Analysis of the Promoter of Emb5 from Zea mays Identifies a Region of 523 bp Responsible for Its Embryo-Specific Activity

The maize Emb5 is an abscisic acid–responsive gene which is specifically expressed in the late embryo during seed maturity. To further dissect and identify the elements specific for its embryo expression pattern, we investigated the activity of the − 1653 bp upstream of the “full-length” promoter region of this gene in transgenic Arabidopsis plants. We first confirmed that the “full-length” promoter could indeed drive the expression of β-glucuronidase reporter gene (GUS) in the transgenic Arabidopsis seed embryo. Subsequently, DNA fragments of ~ 500 bp in length were generated after a series of progressive deletions from positions − 1653 bp to − 1 bp relative to the transcriptional initiation site. These fragments were fused with GUS and introduced into Arabidopsis. Measurement of the GUS activity in the immature seeds isolated from the transgenic plants revealed that the region between positions − 523 bp and − 1 bp, namely ProEm-D, is absolutely required and sufficient for the temporal and embryo-specific expression of GUS with an activity comparable with the full-length Emb5 promoter in Arabidopsis. Therefore, our results clearly demonstrated that the 523 bp ProEm-D can replace the − 1653 bp Emb5 promoter to drive embryo-specific expression in Arabidopsis seed. Because of its small size and strong embryo-specific activity, it could become the promoter of choice in metabolic pathway engineering to transfer multiple genes for the production of valuable pharmaceutical products in seeds, such as polyunsaturated fatty acids found in fish oils, or pro-vitamin A where at least three transgenes are required to assemble the entire metabolic pathways

Deep EST profiling of developing fenugreek endosperm to investigate galactomannan biosynthesis and its regulation

Galactomannans are hemicellulosic polysaccharides composed of a (1 → 4)-linked β-D-mannan backbone substituted with single-unit (1 → 6)-α-linked D-galactosyl residues. Developing fenugreek (Trigonella foenum-graecum) seeds are known to accumulate large quantities of galactomannans in the endosperm, and were thus used here as a model system to better understand galactomannan biosynthesis and its regulation. We first verified the specific deposition of galactomannans in developing endosperms and determined that active accumulation occurred from 25 to 38 days post anthesis (DPA) under our growth conditions. We then examined the expression levels during seed development of ManS and GMGT, two genes encoding backbone and side chain synthetic enzymes. Based on transcript accumulation dynamics for ManS and GMGT, cDNA libraries were constructed using RNA isolated from endosperms at four ages corresponding to before, at the beginning of, and during active galactomannan deposition. DNA from these libraries was sequenced using the 454 sequencing technology to yield a total of 1.5 million expressed sequence tags (ESTs). Through analysis of the EST profiling data, we identified genes known to be involved in galactomannan biosynthesis, as well as new genes that may be involved in this process, and proposed a model for the flow of carbon from sucrose to galactomannans. Measurement of in vitro ManS and GMGT activities and analysis of sugar phosphate and nucleotide sugar levels in the endosperms of developing fenugreek seeds provided data consistent with this model. In vitro enzymatic assays also revealed that the ManS enzyme from fenugreek endosperm preferentially used GDP-mannose as the substrate for the backbone synthesis

PtrWRKY19, a novel WRKY transcription factor, contributes to the regulation of pith secondary wall formation in Populus trichocarpa

WRKY proteins are one of the largest transcription factor families in higher plants and play diverse roles in various biological processes. Previous studies have shown that some WRKY members act as negative regulators of secondary cell wall formation in pith parenchyma cells. However, the regulatory mechanism of pith secondary wall formation in tree species remains largely unknown. In this study, PtrWRKY19 encoding a homolog of Arabidopsis WRKY12 was isolated from Populus trichocarpa. PtrWRKY19 was expressed in all tissues tested, with highest expression in stems, especially in pith. PtrWRKY19 was located in the nucleus and functioned as a transcriptional repressor. Ectopic expression of PtrWRKY19 in an atwrky12 mutant successfully rescued the phenotype in pith cell walls caused by the defect of AtWRKY12, suggesting that PtrWRKY19 had conserved functions for homologous AtWRKY12. Overexpression of PtrWRKY19 in poplar plants led to a significant increase in the number of pith parenchyma cells. qRT-PCR analysis showed that lignin biosynthesis-related genes were repressed in transgenic plants. In transcient reporter assays, PtrWRKY19 was identified to repress transcription from the PtoC4H2 promoter containing the conserved W-box elements. These results indicated that PtrWRKY19 may function as a negative regulator of pith secondary wall formation in poplar

An efficient approach to finding Siraitia grosvenorii triterpene biosynthetic genes by RNA-seq and digital gene expression analysis

<p>Abstract</p> <p>Background</p> <p><it>Siraitia grosvenorii </it>(Luohanguo) is an herbaceous perennial plant native to southern China and most prevalent in Guilin city. Its fruit contains a sweet, fleshy, edible pulp that is widely used in traditional Chinese medicine. The major bioactive constituents in the fruit extract are the cucurbitane-type triterpene saponins known as mogrosides. Among them, mogroside V is nearly 300 times sweeter than sucrose. However, little is known about mogrosides biosynthesis in <it>S. grosvenorii</it>, especially the late steps of the pathway.</p> <p>Results</p> <p>In this study, a cDNA library generated from of equal amount of RNA taken from <it>S. grosvenorii </it>fruit at 50 days after flowering (DAF) and 70 DAF were sequenced using Illumina/Solexa platform. More than 48,755,516 high-quality reads from a cDNA library were generated that was assembled into 43,891 unigenes. De novo assembly and gap-filling generated 43,891 unigenes with an average sequence length of 668 base pairs. A total of 26,308 (59.9%) unique sequences were annotated and 11,476 of the unique sequences were assigned to specific metabolic pathways by the Kyoto Encyclopedia of Genes and Genomes. cDNA sequences for all of the known enzymes involved in mogrosides backbone synthesis were identified from our library. Additionally, a total of eighty-five cytochrome P450 (CYP450) and ninety UDP-glucosyltransferase (UDPG) unigenes were identified, some of which appear to encode enzymes responsible for the conversion of the mogroside backbone into the various mogrosides. Digital gene expression profile (DGE) analysis using Solexa sequencing was performed on three important stages of fruit development, and based on their expression pattern, seven <it>CYP450</it>s and five <it>UDPG</it>s were selected as the candidates most likely to be involved in mogrosides biosynthesis.</p> <p>Conclusion</p> <p>A combination of RNA-seq and DGE analysis based on the next generation sequencing technology was shown to be a powerful method for identifying candidate genes encoding enzymes responsible for the biosynthesis of novel secondary metabolites in a non-model plant. Seven <it>CYP450</it>s and five <it>UDPG</it>s were selected as potential candidates involved in mogrosides biosynthesis. The transcriptome data from this study provides an important resource for understanding the formation of major bioactive constituents in the fruit extract from <it>S. grosvenorii</it>.</p

Metabolic Profiles and cDNA-AFLP Analysis of Salvia miltiorrhiza and Salvia castanea Diel f. tomentosa Stib

Plants of the genus Salvia produce various types of phenolic compounds and tanshinones which are effective for treatment of coronary heart disease. Salvia miltiorrhiza and S. castanea Diels f. tomentosa Stib are two important members of the genus. In this study, metabolic profiles and cDNA-AFLP analysis of four samples were employed to identify novel genes potentially involved in phenolic compounds and tanshinones biosynthesis, including the red roots from the two species and two tanshinone-free roots from S. miltiorrhiza. The results showed that the red roots of S. castanea Diels f. tomentosa Stib produced high contents of rosmarinic acid (21.77 mg/g) and tanshinone IIA (12.60 mg/g), but low content of salvianolic acid B (1.45 mg/g). The red roots of S. miltiorrhiza produced high content of salvianolic acid B (18.69 mg/g), while tanshinones accumulation in this sample was much less than that in S. castanea Diels f. tomentosa Stib. Tanshinones were not detected in the two tanshinone-free samples, which produced high contents of phenolic compounds. A cDNA-AFLP analysis with 128 primer pairs revealed that 2300 transcript derived fragments (TDFs) were differentially expressed among the four samples. About 323 TDFs were sequenced, of which 78 TDFs were annotated with known functions through BLASTX searching the Genbank database and 14 annotated TDFs were assigned into secondary metabolic pathways through searching the KEGGPATHWAY database. The quantitative real-time PCR analysis indicated that the expression of 9 TDFs was positively correlated with accumulation of phenolic compounds and tanshinones. These TDFs additionally showed coordinated transcriptional response with 6 previously-identified genes involved in biosynthesis of tanshinones and phenolic compounds in S. miltiorrhiza hairy roots treated with yeast extract. The sequence data in the present work not only provided us candidate genes involved in phenolic compounds and tanshinones biosynthesis but also gave us further insight into secondary metabolism in Salvia

Transcriptome analysis of Bupleurum chinense focusing on genes involved in the biosynthesis of saikosaponins

<p>Abstract</p> <p>Background</p> <p><it>Bupleurum chinense </it>DC. is a widely used traditional Chinese medicinal plant. Saikosaponins are the major bioactive constituents of <it>B. chinense</it>, but relatively little is known about saikosaponin biosynthesis. The 454 pyrosequencing technology provides a promising opportunity for finding novel genes that participate in plant metabolism. Consequently, this technology may help to identify the candidate genes involved in the saikosaponin biosynthetic pathway.</p> <p>Results</p> <p>One-quarter of the 454 pyrosequencing runs produced a total of 195, 088 high-quality reads, with an average read length of 356 bases (NCBI SRA accession SRA039388). A <it>de novo </it>assembly generated 24, 037 unique sequences (22, 748 contigs and 1, 289 singletons), 12, 649 (52.6%) of which were annotated against three public protein databases using a basic local alignment search tool (E-value ≤1e-10). All unique sequences were compared with NCBI expressed sequence tags (ESTs) (237) and encoding sequences (44) from the <it>Bupleurum </it>genus, and with a Sanger-sequenced EST dataset (3, 111). The 23, 173 (96.4%) unique sequences obtained in the present study represent novel <it>Bupleurum </it>genes. The ESTs of genes related to saikosaponin biosynthesis were found to encode known enzymes that catalyze the formation of the saikosaponin backbone; 246 cytochrome P450 (<it>P450</it>s) and 102 glycosyltransferases (<it>GT</it>s) unique sequences were also found in the 454 dataset. Full length cDNAs of 7 <it>P450</it>s and 7 uridine diphosphate <it>GT</it>s (<it>UGT</it>s) were verified by reverse transcriptase polymerase chain reaction or by cloning using 5' and/or 3' rapid amplification of cDNA ends. Two <it>P450</it>s and three <it>UGT</it>s were identified as the most likely candidates involved in saikosaponin biosynthesis. This finding was based on the coordinate up-regulation of their expression with <it>β-AS </it>in methyl jasmonate-treated adventitious roots and on their similar expression patterns with <it>β-AS </it>in various <it>B. chinense </it>tissues.</p> <p>Conclusions</p> <p>A collection of high-quality ESTs for <it>B. chinense </it>obtained by 454 pyrosequencing is provided here for the first time. These data should aid further research on the functional genomics of <it>B. chinense </it>and other <it>Bupleurum </it>species. The candidate genes for enzymes involved in saikosaponin biosynthesis, especially the <it>P450</it>s and <it>UGT</it>s, that were revealed provide a substantial foundation for follow-up research on the metabolism and regulation of the saikosaponins.</p

A combined functional and structural genomics approach identified an EST-SSR marker with complete linkage to the Ligon lintless-2 genetic locus in cotton (Gossypium hirsutum L.)

<p>Abstract</p> <p>Background</p> <p>Cotton fiber length is an important quality attribute to the textile industry and longer fibers can be more efficiently spun into yarns to produce superior fabrics. There is typically a negative correlation between yield and fiber quality traits such as length. An understanding of the regulatory mechanisms controlling fiber length can potentially provide a valuable tool for cotton breeders to improve fiber length while maintaining high yields. The cotton (<it>Gossypium hirsutum </it>L.) fiber mutation Ligon lintless-2 is controlled by a single dominant gene (<it>Li₂</it>) that results in significantly shorter fibers than a wild-type. In a near-isogenic state with a wild-type cotton line, <it>Li₂</it>is a model system with which to study fiber elongation.</p> <p>Results</p> <p>Two near-isogenic lines of Ligon lintless-2 (<it>Li₂</it>) cotton, one mutant and one wild-type, were developed through five generations of backcrosses (BC₅). An F₂population was developed from a cross between the two <it>Li₂</it>near-isogenic lines and used to develop a linkage map of the <it>Li₂</it>locus on chromosome 18. Five simple sequence repeat (SSR) markers were closely mapped around the <it>Li₂</it>locus region with two of the markers flanking the <it>Li₂</it>locus at 0.87 and 0.52 centimorgan. No apparent differences in fiber initiation and early fiber elongation were observed between the mutant ovules and the wild-type ones. Gene expression profiling using microarrays suggested roles of reactive oxygen species (ROS) homeostasis and cytokinin regulation in the <it>Li₂</it>mutant phenotype. Microarray gene expression data led to successful identification of an EST-SSR marker (NAU3991) that displayed complete linkage to the <it>Li₂</it>locus.</p> <p>Conclusions</p> <p>In the field of cotton genomics, we report the first successful conversion of gene expression data into an SSR marker that is associated with a genomic region harboring a gene responsible for a fiber trait. The EST-derived SSR marker NAU3991 displayed complete linkage to the <it>Li₂</it>locus on chromosome 18 and resided in a gene with similarity to a putative plectin-related protein. The complete linkage suggests that this expressed sequence may be the <it>Li₂</it>gene.</p

Recent advances of metabolomics in plant biotechnology

Biotechnology, including genetic modification, is a very important approach to regulate the production of particular metabolites in plants to improve their adaptation to environmental stress, to improve food quality, and to increase crop yield. Unfortunately, these approaches do not necessarily lead to the expected results due to the highly complex mechanisms underlying metabolic regulation in plants. In this context, metabolomics plays a key role in plant molecular biotechnology, where plant cells are modified by the expression of engineered genes, because we can obtain information on the metabolic status of cells via a snapshot of their metabolome. Although metabolome analysis could be used to evaluate the effect of foreign genes and understand the metabolic state of cells, there is no single analytical method for metabolomics because of the wide range of chemicals synthesized in plants. Here, we describe the basic analytical advancements in plant metabolomics and bioinformatics and the application of metabolomics to the biological study of plants