Zhang, Yizhu , Yang, Jie , Showalter, Allan M. .
Functional characterization of Lysine-rich AGPs by reverse genetics.
Arabinogalactan proteins (AGPs) are a class of hydroxyproline-rich glycoproteins found at the plant cell surface. They are highly glycosylated and are thought to play important roles in plant cell differentiation, cell-cell recognition, embryogenesis and programmed cell death. There are three homologous genes in the lysine-rich AGP subfamily in Arabidopsis : AtAGP17, AtAGP18 and AtAGP19. They are composed of an N-terminal signal peptide, a Pro/Hyp-rich central domain disrupted by a lysine-rich region and a C-terminal GPI anchor addition region.
A T-DNA knockout mutation in AtAGP19, was obtained and examined. Compared to wild type plants, the atagp19 mutant had: 1) lighter green leaves containing less chlorophyll and anthocyanins, 2) rounder leaves, with shorter petioles, 3) shorter and thinner inflorescence stems, 4) slower growth with delayed and reduced flowering, 5) fewer siliques and seeds and 6) fewer lateral roots. Complementation of this mutant with the wild type AtAGP19 gene restored the wild type phenotypes. How a mutation in AtAGP19 can elicit these phenotypic changes is still unknown. In order to begin to address this question, a microarray approach was used to elucidate changes in gene expression associated with the atagp19 mutant in Arabidopsis leaves. Wild type plants were grown together with homozygous atagp19 mutant plants under identical environmental conditions for 14 days. Total RNAs were extracted from wild type and atagp19 mutant leaves using the Qiagen RNeasy Plant Mini Kit and sent to the University of California, Irvine UCI DNA & Protein MicroArray Facility for microarray analysis. RNA expression levels for the ~22,000 genes in both wild type and atagp19 mutant plants were analyzed by ArrayAssist 3.0 (Stratagene). Among these genes, 79 genes are up-regulated more than two-fold and 32 genes are down-regulated more than two-fold. These gene products are predicted to be in the chloroplast, endomembrane system, mitochondria, nucleus, peroxisome and microtubule. Expression levels for several of these genes were further examined by QPCR to confirm the microarray data.
An overexpression approach was also used to elucidate AtAGP17/18/19 function(s). Constructs with EGFP (Enhanced Green Fluorescent Protein)-AtAGP17/18/19 fusion protein under the control of the CaMV35S promoter were introduced into Arabidopsis using Agrobacterium-mediated transformation. T1 seeds were screened on kanamycin-selective media and PCR with vector-specific primers was performed to detect the incorporation of the constructs into the plant genome. T2 plants were used for phenotype analysis. Compared to wild type, transformants which overexpressed AtAGP18 were significantly shorter and highly branched. Construct with only EGFP was also introduced into Arabidopsis as a control and T1 seeds are being screened now.
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1 - Ohio University, Environmental and Plant Biology, 315 Porter Hall, Ohio University, Environmental and Plant Biology, Athens, OH, 45701, US
2 - Ohio University, Environmental and Plant Biology
hyperglycosylated hydoxyproline-rich glycoproteins
Presentation Type: ASPB Minisymposium
Location: International Ballroom/Hilton
Date: Monday, July 9th, 2007
Time: 11:30 AM