Physiological and transcriptional responses of broad bean (Vicia faba L.) leaves to aluminium stress

Aluminium (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 mu M 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 C-13-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 mu M AlCl3 stress were used as testers and cDNA from leaves subjected to 0 mu M 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.