1 MSH1-Derived Epigenetic Breeding Potential in Tomato 1 2 Xiaodong Yang 1, 2 , Hardik Kundariya 2 , Ying-Zhi Xu 2 , Ajay Sandhu 2 , Jiantao Yu 2 , Samuel F. 3 Hutton 3 , Mingfang Zhang 1 , Sally A. Mackenzie 2 4 1 Laboratory of Genetic Resources & Functional Improvement for Horticultural Plants, 5 Department of Horticulture, Zhejiang University, Hangzhou 310029, People’s Republic of 6 China 7 2 Department of Agronomy and Horticulture, University of Nebraska, Lincoln, NE 68588- 8 0660, USA 9 3 Gulf Coast Research and Education Center, Institute of Food and Agricultural Sciences, 10 University of Florida, 14625 CR 672, Wimauma, FL 33598-6101, USA 11 12 13 14 Corresponding Author: 15 Sally Mackenzie 16 N305 Beadle Center 17 University of Nebraska 18 Lincoln, NE 68588-0660 19 Ph 402 472 6997 fax 402 472 3139 20 Email [email protected]21 22 23 24 25 26 27 X. Yang [email protected]28 H. Kundariya [email protected]29 Y.Z. Xu [email protected]30 A. Sandhu [email protected]31 J. Yu [email protected]32 S. Hutton [email protected]33 M. Zhang [email protected]34 S. Mackenzie [email protected]35 Plant Physiology Preview. Published on March 3, 2015, as DOI:10.1104/pp.15.00075 Copyright 2015 by the American Society of Plant Biologists https://plantphysiol.org Downloaded on March 16, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.
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MSH1-Derived Epigenetic Breeding Potential in Tomato 1 2 Xiaodong Yang1, 2, Hardik Kundariya2, Ying-Zhi Xu2, Ajay Sandhu2, Jiantao Yu2, Samuel F. 3 Hutton3, Mingfang Zhang1, Sally A. Mackenzie2 4 1 Laboratory of Genetic Resources & Functional Improvement for Horticultural Plants, 5 Department of Horticulture, Zhejiang University, Hangzhou 310029, People’s Republic of 6 China 7 2Department of Agronomy and Horticulture, University of Nebraska, Lincoln, NE 68588-8 0660, USA 9 3 Gulf Coast Research and Education Center, Institute of Food and Agricultural Sciences, 10 University of Florida, 14625 CR 672, Wimauma, FL 33598-6101, USA 11 12 13 14 Corresponding Author: 15 Sally Mackenzie 16 N305 Beadle Center 17 University of Nebraska 18 Lincoln, NE 68588-0660 19 Ph 402 472 6997 fax 402 472 3139 20 Email [email protected] 21 22 23 24 25 26 27 X. Yang [email protected] 28 H. Kundariya [email protected] 29 Y.Z. Xu [email protected] 30 A. Sandhu [email protected] 31 J. Yu [email protected] 32 S. Hutton [email protected] 33 M. Zhang [email protected] 34 S. Mackenzie [email protected] 35
Plant Physiology Preview. Published on March 3, 2015, as DOI:10.1104/pp.15.00075
Copyright 2015 by the American Society of Plant Biologists
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36 37 Abstract 38 Evidence is compelling in support of naturally occurring epigenetic influence on phenotype 39 expression in land plants, although discerning epigenetic contribution is difficult. 40 Agriculturally important attributes like heterosis, inbreeding depression, phenotypic 41 plasticity and environmental stress response are thought to have significant epigenetic 42 components, but unequivocal demonstration of this is often infeasible. Here we investigate 43 gene silencing of a single nuclear gene, MSH1, in the tomato variety ‘Rutgers’ to effect 44 developmental reprogramming of the plant. The condition is heritable in subsequent 45 generations independent of the MSH1-RNAi transgene. Crossing these transgene-null, 46 developmentally altered plants to the isogenic Rutgers wild type results in progeny lines 47 that show enhanced, heritable growth vigor under both greenhouse and field conditions. 48 This boosted vigor appears to be graft-transmissible and is partially reversed by treatment 49 with the methylation inhibitor 5-azacytidine, implying influence of mobile, epigenetic 50 factors and DNA methylation changes. These data provide compelling evidence for the 51 feasibility of epigenetic breeding in a crop plant. 52
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53 Introduction 54 Epigenetic variation in nature is an underpinning to phenotypic plasticity and adaptive 55 capacity of an organism (Mirouze and Paszkowski 2011; Sahu et al. 2013). Chromatin 56 modifications provide memory of abiotic or biotic stress that has been experienced, 57 priming a biological system to better cope with future recurrence (Dowen et al. 2012; 58 Gimanelli and Roudier 2013). The extent to which epigenetic memory extends 59 transgenerationally has been the subject of vigorous investigation. While it is clear that 60 epigenetic traits can be stably maintained through extremely long lineages (Cubas et al. 61 1999), it is also clear that plant cytosine methylation patterns are highly dynamic (Becker 62 et al. 2011; Schmitz et al. 2011). Consequently, the extent to which epigenetic variation can 63 be directly exploited in meaningful plant or animal breeding strategies remains largely 64 unknown. 65 66 The behavior and stability of genome-wide epigenetic changes in plants has been pursued 67 most powerfully through development of epigenetically modified recombinant inbred lines 68 in Arabidopsis. The approach capitalizes on availability of genetic mutations in the DNA 69 methylation machinery (Reinders et al. 2009; Roux et al. 2011). Studies have confirmed 70 association between plant phenotypic variation and modifications in genome methylation, 71 and have shown stable inheritance of some derived epi-traits over multiple generations 72 (Cortijo et al. 2014). These studies suggest that epigenetic variation should be amenable to 73 plant selection. 74 75
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However, mutation-mediated disruption of the plant methylation machinery has not 76 necessarily produced altered growth behavior that is adaptive or agriculturally 77 advantageous. It is feasible to identify natural epialleles that influence growth behavior. 78 The tomato fruit ripening variant colorless non-ripening (cnr) appears to be a naturally 79 occurring epiallele of CNR, which encodes an SBP-box transcription factor. In the variant, 80 CNR expression is silenced by promoter hypermethylation to inhibit normal fruit ripening 81 (Manning et al., 2006). One strategy for accessing natural epigenetic variation was 82 presented in canola, where recursive selection for variation of respiratory parameters in a 83 genetically fixed doubled haploid line resulted in a range of phenotypic variation suspected 84 to be non-genetic (Hauben et al. 2009). Some emerging phenotypes appeared to be 85 improved in growth performance. Similarly in tomato, transgressive phenotypes in hybrids 86 can be epigenetically influenced (Shivaprasad et al. 2012) and, in Arabidopsis, hybrid vigor 87 phenotypes appear to show some relationship to cytosine methylation behavior of the 88 combined genomes (Greaves et al. 2014). These observations imply that epigenetic 89 variation influencing plant growth may be important agriculturally. 90 91 MutS HOMOLOG 1 (MSH1) encodes a dual targeted protein that localizes to the 92 mitochondrion, where it suppresses illegitimate DNA recombination (Abdelnoor et al. 93 2003; Davila et al. 2011), and to the plastid. Its role in the plastid is less well defined, but 94 mutation of the MSH1 gene in Arabidopsis is known to condition plastid-associated changes 95 in plant growth behavior. The msh1 mutant is markedly altered in growth rate, branching 96 behavior, flowering time, juvenility-maturity growth transition, perennial growth behavior, 97 and abiotic stress response (Xu et al. 2012). This developmental reprogramming (DR) is 98
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accompanied by altered genome methylation, with intensity of altered phenotype showing 99 apparent association with enhanced non-CG hypermethylation of pericentromeric genomic 100 intervals (Virdi et al. 2015). This complex DR phenotype has been recapitulated in several 101 crop species, both monocot and dicot, by RNAi suppression of MSH1, prompting changes in 102 growth rate, tillering, flowering time, leaf morphology, and abiotic stress tolerance (Xu et 103 al. 2012). In each case, the altered phenotype is subsequently inherited independent of 104 RNAi transgene segregation, implicating a non-genetic mechanism (Xu et al. 2012; 105 Santamaria et al. 2014; and unpublished data). 106 107 The DR state conditioned by mutation or silencing of MSH1 is unusual. Crossing of the 108 modified plant, either Arabidopsis msh1 mutant or sorghum MSH1-RNAi suppression line, 109 to its isogenic wild type counterpart produces heritable enhanced growth vigor in 110 subsequent progeny generations (Virdi et al. 2015; Santamaria et al. 2014). The 111 enhancement in vigor is evidenced not only in more rapid growth, earlier flowering and 112 greater above-ground biomass, but in markedly increased seed yield. 113 114 To investigate the potential for exploiting non-genetic variation in a directed crop breeding 115 effort, we introduced an MSH1-RNAi transgene construction to the tomato cultivar 116 ‘Rutgers’. Here we demonstrate that MSH1 suppression results in tomato developmental 117 reprogramming, and that crossing of the modified line to its isogenic Rutgers wild type 118 produces marked enhancement in growth vigor. We show that these growth changes 119 greatly out-perform Rutgers under commercial field production conditions, leading to 120 earlier ripening, higher yields and heat tolerance. The enhancements in growth appear to 121 https://plantphysiol.orgDownloaded on March 16, 2021. - Published by
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be graft-transmissible, and the altered phenotype is partially obviated by exogenous 122 application of a methylase inhibitor, features characteristic of epigenetic influence in the 123 growth changes we observe. 124 125 126
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Results 127 128 MSH1 RNAi suppression in tomato leads to developmental reprogramming 129 Development of the Rutgers MSH1-RNAi transgenic lines was described previously, and 130 lines used in this study were each shown to contain a single copy of the transgene (Sandhu 131 et al. 2007). For the present study we included two independent transformants. 132 Suppression of MSH1 expression consistently resulted in a wide range of altered 133 phenotypes, including changes in leaf morphology, variegation, dwarfing, male sterility, 134 flower development and flower timing (Figure 1; Table 1). All altered traits showed 135 incomplete penetrance and a small (ca. 10%) proportion of the transgenic lines produced a 136 sufficiently severe phenotype that the plants terminated growth or were completely sterile. 137 Over 90% of the transgenic lines produced viable seed. Segregation of the transgene 138 occurred in progeny of transgenic lines, and transgene-null segregants tended to revert 139 phenotypically to a milder range of altered growth and to restored MSH1 transcript levels 140 (Figure 2). However, we observed variation in phenotype in both transgene-positive and 141 transgene-null selections, demonstrating that the altered phenotypes were subsequently 142 inherited independent of the transgene (Table S1). In each cycle of self-pollination, 143 progeny produced a range in phenotype intensity, and these were not subjected to 144 intentional selection. To date, we have maintained these transgene-null variants for up to 8 145 generations by recurrent self-pollination. 146 147 Isogenic crosses using MSH1-RNAi transgene nulls result in enhanced growth 148
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Reciprocal crosses of the transgene-null lines to wild type (Figure 3A) resulted in F1 149 progeny showing normal growth, and F2 progeny (designated epiF2) showing a range of 150 enhanced growth vigor (Figure 3B). The derived epi-lines were maintained on a Rutgers 151
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genetic background. Early growth of the epiF2 plants was more rapid than Rutgers wild 152 type, resulting in taller plants until the 10-week point (Figure 4). At 10 weeks, the epiF2 153 lines slowed vegetative growth and transitioned to flowering, while Rutgers wild type 154 https://plantphysiol.orgDownloaded on March 16, 2021. - Published by
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continued vegetative growth, flowering slightly later (Figure S1). Single plant selections 155 from the broad range of epiF2 phenotypes resulted in both epiF3 families that yielded 156 https://plantphysiol.orgDownloaded on March 16, 2021. - Published by
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higher and a few yielding lower than wild type (Figure S2; Table S2). These results suggest 157 that MSH1-associated non-genetic variation shows at least mild response to selection. 158 159 Enhanced-growth tomato phenotypes show enhanced field performance 160 Epi-lines displayed enhanced seedling growth vigor relative to wildtype, evident within the 161 first 2-3 weeks (Figure S3), and early transition to reproduction (Table S4). EpiF2, epiF3 162 and epiF4 families were grown under Florida field conditions to assess their performance 163 and response to selection. Results in the field were generally similar to the greenhouse, and 164 good correlation was seen between greenhouse and field data for fruit number and plant 165 height (Figure S4). 166 167
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Rutgers wild type plants were similar in photosynthetic rate, but were increased in overall 168 vegetative growth and lower in fruit set than the epi-lines (Figure S5). Rutgers plants 169 produced an average vegetative fresh weight (minus fruit) of 2.35 kg and dry weight of 170 0.38 kg, while the epiF4 plants averaged 1.62 kg in fresh vegetative weight and 0.28 kg dry 171 weight (averaged from 6 plants each). Flower and fruit set showed steady increases in the 172 epiF2, epiF3 and epiF4 over wildtype, with epiF4 productivity consistently the highest 173 (Figure S6). While average fruit size was lower in the epi-lines relative to wild type (Figure 174 S6), overall fruit yield was greater, seed number was greater and fruit sugar content was 175 unchanged (Figure S6). Fruit ripening was more rapid in the epi-lines, resulting in a greater 176 proportion of red fruit at a single harvest time (Figure 3 C,D, Table S4). Because the 177 variation observed was largely associated with growth vigor, and heritability was variable 178 (Table 2), we applied relatively low selection pressure each cycle. Bulking seed taken from 179 the top 50% in the epiF2 to produce an epiF3 bulk, and again in the epiF3 to produce an 180 epiF4 bulk resulted in decreases in variance each cycle and a 35% increase in mean yield 181 over the two rounds of selection (Figure 5, S7). These results imply significant unrealized 182 epigenetic yield potential in this line. Surprisingly, this significant yield advantage was also 183 evident when the wild type and Rutgers epiF3 lines were grown under high heat conditions 184 (Figure 6), providing some indication that the MSH1 effect can enhance abiotic stress 185 tolerance. To date, we have not observed evidence of enhanced biotic stress tolerance in 186 the modified Rutgers line. 187 188 The enhanced growth effect is partially reversed by a methylation inhibitor 189
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The enhanced growth vigor phenotype was partially obviated with the exogenous 190 application of the DNA methylation inhibitor 5-azacytidine (Figure 7). Three separate 191 https://plantphysiol.orgDownloaded on March 16, 2021. - Published by
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experiments testing seedling growth in media supplemented with 30μM inhibitor 192 produced significant changes in epiF4 seedling growth, while only mild change in growth 193 https://plantphysiol.orgDownloaded on March 16, 2021. - Published by
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was observed in wild type plants (Figures7, S8). EpiF4 seedlings growing in the presence 194 of the inhibitor were reduced in plant height relative to the untreated epiF4 control as well 195 as the treated wild type seedlings. A subset of the seedlings from these experiments were 196 transplanted to potting media and carried out to 23 days to monitor subsequent growth 197 behavior. EpiF4 plants that were previously treated with 5-azacytidine were reduced in 198 growth relative to untreated plants following transfer to soil. By 23 days after transplanting 199 (dat), the previously treated epi-F4 plants were reduced 20% in plant height (Figure S8), 200 suggesting that some portion of the growth enhancement can be attributed to altered DNA 201 methylation state in the epiF4 lines. However, previously treated epiF4 plants, nearly 202 equal in height to wild type at transplanting, began to outgrow wild type by 12 dat, 203 showing nearly 60% increase in plant height over untreated wild type (Figure S8). This 204 observation may reflect partial re-establishment of the previous methylation state after 205 removal from 5-azacytidine.Interestingly, previously treated Rutgers wild type plants 206 showed a slight enhancement in growth rate relative to untreated control. This effect might 207 https://plantphysiol.orgDownloaded on March 16, 2021. - Published by
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signal an association between DNA methylation state and mild inbreeding depression 208 (Vergeer et al. 2012), although this has not been investigated further. 209 210 The enhanced growth effects appear to be graft-transmissible in tomato 211 Previous studies of the MSH1 effect in Arabidopsis showed that grafting of wild type 212 Arabidopsis Col-0 scion to the msh1 dwarfed mutant as rootstock resulted in seed progeny 213 with unusual enhanced growth vigor closely resembling epiF3 lines (Virdi et al. 2015). 214 Consequently, we carried out grafting experiments between Rutgers wild type and Rutgers 215 transgenic MSH1-RNAi lines. While we detected no significant growth change in progeny 216 coming from wild type grafted to wild type, progeny from wild type scion grafted to the 217 MSH1-RNAi transgenic line as rootstock showed markedly enhanced early growth rate, 218 resembling the epiF3 effect (Figure 8). As in the case of Arabidopsis, these results further 219 support the hypothesis that enhanced growth vigoris non-genetic and likely includes a 220 mobile signal within the plant. 221 222 RNA-Seq profiling reveals a common process underlying developmental 223 reprogramming caused by MSH1 suppression in Arabidopsis and tomato 224 We conducted RNA-seq in tomato dwarf-DR, mild-DR, epiF3, and wild type plants and in 225 the Arabidopsis T-DNA insertion msh1 mutant dwarf-DR plants in order to identify shared 226 differentially expressed gene responses to MSH1 disruption, and to begin to understand the 227 cellular processes underlying phenotypic diversity observed in these materials. 228 In a previous report (Xu et al, 2012), we identified several differentially expressed genes in 229 the Arabidopsis msh1 mutant, involving multiple developmental and stress response 230
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pathways, by microarray analysis. Here, we show results of comparative studies involving 231 both Arabidopsis and tomato that reveal similar patterns of gene expression change in both 232 species in response to MSH1 disruption. Tomato and Arabidopsis dwarf-DR plants shared 233 https://plantphysiol.orgDownloaded on March 16, 2021. - Published by
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1252 common gene expression changes, which accounted for 46.9% of the observed 234 tomato dwarf-DR plant changes and 12.7% in the Arabidopsis counterpart (Figure 235 S9).These 1252 genes included components in response to stimulus, protein amino acid 236 phosphorylation, cell surface receptor-linked signal transduction, cell division, and 237 development (Table S7). The changes show striking similarity to profiles in Arabidopsis 238 msh1-associated developmental reprogramming (Xu et al. 2012). For instance, similarly to 239 Arabidopsis, we found changes in genes regulating cell division, including cyclin family 240 proteins, and genes involved in cell proliferation and differentiation. Most of these were 241 down-regulated in both tomato and Arabidopsis dwarf-DR plants, consistent with the 242 dwarf phenotype and delayed growth (Table 3). In terms of stress response, alternative 243 oxidase is strongly up-regulated in both Arabidopsis (AT1G32350 AOX1D, AT3G22370 244 AOX1A) and tomato (Soly08g075540.0), consistent with redox changes and organellar 245 electron transport chain perturbation with MSH1 disruption (Table 3). Several biotic and 246 abiotic stress response genes were affected in expression in both plant species, along with 247 phytohormone gene responses influencing auxin, gibberellin, ethylene, cytokinin and 248 brassinosteroid pathways (Table 3). The observed overlaps between tomato and 249 Arabidopsis msh1-dr gene expression patterns allow for confirmation of signature changes 250 underlying the msh1 developmental reprogramming process, a phenomenon that appears 251 to be triggered by plastid perturbation (Xu et al. 2012). 252 253 To investigate the phenotypic range that emerges with MSH1 disruption, we compared 254 gene expression changes in the tomato dwarf-DR plants (containing the MSH1-RNAi 255 transgene and displaying extreme changes in development) with mild-DR plants (MSH1-256
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RNAi transgene-null and displaying mild developmental effects). An 88.7% decrease is 257 observed in gene expression changes in the mild-DR plants relative to dwarf-DR (from 258 2671 in dwarf-DR to 302 in mild-DR), and half of these genes (171) overlap with the dwarf-259 DR profile (Figure S10). These results show that the observed range in the intensity of 260 developmental reprogramming coincides with the magnitude of gene expression changes, 261 and confirm our prediction that the identified signature pathways overlapping between 262 Arabidopsis and tomato underlie the DR phenotype. 263 264 In a comparison of epiF3 plants to wildtype in tomato, using the cutoff of adjusted P<0.05, 265 we identified 437 genes differentially expressed in the enhanced-growth line. Drawing on 266 gene ontology analysis, these genes appeared to be involved predominantly in the 267 vegetative to reproductive phase transition, growth and development, stress response, and 268 cellular ketone metabolic processes (Table S8). The observed gene expression changes 269 agree well with epiF3 phenotypes in tomato of enhanced flowering and flower time 270 changes, early growth vigor, earlier ripening, higher yield and heat tolerance. 271 272 The key regulator gene for flowering Solyc05g053850.2 (SELF PRUNING, homolog of TSF in 273 Arabidopsis), for example, is up- regulated together with another important flowering 274 gene, Solyc07g052700.2 (MADS-box transcription factor 1). The Arabidopsis homolog 275 AGL66 is involved in late stage pollen development and pollen tube growth. We also 276 observed markedly decreased expression of gene Solyc08g082980.2, with an Arabidopsis 277 putative homolog WNK8 involved in the photoperiod flowering pathway and its mutant 278 showing early flowering in Arabidopsis. Expression changes of these genes are likely 279 https://plantphysiol.orgDownloaded on March 16, 2021. - Published by
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associated with the enhanced flowering and flower time changes observed in tomato epi-280 lines (Table S9, Table S10). A group of differentially expressed genes suspected to underlie 281 the observed early growth vigor includes auxin signaling and response genes 282 Solyc07g043610.2 (homolog to Arabidopsis AUXIN RESPONSE FACTOR 6, ARF6), 283 Solyc02g077560.2 (homolog to Arabidopsis AUXIN RESPONSE FACTOR 3, ARF3), cell 284 growth gene Solyc06g049050.2 (homolog to Arabidopsis Expansin A8), and a component 285 of SCF complexes, Solyc06g008710.1 (homolog to Arabidopsis CULLIN-1), which mediates 286 responses to auxin and jasmonic acid. Stress response genes altered in their expression 287 include several heat shock protein genes, which may be relevant to the enhanced heat 288 tolerance observed in our tomato epi-lines (Table S9, Table S10). 289 290 291 292
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Discussion 293 The cultivar ‘Rutgers’ was released in the 1930’s. It was bred for significant vegetative 294 growth, nicely rounded and moderately sized fruits, and slightly delayed harvest 295 (Schermerhorn, 1934). Interestingly, all of these traits were modified in response to MSH1 296 manipulation. By epiF4, populations showed early flowering and fruiting, fruit number was 297 markedly increased and early to mature, and vegetative growth was reduced in favor of 298 fruit production. While fruit size was reduced in response to the increased fruit numbers, 299 Rutgers was not originally bred for large fruit size. Possibly, a variety genetically selected 300 for large fruit size would undergo less of a reduction in response to MSH1 modulation. 301 Rutgers was also not bred specifically for heat tolerance; we presume that the enhanced 302 abiotic stress tolerance is a direct outcome of the MSH1 effect. 303 304 Evidence from this study, and earlier studies in Arabidopsis and sorghum, suggest that 305 MSH1 suppression produces on-genetic changes in the plant. This evidence includes 306 observation of reproducible patterns of cytosine methylation change in the genome of 307 Arabidopsis msh1 mutants and epi-lines (Virdi et al. 2014), parallel changes in tomato and 308 sorghum (Santamaria et al. 2014) phenotype that are inherited independent of the MSH1-309 RNAi transgene, partial reversal of phenotype with 5-azacytidine in Arabidopsis (Virdi et 310 al. 2015) and tomato, and graft-transmissibility of the enhanced growth phenotype in 311 Arabidopsis and tomato. In both tomato and sorghum, enhanced growth in the epi-lines 312 was heritable through multiple generations but, in sorghum, the phenotype appeared to 313 revert back to wild type by the epiF5. This erosion of phenotype may be characteristic of 314 epigenetic traits (Cortijo et al. 2014). What distinguishes the effects of MSH1 suppression 315
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from previous epigenetic studies that involved perturbation of the methylation machinery 316 is that emergent traits reiterate with the same transgenerational behavior across multiple 317 plant species, both for developmental reprogramming and vigor enhancement through 318 crossing. The MSH1enhanced growth vigor is both surprising and potentially exploitable 319 agronomically; whether the effect is analogous to heterosis is unclear. MSH1-derived 320 growth vigor appears uncanny in its resemblance to heterosis, but subsequent heritability 321 over multiple generations (it has been observed to epiF7 in Arabidopsis, to date) and its 322 seeming responsiveness to selection distinguish this phenomenon from classic definitions 323 of heterosis. 324 325 Observation of greater heat tolerance in the MSH1-modified lines, relative to Rutgers wild 326 type, implies that the induced effects might influence abiotic stress response. Earlier 327 transcript profile analysis of the msh1 mutant in Arabidopsis, and in the present study of 328 tomato, show changes in a number of abiotic stress response pathways (Shedge et al. 2010; 329 Xu et al. 2011, Xu et al. 2012). What is intriguing is the marked similarity in gene 330 expression responses to MSH1 disruption in both Arabidopsis and tomato, consistent with 331 our hypothesis of a programmed response. Interestingly, we have not yet observed 332 evidence of effects on biotic stress tolerance, and bacterial spot and virus incidence in the 333 Florida field experiments were similar in both Rutgers wild type and the epi-lines. 334 335 Conspicuous similarity in phenotype and behaviors of different plant species undergoing 336 the MSH1 effect indicates that this is a well-conserved process, and we speculate that the 337 MSH1 effect may participate in plant environmental adaptation. The enhanced growth that 338 https://plantphysiol.orgDownloaded on March 16, 2021. - Published by
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emerges following crossing, direct and reciprocal, arising through grafting, and partially 339 reversed with exogenous 5-azacytidine is presumed to be epigenetic. However, it is not yet 340 clear the extent to which initial developmental reprogramming might actually be a plastid 341 phenomenon. It is possible that early changes in growth rate, branching, flowering time 342 and stress response are conditioned by plastid signals that are distinct from the changes 343 directing growth vigor. Consequently, more work is needed to dissect the various 344 components of the MSH1 effect and to identify the evolutionary process that underlies this 345 unusual multi-functionalization of MSH1. 346 347 348 349 350 351
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Methods 352 Plant materials 353 MSH1 suppression lines in Rutgers background were developed previously (Sandhu et al. 354 2007), and progenies from two independent transformation events (T17and T20) were 355 used in this study. Both lines were confirmed to contain a single transgene copy (Sandhu et 356 al., 2007). Two MSH1-RNAi transgene-null plants each from T17 and T20, showing mild 357 dwarfing phenotype, were crossed with wild type inbred Rutgers reciprocally to generate 358 F1 seeds, and F1 plants were selfed to produce epiF2 families. Progenies from T17 crosses 359 were followed to the epiF4 in both greenhouse and field, while progenies from T20 were 360 followed to the epiF2 in the greenhouse. Plants in the greenhouse were germinated on 361 MetroMix 200 medium (SunGro, USA) and maintained at 26-28°C with 15-h day length and 362 at 20-22.8 °C with 9-h dark periods. Primers Tom-CD1F:5’-CGCAGGTATCACGA-363 GGCAAGTGCTAA-3’ and Intro-PIR (new):5’-GTGTACTCATGTGCATCTGACTTGAC-3’ were 364 used to genotype for the transgene. 365 366 Field trials 367 Field trials were conducted during spring and fall, 2013, at the Gulf Coast Research and 368 Education Center in Florida (27°45‘N, 82°13’W). Seedlings were grown in the greenhouse 369 for 30-40 d and then transplanted to the field. For the spring trial, seed was sown on 370 1/16/2013, and transplants were planted in the field on 3/8/2013; the average ambient 371 temperature was 21°C. For the fall trial, seed was sown on 7/25/2013, and transplanting 372 occurred on 9/14/2013; the average ambient temperature was 21.98°C. For the heat 373 tolerance trial, seed was sown on 7/2/2013, transplanting occurred on 8/12/2013, and 374
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plots were harvested on 10/21/2013; average ambient temperature was 25.2°C, with 375 highest temperature reaching 35°C. All experiments were conducted under commercial 376 production practices, as described by Hutton et al. (2014), except that beds were fumigated 377 with Pic-Clor60 (336 kg per treated hectare). Both spring field (Rutgers control, epiF2 and 378 epiF3) and fall field (Rutgers control, epiF2, epiF3 and epiF4) trials were comprised of 379 three blocks, 6 entries each and 2 replicates (3 x 6 alpha design). Each entry was 380 comprised of 15 plants. 381 Plant height, inflorescence number and fruit number were measured at multiple time 382 points during the growing season. Total fruit from each plant was harvested separately (12 383 plants minimum for each entry), and data were collected for fruit number and total fruit 384 weight, from which average fruit weight was calculated. Twelve fruit selected randomly 385 from each plant were used to measure seed number and Brix (PR-32α, ATAGO Palette). Six 386 representative plants from Rutgers wild type and epiF4 were used to measure above-387 ground biomass. 388 389 Real-time PCR Analysis 390 Total RNA was extracted from leaf tissue of 4-week-old seedlings using TRIzol (Qiagen) and 391 treated with DNAse (Qiagen). Reverse transcription for real-time PCR was performed with 392 the QuantiTect Kit (Qiagen). Quantitative PCR was performed for MSH1 transcripts 393 (Slmsh1-F1 5’-GGACGAAATTGGCTGTTTGG-3’ and Slmsh1-R1 5’-394 ACCGTCAACATATTCAGCTCC-3’) on the icycleriQ system (Bio-Rad) with SYBR Green 395 Supermix (Invitrogen). The tomato gene SlEF (SlEF-F 5’ GATTGGTGGTATTGGAACTGTC-3’ 396 and SlEF-R 5’-AGCTTCGTGGTGCATCTC-3’) was used to normalize transcript levels. 397
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398 Statistical analysis 399 For comparisons between two groups, t-tests were performed. For comparison between 400 multiple groups in field experiments and greenhouse experiments, the data were fit to a 401 linear model with multiple comparisons between means performed using a heteroscedastic 402 consistent covariance estimation (Herberich et al, 2010.). P-values were corrected for 403 multiple tests using the Benjamini-Hochberg method. 404 405 5-azacytidine treatment 406 Seeds of Rutgers wild type and epiF4 were surface-sterilized in 4% sodium hypochlorite, 407 rinsed thoroughly with sterile water and sown in 8-oz clear cups (Fabri-Kal) containing 30 408 ml 0.5M MS medium (SIGMA) supplemented with 1% agar and 0(control) or 30 μM 5-409 azacytidine (SIGMA). These seeds were germinated and grown in tissue culture facilities at 410 24 oC，18-hr day length, and 200 μmol m-2 s-1 light intensity for 14 or 20 days. For long- 411 term observation, 14-day treated plants were transferred to growth medium, and grown 412 under standard conditions in the greenhouse. The experiment was carried out three times, 413 with at least 15 replicates for each treatment per experiment. 414 415 Photosynthetic rate measurement 416 4-week-old seedlings of Rutgers and epiF4 were used for photosynthetic rate 417 measurements. The fully expanded leaves of 15 Rutgers plants and 15 epiF4 plants were 418 measured under saturated photosynthetic photon flux density (1500 μmol/m2/s), at a 419 temperature of 30oC at 400 μmol·mol−1 CO2 in the reference chamber with a Li-6400 420
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Portable Photosynthesis System (Li-Cor). Three replicate measurements were taken at 421 11AM on three continuously sunny days. 422 423 Grafting 424 Tube Grafting was carried out with seedlings at the two- to four-leaf stage following the 425 procedure described by Rivard and Louws (2006). MSH1-RNAi plants with and without 426 transgene were used in the grafting experiments (scion/rootstock): wild type/wild type, 427 wild type/ mild-DR (transgene null) and reciprocal, and wild type/dwarf-DR (transgenic) 428 and reciprocal. Fruits from each grafted plant were harvested separately and derived seed 429 planted as the first progeny. Each grafted combination involved at least two replicates, with 430 the experiment repeated three times. 431 432 RNA-Seq sample collection and data processing 433 For tomato RNA-seq, apical meristem tissues from 4-week-old wild type, epiF3, mild-DR 434 and dwarf-DR plants were used for total RNA extraction. For Arabidopsis RNA-seq, leaf 435 material from 4-week-old plants prior to bolting were used, RNA libraries were 436 constructed as described by TruSeq® RNA Sample Preparation v2 Guide. These libraries 437 were sequenced at a final concentration of 5pM in a Hi-Seq 2500 rapid 100 bp single read 438 run at the Sequencing and Microarray Core Facilities of the University of Nebraska Medical 439 Center. For each sample, three replicates were sequenced and at least 20 million reads per 440 replicate were generated. After sequencing, the adapter sequences and the barcodes were 441 removed. FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) was used 442 to confirm sequencing quality. Bowtie-2.1.0 (Langmead et al, 2009) and Tophat-2.0.10 443
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(Kim D et al, 2013) (with the default parameter) were used to map the reads of tomato 444 samples to gene models in the tomato reference genome ITAG 2.4 445 (ftp://ftp.solgenomics.net/tomato_genome/annotation/ITAG2.4_release/). DESeq2 446 (Anders et al, 2010) was used to identify differentially expressed genes (DEGs) between 447 each mutant and the wild-type control. Raw P-values were adjusted using the Benjamini-448 Hochberg procedure (Benjamini et al, 1995), and a cut-off value of adjusted p< 0.05 was 449 used to identify significant DEGs. Arabidopsis RNA-Seq data were processed similarly using 450 TAIR10 as the reference genome. For cross comparison, BLAST was used to identify 451 orthologs of tomato genes in Arabidopsis, with the match having the lowest E-value used in 452 each case. After corresponding orthologs were identified, lists of differentially expressed 453 genes in tomato msh1-RNAi and Arabidopsis msh1 (compared to their wild-type 454 counterparts) were compared for overlap. DAVID Bioinformatics Resources 6.7 was used 455 for GO function enrichment analysis (Huang DW et al, 2009). 456 457 Accession number 458 The whole RNA-seq dataset including both row data and processed data were deposited on 459 Gene Expression Omnibus (GEO) under accession number GSE65242. All data are available 460 at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE65242 461 462 Acknowledgements 463 We thank Timothy Davis and members of the Tomato Breeding Program at University of 464 Florida for technical assistance with the field experiments, Mon-Ray Shao and Robersy 465 Sanchez for assistance with statistical analysis, Sunil Kumar KR for valuable discussion, 466
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Kamaldeep Virdi for grafting experiment design, and Vikas Shedge for assistance in the 467 design of the methylase inhibitor experiments. We also thank the support staff of the 468 UNMC Sequencing Core Facility for assistance with RNAseq. The research was supported 469 by funding from Syngenta Biotechnology Inc., NSF (IOS 1126935), and internal funding 470 from the UNL IANR. 471 472 Table 1
DR phenotype range
Height Flowering date Fruit number
(cm 15 weeks) (days after germination) (18weeks)
Rutgers 170.2 ± 7.5 48.0 ± 0 12.3 ±1.8
Mild-DR 160.4 ± 4.0 49 ± 0.7 20.8 ±2.4
Dwarf-DR 89.7 ± 8.5 65.5 ± 0.7 1.5 ± 1.0 Data shown are mean ± SE from at least 5 plants, flowering date was documented as the date of first visible mature flower appearance 473 Table 2. Narrow-sense Heritability
Heritability Response Selection differential
epiF3-B1 -0.28 -1.21 4.34 epiF3-B2 0.02 0.07 3.73 epiF3-B3 0.17 0.38 2.30 epiF4-B2 0.40 0.58 1.46 epiF3-B1, epiF3-B2,epiF3-B3,are three plants selected from epiF2 474 for higher yield, epiF4-B2 were selected for high yield form epiF3-B2 475 h²: Heritability R: Response S: Selection differential 476 R= mean of progeny - mean of parental (replanting) 477 S=selected plant - mean of parental 478 h²= R/S 479
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Table 3. Differential expressed genes common between tomato dwarf-DR plants and Arabidopsis dwarf-DR plants (shown as fold change, significant at a false discovery rate of less than 0.05) Tomato
Arabidopsis
ID Fold
change
Annotation ID Fold change
Annotation
Response to stress GO:0006950 Solyc01g104740.2 36.6 Multiprotein bridging
Parsed CitationsAbdelnoor, R.V. Yule R, Elo A, Christensen AC, Meyer-Gauen G, Mackenzie SA. 2003. Substoichiometric shifting in the plantmitochondrial genome is influenced by a gene homologous to MutS. Proc. Natl. Acad. Sci. USA 100: 5968-5973.
PubMed: http://www.ncbi.nlm.nih.gov/pubmed/12730382?dopt=abstractCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Anders, S., and Huber, W. Differential expression analysis for sequence count data. Genome Biol. 2010, 11: R106.Becker, C.Hagmann J, Müller J, Koenig D, Stegle O, Borgwardt K, Weigel D. 2011. Spontaneous epigenetic variation in the Arabidopsisthaliana methylome. Nature 480: 245-249.
CrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Cortijo, S Wardenaar R, Colomé-Tatché M, Gilly A, Etcheverry M, Labadie K, Caillieux E, Hospital F, Aury JM, Wincker P, Roudier F,Jansen RC, Colot V, Johannes F.2014. Mapping the epigenetic basis of complex traits. Science.5, epub ahead of print.
PubMed: http://www.ncbi.nlm.nih.gov/pubmed/24505129?dopt=abstractCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Cubas P, Vincent C, Coen E. 1999. An epigenetic mutation responsible for natural variation in floral symmetry. Nature 401: 157-161.PubMed: http://www.ncbi.nlm.nih.gov/pubmed/10490023?dopt=abstractCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Davila JI, Arrieta-Montiel MP, Wamboldt Y, Cao J, Hagmann J, Shedge V, Xu YZ, Weigel D, Mackenzie SA. 2011. Double-strandbreak repair processes drive evolution of the mitochondrial genome in Arabidopsis. BMC Biol.9:64
PubMed: http://www.ncbi.nlm.nih.gov/pubmed/21951689?dopt=abstractCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Dowen RH, Pelizzola M, Schmitz RJ, Lister R, Dowen JM, Nery JR, Dixon JE, Ecker JR.2012. Widespread dynamic DNA methylationin response to biotic stress. Proc. Natl. Acad. Sci. USA109:E2183-2191.
PubMed: http://www.ncbi.nlm.nih.gov/pubmed/22733782?dopt=abstractCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Greaves IK, Groszmann M, Wang A, Peacock WJ, Dennis ES. 2014. Inheritance of trans-chromosomal methylation patterns fromArabidopsis F1 hybrids. Proc Natl Acad Sci USA 111:2017-22.
CrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Grimanelli D, Roudier F. 2013. Epigenetics and development in plants: green light to convergent innovations. Curr Top Dev Biol.104:189-222.
PubMed: http://www.ncbi.nlm.nih.gov/pubmed/23587242?dopt=abstractCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Hauben M, Haesendonckx B, Standaert E, Van Der Kelen K, Azmi A, Akpo H, Van Breusegem F, Guisez Y, Bots M, Lambert B, LagaB, De Block M. 2009. Energy use efficiency is characterized by an epigenetic component that can be directed through artificialselection to increase yield. Proc Natl Acad Sci USA. 106:20109-14.
PubMed: http://www.ncbi.nlm.nih.gov/pubmed/19897729?dopt=abstractCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Herberich E, Sikorski J, Hothorn T (2010) A Robust Procedure for Comparing Multiple Means under Heteroscedasticity inUnbalanced Designs. PLoS ONE 5(3): e9788. doi:10.1371/journal.pone.0009788
PubMed: http://www.ncbi.nlm.nih.gov/pubmed/20360960?dopt=abstractCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Huang DW, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID BioinformaticsResources. Nature Protoc. 2009; 4(1):44-57.
PubMed: http://www.ncbi.nlm.nih.gov/pubmed/19131956?dopt=abstractCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Hutton SF, Scott JW, Vallad GE. 2014. Association of the fusarium wilt race 3 resistance gene, I-3, on chromosome 7 with increasedsusceptibility to bacterial spot race T4 in tomato. J Amer Soc Hort Sci (in press).
CrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg SL. TopHat2: accurate alignment of transcriptomes in the presence ofinsertions, deletions and gene fusions. Genome Biol. 2013
CrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Virdi KS ,Laurie JD, Xu Y-Z, Yu J-T, Shao M-R, Sanchez R, Feng S-H, Zhai J-X, Kundariya H, WangD, Wamboldt Y, RiethovenJ-JM,Arrieta-Montiel MP,Shedge V, Mackenzie SA. 2015. Arabidopsis MSH1 mutation alters the epigenome to produce heritablehttps://plantphysiol.orgDownloaded on March 16, 2021. - Published by
Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.
changes in plant growth. Nat. Commun . 6386 doi:10.1038/ncomms7386PubMed: http://www.ncbi.nlm.nih.gov/pubmed/25722057?dopt=abstractCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Langmead, B., Trapnell, C., Pop, M., and Salzberg, S.L. Ultrafast and memory-efficient alignment of short DNA sequences to thehuman genome. Genome Biol. 2009, 10: R25.
PubMed: http://www.ncbi.nlm.nih.gov/pubmed/19261174?dopt=abstractCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Manning K, Tor M, Poole M, et al., 2006. A naturally occurring epigenetic mutation in a gene encoding an SBP-box transcriptionfactor inhibits tomato fruit ripening. Nat Genet 38, 948-52.
PubMed: http://www.ncbi.nlm.nih.gov/pubmed/16832354?dopt=abstractCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Mirouze, M. &Paszkowski, J. 2011.Epigenetic contribution to stress adaptation in plants. Curr Op in Plant Biol.14, 267-274.PubMed: http://www.ncbi.nlm.nih.gov/pubmed/21450514?dopt=abstractCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Reinders, J. Wulff BB, Mirouze M, Marí-Ordóñez A, Dapp M, Rozhon W, Bucher E, Theiler G, Paszkowski J.2009. Compromisedstability of DNA methylation and transposon immobilization in mosaic Arabidopsis epigenomes. Genes Dev. 23, 939-950.
PubMed: http://www.ncbi.nlm.nih.gov/pubmed/19390088?dopt=abstractCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Rivard, C. L., and Louws, F. J. 2006.Grafting for Disease Resistance in Heirloom Tomatoes College of Agriculture and LifeSciences, ed. North Carolina Cooperative Extension Service.
CrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Roux, F. Colomé-Tatché M, Edelist C, Wardenaar R, Guerche P, Hospital F, Colot V, Jansen RC, Johannes F. 2011. Genome-wideepigenetic perturbation jump-starts patterns of heritable variation found in nature. Genetics 188: 1015-1017.
PubMed: http://www.ncbi.nlm.nih.gov/pubmed/21596900?dopt=abstractCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Sandhu AP, Abdelnoor RV, Mackenzie SA. 2007. Transgenic induction of mitochondrial rearrangements for cytoplasmic malesterility in crop plants. Proc Natl Acad Sci USA 104:1766-70.
PubMed: http://www.ncbi.nlm.nih.gov/pubmed/17261806?dopt=abstractCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Santamaria RR, Shao M-R, Wang G, Nino-Liu DO, Kundariya H, Wamboldt Y, Dweikat IM, Mackenzie SA. 2014. Implementingselection of MSH1-induced non-genetic variation in Sorghum bicolor as a model for epigenetic breeding in crops. Plos One. DOI:10.1371/journal.pone.0108407
PubMed: http://www.ncbi.nlm.nih.gov/pubmed/25347794?dopt=abstractCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Sahu PP, Pandey G, Sharma N, Puranik S, Muthamilarasan M, Prasad M. 2013. Epigenetic mechanisms of plant stress responsesand adaptation. Plant Cell Rep. 32(8):1151-9.
PubMed: http://www.ncbi.nlm.nih.gov/pubmed/23719757?dopt=abstractCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Schermerhorn, LG. 1934. Scientific breeding gives New Jersey the 'Rutgers' tomato. NJ State Hort Soc News vol 15, no. 6.CrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Schmitz, R.J. Schultz MD, Lewsey MG, O'Malley RC, Urich MA, Libiger O, Schork NJ, Ecker JR. 2011. Transgenerational epigeneticinstability is a source of novel methylation variants. Science 334: 369-373.
PubMed: http://www.ncbi.nlm.nih.gov/pubmed/21921155?dopt=abstractCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Shedge, V., Davila, J., Arrieta-Montiel, M.P., Mohammed, S. & Mackenzie S.A. 2010.Extensive rearrangement of the Arabidopsismitochondrial genome elicits cellular conditions for thermotolerance. Plant Physiol. 152:1960-1970
PubMed: http://www.ncbi.nlm.nih.gov/pubmed/20139171?dopt=abstractCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Shivaprasad PV, Dunn RM, Santos BA, Bassett A, Baulcombe DC. 2012. Extraordinary transgressive phenotypes of hybrid tomatoare influenced by epigenetics and small silencing RNAs. EMBO J. 31:257-266.
PubMed: http://www.ncbi.nlm.nih.gov/pubmed/22179699?dopt=abstractCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Vergeer P, Wagemaker NC, Ouborg NJ. 2012. Evidence for an epigenetic role in inbreeding depression.BiolLett 8:798-801.https://plantphysiol.orgDownloaded on March 16, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.
Vergeer P, Wagemaker NC, Ouborg NJ. 2012. Evidence for an epigenetic role in inbreeding depression.BiolLett 8:798-801.PubMed: http://www.ncbi.nlm.nih.gov/pubmed/22791708?dopt=abstractCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Xu, Y.-Z.Arrieta-Montiel MP, Virdi KS, de Paula WB, Widhalm JR, Basset GJ, Davila JI, Elthon TE, Elowsky CG, Sato SJ, ClementeTE, Mackenzie SA.2011. MutS HOMOLOG1 is a nucleoid protein that alters mitochondrial and plastid properties and plantresponse to high light. Plant Cell 23:3428-3441.
PubMed: http://www.ncbi.nlm.nih.gov/pubmed/21934144?dopt=abstractCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Xu, Y.-Z., Santamaria RR, Virdi KS, Arrieta-Montiel MP, Razvi F, Li S, Ren G, Yu B, Alexander D, Guo L, Feng X, Dweikat IM,Clemente TE, Mackenzie SA. 2012. The chloroplast triggers developmental reprogramming when MUTS HOMOLOG1 issuppressed in plants. Plant Physiol. 159:710-720.
PubMed: http://www.ncbi.nlm.nih.gov/pubmed/22496509?dopt=abstractCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Y. Benjamini and Y. Hochberg. Controlling the false discovery rate: a practical and powerful approach to multiple testing. Journalof the Royal Statistical Society B, 1995, 57:289-300.
CrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
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