High pollen fertility can ensure the yield and efficiency of breeding work, but factors that affect the fertility of pepper pollen have not been studied extensively. In this work, we screened the reduced pollen fertility 1 (rpf1) mutant with reduced pollen fertility and yellow anthers from an EMS-mutagenized pepper population. Through construction of an F2 population, followed by BSA mapping and KASP genotyping, we identified CabHLH1 as a candidate gene for control of this trait. A G→A mutation at a splice acceptor site in CabHLH1 causes a frameshift mutation in the mutant, and the translated protein is terminated prematurely. Previous studies on CabHLH1 have focused on the regulation of flavonoid synthesis. Here, we found that CabHLH1 also has an important effect on pollen fertility. Pollen vigor, anther flavonoid content, and seed number were lower in CabHLH1-silenced pepper plants, whereas anther H2O2 and MDA contents were higher. RNA-Seq analyses showed that expression of the flavonoid synthesis genes DFR, ANS, and RT was significantly reduced in anthers of CabHLH1-silenced plants and rpf1, as was the expression of DTX35, a gene related to pollen fertility and flavonoid transport. Yeast one-hybrid and dual-luciferase reporter assays showed that CabHLH1 can directly bind to the promoters of DTX35 and DFR and activate their expression. These results indicate that CabHLH1 regulates ROS homeostasis by promoting the synthesis of anther flavonoids and acts as a positive regulator of pepper pollen fertility.

中文翻译：

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bHLH1-DTX35/DFR 模块通过促进辣椒中的类黄酮生物合成来调节花粉生育力。

花粉肥力高可以保证育种工作的产量和效率，但影响辣椒花粉肥力的因素尚未得到广泛研究。在这项工作中，我们从 EMS 诱变辣椒种群中筛选了花粉生育力降低和黄色花药的花粉生育力降低 1 (rpf1) 突变体。通过构建 F2 种群，然后进行 BSA 作图和 KASP 基因分型，我们将 CabHLH1 鉴定为控制该性状的候选基因。AG→CabHLH1 中剪接受体位点的突变导致突变体发生移码突变，翻译的蛋白质提前终止。以前对 CabHLH1 的研究主要集中在类黄酮合成的调节上。在这里，我们发现 CabHLH1 对花粉的生育能力也有重要影响。花粉活力，花药黄酮含量，CabHLH1 沉默的辣椒植株的种子数和种子数较低，而花药 H2O2 和 MDA 含量较高。RNA-Seq 分析表明，在 CabHLH1 沉默植物和 rpf1 的花药中，类黄酮合成基因 DFR、ANS 和 RT 的表达显着降低，与花粉育性和类黄酮转运相关的基因 DTX35 的表达也是如此。酵母单杂交和双荧光素酶报告基因分析表明，CabHLH1 可以直接与 DTX35 和 DFR 的启动子结合并激活它们的表达。这些结果表明，CabHLH1 通过促进花药类黄酮的合成来调节 ROS 稳态，并作为辣椒花粉生育力的正调节剂。CabHLH1 沉默植物的花药和 rp​​f1 的 ANS 和 RT 显着降低，与花粉育性和类黄酮转运相关的基因 DTX35 的表达也显着降低。酵母单杂交和双荧光素酶报告基因分析表明，CabHLH1 可以直接与 DTX35 和 DFR 的启动子结合并激活它们的表达。这些结果表明，CabHLH1 通过促进花药类黄酮的合成来调节 ROS 稳态，并作为辣椒花粉生育力的正调节剂。CabHLH1 沉默植物的花药和 rp​​f1 的 ANS 和 RT 显着降低，与花粉育性和类黄酮转运相关的基因 DTX35 的表达也显着降低。酵母单杂交和双荧光素酶报告基因分析表明，CabHLH1 可以直接与 DTX35 和 DFR 的启动子结合并激活它们的表达。这些结果表明，CabHLH1 通过促进花药类黄酮的合成来调节 ROS 稳态，并作为辣椒花粉生育力的正调节剂。