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Acta Agronomica Sinica ›› 2021, Vol. 47 ›› Issue (7): 1239-1247.doi: 10.3724/SP.J.1006.2021.04217

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GhP4H2 encoding a prolyl-4-hydroxylase is involved in regulating cotton fiber development

GAO Lu1,2, XU Wen-Liang1,*()   

  1. 1Hubei Key Laboratory of Genetic Regulation and Integrative Biology / School of Life Sciences, Central China Normal University, Wuhan 430079, Hubei, China
    2Wheat Research Institute, Shanxi Agricultural University, Linfen 041000, Shanxi, China
  • Received:2020-09-22 Accepted:2020-12-01 Online:2021-07-12 Published:2020-12-29
  • Contact: XU Wen-Liang E-mail:wenliangxu@mail.ccnu.edu.cn
  • Supported by:
    This study was supported by the National Natural Science Foundation of China(31970516);This study was supported by the National Natural Science Foundation of China(31671735)

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Abstract:

Arabinogalactan proteins (AGPs) perform crucial roles during cotton fiber development, and they are composed of a hydroxyproline (Hyp)-rich core protein and large type-II arabinogalactan (AG) moieties. AGPs are highly glycosylated proteins which involve two most important post-translational processes. First, the proline residues were hydroxylated by prolyl- 4-hydroxylases (P4Hs), then the Hyps were substituted with large arabinogalactan polysaccharides or small arabino- oligosaccharides by glycosyltransferases (GTs). Our previous work had shown that fiber elongation was repressed when in vitro-cultured cotton ovules were treated with inhibitors of P4Hs, suggesting that P4Hs were involved in cotton fiber development. In this study, GhP4H2 was detected to be relatively highly expressed at fiber elongation stage. Overexpression and RNAi-silencing vectors of GhP4H2 were constructed and transformed into cotton. Phenotypic analysis showed that GhP4H2 overexpressing fibers were significantly shorter compared with the wild type from T1 generation to T3 generation. In addition, AGP content had increased in GhP4H2 overexpression fibers. Immuno dot-blot analysis also showed that carbohydrate moieties of AGP had changed. Moreover, RNA-seq revealed that expression levels of genes encoding hydroxyproline (Hyp)-rich glycoproteins including AGPs were enhanced. Overall, these results indicated that GhP4H2 may regulate fiber development primarily through affecting AGPs composition.

Key words: cotton fiber, arabinogalactan proteins (AGPs), prolyl-4-hydroxylase (P4Hs)

Table 1

Primers used in this study"

引物
Primer		 正向序列
Forward sequence (5'-3')		 反向序列
Reverse sequence (5'-3')
Gh_D12G1409RT TAGAACCCAAGCTGGTCAGG TCGATTTAGACCCTGTCGATG
Gh_D01G1917RT AGATCTCATGCGCCGTCA AGGCAAAGAAAGAAACAAGTGTG
Gh_D07G2484RT TTTGCCTTTTTCTGTGAGCA GCTGGTTGGGATGAACACTT
Gh_D10G0598RT CTATGAGTGTGATTGGCGTACAGGTAA AATCCGTGGTCACGTTATATTTGG
Gh_D08G2475RT TAACCCACCAAGTGGGAAAC TGAGCTTAAGGGTGGTGCAT
Gh_A07G0433RT TGAAGAAGACGAAGAACCATCACCA AGAGCCAGAGCTTTCAACTGATGT
Gh_D05G0131RT TTAAGCCACCCATTGACCTC TCCTGAAAGAACCGGACAAC
Gh_D12G2178RT TCTTTTCCCTGGTGAGTGCT AACGGCAGCTTCAAGATCAG
GhGalT1RT TCCCTCCTCACCTTCGCCATTG CCTTGACGATCAGAAGGCATCCAC
GhGalT4RT GATGATACTTTGAAAATCGTTGCT ATTTTCTTCAAAGTTTCACCGCT
GhGalT6RT TCAGGAGTATGTACCCCAGCTTGC TGCAAATGAAGTCACATGTCGAC
GhGalT7RT CCTCACCAGATCAACAGCCCTCT CCGTTGAAGGCCCTGATGATC
GhP4H2RT ATCGGAATGTGCAAAGAAGG AGCCAGGAAGTTCTGCAGTT
GhUBI1RT CTGAATCTTCGCTTTCACGTTATC GGGATGCAAATCCTTCGTGAAAAC
GhP4H2 RNAiL1 CTTGGATCCGTGCAAAGAAGGGAATTG GGGTCTAGAAATAGCCAGGAAGTTCTG
GhP4H2RNAiL2 GGGGAGCTCGTGCAAAGAAGGGAATTG CTTGCGGCCGCAATAGCCAGGAAGTTCT
GhP4H2OE GGGTCTAGAATGGCTATTGAGAGGATTT CTTGTCGACTCAACATACTTTGCAGCTT

Fig. 1

Phylogenetic analysis of GhP4H2 and expression profile of GhP4H2 A: phylogenetic relationship of GhP4H2 and Arabidopsis P4Hs; B: expression analysis of GhP4H2 in different cotton tissues. 1: root; 2: hypocotyl; 3: cotyledon; 4: leaves; 5: petals; 6: anthers; 7: 15 DPA ovule; 8: 0 DPA fiber (with ovule); 9: 3 DPA fiber (with ovule); 10: 5 DPA fiber; 11: 9 DPA fiber; 12: 15 DPA fiber; 13: 20 DPA fiber. DPA: days post anthesis. Error bar represents the standard deviation."

Fig. 2

Expression analysis of GhP4H2 in wild type and transgenic cotton lines A and B: quantitative RT-PCR analysis of GhP4H2 expression in 5 DPA (A) and 10 DPA (B) fibers from independent transgenic cotton lines and wild type. RiL16 and RiL28 represent two independent GhP4H2-RNAi lines; WT represents the wild type; OEL10 and OEL38 represent two independent GhP4H2 overexpression lines. ** means significant difference at the 0.01 probability level."

Fig. 3

Comparison of mature fibers from transgenic cotton lines and wild type A and B: measurement and statistical analysis of mature fiber length and seed size of the transgenic cotton plants and wild type. ** means significant difference at the 0.01 probability level. Abbreviations of lines name are the same as those given in Fig. 2."

Fig. 4

AGP content in fibers of GhP4H2 transgenic cotton lines and wild type A: halos of different samples from transgenic lines and wild type. B: standard curve; Abscissa: the AGPs concentration; Ordinate: the area of the corresponding halos. C: the AGPs content of different samples from transgenic lines and wild type. 0.1-0.6 (μg μL-1): gum arabic concentration. ** means significant difference at the 0.01 probability level. Abbreviations of lines name are the same as those given in Fig. 2."

Fig. 5

Distribution and abundance of AGP epitopes in GhP4H2 transgenic cotton and wild type -: PBS negative control. +: 1 mol L-1 arabic gum positive control. Abbreviations of lines name are the same as those given in Fig. 2."

Fig. 6

Analysis of differentially expressed genes related cell wall A: major classes of the upregulated cell wall genes in transgenic fibers; B: expression analysis of genes encoding glycoproteins. PRP: proline-rich protein; AGP: arabinogalactan protein; EXT: extensin; PAL: phenylalanine ammonia-lyase. ** means significant difference at the 0.01 probability level. Abbreviations of lines name are the same as those given in Fig. 2."

Fig. 7

Expression analysis of several GhGalT genes * and ** mean significant differences at the 0.05 and 0.01 probability levels, respectively. Abbreviations of lines name are the same as those given in Fig. 2."

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