Identification of novel genes in Arabidopsis involved in secondary cell wall formation using expression profiling and reverse genetics

Forward genetic screens have led to the isolation of several genes involved in secondary cell wall formation. A variety of evidence, however, suggests that the list of genes identified is not exhaustive. To address this problem, microarray data have been generated from tissue undergoing secondary cell wall formation and used to identify genes that exhibit a similar expression pattern to the secondary cell wall-specific cellulose synthase genes IRREGULAR XYLEM1 (IRX1) and IRX3. Cross-referencing this analysis with publicly available microarray data resulted in the selection of 16 genes for reverse genetic analysis. Lines containing an insertion in seven of these genes exhibited a clear irx phenotype characteristic of a secondary cell wall defect. Only one line, containing an insertion in a member of the COBRA gene family, exhibited a large decrease in cellulose content. Five of the genes identified as being essential for secondary cell wall biosynthesis have not been previously characterized. These genes are likely to define entirely novel processes in secondary cell wall formation and illustrate the success of combining expression data with reverse genetics to address gene function.     

An integrative approach to the identification of Arabidopsis and rice genes involved in xylan and secondary wall development

Xylans constitute the major non-cellulosic component of plant biomass. Xylan biosynthesis is particularly pronounced in cells with secondary walls, implying that the synthesis network consists of a set of highly expressed genes in such cells. To improve the understanding of xylan biosynthesis, we performed a comparative analysis of co-expression networks between Arabidopsis and rice as reference species with different wall types. Many co-expressed genes were represented by orthologs in both species, which implies common biological features, while some gene families were only found in one of the species, and therefore likely to be related to differences in their cell walls. To predict the subcellular location of the identified proteins, we developed a new method, PFANTOM (plant protein family information-based predictor for endomembrane), which was shown to perform better for proteins in the endomembrane system than other available prediction methods. Based on the combined approach of co-expression and predicted cellular localization, we propose a model for Arabidopsis and rice xylan synthesis in the Golgi apparatus and signaling from plasma membrane to nucleus for secondary cell wall differentiation. As an experimental validation of the model, we show that an Arabidopsis mutant in the PGSIP1 gene encoding one of the Golgi localized candidate proteins has a highly decreased content of glucuronic acid in secondary cell walls and substantially reduced xylan glucuronosyltransferase activity.     

Identification and characterization of Arabidopsis thaliana genes involved in xylem secondary cell walls

The xylem of higher plants offers support to aerial portions of the plant body and serves as conduit for the translocation of water and nutrients. Terminal differentiation of xylem cells typically involves deposition of thick secondary cell walls. This is a dynamic cellular process accompanied by enhanced rates of cellulose deposition and the induction of synthesis of specific secondary-wall matrix polysaccharides and lignin. The secondary cell wall is essential for the function of conductive and supportive xylem tissues. Recently, significant progress has been made in identifying the genes responsible for xylem secondary cell wall formation. However, our present knowledge is still insufficient to account for the molecular processes by which this complex system operates. To acquire further information about xylem secondary cell walls, we initially focused our research effort on a set of genes specifically implicated in secondary cell wall formation, as well as on loss-of-function mutants. Results from two microarray screens identified several key candidate genes responsible for secondary cell wall formation. Reverse genetic analyses led to the identification of a glycine-rich protein involved in maintaining the stable structure of protoxylem, which is essential for the transport of water and nutrients. A combination of expression analyses and reverse genetics allows us to systematically identify new genes required for the development of physical properties of the xylem secondary wall.     

Tissue-specific localization of gibberellins and expression of gibberellin-biosynthetic and signaling genes in wood-forming tissues in aspen

Bioactive gibberellins (GAs) are known regulators of shoot growth and development in plants. In an attempt to identify where GAs are formed, we have analyzed the expression patterns of six GA biosynthesis genes and two genes with predicted roles in GA signaling and responses in relation to measured levels of GAs. The analysis was based on tangential sections, giving tissue-specific resolution across the cambial region of aspen trees (Populus tremula). Gibberellin quantification by GC/MS-SRM showed that the bioactive GA1 and GA4 were predominantly located in the zone of expansion of xylem cells. Based on co-localization of the expression of the late GA biosynthesis gene GA 20-oxidase 1 and bioactive GAs, we suggest that de novo GA biosynthesis occurs in the expanding xylem. However, expression levels of the first committed GA biosynthesis enzyme, ent-copalyl diphosphate synthase, were high in the phloem, suggesting that a GA precursor(s) may be transported to the xylem. The expression of the GA signaling and response genes DELLA-like1 and GIP-like1 coincided well with sites of high bioactive GA levels. We therefore suggest that the main role of GA during wood formation is to regulate early stages of xylem differentiation, including cell elongation.     

Functional identification of ELO-like genes involved in very long chain fatty acid synthesis in Arabidopsis thaliana

Very long chain fatty acids (VLCFAs) are essential lipid components in many plants. 3-Ketoacyl-CoA synthase (KCS) catalyzes the condensation reaction to form 3-ketoacyl-CoA in VLCFA synthesis. AtELO4 has been reported to be involved in VLCFA synthesis, functioning as a KCS in Arabidopsis. However, no studies on other three AtELO members have been reported. Here, we initially found by real-time PCR in Arabidopsis thaliana (L.) Heynh. that AtELO1, AtELO3, and AtELO4 displayed characteristic expression patterns, but AtELO2 was nearly expressed in any organ. Then the transient expression of ELO-like-eGFP fusions in Arabidopsis green leaf protoplasts showed that AtELO1, AtELO3, and AtELO4 were localized in the endoplasmic reticulum (ER), where VLCFA synthesis took place. Finally, we found that the contents of all fatty acids were decreased by 10–20% in seeds of atelo1 T-DNA insertion mutants. In seeds of Pro35S:AtELO1 plants, the levels of all remaining components, except C20:0 and C20:3, were significantly increased. Taken together, our study revealed biological functions of AtELO members and might lay the foundation for further genetic manipulations to generate oil crops with the high oil content.     

Muscular expressions: profiling genes in complex tissues

Gene-expression profiling has yielded important information about simple systems, but complex tissues have not yet been widely profiled. Four recent studies of mammalian skeletal muscles have added to the catalogs of their gene expression differences, but have yet to lead to better understanding of the molecular processes underlying their physiological differences.     

Cytokinins initiate secondary growth in the Arabidopsis root through a set of LBD genes

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