Mechanisms Of Al-stimulated Citrate Efflux In Rice Bean Roots And The Possible Role Of Artificial Synthetic Polypeptides Expressing In Yeast In Al Resistance

Aluminum (Al) toxicity is one of the most significant constraints limiting plant growth and crop production in acid soils. Some plant species or genotypes have evolved sophisticated mechanisms to cope with Al stress. The efflux of organic acid anions from plant roots is considered as one of the most important mechanisms by which plant detoxifies Al toxicity. Two patterns of Al-stimulated efflux of organic acid anions have been proposed on the basis of the timing of efflux. However, the processes leading to the efflux of organic acid anions in response to Al stress is still unclear. Furthermore, plant tissues are abundant in amino acids and some amino acids with negative charges especially aspartate and glutamate with two carboxylic groups and polypeptides enriching in these amino acids have theoretically the capacity to chelate Al³⁺ strongly and thereby reducing Al toxicty. However, so far there is no evidence that plants or microbes detoxify Al toxicity by exuding such polypeptides. Therefore, the objectives of the present study are to: 1) identify the key step leading to the efflux of citrate from rice bean, a pattern II plant species (there is a lag phase between onset of Al stress and organic acid anions efflux), roots in response to Al stress and 2) preliminary examine the possibility whether the artificial synthetic polypeptides expressing in yeast can enhance Al resistance. The results were as follows: 1. The key step leading to the efflux of citrate from rice bean roots in response to AlstressAluminum (Al) stimulates the specific efflux of citrate from the apices of rice bean (Vigna umbellata [Thunb.] Ohwi & Ohashi) roots. This response was delayed at least 3 h when exposed to 50 μM Al, indicating some inducible process leading to citrate efflux is involved in the roots of rice bean. Thirteen-day P deficiency and 50 uM La treatments failed to induce citrate efflux, indicating that the efflux of citrate was specific to Al stress. We examined the possible reasons responsible for the delayed response using several antagonists of anion channels and citrate carriers, anda kind of protein-synthesis inhibitor, cycloheximide. Results indicated that either anion-channel inhibitors or citrate-carrier inhibitors can inhibit Al-stimulated citrate efflux, with the anthrancene-9-carboxylic acid (an anion-channle inhibitor) and phenylisothiocyanate (a citrate-carrier inhibitor) the most effective inhibitors used. A 6-h pulse of 50 uM Al induced significant increase of citrate content in root apices and release of citrate. However, the increase in citrate content precedes the efflux. Furthermore, the release of citrate was inhibited by A-9-C and PI, indicating the importance of citrate carrier on mitochondrial membrane and anion channle on plasma membrane for Al-stimulated citrate efflux. Twenty-uL of cycloheximide also significantly inhibited Al-stimulated citrate efflux, confirming the gene activation is required for the Al-stimulated citrate efflux. These results indicate that the activation of genes possibly encoding citrate transporters is involved in the Al-stimulated citrate efflux. 2. The role of the artificial synthetic polypeptides in yeast Al resistance.The acidic polypeptides, poly(L-aspartic acid) and poly(L-glutamic acid) and the serum protein, transferring, have been shown to bind Al3+ in a number of biochemical and functional assays. We hypothesized that plants or microbes with ability to exude these polypeptides can effectively bind Al3+ and detoxifying Al toxicity. Here, we preliminary tested the hypothesis. We designed 3 gene fragments expressing mainly L-Asp and L-Glu and some others for insertion. Vector was constructed according to the methanol-inducible Pichia pastoris yeast expression system. The results showed that 3 anticipated polypeptides successfully expressed in Pichia pastoris yeast. However, whether these polypeptides can secrete and their role in enhancing Al resistance has yet to be investigated in the near future.