Yusuke Ito

Monitoring Expression Profiles of Rice Genes under Cold, Drought, and High-Salinity Stresses and Abscisic Acid Application Using cDNA Microarray and RNA Gel-Blot Analyses

To identify cold-, drought-, high-salinity-, and/or abscisic acid (ABA)-inducible genes in rice (Oryza sativa), we prepared a rice cDNA microarray including about 1,700 independent cDNAs derived from cDNA libraries prepared from drought-, cold-, and high-salinity-treated rice plants. We confirmed stress-inducible expression of the candidate genes selected by microarray analysis using RNA gel-blot analysis and finally identified a total of 73 genes as stress inducible including 58 novel unreported genes in rice. Among them, 36, 62, 57, and 43 genes were induced by cold, drought, high salinity, and ABA, respectively. We observed a strong association in the expression of stress-responsive genes and found 15 genes that responded to all four treatments. Venn diagram analysis revealed greater cross talk between signaling pathways for drought, ABA, and high-salinity stresses than between signaling pathways for cold and ABA stresses or cold and high-salinity stresses in rice. The rice genome database search enabled us not only to identify possible known cis-acting elements in the promoter regions of several stress-inducible genes but also to expect the existence of novel cis-acting elements involved in stress-responsive gene expression in rice stress-inducible promoters. Comparative analysis of Arabidopsis and rice showed that among the 73 stress-inducible rice genes, 51 already have been reported in Arabidopsis with similar function or gene name. Transcriptome analysis revealed novel stress-inducible genes, suggesting some differences between Arabidopsis and rice in their response to stress.

Transcriptional Regulatory Networks in Response to Abiotic Stresses in Arabidopsis and Grasses

Various abiotic stresses such as drought, high salinity, high temperature, and low temperature negatively impact plant growth and productivity of crops. Plants have adapted to respond to these stresses at the molecular, cellular, physiological, and biochemical level, enabling them to survive.

The abiotic stress-responsive NAC-type transcription factor OsNAC5 regulates stress-inducible genes and stress tolerance in rice

The transcription factor OsNAC5 in rice is a member of the plant-specific NAC family that regulates stress responses. Expression of OsNAC5 is induced by abiotic stresses such as drought, cold, high salinity, abscisic acid and methyl jasmonic acid. Transactivation assays using rice protoplasts demonstrated that OsNAC5 is a transcriptional activator, and subcellular localization studies using OsNAC5-GFP fusion proteins showed that it is localized to the nucleus. Pull-down assays revealed that OsNAC5 interacts with OsNAC5, OsNAC6 and SNAC1. To analyze the function of OsNAC5 in rice plants, we generated transgenic plants that overexpressed OsNAC5. The growth of these plants was similar to that of control plants, whereas the growth of OsNAC6-overexpressing transgenic plants was retarded. OsNAC5-overexpressing transgenic plants also had improved tolerance to high salinity compared to control plants. By microarray analysis, many stress-inducible genes, including the “late embryogenesis abundant” gene OsLEA3, were upregulated in rice plants that overexpressed OsNAC5. By gel mobility shift assay, OsNAC5 and OsNAC6 were shown to bind to the OsLEA3 promoter. Collectively, our results indicate that the stress-responsive proteins OsNAC5 and OsNAC6 are transcriptional activators that enhance stress tolerance by upregulating the expression of stress-inducible rice genes such as OsLEA3, although the effects of these proteins on growth are different. Furthermore, because OsNAC5 overexpression did not retard growth, OsNAC5 may be a useful gene that can improve the stress tolerance of rice without affecting its growth.

Comprehensive analysis of rice DREB2-type genes that encode transcription factors involved in the expression of abiotic stress-responsive genes

DREB2s (dehydration-responsive element-binding protein 2s) are transcription factors that interact with a cis-acting DRE (dehydration-responsive element)/CRT (C-repeat) sequence and activate the expression of downstream genes involved in water- and heat-shock stress responses and tolerance in Arabidopsis thaliana. In this study, we performed a comprehensive analysis of all five DREB2-type genes in rice (OsDREB2s: OsDREB2A, OsDREB2B, OsDREB2C, OsDREB2E and OsABI4) to determine which of them contribute to plant stress responses. We analysed the expression patterns of these genes under abiotic stress conditions, and we examined the subcellular localisation and transcriptional activation activity of their translational products in protoplasts. Only OsDREB2A and OsDREB2B showed abiotic stress-inducible gene expression. In addition, OsDREB2B showed nuclear specific localisation and the highest transactivation activity. OsDREB2B has functional and non-functional forms of its transcript similar to its orthologues in the grass family, and the functional form of its transcript was markedly increased during stress conditions. We analysed the splicing mechanism of OsDREB2B with transgenic rice that express the non-functional transcript and we found that the non-functional form is not a precursor of the functional form; thus, stress-inducible alternative splicing of pre-mRNA is an important mechanism for the regulation of OsDREB2B. Transgenic Arabidopsis plants overexpressing OsDREB2B showed enhanced expression of DREB2A target genes and improved drought and heat-shock stress tolerance. These results suggest that OsDREB2B is a key gene that encodes a stress-inducible DREB2-type transcription factor that functions in stress-responsive gene expression in rice.

Rice phytochrome-interacting factor-like protein OsPIL1 functions as a key regulator of internode elongation and induces a morphological response to drought stress

The mechanisms for plant growth restriction during stress conditions remains unclear. Here, we demonstrate that a phytochrome-interacting factor-like protein, OsPIL1/OsPIL13, acts as a key regulator of reduced internode elongation in rice under drought conditions. The level of OsPIL1 mRNA in rice seedlings grown under nonstressed conditions with light/dark cycles oscillated in a circadian manner with peaks in the middle of the light period. Under drought stress conditions, OsPIL1 expression was inhibited during the light period. We found that OsPIL1 was highly expressed in the node portions of the stem using promoter-glucuronidase analysis. Overexpression of OsPIL1 in transgenic rice plants promoted internode elongation. In contrast, transgenic rice plants with a chimeric repressor resulted in short internode sections. Alteration of internode cell size was observed in OsPIL1 transgenic plants, indicating that differences in cell size cause the change in internode length. Oligoarray analysis revealed OsPIL1 downstream genes, which were enriched for cell wall-related genes responsible for cell elongation. These data suggest that OsPIL1 functions as a key regulatory factor of reduced plant height via cell wall-related genes in response to drought stress. This regulatory system may be important for morphological stress adaptation in rice under drought conditions.

Folliculin-interacting proteins Fnip1 and Fnip2 play critical roles in kidney tumor suppression in cooperation with Flcn.

Significance The role of FLCN as a tumor suppressor in kidney cancer has been well documented, whereas the functional roles of folliculin (FLCN)-interacting proteins 1 and 2 (FNIP1 and FNIP2) in kidney are unknown. In this study, we demonstrate that double inactivation of Fnip1 and Fnip2 leads to enlarged polycystic kidneys or kidney cancer, which mimics the phenotypes seen in Flcn-deficient kidneys and underscores the significance of Fnip1 and Fnip2 in kidney tumor suppression. Moreover, we found that Fnip1/Fnip2 mRNA ratios differ among organs, which may reflect tissue-specific roles for each Fnip. Our findings define Fnip1 and Fnip2 as critical components of the Flcn complex that are essential for its tumor suppressive function and will aid in the development of novel therapeutics for kidney cancer. Folliculin (FLCN)-interacting proteins 1 and 2 (FNIP1, FNIP2) are homologous binding partners of FLCN, a tumor suppressor for kidney cancer. Recent studies have revealed potential functions for Flcn in kidney; however, kidney-specific functions for Fnip1 and Fnip2 are unknown. Here we demonstrate that Fnip1 and Fnip2 play critical roles in kidney tumor suppression in cooperation with Flcn. We observed no detectable phenotype in Fnip2 knockout mice, whereas Fnip1 deficiency produced phenotypes similar to those seen in Flcn-deficient mice in multiple organs, but not in kidneys. We found that absolute Fnip2 mRNA copy number was low relative to Fnip1 in organs that showed phenotypes under Fnip1 deficiency but was comparable to Fnip1 mRNA copy number in mouse kidney. Strikingly, kidney-targeted Fnip1/Fnip2 double inactivation produced enlarged polycystic kidneys, as was previously reported in Flcn-deficient kidneys. Kidney-specific Flcn inactivation did not further augment kidney size or cystic histology of Fnip1/Fnip2 double-deficient kidneys, suggesting pathways dysregulated in Flcn-deficient kidneys and Fnip1/Fnip2 double-deficient kidneys are convergent. Heterozygous Fnip1/homozygous Fnip2 double-knockout mice developed kidney cancer at 24 mo of age, analogous to the heterozygous Flcn knockout mouse model, further supporting the concept that Fnip1 and Fnip2 are essential for the tumor-suppressive function of Flcn and that kidney tumorigenesis in human Birt–Hogg–Dubé syndrome may be triggered by loss of interactions among Flcn, Fnip1, and Fnip2. Our findings uncover important roles for Fnip1 and Fnip2 in kidney tumor suppression and may provide molecular targets for the development of novel therapeutics for kidney cancer.

Neutrophil-to-lymphocyte ratio is a prognostic marker in bladder cancer patients after radical cystectomy

BackgroundThere is no reliable biomarker for predicting the prognosis of patients who undergo radical cystectomy for bladder cancer. Recent studies have shown that the neutrophil-to-lymphocyte ratio (NLR) could function as a useful prognostic factor in several types of malignancies. This study aimed to assess the usefulness of NLR in bladder cancer.MethodsA total of 74 patients who underwent radical cystectomy in our institutions from 1999 to 2014 were analyzed. The NLR was calculated using the patients’ neutrophil and lymphocyte counts before radical cystectomy. An immunohistochemical analysis was also performed to detect tumor infiltrating neutrophils (CD66b) and lymphocytes (CD8) in bladder cancer specimens.ResultsA univariate analysis showed that the patients with a high NLR (≥2.38; HR = 4.84; p = 0.007), high C-reactive protein level (>0.08; HR = 10.06; p = 0.030), or pathological lymph node metastasis (HR = 4.73; p = 0.030) had a significantly higher risk of cancer-specific mortality. Kaplan-Meier and log-rank tests further revealed that NLR was strongly correlated with overall survival (p = 0.018), but not progression-free survival (p = 0.137). In a multivariate analysis, all of these were found to be independent risk factors (HR = 4.62, 10.8, and 12.35, respectively). The number of CD8-positive lymphocytes was significantly increased in high-grade (p = 0.001) and muscle-invasive (p = 0.012) tumors, in comparison to low-grade and non-muscle-invasive tumors, respectively.ConclusionsThe NLR predicted the prognosis of patients who underwent radical cystectomy and might therefore function as a reliable biomarker in cases of invasive bladder cancer.

Adipocyte‐derived monocyte chemotactic protein‐1 (MCP‐1) promotes prostate cancer progression through the induction of MMP‐2 activity

Obesity is known to be associated with prostate cancer development and progression, but the detailed mechanism is not clear. Monocyte chemotactic protein‐1 (MCP‐1) is secreted from cancer cells, stromal cells, and adipocytes, and it is involved in prostate cancer progression. Here we investigated the biological role of MCP‐1 secreted from adipocytes for prostate cancer cells.

Expression of ArabidopsisDREB1C improves survival, growth, and yield of upland New Rice for Africa (NERICA) under drought

Dehydration-responsive-element-binding protein 1 genes have important roles in response to stress. To improve the drought tolerance of an upland rice cultivar NERICA1, we introduced ArabidopsisAtDREB1C or rice OsDREB1B driven by a stress-inducible rice lip9 promoter. Plants of some transgenic lines survived better than non-transgenic plants under severe drought. AtDREB1C transgenic plants had higher dry weights than non-transgenic plants when grown under moderate drought until the late vegetative growth stage. On the other hand, OsDREB1B transgenic plants had lower dry weights than non-transgenic plants under the same condition. Similar results were obtained under osmotic stress. The AtDREB1C transgenic plants headed earlier, had a larger sink capacity, and had more filled grains than non-transgenic plants. These results suggest that AtDREB1C expressed in NERICA1 improves not only survival under severe drought, but also growth and yield under moderate drought.

Neutrophil-to-lymphocyte ratio predicts prostatic carcinoma in men undergoing needle biopsy

Neutrophil-to-lymphocyte ratio (NLR), a simple marker of systemic inflammatory response, has been demonstrated as an independent prognosticator for some solid malignancies, including prostate cancer. In the present study, we evaluated the role of NLR in men who underwent prostate needle biopsy for their initial diagnosis of prostatic carcinoma. Both complete blood counts and free/total (F/T) prostate-specific antigen (PSA) ratio were examined in a total of 3,011 men in our institution. Of these, 1,207 had a PSA level between 4 and 10 ng/mL, and 357 of 810 who subsequently underwent prostate needle biopsy were found to have prostatic adenocarcinoma. NLR value was significantly higher in men with PSA of ≥ 20 ng/mL than in those with PSA of < 20 ng/mL (p < 0.001). NLR was also significantly higher in men with positive biopsy than in those with negative biopsy (p < 0.001). Using NLR cut-off point of 2.40 determined by the AUROC curve, positive/negative predictive values of NLR alone and NLR combined with F/T PSA ratio (cut-off: 0.15) were 56.6%/60.8% and 80.7%/60.1%, respectively. Multivariate analysis revealed that not only F/T PSA ratio (HR = 3.13) but also NLR (HR = 2.21) was an independent risk factor for prostate cancer. NLR is thus likely elevated in patients with prostate cancer. Accordingly, NLR, with or without combination with F/T PSA ratio, may function as a new biomarker to predict prostate cancer in men undergoing prostate needle biopsy.