Abstract
A Citrus clementina gene, CcGASA4, which is involved in the responses of citrus to stress, was characterized. The gene was induced by Citrus tristeza virus infection, wounding and gibberellic, salicylic and abscisic acid treatments. A qRT-PCR analysis showed that CcGASA4 had a very high basal expression in flowers yet was still able to be further induced independently in giberellic, salicylic and abscisic acid-treated flowers. Subcellular localization analysis revealed that the CcGASA4 protein localized to the cell membrane and nucleus. A yeast two-hybrid analysis and bimolecular fluorescence complementation (BiFC) assays showed that CcGASA4 interacted with two proteins, the large proline-rich protein bag6-A (PRPBAG6-A) and the general negative regulator of transcription subunit 3 (CNOT3). PRPBAG6 has been reported to be involved in disease resistance. Replacing some of CcGASA4's conserved cysteines with alanines (Cys → Ala) abolished the protein’s interaction with CNOT3 but did not show any effect on the protein’s interaction with PRPBAG6-A. Thus, CcGASA4 appears to play multiple roles in Citrus, probably by interacting with different proteins and/or by localizing to different subcellular compartments.
Highlights
Cysteine-rich peptides are well represented among plants and are categorized into different classes based on the number and arrangement of the cysteine residuesElectronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.1 3 Vol.:(0123456789)Plant Growth Regulation (2020) 91:89–99 proteins feature cysteine-disulfide bridges in their distinct three-dimensional structural folds (Silverstein et al 2007)
In our previous gene chip experiments, we found that the citrus homolog of AtGASA4, Cit.11064.1.S1_at, was highly induced in citrus leaves after infection with Citrus tristeza virus (CTV) (Zhang 2010), indicating that GASA4 may play a role in biotic stress responses
A BLAST alignment showed that the similarities between the predicted CcGASA4 protein and the GA-stimulated transcript (GAST)-like proteins from cucumber, grape, cotton and Rosa damascene were greater than 60%
Summary
Plant Growth Regulation (2020) 91:89–99 proteins feature cysteine-disulfide bridges in their distinct three-dimensional structural folds (Silverstein et al 2007). These disulfide bonds may be essential for the interactions of GASA proteins with other proteins (Nahirnak et al 2012a, b). The GASA4 gene was first reported in Arabidopsis (Herzog et al 1995) It is GA-responsive and involved in plant reproduction. The expression pattern of AtGASA4 and the phenotypes of both the loss-of-function and the gain-offunction mutants clearly showed its involvement in bolting, branching, flowering and seed development in Arabidopsis (Rubinovich and Weiss 2010). An analysis of GASA4 mutants indicated that GASA4 regulates floral meristem identity and positively affects both seed size and total seed yield (Roxrud et al 2007)
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