Studies On The Accumulation Mechanisms And Related Synthases Of Toxin In Lathyrus Sativus

Grass pea (Lathyrus sativus L.), a highly drought-tolerant grain legume with long cropping history, is now grown to a lesser extent in the arid and semi-arid regions of the world because of the presence of the toxinβ-N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP) and low levels of sulfur-containing nutrients in seeds. The present study was therefore carried out to further understand mechanisms of P-ODAP accumulation triggered by environmental factors and evaluate the possible relationship between P-ODAP accumulation and cysteine synthases. A detailed knowledge of these may be essential for the development of grass pea new varieties with low toxin and high sulfur-containing amino acids. Firstly, the influences of environmental factors on P-ODAP contents were reviewed in the study. Secondly, the contents ofβ-ODAP, O2·- and H2O2 in the different leaves were analyzed. Finally,β-(isoxazoline-5-on-2-yl)- -L-alanine (BIA), a biosynthetic precursor ofβ-ODAP, was separated, and isoxazoline-5-one, the precuror of BIA, was chemically synthesized. Meanwhile, the cysteine synthase (CSase) family, which were reported to involve in the BIA synthesis, was mainly investigated. In addition, the relationship between BIA and glutathione (GSH) contents was also evaluated. The main results are the following:1 The characteristics of P-ODAP accumulation under both developmental and environmental stresses indicate that its biosynthesis in the tissues may be related with the mobilization of nitrogen caused by senescing processes. Thus, those nitrogen mobilizations may provide the substrates forβ-ODAP synthesis.2 There is a negative correlation between content of P-ODAP and both O2·- and H2O2 in grass pea tissues by using HPLC analysis and histochemical staining. It can therefore be concluded that low level of ROS is necessary for P-ODAP accumulation.3 The separation of BIA from grass pea 3-day old seedlings was carried out using cation exchange chromatography on Dowex 50W(H+). The separation with grass pea seedlings has been shown to be more effective than with pea (Pisum sativum) seedlings in terms of interfering factors, costs and production.4 Isoxazoline-5-one was synthesized by interaction of ethyl propiolate and hydroxylamine in alkaline medium. The results showed that the oxidative side-reactions were inhibited effectively under the protection of nitrogen, and thus the great increase in production was obtained.5 Electrophoretic analysis of grass pea CSase isoenzymes were conducted using native polyacrylamide gel electrophoresis (PAGE) with 5-15% gradient gels combined with an enzyme activity stain. The well separation of four types of isoenzymes was achieved on native PAGE gels. These isoenzymes were designated tentatively asⅠ,Ⅱ,ⅢandⅣbased on the molecular weight, of which I and IV were found for the first time in the present study.6 Activity analysis of both CSase andβ-cyanoalanine synthase (CASase) combined with the patterns of isoenzyme electrophoresis indicated that enzymeⅠwas actually a CASase, whileⅡ,Ⅲand IVall belonged to CSase, of whichⅡandⅢwere major isoenzymes of grass pea CSase family. A great abundance of enzymeⅠwas observed in cotyledon and etiolated seedlings, while a significant increase in enzymeⅡactivity was found in green tissues.The relative abundance of both enzyme III andⅣwas almost unchanged in all tissues.7 The BIA content was found to be positively correlated with activity of CASase, but not CSase. Moreover, the relative synthetic rate of BIA by CASase from isoxazoline-5-one and Cys was nearly ten times greater than those from isoxazoline-5-one and OAS. Therefore, it was reasonable to infer that CASase rather than CSase was responsible for accumulation of BIA during early germination.8 A negative relationship between BIA and GSH contents was found in both 2-and 3-day old grass pea seedlings.Based on the above results, it can therefore conclude that the specific physiological environments in grass pea cells are necessary for synthesis of both BIA andβ-ODAP, and that the low levels of ROS, the relatively high CASase activity and the related substrates may be three essential components of the endogenous environment of theβ-ODAP and BIA-accumulating cells. In these cells, the related substrates may be derived from the degradation of nucleic acids and proteins. In general, low GSH levels are required for cells under low levels of ROS, and thus more Cys is used to synthesize BIA by CASase. This therefore leads to the cells containing the high contents of BIA andβ-ODAP but low levels of ROS and GSH.The cells of grass pea tissues, based on the different physiological states as mentioned above, can be divided into two groups:typeⅠand typeⅡ. TypeⅡcells may be at their early stage of senescence or apoptosis, in which the specific conditions promoting synthesis of BIA andβ-ODAP are generally found. They may be the main contributor to accumulations of BIA andβ-ODAP. TypeⅡcells, however, may be at fast-growing stage with or without ability of synthesis BIA andβ-ODAP, but can accumulateβ-ODAP imported from type I cells.