|拟南芥开花因子 FLK 和 FLC 调控 DELLA 介导的高温花粉发育
|Flowering factors FLK and FLC regulate DELLA-mediated pollen development under high temperature in Arabidopsis
|郭光沁 ; 李晓峰
|Place of Conferral
|高温 high temperature FLK FLK 绒毡层 tapetum 花粉发育 pollen development FLC FLC 赤霉素 gibberellin DELLA DELLA
持续高温使作物产量和粮食安全面临巨大挑战。高温影响植物有性生殖发育 过程，其中花粉发育对高温最为敏感。因此花粉的高温应答机制研究是重要的科 学命题。本课题组前期筛选到一个高温不育和晚花表型的突变体，将其命名为 flk6 (flowering locus k homology domain-6)。图位克隆结合全基因组测序克隆到基因 AT3G04610 发生缺失突变。该基因编码含 3 个 RNA 结合结构域(KH domain)的 蛋白 FLK，调控开花时间。 本论文研究结果如下： 1、已知开花基因 FLK 具有调控高温花粉发育的功能。细胞学观察和数据分 析发现高温诱导野生型的绒毡层细胞提前退化，而功能缺失突变体 flk-6 花药发 育中绒毡层细胞和小孢子分别在第 8 期和第 9 期起始空泡化，引起花粉外壁组分 积累减少，花粉壁塌陷粘连，小孢子退化，最终产生败育的花粉粒。高温诱导的 花粉发育和形成缺陷能够被 FLK 功能互补，恢复 flk-6 的育性表型。 2、利用外源赤霉素处理探究 FLK 调控花粉发育的机制，发现 200 μM GA3 处理后 flk-6 突变体可育角果比例和单角果种子数极显著降低，表明赤霉素信号 可能参与FLK介导的育性调控。生化数据证明FLK与赤霉素受体GIBBERELLIN INSENSITIVE DWARF1 (GID1)竞争结合负调控因子 DELLA 蛋白的 N 端，并干 扰 DELLA 蛋白的 C 端与 SLY1 的相互作用，抑制 DELLA 的泛素化降解，进而 维持 DELLA 蛋白水平，确保高温下正常的花粉发育。 3、通过分析高温下 FLK 调控的开花基因转录水平，发现开花信号通路中的 负调控因子 FLOWERING LOCUS C (FLC)可能参与高温育性调控。遗传分析和 细胞学数据表明 FLC 功能缺失和获得均影响高温下绒毡层的降解、花粉发育、 花粉粒活力和外壁的形态建成。flk-6 flc-3 双突变体的开花时间表型验证了开花 信号通路中 FLC 位于 FLK 的下游发挥作用，而育性表型证明 FLC 位于 FLK 的 上游调控高温花粉发育，且不依赖于下游开花调控因子 FT 和 SOC1 发挥作用。 表明在高温育性和开花时间调控中二者的遗传关系具有差异性。利用显微成像技 术发现 22℃条件下 FLC 维持 DELLA 的蛋白水平，而 29℃条件下 FLC 降低 IV DELLA 的蛋白水平。生化数据表明 FLC 与 SLY1 直接相互作用。 综上所述，本论文系统研究了植物花粉响应高温过程中赤霉素参与调控的分 子机制。揭示了拟南芥开花因子 FLK 和 FLC 通过调节 DELLA 的蛋白水平调控 高温花粉发育，为作物耐高温品种培育和提高作物产量提供理论基础。
Continuous high temperature poses great challenges to crop yield and food security. High temperature affects the process of plant sexual reproduction, in which pollen development has been identified as the most heat-vulnerable stage. Therefore, it is an important scientific proposition to study the mechanism of pollen response to high temperature. A large-scale screening for temperature-related mutants has previously performed, in which the flk-6 (flowering locus k homology domain-6) was isolated with high temperature sterility and late flowering phenotype. With the map-based cloning and whole genome resequencing, a deletion mutation in the gene AT3G04610 was cloned. This gene encodes FLK protein containing three RNA-binding (KH domains) that regulates flowering time. Here, our results are as follows: 1. The known FLOWERING gene FLK has a significant function of regulating pollen development at high temperature. Cytological observation and data analysis showed that high temperature induced premature degeneration of wild-type tapetum cells, while tapetum cells and microspore of flk-6 mutant anthers was vacuolated at stages 8 and 9, respectively, resulting in the reduction of pollen exine components, pollen wall collapse and adhesion, microspore degradation, and abortive pollen grains. Pollen development defects induced by high temperature can be functionally complemented by FLK to restore fertility phenotype of flk-6. 2. The mechanism of FLK-regulated pollen development was explored by exogenous gibberellin treatment. Results showed that the percentage of siliques and seed counts per silique of flk-6 mutant were significantly decreased after 200 μM GA3 treatment, suggesting that gibberellin signaling is involved in FLK-mediated fertility regulation. Biochemical data demonstrated that FLK and GIBBERELLIN (GA) receptor GA-INSENSITIVE DWARF1 (GID1) competed to bind the N-terminal of the VI negative regulator DELLA proteins, interfered with the interaction between the Cterminus of DELLA proteins and SLY1, and inhibited DELLA proteins ubiquitination. In turn, DELLA proteins levels are maintained to ensure normal pollen development at high temperature. 3. Through analyzing the transcripts of other FLOWERING factors regulated by FLK under high temperature, it was found that FLOWERING LOCUS C (FLC), a negative regulatory factor in flowering signaling pathway, may be involved in the regulation of high temperature fertility. Genetic analysis and cytological data indicate that both loss and gain of FLC function affected the degradation of tapetum, pollen development, pollen grains viability and exine morphogenesis at high temperature. The flowering time phenotype of flk-6 flc-3 double mutant verified that FLC was located downstream of FLK in the flowering signaling pathway, and the fertility phenotype proved that FLC was located upstream of FLK to regulate pollen development at high temperature in a downstream flowering regulators FT and SOC1-independent manner. These results indicate that the genetic relationship between high temperature fertility and flowering time pathway is different. Microscopic imaging showed that FLC maintained DELLA protein level at 22℃, while FLC reduced DELLA protein level at 29℃. Biochemical data suggested that FLC directly interacted with SLY1. In summary, this paper systematically studied the molecular mechanism of gibberellin regulating pollen development at high temperature. It was revealed that the flowering factors FLK and FLC regulate pollen development by modulating DELLA proteins levels under high temperature in Arabidopsis. It provides a theoretical basis for the cultivation of high temperature resistant varieties and the improvement of crop yield.
|MOST Discipline Catalogue
|理学 - 生物学 - 细胞生物学
|费琼晖. 拟南芥开花因子 FLK 和 FLC 调控 DELLA 介导的高温花粉发育[D]. 兰州. 兰州大学,2023.
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