Cloning of cucumber LCD and DES gene and their response to abiotic stress

Abstract

Hydrogen sulfide (H2S) has been reported as a significant gasotransmitter, which involved in a range of physiological processes in plant, including growth, development and abiotic stresses resistence. Enzymatic activity assays showed that L-cysteine desulfhydrase (L-CD) was primarily responsible for the generation of endogenous H2S in plant. In this work, we identified two activated L-CDs from cucumber 9930 varieties. Firstly, three L-CD genes, CsaLCD, CsaDES1 and CsaDES2 were acquired from tBLASTn algorithm-based search of NCBI and a cucumber genomic database (http://www.icugi.org), using the Arabidopsis LCD and DES1 protein sequences as the query. The CDS of CsaLCD (Csa2P034800.1) and CsaDES2 (Csa1P574810.1) were amplified successfully by PCR, while the CsaDES1 (Csa1P574800.1) could not be obtained in the case of cDNA as the template. The recombinant proteins His-CsaLCD and His-CsaDES2, which were purified from E.coli BL21 strain, exhibited evident L-CD activity. The mRNA level of CsaLCD was induced by 37 °C, low light or PEG8000 treatments, and CsaDES2 was induced by 4 °C or 37 °C treatments, while CsaDES1 itself has no expression. The CDS of CsaLCD or CsaDES2 with 35S promoter were transformated into lcd/des mutant of Arabidopsis, and both their heterologous expression restored the H2S production rate, as well as enhancing the resistance to osmosis stress of the transgenetic seedlings. Therefore, CsaLCD and CsaDES2 are the main enzymes for endogenous H2S production in cucumber, and play a pivotal role in H2S generation during stresses responses.