ALBERT

Description: SUMMARYMedicago sativa is an excellent pasture legume, but it is very sensitive to aluminium (Al) toxicity. To better understand the mechanism of M. sativa sensitivity to Al, a forward suppression subtractive hybridization (SSH) cDNA library for an Al-sensitive cultivar, M. sativa L. cv. Yumu No. 1 (YM1), under 5 μm Al stress over a 24 h period was constructed to analyse changes in its gene expression in response to Al stress. Sequence analysis for the SSH cDNA library generated 291 high-quantity expression sequence tags (ESTs). Of these, 229 were known as functional ESTs, 137 of which have already been reported as Al response genes, whereas the other 92 were potentially novel Al-associated genes. The up-regulation of known Al resistance-associated genes encoding the transcription factor sensitive to proton rhizotoxicity 1 (STOP1) and malate transporter MsALMT1 (Al-activated malate transporter) as well as genes for antioxidant enzymes was observed. Reverse transcription polymerase chain reaction analysis validated the reliability of the SSH data and confirmed the up-regulated expression of STOP1 and MsALMT1 under 5 μm Al stress. The analysis of physiological changes indicated that hydrogen peroxide (H2O2) and malondialdehyde levels were elevated rapidly under 5 μm Al stress, suggesting that severe oxidative stress occurred in the YM1 roots. The up-regulation of antioxidant-related genes might be an important protective mechanism for YM1 in response to the oxidative stress induced by 5 μm Al toxicity. Al-induced malate exudation was increased drastically during the early period after Al treatment, which might have been due to the up-regulation and function of MsALMT and STOP1. However, malate exudation from the YM1 roots declined quickly during the subsequent period, and a gradual decrease in malate content was simultaneously observed in the YM1 roots. This result is in agreement with the observation that organic acid metabolism-associated enzymes such as phosphoenolpyruvate carboxylase, citrate synthase and malate dehydrogenase were not present in the SSH library. This might be a major reason for the YM1 sensitivity to Al.

Description: SummaryAluminium (Al) toxicity is the major factor-limiting crop productivity in acid soils. In the present study, physiological and transcriptional responses of broad bean leaves to Al stress were investigated. Malondialdehyde (MDA) content, H2O2 content and protein carbonyls (PC) level in leaves were increased after 100 μm AlCl3 stress treatment, whereas the total protein content was decreased, compared with the plants without Al treatment. Stomatal closure in leaves of broad bean was increased after Al stress, suggesting that the photosynthesis rate might be affected by Al stress. The relative citrate secretion in leaves was decreased after Al treatment for 24 h according to the 13C-NMR analysis, indicating that citrate in leaves might be transported to the root to chelate Al3+. To investigate the molecular mechanisms of Al toxicity in leaves of broad bean, a suppression subtractive hybridization (SSH) library was constructed to identify up-regulated genes: cDNA from leaves subjected to 12, 24, 48 and 72 h of 50 and 100 μm AlCl3 stress were used as testers and cDNA from leaves subjected to 0 μm AlCl3 treatment for the same lengths of time as above were used as a driver. The SSH analysis identified 156 non-redundant putative Al stress-responsive expressed sequence tags (ESTs) out of 960 clones. The ESTs were categorized into ten functional groups, which were involved in metabolism (0·21), protein synthesis and protein fate (0·10), photosynthesis and chloroplast structure (0·09), transporter (0·08), cell wall related (0·06), signal transduction (0·05), defence, stress and cell death (0·05), energy (0·03), transcription factor (0·03) and unknown proteins (0·30). The effect of Al treatment on expression of 15 selected genes was investigated by reverse transcription polymerase chain reaction (RT–PCR), confirming induction by Al stress. The results indicated that genes involved in organic acid metabolism, transport, photosynthesis and chloroplast structure, defence, stress and cell death might play important roles under Al stress.