Global epigenetic and metabolic changes accompany the ...27 Abstract 28 To further understand the...
Transcript of Global epigenetic and metabolic changes accompany the ...27 Abstract 28 To further understand the...
Global epigenetic and metabolic changes accompany the
alterations in fruit size and shape of Cucurbita pepo L. intra-
species grafting
Aliki Xanthopoulou 1 , Aphrodite Tsaballa 2 , Ioannis Ganopoulos 3 , Aliki Kapazoglou 1 , Evangellia Avramidou 4
, Filippos Aravanopoulos 4 , Theodoros Moysiadis 1 , Maslin Osathanunkul 5 , Athanasios Tsaftaris 6 , Andreas
Doulis 7 , Apostolos Kalivas 3 , Eirini Sarrou 3 , Stefan Martens 8 , Irini Nianiou- Obeidat 9 , Panagiotis MadesisCorresp. 10
1 Institute of Applied Biosciences, Institute of Applied Biosciences/CERTH, Thermi Thessaloniki, Greece
2 School of Biological Sciences, University of East Anglia, Norwich, UK., School of Biological Sciences, University of East Anglia, Norwich, UK., Norwich,
United Kingdom3 Hellenic Agricultural Organization DEMETER (ex NAGREF), Institute of Plant Breeding and Genetic Resources, Thermi, Thessaloniki, Greece
4 Faculty of Forestry and Natural Environment, Laboratory of Forest Genetics and Tree Breeding, Aristotle University of Thessaloniki, Thessaloniki, Greece
5 Chiang Mai University, Department of Biology, Faculty of Science,, Chiang Mai, Thailand
6 American Farm School, Perrotis College, Thessaloniki, Greece
7 Hellenic Agricultural Organization DEMETER (ex NAGREF),, Institute of Olive Tree, Subtropical Crops and Viticulture, Heraklion, Greece
8 IASMA Research and Innovation Centre, Fondazione Edmund Mach (FEM),, Department of Food Quality and Nutrition Department, San Michele all' Adige,
Italy9 Department of Genetics and Plant Breeding, Aristotle University of Thessaloniki, Thessaloniki, Greece
10 Institute of Applied Biosciences, Institute of Applied Biosciences/CERTH
Corresponding Author: Panagiotis Madesis
Email address: [email protected]
To further understand the impact of grafting on fruit characteristics and to comprehend
the mechanisms involved in graft-induced changes we studied homo- and hetero- grafted
Cucurbita pepo cultivars (cv.) that vary in fruit size and shape. C. pepo cv. ‘Munchkin’
(small fruit) and cv. ‘Big Moose’ (large fruit) as well as cv. ‘Round green’ (round fruit) and
cv. ‘Princess’ (elongated fruit) were homografted and reciprocally heterografted. The
results show significant changes in fruit size when ‘Big Moose’ was grafted onto ‘Munchkin’
rootstocks in comparison to homo-grafted controls. Moderate changes in fruit shape were
observed when grafting of cv. ‘Round green’ and cv. ‘Princess’ were performed. This is the
first report of such phenotypic changes after intra-species/inter-cultivar grafting in
Cucurbitaceae. Additionally, we found significant changes in i) secondary metabolite
profile, ii) global DNA methylation pattern and iii) miRNA expression patterns in grafted
scions and iv) DNA methylation on graft-induced phenotypic changes in grafted plants. Our
results contribute to further understanding graft-induced effects on fruit morphology in
intra-species grafting. Furthermore, our results pave the way for understanding the role of
phenolic metabolites and epigenetic molecular mechanisms on the phenotypic changes
recorded.
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3389v1 | CC BY 4.0 Open Access | rec: 3 Nov 2017, publ: 3 Nov 2017
1 Global epigenetic and metabolic changes accompany the alterations in fruit size and shape
2 of Cucurbita pepo L. intra-species grafting
3 Aliki Xanthopoulou1,2, Aphrodite Tsaballa3, Ioannis Ganopoulos4,, Aliki Kapazoglou2, Evangelia
4 Avramidou5, Filippos A. Aravanopoulos5, Theodoros Moysiadis2, Maslin Osathanunkul9,
5 Athanasios Tsaftaris1,2,8 Andreas G. Doulis6, Apostolos Kalivas4, Eirini Sarrou4, Stefan Martens7,
6 Irini Nianiou- Obeidat1*, Panagiotis Madesis2*
71Aristotle University of Thessaloniki, Department of Genetics and Plant Breeding, Thessaloniki,
8 GR-54124, Greece
92Institute of Applied Biosciences (INAB), CERTH, Thermi-Thessaloniki, GR-57001, Greece
103School of Biological Sciences, University of East Anglia, Norwich, UK.
114Institute of Plant Breeding and Genetic Resources, Hellenic Agricultural Organization
12 DEMETER (ex NAGREF), Thermi, Macedonia, GR-57001, Greece
135Aristotle University of Thessaloniki, Faculty of Forestry and Natural Environment, Laboratory
14 of Forest Genetics and Tree Breeding, GR-54124, Greece
156Hellenic Agricultural Organization DEMETER (ex NAGREF), Institute of Olive Tree,
16 Subtropical Crops and Viticulture, Heraklion, Crete, GR-71003, Greece.
177Department of Food Quality and Nutrition Department, IASMA Research and Innovation
18 Centre, Fondazione Edmund Mach (FEM), Via E. Mach 1, 38010 San Michele all' Adige, (TN),
19 Italy
208Perrotis College, American Farm School. Thessaloniki, GR-57001. Greece
219Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200,
22 Thailand
23
24 Corresponding authors: [email protected] and [email protected]
25
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3389v1 | CC BY 4.0 Open Access | rec: 3 Nov 2017, publ: 3 Nov 2017
27 Abstract
28 To further understand the impact of grafting on fruit characteristics and to comprehend the
29 mechanisms involved in graft-induced changes we studied homo- and hetero- grafted Cucurbita
30 pepo cultivars (cv.) that vary in fruit size and shape. C. pepo cv. ‘Munchkin’ (small fruit) and cv.
31 ‘Big Moose’ (large fruit) as well as cv. ‘Round green’ (round fruit) and cv. ‘Princess’ (elongated
32 fruit) were homografted and reciprocally heterografted. The results show significant changes in
33 fruit size when ‘Big Moose’ was grafted onto ‘Munchkin’ rootstocks in comparison to homo-
34 grafted controls. Moderate changes in fruit shape were observed when grafting of cv. ‘Round
35 green’ and cv. ‘Princess’ were performed. This is the first report of such phenotypic changes
36 after intra-species/inter-cultivar grafting in Cucurbitaceae. Additionally, we found significant
37 changes in i) secondary metabolite profile, ii) global DNA methylation pattern and iii) miRNA
38 expression patterns in grafted scions and iv) DNA methylation on graft-induced phenotypic
39 changes in grafted plants. Our results contribute to further understanding graft-induced effects on
40 fruit morphology in intra-species grafting. Furthermore, our results pave the way for
41 understanding the role of phenolic metabolites and epigenetic molecular mechanisms on the
42 phenotypic changes recorded.
43
44 Keywords: Cucurbitaceae, MSAP methylation sensitive amplified polymorphisms markers,
45 epigenetic, phenolics, miRNAs, fruit morphology
46
47
48
49
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3389v1 | CC BY 4.0 Open Access | rec: 3 Nov 2017, publ: 3 Nov 2017
50 Introduction
51 Grafting is an important cultivation technique in agriculture, employed widely to improve
52 performance of important horticultural crops such as woody fruit trees, grapes and lately on
53 vegetable crops (Goldschmidt 2014; Mudge et al. 2009). It is an ancient technique used for over
54 2,000 years that was first applied in woody species for improving yield and disease resistance
55 (Kubota et al. 2008). Vegetable grafting which originated in the early 20th century, involves
56 mainly Cucurbitaceae and Solanaceae species and has been utilized extensively for enhancing
57 fruit quality, resistance to diseases and tolerance to abiotic stresses (Goldschmidt 2014; Mudge et
58 al. 2009). Grafting was originally employed to avoid the detrimental effects of soil borne
59 pathogens infecting the roots of susceptible plants through the use of resistant rootstocks grafted
60 onto the plant of interest (King et al. 2008; Louws et al. 2010). Selected beneficial rootstocks can
61 provide resistance to bacteria, nematodes, fungi, as well as enhanced tolerance to abiotic stresses
62 such as extreme temperatures, increased salinity, increased calcium and heavy metals in the soil
63 (Colla et al. 2010; King et al. 2008).
64 Whereas horticulturists were trying to improve crops through grafting in order to provide
65 higher biomass performance and resistance in plants’ diseases and biotic stress, the quality
66 (nutritional/organoleptic properties) of product didn’t attract much attention. Nowadays such
67 kind of improvement strategies seem to focus also on some specific compounds contributing to
68 the nutritional value of vegetables, such as polyphenols, carotenoids, volatiles etc. Polyphenols,
69 as products of secondary metabolism, have received much attention the last decades, due to their
70 antioxidant (Valenzuela et al. 2014), antimicrobial (Nascimento et al. 2014), antiviral (Katayama
71 et al. 2013) bioactivities among others. In the past decade, a great number of studies regarding
72 Cucurbitaceae grafting aimed at improving fruit quality and plant vigour of economically
73 important vegetables such as cucumber, melon and watermelon (Cohen et al. 2007). Grafting of
74 watermelon onto selected rootstock genotypes resulted in significant increase of lycopene
75 content in watermelon fruits in addition to enhanced resistance to soil-borne diseases (Mohamed
76 et al. 2012), whereas different rootstock-scion combinations led to alterations in the content of
77 plant nutrients in leaves and fruits of watermelon grafts (Yetisir et al. 2013). Moreover,
78 cucumber and melon grafted onto specific pumpkin rootstocks led to altered organoleptic
79 properties in addition to enhanced tolerance to high salinity (Huang et al. 2013; Orsini et al.
80 2013; Rouphael et al. 2012). Numerous complex processes occur during grafting, including
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3389v1 | CC BY 4.0 Open Access | rec: 3 Nov 2017, publ: 3 Nov 2017
81 formation of the graft union and the common vasculature, transport of information from
82 rootstock to scion and vice versa, as well as the differing effects of scion and rootstock genotype
83 combinations. Thus, it is reasonable to assume multiple molecular mechanisms operating
84 throughout the growth of a grafted plant. These mechanisms include transport of important
85 information molecules such as small RNAs as well as epigenetic effects, such as methylation of
86 DNA, both of which can, in turn, affect gene expression. Trafficking of macromolecules such as
87 proteins and of genetic material or hormones and nutrients, have been demonstrated in many
88 instances between grafting partners (Albacete et al. 2015). Regarding movement of genetic
89 material, an increasing number of reports implicate long distance transport of RNA molecules
90 such as messenger and small RNAs. Movement of RNA molecules over long distances, across
91 plant organs and through the phloem has been well documented (Kalantidis et al. 2008; Kehr &
92 Buhtz 2008; Lough & Lucas 2006; Lucas et al. 2001; Mermigka et al. 2015; Omid et al. 2007;
93 Spiegelman et al. 2013). Similarly, transport of small non-coding RNAs (siRNAs and miRNAs)
94 across graft segments of homo-grafted and hetero-grafted plants has been demonstrated in a
95 number of studies in Arabidopsis, Solanaceae and Cucurbitaceae (Haroldsen et al. 2012; Melnyk
96 et al. 2011; Palauqui et al. 1997).. In many occasions mobile RNA molecules have a
97 transcriptional or post-transcriptional gene silencing (Haroldsen et al. 2012; Melnyk et al. 2011;
98 Palauqui et al. 1997). It is known that miRNAs play crucial roles in all plant biological
99 processes, including growth, vegetative and reproductive development, and response to abiotic
100 and biotic stresses (Barrera-Figueroa et al. 2013; Li et al. 2016; Zhang 2015). MiRNAs are post-
101 transcriptional gene regulators that target mRNAs and either cleave the transcript or inhibit
102 translation (Dong et al. 2013). They target a wide range of transcription factors related to
103 developmental processes as well as genes that code for enzymes involved in metabolic pathways.
104 Several evolutionary conserved families of miRNAs such as the miR156, miR159, miR165/166,
105 mirR171, miR172, control various aspects of development in plants. MiR156 has been
106 associated with changes in leaf biomass as its overexpression led to significant increase in total
107 leaf number and subsequent plant biomass in Arabidopsis (Schwab et al. 2005).
108 Global DNA methylation studies, using either methylation sensitive amplified
109 polymorphisms (MSAP) or locus-specific bisulfite sequencing analyses revealed significant
110 alterations in the DNA methylation pattern of grafted plants after reciprocal inter-species grafting
111 among three Solanaceae species (Wu et al. 2013). In particular, the global DNA methylation
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112 pattern of tomato, eggplant and pepper scions was shown to be extensively altered in two DNA
113 methylation contexts (CG and CHG) upon grafting. Importantly, the graft-induced methylation
114 changes were inherited in the self-pollinated progeny, indicating that grafting can induce
115 epigenetic changes that can be stably transferred to subsequent generations (Wu et al. 2013). In
116 agreement with the study above, our group has recently demonstrated altered DNA methylation
117 patterns upon inter-species hetero-grafting of three Cucurbitaceae species 33. In particular, DNA
118 methylation levels were significantly increased in melon and cucumber scions grafted onto
119 pumpkin rootstocks (Avramidou et al. 2014).
120 Fruit morphology, which includes fruit shape and size, is another important trait of
121 agronomic importance in vegetable species of the Solanaceae and Cucurbitaceae families.
122 However, although studies during the past decades have focused on fruit yield and quality
123 (texture, aroma, flavor, nutrients), resistance to diseases and tolerance to abiotic stresses, little is
124 known about the effect of grafting on fruit morphology and the underlying molecular
125 mechanisms governing this process. A few reports have described an effect on fruit shape upon
126 intra-species grafting in pepper (Capsicum annuum) (Taller et al. 1999; Tsaballa et al. 2012;
127 Yagishita & Hirata 1987). Tsaballa et al. (2012) 35 have demonstrated remarkable effects on fruit
128 shape of the scion partner in a rootstock–scion combination of two different pepper genotypes
129 with different fruit shape. Specifically, grafting of the round-fruit shaped cv. ‘Round’, onto a
130 long-fruit shaped cv. ‘Long’, resulted in substantially elongated fruits in the scion cv. ‘Round’
131 plants (Tsaballa et al. 2012). However, there are no reports, to date, concerning the effects of
132 grafting on fruit morphology in Cucurbitaceae. In order to further investigate grafting in
133 Cucurbitaceae and the effect of rootstock-scion combinations in fruit size and shape we have
134 applied reciprocal hetero-graftings of two different C. pepo cvs., ‘Big Moose’ and ‘Munchkin’,
135 with different fruit sizes as well as two other C. pepo cvs., ‘Princess’ and ‘Round green’, with
136 different fruit shapes. In addition, we have assessed changes in DNA methylation patterns
137 employing comparative MSAP methodology which allows for the documentation of epigenetic
138 alterations between different crosses and between plants bearing fruits of different sizes and
139 morphology. Further, we have analyzed the expression of several miRNAs in order to probe
140 variations of these epigenetic modifiers linked to the grafting process and possibly with grafting-
141 induced fruit morphological changes. Additionally, we determined concentrations of a series of
142 selected secondary metabolites and compared thus produced metabolic profiles between different
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3389v1 | CC BY 4.0 Open Access | rec: 3 Nov 2017, publ: 3 Nov 2017
143 grafting combinations and non-grafted controls. We report here graft-induced changes in fruit
144 size and moderate changes in fruit shape of C. pepo scions accompanied by significant changes
145 in scion genomic DNA methylation, patterns, abundance of specific miRNA molecules and
146 secondary metabolites as compared to non-grafted control tissues.
147
148 Materials and Methods
149 Plant Material
150 Two independent grafting and MSAP experiments were conducted during two
151 consecutive summer periods (2014 and 2015). Samples from 2014 were used for MSAP analysis
152 in relation to fruit size, whereas samples from 2015 were used for MSAP analysis in relation to
153 fruit shape and metabolite profile.
154 Four Cucurbita pepo cultivars were used for grafting; (1) cv. ‘Round green’ (round fruit
155 shape) (2) cv. ‘Princess’ (long fruit shape) (3) cv. ‘Big Moose’ (large fruit size) and (4) cv.
156 ‘Munchkin’ (small fruit size). Seeds were initially sown in small pots that were kept in the
157 greenhouse at 25 °C. Grafting was carried out by the time plants had 2–3 true leaves and a shoot
158 diameter of about 1.5–2.0 mm. In total, four grafting combinations were produced representing
159 two reciprocal cv. combinations as following: (1) cv. ‘Round green’ on cv. ‘Princess’, (2) cv.
160 ‘Princess’ on cv. ‘Round green’, (3) cv. ‘Big Moose’ on cv. ‘Munchkin’ and (4) cv. ‘Munchkin’
161 on cv. ‘Big Moose’. Grafting was performed following the splice-grafting method employing
162 grafting clips (for a review of grafting methods see (Lee et al. 2010)). A total of 4 plants from
163 each grafting combination i.e. rootstock (RS) x scion (SC) were used for scoring for either fruit
164 size or fruit shape as well as for MSAP analysis. Controls included were (1) the homo-grafted
165 plants for example cv. ‘Round green’ grafted onto cv. ‘Round green’ for all four cvs. and (2) the
166 non-grafted s that served as reference state in MSAP comparisons as well (see below). Following
167 grafting, all plants were transferred in the growth chamber under constant temperature of 25 °C,
168 RH 85–95%, light flux 2,000 lux, photoperiod of 16 h light/8 h dark and kept for 2 weeks.
169 Successfully grafted plants were transferred in the greenhouse for an acclimation period of one
170 month, transferred to 7 L pots filled with calcareous clay soil afterwards and allowed to grow in
171 the open while watered regularly with nutrient solution. The same grafting design was used both
172 years.
173
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174 DNA extraction and ISSR molecular analysis
175 DNA extraction from leaves of controls and scions, from plants presenting statistically
176 different phenotypes were used, was performed using NucleoSpin Plant II Kit (Macherey Nagel,
177 Duren, Germany), according to manufacturer's instructions.
178 For the detection of putative intra-cultivar variation, we selected 6 genotypes of each
179 grafting cultivar combinations (72 individuals in total) and we used a set of six Inter Simple
180 Sequence Repeats (ISSR) primers as described previously by Xanthopoulou et al. (2015). PCR
181 reactions and gel electrophoresis were composed according to the authors above.
182
183 Methylation Sensitive Amplification Polymorphism (MSAP) analysis
184 For each sample, MSAP analyses were performed using both EcoRI/HpaII and
185 EcoRI/MspI digests. Digestion of 200-ng aliquots of genomic DNA with either of the two
186 isoschizomers (HpaII, MspI) was carried out in 20 μl containing 1Χ one for all Buffer, 4 U
187 EcoRI (New England Biolabs, Ipswich, MA, USA) and 3 U of either HpaII or MspI enzyme
188 (New England Biolabs, Ipswich, MA, USA) for 3 h at 37°C. Two different adapters, designed to
189 avoid reconstruction of restriction sites, one for the EcoRI sticky end and one for the HpaII/MspI
190 sticky end, were ligated to the DNA after digestion, by adding to each final digestion 5 μl of a
191 mix containing 5 pmol of EcoRI adapter, 50 pmol of HpaII/MspI adapter, 1 mM ATP, 1Χ one
192 for all Buffer, and 1 U of T4 DNA ligase (Invitrogen, CA, USA). The ligation was incubated for
193 3 h at 25°C. The EcoRI adapter consisted of the combination of two primers: 5Ά-
194 CTCGTAGACTGCGTACC-3Ά and 3Ά-CTGACGCATGGTTAA-5Ά. EcoRI and HpaII/MspI
195 adapters are presented in Table S1. Digested and ligated DNA fragments were diluted 5-fold and
196 used as templates for the pre-amplification reaction. Two different pre-amplification reactions
197 were performed using EcoRI/MspI-A and EcoRI/HpaII-A primers in a total volume of 20 μl
198 containing 1X Kapa Taq Buffer, 0.2mM of each dNTP, 2.5 mM MgCl2, 30 ng of each primer
199 EcoRI+A, HpaII/MspI+A, 1U Taq DNA polymerase (Kapa Biosystems, Woburn, MA) and 5 μl
200 of diluted fragments. PCR amplifications were carried out in a BioRad thermocycler for 23
201 cycles of 30 s at 94°C, 30 sec at 56°C, and 1 min at 72°C. Pre-amplified fragments were diluted
202 10-fold and used as templates for the selective amplification. Οne labelled EcoRI and one
203 HpaII/MspI primer, with the same sequences as those used for the pre-amplification but with two
204 and three selective nucleotides respectively at the 3 end, were used in each analysis. PCR was
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205 performed in a total volume of 10 μl including 1X Kapa Taq Buffer 2.5 mM MgCl2, 0.08 mM of
206 each dNTP, 5 ng of labeledEcoRI primer, 30 ng of HpaII/MspI primer, 1 U of Taq DNA
207 polymerase (Kapa Biosystems), and 3 μl of diluted pre-amplified DNA. The selective
208 amplification was carried out using classical AFLP cycling parameters (Vos et al. 1995),
209 employing eight different primer combinations (Table S1).
210
211 MSAP data collection and processing
212 MSAP product mixtures were denatured in formamide at 94°C for 2 min and
213 electrophoretically fractionated on an ABI Prism 3730xl (Applied Biosystems, Carlsbad, CA,
214 USA). In order to reduce the impact of potential size homoplasy only fragments ranging from
215 150 to 500 bases in size were counted (Vekemans et al. 2002). Fragment size was determined
216 using Genemapper v4.0 (Applied Biosystems, Carlsbad, CA, USA) employing an internal
217 standard (GS 500 LIZ, Applied Biosystems) and an initial data matrix was produced with allele
218 presence scored as “1” and absence as “0”.
219 Our scoring initially entailed determining marker presence / absence between a seedling
220 (reference state) and grafted plants of the same cv. even if a single band in one of the two
221 isoschizomer profiles of a single individual was detected. Scions from “homo-” or “hetero-”
222 grafted plants were scored independently and were considered as two different treatment states.
223 The same individual control seedlings were considered as reference states in comparisons
224 involving scions of the same cv. irrespective of rootstock cv. (i.e. irrespective of “homo-” or
225 “hetero-” treatment states). Each methylation state was described with two binary digits (duplets)
226 corresponding to the two isoschizomer profiles. Quadruplets (a series of four binary digits) were
227 subsequently, produced by pooling together duplets from the reference and from the treatment
228 state (two different treatment states in total; “homo-” and “hetero-”) and were assigned to state
229 change events between reference and treatment states. Sixteen alternate quadruplets could thus
230 be produced each grouped according to (Haoa et al. 2004) as de novo methylation (three events),
231 demethylation (three events), other (ambiguous or too complex pattern to be explained; four
232 events) or no change (six events; not specifically enumerated) (Haoa et al. 2004). All data
233 management steps, from initial matrix formation till event change classification and tallying
234 were performed employing an in-house script. Only polymorphic fragments (more than 5%
235 present or absent) were retained for subsequent analysis (MSAP polymorphic loci). Once the
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236 quadruplets of each sample (grafting combination) have been derived (by comparison to
237 respective non-grafted controls) they were categorized according to deduced methylation change
238 event (de-methylation, de novo methylation, no change, other-non classifiable). The number of
239 each one of the four change events -per sample- was converted to percentages against the
240 number of total events of each sample separately. Four different percentages are produced per
241 each sample corresponding to the four different change events. Comparisons and statistical
242 analyses of such percentage across samples could thus be made (Figures 3 and 4). Change event
243 “other” is not portrayed in Figures 3 and 4). Subsequently, statistically significant differences
244 between the two treatment states i.e. “homo-“ vs. “hetero-“ grafted plants were assessed for the
245 MSAP polymorphic loci by means of a t-test employing SPSS (ver. 22). Separate tests were
246 performed for each cv. serving as rootstock (four independent experiments / statistical analyses
247 in total) and the two combinations employing the self cv. as scion.
248
249 miRNA expression profile via stem-loop RT-PCR
250 Total RNA was extracted from leaves (from plants presenting statistically different
251 phenotypes) using the TRIzol® Reagent (Invitrogen, USA) and DNase I was then used to
252 remove DNA. The integrity and purity of the total RNA were evaluated using 2% denaturing gel
253 electrophoresis and NanoDrop DU8000 spectrophotometry (A260/A280 and A260/A230). The
254 expression profiles of five miRNAs were assayed by stem–loop reverse transcription-PCR (RT–
255 PCR). A 200 ng aliquot of total RNA was used for the initiation of the reverse transcription
256 reaction. The stem–loop reverse transcription primers were designed following the method
257 described by (Varkonyi-Gasic et al. 2007). The reverse transcription product is amplified using a
258 miRNA-specific forward primer and a universal reverse primer. The stem–loop reverse
259 transcription reactions were performed by Superscript III reverse transcriptase (Life
260 Technologies, Carlsbad, CA, USA) transcriptase according to the supplier's manual. PCR
261 primers were then added to perform the PCR. Relative quantification was performed using
262 Elongation Factor-1 (EF-1) as the endogenous control reference gene (Obrero et al. 2011). The
263 data were analyzed using the REST software (Pfaffl et al. 2002).
264
265 Phenolic metabolite analysis
266 Fresh collected samples (each sample obtained from pooling fruit material from three
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267 individual plants) were freeze dried (Freeze-dryer Alpha 1-2 LD plus, Christ, Germany; at -
268 24°C), and then pulverized to fine powder. The extraction was performed by mixing 100 mg of
269 freeze-dried fruit sample with 5 mL 80% methanol into 15 mL falcon tube. The samples and
270 solvent were mixed by orbital shaker for 3 h at room temperature and the extraction proceeded
271 overnight at 4 °C in the dark. The resulting solutions were filtered on a 0.22 µm PFTE membrane
272 into a glass vial and analyzed as described below. Three replicates for each sample were done.
273 The analysis of phenolic compounds was performed using the method described previously
274 by (Vrhovsek et al. 2012). Samples were directly injected after extraction. Targeted Ultra
275 Performance Liquid Chromatography was performed on a Waters Acquity UPLC system
276 (Milford, MA, USA) consisting of a binary pump, an online vacuum degasser, an autosampler,
277 and a column compartment. Separation of the phenolic compounds was achieved on a Waters
278 Acquity HSS T3 column 1.8 μm, 100 mm × 2.1 mm (Milford), kept at 40 °C.
279 Mass spectrometry detection was performed on a Waters Xevo TQMS (Milford)
280 instrument equipped with an electrospray (ESI) source. Data processing was performed using the
281 Mass Lynx Target Lynx Application Manager (Waters).
282
283 Results
284
285 Graft-induced changes in fruit size and shape
286 Two C. pepo cultivars differing in fruit size, cv. ‘Munchkin’, a small fruited cultivar, and
287 cv. ‘Big Moose’, a large fruited cultivar (Figure 1A) and two cultivars differing in fruit shape,
288 cv. ‘Round green’, a round fruited cultivar and cv. ‘Princess’, an elongated fruited cultivar
289 (Figure 2A), were used to assess possible graft-induced alterations in fruit morphology in
290 reciprocal hetero-grafting combinations. Fruit morphology of grafted plants was assessed using
291 morphological traits according to UPOV descriptors such as weight, polar diameter and
292 equatorial diameter for the scion fruits from each grafting combination as well as from control
293 (non-grafted). The majority of the fruits produced by the ‘Munchkin’ and ‘Big Moose’ grafting
294 combinations although they had the expected phenotype i.e. they were similar to the fruit
295 phenotype of the non-grafted scion in every case (Figure 1B), they presented statistically
296 significant phenotypic differences with the fruits from the non-grafted or homografted plants in
297 terms of weigh, equatorial and polar diameter. In addition a single grafted ‘Munchkin’ (RS) x
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298 ‘Big Moose’ (SC) plant (termed “with altered fruit size”; Table 1) produced fruits with
299 pronounced morphological differences when compared to the non-grafted ‘Big Moose’ plants
300 (Figure 1C). A Distinct reduction in fruit weight and fruit size in this plant could be observed
301 when compared to the rest of the fruits produced by this grafting combination (Figure 1D).
302 Further, the reduction in fruit weight was approximately 2.6-fold compared to ‘Big Moose’ non-
303 grafted control, more than 3-fold compared to ‘Big Moose’ homografts and 2.4-fold compared to
304 the majority of the ‘Munchkin’ (RS) x ‘Big Moose’ (SC). In addition, a significant decrease in
305 both equatorial and polar fruit diameter of about 2-fold was evidenced in this heterograft as
306 compared to the control non-grafted ‘Big Moose’ and Munchkin’ (RS) x ‘Big Moose’ (SC)
307 plants (Table 1).
308
309 On the other hand the reciprocal combination, ‘Big Moose’ (RS) x ‘Munchkin’ (SC) did
310 not produce any discrete evident morphological changes (i.e. in fruit size) like the one before
311 (Figure 1B-bottom) (Table 1). However, moderate changes statistically significant were observed
312 in fruit shape in the reciprocal combinations of ‘Round green’ and ‘Princess’. ‘Princess’ (RS) x
313 ‘Round green’ (SC) resulted in less round fruits and the reverse combination, ‘Round green’
314 (RS) x ‘Princess’ (SC) led to fruits that were smaller as compared to the control seedlings
315 (Figure 2B). Grafting combinations of ‘Princess’ and ‘Round green’ did not produce extreme
316 differences in terms of morphological traits yet there were statistically significant especially for
317 equatorial and polar diameter when compared to non-grafted or homografted plants (Table 1).
318 To exclude the possibility that fruit morphology changes rise from genetic variation
319 within cultivars or cross-pollination between them since C. pepo is an openly pollinated species,
320 molecular analysis with six polymorphic Inter simple sequence repeat (ISSR) markers was
321 performed according to (Xanthopoulou et al. 2015). No intra-cultivar variation was found
322 among the 72 examined plants suggesting that epigenetic (rather than genetic) changes
323 associated with grafting should be sought as the underlying mechanism inducing morphological
324 alterations.
325
326 Graft-induced changes in scion global DNA methylation pattern
327 The effect of grafting on the pattern of scion DNA methylation pattern was assessed
328 utilizing a comparative MSAP analysis (Supplementary Figure 1). Initially, the MSAP method
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329 can detect the DNA methylation status of 5’-CCGG-3’sites, employing two pairs of
330 isoschizomers EcoRI/HpaIIand EcoRI/MspI that have different sensitivity to methylation at the
331 inner or outer cytosines. For each sample the DNA methylation pattern at 5΄-CCGG-3΄ sites is
332 determined by scoring the presence or absence of marker bands of EcoRI/HpaII and EcoRI /MspI
333 digestion of genomic DNA followed by PCR amplification (Supplementary Figure 1).
334 Differences in PCR products obtained from two different samples reflect different methylation
335 states at the cytosines restriction site. Per sample, methylation patterns are deduced by
336 determining amplification differences between the two pairs of isoschizomers while methylation
337 pattern changes are determined following comparisons between treatments (different grafting
338 combinations) and controls (non-grafted seedlings). We consider the case with four different
339 methylation change events (1. de novo methylation, 2. demethylation, 3. no change, 4. other
340 variation).
341 As a first step, methylation change (MSAP) data were averaged and plot by event and by
342 grafting combination (Figures 3 and 4). In the ‘Munchkin’ (RS) x ‘Big Moose’(SC) combination,
343 which resulted in the significant fruit size reduction, an increase of approximately 5-fold in DNA
344 de novo methylation was observed in the scion leaves compared to the control ‘Big Moose’ non-
345 grafted seedlings (Figure 3A). In addition, an increase of about 2-fold in demethylation was
346 observed in the scions in comparison to the control ‘Big Moose’ non-grafted, whereas 40% of
347 cytosine sites remained unchanged (Figure 3A). In the ‘Big Moose’ (RS) x ‘Munchkin’ (SC)
348 combination an increase in DNA methylation of approximately 2-fold was evidenced in the
349 leaves of the scion plants as compared to the ‘Munchkin’ control seedlings, whereas a change of
350 approximately 8-fold in DNA demethylation levels was detected (Figure 3B). In the ‘Princess’
351 (RS) x ‘Round green’ (SC) graft which resulted to a flat-shaped-fruit, a remarkable increase in de
352 novo DNA methylation of 20-fold is evidenced in the scions as compared to the control ‘Round
353 green’ non-grafted (Figure 4A). A marked increase in DNA demethylation of 12-fold was also
354 observed in the ‘Round green’ scions, as compared to the control ‘Round green’ (Figure 4A).
355 Likewise, in the ‘Round green’ (RS) x ‘Princess’ (SC) graft, which resulted in a smaller fruit
356 compared to the non-grafted seedling, a significant increase in DNA methylation of
357 approximately 16-fold and an increase in DNA demethylation of about 12- fold was observed in
358 ‘Princess’ scions, relative to the control ‘Princess’ non-grafted plants (Figure 4B).
359 As a second step, methylation change data underwent a detailed statistical analysis. The
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360 chi-square test was applied to test the null hypothesis that homografting vs heterografting (four
361 independent comparisons-please see a)-d) below) are homogeneous regarding the methylation
362 event, i.e. the respective theoretical distributions of percentages regarding the categories of the
363 methylation event do not differ statistically significantly. The significance level was set to 0.05.
364 We considered the case with four different methylation event categories (1. de novo methylation,
365 2. demethylation, 3. no change, 4. other variation). A significant result would indicate significant
366 overall difference in the methylation categories’ percentages between homografting and
367 heterografting, and therefore statistically significant difference in how methylation is expressed
368 in these two cases. We performed independently four comparisons, i.e. a) ‘Munchkin’ homo
369 versus ‘Big Moose’ x ‘Munchkin’, b) ‘Big Moose’ homo versus ‘Munchkin’ x ‘Big Moose’, c)
370 ‘Princess’ homo versus ‘Round green’ x ‘Princess’ and d) ‘Round green’ homo versus ‘Princess’
371 x ‘Round green’. The results were a) 46.256, p-value<0.001, b) 49.640, p-value<0.001, c)
372 16.215, p-value=0.001 and d) 29.867, p-value<0.001. Therefore, the null hypothesis of
373 homogeneous methylation behaviour was rejected for all comparisons, i.e. all differences
374 considered were statistically significant. In addition to the above comparisons, for each one of
375 the four cultivars, the goodness of fit test was applied to test the null hypothesis that the
376 theoretical percentages regarding the categories of the methylation event are equal, and by that to
377 actually test if the type of methylation event appears at random within each cultivar. The results
378 were i) 307.668, p-value<0.001, ii) 304.480, p-value<0.001, iii) 248.066, p-value=0.001 and iv)
379 120.224, p-value<0.001. Therefore, the null hypothesis of equal methylation categories’
380 percentages was rejected for all comparisons, i.e. the type of change did not appear at random.
381 The Cochran’s criterion for the validity of all the above chi-square based tests has been checked
382 and was satisfied in all cases.
383 Graft induced changes in miRNA expression
384 As depicted in Figure 5A, significant changes were observed in expression levels of a
385 number of miRNAs in the ‘Munchkin’ and ‘Big Moose’ grafted plants, when compared to the
386 non-grafted controls. In the ‘Munchkin’ (RS) x ‘Big Moose’ (SC) that was the combination in
387 the grafted plant with the reduced fruit size, the expression of miR159 and miR164 was
388 significantly decreased by approximately 4-fold, whereas miR171 was decreased by 2-fold and
389 miR166 by 1.6-fold. On the contrary, non-significant expression changes were recorded in the
390 scions of the ‘Big Moose’ (RS) x ‘Munchkin’ (SC) heterograft, except for miR171, which was
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391 induced by about 5-fold and miR164, which was 1.5 times more abundant in comparison to the
392 control (Figure 5A). A significant induction of about 2-fold, for miR171 was also observed in the
393 ‘Round green’ (RS) x ‘Princess’ (SC) heterograft, which had a mild effect in fruit shape (Figure
394 5B). A reduction of 1.6-fold was evidenced for miR159 in the ‘Round green’ (RS) x ‘Princess’
395 (SC) heterograft (Figure 5B).
396
397 Phenolic metabolite analysis
398 Fruit samples from all grafting combinations were subjected to targeted LC-MS/MS
399 analysis in order to identify the effect and possible changes on the secondary metabolite level in
400 different grafting combinations. In total, seventeen compounds were identified in C. pepo fruit
401 samples from the size differing cultivars and their grafting combinations while eighteen
402 compounds were identified in the shape differing cultivars and their grafting combinations.
403 Compounds detected varied in presence and concentration, as shown in the heatmaps produced
404 (Figures 6A and B). In the majority of samples, kaempferol-3-O-rutinoside (Km3rut), rutin and
405 isorhamnetin-3-O-rutinoside (Iso3rut) were the most abundant compounds, whereas in the
406 cultivars differing in fruit size significant amounts were detected for p-hydroxybenzoic (p-HBA),
407 cinnamic, ferulic and vanilic acid in scion fruits. In the different cultivars, nine (‘Big Moose’,
408 ‘Princess’), ten (‘Munchkin’) and fourteen (‘Round green’) phenolic metabolites were identified
409 (Tables 2 and 3), while several metabolites seem to be influenced by grafting combination.
410 Concerning the fruit cultivars differing in shape, some of the identified metabolites such as
411 Km3rut, quercetin-3-O-glucoside, rutin and Iso3rut, which are present in the control non grafted
412 and the ‘Big Moose’ homograft as long as in the heterograft ‘Munchkin’ (RS) x ‘Big Moose’
413 (SC), were absent in all the rest of grafting samples. On the other hand, the compounds p-HBA,
414 sinapoyl alcohol and arbutin were present only in the fruits obtained from the ‘Munchkin’ non-
415 grafted plants, its homografts, and the heterografts where ‘Munchkin’ cultivar was used as the
416 scion. Furthermore, it became apparent in the case of the heterograft ‘Princess’ (RS) x ‘Round’
417 (SC) that there was a stimulation in most of the identified metabolites in the cultivars with green
418 fruit colour, especially in the category of rutinosides. Both homografts of ‘Princess’ and ‘Round
419 green’ exhibited a limited level of metabolites.
420
421 Discussion
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3389v1 | CC BY 4.0 Open Access | rec: 3 Nov 2017, publ: 3 Nov 2017
422 In the current study we investigated the effect of C. pepo intra-species grafting on scion’s
423 fruit size and shape and its association with global epigenetic and metabolic profiles. The
424 heterograft of two C. pepo cultivars with extremely different fruit size, ‘Munchkin’ (RS) x ‘Big
425 Moose’ (SC) displayed remarkable alterations in fruit size as it resulted in a dramatic decrease in
426 fruit mass and a reduction in both equatorial and polar fruit diameter. This is the first time that
427 intra-species grafting in a Cucurbita species results in changes in fruit morphology. However,
428 grafting has been associated with changes in a number of fruit quality characteristics in the past.
429 Pepper is one of the plants where fruit shape changes attributed to grafting were early recorded
430 and actually found to be inherited to the grafting progenies introducing the whole concept of
431 grafting-induced phenotypic variation) (Taller et al. 1999; Tsaballa et al. 2012; Yagishita &
432 Hirata 1987). (Taller et al. 1999) managed to produce pepper varieties with stable but different
433 fruit characteristics such as fruit shape and flavour from the graft-induced variants of an intra-
434 species grafted plant. In our previous work we have found that grafting-induced fruit shape
435 changes were stably inherited to the grafting progenies when a round-fruited pepper cultivar was
436 grafted on a long-fruited pepper cultivar; scion’s fruits in this case resembled more the fruit
437 shape of the rootstock than that of the scion (Tsaballa et al. 2012). These findings support the
438 already formed idea of graft ‘hybridization’ that involves the movement of genetic material from
439 the rootstock to the scion, a process also called graft transformation and results in changes that
440 are heritable to the scion’s progenies (Goldschmidt 2014; Mudge et al. 2009). It was recently
441 found that whole nuclear genomes can be transferred between plant cells during grafting creating
442 new allopolyploid species (Fuentes et al. 2014). Our results support the notion that grafting is
443 associated with genetic changes in the phenotype of the grafted plants in a way that it could also
444 be used for the introduction of new characteristics in crops.
445 Apart from genetic changes leading to phenotypic changes, agronomical traits such as
446 yield, plant architecture, fruit morphology, fruit quality, as well as the response to abiotic stress
447 or resistance to diseases can be affected substantially by changes in DNA methylation, histone
448 post-translational modifications and miRNAs, all leading to profound changes in gene expression
449 (Dong et al. 2013; Fujimoto et al. 2012; Tsaftaris et al. 2012; Zhong et al. 2013). Likewise, the
450 same traits can be affected by grafting, while increasing evidence has indicated that grafting may
451 induce DNA methylation-pattern alterations. Global DNA methylation patterns were found
452 significantly altered in Solanaceae and Cucurbitaceae inter-species grafting (Avramidou et al.
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453 2014; Wu et al. 2013). In Solanaceae species grafting, scion genomic loci with altered DNA
454 methylation patterns contained genes associated to diverse cellular functions while a series of
455 genes encoding DNA methylation-related enzymes were found to have altered gene expression
456 patterns (Wu et al. 2013). In addition, a portion of DNA methylation changes were found to be
457 inherited in the self-pollinated progeny (Wu et al. 2013). Changes in DNA methylation patterns
458 were also shown in grafts between Brassica juncea and B. oleraceae and it was suggested that
459 they are possibly linked to the phenotypic variability observed although in these species it was
460 found that the changes can be both heritable and reversible in the subsequent generations (Cao et
461 al. 2016). In our study, we have noticed significant changes in the DNA methylation pattern of
462 the grafted plants. In the ‘Munchkin’ (RS) x ‘Big Moose’ (SC) combination which resulted in a
463 dramatic fruit size reduction, de-novo DNA methylation levels were markedly increased,
464 suggesting that graft-associated DNA methylation alterations in certain loci may be responsible
465 for altering fruit shape in the scion. On the other hand, in the reciprocal combination, ‘Big
466 Moose’ (RS) x ‘Munchkin’ (SC), a significant increase in DNA demethylation levels was also
467 detected. Although fruit morphology changes were not evidenced in this combination it is
468 possible that increased DNA demethylation has affected other loci associated with fruit quality,
469 and/or environmental responses. Further experiments are needed in order to elucidate the effect
470 of enhanced DNA demethylation in these grafts. Fruit shape changes on grafted plants was also
471 found to be accompanied by alterations in the DNA methylation patterns of scion leaves as
472 shown by the results of ‘Princess’ (RS) x ‘Round green’ (SC) and the reciprocal graft
473 methylation patterns.
474 Changes in DNA methylation of the graft partners could result in profound changes in
475 gene expression in both scion and rootstock tissues and consequently affect a multitude of
476 cellular pathways. The latter can be associated with distinct phenotypes including shoot
477 architecture, total yield, fruit morphology and quality, and responses to environmental stress. Our
478 data showed that intra-species Cucurbita heterografting altered fruit morphology. In the past we
479 have reported similar results when intra-species grafting in pepper using two cultivars of
480 different shape, cv. ‘Long’ (RS) x cv. ‘Round green’ (SC) resulted in a change in scion’s fruit
481 morphology as the fruits had acquired a more elongated shape compared to the control s and the
482 homografted plants, resembling more the fruit phenotype of the rootstock rather than the scion.
483 Taking into account all the above considerations, it is possible that fruit morphology changes in
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484 inter-species heterografting of Cucurbitaceae could be associated with DNA methylation
485 alterations in scion tissues that in turn affect gene expression programs linked to fruit
486 morphology traits.
487 However, epigenetic changes in gene expression that may result in phenotypic variation
488 are not only produced by changes in DNA methylation but by other epigenetic mechanisms
489 linked to small RNAs. The 21nt small RNA class of miRNAs has been shown to play crucial
490 roles in plant growth and development controlling seed, vegetative, flowering, reproductive and
491 developmental processes and the responses to abiotic and biotic stress (Li et al. 2016; Zhang
492 2015). In addition, changes in miRNA expression have been demonstrated recently to take place
493 during grafting. MiRNA transcriptomic analyses in Cucurbitaceae grafted plants have revealed
494 that groups of conserved miRNAs such as miR156, miR159, miR164, miR165/166, miR171,
495 miR172, were either induced or repressed in inter-species hetero-grafted scions implying a
496 regulatory role of miRNAs in the grafting process (Li et al. 2013). In our experiments, miR159,
497 miR164, miR166 and miR171 showed significant downregulation in the ‘Munchkin’ (RS) x ‘Big
498 Moose’ (SC) heterograft that resulted in the changed, smaller fruit. These results point out that
499 there is a graft-induced miRNA controlled epigenetic mechanism that possibly underlies fruit
500 development in grafted scions. MiR156 targets plant specific transcription factors of the
501 SQUAMOSA-promoter binding protein (SBP) type, or SBP-like (SPL) type, as well as
502 APETALA2 (AP2) which have been associated with flowering time and organ size (Kim et al.
503 2012; Wang et al. 2009; Wang et al. 2008; Zhang et al. 2011). MiRNA 159 has been implicated
504 in floral meristem development by targeting SPL transcription factors which in turn regulate
505 SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), LFY and AP1 genes
506 involved in SAM cell differentiation and floral meristem initiation (Achard et al. 2004). In
507 addition, miR159 mediates floral meristem formation by regulating MYB family transcription
508 factors and in particular gibberillin (GA)-specific MYBs. Loss of function
509 of mir159a and mir159b results in the abnormal expression of MYB33 and MYB65 which
510 influences plant development and decreases cell proliferation in leaves (Allen et al. 2010; Allen
511 et al. 2007). Mir164 controls proper formation and separation of adjacent plant organs by
512 targeting genes coding for the NON APICAL MERISTEM (NAM), ATAF and NAC family of
513 transcription factors. These genes regulate the establishment and maintenance of the shoot apical
514 and axillary meristem during floral development and control the formation of floral organs and
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515 organ boundaries (Aida & Tasaka 2006; Baker et al. 2005; Kusumanjali et al. 2012; Mallory et
516 al. 2004). MiRNA172 targets the transcription factor gene AP2 and regulates floral meristem
517 initiation, flower patterning and floral organ identity (Chen 2004; Li & Zhang 2015). In addition,
518 miR172 which is regulated by FRUITFULL and AUXIN RESPONSE FACTOR positively
519 controls carpel size via repression of AP2 (Ripoll et al. 2015). MiRNA171 targets the GRAS
520 family transcription factors that play a key role in meristem maintenance, shoot development and
521 flowering time (Wang et al. 2010). Overexpression of miR171 in Arabidopsis led to altered plant
522 height, short branching, root elongation, leaf shape and flower structure (Wang et al. 2010).
523 Overexpression of miR171 in rice and barley had an effect on phase transitions and floral
524 meristem determinacy (Curaba et al. 2013; Fan et al. 2015), whereas overexpression of miR171
525 in tomato resulted in taller plants and earlier flowering (Huang et al. 2013). In conclusion all the
526 miRNAs that were found to be downregulated in the grafted plants we used have been shown to
527 be implicated directly or indirectly in processes linked to cell, meristem, flower and fruit
528 development. Changes in expression of such important miRNA molecules may have huge
529 implications in fruit morphology and may be responsible for the altered phenotypes of the
530 grafted plants. It is possible that some of these miRNA expression changes control key-genes in
531 the fruit growth process that result in grafted plant’s fruit phenotypic change.
532 Apart from the transcriptomic level was studied the grafting effect on the qualitative and
533 quantitative profile of secondary metabolites, mainly polyphenols, of C. pepo fruit samples. A
534 fast, efficient and sensitive state of the art targeted LC-MS/MS analysis was used for this
535 purpose. In the samples used, seventeen phenolic metabolites were identified in comparison to
536 (Iswaldi et al. 2013) who reported twenty-five phenolic compounds obtained from two green
537 zucchini varieties. To a certain extend qualitative differences may arise due to different technical
538 issues: extraction methods, plant material used (genotype, tissue), environmental factors
539 (altitude, climate, rainfall), abiotic stress, drought, UV-radiation, salt stress and nutrient
540 availability, harvesting season, and/or postharvest processing of fruits fruits (Boeing et al. 2014;
541 Cheynier et al. 2013; Nayak et al. 2015; Tiwari & Cummins 2013). Five additional metabolites
542 were identified in this study; elculetin, coniferyl and sinapoyl alcohol, arbutin, carnosic acid and
543 carnosol, even as traces. In fact, to the best of our knowledge this is the first report that the last
544 two diterpenes (carnosic acid and carnosol) are detected in C. pepo, except for Rosmarinus and
545 Salvia species (Birtić et al. 2015; Sarrou et al. 2016). The promotive effect of grafting on
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3389v1 | CC BY 4.0 Open Access | rec: 3 Nov 2017, publ: 3 Nov 2017
546 biochemical and nutritional traits of some vegetables such as tomato, watermelon and eggplant
547 has been previously reported (Krumbein & Schwarz 2013; Kyriacou et al. 2015; Nicoletto et al. 2013;
548 Soteriou et al. 2014). In the majority of these reports the observed changes were also indirectly
549 connected to the belated harvest point (due to rootstock effect), the seasonal change and the
550 postharvest storage too. According to the data obtained, the cultivar that is used as the scion to
551 the heterografts plays a crucial role for the biosynthesis of the phenolic metabolites. That became
552 apparent, as compounds that were not present in the control s or the homograft of ‘Big Moose’,
553 were detected in the heterograft of ‘Big Moose’ x ‘Munchkin’, while ‘Munchkin’ contained such
554 phenolic compounds in relative concentrations as well. Thus, the changes observed in the
555 phenolic profile of ‘Big Moose’ (RS) x ‘Munchkin’ (SC) and ‘Munchkin’ (RS) x ‘Big Moose’
556 (SC) are more likely scion-dependent than rootstock-dependent. These observations are in
557 agreement with previous studies on Solanaceae species that also reported the main scion effect
558 on final yield and fruit quality traits in grafted plants, while rootstock effects can alter such
559 characteristics possibly due to changes in vigor and concentration of plant growth regulators
560 induced by the rootstocks (Gisbert et al. 2012; Gisbert et al. 2011; Moncada et al. 2013; Muñoz-
561 Falcón et al. 2008; Passam et al. 2005). Furthermore, the ‘trend’ in the accumulation of phenolics
562 seems to be also genetically controlled as in green fruit C. pepo cultivars and more complex than
563 yellow ones.
564 Previous investigations in Cucurbita inter-species heterografting involving significant
565 DNA methylation changes in melon and cucumber scions grafted onto pumpkin rootstocks has
566 been expanded by the current study (Avramidou et al. 2014).
567
568 Conclusion
569 Herein, for the first time, has been demonstrated that intra-species grafting among C. pepo
570 cultivars induced dramatic changes in scion fruit size and that these changes are accompanied by
571 significant alterations in global DNA methylation patterns of scion genomes, miRNA expression
572 changes, and phenolic profile changes. Moreover, it is shown that intra-species C. pepo grafting
573 induced moderate alterations in fruit shape with concomitant substantial changes in scion DNA
574 methylation and miRNA expression patterns. These investigations contribute to a deeper
575 understanding of grafting in Cucurbitaceae and will shed light to the poorly understood
576 molecular mechanisms underlying in the effect of grafting on fruit morphology. Moreover, they
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577 will help in understanding the association between graft-induced DNA methylation, miRNA
578 expression and altered phenolic profiles in fruits of grafted plants. Taken together, the
579 differential epigenetic marks and phenolic patterns induced by grafting may lead to the
580 development of epi-molecular markers and metabolic predictors for rapid and efficient selection
581 of grafted vegetables with desired traits of agronomical relevance.
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792
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793 Figure Legends
794 Figure 1: Fruits of cv. ‘Munchkin’ and cv. ‘Big Moose’ grafted plants and their controls
795 (A) Control non-grafted ‘Munchkin’ (left) and ‘Big Moose’ (right)
796 (B) Grafted ‘Big Moose’ (SC) on ‘Munchkin’ (RS) (top) and reciprocal combination (bottom)
797 with no change in fruit morphology that is the same as the fruit morphology of the non-grafted
798 scions.
799 (C) Grafted ‘Big Moose’ (SC) on cv. ‘Munchkin’ (RS) with the altered fruit morphology
800 growing on the grafted plant.
801 (D) Grafted ‘Big Moose’ (SC) on cv. ‘Munchkin’ (RS) with the altered fruit morphology (left)
802 next to grafted ‘Big Moose’ (SC) on cv. ‘Munchkin’ (RS) with no alteration (right).
803
804 Figure 2: Fruits, flowers and leaves of grafted plants cv. ‘Round green’, cv. ‘Princess’ and
805 their controls.
806 (A) Control non-grafted ‘Round green’
807 (B) Grafted ‘Round green’ (SC) on ‘Princess’ (RS)
808 (C) Control non-grafted ‘Princess’
809 (D) Grafted ‘Princess’ (SC) on ‘Round green’ (RS)
810
811 Figure 3: Relative (by comparison to non-grafted seedlings) DNA methylation and
812 demethylation levels (percent of total within each sample; averaged) of grafted plants based
813 on MSAP analysis. Relative levels at randomly sampled 5’- CCGG -3’sites with the MSAP
814 marker (A) ‘Munchkin’ x ‘Big Moose’ grafted plants versus ‘Big Moose’ s. B) ‘Big Moose’ x
815 ‘Munchkin’ plants versus ‘Munchkin’ s. C) ‘Munchkin’ x ‘Big Moose’ grafted plant with fruit
816 size changes versus ‘Big Moose’ seedlings. Asterisks indicate values significantly lower than the
817 values of the corresponding seedling controls (P<0.05).y-axis: relative (percent) methylation
818 level.
819
820 Figure 4: Relative (by comparison to non-grafted seedlings) levels at randomly sampled 5’-
821 CCGG -3’sites with MSAP markers (A) ‘Round green’ x ‘Princess grafted plants versus
822 ‘Princess’ seedlings. B) ‘Princess’ x ‘Round green’ plants versus ‘Round green’ seedlings
823 (percent of total within each sample; averaged). Asterisks indicate values significantly lower
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3389v1 | CC BY 4.0 Open Access | rec: 3 Nov 2017, publ: 3 Nov 2017
824 than the values of the corresponding seedling controls (P<0.05).y-axis: relative (percent)
825 methylation level.
826 Figure 5: Quantitative qRT-PCR analyses of selected miRNAs in grafted plants and their
827 non-grafted controls
828 (A) miRNA expression in heterografts of ‘Big moose’ and ‘Munchkin’. B= ‘Big Mouse’, M=
829 ‘Munchkin’.
830 (B) miRNA expression levels in heterografts of ‘Princess’ and ‘Round green’. P= ‘Princess’,
831 R= ‘Round green’
832 Expression values were normalized to those of the reference gene CpEF-1a. Relative expression
833 ratio of each sample was compared to the control group which was assigned arbitrarily the value
834 of 1. Data represent mean values from two independent experiments with standard deviations.
835 Values significantly different (P < 0.05) from the control sample (‘Big Moose’) are marked with
836 an asterisk (*).
837
838 Figure 6: Overlay heat map of metabolite profiles in grafted plants in comparison with
839 non-grafted controls.
840 (A) For the cultivars that differ in fruit size and their grafted plants. On the left the part of the
841 heatmap that corresponds to the graftings and the control that produced small fruits in terms of
842 size (red color), on the right the part of the heatmap that corresponds to the ones that produced
843 large fruit (pink color).
844 (B) For the cultivars that differ in fruit shape and their grafted plants. On the left the part of the
845 heatmap that corresponds to the graftings and the control that produced round fruits (red color),
846 on the right the part of the heatmap that corresponds to the ones that produced elongated fruits
847 (pink color). The metabolites in each square represent the effect of plant grafting on the amount
848 of every metabolite using a false-color scale.
849
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Figure 1(on next page)
Fruits of cv. ‘Munchkin’ and cv. ‘Big Moose’ grafted plants and their controls.
Figure 1: Fruits of cv. ‘Munchkin’ and cv. ‘Big Moose’ grafted plants and their controls (A)
Control non-grafted ‘Munchkin’ (left) and ‘Big Moose’ (right) (B) Grafted ‘Big Moose’ (SC) on
‘Munchkin’ (RS) (top) and reciprocal combination (bottom) with no change in fruit
morphology that is the same as the fruit morphology of the non-grafted scions. (C) Grafted
‘Big Moose’ (SC) on cv. ‘Munchkin’ (RS) with the altered fruit morphology growing on the
grafted plant. (D) Grafted ‘Big Moose’ (SC) on cv. ‘Munchkin’ (RS) with the altered fruit
morphology (left) next to grafted ‘Big Moose’ (SC) on cv. ‘Munchkin’ (RS) with no alteration
(right).
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3389v1 | CC BY 4.0 Open Access | rec: 3 Nov 2017, publ: 3 Nov 2017
Munchkin BigMoose
MunchkinxBigMoose
fruitchangedplant MunchkinxBigMoose
A)
C) D)
MunchkinxBigMoose
BigMoosexMunchkin
B)
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3389v1 | CC BY 4.0 Open Access | rec: 3 Nov 2017, publ: 3 Nov 2017
Figure 2(on next page)
: Fruits, flowers and leaves of grafted plants cv. ‘Round green’, cv. ‘Princess’ and their
controls
Figure 2: Fruits, flowers and leaves of grafted plants cv. ‘Round green’, cv.
‘Princess’ and their controls. (A) Control non-grafted ‘Round green’ (B) Grafted ‘Round
green’ (SC) on ‘Princess’ (RS) (C) Control non-grafted ‘Princess’ (D) Grafted ‘Princess’ (SC) on
‘Round green’ (RS)
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3389v1 | CC BY 4.0 Open Access | rec: 3 Nov 2017, publ: 3 Nov 2017
RoundgreenxPrincessPrincess
Roundgreen PrincessxRoundgreen
A) B)
C) D)
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Figure 3(on next page)
Relative (by comparison to non-grafted seedlings) DNA methylation and demethylation
levels (percent of total within each sample; averaged) of grafted plants based on MSAP
analysis.
Figure 3: Relative (by comparison to non-grafted seedlings) DNA methylation and
demethylation levels (percent of total within each sample; averaged) of grafted
plants based on MSAP analysis. Relative levels at randomly sampled 5’- CCGG -3’sites
with the MSAP marker (A) ‘Munchkin’ x ‘Big Moose’ grafted plants versus ‘Big Moose’ s. B)
‘Big Moose’ x ‘Munchkin’ plants versus ‘Munchkin’ s. C) ‘Munchkin’ x ‘Big Moose’ grafted
plant with fruit size changes versus ‘Big Moose’ seedlings. Asterisks indicate values
significantly lower than the values of the corresponding seedling controls (P<0.05).y-axis:
relative (percent) methylation level.
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3389v1 | CC BY 4.0 Open Access | rec: 3 Nov 2017, publ: 3 Nov 2017
0
5
10
15
20
25
30
35
40
Nochange Demethyla5on De-novomethyla5on
MunchkinxBigMoosegra@edplantsversusBigMooseseed-plants
0
5
10
15
20
25
30
35
Nochange Demethyla4on De-novomethyla4on
BigMoosexMunchkingra?edplantsversusMunchkinseed-plants
0
5
10
15
20
25
30
35
Nochange Demethyla4on De-novomethyla4on
MunchkinxBigMoosegra?edplantwithfruitsizechangesversusBig
Mooseseed-plant
A)
B)
C)
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3389v1 | CC BY 4.0 Open Access | rec: 3 Nov 2017, publ: 3 Nov 2017
Figure 4(on next page)
Relative (by comparison to non-grafted seedlings) levels at randomly sampled 5’- CCGG
-3’sites with MSAP markers
Figure 4: Relative (by comparison to non-grafted seedlings) levels at randomly
sampled 5’- CCGG -3’sites with MSAP markers (A) ‘Round green’ x ‘Princess grafted plants
versus ‘Princess’ seedlings. B) ‘Princess’ x ‘Round green’ plants versus ‘Round green’
seedlings (percent of total within each sample; averaged). Asterisks indicate values
significantly lower than the values of the corresponding seedling controls (P<0.05).y-axis:
relative (percent) methylation level.
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3389v1 | CC BY 4.0 Open Access | rec: 3 Nov 2017, publ: 3 Nov 2017
0
2
4
6
8
10
12
14
16
18
Nochange Demethyla5on De-novomethyla5on
RoundgreenxPrincessgra@edplantsversusPrincessseed-plants
0
5
10
15
20
25
30
Nochange Demethyla4on De-novomethyla4on
PrincessxRoundgreengra?edplantsversusRoundgreenseed-plants
A)
B)
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3389v1 | CC BY 4.0 Open Access | rec: 3 Nov 2017, publ: 3 Nov 2017
Figure 5(on next page)
Quantitative qRT-PCR analyses of selected miRNAs in grafted plants and their non-
grafted controls
Figure 5: Quantitative qRT-PCR analyses of selected miRNAs in grafted plants and
their non-grafted controls (A) miRNA expression in heterografts of ‘Big moose’ and
‘Munchkin’. B= ‘Big Mouse’, M= ‘Munchkin’. (B) miRNA expression levels in heterografts of
‘Princess’ and ‘Round green’. P= ‘Princess’, R= ‘Round green’ Expression values were
normalized to those of the reference gene CpEF-1a. Relative expression ratio of each sample
was compared to the control group which was assigned arbitrarily the value of 1. Data
represent mean values from two independent experiments with standard deviations. Values
significantly different (P < 0.05) from the control sample (‘Big Moose’) are marked with an
asterisk (*).
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3389v1 | CC BY 4.0 Open Access | rec: 3 Nov 2017, publ: 3 Nov 2017
0
0,2
0,4
0,6
0,8
1
1,2
B MXB M BXM
Rela%
vreexpressionra%
o
miR156
0
0,2
0,4
0,6
0,8
1
1,2
B MXB M BXM
miR159
0
0,2
0,4
0,6
0,8
1
1,2
1,4
B MXB M BXM
miR164
0
0,2
0,4
0,6
0,8
1
1,2
B MXB M BXM
miR166
0
1
2
3
4
B MXB M BXM
miR171
A)
B)
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3389v1 | CC BY 4.0 Open Access | rec: 3 Nov 2017, publ: 3 Nov 2017
Figure 6(on next page)
Overlay heat map of metabolite profiles in grafted plants in comparison with non-
grafted controls.
Figure 6: Overlay heat map of metabolite profiles in grafted plants in comparison
with non-grafted controls. (A) For the cultivars that differ in fruit size and their grafted
plants. On the left the part of the heatmap that corresponds to the graftings and the control
that produced small fruits in terms of size (red color), on the right the part of the heatmap
that corresponds to the ones that produced large fruit (pink color). (B) For the cultivars that
differ in fruit shape and their grafted plants. On the left the part of the heatmap that
corresponds to the graftings and the control that produced round fruits (red color), on the
right the part of the heatmap that corresponds to the ones that produced elongated fruits
(pink color). The metabolites in each square represent the effect of plant grafting on the
amount of every metabolite using a false-color scale.
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3389v1 | CC BY 4.0 Open Access | rec: 3 Nov 2017, publ: 3 Nov 2017
A)
B)
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Table 1(on next page)
Data for the three morphological traits in control (non-grafted), homo-grafted and
hetero-grafted pumpkin plants.
Table 1. Data for the three morphological traits in control (non-grafted), homo-grafted and
hetero-grafted pumpkin plants.
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3389v1 | CC BY 4.0 Open Access | rec: 3 Nov 2017, publ: 3 Nov 2017
1 Table 1. Data for the three morphological traits in control (non-grafted), homo-grafted and
2 hetero-grafted pumpkin plants.
Grafts
(rootstock,
scion),
seedlings (cv.)
‘Big
Moose’‘Munchkin’
‘Big Moose’
x ‘Big
Moose’
‘Munchkin’ x
‘Big Moose’
‘Munchkin’ x
‘Munchkin’
‘Big Moose’
x
‘Munchkin’
‘Munchkin’ x
‘Big Moose’
with altered
fruit size
Weight (kg) 9.71±1.39a 0.18±0.04b 11.73±1.92a 8.78±0.83a 0.12±0.02b 0.15±0.03b 3.7ab
Equatorial
diameter (cm)
37.83±2.36
a8.78±0.92b 40.22±3.62a 36.05±2.09a 8.28±0.67b 8.02±1.03b 19.15b
Polar diameter
(cm)
31.89±5.32
a4.09±0.43b 31.94±8.66a 28.85±5.05a 4.12±0.56b 4.47±0.65b 17.26b
Grafts
(rootstock,
scion),
seedlings (cv.)
‘Princess’‘Round
green’
‘Princess’ x
‘Princess’
‘Princess’ x
‘Round
green’
‘Round
green’ x
‘Round
green’
‘Round
green’ x
‘Princess’
Weight (kg) 0.20±0.04a 0.17±0.03a 0.23±0.05a 0.16±0.13a 0.15±0.14a 0.21±0.06a
Equatorial
diameter (cm)
17.83±1.32
a9.22±0.06b 17.4±1.55a 9.34±0.02b 9.18±0.04b 17.21±1.85a
Polar diameter
(cm)5.81±0.06a 10.34±0.3b 5.93±0.24a 10.12±0.32b 9.92±0.12b 6.42±0.12a
3
4 Different letters in the columns indicate significant differences according to Duncans’ test for P< 0.05
Grafts
(rootstock,
scion),
seedlings (cv.)
‘Big Moose’ ‘Munchkin’
‘Big Moose’
x ‘Big
Moose’
‘Munchkin’ x
‘Big Moose’
‘Munchkin’ x
‘Munchkin’
‘Big Moose’
x
‘Munchkin’
‘Munchkin’ x
‘Big Moose’
with altered
fruit size
Weight (kg) 9.71±1.39a 0.18±0.04b 11.73±1.92a 8.78±0.83ab 0.12±0.02b 0.15±0.03b 3.7ab
Equatorial
diameter (cm)37.83±2.36a 8.78±0.92b 40.22±3.62a 36.05±2.09a 8.28±0.67b 8.02±1.03b 19.15b
Polar diameter
(cm)31.89±5.32a 4.09±0.43b 31.94±8.66a 28.85±5.05ab 4.12±0.56b 4.47±0.65b 17.26b
Grafts
(rootstock,
scion),
seedlings (cv.)
‘Princess’‘Round
green’
‘Princess’ x
‘Princess’
‘Princess’ x
‘Round
green’
‘Round
green’ x
‘Round
green’
‘Round
green’ x
‘Princess’
Weight (kg) 0.20±0.04a 0.17±0.03a 0.23±0.05a 0.16±0.13a 0.15±0.14a 0.21±0.06a
Equatorial
diameter (cm)17.83±1.32a 9.22±0.06b 17.4±1.55a 9.34±0.02b 9.18±0.04b
17.21±1.85a
b
Polar diameter
(cm)5.81±0.06a 10.34±0.3b 5.93±0.24a 10.12±0.32b 9.92±0.12b 6.42±0.12ab
5
6 Different letters in the columns indicate significant differences according to Duncans’ test for P< 0.05
7
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3389v1 | CC BY 4.0 Open Access | rec: 3 Nov 2017, publ: 3 Nov 2017
Table 2(on next page)
The effect of grafting on secondary metabolites of pumpkin related with fruit size.
Table 2: The effect of grafting on secondary metabolites of pumpkin related with fruit size.
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3389v1 | CC BY 4.0 Open Access | rec: 3 Nov 2017, publ: 3 Nov 2017
1 Table 2: The effect of grafting on secondary metabolites of pumpkin related with fruit size.
Concentration (mg/100 g DW)
Compound Big MooseBig Moose x
Big Moose
Big Moose
x
Munchkin
MunchkinMunchkin x
Munchkin
Munchkin x
Big Moose
1 p-hydroxybenzoic acid 0.000 0.000 0.575 a 0.666 a 0.492 a 0.000
2 ferulic acid 0.042 b 0.004 b 0.264 a 0.083 b 0.060 b 0.000
3 vanillic acid 0.000 0.000 0.039 a 0.000 0.005 b 0.000
4 chlorogenic acid 0.000 0.134 a 0.000 0.105 b 0.000 0.000
5 esculetin 0.016 a 0.010 ab 0.007 ab 0.000 0.006 ab 0.011 ab
6 coniferyl alcohol 0.134 a 0.000 0.137 a 0.140 a 0.056 bc 0.089 ab
7 sinapyl alcohol 0.000 0.000 0.092 b 0.215 a 0.000 0.000
8 naringenin 0.000 0.000 0.027 0.000 0.000 0.000
9 dihydrokaempferol 0.000 0.000 0.000 0.090 0.000 0.000
10 kaempferol-3-O-rutinoside 1.915 b 3.069 a 0.000 0.000 0.000 1.726 b
11quercetin-3-O-glucoside +
quercetin-3-O-galactoside0.077 b 0.053 c 0.000 0.000 0.000 0.405 a
12 rutin 1.598 b 0.678 c 0.000 0.023 d 0.000 2.434 a
13 isorhamnetin 0.000 0.000 0.000 0.000 0.000 0.019
14 isorhamnetin-3-O-glucoside 0.000 0.187 b 0.000 0.000 0.000 0.326 a
15 isorhamnetin-3-O-rutinoside 18.855 a 15.703 b 0.000 0.000 0.000 14.430 b
16 arbutin 0.000 0.000 0.262 a 0.261 a 0.141 b 0.000
17 carnosol 0.104 b 0.165 a 0.081 bc 0.032 d 0.040 d 0.056 cd
18 carnosic acid 0.396 a 0.431 a 0.135 b 0.104 b 0.030 c 0.134 b
Total identified 23.137 20.434 1.618 1.719 0.831 19.630
2 Different letters in the columns indicate significant differences according to Duncans’ multiple range test performed across all grafting treatments (
3 P< 0.05).
4
5
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3389v1 | CC BY 4.0 Open Access | rec: 3 Nov 2017, publ: 3 Nov 2017
Table 3(on next page)
The effect of grafting on secondary metabolites of pumpkin related with fruit shape.
Table 3: The effect of grafting on secondary metabolites of pumpkin related with fruit shape.
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3389v1 | CC BY 4.0 Open Access | rec: 3 Nov 2017, publ: 3 Nov 2017
1 Table 3: The effect of grafting on secondary metabolites of pumpkin related with fruit shape.
Concentration (mg/100 g DW)
Compound Princess Princess x Princess
Princess x Round
green Round green
Round green x
Round green
Round green x
Princess
1 p-hydroxybenzoic acid 0.671 c 1.073 ab 1.298 a 0.601 c 0.818 bc 1.029 ab
2 cinnamic acid 2.422 a 1.366 b 0.353 c 1.680 b 0.000 1.372 a
3 ferulic acid 2.720 a 0.000 1.888 b 1.494 c 0.086 d 2.805 a
4 vanillic acid 0.818 b 0.000 0.000 0.425 c 0.783 b 1.215 a
5 esculetin 0.006 bc 0.000 0.032 a 0.015 b 0.012 bc 0.006 bc
6 coniferyl alcohol 0.042 b 0.000 0.098 a 0.127 a 0.000 0.000
7 orientin 0.000 0.000 0.038 0.030 0.000 0.000
8 naringenin 0.000 0.000 0.015 0.017 0.000 0.026
9 dihydrokaempferol 0.000 0.017 b 0.000 0.034 a 0.000 0.000
10 kaempferol-3-O-rutinoside 0.000 0.029 b 1.004 a 0.194 b 0.071 b 0.000
11 quercetin 0.000 0.000 0.321 0.000 0.000 0.000
12
quercetin-3-O-glucoside +
quercetin-3-O-galactoside 0.000 0.000 0.416 0.000 0.000 0.000
13 rutin 0.278 c 0.000 21.268 a 2.000 b 0.234 c 0.291 c
14 isorhamnetin 0.000 0.000 0.036 0.000 0.000 0.000
15 isorhamnetin-3-O-glucoside 0.000 0.000 0.202 a 0.066 b 0.000 0.000
16 isorhamnetin-3-O-rutinoside 0.478 cd 0.960 c 16.305 a 9.948 b 0.942 c 0.310 d
17 carnosol 0.052 b 0.002 d 0.151 a 0.039 bc 0.021 bcd 0.006 cd
Total identified 7.487 3.448 43.424 16.671 2.967 7.060
2
3 Different letters in the columns indicate significant differences according to Duncans’ multiple range test performed across all grafting treatments
4 ( P< 0.05).
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3389v1 | CC BY 4.0 Open Access | rec: 3 Nov 2017, publ: 3 Nov 2017