SummaryPlant cell walls represent the most abundant renewable resource on this planet. Despite their great abundance, only 2% of this resource is currently used by humans. Hence, research into the feasibility of using plant cell walls in the production of cost-effective biofuels is desirable. The main bottleneck for using wall materials is the recalcitrance of walls to efficient degradation into fermentable sugars. Manipulation of the wall polysaccharide biosynthetic machinery or addition of wall structure-altering agents should make it possible to tailor wall composition and architecture to enhance sugar yields upon wall digestion for biofuel fermentation. Study of the biosynthetic machinery and its regulation is still in its infancy and represents a major scientific and technical research challenge. Of course, any change in wall structure to accommodate cost-efficient biofuel production may have detrimental effects on plant growth and development due to the diverse roles of walls in the life of a plant. However, the diversity and abundance of wall structures present in the plant kingdom gives hope that this challenge can be met.

SummaryThe plant cell wall is an important factor for determining cell shape, function and response to the environment. Secondary cell walls, such as those found in xylem, are composed of cellulose, hemicelluloses and lignin and account for the bulk of plant biomass. The coordination between transcriptional regulation of synthesis for each polymer is complex and vital to cell function. A regulatory hierarchy of developmental switches has been proposed, although the full complement of regulators remains unknown. Here, we present a protein-DNA network between Arabidopsis transcription factors and secondary cell wall metabolic genes with gene expression regulated by a series of feed-forward loops. This model allowed us to develop and validate new hypotheses about secondary wall gene regulation under abiotic stress. Distinct stresses are able to perturb targeted genes to potentially promote functional adaptation. These interactions will serve as a foundation for understanding the regulation of a complex, integral plant component.

Xyloglucans are the main hemicellulosic polysaccharides found in the primary cell walls of dicots and nongraminaceous monocots, where they are thought to interact with cellulose to form a three-dimensional network that functions as the principal load-bearing structure of the primary cell wall. To determine whether two Arabidopsis thaliana genes that encode xylosyltransferases, XXT1 and XXT2, are involved in xyloglucan biosynthesis in vivo and to determine how the plant cell wall is affected by the lack of expression of XXT1, XXT2, or both, we isolated and characterized xxt1 and xxt2 single and xxt1 xxt2 double T-DNA insertion mutants. Although the xxt1 and xxt2 mutants did not have a gross morphological phenotype, they did have a slight decrease in xyloglucan content and showed slightly altered distribution patterns for xyloglucan epitopes. More interestingly, the xxt1 xxt2 double mutant had aberrant root hairs and lacked detectable xyloglucan. The reduction of xyloglucan in the xxt2 mutant and the lack of detectable xyloglucan in the xxt1 xxt2 double mutant resulted in significant changes in the mechanical properties of these plants. We conclude that XXT1 and XXT2 encode xylosyltransferases that are required for xyloglucan biosynthesis. Moreover, the lack of detectable xyloglucan in the xxt1 xxt2 double mutant challenges conventional models of the plant primary cell wall.

Root hairs are single cells that develop by tip growth and are specialized in the absorption of nutrients. Their cell walls are composed of polysaccharides and hydroxyproline-rich glycoproteins (HRGPs) that include extensins (EXTs) and arabinogalactan-proteins (AGPs). Proline hydroxylation, an early posttranslational modification of HRGPs that is catalyzed by prolyl 4-hydroxylases (P4Hs), defines the subsequent O-glycosylation sites in EXTs (which are mainly arabinosylated) and AGPs (which are mainly arabinogalactosylated). We explored the biological function of P4Hs, arabinosyltransferases, and EXTs in root hair cell growth. Biochemical inhibition or genetic disruption resulted in the blockage of polarized growth in root hairs and reduced arabinosylation of EXTs. Our results demonstrate that correct O-glycosylation on EXTs is essential for cell-wall self-assembly and, hence, root hair elongation in Arabidopsis thaliana.

Tomato (Solanum lycopersicum) is a well-studied model of fleshy fruit development and ripening. Tomato fruit development is well understood from a hormonal-regulatory perspective, and developmental changes in pigment and cell wall metabolism are also well characterized. However, more general aspects of metabolic change during fruit development have not been studied despite the importance of metabolism in the context of final composition of the ripe fruit. In this study, we quantified the abundance of a broad range of metabolites by gas chromatography-mass spectrometry, analyzed a number of the principal metabolic fluxes, and in parallel analyzed transcriptomic changes during tomato fruit development. Metabolic profiling revealed pronounced shifts in the abundance of metabolites of both primary and secondary metabolism during development. The metabolite changes were reflected in the flux analysis that revealed a general decrease in metabolic activity during ripening. However, there were several distinct patterns of metabolite profile, and statistical analysis demonstrated that metabolites in the same (or closely related) pathways changed in abundance in a coordinated manner, indicating a tight regulation of metabolic activity. The metabolite data alone allowed investigations of likely routes through the metabolic network, and, as an example, we analyze the operational feasibility of different pathways of ascorbate synthesis. When combined with the transcriptomic data, several aspects of the regulation of metabolism during fruit ripening were revealed. First, it was apparent that transcript abundance was less strictly coordinated by functional group than metabolite abundance, suggesting that posttranslational mechanisms dominate metabolic regulation. Nevertheless, there were some correlations between specific transcripts and metabolites, and several novel associations were identified that could provide potential targets for manipulation of fruit compositional traits. Finally, there was a strong relationship between ripening-associated transcripts and specific metabolite groups, such as TCA-cycle organic acids and sugar phosphates, underlining the importance of the respective metabolic pathways during fruit development.

Shoot meristems of plants are composed of stem cells that are continuously replenished through a classical feedback circuit involving the homeobox WUSCHEL (WUS) gene and the CLAVATA (CLV) gene signaling pathway. In CLV signaling, the CLV1 receptor complex is bound by CLV3, a secreted peptide modified with sugars. However, the pathway responsible for modifying CLV3 and its relevance for CLV signaling are unknown. Here we show that tomato inflorescence branching mutants with extra flower and fruit organs due to enlarged meristems are defective in arabinosyltransferase genes. The most extreme mutant is disrupted in a hydroxyproline O-arabinosyltransferase and can be rescued with arabinosylated CLV3. Weaker mutants are defective in arabinosyltransferases that extend arabinose chains, indicating that CLV3 must be fully arabinosylated to maintain meristem size. Finally, we show that a mutation in CLV3 increased fruit size during domestication. Our findings uncover a new layer of complexity in the control of plant stem cell proliferation.

(M.P.) Pectin, one of the main components of plant cell wall, is secreted in a highly methylesterified form and is demethylesterified in muro by pectin methylesterase (PME). The action of PME is important in plant development and defense and makes pectin susceptible to hydrolysis by enzymes such as endopolygalacturonases. Regulation of PME activity by specific protein inhibitors (PMEIs) can, therefore, play a role in plant development as well as in defense by influencing the susceptibility of the wall to microbial endopolygalacturonases. To test this hypothesis, we have constitutively expressed the genes AtPMEI-1 and AtPMEI-2 in Arabidopsis (Arabidopsis thaliana) and targeted the proteins into the apoplast. The overexpression of the inhibitors resulted in a decrease of PME activity in transgenic plants, and two PME isoforms were identified that interacted with both inhibitors. While the content of uronic acids in transformed plants was not significantly different from that of wild type, the degree of pectin methylesterification was increased by about 16%. Moreover, differences in the fine structure of pectins of transformed plants were observed by enzymatic fingerprinting. Transformed plants showed a slight but significant increase in root length and were more resistant to the necrotrophic fungus Botrytis cinerea. The reduced symptoms caused by the fungus on transgenic plants were related to its impaired ability to grow on methylesterified pectins.Pectin is a structurally complex polysaccharide that accounts for nearly 35% of the dicot and nongraminaceous monocot primary cell wall. A main component of pectin is homogalacturonan (HGA) consisting of a backbone of 1,4-linked a-D-GalUA units, with variable amounts of methylester in the C 6 position. Pectins are secreted into the cell wall in a highly methylesterified form and, soon thereafter, are deesterified in muro by pectin methylesterase (PME; Brummell and Harpster, 2001;Willats et al., 2001). Demethylesterification produces free carboxyl groups and modifies the pH and charge of the wall, allowing the aggregation of polyuronides into a calcium-linked gel structure and increasing the wall firmness (Willats et al., 2001). In addition, the action of PMEs makes HGA susceptible to degradation by hydrolases such as endopolygalacturonases (endoPGs), contributing to the softening of the cell wall (Brummell and Harpster, 2001;Wakabayashi et al., 2003).Plant PMEs are involved in important physiological processes such as microsporogenesis, pollen growth, pollen separation, seed germination, root development, polarity of leaf growth, stem elongation, fruit ripening, and loss of tissue integrity

Transcriptome analysis based on deep expressed sequence tag (EST) sequencing allows quantitative comparisons of gene expression across multiple species. Using pyrosequencing, we generated over 7 million ESTs from four stages of developing seeds of Ricinus communis, Brassica napus, Euonymus alatus and Tropaeolum majus, which differ in their storage tissue for oil, their ability to photosynthesize and in the structure and content of their triacylglycerols (TAG). The larger number of ESTs in these 16 datasets provided reliable estimates of the expression of acyltransferases and other enzymes expressed at low levels. Analysis of EST levels from these oilseeds revealed both conserved and distinct species-specific expression patterns for genes involved in the synthesis of glycerolipids and their precursors. Independent of the species and tissue type, ESTs for core fatty acid synthesis enzymes maintained a conserved stoichiometry and a strong correlation in temporal profiles throughout seed development. However, ESTs associated with non-plastid enzymes of oil biosynthesis displayed dissimilar temporal patterns indicative of different regulation. The EST levels for several genes potentially involved in accumulation of unusual TAG structures were distinct. Comparison of expression of members from multi-gene families allowed the identification of specific isoforms with conserved function in oil biosynthesis. In all four oilseeds, ESTs for Rubisco were present, suggesting its possible role in carbon metabolism, irrespective of light availability. Together, these data provide a resource for use in comparative and functional genomics of diverse oilseeds. Expression data for more than 350 genes encoding enzymes and proteins involved in lipid metabolism are available at the ‘ARALIP’ website (http://aralip.plantbiology.msu.edu/).