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 for the identification of quantitative trait loci

The genetic dissection of complex traits is one of the most difficult and most important challenges facing science today. We discuss here an integrative approach to quantitative trait loci (QTL) mapping in mice. This approach makes use of the wealth of genetic tools available in mice, as well as the recent advances in genome sequence data already available for a number of inbred mouse strains. We have developed mapping strategies that allow a stepwise narrowing of a QTL mapping interval, prioritizing candidate genes for further analysis with the potential of identifying the most probable candidate gene for the given trait. This approach integrates traditional mapping tools, fine mapping tools, sequence-based analysis, bioinformatics and gene expression.     

Gene trapping in Arabidopsis reveals genes involved in vascular development

The procambium is made up of stem cells that give rise to various vascular cells in plants. To understand the molecular nature of procambium cells, we tried to identify genes that characterize procambium cells using Arabidopsis gene trap lines. Among 26,000 gene trap lines, we found 67 lines in which beta-glucuronidase (GUS) staining occurred along vascular tissues in cotyledons and/or adult leaves. Although four gene trap lines showed procambium-preferential GUS expression, their expression patterns differed from each other during procambium development in root tips and young rosette leaves. Genomic regions flanking the gene trap insertion points in 25 of the 67 lines were determined, including three lines showing preferential GUS staining of the procambium. The three procambium-related genes encoded PINHEAD, katanin and an unknown DUF740 domain-containing protein. We discuss procambium development based on the functions and the differential GUS staining patterns of the procambium-related genes.     

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.     

A genomic approach for the identification and classification of genes involved in cell wall formation and its regulation in Saccharomyces cerevisiae

Using a hierarchical approach, 620 non-essential single-gene yeast deletants generated by EUROFAN I were systematically screened for cell-wall-related phenotypes. By analyzing for altered sensitivity to the presence of Calcofluor white or SDS in the growth medium, altered sensitivity to sonication, or abnormal morphology, 145 (23%) mutants showing at least one cell wall-related phenotype were selected. These were screened further to identify genes potentially involved in either the biosynthesis, remodeling or coupling of cell wall macromolecules or genes involved in the overall regulation of cell wall construction and to eliminate those genes with a more general, pleiotropic effect. Ninety percent of the mutants selected from the primary tests showed additional cell wall-related phenotypes. When extrapolated to the entire yeast genome, these data indicate that over 1200 genes may directly or indirectly affect cell wall formation and its regulation. Twenty-one mutants with altered levels of beta1,3-glucan synthase activity and five Calcofluor white-resistant mutants with altered levels of chitin synthase activities were found, indicating that the corresponding genes affect beta1,3-glucan or chitin synthesis. By selecting for increased levels of specific cell wall components in the growth medium, we identified 13 genes that are possibly implicated in different steps of cell wall assembly. Furthermore, 14 mutants showed a constitutive activation of the cell wall integrity pathway, suggesting that they participate in the modulation of the pathway either directly acting as signaling components or by triggering the Slt2-dependent compensatory mechanism. In conclusion, our screening approach represents a comprehensive functional analysis on a genomic scale of gene products involved in various aspects of fungal cell wall formation.     

A proteomic approach to apoplastic proteins involved in cell wall regeneration in protoplasts of Arabidopsis suspension-cultured cells

To clarify the mechanisms of cell wall construction, we used a proteomic approach to investigate the proteins secreted into cell wall spaces during cell wall regeneration from the protoplasts of Arabidopsis suspension-cultured cells. We focused on cell wall proteins loosely bound to the cell wall architecture and extractable with 1 M KCl solutions from: (i) native suspension cultured cells; (ii) protoplasts that had been allowed to regenerate their cell walls for 1 h; and (iii) protoplasts allowed to regenerate their cell walls for 3 h. We adopted a non-destructive extraction procedure without disrupting cellular integrity, thereby avoiding contamination from cytoplasmic proteins. Using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and matrix-assisted laser desorption ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS), we separated, mapped and identified 71 proteins derived from the native cell wall, and 175 and 212 proteins derived from the 1 and 3 h regenerated protoplasts, respectively. Quite different sets of proteins with differing status of their post-translational modifications, including phosphorylation and glycosylation, were identified in the three protein fractions. This indicated dynamic in muro changes in the cell wall proteins during cell wall regeneration in the protoplasts. The analysis revealed a set of enzymes specifically involved in cell wall expansion and construction in suspension-cultured cells. This approach has also determined a set of cell wall proteins that had not been predicted to be localized in cell wall spaces.     

An evolutionary genomic approach to identify genes involved in human birth timing

Coordination of fetal maturation with birth timing is essential for mammalian reproduction. In humans, preterm birth is a disorder of profound global health significance. The signals initiating parturition in humans have remained elusive, due to divergence in physiological mechanisms between humans and model organisms typically studied. Because of relatively large human head size and narrow birth canal cross-sectional area compared to other primates, we hypothesized that genes involved in parturition would display accelerated evolution along the human and/or higher primate phylogenetic lineages to decrease the length of gestation and promote delivery of a smaller fetus that transits the birth canal more readily. Further, we tested whether current variation in such accelerated genes contributes to preterm birth risk. Evidence from allometric scaling of gestational age suggests human gestation has been shortened relative to other primates. Consistent with our hypothesis, many genes involved in reproduction show human acceleration in their coding or adjacent noncoding regions. We screened >8,400 SNPs in 150 human accelerated genes in 165 Finnish preterm and 163 control mothers for association with preterm birth. In this cohort, the most significant association was in FSHR, and 8 of the 10 most significant SNPs were in this gene. Further evidence for association of a linkage disequilibrium block of SNPs in FSHR, rs11686474, rs11680730, rs12473870, and rs1247381 was found in African Americans. By considering human acceleration, we identified a novel gene that may be associated with preterm birth, FSHR. We anticipate other human accelerated genes will similarly be associated with preterm birth risk and elucidate essential pathways for human parturition.     

Arabidopsis ADC genes involved in polyamine biosynthesis are essential for seed development

Arginine decarboxylase (ADC) is a rate-limiting enzyme that catalyzes the first step of polyamine (PA) biosynthesis in Arabidopsis thaliana. We generated a double mutant deficient in Arabidopsis two ADC genes (ADC1-/- ADC2-/-) and examined their roles in seed development. None of the F2 seedlings from crosses of adc1-1 and adc2-2 had the ADC1-/- ADC2-/- genotype. In addition, some abnormal seeds were observed among the ADC1+/- ADC2-/- and ADC1-/- ADC2+/- siliques. Viable offspring with the ADC1-/- ADC2-/- genotype could not be obtained from the ADC1+/- ADC2-/- and ADC1-/- ADC2+/- plants. These results indicate that AtADC genes are required for production of polyamines that are essential for normal seed development in Arabidopsis.     

Comprehensive approach to genes involved in cell wall modifications in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The plant cell wall is of supermolecular architecture, and is composed of various types of heterogeneous polymers. A few thousand enzymes and structural proteins are directly involved in the construction processes, and in the functional aspects of the dynamic architecture in Arabidopsis thaliana. Most of these proteins are encoded by multigene families, and most members within each family share significant similarities in structural features, but often exhibit differing expression profiles and physiological functions. Thus, for the molecular dissection of cell wall dynamics, it is necessary to distinguish individual members within a family of proteins. As a first step towards characterizing the processes involved in cell wall dynamics, we have manufactured a gene-specific 70-mer oligo microarray that consists of 765 genes classified into 30 putative families of proteins that are implicated in the cell wall dynamics of Arabidopsis. By using this array system, we identified several sets of genes that exhibit organ preferential expression profiles. We also identified gene sets that are expressed differentially at certain specific growth stages of the Arabidopsis inflorescence stem. Our results indicate that there is a division of roles among family members within each of the putative cell wall-related gene families.     

Genetic approach towards the identification of auxin-cytokinin crosstalk components involved in root development

Phytohormones are important plant growth regulators that control many developmental processes, such as cell division, cell differentiation, organogenesis and morphogenesis. They regulate a multitude of apparently unrelated physiological processes, often with overlapping roles, and they mutually modulate their effects. These features imply important synergistic and antagonistic interactions between the various plant hormones. Auxin and cytokinin are central hormones involved in the regulation of plant growth and development, including processes determining root architecture, such as root pole establishment during early embryogenesis, root meristem maintenance and lateral root organogenesis. Thus, to control root development both pathways put special demands on the mechanisms that balance their activities and mediate their interactions. Here, we summarize recent knowledge on the role of auxin and cytokinin in the regulation of root architecture with special focus on lateral root organogenesis, discuss the latest findings on the molecular mechanisms of their interactions, and present forward genetic screen as a tool to identify novel molecular components of the auxin and cytokinin crosstalk.