Senescence and Defense Pathways Contributeto Heterosis1[OPEN]

Rebeca Gonzalez-Bayon,a Yifei Shen,a,b Michael Groszmann,c Anyu Zhu,a Aihua Wang,a

Annapurna D. Allu,a Elizabeth S. Dennis ,a,d W. James Peacock,a,d and Ian K. Greavesa,2,3

aCSIRO Agriculture and Food, Canberra, Australian Capital Territory 2601, AustraliabInstitute of Crop Science & Institute of Bioinformatics, Zhejiang University, Hangzhou 310058, ChinacARC Centre of Excellence for Translational Photosynthesis, Research School of Biology, The AustralianNational University, Canberra, Australian Capital Territory 2601, AustraliadUniversity of Technology Sydney, Sydney, New South Wales 2007, Australia

ORCID IDs: 0000-0002-5015-6156 (M.G.); 0000-0002-3720-5726 (A.Z.); 0000-0002-7850-591X (E.S.D.); 0000-0001-7314-9074 (W.J.P.);0000-0003-3923-9740 (I.K.G.).

Hybrids are used extensively in agriculture due to their superior performance in seed yield and plant growth, yet the molecularmechanisms underpinning hybrid performance are not well understood. Recent evidence has suggested that a decrease in basaldefense response gene expression regulated by reduced levels of salicylic acid (SA) may be important for vigor in certain hybridcombinations. Decreasing levels of SA in the Arabidopsis (Arabidopsis thaliana) accession C24 through the introduction of the SAcatabolic enzyme salicylate1 hydroxylase (NahG) increases plant size, phenocopying the large-sized C24/Landsberg erecta (Ler)F1 hybrids. C24♀ 3 Ler♂ F1 hybrids and C24 NahG lines shared differentially expressed genes and pathways associated withplant defense and leaf senescence including decreased expression of SA biosynthetic genes and SA response genes. Theexpression of TL1 BINDING TRANSCRIPTION FACTOR1, a key regulator in resource allocation between growth anddefense, was decreased in both the F1 hybrid and the C24 NahG lines, which may promote growth. Both C24 NahGlines and the F1 hybrids showed decreased expression of the key senescence-associated transcription factors WRKY53,NAC-CONTAINING PROTEIN29, and ORESARA1 with a delayed onset of senescence compared to C24 plants. The delayin senescence resulted in an extension of the photosynthetic period in the leaves of F1 hybrids compared to the parentallines, potentially allowing each leaf to contribute more resources toward growth.

Hybrid vigor describes the superior performanceof hybrid crop plants relative to their parents in im-portant agronomic traits such as biomass and seedyield (reviewed by Chen, 2013). Hybrids in maize (Zeamays), rice (Oryza sativa), and canola (Brassica napus)have increased seed yield and are used extensively in

agriculture. Hybrid vigor is associated with altera-tions in gene expression profiles resulting from in-teractions between the two parental genomes presentin the one nucleus. Changes in patterns of gene ac-tivity due to dominance, overdominance, and epistasishave been proposed to explain the hybrid vigor phe-notype (reviewed by Fu et al., 2015); how this is ach-ieved on a genomic level is not understood.

In Arabidopsis (Arabidopsis thaliana), F1 hybrids de-velop at a faster rate than parental lines, resulting in anincrease in biomass and seed yield (Groszmann et al.,2014; Zhu et al., 2016; Wang et al., 2017). The increase ingrowth rate can occur at different stages of plant de-velopment and differs among hybrid lines (Groszmannet al., 2014). In C24/Col hybrid seedlings, the increasedgrowth rate was observed at 3–4 d after sowing (DAS;Meyer et al., 2012). The differences in enhanced growthduring different developmental stages highlight theimportance of defining stages of development criticalfor the observed phenotypic vigor. The increase inhybrid biomass may result from altered patterns ofexpression in circadian rhythm genes affecting pho-tosynthetic and other metabolic genes essential forgrowth (Ni et al., 2009; Shen et al., 2012; Groszmannet al., 2014; Miller et al., 2015; Wang et al., 2017).Some hybrids have increased expression levels of

1This work was supported by the ARC Centre of Excellence forTranslational Photosynthesis (grant no. CE1401000015 to M.G.); theNational Collaborative Research Infrastructure Strategy of the Aus-tralian Government (providing The Australian National Universitygrowth facilities and PlantScreen services of the Australian PlantPhenomics Facility); and Longjiang Fan and Zhejiang University(to Y.S.).

2Author for contact: ian.greaves@csiro.au.3Senior author.The 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: IanK. Greaves (ian.greaves@csiro.au).

R.G.-B., M.G., E.S.D., W.J.P., and I.K.G. conceived the original re-search plans; Y.S., R.G.-B., M.G., A.Z., A.W., A.D.A., and I.K.G. per-formed most of the experiments; A.W. provided technical assistanceto I.K.G.; Y.S., R.G.-B., M.G., and I.K.G. designed the experiments andanalyzed the data; I.K.G., E.S.D., and W.J.P. wrote the manuscript.

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genes involved in the biosynthesis, signaling, and trans-port of auxin, a hormone that promotes plant growththrough the regulation of cell proliferation and cell ex-pansion (Perrot-Rechenmann, 2010; Shen et al., 2012;Wang et al., 2017). In F1 hybrids, the changes in auxinbiosynthesis and signaling may result from increasedlevels of PHYTOCHROME INTERACTING FACTOR4(PIF4), which regulates genes in the auxin biosynthesisand signaling pathways (Wang et al., 2017).Some hybrid systems show a decrease in basal ex-

pression of defense response genes in the seedling,which, in the absence of pathogens, may increase re-source allocation to plant growth, potentially contrib-uting to hybrid vigor (Groszmann et al., 2015; Milleret al., 2015; Yang et al., 2017). Conversely, altering thegrowth-defense balance toward defense can result in

hybrid weakness, where hybrids with hyperactivateddefense pathways have reduced growth (Todesco et al.,2010). Hybrids with a decrease in basal defense geneexpression may have an associated decrease in the levelof salicylic acid (SA; Groszmann et al., 2015). Reducedlevels of SA in the Arabidopsis accession C24 using thebacterial degradative enzyme salicylate1 hydroxylase(NahG), phenocopies hybrid vigor including the in-creased plant size, suggesting that SA-regulated path-ways may have an important role in certain hybridcombinations (Groszmann et al., 2015).We determined whether the size increases of F1 hy-

brids and C24 NahG lines result from changes to theexpression level of genes in the same pathways. In bothgenotypes the decrease in SA is associatedwith reducedexpression of basal defense response genes and a delay

Figure 1. C24 NahG lines show vigor during latedevelopment. A, 28 DAS plants of C24, three T3C24 transgenic lines (C24 NahG 2-5, C24 NahG3-3, and C24 NahG 3-4), and C24♀ 3 Ler♂ F1hybrids (F1). B, Rosette diameter of C24, C24NahG 2-5, C24 NahG 3-3, C24 NahG 3-4, andF1 (Supplemental Table S1). Purple, blue, green,and yellow asterisks represent statistical signifi-cance versus C24 (Student’s t test P# 0.01) of F1,C24NahG2-5, C24NahG3-3, andC24NahG3-4,respectively. C, Aerial fresh weight of 42-DAS–oldplants. D, SA concentration in parental, NahG,and F1 hybrid genotypes at 21 DAS. For (C) and(D), asterisks represent statistical significance ver-sus C24 (Student’s t test, P# 0.01). Error bars = SE.

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in senescence, both of which may promote increasedgrowth.

RESULTS

C24 NahG Lines Show Increased Growth at Late Stages ofSeedling Development

Reducing SA levels in Arabidopsis C24 by intro-ducing the bacterial NahG gene into its genome resultsin an increased plant size similar to that of F1 hybridsbetween C24 and Landsberg erecta (Ler; Groszmannet al., 2015). From 0 to 21 DAS, three T3 C24 NahGlines were the same size as C24 (Fig. 1; SupplementalFig. S1). After 21 DAS, all three C24 NahG lines beganto outgrow C24, resulting in an 18% to 36% increase inrosette diameter and a 27% to 45% increase in freshweight relative to C24 (Fig. 1, B and C; SupplementalFig. S1; Supplemental Table S1). Ler has low levels ofSA that are only slightly reduced in the Ler NahG line,and there is only a small increase in plant size (Fig. 1D;Supplemental Fig. S1C). Unlike C24 NahG, the F1 hy-brids are larger than the parental accessions at all de-velopmental stages, suggesting that gene expressionchanges not related to SA also contribute to the hybridvigor phenotype (Supplemental Fig. S1C; SupplementalTable S1).

NahG Alters Gene Expression Patterns in C24 But OnlyMinimally in Ler

Transcriptomes of C24, C24 NahG (T2 independentlines 2 and 3), Ler, Ler NahG, and the F1 hybrid wereanalyzed at 21 DAS to determine which altered path-ways were associated with the size increases in C24NahG and the F1 hybrids. The NahG transgene wasexpressed in the NahG lines (Supplemental Fig. S1D). Aquantity of 6,816 genes (35% of the expressed genes)were differentially expressed between the two parentallines (C24 and Ler) at the 21-DAS time point (Fig. 2A;Supplemental Fig. S2A; Supplemental Table S2; P #0.01; false discovery rate [FDR] # 0.01; fold change $61.2). In the F1 hybrid, 3,371 genes were differentiallyexpressed compared to the average expression of thetwo parents (Mid Parent Value), with ;60% of thesedown-regulated (Fig. 2A; Supplemental Fig. S2A;Supplemental Table S2). Compared to C24, C24 NahG2- and C24 NahG 3-lines had 1,601 and 1,907 differen-tially expressed genes (DEGs), with many present inboth lines (1,386; Fig. 2A; Supplemental Fig. S2B;Supplemental Table S2). Only genes altered in bothC24 NahG lines were used for subsequent compari-sons. Most of the down-regulated C24 NahG DEGswere also down-regulated in the F1 hybrid (75%;Fig. 2B). The F1 hybrid contained many more down-regulated DEGs than C24 NahG, which may reflectchanges in expression of both C24 and Ler alleles in the F1hybrid. There was only a small overlap in up-regulated

DEGs between C24 NahG and the F1 hybrid, indicatingthat if they do share a common mechanism for in-creased size, it is likely due to the shared down-regulated genes (Fig. 2B).

In Ler NahG, only 225 genes were differentiallyexpressed compared to Ler, with 87% (154) of thedown-regulated DEGs overlapping with those of C24NahG (Fig. 2A; Supplemental Fig. S2C; SupplementalTable S2). The difference in the number of DEGs be-tween Ler NahG and C24 NahG compared to theirparents is likely to be a consequence of the higher initiallevel of SA in C24 where there is a greater scope fordown-regulation (Fig. 1D). With already low levels ofSA in Ler, the addition of NahG will have little impacton SA levels and consequent gene expression changes.

Both F1 Hybrids and C24 NahG Plants Have DecreasedExpression of Genes Involved in Defense Pathways

SA is produced through the isochorismate and thePhe ammonia-lyase pathways (for review, see Denancéet al., 2013). At 21 DAS, the F1 hybrids and C24 NahGshow down-regulation of genes in the isochorismate

Figure 2. Transcriptome sequencing of parental, NahG, and F1 hybridlines. A, DEGs (fold change $ 61.2, P # 0.01, FDR # 0.01). B, Venndiagram of overlapping DEGs (compared to C24) between F1 hybridsand C24NahG. Blue represents F1 hybrid DEGs, yellow represents C24NahG DEGs, and light blue represents DEGs present in both samples.

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pathway including ISOCHORISMATE SYNTHASE1,AVRPPHB SUSCEPTIBLE3, and ENHANCED DISEASESUSCEPTIBILITY5 (Fig. 3A). The decrease of expressionof these SA biosynthesis genes is associated with de-creased expression of downstream genes such asWRKYtranscription factors, PATHOGEN RELATED GENES(PR1–5), and ACCELERATED CELL DEATH6 (Fig. 3B).

In LerNahG, the NahG transgene had little influence onSA biosynthetic gene expression (Fig. 3A); however, anumber of defense response pathways and SA genetargets, such asWRKYs, PR1, andACCELERATEDCELLDEATH6, were down-regulated (Fig. 3, B and C). TheNahG transgene had a much greater impact on theextent of down-regulation upon activators of SA and

Figure 3. Altered expression of genes in the SA pathway. A, Expression changes in SA biosynthetic genes ISOCHORISMATESYNTHASE (ICS1) ENHANCED DISEASE SUSCEPTIBILITY5 (EDS5), AVRPPHB SUSCEPTIBLE3 (PBS3), ENHANCED PSEUDO-MONAS SUSCEPTIBILTY1 (EPS1), and PHE AMMONIA-LYASE1 (PAL1). Genes significantly up-regulated or down-regulated arein red or blue, respectively (fold change$61.2, P# 0.01, FDR# 0.01). Genes trending toward up-regulation (fold change$ 1.2)or down-regulation (fold change# 1.2) are in pink or light blue, respectively. Geneswith no change in expression are in black. B,Activators and downstream targets of SA (blocks represent fold change $ 61.2, P # 0.01, FDR # 0.01). C, Enriched GO termsaltered in F1 hybrids and NahG lines. “Shared” are pathways where genes are altered in both F1 hybrid and C24 NahG.“F1 unique” are pathways enriched for genes only altered in the F1 hybrid, whereas “NahG unique” are pathways enriched forgenes only altered in C24 NahG. LerNahG are pathways altered compared to Ler. The GO enrichment analysis of the LerNahGversus Ler is limited by the small number of DEGs. Red represents pathways enriched for genes up-regulated, blue representspathways enriched for genes down-regulated, and white represents pathways that weren’t enriched.

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downstream targets of SA in C24 compared to Ler(e.g. 23 versus 21 log2[fold change]; Supplemental Fig.S3A). The pattern of allelic expression of SA biosyntheticgenes and downstream targets of SA demonstrates thatthe decreased expression of these genes in the F1 hybridis through a reduction in expression of the C24 alleleswith little change in the expression of the Ler alleles(Supplemental Fig. S3B; Supplemental Table S3). Severalother defense-related pathways, including stress re-sponse, biotic stimuli, and programmed cell death, arealso down-regulated (Fig. 3C; Supplemental Table S4).The down-regulation of all these pathways in the F1hybrid and C24 NahG compared to the C24 parentsuggests that changes to these pathways may contributeto the increased growth of F1 hybrids and C24 NahG.

Although many down-regulated genes and pathwayswere shared between the F1 hybrid andC24NahG, thosewith up-regulated expression levels differed between thetwo lines (Fig. 3C). At 21 DAS, there was an enrichmentin photosynthesis (Gene Ontology [GO]:0015979), starchmetabolic processes (GO:0005982), chlorophyll metabolicprocesses (GO:0015994), light reaction (GO:0019684), andcarbon fixation pathways in the F1 hybrids (GO:0015977;Fig. 3C; Supplemental Fig. S4; Supplemental Table S4).Hybrids but not NahG lines had increased expression ofPIF4 and PIF5, genes that have been implicated in hybridvigor through effects on auxin (Fig. 4A; Wang et al.,2017). This difference could result in the larger size ofF1 hybrids compared to C24 NahG at early stages of

development when these genes are highly expressed(Wang et al., 2017). Another difference was that, in theC24 NahG lines, there was an altered level of jasmonicacid (JA)-responsive genes and glucosinolate biosyn-thesis genes that was not present in the F1 hybrid nor inLer NahG lines. Whereas JA-responsive genes showedaltered expression in C24 NahG, no changes wereobserved in JA biosynthetic gene expression or JAhormone levels (Supplemental Fig. S5).

TL1 BINDING TRANSCRIPTION FACTOR1, a MasterRegulator of Defense and Growth, Is Down-Regulatedin F1 Hybrids and C24 NahG

The vigor observed in both F1 hybrids and C24NahGplants is associated with an increase in leaf cell size(Groszmann et al., 2015). Several genes that increase cellsize including EXPANSINs (EXP3, EXP5, and EXP8)and XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASEs (XTH4, XTH7, and XTH17) showedincreased expression in both F1 hybrids and C24 NahG(Fig. 4B; Supplemental Fig. S6). Some of these geneswere also up-regulated in Ler NahG, but not to the ex-tent observed in either C24 NahG or in F1 hybrids,again highlighting the different responses to NahG bythe parental genotypes (Fig. 4; Supplemental Fig. S6).Genes important for limiting cell size were down-regulated in both the F1 hybrid and C24 NahG (e.g.EXTENSIN3; Fig. 4; Supplemental Fig. S6).

In the F1 hybrid, the decrease in SA and in expressionof related defense pathway genes could release moreresources to be allocated to plant growth. TL1 BINDINGTRANSCRIPTION FACTOR1 (TBF1) is a transcriptionfactor that is induced by SA, and regulates resourceallocation between defense and growth (Pajerowska-Mukhtar et al., 2012). TBF1 represses genes associatedwith plant growth, including chloroplast proteins andenhances expression of genes involved in plant defense(Pajerowska-Mukhtar et al., 2012). In F1 hybrids, wefound a .3-fold reduction in TBF1 expression rela-tive to C24, with levels below both parents (Fig. 5A;Supplemental Fig. S7A). Like other SA-responsivegenes (Supplemental Fig. S3B), the reduced expres-sion of TBF1 is mainly through a large decrease in theexpression of the C24 allele, with only a small reductionin the expression of the Ler allele (Fig. 5B; SupplementalTable S3). Downstream targets repressed by TBF1, in-cluding chloroplast-associated genes, were up-regulatedwhile defense genes induced by TBF1 were down-regulated mainly through changes in the expression oftheC24 allele (Fig. 5; Supplemental Fig. S7B; Pajerowska-Mukhtar et al., 2012). In the NahG lines, TBF1 and anumber of TBF1-targeted genes were either down-regulated or up-regulated in C24 NahG but not inLer NahG (Fig. 5A; Supplemental Fig. S7C). The reg-ulation of TBF1 occurs at both transcriptional andtranslational levels (Pajerowska-Mukhtar et al., 2012).A network analysis of the genes regulated by TBF1suggests that TBF1 protein activity is down-regulated

Figure 4. The log2 fold change in gene expression of PIFs and cell sizegenes. A, PIF are up-regulated in F1 hybrids (Waldtest *P# 0.05; **P#

0.01, FDR # 0.01; fold change $ 61.2). B, XTH4, EXP3, and EXTEN-SINS (EXT3) have altered expression in F1 hybrids and C24 NahG.Genes up-regulated are in red, whereas genes down-regulated arein blue.

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in the F1 hybrid and C24 NahG (Supplemental Fig. S8,A and B). This did not occur in Ler NahG, where genesregulated by TBF1 showed only small changes in geneexpression (Supplemental Fig. S8C).

Decreased Expression of SA-Associated Genes IsConsistent with Delayed Senescence in F1 Hybridsand NahG Lines

Senescence is a tightly regulated process that allows aplant to repurpose nutrients from older leaves into thedevelopment of new leaves or to reproductive struc-tures. Senescence can be induced by a number of ex-ternal factors such as biotic and abiotic stress andinternal factors such as developmental age, hormonelevels, and flowering time (reviewed inWoo et al., 2013;Kim et al., 2018). In F1 hybrids and C24 NahG, genesinvolved in programmed cell death (GO:0012501) andcell death (GO:0008219)—both processes associated

with the timing of senescence—were down-regulated(Fig. 3C). SA is a strong driver of senescence and thedecrease in SA levels could alter the onset of senescence(Morris et al., 2000; Zhang et al., 2013, 2017; Zhao et al.,2016). Down-regulated genes included the senes-cence initiating transcription factors WRKY53, NAC-CONTAINING PROTEIN 92/ORESARA1, and NAC-CONTAINING PROTEIN29 (reviewed in Woo et al.,2013; Fig. 6A). EPITHIOSPECIFYING SENESCENCEREGULATOR, a gene known to repress WRKY53 ac-tivity, was up-regulated in the F1 hybrids, consistentwith the observed decrease in WRKY53 expression(Miao and Zentagraf, 2007; Fig. 6A).We analyzed the expression pattern of 3,211 genes

known to be up-regulated during developmental se-nescence and 2,496 genes known to be down-regulatedduring developmental senescence (Supplemental TableS5; Allu et al., 2014). Relative to C24, C24NahG, Ler, LerNahG, and the F1 hybrid displayed a pattern of ex-pression consistent with a delay in senescence (Fig. 6B).

Figure 5. TBF1, a master-switch between de-fense and growth, is down-regulated in F1 hy-brids. A, Altered expression of TBF1 and genesregulated by TBF1 (block represents fold change$61.2, P# 0.01, FDR# 0.01); The up or downsymbol represents a trend for increased expres-sion (red) or decreased expression (blue) in NahG(fold change$61.5; Pajerowska-Mukhtar et al.,2012). B, TBF1 allelic expression in parents andhybrids. Parent columns represent haploid readnumbers for each parent. F1 columns representexpression of either the C24 allele or the Ler al-lele in the F1 hybrid. TBF1 allelic expressionvalues can be found in Supplemental Table S3. C,Genes repressed by TBF1 are up-regulated in F1hybrids (fold change$61.2, P# 0.01, FDR#

0.01). A more extensive list of photosyn-thetic genes repressed by TBF1 can be found inSupplemental Figure S7C.

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Approximately 30% of the genes down-regulated inhybrids are genes known to be up-regulated duringsenescence, while 40% of the F1 hybrid’s up-regulatedgenes are known to be down-regulated during senes-cence (Supplemental Fig. S9). A similar pattern wasobserved in C24 NahG but not Ler NahG, wherethere was no difference in the overlap between genesup-regulated or down-regulated during senescence(Supplemental Fig. S9C). These patterns of expres-sion are consistent with the F1 hybrid and C24 NahGhaving delayed senescence. Hormone levels were alsoaltered in a way consistent with a delay in senescence.Along with the decreased level of SA, we observed astatistically significant decrease in levels of abscisic acid(ABA) at 21 DAS, which is also known to promote se-nescence (Fig. 6C; reviewed in Kim et al., 2017). Thecombination of gene expression patterns and hormonelevels suggested that both the F1 hybrid and the C24NahG plants have delayed senescence.

Under long-day conditions, both C24 NahG and theF1 hybrid had delayed senescence of leaf 7 compared to

C24 (35 DAS; Supplemental Fig. S10). Although LerNahG also had delayed senescence compared to Ler,the delay was less. The delay in senescence was ob-served across the whole rosette with the F1 hybridhaving fewer senescing leaves compared to either par-ent (Supplemental Figs. S10B and S11). Flowering is astrong inducer of senescence and could influence thesenescence patterns. Flowering time (50% of plants hadbolted) for Ler and Ler NahG was similar at 18 DAS.C24 had a flowering time of 31 DAS while the C24NahG and F1 hybrids had a slight delay in floweringtime of 31–34 DAS and 34 DAS, respectively. The sim-ilarity in flowering time amongC24, C24NahG, and theF1 hybrid suggests that flowering time is not respon-sible for the delay in senescence.

To remove any influence of flowering time on se-nescence, we examined senescence under short-dayconditions where flowering is delayed in all genotypes(including the early flowering Ler). Fluorescent imag-ing was used to evaluate chlorophyll degradationand PSII efficiency as markers for the progression of

Figure 6. Gene expression patterns of senescence-related genes and hormones at 21 DAS. A, Tran-scriptome changes in transcription factors importantin the initiation of senescence (blocks representfold change$ 61.2, P # 0.01, FDR# 0.01). Theup or down symbol represents a trend for increasedexpression (red) or decreased expression (blue) inNahG (fold change$61.5). B, Heat map of genesknown to be up-regulated during senescence anddown-regulated during senescence (SupplementalTable S5; Allu et al., 2014). Bars represent log2 foldchange increase (red) or decrease (blue) in ex-pression versus C24. C, Levels of ABA in 21-DASplants. Black asterisk represents statistical signifi-cance versus C24 (P # 0.01 Student’s t test). Errorbars = SE.

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senescence during a dark-induced senescence treat-ment (Bresson et al., 2018). Before the induction of se-nescence, all samples showed the same level of PSIIefficiency values (Fv/Fm; Fig. 7A). As senescence begins,chlorophyll content and PSII efficiency decreases dra-matically in the leaves of the parental lines (C24 andLer), which are considered “dead” by 6–7 d after in-duction (dead = Fv/Fm , 0.3; Woo et al., 2008; Fig. 7A).The F1 hybrid, along with C24 NahG, retains higherlevels of PSII efficiency with a delayed initiation of se-nescence coinciding with a delay in leaf death (Fig. 7A;Supplemental Fig. S12). The average rate of chlorosisper day (chlorophyll loss per leaf area) was 15.56 0.8%(C24), 12.6 6 0.9% (Ler), 10.7 6 0.5% (Ler NahG), 9.5 60.5% (C24 NahG), and 8.3 6 0.3% (F1 hybrid; Fig. 7B).These values demonstrate that altering levels of SA hasa stronger impact on senescence in the C24 genotypethan in the Ler genotype (6% versus 1.9% difference inchlorosis rate between the NahG line and wild type). Totake into account differences in leaf size between geno-types, we combined absolute chlorophyll area (wholeleaf) with PSII efficiency (Fv/Fm) to obtain photosyntheticcapacity of the leaf. The F1 hybrids had greater photo-synthetic capacity than the other genotypes, potentiallycontributing more resources to growth (Fig. 7A).

We examined the impact that delayed senescencehad on leaf longevity in intact rosettes under short-day conditions. We identified three stages of leafdevelopment: “early,” when leaves are increasing insize; a “plateau” stage, where leaves have reachedmaximum size and have the maximum photosyn-thetic potential; and “senescence,” where we beginto see chlorosis (Fig. 7C; Supplemental Fig. S12).Leaves 5 and 6 attained their maximum size in allgenotypes between 40 and 44 DAS (within 5% ofmaximum size). C24 stopped leaf growth at 40 dwhereas Ler, Ler NahG, and the F1 hybrid increasedin size for two more days (42 DAS). C24 NahGshowed the largest extension time for growthreaching maximum size by 44 DAS. C24 retainedmaximum photosynthetic potential for 2–3 d beforeinitiating senescence (Fig. 7C; Supplemental Fig.S12). The F1 hybrid and Ler both plateaued for 7 d,but once senescence began, the rate of chlorosis wasslower in the F1 hybrid (Fig. 7C; Supplemental Fig.S12B). The addition of NahG to C24 resulted in theextension of maximum photosynthetic potential by 14DAS, while in Ler, maximum photosynthetic potentialwas only extended by 4 DAS (Fig. 7C). The extendedperiod of maximum photosynthetic potential could

Figure 7. Dark-induced detached leaf senescence on leaves 13/14 from 53-DAS plants grown in short-day conditions. A,Chlorophyll area (leaf size; standardized to 1), PSII efficiency, and photosynthetic capacity (Fv/Fm 3 absolute chlorophyll area)over several days of dark induction. Black dotted line represents leaves that are considered dead (Fv/Fm # 0.3; Woo et al., 2008).B, Average rate (per day) of chlorosis and reduction in PSII efficiencywas used as ameasure for the progression of senescence. Redand blue asterisks represent statistical significance (Student’s t test, P# 0.01) versus C24 and Ler, respectively. C, Leaf longevity ofleaves 5 and 6 under short-day conditions. Growth phases were split into growth (red), plateau in size and maximum photo-synthetic potential (green), and senescence (yellow). Columns are derived from Supplemental Figure S12. Error bars = SE.

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produce more resources for growth in F1 hybrids andNahG lines.

We investigated whether a delay in senescence oc-curs in Col♀3 Ler♂, a combination where both parentsand hybrids have similar low levels of SA (Groszmannet al., 2015). At 21 DAS, Col♀3 Ler♂ hybrids show nochange in senescence compared to their parents indark treatment (Supplemental Fig. S13A) andWRKY53,EPITHIOSPECIFYING SENESCENCE REGULATOR,ORESARA1, and SENESCENCE-ASSOCIATED GENE12(SAG21) did not show any of the changes in expres-sion observed in the C24/Ler F1 hybrid (SupplementalFig. S13B).

DISCUSSION

Arabidopsis C24/Ler F1 hybrids and C24 NahGboth have increased size compared to the C24 geno-type. We asked whether the size increase of both F1hybrids and C24 NahG resulted from similar changesin gene expression. The two lines had similar changesto a number of developmental processes that couldinfluence growth; however, they also showed changesunique to each genotype.

Both F1 hybrids and C24 NahG had reduced SAconcentrations and low expression of basal defense re-sponse genes. A reduction in expression of defense re-sponse genes at 21 DAS may allocate more resources togrowth than to defense (Brown, 2002; Tian et al., 2003;Brown and Rant, 2013; Denancé et al., 2013; Huot et al.,2014). Certain hybrids of Arabidopsis have reducedsize because of a hyper-activated defense response(Alcázar et al., 2009; Chae et al., 2014; Todesco et al.,2014; �Swiadek et al., 2017) and hybrids that have bio-mass vigor have decreased defense gene expression,presumably allowingmore resources to be allocated forgrowth (Groszmann et al., 2015; Miller et al., 2015). TheF1 hybrid and C24 NahG had a decreased expression ofTBF1, a transcription factor that is a master-switch al-locating resources between growth and defense pro-cesses (Pajerowska-Mukhtar et al., 2012; Xu et al., 2017).The decreased expression of TBF1 correlated withincreased expression of growth promoting genes anddecreased expression of defense pathway genes sug-gesting that both F1 hybrids and C24 NahG, through aSA-driven change in TBF1, may have altered resourceallocation toward plant growth.

The decrease in basal defense response genes mayimpact the F1 hybrid’s ability to respond to biotic andabiotic conditions. However this does not seem to bethe case in C24-derived hybrids that respond normallyto either biotic or abiotic stress conditions, suggestingthat the SA-related changes are reversible (Rohde et al.,2004; Groszmann et al., 2015; Yang et al., 2015). Thesimilarity between F1 hybrids and C24 NahG did notextend to LerNahG. LerNahG lines did have decreasedexpression of a number of SA-related defense responsegenes, but the reduction in expression compared to Lerwas much smaller than that observed for the C24 NahG

system. Ler NahG also lacked changes in expression ofSA biosynthetic genes and TBF1, suggesting that unlikeC24 NahG, Ler NahG does not have any change in itsresource allocation. These differences may reflect whyC24NahG shows F1-like vigorwhereas LerNahG showsonly a slight increase in biomass compared to Ler.

A difference between the F1 hybrids and the C24NahG lines was the developmental timing at whichvigor was observed. F1 hybrids displayed increasedvigor at all time points, while C24 NahG only displayedvigor in later stages of development. Recent publica-tions suggest that the early vigor of F1 hybrids may bedue to increased auxin (Perrot-Rechenmann, 2010; Shenet al., 2012; Groszmann et al., 2015; Wang et al., 2017).F1 hybrids show increased expression levels of PIF4at 3 DAS, which may up-regulate auxin biosynthetic(YUCCA8) and signaling genes (indole-3-acetic acidinducible 29), resulting in increased auxin and growth(Wang et al., 2017). Therefore, auxin may contribute tothe phenotypic increase in biomass observed at earliertime points in F1 hybrids (3–14 DAS), whereas SA maycontribute to the phenotype at later time points (.21DAS)in both F1 hybrids and C24 NahG.

Many of the transcription factors that regulate senes-cence were down-regulated in both C24 NahG and F1hybrids, including WRKY53, ORESARA1, NAC016, andNTL4, as were many downstream senescence-associatedgenes (reviewed in Woo et al., 2013). Alterations in theexpression of these genes was supported by decreases inhormones known to promote senescence including SAand ABA (reviewed in Jibran et al., 2013). Recently Songet al. (2018) demonstrated a decrease in ethylene in Ara-bidopsisC24/Col hybrids,which canpromote senescence(reviewed by Jibran et al., 2013; Song et al., 2018). Thesedata strongly support the phenotypic observations ofdelayed senescence in F1 hybrids. The delay in senescencewas not linked to flowering time changes as both the F1hybrid and the C24 parent flowered at similar times. Thechange in senescence could be linked to the lower level ofSA and its impact on senescence-associated transcriptionfactors (Miao et al., 2004; Miao and Zentgraf, 2007). Manyof the defense genes down-regulated in the F1 hybrid andC24 NahG are also implicated in developmental senes-cence including SENESCENCE-ASSOCIATED GENE12(SAG12), SAG13, and NITRILASE2 (Quirino et al., 1999).

The increased longevity of the leaves in F1 hybridsresulted in an extension in the period of photosynthesis.This change in photosynthesis along with the observedchange in gene expression related to resource allocationmay explain the increased size of F1 hybrids and C24NahG. Ler NahG also showed a delay in senescence;however, the difference between Ler and Ler NahG insenescence wasmuch smaller than that for C24 and C24NahG. This difference along with the absence of ex-pression changes in TBF1 and associated genes mayexplain why Ler and Ler NahG are of similar size.

In Col NahG lines, decreased SA levels and delayedsenescence correlates with an increase in biomass andseed yield (Abreu and Munné-Bosch, 2009). The asso-ciation between delayed senescence and seed yield also

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occurs in other plant species. A number of crops havestay-green mutants in which the plants retain chlorophylland photosynthetic activity in late stages of repro-ductive development (Hörtensteiner, 2009). One of thehighest yielding maize varieties, FS854, is a stay-greenmutant (Thomas and Howarth, 2000). In rice the highyield of the stay-green mutant SNU-SG-1 is correlatedwith a delay in senescence during late development (Yooet al., 2007). Stay-green mutants in durum wheat(Triticum durum) also showed a delay in senescenceand an extension of photosynthesis in flag leavesalong with an increase in seed weight and grain yield(Spano et al., 2003).In a Chinese hybrid wheat variety, the high yield of

the hybrid was related to higher levels of CO2 assimi-lation and PSII activity compared to the parents in latestages of leaf development (Yang et al., 2007). In superhigh-yield hybrid rice, a delay in senescence correlateswith higher levels of photosynthetic activity duringmid to late development and increased seed yieldcompared to parents (Zhang et al., 2007; Chang et al.,2016). Ear leaves of somemaize hybrids show a delay insenescence compared to the parents with an increase inphotosynthetic functions during mid to late stages ofgrowth, which correlates with increased leaf size andyield (Song et al., 2016).

CONCLUSION

In the hybrid nucleus, interactions between the twoparental genomes and epigenomes alter the expressionof some genes, contributing to hybrid vigor. A reduc-tion in the level of SA in transgenic C24 NahG leads toincreased growth and decreased expression levels ofbasal defense response genes. Parallel changes occur inC24/Ler hybrids, which have decreased levels of SA.In both systems, the decrease in SA and associatedchanges in defense response gene expression mayaffect resource allocation through the action of TBF1,which facilitates resource distribution between growthand defense. The decrease in SA is correlated with adelay in senescence that extends the period of photo-synthesis per leaf increasing energy resources availablefor continued growth. The change in resource allocationand the delayed onset of senescence may be importantcontributors to the development of heterosis.

MATERIALS AND METHODS

Plant Material

Wild type Arabidopsis (Arabidopsis thaliana) C24 and Ler, two independentT2 C24 NahG lines (C24 NahG 2 and C24 NahG 3), a T2 Ler NahG line, andC24♀ 3 Ler♂ F1 hybrids were used for transcriptome sequencing. For senes-cence and hormone experiments, three T3 C24 NahG lines—C24 NahG 2-5(derived from C24 NahG 2), C24 NahG 3-3, and C24 NahG 3-4 (derived fromC24 NahG 3)—were used to compare to parents and F1 hybrids. Hybrid seedwas generated through hand pollination. Seeds were sterilized and stratified at4°C for 3 d and then grown on Gamborg’s B-5 Basal Media (G5893-10L; Sigma-Aldrich), pH 5.7 (KOH), 0.6% w/t agar. Hybrids were always grown with

parental lines on the same plate. Transgenic NahG lines were always grownwithwild type parents on the same plate. At 14 DAS, plants were transferred intoDebco seed raising mix supplemented with 1-g/L Osmocote extract mini con-trolled release fertilizer. Positioning of genotypes was randomized across trays.For long-day conditions, plants were grown under 16 h-light/8-h dark at 21°C.For short-day experiments, plants were grown under 8-h light/16-h dark at 21°C.Light intensity for all experiments was between 120 and 180 mmol$m-2.s21.

RNA Isolation and Transcriptome Sequencing

21-DAS plants were snap-frozen in liquid N. Frozen material was ground ina mortar and pestle and RNA was extracted using the Qiagen Plant RNeasymini kit (74904; Qiagen). Total RNA was sent to the Australian Genomics Re-source Facility. Transcriptome libraries were made by the Australian GenomicResource Facility via the manufacturer’s instructions and sequenced on anIllumina Hiseq-2500 using 100-basepair pair-ended sequencing. The tran-scriptome data were done in two separate experiments. Three biologicalreplicates were sequenced for C24—Ler, T2 Ler NahG, and C24♀ 3 Ler♂hybrids—with each biological replicate being a pool of 6–10 plants. Two bio-logical replicates were sequenced for T2 C24NahG 2, C24NahG 3, and a secondset of parental C24, each being a pool of 6–10 plants.

Data Analysis

Statistically significant differences in plant growth, hormone levels, leafgrowth (chlorophyll area), rates of chlorosis, and the reduction in PSII efficiencywere tested using Student’s t test. Plant growth was defined by measuring therosette diameter every 7 d for 42 d. After 42 d, aerial tissue was collected andweighed. Rosette diameter was measured on RGB photos using the softwareImageJ (National Institutes of Health).

The mRNA fastq files were first run through FASTQC to check quality.Libraries were mapped with STAR version 2.5.3 using the TAIR10 ge-nome and the araport11 annotation. Default settings were used apart from–alignTranscriptsPerReadNmax 500000 and –quantMode TranscriptomeSAMGeneCounts. The R package DESEQ2 was used to standardize reads betweensamples and to determine DEGs using a Wald test. Statistical significance wasdefined as 61.2 fold change, P value # 0.01, and a Benjamini-HochbergP-adjusted value # 0.01 (FDR). Genes were only considered expressed andanalyzed if one genotype had an average normalized read fragment count$ 30.Statistical tests presented in all heatmaps and log2 fold-change gene expressiongraphs refer to the above statistical tests.

Functional categorization of DEGs was carried out as described inGroszmann et al. (2015). Briefly, DEG underwent an enrichment analysis usingagriGO (bioinfo.cau.edu.cn/agriGO/). The full expressed gene list was used asthe background. REViGO was then used to account for redundancies withsettings described in Groszmann et al. (2015). For the heat maps in this paper,gene expression changes .3 were maximized to 3.

Chloroplast-associated genes repressed by SA through a TBF1-dependentmanner were obtained from Supplemental Table S1 of Pajerowska-Mukhtaret al. (2012). The gene list was run through agriGO using TAIR9 as a back-ground reference.

Single Nucleotide Polymorphism Analysis

Single nucleotide polymorphism (SNP) analysiswas carried out as describedin Zhu et al. (2016). The number of read counts for each allele was defined bySAMtools mpileup (SAMtools 1.3.1). Replicates were combined for this anal-ysis. Only positions of known SNPs between C24 and Col or between Ler andCol were reported using the –l option. SNPs were obtained from the 1001Genome Project (www.1001genomes.org). If the same SNP was present inboth C24 and Ler, it was excluded from the analysis. The BEDtools “intersect”function was used to place SNPs within genes followed by the BEDtools“groupby” function to combine reads numbers of all SNPs within a gene. Theresulting read numbers were normalized to the highest sample. Only geneswith at least three SNPs present were used for the analysis. Allele ratios werethen compared between the parents and the F1 hybrid.

Senescence-Associated Gene Analysis

Senescence-associated genes were obtained from Allu et al. (2014).Only genes where one sample had at least 30 read fragments were used

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(Supplemental Table S5). Log2 fold change compared to C24 was producedfor the samples. Hierarchal clustering of the samples was then done usingMorpheus (https://software.broadinstitute.org/morpheus/). Venn diagramswere produced using Venn diagram plotter (https://omics.pnl.gov/software/venn-diagram-plotter).

TBF1 Protein Activity

The regulatory networks were reverse-engineered by ARACNe frommultiple RNA-seq data sets in seedling Arabidopsis. ARACNe was runwith 100 bootstrap iterations using all probe clusters (Margolin et al., 2006;Groszmann et al., 2015; Wang et al., 2015, 2017). Parameters were set to 0.2 dataprocessing inequality tolerance. Function aracne2regulon was used to generateregulon objects from networks reverse-engineered with the ARACNe algo-rithm. This step took two arguments as input: the ARACNe output file, and theexpression data-set used by ARACNe to reverse engineer the network. Geneexpression signatures (GES) were identified by filtering for DEGs using thefunction “rowTtest,” which is included in the Viper package that efficiently per-forms Student’s t test for each row of a dataset. The “rowTtest” function took an“ExpressionSet” object as argument and produces a list object containing theStudent’s t test’s P value that by default is estimated by a two-tail test. ThemsVIPER analysis is performed by the “msVIPER” function (Alvarez et al., 2016).It requires a GES, regulon object, and null model as arguments, and produces anobject of class “msVIPER,” containing the GES, regulon, and estimated en-richment, including the Normalized Enrichment Score and P value, as output.

Reverse-Transcription Quantitative PCR

For reverse-transcription quantitative PCR, aerial tissue of 21 DAS weresnap-frozen in liquid N and ground in a mortar and pestle. Total RNA wasextracted using the Spectrum Plant Total RNA Kit (STRN250; Sigma-Aldrich).Genomic DNA was digested from 2 mg of total RNA using DNase I followingthe manufacturer’s instructions (18068015; Invitrogen). cDNA was then syn-thesized from the RNA using Superscript III reverse transcriptase following themanufacturer’s instructions (18080044; Invitrogen). Then, 20 mL of cDNA wasdiluted with 380 mL of water with 5 mL used per reaction. Quantitative PCRwas performed using SYBR green and Platinum Taq Polymerase (10966026;Invitrogen). For quantitative PCR, 2–4 technical replicates were used with atleast three biological replicates. Reactions were carried out on a thermocycler(Corbett) using the following conditions: 45 cycles at 94°C for 10min; 95°C for20 s; 58°C for 20 s; and 72°C for 20 s. Gene transcripts were normalized to thecontrol gene At4g24610. Primer sequences can be found in SupplementalTable S6.

Detached Leaf Senescence

Leaveswere collectedat either 5weeks (35–38DAS)or after 7weeks (51–53DAS)from plants grown under short-day conditions. For 5-week-old plants, all theleaves from a rosette were detached and placed in a 350-3 250-mm square petridish containing two pieces of Whatman paper. Thirty-five milliliters of steril-ized water were added to each petri dish. Detached leaves were placed in thepetri dish and wrapped in foil and kept at room temperature for several days.Photos and trayscan images were taken each day at the same time until 12 d ofdark treatment. For 7-week-old plants, leaves 13 and 14 were collected andplaced in dishes as described above.

Developmental Senescence

Plantswere grownonGamborgsmediaplates (see above) until 14DASwhenplants were transferred to soil. Under long-day conditions, leaf 7 was collectedfrom several plants at three time points: 28 DAS, 35 DAS, and 42 DAS. Forsenescence on the whole plant rosette, the number of partial and fully senescedleaves was counted once a week up until 49 DAS. Assigned classifications of“partial senescence”were considered when yellowing was present over 20% to50% of the leaf; “full senescence”was deemed when the whole leaf was yellow.For analyzing the differences in senescence and leaf longevity of leaf 5/6 ofintact rosettes, plants were propagated as above. Once in soil, plastic tags werepositioned under leaves 5/6 to avoid premature senescence triggers due tocontact with the soil. To avoid shading-induced senescence triggers fromoverlapping leaves and to expose leaves 5/6 for over-head imaging, later de-veloping leaves were kept aside using wooden toothpicks.

Fluorescence Imaging

Chlorophyll distribution and Fv/Fm ratio, reflecting the maximal quantumyield of PSII photochemistry, were measured using the PlantScreen system(Photon System Instruments; www.psi.cz). Measured basal fluorescence (F0)andmaximum fluorescence (Fm) and calculated Fv/Fm ([Fm-F0]/Fm) values fromimages were derived using a FluorCam 7 (version 1.5.0.46; Photon System In-struments). Images of leaves from intact rosettes were taken 1 h predawn toensure leaves were fully dark-adapted. Leaf growth was defined as the chlo-rophyll area produced using the FluorCam 7 (Photon System Instruments).Healthy presenescing leaves had Fv/Fm $ 0.83, indicating settings for thelight pulses were correctly calibrated. All leaves were imaged from theiradaxial side. False-color images of Fm and Fv/Fm allowed the visualization ofthe spatio-temporal progression of senescence in the leaves by observing thereduction in fluorescent area. A 24-color card (www.cameratrax.com) waspresent in each imaging batch and used to color-standardize all RGB imagesusing an in-house script.

Hormone Analysis

Twenty-one DAS plants grown under long-day conditions were col-lected and frozen in liquid N. Seven replicates were prepared for C24: C24NahG 3-3, Ler, Ler NahG, and C24♀ 3 Ler♂. Each replicate contained twoplants. Material was ground in liquid N and 100 mg of the ground tissuewas used for the subsequent analysis. Hormone samples were extracted asdescribed in Xu et al. (2016). Hormone samples and standards (5 mL) wereinjected onto a Zorbax Eclipse 1.8 mm XDB-C18 2.1 3 50-mm column(Agilent). Solvent A consisted of 0.1% aqueous formic acid (v/v), andsolvent B consisted of methanol with 0.1% formic acid (v/v). The planthormones were eluted with a linear gradient from 10% to 50% solvent Bover 8 min and 50% to 70% solvent B from 8 min to 12 min (then held at70% from 12 min to 20 min) at a flow rate of 200 mL/min. Solvents wereliquid chromatography–mass spectrometry grade from Fisher Chemical.The eluted hormones from the column were introduced to the mass spec-trometer via a heated electrospray ionization-II probe and analyzed usingQ-Exactive Plus (HESI-II; Thermo Fisher Scientific). The heated electro-spray ionization negative ion polarity parameters were as follows: Theelectrospray voltage was 2.5 kV, and the ion transfer tube temperature was250°C. The vaporizing temperature and the S-lens radio frequency levelwere 300°C and 50 V, respectively. The sheath gas flowwas 45 Lmin21 of N,10 L min21 of auxiliary gas, and 2 L min21 of sweep gas, respectively.Targeted parallel reaction monitoring was acquired in the quadrupole-Orbitrap mass spectrometer over the mass range m/z 100–1,500 with amass resolution of 17,500 at 1.0 microscan. Supplemental Table S7 showsthe tandem mass spectrometry acquisition parameters. The AutomaticGain Control target value was set at 1.0E+05 counts, maximum accumu-lation time was 50 ms, and the isolation window was set at m/z 4.0. Datawere acquired and analyzed using the software Xcalibur (4.0; ThermoFisher Scientific).

Accession Numbers

Sequencing data has been deposited in the Gene Expression Omnibusdatabase (accession no. GSE113989).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Growth rates of NahG lines.

Supplemental Figure S2. DEGs.

Supplemental Figure S3. Changes in SA-related genes.

Supplemental Figure S4. Increased expression of photosynthetic genes at21 DAS.

Supplemental Figure S5. Pathways unique to C24 NahG.

Supplemental Figure S6. Cell EXP gene expression in F1 hybrids andNahG lines based on Log2 fold change.

Supplemental Figure S7. TBF1 targeted chloroplast genes are up-regulatedin F1 hybrids.

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Supplemental Figure S8. TBF1 activity is down in F1 hybrids and C24NahG, but not in Ler NahG.

Supplemental Figure S9. Overlap between senescence-associated geneswith DEGs in F1 hybrids.

Supplemental Figure S10. Senescence patterns under long-day conditions(16-h light/8-h dark).

Supplemental Figure S11. Dark-induced senescence patterns in C24/Lerhybrids under short-day conditions.

Supplemental Figure S12. Leaf longevity under short-day conditions.

Supplemental Figure S13. Senescence is unchanged in Col♀ 3 Ler♂ F1hybrids.

Supplemental Table S1. Rosette diameter of plants over development.

Supplemental Table S2. Complete list of DEGs.

Supplemental Table S3. Table of allelic expression of SA-related genes andTBF1-related genes.

Supplemental Table S4. GO annotations for DEGs.

Supplemental Table S5. Genes that respond to senescence.

Supplemental Table S6. Primer sequences used for real-time PCR.

Supplemental Table S7. Mass spectroscopy acquisition parameters.

ACKNOWLEDGMENTS

We thank Ming-Bo Wang and Chris Helliwell for commenting on themanuscript. We thank Thy Truong from the RSB/RSC Mass SpectrometryFacility for help with the liquid chromatography–mass spectrometry. We thankLi Wang for helping supply seed.

Received October 1, 2018; accepted January 17, 2019; published February 1,2019.

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