The tapetum is a special tissue within the stamen that supplies nutrients to support microspore differentiation. Many genes have been identified to function in tapetum differentiation (Astrand et al., 2021). Ubiquitin C-terminal hydrolase (UCH) is a type of cysteine protease involved in protein deubiquitination process (Mevissen and Komander, 2017) and plays roles in gonadal transformation and spermatogenesis in animals (Luo et al., 2009; Kwon et al,. 2004; Wang et al., 2006). OsUCH3 (Os02g43760) was highly expressed in early rice stamen cells (Chen et al., 2015), the OsUCH3 expression was first observed in all cells of anther primordium and then gradually concentrated into the germ cells and tapetum cells, and OsUCH3 protein exhibits stronger ubiquitin C-terminal hydrolase enzymatic activity (Wang et al., 2018). These data suggested that OsUCH3 is likely associated with stamen development.

To explore whether OsUCH3 plays roles in rice stamen development, we constructed transgenic lines to down-regulate OsUCH3 expression through RNAi vector as described previously with primers (ccACTAGTATGGAGGATGCTCATTCC and TcGGATCCCACAACTTTCGAAAGAGC) (Liu et al., 2005) (Figure 1a–g) and the OsUCH3-targeting CRISPR-Cas9 mutant lines (Figure 1h). CRISPR-Cas9 osuch3 mutant lines exhibited the same male-sterile phenotype as OsUCH3R.

In observation of the overall morphology, there was no significant difference at vegetable development (Figure 1a). However, low seed setting rate was observed in the RNAi lines (Figure 1b) and CRISPR-Cas9 osuch3 mutant (Figure 1h). Alexander staining showed that OsUCH3R lines had abnormal pollen (Figure 1d) with the latter being indicated by the significant reduction in pollen grain number (Figure 1e, g). The RT-qPCR results revealed that OsUCH3 expression levels were significantly reduced in OsUCH3R transgenic rice (Figure 1f).

To examine whether OsUCH3 affected stamen development, we pollinated OsUCH3R pistils with wild-type pollen grains, which rescued the sterility of OsUCH3R. The result suggested that the low seed setting rate resulted mainly from down-expression of OsUCH3 (Figure 1i). Morphological analysis revealed abnormal tapetum differentiation and locule collapse were observed in the OsUCH3R lines from stage 5 (S5) through 12 (S12) (Figure 1j). This observation suggested that the downregulation of OsUCH3 caused a defect in the tapetum differentiation and stamen development.

In contrast to the degeneration of the tapetum that was observed in the WT from stages 7 (S7) to 9 (S9) (Figure 1k–m), the tapetum in the OsUCH3R lines was not properly degenerated during these stages (Figure 1n–p) as revealed by transmission electron microscopy (TEM) analysis. In addition, TEM observations revealed that the secondary cell walls of the endothecium cells were thinner in the OsUCH3R lines than in the wild type (Figure 1p, red arrowhead). We found green fluorescence in wild-type tapetal cells from stages 7 to 9 (Figure 1q-t) using the terminal deoxynucleotidyl transferase-mediated TdT-mediated dUTP Nick-End Labeling (TUNEL) assay (Chen et al., 2018). No such TUNEL signals were detected in OsUCH3R tapetal cells during the same developmental period (Figure 1u–x). These results suggested that DNA degradation during tapetum differentiation after stage 7 was abnormal in OsUCH3R.

Numerous genes were known being involved in tapetum differentiation (Chen et al., 2018). GAMYB4 was up-regulated while the expression of YY1, OsCP1 and UDT1 exhibited no change in the OsUCH3R lines (Figure 1y). OsUCH3 is lower expressed in shoot tips and leaf primordia. While no differential expression of SAM genes OsWOX3, the expression of OSH1 and OsWUS was significantly decreased in the OsUCH3R lines (Figure 1y). These changes demonstrated the link between abnormal tapetum differentiation in the OsUCH3R lines with the changes in the expression levels of known tapetum genes.

The phenotypes we found in OsUCH3R lines and CRISPR mutants demonstrated that tapetal differentiation is sensitive to the change of expression level of OsUCH3 and protein deubiquitination is an importance factor for proper tapetum differentiation. This finding provided a new opportunity not only in deciphering the regulatory network of tapetum differentiation at protein level, but in manipulating male sterility for agricultural applications.

Accession numbers

Sequence data from this article can be found in the GenBank/EMBL data library.

中文翻译：

---

水稻绒毡层分化对 OsUCH3（一种泛素 C 末端水解酶）的下调敏感

绒毡层是雄蕊内的特殊组织，提供营养以支持小孢子分化。许多基因已被鉴定在绒毡层分化中发挥作用（Astrand等人，  2021）。泛素C末端水解酶（UCH）是一种参与蛋白质去泛素化过程的半胱氨酸蛋白酶（Mevissen和Komander，  2017），在动物性腺转化和精子发生中发挥作用（Luo等，  2009；Kwon等，  2004 ） ；王等人，  2006）。OsUCH3 (Os02g43760) 在早期水稻雄蕊细胞中高表达 (Chen et alOsUCH3蛋白首先在花药原基的所有细胞中观察到表达，然后逐渐集中到生殖细胞和绒毡层细胞中，且OsUCH3蛋白表现出更强的泛素C端水解酶活性(Wang et al . ,  2018 )  。这些数据表明OsUCH3可能与雄蕊发育有关。

为了探索OsUCH3是否在水稻雄蕊发育中发挥作用，我们构建了转基因品系，通过 RNAi 载体下调OsUCH3 的表达，如前所述，使用引物（ccACTAGTATGGAGGATGCTCATTCC 和 TcGGATCCCACAACTTTCGAAAGAGC）（Liu等，  2005）（图 1a-g）和OsUCH3靶向 CRISPR-Cas9 突变株系（图 1h）。CRISPR-Cas9 osuch3突变系表现出与OsUCH3R相同的雄性不育表型。

从整体形态观察来看，蔬菜发育没有显着差异（图1a）。然而，在 RNAi 系（图 1b）和 CRISPR-Cas9 osuch3突变体（图 1h）中观察到结籽率较低。亚历山大染色显示OsUCH3R系具有异常花粉（图 1d），后者表现为花粉粒数量显着减少（图 1e，g）。RT-qPCR 结果显示OsUCH3R转基因水稻中OsUCH3表达水平显着降低（图 1f）。

为了检查OsUCH3是否影响雄蕊发育，我们用野生型花粉粒给OsUCH3R雌蕊授粉，这挽救了OsUCH3R的不育性。结果表明，结籽率低主要是由于OsUCH3的表达下调所致（图 1i）。形态学分析显示，从第 5 阶段 (S5) 到第 12 阶段 (S12)，OsUCH3R系中观察到异常绒毡层分化和小室塌陷（图 1j）。这一观察结果表明OsUCH3的下调导致绒毡层分化和雄蕊发育缺陷。

与在 WT 中从阶段 7 (S7) 到 9 (S9) 观察到的绒毡层退化相反（图 1k-m），OsUCH3R 系中的绒毡层在这些阶段没有适当退化（图 1n- p） ）如透射电子显微镜（TEM）分析所示。此外，TEM 观察表明， OsUCH3R系的内皮细胞次生细胞壁比野生型更薄（图 1p，红色箭头）。我们使用末端脱氧核苷酸转移酶介导的T dT 介导的 d U TP Nick - E nd L标签 (TUNEL) 测定法，在第 7 至 9 阶段的野生型绒毡层细胞中发现了绿色荧光（图 1q-t）（ Chen等人，  2018）。在同一发育时期，在OsUCH3R绒毡层细胞中未检测到此类 TUNEL 信号（图 1u-x）。这些结果表明，OsUCH3R中第 7 阶段后绒毡层分化过程中的 DNA 降解是异常的。

已知许多基因参与绒毡层分化（Chen等人，  2018）。GAMYB4上调，而YY1、OsCP1和UDT1的表达在OsUCH3R系中没有表现出变化（图 1y）。OsUCH3在茎尖和叶原基中表达较低。虽然 SAM 基因OsWOX3没有差异表达，但OsUCH3R系中OSH1和OsWUS的表达显着降低（图 1y）。这些变化证明了OsUCH3R中异常绒毡层分化之间的联系与已知绒毡层基因表达水平的变化相符。

我们在OsUCH3R系和 CRISPR 突变体中发现的表型表明，绒毡层分化对OsUCH3表达水平的变化敏感，并且蛋白质去泛素化是绒毡层正常分化的重要因素。这一发现不仅为破译蛋白质水平上绒毡层分化的调控网络提供了新的机会，而且为农业应用中操纵雄性不育提供了新的机会。

入藏号

本文的序列数据可以在 GenBank/EMBL 数据库中找到。