Homologous cloning, characterization and expression of a new halophyte phytochelatin synthase gene in Suaeda salsa

The halophyte Suaeda salsa can grow in heavy metal-polluted areas along intertidal zones having high salinity.Since phytochelatins can effectively chelate heavy metals,it was hypothesized that S.salsa possessed a phytochelatin synthase(PCS) gene.In the present study,the cDNA of PCS was obtained from S.salsa(designated as SsPCS) using homologous cloning and the rapid amplification of cDNA ends(RACE).A sequence analysis revealed that SsPCS consisted of 1 916 bp nucleotides,encoding a polypeptide of 492 amino acids with one phytochelatin domain and one phytochelatin C domain.A similarity analysis suggested that SsPCS shared up to a 58.6%identity with other PCS proteins and clustered with PCS proteins from eudicots.There was a new kind of metal ion sensor motif in its C-terminal domain.The SsPCS transcript was more highly expressed in elongated and fibered roots and stems(P<0.05) than in leaves.Lead and mercury exposure significantly enhanced the mRNA expression of SsPCS(P<0.05).To the best of our knowledge,SsPCS is the second PCS gene cloned from a halophyte,and it might contain a different metal sensing capability than the first PCS from Thellungiella halophila.This study provided a new view of halophyte PCS genes in heavy metal tolerance.

Enhanced Cadmium Accumulation in Transgenic Tobacco Expressing the Phytochelatin Synthase Gene of Cynodon dactylon L

Bermudagrass （Cynodon dactylon L. cv. Goldensun） is highly resistant to and accumulates large amounts of cadmium （Cd）. A phytochelatin synthase （PCS） cDNA （CdPCS1） was isolated from this grass by rapid amplification of cDNA ends. The putative CdPCS1 protein shared a high homology with PCS from other plants, with 79% homology at the N-terminal and 47% homology at the C-terminah However, 16 Cys residues were found at the C-terminal of CdPCS1, and among these residues, three positions were different from other PCS proteins. Semiquantitative reverse transcription-polymerase chain reaction analysis showed that Cd stress induced CdPCS1 expression in both roots and leaves in Bermudagrass. We verified that CdPCS1 plays an important role in Cd tolerance in yeast cells by expressing the gene in ABDE1, a Cdsensitive mutant. CdPCS1 was then introduced into tobacco plants. The phytochelatin level in some transgenic tobacco lines increased 3.88-fold more than in wild type plants and Cd accumulation in these transgenic plants was enhanced 3.21-fold accordingly. The results suggested that CdPCS1 could be used as a gene element for phytoremediation in the future.

RNA Interference-mediated Silencing of Phytochelatin Synthase Gene Reduce Cadmium Accumulation in Rice Seeds

Phytochelatins （PCs） play an important role in heavy metal resistance and accumulation. To reduce the accumulation of cadmium （Cd） in rice seeds, the expression of phytochelatin synthase （PCS） gene OsPCS1 was suppressed by RNA interference （RNAi）. A hairpin construct of a PCS fragment was designed in the pRNAi-OsPCS1 under the control of ZMM1, a seed-specific promoter from maize. The construct was introduced into rice （japonica） through Agrobacterium tumefaciens. The RNAi rice plantlets were selected and cultivated in pots exposured to 10 mg/kg Cd. The transcriptional level of OsPCS1 declined in seeds of some RNAi rice compared to the wild type. As a result Cd accumulation was reduced by about half in the seeds of RNAi rice. As expected, no apparent difference of growth appeared between RNAi and wild-type plants. The results suggest that this new approach can be used to control heavy metal accumulation in crops.

Root cell walls and phytochelatins in low-cadmium cultivar of Brassica parachinensis

‘Lvbao-701’ is a cultivar of Chinese flowering cabbage(Brassica parachinensis) that exhibits low cadmium(Cd) accumulation and high Cd tolerance.In this study, this cultivar was compared with a high-Cd accumulating cultivar, ‘Chixin-4’, to characterize the mechanisms influencing Cd accumulation in B. parachinensis. Root cell walls were isolated by dissolving the cytoplasm with an organic solvent, and root Cd and phytochelatin(PC) contents were analyzed. In addition, a PC synthase gene fragment was cloned and expressed under Cd stress conditions. The proportions of Cd bound to root cell walls were higher in the ‘Lvbao-701’ plants(68.32%–76.80%) than in the ‘Chixin-4’ plants(35.36%–54.18%) after exposure to Cd stress. The proportions of Cd bound to root cell walls measured using cell walls isolated with a non-destructive method were higher than those obtained using a conventional method that required grinding and centrifugation. Exposure to Cd stress induced the PC production and resulted in higher PC contents in the ‘Lvbao-701’ plants than in the ‘Chixin-4’ plants. Cloning and expression analysis of a B. parachinensis PC synthase cDNA fragment indicated that PC synthase gene expression was induced by Cd and occurred mainly in the roots of both ‘Lvbao-701’ and ‘Chixin-4’ plants. However, the PC synthase gene expression level was higher in the‘Lvbao-701’ roots than in the ‘Chixin-4’ roots. Therefore, a higher abundance of Cd in the root cell walls of ‘Lvbao-701’ and up-regulated PC production in the roots are probably the main reasons why ‘Lvbao-701’ exhibits lower Cd translocation to the shoots and higher tolerance to Cd than ‘Chixin-4’.