Expression profiling of Crambe abyssinica under arsenate stress identifies genes and gene networks involved in arsenic metabolism and detoxification

Abstract

Background: Arsenic contamination is widespread throughout the world and this toxic metalloid is known to cause cancers of organs such as liver, kidney, skin, and lung in human. In spite of a recent surge in arsenic related studies, we are still far from a comprehensive understanding of arsenic uptake, detoxification, and sequestration in plants. Crambe abyssinica, commonly known as 'abyssinian mustard', is a non-food, high biomass oil seed crop that is naturally tolerant to heavy metals. Moreover, it accumulates significantly higher levels of arsenic as compared to other species of the Brassicaceae family. Thus, C. abyssinica has great potential to be utilized as an ideal inedible crop for phytoremediation of heavy metals and metalloids. However, the mechanism of arsenic metabolism in higher plants, including C. abyssinica, remains elusive.

Results: To identify the differentially expressed transcripts and the pathways involved in arsenic metabolism and detoxification, C. abyssinica plants were subjected to arsenate stress and a PCR-Select Suppression Subtraction Hybridization (SSH) approach was employed. A total of 105 differentially expressed subtracted cDNAs were sequenced which were found to represent 38 genes. Those genes encode proteins functioning as antioxidants, metal transporters, reductases, enzymes involved in the protein degradation pathway, and several novel uncharacterized proteins. The transcripts corresponding to the subtracted cDNAs showed strong upregulation by arsenate stress as confirmed by the semi-quantitative RT-PCR.

Conclusions: Our study revealed novel insights into the plant defense mechanisms and the regulation of genes and gene networks in response to arsenate toxicity. The differential expression of transcripts encoding glutathione-S-transferases, antioxidants, sulfur metabolism, heat-shock proteins, metal transporters, and enzymes in the ubiquitination pathway of protein degradation as well as several unknown novel proteins serve as molecular evidence for the physiological responses to arsenate stress in plants. Additionally, many of these cDNA clones showing strong upregulation due to arsenate stress could be used as valuable markers. Further characterization of these differentially expressed genes would be useful to develop novel strategies for efficient phytoremediation as well as for engineering arsenic tolerant crops with reduced arsenic translocation to the edible parts of plants.

Figure 1

Arsenic accumulation in C. abyssinica and other Brassica species seedlings grown in MS medium with different concentrations of As. Total As contents in shoot tissues of C. abyssinica (Ca) compared with Brassica napus (Bn), B. compestris cv Turkey (Bc Turkey), B. compestris cv yellow sarson (Bc YS), B. oleracea (Bo) and B. rapa (Br).

Figure 2

Effect of various concentrations of arsenate on fresh biomass accumulation in C. abyssinica seedlings. Fresh weight shown here is the average of 20 seedlings.

Figure 3

Colony Array for differential screening of subtracted cDNA clones. Membranes containing colonies expressing subtracted cDNAs hybridized with ³²P-labeled forward subtracted probe (A) and reverse subtracted probe (B). Dotted arrows indicate false positives and solid arrows indicate positive colonies.

Figure 4

Semi-quantitative RT-PCR analysis of the C. abyssinica transcripts corresponding to the subtracted cDNAs after 6, 12 and 24 hrs of arsenate treatments. The details about the subtracted cDNAsequences identified by their serial numbers are given in Table 1. The number of optimized PCR cycles used for amplification for each cDNA clone is written on the right hand side of the panel for each cDNA. Actin2 gene, ACT2, was used as an internal control for equal loading of cDNA template of each clone.

Figure 5

Semi-quantitative RT-PCR analysis of the C. abyssinica cDNAs in response to phosphate deficiency. Phosphate deficiency at 6, 12 and 24 hrs for short term (A), and 3 and 7 days for long-term exposure (B). Sequences are identified by their serial numbers as shown in Table 1. The number of optimized PCR cycles used for amplification for each cDNA clone is written on the right hand side of the panel for each cDNA. Actin2 gene, ACT2, was used as an internal control for equal loading of cDNA template of each clone.