• Flavio Nguyen posted an update 1 year, 5 months ago

    Here, we refer to both genuine chitinases and their homologs collectively as chitinase-like proteins. Chitinases catalyze cleavage of b-1,4-glycoside bonds of chitin and are organized in five classes, which can be distinguished on the basis of sequence similarity. Classes I, II, and IV belong to glycoside hydrolase family 19, found primarily in plants, whereas Classes III and V belong to glycoside hydrolase family 18 present in various types of organisms. The Class I chitinases are found in both monocots and dicots, while classes II and IV are found mainly in dicots. Class I and IV chitinases contain a highlyconserved cysteine-rich domain – the chitin binding domain – at the N- terminal region, but there are two characteristic deletions in the main catalytic domain in Class IV chitinases. Because chitin is the major component of fungal cell walls, chitinases are classic pathogenesis-related proteins involved in non-host-specific defense. Plants also contain chitinase-like proteins that are not induced by pathogens or stresses. In many cases, these chitinase-like proteins have been shown to lack detectable chitinase activity. Chitinase-like proteins may play an important role during normal plant growth and development. For example, AtCTL1 is constitutively expressed in many organs of Arabidopsis. Mutations of AtCTL1 lead to ectopic deposition of lignin in the secondary cell wall, reduction of root and hypocotyl lengths, and increased numbers of root hairs. It was suggested that this gene could be involved in root expansion, Temozolomide distributor cellulose biosynthesis, and responses to several environmental stimuli. In particular, coexpression of some CTLs with secondary cell wall cellulose synthases was reported. It has been suggested that these chitinase-like proteins could take part in cellulose biosynthesis and play a key role in establishing interactions between cellulose microfibrils and hemicelluloses. The xylan-type secondary wall is the most common secondary wall in land plants and is characteristically rich in cellulose, xylan, and lignin. Compared to typical xylan-type secondary walls, gelatinous layers are enriched in cellulose, have a higher degree of cellulose crystallinity, larger crystallites, and a distinctive set of matrix polysaccharides. Presumably, cellulose synthase genes have a significant role in gelatinous cell wall formation, but the expression patterns of the complete flax CESA family has not been described to date. It is known that at least three isoforms of CESAs comprise the cellulose synthase rosette: CESA1, CESA3, and CESA6 are required for cellulose biosynthesis in primary cell walls, whereas CESA4, CESA7, and CESA8 are required for cellulose biosynthesis during secondary wall deposition. Flax is a useful model for comparative studies of cell wall development: different parts of the flax stem form a primary cell wall, xylan type secondary cell wall, or gelatinous cell wall; these stem parts may be separated and analyzed by diverse approaches, including functional genomics. Furthermore, the publication of a flax whole genome assembly facilitates a thorough study of key gene families. In the present study, we measured expression of all predicted LusCTL genes of the GH19 family in various tissues including those that produce gelatinous-type and xylan-type cell walls. We also described the LusCESA gene family and measured expression of its transcripts in comparison to LusCTLs. Phylogenetic analysis of LusCTL and LusCESA genes identified distinct groups of LusCTL genes that were expressed preferentially at specific stages of bast fiber gelatinous cell wall development. Certain fibers of many plant species form G-type cell walls, which are rich in crystalline cellulose. Expression of CTLs has been previously reported to be enriched during development of Gtype cell walls, along with specific FLAs, LTPs and BGALs. In this work, we analyzed expression of all LusCTL genes of GH 19 in different flax tissues and compared this expression with LusCESAs and to their inferred phylogenies. In the flax genome, 16 predicted LusCESAs were identified. Previously only partial sequences of some flax CESAs were published. All 16 flax CESAs could be placed in discrete clades with Arabidopsis and Populus CESA homologs. We generally numbered LusCESAs in a way that reflects the association of each flax gene with its nearest relative in the Arabidopsis genome, as was done for CESAs of Populus. Following this pattern, the LusCESA6A-F genes we named as a group, similar to PtiCESA6A-F and were not distinguished as CESA2/9/5/6 as in Arabidopsis clade. Most of the flax and Populus CESA genes are present as pairs of paralogs in their respective genomes, although there were three LusCESA3 genes for only two Populus genes and one Arabidopsis gene. AtCESA1 and AtCESA10 were represented by only one pair of genes in flax. It is well established that proteins encoded by different sets of three CESA genes are required for cellulose synthesis during primary and secondary wall formation, respectively. The functional relationships of the various paralogs of LusCESAs are presently unclear. According to the data obtained here, secondary cell wall LusCESA4, LusCESA7-A, B and LusCESA8-A, B were highly expressed both in the xylem cells with lignified cell walls and in the phloem fibers with thick gelatinous cell wall. This suggests that phloem fibers and xylem may use similar, rather than specialized rosettes. This is consistent with observations from poplar showing only minor differences in expression of cellulose biosynthetic genes in tension wood as compared to normal wood. The different properties of gelatinous and xylan type cell walls are therefore likely determined not by CESAs, but by other proteins associated with cellulose synthesis, which could include specific CTLs. CTL2 is strongly upregulated during secondary wall formation in interfascicular fibers in A. thaliana. Reduction in crystalline cellulose content in ctl1 ctl2 mutants was demonstrated, leading to the to the suggestion that AtCTLs are involved in cellulose assembly. Furthermore, in P. trichocarpa, expression of chitinase genes related to AtCTL1, AtCTL2, and GhCTLVII are highly correlated with secondary wall formation of xylem. It has therefore been proposed that CTL1 and CTL2 work in conjunction with primary- and secondary-cell wall CESAs, respectively. One of the hypotheses for CTL1/2 function is regulation of cellulose assembly and of interaction with hemicelluloses via binding to emerging cellulose microfibrils. However, the mechanism of CTL action in cell wall biosynthesis as well as substrates of catalytic activity remains unknown.