Yongxiong Yu

Phosphorylation and Interaction with the 14-3-3 Protein of the Plasma Membrane H+-ATPase are Involved in the Regulation of Magnesium-Mediated Increases in Aluminum-Induced Citrate Exudation in Broad Bean (Vicia faba. L)

Several studies have shown that external application of micromolar magnesium (Mg) can increase the resistance of legumes to aluminum (Al) stress by enhancing Al-induced citrate exudation. However, the exact mechanism underlying this regulation remains unknown. In this study, the physiological and molecular mechanisms by which Mg enhances Al-induced citrate exudation to alleviate Al toxicity were investigated in broad bean. Micromolar concentrations of Mg that alleviated Al toxicity paralleled the stimulation of Al-induced citrate exudation and increased the activity of the plasma membrane (PM) H(+)-ATPase. Northern blot analysis shows that a putative MATE-like gene (multidrug and toxic compound extrusion) was induced after treatment with Al for 4, 8 and 12 h, whereas the mRNA abundance of the MATE-like gene showed no significant difference between Al plus Mg and Al-only treatments during the entire treatment period. Real-time reverse transcription-PCR (RT-PCR) and Western blot analyses suggest that the transcription and translation of the PM H(+)-ATPase were induced by Al but not by Mg. In contrast, immunoprecipitation suggests that Mg enhanced the phosphorylation levels of VHA2 and its interaction with the vf14-3-3b protein under Al stress. Taken together, our results suggest that micromolar concentrations of Mg can alleviate the Al rhizotoxicity by increasing PM H(+)-ATPase activity and Al-induced citrate exudation in YD roots. This enhancement is likely to be attributable to Al-induced increases in the expression of the MATE-like gene and vha2 and Mg-induced changes in the phosphorylation levels of VHA2, thus changing its interaction with the vf14-3-3b protein.

Transcriptional and physiological changes of alfalfa in response to aluminium stress

Medicago 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 μ m 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 μ m Al stress. The analysis of physiological changes indicated that hydrogen peroxide (H 2 O 2 ) and malondialdehyde levels were elevated rapidly under 5 μ m 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 μ m 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.

Changes in the Activity and Transcription of Antioxidant Enzymes in Response to Al Stress in Black Soybeans

In this study, the effects of Al stress on the activity and transcription of antioxidant enzymes were investigated in an acid-resistant black soybean (RB) and an acid-sensitive black soybean (SB) under hydroponic conditions to further clarify the role of antioxidant enzymes in the plant’s response to Al stress. The results indicated that oxidative stress was induced in the roots and leaves of RB and SB and that the stress level was higher in SB than in RB. Changes in the catalase (CAT) activity in response to Al stress occurred faster in RB roots and leaves than in SB. As the duration of Al stress increased, the peroxidase (POD) activity was enhanced more pronouncedly in RB roots and leaves than in SB. The activity of superoxide dismutase (SOD) in the roots and leaves of RB and SB was not responsive to Al stress. A high transcription level of a selected POD gene was detected in RB leaves, but no transcription of this POD gene was observed in SB leaves under Al stress. Moreover, the transcription level of this POD gene was higher in RB roots than in SB roots. Under Al stress, the transcription of two selected SOD genes showed an increasing trend in RB but decreased in SB. Furthermore, the transcription levels of these two selected SOD genes were always higher in RB than in SB. The above results suggest that not only does RB have a higher level of antioxidant enzyme activities but also that antioxidant enzyme genes can be upregulated by Al stress. This may be an important mechanism for RB to deal with oxidative stress induced by Al toxicity.

Physiological and molecular responses of broad bean (Vicia faba L.) to aluminum stress

In this study, the responses of broad bean cultivars resistant (YD) and sensitive (AD) to aluminum (Al) stress were investigated at physiological and molecular levels. The results showed that Al induced more citrate exudation in YD roots than that in AD roots, suggesting that citrate exudation is involved in broad bean Al resistance. The analyses for oxidative stress levels and antioxidant enzyme activities indicated that YD had a strong ability to cope with the oxidative stress induced by Al. To investigate the molecular responses of broad bean to Al stress further, a forward suppression subtractive hybridization cDNA library was constructed to identify Al-responsive genes in YD roots treated with 50-μM Al for a 24-h period. Of the obtained 162 high-quality ESTs, genes related to antioxidant enzymes including copper-zinc superoxide dismutase (SOD), class III peroxidase (POD) and germin-like protein (GEP) were up-regulated. Higher transcription levels of SOD and POD were observed in YD but not in AD roots, which is in agreement with the enhanced activities of antioxidant enzymes in YD roots under Al stress conditions. Furthermore, the up-regulated expression of vha2, encoding a plasma membrane (PM) H+-ATPase, and 14-3-3b in YD roots under Al stress were also detected and confirmed by RT-PCR analysis. Western and immunoprecipitation analyses indicated that Al-enhanced expressions and interactions of the PM H+-ATPase and 14-3-3 proteins might be involved in the regulation of citrate secretion in YD roots under Al stress.

Aluminium-inhibited NO3- uptake is related to Al-increased H2O2 content and Al-decreased plasma membrane ATPase activity in the root tips of Al-sensitive black soybean

In this study, Al-sensitive black soybean (Glycine max (L.) Merr.) specimens were treated in Hoagland solutions containing 50-400µM Al for 1-4 days. The measurement for NO3- uptake showed that the NO3- uptake decreased gradually as the Al concentration and treatment time increased, suggesting that Al stress significantly reduced the NO3- uptake by soybean. Under 100-µM Al stress for 4 days, the plasma membrane (PM) ATPase activity (inorganic phosphate (Pi) release), H+ pump activity, phosphorylation of PM ATPase and its interaction with 14-3-3 protein in soybean root tips were all smaller than those in the root tips of control plants. The addition of 150µM Mg2+ in Al treatment solutions significantly alleviated the Al inhibition of NO3- uptake in soybean. The presence of Mg2+ in a 100-µM Al solution pronouncedly enhanced PM ATPase activity, H+ pump activity, phosphorylation of PM ATPase and its interaction with 14-3-3 protein in soybean root tips. The application of 2mM ascorbic acid (AsA, an H2O2 scavenger) in Al treatment solutions significantly decreased Al-inhibited NO3- uptake in soybean. The cotreatment of soybeans with 2mM AsA and 100µM Al significantly reduced H2O2 accumulation and increased the PM ATPase activity, H+ pump activity, phosphorylation of PM H+-ATPase and its interaction with 14-3-3 protein in soybean root tips. The evidence suggested that Al-inhibited NO3- uptake is related to Al-increased H2O2 content and Al-decreased phosphorylation of PM ATPase and its interaction with 14-3-3 protein as well as PM ATPase activity in the root tips of soybean.