Background Cuticular wax plays important role in protecting plants from drought stress. In Arabidopsis WRI4 improves drought tolerance by regulating the biosynthesis of fatty acids and cuticular wax. Cyperus esculentus (yellow nutsedge) is a tough weed found in tropical and temperate zones as well as in cooler regions. In the current study, we report the molecular cloning of a WRI4-like gene from Cyperus esculentus and its functional characterization in Arabidopsis. Results Using RACE PCR, full-length WRI-like gene was amplified from yellow nutsedge. Phylogenetic analyses and amino acid comparison suggested it to be a WRI4-like gene. According to the tissue-specific expression data, the highest expression of WRI4-like gene was found in leaves, followed by roots and tuber. Transgenic Arabidopsis plants expressing nutsedge WRI4-like gene manifested improved drought stress tolerance. Transgenic lines showed significantly reduced stomatal conductance, transpiration rate, chlorophyll leaching, water loss and improved water use efficiency (WUE). In the absence of drought stress, expression of key genes for fatty acid biosynthesis was not significantly different between transgenic lines and WT while that of cuticular wax biosynthesis genes was significantly higher in transgenic lines than WT. The PEG-simulated drought stress significantly increased expression of key genes for fatty acid as well as wax biosynthesis in transgenic Arabidopsis lines but not in WT plants. Consistent with the gene expression data, cuticular wax load and deposition was significantly higher in stem and leaves of transgenic lines compared with WT under control as well as drought stress conditions. Conclusions WRI4-like gene from Cyperus esculentus improves drought tolerance in Arabidopsis probably by promoting cuticular wax biosynthesis and deposition. This in turn lowers chlorophyll leaching, stomatal conductance, transpiration rate, water loss and improves water use efficiency under drought stress conditions. Therefore, CeWRI4-like gene could be a good candidate for improving drought tolerance in crops.

The extracellular matrix (ECM) plays an important role in cell to cell signaling pathways. Our goal is to provide a full laboratory guide for students to study gene expression in zebrafish embryos by in situ hybridization. Prior to our study, the laboratory had observed disorganized and shortened cilia in cells that are important for cell signaling in the pronephric duct and neural tube floor plate of the zebrafish embryo. Ciliogenesis depends on a master transcriptional regulator, foxj1a, whose mRNA expression can be monitored through in situ hybridization and microscopic imaging. Knockdown morpholino-injected, control mismatched morpholino-injected, and uninjected embryos were fixed to determine if foxj1a transcription is qualitatively affected by ECM gene knockdown. Our results showed that the knockdown embryos portrayed an inconsistent foxj1a signal strength along the length of the pronephric duct, when compared to analysis of control mismatched and wild-type uninjected embryos. We created this manuscript for other students to observe how ECM gene knockdown can affect foxj1a mRNA expression, but also to give them a guide to the tools they would need to explore their own genes of interest, in zebrafish or in many other organisms and tissues.

In this study we cloned a WRI1-like gene from yellow nutsedge. Conserved domain and phylogenetic analyses indicated it to be a WRI3/4-like gene. Arabidopsis plants transformed with WRI3/4-like gene showed significantly improved tolerance to both PEG-simulated drought stress and real dehydration compared with the wild type. Quantitative RT-PCR indicated that, under unstressed conditions, the expressions of key genes involved in fatty acid biosynthesis was not significantly different between wild type (WT) and transgenic lines, while the expressions of genes involved in cuticular wax biosynthesis was significantly higher in transgenic lines compared with the wild type. The PEG treatment slightly decreased the expression of above mentioned genes in WT plants while it was significantly increased in transgenic lines compared with their respective unstressed control. Without PEG treatment, the expression of TAG1, the gene involved in triacylglycerol (TAG) accumulation, was 10-40% lower in the transgenic lines than that in the wild type. However, after PEG treatment, the expression of TAG1 was slightly decreased in the wild type, while in the transgenic lines its expression was decreased by 20-70% compared with unstressed transgenic lines and was highly significantly lower than that in the wild type. The cuticular wax content in Arabidopsis leaves was significantly higher in the transgenic lines than that in the wild type, while the oil content was not significantly different.

Using RACE PCR, full length WRI1-like gene was amplified from yellow nutsedge. Conserved domain and phylogenetic analyses suggested it as WRI3/4-like gene. Tissue-specific expression data showed the highest expression in leaves, followed by roots while the lowest expression was detected in tuber. Transgenic Arabidopsis plants expressing nutsedge WRI3/4-like gene showed significantly improved tolerance to both PEG-simulated drought stress and real dehydration, compared with the wild type (WT). Under normal growth conditions, the expressions of key fatty acid biosynthesis genes was not significantly different between WT and transgenic lines, while the expressions of genes involved in cuticular wax biosynthesis was significantly higher in transgenic lines compared with the WT. The PEG-simulated drought stress did not induce any significant change in the expression of fatty acid and wax biosynthesis genes in WT plants, while the expression of fatty acid and wax biosynthesis genes was significantly increased in transgenic lines compared with WT as well as unstressed transgenic control. The expression of TAG1, the gene involved in triacylglycerol (TAG) accumulation, was significantly lower in the transgenic lines than that in the WT in normal growth conditions. Drought stress slightly decreased the expression of TAG1 in the WT, but significantly lowered it in transgenic lines compared with its unstressed transgenic control and WT. Consistent with gene expression data, the cuticular wax content in Arabidopsis leaves was significantly higher in the transgenic lines than in the WT, while the oil content was not significantly different. Our results indicated that WRI3/4-like gene from Cyperus esculentus improves drought tolerance in Arabidopsis probably by promoting cuticular wax biosynthesis and, hence, could be a valuable target for improving drought tolerance in crops through recombinant DNA technology.