Summary The rice bean (Vigna umbellata) root apex specifically secretes citrate through expression activation of Vigna umbellata Multidrug and Toxic Compound Extrusion 1 (VuMATE1) under aluminum (Al3+) stress. However, the underlying mechanisms regulating VuMATE1 expression remain unknown. We isolated and characterized a gene encoding Sensitive to Proton Rhizotoxicity1 (STOP1)‐like protein, VuSTOP1, from rice bean. The role of VuSTOP1 in regulating VuMATE1 expression was investigated using the yeast one‐hybrid assay. We characterized the function of VuSTOP1 in Al3 + ‐ and H+‐tolerance using in planta complementation assays. We demonstrated that VuSTOP1 has transactivation potential. We found that VuSTOP1 expression is inducible by Al3+ and H+ stress. However, although VuSTOP1 binds to the promoter of VuMATE1, the inconsistent tissue localization patterns of VuSTOP1 and VuMATE1 preclude VuSTOP1 as the major factor regulating VuMATE1 expression. In addition, when a protein translation inhibitor increased expression of VuSTOP1, VuMATE1 expression was inhibited. In planta complementation assay demonstrated that VuSTOP1 could fully restore expression of genes involved in H+ tolerance, but could only partially restore expression of AtMATE. We conclude that VuSTOP1 plays a major role in H+ tolerance, but only a minor role in Al3+ tolerance. The differential transcriptional regulation of VuSTOP1 and VuMATE1 reveals a complex regulatory system controlling VuMATE1 expression.

Acyl Activating Enzyme3 (AAE3) was identified to be involved in the catabolism of oxalate, which is critical for seed development and defense against fungal pathogens. However, the role of AAE3 protein in abiotic stress responses is unknown. Here, we investigated the role of rice bean (Vigna umbellata) VuAAE3 in Al tolerance. Recombinant VuAAE3 protein has specific activity against oxalate, with K m = 121 6 8.2 mM and V max of 7.7 6 0.88 mmol min 21 mg 21 protein, indicating it functions as an oxalyl-CoA synthetase. VuAAE3-GFP localization suggested that this enzyme is a soluble protein with no specific subcellular localization. Quantitative reverse transcription-PCR and VuAAE3 promoter-GUS reporter analysis showed that the expression induction of VuAAE3 is mainly confined to rice bean root tips. Accumulation of oxalate was induced rapidly by Al stress in rice bean root tips, and exogenous application of oxalate resulted in the inhibition of root elongation and VuAAE3 expression induction, suggesting that oxalate accumulation is involved in Al-induced root growth inhibition. Furthermore, overexpression of VuAAE3 in tobacco (Nicotiana tabacum) resulted in the increase of Al tolerance, which was associated with the decrease of oxalate accumulation. In addition, NtMATE and NtALS3 expression showed no difference between transgenic lines and wildtype plants. Taken together, our results suggest that VuAAE3-dependent turnover of oxalate plays a critical role in Al tolerance mechanisms.

Formate dehydrogenase (FDH) is involved in various higher plant abiotic stress responses. Here, we investigated the role of rice bean (Vigna umbellata) VuFDH in Al and low pH (H + ) tolerance. Screening of various potential substrates for the VuFDH protein demonstrated that it functions as a formate dehydrogenase. Quantitative reverse transcription-PCR and histochemical analysis showed that the expression of VuFDH is induced in rice bean root tips by Al or H + stresses. Fluorescence microscopic observation of VuFDH-GFP in transgenic Arabidopsis plants indicated that VuFDH is localized in the mitochondria. Accumulation of formate is induced by Al and H + stress in rice bean root tips, and exogenous application of formate increases internal formate content that results in the inhibition of root elongation and induction of VuFDH expression, suggesting that formate accumulation is involved in both H + -and Al-induced root growth inhibition. Over-expression of VuFDH in tobacco (Nicotiana tabacum) results in decreased sensitivity to Al and H + stress due to less production of formate in the transgenic tobacco lines under Al and H + stresses. Moreover, NtMATE and NtALS3 expression showed no changes versus wild type in these over-expression lines, suggesting that herein known Al-resistant mechanisms are not involved. Thus, the increased Al tolerance of VuFDH over-expression lines is likely attributable to their decreased Al-induced formate production. Taken together, our findings advance understanding of higher plant Al toxicity mechanisms, and suggest a possible new route toward the improvement of plant performance in acidic soils, where Al toxicity and H + stress coexist.

Significant secretion of citrate from root apex of rice bean (Vigna umbellata) is delayed by several hours under aluminium (Al) stress. However, the molecular basis of regulation of VuMATE1, a gene encoding an Al-activated citrate transporter, remains unclear. In this study, we used suppression subtractive hybridization together with reverse northern blot analysis and qRT-PCR to identify genes with altered transcript levels in the root apex after treatment with low (5 μM) or high (25 μM) concentration of AlCl3 for a short time (4 h). We found that in addition to VuMATE1, 393 genes showed an early response to Al. Among functionally annotated genes, those related to 'metabolism and energy', 'signal transduction and transcription' and 'transport' was predominantly up-regulated, whereas those associated with 'protein translation, processing and degradation' was predominantly down-regulated. Comparative analysis of transcriptional profiles highlighted candidate genes associated with citrate secretion and revealed several new aspects of the molecular processes underlying Al toxicity and tolerance. Based on the data, it is proposed that metabolic changes represent adaptive mechanisms to Al stress, whereas inhibition of both cell elongation and cell division underlies Al-induced root growth inhibition.

We investigated if elevated CO2 could alleviate the negative effect of high temperature on fruit yield of strawberry (Fragaria × ananassa Duch. cv. Toyonoka) at different levels of nitrogen and also tested the combined effects of CO2, temperature and nitrogen on fruit quality of plants cultivated in controlled growth chambers. Results show that elevated CO2 and high temperature caused a further 12% and 35% decrease in fruit yield at low and high nitrogen, respectively. The fewer inflorescences and smaller umbel size during flower induction caused the reduction of fruit yield at elevated CO2 and high temperature. Interestingly, nitrogen application has no beneficial effect on fruit yield, and this may be because of decreased sucrose export to the shoot apical meristem at floral transition. Moreover, elevated CO2 increased the levels of dry matter-content, fructose, glucose, total sugar and sweetness index per dry matter, but decreased fruit nitrogen content, total antioxidant capacity and all antioxidant compounds per dry matter in strawberry fruit. The reduction of fruit nitrogen content and antioxidant activity was mainly caused by the dilution effect of accumulated non-structural carbohydrates sourced from the increased net photosynthetic rate at elevated CO2. Thus, the quality of strawberry fruit would increase because of the increased sweetness and the similar amount of fruit nitrogen content, antioxidant activity per fresh matter at elevated CO2. Overall, we found that elevated CO2 improved the production of strawberry (including yield and quality) at low temperature, but decreased it at high temperature. The dramatic fluctuation in strawberry yield between low and high temperature at elevated CO2 implies that more attention should be paid to the process of flower induction under climate change, especially in fruits that require winter chilling for reproductive growth.

Aluminum (Al)-induced organic acid secretion from the root apex is an important Al resistance mechanism. However, it remains unclear how plants fine-tune root organic acid secretion which can contribute significantly to the loss of fixed carbon from the plant. Here, we demonstrate that Al-induced citrate secretion from the rice bean root apex is biphasic, consisting of an early phase with low secretion and a later phase of large citrate secretion. We isolated and characterized VuMATE2 as a possible second citrate transporter in rice bean functioning in tandem with VuMATE1, which we previously identified. The time-dependent kinetics of VuMATE2 expression correlates well with the kinetics of early phase root citrate secretion. Ectopic expression of VuMATE2 in Arabidopsis resulted in increased root citrate secretion and Al resistance. Electrophysiological analysis of Xenopus oocytes expressing VuMATE2 indicated VuMATE2 mediates anion efflux. However, the expression regulation of VuMATE2 differs from VuMATE1. While a protein translation inhibitor suppressed Al-induced VuMATE1 expression, it releases VuMATE2 expression. Yeast one-hybrid assays demonstrated that a previously identified transcription factor, VuSTOP1, interacts with the VuMATE2 promoter at a GGGAGG cis-acting motif. Thus, we demonstrate that plants adapt to Al toxicity by fine-tuning root citrate secretion with two separate root citrate transport systems.

Relying on Al-activated root oxalate secretion, and internal detoxification and accumulation of Al, buckwheat is highly Al resistant. However, the molecular mechanisms responsible for these processes are still poorly understood. It is well-known that root apex is the critical region of Al toxicity that rapidly impairs a series of events, thus, resulting in inhibition of root elongation. Here, we carried out transcriptome analysis of the buckwheat root apex (0–1 cm) with regards to early response (first 6 h) to Al stress (20 μM), which is crucial for identification of both genes and processes involved in Al toxicity and tolerance mechanisms. We obtained 34,469 unigenes with 26,664 unigenes annotated in the NCBI database, and identified 589 up-regulated and 255 down-regulated differentially expressed genes (DEGs) under Al stress. Functional category analysis revealed that biological processes differ between up- and down-regulated genes, although ‘metabolic processes’ were the most affected category in both up- and down-regulated DEGs. Based on the data, it is proposed that Al stress affects a variety of biological processes that collectively contributes to the inhibition of root elongation. We identified 30 transporter genes and 27 transcription factor (TF) genes induced by Al. Gene homology analysis highlighted candidate genes encoding transporters associated with Al uptake, transport, detoxification, and accumulation. We also found that TFs play critical role in transcriptional regulation of Al resistance genes in buckwheat. In addition, gene duplication events are very common in the buckwheat genome, suggesting a possible role for gene duplication in the species’ high Al resistance. Taken together, the transcriptomic analysis of buckwheat root apex shed light on the processes that contribute to the inhibition of root elongation. Furthermore, the comprehensive analysis of both transporter genes and TF genes not only deep our understanding on the responses of buckwheat roots to Al toxicity but provide a good start for functional characterization of genes critical for Al tolerance.

Transcriptional regulation is important for plants to respond to toxic effects of aluminium (Al). However, our current knowledge to these events is confined to a few transcription factors. Here, we functionally characterized a rice bean (Vigna umbellata) NAC‐type transcription factor, VuNAR1, in terms of Al stress response. We demonstrated that rice bean VuNAR1 is a nuclear‐localized transcriptional activator, whose expression was specifically upregulated by Al in roots but not in shoot. VuNAR1 overexpressing Arabidopsis plants exhibit improved Al resistance via Al exclusion. However, VuNAR1‐mediated Al exclusion is independent of the function of known Al‐resistant genes. Comparative transcriptomic analysis revealed that VuNAR1 specifically regulates the expression of genes associated with protein phosphorylation and cell wall modification in Arabidopsis. Transient expression assay demonstrated the direct transcriptional activation of cell wall‐associated receptor kinase 1 (WAK1) by VuNAR1. Moreover, yeast one‐hybrid assays and MEME motif searches identified a new VuNAR1‐specific binding motif in the promoter of WAK1. Compared with wild‐type Arabidopsis plants, VuNAR1 overexpressing plants have higher WAK1 expression and less pectin content. Taken together, our results suggest that VuNAR1 regulates Al resistance by regulating cell wall pectin metabolism via directly binding to the promoter of WAK1 and induce its expression.