Abstract
Expression of the late embryogenesis abundant (LEA) gene is usually associated with plant response to dehydration. In this study, a drought-tolerant genotype was screened from 48 accessions of Tibetan hulless barley (Hordeum vulgare ssp. vulgare). By using virus-induced gene silencing, the influence of two LEA genes (HVA1 and Dhn6) on drought tolerance of Tibetan hulless barley was investigated. Results of quantitative real-time PCR indicated that the relative expression levels of HVA1 and Dhn6 in silenced plants were significantly reduced compared with control plants. Both HVA1-silenced and Dhn6-silenced plants showed a consequently lower survival rate than control plants under drought stress. However, only HVA1-silenced plants exhibited a significantly higher water loss rate (WLR). These results suggested that HVA1 and Dhn6 might participate in adaptive responses to water deficit in different ways. Vegetative growth of HVA1-silenced plants was significantly retarded even under optimal growth conditions, and their biomass accumulation was also much lower than that of the controls. These results indicate that HVA1 might play a role in vegetative growth of Tibetan hulless barley.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Abba S, Ghignone S, Bonfante P (2006) A dehydration-inducible gene in the truffle Tuber borchii identifies a novel group of dehydrins. BMC Genomics 7:39–53. doi:10.1186/1471-2164-7-39
Araus JL, Slafer GA, Reynolds MP, Royo C (2002) Plant breeding and drought in C-3 cereals: what should we breed for? Ann Bot 89:925–940. doi:10.1093/aob/mcf049
Babu RC, Zhang J, Blum A, Ho THD, Wu R, Nguyen HT (2004) HVA1, a LEA gene from barley confers dehydration tolerance in transgenic rice (Oryza sativa L.) via cell membrane protection. Plant Sci 166:855–862. doi:10.1016/j.plantsci.2003.11.023
Bahieldin A, Mahfouz HT, Eissa HF, Saleh OM, Ramadan AM, Ahmed IA, Dyer WE, El-Itriby HA, Madkour MA (2005) Field evaluation of transgenic wheat plants stably expressing the HVA1 gene for drought tolerance. Physiol Plant 123:421–427. doi:10.1111/j.1399-3054.2005.00470.x
Baulcombe DC (1999) Fast forward genetics based on virus induced gene silencing. Curr Opin Plant Biol 2:109–113. doi:10.1016/S1369-5266(99)80022-3
Bies-Ethève N, Gaubier-Comella P, Debures A, Lasserre E, Jobet E, Raynal M, Cooke R, Delseny M (2008) Inventory, evolution and expression profiling diversity of the LEA (late embryogenesis abundant) protein gene family in Arabidopsis thaliana. Plant Mol Biol 67:107–124. doi:10.1007/s11103-008-9304-x
Bray EA (1997) Plant responses to water deficit. Trends Plant Sci 2:48–54. doi:10.1016/S1360-1385(97)82562-9
Bray EA, Bailey-Serres J, Weretilnyk E (2000) Responses to abiotic stresses. In: Gruissem W, Buchannan B, Jones R (eds) Biochemistry and molecular biology of plants. American Society of Plant Physiologists, Rockville, pp 1158–1249
Browne JA, Dolan KM, Tyson T, Goyal K, Tunnacliffe A, Burnell AM (2004) Dehydration-specific induction of hydrophilic protein genes in the anhydrobiotic nematode Aphelenchus avenae. Eukaryot Cell 3:966–975. doi:10.1128/EC.3.4.966-975.2004
Burch-Smith TM, Anderson JC, Martin GB, Dinesh-Kumar SP (2004) Applications and advantages of virus-induced gene silencing for gene function studies in plants. Plant J 39:734–746. doi:10.1111/j.1365-313X.2004.02158.x
Campbell SA, Close TJ (1997) Dehydrins: genes, proteins, and associations with phenotypic traits. New Phytol 137:61–74. doi:10.1046/j.1469-8137.1997.00831.x
Chandler PM, Robertson M (1994) Gene expression regulated by abscisic acid and its relation to stress tolerance. Annu Rev Plant Physiol Plant Mol Biol 45:113–141. doi:10.1146/annurev.pp.45.060194.000553
Chauhan H, Khurana P (2011) Use of doubled haploid technology for development of stable drought tolerant bread wheat (Triticum aestivum L.) transgenics. Plant Biotechnol J 9:408–417. doi:10.1111/j.1467-7652.2010.00561.x
Clarke JM, McCaig TN (1982) Evaluation of techniques for screening for drought resistance in wheat. Crop Sci 22:503–506. doi:10.2135/cropsci1982.0011183X002200030015x
Dalal M, Tayal D, Chinnusamy V, Bansal KC (2009) Abiotic stress and ABA-inducible group 4 LEA from Brassica napus plays a key role in salt and drought tolerance. J Biotechnol 139:137–145. doi:10.1016/j.jbiotec.2008.09.014
Demmig-Adams B, Adams WW III (1992) Photoprotection and other responses of plants to high light stress. Annu Rev Plant Physiol Plant Mol Biol 43:599–626. doi:10.1146/annurev.pp.43.060192.003123
Dombrowski JE, Martin RC (2009) Evaluation of reference genes for quantitative RT-PCR in Lolium temulentum under abiotic stress. Plant Sci 176:390–396. doi:10.1016/j.plantsci.2008.12.005
Dure L III, Chlan C (1981) Developmental biochemistry of cottonseed embryogenesis and germination. XII. Purification and properties of principal storage proteins. Plant Physiol 68:180–186. doi:10.1104/pp.68.1.180
Dure L III, Galau GA (1981) Developmental biochemistry of cottonseed embryogenesis and germination. XIII. Regulation of biosynthesis of principal storage proteins. Plant Physiol 68:187–194. doi:10.1104/pp.68.1.187
Dure L III, Greenway SC, Galau GA (1981) Developmental biochemistry of cottonseed embryogenesis and germination: changing messenger ribonucleic acid populations as shown by in vitro and in vivo protein synthesis. Biochemistry 20:4162–4168. doi:10.1021/bi00517a033
Dure L III, Crouch M, Harada J, Ho THD, Mundy J, Quatrano R, Thomas T, Sung ZR (1989) Common amino acid sequence domains among the LEA proteins of higher plants. Plant Mol Biol 12:475–486. doi:10.1007/BF00036962
Fu D, Huang B, ** bentgrass for improving drought tolerance. Plant Cell Rep 26:467–477. doi:10.1007/s00299-006-0258-7
Garay-Arroyo A, Colmenero-Flores JM, Garciarrubio A, Covarrubias AA (2000) Highly hydrophilic proteins in prokaryotes and eukaryotes are common during conditions of water deficit. J Biol Chem 275:5668–5674. doi:10.1074/jbc.275.8.5668
Godge MR, Purkayastha A, Dasgupta I, Kumar PP (2008) Virus-induced gene silencing for functional analysis of selected genes. Plant Cell Rep 27:209–219. doi:10.1007/s00299-007-0460-2
Guo P, Baum M, Grando S, Ceccarelli S, Bai G, Li R, von Korff M, Varshney RK, Graner A, Valkoun J (2009) Differentially expressed genes between drought-tolerant and drought-sensitive barley genotypes in response to drought stress during the reproductive stage. J Exp Bot 60(12):3531–3544. doi:10.1093/jxb/erp194
Hartl M, Merker H, Schmidt DD, Baldwin IT (2008) Optimized virus-induced gene silencing in Solanum nigrum reveals the defensive function of leucine aminopeptidase against herbivores and the shortcomings of empty vector controls. New Phytol 179:356–365. doi:10.1111/j.1469-8137.2008.02479.x
Holzberg S, Brosio P, Gross C, Pogue GP (2002) Barley stripe mosaic virus-induced gene silencing in a monocot plant. Plant J 30:315–327. doi:10.1046/j.1365-313X.2002.01291.x
Hundertmark M, Hincha DK (2008) LEA (Late Embryogenesis Abundant) proteins and their encoding genes in Arabidopsis thaliana. BMC Genomics 9:118–139. doi:10.1186/1471-2164-9-118
Ingram J, Bartels D (1996) The molecular basis of dehydration tolerance in plants. Annu Rev Plant Physiol Plant Mol Biol 47:377–403. doi:10.1146/annurev.arplant.47.1.377
Kumagai MH, Donson J, Della-Cioppa G, Harvey D, Hanley K, Grill LK (1995) Cytoplasmic inhibition of carotenoid biosynthesis with virus-derived RNA. Proc Natl Acad Sci USA 92:1679–1683. doi:10.1073/pnas.92.5.1679
Lal S, Gulyani V, Khurana P (2008) Overexpression of HVA1 gene from barley generates tolerance to salinity and water stress in transgenic mulberry (Morus indica). Transgenic Res 17:651–663. doi:10.1007/s11248-007-9145-4
Liu Y, Schiff M, Dinesh-Kumar SP (2002) Virus-induced gene silencing in tomato. Plant J 31:777–786. doi:10.1046/j.1365-313X.2002.01394.x
Long XY, Wang JR, Ouellet T, Rocheleau H, Wei YM, Pu ZE, Jiang QT, Lan XJ, Zheng YL (2010) Genome-wide identification and evaluation of novel internal control genes for Q-PCR based transcript normalization in wheat. Plant Mol Biol 74:307–311. doi:10.1007/s11103-010-9666-8
Mtwisha L, Brandt W, McCready S, Lindsey GG (1998) HSP 12 is a LEA-like protein in Saccharomyces cerevisiae. Plant Mol Biol 37:513–521. doi:10.1023/A:1005904219201
NDong C, Danyluk J, Wilson KE, Pocock T, Huner NP, Sarhan F (2002) Cold-regulated cereal chloroplast late embryogenesis abundant-like proteins: molecular characterization and functional analyses. Plant Physiol 129:1368–1381. doi:10.1104/pp.001925
Nicot N, Hausman JF, Hoffmann L, Evers D (2005) Housekee** gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress. J Exp Bot 56(421):2907–2914. doi:10.1093/jxb/eri285
Olvera-Carrillo Y, Campos F, Reyes JL, Garciarrubio A, Covarrubias AA (2010) Functional analysis of the group 4 late embryogenesis abundant proteins reveals their relevance in the adaptive response during water deficit in Arabidopsis. Plant Physiol 154:373–390. doi:10.1104/pp.110.158964
Puhakainen T, Hess MW, Makela P, Svensson J, Heino P, Palva ET (2004) Overexpression of multiple dehydrin genes enhances tolerance to freezing stress in Arabidopsis. Plant Mol Biol 54:743–753. doi:10.1023/B:PLAN.0000040903.66496.a4
Qian G (2007) Genotypic variability in sequences and expression of LEA2/LEA3 genes in Tibetan Hulless Barley, Hordeum vulgare ssp. Vulgare, associated with resistance to water deficit. Dissertation, Chengdu Institute of Biology, the Chinese Academy of Sciences
Qian G, Han ZX, Zhao T, Deng GB, Pan ZF, Yu MQ (2007) Genotypic variability in sequence and expression of HVA1 gene in Tibetan hulless barley, Hordeum vulgare ssp. vulgare, associated with resistance to water deficit. Aust J Agric Res 58:425–431. doi:10.1071/AR06300
Ratcliff F, Harrison BD, Baulcombe DC (1997) A similarity between viral defense and gene silencing in plants. Science 276:1558–1560. doi:10.1126/science.276.5318.1558
Ristic Z, Jenks MA (2002) Leaf cuticle and water loss in maize lines differing in dehydration avoidance. J Plant Physiol 159:645–651. doi:10.1078/0176-1617-0743
Robertson M, Chandler PM (1994) A dehydrin cognate protein from pea (Pisum sativum L.) with an atypical pattern of expression. Plant Mol Biol 26:805–816. doi:10.1007/BF00028850
Rohila JS, Jain RK, Wu R (2002) Genetic improvement of Basmati rice for salt and drought tolerance by regulated expression of a barley Hva1 cDNA. Plant Sci 163:525–532. doi:10.1016/S0168-9452(02)00155-3
Seckler D, Amarasinghe U, Molden D, de Silva R, Barker R (1998) World water demand and supply, 1990 to 2025: scenarios and issues. Colombo, Sri Lanka: International Water Management Institute (IIMI); IWMI. vi, 40p. (IIMI Research Report 19/IWMI Research Report 19). doi:10.3910/2009.019
Seckler D, Barker R, Amarasinghe UA (1999) Water scarcity in the twenty-first century. Int J Water Resour Dev 15:29–42. doi:10.1080/07900629948916
Sivamani E, Bahieldin A, Wraith JM, Al-Niemi T, Dyer WE, Ho THD, Qu R (2000) Improved biomass productivity and water use efficiency under water deficit conditions in transgenic wheat constitutively expressing the barley HVA1 gene. Plant Sci 155:1–9. doi:10.1016/S0168-9452(99)00247-2
Solomon A, Salomon R, Paperna I, Glazer I (2000) Desiccation stress of entomopathogenic nematodes induces the accumulation of a novel heat-stable protein. Parasitology 121:409–416. doi:10.1017/S0031182099006563
Stacy RAP, Aalen RB (1998) Identification of sequence homology between the internal hydrophilic repeated motifs of group 1 late-embryogenesis-abundant proteins in plants and hydrophilic repeats of the general stress protein GsiB of Bacillus subtilis. Planta 206:476–478. doi:10.1007/s004250050424
Suppunova T, Krugman T, Fahima T, Chen G, Shams I, Korol A, 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. doi:10.1111/j.1365-3040.2004.01237.x
Thomashow MF (1998) Role of cold-responsive genes in plant freezing tolerance. Plant Physiol 118:1–8. doi:10.1104/pp.118.1.1
Wan H, Zhao Z, Qian C, Sui Y, Malik AA, Chen J (2010) Selection of appropriate reference genes for gene expression studies by quantitative real-time polymerase chain reaction in cucumber. Anal Biochem 399:257–261. doi:10.1016/j.ab.2009.12.008
Wu CJ, Jia LL, Goggin F (2011) The reliability of virus-induced gene silencing experiments using tobacco rattle virus in tomato is influenced by the size of the vector control. Mol Plant Pathol 12(3):299–305. doi:10.1111/J.1364-3703.2010.00669.X
**ao B, Huang Y, Tang N, **ong L (2007) Over-expression of a LEA gene in rice improves drought resistance under the field conditions. Theor Appl Genet 115:35–46. doi:10.1007/s00122-007-0538-9
Xu D, Duan X, Wang B, Hong B, Ho T-HD, Wu R (1996) Expression of a late embryogenesis abundant protein gene, HVA1, from barley confers tolerance to water deficit and salt stress in transgenic rice. Plant Physiol 110:249–257. doi:10.1104/pp.110.1.249
Yang GF, Peng HX, Li CA, Yin HF (2001) Analysis of hydrothermal factors and presentation of ecological recovery measures in Northwest China. Northwest Geol 34:9–15. doi:cnki:SCN:61-1174.0.2001-04-00
Acknowledgments
We thank Chengdu Institute of Biology, Chinese Academy of Sciences for the Senior Research Fellowship award. Prof. Daowen Wang of Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Bei**g, are kindly acknowledged for providing BSMV vectors. We sincerely acknowledge Dr. Bin Li for technical expertise and advice on BSMV inoculation, and Dr. Gang Qian for thoughtful discussion during the preparation of this manuscript. We also wish to thank the two anonymous reviewers for helpful comments that improved the manuscript. This work was supported by Major transgenic program (2008ZX08002-002), Ministry of Agriculture, P. R. China and Special Basic Research Funds of the Ministry of Science and Technology of China (No. 2006FY110700).
Author information
Authors and Affiliations
Corresponding authors
Additional information
Junjun Liang and Guangbing Deng contributed equally to this paper.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Liang, J., Deng, G., Long, H. et al. Virus-induced silencing of genes encoding LEA protein in Tibetan hulless barley (Hordeum vulgare ssp. vulgare) and their relationship to drought tolerance. Mol Breeding 30, 441–451 (2012). https://doi.org/10.1007/s11032-011-9633-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11032-011-9633-3