Arabinogalactan proteins (AGPs) are involved in many aspects of plant development. In this study, biochemical and genetic approaches demonstrated that AGPs are abundant in developing fibers and may be involved in fiber initiation and elongation. To further investigate the role of AGPs during fiber development, a fasciclin-like arabinogalactan protein gene (GhFLA1) was identified in cotton (Gossypium hirsutum). Overexpression of GhFLA1 in cotton promoted fiber elongation, leading to an increase in fiber length. In contrast, suppression of GhFLA1 expression in cotton slowed down fiber initiation and elongation. As a result, the mature fibers of the transgenic plants were significantly shorter than those of the wild type. In addition, expression levels of GhFLAs and the genes related to primary cell wall biosynthesis were remarkably enhanced in the GhFLA1 overexpression transgenic fibers, whereas the transcripts of these genes were dramatically reduced in the fibers of GhFLA1 RNA interference plants. An immunostaining assay indicated that both AGP composition and primary cell wall composition were changed in the transgenic fibers. The levels of glucose, arabinose, and galactose were also altered in the primary cell wall of the transgenic fibers compared with those of the wild type. Together, our results suggested that GhFLA1 may function in fiber initiation and elongation by affecting AGP composition and the integrity of the primary cell wall matrix.

Di19 (drought-induced protein19) family is a novel type of Cys2/His2 zinc-finger proteins. In this study, Arabidopsis Di19-3 was functionally characterized. The experimental results revealed that AtDi19-3 is a transcriptional activator, and could bind to the TACA(A/G)T sequence. AtDi19-3 expression in plants was remarkably induced by NaCl, mannitol and abscisic acid (ABA). T-DNA insertion mutation of AtDi19-3 results in an increase in plant tolerance to drought and high salinity stresses and ABA, whereas overexpression of AtDi19-3 leads to a drought-, salt- and ABA-sensitive phenotype of the transgenic plants. In the presence of NaCl, mannitol or ABA, rates of seed germination and cotyledon greening in Atdi19-3 mutant were higher, but in AtDi19-3 overexpression transgenic plants were lower than those in wild type. Roots of Atdi19-3 mutant seedlings were longer, but those of AtDi19-3 overexpression transgenic seedlings were shorter than those of wild type. Chlorophyll and proline contents in Atdi19-3 mutant were higher, but in AtDi19-3 overexpression seedlings were lower than those in wild type. Atdi19-3 mutant showed greater drought-tolerance, whereas AtDi19-3 overexpression transgenic plants exhibited more drought-sensitivity than wild type. Furthermore, expression of the genes related to ABA signaling pathway was altered in Atdi19-3 mutant and AtDi19-3 transgenic plants. These data suggest that AtDi19-3 may participate in plant response to drought and salt stresses in an ABA-dependent manner.

The CBL-CIPK signaling pathway represents a central and critical signaling system involved in plant response to abiotic stress and hormone signaling. In this study, we focused on AtCIPK6 gene, which have been shown to be required for development and salt tolerance in Arabidopsis, but the transcriptional regulatory mechanism of AtCIPK6 gene and whether it plays a role in ABA signaling is still unknown. Here, we showed that in addition to its expression induced by salt and drought stress, the transcripts of AtCIPK6 gene were largely accumulated in abscisic acid treated seedling, compared to basal level expression. Moreover, promoter sequence analysis revealed that there are some elements involved in stress and hormone response detected in the promoter region of AtCIPK6. AtCIPK6p:GUS transgenic assays unraveled that AtCIPK6 promoter is salt/osmotic stress- and ABA-inducible. Overexpression of AtCIPK6 gene in Arabidopsis increased plant tolerance to salt stress, but sensitivity to ABA. Our results contribute to the understanding of transcriptional regulatory mechanism of AtCIPK6 gene and may also provide a good stress-inducible promoter candidate for transgenic engineering.

Summary Aquaporins are thought to be associated with water transport and play important roles in cotton (Gossypium hirsutum) fibre elongation. Among aquaporins, plasma membrane intrinsic proteins (PIPs) constitute a plasma‐membrane‐specific subfamily and are further subdivided into PIP1 and PIP2 groups. In this study, four fibre‐preferential GhPIP2 genes were functionally characterized. The selective interactions among GhPIP2s and their interaction proteins were studied in detail to elucidate the molecular mechanism of cotton fibre development. GhPIP2;3 interacted with GhPIP2;4 and GhPIP2;6, but GhPIP2;6 did not interact with GhPIP2;4. Coexpression of GhPIP2;3/2;4 or GhPIP2;3/2;6 resulted in a positive cooperative effect which increased the permeability coefficient of oocytes, while GhPIP2;4/2;6 did not. GhBCP2 (a blue copper‐binding protein) inhibited GhPIP2;6 water channel activity through their interaction. Overexpression of GhPIP2 genes in yeast induced longitudinal growth of the host cells. By contrast, knockdown of expression of GhPIP2 genes in cotton by RNA interference markedly hindered fibre elongation. In conclusion, GhPIP2 proteins are the primary aquaporin isoforms in fibres. They selectively form hetero‐oligomers in order to regulate their activities to meet the requirements for rapid fibre elongation.

Arabinogalactan proteins (AGPs) are highly glycosylated proteins that play pivotal roles in diverse developmental processes in plants. Type-II AG glycans, mostly O-linked to the hydroxyproline residues of the protein backbone, account for up to 95% w/w of the AGP, but their functions are still largely unclear. Cotton fibers are extremely elongated single-cell trichomes on the seed epidermis; however, little is known of the molecular basis governing the regulation of fiber cell development. Here, we characterized the role of a CAZy glycosyltransferase 31 (GT31) family member, GhGalT1, in cotton fiber development. The fiber length of the transgenic cotton overexpressing GhGalT1 was shorter than that of the wild type, whereas in the GhGalT1-silenced lines there was a notable increase in fiber length compared with wild type. The carbohydrate moieties of AGPs were altered in fibers of GhGalT1 transgenic cotton. The galactose: arabinose ratio of AG glycans was higher in GhGalT1 overexpression fibers, but was lower in GhGalT1-silenced lines, compared with that in the wild type. Overexpression of GhGalT1 upregulates transcript levels of a broad range of cell wall-related genes, especially the fasciclin-like AGP (FLA) backbone genes. An enzyme activity assay demonstrated that GhGalT1 is a β-1,3-galactosyltransferase (β-1,3-GalT) involved in biosynthesis of the β-1,3-galactan backbone of the type-II AG glycans of AGPs. We also show that GhGalT1 can form homo- and heterodimers with other cotton GT31 family members to facilitate AG glycan assembly of AGPs. Thus, our data demonstrate that GhGalT1 influences cotton fiber development via controlling the glycosylation of AGPs, especially FLAs.

TCP proteins are plant-specific transcription factors (TFs), and perform a variety of physiological functions in plant growth and development. In this study, 74 non-redundant TCP genes were identified in upland cotton (Gossypium hirsutum L.) genome. Cotton TCP family can be classified into two classes (class I and class II) that can be further divided into 11 types (groups) based on their motif composition. Quantitative RT-PCR analysis indicated that GhTCPs display different expression patterns in cotton tissues. The majority of these genes are preferentially or specifically expressed in cotton leaves, while some GhTCP genes are highly expressed in initiating fibers and/or elongating fibers of cotton. Yeast two-hybrid results indicated that GhTCPs can interact with each other to form homodimers or heterodimers. In addition, GhTCP14a and GhTCP22 can interact with some transcription factors which are involved in fiber development. These results lay solid foundation for further study on the functions of TCP genes during cotton fiber development.

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Homeodomain-leucine zipper (HD-Zip) proteins are transcriptional factors involved in plant development. In this study, one cDNA clone (Gossypium hirsutum homeobox1, designated GhHB1) encoding HD-Zip protein was isolated from a cotton root cDNA library. The GhHB1 cDNA is 1132 bp in length, including an 828 bp open reading frame that encodes a peptide with 275 amino acids, and 5'/3'untranslated regions. The predicted GhHB1 protein containing a homeodomain and a leucine-rich zipper motif shares relatively high identity with other plant HD-Zip proteins. Analysis using quantitative real-time RT-PCR indicated that the GhHB1 gene is predominantly expressed in roots and hypocotyls. Furthermore, GhHB1 transcripts were largely accumulated in early root development, and significantly reduced to very low levels as roots further developed, suggesting that the gene might function in the early development of roots. Under treatment with 1% NaCl, the expression level of the GhHB1 gene was dramatically increased in roots. Likewise, GhHB1 activity in roots was up-regulated by abscisic acid. These results imply that GhHB1 might play an important role in response to salt stress and to abscisic acid signaling.