Dataset Information

Characterization and Activity Analyses of the FLOWERING LOCUS T Promoter in Gossypium Hirsutum.

ABSTRACT: Flowering transition is a crucial development process in cotton (Gossypium hirsutum L.), and the flowering time is closely correlated with the timing of FLOWERING LOCUS T (FT) expression. However, the mechanism underlying the coordination of various cis-regulatory elements in the FT promoter of cotton has not been determined. In this study, a 5.9-kb promoter of FT was identified from cotton. A bioinformatics analysis showed that multiple insertion-deletion sites existed in the 5.9-kb promoter. Different expression levels of a reporter gene, and the induction by sequential deletions in GhFT promoter, demonstrated that 1.8-kb of the GhFT promoter was stronger than 4.2-, 4.8-, and 5.9-kb promoter fragments. The binding sites of the CONSTANS (CO) and NUCLEAR FACTOR Y transcription factors were located within the 1.0-kb sequence upstream of the FT transcription start site. A large number of repeat segments were identified in proximal promoter regions (-1.1 to -1.4 kb). A complementation analysis of deletion constructs between 1.0 and 1.8 kb of G. hirsutum, Gossypium arboretum, and Gossypium raimondii FT promoters revealed that the 1.0-kb fragment significantly rescued the late-flowering phenotype of the Arabidopsis FT loss-of-function mutant ft-10, whereas the 1.8-kb promoter only slightly rescued the late-flowering phenotype. Furthermore, the conserved CORE motif in the cotton FT promoter is an atypical TGTG(N2-3)ATG, but the number of arbitrary bases between TGTG and ATG is uncertain. Thus, the proximal FT promoter region might play an important role affecting the activity levels of FT promoters in cotton flowering.

SUBMITTER: Sang N

PROVIDER: S-EPMC6801411 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Characterization and Activity Analyses of the FLOWERING LOCUS T Promoter in Gossypium Hirsutum.

Sang Na N Cai Darun D Li Chao C Sun Yuqiang Y Huang Xianzhong X

International journal of molecular sciences 20190926 19

Flowering transition is a crucial development process in cotton (Gossypium hirsutum L.), and the flowering time is closely correlated with the timing of FLOWERING LOCUS T (FT) expression. However, the mechanism underlying the coordination of various cis-regulatory elements in the FT promoter of cotton has not been determined. In this study, a 5.9-kb promoter of FT was identified from cotton. A bioinformatics analysis showed that multiple insertion-deleti ...[more]

PMID: 31561427

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Project description:Apoplastic ascorbate oxidase (AO) plays significant roles in plant cell growth. However, the mechanism of underlying the transcriptional regulation of AO in Gossypium hirsutum remains unclear. Here, we obtained a 1,920-bp promoter sequence from the Gossypium hirsutum ascorbate oxidase (GhAO1) gene, and this GhAO1 promoter included a number of known cis-elements. Promoter activity analysis in overexpressing pGhAO1::GFP-GUS tobacco (Nicotiana benthamiana) showed that the GhAO1 promoter exhibited high activity, driving strong reporter gene expression in tobacco trichomes, leaves and roots. Promoter 5'-deletion analysis demonstrated that truncated GhAO1 promoters with serial 5'-end deletions had different GUS activities. A 360-bp fragment was sufficient to activate GUS expression. The P-1040 region had less GUS activity than the P-720 region, suggesting that the 320-bp region from nucleotide -720 to -1040 might include a cis-element acting as a silencer. Interestingly, an auxin-responsive cis-acting element (TGA-element) was uncovered in the promoter. To analyze the function of the TGA-element, tobacco leaves transformed with promoters with different 5' truncations were treated with indole-3-acetic acid (IAA). Tobacco leaves transformed with the promoter regions containing the TGA-element showed significantly increased GUS activity after IAA treatment, implying that the fragment spanning nucleotides -1760 to -1600 (which includes the TGA-element) might be a key component for IAA responsiveness. Analyses of the AO promoter region and AO expression pattern in Gossypium arboreum (Ga, diploid cotton with an AA genome), Gossypium raimondii (Gr, diploid cotton with a DD genome) and Gossypium hirsutum (Gh, tetraploid cotton with an AADD genome) indicated that AO promoter activation and AO transcription were detected together only in D genome/sub-genome (Gr and Gh) cotton. Taken together, these results suggest that the 1,920-bp GhAO1 promoter is a functional sequence with a potential effect on fiber cell development, mediated by TGA-element containing sequences, via the auxin-signaling pathway.

Project description:Background:Genetically modified cotton accounts for 64% of the world's cotton growing area (22.3 million hectares). The genome sequencing of the diploid cotton progenitors Gossypium raimondii and Gossypium arboreum as well as the cultivated Gossypium hirsutum has provided a wealth of genetic information that could be exploited for crop improvement. Unfortunately, gene functional characterization in cotton is lagging behind other economically important crops due to the low efficiency, lengthiness and technical complexity of the available stable transformation methods. We present here a simple, fast and efficient method for the transient transformation of G. hirsutum that can be used for gene characterization studies. Results:We developed a transient transformation system for gene characterization in upland cotton. Using ?-glucuronidase as a reporter for Agrobacterium-mediated transformation assays, we evaluated multiple transformation parameters such as Agrobacterium strain, bacterial density, length of co-cultivation, chemicals and surfactants, which can affect transformation efficiency. After the initial characterization, the Agrobacterium EHA105 strain was selected and a number of binary constructs used to perform gene characterization studies. 7-days-old cotton seedlings were co-cultivated with Agrobacterium and transient gene expression was observed 5 days after infection of the plants. Transcript levels of two different transgenes under the control of the cauliflower mosaic virus (CaMV) 35S promoter were quantified by real-time reverse transcription PCR (qRT-PCR) showing a 3-10 times increase over the levels observed in non-infected controls. The expression patterns driven by the promoters of two G. hirsutum genes as well as the subcellular localization of their corresponding proteins were studied using the new transient expression system and our observations were consistent with previously published results using Arabidopsis as a heterologous system. Conclusions:The Agrobacterium-mediated transient transformation method is a fast and easy transient expression system enabling high transient expression and transformation efficiency in upland cotton seedlings. Our method can be used for gene functional studies such as promoter characterization and protein subcellular localization in cotton, obviating the need to perform such studies in a heterologous system such as Arabidopsis.

Dataset Information

Characterization and Activity Analyses of the FLOWERING LOCUS T Promoter in Gossypium Hirsutum.

Publications

Characterization and Activity Analyses of the <i>FLOWERING LOCUS T</i> Promoter in <i>Gossypium Hirsutum</i>.

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