Kanwarpal S. Dhugga

Cellulose synthesis in maize: isolation and expression analysis of the cellulose synthase (CesA) gene family

Stalk lodging in maize results in significant yield losses. We have determined that cellulose per unit length of the stalk is the primary determinant of internodal strength. An increase in cellulose concentration in the wall might allow simultaneous improvements in stalk strength and harvest index. Cellulose formation in plants can be perturbed by mutations in the genes involved in cellulose synthesis, post-synthetic cellulose alteration or deposition, N-glycosylation, and some other genes with as yet unknown functions. We have isolated 12 members of the cellulose synthase (CesA) gene family from maize. The genes involved in primary wall formation appear to have duplicated relatively independently in dicots and monocots. The deduced amino acid sequences of three of the maize genes, ZmCesA10–12, cluster with the Arabidopsis CesA sequences that have been shown to be involved in secondary wall formation. Based on their expression patterns across multiple tissues, these three genes appear to be coordinately expressed. The remaining genes show overlapping expression to varying degrees with ZmCesA1, 7, and 8 forming one group, ZmCesA3 and 5 a second group, and ZmCesA2 and 6 exhibiting independent expression of any other gene. This suggests that the varying levels of coexpression may just be incidental except in the case of ZmCesA10–12, which may interact with each other to form a functional enzyme complex. Isolation of the expressed CesA genes from maize and their association with primary or secondary wall formation has made it possible to test their respective roles in cellulose synthesis through mutational genetics or transgenic approaches. This information would be useful in improving stalk strength.

Combining expression and comparative evolutionary analysis. The COBRA gene family

Plant cell shape is achieved through a combination of oriented cell division and cell expansion and is defined by the cell wall. One of the genes identified to influence cell expansion in the Arabidopsis (Arabidopsis thaliana) root is the COBRA (COB) gene that belongs to a multigene family. Three members of the AtCOB gene family have been shown to play a role in specific types of cell expansion or cell wall biosynthesis. Functional orthologs of one of these genes have been identified in maize (Zea mays) and rice (Oryza sativa; Schindelman et al., 2001; Li et al., 2003; Brown et al., 2005; Persson et al., 2005; Ching et al., 2006; Jones et al., 2006). We present the maize counterpart of the COB gene family and the COB gene superfamily phylogeny. Most of the genes belong to a family with two main clades as previously identified by analysis of the Arabidopsis family alone. Within these clades, however, clear differences between monocot and eudicot family members exist, and these are analyzed in the context of Type I and Type II cell walls in eudicots and monocots. In addition to changes at the sequence level, gene regulation of this family in a eudicot, Arabidopsis, and a monocot, maize, is also characterized. Gene expression is analyzed in a multivariate approach, using data from a number of sources, including massively parallel signature sequencing libraries, transcriptional reporter fusions, and microarray data. This analysis has revealed that the expression of Arabidopsis and maize COB gene family members is highly developmentally and spatially regulated at the tissue and cell type-specific level, that gene superfamily members show overlapping and unique expression patterns, and that only a subset of gene superfamily members act in response to environmental stimuli. Regulation of expression of the Arabidopsis COB gene family members has highly diversified in comparison to that of the maize COB gene superfamily members. We also identify BRITTLE STALK 2-LIKE 3 as a putative ortholog of AtCOB.

Plant Cell Wall Matrix Polysaccharide Biosynthesis

The wall of an expanding plant cell consists primarily of cellulose microfibrils embedded in a matrix of hemicellulosic and pectic polysaccharides along with small amounts of structural and enzymatic proteins. Matrix polysaccharides are synthesized in the Golgi and exported to the cell wall by exocytosis, where they intercalate among cellulose microfibrils, which are made at the plasma membrane and directly deposited into the cell wall. Involvement of Golgi glucan synthesis in auxin-induced cell expansion has long been recognized; however, only recently have the genes corresponding to glucan synthases been identified. Biochemical purification was unsuccessful because of the labile nature and very low abundance of these enzymes. Mutational genetics also proved fruitless. Expression of candidate genes identified through gene expression profiling or comparative genomics in heterologous systems followed by functional characterization has been relatively successful. Several genes from the cellulose synthase-like (Csl) family have been found to be involved in the synthesis of various hemicellulosic glycans. The usefulness of this approach, however, is limited to those enzymes that probably do not form complexes consisting of unrelated proteins. Nonconventional approaches will continue to incrementally unravel the mechanisms of Golgi polysaccharide biosynthesis.

Cell Wall Reactive Proteins in the Coat and Wall of Maize Pollen POTENTIAL ROLE IN POLLEN TUBE GROWTH ON THE STIGMA AND THROUGH THE STYLE

The surface of a pollen grain consists of an outermost coat and an underlying wall. In maize (Zea mays L.), the pollen coat contains two major proteins derived from the adjacent tapetum cells in the anthers. One of the proteins is a 35-kDa endoxylanase (Wu, S. S. H., Suen, D. F., Chang, H. C., and Huang, A. H. C. (2002) J. Biol. Chem. 277, 49055–49064). The other protein of 70 kDa was purified to homogeneity and shown to be a β-glucanase. Its gene sequence and the developmental pattern of its mRNA differ from those of the known β-glucanases that hydrolyze the callose wall of the microspore tetrad. Mature pollen placed in a liquid medium released about nine major proteins. These proteins were partially sequenced and identified via GenBank™ data bases, and some had not been previously reported to be in pollen. They appear to have wall-loosening, structural, and enzymatic functions. A novel pollen wall-bound protein of 17 kDa has a unique pattern of cysteine distribution in its sequence (six tandem repeats of CX3CX10–15) that could chelate cations and form signal-receiving finger motifs. These pollen-released proteins were synthesized in the pollen interior, and their mRNA increased during pollen maturation and germination. They were localized mainly in the pollen tube wall. The pollen shell was isolated and found to contain no detectable proteins. We suggest that the pollen-coat β-glucanase and xylanase hydrolyze the stigma wall for pollen tube entry and that the pollen secrete proteins to loosen or become new wall constituents of the tube and to break the wall of the transmitting track for tube advance.

Building the wall: genes and enzyme complexes for polysaccharide synthases.

The complete sequence of the Arabidopsis genome has revealed a total of 40 cellulose synthase (CesA) and cellulose synthase-like (Csl) genes. Recent studies suggest that each CESA polypeptide contains only one catalytic center, and that two or more polypeptides from different genes might be needed to form a functional cellulose synthase complex.

The response of the maize nitrate transport system to nitrogen demand and supply across the lifecycle

An understanding of nitrate (NO3-) uptake throughout the lifecycle of plants, and how this process responds to nitrogen (N) availability, is an important step towards the development of plants with improved nitrogen use efficiency (NUE). NO3- uptake capacity and transcript levels of putative high- and low-affinity NO3- transporters (NRTs) were profiled across the lifecycle of dwarf maize (Zea mays) plants grown at reduced and adequate NO3-. Plants showed major changes in high-affinity NO3- uptake capacity across the lifecycle, which varied with changing relative growth rates of roots and shoots. Transcript abundances of putative high-affinity NRTs (predominantly ZmNRT2.1 and ZmNRT2.2) were correlated with two distinct peaks in high-affinity root NO3- uptake capacity and also N availability. The reduction in NO3- supply during the lifecycle led to a dramatic increase in NO3- uptake capacity, which preceded changes in transcript levels of NRTs, suggesting a model with short-term post-translational regulation and longer term transcriptional regulation of NO3- uptake capacity. These observations offer new insight into the control of NO3- uptake by both plant developmental processes and N availability, and identify key control points that may be targeted by future plant improvement programmes to enhance N uptake relative to availability and/or demand.

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.

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

BackgroundThe 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.ResultsThe 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 β-expansins, UDP-Xylose synthase and UDP-Glucose pyrophosphorylase, some xyloglucan endotransglycosylases/hydrolases, genes involved in monolignol biosynthesis, and NAM and MYB transcription factor genes.ConclusionsThe 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.

Biosynthesis of non-cellulosic polysaccharides of plant cell walls.

Enzymes that make the polymer backbones of plant cell wall polysaccharides have proven to be recalcitrant to biochemical purification. Availability of mutational genetics and genomic tools paved the way for rapid progress in identifying genes encoding various cell wall glycan synthases. Mutational genetics, the primary tool used in unraveling cellulose biosynthesis, was ineffective in assigning function to any of the hemicellulosic, polymerizing glycan synthases. A combination of comparative genomics and functional expression in a heterologous system allowed identification of various cellulose synthase-like (Csl) sequences as being involved in the formation of β-1,4-mannan, β-1,4-glucan, and mixed-linked glucan. A number of xylose-deficient mutants have led to a variety of genes, none of which thus far possesses the motifs known to be conserved among polymerizing β-glycan synthases. Except for xylan synthase, which appears to be an agglomerate of proteins just like cellulose synthase, Golgi glycan synthases already identified suggest that the catalytic polypeptide by itself is sufficient for enzyme activity, most likely as a homodimer. Several of the Csl genes remain to be assigned a function. The possibility of the involvement of various Csl genes in making more than one product remains.

Endo-(1,4)-β-Glucanase gene families in the grasses: temporal and spatial Co-transcription of orthologous genes1

BackgroundEndo-(1,4)-β-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)-β-glucosyl residues, and wall loosening during cell elongation.ResultsThe endo-(1,4)-β-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)-β-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)-β-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)-β-glucanase family and other genes known to be involved in cell wall loosening and cell expansion, such as expansins and xyloglucan endotransglycosylases.ConclusionsThe 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.