Chinese Bulletin of Botany ›› 2020, Vol. 55 ›› Issue (2): 137-146.doi: 10.11983/CBB19109

• EXPERIMENTAL COMMUNICATIONS • Previous Articles     Next Articles

Cloning of Wheat TaLCD Gene and Its Regulation on Osmotic Stress

Zhang Yang,Liu Huajie,Xue Ruili,Li Haixia,Li Hua()   

  1. College of Life Sciences, Henan Agricultural University, Zhengzhou 450002, China
  • Received:2019-06-14 Accepted:2019-11-05 Online:2020-02-12 Published:2020-03-01
  • Contact: Li Hua E-mail:lihuahnnd@163.com

Abstract:

Cysteine desulphydrase (CDes) can degrade cysteine (Cys) to form hydrogen sulfide (H2S). In this study, L-cysteine desulphydrase TaLCD from wheat (Triticum aestivum) was cloned and overexpressed in Arabidopsis thaliana. The effects of TaLCD on seed germination and root growth under osmotic stress in TaLCD overexpressing plants were examined, and the response to drought stress was also investigated. The results showed that, the seed germination rate of TaLCD overexpressing plants under salt treatment was remarkably higher than the wild type (WT), and the root length of TaLCD overexpressing plants was obviously longer when exposed to mannitol. Moreover, overexpression of TaLCD distinctly increased plant drought resistance. In addition, TaLCD overexpressing plants were more sensitive to ABA, with decreased seed germination rate and root length under ABA treatment. Furthermore, the expression of stress-responsive genes (COR47, RD29A, RAB18 and RD22) and ABA signaling pathway-related genes (NCED3, HAB1, HAB2, ABI1, ABI2 and ABF2) was significantly higher in TaLCD overexpressing plants than that in WT under drought stress. These results suggest that TaLCD enhances the drought and salt tolerance of plants possibly by ABA signaling pathway.

Key words: TaLCD, osmotic stress, ABA, germination and growth

Table 1

Primers used for qRT-PCR analysis"

Gene name Primer sequences
Forward (5'-3') Reverse (5'-3')
TaDCD GAGGAAGGACGGAAGCCATAT TCAGGGTCATCGCAAACAGAG
TaLCD TCCATTACGCCTACGGAGCAG CAAGCCGGACCTTACGACCA
ABF2 ATCAGAAGGGATAGGGAAGAGTAAT TTGGTCTGCCGTGAATATCTGT
HAB1 GTGTTCTCGCCATGTCTAGGTC CTATTTCGCAGACTTCTTGGTTG
HAB2 GCAGAAGTCCTTATTGCGAGTC CTCAGAAGTTGCCACCTCCATA
ABI1 TGACGGCTGTGAAGAGAGTA CCATCTCACACGCTTCTTCA
ABI2 ATTCAGACCATTCACTGACCCTC GCTCCGTCGCCAGAACAAG
NCED3 TGGCTTCTTTCACGGCAAC ACAACAATGGCGGGAGAGTTT
COR47 GAGGTTACGGATCGTGGAT GAGCTGTTGGATCGGTGA
RAB18 ATGGCGTCTTACCAGAACCGTCCA TACCCTTGGCCACCTGATCC
RD29A GTTACTGATCCCACCAAAGAAGA GGAGACTCATCAGTCACTTCCA
RD22 AGGGCTGTTTCCACTGAGG CACCACAGATTTATCGTCAGACA
Actin2 TACCCGATGGGCAAGTCA TGCTCATACGGTCAGCGATA

Figure 1

Sequence analysis of TaLCD (A) Comparison of the derived amino acid sequences of TaLCD in Triticum aestivum with other 4 species (Aegilops tauschii, Brachypodium distachyon, Setaria italica, and Oryza brachyantha); (B) Phylogenetic analysis of LCD in five species; (C) Schematic structures of TaLCD, black box indicates Aminotran_5 domain."

Figure 2

The expression of TaLCD and enzyme activity of TaLCD in wheat under NaCl and PEG treatments (A) The expression of TaLCD under NaCl treatment; (B) The expression of TaLCD under PEG treatment; (C) The enzyme activity of TaLCD under NaCl treatment; (D) The enzyme activity of TaLCD under PEG treatment. * indicate significant differences (P<0.05)."

Figure 3

Expression of TaLCD and enzyme activity of TaLCD in transgenic Arabidopsis (A) RT-PCR; (B) qRT-PCR; (C) Enzyme activity of TaLCD under NaCl treatment. Marker: DNA marker; WT: Wild type; 2-8, 3-4, 5-5, and 12-4: TaLCD transgenic lines; * indicate significant differences (P<0.05)."

Figure 4

Seed germination rate of Arabidopsis lines expressing TaLCD under different concentrations of NaCl treatment WT: Wild type; OX-LCD-1 and OX-LCD-2: Transgenic lines; * indicate significant differences (P<0.05)."

Figure 5

Root growth of Arabidopsis lines expressing TaLCD under different concentrations of mannitol treatment (A) Phenotype of root growth (Bars=1 cm); (B) Root elongation measurements. WT: Wild type; OX-LCD-1 and OX-LCD-2: Transgenic lines; * indicate significant differences (P<0.05)."

Figure 6

Drought resistance of Arabidopsis lines expressing TaLCD (A) Phenotype of three week-old wild type (WT), OX-LCD-1 and OX-LCD-2 plants after drought treatment (Bars=2 cm); (B) Leaf water loss"

Figure 7

Seed germination and growth of Arabidopsis lines expressing TaLCD under different concentrations of ABA treatment (A) Phenotype on the 10th day (Bars=1 cm); (B) Seed germination rate after 4 d incubation; (C) Phenotype of root growth on the 10th day (Bars=0.4 cm); (D) Root length on the 10th day. WT: Wild type; ABA: Abscisic acid; * indicate significant differences (P<0.05)."

Figure 8

The expression of stress response genes and ABA signaling related genes in Arabidopsis lines expressing TaLCD under drought stress (A)-(D) Stress response genes; (E)-(J) ABA signaling related genes. WT: Wild type; OX-LCD-2: Transgenic lines"

[1] 侯智慧, 刘菁, 侯丽霞, 李希东, 刘新 ( 2011). H2S可能作为H2O2的下游信号介导茉莉酸诱导的蚕豆气孔关闭. 植物学报 46, 396-406.
[2] 刘菁, 侯智慧, 赵方贵, 刘新 ( 2011). H2S介导ABA诱导蚕豆气孔运动的生理机制研究. 西北植物学报 31, 298-304.
[3] 单长卷, 周岩 ( 2011). 外源硫化氢对水分胁迫下玉米种子萌发和生长的影响. 广东农业科学 38(20), 28-30.
[4] Chen J, Wang WH, Wu FH, You CY, Liu TW, Dong XJ, He JX, Zheng HL ( 2013). Hydrogen sulfide alleviates aluminum toxicity in barley seedlings. Plant Soil 362, 301-318.
[5] Christou A, Manganaris GA, Papadopoulos I, Fotopoulos V ( 2013). Hydrogen sulfide induces systemic tolerance to salinity and non-ionic osmotic stress in strawberry plants through modification of reactive species biosynthesis and transcriptional regulation of multiple defence pathways. J Exp Bot 64, 1953-1966.
[6] Dooley FD, Nair SP, Ward PD ( 2013). Increased growth and germination success in plants following hydrogen sulfide administration. PLoS One 8, e62048.
[7] Fang HH, Liu ZQ, Long YP, Liang YL, Jin ZP, Zhang LP, Liu DM, Li H, Zhai JX, Pei YX ( 2017). The Ca 2+/CaM2- binding transcription factor TGA3 elevates LCD expression and H2S production to bolster Cr 6+ tolerance in Arabidopsis. Plant J 91, 1038-1050.
[8] García-Mata C, Lamattina L ( 2010). Hydrogen sulphide, a novel gasotransmitter involved in guard cell signaling. New Phytol 188, 977-984.
[9] Guo HM, Xiao TY, Zhou H, Xie YJ, Shen WB ( 2016). Hydrogen sulfide: a versatile regulator of environmental stress in plants. Acta Physiol Plant 38, 16.
[10] Jia HL, Hu YF, Fan TT, Li JS ( 2015). Hydrogen sulfide modulates actin-dependent auxin transport via regulating ABPs results in changing of root development in Arabidopsis. Sci Rep 5, 8251.
[11] Jin ZP, Shen JJ, Qiao ZJ, Yang GD, Wang R, Pei YX ( 2011). Hydrogen sulfide improves drought resistance in Arabidopsis thaliana. Biochem Biophys Res Commun 414, 481-486.
[12] Jin ZP, Xue SW, Luo YA, Tian BH, Fang HH, Li H, Pei YX ( 2013). Hydrogen sulfide interacting with abscisic acid in stomatal regulation responses to drought stress in Arabidopsis. Plant Physiol Biochem 62, 41-46.
[13] Letunic I, Doerks T, Bork P ( 2015). SMART: recent updates, new developments and status in 2015. Nucleic Acids Res 43, D257-D260.
[14] Li CJ, Liu ZJ, Zhang QR, Wang RZ, Xiao LT, Ma H, Chong K, Xu YY ( 2012). SKP1 is involved in abscisic acid signaling to regulate seed germination, stomatal opening and root growth in Arabidopsis thaliana. Plant Cell Environ 35, 952-965.
[15] Li H, Gao MQ, Xue RL, Wang D, Zhao HJ ( 2015). Effect of hydrogen sulfide on D1 protein in wheat under drought stress. Acta Physiol Plant 37, 225.
[16] Li H, Li M, Wei XL, Zhang X, Xue RL, Zhao YD, Zhao HJ ( 2017). Transcriptome analysis of drought-responsive genes regulated by hydrogen sulfide in wheat ( Triticum aestivum L.) leaves. Mol Genet Genomics 292, 1091-1110.
[17] Ma DY, Ding HN, Wang CY, Qin HX, Han QX, Hou JF, Lu HF, Xie YX, Guo TC ( 2016). Alleviation of drought stress by hydrogen sulfide is partially related to the abscisic acid signaling pathway in wheat. PLoS One 11, e0163082.
[18] Papenbrock J, Riemenschneider A, Kamp A, Schulz- Vogt HN, Schmidt A ( 2007). Characterization of cysteine-degrading and H2S-releasing enzymes of higher plants—from the field to the test tube and back. Plant Biol 9, 582-588.
[19] Shen JJ, Qiao ZJ, Xing TJ, Zhang LP, Liang YL, Jin ZP, Yang GD, Pei YX ( 2012). Cadmium toxicity is alleviated by AtLCD and AtDCD in Escherichia coli. J Appl Microbiol 113, 1130-1138.
[20] Shi HT, Ye TT, Chan ZL ( 2013). Exogenous application of hydrogen sulfide donor sodium hydrosulfide enhanced multiple abiotic stress tolerance in bermudagrass (Cynodon dactylon(L). Pers.). Plant Physiol Biochem 71, 226-234.
[21] Shi HT, Ye TT, Han N, Bian HW, Liu XD, Chan ZL ( 2015). Hydrogen sulfide regulates abiotic stress tolerance and biotic stress resistance in Arabidopsis. J Integr Plant Biol 57, 628-640.
[22] Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S ( 2011). MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28, 2731-2739.
[23] Wang YQ, Li L, Cui WT, Xu S, Shen WB, Wang R ( 2012). Hydrogen sulfide enhances alfalfa ( Medicago sativa) tolerance against salinity during seed germination by nitric oxide pathway. Plant Soil 351, 107-119.
[24] Xie YJ, Lai DW, Mao Y, Zhang W, Shen WB, Guan RZ ( 2013). Molecular cloning, characterization, and expression analysis of a novel gene encoding L-cysteine desulfhydrase from Brassica napus. Mol Biotechnol 54, 737-746.
[25] Zhang H, Hu LY, Hu KD, He YD, Wang SH, Luo JP ( 2008). Hydrogen sulfide promotes wheat seed germination and alleviates oxidative damage against copper stress. J Integr Plant Biol 50, 1518-1529.
[26] Zhang H, Hu LY, Li P, Hu KD, Jiang CX, Luo JP ( 2010a). Hydrogen sulfide alleviated chromium toxicity in wheat. Biol Plantarum 54, 743-747.
[27] Zhang H, Hu SL, Zhang ZJ, Hu LY, Jiang CX, Wei ZJ, Liu J, Wang HL, Jiang ST ( 2011). Hydrogen sulfide acts as a regulator of flower senescence in plants. Postharvest Biol Technol 60, 251-257.
[28] Zhang H, Jiao H, Jiang CX, Wang SH, Wei ZJ, Luo JP, Jones RL ( 2010b). Hydrogen sulfide protects soybean seedlings against drought-induced oxidative stress. Acta Physiol Plant 32, 849-857.
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[1] Lu Zhong-shu. Plant Growth Regutators in Relation to Plant Water Status[J]. Chinese Bulletin of Botany, 1985, 3(04): 1 -6 .
[2] Li Da Jue;Han Yun-zhou and Wan Li-ping. Studies on Germplasm Collections of Carthamus tinctorius IV Screening of the characterization of Seed Domancy[J]. Chinese Bulletin of Botany, 1990, 7(02): 50 -52 .
[3] . [J]. Chinese Bulletin of Botany, 1999, 16(增刊): 45 -46 .
[4] Yang Hong-yuan. Basic Principle and Method of Fluorescence Microscopy[J]. Chinese Bulletin of Botany, 1984, 2(06): 45 -48 .
[5] LU Jin-Yao;LUO Ai-Ling and LIANG Zheng. Some Improvement of TD-PAGE Technology[J]. Chinese Bulletin of Botany, 1998, 15(03): 69 -72 .
[6] LI Ling-Hao and CHEN Zuo-Zhong. The Global Carbon Cycle in Grassland Ecosystems and Its Responses to Global Change I . Carbon Flow Compartment Model, Inputs and Storage[J]. Chinese Bulletin of Botany, 1998, 15(02): 14 -22 .
[7] Huanhuan Xu, Jian Kang, Mingxiang Liang. Research Advances in the Metabolism of Fructan in Plant Stress Resistance[J]. Chinese Bulletin of Botany, 2014, 49(2): 209 -220 .
[8] . [J]. Chinese Bulletin of Botany, 2013, 48(1): 4 -5 .
[9] . [J]. Chinese Bulletin of Botany, 1996, 13(专辑): 45 .
[10] SHU Qun-Fang;ZHOU Lu;LI Wen-Bin;ZHANG LI-Ming and SUN Yong-Ru. Study on Gel Electrophoresis of Protein from Plant and Our Improved Methods[J]. Chinese Bulletin of Botany, 1998, 15(06): 73 -78 .