Identification and evolution of a diterpenoid phytoalexin oryzalactone biosynthetic gene in the genus Oryza.

The natural variation of plant-specialized metabolites represents the evolutionary adaptation of plants to their environments. However, the molecular mechanisms that account for the diversification of the metabolic pathways have not been fully clarified. Rice plants resist attacks from pathogens by accumulating diterpenoid phytoalexins. It has been confirmed that the composition of rice phytoalexins exhibits numerous natural variations. Major rice phytoalexins (momilactones and phytocassanes) are accumulated in most cultivars, although oryzalactone is a cultivar-specific compound. Here, we attempted to reveal the evolutionary trajectory of the diversification of phytoalexins by analyzing the oryzalactone biosynthetic gene in Oryza species. The candidate gene, KSLX-OL, which accounts for oryzalactone biosynthesis, was found around the single-nucleotide polymorphisms specific to the oryzalactone-accumulating cultivars in the long arm of chromosome 11. The metabolite analyses in Nicotiana benthamiana and rice plants overexpressing KSLX-OL indicated that KSLX-OL is responsible for the oryzalactone biosynthesis. KSLX-OL is an allele of KSL8 that is involved in the biosynthesis of another diterpenoid phytoalexin, oryzalexin S and is specifically distributed in the AA genome species. KSLX-NOL and KSLX-bar, which encode similar enzymes but are not involved in oryzalactone biosynthesis, were also found in AA genome species. The phylogenetic analyses of KSLXs, KSL8s, and related pseudogenes (KSL9s) indicated that KSLX-OL was generated from a common ancestor with KSL8 and KSL9 via gene duplication, functional differentiation, and gene fusion. The wide distributions of KSLX-OL and KSL8 in AA genome species demonstrate their long-term coexistence beyond species differentiation, suggesting a balancing selection between the genes.

Regulation of the plastochron by three many-noded dwarf genes in barley.

The plastochron, the time interval between the formation of two successive leaves, is an important determinant of plant architecture. We genetically and phenotypically investigated many-noded dwarf (mnd) mutants in barley. The mnd mutants exhibited a shortened plastochron and a decreased leaf blade length, and resembled previously reported plastochron1 (pla1), pla2, and pla3 mutants in rice. In addition, the maturation of mnd leaves was accelerated, similar to pla mutants in rice. Several barley mnd alleles were derived from three genes-MND1, MND4, and MND8. Although MND4 coincided with a cytochrome P450 family gene that is a homolog of rice PLA1, we clarified that MND1 and MND8 encode an N-acetyltransferase-like protein and a MATE transporter-family protein, which are respectively orthologs of rice GW6a and maize BIGE1 and unrelated to PLA2 or PLA3. Expression analyses of the three MND genes revealed that MND1 and MND4 were expressed in limited regions of the shoot apical meristem and leaf primordia, but MND8 did not exhibit a specific expression pattern around the shoot apex. In addition, the expression levels of the three genes were interdependent among the various mutant backgrounds. Genetic analyses using the double mutants mnd4mnd8 and mnd1mnd8 indicated that MND1 and MND4 regulate the plastochron independently of MND8, suggesting that the plastochron in barley is controlled by multiple genetic pathways involving MND1, MND4, and MND8. Correlation analysis between leaf number and leaf blade length indicated that both traits exhibited a strong negative association among different genetic backgrounds but not in the same genetic background. We propose that MND genes function in the regulation of the plastochron and leaf growth and revealed conserved and diverse aspects of plastochron regulation via comparative analysis of barley and rice.

Pre-treatment serum protein levels predict survival of non-small cell lung cancer patients without durable clinical benefit by PD-1/L1 inhibitors.

While PD-1/L1 inhibitors are characterized by durable tumor control, they also prolong survival without prolongation of progression-free survival (PFS) in part of patients. However, little is known about the factors and mechanisms involved in this. Between December 2015 and September 2018, 106 patients with advanced non-small cell lung cancer treated with ICI monotherapy were enrolled in a prospective-observational study. Sixty-nine of whom progressed or died within 6 months after ICI initiation were defined as patients without durable clinical benefit (NDBs). Clinical factors and 39 serum proteins before ICI initiation and at the time of progressive disease (PD) were explored for an association with overall survival (OS) and OS after PD (OS-PD). As a result, median PFS, OS, and OS-PD were 44 days [95% confidence interval (CI): 39-56), 211 days (95% CI: 158-425), and 193 days (95% CI: 118-349), respectively. By multivariate analysis for OS, CRP (> 1.44 mg/dl) [HR 2.59 (95% CI:1.33-5.04), P = 0.005] and follistatin (> 685 pg/ml) [HR 2.29 (95% CI:1.12-4.69), P = 0.023] before ICI initiation were significantly predictive. Notably, no serum protein at the time of PD was predictive for OS-PD. There were also no serum predictive factors of OS in the 33 patients with durable clinical benefit. In conclusion, serum levels of CRP and follistatin before ICI initiation, not at the time of PD, are predictive for OS in NDBs, suggesting long-term survivor in NDBs are predetermined by the immune status before ICI initiation.

A nonproteinogenic amino acid, ß-tyrosine, accumulates in young rice leaves via long-distance phloem transport from mature leaves.

Oryza sativa L. ssp. japonica cv. Nipponbare produces a nonproteinogenic amino acid (3R)-ß-tyrosine from l-tyrosine by tyrosine aminomutase (OsTAM1). However, physiological and ecological function(s) of ß-tyrosine have remained obscure. Often an improved understanding of metabolite localization and transport can aid in design of experiments to test physiological functions. In the current study, we investigated the distribution pattern of ß-tyrosine in rice seedlings and found that ß-tyrosine is most abundant in the youngest leaves. Based upon observations of high TAM1 activity in mature leaves, we hypothesized that ß-tyrosine is transported from mature leaves to young leaves. Patterns of predominant mature synthesis and young leaf accumulation were supported by stable isotope studies using labeled ß-tyrosine and the removal of mature leaves. Stem exudate analyses was also consistent with ß-tyrosine transport through phloem. Thus, we identify young leaves as a key target in efforts to understand the biological function(s) of ß-tyrosine in rice.

Genome-wide analysis of spatiotemporal expression patterns during rice leaf development.

BACKGROUND: Rice leaves consist of three distinct regions along a proximal-distal axis, namely the leaf blade, sheath, and blade-sheath boundary region. Each region has a unique morphology and function, but the genetic programs underlying the development of each region are poorly understood. To fully elucidate rice leaf development and discover genes with unique functions in rice and grasses, it is crucial to explore genome-wide transcriptional profiles during the development of the three regions. RESULTS: In this study, we performed microarray analysis to profile the spatial and temporal patterns of gene expression in the rice leaf using dissected parts of leaves sampled in broad developmental stages. The dynamics in each region revealed that the transcriptomes changed dramatically throughout the progress of tissue differentiation, and those of the leaf blade and sheath differed greatly at the mature stage. Cluster analysis of expression patterns among leaf parts revealed groups of genes that may be involved in specific biological processes related to rice leaf development. Moreover, we found novel genes potentially involved in rice leaf development using a combination of transcriptome data and in situ hybridization, and analyzed their spatial expression patterns at high resolution. We successfully identified multiple genes that exhibit localized expression in tissues characteristic of rice or grass leaves. CONCLUSIONS: Although the genetic mechanisms of leaf development have been elucidated in several eudicots, direct application of that information to rice and grasses is not appropriate due to the morphological and developmental differences between them. Our analysis provides not only insights into the development of rice leaves but also expression profiles that serve as a valuable resource for gene discovery. The genes and gene clusters identified in this study may facilitate future research on the unique developmental mechanisms of rice leaves.

Computed tomography findings for predicting the future occurrence of pancreatic cancer: value of pancreatic volumetry.

BACKGROUND: The features of pancreatic parenchyma that tend to progress towards pancreatic cancer (PC) are unknown. We performed volumetry of the pancreas in PC patients using computed tomography (CT) scans acquired before detection of PC, and investigated whether CT findings of pancreatic parenchyma could predict the future occurrence of PC. METHODS: Between April 2009 and March 2017, a total of 3769 patients underwent abdominal contrast-enhanced CT, the scans of which were archived as digital images. Among them, 15 PC patients underwent abdominal CT 6-120 months before diagnosis of PC. This retrospective study compared the 15 PC patients (PC group) with 15 propensity score-matched subjects without PC (non-PC group). Pancreatic volumetry and radiological findings were compared between the two groups. RESULTS: There were significant differences between the PC and non-PC groups in the volume of the main pancreatic duct (MPD) plus any cystic lesion (P = 0.007), volume of the MPD plus any cystic lesion/body surface area (BSA; P = 0.009), MPD diameter (P = 0.011), and MPD diameter/BSA (P = 0.013). Univariate analysis revealed volume of MPD plus any cystic lesion/BSA ≥ 0.53 mL/m2 (odds ratio [OR] 38.50, P = 0.002), volume of pancreatic parenchyma/BSA < 27.0 mL/m2 (OR 12.25, P = 0.030), and MPD diameter/BSA ≥ 1.0 mm/m2 (OR 13.00, P = 0.006) as significant risk factors for PC. CONCLUSIONS: Quantification of the volume of MPD plus any cystic lesion/BSA, volume of pancreatic parenchyma/BSA, and MPD diameter/BSA on pre-diagnosis CT were useful for predicting the future occurrence of PC.

Lineage-specific gene acquisition or loss is involved in interspecific hybrid sterility in rice.

Understanding the genetic basis of reproductive barriers between species has been a central issue in evolutionary biology. The S1 locus in rice causes hybrid sterility and is a major reproductive barrier between two rice species, Oryza sativa and Oryza glaberrima The O. glaberrima-derived allele (denoted S1g) on the S1 locus causes preferential abortion of gametes with its allelic alternative (denoted S1s) in S1g/S1s heterozygotes. Here, we used mutagenesis and screening of fertile hybrid plants to isolate a mutant with an allele, S1mut, which does not confer sterility in the S1mut/S1g and S1mut/S1s hybrids. We found that the causal mutation of the S1mut allele was a deletion in the peptidase-coding gene (denoted "SSP") in the S1 locus of O. glaberrima No orthologous genes of SSP were found in the O. sativa genome. Transformation experiments indicated that the introduction of SSP in carriers of the S1s allele did not induce sterility. In S1mut/S1s heterozygotes, the insertion of SSP led to sterility, suggesting that SSP complemented the loss of the functional phenotype of the mutant and that multiple factors are involved in the phenomenon. The polymorphisms caused by the lineage-specific acquisition or loss of the SSP gene were implicated in the generation of hybrid sterility. Our results demonstrated that artificial disruption of a single gene for the reproductive barrier creates a "neutral" allele, which facilitates interspecific hybridization for breeding programs.

Novel endoscopic ultrasonography imaging technique for visualizing microcirculation without contrast enhancement in subepithelial lesions: Prospective study.

OBJECTIVES: Detective flow imaging for endoscopic ultrasonography (DFI?EUS) is a new imaging modality developed for detecting fine vessels without using ultrasound contrast agents. This study aimed to evaluate its utility by comparing it with a type of directional power Doppler (eFLOW) for subepithelial lesions (SELs). METHODS: Between January 2019 and January 2020, 28 patients with SELs undergoing DFI?EUS and eFLOW?EUS were enrolled. DFI?EUS and eFLOW?EUS assessing the vascularity in SELs were compared in terms of the rates of identification of intratumoral vessels. We also investigated how large vessels were depicted in both modalities based on surgical specimens as well as the detection rates of intratumoral vessels in gastrointestinal stromal tumors (GISTs) and non?GISTs using either DFI?EUS or eFLOW?EUS. RESULTS: Among 28 patients, 23 with pathological confirmation by EUS?guided fine?needle aspiration biopsy (EUS?FNAB) specimens were included. Of those 23 patients, the 10 who underwent surgical resection were selected for analysis. The rate of detection of intratumoral vessels in SELs was significantly higher on DFI?EUS (80%) than on eFLOW?EUS (30%) (P\xA0=\xA00.03). Comparison with surgical specimens revealed that detection rate for vessels with maximum size of less 1000\xA0µm was higher in DFI?EUS (66%) than that in eFLOW?EUS (0%). GIST patients had significantly higher positive rates (90%) of intratumoral vessels than non?GIST patients (31%) on DFI in 23 cases including EUS?FNAB specimens (P\xA0=\xA00.045). CONCLUSIONS: Detective flow imaging?EUS is more sensitive for depicting intratumoral vessels than eFLOW?EUS. Evaluation of intratumoral vessels on DFI?EUS is useful for identifying GISTs without contrast agents.

Factors associated with quality of dreams during general anesthesia: a prospective observational study.

Patients frequently report having dreams during general anesthesia, and the dreams are often reported to be pleasant dreams. However, factors associated with the quality of dreams during general anesthesia have not been clarified. The aim of this study was to determine the relationships between the quality of dreams during general anesthesia and perioperative factors. This prospective observational study included patients scheduled for elective surgery under general anesthesia. Preoperative mental status was assessed by the Hospital Anxiety and Depression Scale (HADS). A postoperative interview was carried out in the operating room after recovery from general anesthesia. Dreams and awareness during general anesthesia were assessed by a modified Brice interview. The quality of dreams was classified in accordance with the patient's own opinion as pleasant, indifferent, or unpleasant. A total of 1100 patients were included in the study, and 293 (25.4%) of the patients reported having dreams during anesthesia. Half of the patients who experienced dreams during anesthesia (50.2%, 147/293 patients) reported having a pleasant dream. Multivariate logistic regression analysis revealed that only HADS-depression score of less than 11 was related to pleasant dreams (OR: 3.3 [95% CI 1.3-10.0]).

Adipophilin expression in cutaneous malignant melanoma is associated with high proliferation and poor clinical prognosis.

Adipophilin (ADP) is a primary protein component of lipid droplets (LDs). For more than half a century, certain types of cancer cells have been known to contain LDs in their cytoplasm. However, the pathological significance of ADP or LDs in cancer remains unclear. In the present study, we investigated the association between ADP and other pathological characteristics in cutaneous malignant melanomas to clarify the role of ADP in melanoma cells. We immunostained whole paraffin sections of primary cutaneous melanomas obtained from 90 cases for ADP, after which we analyzed the correlation between ADP immunohistochemistry (IHC) and patient survival data. We also studied the relationship between the ADP IHC score and in situ hybridization (ISH) score of ADP mRNA, and the Ki67-labeling index (Ki67-LI) by using tissue microarrays consisting of 74 primary cutaneous malignant melanomas, 19 metastasizing melanomas, and 29 melanocytic nevi. Finally, we analyzed the relationship between ADP expression and cell proliferation in cutaneous melanoma cell lines. We found that high ADP expression was associated with poor metastasis-free survival, disease-specific survival, and overall survival rates of patients with cutaneous melanomas (P < 0.05). By linear regression analysis, ADP IHC was correlated with increasing ADP mRNA ISH H-scores and Ki67-LI scores in melanocytic lesions (P < 0.01). ADP IHC and ADP ISH H-scores and Ki67-LI scores were greater in pT3-4 melanomas than in pT1-2 melanomas. In cell-based assays, cells with increased ADP expression showed higher proliferation rates compared with those of low-ADP cells. Thus, ADP expression in malignant melanoma was significantly associated with high cell proliferation and poor clinical prognosis. Our results thus indicate a significant association between ADP and melanoma progression, and we propose that ADP may be a novel marker of aggressive cutaneous melanoma with a lipogenic phenotype.

Optimization of therapeutic strategy for p16-positive oropharyngeal squamous cell carcinoma: Multi-institutional observational study based on the national Head and Neck Cancer Registry of Japan.

BACKGROUND: Although the American Joint Committee on Cancer TNM classification has been amended to include human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OPSCC) as an independent entity, to the authors' knowledge the optimized de-escalating treatment modality has not been established to date. METHODS: The authors conducted a retrospective, nationwide, observational study in patients with HPV-related OPSCC who were treated from 2011 to 2014 in Japan to determine the best treatment modality. RESULTS: A total of 688 patients who were newly diagnosed with HPV-related OPSCC who were treated with curative intent at 35 institutions and had coherent clinical information and follow-up data available were included in the current study. In patients with T1-T2N0 disease (79 patients), both the 3-year recurrence-free survival and overall survival (OS) rates were 100% in the group treated with radiotherapy (RT) as well as the group receiving concurrent chemoradiotherapy (CCRT). The 3-year OS rates were 94.4% (for patients with T1N0 disease) and 92.9% (for patients with T2N0 disease) among the patients treated with upfront surgery. In patients with stage I to stage II HPV-related OPSCC, the 5-year recurrence-free survival and OS rates were 91.4% and 92%, respectively, in the patients treated with CCRT with relatively high-dose cisplatin (≥160 mg/m2 ; 114 patients) and 74.3% and 69.5%, respectively, in the patients treated with low-dose cisplatin (<160 mg/m2 ; 17 patients). CONCLUSIONS: Despite it being a retrospective observational trial with a lack of information regarding toxicity and morbidity, the results of the current study demonstrated that patients with T1-T2N0 HPV-related OPSCC could be treated with RT alone because of the equivalent outcomes of RT and CCRT, and patients with stage I to stage II HPV-related OPSCC other than those with T1-T2N0 disease could be treated with CCRT with cisplatin at a dose of ≥160 mg/m2 .

Jasmonate regulates juvenile-to-adult phase transition in rice.

Juvenile-to-adult phase transition is an important shift for the acquisition of adult vegetative characteristics and subsequent reproductive competence. We identified a recessive precocious (pre) mutant exhibiting a long leaf phenotype in rice. The long leaf phenotype is conspicuous in the second to the fourth leaves, which are juvenile and juvenile-to-adult transition leaves. We found that morphological and physiological traits, such as midrib formation, shoot meristem size, photosynthetic rate and plastochron, in juvenile and juvenile-to-adult transition stages of the pre mutant have precociously acquired adult characteristics. In agreement with these results, expression patterns of miR156 and miR172, which are microRNAs regulating phase change, support the accelerated juvenile-to-adult phase change in the pre mutant. The mutated gene encodes an allene oxide synthase (OsAOS1), which is a key enzyme for the biosynthesis of jasmonic acid (JA). The pre mutant showed a low level of JA and enhanced sensitivity to gibberellic acid, which promotes the phase change in some plant species. We also show that prolonged plastochron in the pre mutant is caused by accelerated PLASTOCHRON1 (PLA1) function. The present study reveals a substantial role of JA as a negative regulator of vegetative phase change.

LEAF LATERAL SYMMETRY1, a Member of the WUSCHEL-RELATED HOMEOBOX3 Gene Family, Regulates Lateral Organ Development Differentially from Other Paralogs, NARROW LEAF2 and NARROW LEAF3 in Rice.

In several eudicot species, one copy of each member of the WUSCHEL-RELATED HOMEOBOX (WOX) gene family, WOX1 and WOX3, is redundantly or differentially involved in lateral leaf outgrowth, whereas only the WOX3 gene regulating the lateral domain of leaf development has been reported in grass. In this study, we show that a WOX3 gene, LEAF LATERAL SYMMETRY1 (LSY1), regulates lateral leaf development in a different manner ftom that of other duplicated paralogs of WOX3, NARROW LEAF2 (NAL2)/NAL3, in rice. A loss-of-function mutant of LSY1 exhibited an asymmetrical defect from early leaf development, which is different from a symmetric defect in a double loss-of-function mutant of NAL2/3, whereas the expression of both genes was observed in a similar domain in the margins of leaf primordia. Unlike NAL2/3, overexpression of LSY1 produced malformed leaves whose margins were curled adaxially. Expression domains and the level of adaxial/abaxial marker genes were affected in the LSY1-overexpressing plants, indicating that LSY1 is involved in regulation of adaxial-abaxial patterning at the margins of the leaf primordia. Additive phenotypes in some leaf traits of lsy1 nal2/3 triple mutants and the unchanged level of NAL2/3 expression in the lsy1 background suggested that LSY1 regulates lateral leaf development independently of NAL2/3. Our results indicated that all of the rice WOX3 genes are involved in leaf lateral outgrowth, but the functions of LSY1 and NAL2/3 have diverged. We propose that the function of WOX3 and the regulatory mode of leaf development in rice are comparable with those of WOX1/WOX3 in eudicot species.

Barley NARROW LEAFED DWARF1 encoding a WUSCHEL-RELATED HOMEOBOX 3 (WOX3) regulates the marginal development of lateral organs.

Barley (Hordeum vulgare L.) is the fourth most-produced cereal in the world and is mainly utilized as animal feed and malts. Recently barley attracts considerable attentions as healthy food rich in dietary fiber. However, limited knowledge is available about developmental aspects of barley leaves. In the present study, we investigated barley narrow leafed dwarf1 (nld1) mutants, which exhibit thin leaves accompanied by short stature. Detailed histological analysis revealed that leaf marginal tissues, such as sawtooth hairs and sclerenchymatous cells, were lacked in nld1, suggesting that narrowed leaf of nld1 was attributable to the defective development of the marginal regions in the leaves. The defective marginal developments were also appeared in internodes and glumes in spikelets. Map-based cloning revealed that NLD1 encodes a WUSCHEL-RELATED HOMEOBOX 3 (WOX3), an ortholog of the maize NARROW SHEATH genes. In situ hybridization showed that NLD1 transcripts were localized in the marginal edges of leaf primordia from the initiating stage. From these results, we concluded that NLD1 plays pivotal role in the increase of organ width and in the development of marginal tissues in lateral organs in barley.

The rice FISH BONE gene encodes a tryptophan aminotransferase, which affects pleiotropic auxin-related processes.

Auxin is a fundamental plant hormone and its localization within organs plays pivotal roles in plant growth and development. Analysis of many Arabidopsis mutants that were defective in auxin biosynthesis revealed that the indole-3-pyruvic acid (IPA) pathway, catalyzed by the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA) and YUCCA (YUC) families, is the major biosynthetic pathway of indole-3-acetic acid (IAA). In contrast, little information is known about the molecular mechanisms of auxin biosynthesis in rice. In this study, we identified a auxin-related rice mutant, fish bone (fib). FIB encodes an orthologue of TAA genes and loss of FIB function resulted in pleiotropic abnormal phenotypes, such as small leaves with large lamina joint angles, abnormal vascular development, small panicles, abnormal organ identity and defects in root development, together with a reduction in internal IAA levels. Moreover, we found that auxin sensitivity and polar transport activity were altered in the fib mutant. From these results, we suggest that FIB plays a pivotal role in IAA biosynthesis in rice and that auxin biosynthesis, transport and sensitivity are closely interrelated.

Wide genetic variation in phenolic compound content of seed coats among black soybean cultivars.

Black soybeans have been used as a food source and also in traditional medicine because their seed coats contain natural phenolic compounds such as proanthocyanidin and anthocyanin. The objective of this research is to reveal the genetic variation in the phenolic compound contents (PCCs) of seed coats in 227 black soybean cultivars, most of which were Japanese landraces and cultivars. Total phenolics were extracted from seed coats using an acidic acetone reagent and the proanthocyanidin content, monomeric anthocyanin content, total flavonoids content, total phenolics content, and radical scavenging activity were measured. The cultivars showed wide genetic variation in PCCs. Each of the contents was highly correlated with one another, and was closely associated with radical scavenging activity. PCCs were also moderately associated by flowering date but not associated by seed weight. Cultivars with purple flowers had a tendency to produce higher PCCs compared with cultivars with white flowers, suggesting that the W1 locus for flower color can affect phenolic compound composition and content. Our results suggest that developing black soybean cultivars with high functional phenolic compounds activity is feasible.

Change of shoot architecture during juvenile-to-adult phase transition in soybean.

Juvenile-to-adult phase change is an indispensable event which guarantees a successful life cycle. Phase change has been studied in maize, Arabidopsis and rice, but is mostly unknown in other species. Soybean/Fabaceae plants undergo drastic changes of shoot architecture at the early vegetative stage including phyllotactic change and leaf type alteration from simple to compound. These characteristics make soybean/Fabaceae plants an interesting taxon for investigating vegetative phase change. Following the expansion of two cotyledons, two simple leaves simultaneously emerge in opposite phyllotaxy. The phyllotaxy of the third and fourth leaves is not fixed; both opposite and distichous phyllotaxis are observed within the same population. Leaves were compound from the third leaf. But the third leaf was rarely simple. Morphological and quantitative changes in early vegetative phase were recognized in leaf size, leaf shape, number of trichomes, stipule size and shape, and shoot meristem shape. Two microRNA genes, miR156 and miR172, are known to be associated with vegetative phase change. Examination of the expression level revealed that miR156 expression was high in the first two leaves and subsequently down-regulated, and that of miR172 showed the inverse expression pattern. These expression patterns coincided with the case of other species. Taken all data together, the first and second leaves represent juvenile phase, the fifth and upper leaves adult phase, and the third and fourth leaves intermediate stage. Further investigation of soybean phase change would give fruitful understandings on plant development.

Rice slender leaf 1 gene encodes cellulose synthase-like D4 and is specifically expressed in M-phase cells to regulate cell proliferation.

Cellulose synthase-like (CSL) genes are predicted to catalyse the biosynthesis of non-cellulosic polysaccharides such as the ß-D-glycan backbone of hemicelluloses and are classified into nine subfamilies (CSLA-CSLH and CSLJ). The CSLD subfamily is conserved in all land plants, and among the nine CSL subfamilies, it shows the highest sequence similarity to the cellulose synthase genes, suggesting that it plays fundamental roles in plant development. This study presents a detailed analysis of slender leaf 1 (sle1) mutants of rice that showed rolled and narrow leaf blades and a reduction in plant height. The narrow leaf blade of sle1 was caused by reduced cell proliferation beginning at the P3 primordial stage. In addition to the size reduction of organs, sle1 mutants exhibited serious developmental defects in pollen formation, anther dehiscence, stomata formation, and cell arrangement in various tissues. Map-based cloning revealed that SLE1 encodes the OsCSLD4 protein, which was identified previously from a narrow leaf and dwarf 1 mutant. In situ hybridization experiments showed that OsCSLD4 was expressed in a patchy pattern in developing organs. Double-target in situ hybridization and quantitative RT-PCR analyses revealed that SLE1 was expressed specifically during the M-phase of the cell cycle, and suggested that the cell-cycle regulation was altered in sle1 mutants. These results suggest that the OsCSLD4 protein plays a pivotal role in the M phase to regulate cell proliferation. Further study of OsCSLD4 is expected to yield new insight into the role of hemicelluloses in plant development.

Natural variation of diterpenoid phytoalexins in rice: Aromatic diterpenoid phytoalexins in specific cultivars.

Rice (Oryza sativa L.) plants accumulate antimicrobial compounds known as phytoalexins in response to pathogen attack. To date, more than 20 compounds have been isolated as phytoalexins from rice, mostly diterpenoids. However, the quantitative analysis of diterpenoid phytoalexins in various cultivars has revealed that the cultivar 'Jinguoyin' does not accumulate these compounds at detectable concentrations. Therefore, in this study, we attempted to detect a new class of phytoalexins from Bipolaris oryzae infected leaves of 'Jinguoyin'. We detected five compounds in the leaves of the target cultivar, whereas these compounds were not detected in the leaves of 'Nipponbare' or 'Kasalath', which are representative cultivars of the japonica and indica subspecies. Subsequently, we isolated these compounds from ultraviolet (UV)-light-irradiated leaves and determined their structures by spectroscopic analysis and the crystalline sponge method. All the compounds were diterpenoids containing a benzene ring and were detected from the pathogen-infected rice leaves for the first time. Because the compounds showed antifungal activity against B. oryzae and Pyricularia oryzae, we propose that they function as phytoalexins in rice and named them abietoryzins A-E. The abietoryzins tended to accumulate at high concentrations in cultivars that accumulated low levels of known diterpenoid phytoalexins after UV-light irradiation. Of the total of 69 cultivars in the WRC, 30 cultivars accumulated at least one of the abietoryzins, and, in 15 cultivars, the amounts of some abietoryzins were the highest among those of the analyzed phytoalexins. Therefore, abietoryzins are a major phytoalexin group in rice, although their presence has, to date, been overlooked.

Measurements of Antibacterial Activity of Seed Crude Extracts in Cultivated Rice and Wild Oryza Species.

Seeds are continuously exposed to a wide variety of microorganisms in the soil. In addition, seeds contain large amounts of carbon and nitrogen sources that support initial growth after germination. Thus, seeds in the soil can easily promote microbial growth, and seeds are susceptible to decay. Therefore, seed defense against microorganisms is important for plant survival. Seed-microbe interactions are also important issues from the perspective of food production, in seed quality and shelf life. However, seed-microbe interactions remain largely unexplored. In this study, we established a simple and rapid assay system for the antibacterial activity of rice seed crude extracts by colorimetric quantification methods by the reduction of tetrazolium compound. Using this experimental system, the diversity of effects of rice seed extracts on microbial growth was analyzed using Escherichia coli as a bacterial model. We used collections of cultivated rice, comprising 50 accessions of Japanese landraces, 52 accessions of world rice core collections, and of 30 wild Oryza accessions. Furthermore, we attempted to find genetic factors responsible for the diversity by genome-wide association analysis. Our results demonstrate that this experimental system can easily analyze the effects of seed extracts on bacterial growth. It also suggests that there are various compounds in rice seeds that affect microbial growth. Overall, this experimental system can be used to clarify the chemical entities and genetic control of seed-microbe interactions and will open the door for understanding the diverse seed-microbe interactions through metabolites.