Cloning of Wheat TaLCD Gene and Its Regulation on Osmotic Stress

doi:10.11983/CBB19109

Abstract

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

Yang Zhang,Huajie Liu,Ruili Xue,Haixia Li,Hua Li. Cloning of Wheat TaLCD Gene and Its Regulation on Osmotic Stress[J]. Chinese Bulletin of Botany, 2020, 55(2): 137-146.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB19109

https://www.chinbullbotany.com/EN/Y2020/V55/I2/137

Figures/Tables 9

Table 1 Primers used for qRT-PCR analysis

Gene name	Primer sequences
Gene name	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

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