Hisashi Koiwa

Regulation of protease inhibitors and plant defense

Protease inhibitors are an important element of the plant defense response to insect predation, and may also act to restrict infection by some nematodes. Production of these inhibitors is highly regulated by a signal transduction pathway that is initiated by predation and transduced as a wound response. Local and systemic extracellular inducers of the signal pathway are released by injury. Current evidence suggests that the production of the inhibitors occurs via the octadecanoid pathway, which catalyzes the breakdown of linolenic acid and the formation of jasmonic acid to induce protease inhibitor gene expression.

AtHKT1 is a salt tolerance determinant that controls Na^+ entry into plant roots

Two Arabidopsis thaliana extragenic mutations that suppress NaCl hypersensitivity of the sos3–1 mutant were identified in a screen of a T-DNA insertion population in the genetic background of Col-0 gl1 sos3–1. Analysis of the genome sequence in the region flanking the T-DNA left border indicated that sos3–1 hkt1–1 and sos3–1 hkt1–2 plants have allelic mutations in AtHKT1. AtHKT1 mRNA is more abundant in roots than shoots of wild-type plants but is not detected in plants of either mutant, indicating that this gene is inactivated by the mutations. hkt1–1 and hkt1–2 mutations can suppress to an equivalent extent the Na+ sensitivity of sos3–1 seedlings and reduce the intracellular accumulation of this cytotoxic ion. Moreover, sos3–1 hkt1–1 and sos3–1 hkt1–2 seedlings are able to maintain [K+]int in medium supplemented with NaCl and exhibit a substantially higher intracellular ratio of K+/Na+ than the sos3–1 mutant. Furthermore, the hkt1 mutations abrogate the growth inhibition of the sos3–1 mutant that is caused by K+ deficiency on culture medium with low Ca2+ (0.15 mM) and <200 μM K+. Interestingly, the capacity of hkt1 mutations to suppress the Na+ hypersensitivity of the sos3–1 mutant is reduced substantially when seedlings are grown in medium with low Ca2+ (0.15 mM). These results indicate that AtHKT1 is a salt tolerance determinant that controls Na+ entry and high affinity K+ uptake. The hkt1 mutations have revealed the existence of another Na+ influx system(s) whose activity is reduced by high [Ca2+]ext.

Transcriptional Regulation of Sorghum Defense Determinants against a Phloem-Feeding Aphid

When attacked by a phloem-feeding greenbug aphid (Schizaphis graminum), sorghum (Sorghum bicolor) activates jasmonic acid (JA)- and salicylic acid (SA)-regulated genes, as well as genes outside known wounding and SA signaling pathways. A collection of 672 cDNAs was obtained by differential subtraction with cDNAs prepared from sorghum seedlings infested by greenbug aphids and those from uninfested seedlings. Subsequent expression profiling using DNA microarray and northern-blot analyses identified 82 transcript types from this collection responsive to greenbug feeding, methyl jasmonate (MeJA), or SA application. DNA sequencing analyses indicated that these encoded proteins functioning in direct defense, defense signaling, oxidative burst, secondary metabolism, abiotic stress, cell maintenance, and photosynthesis, as well as proteins of unknown function. In response to insect feeding, sorghum increased transcript abundance of numerous defense genes, with some SA-dependent pathogenesis-related genes responding to greenbug more strongly than to SA. In contrast, only weak induction of MeJA-regulated defense genes was observed after greenbug treatment. However, infestation tests confirmed that JA-regulated pathways were effective in plant defense against greenbugs. Activation of certain transcripts exclusively by greenbug infestation was observed, and may represent unique signal transduction events independent of JA- and SA-regulated pathways. Results indicate that plants coordinately regulate defense gene expression when attacked by phloem-feeding aphids, but also suggest that aphids are able to avoid triggering activation of some otherwise potentially effective plant defensive machinery, possibly through their particular mode of feeding.

Specific interactions between Dicer-like proteins and HYL1/DRB- family dsRNA-binding proteins in Arabidopsis thaliana

Proteins that specifically bind double-stranded RNA (dsRNA) are involved in the regulation of cellular signaling events and gene expression, and are characterized by a conserved dsRNA-binding motif (dsRBM). Here we report the biochemical properties of nine such gene products, each containing one or two dsRBMs: four ArabidopsisDicer-like proteins (DCL1-4), ArabidopsisHYL1 and four of its homologs (DRB2, DRB4, DRB5 and OsDRB1). DCL1, DCL3, HYL1 and the four HYL1 homologs exhibit significant dsRNA-binding activity, indicating that these proteins are involved in RNA metabolism. The dsRBMs from dsRBM-containing proteins (dsRBPs) also function as a protein–protein interaction domain and homo- and heterodimerization are essential for biological functioning of these proteins. We show that DRB4 interacts specifically with DCL4, and HYL1 most strongly interacts with DCL1. These results indicate that each HYL1/DRB family protein interacts with one specific partner among the four Dicer-like proteins. Localization studies using GFP fusion proteins demonstrate that DCL1, DCL4, HYL1 and DRB4 localize in the nucleus, while DRB2 is present in the cytoplasm. Subcellular localizations of HYL1, DRB4, DCL1 and DCL4 further strengthen the notion that HYL1 and DCL1, and DRB4 and DCL4, exist as complexes. The presented data suggest that each member of the HYL1/DRB protein family may individually modulate Dicer function through heterodimerization with a Dicer-like protein in vivo.

A genomics approach towards salt stress tolerance

Abiotic stresses reduce plant productivity. We focus on gene expression analysis following exposure of plants to high salinity, using salt-shock experiments to mimic stresses that affect hydration and ion homeostasis. The approach includes parallel molecular and genetic experimentation. Comparative analysis is employed to identify functional isoforms and genetic orthologs of stress-regulated genes common to cyanobacteria, fungi, algae and higher plants. We analyze global gene expression profiles monitored under salt stress conditions through abundance profiles in several species: in the cyanobacterium SynechocystisPCC6803, in unicellular (Saccharomyces cerevisiae) and multicellular (Aspergillus nidulans) fungi, the eukaryotic alga Dunaliella salina, the halophytic land plant Mesembryanthemum crystallinum , the glycophytic Oryza sativa and the genetic model Arabidopsis thaliana. Expanding the gene count, stress brings about a significant increase of transcripts for which no function is known. Also, we generate insertional mutants that affect stress tolerance in several organisms. More than 400 000 T-DNA tagged lines of A. thaliana have been generated, and lines with altered salt stress responses have been obtained. Integration of these approaches defines stress phenotypes, catalogs of transcripts and a global representation of gene expression induced by salt stress. Determining evolutionary relationships among these genes, mutants and transcription profiles will provide categories and gene clusters, which reveal ubiquitous cellular aspects of salinity tolerance and unique solutions in multicellular species.

OSM1/SYP61: A Syntaxin Protein in Arabidopsis Controls Abscisic Acid–Mediated and Non-Abscisic Acid–Mediated Responses to Abiotic Stress

To identify the genetic loci that control salt tolerance in higher plants, a large-scale screen was conducted with a bialaphos marker–based T-DNA insertional collection of Arabidopsis ecotype C24 mutants. One line, osm1 (for osmotic stress–sensitive mutant), exhibited increased sensitivity to both ionic (NaCl) and nonionic (mannitol) osmotic stress in a root-bending assay. The osm1 mutant displayed a more branched root pattern with or without stress and was hypersensitive to inhibition by Na+, K+, and Li+ but not Cs+. Plants of the osm1 mutant also were more prone to wilting when grown with limited soil moisture compared with wild-type plants. The stomata of osm1 plants were insensitive to both ABA-induced closing and inhibition of opening compared with wild-type plants. The T-DNA insertion appeared in the first exon of an open reading frame on chromosome 1 (F3M18.7, which is the same as AtSYP61). This insertion mutation cosegregated closely with the osm1 phenotype and was the only functional T-DNA in the mutant genome. Expression of the OSM1 gene was disrupted in mutant plants, and abnormal transcripts accumulated. Gene complementation with the native gene from the wild-type genome completely restored the mutant phenotype to the wild type. Analysis of the deduced amino acid sequence of the affected gene revealed that OSM1 is related most closely to mammalian syntaxins 6 and 10, which are members of the SNARE superfamily of proteins required for vesicular/target membrane fusions. Expression of the OSM1 promoter::β-glucuronidase gene in transformants indicated that OSM1 is expressed in all tissues except hypocotyls and young leaves and is hyperexpressed in epidermal guard cells. Together, our results demonstrate important roles of OSM1/SYP61 in osmotic stress tolerance and in the ABA regulation of stomatal responses.

Salt tolerance of Arabidopsis thaliana requires maturation of N-glycosylated proteins in the Golgi apparatus

Protein N-glycosylation in the endoplasmic reticulum (ER) and in the Golgi apparatus is an essential process in eukaryotic cells. Although the N-glycosylation pathway in the ER has been shown to regulate protein quality control, salt tolerance, and cellulose biosynthesis in plants, no biological roles have been linked functionally to N-glycan modifications that occur in the Golgi apparatus. Herein, we provide evidence that mutants defective in N-glycan maturation, such as complex glycan 1 (cgl1), are more salt-sensitive than wild type. Salt stress caused growth inhibition, aberrant root-tip morphology, and callose accumulation in cgl1, which were also observed in an ER oligosaccharyltransferase mutant, staurosporin and temperature sensitive 3a (stt3a). Unlike stt3a, cgl1 did not cause constitutive activation of the unfolded protein response. Instead, aberrant modification of the plasma membrane glycoprotein KORRIGAN 1/RADIALLY SWOLLEN 2 (KOR1/RSW2) that is necessary for cellulose biosynthesis occurred in cgl1 and stt3a. Genetic analyses identified specific interactions among rsw2, stt3a, and cgl1 mutations, indicating that the function of KOR1/RSW2 protein depends on complex N-glycans. Furthermore, cellulose deficient rsw1-1 and rsw2-1 plants were also salt-sensitive. These results establish that plant protein N-glycosylation functions beyond protein folding in the ER and is necessary for sufficient cell-wall formation under salt stress.

Cowpea bruchid Callosobruchus maculatus uses a three-component strategy to overcome a plant defensive cysteine protease inhibitor.

The soybean cysteine protease inhibitor, soyacystatin N (scN), negatively impacts growth and development of the cowpea bruchid, Callosobruchus maculatus[Koiwa et al. (1998) Plant J 14: 371–379]. However, the developmental delay and feeding inhibition caused by dietary scN occurred only during the early developmental stages (the 1st, 2nd and 3rd instars) of the cowpea bruchid. The 4th instar larvae reared on scN diet (adapted) exhibited rates of feeding and development which were comparable to those feeding on an scN‐free diet (unadapted) prior to pupation. Total gut proteolytic capacity at this larval stage significantly increased in the scN‐adapted insects. The elevated enzymatic activity was attributed to a differential expression of insect gut cysteine proteases (representing the major digestive enzymes), and of aspartic proteases. scN degradation by the gut extract was observed only in adapted bruchids, and this activity appeared to be a combined effect of scN‐induced cysteine and aspartic proteases. Thirty cDNAs encoding cathepsin L‐like cysteine proteases were isolated from insect guts, and they were differentially regulated by dietary scN. Our results suggest that the cowpea bruchid adapts to the challenge of scN by qualitative and quantitative remodelling of its digestive protease complement, and by activating scN‐degrading protease activity.

The STT3a Subunit Isoform of the Arabidopsis Oligosaccharyltransferase Controls Adaptive Responses to Salt/Osmotic Stress

Arabidopsis stt3a-1 and stt3a-2 mutations cause NaCl/osmotic sensitivity that is characterized by reduced cell division in the root meristem. Sequence comparison of the STT3a gene identified a yeast ortholog, STT3, which encodes an essential subunit of the oligosaccharyltransferase complex that is involved in protein N-glycosylation. NaCl induces the unfolded protein response in the endoplasmic reticulum (ER) and cell cycle arrest in root tip cells of stt3a seedlings, as determined by expression profiling of ER stress–responsive chaperone (BiP-GUS) and cell division (CycB1;1-GUS) genes, respectively. Together, these results indicate that plant salt stress adaptation involves ER stress signal regulation of cell cycle progression. Interestingly, a mutation (stt3b-1) in another Arabidopsis STT3 isogene (STT3b) does not cause NaCl sensitivity. However, the stt3a-1 stt3b-1 double mutation is gametophytic lethal. Apparently, STT3a and STT3b have overlapping and essential functions in plant growth and developmental processes, but the pivotal and specific protein glycosylation that is a necessary for recovery from the unfolded protein response and for cell cycle progression during salt/osmotic stress recovery is associated uniquely with the function of the STT3a isoform.

Arabidopsis vegetative storage protein is an anti-insect acid phosphatase

Indirect evidence previously suggested that Arabidopsis (Arabidopsis thaliana) vegetative storage protein (VSP) could play a role in defense against herbivorous insects. To test this hypothesis, other AtVSP-like sequences in Arabidopsis were identified through a Basic Local Alignment Search Tool search, and their transcriptional profiles were investigated. In response to methyl jasmonate application or phosphate starvation, AtVSP and AtVSP-like genes exhibited differential expression patterns, suggesting distinct roles played by each member. Arabidopsis VSP2 (AtVSP2), a gene induced by wounding, methyl jasmonate, insect feeding, and phosphate deprivation, was selected for bacterial expression and functional characterization. The recombinant protein exhibited a divalent cation-dependent phosphatase activity in the acid pH range. When incorporated into the diets of three coleopteran and dipteran insects that have acidic gut lumen, recombinant AtVSP2 significantly delayed development of the insects and increased their mortality. To further determine the biochemical basis of the anti-insect activity of the protein, the nucleophilic aspartic acid-119 residue at the conserved DXDXT signature motif was substituted by glutamic acid via site-directed mutagenesis. This single-amino acid alteration did not compromise the proteins secondary or tertiary structure, but resulted in complete loss of its acid phosphatase activity as well as its anti-insect activity. Collectively, we conclude that AtVSP2 is an anti-insect protein and that its defense function is correlated with its acid phosphatase activity.