Department of Biology and Biotechnology, Sapienza University of Rome, P.le A. Moro 5, 00185, Rome, Italy.
Department of Bioscience, University of Milan, Milan, Italy.
BMC Plant Biol. 2018 Dec 17;18(1):356. doi: 10.1186/s12870-018-1571-3.
In 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.
We 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.
Overall, 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.
在许多植物中，脯氨酸在花粉中强烈积累，而拟南芥中脯氨酸合成的破坏导致小孢子发育的败育。到目前为止，还不清楚是脯氨酸的局部生物合成还是运输决定了可育花粉的发育成功。
我们分析了脯氨酸生物合成基因 PYRROLINE-5-CARBOXYLATE SYNTHETASE 1 和 2（P5CS1 和 P5CS2）在拟南芥花药中的表达模式，这两种同工型在发育中的小孢子和花粉粒中强烈表达，但在周围的孢子组织中表达不一致。我们在 p5cs1/p5cs1 p5cs2/P5CS2 突变体背景下，在 Cauliflower Mosaic Virus（CaMV）35S 启动子、绒毡层特异性 LIPID TRANSFER PROTEIN 12（Ltp12）启动子或花粉特异性 At5g17340 启动子的控制下，引入了 P5CS2 的额外拷贝，以确定脯氨酸生物合成可以在哪个部位恢复脯氨酸缺乏的小孢子的育性。这些启动子的特异性通过 β-葡萄糖醛酸酶（GUS）分析以及花粉粒和阶段 9/10 花药中直接脯氨酸测量来证实。P5CS2 在 At5g17340 启动子的控制下表达完全挽救了突变体花粉的脯氨酸含量和正常形态和育性。相比之下，由 Ltp12 或 CaMV35S 启动子驱动的 P5CS2 的表达仅导致花粉发育部分恢复，对花粉育性几乎没有影响。
总的来说，我们的结果表明，脯氨酸的运输不能满足雄性生殖细胞的需求。花粉的发育和育性依赖于小孢子发育晚期和成熟花粉粒中局部脯氨酸的生物合成。
