Identification of lead-regulated genes by suppression subtractive hybridization in the heavy metal accumulator Sesbania drummondii

Abstract

Heavy metal contamination of soils is of widespread occurrence as a result of human, agricultural and industrial activities. Among heavy metals, lead is a potential pollutant that readily accumulates in soils and sediments. Although lead is not an essential element for plants, it gets easily absorbed and accumulated in Sesbania drummondii, which exhibits a significant level of tolerance to lead. The response of a metal tolerant plant to heavy metal stress involves a number of biochemical and physiological pathways. To investigate the overall molecular response of a metal-tolerant plant to lead exposure, suppression subtractive hybridization (SSH) was used to construct a cDNA library enriched in lead induced mRNA transcripts from lead-tolerant Sesbania. Screening the library by reverse Northern analysis revealed that between 20 and 25% of clones selected from the library were differentially regulated in lead treated plants. After differential screening, we isolated several differentially expressed cDNA clones, including a type 2 metallothionein (MT) gene which is involved in detoxification and homeostasis and shown to be differentially regulated in lead treated plants. The data from the reverse Northern analysis was further confirmed with conventional Northern analysis of a select group of genes including MT, ACC synthase/oxidase, cold-, water stress-, and other abiotic stress-induced genes, which are up-regulated rapidly in response to lead treatment. The mRNA levels of MT increased substantially after lead treatment indicating a potential role for it under lead stress in Sesbania. The present results show that SSH can serve as an effective tool for isolating genes induced in response to lead heavy metal tolerance in Sesbania. A better understanding of lead induced gene expression in Sesbania should help select candidates associated with remediation of heavy metal toxicity. The possible link between this result and the heavy-metal response of plants is discussed.

Differential expression of expressed sequence tags in alfalfa roots under aluminum stress

“…By comparing the abundance of a housekeeping gene b-tubulin before and after subtraction, we found the subtraction efficiency of the libraries was high. SSH based on suppression PCR is a good method to find genes differentially expressed in two groups and was used to find genes related to heavy metal tolerance and accumulation in plants in some studies (Srivastava et al 2007;Lewandowska et al 2007;Huang et al 2009a, b).…”

Pb-Stress Induced Oxidative Stress Caused Alterations in Antioxidant Efficacy in Two Groundnut (<i>Arachis hypogaea</i> L.) Cultivars

“…GSH is the direct precursor of phytochelatins (PCs) in a reaction catalyzed by phytochelatin synthase (PCS). It is known that the metal stress alleviates the depletion of GSH [60], and enhance PCs synthesis, resulting in an increased metal tolerance of plants [71]- [73]. Nouairi et al [71], reported that the level of PC-SH in tolerant plants was significantly increased under Cd stress in Brassica juncea.…”

Molecular cloning and characterization of phosphorus starvation responsive genes in common bean (Phaseolus vulgaris L.)

“…The cDNAs generated from plants at low P level served as "testers", while cDNAs generated from plants at high P level were used as "drivers". cDNA digestion, adaptor ligation, hybridization, and PCR ampliWcation were done according to the protocol described earlier (Srivastava et al 2007). Subtracted PCR ampliWed cDNAs were cloned into pCRscript vector using TOPO TA Cloning kit (Invitrogen, USA).…”

“…In this technique diVerentially expressed genes can be normalized and enriched over 1000-fold in a single round of hybridization (Diatchenko et al 1996). SSH has successfully been used on various plant species to isolate genes involved in responses to biotic and abiotic stresses (Zhang et al 2005;Srivastava et al 2007;Venkatachalam et al 2007). In the present investigation, an attempt was made to isolate P starvation-regulated genes from a P-eYcient common bean genotype (G19833) using SSH analysis.…”