Wheat (Triticum aestivum L.) is one of the most widely grown crop and essential for global food security, and its planting area is determined by the developmental characteristics of wheat cultivars (Reynolds et al., 2011). Vernalization is a crucial phase and also a complicated process regulated by many genes in wheat development process. Until now, four genes (VRN1, VRN2, VRN3 and VRN-D4) have been cloned (Distelfeld et al., 2009; Kippes et al., 2014; Jin and Wei, 2016; Muterko et al., 2016). VRN1 encodes a MADS-box transcriptional factor and the dominant VRN-A1 allele exhibits spring growth habit (Tanaka et al., 2018). VRN2, including two repeated genes ZCCT1 and ZCCT2 that encode proteins carrying putative a zine finger and a CCT domain, acts as long day repressors of flowering and is down-regulated by vernalization (Kippes et al., 2016). VRN3 is highly similar to Arabidopsis protein flowering locus t (FT) and also a flowering promoting gene (Jin and Wei, 2016). According to the current research results of VRN genes, the genotype between spring and winter wheat variety can be distinguished. Yuan reported that the genotypes among semi-winter, winter and strong winter were not distinguished by the allelic composition of VRN1. For example, the genotype of YM49-198 (semi-winter), J841 (winter), FM (strong winter) was van-A1, vrn-B1, vrn-D1, respectively (Yuan et al., 2008), so it is necessary to further explore new genes or molecular markers related to vernalization (Huang et al., 2018).

Plants have evolved multiple physiological and biochemical strategies at gene or protein level to adapt unfavorable environmental state during developmental process. Under low-temperature, multiple genes are induced and their expression products include both regulatory and functional proteins (Kosova et al., 2014). These proteins directly participate in the resistance to low-temperature, such as dehydrin (DHN) (Hossain et al., 2013). Dehydrin, also was known as LEA II protein, which was composed of approximately 82-575 amino acids. The dehydrin protein typically has a conserved lysine-rich K fragment composed of 15 amino acids (EKKGIMDKIKEKLPG), S fragment composed of a series of serine residues and Y fragment composed of conservative motifs T/VDEYGNP (Banerjee and Roychoudhury, 2016; Hill et al., 2016). According to the difference in amino acid sequence of Y, S and K fragments, dehydrin proteins also can be divided into 5 categories: Kn, SKn, KnS, YsKn and YnSK2 (Allagulova et al., 2003). Among them, SKn, KnS and YsKn are acidic proteins, YnSK2 is a neutral and basic protein, Kn is an acidic and neutral protein (Shekhawat et al., 2011; Wang et al., 2014). YnSK2 can be induced by drought and Abscisic acid (ABA) (Suprunova et al., 2004; Binott et al., 2017); SKn can be induced by low temperature, but it is less expressed under drought and ABA (Fowler et al., 2001), Kn, KnS and YsKn can be induced by low temperature (Ismail et al., 1999; Kosova et al., 2010; Archambault and Stromvik, 2012). Therefore, the different conserved sequences of dehydrin determine its acidity and alkalinity, and further determine its function.

With the rapid application of high-throughput sequencing technologies, many genes have been isolated and cloned from wheat. But how to validate gene functions has become a bottleneck because of the lower genetic transformation efficiency comparing with Arabidopsis and rice, due to its complicated and huge genome and polyploidy characteristics. Nowadays, Virus-induced gene silencing (VIGS) has been developed as an effective transgenic technology for determining gene functions in dicot and monocot cereals (Ma et al., 2012), which only partial gene sequence is sufficient for silencing gene (Senthil-Kumar and Mysore, 2011). The function of hundreds of plant genes involved in defense response pathways, plant development, and metabolism have been identified by VIGS (Guo et al., 2010; Rivas et al., 2014).

We had conducted a high-throughput transcriptome sequencing analysis using winter wheat cultivar Jing841 (J841) and spring wheat cultivar Liaochun10 (LC10) as experimental materials under vernalization and non-vernalization treatments (Feng et al., 2016), and obtained many differentially expressed genes; among them, a differentially expressed candidate Unigene1312 encoding Dehydrin protein was selected and temporarily named Tadhn1312. Expression analysis showed that Tadhn1312 was expressed in LC10 and J841, but the expression level in J841 was higher than that in LC10 under vernalization treatment, indicating that the gene was responsive to vernalization treatment. In the present study, we employed the Barley Stripe Mosaic Virus (BSMV) based VIGS system for the primary function verification of Tadhn1312 gene

1 Results and Analysis

1.1 Phenotypes of wheat leaves after the inoculation with BSMV Virus

Virus-Induced Gene Silencing is a powerful reverse genetics approach for knocking down target genes expression to study their functions and is widely used in the regulation of plant metabolism, growth, and development (Guo et al., 2010). A 242 bp coding sequence of Tadhn1312 gene was amplified and used to construct the BSMV vector, and designated as BSMV: Tadhn1312 (Figure 1). When wheat plants were at two-leaf stage, BSMV: Tadhn1312 was used to inoculate wheat leaves, BSMV: TaPDS as the positive control and BSMV: 00 as the negative control.

The second leaves of wheat plants appeared virus symptom and chlorotic phenotype at 7 days after inoculated with BSMV: TaPDS and BSMV: Tadhn1312; and the obvious photo bleaching phenotype were shown at 14 days, indicating that the virus had successfully infected wheat leaves. At 21 days, the third leaves showed radial banded white spots or the entire leaves showed photo bleaching (Figure 2A). As time went by, the phenotype gradually subsided, but the young leaves showed mottled chlorotic phenotype at 28 days. Obvious bleaching lasted for 35 days after inoculation. The leaves of negative control BSMV: 00 had no bleaching phenotype. Among 50 wheat leaves infected by BSMV: TaPDS and BSMV: Tadhn1312, respectively, 32 and 29 leaves showed chlorosis with silencing efficiencies of 64% and 58% and reached the significant level (Figure 2B), indicating that the VIGS system is effective.

1.2 Chlorophyll content

Chlorophyll content was measured every 7 days after the inoculation. The result showed that the chlorophyll content of leaves with BSMV: TaPDS inoculation decreased at 7 days, and reached the minimum level at 21 days, accounted for 43.8% of BSMV: 00, the difference was extremely significant. At 28 d, the chlorophyll content gradually increased, but was still lower than that of negative controls BSMV: 00 (Figure 3A).

1.3 Transcription level of target gene after BSMV recombinant vector inoculation

qRT-PCR was used to measure the Tadhn1312 and TaPDS transcript abundances at different time after inoculation. The relative expression level of TaPDS rapidly decreased at 7 days, and continued to decrease and reached the lowest level at 21 days. At 28 days, the transcript level TaPDS gradually increased, and recovered to about half of the initial level at 35 days, indicating that the expression of TaPDS gene had been significantly inhibited. The expression of target gene Tadhn1312 exhibited similar expression trend as TaPDS after BSMV virus inoculation. The transcript level of Tadhn1312 was rapidly decreased at 7 days, and reached the minimum value at 21 days, accounting for 27% of that of negative control and reached the significant level, suggesting that the target gene Tadhn1312 was inhibited successfully (Figure 3B).

1.4 Spike differentiation Observation after Inoculation with BSMV: Tadhn1312

Wheat spike differentiation is affected by various factors, including cultivar, illumination and temperature. The double ridge stage is an important indicator for passing through vernalization process (Baloch et al., 2003). The spike differentiation of negative control plants (BSMV: 00) and BSMV: Tadhn1312 remain single ridge stage at 14 d after BSMV virus inoculation. At 21 days, the spike differentiation of negative control plants (BSMV: 00) entered the early double ridge stage, while the spike differentiation of plants inoculated with BSMV: Tadhn1312 still remained in single ridge stage. When the spike differentiation of plants inoculated with BSMV: Tadhn1312 entered into the early double ridge stage at 28 days, the spike differentiation of control plants inoculated with BSMV: 00 has been in the middle of the double edges (Figure 4). The result showed that the silencing of Tadhn1312 prolonged the spike differentiation process, illustrating Tadhn1312 gene was involved in the spike differentiation process of wheat directly or indirectly.

2 Discussion

As a hydrophilic protein with high thermal stability, dehydrin widely exists in plants and can protect intra-cellular proteins and membrane structure from damage (Abedini et al., 2017). In recent years, dehydrin has attracted much attention due to its protective effect on plants under abiotic stress (Ramakrishna and Gokare, 2011; Yu and Yang, 2016). The expression of CaDHN1 from pepper was markedly upregulated in response to cold, salt, osmotic stresses and salicylic acid (SA) treatment, while the silencing of CaDHN1 using the virus-induced gene silencing (VIGS) technique led to decreased tolerance to cold, salt and osmotic induced stresses (Chen et al., 2015). In another work, seven dehydrin genes were up-regulated under low-temperature stress in loquat, and the expression of dehydrin gene in the cultivars with strong cold-resistance was greater compared to those with weak cold resistance (Xu et al., 2014). The transgenic Arabidopsis plants with over-expressed IpDHN gene showed a significant enhancement in tolerance to salt/drought stresses, less accumulation of hydrogen peroxide (H₂O₂) and the superoxide radical (O-2(-)), accompanied by increasing activity of the antioxidant enzyme system in vivo (Zhang et al., 2018). There are many reports about dehydrin related to abiotic or biotic stress (Guo et al., 2000; Sun et al., 2000; Kumar et al., 2014), while there are fewer literatures about dehydrin related to wheat development. Sequencing analysis showed that Tadhn1312 was highly similar with T.durum Dehydrin mRNA pTd38 and TaCOR80, with 99% and 96% identity, respectively. Kobayashi et al. reported that COR genes were up-regulated and the COR proteins were accumulated more in winter cultivar than spring one in the cold acclimation process (Kobayashi et al., 2005).

Vernalization is a periodic characteristic, and the vernalization treatment duration and optimum temperature of wheat varieties with different developmental patterns are different. Yin et al. reported that spring wheat cultivars could accomplish the transition from the vegetative to the reproductive phase under non-vernalization condition and finish the spike differentiation process; the spike differentiation was still at the single ridge stage in winter wheat cultivar BJ No.10 and semi-winter cultivar ZM 9405 under non-vernalization after sowing 62-d, 48 d, respectively, indicating that winter wheat and semi-winter cultivars could flower and head normally only after vernalization treatment (Yin et al., 2017). The VIGS technique is conducted to silence target genes by using the plant’s RNAi-mediated antiviral defense mechanism (Lee et al., 2017). After constructing recombinant BSMV infected wheat leaves in this study, the transcript level of the target gene decreased sharply, imaging that BSMV had successfully silenced the endogenous gene. The spike differentiation process caused by silencing of the Tadhn1312 gene was later than that of the negative control, indicating that Tadhn1312 has a promoting effect on wheat developmental process. While the silencing duration caused by VIGS can only be maintained for approximately 30 days, which may bring difficulties for long-term observation of plant phenotype, but VIGS technique still has incomparable advantages than other gene function research methods.

3 Materials and Methods

3.1 Plant materials and treatment

The spring wheat cultivar Liaochun10 (LC10) was used in this study, provided by the National Engineering Research Center for Wheat. Plump seeds were placed on moist filter paper in petri dishes and placed in the greenhouse at (25±2)°C with 16 h/8 h day/night photoperiod cycles and photon flux density of approximately 300µmol/ (m^-2·s^-1) and 60% relative humidity. After germination, seeds were placed in the vernalization box for 30 days and then put into pots containing sterilized vermiculite, the growth conditions were the same as above. The seedlings with consistent growth state were selected at the two-leaf stage for inoculation; leaf samples were picked every 7 days after inoculation and immediately frozen in the liquid nitrogen.

3.2 BSMV Vector construction, in-vitro transcription and inoculation

A conserved cDNA fragment 242 bp of Tadhn1312 was amplified and used to construct the BSMV recombinant vector. The BSMV vectors, including a, b and g tripartite genome, were transcribed into RNA in vitro by T7 RNA polymerase. Equal amount of the three RNA was inoculated into wheat leaves. Target genes were cloned into BSMV-g through the NheI restriction site. TaPDS is essential in the carotenoid pigment biosynthetic pathway and the suppression of its activity results in photolysis of chlorophyll, also referred to as photobleaching in the affected tissues.

The recombinant plasmids of BSMV:00, BSMV:TaPDS, BSMV:Tadhn1312, and BSMV-α were linearized with Mlu I digestion, the BSMV- β backbone was linearized via Spe I, at 37°C for 4 h. The linearizing products were purified and transcribed in vitro using the RiboMAX™ Large Scale RNA Production System-T7 kit (Promega, USA) as the manufacturer’s instructions. The RNA-α, RNA-β, and RNA-γ (or its derivative) transcripts were mixed in a 1:1:1 ratio, and diluted with nine volumes of DEPC water. In addition, 12 volumes of 2 × GKP buffer were added to the diluted transcript mixture for subsequent inoculation. Plants with consistent growth were chosen for virus inoculation at the two-leaf stage using 10 mL transcript mixtures per leaf. After inoculation, BSMV-inoculated leaves were fog-sprayed with RNase-free water and covered with plastic film for 3 days and cultured in an illumination incubator (16 h light/8 h dark photoperiod at 25°C). The control plants BSMV: 00 and BSMV: TaPDS were used as negative control and positive control, respectively (Wang et al., 2010).

3.3 Determination of chlorophyll content

Chlorophyll meter SPAD-502 (Konica Minolta sensing Inc., Japan) was used to determine the chlorophyll content of wheat leaves. Two leaves were measured per plant. Each leaf was measured 5 to 6 times from the leaf base to the tip and 10 samples were randomly selected for each treatment.

3.4 RNA extraction and reverse transcription

Total RNA was extracted from wheat leaves using RNAiso Plus kit (TaKaRa, Dalian, China), solubilized with DEPC-treated ddH₂O and stored at -80°C. The integrity of the RNA was assessed by 1.0% agarose gel electrophoresis. The first strand of cDNA was synthesized using the Prime Script™ RT reagent Kit with gDNA Eraser (TaKaRa, Dalian, China) as the manufacturer’s instructions.

3.5 Quantitative Real-time PCR (qRT-PCR)

qRT-PCR was performed using the first-strand cDNA as template using 2×SYBRRPremix Ex Taq^TM (TaKaRa, Dalian China). The reaction contained 10 µL of 2 × SYBR, 10 µL of Premix Ex Taq TMII, 1 µL of cDNA template, 2 µL of real-time PCR primers (10 µM) ( Table 1), and ddH₂O supplemented to 20 µL. Reaction conditions were as follows: 95°C for 30 s; 95°C for 5 s and 60°C for 30 s, circulating 40 times. Fluorescence signals were collected during the reaction steps at 60°C; 3 repeat samples were set for each sample to correct the copy number of PCR templates. β-actin (GenBank accession no. AB181991) was used as internal control (Liu et al., 2016). The 2^-ΔΔCt method used to calculate relative changes in gene expression determined from qRT-PCR experiments (Livak and Schmittgen, 2016). Each value is the mean±standard deviation of three independent biological replicates. Asterisks indicate significant differences (p<0.05). BSMV: 00 and BSMV: TaPDS were used as a negative control and a positive control, respectively.

3.6 Observing the process of spike differentiation

Spike differentiation of inoculated and control wheat plants was observed at 14 days, 21 days and 28 days after virus inoculation and three samples were observed for each time. The spike differentiation stage for each main shoot was determined using a binocular stereoscopic microscope (OLYMPUS SZX12) with magnification of 40×. All primers used in the study are listed in Table 1.

3.7 Statistical analysis

All data were subjected to analysis of variance using the SAS 9.3 program. And the significant difference among group means tested by using Duncan’s multiple range tests at 0.05 (p < 0.05) level.

Authors’ contributions

The work presented here was carried out in collaboration between all authors. ZPY , WTC and WL defined the research theme and co-designed experiments, and discussed analyses. ZPY designed methods, experiments, and wrote this paper. ZPY and QX carried out the laboratory experiments and analyzed the data. WGR worked on experiments design and obtaining test data. WL provide financial support. All authors read and approved the final manuscript.

Acknowledgments

This study was supported by Henan Scientific and Technological Research Program (182102111101).

References

Abedini R., Golmohammadi F.G., PishkamRad R., Pourabed E., Jafarnezhad A., Shobbar Z.S., and Shahbazi M., 2017, Plant dehydrins: shedding light on structure and expression patterns of dehydrin gene family in barley,Journal of Plant Research.,130: 747-763.

https://doi.org/10.1007/s10265-017-0941-5
PMid:28389925

Allagulova C.R., Gimalov F.R., Shakirova F.M., and Vakhitov V.A., 2003, The plant dehydrins: Structure and putative functions, Biochemistry-Moccow., 68: 945-951
https://doi.org/10.1023/A:1026077825584
PMid:14606934

Archambault A., and Stromvik M.V., 2012, The Y-segment of novel cold dehydrin genes is conserved and codons in the PR-10 genes are under positive selection in Oxytropis (Fabaceae) from contrasting climates, Mol Genet Genomics., 287: 123-142
https://doi.org/10.1007/s00438-011-0664-6
PMid:22183143

Baloch D.M., Karow R.S., Marx E., Kling J.G., and Witt M.D., 2003, Vernalization studies with Pacific Northwest wheat, Agron J., 95: 1201-1208
https://doi.org/10.2134/agronj2003.1201

Banerjee A., and Roychoudhury A., 2016, Group II late embryogenesis abundant (LEA) proteins: structural and functional aspects in plant abiotic stress, Plant Growth Regul., 79: 1-17
https://doi.org/10.1007/s10725-015-0113-3

Binott J.J., Owuoche J.O., and Bartels D., 2017, Physiological and molecular characterization of Kenyan barley (Hordeum vulgare L.) seedlings for salinity and drought tolerance, Euphytica., 213: 7
https://doi.org/10.1007/s10681-017-1924-2

Chen R.G., Jing H., Guo W.L., Wang S.B., Ma F., Pan B.G., and Gong Z.H., 2015, Silencing of dehydrin CaDHN1 diminishes tolerance to multiple abiotic stresses in Capsicum annuum L.Plant cell reports., 34: 2189-2200.
https://doi.org/10.1007/s00299-015-1862-1
PMid:26408144

Distelfeld A., Tranquilli G., Li C.X.,Yan L.L., and Dubcovsky J., 2009, Genetic and Molecular Characterization of the VRN2 Loci in Tetraploid Wheat, Plant physiol., 149: 245-257
https://doi.org/10.1104/pp.108.129353
PMid:19005084 PMCid:PMC2613703

Feng Y.L,, Zhao Y.Y., Wang K.T., LI Y.C., Wang X., and Yin J., 2016, Identification of vernalization responsive genes in the winter wheat cultivar Jing841 by transcriptome sequencing, Journal of Genetics., 95(4): 957-964
https://doi.org/10.1007/s12041-016-0724-0
PMid:27994195

Fowler D.B., Breton G., Limin A.E., Mahfoozi S., and Sarhan F., 2001, Photoperiod and temperature interactions regulate low-temperature-induced gene expression in barley, Plant Physiol., 127: 1676-1681
https://doi.org/10.1104/pp.010483
PMid:11743112 PMCid:PMC133572

Guo X.Y., Zhang L., Zhu J.B., Liu H.L., and Wang A.Y., 2000, Cloning and characterization of SiDHN, a novel dehydrin gene from Saussurea involucrata Kar. et Kir. that enhances cold and drought tolerance in tobacco, PLANT Sci., 256: 160-169
https://doi.org/10.1016/j.plantsci.2016.12.007
PMid:28167030

Guo Y.S., Huang C.J., Xie Y., Song F.M., and Zhou X.P., 2010, A tomato glutaredoxin gene SlGRX1 regulates plant responses to oxidative, drought and salt stresses, Planta., 232: 1499-1509
https://doi.org/10.1007/s00425-010-1271-1
PMid:20862491

Hill W., Jin X.L., and Zhang X.H., 2016, Expression of an arctic chickweed dehydrin, CarDHN, enhances tolerance to abiotic stress in tobacco plants, Plant Growth Regul., 80:323-334
https://doi.org/10.1007/s10725-016-0169-8

Hossain Z., Khatoon A., and Komatsu S., 2013, Soybean proteomics for unraveling abiotic stress response mechanism, J Proteome Res., 12: 4670-4684
https://doi.org/10.1021/pr400604b
PMid:24016329

Huang M., Mheni N., Brown-Guedira G., McKendry A., Griffey C., Van S.,anford D., Costa J., and Sneller C., 2018, Genetic analysis of heading date in winter and spring wheat, Euphytica., 214: 128
https://doi.org/10.1007/s10681-018-2199-y

Ismail A.M., Hall A.E., and Close T.J., 1999, Allelic variation of a dehydrin gene cosegregates with chilling tolerance during seedling emergence, P Natl Acad Sci USA, 96: 13566-13570
https://doi.org/10.1073/pnas.96.23.13566
PMid:10557361 PMCid:PMC23988

Jin F.F., and Wei L., 2016,The Expression Patterns of Three VRN Genes in Common Wheat (Triticum aestivum L.) in Response to Vernalization, Cereal Res Commun., 44: 1-12
https://doi.org/10.1556/0806.43.2015.041

Kippes N., Zhu J., Chen A., Vanzetti L., Lukaszewski A., Nishida H., Kato K.., Dvorak J., and Dubcovsky J., 2014, Fine mapping and epistatic interactions of the vernalization gene VRN-D4 in hexaploid wheat, Mol Genet Genomics., 289: 47-62
https://doi.org/10.1007/s00438-013-0788-y
PMid:24213553 PMCid:PMC3916708

Kippes N., Chen A., Zhang X.Q., Lukaszewski A.J., and Dubcovsky J., 2016, Development and characterization of a spring hexaploid wheat line with no functional VRN2 genes, Theor Appl Genet., 129: 1417-1428
https://doi.org/10.1007/s00122-016-2713-3
PMid:27112150 PMCid:PMC4909811

Kobayashi F., Takumi S., Kume S., Ishibashi M., Ohno R., Murai K., and Nakamura C., 2005, Regulation by Vrn-1/Fr-1 chromosomal intervals of CBF-mediated Cor/Lea gene expression and freezing tolerance in common wheat, J Exp Bot., 56: 887-895
https://doi.org/10.1093/jxb/eri081
PMid:15668223

Kosova K., Prasil I.T., Prasilova P., Vitamvas P., and Chrpova J., 2010, The development of frost tolerance and DHN5 protein accumulation in barley (Hordeum vulgare) doubled haploid lines derived from Atlas 68 x Igri cross during cold acclimation, J Plant Physiol., 167: 343-350
https://doi.org/10.1016/j.jplph.2009.09.020
PMid:19962784

Kosova K., Vitamvas P., and Prasil I.T., 2014, Proteomics of stress responses in wheat and barley-search for potential protein markers of stress tolerance, Front Plant Sci., 5: 711
https://doi.org/10.3389/fpls.2014.00711
PMid:25566285 PMCid:PMC4263075

Kumar M., Lee S.C., Kim J.Y., Kim S.J., Aye S.S., and Kim S.R., 2014, Over-expression of Dehydrin Gene, OsDhn1, Improves Drought and Salt Stress Tolerance through Scavenging of Reactive Oxygen Species in Rice (Oryza sativa L.), J Plant Biol., 57: 383-393
https://doi.org/10.1007/s12374-014-0487-1

Lee J., Cao D.V., Kim J., Pamplona R. S., Ahn J., and Cho, S. K., 2017, Development of a virus-induced gene silencing (VIGS) system for Spinacia oleracea L, In Vitro Cellular & Developmental Biology-Plant., 53: 97-103
https://doi.org/10.1007/s11627-017-9806-9

Liu G.Y., Wu Y.F., Xu M.J., Gao T., Wang P.F., Wang L.N., Guo T.C., and Kang G.Z., 2016, Virus-Induced Gene Silencing Identifies an Important Role of the TaRSR1 Transcription Factor in Starch Synthesis in Bread Wheat, INT J MOL SCI., 17:10
https://doi.org/10.3390/ijms17101557
PMid:27669224 PMCid:PMC5085620

Livak K.J., and Schmittgen T.D., 2016, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)), METHODS., 25: 402-408
https://doi.org/10.1006/meth.2001.1262
PMid:11846609

Ma M., Yan Y., Huang L., Chen M.S., and Zhao H.X., 2012,Virus-induced gene-silencing in wheat spikes and grains and its application in functional analysis of HMW-GS-encoding genes, BMC Plant Biol., 12: 141
https://doi.org/10.1186/1471-2229-12-141
PMid:22882902 PMCid:PMC3462119

Muterko A., Kalendar R., and Salina E., 2016, Allelic variation at the VERNALIZATION-A1, VRN-B1, VRN-B3, and PHOTOPERIOD-A1 genes in cultivars of Triticum durum Desf, Planta., 244: 1253-1263
https://doi.org/10.1007/s00425-016-2584-5
PMid:27522649

Ramakrishna A., and Gokare A.R., 2011, Influence of abiotic stress signals on secondary metabolites in plants, Plant Signaling Behavior 6: 1720-1731
https://doi.org/10.4161/psb.6.11.17613
PMid:22041989 PMCid:PMC3329344

Reynolds M., Bonnett D., Chapman S.C., Furbank R.T., Manes Y., Mather D.E., and Parry M.A.J., 2011, Raising yield potential of wheat. I. Overview of a consortium approach and breeding strategies,J Exp Bot., 62:439-453
https://doi.org/10.1093/jxb/erq311
PMid:20952629

Rivas S., Rougon-Cardoso A., Smoker M., Schauser L., Yoshioka H., and Jones J.D.G., 2004, CITRX thioredoxin interacts with the tomato Cf-9 resistance protein and negatively regulates defense, EMBO J, 23:2156-2165
https://doi.org/10.1038/sj.emboj.7600224
PMid:15131698 PMCid:PMC424418

Senthil-Kumar M., and Mysore K.S., 2011, New dimensions for VIGS in plant functional genomics, TRENDS Plant Sci., 16: 656-665
https://doi.org/10.1016/j.tplants.2011.08.006
PMid:21937256

Shekhawat U.K.S., Srinivas L., and Ganapathi T.R., 2011, MusaDHN-1, a novel multiple stress-inducible SK3-type dehydrin gene, contributes affirmatively to drought- and salt-stress tolerance in banana, Planta., 234: 915-932
https://doi.org/10.1007/s00425-011-1455-3
PMid:21671068

Sun J., Nie L.Z., Sun G.Q., Guo J.F., and Liu Y.Z., 2000, Cloning and characterization of dehydrin gene from Ammopiptanthus mongolicus, Mol Biol Rep., 40:2281-2291
https://doi.org/10.1007/s11033-012-2291-7
PMid:23212615

Suprunova T., Krugman T., Fahima T., Chen G., Shams I., Korol A., and Nevo E., 2004, Differential expression of dehydrin genes in wild barley, Hordeum spontaneum, associated with resistance to water deficit, Plant Cell Environ., 27: 1297-1308
https://doi.org/10.1111/j.1365-3040.2004.01237.x

Tanaka C., Itoh T., Iwasaki Y., Mizuno N., Nasuda S., and Murai K., 2018, Direct interaction between VRN1 protein and the promoter region of the wheat FT gene, Genes Genet Syst., 93: 25-29
https://doi.org/10.1266/ggs.17-00041
PMid:29343669

Wang X.G., Zhu H.L., Shao Y., Chen A.J., Ma Y.Z., Luo Y.B., and Zhu B.Z., 2010, Analysis of gene functions by a syringe infiltration method of VIGS, RUSS J Plant Physl., 57: 590-597
https://doi.org/10.1134/S1021443710040199

Wang Y.Z., Xu H.B., Zhu H.L., Tao Y., Zhang G.X., Zhang L.X., Zhang C.Q., Zhang Z.Z., and Ma Z.Q., 2014, Classification and expression diversification of wheat dehydrin genes, Plant Sci., 214: 113-120
https://doi.org/10.1016/j.plantsci.2013.10.005

PMid:24268169

Xu H., Yang Y., Xie L., Li X.Y., Feng C., Chen J.W., and Xu C.J., 2014, Involvement of multiple types of dehydrins in the freezing response in loquat (Eriobotrya japonica), PLoS One., 1: 1-11
https://doi.org/10.1371/journal.pone.0087575
PMid:24498141 PMCid:PMC3909202

Yin J., Miao G.Y., and Yin F., 2017, Wheat thermo-photoperiod development and its molecular basis, Science Press., pp: 40

Yu Y., and Yang Z.J., 2016, The isolation and characterization of a putative dehydrin gene in Triticum aestivum L. Biochemical Systematics and Ecology, Biochem Syst Ecol., 6: 173-180
https://doi.org/10.1016/j.bse.2016.04.003

Yuan X.Y., Li Y.C., Meng F.R., Yan Y.T., and Yin J., 2008, Composition and Characteristic of the VRN1 Gene in Nine Wheat (Triticum aestivum L.) Cultivars with Different Vernalization Traits, Plant physiology communication., 44: 699-704

Zhang H., Zheng J.X., Su H.X., Xia K.F., Jian H.G., and Zhang M., 2018, Molecular Cloning and Functional Characterization of the Dehydrin (IpDHN) Gene From Ipomoea pescaprae, Frontiers in Plant Science., 9, 1454
https://doi.org/10.3389/fpls.2018.01454
PMid:30364314 PMCid:PMC6193111