Spatial gradients in cell wall composition and transcriptional profiles along elongating maize internodes.

BACKGROUND: The elongating maize internode represents a useful system for following development of cell walls in vegetative cells in the Poaceae family. Elongating internodes can be divided into four developmental zones, namely the basal intercalary meristem, above which are found the elongation, transition and maturation zones. Cells in the basal meristem and elongation zones contain mainly primary walls, while secondary cell wall deposition accelerates in the transition zone and predominates in the maturation zone. RESULTS: The major wall components cellulose, lignin and glucuronoarabinoxylan (GAX) increased without any abrupt changes across the elongation, transition and maturation zones, although GAX appeared to increase more between the elongation and transition zones. Microarray analyses show that transcript abundance of key glycosyl transferase genes known to be involved in wall synthesis or re-modelling did not match the increases in cellulose, GAX and lignin. Rather, transcript levels of many of these genes were low in the meristematic and elongation zones, quickly increased to maximal levels in the transition zone and lower sections of the maturation zone, and generally decreased in the upper maturation zone sections. Genes with transcript profiles showing this pattern included secondary cell wall CesA genes, GT43 genes, some ß-expansins, UDP-Xylose synthase and UDP-Glucose pyrophosphorylase, some xyloglucan endotransglycosylases/hydrolases, genes involved in monolignol biosynthesis, and NAM and MYB transcription factor genes. CONCLUSIONS: The data indicated that the enzymic products of genes involved in cell wall synthesis and modification remain active right along the maturation zone of elongating maize internodes, despite the fact that corresponding transcript levels peak earlier, near or in the transition zone.

Endo-(1,4)-ß-glucanase gene families in the grasses: temporal and spatial co-transcription of orthologous genes.

BACKGROUND: Endo-(1,4)-ß-glucanase (cellulase) glycosyl hydrolase GH9 enzymes have been implicated in several aspects of cell wall metabolism in higher plants, including cellulose biosynthesis and degradation, modification of other wall polysaccharides that contain contiguous (1,4)-ß-glucosyl residues, and wall loosening during cell elongation. RESULTS: The endo-(1,4)-ß-glucanase gene families from barley (Hordeum vulgare), maize (Zea mays), sorghum (Sorghum bicolor), rice (Oryza sativa) and Brachypodium (Brachypodium distachyon) range in size from 23 to 29 members. Phylogenetic analyses show variations in clade structure between the grasses and Arabidopsis, and indicate differential gene loss and gain during evolution. Map positions and comparative studies of gene structures allow orthologous genes in the five species to be identified and synteny between the grasses is found to be high. It is also possible to differentiate between homoeologues resulting from ancient polyploidizations of the maize genome. Transcript analyses using microarray, massively parallel signature sequencing and quantitative PCR data for barley, rice and maize indicate that certain members of the endo-(1,4)-ß-glucanase gene family are transcribed across a wide range of tissues, while others are specifically transcribed in particular tissues. There are strong correlations between transcript levels of several members of the endo-(1,4)-ß-glucanase family and the data suggest that evolutionary conservation of transcription exists between orthologues across the grass family. There are also strong correlations between certain members of the endo-(1,4)-ß-glucanase family and other genes known to be involved in cell wall loosening and cell expansion, such as expansins and xyloglucan endotransglycosylases. CONCLUSIONS: The identification of these groups of genes will now allow us to test hypotheses regarding their functions and joint participation in wall synthesis, re-modelling and degradation, together with their potential role in lignocellulose conversion during biofuel production from grasses and cereal crop residues.

A customized gene expression microarray reveals that the brittle stem phenotype fs2 of barley is attributable to a retroelement in the HvCesA4 cellulose synthase gene.

The barley (Hordeum vulgare) brittle stem mutants, fs2, designated X054 and M245, have reduced levels of crystalline cellulose compared with their parental lines Ohichi and Shiroseto. A custom-designed microarray, based on long oligonucleotide technology and including genes involved in cell wall metabolism, revealed that transcript levels of very few genes were altered in the elongation zone of stem internodes, but these included a marked decrease in mRNA for the HvCesA4 cellulose synthase gene of both mutants. In contrast, the abundance of several hundred transcripts changed in the upper, maturation zones of stem internodes, which presumably reflected pleiotropic responses to a weakened cell wall that resulted from the primary genetic lesion. Sequencing of the HvCesA4 genes revealed the presence of a 964-bp solo long terminal repeat of a Copia-like retroelement in the first intron of the HvCesA4 genes of both mutant lines. The retroelement appears to interfere with transcription of the HvCesA4 gene or with processing of the mRNA, and this is likely to account for the lower crystalline cellulose content and lower stem strength of the mutants. The HvCesA4 gene maps to a position on chromosome 1H of barley that coincides with the previously reported position of fs2.

Artificial trans-acting siRNAs confer consistent and effective gene silencing.

Manipulating gene expression is critical to exploring gene function and a useful tool for altering commercial traits. Techniques such as hairpin-based RNA interference, virus-induced gene silencing, and artificial microRNAs take advantage of endogenous posttranscriptional gene silencing pathways to block translation of designated transcripts. Here we present a novel gene silencing method utilizing artificial trans-acting small interfering RNAs in Arabidopsis (Arabidopsis thaliana). Replacing the endogenous small interfering RNAs encoded in the TAS1c gene with sequences from the FAD2 gene silenced FAD2 activity to levels comparable to the fad2-1 null allele in nearly all transgenic events. Interestingly, exchanging the endogenous miR173 target sequence in TAS1c with an miR167 target sequence led to variable, inefficient silencing of FAD2, suggesting a specific requirement for the miR173 trigger for production of small interfering RNAs from the TAS1c locus.

Conserved noncoding genomic sequences associated with a flowering-time quantitative trait locus in maize.

Flowering time is a fundamental trait of maize adaptation to different agricultural environments. Although a large body of information is available on the map position of quantitative trait loci for flowering time, little is known about the molecular basis of quantitative trait loci. Through positional cloning and association mapping, we resolved the major flowering-time quantitative trait locus, Vegetative to generative transition 1 (Vgt1), to an approximately 2-kb noncoding region positioned 70 kb upstream of an Ap2-like transcription factor that we have shown to be involved in flowering-time control. Vgt1 functions as a cis-acting regulatory element as indicated by the correlation of the Vgt1 alleles with the transcript expression levels of the downstream gene. Additionally, within Vgt1, we identified evolutionarily conserved noncoding sequences across the maize-sorghum-rice lineages. Our results support the notion that changes in distant cis-acting regulatory regions are a key component of plant genetic adaptation throughout breeding and evolution.

Monitoring genome-wide changes in gene expression in response to endogenous cytokinin reveals targets in Arabidopsis thaliana.

Cytokinins have been implicated in developmental and growth processes in plants including cell division, chloroplast biogenesis, shoot meristem initiation and senescence. The regulation of these processes requires changes in cytokinin-responsive gene expression. Here, we induced the expression of a bacterial isopentenyl transferase gene, IPT, in transgenic Arabidopsis thaliana seedlings to study the regulation of genome-wide gene expression in response to endogenous cytokinin. Using MPSS (massively parallel signature sequencing) we identified 823 and 917 genes that were up- and downregulated, respectively, following 24 h of IPT induction. When comparing the response to cytokinin after 6 and 24 h, we identified different clusters of genes showing a similar course of regulation. Our study provides researchers with the opportunity to rapidly assess whether genes of interest are regulated by cytokinins.

Genome-wide gene expression profiling in Arabidopsis thaliana reveals new targets of abscisic acid and largely impaired gene regulation in the abi1-1 mutant.

The phytohormone abscisic acid (ABA) plays important regulatory roles in many plant developmental processes including seed dormancy, germination, growth, and stomatal movements. These physiological responses to ABA are in large part brought about by changes in gene expression. To study genome-wide ABA-responsive gene expression we applied massively parallel signature sequencing (MPSS) to samples from Arabidopsis thaliana wildtype (WT) and abi1-1 mutant seedlings. We identified 1354 genes that are either up- or downregulated following ABA treatment of WT seedlings. Among these ABA-responsive genes, many encode signal transduction components. In addition, we identified novel ABA-responsive gene families including those encoding ribosomal proteins and proteins involved in regulated proteolysis. In the ABA-insensitive mutant abi1-1, ABA regulation of about 84.5% and 6.9% of the identified genes was impaired or strongly diminished, respectively; however, 8.6% of the genes remained appropriately regulated. Compared to other methods of gene expression analysis, the high sensitivity and specificity of MPSS allowed us to identify a large number of ABA-responsive genes in WT Arabidopsis thaliana. The database given in our supplementary material (http://jcs.biologists.org/supplemental) provides researchers with the opportunity to rapidly assess whether genes of interest may be regulated by ABA. Regulation of the majority of the genes by ABA was impaired in the ABA-insensitive mutant abi1-1. However, a subset of genes continued to be appropriately regulated by ABA, which suggests the presence of at least two ABA signaling pathways, only one of which is blocked in abi1-1.

Insertion-deletion polymorphisms in 3' regions of maize genes occur frequently and can be used as highly informative genetic markers.

Single-nucleotide polymorphisms (SNPs) are the most frequent variations in the genome of any organism. SNP discovery approaches such as resequencing or data mining enable the identification of insertion deletion (indel) polymorphisms. These indels can be treated as biallelic markers and can be utilized for genetic mapping and diagnostics. In this study 655 indels have been identified by resequencing 502 maize (Zea mays) loci across 8 maize inbreds (selected for their high allelic variation). Of these 502 loci, 433 were polymorphic, with indels identified in 215 loci. Of the 655 indels identified, single-nucleotide indels accounted for more than half (54.8%) followed by two- and three-nucleotide indels. A high frequency of 6-base (3.4%) and 8-base (2.3%) indels were also observed. When analysis is restricted to the B73 and Mol7 genotypes, 53% of the loci analyzed contained indels, with 42% having an amplicon size difference. Three novel miniature inverted-repeat transposable element (MITE)-like sequences were identified as insertions near genes. The utility of indels as genetic markers was demonstrated by using indel polymorphisms to map 22 loci in a B73 x Mo17 recombinant inbred population. This paper clearly demonstrates that the resequencing of 3' EST sequence and the discovery and mapping of indel markers will position corresponding expressed genes on the genetic map.

DNA array profiling of gene expression changes during maize embryo development.

We are using DNA microarray-based gene expression profiling to classify temporal patterns of gene expression during the development of maize embryos, to understand mRNA-level control of embryogenesis and to dissect metabolic pathways and their interactions in the maize embryo. Genes involved in carbohydrate, fatty acid, and amino acid metabolism, the tricarboxylic acid (TCA) cycle, glycolysis, the pentose phosphate pathway, embryogenesis, membrane transport, signal transduction, cofactor biosynthesis, photosynthesis, oxidative phosphorylation and electron transfer, as well as 600 random complementary DNA (cDNA) clones from maize embryos, were arrayed on glass slides. DNA arrays were hybridized with fluorescent dye-labeled cDNA probes synthesized from kernel and embryo poly(A)(+)RNA from different stages of maize seed development. Several characteristic developmental patterns of expression were identified and correlated with gene function. Patterns of coordinated gene expression in the TCA cycle and glycolysis were analyzed in detail. The steady state level of poly(A)(+) RNA for many genes varies dramatically during maize embryo development. Expression patterns of genes coding for enzymes of fatty acid biosynthesis and glycolysis are coordinately regulated during development. Genes of unknown function may by assigned a hypothetical role based on their patterns of expression resembling well characterized genes. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s10142-002-0046-6.