Involvement of S-type anion channels in disease resistance against an oomycete pathogen in Arabidopsis seedlings.

Pharmacological indications suggest that anion channel-mediated plasma membrane (PM) anion efflux is crucial in early defense signaling to induce immune responses and programmed cell death in plants. Arabidopsis SLAC1, an S-type anion channel required for stomatal closure, is involved in cryptogein-induced PM Cl- efflux to positively modulate the activation of other ion fluxes, production of reactive oxygen species and a wide range of defense responses including hypersensitive cell death in tobacco BY-2 cells. We here analyzed disease resistance against several pathogens in multiple mutants of the SLAC/SLAH channels of Arabidopsis. Resistance against a biotrophic oomycete Hyaloperonospora arabidopsidis Noco2 was significantly enhanced in the SLAC1-overexpressing plants than in the wild-type, while that against a bacteria Pseudomonas syringae was not affected significantly. Possible regulatory roles of S-type anion channels in plant immunity and disease resistance against bacterial and oomycete pathogens is discussed.

Functional characterization of an aluminum (Al)-inducible transcription factor, ART2, revealed a different pathway for Al tolerance in rice.

High aluminum (Al) tolerance in rice (Oryza sativa) is controlled by a Cys2His2-type zinc finger transcription factor ART1 (Al resistance transcription factor 1). There are five close homologs of ART1 in the rice genome, but the role of these homologs is unknown. We functionally characterized one of the ART1 homologs, ART2, in terms of tissue and spatial expression, subcellular localization, transcriptional activation activity, and phenotypic analysis of the knockout lines. ART2 was localized to the nucleus and showed a transcriptional activation potential in yeast. ART2 was mainly expressed in the roots, but the expression level was much lower than that of ART1. The ART2 expression was rapidly induced by Al in the roots of the wild-type rice, but not in art1 mutant. Knockout of ART2 resulted in increased sensitivity to Al toxicity, but did not alter sensitivity to different pH values. Expression profile analysis by RNA-sequencing showed that ART2 was not involved in activation of genes regulated by ART1; however, four genes seems to be regulated by ART2, which are implicated in Al tolerance. These results indicate that ART1 and ART2 regulate different pathways leading to Al tolerance, and ATR2 plays a supplementary role in Al tolerance in rice.

Expression of barley Glutathione S-Transferase13 gene reduces accumulation of reactive oxygen species by trichothecenes and paraquat in Arabidopsis plants.

Glutathione S-transferases (GSTs) play an important role in the detoxification of reactive oxygen species (ROS) and toxic compounds. We found that the barley phi class GST (HvGST13) gene is upregulated by trichothecene phytotoxin produced by the fungal pathogen Fusarium graminearum in barley. Trichothecene phytotoxins such as DON and T-2 toxin induce accumulation of ROS and cell death in plants. It is known that the death of host cells contributes to the virulence of F. graminearum during the later stages of infection. To characterize the role of the HvGST13 gene, we generated Arabidopsis plants in which HvGST13 was overexpressed. Growth inhibition by DON and T-2 toxin was significantly alleviated in the HvGST13ox Arabidopsis plants compared with the wild type. Accumulation of ROS and cell death apparently decreased in HvGST13ox Arabidopsis plants treated with trichothecene. Paraquat herbicide is well known to induce the generation of ROS in plants. Paraquat-induced growth retardation was also suppressed in the HvGST13ox Arabidopsis plants compared with wild type. The inoculation of F. graminearum causes disease symptoms that are markedly decreased in HvGST13ox Arabidopsis plants compared to those in the wild type. Therefore, the HvGST13 gene suppressed the phytotoxic activity of trichothecenes in plants, possibly by the scavenging of ROS.

The Plant-Specific RAB5 GTPase ARA6 is Required for Starch and Sugar Homeostasis in Arabidopsis thaliana.

Endosomal trafficking plays integral roles in various eukaryotic cell activities. In animal cells, a member of the RAB GTPase family, RAB5, is a key regulator of various endosomal functions. In addition to orthologs of animal RAB5, plants harbor the plant-specific RAB5 group, the ARA6 group, which is conserved in land plant lineages. In Arabidopsis thaliana, ARA6 and conventional RAB5 act in distinct endosomal trafficking pathways; ARA6 mediates trafficking from endosomes to the plasma membrane, whereas conventional RAB5 acts in endocytic and vacuolar trafficking pathways. ARA6 is also required for normal salt and osmotic stress tolerance, although the functional link between ARA6 and stress tolerance remains unclear. In this study, we investigated ARA6 function in stress tolerance by monitoring broad-scale changes in gene expression in the ara6 mutant. A comparison of the expression profiles between wild-type and ara6-1 plants revealed that the expression of the Qua-Quine Starch (QQS) gene was significantly affected by the ara6-1 mutation. QQS is involved in starch homeostasis, consistent with the starch content decreasing in the ara6 mutants to approximately 60% of that of the wild-type plant. In contrast, the free and total glucose content increased in the ara6 mutants. Moreover, the proliferation of Pseudomonas syringae pv. tomato DC3000 was repressed in ara6 mutants, which could be attributed to the elevated sugar content. These results suggest that ARA6 is responsible for starch and sugar homeostasis, most probably through the function of QQS.

Comparative genome-wide transcriptional analysis of Al-responsive genes reveals novel Al tolerance mechanisms in rice.

Rice (Oryza sativa) is the most aluminum (Al)-tolerant crop among small-grain cereals, but the mechanism underlying its high Al resistance is still not well understood. To understand the mechanisms underlying high Al-tolerance, we performed a comparative genome-wide transcriptional analysis by comparing expression profiling between the Al-tolerance cultivar (Koshihikari) and an Al-sensitive mutant star1 (SENSITIVE TO AL RHIZOTOXICITY 1) in both the root tips and the basal roots. Exposure to 20 µM AlCl(3) for 6 h resulted in up-regulation (higher than 3-fold) of 213 and 2015 genes including 185 common genes in the root tips of wild-type and the mutant, respectively. On the other hand, in the basal root, genes up-regulated by Al were 126 and 2419 including 76 common genes in the wild-type and the mutant, respectively. These results indicate that Al-response genes are not only restricted to the root tips, but also in the basal root region. Analysis with genes up- or down-regulated only in the wild-type reveals that there are other mechanisms for Al-tolerance except for a known transcription factor ART1-regulated one in rice. These mechanisms are related to nitrogen assimilation, secondary metabolite synthesis, cell-wall synthesis and ethylene synthesis. Although the exact roles of these putative tolerance genes remain to be examined, our data provide a platform for further work on Al-tolerance in rice.

Identification of a cis-acting element of ART1, a C2H2-type zinc-finger transcription factor for aluminum tolerance in rice.

Rice (Oryza sativa) is one of the most aluminum (Al)-tolerant species among small-grain cereals. Recent identification of a transcription factor AL RESISTANCE TRANSCRIPTION FACTOR1 (ART1) revealed that this high Al tolerance in rice is achieved by multiple genes involved in detoxification of Al at different cellular levels. ART1 is a C2H2-type zinc-finger transcription factor and regulates the expression of 31 genes in the downstream. In this study, we attempted to identify a cis-acting element of ART1. We used the promoter region of SENSITIVE TO AL RHIZOTOXICITY1, an Al tolerance gene in the downstream of ART1. With the help of gel-shift assay, we were able to identify the cis-acting element as GGN(T/g/a/C)V(C/A/g)S(C/G). This element was found in the promoter region of 29 genes among 31 ART1-regulated genes. To confirm this cis-acting element in vivo, we transiently introduced this element one or five times tandemly repeated sequence with 35S minimal promoter and green fluorescent protein reporter together with or without ART1 gene in the tobacco (Nicotiana tabacum) mesophyll protoplasts. The results showed that the expression of green fluorescent protein reporter responded to ART1 expression. Furthermore, the expression increased with repetition of the cis-acting element. Our results indicate that the five nucleotides identified are the target DNA-binding sequence of ART1.

DEAR1, a transcriptional repressor of DREB protein that mediates plant defense and freezing stress responses in Arabidopsis.

Plants have evolved intricate mechanisms to respond and adapt to a wide variety of biotic and abiotic stresses in their environment. The Arabidopsis DEAR1 (DREB and EAR motif protein 1; At3g50260) gene encodes a protein containing significant homology to the DREB1/CBF (dehydration-responsive element binding protein 1/C-repeat binding factor) domain and the EAR (ethylene response factor-associated amphiphilic repression) motif. We show here that DEAR1 mRNA accumulates in response to both pathogen infection and cold treatment. Transgenic Arabidopsis overexpressing DEAR1 (DEAR1ox) showed a dwarf phenotype and lesion-like cell death, together with constitutive expression of PR genes and accumulation of salicylic acid. DEAR1ox also showed more limited P. syringae pathogen growth compared to wild-type, consistent with an activated defense phenotype. In addition, transient expression experiments revealed that the DEAR1 protein represses DRE/CRT (dehydration-responsive element/C-repeat)-dependent transcription, which is regulated by low temperature. Furthermore, the induction of DREB1/CBF family genes by cold treatment was suppressed in DEAR1ox, leading to a reduction in freezing tolerance. These results suggest that DEAR1 has an upstream regulatory role in mediating crosstalk between signaling pathways for biotic and abiotic stress responses.

Salicylic acid and a chitin elicitor both control expression of the CAD1 gene involved in the plant immunity of Arabidopsis.

The Arabidopsis mutant cad1 (constitutively activated cell death 1) shows a phenotype that mimics hypersensitive response (HR)-like cell death. The CAD1 gene, which encodes a protein containing a domain with significant homology to the MACPF (membrane attach complex and perforin) domain of complement components and perforin, is likely to control plant immunity negatively and has a W-box cis-element in its promoter region. We found that expression of the CAD1 gene and other W-box containing genes, such as NPR1 and PR2, was promoted by salicylic acid (SA) and benzothiadiazole (BTH) as a SA agonist. The CAD1 gene was also stimulated by a purified chitin oligosaccharide elicitor (degree of polymerization = 8). This latter control was not under SA, because CAD1 expression was not suppressed in 35SnahG transgenic plants, which are unable to accumulate SA. These expression profiles were confirmed by promoter analysis using pCAD1::GUS transgenic plants. The CAD1 expression promoted by BTH and the chitin elicitor was not suppressed in the npr1 mutant, which is insensitive to SA signaling. These results indicate that the CAD1 gene is regulated by two distinct pathways involving SA and a chitin elicitor: viz., SA signaling mediated through an NPR1-independent pathway, and chitin elicitor signaling, through an SA-independent pathway. Three CAD1 homologs that have multiple W-box elements in their promoters were also found to be under the control of SA.

The Arabidopsis gene CAD1 controls programmed cell death in the plant immune system and encodes a protein containing a MACPF domain.

To clarify the processes involved in plant immunity, we have isolated and characterized a single recessive Arabidopsis mutant, cad1 (constitutively activated cell death 1), which shows a phenotype that mimics the lesions seen in the hypersensitive response (HR). This mutant shows spontaneously activated expression of pathogenesis-related (PR) genes, and leading to a 32-fold increase in salicylic acid (SA). Inoculation of cad1 mutant plants with Pseudomonas syringae pv tomato DC3000 shows that the cad1 mutation results in the restriction of bacterial growth. Cloning of CAD1 reveals that this gene encodes a protein containing a domain with significant homology to the MACPF (membrane attack complex and perforin) domain of complement components and perforin proteins that are involved in innate immunity in animals. Furthermore, cell death is suppressed in transgenic cad1 plants expressing nahG, which encodes an SA-degrading enzyme. We therefore conclude that the CAD1 protein negatively controls the SA-mediated pathway of programmed cell death in plant immunity.

Knock-out of the plastid ribosomal protein S21 causes impaired photosynthesis and sugar-response during germination and seedling development in Arabidopsis thaliana.

To clarify the mechanism of sugar-response of higher plants, the ghs1 (glucose hypersensitive) mutant of Arabidopsis was isolated and characterized. The ghs1 mutant had an increased sensitivity to glucose, showing a dramatic inhibition of chlorophyll synthesis and developmental arrest of leaves when grown on medium containing more than 5% glucose; the wild type required exposure to 7% glucose to show the same response. The ghs1 mutant is a single recessive loss-of-function mutation caused by a T-DNA insertion in the GHS1 gene (At3g27160), which encodes the plastid 30S ribosomal protein S21. The mutant showed: (1) reduction in the translation product but not the transcript for plastid-encoded rbcL, (2) reduction in photosynthetic activity monitored with pulse-amplitude modulated fluorometry, (3) impaired chloroplast development, as observed by electron microscopy. These results indicate that the deficiency of such chloroplast functions as photosynthetic activity observed in the ghs1 mutant is caused by impaired plastid protein synthesis associated with loss of ribosomal S21 protein. Relationships between the GHS1 gene and sugar-response are discussed.