Abstract
BackgroundIn many plants, the amino acid proline is strongly accumulated in pollen and disruption of proline synthesis caused abortion of microspore development in Arabidopsis. So far, it was unclear whether local biosynthesis or transport of proline determines the success of fertile pollen development.ResultsWe analyzed the expression pattern of the proline biosynthetic genes PYRROLINE-5-CARBOXYLATE SYNTHETASE 1 & 2 (P5CS1 & 2) in Arabidopsis anthers and both isoforms were strongly expressed in developing microspores and pollen grains but only inconsistently in surrounding sporophytic tissues. We introduced in a p5cs1/p5cs1 p5cs2/P5CS2 mutant background an additional copy of P5CS2 under the control of the Cauliflower Mosaic Virus (CaMV) 35S promoter, the tapetum-specific LIPID TRANSFER PROTEIN 12 (Ltp12) promoter or the pollen-specific At5g17340 promoter to determine in which site proline biosynthesis can restore the fertility of proline-deficient microspores. The specificity of these promoters was confirmed by β-glucuronidase (GUS) analysis, and by direct proline measurement in pollen grains and stage-9/10 anthers. Expression of P5CS2 under control of the At5g17340 promoter fully rescued proline content and normal morphology and fertility of mutant pollen. In contrast, expression of P5CS2 driven by either the Ltp12 or CaMV35S promoter caused only partial restoration of pollen development with little effect on pollen fertility.ConclusionsOverall, our results indicate that proline transport is not able to fulfill the demand of the cells of the male germ line. Pollen development and fertility depend on local proline biosynthesis during late stages of microspore development and in mature pollen grains.
Highlights
In many plants, the amino acid proline is strongly accumulated in pollen and disruption of proline synthesis caused abortion of microspore development in Arabidopsis
The promoters of P5CS1 and P5CS2 are active in microspores and pollen grains at late stages of pollen development As a first step to establish where the proline required for pollen development and fertility is synthesized, we analyzed the pattern of GUS activity under the control of the P5CS1 and P5CS2 promoter regions in Arabidopsis anthers at different stages of development (Fig. 1, Additional file 1: Figure S1)
Cauliflower mosaic virus 35S transcript (CaMV35S) promoter-driven sporophytic expression of P5CS2 does not rescue the abnormalities of p5cs1/p5cs2 pollen In an attempt to complement the aberrant pollen development of p5cs sesquimutants by overexpression of P5CS2, we introduced a transgenic copy of P5CS2 under the control of the CaMV35S promoter, which was reported to drive gene expression in most tissues and organs of Arabidopsis, not in developing microspores and pollen grains [32]
Summary
The amino acid proline is strongly accumulated in pollen and disruption of proline synthesis caused abortion of microspore development in Arabidopsis. The importance of proline for pollen fertility has been recently highlighted by the observation that disruption of proline synthesis in Arabidopsis caused infertility by abortion during gametophyte development [1,2,3]. These findings provided a functional link between fertility and the accumulation of high levels of free proline under non-stressed conditions in floral organs - . The differential localization of these two enzymes and the observation that functional P5CS expression is essential for reproduction (see below) suggested that synthesis from glutamate is the only functional pathway for proline biosynthesis in Arabidopsis [2, 3, 13]
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