Abstract

   Grapevine is an important and extensively grown fruit crop, which is
   severely hampered by drought worldwide. So, comprehending the impact of
   drought on grapevine genetic resources is necessary. In the present
   study, RNA-sequencing was executed using cDNA libraries constructed
   from both drought-stress and control plants. Results generated 12,451
   differentially expressed genes (DEGs), out of which 8,021 genes were
   up-regulated, and 4,430 were down-regulated. Further physiological and
   biochemical investigations were also performed to validate the
   biological processes associated with the development of grapevine in
   response to drought stress. Results also revealed that decline in the
   rate of stomatal conductance, in turn, decrease the photosynthetic
   activity and CO[2] assimilation in the grapevine leaves. Reactive
   oxygen species, including stress enzymes and their related proteins,
   and secondary metabolites were also activated in the present study.
   Likewise, various hormones also induced in response to drought stress.
   Overall, the present study concludes that these DEGs play both positive
   and negative roles in drought tolerance by regulating various
   biological pathways of grapevine. Nevertheless, our findings have
   provided valuable gene information for future studies of abiotic stress
   in grapevine and various other fruit crops.

Introduction

   Grapevine (Vitis vinifera L.) is an important crop, having 7.8 million
   hectares of cultivated land with an annual production of 67.6 million
   tons worldwide^[40]1. The climate change has noticeable effects on the
   survival and productivity of fruit plants^[41]2. Hence, the growth of
   grapevine is consequently, affected by abiotic stress, such as drought
   and salinity. Among these, drought has deleterious impacts on
   viticulture around the world^[42]3,[43]4. Globally, 45% of the
   agricultural terrains are under constant/periodic water
   scarcities^[44]5, causing nearly 50% of yield losses. Plants as sessile
   organisms can make versatile vicissitudes in physiology and morphology
   that allow them to endure environmental stress. However, these
   adaptations are derisory to recover physiological water potential in
   the cell^[45]6. Plant response to these minimal water conditions is
   mediated by expression of numerous genes encoding stress-related
   proteins, enzymes and metabolites functioning in the various pathways
   of cell metabolism^[46]7. The genes induced by osmotic stress in plants
   are categorized into two groups, such as functional proteins and
   regulatory proteins^[47]8,[48]9. In previous studies, several salient
   genes were identified in grapevine genome in response to biotic and
   abiotic stresses, 59 similar genes encoding putative WRKY proteins were
   identified from the available database^[49]10. Similarly, plant
   pathogenesis-related proteins were believed to be involved in plant
   defense and are usually induced during biotic and abiotic
   stresses^[50]11. In addition, over-expression of AtHSP70 has
   demonstrated its contribution in enhancing tolerance to abiotic
   stress^[51]12. Transcriptomic analysis of grapevine during drought
   stress is of vital importance whose results could provide a
   defense-related gene information, which offers a foundation for further
   development of drought-resistant grapevine cultivars.

   Water scarcity is not the only threat to viticulture productivity, but
   also for wine quality^[52]13,[53]14. Schultz proposed that an increase
   in environmental temperature due to rise in atmospheric CO[2] is a
   primary cause of water shortages for viticulture^[54]15. Grapevine
   possesses distinct molecular machinery which adjusts the circulation of
   water to leaf and then to the atmosphere by vessel anatomy^[55]16,
   stomatal conductance^[56]17 and aquaporin^[57]18. Consequently, the
   sluggish leaf and shoot growth, elongation of tendrils, restrained
   internodes extension, leaf augmentation, a decline of an average
   diameter of xylem vessels and a minor stimulation in root growth under
   drought are observed in grapevine^[58]16.

   RNA-seq is a deep-sequencing technology to acquire transcriptomic
   profiling of both model and non-model plants. In a single assay, this
   approach reveals the detection of unique genes, transcript information,
   allele-specific gene expression and single nucleotide variants without
   the availability of ESTs and gene annotations. Moreover, transcriptome
   data have also been utilized in defining large-scale genes governing
   the complex interaction and metabolic processes of the plants under
   stress^[59]19. In addition, qRT-PCR enables the quantification of
   absolute gene expression, which allows researchers to validate the
   RNA-seq results. Thus, the advantage of this technique ought to be
   manipulated in the crop production, especially the countries suffered
   more losses from adverse environmental conditions. we have effectively
   implicated this technique in our previous study of different
   fertilization trials in grapevine^[60]20. However, drought-regulated
   stress-response in grapevine has not been investigated in detail so
   far. Therefore, the aim of this study is to elucidate the physiological
   responses of grapevine to drought stress and further identify and
   analyze the DEGs in various biological pathways to gain insight into
   grapevine defense response to drought stress.

Results

   The sequence data obtained from the Illumina deep-sequencing was
   submitted to Short Read Archive (SRA) database at NCBI under accession
   number SAMN04914490. After filtering, raw data yielded 42.47 and 53.05
   million clean reads in control and drought-stressed leaf samples,
   respectively. The sequence alignment (soap2/SOAPaligner;
   [61]http://soap.genomics.org.cn) to the grapevine reference genome,
   allowed two base mismatch. The total mapped reads (73.44%) were
   corresponding to unique (72.01%) and multiple (1.44%) genomic positions
   (Supplementary Table [62]S1).

   In the current study, the sum of 12,451 DEGs was expressed under
   drought stress (|log[2]Ratio| ≥ 1) and false discovery rate
   (FDR ≤ 0.001); whereas, 8,021 (64.43%) were up-regulated and 4430
   (35.57%) were down-regulated (Supplementary Table [63]S2).

Gene ontology (GO) and KEGG analysis of differentially-expressed genes

   A total of 12,451 DEGs were subjected to GO and KEGG for annotation.
   All the DEGs were assigned to different groups based on their
   functional properties, such as biological process (21), cellular
   component (16) and molecular functions (14). Under the broad category
   of “Biological process”, 4,537 transcripts were assigned to ‘metabolic
   processes’ (GO: 0008152), followed by ‘cellular process’ which consists
   of 3,632 transcripts (GO: 0009987) and 3,185 transcripts were involved
   in ‘single organism process’ (GO: 0044699). Similarly, under “Cellular
   component” category, 3,247 transcripts were assigned to ‘cell’ and
   ‘cell part’ (GO: 0005623, and GO: 0044464), followed by 2,358
   transcripts in ‘organelle’ (GO: 0043226). Further, under “Molecular
   functions” category, 4,291 transcripts were in ‘catalytic activities’
   (GO: 0003824), and 3,497 transcripts were assigned to ‘binding
   activities’ (GO: 0005488; Supplementary Table [64]S3; Figure [65]S1 ).

   Several DEGs from the current study were subjected to KEGG annotation
   for further characterization of transcripts, where 12,451 transcripts
   were allotted to 306 KEGG pathways. Our results revealed that the
   highest numbers of transcripts (1,425) were involved in the “Metabolic
   pathway” (1,257 up-regulated, 168 down-regulated), followed by
   “Biosynthesis of secondary metabolites”, which consists of 1,160
   transcripts (681 up-regulated, 479 down-regulated), then 756
   transcripts were recorded in “Plant-pathogen interaction pathway” (487
   up-regulated, 269 down-regulated), while lowest transcripts (21) were
   recorded in “Sesquiterpenoid and triterpenoid biosynthesis pathway” in
   which 14 transcripts were up-regulated and 7 transcripts were
   down-regulated (Supplementary Table [66]S4).

Chlorophyll degradation and photosynthetic competencies under drought stress

   The results of chlorophyll (chl) estimation unveiled that 34.88%
   decrease of chl-a content in drought treated grapevine leaf
   (0.28 ± 0.06 mg g^−1) when compared with that of control plant leaf
   (0.43 ± 0.11 mg g^−1). Similarly, 21.92% decrease in chlb of leaf
   exposed to drought stress (0.57 ± 0.04 mg g^−1) compared to control
   leaf (0.73 ± 0.06 mg g^−1). In the same way, photosynthesis rate was
   also decreased by 32.20% in drought treatment (16.08 ± 0.75 µmole m^2−
   sec^−1) when compared to control (23.67 ± 0.81 µmole m^−2 sec^−1).
   Moreover, stomatal conductance and CO[2] assimilation rate also showed
   a significant reduction by 40.00% (0.11 ± 0.04) and 44.44% (5 ± 0.03)
   in drought treated grapevine leaves compared to that of control
   (Table [67]1).

Table 1.

   Comparison of physiological and biochemical parameters in grapevine
   leaves in response to drought stress.
   Physiological and biochemical parameters Control Drought treatment
   Range of increasing %
   Chlorophyll contents (mg g^−1) 1.16 ± 0.08 0.85 ± 0.09 −26.72
   Chla contents (mg g^−1) 0.43 ± 0.11 0.28 ± 0.06 −34.88
   Chlb contents (mg g^−1) 0.73 ± 0.06 0.57 ± 0.04 −21.92
   Photosynthesis activity (µmole m^−2sec^−1) 23.67 ± 0.81 16.08 ± 0.75
   −32.20
   Stomatal conductance (µmole m^−2sec^−1) 0.15 ± 0.03 0.09 ± 0.02 40.00
   Net CO2 assimilation (µmole m^−2sec^−1) 9 ± 0.03 5 ± 0.03 44.44
   MDA contents (nmol/g) 5.35 ± 0.21 8.61 ± 0.25 60.93
   SOD activity (U/g/min) 371.56 ± 10.21 650.85 ± 15.7 75.16
   POD activity (U/g/min) 18.23 ± 0.97 43.9 ± 1.01 140.81
   CAT activity (U/g/min) 6.32 ± 1.21 19.01 ± 0.99 200.79
   Proline (ng/g FW) 1.124 ± 0.04 1.711 ± 0.05 52.37
   [68]Open in a new tab

   In the grapevine transcriptome, 23 DEGs involving in chlorophyll
   metabolic pathway responded differently to drought stress compared with
   control, of which 13 transcripts were up-regulated, and 10 transcripts
   were down-regulated. However, out of 23 transcripts functioning in chl
   synthesis and degradation, 9 transcripts involved in chla synthesis
   (Glutamate tRNA Ligase; Radical S-adenosyl methionine domain-containing
   protein1; Protoporphyrinogen oxidase; Dehydrogenase/reductase SDR
   family member; Protochlorophyllide oxidoreductase, and four transcripts
   of Short chain dehydrogenase, TIC32) were significantly up-regulated;
   whereas, 6 transcripts (2 transcripts of HemA, Glutamate tRNA reductase
   1; Proporphynogen oxidase 1; 2 transcripts of CHLH, Magnesium chelatase
   H subunit; Protochlorophyllide oxidoreductase and Short chain
   dehydrogenase) were significantly down-regulated. Meanwhile, 1
   transcript (Chlorophyllide a oxygenase; CAO) was significantly
   down-regulated, but 1 transcript (Chlorophyll (ide) b reductase NYC1;
   CBR) was up-regulated by the drought treatment in the chl cycling
   process. Whereas, 3 transcripts (Chlorophyllase-II, Pheophorbide a
   oxygenase, and Protochlorophyllide-dependent translocon component 52)
   were significantly up-regulated and 3 transcripts of Chlorophyllase-I
   were down-regulated during the chl degradation process (Table [69]2,
   Fig. [70]1). The expression level of VIT_08s0007g08540.t01
   (307.93–106.65 RPKM) and VIT_19s0014g03160.t01 (1360.37–307.58 RPKM)
   revealed high profusion in chla synthesis pathway. Moreover, in the
   phytochromobilin synthesis, the expression of Ferrochelatase-2
   (VIT_07s0031g03200.t01, |log[2]FC| = 3.032), Heme oxygenase 1
   (VIT_11s0016g05300.t01, |log[2]FC| = 2.403), Heme oxygenase 2
   (VIT_18s0001g11040.t01, |log[2]FC| = 2.249) and
   Phytochromobilin:ferredoxin oxidoreductase (VIT_06s0009g03770.t01,
   |log[2]FC| = 1.705) was also induced by drought stress (Supplementary
   Table [71]S5).

Table 2.

   List of differentially-expressed genes related to chlorophyll
   degradation and photosynthesis in response to drought stress.
   Trait name Description No. of up-regulated No. of down-regulated sum
   Chlorophyll Metabolism Chlorophyll a synthesis 9 6 15
   Chlorophyll cycle 1 1 2
   Chlorophyll degradation 3 3 6
   Photosystem II psbB 0 2 2
   psbC 0 2 2
   psbW 0 1 1
   Photosystem I psaB 0 2 2
   Cytochrome b6-f complex petA 0 1 1
   petC 1 2 3
   Phtosynthetic electron transport petF 0 2 2
   petH 2 0 2
   F-type ATPase ATPF1B 0 1 1
   ATPF1A 0 1 1
   ATPF1G 0 1 1
   ATPF0C 0 1 1
   Photosynthesis-antenna proteins LHCB1 0 1 1
   LHCB2 0 1 1
   LHCB3 0 1 1
   LHCB6 0 1 1
   [72]Open in a new tab

   psbB, Photosystem II CP47 chlorophyll apoprotein gene; psbC,
   Photosystem II CP43 chlorophyll apoprotein gene; psbW, Photosystem II
   reaction center W protein; psaB, photosystem I P700 apoprotein A2 gene;
   petA, cytochrome f; petC, cytochrome b6-f complex iron-sulfur subunit
   1; petF, ferredoxin-3; petH, ferredoxin–NADP reductase, leaf-type
   isozyme; ATPF1B, ATP synthase CF1 beta; ATPF1A, ATP synthase CF1 alpha;
   ATPF1G, ATP synthase gamma; LHCB1, chlorophyll a-b binding protein of
   LHCII; LHCB2, light harvesting chlorophyll A/B binding protein; LHCB3,
   light-harvesting chlorophyll binding protein 3 gene; LHCB6, chlorophyll
   a-b binding protein CP24 10 A.

Figure 1.

   [73]Figure 1
   [74]Open in a new tab

   Chlorophyll metabolic pathway in drought-stress grapevine leaves. GLTL,
   Glutamate tRNA ligase; HemA, Glutamate tRNA reductase 1; GSA,
   Glutamate-1-semialdehyde; ALAD, Delta-aminolevulinic acid dehydrates;
   PBGD, porphobilinogen deaminase; UROS, Uroporphyrinogen III synthase;
   RMA1, Radical S-adenosyl methionine domain-containing protein 1; PPOX1;
   Proporphynogen oxidase 1; PPOX, Proporphynogen oxidase; UROD,
   Uroporphyrinogen III decarboxylase; CHLH, Magnesium chelatase H
   subunit; CHL1, Magnesium-chelatase I subunit; CHLD, Magnesium chelatase
   D subunit; CHLM, Mg-proto IX methyltransferase; CRD1, Mg-protophyrin IX
   monomethylester (oxidative) cyclase; POR, Protochlorophyllide
   oxidoreductase; DHR, Dehydrogenase/reductase SDR family member; SCD,
   Short chain dehydrogenase, TIC32; CHLG, CAO; Chlorophyllide a
   oxygenase; CBR, Chlorophyll(ide) b reductase NYC1; CLH1,
   Chlorophyllase-I; CLH2, Chlorophyllase-II; PAO, Pheophorbide a
   oxygenase; PDT, Protochlorophyllide-dependent translocon component 52.

   In grapevine transcription analysis, a sum of 23 DEGs related to
   photosynthesis pathway, including PSII (5), PSI (2), cytochrome b6-f
   complex (4), photosynthetic electron transport (4), F-type ATPase (4),
   photosynthesis-antenna proteins (4) were sensitive to drought stress.
   In PSII (5 DEGs), which includes psbB (2), psbCs (2) and psbW (1) and
   all 5 DEGs were found to be significantly down-regulated. psbC
   (VIT_00s0396g00010.t01, 280.56–1.32 RPKM) possessed the high expression
   abundance. Moreover, 2 psaBs in PSI and 3 transcripts related to
   cytochrome b6-f complex (petA and petC) revealed a significant
   reduction in their expression levels, perhaps 1 transcript of petC were
   found to be increased with the control group. Similarly, 4 genes
   involved in the photosynthetic electron transport unveiled that, two
   transcripts of petF (VIT_12s0035g00270.t01 and VIT_06s0080g00410.t01)
   were down-regulated and two transcripts of petH (VIT_04s0023g03510.t01
   and VIT_10s0003g04880.t01) were up-regulated when compared with
   control. In addition, the F-type ATPase-related genes (ATPF1B, ATPF1A,
   ATPF1G and ATPF0C) and photosynthesis-antenna proteins-related genes
   (LHCB1, LHCB2, LHCB3 and LHCB6) were found to be significantly
   down-regulated in drought treated leaves (Table [75]2, Supplementary
   Table [76]S6).

ROS system under drought stress

   The Malondialdehyde activity was increased significantly (60.93%) in
   drought treatment (8.61 ± 0.25 nmol g^−1) compared to control
   (5.35 ± 0.21 nmol g^−1). A significant increase in the activity of
   superoxide dismutase (75.16%), peroxidase (140.81%) and catalase
   (200.79%) was observed in drought-responsive grapevine in comparison
   with control (Table [77]1). In the transcriptomic analysis, one NADPH
   respiratory oxidase and five amine oxidases functioning in the ROS
   synthesis process were significantly up-regulated in drought treated
   grapevine leaf samples. In ROS scavenging system, 60 DEGs were
   identified and categorized into Fe superoxide dismutase (2
   transcripts), peroxidase (6 transcripts), catalase (3 transcripts),
   glutathione-ascorbate cycle (9 transcripts), glutathione peroxidase (1
   transcript), glutathione S-transferase (26 transcripts),
   peroxiredoxin/thioredoxin pathway (8 transcripts), alternative oxidases
   (3 transcripts) and polyphenol oxidase (2 transcripts) (Fig. [78]2;
   Supplementary Table [79]S7).

Figure 2.

   [80]Figure 2
   [81]Open in a new tab

   Reactive oxygen species (ROS) scavenging pathway in plants. (a) The
   ascorbate-glutathione (AsA-GSH) cycle, (b) The glutathione peroxidase
   (GPX) cycle. SOD (superoxide dismutase) initiate the line of defense by
   converting O[2] ^− into H[2]O[2], which is further detoxified by CAT
   (catalses), APX (ascorbate peroxidases (APX) and GPX (glutathione
   ascorbate). Abbreviations: DHA, dehydroascorbate; GSH, glutathione;
   GSSG, oxidized glutathione; GR, glutathione reductase; MDAR,
   monodehydroascorbate reductase; DHAR, dehydroascorbate reductase.

   In our findings, two Fe-SODS were up-regulated, but both genes showed
   low expression abundance. In contrast, 2 Cu/Zn-SOD were significantly
   down-regulated (|log2FC| < 1), CAT (3 transcripts) and POD (2
   transcripts) were significantly up-regulated. All three up-regulated
   CAT transcripts (VIT_18s0122g01320.t01, from 2888.01 to 358.79 RPKM;
   VIT_00s0698g00010.t01, from 428.21 to 106.90 RPKM;
   VIT_04s0044g00020.t01, from 767.21 to 263.73 RPKM) showed high
   expression abundance; whereas, all up/down-regulated POD genes showed
   moderate to low expression abundance. Furthermore, 9 GSH-AsA (5
   up-regulated, 4 down-regulated), 27 GPX-pathway (23 up-regulated, 4
   down-regulated), eight Prx/Trx (5 up-regulated, 3 down-regulated),
   three AOX (2 up-regulated, 1 down-regulated) and two PPO
   (down-regulated) genes were identified in response to drought stress,
   (Table [82]3, Supplementary Table [83]S7).

Table 3.

   List of differentially-expressed genes related to ROS system in
   response to drought stress.
   Trait name Description No. of up-regulated No. of down-regulated sum
   ROS synthesis Rboh 1 1 2
   AO 5 0 5
   ROS scavenging Fe-SOD 2 0 2
   POD 2 4 6
   CAT 3 0 3
   GSH-AsA cycle MDAR 1 0 1
   DHAR 1 0 1
   GR 1 0 1
   Grx 2 4 6
   GPX pathway GPX 1 0 1
   GST 22 4 26
   Prx/Trx Prx 0 1 1
   Trx 5 2 7
   Cyanide-resistant respiration AOX 2 1 3
   Copper-containing enzymes PPO 0 2 2
   [84]Open in a new tab

   Rboh, respiratory burst oxidase; AO, amine oxidase; Fe-SOD, Fe
   superoxide dismutase; POD, peroxidase; CAT, catalase, APX, ascorbate
   peroxidase; MDAR, monodehydroascorbate reductase; DHAR,
   dehydroascorbate reductase; GR, glutathione reductase; Grx,
   glutaredoxin; GPX, glutathione peroxidase; GST, glutathione S
   transferase; Prx, peroxiredoxin; Trx, thioredoxin; AOX, alternative
   oxidase, PPO, polyphenol oxidase.

Plant hormone signal transduction pathway under drought stress

   The hormonal level, including auxin, was increased in drought treatment
   (1.626 ± 0.03 ng g^−1 FW) compared to control (1.373 ± 0.02 ng g^−1
   FW). A similar trend was observed in abscisic acid that is
   0.908 ± 0.01, and 0.257 ± 0.01 ng g^−1 FW for drought and control
   treatments, respectively. In the same way, jasmonic acid in drought
   treatment sample was 1.67 ± 0.05 ng g^−1 FW, whereas, in control, it
   was found to be 1.451 ± 0.03 ng g^−1 FW. Further gibberellic acid (GA)
   in treated and control sample was recorded to be 1.671 ± 0.02, and
   1.53 ± 0.02 ng g^−1 FW, respectively. Alike brassinosteroid also showed
   1.091 ± 0.01, and 1.073 ± 0.01 ng g^−1 FW for drought and control
   treatment samples, respectively (Fig. [85]3). In grapevine
   transcriptome, several DEGs related to AUX, GA, ABA, JA, ET (ethylene),
   and BR were found in signal transduction pathways in drought stressed
   grapevine leaves. Under AUX signaling, three genes (down-regulated)
   related to auxin transport, eleven auxin-response factors (7
   up-regulated and 4 down-regulated) involved in the transcriptional
   repressors were detected. Moreover, fifteen genes in auxin induced and
   responsive proteins (2 up-regulated and 13 down-regulated), six IAA
   synthetase (GH3; 1 up-regulated and 5 down-regulated) and seventeen
   genes related to auxin and IAA-induced proteins (SAUR; 5 up-regulated
   and 12 down-regulated) were perceived in grapevine under drought
   stress. Two natural receptors were up-regulated while four DELLA
   proteins were down-regulated in the GA under drought stress. Moreover,
   3 ABA-responsive proteins (down-regulated), two SNF1-related protein
   kinases 2 (SnRK2; 1 up- and 1 down-regulated), 3 PP2C group
   (up-regulated) genes and 6 transcription factors (ABF, up-regulated)
   were involved in the abscisic acid pathway. Six transcripts of
   jasmonate-ZIM-domain proteins (one up-regulated and 5 down-regulated)
   and single jasmonoyl isoleucine conjugate synthase 1 (up-regulated)
   were found in JA hormonal signaling. Moreover, 12-oxophytodienoate
   reductase 2-like (up-regulated), linoleate 13S-lipoxygenase 2-1
   (up-regulated) and allene oxide synthase (down-regulated) were
   identified in JA pathway under drought stress. Three
   ethylene-responsive transcriptional factors (3 up-regulated) being
   crucial to ET, five ethylene response factor (down-regulated) and three
   ACC oxidases (up-regulated) were perceived in drought-treated grapevine
   leaves. In BRs, two transcripts related to BRASSINOSTEROID INSENSITIVE1
   (up-regulated), ten (down-regulated) brassinosteroid-regulated proteins
   (BRU1) and 9 (down-regulated) D-type cyclins were functioning in plant
   hormone signal transduction pathway under drought stress conditions
   (Supplementary Table [86]S8).

Figure 3.

   [87]Figure 3
   [88]Open in a new tab

   The activities of different hormone, including IAA (indole-acetic
   acid), ABA (abscisic acid), JA (jasmonic acid), GA (gibberellic acid)
   and BR (brassinsteroid) in control and drought treatment.

Proline metabolism under drought stress

   The proline level showed a significant increase in grapevine leaves
   responding to drought stress (1.711 ± 0.05 ng g^−1 FW) as compared with
   control plant leaves (1.624 ± 0.04 ng g^−1 FW; Table [89]1). In
   transcriptomic analysis, a total of 18 DEGs, including
   pyroline-5-carboxylate synthetase, proline dehydrogenase, Proline
   methyltransferase ϒ-Glutamyl kinase, Glutamic-ϒ-semialdehye
   dehydrogenase, Pyrroline-5-carboxyate dehydrogenase, Prolyl hydroxylase
   (4 transcripts), Acetyl-CoA: glutamate N-acetyl transferase 2
   transcripts), N-Acetylglutamate kinase, Acetyl glutamic-ϒ-semialdehyde
   dehydrogenase, Acetyl ornithine aminotransferase, Acetyl ornithine
   deacetylase (2 transcripts), Arginino succinate lyase (ASL) and
   Arginase were significantly up-regulated functioning in the proline
   synthesis and metabolism pathway in drought treatment compared to
   control (Fig. [90]4, Supplementary Table [91]S9).

Figure 4.

   [92]Figure 4
   [93]Open in a new tab

   Differential expressions of genes during biosynthesis and degradation
   of proline in response to drought stress. Given numbers represents the
   individual genes catalyzing specific reactions. P5CS,
   pyroline-5-carboxylate synthetase; ARG, arginase; δ-AOT;
   ornithine-δ-aminotransferase; P5CR, pyrroline 5-carboxylate reductase;
   PDH, Proline dehydrogenase; P5CDH, Pyrroline-5-carboxyate
   dehydrogenase.

Biosynthesis of secondary metabolites under drought stress

   In the transcriptomic study, 70 secondary metabolites related genes
   linked with shikimate acid (9), alkaloid (2), anthocyanin (32), lignin
   (19) and terpenoid (8) were recognized under drought treated grapevine
   leaves.

   Shikimate acid (SA) pathway possessed one up-regulated
   3-deoxy-D-arabino-heptulosonate-7-phosphate synthase 03, two
   down-regulated 3-dehydroquinate dehydratase/shikimate dehydrogenase,
   one down-regulated shikimate kinase one up-regulated chorismate
   synthase-1, two down-regulated anthranilate phosphoribosyltransferase
   (AnPRT) and both up-regulated indole-3-glycerol phosphate synthase
   (IGPS) and tryptophan synthase beta chain 1 (TS1), respectively.

   In alkaloid biosynthetic pathway, genes related to strictosidine
   synthase 3 and D-amino-acid transaminase were down-regulated. Out of 33
   genes in anthocyanin biosynthesis, 8 genes related phenylalanine
   ammonia-lyase (4-up-regualted and 4-down-regulated), one
   trans-cinnamate 4-monooxygenase (down-regulated), two 4-coumarate–CoA
   ligase-like 9 (up and down-regulated), 13 stilbene synthase (6
   up-regulated and 7 down-regulated), 3 flavonol synthase/flavanone
   3-hydroxylase (one up-regulated and 2 down-regulated), one
   1-aminocyclopropane-1-carboxylate oxidase 5 (down-regulated), two
   dihydroflavonol-4-reductase (down-regulated), one anthocyanidin
   reductase (up-regulated) and one anthocyanidin 3-O-glucosyltransferase
   2 (down-regulated) were observed, (Table [94]4, Fig. [95]5,
   Supplementary Table [96]S10).

Table 4.

   Elucidation on differential expression of genes related to secondary
   metabolites under drought stress.
   Trait name Description No. of up-regulated No. of down-regulated sum
   Shikimate acid pathway DAHPS3 1 0 1
   B3D/SDH 0 2 2
   SHK 0 1 1
   CS1 1 0 1
   AnPRT 0 2 2
   IGPS 1 0 1
   TS 1 0 1
   Alkaloids biosynthetic pathway STR3 0 1 1
   DAT 0 1 1
   Anthocyanin biosynthetic pathway PAL 4 4 8
   TC4M 0 1 1
   STS 6 7 13
   4CL 1 1 2
   F3D 1 0 1
   FLSI 1 2 3
   DFR 0 2 2
   UFGT 0 1 1
   ANR 1 0 1
   Lignin biosynthetic pathway SOH 1 0 1
   CA3M 0 2 2
   COM 2 0 2
   CCR1 1 1 2
   CAD1 1 0 1
   POD 2 5 7
   LAC 1 3 4
   Terpenoid biosynthetic pathway HMGS 1 1 2
   DXPS 1 1 2
   IPI2 1 0 1
   TSE 1 0 1
   SED 0 2 2
   [97]Open in a new tab

   DAHPS3, 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase 03;
   B3D/SDH, bifunctional 3-dehydroquinate dehydratase/shikimate
   dehydrogenase; SHK, shikimate kinase; CS1, chorismate synthase 1;
   AnPRT, anthranilate phosphoribosyltransferase; IGPS, indole-3-glycerol
   phosphate synthase; TS, tryptophan synthase beta chain 1; STR3;
   strictosidine synthase 3; DAT, D-amino-acid transaminase; PAL,
   phenylalanine ammonia-lyase; TC4M, Trans-cinnamate 4-monooxygenase;
   STS, stilbene synthase; 4CL, 4-coumarate–CoA ligase; F3D, flavanone
   3-dioxygenase; FLS1, flavonol synthase/flavanone 3-hydroxylase; DFR,
   dihydroflavonol-4-reductase; UFGT; anthocyanidin
   3-O-glucosyltransferase 2; ANR, anthocyanidin reductase; SOH, shikimate
   O-hydroxycinnamoyltransferase; CA3M, caffeic acid
   3-O-methyltransferase; COM, caffeoyl-CoA O-methyltransferase; CCR1,
   cinnamoyl-CoA reductase 1; CAD1; cinnamyl alcohol dehydrogenase 1; POD;
   Peroxidase; LAC; laccase; HMGS, hydroxymethylglutaryl-CoA synthase;
   DXPS, 1-deoxy-D-xylulose-5-phosphate synthase; IPI2, isopentenyl
   diphosphate isomerase II; TSE, terpene synthase; SED, squalene
   epoxidase.

Figure 5.

   [98]Figure 5
   [99]Open in a new tab

   Differential expression of genes related to secondary metabolites under
   drought stress. DAHPS3, 3-deoxy-D-arabino-heptulosonate-7-phosphate
   synthase 03; B3D/SDH, bifunctional 3-dehydroquinate
   dehydratase/shikimate dehydrogenase; SHK, shikimate kinase; CS1,
   chorismate synthase 1; AnPRT, anthranilate phosphoribosyltransferase;
   IGPS, indole-3-glycerol phosphate synthase; TS, tryptophan synthase
   beta chain 1; STR3; strictosidine synthase 3; DAT, D-amino-acid
   transaminase; PAL, phenylalanine ammonia-lyase; TC4M, Trans-cinnamate
   4-monooxygenase; STS, stilbene synthase; 4CL, 4-coumarate–CoA ligase;
   F3D, flavanone 3-dioxygenase; FLS1, flavonol synthase/flavanone
   3-hydroxylase; DFR, dihydroflavonol-4-reductase; UFGT; anthocyanidin
   3-O-glucosyltransferase 2; ANR, anthocyanidin reductase; SOH, shikimate
   O-hydroxycinnamoyltransferase; CA3M, caffeic acid
   3-O-methyltransferase; COM, caffeoyl-CoA O-methyltransferase; CCR1,
   cinnamoyl-CoA reductase 1; CAD1; cinnamyl alcohol dehydrogenase 1; POD;
   Peroxidase; LAC; laccase.

   In grapevine transcriptome, 21 differentially expressed genes were
   identified in lignin biosynthesis, which were involved in drought
   stress. It includes; 9 up-regulated genes related to shikimate
   O-hydroxycinnamoyltransferase, aldehyde 5-hydroxylase, two caffeoyl-CoA
   O-methyltransferase, cinnamoyl-CoA reductase 1, cinnamyl alcohol
   dehydrogenase 1, two peroxidase and laccase; whereas, 12 DEGs were
   down-regulated including, two caffeic acid 3 O-methyltransferase, one
   cinnamoyl-CoA reductase 1, five peroxidases, three laccase transcripts.

   Further, eight genes were identified to be involved in terpenoid
   biosynthesis from which hydroxymethylglutaryl-CoA synthase,
   1-deoxy-D-xylulose 5-phosphate reductoisomerase, isopentenyl
   diphosphate isomerase II and terpene synthase were up-regulated, while
   hydroxymethylglutaryl-CoA synthase, 1-deoxy-D-xylulose-5-phosphate
   synthase and two squalene epoxidase were down-regulated in
   drought-stressed grapevine leaves (Table [100]4, Fig. [101]5,
   Supplementary Table [102]S10).

Heat shock protein (HSP) and pathogenesis-related protein (PR) in response to
drought stress

   The results revealed that 49 DEGs were identified in HSPs, including 1
   HSP101 (down-regulated), 3 HSP90 (1 up-regulated and 2 down-regulated),
   2 HSP70 (1 up-regulated, 1 down-regulated), 18 sHSPs (12 up-regulated
   and 6 down-regulated), 20 other HSP genes (17 up-regulated and 3
   down-regulated) and 5 heat-stress transcription factors (4 up-regulated
   and 1 down-regulated). The high molecular weight HSPs (HMW HSPs),
   including HSP90s and HSP70s were also found to be up-regulated in our
   findings. One up-regulated transcript of HSP70s was expressed at higher
   abundance level (VIT_17s0000g03310.t01; 650.81 to 532.18 RPKM),
   compared with other HMW HSPs. The up-regulated, VIT_16s0098g01060.t01
   (from 706.59 to 1.98 RPKM) from sHSPs and VIT_14s0060g01490.t01 (from
   363.93 to 355.88 RPKM) from other HSPs, expressed at moderate
   abundances, but remaining other HSPs, along with sHSP, and heat-stress
   transcription factors expressed at lower abundances (Table [103]5,
   Supplementary Table [104]S11).

Table 5.

   List of differentially-expressed genes related to heat-shock proteins
   (HSPs) and pathogens resistance (PRs) proteins in grapevine perceived
   during drought stress.
   Trait name Description No. of up-regulated No. of down-regulated sum
   Heat shock proteins HSP101 0 1 1
   HSP90 1 2 3
   HSP70 1 1 2
   small HSP 12 6 18
   other HSP 17 3 20
   heat-stress transcription factor 4 1 5
   PR-1 pathogenesis-related protein 2 4 6 10
   PR-2 Beta-1,3-glucanase 4 5 9
   PR-3, 4, 8, 11 chitinase 4 15 19
   PR-5 Thaumatin-like protein 6 8 14
   PR-10 Pathogenesis-related protein 10 2 2 4
   PR-14 lipid transfer protein 5 5 10
   PR-15 germin-like protein 2 2 2 4
   PTI transcriptional activator 2 2
   dirigent protein 3 7 10
   proline related protein 6 6 12
   [105]Open in a new tab

   In this study, 72 DEGs encoding pathogenesis-related proteins were
   identified, including 10 pathogenesis-related protein PR-1 (4
   up-regulated, 6 down-regulated), 9 Beta-1,3-glucanase (PR2; 4
   up-regulated, 5 down-regulated), 19 chitinase (4 up-regulated, 15
   down-regulated), 14 thaumatin-like protein (PR5; 6 up-regulated, 8
   down-regulated), 4 Pathogenesis-related protein-10 (2 up-regulated, 2
   down-regulated), 10 non-specific lipid-transfer protein (PR14; 5
   up-regulated, 5 down-regulated), 4 Germin-like protein 2 (2
   up-regulated, 2 down-regulated), and 2 pathogenesis-related
   transcription factors (2 down-regulated) to code disease resistance
   proteins. Conversely to HSPs, most of the PR showed down-regulation in
   grapevine leaves under drought stress. Moreover, 4 up-regulated
   transcripts, including PR1 (VIT_03s0088g00890.t01, |log[2]FC| = 8.75),
   chitinase (VIT_05s0094g00320.t01, |log[2]FC| = 8.29), thaumatin-like
   protein (VIT_02s0025g04290.t01, |log[2]FC| = 3.84) and
   Pathogenesis-related protein-10 (VIT_05s0077g01600.t01,
   |log[2]FC| = 8.31) were only expressed in treatment group.
   Additionally, 10 dirigent proteins (3 up, 7 down-regulated) and 12
   proline related proteins (6 up, 6 down-regulated) were also recorded
   from this study (Table [106]5, Supplementary Table [107]S11).

qRT-PCR validation of DEGs from Illumina RNA-Seq

   In order to investigate the accuracy and reproducibility, 16 DEGs were
   selected from RNA-Seq results for quantitative real-time PCR; these
   transcripts represent all the major up/down-regulated functions that
   were identified in our transcriptome data including, metabolism,
   hormone signaling, disease resistance and regulatory proteins. The gene
   function, primer sequence, RPKM, Log[2] values and qRT-PCR results are
   presented in Fig. [108]6; Supplementary Table [109]S12. The qRT-PCR
   findings of 16 (8 up-regulated and 8 down-regulated) selected genes
   were consistent with the RNA-seq results, revealing the accuracy and
   reliability of our RNA-seq results.

Figure 6.

   [110]Figure 6
   [111]Open in a new tab

   Verification of relative expression levels of DEGs by qRT-PCR. Error
   bars indicate standard deviation from 3 technical replicates of
   RT-qPCR. Expression patterns of 16 DEGs selected from different
   elucidated pathways by qRT-PCR (blue bar) and RNA-Seq (red dot). (1)
   Seq ID: VIT_07s0151g00110.t01 (Chlorophyllase-1), (2) Seq ID:
   VIT_00s0396g00010.t01 (psbC; Photosystem II CP43 chlorophyll apoprotein
   gene), (3) Seq ID: VIT_00s2608g00020.t01 (psbB; Photosystem II CP47
   chlorophyll apoprotein gene), (4) Seq ID: VIT_18s0001g08620.t01 (psaB;
   photosystem I P700 apoprotein A2 gene), (5) Seq ID:
   VIT_19s0027g01930.t01 (peroxiredoxin (Prx), (6) Seq ID:
   VIT_11s0037g00940.t01 (S-adenosylmethionine decarboxylase proenzyme),
   (7) Seq ID: VIT_12s0055g01020.t01 (peroxidase N1-like), (8) Seq ID:
   VIT_18s0001g08550.t01 (squalene monooxygenase), (9) Seq ID:
   VIT_06s0061g00790.t01 (Pheophorbide a oxygenase), (10) Seq ID:
   VIT_17s0000g06130.t01 (glutathione S-transferase U9), (11) Seq ID:
   VIT_04s0023g03230.t01 (auxin-induced protein 15A-like), (12) Seq ID:
   VIT_01s0146g00350.t01 (BRASSINOSTEROID INSENSITIVE 1-associated
   receptor kinase 1), (13) Seq ID: VIT_01s0011g00480.t01 (glutamate
   5-kinase), (14) Seq ID: VIT_07s0129g00460.t01 (prolyl 4-hydroxylase 9),
   (15) Seq ID: VIT_16s0039g01360.t01 (phenylalanine ammonia-lyase), (16)
   Seq ID: VIT_03s0088g00710.t01 (pathogenesis-related protein PR-1).

Discussion

   Drought stress can subdue the plant growth by hindering many
   physiological processes of plants. Chlorophylls (chls) are the
   principal light-absorbing pigments and key components of photosynthesis
   in plants. The physiological and transcriptomic studies of grapevine
   leaves responding to drought stress have revealed that chl contents
   were incredibly decreased, which in turn, inhibited the photosynthetic
   activity. Similarly, the reduction in chl contents has been reported in
   corn and chickpea in response to drought stress^[112]21,[113]22.
   Moreover, transcriptomic data demonstrated that drought stress
   suppressed the chl biosynthesis process by inhibiting the activity of
   key enzymes, such as HemA (Glutamyl-tRNA reductase 1) and CHLH
   (Magnesium chelatase H subunit), which play an essential role in chla
   synthesis process^[114]23. Furthermore, in the chl cycle, the
   oxygenation reactions of chlorophyll(ide) a to chlorophyll(ide) b were
   catalyzed by chlorophyllide a oxygenase (CAO)^[115]24, whose activity
   likewise was decreased under drought stress, suggesting the inhibition
   of chl cycle. In contrast, the chlorophyll(ide) b to a conversion is
   catalyzed by chlorophyll(ide) b reductase NYC1 (CBR) and its activity
   was up-regulated, suggesting that chl cycle process was also suppressed
   by the drought treatment^[116]25. Furthermore, PAO (pheophorbide a
   oxygenase) is regarded as a vital chl catabolic enzyme^[117]26,[118]27,
   and contributed well in chl deprivation process as its activity was
   enhanced under drought stress (Fig. [119]1 Supplementary
   Table [120]S5). Buchert^[121]28 and Du^[122]29 have demonstrated the
   key role of PAO as an important chl degradation enzyme investigated
   during senescence of broccoli and banana, respectively.

   Meantime, the photosynthetic activity, stomatal conductance, and CO[2]
   assimilation rate were significantly decreased in drought treated
   grapevine leaves as compared to control. Similar findings have also
   been reported in grapevine under Cu and drought
   stresses^[123]30,[124]31. Moreover, the photosynthesis-related genes,
   involved in PSII, PSI, cytochrome b6-f complex, photosynthetic electron
   transport, F-type ATPase and photosynthesis-antenna proteins were
   significantly down-regulated by drought stress, yet the extent of
   light-harvesting proteins (CP47, CP43), which binds the chla molecules
   was found down-regulated by drought stress (Supplementary
   Table [125]S6). Furthermore, PsaB is anticipated as the heart of PSI
   that binds P700 special chlorophyll pair^[126]32 was also
   down-regulated by drought stress in our findings. Finally, drought
   stress gradually decreased the activities of PSII electron transport
   and light-harvesting complex (photosynthesis-antenna proteins).
   Stomatal closure is known to reduce the CO[2] absorption which limits
   the photosynthetic activity in plants under drought stress
   environment^[127]33–[128]35. In the present study, the results of
   physiology and transcriptome analysis revealed that the decline in
   photosynthetic process was primarily connected with the chl
   degradation, which indicates that drought stress has direct influence
   on the photosynthesis process in plants.

   ROS is a universal response of the plants to counter environmental
   stress to prevent oxidative damage. Several studies have already been
   performed to investigate the importance of MDA under oxidative stress
   in different crops, such as wheat (Triticum aestivum) and oilseed rape
   (Brassica napus), proposed that MDA was accumulated by drought
   stress^[129]36–[130]38. On the contrary, plants have the ability to
   accrue different antioxidative enzymes to confer drought severity,
   while similar investigations in olive^[131]39 and wheat^[132]37 support
   our findings of enhanced activity of ROS enzymes against elevated
   levels of MDA. The transcriptomic investigation revealed that one NADPH
   oxidase and five amine oxidases were remarkably up-regulated, while
   both have a crucial role in the ROS synthesis and accumulation under
   various stress environments^[133]40. SODs are regarded as the first
   line of defense against ROS which has two isozymes Fe-SOD and Cu/Zn-SOD
   in plant chloroplast^[134]40. It is worth mentioning that Fe-SODs was
   up-regulated, but Cu/Zn-SODs was down-regulated, which is in agreement
   with our previous findings in grapevine under Cu stress
   conditions^[135]30. Other enzymes, including CAT, POD, GSH-AsA cycle,
   PPO, GST, AO, MDHAR, DHAR and GR also possess drought-responsive
   antioxidative defense system in grapevine^[136]41. Perhaps,
   non-enzymatic antioxidants, such as glutathione and proline also
   assisted in enhancing the ROS scavenging system in drought treated
   grapevine, which is consistent with the ROS scavenging system
   investigated in the V. vinifera and S. lycopersicum under drought
   stress^[137]42,[138]43. The observed higher antioxidant capacity,
   increased activity of ROS enzymes and enhanced expression of
   genes-related to ROS system seems to be a mechanism operative in plant
   tolerance to drought stress.

   Drought stress causes dehydration in plant cells. Plant hormones, such
   as abscisic acid, auxin, gibberellin, ethylene, jasmonic acid and
   brassinosteroid accumulate under dehydration conditions and play a
   major role in stress tolerance in plants^[139]44. In Arabidopsis, ABA
   activates the subclass III protein kinases of SnRK2 family, which
   further facilitate the regulation of stomatal conductance to optimize
   plant water status through guard cells^[140]45,[141]46, favor our
   findings of increased activity of SRK2I protein kinase in
   drought-treated grapevine. The activation of PP2C genes in grapevine
   responding to drought stress proposed that PP2C has its primary role in
   stress tolerance, especially in regulating stomatal responses to cope
   transpiration losses^[142]47. The AUX gene family includes early
   response AUX genes, Aux/IAA, GH3 and SAUR and the regulators of AUX
   genes, ARF, while their activities were down-regulated in our findings.
   ARFs are also the important abiotic stress-responsive genes and have
   their crucial role in physiological processes of fruit plants. Wang et
   al.^[143]44 investigated the AUX gene family in sorghum (Sorghum
   bicolor) and specified that most of these genes were induced by the
   exogenous application of IAA under drought stress conditions. Moreover,
   GA activity and the accumulation of DELLA proteins were up-regulated in
   drought treatment, while similar findings in Arabidopsis have suggested
   that DELLA proteins restrain the plant growth and promote survival
   under drought stress^[144]48. JA biosynthesis and signaling together
   with ABA and other hormones have been extensively studied in many
   crops. In current investigations, JA amino acid conjugate (JAR1) was
   up-regulated, while JAR1 are enduringly present in the plant leaves and
   together with ABA induce the stomatal closure under osmotic stress,
   have been extensively studied in Arabidopsis ^[145]49. Interestingly,
   jasmonate-zim domain proteins (JAZ) were significantly down-regulated,
   which was observed up-regulated in another study in rice^[146]50, may
   be because of prolonged duration of drought stress. Moreover, the
   activity of AOS and LOX were increased, which is similar to the
   findings of Leng et al.^[147]36 in V. vinifera. Ethylene is an
   important stress hormone because its synthesis is induced under
   different oxidative environments. Under drought stress, the synthesis
   of ethylene precursor 1-aminocyclopropane-1-carboxylate oxidase was
   up-regulated in grapevine, which can stimulate plant development and
   functioning by prompting the diffusion possibility of ABA to its active
   site^[148]51,[149]52. Furthermore, the expressions of the
   ethylene-related regulatory genes (ETR1 and CTR1) were increased in our
   findings, suggesting their functional role in ethylene biosynthesis as
   described by Schachtman and Goodger^[150]53. BRs are the only plant
   steroids, which elicit the expression of many genes, especially during
   stress environments. Brassinosteroid Insensitive 1 (BRI1) was
   up-regulated in our findings, which is known to play the key role in
   plant growth, morphogenesis and response to drought stress. Feng, et
   al.^[151]54 created RNAi mutants for bdBRI1 in Brachypodium distachyon
   and suggested that this gene produces a dwarf phenotype with enhanced
   tolerance towards drought stress. BR signal transduction, from cell
   surface perception to activation of specific nuclear genes will be
   interesting to investigate in the future.

   Plants cope with environmental stress by the accumulation of certain
   compatible osmolytes, such as proline, which is known to induce
   drought-tolerance in plants^[152]55 and up-regulation of all the genes
   related to proline metabolism is the clear evidence of induced
   grapevine tolerance in our study. Proline biosynthesis commenced with
   the phosphorylation of glutamate, which then converted into
   gulatamic-ϒ-semialdehyde by Pyroline-5-carboxylate synthetase
   (up-regulated). Similarly, arginine is converted into orthinine by
   arginase (up-regulated) and then into GSA by the
   ornithine-δ-aminotransferase (not-detected). GSA is then converted into
   pyrroline 5-carboxylate (P5C) by spontaneous cyclization. Finally,
   proline is synthesized from the P5C by P5C reductase (P5CR)
   enzyme^[153]55,[154]56. In proline degradation pathway, proline is
   re-converted into P5C by Proline dehydrogenase (PDH; up-regulated) and
   then into glutamate by Pyrroline-5-carboxylate dehydrogenase (P5CDH;
   up-regulated). Thus PDH and P5CDH are believed to be most important
   enzymes in proline degradation to glutamate^[155]57,[156]58. Hence,
   proline metabolism may regulate the gene expression during drought
   stress.

   In higher plants, accumulation of various secondary metabolites, such
   as amino acids, carbohydrates and lipids occur when a plant is
   subjected to environmental stress^[157]59. Shikimate pathway not only
   acts as a bridge between central and secondary metabolism but also
   serves as a precursor for other secondary metabolites^[158]60.
   Additionally, Tyr is a precursor of IAA and initiate the synthesis of
   indole alkaloids and isoquinoline alkaloids, which prevent plants from
   oxidative stress^[159]61. Phe is considered as the precursor of
   secondary metabolites family, and PAL participates in phenylpropanoid
   biosynthesis; a key step towards biosynthesis of stilbenes, flavonoids,
   lignins and various other compounds^[160]62. STS (stilbene synthase)
   catalyzes the initial step of flavonoid biosynthesis pathway, which has
   the protective function during drought stress^[161]63. Overall, 4 PAL
   and 6 STS were significantly up-regulated in our findings, proposing
   the innate link with drought stress. The respective, up and
   down-regulation of 1-deoxy-D-xylulose 5-phosphate reductoisomerase and
   1-deoxy-D-xylulose-5-phosphate synthase can act as a rate limiting
   enzymes in MEP pathway, also found in Cu-stressed grapevine
   leaves^[162]30. Dimethylallyl diphosphate and isopentenyl diphosphate
   are the universal 5 carbon precursors found in terpenoid synthesis. It
   has been reported that one isopentenyl-diphosphate isomerase II can
   catalyze isopentenyl diphosphate to form dimethylallyl diphosphate and
   one terpene synthase^[163]64,[164]65, while both were up-regulated in
   our findings. The down-regulation of most of the genes related to
   anthocyanin, lignin and terpenoid biosynthesis have elucidated the
   negative role of drought stress on an accumulation of secondary
   metabolites in grapevine leaves.

   HSPs are ubiquitous stress-related proteins that act as molecular
   chaperone, HSP members participate in the protein synthesis, folding,
   aggregation and transportation from the cytoplasm to different
   intracellular compartments^[165]66,[166]67. In the present study,
   majority of HSP genes were up-regulated (35), however genes related to
   sHSP along with heat stress transcription factors and other HSP were
   found to be predominant in their expression, in contrast, it was noted
   that drought has also significantly suppressed few genes (14) related
   to HSP. Heat-shock protein 70 (Hsp70) proteins are one of the large
   families of highly conserved molecular chaperones and are extensively
   found in almost all organisms. HSP70 is one of the highly conserved
   protein, known to induce during environmental stress, for example, in
   Erianthus arundinaceus, HSP70 was expressed 7-fold higher under
   drought-stressed condition^[167]68. sHSPs are plant tolerant proteins
   generally induced upon abiotic stress^[168]69. In the same way, Vasquez
   reported that expression of HSP70 and sHSP was higher during stress
   conditions in pine tree, indicating their major role in mitigating the
   drought stress^[169]70. Therefore, transcriptional analysis of the
   present study revealed that HSP also play a fundamental role in defense
   mechanism of grapevine during drought stress. In addition,
   pathogenesis-related (PR) proteins are derived from plant allergens and
   act as defense-responsive proteins by increasing their expression under
   pathogen attack and variable stress environments. Depending on the
   functions and properties, PR-proteins are classified into 17 families,
   such as beta-1,3-glucanases, chitinases, thaumatin-like proteins,
   peroxidases, small proteins (defensins and thionins) and lipid transfer
   proteins (LTPs)^[170]11,[171]71. Most of the PR-proteins were
   down-regulated in our study, suggesting that drought stress posed a
   negative effect on PR-proteins defense response. Contrarily, most of
   the genes related to dirigent-proteins (DIR), play a role in lignin
   formation and proline-related proteins were up-regulated, suggesting
   their possible defensive role in grapevine in response to drought
   stress.

Conclusion

   Our results have provided substantial shreds of evidence to demonstrate
   that grapevine adaptation to drought stress is a multi-step component
   system consisting of several genes that regulate various pathways. Out
   of 12,451 DEGs, 8021 transcripts were up-regulated and 4,430
   transcripts were down-regulated. Nearly 2 fold up-regulations of DEGs
   have clearly indicated their defense-related role in grapevine
   responding to drought stress. The significant increase in the activity
   of ROS enzymes and hormones level revealed the defensive function of
   these enzymes and hormones during drought stress in grapevine leaves.
   Transcription analysis unveils that drought has affected the overall
   physiology of the grapevine; however, the regulatory mechanism of
   certain key genes like chlorophyll synthesis, ROS system, HSPs and
   other defense-related pathways assist grapevine in mitigating drought
   severity.

Materials and Methods

Plant material and drought treatments

   Two-year old grapevine (V. vinifera cv. ‘Summer Black’) pot grown
   plants were selected as experimental material which were grown in
   standard greenhouse condition (25 ± 5 °C) under 16-h light/8-h dark
   photoperiod and 65% relative humidity (RH) at the Nanjing Agricultural
   University-Nanjing, China. Grapevine plants were subjected to drought
   with an interval of 20 days against control, each with three biological
   replicates. The fourth unfolded leaf from the shoot apex was collected
   from each replicates of both control and drought treatment with the
   interval of 0 and 20th day, respectively, and the three samples were
   mixed to make one composite sample. After harvesting, the samples were
   immediately put in liquid nitrogen and then stored at −80 °C until
   analysis.

Determination of important biochemistry and physiology-related traits

   The chlorophyll a and b contents was determined using spectrophotometer
   at 663 and 645 nm, respectively as briefly explained by Leng, et
   al.^[172]26. Photosynthesis activity, stomatal conductance and CO[2]
   assimilation rate were carried out on mature leaf between 4^th to 7^th
   nodes from the shoot base for both control and drought treatment;
   between 9:00–11:00 AM measured using LI-COR (LI-6400XT, Germany) meter
   as described by Tombesi et al. (2015). Malondialdehyde (MDA) contents
   were quantified by using thiobartiburic acid. The activities of
   antioxidant enzymes (SOD, POD and CAT) were measured using the method
   briefly described by Haider, et al.^[173]72. The activities of
   indole-acetic acid (IAA), abscisic acid (ABA), jasmonic acid (JA),
   gibberellic acid (GA) and brassinosteroid (BR) were measured following
   the method of Tombesi, et al.^[174]31. Three technical repeats were
   generated for all the quantifications. Data was subjected to one-way
   analysis of variance (ANOVA) at p < 0.05, using MINITAB (ver. 16) and
   represented as mean ± standard deviation (SD).

RNA extraction, cDNA library construction and Illumina deep sequencing

   Total RNA from leaf samples of both control and drought-stressed were
   extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) (1%
   agarose gel buffered by Tris–acetate-EDTA was run to indicate the
   integrity of the RNA.) and subsequently used for mRNA purification and
   library construction with the Ultra™ RNA Library Prep Kit for Illumina
   (NEB, USA) following the manufacturer’s instructions. The samples were
   sequenced on an Illumina HiseqTM2500 for 48 h.

Analysis of gene expression level, gene ontology (GO) and Kyoto encyclopedia
of genes and genomics (KEGG)

   After adaptor trimming and quality trimming, the clean reads were
   mapped to the V. vinifera transcriptome using Bowtie 1.1.2. Then, Sam
   tools and BamIndexStats.jar were used to calculate the gene expression
   level, and reads per kilobase per million (RPKM) value was computed
   from SAM files^[175]73. Gene expression differences between log[2] and
   early stationary phase were obtained by MARS (MA-plot-based method with
   Random Sampling model), a package from DEGseq. 3.3 (Leng et al., 2015).
   We simply defined genes with at least 2-fold change between two samples
   and FDR (false discovery rate) less than 0.001 as differential
   expressed genes. Transcripts with |log2FC| < 1 were assumed to have no
   change in their expression levels. The gene ontology (GO) enrichment
   (p-value < 0.05) was investigated by subjecting all DEGs to GO database
   ([176]http://www.geneontology.org/) in order to further classify genes
   or their products into terms (molecular function, biological process
   and cellular component) helpful in understanding genes biological
   functions. Kyoto encyclopedia of genes and genomics (KEGG; the major
   public pathway-related database) was used to perform pathway enrichment
   analysis of DEGs^[177]74.

Illumina RNA-seq results validation by qRT-PCR

   In order to validate the Illumina RNA-seq results, drought-stressed
   grapevine leaf samples of each collection were applied to qRT-PCR
   analysis. Total RNA of the collected samples was extracted following
   the above mentioned method, and then was reverse-transcribed using the
   PrimeScript RT Reagent Kit with gDNA Eraser (Takara, Dalian, China),
   following the manufacturers’ protocol. Gene specific qRT-PCR primers
   were designed using Primer3 software ([178]http://primer3.ut.ee/), for
   20 selected genes with the sequence data in 3′UTR (Table [179]S12).
   qRT-PCR was carried out using an ABI PRISM 7500 real-time PCR system
   (Applied Biosystems, USA). Each reaction contains 10 µl 2 × SYBR Green
   Master Mix Reagent (Applied Biosystems, USA), 2.0 µl cDNA sample, and
   400 nM of gene-specific primer in a final volume of 20 µl. PCR
   conditions were 2 min at 95 °C, followed by 40 cycles of heating at
   95 °C for 10 s and annealing at 60 °C for 40 s. A template-free control
   for each primer pair was set for each cycle. All PCR reactions were
   normalized using the Ct value corresponding to the Grapevine UBI gene.
   Three biological replicates were generated and three measurements were
   performed on each replicate.

Electronic supplementary material

   [180]Supplementary: Figure S1^ (3.3MB, xls)
   [181]Supplementary: Table S1^ (19KB, xls)
   [182]Supplementary: Table S2^ (2.6MB, xls)
   [183]Supplementary: Table S3^ (26.5KB, xls)
   [184]Supplementary: Table S4^ (56KB, xls)
   [185]Supplementary: Table S5^ (24.5KB, xls)
   [186]Supplementary: Table S6^ (27.5KB, xls)
   [187]Supplementary: Table S7^ (33.5KB, xls)
   [188]Supplementary: Table S8^ (54KB, xls)
   [189]Supplementary: Table S9^ (33KB, xls)
   [190]Supplementary Table S10^ (33.5KB, xls)
   [191]Supplementary Table S11^ (51.5KB, xls)
   [192]Supplementary Table S12^ (23.5KB, xls)

Acknowledgements