Joseph E. Varner

Plant cell wall architecture

Joseph E. Varner and Liang-Shiou Lin Institute of Molecular Biology Academia Sinica Nankang, Taipei 11529 Republic of China and Department of Biology Washington University St. Louis, Missouri 63130 Introduction Several hundred, perhaps a few thousand, genes are re- quired uniquely for the specification of the enzymes, lec- tins, structural proteins, polysaccharides, lignins, and waxes that make up the nonaqueous portion of the walls of the many cell types that constitute a whole plant. Addi- tional unique genes are most likely required to specify the agents for transient changes-for example, rapid efflux and/or influx of calcium ions, protons, and perhaps ascor- bic acid-that may be required in growing walls. The wall laid down while a cell is increasing in volume is the pri- mary wail. Layers laid down after the cell has stopped growing comprise the secondary wall. The cell walls of a plant collectively constitute its form. The form of any given organ is determined by the planes of division of the dividing cells and the ratio of longitudinal to radial growth of the growing cells. The walls of adjacent cells are glued together by a middle lamella made mainly of pectic substances. Intrusive growth of, for example, a fiber cell or a pollen tube may separate adjacent cells, and disruptive growth of lateral roots may destroy existing cells, but at no stage in plant development do cells crawl over one another. In an enlarging organ the cells of adja- cent cell layers may have different rates of cell division- in the oat coleoptile epidermal cells undergo no divisions while the subepidermal cells divide twice-yet, obviously, all cell layers must grow at the same rate. Sliding between cell layers does not occur, and cell wall thinning generally does not occur. Thus, adjacent cells dividing at different rates and having different functions must nonetheless coordinate the synthesis, secretion, and assembly of their respective wall components. Although we know few of the details, the processes of synthesis and secretion of wall components are broadly outlined. Assembly of these components into a growing wall is poorly understood. During cell division, as the daughter nuclei take up their new positions, a cell plate arises de novo between them until the existing walls are connected. The components first laid down in the cell plate are not known. It is conceivable that the addition of noncellulosic components to an existing wall is entirely by self-assembly, with each component designed to fit ap- propriately with others. Cellulose microfibrils are of neces- sity laid down by apposition on the inner surface of the existing wall. The matrix components- hemicelluloses, pectins, and proteins-are added by intussusception and

An Alternative Methylation Pathway in Lignin Biosynthesis in Zinnia

S-Adenosyl-L-methionine:trans-caffeoyl-coenzyme A 3-O-methyltransferase (CCoAOMT) is implicated in disease resistant response, but whether it is involved in lignin biosynthesis is not known. We isolated a cDNA clone for CCoAOMT in differentiating tracheary elements (TEs) induced from Zinnia-isolated mesophyll cells. RNA gel blot analysis showed that the expression of the CCoAOMT gene was markedly induced during TE differentiation from the isolated mesophyll cells. Tissue print hybridization showed that the expression of the CCoAOMT gene is temporally and spatially regulated and that it is associated with lignification in xylem and in phloem fibers in Zinnia organs. Both CCoAOMT and caffeic acid O-methyltransferase (COMT) activities increased when the isolated Zinnia mesophyll cells were cultured, whereas only CCoAOMT activity was markedly enhanced during lignification in the in vitro-differentiating TEs. The induction pattern of the OMT activity using 5-hydroxyferuloyl CoA as substrate during lignification was the same as that using caffeoyl CoA. Taken together, the results indicate that CCoAOMT is associated with lignification during xylogenesis both in vitro and in the plant, whereas COMT is only involved in a stress response in vitro. We propose that CCoAOMT is involved in an alternative methylation pathway in lignin biosynthesis. In Zinnia in vitro-differentiating TEs, the CCoAOMT mediated methylation pathway is dominant.

Tissue-Specific Expression of Cell Wall Proteins in Developing Soybean Tissues.

Cell wall hydroxyproline-rich glycoproteins (HRGPs) and glycine-rich proteins (GRPs) were examined at the protein and at the mRNA levels in developing soybean tissues by tissue print immunoblots and RNA blots. In young soybean stems, HRGPs are expressed most heavily in cambium cells, in a few layers of cortex cells surrounding primary phloem, and in some parenchyma cells around the primary xylem, whereas GRPs are highly expressed in the primary xylem and also in the primary phloem. In older soybean stems, HRGP genes are expressed exclusively in cambium cells and GRP genes are most heavily expressed in newly differentiated secondary xylem cells. Similar expression patterns of HRGPs and of GRPs were found in soybean petioles, seedcoats, and young hypocotyls, and also in bean petioles and stems. HRGPs and GRPs become insolubilized in soybean stem cell walls. Three major HRGP mRNAs and two major GRP mRNAs accumulate in soybean stems. Soluble HRGPs are abundant in young hypocotyl apical regions and young root apical regions, whereas in hypocotyl and root mature regions, soluble HRGPs are found only in a few layers of cortex cells surrounding the vascular bundles. GRPs are specifically localized in primary xylem cell walls of young root. These results show that the gene expression of HRGPs and GRPs is developmentally regulated in a tissue-specific manner. In soybean tissues, HRGPs are most heavily expressed in meristematic cells and in some of those cells that may be under stress, whereas GRPs are expressed in all cells that are or are going to be lignified.

An extracellular matrix protein in plants: characterization of a genomic clone for carrot extensin.

Extensins are hydroxyproline‐rich glycoproteins found in many plant cell walls as a major protein component. The peptide Ser‐Hyp‐Hyp‐Hyp‐Hyp is abundant in the extensins. Using extensin cDNA clones as probes, we isolated six different clones from carrot genomic libraries. One of the genomic clones, pDC5A1, was characterized and found to contain an open reading frame encoding extensin and a single intron in the 3′‐non‐coding region. The derived amino acid sequence contains a signal peptide sequence and 25 Ser‐Pro‐Pro‐Pro‐Pro repetitive sequences. Two extensin transcripts were found corresponding to pDC5A1 with different 5′ start sites. These transcripts increase in abundance after wounding. This is consistent with the reported extensin accumulation in the cell wall upon wounding.

Induction of cysteine and serine proteases during xylogenesis in Zinnia elegans

The terminal process of xylogenesis, autolysis, is essential for the formation of a tubular system for conduction of water and solutes throughout the whole plant. Several hydrolase types are implicated in autolysis responsible for the breakdown of cytoplasm. Here, we characterize p48h-17 cDNA from in vitro tracheary elements (TEs) of Zinnia elegans which encodes a preproprotein similar to papain. The putative mature protein, a cysteine protease, has a molecular mass of 22,699 Da with a pI of 5.7. DNA gel blot analysis indicated that p48h-17 is likely encoded by one or two genes. The p48h-17 mRNA accumulated markedly in in vitro differentiating TEs, whereas it appeared not to be induced in response to senescence and wounding in the leaves or H2O2 challenge in the cultured mesophyll cells. In stems, the expression of the p48h-17 gene was preferentially associated with differentiating xylem. Activity gel assays demonstrated that a cysteine and a serine protease, which had apparent molecular masses of 20 kDa and 60 kDa, respectively, were markedly induced during in vitro TE differentiation. The cysteine protease activity was also preferentially present in the xylem of Zinnia stems. Transient expression of the p48h-17 cDNA in tobacco protoplasts resulted in the production of a 20 kDa cysteine protease. Taken together, the results indicate that the p48h-17 gene appears to be preferentially associated with xylogenesis, and both the cysteine and serine proteases might be involved in autolysis during xylogenesis.

Differential Expression of Two O-Methyltransferases in Lignin Biosynthesis in Zinnia elegans

Caffeic acid 3–O-methyltransferase (CAOMT) and caffeoyl-coenzyme A 3–O-methyltransferase (CCoAOMT) are involved in different methylation pathways in lignin biosynthesis. We previously showed that only the CCoAOMT was markedly induced during lignification in in vitro differentiating tracheary elements (TEs) of Zinnia elegans. To further examine the expression patterns of CAOMT in lignification, we isolated a cDNA clone for Zinnia CAOMT. RNA gel blot analysis showed that the expression of the CAOMT gene did not correlate well with lignification during in vitro TE differentiation from Zinnia-isolated mesophyll cells. Tissue-print hybridization showed that, in the young internodes, the CAOMT mRNA signal was much more evident in phloem fibers than in xylem, whereas the CCoAOMT mRNA signal was predominantly present in differentiating xylem regions. In the older internode, both the CAOMT and CCoAOMT mRNAs markedly accumulated in phloem fibers and differentiating xylem regions. Immunocytochemical localization showed that the CAOMT protein staining was much more evident in phloem fibers and xylem fibers than in xylem TEs. These results indicate that the expression of these two O-methyltransferases is differentially regulated during lignification in different cell types in Zinnia. We suggest that all the intermediates in the CAOMT-mediated methylation pathway might become substrates for the CCoAOMT-mediated methylation pathway after coenzyme A ligation when these two pathways occur in the same lignifying cell types.

Enzyme synthesis in the cotyledons of germinating seeds

Abstract It has been shown that the increase in the activities of a phosphatase and an amylase in the cotyledons of germinating peas is the result of a process which is energy dependent and inhibited by chloramphenicol and p -fluoro-phenylalanine. It is concluded that these increased activities represent a net synthesis of enzyme-proteins. The phosphatase studied is rather specific for adenosine triphosphate and adenosine diphosphate and is maximally activated by cadmium ions.

Insolubilization of hydroxyproline-rich cell wall glycoprotein in aerated carrot root slices

The hydroxyproline-rich glycoprotein of plant cell walls is secreted from the cytoplasm as a soluble monomer which slowly becomes insolubilized. A tyrosine derivative, isodityrosine, is formed in the cell wall during this insolubilization and could serve as a protein-protein crosslink. Glycoprotein insolubilization is inhibited by peroxidase inhibitors and free radical scavengers, the most effective of which is L-ascorbate. These data support a hypothesis that the hydroxyproline-rich cell wall glycoprotein forms a covalently crosslinked wall network under the control of an extracellular peroxidase/ascorbate oxidase system.

3,4-Dehydroproline Inhibits Cell Wall Assembly and Cell Division in Tobacco Protoplasts

We investigated the function of cell wall hydroxyproline-rich glycoproteins by observing the effects of a selective inhibitor of prolyl hydroxylase, 3,4-dehydro-L-proline (Dhp), on wall regeneration by Nicotiana tabacum mesophyll cell protoplasts. Protoplasts treated with micromolar concentrations of Dhp do not develop osmotic stability and do not initiate mitosis. The architecture of regenerated cell walls was examined using deep-etch, freeze-fracture electron microscopy of rapidly frozen tobacco cells. Untreated protoplasts assemble a dense fibrillar cell wall consisting of laterally associating subelementary fibrils. In contrast, treatment of protoplasts with Dhp alters the structure of the regenerated wall fibrils in several ways: first, the microfibrils are coated with globular knobs; second, some larger fiber bundles have an open ribbon-like appearance; and third, the smallest subelementary fibrils were not visible. Tobacco cells develop an abnormal morphology as a consequence of this abnormal cell wall structure. Thus, inhibition of prolyl hydroxylase results in the regeneration of a cell wall with abnormal structural and functional properties. These data provide experimental evidence that hydroxyproline-rich glycoproteins are important for the structural integrity of primary cell walls and for the correct assembly of other wall polymers, and that wall structure is an important regulator of cell division and cell morphology.

Tomato extensin and extensin-like cDNAs: structure and expression in response to wounding

Two tomato cDNA libraries were synthesized from poly(A)+ RNAs isolated from unwounded and wounded tomato stems. These cDNA libraries were packaged in λgt10 and screened by in situ plaque hybridization with a tomato extensin gene clone (pTom 5.10). Several cDNA clones were identified and isolated from both libraries in this manner and subjected to restriction enzyme digestion, Southern gel blot hybridization, RNA gel blot hybridization, and DNA sequence analyses. From these analyses, the various cDNA clones were found to fall into one of five distinct classes (classes I–V). Class I clones hybridized to a 4.0 kb mRNA which accumulated markedly after wounding and encoded an extensin characterized largely by Ser-(Pro)4-Ser-Pro-Ser-(Pro)4-(Tyr)3-Lys repeats. Class II clones hybridized to a 2.6 kb mRNA which showed no accumulation following wounding and encoded an extensin containing Ser-(Pro)4-Ser-Pro-Ser-(Pro)4-Thr-(Tyr)1–3-Ser repeats. Class III clones hybridized to a 0.6 kb mRNA which greatly accumulated in response to wounding and encoded a glycine-rich protein (GRP) with (Gly)2–6-Tyr-Pro and(Gly)2–6-Arg repeats. Class IV clones contained both class I and class III DNA sequences and consequently hybridized to both the 4.0 kb and the 0.6 kb wound-accumulating mRNAs; these clones encoded a portion of a GRP sequence on one DNA strand and encoded a portion of an extensin sequence on the other DNA strand. Class V clones hybridized to a 2.3 kb mRNA which decreased following wounding and encoded a GRP sequence characterized by (Gly)2–5-Arg repeats.