Qouta, Lolita Abdulla
The biochemistry and molecular biology of intercellular adhesion in plant tissue culture.
PhD thesis, University of Glasgow.
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The adhesion between neighbouring plant cells is established as cells are formed during cytokinesis through the middle lamella that is made principally of pectins and proteins. Pectins are secreted into the cell wall in a highly methylesterified form and subsequently de-esterified in muro by pectin methyl esterase (PME, E.C. 3.1.11). The present study reports on the biochemical characterization and immunochemical analyses of phosphate buffer/EDTA pectic extracts associated with cell-cell adhesion in suspension cultures of wild type (WT), salt tolerant (HHS) cell lines and synchronized Arabidopsis suspension cultures. Using the synchronized cultures, The PME-mediated configuration of pectins at the onset of adhesion during cytokinesis, was assessed through the analysis of the expression patterns of the PME isoforms annotated to be expressed throughout the cell cycle
The wild type Arabidopsis seemed to maintain the intercellular adhesion through the gelling of the highly methylated JIM7 recognized homogalacturonans that were shown to be abundant in the primary cell walls, middle lamellae and cellular junctions, possibly due to the hydrophobic interactions between the methoxy groups. The rhamnogalacturonan-I fraction was rich in arabinan side chains reflecting the proliferative state of the cells. The increase in arabinan content was accompanied by a reduction in the galactan content 4 days after subculturing. The cell walls of salt tolerant Arabidopsis contained the JIM7 and LM7recognized epitopes along with a high degree of branching of rhamnogalacturonan-I carrying galactans and arabinans as side chains. The change in the detected epitopes is thought to play a role in the ability of the cells to withstand the high osmotic pressure and increase the in the level of adhesion between cells. The JIM5 low methylesterified HGs were less abundant in both cultures, and the absence of the 2F4 antibody recognizing the Ca2+ egg boxes could be attributed to the scarce amounts of Ca2+ present in the culturing medium
The immunochemical studies of the pectin extracted from the synchronized Arabidopsis suspension cultures after washing out aphidicolin indicated that the recognition of both of JIM7 and JIM5 varied in parallel during the cell cycle, whereas, the recognition of arabinan increased during the cell division. The sequence and phylogenetic analysis of ten PME isoforms that were annotated to be expressed at one or more phases of the cell cycle of synchronized Arabidopsis thaliana suspension cultures (Menges and Murray, 2002 and 2003), revealed that only five of these genes could be PMEs. The genes At4g02330, At1g02810, At2g26440, and At2g47550 were thought to be of type II PMEs which have a pre-pro-catalytic domains and At5g47500 is a type I PME that lack the pro-region. The amino acid sequence of At4g12390 showed similarities with the N-terminal pro-peptides of plant PME and invertase inhibitors.
The expression of several PME genes was studied in suspension cultures of Arabidopsis thaliana synchronised using aphidicolin. Semi-quantitative PCR experiments showed that the expression of At5g47500 transcript was always detected during M phase of the cell cycle. The rest of the genes failed to show consistent patterns of expression. Northern blots revealed that mRNA coding for At5g47500 decreases during S and G2 phases and accumulates during the M phase of the cell cycle. Our results suggest that this PME isoform is involved in the modulations of the cell walls as the cells are going through division and cytokinesis.
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