Integrating Omics and Alternative Splicing Reveals · Integrating Omics and Alternative Splicing...

Integrating Omics and Alternative Splicing RevealsInsights into Grape Response to High Temperature1[OPEN]

Jianfu Jiang2, Xinna Liu2, Chonghuai Liu2, Guotian Liu, Shaohua Li*, and Lijun Wang*

Zhengzhou Fruit Research Institute (J.J., C.L.), Chinese Academy of Agricultural Sciences, Zhengzhou 450009,China; and Institute of Botany (X.L., G.L., S.L., L.W.), Chinese Academy of Sciences, Beijing 100093, China

ORCID ID: 0000-0001-5973-6053 (L.W.).

Heat stress is one of the primary abiotic stresses that limit crop production. Grape (Vitis vinifera) is a cultivated fruit with higheconomic value throughout the world, with its growth and development often influenced by high temperature. Alternativesplicing (AS) is a widespread phenomenon increasing transcriptome and proteome diversity. We conducted high-temperaturetreatments (35°C, 40°C, and 45°C) on grapevines and assessed transcriptomic (especially AS) and proteomic changes in leaves.We found that nearly 70% of the genes were alternatively spliced under high temperature. Intron retention (IR), exon skipping,and alternative donor/acceptor sites were markedly induced under different high temperatures. Among all differential ASevents, IR was the most abundant up- and down-regulated event. Moreover, the occurrence frequency of IR events at 40°C and45°C was far higher than at 35°C. These results indicated that AS, especially IR, is an important posttranscriptional regulatoryevent during grape leaf responses to high temperature. Proteomic analysis showed that protein levels of the RNA-bindingproteins SR45, SR30, and SR34 and the nuclear ribonucleic protein U1A gradually rose as ambient temperature increased, whichrevealed a reason why AS events occurred more frequently under high temperature. After integrating transcriptomic andproteomic data, we found that heat shock proteins and some important transcription factors such as MULTIPROTEINBRIDGING FACTOR1c and HEAT SHOCK TRANSCRIPTION FACTOR A2 were involved mainly in heat tolerance in grapethrough up-regulating transcriptional (especially modulated by AS) and translational levels. To our knowledge, these resultsprovide the first evidence for grape leaf responses to high temperature at simultaneous transcriptional, posttranscriptional, andtranslational levels.

Heat stress is one of the main abiotic stresses limitingcrop production worldwide. Global warming is pre-dicted to be accompanied by more frequent and pow-erful extreme temperature events (Lobell et al., 2008).Therefore, a better understanding of the response ofplants to heat stress is essential for improving their heattolerance. In general, heat stress is a complex function ofintensity (temperature in degrees), duration, and rate ofincrease in temperature. At very high temperatures, acatastrophic collapse of cellular organizationmay occurwithin minutes, which results in severe cellular injuryand even cell death (Wahid et al., 2007). At moder-ately high temperatures, direct injuries include protein

denaturation and aggregation and the increased fluid-ity of membrane lipids. Indirect or slower heat injuriesinclude inactivation of enzymes, inhibition of proteinsynthesis, protein degradation, and loss of membraneintegrity. These injuries eventually lead to a decline ofnet photosynthetic rate, reduced ion flux, production oftoxic compounds and reactive oxygen species, and in-hibition of growth. Actually, plants are not passivelydamaged by heat stress but respond to temperaturechanges by reprogramming their composition of certaintranscripts, proteins, andmetabolites. Such changes areaimed at establishing a new steady-state balance of bi-ological processes that enable the organism to function,survive, and even reproduce at a higher temperature(Bokszczanin, 2013).

In general, most previous studies about heat stressin plants have focused on physiological changes (Bitaand Gerats, 2013). With the advancement of biotech-nology, more and more studies have investigatedtranscriptomic or proteomic changes. Lim et al. (2006)found that the expression of 165 genes changed, espe-cially those of heat shock proteins (HSPs), in Arabi-dopsis (Arabidopsis thaliana) suspension cells duringacclimation at a moderate heat. With cDNA micro-arrays and semiquantitative RT-PCR techniques, Franket al. (2009) found that HSP70, HSP90, and the heatshock transcription factors (HSF) HSFA2 and HSFA3were important to tomato (Solanum lycopersicum)

1 This work was supported by the Agricultural Science and Tech-nology Innovation Program (grant no. CAAS-ASTIP-2016-ZFRI toC.L.) and the National Natural Science Foundation of China (grantno. 31270718 to L.W.).

2 These authors contributed equally to the article.* Address correspondence to [email protected] or [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:Lijun Wang ([email protected]).

L.W., S.L., and C.L. conceived and designed the experiments; J.J.and X.L. performed the experiments; G.L. provided technical assis-tance to J.J. and X.L.; L.W. analyzed the data and wrote the article.

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microspore resistance to heat stress. González-Schainet al. (2016) conducted genome-wide transcriptomicanalyses during anthesis and revealed new insights intothe molecular basis of heat stress responses in tolerantand sensitive rice (Oryza sativa) varieties. In addition,proteomic responses to heat stress have been studied insome species, and various organs have been analyzed(Kosová et al., 2011). The accumulation or decrease ofspecific proteins has been documented by comparisonof heat-sensitive and heat-tolerant vegetative tissues,and the significance of certain factors for conferringthermotolerance has been confirmed by genetic engi-neering (Grover et al., 2013). Although gene expressionanalyses have offered insights into the mechanismsunderlying the high-temperature stress response, thereis frequently a poor correlation between transcript andprotein levels in data from heat-treated samples. In re-cent years, a new technique termed iTRAQ (isobarictags for relative and absolute quantitation) has beenapplied for proteomic quantification to overcome someof the limitations of two-dimensional gel-based tech-niques. This technique has a high degree of sensitivity.The amine-specific isobaric reagents of iTRAQ allowthe identification and quantification of up to eight dif-ferent samples simultaneously (Sankaranarayananet al., 2013). Therefore, it is possible to integrate tran-scriptomic and proteomic data to reveal the response ofplants to heat stress (Wang et al., 2015).Grape (Vitis vinifera) is a popular cultivated fruit

throughout the world and represents one of the mostimportant crops with a high economic value. Extremeweather events and temperature shifts create an inim-ical environment for grape crops, inhibiting physio-logical activities in plants and resulting in reduced yieldand quality. High-temperature events are occurringmore often, and the maximummidday air temperatureis often 40°C, even exceeding 45°C, in many regions,which causes huge economic losses (Salazar-Parra et al.,2010; Pillet et al., 2012). Although our understanding ofthe response and adaptation of grape to high temper-ature is limited and has focused mostly on grape mor-phological and physiological changes, increasing dataon mRNA expression and the proteome during heatstress responses in grape have provided novel insightsinto the underlying molecular mechanism. Tran-scriptomic analysis of grape leaves under heat stressand subsequent recovery showed that the effect of heatstress and recovery on grape appears to be associatedwith multiple processes and mechanisms, includingstress-related genes, transcription factors, and metab-olism (Liu et al., 2012). Carbonell-Bejerano et al. (2013)revealed that the establishment of a thermotoleranceresponse in berries under high temperatures wasmarked by the induction of HSPs and the repression oftransmembrane transporter-encoding transcripts usingthe GrapeGen GeneChip. George et al. (2015) reportedon proteome changes in cv Cabernet Sauvignon grapecells exposed to sudden high-temperature stresses,with more than 2,000 proteins identified and quantifiedusing spectral counting of the entire data set. Wu et al.

(2015) investigated changes of fruit quality and proteinexpression profiles of grape berries upon hot watertreatment during the subsequent 45 d of cold storage,finding immediate increased expression of proteinsassociated with carbohydrate and energy metabolism.Liu et al. (2014) combined a physiological analysis withiTRAQ-based proteomics of cv Cabernet Sauvignongrape leaves subjected to 43°C stress for 6 h. Theyproposed that some proteins related to the electrontransport chain of photosynthesis, antioxidant en-zymes, HSPs, and other stress response proteins, aswell as glycolysis, may play key roles in enhancinggrapevine adaptation to and recovery capacity fromheat stress. However, these transcriptomic and pro-teomic studies were not conducted on the same grapematerials, so it is difficult to compare their results. Inaddition, the Affymetrix Gene-Chip Vitis vinifera(Grape) Genome Array contains only 15,700 probe sets,which is about half of the grape genome size, and thenumber of proteins identified in these experiments waslower. Therefore, it is difficult to reveal a completepicture of thermotolerance mechanisms in grape fromthe previous experiments.

In this study, based on high-throughput sequencingand the iTRAQ labeling technique, we assessed pro-teomic and transcriptomic changes in grape leavesunder four different temperature regimes in order touncover the cellular responses to sudden temperaturechanges and characterize the differentially expressedproteins, genes, and pathways. As a result, a new insightinto the response of grape leaves to high temperaturewas revealed after the integration of transcriptomic andproteomic analyses.

RESULTS

Heat Injury in Grape Leaves Exposed to DifferentHigh Temperatures

Photosynthesis is a sensitive biological process tohigh temperature in plants (Ashraf and Harris, 2013).Heat injury may be evaluated rapidly by the chloro-phyll a fluorescence transient (O-J-I-P test) through in-vestigating changes in the electron transport chain ofPSII. The parameter Fv/Fm represents the potentialmaximum quantum yield of primary photochemistryand was thought to be suitable for evaluating heat in-jury in grape leaves (Xu et al., 2014). In this study, be-fore temperature treatments, Fv/Fm values of grapeleaves ranged from 0.78 to 0.81 (data not shown), whichsuggested that these grape leaves were healthy. After35°C treatment for 2 h, Fv/Fm values of grape leaves didnot show significant (P, 0.05, the same as below) signof change compared with the control group (at 25°C).When heat treatmentwas 40°C or 45°C for 2 h, the Fv/Fmvalues declined significantly (P , 0.05). Moreover, thehigher the treatment temperature, the lower the Fv/Fmvalues (Fig. 1). These results suggested that grape leaveswere not inhibited or injured by 35°C for 2 h but that they

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were injured by 40°C or 45°C for 2 h. These results werevery similar to previous findings (Sorkel, 2006; Greerand Weedon, 2012).

Transcriptome Sequencing and de Novo Assembly

AcDNA librarywas constructed using equal amountsof RNA extracted from grape leaves that had been ex-posed to four different temperatures (25°C, 35°C, 40°C,and 45°C). To characterize the grape transcriptome, thecDNA library was subjected to paired-end read se-quencing using the Illumina HiSeq2000 platform. Afterremoving reads of low quality, adaptor sequences, orreads with greater than 5% ambiguous nucleotides, atotal of 453,786,102 clean paired-end reads were pro-duced consisting of 68,067,915,300 nucleotides (68.1Gb), with an average GC content of 46.5%. These high-quality reads were then de novo assembled using theTrinity program, resulting in 397,101 contigs with anaverage length of 820 nucleotides and an N50 length of1,340 nucleotides. Based on the paired-end sequenceinformation, the contigs were further assembled to give314,660 unigenes, which accounted for 205,090,426nucleotides (205.1 Mb), with an average length of651 nucleotides. Of these unigenes, 117,055 (37.2%)were longer than 500 nucleotides, 46,687 (14.8%)were longer than 1,000 nucleotides, 25,797 (8.2%) werelonger than 1,500 nucleotides, and 15,656 (5%) werelonger than 2,000 nucleotides. Each treatment had threereplicates, as shown in Supplemental Figure S1, withdifferent replicates having almost unanimous results.

Functional Annotation of Unigenes

The assembled unigene sequenceswere used to searchpublic databases, including the NCBI nonredundant

protein database, the Swissprot protein database, theGene Ontology (GO) database, the Clusters of Ortholo-gous Groups database, and the Kyoto Encyclopedia ofGenes and Genomes database, using the BLASTX algo-rithm with an E value threshold of 1e25. In total, 163,216(51.9%) unigenes were matched to a sequence in at leastone of the above-mentioned databases and more than9,392 unigenes matched more than 80% of annotatedgenes in the nonredundant protein and Swissprot non-redundant protein databases, representing almost thewhole length. In terms of E value distribution, 37.4% ofthe homology ranged between 1e25 and 1e245, while amajority of the sequences (62.5%) showed a threshold Evalue of less than 1e245, indicating strong homology.Among the unigenes annotated using the nonredundantprotein database, 74.6% had more than 60% similaritywith the corresponding gene sequence. There weremorethan 10,000 differentially expressed transcripts betweendifferent treatments according to the criteria of foldchange ($2), false discovery rate (FDR; #0.01), and P,0.05 (Supplemental Fig. S2). For annotated genes map-ped to the grape genome, we used MapMan software toconduct functional categorization. As shown in Figure 2,35°C treatment of grape leaves resulted in differentialexpression of only 252 genes compared with 25°C.However, there were 4,551 and 3,490 differentiallyexpressed genes under 40°C and 45°C, respectively.These genes were associated with biological processesthat were related mainly to protein, RNA, stress, andsignaling. Examples of these genes include HSP90.1,HSP23.6, HSP101, HSFA2, and ribonucleoprotein familyproteins U2 and U3.

The Influence of Different Temperatures on the Regulationof Genes at the Alternative Splicing Level

Alternative splicing (AS) is an important style ofposttranscriptional regulation (Syed et al., 2012). A fewstudies have explored the relationship between AS andtemperature change in plants (Capovilla et al., 2015).Therefore, we further analyzed the patterns of heat-induced AS events from the RNA sequencing data. Inthis study, AS events including exon skipping (ES),intron retention (IR), mutually exclusive exons (MXEs),and alternative 39 and 59 splice sites (A3SS and A5SS)were detected and quantified using rMATS withself-assembled transcript annotation (version 3.0.9;Shen et al., 2014). The inclusion level of each candi-date splicing event was calculated using reads map-ped to splicing junctions (for detailed information,see “Materials and Methods”). At the normal growthtemperature (25°C), about 23.6% of the total assem-bled genes appeared as AS events in the grape leaves,while the 35°C, 40°C, and 45°C, treatments resulted inAS occurrence of 68.2% genes (data not shown). In theAS events, the number of IR events was largest, fol-lowed by ES events, then by A5SS and A3SS, and withthe number of MXE events lowest (SupplementalTable S1).

Figure 1. Photosynthetic efficiency of grape leaves under differenttemperatures (25°C, 35°C, 40°C, and 45°C). Each Fv/Fm value repre-sents the mean 6 SD of five replicates. Different letters indicate sig-nificant differences (P, 0.05) among Fv/Fm values of four temperaturetreatments.

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Based on the data described above, differentialsplicing events were selected with FDR # 0.05. Asshown in Figure 3 and Supplemental Table S2, a hightemperature of 35°C resulted in the occurrence of206 significant AS events compared with 25°C. Ninety-eight AS events were up-regulated, while 108 AS eventswere down-regulated. At 40°C, significant AS eventsincreased to 1,075. Of these, 584 AS events wereup-regulated, in which IR events (334) dominated,while 491 AS events were down-regulated, in which IRand ES events were most common, reaching 115 and269, respectively. When grape leaves were held at 45°Cfor 2 h, 748 AS events occurred significantly, in which476 AS events were up-regulated (413 IR events) and272 AS events were down-regulated. These results

indicated that IR is the most abundant up-regulated ASevent in the response of grape leaves to high tempera-ture, with IR events at 40 and 45°C far more abundantthan those at 35°C. With increased temperatures,up-regulated IR events increased gradually, and up- ordown-regulated ES, A5SS, and A3SS events rosefrom 35°C to 40°C but then declined at 45°C. Weidentified overrepresented GO terms (biological pro-cesses) among functionally annotated genes with IRevents (Supplemental Data Sets S1 and S2) and ESevents (Supplemental Data Sets S3 and S4). The resultsrevealed that these differentially spliced genes wereinvolved in several biological processes, includingresponses to abiotic stimuli and RNA processing,suggesting that high temperature may impact biologi-cal processes through changing pre-mRNA splicing.In particular, the response-to-heat or stress-stimulusfunctional category was markedly increased among thedifferentially spliced genes at 40°C and 45°C. Indeed,further analysis using MapMan software suggested thatgenes with aberrant splicing in grape leaves with hightemperatures of 40°C and 45°Cwere involved in variousstress response pathways, including heat shock proteinpathways, proteolytic pathways, transcription regula-tion of stress responses, and secondary metabolites(Figs. 4 and 5). Interestingly, there were almost no IRevents for HSP genes at 35°C. However, those geneswith up-regulation of IR events at 40°C or 45°C wereinvolved mainly in protein folding, such as HSP21,HSC70.2, HSC70-5, HSC70.7, HSP90.1 (HSP81.1 andHSP83), HSP101, HSFB1, HSFA2, HSF4, and HSFA6B.In addition, those down-regulated genes in ES events at40°C or 45°C were involved mostly in proteolysis, suchas ATMC5 (Cys-type endopeptidase), DegP protease,FtsH protease9 (metallopeptidase), FtsH protease1,FtsH protease11, UPL7 (ubiquitin-protein ligase), andUBP5 (ubiquitin-specific protease).

Figure 3. Summary of all genes with significantly differential ASevents in grape leaves under each high-temperature (35°C, 40°C, and45°C) treatment compared with the control (25°C). Up indicatesup-regulation, and Down indicates down-regulation.

Figure 2. Main biology processes of differentially expressed genes under 35°C, 40°C, and 45°C compared with 25°C in grapeleaves. The x axis indicates the number of up-regulated (right) and down-regulated (left) genes.








Protein Responses to High Temperature in Grape LeavesRevealed by iTRAQ Analysis

To reveal whether different temperatures had dis-tinct effects on protein expression, we performed aniTRAQ analysis of grape leaves treated with 25°C,35°C, 40°C, and 45°C. Proteins extracted fromthese leaves were labeled with iTRAQ (114 for 25°Ctreatment, 115 for 35°C treatment, 116 for 40°C treat-ment, and 117 for 45°C treatment) after being digestedwith trypsin. To improve proteome coverage, high-pHreverse-phase HPLC was used for peptide mixturefractionation. The selected fractionswere analyzedwithnano-liquid chromatography-tandem mass spectrom-etry (LC-MS/MS) using a rapid-separation liquidchromatography system (interfacedwith a Q Exactivedevice [Thermo Fisher Scientific]; Supplemental Fig.S3). A total of 524,912 high-resolution tandem massspectra acquired from 20 3 3 LC-MS/MS runs weresearched using Andromeda integrated in MaxQuant(version 1.3.5.8; Cox and Mann, 2008; Cox et al.,2011) against the UniProt grape protein database(UP000009183; 29,907 sequences). In total, 6,482 non-redundant UniProt-annotated proteins were identifiedwith at least two unique peptides (protein and peptideFDR, 0.01), and among them, 5,108 (78.80%) proteins

were commonly identified in three biological repeats(Supplemental Fig. S4). The iTRAQ reporter ion intensityof protein in three biological repeats was further inte-grated,with the filter of minimum reporter precursorintensity fraction = 0.75, total intensity of certainprotein . 10,000, and unique peptide $ 2, and the5,651 proteins were eventually quantified. When set-ting a quantification ratio . 1.3 as the up-regulatedthreshold and a ratio , 0.7 as the down-regulatedthreshold and P , 0.05, 808 differentially expressedproteins were obtained under the different high tem-peratures compared with the control (25°C), as shownin Figure 6. Under 35°C, 40°C, and 45°C, 110, 204, and318 proteins were up-regulated, respectively, in which24 proteins were co-up-regulated. There were 31common up-regulated proteins between 35°C and40°C, 58 between 35°C and 45°C, and 110 be-tween 40°C and 45°C. Under 35°C, 40°C, and 45°Ctreatments, 204, 195, and 95 proteins were down-regulated, respectively, in which 27 proteins wereco-down-regulated. In addition, there were 63 commondown-regulated proteins between 35°C and 40°C,44 between 35°C and 45°C, and 63 between 40°C and45°C.

Figure 4. A network generated by MapMan indicates that genes with up- and down-regulated IR splicing events are in-volved in various stress response pathways in grape leaves under different high temperatures (35°C, 40°C, and 45°C)compared with 25°C.


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Functional Classification and Enrichment Analysis ofDifferentially Expressed Proteins under DifferentHigh Temperatures

We further conducted functional classification andenrichment analyses of these differentially expressedproteins. The up-regulated biological pathways at35°C included sugar-mediated signaling pathway,photosynthesis, protein targeting to mitochondria,and purine ribonucleoside monophoshate biosyn-thetic processes. The down-regulated biological path-ways at 35°C included aromatic amino acid familymetabolism, sterol biosynthesis, phenylpropanoid me-tabolism, response to light intensity/UV, establishmentof protein localization to membrane, and flavonoidbiosynthesis (Supplemental Fig. S5). Different fromthe 35°C treatment, the up-regulated biological path-ways at 40°C were primarily responses to temperaturestimulus/heat/high light intensity/reactive oxygenspecies/hydrogen peroxide, heat acclimation, anda-amino acid catabolic process. The down-regulatedbiological pathways at 40°C were mainly photosyn-thesis, aromatic amino acid family metabolism, sterol

biosynthesis, phenylpropanoid metabolism, responsesto UV light, pigment biosynthesis, formation of trans-lation preinitiation complexes, hyperosmotic salinityresponses, and starch biosynthesis (SupplementalFig. S6). The up-regulated biological pathways at45°C were mostly involved in mRNA splicing viaspliceosomes, sesquiterpenoid metabolism, chloro-phyll biosynthesis, cellular responses to heat, cellularprotein complex disassembly, sugar-mediated sig-naling pathways, and aromatic amino acid familymetabolism (Supplemental Fig. S7A). The down-regulated biological pathways at 45°C were involvedin responses to UV light, Cys-type peptidase activity,phosphopantetheine attachment site binding involvedin fatty acid biosynthesis, chloroplast thylakoid lumen,cytoplasmic vesicle membrane, and protein phosphor-ylated amino acid binding (Supplemental Fig. S7B). Aswe knew from these results, biological processes ofco-up-regulated proteins at 40°C and 45°C comparedwith the control involved responses to temperaturestimuli or heat, mRNA splicing, splicesomal complexassembly, and photosynthesis. Moreover, those proteinsthat were up-regulated with increasing temperature

Figure 5. A network generated by MapMan indicates that genes with up- and down-regulated ES splicing events are involved invarious stress response pathways in grape leaves under different high temperatures (35°C, 40°C, and 45°C) compared with 25°C.










accounted for ;16% of total differentially expressedproteins. They were involved in responses to high lightintensity, protein processing in the endoplasmic reticu-lum (HSP17.4, HSP17.6II, HSP23.6, HSP70, HAFA2,HSP21, HSP18.2, and ATHSP22.0), and RNA splicing(U1A, SR33, SR45, SC35, SR34, and SR30; Fig. 7).

Integration of Transcriptomic and Proteomic Data

It is not certain that changes in gene expressionindicate corresponding protein changes. Integrativeanalysis of the proteome combined with a compre-hensive transcriptome database provides an importantverification tool for the expression of key genes. Weconstructed splicing transcriptomic data, integratedtranscriptomic and proteomic data, and then analyzedthe regulation of the changes in gene expression withchanges in protein expression. Specifically, those genesand proteins whose change trends were similar wereexamined. As shown in Figure 8, compared with 25°C,four, 50, and 49 co-up-regulated genes and proteins

at 35°C, 40°C, and 45°C, respectively, were found.Only one co-up-regulated gene and protein (i.e.MULTIPROTEIN-BRIDGING FACTOR1c [MBF1c])was identified under the three temperatures. Therewere 36 co-up-regulated genes and proteins between40°C and 45°C, which included HSPs (HSP40, HSP70.4,HSP22, HSP18.1, HSP17.6C, HSP17.4, HSP23.6, HSP90.1,HSP101, HSP17.6, and HSP70-1), Ser/Arg-rich splic-ing factor SR45a, GATA transcription factor, PDX1.2,ABC transporter, Gal-binding protein, 59-adenylylsulfatereductase1, F-box protein, translation initiation fac-tor SUI1 family protein, Ser acetyltransferase1, andT-complex protein11 (Table I). For these genes, RNAsequencing results were validated using qRT-PCR(Supplemental Fig. S8). However, no co-down-regulatedgene and proteinwas found under the three temperaturetreatments. Only one down-regulated gene and protein(VIT_14s0036g00070.1) was identified under 40°C and45°C, which was not named andmay be associated withdefense. Overall, proteins that exhibited notable changesin their expression levels did not always have a corre-sponding alteration in transcript levels, indicating that

Figure 7. Functional classification andenrichment analysis of up-regulatedproteins with the increasing of temper-ature from 25°C to 45°C in grape leaves.

Figure 6. Venn diagrams of differentiallyexpressed proteins in grape leaves under35°C,40°C, and45°Ccomparedwith 25°Catfold change greater than 1.3 (up-regulation)and less than 0.7 (down-regulation).


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posttranscriptional and posttranslational regulationmayplay important roles in the grape response to heat.From the above data, one of the important changes

of the transcriptome under different temperatures wasAS, in which IR events occurred most. We integratedsplicing transcriptome and protein expression dataand found 14 co-up-regulated proteins and IR eventsunder different temperatures (Fig. 8). Co-up-regulatedproteins and IR events between 40°C and 45°C includedHSFA2, HSP90.1, HSP25.3, clpB1 (HSP101), andHSP70.4 (Table II). For 40°C and 45°C, HSFA2 pro-duced two transcripts with IR events that are shown inFigure 9. The model graphs of the IR events of thesefour HSP genes are shown in Supplemental Figures S9to S12. These results also are validated by qRT-PCR inSupplemental Figure S13. However, there were noco-up-regulated proteins and IR events between 35°Cand 45°C or between 35°C and 40°C.

DISCUSSION

Photosynthesis may be inhibited in grapevines underhigh temperature. The O-J-I-P test is a powerful tool forevaluating photosynthetic activity in grape leaves (Xuet al., 2014). We investigated the chlorophyll a fluores-cence parameter Fv/Fm in grape leaves under differenthigh temperatures (35°C, 40°C, and 45°C) versus acontrol temperature (25°C). The results indicated that40°C and 45°C treatments inhibited leaf photosynthesisbut 35°C had very little influence (Fig. 1). Comparedwith the control at 25°C, 35°C treatment resulted inabout 300 genes changing significantly, but 40°C and45°C led to significant changes of about 4,000 genes(Fig. 2). These results also indicated that 35°C hadalmost no influence on grape leaves; however, 40°Cand 45°C treatments influenced or damaged grapeleaves. Integrated analyses at the transcriptomic andproteomic levels were performed to understand the

underlying mechanisms of cellular responses, and sig-nificant insights into gene functions were revealed.

HSPs Are Involved in Heat Tolerance in Grape Leavesthrough Up-Regulating Transcriptional andTranslational Levels

Heat stress often causes protein denaturation inplants. Therefore, maintaining proteins in their func-tional conformations is particularly important for plantsurvival under heat stress (Lee and Vierling, 2000).High temperature can trigger some mechanisms ofdefense such as the production of HSPs, which areclassified into fivemajor families: HSP100 (Clp), HSP90,HSP70 (DnaK), HSP60 (chaperonin and GroEL), andsmall HSPs (sHSPs). Protein folding is mainlymediatedby HSP90s, HSP70s, and HSP60s, whereas sHSPs areinvolved in protection against protein aggregation andHSP100s in the resolubilization of protein aggregates(Kotak et al., 2007). Changes in gene expression at thetranscriptional and/or translational level are thought tobe a fundamental mechanism in plant response to en-vironmental stresses. While previous studies focusedmostly onHSP change at the transcriptional level underheat stress, very little evidence was reported at bothtranscriptional and translational levels. In this study,we first found that 11 HSPs were co-up-regulated at thetranscriptional and translational levels in grape leavesunder only 40°C and 45°C high temperatures comparedwith the control (Fig. 8; Table I). They mainly includedsHSPs (HSP17.4, HSP17.6, HSP17.6C, HSP18.1, HSP22,HSP23.6, HSP40, and HSP25.3), HSP70 (HSP70-1 andHSP70-4), HSP90-1, and HSP101. These results furthercorroborated previous findings showing that HSPswere not induced by a temperature such as 35°C(Vierling, 1991; Tanaka et al., 2000).

The sHSPs cannot refold nonnative proteins, but theycan bind to partially folded or denatured substrateproteins, preventing irreversible unfolding or incorrectprotein aggregation. In rice seedlings, the expression of

Figure 8. Venn diagram analysis of co-up-regulated genes and proteins (left) andco-up-regulated genes with IR events andproteins (right) in grape leaves under 35°C,40°C, and 45°C compared with 25°C.








HSP17.4 was up-regulated during the seedling and an-thesis stages in response to heat stress (Chen et al., 2014). InArabidopsis, up-regulated levels ofHSP17.6were detectedunder heat stress (Sagor et al., 2013). Györgyey et al.(1991) isolated cDNA clones (MsHSP18-1) from alfalfa(Medicago sativa) and found that MsHSP18-1 transcrip-tion was induced by elevated temperature. ThemRNA levels of HSP22 cDNAs increased after hot watertreatment in grapefruit (Citrus 3 paradisi) peel tissue(Rozenzvieg et al., 2004). Three-week-old Arabidopsisplants had increased expression of AtHSP23.6 followingexposure to heat stress (Rhoads et al., 2005). Liu et al. (2012)found the transcriptional levels of HSP17.6, HSP22, andHSP40 to be increased significantly in grape leaves under ahigh temperature (45°C). This study identified sevenco-up-regulated sHSPs at transcriptional and translationallevels in grape leaves under 40°C and 45°C. They wereHSP17.4, HSP17.6, HSP17.6C, HSP18.1, HSP22, HSP23.6,HSP25.3, and HSP40 and may play important roles inthe heat tolerance of grape.

In almost all organisms, HSP70 functions as a cha-perone for newly synthesized proteins to prevent theiraccumulation as aggregates, and so they fold in aproper way during the transfer to their final location. Astudy on Arabidopsis indicated the necessity of HSP70in the stroma of the chloroplast for the differentiation ofgerminating seeds and their heat tolerance (Su and Li,2008). There have been some studies on HSP70 at thetranscriptional and translational levels. Experimentsusing the mRNA transcription inhibitor (actidione D)or protein synthesis inhibitor (cycloheximide) demon-strated that the accumulation of HSP70 by heat accli-mation was controlled at both transcriptional andtranslational levels. The class HSP90 shares roles withother classes. As a molecular chaperone, HSP90 canbind HSP70 in many chaperone complexes and hasan important role in signaling protein function andtrafficking (Pratt and Toft, 2003). In Arabidopsis, therewere some indications that cytoplasmic HSP90 inhibi-ted HSFs in the absence of heat stress, but under heat

Table I. Co-up-regulated genes and proteins with differential expression in grape leaves only under 40°C and 45°C compared with 25°C

Gene Identifier Description

VIT_01s0011g04820.1 Chaperone DnaJ domain-containing proteinVIT_10s0003g00260.1 DNAJ heat shock family proteinVIT_08s0007g00130.1 Heat shock 70-kD protein4VIT_12s0035g01910.1 22-kD heat shock protein (AtHsp22.0)VIT_13s0019g03000.1 18.1-kD class I heat shock protein (18.1-kD heat shock protein; AtHsp18.1)VIT_13s0019g03090.1 17.6-kD class I heat shock protein3 (17.6-kD heat shock protein3; AtHsp17.6C)VIT_13s0019g02740.1 18.1-kD class I heat shock protein (18.1-kD heat shock protein; AtHsp18.1)VIT_13s0019g02780.1 18.1-kD class I heat shock protein (18.1-kD heat shock protein; AtHsp18.1)VIT_13s0019g02840.1 17.4-kD class I heat shock protein (17.4-kD heat shock protein1; AtHsp17.4A)VIT_13s0019g03170.1 18.1-kD class I heat shock protein (18.1-kD heat shock protein; AtHsp18.1)VIT_16s0022g00510.1 23.6-kD heat shock protein, mitochondrial (AtHsp23.6)VIT_16s0050g01150.1 Heat shock protein90-1 (AtHSP90.1, AtHsp90-1; heat shock protein81-1, Hsp81-1; heat shock protein83)VIT_17s0000g07190.1 Chaperone protein ClpB1 (ATP-dependent Clp protease ATP-binding subunit ClpB homolog 1)(Heat shock

protein101)VIT_18s0041g00280.1 T-complex protein11VIT_18s0089g01270 22-kD heat shock protein (AtHsp22.0)VIT_19s0090g00420.1 Translation initiation factor SUI1 family proteinVIT_19s0015g00130.1 Ser acetyltransferase1, chloroplastic (AtSAT-1; EC 2.3.1.30; AtSERAT2;1; SAT-p)VIT_19s0085g01050.1 17.6-kD class I heat shock protein3 (17.6-kD heat shock protein3; AtHsp17.6C)VIT_02s0154g00480.1 23.6-kD heat shock protein, mitochondrial (AtHsp23.6)VIT_04s0008g01490.1 17.6-kD class II heat shock protein (17.6-kD heat shock protein; AtHsp17.6)VIT_04s0008g01500.1 17.6-kD class II heat shock protein (17.6-kD heat shock protein; AtHsp17.6)VIT_04s0008g01520.1 17.6-kD class II heat shock protein (17.6-kD heat shock protein; AtHsp17.6)VIT_04s0008g01530.1 17.6-kD class II heat shock protein (17.6-kD heat shock protein; AtHsp17.6)VIT_04s0008g01590.1 17.6-kD class II heat shock protein (17.6-kD heat shock protein; AtHsp17.6)VIT_04s0008g01510.1 17.6-kD class II heat shock protein (17.6-kD heat shock protein; AtHsp17.6)VIT_06s0004g04470.1 Heat shock 70-kD protein1VIT_06s0004g06010.1 Ser/Arg-rich splicing factor SR45aVIT_07s0005g01080.1 GATA transcription factorVIT_08s0058g00210.1 17.6-kD class I heat shock protein3 (17.6-kD heat shock protein3; AtHsp17.6C)VIT_08s0007g00130.1 Heat shock 70-kD protein4VIT_08s0007g04000.1 Response to oxidative stressVIT_09s0002g04490 Pyridoxal 59-phosphate synthase-like subunit PDX1.2 (AtPDX1.2, AtPDX1;3)VIT_09s0018g00310 ABC transporterVIT_00s0494g00030.1 Gal-binding proteinVIT_00s1490g00010.1 59-Adenylylsulfate reductase1, chloroplasticVIT_00s0181g00080.1 F-box proteinVIT_16s0098g01060.1 25.3-kD heat shock protein, chloroplastic (AtHsp25.3)


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stress this role was suspended temporarily, so thatHSFs were active (Yamada et al., 2007). Under normalconditions, HSF1 activity was suppressed by interac-tion with HSP70 and HSP90 (HSP70/90; Anckar andSistonen, 2011). Heat stress treatment caused the dis-sociation of HSP70/90 from HSFs because denaturedproteins competitively interacted with the HSPs.One unique function of HSP100 was the reactivation ofaggregated proteins (Parsell and Lindquist, 1993) byresolubilization of nonfunctional protein aggregates,and it also helped to degrade irreversibly damagedpolypeptides (Bösl et al., 2006). HSP100 activation re-quired the simultaneous binding of multiple HSP70partners, restricting high HSP100 activity to the surfaceof protein aggregates and ensuring HSP100 substratespecificity. Heat shock induced an increase in HSP70,HSP90, and HSP101 protein expression in creepingbentgrass (Agrostis stolonifera; Wang et al., 2014). In ourstudy, HSP70.1, HSP70.4, HSP90.1 and HSP101 wereco-up-regulated at transcriptional and translationallevels under high temperatures, indicating that they canform HSP70/90 and HSP100/70 complexes. Therefore,these HSPs likely played crucial roles in protectinggrape leaves from heat stress. Apparently, separatetranscriptomic or proteomic analysis was not sufficientto understand the underlying genetic and molecularmechanisms.

AS Acts as an Important Posttranscriptional Regulation inGrape Leaf Response to High Temperature, Especially forProtein Folding and Proteolysis

Plants tightly respond to temperature changes byreorganizing their gene transcription and proteintranslation and acquire a defense capability. Whiledifferent levels of gene expression are known to be re-sponsive to heat stress, gene regulation at the post-transcriptional level is less understood. Recently,increasing evidence has shown that AS is a criticalposttranscriptional event and plays an important role inplant stress responses (Staiger and Brown, 2013). Re-cent studies using massively parallel RNA sequencingrevealed that a large percentage of genes in Arabidopsisundergo AS, which potentially could significantlyincrease the plasticity of the transcriptome and proteomediversity (Mastrangelo et al., 2012). AS is a process inwhich two or more different transcripts are generated

from the same pre-mRNA molecule using differentsplicing sites. Although AS of some stress-responsivegenes has been reported, large-scale or genome-widestudies of AS dynamics under heat stress conditionshave not been conducted. In this study, high tempera-ture caused AS changes of about 70% of the genes ingrape leaves, indicating that AS may be an importantstyle of posttranscriptional regulation in grape leaf re-sponse to high temperature. These marked AS changesunder high temperature could provide molecular plas-ticity for the plants to adapt to stress conditions. How-ever, whether AS can be differentially regulated remainsunclear. In this study, according to the criterion (FDR#0.01) for differential splicing events, compared with25°C, pre-mRNA splicing responded rapidly in grapeleaves at 35°C, and 206 differential splicing events oc-curred. When temperature changed to 40°C, differentialsplicing events reached 1,075. Under 45°C, total differ-ential splicing events were 748 (Fig. 3; SupplementalTable S2). These results indicated that pre-mRNA splic-ing in grape cells responded drastically when tempera-ture reached 40°C.However, increasing temperature didnot further raise the occurrence of differential splicingevents, which may be associated with more seriousdamage at 45°C than 40°C to grape leaves. This resultalso implies that it may be relatively difficult for grapeleaves to modulate gene function under serious stress at45°C. It is possible that 45°C may cause the decrease ofenhancer activity or the increase of repressor activity forAS, allowing splicing regulation of specific gene tran-scripts to become less active. Our RNA sequencing datashowed that MXE events occurred only for a few, whileES, IR, A5SS, and A3SS events are found universallywith transcripts under ambient temperature (25°C) ingrape leaf cells. When grape leaves were at differenthigh temperatures, the AS events were differentiallyregulated,with IR events themost abundant followedbyES events (Fig. 3; Supplemental Table S2). Therefore, IRandES events are themost important posttranscriptionalregulation at the AS level during the response of grapecells to heat stress. Moreover, with the increasing tem-perature, the occurrence of up-regulated IR events in-creased (Fig. 4).

The processing of AS depends on a large ribonucle-oprotein complex, the spliceosome, which consisted ofa core of five small nuclear ribonucleic proteins, calledU1, U2, U4, U5, and U6. U1 and U2 recognize spe-cific sequences in the intronic region. Conformationalchanges of the spliceosome and sequential phosphodi-ester transfer reactions remove the introns and mediatethe joining of exons (Capovilla et al., 2015). The choiceof splice sites is directed to a large extent by RNA-binding proteins, predominantly Ser/Arg (SR) pro-teins and heterogenous nuclear ribonucleoproteins,which promote and block binding of the spliceosome,respectively. SR proteins recognize splicing enhancersand recruit U2 auxiliary factor and U1 (Kornblihtt et al.,2013). High temperature promotes the expression ofactive isoforms of the splicing factor SR30 (Filichkinet al., 2010) but reduces the active isoform of SR34

Table II. Co-up-regulated IR events and proteins with differential ex-pression in grape leaves under 40°C and 45°C compared with 25°C

Gene Identifier Description

VIT_16s0050g01150.1 Heat shock protein90.1VIT_16s0098g01060.1 25.3-kD heat shock protein,

chloroplastic (AtHsp25.3)VIT_17s0000g07190.1 Chaperone protein ClpB1 (HSP101)VIT_04s0008g01110.1 Heat stress transcription factor A-2

(AtHsfA2, AtHsf-04)VIT_08s0007g00130.1 Heat shock 70-kD protein4








(Lazar and Goodman, 2000). Palusa et al. (2007)reported that additional transcripts appeared or theexpression level decreased in SR30 and SR1/SR34 inheat-treated Arabidopsis seedlings. Ous study showedthat protein levels of SR45, SR30, SR34, and U1A ingrape leaves rose gradually as ambient temperatureincreased (Fig. 7). This result may explain why moreAS events occurred under high temperatures in grapeleaves. The AS pattern of several SR proteins has beenshown to have obviously changed under variousabiotic stress conditions, including temperature stress,high salinity, and high-light irradiation (Isshiki et al.,2006; Yamaguchi-Shinozaki and Shinozaki, 2006; Duque,2011). However, in our study, SR genes themselves didnot occur significantly in AS events. A few studies alsoshowed that overexpression of certain splicing factors

could increase plant tolerance to salt and other stresses(Chang et al., 2014). We predict that regulating theexpression of some SR genes or other splicing factors mayincrease plant tolerance to high temperature by enhancingthe correct splicing of heat tolerance genes.

If the increase in AS is merely a nonspecific conse-quence of stress damage, a random distribution wouldbe expected among genes. However, our data, alongwith previous reports, demonstrated that the genesassociated with stress responses tended to be alterna-tively spliced under stress conditions (Figs. 4 and 5). It isknown that some abiotic stresses can activate theexpression of a large number of plant stress-responsivegenes that are not expressed or are expressed at lowerlevels under normal nonstressful conditions (Xionget al., 2002; Yamaguchi-Shinozaki and Shinozaki, 2006).

Figure 9. Characterization of two HSFA2 transcripts with differential IR events in grape leaves under 40°C and 45°C comparedwith 25°C. A, Sashimi plots (stand alone) for alternatively spliced exon and flanking exons in samples. Per-base expression isplotted on the y axis of the Sashimi plot, with genomic coordinates on the x axis. Arcs represent splice junctions connecting exonsand display the number of reads split across the junction (junction depth). Each treatment has three replicates. 25°C-1, -2, and -3represent three replicates of the control 25°C treatment for 2 h. 40°C-1, -2, and -3 represent three replicates of the 40°C treatmentfor 2 h. 45°C-1, -2, and -3 represent three replicates of the 45°C treatment for 2 h. mRNA isoforms quantified are shown at bottom(exons in black and introns as lines with arrowheads). B, Semiquantitative RT-PCR analysis of the expression of HSFA2 IR splicevariants in grape leaves under 25°C, 35°C, 40°C, and 45°C for 2 h. The forward and reverse primers were designed from theupstream and downstream exons of the retention intron based on each IR event, respectively.


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With the simultaneous production of a large amount ofthese stress-inducible pre-mRNAs, cells would need toimmediately recruit a significant amount of splicingand other factors for their cotranscriptional or post-transcriptional processing. In our study, high temper-ature resulted in many HSPs occurring as AS of theIR type: for example, CPHSC70-2, HSC70-7, HSP90.1,HSP101, HOT1, HSP21, CPN20, and HSC70-5 (Fig. 4;Supplemental Data Set S1). These results also demon-strated that IR could be a key AS that results in theup-regulation of HSPs at the transcriptional level.To date, there have only been a few reports about AS

ofHSPs in plants. Lund et al. (2001) observed that formsof HSP22 resulted from alternative intron splicing inmaize (Zea mays). Zhang et al. (2011) reported that13 HSP genes had one transcript each, two genes hadtwo transcripts each, and two had three transcripts eachin bovine. Heat stress-AS occurs in transcripts for spe-cific functions. In this study, heat stress-induced IRevents are mainly for protein folding (HSPs) and heatstress-induced ES events are mainly for protein degra-dation (peptidases; Figs. 4 and 5). These results sug-gested that the occurrence of differential AS eventsunder high temperature was not a random process.Rather, it was heavily influenced by heat stress.Our study showed that HSP90.1, HSP25.3, HSP101,and HSP70.4 transcription in grape leaves underboth 40°C and 45°C generated splicing variants of IR(Supplemental Figs. S9–S12). Moreover, there wereco-up-regulated IR events and proteins with differen-tial expression for the four genes under 40°C and 45°C(Table II). However, it is possible that these HSPIR transcripts were degraded posttranscriptionally.Kalyna et al. (2012) suggested that AS frequently gen-erates nonsense mRNA with premature terminationcodons (PTCs), which are rapidly degraded by thecellular nonsense-mediated mRNA decay machinery inmany cases. This study also showed that transcriptscontaining PTCs are often readily detectable and cancontribute significantly to steady-state transcript levelsof genes. This type of AS is often referred to as unpro-ductive AS for adaptation to developmental demandsand/or plant responses to environmental stresses(Reddy et al., 2013). Some PTC+ mRNAs escapenonsense-mediated mRNA decay and produce trun-cated proteins that may be missing key functional do-mains (Kalyna et al., 2012). In this study, we detected IRtranscripts of these HSPs by RT-PCR (SupplementalFig. S13), and transcript expression levels of theseHSPsincreased (Table II). In addition, we found that intronsequences of these IR transcripts of HSPs included ter-mination codons (Supplemental Data Set S5). There-fore, it is impossible for IR transcripts of these HSPs totranslate into HSPs, but it is possible that they encodedsome truncated proteins that function in the response ofplants to the environment (Liu et al., 2013). In ourproteomics data presented here, peptides correspond-ing to intron sequences of IR transcripts were notdetected. Therefore, we cannot confirm that these IRtranscripts were translated into truncated proteins.

These results indicated that IR events of these HSPsshould contribute to the increase of HSP transcription.However, the translation of these HSPs was conservedand the increase of HSP proteins under 40°C and 45°Cshould result from fully spliced transcripts.

Some Important Transcript Factors Could Be Involved inHeat Tolerance in Grape Leaves through Up-Regulation atTranscriptional and Translational Levels

In this study, using iTRAQ analysis, protein changesacross four different temperatures were studied toelucidate a response network specific to heat stress ingrape leaves (Fig. 6; Supplemental Figs. S4–S7). The ASand the proteomic data were integratively analyzed.The results revealed that co-up-regulated genes at IRsplicing and protein translation under different hightemperatures includedHSFA2 (Fig. 9; Table II). A studyin Arabidopsis showed that HSFA2 plays an importantrole among the 21 HSFs in both basal and acquiredthermotolerance (von Koskull-Döring et al., 2007). Re-cently, it was reported that high temperature inducesHSFA2 to generate a splice variant in Arabidopsis(Sugio et al., 2009), suggesting that high temperaturealso regulates HSFA2 expression at the posttranscrip-tional level in plants. Liu et al. (2013) suggested a novelmechanism underlying the self-regulation of HSFA2expression through a small truncated HSFA2 isoformencoded by this new splice variant. Cheng et al. (2015)found that OsHSFA2d RNA could be alternativelyspliced within the conserved introns of the DNA-binding domain coding region and that these splicingevents could introduce one or two small exons withpremature stop codons, resulting in three AS products,OsHSFA2dI,OsHSFA2dII, andOsHSFA2dIII. A similarphenomenon also was observed for HSF1 in alfalfa(He et al., 2007), HSFA1d and HSFA2 in Arabidopsis(Sugio et al., 2009), and HSFA2a2 in the genus Pota-mogeton (Amano et al., 2012). These findings indicatethat the AS of the HSF intron might serve as a con-served mechanism at the posttranscriptional levelfor HSF expression regulation in plants. AlthoughPotenza et al. (2015) and Vitulo et al. (2014) reportedthe AS occurring in grape, IR events of HSFA2 ex-pression in grape were not reported. Our study foundthat HSFA2 expression in grape leaves under 40°Cand 45°C generated two IR splicing variants (Fig. 9),which could introduce two new truncated proteins.ES and A3SS events of HSFA2 also were detected;however, they exhibited no significant differencesamong different high temperatures (35°C, 40°C, and45°C) compared with the control temperature(25°C). Therefore, IR should be an important post-transcriptional regulation for HSFA2, which makeHSFA2 play a key function in the grape response tohigh temperature.

In our study, in addition to HSPs, co-up-regulatedgenes and proteins between 40°C and 45°C also in-cluded the GATA transcription factor (Table I). GATA











can specifically bind 59-GATA-39 or 59-GAT-39 motifswithin gene promoters and may be involved in theregulation of some light-responsive genes. Only oneco-up-regulated gene and protein (MBF1c) was foundunder 35°C, 40°C, and 45°C treatments (Fig. 8). MBF1 isvery interesting, as it is a highly conserved protein thatfunctions as a non-DNA-binding transcriptional coac-tivator involved in different developmental and meta-bolic pathways in different organisms, ranging fromyeast to humans (Takemaru et al., 1998; Kabe et al.,1999; Liu et al., 2003). At present, there are only a fewstudies of MBF1. In the model plant Arabidopsis, MBF1was encoded by three different genes, MBF1a, MBF1b,and MBF1c (Tsuda et al., 2004). Suzuki et al. (2008)reported that MBF1c protein accumulated rapidlyand was localized to nuclei during heat stress in Ara-bidopsis. It was required for thermotolerance andfunctions upstream of salicylic acid (SA), trehalose, eth-ylene, and PATHOGENESIS-RELATED PROTEIN1. Incontrast, MBF1c was not required for the expressionof transcripts encoding HSFA2 and various HSPs.However, Suzuki et al. (2011) suggested that MBF1c inArabidopsis functions as a transcriptional regulatorthat binds DNA and controls the expression of 36 dif-ferent transcripts during heat stress, including theimportant transcriptional regulator DRE-BINDINGPROTEIN2A, two HSFs (HSFB2B and HSFB2A),and several zinc finger proteins. Moreover, the DNA-binding domain of MBF1c had a dominant-negativeeffect on heat tolerance when constitutively expressedin plants. Yan et al. (2014) isolated a full-length MBF1cDNA sequence (VvMBF1) from V. vinifera 3 Vitisamurensis that was up-regulated in the leaves of grapeplants following both drought and abscisic acid treat-ments. Carbonell-Bejerano et al. (2013) reported thatberryMBF1a andMBF1cwere up-regulated under hightemperature when grape ‘Muscat Hamburg’ plantsgrowing from cuttings were exposed to two differentenvironmental regimes, 20°C/15°C (day/night) and30°C/25°C (day/night), at the onset of ripening(veraison). Wang and Li (2006a) reported that exoge-nous SA pretreatment decreased thiobarbituric acid-reactive substances content and relative electrolyteleakage in grape leaves under heat stress, indicatingthat SA can induce intrinsic heat tolerance in grape. Inaddition, heat acclimation (38°C) induced an increase offree SA concentration in grape leaves (Wang and Li,2006b). In this study, high temperature under 40°C and45°C increased the expression of SALICYLIC ACID-BINDING PROTEIN2 by 860- and 3,100-fold, respec-tively, compared with the control (data not shown).These results indicated that it was possible that MBF1cmodulates heat tolerance through controlling the SAconcentration in grape. In addition, SA was recentlyproposed to play an important role in thermotolerancein other plant species (Larkindale and Knight, 2002;Martel and Qaderi, 2016). Previous studies have shownthat HSFs play an important role in thermotolerance inplants and other organisms, regulating HSPs as well asdifferent acclimation and detoxification proteins (von

Koskull-Döring et al., 2007). Therefore, MBF1c shouldbe a key regulator that controls the HSF-HSP pathwayand the SA pathway in the thermotolerance of grape.Among different temperature treatments, there is nooccurrence of differential AS in the MBF1c gene.Therefore, MBF1c could be involved in heat tolerance ingrape leaves through up-regulating transcriptional andtranslational levels.

In summary, we have provided data to suggest thatgrape leaves respond rapidly to temperature greaterthan 35°C simultaneously at transcriptional, post-transcriptional, and translational levels. AS was animportant posttranscriptional regulation, and IR eventswere the most abundant up- and down-regulated ASevents followed by ES. Proteomic analyses showed thatprotein levels of RNA-binding proteins SR45, SR30,and SR34 and nuclear ribonucleic protein U1A ingrape leaves gradually rose as ambient temperaturesincreased. These results also explained why AS eventsoccurred more under high temperature in grape leaves.HSPs were involved in heat tolerance in grape leavesthrough up-regulating mRNA levels, IR event occur-rence, and translational levels. The important transcriptfactor MBF1c was involved in heat tolerance in grapethrough up-regulating transcriptional and translationallevels, but HSFA2 was involved in heat tolerance bymodulating AS. Further investigations are needed todetermine how temperature signaling regulates splicingactivity and how the splicing machinery distinguishestranscript species.

MATERIALS AND METHODS

Plant Materials and Heat Treatments

One-year-old ‘Jingxiangyu’ grapevines (Vitis vinifera) were planted inpots, then grown in a greenhouse at 70% to 80% relative humidity at 18°C to25°C, with the maximum photosynthetically active radiation at approximately1,000 mmol photons m22 s21. When the sixth leaves (from base to apex) ofgrapevines became mature, all grapevines were divided into four groups andacclimated for 2 d in a controlled-environment room (70% average relativehumidity, 25°C/18°C [12-h/12-h] day/night cycle, and photosynthetically ac-tive radiation at 800 mmol m22 s21). On day 3, the grapevines were subjected tothe following treatments: (1) the plants of the control group were maintainedat the optimal day/night temperature (25°C/18°C) in the above growth room;(2) the plants of the treatment groups were exposed to 35°C, 40°C, or 45°C from11:30 AM to 1:30 PM (the conditions were the same as the control, except fortemperature). The fourth to sixth leaves (from base to apex) of each plant weredetached at 1:30 PM (the end of the heat stress treatment). Each biological rep-licate included three plants, and three replicates were used for both the threetreatments and the control. Leaves were frozen in liquid nitrogen immediatelyand stored at 280°C for further analysis.

Analysis of Chlorophyll Fluorescence Parameters

The O-J-I-P test was measured by a Handy Plant Efficiency Analyzer(Hansatech Instruments) after the leaves (using the fifth leaf from the base) wereadapted for 15 min in the dark when heat treatments ended. Five independentreplicates were used in three treatments and controls, and each replicate con-sisted of a plant. The chlorophyll a fluorescence transient was induced by asaturating photon flux density at 3,000 mmol photons m22 s21, provided by anarray of six light-emitting diodes (peak of 650 nm). The fluorescence signalswere recorded within a time span from 10 ms to 1 s, with a data acquisition rateof 10 ms for the first 2 ms and every 1 ms thereafter. The parameters Fv and Fm


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were measured, and Fv/Fm, which represents the intrinsic efficiency of PSIIphotochemistry (the ratio of variable and maximum fluorescence of PSII in thedark-adapted state), was calculated.

RNA Isolation and mRNA Sequencing

Total RNAwas isolated from grape leaves by use of the RNAprep Pure PlantKit (polysaccharides and polyphenolics rich; Tiangen) and quantified with theNanodrop system (Thermo Fisher Scientific). DNase digestion was performedduring total RNA isolation to remove genomic DNA.A total of 3mg of RNApersample was used as input material for the RNA sample preparations. Se-quencing libraries were generated using the NEBNext Ultra Directional RNALibrary Prep Kit for Illumina (New England Biolabs) following the manufac-turer’s recommendations, and index codeswere added to attribute sequences toeach sample. Briefly, mRNA was purified from total RNA using poly(T) oligo-attached magnetic beads. Fragmentation was carried out using divalent cationsunder elevated temperature in NEBNext First Strand Synthesis Reactionbuffer (53). First-strand cDNAwas synthesized using random hexamer primerand M-MuLV reverse transcriptase (RNaseH2). Second-strand cDNA syn-thesis was subsequently performed using DNA Polymerase I and RNase H. Inthe reaction buffer, deoxyribonucleotide triphosphates with dTTP werereplaced by dUTP. Remaining overhangs were converted into blunt ends viaexonuclease/polymerase activities. After adenylation of 39 ends of DNA frag-ments, NEBNext Adaptor with hairpin loop structure was ligated to prepare forhybridization. In order to select cDNA fragments with the right length, the li-brary fragments were purified with the AMPure XP system (Beckman Coulter).Then, 3 mL of USER Enzyme (New England Biolabs) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°Cbefore PCR. Then, PCR was performed with Phusion High-Fidelity DNApolymerase, Universal PCR primers, and Index (X) Primer. Finally, productswere purified (AMPure XP system), and library quality was assessed on anAgilent Bioanalyzer 2100 system. The clustering of the index-coded sampleswas performed on a cBot Cluster Generation System using TruSeq PE ClusterKit version 3-cBot-HS (Illumina) according to the manufacturer’s instructions.After cluster generation, the library preparationswere sequenced on the IlluminaHiSeq platform, and paired-end reads were generated.

Sequencing Data Processing and DifferentialExpression Analysis

Adaptor sequences and low-quality sequences were first removed, and thenclean reads were mapped to the grape reference genome (IGGP_12x) usingTopHat (version 2.1.0) with Ensembl gene annotation as transcript index andfr-firststrand given to –library-type (Kim et al., 2013). New transcripts wereassembled using Cufflinks (version 2.2.1; Trapnell et al., 2013). Raw read countsfor each gene were calculated using HTSeq (version 0.6.0; Anders et al., 2015).Gene expression normalization among samples was performed usingDESeq2. Then, differentially expressed genes were screened with foldchange $ 2, FDR # 0.01, and P , 0.05 (Love et al., 2014).

AS Analysis

AS events including ES, IR, MXE, A5SS, and A3SS were detected andquantified using rMATS with self-assembled transcript annotation (version3.0.9; Shen et al., 2014). In brief, AS analysis was performed as shown inSupplemental Figure S14. IR is used as an example to illustrate the method ofAS events. First, the exon inclusion level was represented by the count ofuniquely mapped reads to the IR isoform or the ES isoform. The count of readsmapped to the IR isoform was represented by I. The count of reads mapped tothe ES isoform was represented by S. Then, total count reads mapped to the IRisoform or ES isoform would be I + S, denoted as n. Second, as both isoformshad different lengths, isoform-specific read counts were normalized beforecalculating the intron inclusion levels by the lengths of isoform-specific effectivesegments. For a segment with length l and reads with length r, the effectivelength equaled the number of unique reads with intervals of l – r + 1. For IR,the effective length of the IR isoformwas set as ll and that of the ES isoform asls. After normalizing by effective length, the intron inclusion level could bedenoted as a = (I/ll)/(I/ll + S/ls). Then, the proportion of reads from the IRisoform would be P = lla/[lla + ls(1 2 a)]. Third, the comparative model tofilter out differential IR events was constructed using rMATS. Other types ofAS events also can be modeled by this framework, with details illustrated inSupplemental Figure S15 from Shen et al. (2014). The inclusion level of each

candidate splicing event was calculated using reads mapped to splicingjunctions. Differential splicing events under 35°C, 40°C, and 45°C comparedwith 25°Cwere selected with FDR# 0.01. Sashimi plots used to show splicingevents were generated by MISO (version 0.5.3; Katz et al., 2010).

Protein Sample Preparation

Grape samples (approximately 2 g each)were ground in liquid nitrogen, andthe resulting powder was precipitated with 10% (w/v) TCA in cold acetonecontaining 0.07% (v/v) b-mercaptoethanol at 220°C overnight. Protein mix-tures were denatured using 6 M guanidine-HCl in 50 mM Tris-HCl, pH 8, andthen in-solution digested. Disulfide bonds were reduced by the addition of20 mM DTT and incubation at 60°C for 30 min. After cooling to room temper-ature, 40 mM iodoacetamide was added, and the samples were kept in the darkfor 1 h to block free Cys. The samples were dialyzed against 2 M urea and 50 mM

NH4HCO3 for 2 h twice. Trypsin was added to the samples at 1:50 (w/w,trypsin:sample) and incubated at 37°C overnight. The digested peptides werefurther filtered with Amicon ultra centrifugal filters (10-kD MWKO; MerckMillipore) and were dried under vacuum extensively to remove residual pri-mary amine from reagents. Samples were labeled with iTRAQ 4plex reagent(ABSciex) according tomanufacturer’s instructions and combined after labelingand dried.

Analysis of High-pH Reverse-Phase HPLC andNano-LC-MS/MS

iTRAQ-labeled total lysate (400 mg) was solubilized in 200 mL of buffer A(20 mM ammonium formate, pH 10) and separated on an Xbridge column(Waters; C18; 3.5mm, 2.13 150mm) using a linear gradient of 1% Bmin21, thenfrom 2% to 45% B (20 mM ammonium formate in 90% acetonitrile, pH 10) at aflow rate of 200 mL min21 using the same Gilson system as described above.Fractions were collected at 1-min intervals and dried under vacuum.

Nano-LC-MS/MS was performed using a Dionex rapid-separation liquidchromatography system interfaced with a QExactive HF (Thermo Fisher Sci-entific). Samples were loaded onto a self-packed 100-mm 3 2-cm trap packedwith Magic C18AQ, 5 mm, 200 A (Michrom Bioresources), and washed withbuffer A (0.2% formic acid) for 5 min at a flow rate of 10 mLmin21. The trap wasbrought in line with the homemade analytical column (Magic C18AQ; 3 mm,200 A, 75 mm3 50 cm), and peptides were fractionated at 300 nL min21 with amultistep gradient (4% to 15% buffer B [0.16% formic acid and 80% acetonitrile]during 20 min, then 15%–25% B during 40 min, followed by 25%–50% B for30 min). Mass spectrometry data were acquired using a data-dependent ac-quisition procedure with a cyclic series of a full scan acquired with a resolutionof 120,000 followed by tandem mass spectrometry (MS/MS) scans (30% colli-sion energy in theHCD cell) with resolution of 30,000 of the 20most intense ionswith dynamic exclusion duration of 30 s.

Protein Data Analysis

iTRAQ quantification was performed with MaxQuant software version1.5.3.8 (Cox and Mann, 2008; Cox et al., 2011) using a grape database (UniProtUP000009183; 29,907 protein sequences). MS/MS searches for the proteomedata sets were performed with the following parameters: oxidation of Met andiTRAQ 4plex label on the N terminus of peptides as variable modifications;iTRAQ 4plex label on Lys as fixed modification; and carbamidomethylation asfixed modification. Trypsin/P was selected as the digestion enzyme, and amaximum peptide charge was set to six and two missed cleavages per peptide.Values of 67 and 20 ppm were used as tolerances for precursor and productions, respectively. Peptide and protein FDRs were set to 1%. Only proteins withat least two peptides were considered as reliably identified. Labeled proteinquantification was switched on, and unique and razor peptides were consid-ered for quantification with a minimum ratio count of 2. MS/MS identificationswere transferred by b and y ions. The quantification was based on the extractedreporter intensity. The ratio between samples was calculated using reporter ionintensity and normalized to the median ratio of all identified spectra that fitcertain criteria: peptide belongs to the grape database, and peptide FDR# 0.01.Pairwise median ratios (35°C/25°C, 40°C/25°C, and 45°C/25°C) of individualproteins were calculated by the Doby package under the R environment usingall spectra belonging to a protein that fit the criteria above and the sum of re-porter ion intensity of both channels greater than 10,000. After the R analysis,







proteins were further filtered by the number of unique peptides ($1) andMS/MS counts ($2) matched to each protein.

GO Analysis

GO analyses were performed using in-house scripts. The significance ofenrichment of differentially expressed genes or differentially spliced genes ineach GO term were determined at a threshold of adjusted P # 0.05 usingFisher’s exact test together with the Benjamini-Hochberg FDR procedure. Vi-sualizations of enriched GO terms were generated using R and Excel.

Quantitative Reverse Transcription-PCR Analysis

cDNA (20 mL volume) syntheses were performed using 800 ng of total RNA,an oligo(dT), and the HiScript Q RT SuperMix for qPCR (+gDNA wiper) Kit(Vazyme). qRT-PCR analysis involved the use of the AceQ qPCR SYBR GreenMaster Mix (without ROX; Vazyme). Primers designed by DNAMAN 8.0(Adobe) are listed in Supplemental Table S3. qRT-PCR was performed in tripli-cate. VvACT2 (EC969944) was used as an internal control for normalization.

RT-PCR Analysis of IR Splicing

The RT-PCR splicing analysis of selected genes (HSP90-1, HSP25.3,HSP101, HSP70-4, and HSFA2) was performed in a total volume of 20 mL ofcDNA. The first-strand cDNA was used for RT-PCR assays. The cyclingconditions were 98°C for 3 min, followed by 35 cycles of a 10-s denaturationat 98°C, a 5-s annealing at 58°C to 61°C, and a 90-s extension at 72°C, with afinal 2-min extension at 72°C. The PCR amplicons were analyzed using 2%agarose electrophoresis. The amount of first-strand cDNA used for theRT-PCR was determined by the expression of VvACTIN7. The forward andreverse primers were designed from the upstream and downstream exons ofthe introns based on each IR event, respectively. These primers are listed inSupplemental Table S4. The resulting RT-PCR products were cloned intopMD18T vector (Takara) and sequenced. All PCRs were performed usingEx-Taq polymerase (Takara).

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL datalibraries under GEO accession number GSE89113.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Cluster analysis for all transcripts in grape leavesunder four different temperatures (25°C, 35°C, 40°C, and 45°C).

Supplemental Figure S2. Summary number of differentially expressedtranscripts in grape leaves under 35°C, 40°C, and 45°C compared with25°C at a fold change greater than 2 and FDR less than 0.05.

Supplemental Figure S3. Flow chart of RNA sequencing and quantitativeproteome for grape leaves.

Supplemental Figure S4. Venn diagram of nonredundant UniProt-annotated proteins identified with at least two unique peptides (proteinand peptide FDR , 0.01) in grape leaves.

Supplemental Figure S5. Up- and down-regulated biological pathways ofdifferentially expressed proteins in grape leaves under 35°C comparedwith 25°C.

Supplemental Figure S6. Up- and down-regulated biological pathways ofdifferentially expressed proteins in grape leaves under 40°C comparedwith 25°C.

Supplemental Figure S7. Up- and down-regulated biological pathways ofdifferentially expressed proteins in grape leaves under 45°C comparedto 25°C.

Supplemental Figure S8. Linear correlation analysis (r2 = 0.79) betweenqRT-PCR and RNA-sequencing results for 35 genes in grape leaves un-der four temperatures (25°C, 35°C, 40°C, and 45°C).

Supplemental Figure S9. Sashimi plots of HSP90.1 transcripts with an IRevent in grape leaves under 40°C and 45°C compared with 25°C.


Supplemental Figure S11. Sashimi plots of HSP101 transcripts with an IRevent in grape leaves under 40°C and 45°C compared with 25°C.


Supplemental Figure S13. RT-PCR analysis of IR splice variants ofHSP90.1, HSP25.3, HSP101, and HSP70.4 in grape leaves under 25°C,35°C, 40°C, and 45°C for 2 h.

Supplemental Figure S14. Flow chart of AS analysis for grape leaves un-der different temperatures.

Supplemental Figure S15. Schematic diagrams illustrating the read countsand effective lengths of different categories of AS events.

Supplemental Table S1. Statistics of all AS events in grape leaves under35°C, 40°C, and 45°C compared with 25°C.

Supplemental Table S2. Statistics of all genes with significantly differentAS events in grape leaves under 35°C, 40°C, and 45°C compared with25°C.

Supplemental Table S3. qRT-PCR primers used in this study.

Supplemental Table S4. Primers of RT-PCR splicing analysis used in thisstudy.

Supplemental Data Set S1. Enriched analysis of genes with up-regulatedIR events in grape leaves under each high-temperature (35°C, 40°C, and45°C) treatment compared with the control (25°C).

Supplemental Data Set S2. Enriched analysis of genes with down-regulated IR events in grape leaves under each high-temperature(35°C, 40°C, and 45°C) treatment compared with the control (25°C).

Supplemental Data Set S3. Enriched analysis of genes with up-regulatedES events in grape leaves under each high-temperature (35°C, 40°C, and45°C) treatment compared with the control (25°C).

Supplemental Data Set S4. Enriched analysis of genes with down-regulated ES events in grape leaves under each high-temperature(35°C, 40°C, and 45°C) treatment compared with the control (25°C).

Supplemental Data Set S5. Sequence analysis of IR transcripts and corre-sponding proteins of HSP90.1, HSP25.3, HSP101, HSP70.4, and HSFA2genes in grape leaves under 40°C and 45°C.

ACKNOWLEDGMENTS

We thank Baicheng Wang (Institute of Botany, Chinese Academy ofSciences), Douglas D. Archbold (Department of Horticulture, University ofKentucky), Yuehua Cui (Department of Statistics and Probability, MichiganState University), and Xiaoting Qi (College of Life Science, Capital NormalUniversity) for critical reading and valuable suggestions and Dr. TiancongLu (ProteinWorld Biotechnology) for good advice regarding proteomicsanalysis.

Received September 2, 2016; accepted December 22, 2016; published January 3,2017.

LITERATURE CITED

Amano M, Iida S, Kosuge K (2012) Comparative studies of thermotol-erance: different modes of heat acclimation between tolerant and in-tolerant aquatic plants of the genus Potamogeton. Ann Bot (Lond) 109:443–452

Anckar J, Sistonen L (2011) Regulation of HSF1 function in the heat stressresponse: implications in aging and disease. Annu Rev Biochem 80:1089–1115

Anders S, Pyl PT, Huber W (2015) HTSeq: a Python framework to workwith high-throughput sequencing data. Bioinformatics 31: 166–169


Jiang et al.





























Ashraf M, Harris PJC (2013) Photosynthesis under stressful environments:an overview. Photosynthesis 51: 163–190

Bita CE, Gerats T (2013) Plant tolerance to high temperature in a changingenvironment: scientific fundamentals and production of heat stress-tolerant crops. Front Plant Sci 4: 273

Bokszczanin KL (2013) Perspectives on deciphering mechanisms under-lying plant heat stress response and thermotolerance. Front Plant Sci 4:315

Bösl B, Grimminger V, Walter S (2006) The molecular chaperone Hsp104: amolecular machine for protein disaggregation. J Struct Biol 156: 139–148

Capovilla G, Pajoro A, Immink RGH, Schmid M (2015) Role of alternativepre-mRNA splicing in temperature signaling. Curr Opin Plant Biol 27:97–103

Carbonell-Bejerano P, Santa María E, Torres-Pérez R, Royo C, LijavetzkyD, Bravo G, Aguirreolea J, Sánchez-Díaz M, Antolín MC, Martínez-Zapater JM (2013) Thermotolerance responses in ripening berries ofVitis vinifera L. cv Muscat Hamburg. Plant Cell Physiol 54: 1200–1216

Chang CY, Lin WD, Tu SL (2014) Genome-wide analysis of heat-sensitivealternative splicing Physcomitrella patens. Plant Physiol 165: 826–840

Chen X, Lin S, Liu Q, Huang J, Zhang W, Lin J, Wang Y, Ke Y, He H (2014)Expression and interaction of small heat shock proteins (sHsps) in rice inresponse to heat stress. Biochim Biophys Acta 1844: 818–828

Cheng Q, Zhou Y, Liu Z, Zhang L, Song G, Guo Z, Wang W, Qu X, Zhu Y,Yang D (2015) An alternatively spliced heat shock transcription factor,OsHSFA2dI, functions in the heat stress-induced unfolded protein re-sponse in rice. Plant Biol (Stuttg) 17: 419–429

Cox J, Mann M (2008) MaxQuant enables high peptide identification rates,individualized p.p.b.-range mass accuracies and proteome-wide proteinquantification. Nat Biotechnol 26: 1367–1372

Cox J, Neuhauser N, Michalski A, Scheltema RA, Olsen JV, Mann M(2011) Andromeda: a peptide search engine integrated into the Max-Quant environment. J Proteome Res 10: 1794–1805

Duque P (2011) A role for SR proteins in plant stress responses. Plant SignalBehav 6: 49–54

Filichkin SA, Priest HD, Givan SA, Shen R, Bryant DW, Fox SE, WongWK, Mockler TC (2010) Genome-wide mapping of alternative splicingin Arabidopsis thaliana. Genome Res 20: 45–58

Frank G, Pressman E, Ophir R, Althan L, Shaked R, Freedman M, Shen S,Firon N (2009) Transcriptional profiling of maturing tomato (Solanumlycopersicum L.) microspores reveals the involvement of heat shockproteins, ROS scavengers, hormones, and sugars in the heat stress re-sponse. J Exp Bot 60: 3891–3908

George IS, Pascovici D, Mirzaei M, Haynes PA (2015) Quantitative pro-teomic analysis of Cabernet Sauvignon grape cells exposed to thermalstresses reveals alterations in sugar and phenylpropanoid metabolism.Proteomics 15: 3048–3060

González-Schain N, Dreni L, Lawas LMF, Galbiati M, Colombo L, HeuerS, Jagadish KSV, Kater MM (2016) Genome-wide transcriptome anal-ysis during anthesis reveals new insights into the molecular basis of heatstress responses in tolerant and sensitive rice varieties. Plant Cell Physiol57: 57–68

Greer DH, Weedon MM (2012) Modelling photosynthetic responses totemperature of grapevine (Vitis vinifera cv. Semillon) leaves on vinesgrown in a hot climate. Plant Cell Environ 35: 1050–1064

Grover A, Mittal D, Negi M, Lavania D (2013) Generating high tempera-ture tolerant transgenic plants: achievements and challenges. Plant Sci205-206: 38–47

Györgyey J, Gartner A, Németh K, Magyar Z, Hirt H, Heberle-Bors E,Dudits D (1991) Alfalfa heat shock genes are differentially expressedduring somatic embryogenesis. Plant Mol Biol 16: 999–1007

He ZS, Xie R, Zou HS, Wang YZ, Zhu JB, Yu GQ (2007) Structure andalternative splicing of a heat shock transcription factor gene, MsHSF1, inMedicago sativa. Biochem Biophys Res Commun 364: 1056–1061

Isshiki M, Tsumoto A, Shimamoto K (2006) The serine/arginine-richprotein family in rice plays important roles in constitutive and alterna-tive splicing of pre-mRNA. Plant Cell 18: 146–158

Kabe Y, Goto M, Shima D, Imai T, Wada T, Morohashi Ki, Shirakawa M,Hirose S, Handa H (1999) The role of human MBF1 as a transcriptionalcoactivator. J Biol Chem 274: 34196–34202

Kalyna M, Simpson CG, Syed NH, Lewandowska D, Marquez Y, Kusenda B,Marshall J, Fuller J, Cardle L, McNicol J, et al (2012) Alternative splicingand nonsense-mediated decay modulate expression of important regulatorygenes in Arabidopsis. Nucleic Acids Res 40: 2454–2469

Katz Y, Wang ET, Airoldi EM, Burge CB (2010) Analysis and design ofRNA sequencing experiments for identifying isoform regulation. NatMethods 7: 1009–1015

Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg SL (2013)TopHat2: accurate alignment of transcriptomes in the presence of in-sertions, deletions and gene fusions. Genome Biol 14: R36

Kornblihtt AR, Schor IE, Alló M, Dujardin G, Petrillo E, Muñoz MJ(2013) Alternative splicing: a pivotal step between eukaryotic tran-scription and translation. Nat Rev Mol Cell Biol 14: 153–165

Kosová K, Vítámvás P, Prášil IT, Renaut J (2011) Plant proteome changesunder abiotic stress: contribution of proteomics studies to understand-ing plant stress response. J Proteomics 74: 1301–1322

Kotak S, Larkindale J, Lee U, von Koskull-Döring P, Vierling E, ScharfKD (2007) Complexity of the heat stress response in plants. Curr OpinPlant Biol 10: 310–316

Larkindale J, Knight MR (2002) Protection against heat stress-inducedoxidative damage in Arabidopsis involves calcium, abscisic acid, eth-ylene, and salicylic acid. Plant Physiol 128: 682–695

Lazar G, Goodman HM (2000) The Arabidopsis splicing factor SR1 isregulated by alternative splicing. Plant Mol Biol 42: 571–581

Lee GJ, Vierling E (2000) A small heat shock protein cooperates with heatshock protein 70 systems to reactivate a heat-denatured protein. PlantPhysiol 122: 189–198

Lim CJ, Yang KA, Hong JK, Choi JS, Yun DJ, Hong JC, Chung WS, LeeSY, Cho MJ, Lim CO (2006) Gene expression profiles during heat ac-climation in Arabidopsis thaliana suspension-culture cells. J Plant Res 119:373–383

Liu GT, Ma L, Duan W, Wang BC, Li JH, Xu HG, Yan XQ, Yan BF, Li SH,Wang LJ (2014) Differential proteomic analysis of grapevine leaves byiTRAQ reveals responses to heat stress and subsequent recovery. BMCPlant Biol 14: 110

Liu GT, Wang JF, Cramer G, Dai ZW, Duan W, Xu HG, Wu BH, Fan PG,Wang LJ, Li SH (2012) Transcriptomic analysis of grape (Vitis vinifera L.)leaves during and after recovery from heat stress. BMC Plant Biol 12: 174

Liu J, Sun N, Liu M, Liu J, Du B, Wang X, Qi X (2013) An autoregulatoryloop controlling Arabidopsis HsfA2 expression: role of heat shock-induced alternative splicing. Plant Physiol 162: 512–521

Liu QX, Jindra M, Ueda H, Hiromi Y, Hirose S (2003) Drosophila MBF1 isa co-activator for Tracheae Defective and contributes to the formation oftracheal and nervous systems. Development 130: 719–728

Lobell DB, Burke MB, Tebaldi C, Mastrandrea MD, Falcon WP, NaylorRL (2008) Prioritizing climate change adaptation needs for food securityin 2030. Science 319: 607–610

Love MI, Huber W, Anders S (2014) Moderated estimation of foldchange and dispersion for RNA-seq data with DESeq2. Genome Biol15: 550

Lund AA, Rhoads DM, Lund AL, Cerny RL, Elthon TE (2001) In vivomodifications of the maize mitochondrial small heat stress protein,HSP22. J Biol Chem 276: 29924–29929

Martel AB, Qaderi MM (2016) Does salicylic acid mitigate the adverseeffects of temperature and ultraviolet-B radiation on pea (Pisum sativum)plants? Environ Exp Bot 122: 39–48

Mastrangelo AM, Marone D, Laidò G, De Leonardis AM, De Vita P (2012)Alternative splicing: enhancing ability to cope with stress via tran-scriptome plasticity. Plant Sci 185-186: 40–49

Palusa SG, Ali GS, Reddy AS (2007) Alternative splicing of pre-mRNAs ofArabidopsis serine/arginine-rich proteins: regulation by hormones andstresses. Plant J 49: 1091–1107

Parsell DA, Lindquist S (1993) The function of heat-shock proteins in stresstolerance: degradation and reactivation of damaged proteins. Annu RevGenet 27: 437–496

Pillet J, Egert A, Pieri P, Lecourieux F, Kappel C, Charon J, Gomès E,Keller F, Delrot S, Lecourieux D (2012) VvGOLS1 and VvHsfA2 areinvolved in the heat stress responses in grapevine berries. Plant CellPhysiol 53: 1776–1792

Potenza E, Racchi ML, Sterck L, Coller E, Asquini E, Tosatto SCE, VelascoR, Van de Peer Y, Cestaro A (2015) Exploration of alternative splicingevents in ten different grapevine cultivars. BMC Genomics 16: 706

Pratt WB, Toft DO (2003) Regulation of signaling protein function andtrafficking by the hsp90/hsp70-based chaperone machinery. Exp BiolMed (Maywood) 228: 111–133

Reddy ASN, Marquez Y, Kalyna M, Barta A (2013) Complexity of thealternative splicing landscape in plants. Plant Cell 25: 3657–3683





Rhoads DM, White SJ, Zhou Y, Muralidharan M, Elthon TE (2005) Al-tered gene expression in plants with constitutive expression of a mito-chondrial small heat shock protein suggests the involvement ofretrograde regulation in the heat stress response. Physiol Plant 123: 435–444

Rozenzvieg D, Elmaci C, Samach A, Lurie S, Porat R (2004) Isolation offour heat shock protein cDNAs from grapefruit peel tissue and charac-terization of their expression in response to heat and chilling tempera-ture stresses. Physiol Plant 121: 421–428

Sagor GHM, Berberich T, Takahashi Y, Niitsu M, Kusano T (2013) Thepolyamine spermine protects Arabidopsis from heat stress-induceddamage by increasing expression of heat shock-related genes. Trans-genic Res 22: 595–605

Sankaranarayanan S, Jamshed M, Samuel MA (2013) Proteomics ap-proaches advance our understanding of plant self-incompatibility re-sponse. J Proteome Res 12: 4717–4726

Salazar-Parra C, Aguirreolea J, Sanchez-Diaz M, Irigoyen JJ, Morales F(2010) Effects of climate change scenarios on Tempranillo grapevine(Vitis vinifera L.) ripening: response to a combination of elevated CO2

and temperature, and moderate drought. Plant Soil 337: 179–191Shen S, Park JW, Lu ZX, Lin L, Henry MD, Wu YN, Zhou Q, Xing Y (2014)

rMATS: robust and flexible detection of differential alternative splicingfrom replicate RNA-Seq data. Proc Natl Acad Sci USA 111: E5593–E5601

Sorkel K (2006) Thermostability of photosynthesis of Vitis aestivalis andVitis vinifera. J Am Soc Hortic Sci 131: 476–483

Staiger D, Brown JWS (2013) Alternative splicing at the intersection ofbiological timing, development, and stress responses. Plant Cell 25:3640–3656

Su PH, Li HM (2008) Arabidopsis stromal 70-kD heat shock proteins areessential for plant development and important for thermotolerance ofgerminating seeds. Plant Physiol 146: 1231–1241

Sugio A, Dreos R, Aparicio F, Maule AJ (2009) The cytosolic proteinresponse as a subcomponent of the wider heat shock response inArabidopsis. Plant Cell 21: 642–654

Suzuki N, Bajad S, Shuman J, Shulaev V, Mittler R (2008) The tran-scriptional co-activator MBF1c is a key regulator of thermotolerance inArabidopsis thaliana. J Biol Chem 283: 9269–9275

Suzuki N, Sejima H, Tam R, Schlauch K, Mittler R (2011) Identificationof the MBF1 heat-response regulon of Arabidopsis thaliana. Plant J 66:844–851

Syed NH, Kalyna M, Marquez Y, Barta A, Brown JWS (2012) Alternativesplicing in plants: coming of age. Trends Plant Sci 17: 616–623

Takemaru K, Harashima S, Ueda H, Hirose S (1998) Yeast coactivatorMBF1 mediates GCN4-dependent transcriptional activation. Mol CellBiol 18: 4971–4976

Tanaka Y, Nishiyama Y, Murata N (2000) Acclimation of the photosyn-thetic machinery to high temperature in Chlamydomonas reinhardtii re-quires synthesis de novo of proteins encoded by the nuclear andchloroplast genomes. Plant Physiol 124: 441–449

Trapnell C, Hendrickson DG, Sauvageau M, Goff L, Rinn JL, Pachter L(2013) Differential analysis of gene regulation at transcript resolutionwith RNA-seq. Nat Biotechnol 31: 46–53

Tsuda K, Tsuji T, Hirose S, Yamazaki K (2004) Three Arabidopsis MBF1homologs with distinct expression profiles play roles as transcriptionalco-activators. Plant Cell Physiol 45: 225–231

Vierling E (1991) The roles of heat shock proteins in plants. Annu Rev PlantPhysiol Plant Mol Biol 42: 579–620

Vitulo N, Forcato C, Carpinelli EC, Telatin A, Campagna D, D’Angelo M,Zimbello R, Corso M, Vannozzi A, Bonghi C, et al (2014) A deepsurvey of alternative splicing in grape reveals changes in the splicingmachinery related to tissue, stress condition and genotype. BMC PlantBiol 14: 99

von Koskull-Döring P, Scharf KD, Nover L (2007) The diversity of plantheat stress transcription factors. Trends Plant Sci 12: 452–457

Wahid A, Gelani S, Ashraf M, Foolad M (2007) Heat tolerance in plants: anoverview. Environ Exp Bot 61: 199–223

Wang K, Zhang X, Goatley M, Ervin E (2014) Heat shock proteins in re-lation to heat stress tolerance of creeping bentgrass at different N levels.PLoS ONE 9: e102914

Wang LJ, Li SH (2006a) Salicylic acid-induced heat or cold tolerance inrelation to Ca2+ homeostasis and antioxidant systems in young grapeplants. Plant Sci 170: 685–694

Wang LJ, Li SH (2006b) Thermotolerance and related antioxidative enzymeactivities induced by heat acclimation or salicylic acid in grape leaves(Vitis vinifera L. cv. Jingxiu). Plant Growth Regul 48: 137–144

Wang XC, Li Q, Jin X, Xiao GH, Liu GJ, Liu NJ, Qin YM (2015) Quanti-tative proteomics and transcriptomics reveal key metabolic processesassociated with cotton fiber initiation. J Proteomics 114: 16–27

Wu Z, Yuan X, Li H, Liu F, Wang Y, Li J, Cai H, Wang Y (2015) Heat ac-climation reduces postharvest loss of table grapes during cold storage:analysis of possible mechanisms involved through a proteomic ap-proach. Postharvest Biol Technol 105: 26–33

Xiong L, Schumaker KS, Zhu JK (2002) Cell signaling during cold,drought, and salt stress. Plant Cell (Suppl) 14: S165–S183

Xu H, Liu G, Liu G, Yan B, Duan W, Wang L, Li S (2014) Comparison ofinvestigation methods of heat injury in grapevine (Vitis) and assessment toheat tolerance in different cultivars and species. BMC Plant Biol 14: 156

Yamada K, Fukao Y, Hayashi M, Fukazawa M, Suzuki I, Nishimura M(2007) Cytosolic HSP90 regulates the heat shock response that is re-sponsible for heat acclimation in Arabidopsis thaliana. J Biol Chem 282:37794–37804

Yamaguchi-Shinozaki K, Shinozaki K (2006) Transcriptional regulatorynetworks in cellular responses and tolerance to dehydration and coldstresses. Annu Rev Plant Biol 57: 781–803

Yan Q, Hou H, Singer SD, Yan X, Guo R, Wang X (2014) The grapeVvMBF1 gene improves drought stress tolerance in transgenic Arabi-dopsis thaliana. Plant Cell Tissue Organ Cult 118: 571–582

Zhang B, Peñagaricano F, Driver A, Chen H, Khatib H (2011) Differentialexpression of heat shock protein genes and their splice variants in bo-vine preimplantation embryos. J Dairy Sci 94: 4174–4182


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