Bunichi Ezaki

Aluminum-Induced Gene Expression and Protein Localization of a Cell Wall-Associated Receptor Kinase in Arabidopsis

Here, we report the aluminum (Al)-induced organ-specific expression of a WAK1 (cell wall-associated receptor kinase 1) gene and cell type-specific localization of WAK proteins in Arabidopsis. WAK1-specific reverse transcriptase-polymerase chain reaction analysis revealed an Al-induced WAK1 gene expression in roots. Short- and long-term analysis of gene expression in root fractions showed a typical “on” and “off” pattern with a first peak at 3 h of Al exposure followed by a sharp decline at 6 h and a complete disappearance after 9 h of Al exposure, suggesting the WAK1 is a further representative of Al-induced early genes. In shoots, upon root Al exposure, an increased but stable WAK1 expression was observed. Using confocal microscopy, we visualized Al-induced closure of leaf stomata, consistent with previous suggestions that the Al stress primarily experienced in roots associated with the transfer of root-shoot signals. Elevated levels of WAK protein in root cells were observed through western blots after 6 h of Al exposure, indicating a lag time between the Al-induced WAK transcription and translation. WAK proteins are localized abundantly to peripheries of cortex cells within the elongation zone of the root apex. In these root cells, disintegration of cortical microtubules was observed after Al treatment but not after the Al analog lanthanum treatments. Tip-growing control root hairs, stem stomata, and leaf stomatal pores are characterized with high amounts of WAKs, suggesting WAKs are accumulating at plasma membrane domains, which suffer from mechanical stress and lack dense arrays of supporting cortical microtubules. Further, transgenic plants overexpressing WAK1 showed an enhanced Al tolerance in terms of root growth when compared with the wild-type plants, making the WAK1 one of the important candidates for plant defense against Al toxicity.

Mechanism of Gene Expression of Arabidopsis Glutathione S -Transferase, AtGST1 , and AtGST11 in Response to Aluminum Stress

The gene expression of two Al-induced Arabidopsis glutathione S-transferase genes, AtGST1 and AtGST11, was analyzed to investigate the mechanism underlying the response to Al stress. An approximately 1-kb DNA fragment of the 5′-upstream region of each gene was fused to a β-glucuronidase (GUS) reporter gene (pAtGST1::GUS and pAtGST11::GUS) and introduced into Arabidopsis ecotype Landsberg erecta. The constructed transgenic lines showed a time-dependent gene expression to a different degree in the root and/or leaf by Al stress. The pAtGST1::GUS gene was induced after a short Al treatment (maximum expression after a 2-h exposure), while the pAtGST11::GUS gene was induced by a longer Al treatment (approximately 8 h for maximum expression). Since the gene expression was observed in the leaf when only the root was exposed to Al stress, a signaling system between the root and shoot was suggested in Al stress. A GUS staining experiment using an adult transgenic line carrying the pAtGST11::GUS gene supported this suggestion. Furthermore, Al treatment simultaneously with various Ca depleted conditions in root region enhanced the gene expression of the pAtGST11::GUS in the shoot region. This result suggested that the degree of Al toxicity in the root reflects the gene response of pAtGST11::GUS in the shoot via the deduced signaling system. Both transgenic lines also showed an increase of GUS activity after cold stress, heat stress, metal toxicity, and oxidative damages, suggesting a common induction mechanism in response to the tested stresses including Al stress.

Aluminum tolerance associated with enhancement of plasma membrane H+-ATPase in the root apex of soybean

Abstract Seventeen soybean cultivars were screened to discern differences in aluminum (Al) sensitivity. The Sowon (Al-tolerant) and Poongsan (Al-sensitive) cultivars were selected for further study by simple growth measurement. Aluminum-induced root growth inhibition was significantly higher in the Poongsan cultivar than in the Sowon cultivar, although the differences depended on the Al concentration (0, 25, 50, 75 or 100 μmol L–1) and the amount of exposure (0, 3, 6, 12 or 24 h). Damage occurred preferentially in the root apex. High-sensitivity growth measurements using India ink implicated the central elongation zone located 2–3 mm from the root apex. The Al content was lower 0–5 mm from the root apices in the Sowon cultivar than in the apices of the Poongsan cultivar when exposed to 50 μmol L–1 Al for 12 h. Furthermore, the citric acid exudation rate was more than twofold higher in the Sowon cultivar. Protein production of plasma membrane (PM) H+-ATPase from the root apices (0–5 mm) was upregulated in the presence of Al for 24 h in both cultivars. This activity, however, decreased in both cultivars treated with Al and the Poongsan cultivar was more severely affected. We propose that Al-induced growth inhibition is correlated with changes in PM H+-ATPase activity, which is linked to the exudation of citric acid in the root apex.

Possible involvement of GDI1 protein, a GDP dissociation inhibitor related to vesicle transport, in an amelioration of zinc toxicity in Saccharomyces cerevisiae

The GDI1 protein related vesicle transport system was studied to investigate the possibility that an exclusion of toxic zinc (Zn) from the cytoplasm ameliorates Zn toxicity in Saccharomyces cerevisiae (yeast). A temperature‐sensitive gdi1 mutant (originally called sec19), in which the GDP dissociation inhibitor becomes inactive at the non‐permissive temperature (37 °C), was more sensitive to Zn than its parental GDI1 strain at 32 °C (a moderately non‐permissive temperature). The relative efflux of cytoplasmic Zn in the gdi1 mutant was lower than that in the control strain. Treatment with a vesicle transport‐specific inhibitor, Brefeldin A, caused an increase of Zn sensitivity and a decrease of Zn efflux in these strains. It is therefore suggested that the GDI1‐related vesicle transport system contributes to Zn tolerance in yeast. Furthermore, changes in the number of Zn‐specific fluorescent granules (zincosomes) were observed by zinquin staining in the mutant cells under Zn treatment at 32 °C and 37 °C. We concluded that the GDI1 protein is implicated in control of vesicle numbers. Collectively, the results suggest that the GDI1protein is involved in Zn efflux via small vesicle trafficking and contributes to the control of cytoplasmic Zn content, allowing yeast to survive in the presence of toxic Zn. Copyright

Physiology and biochemistry of aluminum toxicity and tolerance in crops

Achieving sustainable food production to feed the increasing population of the problematic lands of the world is an enormous challenge. Aluminum (Al) toxicity in the acid soil is a major worldwide problem. Liming and nutrient management technologies are worthless due to high lime requirement, and the effect of liming does not persist for long. Besides this, conventional breeding is useful to manage Al toxicity as some plants have evolved mechanisms to cope with Al toxicity in acid soil. Therefore, understanding of Al tolerance mechanisms is prime necessity for improving Al tolerance in crops. Al resistance mechanisms include mainly Al avoidance (Al exclusion) and/or Al tolerance (detoxification of Al inside the cell) mechanisms. In this chapter, we summarize Al behavior in plant root cell. We include recent findings of Al resistance mechanisms and Al-resistant genes which can be useful to produce cultivars adapted to acid soils.

An S-adenosyl methionine synthetase (SAMS) gene from Andropogon virginicus L. confers aluminum stress tolerance and facilitates epigenetic gene regulation in Arabidopsis thaliana

Candidate clones which conferred Al tolerance to yeast transformants (TFs) were obtained from a cDNA library derived from a highly Al-tolerant poaceae, Andropogon virginicus L. One such clone, AL3A-4, encoded an S-adenosyl methionine synthetase (SAMS) gene. A full-length cDNA was obtained by 5′-RACE, designated AvSAMS1, and introduced into Arabidopsis thaliana to investigate its biological functions under Al stress. Two TF plant lines both showed higher tolerance than the Col-0 ecotype (non-TF) not only for Al stress, but also for Cu, Pb, Zn and diamide stresses, suggesting the AvSAMS1 was a multiple tolerance gene. More than 40 of A. thaliana Al response-genes (Al induced genes and Al repressed genes) were selected from microarray results and then used for investigations of DNA or histone methylation status under Al stress in Col-0 and the AvSAMS1 TF line. The results indicated that Al stress caused alterations of methylation status in both DNA and histone H3 (H3K4me3 and H3K9me3) and that these alterations were different between the AvSAMS1 TF and Col-0, suggesting the differences were AvSAMS1-gene dependent. These results suggested the existence of AvSAMS1-related epigenetic gene-regulation under Al stress.

Role of N-terminal His-rich domain of Oscillatoria brevis Bxa1 in both Ag(I)/Cu(I) and Cd(II)/Zn(II) tolerance.

A CPx-ATPase (named Bxa1) is induced in the cyanobacterium Oscillatoria brevis upon exposure to multiple heavy metal ions. The function of the bxa1 gene was examined by heterologous expression in both Saccharomyces cerevisiae (yeast) and Escherichia coli. Expression of bxa1 in E. coli caused Ag, Cd and Zn tolerance, but in yeast became sensitive to those metals. To reveal the role of the N-terminal His-rich domain (first 35 amino acids) of Bxa1, we constructed E. coli and yeast transformants carrying the bxa1 (Δ35bxa1). The E. coli transformant with Δ35bxa1 was sensitive to heavy metals. On the other hand, the yeast Δ35bxa1 transformant increased heavy-metal tolerance than bxa1 transformant. Fluorescence microscopy suggested that the two fusion proteins Bxa1::mGFP and Δ35Bxa1::mGFP are mainly localized in yeast endoplasmic reticulum (ER). These results imply that the function of Bxa1 was lost by the N-terminus deletion in both E. coli and yeast transformants. This is the first report that the His-rich domain in O. brevis Bxa1 is essential not only to monovalent (Ag+ and Cu+) but also to divalent (Cd2+ and Zn2+) heavy metal tolerance. Moreover, we clarified the toxicity mechanism against Cd using yeast transformants.

Aluminium tolerance caused by phosphate starvation in cultured tobacco cells

The inhibitive effect of aluminium (Al) on the growth of suspension-cultured cells of a nonchlorophyllic cell line of Nicotiana tabacum was estimated by the relative growth of Al- treated to that of untreated control cells during post-treatment culture. Compared with normal cells, phosphate (Pi)-starved cells were more tolerant to Al and the tolerance increased in parallel with decrease in total phosphorus (P) content in the cells. Pi-starved cells exhibited lower net accumulation of Al than normal cells. Both normal and Pi-starved cells treated with Al released Al from the cells into medium, but the extrusion rate was higher in Pi-starved cells. Thus, the Al tolerance exhibited by Pi-starved cells was in part attributed to a decrease in net Al accumulation based on Al efflux. Changes in the pattern of protein synthesis in response to the Al treatment were investigated. The expression of extracellular proteins was enhanced by Al in both normal and Pi- starved cells. Furthermore, most of these proteins were enhanced by Pi starvation. Thus, Al and Pi starvation cause common changes in protein expression. These results suggest that the proteins induced by both Al and Pi starvation may play important roles in the mechanism of Al tolerance.