Optimized combination methods for exploring and verifying disease-resistant transcription factors in melon.

A large amount of omics data and number of bioinformatics tools has been produced. However, the methods for further exploring omics data are simple, in particular, to mine key regulatory genes, which are a priority concern in biological systems, and most of the specific functions are still unknown. First, raw data of two genotypes of melon (susceptible and resistant) were obtained by transcriptome analysis. Second, 391 transcription factors (TFs) were identified from the plant transcription factor database and cucurbit genomics database. Then, functional enrichment analysis indicated that these genes were mainly annotated in the process of transcription regulation. Third, 243 and 230 module-specific TFs were screened by weighted gene coexpression network analysis and short time series expression miner, respectively. Several TF genes, such as WRKYs and bHLHs, were regarded as key regulatory genes according to the values of significantly different modules. The coexpression network showed that these TF genes were significant correlated with resistance (R) genes, such as DRP2, RGA3, DRP1 and NB-ARC. Fourth, cis-acting element analysis illustrated that these R genes may bind to WRKY and bHLH. Finally, the expression of WRKY genes was verified by quantitative reverse transcription PCR (RT-qPCR). Phylogenetic analysis was carried out to further confirm that these TFs may play a critical role in Curcurbitaceae disease resistance. This study provides a new optimized combination strategy to explore the functions of TFs in a wide spectrum of biological processes. This strategy may also effectively predict potential relationships in the interactions of essential genes.

A study of leaf-senescence genes in rice based on a combination of genomics, proteomics and bioinformatics.

Leaf senescence is a highly complex, genetically regulated and well-ordered process with multiple layers and pathways. Delaying leaf senescence would help increase grain yields in rice. Over the past 15 years, more than 100 rice leaf-senescence genes have been cloned, greatly improving the understanding of leaf senescence in rice. Systematically elucidating the molecular mechanisms underlying leaf senescence will provide breeders with new tools/options for improving many important agronomic traits. In this study, we summarized recent reports on 125 rice leaf-senescence genes, providing an overview of the research progress in this field by analyzing the subcellular localizations, molecular functions and the relationship of them. These data showed that chlorophyll synthesis and degradation, chloroplast development, abscisic acid pathway, jasmonic acid pathway, nitrogen assimilation and ROS play an important role in regulating the leaf senescence in rice. Furthermore, we predicted and analyzed the proteins that interact with leaf-senescence proteins and achieved a more profound understanding of the molecular principles underlying the regulatory mechanisms by which leaf senescence occurs, thus providing new insights for future investigations of leaf senescence in rice.

uORF-mediated translation allows engineered plant disease resistance without fitness costs.

Controlling plant disease has been a struggle for humankind since the advent of agriculture. Studies of plant immune mechanisms have led to strategies of engineering resistant crops through ectopic transcription of plants' own defence genes, such as the master immune regulatory gene NPR1 (ref. 1). However, enhanced resistance obtained through such strategies is often associated with substantial penalties to fitness, making the resulting products undesirable for agricultural applications. To remedy this problem, we sought more stringent mechanisms of expressing defence proteins. On the basis of our latest finding that translation of key immune regulators, such as TBF1 (ref. 3), is rapidly and transiently induced upon pathogen challenge (see accompanying paper), we developed a 'TBF1-cassette' consisting of not only the immune-inducible promoter but also two pathogen-responsive upstream open reading frames (uORFsTBF1) of the TBF1 gene. Here we demonstrate that inclusion of uORFsTBF1-mediated translational control over the production of snc1-1 (an autoactivated immune receptor) in Arabidopsis thaliana and AtNPR1 in rice enables us to engineer broad-spectrum disease resistance without compromising plant fitness in the laboratory or in the field. This broadly applicable strategy may lead to decreased pesticide use and reduce the selective pressure for resistant pathogens.

Ribosome Biogenesis in Plants: From Functional 45S Ribosomal DNA Organization to Ribosome Assembly Factors.

The transcription of 18S, 5.8S, and 18S rRNA genes (45S rDNA), cotranscriptional processing of pre-rRNA, and assembly of mature rRNA with ribosomal proteins are the linchpins of ribosome biogenesis. In yeast (Saccharomyces cerevisiae) and animal cells, hundreds of pre-rRNA processing factors have been identified and their involvement in ribosome assembly determined. These studies, together with structural analyses, have yielded comprehensive models of the pre-40S and pre-60S ribosome subunits as well as the largest cotranscriptionally assembled preribosome particle: the 90S/small subunit processome. Here, we present the current knowledge of the functional organization of 45S rDNA, pre-rRNA transcription, rRNA processing activities, and ribosome assembly factors in plants, focusing on data from Arabidopsis (Arabidopsis thaliana). Based on yeast and mammalian cell studies, we describe the ribonucleoprotein complexes and RNA-associated activities and discuss how they might specifically affect the production of 40S and 60S subunits. Finally, we review recent findings concerning pre-rRNA processing pathways and a novel mechanism involved in a ribosome stress response in plants.

Expression, purification and structural characterization of resveratrol synthase from Polygonum cuspidatum.

Polygonum cuspidatum, an important medicinal plant in China, is a rich source of resveratrol compounds, and its synthesis related resveratrol synthase (RS) gene is highly expressed in stems. The sequence of the resveratrol synthase was amplified with specific primers. Sequence comparison showed that it was highly homologous to the STSs. The RS gene of Polygonum cuspidatum encodes 389 amino acids and has a theoretical molecular weight of 42.4 kDa, which is called PcRS1. To reveal the molecular basis of the synthesized resveratrol activity of PcRS1, we expressed the recombinant protein of full-length PcRS1 in Escherichia coli, and soluble protein products were produced. The collected products were purified by Ni-NTA chelation chromatography and appeared as a single band on SDS-PAGE. In order to obtain higher purity PcRS1, SEC was used to purify the protein and sharp single peak, and DLS detected that the aggregation state of protein molecules was homogeneous and stable. In order to verify the enzyme activity of the high-purity PcRS1, the reaction product was detected at 303 nm. By predicting the structural information of monomer PcRS1 and PcRS1 ligand complexes, we analyzed the ligand binding pocket and protein surface electrostatic potential of the complex, and compared it with the highly homologous STSs protein structures of the iso-ligand. New structural features of protein evolution are proposed. PcRS1 obtained a more complete configuration and the optimal orientation of the active site residues, thus improving its catalytic capacity in resveratrol synthesis.

Thylakoid localized bestrophin-like proteins are essential for the CO2 concentrating mechanism of Chlamydomonas reinhardtii.

The green alga Chlamydomonas reinhardtii possesses a CO2 concentrating mechanism (CCM) that helps in successful acclimation to low CO2 conditions. Current models of the CCM postulate that a series of ion transporters bring HCO3- from outside the cell to the thylakoid lumen, where the carbonic anhydrase 3 (CAH3) dehydrates accumulated HCO3- to CO2, raising the CO2 concentration for Ribulose bisphosphate carboxylase/oxygenase (Rubisco). Previously, HCO3- transporters have been identified at both the plasma membrane and the chloroplast envelope, but the transporter thought to be on the thylakoid membrane has not been identified. Three paralogous genes (BST1, BST2, and BST3) belonging to the bestrophin family have been found to be up-regulated in low CO2 conditions, and their expression is controlled by CIA5, a transcription factor that controls many CCM genes. YFP fusions demonstrate that all 3 proteins are located on the thylakoid membrane, and interactome studies indicate that they might associate with chloroplast CCM components. A single mutant defective in BST3 has near-normal growth on low CO2, indicating that the 3 bestrophin-like proteins may have redundant functions. Therefore, an RNA interference (RNAi) approach was adopted to reduce the expression of all 3 genes at once. RNAi mutants with reduced expression of BST1-3 were unable to grow at low CO2 concentrations, exhibited a reduced affinity to inorganic carbon (Ci) compared with the wild-type cells, and showed reduced Ci uptake. We propose that these bestrophin-like proteins are essential components of the CCM that deliver HCO3- accumulated in the chloroplast stroma to CAH3 inside the thylakoid lumen.

Mechanistic insights from plant heteromannan synthesis in yeast.

Heteromannan (HM) is one of the most ancient cell wall polymers in the plant kingdom, consisting of ß-(1-4)-linked backbones of glucose (Glc) and mannose (Man) units. Despite the widespread distribution of HM polysaccharides, their biosynthesis remains mechanistically unclear. HM is elongated by glycosyltransferases (GTs) from the cellulose synthase-like A (CSLA) family. MANNAN-SYNTHESIS RELATED (MSR) putative GTs have also been implicated in (gluco)mannan synthesis, but their roles have been difficult to decipher in planta and in vitro. To further characterize the products of the HM synthases and accessory proteins, we chose a synthetic biology approach to synthesize plant HM in yeast. The expression of a CSLA protein in Pichia pastoris led to the abundant production of plant HM: up to 30% of glycans in the yeast cell wall. Based on sequential chemical and enzymatic extractions, followed by detailed structural analyses, the newly produced HM polymers were unbranched and could be larger than 270 kDa. Using CSLAs from different species, we programmed yeast cells to produce an HM backbone composed exclusively of Man or also incorporating Glc. We demonstrate that specific MSR cofactors were indispensable for mannan synthase activity of a coffee CSLA or modulated a functional CSLA enzyme to produce glucomannan instead of mannan. Therefore, this powerful platform yields functional insight into the molecular machinery required for HM biosynthesis in plants.

Transposable elements drive rapid phenotypic variation in Capsella rubella.

Rapid phenotypic changes in traits of adaptive significance are crucial for organisms to thrive in changing environments. How such phenotypic variation is achieved rapidly, despite limited genetic variation in species that experience a genetic bottleneck is unknown. Capsella rubella, an annual and inbreeding forb (Brassicaceae), is a great system for studying this basic question. Its distribution is wider than those of its congeneric species, despite an extreme genetic bottleneck event that severely diminished its genetic variation. Here, we demonstrate that transposable elements (TEs) are an important source of genetic variation that could account for its high phenotypic diversity. TEs are (i) highly enriched in C. rubella compared with its outcrossing sister species Capsella grandiflora, and (ii) 4.2% of polymorphic TEs in C. rubella are associated with variation in the expression levels of their adjacent genes. Furthermore, we show that frequent TE insertions at FLOWERING LOCUS C (FLC) in natural populations of C. rubella could explain 12.5% of the natural variation in flowering time, a key life history trait correlated with fitness and adaptation. In particular, we show that a recent TE insertion at the 3' UTR of FLC affects mRNA stability, which results in reducing its steady-state expression levels, to promote the onset of flowering. Our results highlight that TE insertions can drive rapid phenotypic variation, which could potentially help with adaptation to changing environments in a species with limited standing genetic variation.

Genome-Wide Analysis of BpYABs and Function Identification Involving in the Leaf and Silique Development in Transgenic Arabidopsis.

YABs play an important role in the leaf development of the paper mulberry (Broussonetia papyrifera) and of the heterophylly. Thus, we investigated the function of BpYABs. Gene cloning, phylogenetic analysis, motif identification, subcellular localization, transactivation activity assay, qRT-PCR, in situ hybridization, and ectopic expression were used in our study. Six BpYABs were isolated, and four of them had transcriptional activity. BpYAB1, BpYAB3, BpYAB4, and BpYAB5 were localized to the nucleus. BpYAB1 was only expressed in the flower, while BpYAB6 was not expressed in any detected tissues; the four remaining BpYABs were expressed in the bud, leaf and flower, and their expression level decreased with leaf development. Further in situ hybridization showed that BpYAB3 and BpYAB5 were expressed in the vascular tissues and lamina, but neither showed the adaxial-abaxial polarity distribution pattern in the mature leaf lamina. Ectopic expression of BpYAB2, BpYAB3, BpYAB4 and BpYAB5 induced increased expression of AtWOX1 and caused the leaf of Arabidopsis to become smaller and curl downwards. Ectopic expression also led to shorter siliques and smaller seeds, but not for BpYAB5. These results suggest that BpYABs have functional divergency and redundancy in regulating leaf and silique development.

Mechanisms of evolved herbicide resistance.

The widely successful use of synthetic herbicides over the past 70 years has imposed strong and widespread selection pressure, leading to the evolution of herbicide resistance in hundreds of weed species. Both target-site resistance (TSR) and nontarget-site resistance (NTSR) mechanisms have evolved to most herbicide classes. TSR often involves mutations in genes encoding the protein targets of herbicides, affecting the binding of the herbicide either at or near catalytic domains or in regions affecting access to them. Most of these mutations are nonsynonymous SNPs, but polymorphisms in more than one codon or entire codon deletions have also evolved. Some herbicides bind multiple proteins, making the evolution of TSR mechanisms more difficult. Increased amounts of protein target, by increased gene expression or by gene duplication, are an important, albeit less common, TSR mechanism. NTSR mechanisms include reduced absorption or translocation and increased sequestration or metabolic degradation. The mechanisms that can contribute to NTSR are complex and often involve genes that are members of large gene families. For example, enzymes involved in herbicide metabolism-based resistances include cytochromes P450, GSH S-transferases, glucosyl and other transferases, aryl acylamidase, and others. Both TSR and NTSR mechanisms can combine at the individual level to produce higher resistance levels. The vast array of herbicide-resistance mechanisms for generalist (NTSR) and specialist (TSR and some NTSR) adaptations that have evolved over a few decades illustrate the evolutionary resilience of weed populations to extreme selection pressures. These evolutionary processes drive herbicide and herbicide-resistant crop development and resistance management strategies.

Genome-wide association mapping for protein, oil and water-soluble protein contents in soybean.

As a globally important legume crop, soybean provides excellent sources of protein and oil for human and livestock nutrition. Improving seed protein and oil contents has always been an important objective in soybean breeding. Water-soluble protein plays a significant role in the processing and efficacy of soybean protein. Here, a genome-wide association study (GWAS) of seed compositions (protein, oil, and water-soluble protein contents) was conducted using 211 diverse soybean accessions genotyped with a 355 K SoySNP array. Three, four, and five QTLs were identified related to the protein, oil, and water-soluble protein contents, respectively. Furthermore, five QTLs (qPC-15-1, qOC-8-1, qOC-12-1, qOC-20-1 and qWSPC-8-1) were detected in multiple environments. Analysis of the favorable alleles for oil and water-soluble protein contents showed that qOC-8-1 (qWSPC-8-1) exerted inverse effects on oil and water-soluble protein synthesis. Relative expression analysis suggested that Glyma.15G049200 in qPC-15-1 affects protein synthesis and Glyma.08G107800 in qOC-8-1 and qWSPC-8-1 might be involved in oil and water-soluble protein synthesis, producing opposite effects. The candidate genes and significant SNPs detected in the present study will allow a deeper understanding of the genetic basis for the regulation of protein, oil and water-soluble protein contents and provide important information that could be utilized in marker-assisted selection for soybean quality improvement.

The tobacco phosphatidylethanolamine-binding protein NtFT4 simultaneously improves vitality, growth, and protein yield in human cells.

The production of biopharmaceutical proteins in mammalian cells by transient expression or stable transformation requires robust and viable cells. Cell line engineering must therefore balance improved cell growth and viability with high productivity. We tested the ability of nonmammalian phosphatidylethanolamine-binding proteins to enhance cell proliferation in monolayers and suspension cultures. The tobacco protein NtFT4 improved the proliferation of multiple human cell lines. Viable cell density is usually impaired by efficient transfection, but we found that the number of HEK-293TNtFT4 cells at the peak of protein expression was twice that of standard HEK-293T cells, and the antibody yield increased by approximately one-third. Improved growth and viability were observed in different cell lines, in different culture media, and also after transient transfection, suggesting the beneficial trait is consistent and transferable. Additional modifications could boost the productivity of high-density HEK-293TNtFT4 cells even further as we showed for a fluorescent marker protein and recombinant antibody expressed in monolayer cultures. The HEK-293TNtFT4 cell line provides a new human model platform that increases cell proliferation, also achieving a fundamental improvement in recombinant protein expression.

Efficient expression and purification of soluble HarpinEa protein by translation initiation region codon optimization.

HarpinEa protein can stimulate plants to produce defense responses to resist the attack of pathogens, improve plant immune resistance, and promote plant growth. This has extremely high application value in agriculture. To efficiently express soluble HarpinEa protein, in this study, we expressed HarpinEa protein with a 6× His-tag in Escherichia coli BL21 (DE3). Because of the low level of expression of HarpinEa protein in E. coli, three rounds of synonymous codon optimization were performed on the +53 bp of the translation initiation region (TIR) of HarpinEa. Soluble HarpinEa protein after optimization accounted for 50.3% of the total soluble cellular protein expressed. After purification using a Ni Bestarose Fast Flow column, the purity of HarpinEa protein exceeded 95%, and the yield reached 227.5 mg/L of culture medium. The purified HarpinEa protein was sensitive to proteases and exhibited thermal stability. It triggered visible hypersensitive responses after being injected into tobacco leaves for 48 h. Plants treated with HarpinEa showed obvious growth-promoting and resistance-improving performance. Thus, the use of TIR synonymous codon optimization successfully achieved the economical, efficient, and soluble production of HarpinEa protein.

Expression studies of stress responsive genes in cotton Gossypium hirsutum L.

BACKGROUND: Cotton is the world's richest source of natural fiber. Meanwhile cotton plant is top ranked stress sensitive plant thereby affecting its yield and fiber quality. But, in climate change scenario, fiber yield and quality are being affected due to environmental stresses, especially heat, drought and salinity. Present study is aimed to identify cotton genotype harboring prominently expressed stress responsive genes. METHODS: Four cotton genotypes (IUB-13, IUB-222, IUB-09 and MM-58) were evaluated under drought and salinity stress for yield traits and expression of different stress responsive genes (GhWRKY3, GhDREB2 and GhRDR6). RESULTS: Pronounced expression of GhWRKY3, GhDREB2and GhRDR6 was observed in cotton variety IUB-13 in stress condition (drought and salinity) as compared to control followed by IUB-222 which revealed that these genotypes might possess substantial potential to cope with environmental hazards encountered in growing season CONCLUSION: Utilization of cotton genotypes i.e., IUB-13 and IUB-222 in cotton breeding program can be very much fruitful for developing cotton genotypes adoptable to climate change.

Regulation of ß-amylase synthesis: a brief overview.

BACKGROUND: The major activity of ß-amylase (BMY) is the production of maltose by the hydrolytic degradation of starch. BMY is found to be produced by some plants and few microorganisms only. The industrial importance of the enzyme warrants its application in a larger scale with the help of genetic engineering, for which the regulatory mechanism is to be clearly understood. RESULTS AND CONCLUSION: In plants, the activities of BMY are regulated by various environmental stimuli including stress of drought, cold and heat. In vascular plant, Arabidopsis sp. the enzyme is coded by nine BAM genes, whereas in most bacteria, BMY enzymes are coded by the spoII gene family. The activities of these genes are in turn controlled by various compounds. Production and inhibition of the microbial BMY is regulated by the activation and inactivation of various BAM genes. Various types of transcriptional regulators associated with the plant- BMYs regulate the production of BMY enzyme. The enhancement in the expression of such genes reflects evolutionary significance. Bacterial genes, on the other hand, as exemplified by Bacillus sp and Clostridium sp, clearly depict the importance of a single regulatory gene, the absence or mutation of which totally abolishes the BMY activity.

Primary transcripts of microRNAs encode regulatory peptides.

MicroRNAs (miRNAs) are small regulatory RNA molecules that inhibit the expression of specific target genes by binding to and cleaving their messenger RNAs or otherwise inhibiting their translation into proteins. miRNAs are transcribed as much larger primary transcripts (pri-miRNAs), the function of which is not fully understood. Here we show that plant pri-miRNAs contain short open reading frame sequences that encode regulatory peptides. The pri-miR171b of Medicago truncatula and the pri-miR165a of Arabidopsis thaliana produce peptides, which we term miPEP171b and miPEP165a, respectively, that enhance the accumulation of their corresponding mature miRNAs, resulting in downregulation of target genes involved in root development. The mechanism of miRNA-encoded peptide (miPEP) action involves increasing transcription of the pri-miRNA. Five other pri-miRNAs of A. thaliana and M. truncatula encode active miPEPs, suggesting that miPEPs are widespread throughout the plant kingdom. Synthetic miPEP171b and miPEP165a peptides applied to plants specifically trigger the accumulation of miR171b and miR165a, leading to reduction of lateral root development and stimulation of main root growth, respectively, suggesting that miPEPs might have agronomical applications.

Overexpression of stilbene synthase genes to modulate the properties of plants and plant cell cultures.

Plant stilbenes have attracted special attention as they possess valuable health benefits and improve plant resistance to environmental stresses. Stilbenes are synthesized via the phenylpropanoid pathway, where stilbene synthase (STS, EC 2.3.1.95) directly catalyzes the formation of t-resveratrol (monomeric stilbene). This review discusses the features of using STS genes in genetic engineering and plant biotechnology with the purpose to increase plant resistance to environmental stresses and to modify secondary metabolite production.

Third DWF1 paralog in Solanaceae, sterol Δ24-isomerase, branches withanolide biosynthesis from the general phytosterol pathway.

A large part of chemodiversity of plant triterpenes is due to the modification of their side chains. Reduction or isomerization of double bonds in the side chains is often an important step for the diversification of triterpenes, although the enzymes involved are not fully understood. Withanolides are a large group of structurally diverse C28 steroidal lactones derived from 24-methylenecholesterol. These compounds are found in the Indian medicinal plant Withania somnifera, also known as ashwagandha, and other members of the Solanaceae. The pathway for withanolide biosynthesis is unknown, preventing sustainable production via white biotechnology and downstream pharmaceutical usages. In the present study, based on genome and transcriptome data we have identified a key enzyme in the biosynthesis of withanolides: a DWF1 paralog encoding a sterol Δ24-isomerase (24ISO). 24ISO originated from DWF1 after two subsequent duplication events in Solanoideae plants. Withanolides and 24ISO appear only in the medicinal plants in the Solanoideae, not in crop plants such as potato and tomato, indicating negative selection during domestication. 24ISO is a unique isomerase enzyme evolved from a reductase and as such has maintained the FAD-binding oxidoreductase structure and requirement for NADPH. Using phylogenetic, metabolomic, and gene expression analysis in combination with heterologous expression and virus-induced gene silencing, we showed that 24ISO catalyzes the conversion of 24-methylenecholesterol to 24-methyldesmosterol. We propose that this catalytic step is the committing step in withanolide biosynthesis, opening up elucidation of the whole pathway and future larger-scale sustainable production of withanolides and related compounds with pharmacological properties.

Development of a cell-free protein synthesis system for practical use.

Conventional cell-free protein synthesis systems had been the major platform to study the mechanism behind translating genetic information into proteins, as proven in the central dogma of molecular biology. Albeit being powerful research tools, most of the in vitro methods at the time failed to produce enough protein for practical use. Tremendous efforts were being made to overcome the limitations of in vitro translation systems, though mostly with limited success. While great knowledge was accumulated on the translation mechanism and ribosome structure, researchers rationalized that it may be impossible to fully reconstitute such a complex molecular process in a test tube. This review will examine how we have solved the difficulties holding back progress. Our newly developed cell-free protein synthesis system is based on wheat embryos and has many excellent characteristics, in addition to its high translation activity and robustness. Combined with other novel elementary technologies, we have established cell-free protein synthesis systems for practical use in research and applied sciences.

Genes Associated with Foliar Resistance to Septoria Nodorum Blotch of Hexaploid Wheat (Triticum aestivum L.).

The genetic control of host response to the fungal necrotrophic disease Septoria nodorum blotch (SNB) in bread wheat is complex, involving many minor genes. Quantitative trait loci (QTL) controlling SNB response were previously identified on chromosomes 1BS and 5BL. The aim of this study, therefore, was to align and compare the genetic map representing QTL interval on 1BS and 5BS with the reference sequence of wheat and identify resistance genes (R-genes) associated with SNB response. Alignment of QTL intervals identified significant genome rearrangements on 1BS between parents of the DH population EGA Blanco, Millewa and the reference sequence of Chinese Spring with subtle rearrangements on 5BL. Nevertheless, annotation of genomic intervals in the reference sequence were able to identify and map 13 and 12 R-genes on 1BS and 5BL, respectively. R-genes discriminated co-located QTL on 1BS into two distinct but linked loci. NRC1a and TFIID mapped in one QTL on 1BS whereas RGA and Snn1 mapped in the linked locus and all were associated with SNB resistance but in one environment only. Similarly, Tsn1 and WK35 were mapped in one QTL on 5BL with NETWORKED 1A and RGA genes mapped in the linked QTL interval. This study provided new insights on possible biochemical, cellular and molecular mechanisms responding to SNB infection in different environments and also addressed limitations of using the reference sequence to identify the full complement of functional R-genes in modern varieties.