<i>Wrinkled</i>1 (WRI1) Homologs, AP2-Type Transcription Factors Involving Master Regulation of Seed Storage Oil Synthesis in Castor Bean (<i>Ricinus communis</i>L.)

Among APETALA2 (AP2)-type plant specific transcription factor family, WRINKLED1 (WRI1), has appeared to be a master gene transcriptionally regulating a set of carbon metabolism- and fatty acid synthesis (FAS)-related genes responsible for seed specific triacylglycerols (TAGs) storage in oil plants. B3 type transcription factors, such as ABI3 and FUS3, are known to be involved in seed development, such as seed storage protein synthesis and maturation. Based on the recent whole genome sequence data of castor bean (Ricinus communis L.), putative WRI1 homologs (RcWRI1, RcWRI2) specifically expressed in castor bean seed have been identified by comparing organ specific expression profiles among seed development-related transcription factors, seed storage specific genes (Ricin, RcOleosin) and a set of FAS genes including genes for sucrose synthase (RcSUS2), biotin carboxyl carrier protein (a subunit of acetyl-CoA carboxylase, RcBCCP2) and ketoacyl-acyl carrier protein synthase (RcKAS1). Immunoreactive signals with WRI1, FUS3 and ABI5-related polypeptides were also detected in seed specifically, consistent with the expression profiles of seed development-related genes. The WRI1 binding consensus sites, [CnTnG](n)(7)[CG], designated as the AW-box, were found at the promoter region of RcBCCP2 and RcKAS1. Thus, RcWRI1 possibly play a pivotal role in seed specific TAGs storage during seed development by directly activating FAS -related genes.

Effect of Intertidal Elevation at Tsuyazaki Cove,Fukuoka,Japan on Survival Rate of Horseshoe Crab Tachypleus tridentatus Eggs

Physical factors affecting the survival of Tachypleus tridentatus eggs were investigated by translocating their eggs between the high intertidal zone and the low intertidal zone of a known spawning site.The mean egg survival rates per day were highest in the mid intertidal zone(45.1%±25.4%)and the lowest in the low intertidal zone(13.3%±27.6%).Differences in the elevation,air exposure time,and water content of the spawning ground were significant factors determining the egg survival rates.Excessive or insufficient air exposure time resulted in inadequate water content at higher and lower intertidal zones and could reduce egg survival.On the other hand,moderate saturation and dehydration were repeated with each tidal movement in the mid intertidal zone.This dynamic is considered as one of the crucial factors for the survival of eggs and is considered optimal for spawning.Therefore,the protection of the mid intertidal zone is imperative for maximizing the egg survival rate in Tsuyazaki Cove where almost all suitable nesting sites have disappeared due to coastal development.By protecting these optimal sites for spawning and recovering other optimal sites on suitable beaches,a positive contribution can be made to future management and conservation.The study also suggests that translocating eggs from marginal to optimal spawning sites might be a recovery strategy for this globally endangered species.

Hsp90 and reactive oxygen species regulate thermotolerance of rice seedlings via induction of heat shock factor A2 (OsHSFA2) and galactinol synthase 1 (OsGolS1)

Heat stress induces expression of a set of thermotolerance-related genes in plants. We focused on rice (Oryza sativa L.) homologs of the gene family that encodes galactinol synthase (OsGolS), which is closely related to the Arabidopsis thaliana galactinol synthase (AtGolS) family whose expression is induced under various stresses. OsGolS1 was highly up-regulated compare to the level of OsGolS2 in re- sponse to heat stress. Interestingly, OsGolS1 was also up-regulated by treatment with the Hsp90 inhibitor, geldanamycin (GDA). Expression profiles of OsGolS1 were correlated to those of OsHsfA2 under the GDA treatments. Treatment with GDA increased expression of OsHsfA2, but marginally increased or did not change OsHsfA1 expression. Notably, gel shift assay indicated that OsHsfA2 binds directly to OsGolS1 promoter region and that OsHsfA1 also binds to the promoter regions of OsHsfA2. Both OsHsfA2 and OsGolS1 were dramatically induced in response to heat stress. Accordingly, galactinol and raffinose contents in rice seedlings increased significantly following the induction of OsGolS1. Pre-treatment of rice seedlings with raffinose or GDA improved their thermotolerance. These results suggest that OsGolS1 plays an important role in response to heat stress, possibly via the transcription cascade of OsHsfA1-OsHsfA2 that leads to galactinol and raffinose accumulation, and that the increased content of these carbohydrates is a key response factor for rice seedlings to enhance thermotolerance.

The Sucrose Starvation Signal Mediates Induction of Autophagy- and Amino Acid Catabolism-Related Genes in Cowpea Seedling

In higher plants, autophagy is bulk degradation process in vacuole necessary for survival under nutrient-limited conditions and plays important roles in senescence, development and pathogenic response, etc. Cowpea is one of the most important legume crops in semi-aride region, which is highly tolerant to drought stress. Changes of photoassimilate status by drought stress and/or sink-source balance appeared to affect autophagy and senescence of leaf in cowpea. Accordingly, we focused on roles of sucrose signal in autophagy and amino acid recycling in cowpea. Effects of starvation stress on the expression of autophagy-related genes (ATGs) and amino acid catabolism-related genes in cowpea [Vigna unguiculata (L.) Walp] were examined by Reverse transcription-polymerase chain reaction (RT-PCR) and anti-ATG8i specific antibody. Sucrose starvation stress enhanced the expression levels of VuATG8i, VuATG8c and VuATG4 incowpea seedlings. The expressions of amino acid catabolism related genes, such as asparagine synthase (VuASN1), proline dehydrogenase1 (VuProDH) and branched chain amino acid transaminase (VuBCAT2), are also up-regulated under the sucrose starvation. In contrast, high sucrose condition suppressed autophagy and the expressions of ATGs. These results indicate that sucrose starvation stress stimulates both autophagy and amino acid catabolism by regulation of ATGs and VuBCAT2. It is conceivable that sucrose starvation stress enhances autophagy in cowpea, possibly via branched chain amino acid level regulated by the starvation-induced BCAT.

Phylogenetic Analysis of Frequent Recombinant Region in Micropeptin Biosynthetic Gene Cluster among the Genus Microcystis

The gene cluster for the biosynthetic of a nonribosomal peptide, cyanopeptolins and micropeptin (MCN), was identified in Microcystis strains and halogenated MCN-producing Microcystis were found to possess the halogenase gene, mcnD, between nonribosomal peptide synthetase genes, mcnC and mcnE. A comparative sequence analysis of the mcn gene cluster between halogenated and non-halogenated MCN-producing strains revealed mosaic sequence traces from mcnD in the non-coding region between mcnC and mcnE in the latter strains. A phylogenetic analysis based on a 170-bp non-coding region including the mcnD traces suggests that the recombination events occurred in a particular region of the Microcystis’ mcn gene. This study provides novel insight into the ecological patterning of widespread Microcystis species.

A CIN-like TCP transcription factor(LsTCP4)having retrotransposon insertion associates with a shift from Salinas type to Empire type in crisphead lettuce(Lactuca sativa L.)

To improve several agronomic traits in crisphead lettuce(Lactuca sativa L.)under high-temperature growth conditions,we investigated the correlation among those traits in multiple cultivars and performed genetic mapping of their causal genes.In a field cultivation test of Empire type(serrated leaf)and Salinas type(wavy leaf)cultivars,Empire type cultivars showed increased tipburn susceptibility and late bolting compared with Salinas type cultivars.We attempted genetic mapping of leaf shape and bolting time by ddRAD-seq using the F2 population derived from a cross between‘VI185’(Empire type)and‘ShinanoGreen’(Salinas type).These analyses suggested that both traits are controlled by a single locus in LG5.Genotyping of 51 commercial lettuce cultivars with a tightly linked marker(LG5_v8_252.743Mbp)at this locus showed an association between its genotype and the serrated leaf phenotype.By further fine mapping and transcriptome analysis,a gene encoding putative CIN-like TCP transcription factor was determined to be a candidate gene at this locus and was designated as LsTCP4.An insertion of retrotransposable element was found in the allele of‘VI185’,and its transcript level in the leaves was lower than that in‘ShinanoGreen’.Because shapes of leaf epidermal cells in‘VI185’were similar to those in the TCP family mutant of Arabidopsis thaliana,the leaf shape phenotype was likely caused by reduced expression of LsTCP4.Furthermore,because it is known that the TCP family protein also controls flowering time via interaction with FT in A.thaliana,it was highly possible that LsTCP4 gave pleiotropic effects on both leaf shape and bolting time in lettuce.

A Flower Specific Calcineurin B-Like Molecule (CBL)-Interacting Protein Kinase (CIPK) Homolog in Tomato Cultivar Micro-Tom (<i>Solanum lycopersicum</i>L.)

Floral and reproductive organs of higher plants are relatively sensitive to biotic and abiotic stresses compared with the vegetative organs. Calcineurin B-like molecule (CBL) interacting protein kinase (CIPK) has appeared to be involved in acquired tolerance and acclimation under environmental stresses such as salinity, drought and chilling. Semi-quantitative RT-PCR using the vegetative and reproductive organs of tomato Micro-Tom (Solanum lycopersicum L.) at the various developmental stages indicated that SlCIPK2 was expressed specifically in the floral organ. An anti-CIPK specific antibody recognized the recombinant SlCIPK2 specifically and cross-reacted to a CIPK-related polypeptide at a significant level in flower, particularly in stamen. The flower specific CIPK was tightly associated with the microsomes. In vitro pull-down assay of the recombinant SlCIPK2 showed that SlCIPK2 interacts with SlCBLs and stress-responsive transcription factors, SlERF7, SlCBF1 and SlAREB1. The present data suggested that the flower-specific CIPK, SlCIPK2, was involved in calcium signaling in tomato via CBLs and stress tolerance possibly mediated by the stress-responsive transcription factors in stamen.

Effects of rhizobacteria Paenibacillus polymyxa APEC136 and Bacillus subtilis APEC170 on biocontrol of postharvest pathogens of apple fruits

In this study, plant growth-promoting rhizobacteria（PGPR） were evaluated as potential biocontrol agents against postharvest pathogens of apple fruits. In vitro bioassays revealed that, out of 30 isolates screened, isolates APEC136 and APEC170 had the most significant inhibitory effects against the mycelial growth of several fungal pathogens. Analysis of 16 S ribosomal RNA（rR NA） sequences identified the two effective isolates as Paenibacillus polymyxa and Bacillus subtilis, respectively. The two strains showed greater growth in brain-heart infusion broth than in other growth media. Treatment of harvested apples with suspensions of either strain reduced the symptoms of anthracnose disease caused by two fungal pathogens, Colletotrichum gloeosporioides and Colletotrichum acutatum, and white rot disease caused by Botryosphaeria dothidea. Increased productions of amylase and protease by APEC136, and increased productions of chitinase, amylase, and protease by APEC170 might have been responsible for inhibiting mycelial growth. The isolates caused a greater reduction in the growth of white rot than of anthracnose. These results indicate that the isolates APEC136 and APEC170 are promising agents for the biocontrol of anthracnose and white rot diseases in apples after harvest, and suggest that these isolates may be useful in controlling these diseases under field conditions.

Immunosuppression induced by expression of a viral RNase enhances susceptibility of Plutella xylostella to microbial pesticides

Polydnaviruses are a group of insect DNA viruses and are characterized in their segmented genome that is located in the chromosome（s） of host wasps. A polydnavirus, Cotesiaplutellae bracovirus （CpBV）, encodes a viral ribonuclease （RNase） T2 in a specific segment #3 （CpBV-S3）. This study tested its effect on gene expression associated with host immune responses in the diamondback moth, Plutella xylostella. Micro-inj ection of CpBV- $3 into nonparasitized larvae induced expression of its two encoded genes, CpBV-ORF301 （= CpBV-RNase T2） and CpBV-ORF302. In response to a bacterial challenge, four antimi- crobial peptide genes （hemolin, gloverin, cecropin and lysozyme） and six phenoloxidase （PO）-associated genes （proPO-activating proteinase, PO, serine proteinase homolog and serpins 1-3） were up-regulated in their expressions. However, the transient expression of CpBV-S3 suppressed the expressions of cecropin, PO and serpin 1. Double-stranded RNA specific to the viral RNase T2 could specifically knockdown the viral gene expression and restored the three gene expressions suppressed in the larvae injected with CpBV-S3. The inhibitory activity of the viral RNase T2 on the target genes was further proven by the suppression of PO activation in response to bacterial challenge in the larvae injected with CpBV-S3. This immunosuppression by the expression of the viral RNase T2 resulted in significant increase of pathogen susceptibility ofP. xylostella against Bacillus thuringiensis or baculovirus infection.

Baculoviral p94 homologs encoded in Cotesia plutellae bracovirus suppress both immunity and development of the diamondback moth, Plutellae xylostella

Polydnaviruses （PDVs） are a group of insect DNA viruses, which exhibit a mutual symbiotic relationship with their specific host wasps. Moreover, most encapsidated genes identified so far in PDVs share homologies with insect-originated genes, but not with virus-originated genes. In the meantime, PDVs associated with 2 wasp genera Cotesia and Glytapanteles encode some genes presumably originated from other viruses. Cotesia plutellae bracovirus （CpBV） encodes 4 genes homologous to baculoviral p94： CpBV- E94k1, CpB V-E94k2, CpB V-E94k3, and CpB V-E94k4. This study was conducted to predict the origin of CpB V-E94ks by comparing their sequences with those ofbaculoviral orthologs and to determine the physiological functions by their transient expressions in nonparasitized larvae and subsequent specific RNA interference. Our phylogenetic analysis indicated that CpBV-E94ks were clustered with other E94ks originated from different PDVs and shared high similarity with betabaculoviral p94s. These 4 CpBV genes were expressed during most developmental stages of the larvae of Plutella xylostella parasitized by C. plutellae. Expression of these 4 E94ks was mainly detected in hemocytes and fat body. Subsequent functional analysis by in vivo transient expression showed that all 4 viral genes significantly inhibited both host immune and developmental processes. These results suggest that CpB 11- E94ks share an origin with betabaculoviral p94s and play parasitic roles in suppressing host immune and developmental processes.

Three metabolites from an entomopathogenic bacterium, Xenorhabdus nematophila, inhibit larval development of Spodoptera exigua （Lepidoptera： Noctuidae） by inhibiting a digestive enzyme, phospholipase A2

An entomopathogenic bacterium, Xenorhabdus nematophila, has been known to induce significant immunosuppression of target insects by inhibiting immune-associated phospholipase A2 （PLA2）, which subsequently shuts down biosynthesis of eicosanoids that are critical in immune mediation in insects. Some metabolites originated from the bacterial culture broth have been identified and include benzylideneacetone, proline-tyrosine and acetylated phenylalanine-glycine-valine, which are known to inhibit enzyme activity of PLA2 extracted from hemocyte and fat body. This study tested their effects on digestive PLA2 of the beet armyworm, Spodoptera exigua. Young larvae fed different concentrations of the three metabolites resulted in significant adverse effects on larval development even at doses below 100 #g/mL. In particular, they induced significant reduction in digestive efficiency of ingested food. All three metabolites significantly inhibited catalytic activity of digestive PLA2 extracted from midgut lumen of the fifth instar larvae at a low micromolar range. These results suggest that the inhibitory activities of the three bacterial metabolites on digestive PLA2 of S. exigua midgut may explain some of their oral toxic effects.