muts homolog1-derived epigenetic breeding · muts homolog1-derived epigenetic breeding potential in...

MutS HOMOLOG1-Derived Epigenetic BreedingPotential in Tomato1[OPEN]

Xiaodong Yang, Hardik Kundariya, Ying-Zhi Xu, Ajay Sandhu, Jiantao Yu, Samuel F. Hutton,Mingfang Zhang, and Sally A. Mackenzie*

Laboratory of Genetic Resources and Functional Improvement for Horticultural Plants, Department ofHorticulture, Zhejiang University, Hangzhou 310029, People’s Republic of China (X.Y., M.Z.); Department ofAgronomy and Horticulture, University of Nebraska, Lincoln, Nebraska 68588–0660 (X.Y., H.K., Y.-Z.X., A.S.,J.Y., S.A.M.); and Gulf Coast Research and Education Center, Institute of Food and Agricultural Sciences,University of Florida, Wimauma, Florida 33598–6101 (S.F.H.)

ORCID IDs: 0000-0002-4971-2362 (X.Y.); 0000-0002-1635-4861 (S.F.H.); 0000-0003-2077-5607 (S.A.M.).

Evidence is compelling in support of a naturally occurring epigenetic influence on phenotype expression in land plants,although discerning the epigenetic contribution is difficult. Agriculturally important attributes like heterosis, inbreedingdepression, phenotypic plasticity, and environmental stress response are thought to have significant epigenetic components,but unequivocal demonstration of this is often infeasible. Here, we investigate gene silencing of a single nuclear gene, MutSHOMOLOG1 (MSH1), in the tomato (Solanum lycopersicum) ‘Rutgers’ to effect developmental reprogramming of the plant.The condition is heritable in subsequent generations independent of the MSH1-RNA interference transgene. Crossing thesetransgene-null, developmentally altered plants to the isogenic cv Rutgers wild type results in progeny lines that show enhanced,heritable growth vigor under both greenhouse and field conditions. This boosted vigor appears to be graft transmissible andis partially reversed by treatment with the methylation inhibitor 5-azacytidine, implying the influence of mobile, epigeneticfactors and DNA methylation changes. These data provide compelling evidence for the feasibility of epigenetic breeding in acrop plant.

Epigenetic variation in nature is an underpinning tophenotypic plasticity and the adaptive capacity of anorganism (Mirouze and Paszkowski, 2011; Sahu et al.,2013). Chromatin modifications provide a memory ofabiotic or biotic stress that has been experienced,priming a biological system to better cope with futurerecurrence (Dowen et al., 2012; Grimanelli and Roudier,2013). The extent to which epigenetic memory extendstransgenerationally has been the subject of vigorousinvestigation. While it is clear that epigenetic traits canbe stably maintained through extremely long lineages(Cubas et al., 1999), it is also clear that plant cytosinemethylation patterns are highly dynamic (Becker et al.,2011; Schmitz et al., 2011). Consequently, the extent towhich epigenetic variation can be directly exploited inmeaningful plant or animal breeding strategies remainslargely unknown.

The behavior and stability of genome-wide epi-genetic changes in plants have been pursued mostpowerfully through the development of epigeneticallymodified recombinant inbred lines in Arabidopsis(Arabidopsis thaliana). The approach capitalizes on theavailability of genetic mutations in the DNA methyl-ation machinery (Reinders et al., 2009; Roux et al.,2011). Studies have confirmed an association be-tween plant phenotypic variation and modifications ingenome methylation and have shown a stable in-heritance of some derived epi-traits over multiplegenerations (Cortijo et al., 2014). These studies suggestthat epigenetic variation should be amenable to plantselection.

However, mutation-mediated disruption of theplant methylation machinery has not necessarily pro-duced altered growth behavior that is adaptive oragriculturally advantageous. It is feasible to identifynatural epialleles that influence growth behavior. Thetomato (Solanum lycopersicum) fruit-ripening variantcolorless nonripening (cnr) appears to be a naturallyoccurring epiallele of CNR, which encodes an SBP-boxtranscription factor. In the variant, CNR expressionis silenced by promoter hypermethylation to inhibitnormal fruit ripening (Manning et al., 2006). One strat-egy for accessing natural epigenetic variation waspresented in canola (Brassica napus), where recursiveselection for the variation of respiratory parameters in

1 This work was supported by Syngenta Biotechnology, Inc., theNational Science Foundation (grant no. IOS 1126935), and the Uni-versity of Nebraska (Lincoln).

* Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Sally A. Mackenzie ([email protected]).

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a genetically fixed doubled haploid line resulted in arange of phenotypic variations suspected to be non-genetic (Hauben et al., 2009). Some emerging pheno-types appeared to be improved in growth performance.Similarly, in tomato, transgressive phenotypes in hy-brids can be epigenetically influenced (Shivaprasadet al., 2012), and in Arabidopsis, hybrid vigor pheno-types appear to show some relationship to the cytosinemethylation behavior of the combined genomes (Greaveset al., 2014). These observations imply that epigeneticvariation influencing plant growth may be importantagriculturally.MutS HOMOLOG1 (MSH1) encodes a dual-targeted

protein that localizes to the mitochondrion, where itsuppresses illegitimate DNA recombination (Abdelnooret al., 2003; Davila et al., 2011), and to the plastid. Itsrole in the plastid is less well defined, but mutation ofthe MSH1 gene in Arabidopsis is known to conditionplastid-associated changes in plant growth behavior.The msh1 mutant is markedly altered in growth rate,branching behavior, flowering time, juvenility-maturitygrowth transition, perennial growth behavior, and abi-otic stress response (Xu et al., 2012). This developmentalreprogramming (DR) is accompanied by altered ge-nome methylation, with the intensity of the alteredphenotype showing an apparent association with anenhanced non-CG hypermethylation of pericentromericgenomic intervals (Virdi et al., 2015). This complex DRphenotype has been recapitulated in several crop spe-cies, both monocot and dicot, by RNA interference(RNAi) suppression of MSH1, prompting changes ingrowth rate, tillering, flowering time, leaf morphology,and abiotic stress tolerance (Xu et al., 2012). In each case,the altered phenotype is subsequently inherited inde-pendently of RNAi transgene segregation, implicating anongenetic mechanism (Xu et al., 2012; Santamaria et al.,2014).The DR state conditioned by the mutation or si-

lencing of MSH1 is unusual. Crossing of the modifiedplant, either the Arabidopsis msh1 mutant or the sor-ghum (Sorghum bicolor) MSH1-RNAi suppression line,to its isogenic wild-type counterpart produces herita-ble enhanced growth vigor in subsequent progenygenerations (Santamaria et al., 2014; Virdi et al., 2015).The enhancement in vigor is evidenced not only inmore rapid growth, earlier flowering, and greateraboveground biomass but in markedly increased seedyield.To investigate the potential for exploiting nongenetic

variation in a directed crop breeding effort, we intro-duced an MSH1-RNAi transgene construct to the to-mato ‘Rutgers.’ Here, we demonstrate that MSH1suppression results in tomato developmental reprog-ramming and that crossing of the modified line to itsisogenic cv Rutgers wild type produces marked en-hancement in growth vigor. We show that these growthchanges greatly outperform cv Rutgers under com-mercial field production conditions, leading to earlierripening, higher yields, and heat tolerance. The en-hancements in growth appear to be graft transmissible,

and the altered phenotype is partially obviated by theexogenous application of a methylase inhibitor, featurescharacteristic of an epigenetic influence in the growthchanges we observe.

RESULTS

MSH1-RNAi Suppression in Tomato Leads toDevelopmental Reprogramming

Development of the cv Rutgers MSH1-RNAi trans-genic lines was described previously, and lines used inthis study were each shown to contain a single copy ofthe transgene (Sandhu et al., 2007). For this study, weincluded two independent transformants. Suppressionof MSH1 expression consistently resulted in a widerange of altered phenotypes, including changes in leafmorphology, variegation, dwarfing, male sterility,flower development, and flower timing (Fig. 1; Table I).All altered traits showed incomplete penetrance,and a small (approximately 10%) proportion of thetransgenic lines produced a sufficiently severe phe-notype that the plants terminated growth or werecompletely sterile. Over 90% of the transgenic linesproduced viable seed. Segregation of the transgeneoccurred in the progeny of transgenic lines, andtransgene-null segregants tended to revert phenotypi-cally to a milder range of altered growth and to re-stored MSH1 transcript levels (Fig. 2). However, weobserved variation in phenotype in both transgene-positive and transgene-null selections, demonstratingthat the altered phenotypes were subsequently inher-ited independent of the transgene (Supplemental TableS1). In each cycle of self-pollination, progeny produced

Figure 1. A to F, Tomato MSH1-RNAi lines displaying variant phe-notypes: wild-type cv Rutgers (A), mild-DR (transgene null; B), varie-gated-DR (transgenic; C), medium-DR (transgenic; D), dwarf-DR(transgene null; E), and bushy-DR (transgenic; F). G, Leaf morphologychanges in the tomato MSH1-RNAi lines shown above.

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a range of phenotype intensity, and these were not sub-jected to intentional selection. To date, we have main-tained these transgene-null variants for up to eightgenerations by recurrent self-pollination.

Isogenic Crosses Using MSH1-RNAi Transgene NullsResult in Enhanced Growth

Reciprocal crosses of the transgene-null lines to thewild type (Fig. 3A) resulted in F1 progeny showingnormal growth and F2 progeny (designated epiF2)showing a range of enhanced growth vigor (Fig. 3B).The derived epi-lines were maintained on a cvRutgers genetic background. Early growth of theepiF2 plants was more rapid than that of the cvRutgers wild type, resulting in taller plants until the10-week point (Fig. 4). At 10 weeks, the epiF2 linesslowed vegetative growth and transitioned to flow-ering, while the cv Rutgers wild type continued veg-etative growth, flowering slightly later (SupplementalFig. S1). Single plant selections from the broad range ofepiF2 phenotypes resulted in epiF3 families that yieldedhigher and a few yielding lower than the wild type(Supplemental Fig. S2; Supplemental Table S2). Theseresults suggest that MSH1-associated nongenetic vari-ation shows at least a mild response to selection.

Enhanced-Growth Tomato Phenotypes Show EnhancedField Performance

Epi-lines displayed enhanced seedling growth vigorrelative to the wild type, evident within the first 2 to3 weeks (Supplemental Fig. S3), and early transition toreproduction (Supplemental Table S3). EpiF2, epiF3,and epiF4 families were grown under Florida fieldconditions to assess their performance and response toselection. Results in the field were generally similar tothose in the greenhouse, and good correlation was seenbetween greenhouse and field data for fruit numberand plant height (Supplemental Fig. S4; SupplementalTable S4).

The cv Rutgers wild-type plants were similar inphotosynthetic rate but were increased in overallvegetative growth and lower in fruit set than the epi-lines (Supplemental Fig. S5). The cv Rutgers plantsproduced an average vegetative fresh weight (minusfruit) of 2.35 kg and dry weight of 0.38 kg, while the

epiF4 plants averaged 1.62 kg in fresh vegetativeweight and 0.28 kg dry weight (averaged from sixplants each). Flower and fruit set showed steady in-creases in epiF2, epiF3, and epiF4 over the wild type,with epiF4 productivity consistently the highest(Supplemental Fig. S6). While average fruit size waslower in the epi-lines relative to the wild type(Supplemental Fig. S6), overall fruit yield was greater,seed number was greater, and fruit sugar contentwas unchanged, obviating the greenhouse effects(Supplemental Fig. S6; Supplemental Table S5). Fruitripening was more rapid in the epi-lines, resulting in agreater proportion of red fruit at a single harvest time(Fig. 3, C and D; Supplemental Table S3). Because thevariation observed was largely associated with growthvigor and heritability was variable (Table II), we ap-plied relatively low selection pressure in each cycle.Bulking seed taken from the top 50% in epiF2 to pro-duce an epiF3 bulk, and again in epiF3 to produce anepiF4 bulk, resulted in decreases in variance in eachcycle and a 35% increase in mean yield over the tworounds of selection (Fig. 5; Supplemental Fig. S7).These results imply significant unrealized epigeneticyield potential in this line. Surprisingly, this significant

Table I. DR phenotype range

Data shown are means 6 SE from at least five plants. Flowering date was documented as the date of firstvisible mature flower appearance.

Plant Height (15 Weeks) Flowering Date Fruit No. (18 Weeks)

cm d after germination

cv Rutgers 170.2 6 7.5 48.0 6 0 12.3 6 1.8Mild-DR 160.4 6 4.0 49 6 0.7 20.8 6 2.4Dwarf-DR 89.7 6 8.5 65.5 6 0.7 1.5 6 1.0

Figure 2. Quantitative real-time PCR analysis of derived tomato lines.Relative MSH1 transcript levels in variant phenotype tomato plantswith (+) and without (2) transgene are shown. Values are means 6 SE

of three biological replicates.

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yield advantage was also evident when the wild-typeand cv Rutgers epiF3 lines were grown under high-heatconditions (Fig. 6; Supplemental Table S6), providingsome indication that theMSH1 effect can enhance abioticstress tolerance. To date, we have not observed evidenceof enhanced biotic stress tolerance in the modified cvRutgers line.

The Enhanced Growth Effect Is Partially Reversed by aMethylation Inhibitor

The enhanced growth vigor phenotype was par-tially obviated with the exogenous application of theDNA methylation inhibitor 5-azacytidine (Fig. 7). Threeseparate experiments testing seedling growth in me-dium supplemented with 30 mM inhibitor producedsignificant changes in epiF4 seedling growth, whileonly a mild change in growth was observed in wild-type plants (Fig. 7; Supplemental Fig. S8). EpiF4seedlings growing in the presence of the inhibitor werereduced in plant height relative to the untreated epiF4control as well as the treated wild-type seedlings. Asubset of the seedlings from these experiments weretransplanted to potting medium and observed for23 d to monitor subsequent growth behavior. EpiF4plants that were previously treated with 5-azacytidinewere reduced in growth relative to untreated plantsfollowing transfer to soil. By 23 d after transplanting,the previously treated epiF4 plants were reduced 20%in plant height (Supplemental Fig. S8), suggesting thatsome portion of the growth enhancement can be at-tributed to the altered DNA methylation state in theepiF4 lines. However, previously treated epiF4 plants,nearly equal in height to the wild type at trans-planting, began to outgrow the wild type by 12 d aftertransplanting, showing a nearly 60% increase in plantheight over the untreated wild type (Supplemental Fig.S8). This observation may reflect partial reestablish-ment of the previous methylation state after removalfrom 5-azacytidine. Interestingly, previously treated cvRutgers wild-type plants showed a slight enhancementin growth rate relative to the untreated control.This effect might signal an association between DNAmethylation state and mild inbreeding depression

Figure 3. Breeding strategy and phenotypes of epi-lines. A, Schematicrepresentation of the breeding strategy. B, Representative epiF2 plantgrown in the greenhouse with the wild type at 16 weeks. C, Repre-sentative cv Rutgers wild-type plants at 21 weeks in the field. D,Sample epiF3 plants at 21 weeks in the field. Photographs for C and Dwere taken on the same day.

Figure 4. Enhanced seedling vigor and reproductive growth in the epi-lines. Plant growth rate (A) and inflorescence accu-mulation (B) in epiF3 and the cv Rutgers wild type grown in the greenhouse are shown. Error bars correspond to the SE ofbiological replicates (n = 20).










(Vergeer et al., 2012), although this has not been in-vestigated further.

The Enhanced Growth Effects Appear To Be GraftTransmissible in Tomato

Previous studies of the MSH1 effect in Arabidopsisshowed that grafting of wild-type Arabidopsis Co-lumbia-0 scion to the msh1 dwarfed mutant as root-stock resulted in seed progeny with unusual enhancedgrowth vigor closely resembling epiF3 lines (Virdiet al., 2015). Consequently, we carried out graftingexperiments between the cv Rutgers wild type andcv Rutgers transgenic MSH1-RNAi lines. While wedetected no significant growth change in progeny

coming from the wild type grafted to the wild type,progeny from the wild-type scion grafted to theMSH1-RNAi transgenic line as rootstock showedmarkedly enhanced early growth rate, resembling theepiF3 effect (Fig. 8). As in the case of Arabidopsis,these results further support the hypothesis that en-hanced growth vigor is nongenetic and likely includesa mobile signal within the plant.

RNA-Sequencing Profiling Reveals a Common ProcessUnderlying the Developmental Reprogramming Causedby MSH1 Suppression in Arabidopsis and Tomato

We conducted RNA sequencing (RNA-seq) in to-mato dwarf-DR, mild-DR, epiF3, and wild-type plantsand in the Arabidopsis transfer DNA insertion msh1mutant dwarf-DR plants in order to identify shareddifferentially expressed gene responses to MSH1 dis-ruption and to begin to understand the cellular pro-cesses underlying the phenotypic diversity observed inthese materials.

In a previous report (Xu et al., 2012), we identifiedseveral differentially expressed genes in the Arabi-dopsis msh1 mutant, involving multiple developmen-tal and stress response pathways, by microarrayanalysis. Here, we show the results of comparativestudies involving both Arabidopsis and tomato thatreveal similar patterns of gene expression change in

Table II. Narrow-sense heritability

epiF3-B1, epiF3-B2, and epiF3-B3 are three plants selected fromepiF2 for higher yield, and epiF4-B2 was selected for high yield fromepiF3-B2. Response data = mean of progeny 2 mean of parental(replanting); selection differential data = selected plant 2 mean ofparental plant; heritability data = response/selection differential.

Plant Heritability Response Selection Differential

epiF3-B1 20.28 21.21 4.34epiF3-B2 0.02 0.07 3.73epiF3-B3 0.17 0.38 2.30epiF4-B2 0.40 0.58 1.46

Figure 5. Enhanced total fruit yield and fruitnumber in epi-lines. A, Total fruit yield from asingle epiF4 plant (right) compared with wild-type cv Rutgers (left). B, Total fruit yield of epi-line and wild-type plants from the fall fieldexperiments. Bars represent means 6 SE (n .30). C, Total fruit number from epi-line andwild-type plants grown in the fall field exper-iments. Bars represent means 6 SE (n . 30).epiF3 plants were selected from epiF2 for highyield, and epiF4 plants were selected fromepiF3 for high yield. epiF3 Bulk and epiF4Bulk samples were derived by selecting theepiF2 and epiF3 plants representing the upper50% in yield, respectively. All data were col-lected from 22-week-old plants. Different lettersrepresent significant differences at P , 0.05.


Yang et al.



both species in response to MSH1 disruption. Tomatoand Arabidopsis dwarf-DR plants shared 1,252 com-mon gene expression changes, which accounted for46.9% of the observed tomato dwarf-DR plant changesand 12.7% in the Arabidopsis counterpart (SupplementalFig. S9). These 1,252 genes included components in re-sponse to stimulus, protein amino acid phosphorylation,cell surface receptor-linked signal transduction, cell di-vision, and development (Supplemental Table S7). Thechanges show striking similarity to profiles in Arabi-dopsis msh1-associated developmental reprogramming(Xu et al., 2012). For instance, similar to Arabidopsis,we found changes in genes regulating cell division,including cyclin family proteins, and genes involvedin cell proliferation and differentiation. Most of thesewere down-regulated in both tomato and Arabidopsisdwarf-DR plants, consistent with the dwarf phenotypeand delayed growth (Table III). In terms of stress

response, alternative oxidase is strongly up-regulated inboth Arabidopsis (AT1G32350, AOX1D; and AT3G22370,AOX1A) and tomato (Soly08g075540.0), consistentwith redox changes and organellar electron transportchain perturbation with MSH1 disruption (Table III).Several biotic and abiotic stress response genes wereaffected in expression in both plant species, along withphytohormone gene responses influencing auxin, GA,ethylene, cytokinin, and brassinosteroid pathways(Table III). The observed overlaps between tomato andArabidopsis gene expression patterns allow for theconfirmation of signature changes underlying the msh1developmental reprogramming process, a phenome-non that appears to be triggered by plastid perturba-tion (Xu et al., 2012).

To investigate the phenotypic range that emergeswith MSH1 disruption, we compared gene expressionchanges in the tomato dwarf-DR plants (containing theMSH1-RNAi transgene and displaying extreme changesin development) with those in mild-DR plants (MSH1-RNAi transgene null and displaying mild develop-mental effects). An 88.7% decrease is observed in geneexpression changes in the mild-DR plants relative todwarf-DR (from 2,671 in dwarf-DR to 302 in mild-DR),and half of these genes (171) overlap with the dwarf-DRprofile (Supplemental Fig. S10). These results show thatthe observed range in the intensity of developmentalreprogramming coincides with the magnitude of geneexpression changes and confirm our prediction that theidentified signature pathways overlapping betweenArabidopsis and tomato underlie the DR phenotype.

In a comparison of epiF3 plants with the wild typein tomato, using the cutoff of adjusted P , 0.05, weidentified 437 genes differentially expressed in theenhanced-growth line. Drawing on Gene Ontologyanalysis, these genes appeared to be involved pre-dominantly in the vegetative-to-reproductive phasetransition, growth and development, stress response,and cellular ketone metabolic processes (SupplementalTable S8). The observed gene expression changes agreewell with the epiF3 phenotypes in tomato of enhancedflowering and flower time changes, early growthvigor, earlier ripening, higher yield, and heat tolerance.

Figure 6. EpiF2 heat tolerance trial. Heat tolerance was assessed in thefield at Wimauma, Florida (27˚45934.299N, 82˚13931.599W), during thesummer of 2013 (transplanting August 12, 2013/harvesting October21, 2013). Average ambient temperature during the experiment was25.2˚C. Total fruit yields, including both red and green fruits, from thewild type, epiF2 lines, FL8059 (heat-sensitive variety), and FL8044(heat-tolerant variety) are shown. Bars represent means 6 SE (n = 15).EpiF2 versus wild-type differences in red fruit yield (P, 0.01) and totalfruit yield (P , 0.01) were significant, but no significant difference wasseen in green fruit yield. Similar results were seen in FL8059 andFL8044 comparisons (Student’s t test).

Figure 7. Methylation inhibitor influence on growth vigor of epi-line seedlings. A, Phenotype of the cv Rutgers wild type andthe epiF4 line treated with the methylation inhibitor 5-azacytidine (2 weeks after treatment). B, Plant height measurements ofwild-type (WT) and epi-line plants treated with 5-azacytidine. Bars represent means 6 SE; n = 10. Data are from three inde-pendent experiments, with plants treated for 2 weeks in replicate 1 (Rep1) and Rep2 and 3 weeks in Rep3.











The key regulator gene for flowering, Solyc05g053850.2(SELF PRUNING; a homolog of Twin Sister of FLOW-ERING LOCUS T in Arabidopsis), for example, is up-regulated together with another important floweringgene, Solyc07g052700.2 (MADS-BOX TRANSCRIPTIONFACTOR1). The Arabidopsis homolog AGAMOUS-LIKE66 is involved in late-stage pollen development andpollen tube growth. We also observed markedly de-creased expression of Solyc08g082980.2, with an Ara-bidopsis putative homolog, WITH NO LYSINE (K)KINASE8, involved in the photoperiod floweringpathway and its mutant showing early flowering inArabidopsis. Expression changes of these genes arelikely associated with the enhanced flowering andflower time changes observed in tomato epi-lines(Supplemental Tables S9 and S10). A group of differ-entially expressed genes suspected to underlie theobserved early growth vigor includes auxin signalingand response genes Solyc07g043610.2 (homolog toArabidopsis AUXIN RESPONSE FACTOR6 [ARF6]),Solyc02g077560.2 (homolog to Arabidopsis ARF3), thecell growth gene Solyc06g049050.2 (homolog to Ara-bidopsis EXPANSIN A8), and a component of SCF (forSkp, Cullin, F-box-containing complex) complexes,Solyc06g008710.1 (homolog to Arabidopsis CULLIN1),which mediates responses to auxin and jasmonic acid.Stress response genes altered in their expression in-clude several heat shock protein genes, which may berelevant to the enhanced heat tolerance observed inour tomato epi-lines (Supplemental Tables S9 and S10).

DISCUSSION

The cv Rutgers was released in the 1930s. It was bredfor significant vegetative growth, nicely rounded andmoderately sized fruits, and slightly delayed harvest(Schermerhorn, 1934). Interestingly, all of these traitswere modified in response to MSH1 manipulation. By

epiF4, populations showed early flowering and fruiting,the fruit was markedly increased in number and earlyto mature, and vegetative growth was reduced in favorof fruit production. While fruit size was reduced in re-sponse to the increased fruit numbers, cv Rutgers wasnot originally bred for large fruit size. Possibly, a varietygenetically selected for large fruit size would undergoless of a reduction in response to MSH1 modulation.The cv Rutgers also was not bred specifically for heattolerance; we assume that the enhanced abiotic stresstolerance is a direct outcome of the MSH1 effect.

Evidence from this study, and earlier studies inArabidopsis and sorghum, suggest that MSH1 suppres-sion produces nongenetic changes in the plant. This ev-idence includes the observation of reproducible patternsof cytosine methylation change in the genome of Arabi-dopsis msh1 mutants and epi-lines (Virdi et al., 2015),parallel changes in tomato and sorghum (Santamariaet al., 2014) phenotypes that are inherited independent ofthe MSH1-RNAi transgene, partial reversal of the phe-notype with 5-azacytidine in Arabidopsis (Virdi et al.,2015) and tomato, and graft transmissibility of the en-hanced growth phenotype in Arabidopsis and tomato. Inboth tomato and sorghum, enhanced growth in the epi-lines was heritable through multiple generations, but insorghum, the phenotype appeared to revert back to thewild type by epiF5. This erosion of phenotype may becharacteristic of epigenetic traits (Cortijo et al., 2014).What distinguishes the effects of MSH1 suppressionfrom previous epigenetic studies that involved pertur-bation of the methylation machinery is that emergenttraits reiterate with the same transgenerational behavioracross multiple plant species, both for developmentalreprogramming and vigor enhancement through cross-ing. TheMSH1 enhanced growth vigor is both surprisingand potentially exploitable agronomically; whether theeffect is analogous to heterosis is unclear.MSH1-derivedgrowth vigor appears uncanny in its resemblance toheterosis, but subsequent heritability over multiple

Figure 8. Graft transmission of theenhanced-growth phenotype in to-mato. A, First generation progeny of agrafted cv Rutgers wild-type scion ontransgenic dwarf-DR rootstock (right)and a wild-type scion on a wild-typerootstock control (left). The photographshows 7-week-old plants. B and C, Thecv Rutgers wild-type scion on trans-genic dwarf-DR rootstock progenyplants display greater plant height, ev-ident at 7 weeks (Student’s t test, a ,0.05; B), and higher fruit yields (Stu-dent’s t test, a , 0.05; C) in the green-house. Fruit were harvested at 14 weeks.Bars represent means6 SE. For fruit yield,n = 4; for plant height, n = 12.


Yang et al.





generations (to date, it has been observed to epiF7 inArabidopsis) and its seeming responsiveness to selec-tion distinguish this phenomenon from classic defini-tions of heterosis.Observation of greater heat tolerance in the MSH1-

modified lines, relative to the cv Rutgers wild type,implies that the induced effects might influence the

abiotic stress response. An earlier transcript profileanalysis of the msh1 mutant in Arabidopsis, and thisstudy of tomato, show changes in a number of abioticstress response pathways (Shedge et al., 2010; Xu et al.,2011, 2012). What is intriguing is the marked similarityin gene expression responses to MSH1 disruption inboth Arabidopsis and tomato, consistent with our

Table III. Differential expressed genes common between tomato dwarf-DR plants and Arabidopsis dwarf-DR plants

Fold change data are significant at a false discovery rate of less than 0.05.

Tomato Arabidopsis

Identifier Fold Change Annotation Identifier Fold Change Annotation

Response to stress, GO:0006950Solyc01g104740.2 36.6 Multiprotein bridging factor1 AT3G24500 2.4 Multiprotein bridging factor1CSolyc08g075540.2 29.4 Alternative oxidase AT1G32350 5.9 Alternative oxidase1DSolyc08g075540.2 29.4 Alternative oxidase AT3G22370 3.7 Alternative oxidase1ASolyc08g078710.1 25.8 Heat shock protein AT5G51440 16.3 HSP20-like chaperone superfamily

proteinSolyc02g079180.1 5.4 Heat shock transcription factor1 AT5G03720 3.1 Heat shock transcription factor A3Solyc09g014990.2 3.4 WRKY-like transcription factor AT2G38470 7 WRKY DNA-binding protein33Solyc01g103600.2 1.8 Protein TIFY (for TIF[F/Y]) 3A AT1G19180 13.7 Jasmonate-zim-domain protein1

Protein modification process, GO:0006464Solyc11g010150.1 3.7 Ser/Thr protein kinase AT1G78290 2.5 SNF1-related protein kinase2

family proteinSolyc03g114160.1 3 U-box domain-containing protein AT1G60190 9.2 PUB19 (a plant U-box armadillo

repeat protein)Solyc10g049630.1 2.4 Ser/Thr protein phosphatase2C AT4G33920 4.1 Probable protein phosphatase2C63Solyc10g083470.1 1.7 E3 ubiquitin ligase AT4G12570 21.5 Ubiquitin protein ligase5Solyc01g094660.2 1.4 Receptor-like protein kinase AT2G45910 2.4 U-box domain-containing protein

kinase family proteinSolyc12g010450.1 1.3 Protein phosphatase2C AT2G25620 2.3 Probable protein phosphatase2C2Solyc01g034020.2 21.2 Ser/Thr-protein phosphatase AT3G58500 21.4 Protein phosphatase2ASolyc11g065190.1 21.6 Ubiquitin-conjugating enzyme E2-

like proteinAT3G20060 21.7 Ubiquitin-conjugating enzyme19

Solyc06g008780.1 25 Auxin F-box protein 5 AT4G03190 27.1 Glucose Repressed Repression1-like protein1

Developmental process, GO:0032502Solyc04g007000.1 2.4 Ethylene-responsive transcription

factor4AT1G13260 2 AP2/B3 domain transcription factor

Solyc06g008870.2 2.4 GIBBERELLIN INSENSITIVEDWARF1-like GA receptor

AT3G63010 2.3 a/b-Hydrolase superfamily protein

Solyc01g103600.2 1.8 Protein TIFY (for TIF[F/Y]) 3A AT1G19180 13.7 Jasmonate-zim-domain protein1Solyc06g069430.2 1.6 MADS-box transcription factor AT5G60910 2.6 Agamous-like8Solyc10g078370.1 21.5 Auxin efflux carrier family protein AT1G73590 22.2 Auxin efflux carrier family proteinSolyc08g079100.2 21.6 YABBY protein AT4G00180 23.3 Plant-specific transcription factor

YABBY family proteinSolyc07g043620.2 21.8 Auxin response factor6 AT1G30330 22.6 Auxin response factor6Solyc03g006880.2 22.2 GA 20-oxidase1 AT4G25420 27.2 GA receptor GIBBERELLIN

INSENSITIVE DWARF1 L2Solyc08g082980.2 237.4 Ser/Thr protein kinase AT5G41990 21.5 Ser/Thr-protein kinase WITH NO

LYSINE (K) KINASE 8Cell division, GO:0007049

Solyc11g069500.1 21.3 Auxin response factor16 AT2G28350 21.7 Auxin response factor10Solyc08g077550.2 21.3 Novel plant Soluble NSF

Attachment Protein Receptor11AT2G35190 21.7 Novel plant Soluble NSF

Attachment Protein Receptor11Solyc02g071590.1 21.6 a,a-Trehalose-phosphate synthase AT1G78580 21.8 Trehalose-6-phosphate synthaseSolyc11g005090.1 21.6 Cyclin A1 (mitotic-specific cyclin

A)AT1G44110 22.5 Cyclin-A1-1

Solyc01g009040.2 21.6 Cyclin B1 AT5G06150 22.5 Cyclin-B1-2Solyc12g088650.1 21.6 Cyclin D AT4G34160 22.7 Cyclin-D3-1Solyc05g014370.2 21.6 Mitotic spindle checkpoint protein

MAD2AT3G25980 22.1 Mitotic arrest-deficient2

Solyc06g053760.2 21.7 Syntaxin AT1G08560 21.9 Syntaxin of plants111





hypothesis of a programmed response. Interestingly,we have not yet observed evidence of the effects onbiotic stress tolerance, and bacterial spot and virusincidence in the Florida field experiments were similarin both the cv Rutgers wild type and the epi-lines.

The conspicuous similarity in phenotype and be-haviors of different plant species undergoing theMSH1 effect indicates that this is a well-conservedprocess, and we speculate that the MSH1 effect mayparticipate in plant environmental adaptation. Theenhanced growth that emerges following crossing,direct and reciprocal, arising through grafting, andpartially reversed with exogenous 5-azacytidine, ispresumed to be epigenetic. However, it is not yet clearto what extent the initial developmental reprogram-ming might actually be a plastid phenomenon. It ispossible that early changes in growth rate, branching,flowering time, and stress response are conditioned byplastid signals that are distinct from the changesdirecting growth vigor. Consequently, more work isneeded to dissect the various components of the MSH1effect and to identify the evolutionary process that un-derlies this unusual multifunctionalization of MSH1.

MATERIALS AND METHODS

Plant Materials

MSH1 suppression lines in the tomato (Solanum lycopersicum) ‘Rutgers’background were developed previously (Sandhu et al., 2007), and progenyfrom two independent transformation events (T17 and T20) were used in thisstudy. Both lines were confirmed to contain a single transgene copy (Sandhuet al., 2007). Two MSH1-RNAi transgene-null plants each from T17 and T20,showing a mild dwarfing phenotype, were crossed with wild-type inbred cvRutgers reciprocally to generate F1 seeds, and F1 plants were selfed to pro-duce epiF2 families. Progeny from T17 crosses were followed to epiF4 in boththe greenhouse and the field, while progeny from T20 were followed to epiF2in the greenhouse. Plants in the greenhouse were germinated on MetroMix 200medium (SunGro) and maintained at 26°C to 28°C with a 15-h day length andat 20°C to 22.8°C with a 9-h dark period. Primers Tom-CD1F (59-CGCAGG-TATCACGAGGCAAGTGCTAA-39) and Intro-PIR (new; 59-GTGTACTCA-TGTGCATCTGACTTGAC-39) were used to genotype the transgene.

Field Trials

Field trials were conducted during spring and fall, 2013, at the Gulf CoastResearch and Education Center in Florida (27°459N, 82°139W). Seedlings weregrown in the greenhouse for 30 to 40 d and then transplanted to the field. Forthe spring trial, seed was sown on January 16, 2013, and transplants wereplanted in the field on March 8, 2013; the average ambient temperature was21°C. For the fall trial, seed was sown on July 25, 2013, and transplanted onSeptember 14, 2013; the average ambient temperature was 21.98°C. For theheat tolerance trial, seed was sown on July 2, 2013, transplanted on August 12,2013, and plots were harvested on October 21, 2013; the average ambienttemperature was 25.2°C, with highest temperature reaching 35°C. All exper-iments were conducted under commercial production practices, as describedby Hutton et al. (2014), except that beds were fumigated with Pic-Clor60 (336kg per treated hectare). Both spring field (cv Rutgers control, epiF2, and epiF3)and fall field (cv Rutgers control, epiF2, epiF3, and epiF4) trials were com-posed of three blocks, six entries each, and two replicates (three 3 six a-design).Each entry was composed of 15 plants.

Plant height, inflorescence number, and fruit number were measured atmultiple time points during the growing season. Total fruit from each plant washarvested separately (12 plants minimum for each entry), and data werecollected for fruit number and total fruit weight, from which average fruitweight was calculated. Twelve fruit selected randomly from each plant were

used to measure seed number and Brix (PR-32a; ATAGO Palette). Six repre-sentative plants from the cv Rutgers wild type and epiF4 were used to mea-sure aboveground biomass.

Real-Time PCR Analysis

Total RNA was extracted from leaf tissue of 4-week-old seedlings usingTRIzol (Qiagen) and treated with DNase (Qiagen). Reverse transcription forreal-time PCR was performed with the QuantiTect Kit (Qiagen). QuantitativePCR was performed for MSH1 transcripts (Slmsh1-F1, 59-GGACGAAATTG-GCTGTTTGG-39; and Slmsh1-R1, 59-ACCGTCAACATATTCAGCTCC-39) onthe iCycler iQ system (Bio-Rad) with SYBR Green Supermix (Invitrogen). Thetomato gene S. lycopersicum Elongation Factor (SlEF; SlEF-F, 59-GATTGGTG-GTATTGGAACTGTC-39; and SlEF-R, 59-AGCTTCGTGGTGCATCTC-39) wasused to normalize transcript levels.

Statistical Analysis

For comparisons between two groups, Student’s t tests were performed. Forcomparison between multiple groups in field experiments and greenhouseexperiments, the data were fit to a linear model with multiple comparisonsbetween means performed using a heteroscedastic consistent covariance es-timation (Herberich et al., 2010). P values were corrected for multiple testsusing the Benjamini-Hochberg method (Benjamini and Hochberg, 1995).

5-Azacytidine Treatment

Seeds of the cv Rutgers wild type and epiF4 were surface sterilized in 4% (v/v)sodium hypochlorite, rinsed thoroughly with sterile water, and sown in 8-ounceclear cups (Fabri-Kal) containing 30 mL of 0.5 M Murashige and Skoog medium(Sigma) supplemented with 1% (w/v) agar and 0 (control) or 30 mM 5-azacytidine(Sigma). These seeds were germinated and grown in tissue culture facilities at24°C, 18-h day length, and 200 mmol m22 s21 light intensity for 14 or 20 d. Forlong-term observation, 14-d-treated plants were transferred to growth medium andgrown under standard conditions in the greenhouse. The experiment was carriedout three times, with at least 15 replicates for each treatment per experiment.

Photosynthetic Rate Measurement

Four-week-old seedlings of cv Rutgers and epiF4 were used for photo-synthetic rate measurements. The fully expanded leaves of 15 cv Rutgers plantsand 15 epiF4 plants weremeasured under saturated photosynthetic photon fluxdensity (1,500 mmol m22 s21) at a temperature of 30°C at 400 mmol mol21 CO2in the reference chamber with a Li-6400 Portable Photosynthesis System (Li-Cor). Three replicate measurements were taken at 11 AM on three continuouslysunny days.

Grafting

Tube grafting was carried out with seedlings at the two- to four-leaf stagefollowing the procedure described by Rivard and Louws (2006). MSH1-RNAiplants with and without transgene were used in the grafting experiments(scion/rootstock): wild type/wild type, wild type/mild-DR (transgene null)and reciprocal, and wild type/dwarf-DR (transgenic) and reciprocal. Fruitsfrom each grafted plant were harvested separately, and derived seed wasplanted as the first progeny. Each grafted combination involved at least tworeplicates, with the experiment repeated three times.

RNA-seq Sample Collection and Data Processing

For tomato RNA-seq, apical meristem tissue from 4-week-old wild-type,epiF3, mild-DR, and dwarf-DR plants was used for total RNA extraction. ForArabidopsis (Arabidopsis thaliana) RNA-seq, leaf material from 4-week-oldplants prior to bolting was used, and RNA libraries were constructed as de-scribed in the TruSeq RNA Sample Preparation v2 Guide. These libraries weresequenced at a final concentration of 5 pM in a Hi-Seq 2500 rapid 100-bpsingle-read run at the Sequencing and Microarray Core Facilities (Universityof Nebraska Medical Center). For each sample, three replicates were se-quenced, and at least 20 million reads per replicate were generated. Aftersequencing, the adapter sequences and the barcodes were removed. FastQC


Yang et al.



(http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) was used toconfirm sequencing quality. Bowtie 2.1.0 (Langmead et al., 2009) and Tophat2.0.10 (Kim et al., 2013; with the default parameter) were used to map the readsof tomato samples to gene models in the tomato reference genome ITAG 2.4(ftp://ftp.solgenomics.net/tomato_genome/annotation/ITAG2.4_release/).DESeq2 (Love et al., 2014) was used to identify differentially expressed genesbetween each mutant and the wild-type control. Raw P values were adjustedusing the Benjamini-Hochberg procedure (Benjamini and Hochberg, 1995),and a cutoff value of adjusted P , 0.05 was used to identify significantdifferentially expressed genes. Arabidopsis RNA-seq data were processed simi-larly using The Arabidopsis Information Resource 10 as the reference genome. Forcross comparison, BLAST was used to identify orthologs of tomato genes inArabidopsis, with the match having the lowest E value used in each case. Aftercorresponding orthologs were identified, lists of differentially expressed genes intomato msh1-RNAi and Arabidopsis msh1 (compared with their wild-typecounterparts) were compared for overlap. DAVID Bioinformatics Resources 6.7was used for Gene Ontology function enrichment analysis (Huang et al., 2009).

The whole RNA-seq data set including both raw data and processed datawas deposited in the Gene Expression Omnibus under accession numberGSE65242. All data are available at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE65242.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Changes in epi-line maturation.

Supplemental Figure S2. Spring field experiments yield result.

Supplemental Figure S3. Enhancement shown in seedling of epi-line.

Supplemental Figure S4. Correlation between greenhouse and field data.

Supplemental Figure S5. Growth parameters for wild-type and epi F4lines.

Supplemental Figure S6. Fruit features in the wild type and epi-lines.

Supplemental Figure S7. Selection response in epi-lines.

Supplemental Figure S8. Methylation inhibitor treatment.

Supplemental Figure S9.Differential expression genes in DR-dwarf plants.

Supplemental Figure S10. Differential expression genes in tomato dwarf-DR plants and mild-DR plants.

Supplemental Table S1. The DR phenotype is transgene independent.

Supplemental Table S2. Greenhouse yield trial data.

Supplemental Table S3. Red fruit proportion at each harvest time.

Supplemental Table S4. epiF4 greenhouse yield trial data.

Supplemental Table S5. Greenhouse trial soluble sugar content.

Supplemental Table S6. Heat tolerance field trial data summary.

Supplemental Table S7. GO analysis of dwarf-DR plants.

Supplemental Table S8. GO analysis of epiF3 plants.

Supplemental Table S9. Sample up-regulated genes in epiF3.

Supplemental Table S10. Sample down-regulated genes in epiF3.

ACKNOWLEDGMENTS

We thank Timothy Davis and members of the Tomato Breeding Program(University of Florida) for technical assistance with the field experiments, andother laboratory members (University of Nebraska), including Mon-Ray Shaoand Robersy Sanchez for assistance with statistical analysis, Sunil Kumar forvaluable discussion, Kamaldeep Virdi for grafting experiment design, andVikas Shedge for assistance in the design of the methylase inhibitor experi-ments. We also thank the support staff (University of Nebraska MedicalCenter Sequencing Core Facility) for assistance with RNA-seq.

Received January 20, 2015; accepted February 26, 2015; published March 3,2015.

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