Expression And Functional Characterization Of Cotton P4Hs

Cotton is one of the most important economic crops in the world. It is of cardinal importance to improve cotton fiber yield and quality. Cotton fibers are single-celled extensions of the seed coat epidermis. Cotton fiber is composed of over 90% cellulose, little xylan, lignin and glycoproteins. Our previous studies showed that a large number of Arabinogalactan proteins (AGPs) play critical roles in fiber development. AGP consists of a protein backbone and large amount of polysaccharide which occupy over 90% of the molecular mass of the total protein. They undergo extensive posttranslational modification, including proline hydroxylation to hydroxyproline by prolyl-4-hydroxylase (P4Hs), then hydroxyproline was glycosylated by glycosyltransferases. We propose P4Hs play very important roles in AGP functionality. In this study, three cDNAs encoding P4Hs were isolated from cotton cDNA library, named GhP4Hs, the main results are as follows:1. P4Hs are involved in fiber elongationIn vitro ovule culture was used to study the effect of P4Hs inhibitor on fiber development. A certain concentration of P4Hs inhibitor α-α-dipyridyl(DP) and ethy-3,4-dihydroxybenzoate(EDHB) was added to the medium, The growth assay showed that inhibition of proline hydroxylation could suppress cotton fiber elongation, implying P4Hs were involved in the elongation of cotton fiber.2. Phylogenetic analysis of GhP4HsPhylogenetic analysis of P4Hs from cotton and Arabidopsis revealed that cotton GhP4H2 and Arabidopsis At2g17720 (P4H5) were in the same subclade, suggesting that they were phylogenetically closed. And the cotton GhP4Hl and the AtP4H2 were in the same subclade, while cotton GhP4H3 and AtP4H10 were in the same subclade, which showed that they were very closed.3. Expression analysis of three GhP4H genesRT-PCR analysis of expression of GhP4H3 and GhP4Hl showed that they were expressed in all the tissues examined especially in 0 dpa fiber to 15 dpa fibers. GhP4H2 was highly expressed in hypocotyls and 6 dpa fibers. The expression analysis suggested that GhP4Hs may function in cotton fiber development.4. Characterization of Arabidopsis p4h5 mutantPrevious studies showed that root hairs in Arabidopsis p4h5 mutant were much shorter and sparser compared with the wild type.Also the seedlings of p4h5 mutant grow dwarf and the roots were remarkably shorter, leaf blades were smaller in absence of sucrose. However, the seedlings of p4h5 mutant exhibited no obvious difference in 3% sucrose.In this study, hand-cut sections of 6-week-old stem was stained with β-Yariv reagent which specifically binds to AGPs, the staining was more strong in wild type than in the mutant. And AGP content of p4h5 mutant was less. Moreover, Western Blot and Dot Blot analyses revealed MAC207-bound AGPs were less in the p4h5 mutant.The results suggested that mutation in AtP4H5 affected the AGP biosynthesis.5. Expression of GhP4Hs gene in p4h5 mutantWe expressed GhP4H1 in p4h5 mutant to see if they perform similar functions, the results showed that the root hair defects of p4h5 mutant could be partially rescued by expression of GhP4H1. And the dwarf seedings in absence of sucrose can be completely rescued by expression of GhP4H1. The results indicated that P4H1 was a functional homolog of Arabidopsis P4H5.6. Expression of GhP4H1 in wild type Arabidopsis.We expressed GhP4H1 in wild type Arabidopsis to investigate its possible function. The transgenic seedlings displayed very obvious phenotype including smaller leaves,shorter roots compared with the wild type Meanwhile, the AGP content of the transgenic plants were more than that of the wild type. Dot Blot and Western Blot analysis also revealed that MAC207-bound AGPs were more than that of the wild type. The results suggested that GhP4H1 may be involved in the synthesis of AGPs.7. Cotton transformationWe transformed GhP4H1 and GhP4H2 overexpression vectors into cotton. Four lines of GhP4H2 overexpression transgenic cotton respectively were obtained. The transgenen PCR check showed that 80% transgenic plants were positive in the T1 generation. The phenotypic analyses are currently underway.We also transformed GhP4H1 and GhP4H2 RNAi-silencing vectors to cotton, Five lines and seven lines of transgenic cotton respectively were acquired. Transgene PCR check showed that about 60% transgenic plants were positive. Now the Tl generation of transgenic cotton plants are planted in field. The phenotypic analyses remain to be determined.