Transcriptional switch for programmed cell death inpith parenchyma of sorghum stemsMasaru Fujimotoa,1, Takashi Sazukab, Yoshihisa Odac,d, Hiroyuki Kawahigashie, Jianzhong Wue, Hideki Takanashia,Takayuki Ohnishia,2, Jun-ichi Yonedaa,3, Motoyuki Ishimorif, Hiromi Kajiya-Kanegaef, Ken-ichiro Hibarag,4,Fumiko Ishizunah,5, Kazuo Ebinei,j, Takashi Uedai,j,k, Tsuyoshi Tokunagal, Hiroyoshi Iwataf, Takashi Matsumotoe,6,Shigemitsu Kasugam, Jun-ichi Yonemarue,7, and Nobuhiro Tsutsumia,7

aLaboratory of Plant Molecular Genetics, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan; bBioscienceand Biotechnology Center, Nagoya University, Nagoya, Aichi 464-8601, Japan; cCenter for Frontier Research, National Institute of Genetics, Mishima,Shizuoka 411-8540, Japan; dDepartment of Genetics, SOKENDAI (The Graduate University for Advanced Studies), Mishima, Shizuoka 411-8540, Japan;eNational Agriculture and Food Research Organization (NARO), Institute of Crop Science, Tsukuba, Ibaraki 305-8602, Japan; fLaboratory of Biometry andBioinformatics, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan; gLaboratory of Plant Breeding andGenetics, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan; hTechnology Advancement Center, GraduateSchool of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan; iDivision of Cellular Dynamics, National Institute for Basic Biology,Okazaki, Aichi 444-8585, Japan; jDepartment of Basic Biology, SOKENDAI (The Graduate University for Advanced Studies), Okazaki, Aichi 444-8585, Japan;kPrecursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), Kawaguchi, Saitama 332-0012, Japan;lEARTHNOTE Co. Ltd., Nago, Okinawa 905-1152, Japan; and mFaculty of Agriculture, Shinshu University, Minamiminowa, Nagano 399-4598, Japan

Edited by Richard A. Dixon, University of North Texas, Denton, TX, and approved August 7, 2018 (received for review May 5, 2018)

Pith parenchyma cells store water in various plant organs. Thesecells are especially important for producing sugar and ethanol fromthe sugar juice of grass stems. In many plants, the death of pithparenchyma cells reduces their stemwater content. Previous studiesproposed that a hypothetical D gene might be responsible for thedeath of stem pith parenchyma cells in Sorghum bicolor, a promis-ing energy grass, although its identity and molecular function areunknown. Here, we identify the D gene and note that it is locatedon chromosome 6 in agreement with previous predictions. Sorghumvarieties with a functional D allele had stems enriched with dry,dead pith parenchyma cells, whereas those with each of six inde-pendent nonfunctional D alleles had stems enriched with juicy, liv-ing pith parenchyma cells. D expression was spatiotemporallycoupled with the appearance of dead, air-filled pith parenchymacells in sorghum stems. Among D homologs that are present inflowering plants, Arabidopsis ANAC074 also is required for thedeath of stem pith parenchyma cells. D and ANAC074 encode pre-viously uncharacterized NAC transcription factors and are sufficientto ectopically induce programmed death of Arabidopsis culture cellsvia the activation of autolytic enzymes. Taken together, these re-sults indicate that D and its Arabidopsis ortholog, ANAC074, aremaster transcriptional switches that induce programmed death ofstem pith parenchyma cells. Thus, targeting the D gene will providean approach to breeding crops for sugar and ethanol production.

sorghum | stem | pith parenchyma | programmed cell death |NAC transcription factor

Vascular plant stems contain three main types of tissues: theoutermost dermal tissue protects the internal tissues, vas-

cular tissue transports nutrients and water, and ground tissuestores nutrients and water. The ground tissue is generally clas-sified into cortex and pith tissues. The latter consists primarily ofparenchyma cells with thin primary cell walls. In monocots, pa-renchyma cells fill the large space between dermal and vasculartissues, whereas in dicots they fill the large space inside the ringof vascular tissue (1). In many flowering plants, the majority ofstem pith parenchyma cells die, which leads to the formation ofair-filled cavities in the stem (2). The death of stem pith pa-renchyma cells has several effects in plants. It facilitates thegrowth of other tissues and organs in bean, tomato, buckwheat,and maize (3, 4); promotes water conservation during drought intomato (5); and establishes space for gas exchange betweenwaterlogged and nonwaterlogged tissues in sunflower and rice (6,7), while it reduces stem strength and the resistance to stemlodging and stem rot disease in maize (8). Although the death of

stem pith parenchyma cells has long been considered to be a typeof programmed cell death (PCD) (2), genetic and molecularmechanisms of the death of these cells remain largely unknown.

Significance

Sugar and ethanol productivity from the sugar juice of grassstems depends on their water content. Pith parenchyma cellsfunction as a water storage tissue in plant stems, and the deathof these cells reduces stem water content. In this study, weidentified a gene, long referred to as D, in a promising energygrass, Sorghum bicolor, that is responsible for reducing stemwater content. D and its Arabidopsis ortholog encode mastertranscriptional switches that induce programmed death of stempith parenchyma cells by activating autolytic enzymes. Identi-fying D as the gene involved in programmed death of plant pithparenchyma cells will provide an approach to breeding crops forsugar and ethanol production.

Author contributions: M.F., J.-i. Yonemaru, and N.T. designed research; M.F., T.S., Y.O.,H.K., J.W., H.T., T.O., J.-i. Yoneda, K.-i.H., F.I., T.M., S.K., and J.-i. Yonemaru performedresearch; T.S., Y.O., K.E., T.U., and T.T. contributed new reagents/analytic tools; M.F., Y.O.,H.K., M.I., H.K.-K., H.I., and J.-i. Yonemaru analyzed data; and M.F., Y.O., and H.T. wrotethe paper.

Conflict of interest statement: J.-i. Yonemaru, H.K., T.M., N.T., and S.K. are listed on apatent based on this work (Japanese patent P2015-025241), which was filed by NARO.

This article is a PNAS Direct Submission.

Published under the PNAS license.

Data deposition: Raw transcript profiling data in this study have been deposited in theDNA Data Bank of Japan DDBJ Sequence Read Archive database, https://www.ddbj.nig.ac.jp/dra/index.html (accession code DRA004095).1Present address: Social Cooperation Laboratory Genomics Breeding, Graduate School ofAgricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan.

2Present address: Center for Education and Research of Community Collaboration,Utsunomiya University, Utsunomiya, Tochigi 321-8505, Japan.

3Present address: Research and Development Department, EARTHNOTE Co. Ltd., Nago,Okinawa 905-1152, Japan.

4Present address: School of Agricultural Regional Vitalization, Kibi International University,Minamiawaji, Hyogo 656-0484, Japan.

5Present address: Department of Human Life Science and Design, Faculty of ContemporaryHuman Life Science, Tokyo Kasei Gakuin University, Machida, Tokyo 194-0292, Japan.

6Present address: Department of Bioscience, Tokyo University of Agriculture, Tokyo 156-8502,Japan.

7To whom correspondence may be addressed. Email: yonemaru@affrc.go.jp or atsutsu@mail.ecc.u-tokyo.ac.jp.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1807501115/-/DCSupplemental.

Published online August 27, 2018.

www.pnas.org/cgi/doi/10.1073/pnas.1807501115 PNAS | vol. 115 | no. 37 | E8783–E8792

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Sorghum (Sorghum bicolor) is a monocot grass that can beused for food, feed, fiber, and fuel production. Sorghum has be-come the fifth most cultivated cereal crop in the world (9). Com-mon sorghum varieties include grain sorghum, forage sorghum,biomass sorghum, and sweet sorghum (10). Sweet sorghum varie-ties produce sugar juice in the stems, which can be used for sugarand ethanol production (9). In sweet sorghum varieties, as in sug-arcane, the sugar yield increases with increasing stem water contentand sugar concentration, both of which are targets of breedingprograms (11). Sorghum varieties are often classified into twogroups according to their stem water content: juicy-stem varietiesand dry-stem varieties (9, 12). Stem tissues in juicy-stem varietiesare generally rich in fresh pith parenchyma cells, whereas stemtissues in dry-stem varieties largely contain dead pith parenchymacells the cellular spaces of which are filled with air (4). Therefore,juicy- and dry-stem traits of sorghum are tightly coupled with theabundance of living and dead pith parenchyma cells, respectively.For a century, a single gene, long referred to as D, has been

proposed to be responsible for determining the juicy- and dry-stem traits of sorghum (13, 14). This has led us to speculate thatD may be involved in determining the death of pith parenchymacells in sorghum stems. Given that cell death is widely observedin pith parenchyma of flowering plant stems, we speculated thatD may have a similar role in other flowering plants. In previousattempts to identify the sorghum D gene, quantitative trait loci(QTL) studies of crosses between juicy- and dry-stem varietiesmapped the D locus to a region on chromosome 6 (15–17).Recent genome-wide association studies of diverse germplasmsidentified a major QTL for stem water content in the same re-gion of chromosome 6 (18, 19). Despite these advances, thesorghum D gene continued to elude identification.Here, we used positional cloning to identify the sorghum D

gene. We identified D as a single gene encoding a NAC tran-scription factor that controls the expression of genes involved inplant PCD. Our data show that D and its Arabidopsis ortholog,ANAC074, are master switches that induce PCD in the stem pithparenchyma. These results provide an approach to breedingcrops for sugar and energy production.

ResultsPositional Cloning Identified D as a Gene Encoding an NAC FamilyProtein. To identify the D gene in sorghum, we used positionalcloning and the F2 population from a cross between a dry-stemvariety [Senkinshiro (SKS)] and a juicy-stem variety [Nakei MS-3B(MS3B)]. The difference in stem water contents between SKS andMS3B is readily observed by squeezing the stems (Movie S1). At30 d after heading (DAH), the amount of juice squeezed from thestems was ∼8 times higher in MS3B than in SKS (Fig. 1A). Thesugar concentration in the juice squeezed from MS3B and SKSstems at 30 DAH did not significantly differ (Fig. 1B), suggestingthat the sugar content in MS3B stems is ∼7 times higher than thatin SKS stems (Fig. 1C). Cross-sections of the third internodes inSKS and MS3B stems at 9 wk after planting (9 WAP) revealed dry(white color) and moist (pale color) pith parenchyma, respectively(Fig. 1D, Upper). Most of the SKS pith parenchyma cells are filledwith air bubbles, whereas the MS3B pith parenchyma cells retaintheir cellular contents (Fig. 1D, Lower). These differences werealso observed in leaf midribs and their cross-sections (SI Appendix,Fig. S1), as described previously (15, 17). Consistent with theseresults, the air porosity of stem tissue (the relative amount ofspace occupied by air in a given volume of stem tissue) in the thirdinternode of SKS at 9 WAP was ∼8 times higher than that ofMS3B (Fig. 1E). These observations suggest that most of the pithparenchyma cells in MS3B stems are alive, whereas almost all ofthose in SKS stems are dead and filled with air. Hoechst33342 staining of stem pith parenchyma cells in the third in-ternode at 9 WAP revealed that most of the MS3B cells hadnuclei, whereas most of the SKS cells lacked nuclei (Fig. 1 F and

G), confirming that the latter were dead. Staining with Evans blue,an indicator of cell death, confirmed that dead pith parenchymacells were present only in SKS stems (Fig. 1H). Minor Evans blue

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Fig. 1. Phenotypes of dry- and juicy-stem sorghum varieties. (A) Stem juicecontent in Senkinshiro (SKS, dry-stem variety) and Nakei-MS3B (MS3B, juicy-stem variety) at 30 DAH. Error bars show SD (n = 5, ***P < 0.001). (B) Sugarconcentration (Brix value) in stem juice of SKS and MS3B at 30 DAH. Errorbars show SD (n = 5). (C) Stem sugar content of SKS and MS3B at 30 DAH.Error bars show SD (n = 5, ***P < 0.001). (D) Whole and magnified cross-sections of the third internodes in SKS and MS3B stems at 9 WAP. White-colored tissues in the cross-sections (Upper) indicate dry pith parenchyma.Darker cells (Lower) indicate dead air-filled pith parenchyma cells. Red andblue arrowheads mark representative examples of air bubbles and cellularcontents, respectively. (E) Air porosity of the third internodes in SKS andMS3B stems at 9 WAP. Error bars show SD (n = 3, **P < 0.01). (F) Hoechst33342-stained vertical sections of the third internodes in SKS and MS3Bstems at 9 WAP. Arrowheads indicate nuclei. (G) Percentage of pith paren-chyma cells with Hoechst 33342-stained nuclei (calculated from six 0.25-mm2

images containing ∼24–40 cells) in the third internodes of SKS and MS3Bstems at 9 WAP. Error bars show SD (n = 6, ***P < 0.001). (H) Evans blue-stained cross-section images of the first internodes of SKS and MS3B stems at11 WAP. Insets display magnified images of a vascular bundle. White ar-rowheads indicate tracheary elements. (Scale bars: 5 mm and 100 μm inUpper and Lower, respectively, of D; 100 μm in F; and 2 mm and 50 μm inLower Right and Upper Right, respectively, in H.)

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staining was observed in MS3B stems due to the presence oftracheary elements, which are dead empty cells that form water-conducting xylem vessels (Fig. 1H, arrowheads). These data clearlyindicate that SKS and MS3B stems are enriched in dead and livingpith parenchyma cells, respectively, consistent with their dry- andjuicy-stem traits. These results show that SKS and MS3B stems arewell suited for studies of the D gene, which determines the dry- orjuicy-stem trait. Next, we crossed SKS andMS3B and analyzed thesegregation of dry- and juicy-stem traits in the F2 population. F2plants (n = 222) had a 3:1 ratio of dry (n = 168, 75.7%) to juicy(n = 54, 24.3%) stem phenotypes, suggesting that the dry stemwith dead pith parenchyma cells is a dominant trait, whereas thejuicy stem with live pith parenchyma cells is a recessive trait.An initial rough mapping of 54 F2 juicy-stem individuals local-

ized D within a 4.33-Mb region (from 49.7 to 54.0 Mb) on chro-mosome 6 (Fig. 2A). Additional mapping of 1,000 F2 individualsnarrowed theD locus to a 185-kb region (from 51.788 to 51.973Mb)on chromosome 6 (Fig. 2A). We sequenced bacterial artificialchromosome (BAC) clones from the 185-kb regions of both SKSand MS3B (Fig. 2A) and used the results to design six single-nucleotide polymorphism/insertion-deletion (SNP/InDel) mark-ers (Fig. 2B). Fine mapping with 1,925 F2 individuals and the SNP/InDel markers confined the D locus to an 18.99-kb region (from51.794 to 51.808 Mb) on chromosome 6 (Fig. 2B). Analysis of theSKS and MS3B BAC clones and reference genome sequencescorresponding to the 18.99-kb region identified only one gene,Sobic.006G147400 (Fig. 2B). This gene contains three exons andencodes a protein with homology to transcription factors con-taining a plant-specific DNA-binding domain (Fig. 2B), referredto as the NAM/ATAF/CUC (NAC) domain (20, 21).Quantitative RT-PCR analysis indicated that the levels and

patterns of Sobic.006G147400 allele expression in SKS andMS3B were similar (SI Appendix, Fig. S2A). However, a com-parison of SKS and MS3B sequences revealed that the poly-morphism closest to the start codon in the MS3B allele created apremature stop codon within the conserved NAC domain (Fig.2C and SI Appendix, Fig. S2B). Sequence analysis of theSobic.006G147400 promoter and gene regions in 13 cultivars(four dry-stem cultivars and nine juicy-stem cultivars) indicatedthat there were at least four alleles (Fig. 2C). All four of the dry-stem cultivars shared an allele encoding a functional protein(Fig. 2C). By contrast, the nine juicy-stem cultivars had threetypes of nonfunctional alleles, which harbored a premature stopcodon in the first exon, a frameshift insertion in the second exon, orminiature inverted-repeat transposable element (MITE)-like ele-ments that replaced the genomic region including the first andsecond exons (Fig. 2C). Exon sequence analysis of 93 germplasmswith various stem juice sugar concentrations identified three addi-tional nonfunctional alleles in the juicy-stem varieties (Dataset S1).These results clearly show that the presence and absence of func-tional Sobic.006G147400 is exclusively coupled with the juicy-and dry-stem traits, respectively. This strongly suggests thatSobic.006G147400 is theD gene that determines stem water contentin sorghum. Hereafter, we refer to Sobic.006G147400 as D.To determine the historical origin of juicy-stem varieties with

nonfunctional D alleles, we compared the distribution of func-tional and nonfunctional D alleles in a geographical originanalysis of 93 germplasms from Asia and Africa. These resultsindicated that functional and nonfunctional D alleles were widelydistributed in Asian and African germplasms (SI Appendix, Fig.S3A and Dataset S1). Among five nonfunctional D alleles inAsian and African germplasms, only two types (exon I and IIdeletions and exon I stop codon) were found in African germ-plasms (SI Appendix, Fig. S3B). Some of these arose in geo-graphical areas near where sorghum is proposed to have beeninitially domesticated around 4000–3000 BC (9, 22) (SI Appen-dix, Fig. S3B). Thus, these two types of alleles are candidates for

ancient nonfunctional D alleles that were selected during thecourse of sorghum domestication.A BLAST search identified D homologs in Arabidopsis thali-

ana, grape vine (Vitis vinifera), and rice (Oryza sativa), but not inlycophyte (Selaginella moellendorffii) or moss (Physcomitrellapatens) (SI Appendix, Fig. S4). Phylogenetic analysis indicatedthat D and its homologs belong to an uncharacterized NACprotein subfamily, named the D subfamily, which is close to theNAC1 subfamily involved in lateral root development (23) (SIAppendix, Fig. S4). These results suggest that D subfamily pro-teins are conserved in flowering plants, but their functionsremained unknown.

D Is Specifically Expressed in Drying Pith Parenchyma Cells of Stems.To further explore the relationship between D and stem watercontent, we compared the expression patterns of D in reproductivestems of a dry-stem variety (74LH3213) and its nearly isogenic lined-NIL. The latter possesses a genomic fragment (∼3.5–13.7 Mb)from SIL-05 (a juicy-stem variety) that lacks the first and second

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Fig. 2. Identification of D in sorghum. (A) Rough mapping of the D locus. Redbidirectional arrows show the candidate region of the D locus. Blue verticalbars showmolecular marker positions. Dark red and blue horizontal lines showSKS and MS3B-derived BAC clone contigs, respectively. (B) Fine mapping of theD locus. Blue vertical bars showmolecular marker positions. Light gray and redboxes show untranslated and coding regions of D (Sobic.006G147400), re-spectively. Light blue lines show NAC domain-coding regions. (C) Gene struc-tures of D alleles in 13 sorghum cultivars. Blue and green dashed lines indicatedeletion regions in juicy-stem varieties. Dark blue boxes mark MITE-like ele-ments. Blue arrows indicate nonfunctional mutation positions.

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exons of D in a 74LH3213 genetic background (Fig. 3A). In74LH3213 stems at 8 WAP, white pith parenchyma containingdead, air-filled cells was preferentially located in the panicle baseand in the second to sixth internodes (Fig. 3B).D expression levelswere highest in the third and fourth internodes (∼120 and 60 timeshigher than that in the first internode of 74LH3213, respectively)(Fig. 3C), where the greatest formation of white pith parenchyma

was observed (Fig. 3B). D expression was much lower in the sec-ond, fifth, and sixth internodes (∼19, 19, and 3 times higher, re-spectively, than that in the first internode of 74LH3213) (Fig. 3C).D expression was lowest in the first and seventh internodes (Fig.3C), where white pith parenchyma was not observed (Fig. 3B). Inthe fifth internode of 74LH3213 stems at 8 WAP (Fig. 3D), themean expression level ofD in the region with white pith parenchyma

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Fig. 3. D expression during the formation of dry pith parenchyma in sorghum stems. (A) Graphical presentation of 74LH3213 and d-NIL genotypes. Gray andblue boxes indicate homozygous regions from 74LH3213 and SIL-05, respectively. (B) Positions and cross-sections of panicle base (PB) and each stem internode(IN) of 74LH3213 and d-NIL at 8 WAP. (C) Quantitative RT-PCR analysis of relative DmRNA levels (normalized with respect to actin mRNA levels) in PB and eachIN of 74LH3213 (red bars) and d-NIL (blue bars) at 8 WAP. The expression level in the first internode (IN1) of 74LH3213 was defined as 1.00. Error bars show SD(n = 3). (D) Image of the fifth internode cross-section of 74LH3213 at 8 WAP, which was used to examine D expression. Total RNA was extracted from eachportion within the red, orange, and blue rectangles (regions 1–3, respectively). (E) Quantitative RT-PCR analysis of relative D mRNA levels (normalized withrespect to actin mRNA levels) in the three regions as shown in D. The expression level in region 2 was defined as 1.00. Error bars show SD (n = 3, *P < 0.005,**P < 0.0001). (F) Cross-sections of the second stem internodes of 74LH3213 at 7–11 WAP. (G) Quantitative RT-PCR analysis of relative D mRNA levels(normalized with respect to actin mRNA levels) in the second stem internodes of 74LH3213 at 7–11 WAP. The expression level at 7 WAP was defined as 1.0.Error bars show SD (n = 3). (H) In situ RNA hybridization with D antisense probe (Left) and sense probe (Right, the negative control) in cross-sections of thesecond stem internode of 74LH3213 at 8 WAP. Arrowheads indicate cells with violet staining derived from transcript-specific hybridization. (Scale bars: 10 cmand 5 mm in Upper and Lower, respectively, of B; 5 mm in D and F; and 300 μm in H.)

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was ∼10 times higher than that in regions without whitepith parenchyma (Fig. 3E). By contrast, white pith parenchymawas not observed anywhere in the stem of d-NIL at 8 WAP(Fig. 3B). D expression in each internode of d-NIL stems was notdetected or was much lower than D expression in 74LH3213 stems(Fig. 3C). White pith parenchyma in the second internode of74LH3213 stems emerged at 8 WAP, proliferated during 9 and 10WAP, and became saturated at 11 WAP (Fig. 3F). D expressionlevels slightly increased at 8 WAP, peaked at 9 WAP, andthereafter decreased to a level comparable with that at 7 WAP(the stage before white pith parenchyma emerged) (Fig. 3G). Insitu hybridization analysis of 74LH3213 stems at 8 WAP showedthat D was expressed in pith parenchyma cells within the secondinternode portion just before the appearance of white pith pa-renchyma (Fig. 3H, arrowheads). These results strongly indicatethat D expression is spatiotemporally coupled with the formationof dead, air-filled pith parenchyma cells in sorghum stems.

D Is Involved in Cell Death of Stem Pith Parenchyma. A mutant com-plementation assay is the best way to evaluate whether D is in-volved in cell death in stem pith parenchyma. However, methodsfor transforming sorghum are not well developed. Therefore, weused Arabidopsis for these studies because it is easily transformableand has a single D homolog, ANAC074 (SI Appendix, Fig. S4),which shows 42% amino acid identity (52% similarity) with D. Inpreparation for a mutant complementation assay, we identified theArabidopsis tissue with high ANAC074 expression and character-ized loss-of-function mutant phenotypes in this tissue. The ex-pression level of ANAC074 in 65-d-old inflorescence stems wassignificantly higher than the expression levels in 10-d-old seedlings,young and mature rosette leaves, flower buds, flowers, 50- and 65-d-old hypocotyls, and 35- and 50-d-old inflorescence stems (Fig.4A). The activity of a β-glucuronidase (GUS) reporter expressed

under the control of a 2.45-kb ANAC074 promoter was detected inflowers (SI Appendix, Fig. S5A), xylem of 65-d-old hypocotyls (SIAppendix, Fig. S5B), and xylem and degenerating pith parenchymaof 65-d-old inflorescence stems (Fig. 4B). These results suggest thatANAC074 is preferentially expressed in flowers, older hypocotyls,and older inflorescence stems. In pith parenchyma cells of olderinflorescence stems, green fluorescent protein (GFP) fusions ofANAC074 expressed under the control of the ANAC074 promoterwere localized to the nucleus (Fig. 4C), providing further evidencethat ANAC074 is expressed in these cells.Next, we examined whether pith parenchyma cells in inflo-

rescence stems were alive or dead in wild-type and anac074mutant plants (SI Appendix, Fig. S6A). In 75-d-old senescentinflorescence stems of wild-type plants, most pith parenchymacells were stained by Evans blue (Fig. 4D), indicating that theywere dead. By contrast, in 75-d-old senescent inflorescence stemsof mutant anac074 plants lacking detectable ANAC074 expres-sion (SI Appendix, Fig. S6B, Left), only xylem vessels werestained (Fig. 4D), indicating that death was suppressed in pithparenchyma cells but not in tracheary elements. Expression ofANAC074 under the control of the ANAC074 promoter (SIAppendix, Fig. S6B, ANAC074pro::ANAC074-GFP) restored celldeath in the pith parenchyma of anac074 inflorescence stems(Fig. 4D). This clearly demonstrates that ANAC074 is requiredfor cell death in stem pith parenchyma. D expression under thecontrol of the ANAC074 promoter (SI Appendix, Fig. S6B,ANAC074pro::D-GFP) similarly complemented the phenotypeof inflorescence stems in mutant anac074 plants (Fig. 4D),confirming the involvement of D in cell death of stem pith pa-renchyma. These results indicate that the functions of sorghum Dare compatible with those of Arabidopsis ANAC074. We alsoinvestigated the effect of expressing the rice D homolog, OsD (SIAppendix, Fig. S4), under the control of the ANAC074 promoter.

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Fig. 4. Developmental functions of ANAC74 in Arabidopsis. (A) Quantitative RT-PCR analysis of ANAC074 mRNA levels (normalized with respect to UBQ10mRNA levels) in seedlings, young leaves, mature leaves, flower buds, and flowers in 50- and 65-d-old hypocotyls and in 35-, 50-, and 65-d-old inflorescencestems. The expression level in 10-d-old seedlings was defined as 1.00. Error bars show SD (n = 3). Different letters indicate statistically significant differences(Tukey’s honestly significant difference; α = 0.05). (B) Histochemical staining of Arabidopsis expressing the GUS reporter gene under control of the ANAC074promoter. Left and Right show images of 65-d-old inflorescence stem and its cross-section, respectively. (C) Subcellular localization of ANAC074-GFP in pithparenchyma cells of 65-d-old inflorescence stem. Arrowheads indicate nuclei. (D) Evans blue-stained 75-d-old inflorescence stem cross-section images of wild-type, anac074, and anac074 expressing ANAC074-GFP, D-GFP, or OsD-GFP under control of the ANAC074 promoter and terminator regions (anac074ANAC074pro::ANAC074-GFP, anac074 ANAC074pro::D-GFP, and anac074 ANAC074pro::OsD-GFP). White arrowheads and red asterisks indicate trachearyelements and pith parenchyma, respectively. (Scale bars: 1 mm and 200 μm in Left and Right of B; 100 μm in C; and 200 μm in D.)

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OsD has 70% identity (76% similarity) with D and 41% identity(50% similarity) with ANAC074. OsD expression (SI Appendix,Fig. S6B, ANAC074pro::OsD-GFP) also restored cell death inpith parenchyma of anac074 inflorescence stems (Fig. 4D).These results suggest that at least three D subfamily members,sorghum D, Arabidopsis ANAC074, and rice OsD, share over-lapping functions involved in cell death.

D Expression Is Sufficient to Induce Cell Death in Arabidopsis CultureCells.To characterize the cellular function of D, we compared theeffects of ectopic expression of D, ANAC074, OsD, and ArabidopsisNAC1 in Arabidopsis culture cells. NAC1 encodes a NACprotein belonging to the NAC1 subfamily, which is phylogenet-ically close to, but distinct from, the D subfamily (SI Appendix,Fig. S4). Ectopic expression was performed using a conditionalexpression system that combined the human estrogen receptorwith the bacterial repressor LexA (24). We established LexA::D,LexA::ANAC074, LexA::OsD, and LexA::NAC1 cell lines condi-tionally expressing D, ANAC074, OsD, and NAC1, respectively,when estrogen was added to the cell cultures (SI Appendix, Fig.S7). Estrogen did not affect nuclei and plastids in wild-type cells

(Fig. 5A), and there was no significant difference between thepercentage of cells lacking plastids and nuclei in the presence andabsence of estrogen (SI Appendix, Fig. S8, dark orange squaresand light orange triangles). By contrast, at 48 h after estrogenaddition, many of the cells expressing LexA::D, LexA::ANAC074,and LexA::OsD lacked nuclei and plastids (Fig. 5A, arrowheads).The percentage of cells lacking plastids and nuclei reached 55, 24,and 33% at 48 h after estrogen addition to LexA::D, LexA::ANAC074, and LexA::OsD cell lines, respectively (SI Appendix,Fig. S8, dark red, dark blue, and dark green squares, respectively),whereas the percentage of cells lacking plastids and nuclei in thesecell lines did not increase in the absence of estrogen (SI Appendix,Fig. S8, light red, light blue, light green triangles, respectively). InLexA::NAC1 cell lines, estrogen did not affect nuclei and plastids(Fig. 5A), similar to the observations for wild-type cells, and didnot increase the percentage of cells lacking plastids and nuclei (SIAppendix, Fig. S8, dark purple squares).The loss of nuclei and plastids suggests that expression of D,

ANAC074, or OsD induced cell death. At 48 h after estrogenaddition, Evans blue stain marked cells lacking nuclei andplastids in LexA::D, LexA::ANAC074, and LexA::OsD cell lines

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Fig. 5. Cellular effects of estrogen-induced overexpression of D and ANAC074. (A) Bright-field (BF) and fluorescent images of DAPI-stained Arabidopsis wild-type culture cells and cells harboring LexA::D, LexA::ANAC074, LexA::OsD, LexA::NAC1, and LexA::VND6 with or without estrogen immediately after (0 h) and48 h after addition of estrogen. Arrowheads indicate cells without plastids and DAPI-stained nuclei. (B) Bright-field images of Evans blue-stained wild-type,LexA::D, LexA::ANAC074, LexA::OsD, LexA::NAC1, and LexA::VND6 cells with or without estrogen. All images were acquired at 48 h after the addition ofestrogen. (C) Percentage of Evans blue-stained cells immediately after (0 h) and 48 h after the addition of estrogen. −E and +E indicate culture conditionswithout and with estrogen, respectively. Error bars show SD (n = 3). Different letters indicate statistically significant differences (Tukey’s honestly significantdifference; α = 0.05). (D) BF and fluorescent images of WGA-stained wild-type, LexA::D, LexA::ANAC074, LexA::OsD, LexA::NAC1, and LexA::VND6 cells with orwithout estrogen. All images were acquired at 96 h after the addition of estrogen. Arrowheads indicate cells with WGA-stained secondary cell walls. (Scalebars: 50 μm in A, B, and D.)

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(Fig. 5B, Lower), indicating that they were dead. By contrast,in the absence of estrogen, these cell lines were rarely stainedwith Evans blue (Fig. 5B, Upper). Wild-type cells and LexA::NAC1 cell lines were rarely stained with Evans blue in eitherthe presence or absence of estrogen. The percentage of deadcells in LexA::D, LexA::ANAC074, and LexA::OsD cell lines at48 h after estrogen addition was ∼65, 40, and 44%, respectively(Fig. 5C), whereas the percentage of dead cells in these celllines in the absence of estrogen was ∼10%, which was com-parable with that in wild-type cells and LexA::NAC1 cell lines(Fig. 5C). These results strongly suggest that expression of D,ANAC074, or OsD is sufficient to induce cell death in Arabi-dopsis cells, but NAC1 expression is not sufficient. These re-sults also show that D, ANAC074, and OsD are functionallydistinct from NAC1.NAC transcription factors belong to a large protein superfamily

(21, 25). Some NAC transcription factors, such as the ArabidopsisVNS subfamily protein VND6 (SI Appendix, Fig. S4), can inducePCD (26). To compare D and VNS subfamily proteins, we prepareda LexA::VND6 cell line in which PCD and secondary cell-wall de-position can be induced by estrogen (27). Loss of nuclei and plastidsand Evans blue staining were observed in LexA::VND6 cells spe-cifically after estrogen addition (Fig. 5 A–C and SI Appendix, Fig.S8, dark ocher squares), suggesting that these cells had undergonePCD. By contrast, secondary cell walls, which can be stained withAlexa Fluor 594-conjugated wheat germ agglutinin (WGA), wereformed only in LexA::VND6 cells after estrogen addition, but not inLexA::D, LexA::ANAC074, LexA::OsD, LexA::NAC1, or wild-typecells (Fig. 5D). These results indicate that D, ANAC074, andOsD specifically induce cell death but do not induce secondary cell-wall formation, unlike VND6. These results also highlight differ-ences between D and VND6.

D and ANAC074 Activate Expression of PCD-Executing Enzymes.Next,we investigated the transactivation activities of D and ANAC074. InArabidopsis culture cells, GFP-fused D or GFP-fused ANAC074localized to the nucleus before cell death (Fig. 6A), consistent withthe subcellular localization pattern of transcription factors. Ourtransactivation assay in yeast cells showed that D or its C-terminalhalf fused with the GAL4 DNA-binding domain (GAL4 BD) acti-vated the expression of reporter genes (Fig. 6B), and ANAC074 orits C-terminal half fused with GAL4 BD showed similar results (Fig.6B). These results indicate that D and ANAC074 have trans-activation activity. Transcript profiling of the LexA::D and LexA::ANAC074 cell lines (Dataset S2, A and B) showed that induction ofD or ANAC074 selectively up-regulated the expression of genes an-notated with cysteine-type endopeptidase activity in Gene Ontologyterms (SI Appendix, Fig. S9). Most of these genes encode PCD-related cysteine peptidases such as papain-like peptidases, type-IImetacaspase, and vacuolar processing enzymes (28) (Fig. 6C).Induction of D or ANAC074 also up-regulated the expression ofother genes encoding autolytic enzymes, which generally functionto execute PCD, including an aspartic endopeptidase (PASPA3), aserine carboxypeptidase-like acyltransferase (SCPL48), a bifunc-tional nuclease (BFN1), and a ribonuclease (RNS3) (29) (Fig.6C). These results suggest that D and ANAC074 activate the ex-pression of PCD-executing genes.To confirm that D and ANAC074 directly target PCD-related

genes, we performed two experiments using the 5′-upstream re-gion of CEP1, a gene that encodes a cysteine peptidase and is up-regulated by expression of D or ANAC074 in Arabidopsis culturecells (Fig. 6C). One experiment was a transactivation assay thatused Arabidopsis culture cell protoplasts and a firefly luciferasereporter gene under the control of the CEP1 5′-upstream region.Both D and ANAC074 significantly up-regulated expression of thereporter gene (Fig. 6D). The other experiment was a yeast one-hybrid assay for CEP1, which showed that both D and ANAC074specifically bound to the CEP1 5′-upstream region (SI Appendix,

Fig. S10). These results strongly suggest that D and ANAC074 aretranscription factors that directly activate expression of the PCD-related gene CEP1.Finally, we determined the expression patterns of PCD-executing

genes in sorghum stems. Dying pith parenchyma cells were expec-ted to be abundant in the third internode of 74LH3213 stems at 8WAP (Fig. 3B), and we detected the expression of several PCD-executing genes including sorghum homologs of CEP1 and XCP1family peptidases, type-II metacaspases, PASPA3, SCPL48, BFN1,and RNS3 (Fig. 7). By contrast, there were no dying pith paren-chyma cells in the third internode of d-NIL stems (Fig. 3B), and theexpression of these PCD-executing gene homologs either was notdetected or was detected at much lower levels than in74LH3213 stems (Fig. 7). These results suggest that PCD is muchmore active in 74LH3213 stems than in d-NIL stems. D was spe-cifically expressed in 74LH3213 stems but not in d-NIL stems (Fig.7). These combined results further support our observation that Ddirectly activates the expression of PCD-executing enzymes.

DiscussionFunctions of D Subfamily Proteins in Stem Pith Parenchyma PCD. Thegene responsible for determining dry-stem and juicy-stem traits ofsorghum has long been referred to as D. In this study, we identifiedan uncharacterized gene, Sobic.006G147400, as a candidate for D.We could not identify any other genes within the genomic region forD determined by our fine mapping. Sobic.006G147400 was notfunctional in any of the sorghum varieties with the juicy-stem trait,whereas it was functional in all sorghum varieties with the dry-stemtrait. This is consistent with the fact that the dry-stem trait isdominant. Our data strongly suggest that Sobic.006G147400 is D.D encodes a NAC transcription factor that is conserved among

flowering plants. Our phylogenic analysis indicated that D proteinsbelong to a subfamily that is close to the NAC1 subfamily withinthe NAC superfamily. We demonstrated that ANAC074, an Ara-bidopsis ortholog of D, was preferentially expressed in flowers, olderhypocotyls, and older inflorescence stems and that ANAC074 isnecessary and sufficient to induce cell death, at least in pith pa-renchyma of older inflorescence stems. Our experiments with Ara-bidopsis culture cells indicated that ANAC074 expression wassufficient to ectopically induce cell death, and these dead cells lostall visible cellular organelles such as nuclei and plastids. This sug-gests that ANAC074 induces PCD but not necrosis, in which cellsretain cellular organelles even after death (30). The loss of DAPI-stained nuclei (Fig. 5A) indicates that genomic DNA is degraded,and strongly supports the proposal that ANAC074 expression in-duces PCD. Our transcriptome analysis indicated that ANAC074exclusively induced the expression of a range of PCD-executinggenes that encode conserved autolytic enzymes, at least one ofwhich is a direct target of ANAC074. These combined results in-dicate that ANAC074 is the master transcription factor that inducesPCD in Arabidopsis.Our data indicate that the sorghum D gene, like Arabidopsis

ANAC074, encodes the master transcription factor for PCD. Dcomplements mutant anac074 plant phenotypes and inducesectopic PCD in Arabidopsis culture cells by up-regulating theArabidopsis PCD-executing enzymes. In sorghum stems, D isresponsible for the formation of dead, air-filled pith parenchymacells. Taken together, these results suggest that D triggers PCDin pith parenchyma of sorghum stems. In this scenario, PCD isspecifically induced in dry-stem varieties expressing functional D,which reduces their water content and leads to the dry-stem trait.By contrast, juicy-stem varieties have nonfunctional D, do notinduce PCD in the stem pith parenchyma, and retain waterwithin protoplasts of pith parenchyma cells, leading to the juicy-stem trait. We cannot exclude the possibility that genes otherthan D induce PCD in the stems of sorghum varieties that werenot investigated in this study. However, given that PCD in stempith parenchyma is widely observed in flowering plants, and that

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AT1G79340/AtMC4 AT1G79340/AtMC4AT1G79330/AtMC5* AT1G79330/AtMC5AT1G79320/AtMC6 AT1G79320/AtMC6

AT1G79310/AtMC7* AT1G79310/AtMC7AT1G16420/AtMC8 AT1G16420/AtMC8

AT5G04200/AtMC9* AT5G04200/AtMC9

AT2G25940/αVPE* AT2G25940/αVPEAT1G62710/βVPE AT1G62710/βVPEAT4G32940/γVPE AT4G32940/γVPEAT3G20210/δVPE AT3G20210/δVPE

AT1G02170/AtMC1 AT1G02170/AtMC1AT4G25110/AtMC2 AT4G25110/AtMC2AT5G64240/AtMC3 AT5G64240/AtMC3

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AT1G11910/PASPA1AT1G62290/PASPA2 AT1G62290/PASPA2

AT4G04460/PASPA3* AT4G04460/PASPA3*

AT1G11910/PASPA1

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LexA::ANAC0+ Estr / -Es

AT5G50260/CEP1* AT5G50260/CEPAT3G48350/CEP2 AT3G48350/CEAT3G48340/CEP3* AT3G48340/CEP

AT4G35350/XCP1* AT4G35350/XCPAT1G20850/XCP2 AT1G20850/XCP

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AT1G14210 AT1G14210AT1G14220 AT1G14220

RNS3-typeribonucleases

AT5G22980/SCPL47AT3G45010/SCPL48*AT3G10410/SCPL49

SCPL48-type serinecarboxypeptidase-like

acyltransferases

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BFN1-typenucleases

AT1G11190/BFN1* AT1G11190/BFN1AT1G68290/ENDO2 AT1G68290/ENDO2

C

Fig. 6. Molecular functions of D and ANAC074. (A) Localization of D-GFP and ANAC074-GFP in nuclei (arrowheads) of Arabidopsis culture cells. (B) Trans-activation assay of D and ANAC074 in yeast cells. (Upper) Effectors and reporters used in this assay. The GAL4 BD and its fusions of the N- and/or C-terminalhalf of D or ANAC074 serve as effectors. HIS3 and ADE2 genes under control of GAL4-binding sites serve as reporters. (Lower) Growth of yeast cells expressingeach effector in the presence or absence of histidine and adenine. (C) Changes in the expression levels of Arabidopsis genes encoding PCD-related peptidases,acyltransferases, and nucleases. Color scale indicates fold-changes in gene expression level (on a log-2 scale). Asterisks represent statistically significantchanges. (D) Luciferase (LUC)-based transactivation assay in Arabidopsis culture cell protoplasts. (Upper) Schematic of the 555-bp CEP1/AT5G50260 5′-upstream genomic region, CEP1pro(−555), used in this assay. The position of the start codon (ATG) of CEP1/AT5G50260 is numbered as +1. (Lower) Bargraphs represent the relative activities of firefly LUC in Arabidopsis culture cell protoplasts transfected with the indicated combinations of the effector andreporter constructs. LUC activity in each protoplast was normalized with respect to Renilla luciferase activity from the cotransfected internal control construct,and are presented as relative LUC activity. Cauliflower mosaic virus 35S promoter (CaMV35Spro) and terminator (CaMV35Ster) serve as the regulatory ele-ments of D or ANAC074 in the effectors. CEP1pro(−555) or CaMV35Spro and CaMV35Ster serve as the regulatory elements of LUC in the reporters. Error barsshow SD (n = 5). Relative LUC activity in protoplasts transfected with the effector construct harboring CaMV35Spro::::CaMV35Ster (the only regulatory el-ements) and the reporter construct harboring CEP1pro(−555)::LUC::CaMV35Ster was defined as 1.0. Different letters indicate statistically significant differ-ences (Tukey’s honestly significant difference; α = 0.05). (Scale bars: 10 μm in A.)

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D is widely conserved in flowering plants, it is reasonable toconsider that D is the common determinant of PCD in stem pithparenchyma. Thus, our study revealed the molecular and cellularmechanism determining stem water content in sorghum andpossibly other grasses, crops, and vegetables.

Independent Evolution of Two Master Switches to Induce PCD in Plants.NAC is a large superfamily of transcription factor proteins (21).Some VNS subfamily proteins, like some D subfamily proteins,function as master switches for PCD (26, 31, 32). We showed that aVNS subfamily protein, VND6, induces cell death in Arabidopsisculture cells (Fig. 5 A–C and SI Appendix, Fig. S8). However, twolines of evidence suggest that the PCD-inducing activity of D sub-family proteins evolved independently of VNS subfamily proteins.First, in our phylogenic tree (SI Appendix, Fig. S4), D subfamily

proteins form a clade close to NAC1 subfamily proteins, which isseparate from the clade corresponding to VNS subfamily proteins.This is consistent with another study that classified plant NACsuperfamily proteins into six groups (25), in which groups I and IIcorresponded to VNS and D subfamily proteins, respectively.Other group II subfamilies include NAC1 subfamily proteins in-volved in lateral root formation (23), CUC subfamily proteinsinvolved in shoot apical meristem formation (33, 34), and OREsubfamily proteins involved in leaf senescence (35, 36). Theseproteins have not been reported to induce PCD. Even ArabidopsisNAC1, which is phylogenetically closest to the D subfamily, doesnot induce ectopic PCD in Arabidopsis culture cells (Fig. 5 A–Cand SI Appendix, Fig. S8). Thus, among these functionally differ-entiated group II NAC subfamily proteins, so far, only D sub-family proteins—at least those in sorghum, Arabidopsis, and rice—have been shown to have the ability to induce PCD.Second, VNS subfamily proteins are conserved from mosses to

flowering plants and are involved in the differentiation of water-conductive and supporting tissues such as hydroid cells, xylemvessels, and xylem fibers (31). Consistent with this, most of thestudied VNS members activate genes related to cell-wall for-mation and PCD (26, 31, 37). By contrast, D subfamily proteinsare conserved only in flowering plants and specifically inducePCD without activating cell-wall formation. Furthermore, unlikeVNS subfamily proteins, D subfamily proteins do not appear to

be involved in the differentiation of xylem vessels because theirloss of function does not affect the PCD of stem tracheary elements,at least in sorghum and Arabidopsis. The differences between thefunctions of VNS and D subfamily proteins are consistent with thedifferences between cellular properties of xylem vessels and deadpith parenchyma cells. The cell walls of dead pith parenchymacells are generally thinner and contain less cell-wall–reinforcingmaterials, such as lignin, than those of xylem vessels (38–40).Taken together, these results suggest that D subfamily proteinsevolved to induce cell death in stem tissue after vascular plantsacquired a stem structure, which subsequently resulted in the di-vergence of stem function.

Potential Agricultural and Industrial Applications of D. PCD in pithparenchyma of flowering plant stems has been proposed to reducestem water content (4), facilitate nutrient translocation from stems tosink organs (3, 4), and provide drought or waterlogging stress tol-erance (5–7). Our results demonstrate that sorghum D induces PCDin pith parenchyma, which reduces stem water content, and thatnonfunctional alleles of D in juicy-stem varieties enhance high stemwater content. Some of these D alleles originated in the area wheresorghum domestication first occurred. On the other hand, in sor-ghum, as in maize, the death of stem tissues reduces stem strength,which increases the susceptibility to stem lodging and stem rot dis-ease (41–44). Therefore, nonfunctional alleles of D may have beenthe targets of artificial selection because they improve stem watercontent and stem strength in sorghum domestication and breeding.Genome editing technologies can be used to modulate the ac-

tivity of endogenous D in breeding programs for a range of cropsand vegetables. It may be possible to alter the water and nutrientcontents in stems and enhance plant tolerance for drought,waterlogging, lodging, and disease. For example, suppressing en-dogenous D expression in plants that are not currently suitable forsugar or ethanol production may increase their stem water andsugar contents. Conversely, activating D in crops and vegetables atthe appropriate time and in the appropriate organ also may in-crease their sink organ productivity and drought or waterloggingstress tolerance. A search for chemicals that inhibit or enhance theactivity of D should offer new approaches to the breeding ofuntransformable plants. Thus, the discovery of the involvement of

0.001 NDND NDND 0.0010.016

0.547

0.225NDND

0.206

Sobic.006G147400D CEP1 family peptidase homologs XCP1 family peptidase homologs

Sobic.001G058800 Sobic.003G395000 Sobic.005G151700 Sobic.007G172100 Sobic.007G195500 Sobic.007G195500 Sobic.003G443200 Sobic.009G008800

** ** ** * ***

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d-NIL74LH3213

0.178NDND

0.675

0.198

0.691

0.003 0.001ND

Type II Metacaspase homologs PASPA3 homologsSobic.003G325500 Sobic.009G183800 Sobic.009G183900 Sobic.009G184000 Sobic.004G085800 Sobic.004G187200 Sobic.009G234800 Sobic.003G247100

** * ** ** ** **

00.20.40.60.81.01.21.4

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d-NIL74LH3213

0.006

0.326

0.004

0.488

0.145

0.492

NDND NDND

SCPL48 homologs BFN1 homologs RNS3 homologsSobic.004G010000 Sobic.002G301200 Sobic.006G232500 Sobic.003G087200 Sobic.003G087300 Sobic.007G137700 Sobic.002G385500 Sobic.002G278700

*** ** ** ** ** **

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d-NIL74LH3213

Fig. 7. Expression levels of PCD-related genes in 74LH3123 and d-NIL stems. Quantitative RT-PCR analysis of relative mRNA levels (normalized with respect toactin mRNA levels) of D, CEP1 family peptidase homologs, XCP1 family peptidase homologs, type II metacaspases homologs, PASPA3 homologs, SCPL48homologs, BFN1 homologs, and RNS3 homologs in the third internodes of 74LH3123 and d-NIL stems at 8 WAP. The expression level of each gene in74LH3123 was defined as 1.00. Error bars show SD (n = 3, *P < 0.05, **P < 0.005, ***P < 0.001).

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the D gene in the programmed death of stem pith parenchymacells will broadly contribute to agriculture and industry.

Materials and MethodsDetails for plant materials, calculation of stem juice content, measurement ofsugar concentration (Brix), calculation of stem sugar content, air porositymeasurement, map-based cloning, phylogenetic analysis, polymorphism anal-ysis, quantitative RT-PCR, plasmid construction, analysis of amino acid sequencehomology, Arabidopsis transformation, in situ hybridization, histochemical GUSstaining, microscopic observations, transcriptional activation assay in yeast cells,luciferase-based transactivation assay in Arabidopsis protoplasts, yeast one-hybrid assay, 3′-tag digital gene expression profiling, gene ontology analysis,statistical analysis, and data availability are described in SI Appendix, Materialsand Methods. Nucleotide primers used in this study are listed in Dataset S3.

ACKNOWLEDGMENTS. We thank N.-H. Chua (Rockefeller University),U. Grossniklaus (University of Zurich), and The Nottingham Arabidopsis StockCentre for providing the pER8 vector, the pER8-modified pMDC7 vector, andthe Arabidopsis mutant (GK_224H04), respectively; T. Ando, T. Mizubayashi,H. Kanamori, S. H. Choi (NARO), Y. Hirata, S. Lin, T. Hidaka, Y. Sano, M. Ueda,K. Bou, and Y. Tamura (The University of Tokyo) for technical assistance; andT. Fujiwara and M. Tanaka (The University of Tokyo) for assistance with theluciferase-based transactivation assay. This work was supported by grantsfrom the Ministry of Agriculture, Forestry, and Fisheries of Japan (Genomicsfor Agricultural Innovation, Grants QTL5503 and QTL5506); the JST [CoreResearch for Evolutional Science and Technology (CREST), Grant JPMJCR12B5and PRESTO, Grant JPMJPR11B3]; the Ministry of Education, Culture, Sports,Science and Technology of Japan KAKENHI (Grant 16H01247); and the JapanSociety for the Promotion of Science KAKENHI (Grants 16H06172 and17H05019).

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