (Seo et al., 2012).A DEAD-box RNA helicase, named low expression of os-motically responsive genes 4 (LOS4) is important for nuclearmRNA export and participates in the response to cold stress(Gong et al., 2005). Regulator of CBF gene expression 1(RCF1), a pre-mRNA splicing factor, is essential for theproper splicing of COR pre-mRNAs under cold stress (Guanet al., 2013b). Another pre-mRNA splicing factor, Stabilized 1(STA1), is upregulated by cold stress and required for mRNAprocessing of COR genes (Lee et al., 2006a).

Epigenetic regulation

Cold stress can induce changes in chromatin structure andthereby affect gene expression (Kim et al., 2015). Increasing

evidence indicates that epigenetic regulators, especiallyhistone deacetylases, participate in the transcriptional reg-ulation of COR genes. In Arabidopsis, the histone deacety-lase HDA6 is required for cold acclimation and freezingtolerance (To et al., 2011). Histone acetyltransferase GCN5positively regulates freezing tolerance in Arabidopsis (Vla-chonasios et al., 2003). FVE functions as a component of ahistone deacetylase (HDAC) complex to regulate cold re-sponses (Kim et al., 2004a). HOS15, a WD40-repeat do-main-containing protein, associates with HD2Cs to regulatecold tolerance through histone deacetylation (Park et al.,2018; Zhu et al., 2008). The RNA-directed DNAmethylation4 (RDM4) is important for Pol II-mediated transcription ofCBFs and for cold tolerance in Arabidopsis (Chan et al.,2016). Long noncoding RNAs (lncRNAs) are a set of RNAsthat do not encode proteins (Collins et al., 2011). Recently,SVALKA, a cold-responsive lncRNA located near the CBF1locus, was shown to repress CBF1 expression and freezingtolerance (Kindgren et al., 2018). These findings demon-strate that epigenetic regulation is an important mechanismfor plant responses to cold stress. Nevertheless, many mo-lecular details remain unknown.

The heat stress response in plants

Once plants sense high temperatures, they immediately in-itiate a series of defense mechanisms—the heat stress re-sponse (HSR)—that protects against the damage caused byheat stress. Current knowledge of plant response to heatstress, mostly obtained from studies with Arabidopsis, issummarized below.

The function of HsfA1s in plant tolerance to heat stress

The heat stress transcription factors (Hsfs) belong to a familyof transcription factors conserved throughout eukaryotes,and they bind to downstream genes encoding transcriptionfactors, enzymes, and chaperone proteins (Ohama et al.,2017). Heat stress transcription factor A1s (HsfA1s) appearto be critical “master regulators” of thermotolerance (Figure5) (Liu et al., 2011; Mishra et al., 2002; Nishizawa-Yokoi etal., 2011; Yoshida et al., 2011). HsfA1s activate the ex-pression of heat stress-responsive genes, including HsfA7s,HsfA2, HsfBs,Multiprotein bridging factor 1c (MBF1c), andDREB2A, which in turn modulate the synthesis of chaper-ones and enzymes involved in degradation of unfoldedproteins and in ROS scavenging (Ohama et al., 2017;Yoshida et al., 2011). For example, HsfA1 induces miR398,which targets ROS-scavenging genes. The resulting ROSoverproduction, in turn, activates HsfA1 expression (Guan etal., 2013a). In addition, a recent study showed that inchloroplasts, tocopherols (Vitamin E) positively regulate thebiogenesis of miRNAs such as miR398 and promote ther-

18 Gong, Z., et al. Sci China Life Sci

motolerance (Fang et al., 2019). HsfA1s also regulate theexpression of ONSEN, a copia-like retrotransposon, and heatstress memory, a process where plants memorize a prior mildheat exposure and acquire the ability to tolerate further se-vere heat stress (Cavrak et al., 2014; Crisp et al., 2016; Se-daghatmehr et al., 2019). DREB2A is an important regulatorin Arabidopsis thermotolerance regulated by transcriptionfactors other than HsfA1s, including Jungerun-nen1 (JUB1),Growth regulating factor 7 (GRF7), and MBF1c (Kim et al.,2012; Suzuki et al., 2011; Wu et al., 2012). Furthermore,DNA polymerase II subunit B3-1 (DPB3-1) and Nuclearfactor Y (NF-YB3) are induced by heat stress, and NF-YB3protein is translocated from the cytosol into the nucleusunder heat stress. DPB3-1, NF-YB3, and NF-YA2 form atrimer and promote DREB2A activity (Sato et al., 2014). Theamount of active HsfA1s is tightly regulated by high tem-perature. The expression of HsfA1s is regulated by heat-re-sponsive transcription factors, including HsfBs and NAC019(Guan et al., 2014; Ikeda et al., 2011). The DNA bindingactivity of HsfA1a is negatively regulated by the proteinkinase Cyclin-dependent kinase CDC2 and positively regu-lated by the protein kinase CBK3 (Liu et al., 2008; Reindl etal., 1997). PP7, a calmodulin-binding protein phosphatase,interacts with HsfA1a, suggesting that PP7 may modulateHsfA1a activity (Liu et al., 2007). Sumoylation was shownto inhibit HsfA2 activity (Cohen-Peer et al., 2010; Rytz et al.,2018). Moreover, HsfA1d, HsfA2, and HsfB2b were foundto be sumoylated in a SUMO proteomics analysis (Miller etal., 2010). Thus, it is possible that HsfA1 activity is regulatedby sumoylation under heat stress conditions. Heat shockprotein 70 (HSP70) and HSP90 interact with HsfA1 andrepress its transactivation activity and nuclear localization,thus inhibiting HsfA1 activity under normal conditions.Upon heat stress, HsfA1 is released from HSP70/90 and isthus activated (Hahn et al., 2011; Yamada et al., 2007).In addition to the HsfA1-dependent pathways, emerging

evidence suggests that some genes regulate thermotoleranceindependently of HsfA1s. For example, bZIP28, encoding anendoplasmic reticulum (ER) membrane-localized protein, isinduced by heat stress (Gao et al., 2008). Under unstressedconditions, the ER-localized chaperone protein BiP3 binds tobZIP28 and retains it in the ER. Heat stress appears to inducethe proteolytic cleavage of bZIP28, which then moves fromthe ER to the nucleus and induces heat stress-responsivegene expression (Gao et al., 2008; Sun et al., 2013). bZIP28,along with bZIP60, also regulates the heat response duringthe reproductive stage in Arabidopsis (Zhang et al., 2017b).Other HsfA transcription factors, including HsfA5, HsfA4,and HsfA9, modulate the expression of heat stress-re-sponsive genes in an HsfA1-independent manner (Baniwalet al., 2007; Kotak et al., 2007b; von Koskull-Döring et al.,2007). HsfA5 forms a dimer with HsfA4 and represses thelatter’s activity (Baniwal et al., 2007). More recently, the

circadian clock proteins Reveille4/8 (REV4/8) were re-ported to mediate expression of the first wave of heatshock-induced genes (e.g., Ethylene responsive factor 53/54, ERF53/54) and to positively regulate thermotolerancein Arabidopsis (Li et al., 2019a). Despite these findings,little is known about other potential components of HsfA1-independent pathways.

Protein homeostasis

High temperatures can cause accumulation of unfoldedproteins, which are toxic to plant cells. To survive heat stress,plants must renature or degrade these unfolded proteins. Aspart of the heat response, HSF transcription factors rapidlyinduce the expression of HSPs. The molecular chaperonesencoded by these genes play important roles in maintainingprotein quality by renaturing denatured proteins under heatstress conditions (Kotak et al., 2007a). In Arabidopsis,HSP100, HSP90, HSP70, HSP60, and small HSPs (sHSPs)have been shown to function in thermotolerance (Kotak etal., 2007a). HSP100 is involved in resolubilizing proteinaggregates during heat stress (Lee et al., 2007). sHSP21 isimportant for chloroplast development under heat stress(Zhong et al., 2013). In rice, OsHSP101 is a positive reg-ulator of long-term acquired thermotolerance (Lin et al.,2014). Aside from the general molecular chaperone activityof HSPs, their specific functions and targets remain elusive.Other cellular components that deal with denatured proteinsare also induced by heat stress. In rice, a major quantitativetrait locus Thermotolerance 1 (TT1) was cloned from Afri-can rice (Oryza glaberrima). TT1 encodes a 26S proteasomeα2 subunit that enhances the degradation efficiency of pro-teasomes, thereby facilitating the elimination and recyclingof cytotoxic denatured proteins (Li et al., 2015).Autophagy is an intracellular quality control system that

removes nonfunctional proteins and damaged cellular com-ponents. This homeostatic pathway is important for the en-ergy balance of plant cells (Liu and Bassham, 2012).Emerging evidence shows that autophagy is involved in plantresponses to various biotic and abiotic stresses, includingheat stress. Impairment of autophagy in Arabidopsis andtomato causes aggregated proteins to accumulate, leading todecreased heat stress tolerance (Zhou et al., 2013; Zhou etal., 2014b). A recent study showed that autophagy resetscellular memory of heat stress in Arabidopsis. Autophagy isinduced by thermopriming and remains high during ther-morecovery, resulting in the degradation of thermomemory-associated HSPs at the later stages of the thermorecoveryphase (Sedaghatmehr et al., 2019). Although limited pro-gress has been made in understanding the regulation ofprotein homeostasis during heat stress, this topic meritsfurther study.

19Gong, Z., et al. Sci China Life Sci

ROS homeostasis

ROS, such as H2O2, O2–, and 1O2, are produced in chlor-

oplasts and mitochondria during heat stress. Because ROSare produced soon after the onset of heat stress, they functionas early messengers to activate the stress response (Banti etal., 2010; Königshofer et al., 2008; Volkov et al., 2006).Plants treated with H2O2 exhibit enhanced thermotolerance,and mutations of ATRBOHB and ATRBOHD, which encodeNADPH oxidases involved in ROS production, cause defectsin thermotolerance (Larkindale et al., 2005; Larkindale andHuang, 2004). By contrast, excessive ROS are harmful andcan result in cell death. Therefore, ROS homeostasis istightly controlled in plants.Ascorbate peroxidases (APXs) and catalases (CATs) are

two types of ROS-scavenging enzymes that are necessary fordetoxification of ROS (Baxter et al., 2014). Mutation ofAPX2 or CAT2 causes decreased heat stress tolerance inArabidopsis (Vanderauwera et al., 2011). ZAT12, a zincfinger protein, is required for expression of APXs and re-sponds to heat stress, along with ZAT family members ZAT7and ZAT10 (Davletova et al., 2005; Rizhsky et al., 2004).

Copper/zinc superoxide dismutase 1 (CSD1), CSD2, andCopper chaperone for SOD1 (CCS) are key ROS scavengersand regulate thermotolerance in Arabidopsis (Guan et al.,2013a). In rice, the transcription factor SNAC3 is induced byheat stress and prevents damage caused by ROS by activat-ing the expression of ROS-scavenging genes (Fang et al.,2015). OsANN1 is a calcium-binding annexin protein thatcan promote the activities of superoxide dismutase and cat-alase, thereby enhancing rice thermotolerance (Qiao et al.,2015). The RING finger E3 ligase OsHTAS confers heattolerance by maintaining H2O2 homeostasis (Liu et al.,2016). These results suggest that plants acquire thermo-tolerance by activating the ROS scavenging system.

Key scientific questions that remain to be addressed fortemperature stress tolerance

(1) How cold stress regulates CAMTA activity is un-known, and the connections between CAMTAs and Ca2+

signaling and other cold-sensing components require furtherstudies.(2) Ubiquitination is an important mechanism for reg-

Figure 5 Regulatory network of the plant response to heat stress. Heat stress changes membrane fluidity, which may be sensed by proteins, such as Ca2+

channels and receptor-like kinases, localized at the plasma membrane. HsfA1 transcription factors are master regulators of plant tolerance to heat stress. Theactivity of HsfA1s is repressed by HSP70/90 under unstressed conditions. HsfA1s are activated by heat, and they target downstream transcription factors(such as DREB2A and Hsfs), microRNAs, and ONSEN to induce the expression of heat stress-responsive genes, which are important for ROS scavenging,protein homeostasis, and heat stress memory. In addition, some genes, including bZIP28, HsfAs, and REV4/8, regulate the heat stress response independentlyof HsfA1s.

20 Gong, Z., et al. Sci China Life Sci

ulating plant cold responses. Additional E3 ubiquitin ligasesinvolved in cold stress regulation await identification.(3) The molecular details of epigenetic regulation for plant

responses to cold stress require further studies.(4) Additional components of HsfA1-independent path-

ways regulating plant thermotolerance need to be identified.(5) The biological function of autophage in protein

homeostasis during heat stress merits further studies.

Molecular mechanisms of plant response to heavymetal stress

In a broad sense, heavy metals are defined as metals withdensities higher than 5 g cm–3. According to this definition,53 naturally occurring metals have been classified as heavymetals. At high concentrations, heavy metals are all toxic toplants, but some of them, e.g., zinc (Zn), iron (Fe), andcopper (Cu), are essential nutrients at low concentrations,and some others, e.g., cadmium (Cd), chromium (Cr) andlead (Pb), are harmful to plants even at very low con-centrations. Those heavy metals exhibiting toxicity to plantsat low concentrations are commonly referred to as heavymetals. In addition, arsenic (As) is as toxic as heavy metals toplants and other organisms, so it is also commonly con-sidered as a heavy metal or metalloid (Bothe, 2011).

Cadmium

Cadmium is considered to be one of the most dangerousheavy metal elements to human health. Excessive accumu-lation of Cd in foods will increase the risk of cancer, kidneytoxicity and other diseases (Amzal et al., 2009; Jablonska etal., 2017; Larsson et al., 2015). Cd is transferred from the soilto the root, and then transported over long distances to theshoots in plants. The uptake of Cd by plant roots is generallythrough transporter proteins evolved for the uptake of es-sential elements with similar chemical properties to Cd, suchas Fe, Zn and Mn (Clemens et al., 2013). Taking rice for aninstance, the uptake of Cd is predominantly mediated byOsNRAMP5 (Figure 6), an important plasma membrane-localized transporter for Mn, Fe and Zn. Knockout of OsN-RAMP5 could decrease grain Cd concentrations by up to90%, though Mn, Fe and Zn were also significantly de-creased (Ishikawa et al., 2012; Sasaki et al., 2012). The re-markable decrease of grain Cd by knockout of OsNRAMP5was soon used for generating low Cd rice by the CRISPR/CAS9 system, although whether such mutations result inyield loss for all varieties remains unclear (Ishikawa et al.,2012; Sasaki et al., 2012; Tang et al., 2017). OsCd1 is an-other transporter in Cd uptake, which belongs to major fa-cilitator superfamily (Yan et al., 2019). Compared with wild-type rice plants, the Cd concentration in the shoots and roots

of oscd1 mutant was significantly lower under Cd treatment.Interestingly, OsCd1 is diverged between indica and japonicasubspecies, and the indica allele OsCd1V449 reduces thegrain Cd contents by ~50% on average compared with thejaponica allele OsCd1D449, suggesting a potential for mar-ker-assisted selection of low Cd rice (Yan et al., 2019).After it enters root cells, Cd is unloaded to the xylem for

long-distance transport to the shoot by some heavy metalATPases, which normally function as Zn efflux transporters.In Arabidopsis, Cd unloading to the xylem of roots is ful-filled by AtHMA2 and AtHMA4 (Hussain et al., 2004), andloss of function of these two proteins could result in a 98%decrease of Cd transportation from root to shoot (Wong andCobbett, 2009). A Cd hyperaccumulator, Arabidopsis hel-leri, accumulates high concentrations of Cd through over-expression of AhHMA4, an AtHMA4 ortholog, as a result ofcis-element changes and gene triplication, which representadaptive genetic variations for local adaptation (Hanikenneet al., 2008). In rice, long-distance transportation of Cd isalso mediated by a single AtHMA4 ortholog, OsHMA2(Nocito et al., 2011; Satoh-Nagasawa et al., 2012).Compartmentalization is another strategy for limiting

toxicity of Cd. HMA3, a tonoplast transporter, plays a keyrole in the sequestration of cadmium into vacuoles and inlimiting translocation from the roots to shoots in many plantspecies. Loss-of-function of HMA3 either in Arabidopsis orrice results in significantly increased Cd concentration inshoots (Hussain et al., 2004; Yan et al., 2016), while over-expression of OsHMA3 causes remarkable decreases in Cdaccumulation in rice grains (Sasaki et al., 2014). Interest-ingly, natural variations of shoot Cd concentration werefound to be controlled by HMA3s in many plant species. InArabidopsis, 10 major haplotypes of AtHMA3were observedwith five active haplotypes contributing to low leaf Cd andfive inactive haplotypes contributing to high leaf Cd (Chao etal., 2012). In rice, the polymorphisms in OsHMA3 promoterwere also found to drive natural variation of grain Cd ac-cumulation between Indica and Japonica (Liu et al., 2020).Furthermore, in the cadmium/zinc hyperaccumulator Sedumplumbizincicola, SpHMA3 was revealed as a key detox-ification gene which improves the development of youngleaves of plants grown in Cd-contaminated soils (Liu et al.,2017a). Metal chelators also play crucial roles for the com-partmentalization of Cd in vacuoles or cell walls and redu-cing toxicity of Cd on essential organelles or macromol-ecules. Recently, a defensin-like protein, Cadmium Accu-mulation in Leaf 1 (CAL1), was identified to play an im-portant role in restricting long-distance Cd transport of rice(Luo et al., 2018). CAL1 encodes a peptide localized on cellwalls and is predominantly expressed in root exodermis andxylem parenchyma cells. It is capable of chelating Cd in thecytosol and can be secreted to the cell wall apoplast andxylem. Mutants of OsCAL1 are more sensitive to Cd stress

21Gong, Z., et al. Sci China Life Sci

and have significantly reduced concentrations of cadmium inrice leaves compared with wild-type plants (Luo et al.,2018).Moreover, some phytohormones, such as Jasmonic acid

(JA), abscisic acid (ABA), and salicylic acid (SA), are alsothought to be involved in plant responses to Cd stress (Fan etal., 2014; Guo et al., 2016; Lei et al., 2020; Tao et al., 2019).Lei et al. (Lei et al., 2020) revealed that Cd-induced JArestricts Cd accumulation and lessens Cd toxicity in Arabi-dopsis through a JA signaling pathway. Exogenous MeJAlargely decreased the Cd concentration in shoot and root cellsap, alleviating Cd toxicity in the shoots. Whether otherphytohormones are involved in Cd detoxification or adap-tation remains an open question.

Arsenic

Arsenic is another typical heavy metal that could cause se-vere damages to plants and human health. But unlike Cdwhich exists in soils predominantly as a divalent cation, Asexists in soil in many isoforms, such as arsenate (As(V)),arsenite (As(III)) and organic arsenic forms. Therefore,plants have evolved different mechanisms for dealing withdifferent arsenic species. Among the various arsenic species,the two inorganic arsenic forms, As(III) and As(V), havebeen studied the most, and the mechanisms of detoxificationof these two arsenic species are relatively better understood.As(V) can be readily taken up and transported by plant

roots via phosphate transporters as it is chemically similar tophosphate, while As(III) is mainly ingested by plants viasilicic acid transporters and aquaporin channels (Cao et al.,2019; Ma et al., 2008; Wang et al., 2016; Xu et al., 2015). In

rice, OsPT4 and OsPT8 were found to contribute to bothphosphate and As(V) uptake and transport, and their mutantsare resistant to As(V) than wild-type plants (Wang et al.,2016; Ye et al., 2017). In Arabidopsis, Nodulin-26-like in-trinsic proteins (NIPs) were widely found to be involved inAs(III) uptake and transport. For example, the loss-of-function mutants of NIP1;1 and NIP3;1 both display As(III)tolerance and accumulate less As in shoots compared withwild-type plants (Kamiya et al., 2009; Xu et al., 2015).NIP7;1 was also found to affect As(III) uptake and seed Asdeposition by hindering As long-distance transport (Lindsayand Maathuis, 2016). Arsenite uptake and transport in rice isalso mediated by aquaporin channels, such as Lsi1 and Os-NIP3;2 (Chen et al., 2017a; Ma et al., 2008).After being taken up, As(V) is often reduced to As(III)

with the help of As reductase in plants (Chao et al., 2014;Sánchez-Bermejo et al., 2014; Shi et al., 2016; Xu et al.,2017). By using the genome-wide association approach,Chao et al. (Chao et al., 2014) identified a gene namedHAC1that controls natural variation in leaf arsenic contents inworld-wide A. thaliana accessions, and they uncovered thatthis gene encodes a previously uncharacterized arsenate re-ductase. HAC1 is mainly expressed in root hairs, epidermis,and pericycle cells of roots. The reduction of As(V) to As(III) by HAC1 facilities efflux of arsenic from root to soil andthus the presence of HAC1 reduces As accumulation inplants (Chao et al., 2014). Almost at the same time, anotherallele of HAC1 named ATQ1 was also identified by an in-dependent group based on arsenate resistance (Sánchez-Bermejo et al., 2014). In rice, the HAC1 orthologs OsH-AC1;1, OsHAC1;2 and OsHAC4 also function as As(V)reductases and control As accumulation. Similar to HAC1,

Figure 6 The detoxification system for heavy metals in plants. Different molecules are presented in different shapes and colors as indicated. Arrows showthe flow of the small molecules.

22 Gong, Z., et al. Sci China Life Sci

OsHAC1;1,OsHAC1;2 andOsHAC4 are highly expressed inepidermis and are induced by arsenate exposure. Over-expression of either OsHAC1;1 or OsHAC1;2 reduced Asaccumulation and enhanced As(V) tolerance, suggesting apotential use of these genes in engineering low As rice (Shi etal., 2016; Xu et al., 2017). Recently, a new arsenate reductasegene was identified in the arsenic hyperaccumulating fernPteris vittata, which was named Glutathiones-Transferase(PvGSTF1). This gene together with another two genes,PvGAPC1 and PvOCT4, were reported to regulate arsenictolerance in a bacterial-like mechanism (Cai et al., 2019).As (III) can also be compartmentalized in vacuoles after it

is chelated by thiol compounds. Such compartmentalizationis important for further As detoxification and for decreasingthe translocation of As from roots to shoots. ABCC-typetransporters, i.e., ABCC1 and ABCC2 in A. thaliana, werefirst identified as phytochelatin transporters that function insequestering As–PC into the vacuole (Song et al., 2010).Their orthologs in rice were found to play a similar role.Knockout of OsABCC1 resulted in increased As sensitivityand higher As content in rice grains (Song et al., 2014). Thephytochelatin synthase OsPCS1 also plays a crucial role inreducing arsenic levels in rice grains, as OsABCC1 pre-ferentially cooperates with OsPCS1 to sequester As (Hayashiet al., 2017). Moreover, Arsenic Compounds Resistance 3(ACR3), an As(III) antiporter, may also mediate As(III) se-questration in plants. In P. vittata, PvACR3 and PvACR3;1are both localized on tonoplast membranes and function insequestrating As(III) into the vacuole (Wang et al., 2018a).Additionally, ectopic expression of PvACR3;1 in rice notonly led to lower As in the shoots, but also reduced Asaccumulation in the grains under As exposure, which was ofsignificance to engineering new low-As rice (Chen et al.,2019b).

Key scientific questions that remain to be addressed forheavy metal tolerance

(1) Are there specific sensors in plants for different heavymetals? If yes, what are they?(2) How do we limit heavy metal accumulation in crops

without reducing the uptake of essential mineral nutrients (e.g., P, Fe, Zn and Mn)?

Plant nutrient use efficiency

Dynamic nutrient status impacted by varied crop cultiva-tion and climate change

Crop nutrient status has been generally evaluated by mea-suring the content of various nutrients in the plant and byobserving nutrient-specific symptoms (Dobermann andFairhurst, 2000). However, more accurate and sensitive

strategies are needed to assess nutrient status and its effect onplant development and yield under the highly dynamic soiland environmental conditions in the field. For instance,color-based visual reporter systems driven by promoters re-sponsive to specific nutrients could be used for monitoringthe nutrient status by the naked eye and for accurate asses-sement by spectral reflectance (Li et al., 2014). Co-expres-sion analysis of comprehensive transcriptome data of cropplant grown under field conditions could allow the identifi-cation of regulatory networks that respond to specific nu-trient deficiencies and the dynamics of nutrient availabilityinside the plant at different stages of development (Takehisaand Sato, 2019). From these networks, indicator gene setsthat allow for more accurate examination of the status ofspecific nutrients can be identified and used to monitor nu-trient status throughout the growth cycle of crops under fieldconditions (Takehisa and Sato, 2019). Ionomic analysis isalso an important technology to follow nutrient status ofplants during their growth cycle in the field. Most often thestatus of single elements has been used to explore geneticdiversity in a given plant species to identify QTLs involvedin nutrient uptake and utilization (Baxter, 2015). However,given the large amount of data generated by ionomic ana-lysis, it should be possible to search for complex QTLs thatinfluence more than one element and that may appear asminor or undetectable QTLs if one single element is con-sidered in the analysis (Baxter, 2015). Ionomics togetherwith indicator genes could provide a molecular frameworkfor further elucidating the dynamics of nutrient requirementsthroughout the growth of crop plants in field conditions andfor designing more precise breeding strategies and moreeffective fertilization schemes.It will also be important to determine how much the cli-

mate change will directly or indirectly impact plant nutrition.For instance, it has been shown that nitrogen content declinesin plants grown at elevated CO2 concentrations (Taub andWang, 2008). This might be due to an apparent misconcep-tion that the photorespiratory pathway in most plants dis-sipates over 30% of the photosynthate as waste heat and thatthis futile cycle has continued as an evolutionary dead-endeven in the absence of a selective pressure to retain thispathway in plants. It has recently been proposed that pho-torespiration stimulates the production of malate in chlor-oplasts and generates reductants for nitrate assimilation(Busch et al., 2018). Moreover, binding of Mn instead of Mgto Rubisco could drive a photorespiratory pathway that in-creases the energetic efficiency of photosynthesis (Bloomand Lancaster, 2018). Therefore, photorespiration makesplants particularly suited to use soil nitrate as a nitrogensource, as a few other organisms which can afford the energyto assimilate this low-energy inorganic nitrogen form intoamino acids or other organic forms (Eisenhut et al., 2019).How an increase in atmospheric CO2 will impact photo-

23Gong, Z., et al. Sci China Life Sci

respiration and N assimilation, particularly in ammonium ornitrate preferred C3 plants, remains to be determined.Recently, it has been shown that the rice GROWTH-

REGULATING FACTOR 4 (GRF4) and the growth inhibitorDELLA, characteristic of the green revolution varieties wereshown to have opposite roles in regulating growth, andcarbon and nitrogen metabolism by physical interaction (Liet al., 2018a). In addition, miR396ef-GRF4/6/8-GIF1/2/3modules have been shown to regulate rice seed and panicledevelopment, making this miRNA a promising target forbreeding rice varieties that require less nitrogen fertilizers(Zhang et al., 2020b). Thus, modulation of plant growth andmetabolic co-regulation may enable novel breeding strate-gies for sustainable agriculture.

Root-microbiota association in favor of nutrient acquisi-tion

An increasing amount of information has placed the asso-ciation of plants with soil microorganism as a major playerfavoring their tolerance to abiotic and biotic stress. Asso-ciation with beneficial microbes enhances nutrient uptakeand assimilation, and increases resistance to pests and dis-eases, drought, and salinity (Compant et al., 2019). However,the mechanisms of perception and signaling of nutrientavailability by associating with specific sets of microbesremain to be determined. Interestingly, it has been recentlyshown that key regulators of nutrient sensing pathways alsoplay an important role in defining the integration of themicrobiomes that associate with plants. For instance, it wasreported that association with microbes can enhance thephosphate starvation response (PSR) in Arabidopsis by di-rectly enhancing the activity of PHR1, the master regulatorof the PSR response (Bustos et al., 2010; Rubio et al., 2001).Meanwhile, mutations in PHR1 influence root microbiomecomposition in plants grown in a phosphate (Pi)-repleted soil(Castrillo et al., 2017). These authors also found that PHR1 isa direct regulator of a relevant set of jasmonic acid-re-sponsive plant immune system genes in Pi-deprived plants(Castrillo et al., 2017). Therefore, PSR and immune systemoutputs are directly integrated by PHR1. Moreover, it wasalso reported that Arabidopsis lines carrying mutations in thephosphate signaling network genes exhibited altered rootfungal communities characterized by the depletion of thechytridiomycete taxon Olpidium brassicae specifically un-der P-replete conditions (Fabiańska et al., 2019). These re-sults suggest that nutrient availability in the soil, as well asgenes involved in the nutrient signaling pathways, playsimportant roles in determining the association with soil mi-crobes. In another study, the superior nitrogen use efficiencyof Indica rice is related to the association with specific mi-crobiomes (Zhang et al., 2019). Indica-enriched bacterialtaxa were found to be more diverse, which contains more

genera with nitrogen metabolism functions. Using associa-tion and genetic approaches, the authors provided evidencethat NRT1.1B, known as a nitrate sensor/transporter, is as-sociated with the selective recruitment of bacterial taxa inIndica rice (Zhang et al., 2019). Since natural variation inNRT1.1B explained only 29% of the differential abundancein root microbiota between Indica and Japonica rice, it willbe interesting to identify additional key genes and pathwaysinvolved in the selective recruitment of microbiomes that canfavor rice performance under field conditions. By using acombination of a novel approach that measures microbialsulfatase activity in the soil and a genome-wide associationanalysis, the authors demonstrated that camalexin productionis required for the establishment of plant microbio interac-tion in the root (Koprivova et al., 2019). Two alleles of theCYP71A27 cytochrome P450 were identified among Ara-bidopsis accessions that differed in camalexin synthesis andfresh weight gain in response to Pseudomonas sp. CH267(Koprivova et al., 2019).Assessments of leaf and shoot microbiomes have been

made for an increasing number of plant species includingArabidopsis, rice, barley, grapevine, lettuce, potato, tomato,sugarcane and several tree species. However, the use ofdifferent sampling protocols, primers, and sequencing pipe-lines makes it difficult for a direct comparison of the resultsobtained by different research groups. Nevertheless, thesestudies conclusively demonstrated that the plant bacterialmicrobiome is composed of a few dominant phyla, mainlyProteobacteria, Actinobacteria, and Bacteroidetes, and to alesser extent, Firmicutes. These topics, as well as an in-depthdescription of the functional adaptation of the microbiome tothe plant environment, the driving factors that orchestrate theestablishment of plant microbiomes and the importance ofthe microbiota on plant fitness have been presented in a re-view by Muller and colleagues (Müller et al., 2016). Morerecently, it was shown in Arabidopsis that although the leafand root microbiomes are specialized in their respective ni-ches, there is a taxonomic and genome-encoded functionalcapability overlap between the microbiomes of the two or-gans (Bai et al., 2015). Moreover, the microbiome seems tobe highly dynamic during the plant growth cycle; for in-stance, the rice root microbiome is highly variable duringvegetative stages but is more stable during the reproductivestage (Zhang et al., 2018a). The pattern of root microbiomeshift is also influenced by the rice genotype and geographicallocation (Zhang et al., 2018a). A recent study showed thetriterpene produced by Arabidopsis roots can shape the rootassociated microbiome. The authors found that mutations inthree metabolic pathways of triterpens synthesis disruptedthe biosynthesis of compounds and led to assembly of dif-ferent root microbiota compared to that of wild type (Huanget al., 2019). Moreover, these triterpens can selectively reg-ulate the growth of bacteria from different taxa by acting as

24 Gong, Z., et al. Sci China Life Sci

antibiotics or proliferating agents. From these types of ge-netic/biochemical studies, it is becoming clear that the vastmetabolic diversity of natural compounds produced by plantsmay provide a basis for selectively promoting, inhibiting,and recruiting specific microbes to enable the shaping ofmicrobial communities tailored to the needs of the host, andthis may in part explain the existence of plant-specializedmetabolism. Altogether, the results reported in these studiesconfirm that the host genotype has a considerable impact onroot microbiota establishment under fixed environmentalconditions. The links between plant genotype and root mi-crobiota membership established in these studies will informbreeding schemes to improve nutrient availability in therhizosphere and root acquisition efficiency, which will helpto define the strategies to design synthetic communities tofavor yield improvement in different crops.

Molecular mechanisms of interaction among the majornutrients

As mentioned earlier, most studies on abiotic stress havebeen carried out using single types of nutritional or en-vironmental stresses; however, plants in nature have to si-multaneously face highly variable levels of multiplenutrients as well as the presence of toxic levels of severalions. Acid soils are a good example of the complexity ofsignals that the plants need to perceive and integrate foractivating adequate responses to survive and reproduce. Inacid soils, plants face low pH, low levels of Pi availability,toxic levels of Al3+, elevated levels of Fe3+, and a variablelevel of N. How does the plant integrate all these signals toactivate the most appropriate developmental programs andsustain cellular homeostasis? Implemented mechanismsmust involve the integration of signaling pathways intocomplex regulatory networks that allow the plant to globallyadjust the expression of its genome. For instance, the im-portance of N/P interaction has long been recognized;however, the mechanisms by which plants integrate N and Psignaling into an interconnected regulatory network are stillunraveled. In plants, nitrate and Pi are the major sources of Nand P that also act as signal molecules (Hu and Chu, 2020).Nitrate and Pi signaling pathways have been well char-acterized in Arabidopsis and rice. In Arabidopsis, nitrate issensed by the nitrate transceptor AtNRT1.1 and then a low Nresponse is mediated by the central transcription factor NIN-like Protein 7 (AtNLP7) (Ho et al., 2009), who activates geneexpression by binding to nitrate-responsive cis-elements.AtNRT1.1 triggers an increase of cytoplasmic Ca2+ levels,which in turn activates Ca2+-sensor protein kinases thatphosphorylate AtNLP7. Phosphorylated AtNLP7 enters thenucleus where it activates the expression of key N pathwaygenes, thus relaying the nitrate signal from the cytoplasm tothe nucleus (Marchive et al., 2013). In the case of Pi sig-

naling, the MYB-CC transcription factor AtPHR1 (in Ara-bidopsis) and OsPHR2 (in rice) act as master regulators thatcontrol the expression of phosphate starvation-induced (PSI)genes (Rubio et al., 2001; Zhou et al., 2008). In both Ara-bidopsis and rice, the proteins containing the SYG1/Pho81/XPR1 domain can form an interaction with AtPHR1 andOsPHR2 respectively in a Pi-concentration-dependentmanner, which blocks their transcriptional activity (Puga etal., 2014; Wang et al., 2014). In rice, the repressor proteinOsSPX4, which is degraded under Pi starvation, can interactwith OsPHR2 to block its cytoplasmic-nuclear shuttling (Lvet al., 2014). Thus, the OsSPX1-OsSPX4–OsPHR2 complexexplains the mechanism of Pi starvation signal transductionin rice (Lv et al., 2014). Although it had been proposed thatPi itself mediated the protein-protein interaction of AtPHR1/OsPHR2 with SPX proteins, more recent studies showed thatSPX proteins directly bind inositol polyphosphates (InsPs) atphysiological concentrations, suggesting that InsPs ratherthan Pi are the true signal molecules that are sensed andtrigger the phosphorus signaling pathway (Dong et al., 2019;Wild et al., 2016).The mutual influences of N and P, where a shortage of

either nutrient generally has a negative impact on the uptakeof the other, have long been known. However, the mechan-isms underlying this phenomenon have only very recentlystarted to be unraveled. A study of an NLP-dependent tran-scriptional cascade revealed that plants also use negativeregulators to control transcription of genes related to nitro-gen use during nitrate responses. The NITRATE-IN-DUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRE-SSOR1 (NIGT1) family of proteins directly suppress ex-pression of the high-affinity nitrate transporter NRT2 genes.The expression of AtNIGT1/HHO genes is independentlyactivated by both NLP and PHR family transcription factors(Kiba et al., 2018; Maeda et al., 2018). Expression of At-NIGT1 is either induced by nitrate, or by Pi starvation butonly under high nitrate conditions. NIGT1 also targets therepressor genes of the PSR, including AtSPX1/2/4 and At-PHO2, thus activating Pi utilization (Kiba et al., 2018).A recent study revealed that the nitrate sensor OsNRT1.1B

plays an essential role in activating Pi utilization (Hu et al.,2019), further supporting the notion of a direct interactionbetween N and P signaling pathways. In rice, OsNRT1.1Band OsPHR2 represent the central elements for activating Nand Pi signaling pathways. OsNRT1.1B can interact withOsSPX4, a negative regulator of OsPHR2, and promotes itsdegradation (Hu et al., 2019). These results revealed theexistence of an OsNRT1.1B-OsSPX4-OsPHR2 signalingmodule that transduces the nitrate signal to activate the PSR.This regulatory module integrates the N and P signalingpathways into a single regulatory network with several ne-gative regulatory loops that allow a balance between N and Puptake and utilization depending on the physiological status

25Gong, Z., et al. Sci China Life Sci

of the plant. Recent studies illustrate the complexity of theregulatory networks that integrate the plant responses to theavailability of different nutrients. Probably these networkswill become more complex when the signaling pathways areintegrated with the uptake and assimilation of other nutrients,such as Ca2+ (directly participates in N signaling), Mg2+

(required for chlorophyll biosynthesis), and Mg2+/Mn2+

(regulate photosynthesis and photorespiration).In the case of acid soils, alterations in Arabidopsis root

development have been reported in response to Al3+, low pH,and low Pi availability. Genetic approaches led to the iden-tification of a regulatory node which is composed of twotranscription factors, SENSITIVE TO PROTON RHIZO-TOXICITY (STOP1) and the ALUMINUM ACTIVATEDMALATE TRANSPORTER 1 (ALMT1) (Gutiérrez-Alaníset al., 2017). It has been shown that upon environmentalstimuli, STOP1 binds to the promoter of ALMT1 to activateits transcription leading to an increased exudation of malateinto the apoplast of root tip cells (Balzergue et al., 2017).Malate has been proven to prevent the toxic effect of Al3+ bypreventing Al3+ entry into the root cells, and solubilize in-soluble forms of Pi. In the case of primary root inhibition bylow Pi, malate interacts with Fe3+ to facilitate a redox cyclemediated by the ferroxidases LPR1/LPR2, which producesreactive oxygen species that triggers the expression of thesignaling peptide CLE14 to promote the differentiation ofcells in the primary root meristem (Balzergue et al., 2017;Mora-Macías et al., 2017). Since Fe3+ and Pi easily react toform insoluble compounds and the level of Pi and Fe3+

availability is interdependent, it is clear that the balancebetween these two ions will determine whether primary rootis inhibited or not. Interestingly, this Arabidopsis root re-sponse has been shown to be dependent on light; if Arabi-dopsis seedlings are germinated in the dark or root exposureto light is prevented, the inhibition of primary root by low Piis less evident (Zheng et al., 2019). This suggests that pri-mary root response to low Pi is most relevant during seedgermination and the early penetration to soil, in which theseedling is primarily exposed to a homogeneous distributionof Pi in the soil solution. This early response could play animportant role in determining the root architecture of olderplants when the root has completely penetrated the soil and isexposed to an uneven distribution of Pi in the soil. As theactivity of STOP1 is regulated at the posttranscriptional le-vel, how Arabidopsis integrates different environmentalsignals to modulate the activity of this transcription factorremains to be determined.

Impact of nutrient status on resistance to pests and dis-eases

The nutrient status of plants has also been reported to have animpact on other types of environmental stresses and on the

resistance to pests and diseases. How these responses areintegrated are still largely unknown, but recent studies beginto provide clues on the interaction of nutritional and othersignaling pathways. For instance, it has been reported thatplants experiencing Pi deficiency induce jasmonic acid (JA)biosynthesis and enhance defense against insect herbivory. Pideficiency triggered increased resistance to Spodoptera lit-toralis in Pi-deprived wild type Arabidopsis, tomato (Sola-num lycopersicum) and Nicotiana benthamiana, revealingthat the link between Pi deficiency and enhanced insect re-sistance is conserved (Khan et al., 2016). Interestingly, theearly increase in JA synthesis but not the increase observedat later stages of Pi-deprived in Arabidospsis seedlings isdependent on PHR1 (Khan et al., 2016), suggesting that thenetworks coordinating the induction of herbivory resistanceand PSR involve PHR1-dependent and -independent sig-naling pathways. As mentioned above, the PHR1-dependentPSR signaling pathway partially modulates the plant immuneresponse reaction (including disease resistance and the plant-microbiome association). Thus, PHR1 function as a centralnode, integrating nutritional signals (including interactionswith N status), immunity, and herbivory responses. It is alsointeresting how nutritional signaling pathways can be hi-jacked by plant pests. This also highlights the global im-portance of nutrient signaling pathways in the health andproductivity of plants. It has been reported that N signalingvia NLA induces salicylic acid and immunity against bac-terial pathogens (Yaeno and Iba, 2008). Recently, an im-portant regulatory role of miR827 and its NLA target genewere revealed (Hewezi et al., 2016). The nematode Hetero-derata schachtii induces the expression of miR827, whichdownregulates its NLA and downstream defense genes. Re-ducing the level of miR827 by RNA mimicry or expressingan NLA transcript lacking the target site for this microRNAresulted in increased resistance to the nematode (Hewezi etal., 2016).

Key scientific questions that remain to be addressed fornutrient use efficiency

(1) There still is no molecular framework for elucidatingdynamic nutrient status impacted by varied soil fertility andcultivation.(2) We need to understand the genetic control of root-

microbiota association under varied soil management in fa-vor of nutrient acquisition and root health.(3) How do we modulate plant growth and metabolic co-

regulation for enhancing nutrient use efficiency in the face ofclimate change?(4) Regulatory networks need to be built for understanding

the impact of nutrient status on plant resistance to pests anddiseases.(5) Precise breeding strategies need to be developed for

26 Gong, Z., et al. Sci China Life Sci

maximizing nutrient use efficiency and minimizing the ac-cumulation of harmful substances.

Summary and perspectives

As a result of global climate change, episodes of environ-mental stress are becoming more frequent and are lastinglonger, with significant adverse effects on plant growth, plantdevelopment, and crop yield. As sessile organisms, plantshave evolved sophisticated systems to withstand abioticstresses. Although some key components of plant responsesto stresses have been identified, our knowledge of the re-sponse network is limited.Important details about the downstream events of abiotic

stress signaling have been revealed over the past few years,yet there is still limited information on how plant cells per-ceive these stress signals in the first place. The few candidatestress sensors identified recently provide new opportunitiesto gain insights into stress signal perception. Revealing thestructures of these proteins and identifying their immediatedownstream components will help us to understand theirmodes of action. New research on stress signaling at theinterface of lipid membranes, cell walls, and their connectionto early events involved in transient Ca2+ increases is ofparticular interest. With the availability and application ofnew technologies, it is expected that the field of stress sen-sing will soon catch up with other areas of stress signaltransduction.It is not well understood how plants respond to different

stress stimuli coordinately and how plants respond to stressesin different cell and tissue types and how the responses indifferent cells and tissues are coordinated and integrated viaintercellular communication and long distance signals. Forexample, which cell layers in roots are the various sodiumtransporters localized? What are the functional differences ofthese transporters in root versus shoot? Are these transpor-ters localized uniformly in the cells or are they asymme-trically distributed within a cell? Answering these questionsis important for manipulating these genes for improvingplant salt tolerance.Studies in Arabidopsis have provided substantial under-

standing of stress-response mechanisms. In crops, however,systemic studies on the molecular mechanisms of stresstolerance are still lagging. Recent advances in genome se-quencing, assembly and annotation have facilitated the stu-dies of crops in many aspects including stress tolerance. Inaddition, collection and analysis of germplasms of the majorcrops have accelerated the discovery of stress tolerancegenes and alleles by GWAS. It is expected that GWAS willlead to the discovery of more stress tolerance genes andperhaps even novel stress tolerance mechanisms in crops inthe next few years. However, in addition to collecting re-

presentative germplasms, high-throughput phenotyping toolsneed to be developed and stress tolerance parameters usedfor phenotyping need to be carefully considered (Morton etal., 2019). Compared to Arabidopsis, crops are often morecomplex and likely to have additional mechanisms to copewith stress, and different crop species may possess distinct aswell as common genes and mechanisms for stress response.Therefore, more in-depth studies in stress tolerance in cropswill uncover novel genes and mechanisms for improvementof stress tolerance in economically important plant species.A plant’s response to stresses can disrupt the balance be-

tween energy harvest and energy consumption, reducing theplant’s capacity for growth. For instance, the constitutiveoverexpression of CBFs adversely affects plant growth undernormal growth conditions (Gilmour et al., 2000; Jaglo-Ot-tosen et al., 1998). The “sacrifice-for-survival” strategytemporarily slows root growth to ensure that normal growthresumes once chilling stress terminates (Hong et al., 2017).More information about the mechanisms underlying thetrade-off between plant growth and stress tolerance will helpus develop crops that can sustain growth and yield in adverseenvironments.Plants have evolved precise mechanisms to perceive

multiple and fluctuating environmental cues. To be able tocomprehend these mechanisms, we need a greater under-standing of the crosstalk between stress responses and othersignaling pathways (e.g., those for light, hormones, humid-ity, temperature, nutrients, and pathogens). Understandingthe regulatory networks that communicate plant nutritionstatus with biotic and abiotic stresses will provide newavenues for designing new breeding programs to generatecrop varieties more resilient to adverse climactic conditionswith high yield and quality of the end product. Currently,many studies focus on the mechanisms of stress tolerance atthe vegetative stage of plant development. However, re-productive development and fertilization are also sensitive tostress conditions, often leading to more serious losses of cropyield (Bac-Molenaar et al., 2015; Barnabás et al., 2008; Penget al., 2004; Zhang et al., 2016b). Therefore, we need toextend our knowledge about plant stress responses to thereproductive stages of development.

Compliance and ethics The author(s) declare that they have no conflictof interest.

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