To further understand the process of Al-induced citrate secretion from soybean roots, the effect of protein synthesis inhibitor, anion channel blockers, and citrate carrier inhibitors on Al-induced citrate exudation was investigated in Al-resistant soybean cultivar PI 416937. Citrate exudation from roots increased with the increase of Al concentration from 10 to 50 μM and initiated after 4 h of Al exposure. Protein synthesis inhibitor, cycloheximide (CHM; 25 μM) completely inhibited Al-induced citrate secretion during 12-h exposure, suggesting that novel protein synthesis was necessary in Al-induced citrate efflux. Also both anion channel blocker anthracene-9-carboxylic acid (A-9-C) and citrate carrier inhibitor mersalyl acid (Mersalyl) significantly reduced citrate secretion, suggesting that both anion channels in plasma membrane and citrate carriers in mitochondria membrane were the rate limiting factors of Al dependent citrate release. However, Al-induced citrate secretion was insensitive to anion channel blockers phenylglyoxal (PG), 4,4′-diisothiocyanostibene-2,2′-disulfonat (DIDS) and citrate carrier inhibitor pyridoxal 5′-P (PP).

The concentration of soluble aluminium (Al) in the soil solution increases at low pH and the prevalence of toxic Al3+ cations represent the main factor limiting plant growth on acid soils. Citrate secretion from roots is an important Al-tolerance mechanism in many species including soybean. We isolated mitochondria from the roots of an Al-resistant soybean (Glycine max L.) cv. Jiyu 70 to investigate the relationship between citrate metabolism and Al-induced citrate secretion. Spectrophotometric assays revealed that the activities of mitochondrial malate dehydrogenase and citrate synthase increased and aconitase decreased with increasing of Al concentration (0–50 µM) and duration of Al treatment (30 µM Al, 0.5–9 h). Al-induced citrate secretion was inhibited by the citrate synthase inhibitor suramin, and enhanced by the aconitase inhibitor fluorocitric acid. Mersalyl acid, an inhibitor of a citrate carrier located in mitochondria membrane, also suppressed Al-induced citrate secretion. Transcript level of the mitochondrial citrate synthase gene increased in soybean roots exposed to Al, whereas expression of aconitase showed no significant difference. Expression of Gm-AlCT, a gene showing homology to Al-activated citrate transporters was also induced after 4 h in Al treatment. The Al-dependent changes in activity and expression of these enzymes are consistent with them supporting the sustained release of citrate from soybean roots.

Abscisic acid (ABA) plays an important role in mediating some biotic and abiotic stresses. In the present study, to better understand the relationship of ABA production and Aluminum (Al)-resistance in plants, Al-resistance genotype (Jiyu70) of soybean was adopted to investigate the accumulation and transport of ABA in plants exposed to Al. Results showed that exogenous application of ABA and ABA synthesis inhibitor-fluridone respectively increased and reduced endogenous ABA content in root apices of soybean, and results in the corresponding reduction and aggravation of Al toxicity. Increasing of either Al concentration (0–50 μM) or treatment duration (0–12 h, 30 μM Al) cause a higher inhibition of root elongation and ABA accumulation in root apices of soybean. Al-induced enhancement of endogenous ABA production not only was in roots but also in leaves, whereas La3+ (behaves similarly as Al3+ at the level of cell surface) only increased ABA accumulation in roots. In split-root experiments, Al treatment in two parts of roots (Part A, + Al; Part B, + Al) both decreased root elongation and increased ABA accumulation in root apices of soybean. Whereas when only part A of roots was exposed to Al (Part A, + Al; Part B, -Al), endogenous ABA content in root apices increased in part A but inversely in part B, but root elongation inhibition only was found in part A. Using [3H]-ABA radioisotope technique, it was found that [3H]-ABA can transport at the rate of more than 3.2 cm·min−1 in the whole plants, and this can be accelerated by Al supply. In addition, [3H]-ABA tended to distribute in the root part under Al stress. Together, these results suggest that ABA may play an important role in regulating Al resistance of soybean as an Al-stress signal.

Aluminum(Al)-induced organic acid anions secretion is known as one of important Al-resistance mechanisms in plants.Abscisic acid(ABA)plays an important role in mediating some biotic and abiotic stresses.A series of experiments including split-root experiment and time course experiment were carried out to clarify whether ABA is involved in the process of Al-induced secretion of citrate from soybean roots.The results are as follows: 1)ABA accumulation in Al-induced soybean roots is increased under the exogenous ABA application,while ABA accumulation is inhibited under the Fluridone(ABA biosynthesis inhibitor).However,the both applications no effects on either Al induced citrate exudation,or the Al induced increase of citrate content and citrate synthase activity.2)In the split-root experiment(the whole roots of soybean plant were separated equally to Part A and Part B),endogenous ABA content of root apices is increased in part A,while the content is decreased in part B when roots of part A are exposed to Al(Part A,+Al;Part B,-Al),and Al-induced citrate secretion is only found in part A.3)The amount of citrate secretion of Al-tolerant cultivar is higher than that of Al-sensitive cultivar,while the ABA contents of roots of both cultivars have no differences under same Al stress.4)In the time course experiment,both citrate secretion rate and endogenous ABA content are increased with increase of Al treatment time during 0-12 h.During the next 6 h pulse(12-18 h)without Al stress,endogenous ABA is dropped quickly,while citrate secretion rate is increased.The above results suggest that endogenous ABA in soybean roots is not involved in modulating Al-induced citrate exudation.