The rice cytosolic pentatricopeptide repeat protein OsPPR2-1 regulates OsGLK1 to control tapetal development and programmed death

Shaoyan Zheng State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Sciences, South China Agricultural University Jingfang Dong Guangdong Key Laboratory of New Technology in Rice Breeding, Rice Research Institute, Guangdong Academy of Agricultural Sciences Jingqin Lu State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Sciences, South China Agricultural University Jing Li State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Sciences, South China Agricultural University Dagang Jiang State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Sciences, South China Agricultural University Simiao Ye State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Sciences, South China Agricultural University Wenli Bu State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Sciences, South China Agricultural University Zhenlan Liu State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Sciences, South China Agricultural University Hai Zhou State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Sciences, South China Agricultural University Chuxiong Zhuang (  [email protected] ) State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Sciences, South China Agricultural University Article

Keywords: OsPPR2-1, , PCD, plastid, OsGLK1

Posted Date: January 29th, 2021

DOI: https://doi.org/10.21203/rs.3.rs-131663/v1

License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License 1 The rice cytosolic pentatricopeptide repeat protein OsPPR2-1 2 regulates OsGLK1 to control tapetal plastid development and 3 programmed cell death 4 5 Shaoyan Zheng1,3,4, Jingfang Dong2,4, Jingqin Lu1,3,4, Jing Li1,3, Dagang Jiang1,3, Simiao 6 Ye1,3, Wenli Bu1,3, Zhenlan Liu1,3, Hai Zhou1,3, Chuxiong Zhuang1,3,* 7 8 1State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, 9 South China Agricultural University, Guangzhou 510642, China 10 2Guangdong Key Laboratory of New Technology in Rice Breeding, Rice Research Institute, 11 Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China 12 3Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou 510642, China 13 4These authors contributed equally to this article. 14 15 *Correspondence: Chuxiong Zhuang ([email protected]) 16 17 18 19 ABSTRACT 20 Most pentatricopeptide repeat (PPR) proteins localize to or mitochondria, where 21 they participate in RNA metabolism and post-transcriptionally regulate gene 22 expression. However, whether PPR proteins regulate the expression of nucleus-encoded 23 genes remains unclear. Here, we uncovered a function for the rice (Oryza. sativa L.) PPR 24 protein OsPPR2-1 (Os02g0110400) in development and showed that, in contrast to 25 most other PPR proteins, OPPR2-1 resides in the . Downregulating OsPPR2-1 26 expression led to abnormal plastid development in tapetal cells, prolonged programmed 27 cell death (PCD), prolonged tapetum degradation, and significantly reduced pollen fertility. 28 Transcriptome analysis revealed that the expression of OsGOLDEN-LIKE 1 (OsGLK1), 29 encoding a transcription factor that regulates plastid development and maintenance, was 30 significantly higher in plants with downregulated OsPPR2-1 expression compared to the 31 wild type. Moreover, OsPPR2-1 bound to the OsGLK1 mRNA in RNA 32 immunoprecipitation and RNA-electrophoretic mobility shift assays. An in vitro cleavage 33 assay showed that OsPPR2-1 could degrade the OsGLK1 mRNA. Notably, knockdown of 34 OsGLK1 partially restored pollen fertility in OsPPR2-1-knockdown plants and OsGLK1- 35 overexpressing plants showed abnormal tapetum and plastid development, similar to the 36 OsPPR2-1-knockdown plants. Together, our findings demonstrate that OsPPR2-1 37 regulates OsGLK1 expression, thereby controlling plastid development and PCD in the 38 tapetum. 39 40 Key words: OsPPR2-1, tapetum, PCD, plastid, OsGLK1 41 42 43 44 INTRODUCTION 45 Pentatricopeptide repeat (PPR) proteins are ubiquitous in eukaryotes but are rarely 46 found in prokaryotes1. More than 600 PPR genes have been identified in Arabidopsis 47 thaliana, accounting for approximately 2% of the total number of Arabidopsis genes. In 48 addition, 435 and 441 non-redundant PPR genes have been identified in japonica and 49 indica rice (Oryza sativa), respectively2-6. 50 PPR proteins contain tandem repeats of 35-amino-acid motifs (PPR motifs), with 2– 51 26 PPR motifs per PPR protein7. PPR proteins are classified into the P-type and PLS-type 52 subfamilies based on the sequence characteristics of their PPR motifs. Almost half of PPR 53 family proteins are P-type subfamily members, most of which contain only typical PPR 54 motifs. P-type PPR proteins contain arrays of P motifs comprising the usual 35 amino acids, 55 whereas PLS-type subfamily members contain S (short, 31 amino acids) and L (long, 35– 56 36 amino acids) motifs as well as P motifs. Based on their C-terminal conserved domains, 57 PLS-type subfamily proteins can be divided into PLS, E, E+, and DYW subgroups2. P-type 58 PPR proteins usually function in RNA splicing, RNA stabilization, and activation of 59 translation8. Some PPR proteins have a small MutS-related domain (SMR) at the C- 60 terminus; this domain could be involved in protein–RNA interactions and PPR-SMR 61 proteins in plants have RNA endonuclease activity9. 62 The PPR protein family in rice is large, and most of these proteins localize to plastids 63 (), mitochondria, or the nucleus4,5. In rice, several -targeted PPR 64 proteins function in leaf development; among them, WHITE STRIPE LEAF (WSL)10, 65 WSL411, and plastid TRANSCRIPTIONALLY ACTIVE CHROMOSOME PROTEIN 2 66 (OspTAC2)12 are essential for RNA splicing of chloroplast gene transcripts during leaf 67 development. In addition, some PPR members can restore male fertility or regulate plant 68 growth and development by regulating the mitochondrial RNA metabolism or participates 69 in mitochondrial RNA editing13-16. The nucleus-localized PPR protein OsNPPR3 functions 70 in starch biosynthesis and seed vigor by affecting the expression and splicing of nuclear 71 and mitochondrial genes17. The above studies mainly focused on the regulation of 72 mitochondrial- or chloroplast-encoded genes by PPR proteins located in mitochondria, 73 plastids, or the nucleus. Despite these advances, the roles of cytosolic PPR proteins remain 74 largely unclear, and how P-type PPR proteins are involved in regulating nuclear genes 75 remains to be elucidated. 76 Here, we determined the function of the PPR protein-encoding gene OsPPR2-1 in rice. 77 OsPPR2-1 encodes a P- type PPR protein with 16 PPR motifs that localizes to the 78 and functions in rice pollen development by regulating the expression of the nuclear gene 79 OsGOLDEN-LIKE 1 (OsGLK1). Our findings suggest that OsPPR2-1 regulates the 80 expression of OsGLK1 to control plastid development in the tapetum, thereby affecting 81 pollen development in rice. 82 83 RESULTS 84 OsPPR2-1 Is a Cytosol-Localized PPR Protein 85 In our previous research, we observed that the PPR protein-encoding gene OsPPR2- 86 1 (Os02g0110400, NM_001052184) is downregulated in thermo-sensitive male sterile 87 lines18. This reduced expression suggested that OsPPR2-1 may be related to pollen 88 development. To characterize the expression pattern of OsPPR2-1 in rice anthers, we 89 measured its transcript levels by quantitative real-time PCR (qRT-PCR). OsPPR2-1 was 90 expressed in various anther stages, with the highest transcript levels detected in stage 10 91 and stage 11 anthers (Fig. 1a). The cytological descriptions of the different stages of rice 92 anther development used in this study were based on those of Zhang et al19. To further 93 analyze the spatiotemporal expression patterns of OsPPR2-1 during anther development, 94 we performed RNA in situ hybridization. In situ signals were mainly observed in the tapetal 95 cells of anthers from stage 8 to stage 10, with the highest level detected in stage 10 anthers 96 (Fig. 1b). 97 To analyze the subcellular localization of OsPPR2-1, we fused the green fluorescent 98 protein (GFP) sequence to OsPPR2-1 and expressed the fusion protein (OsPPR2-1-GFP) 99 under the control of the CaMV 35S promoter. We transformed this construct into sheath 100 protoplasts of rice cv. Zhonghua11 (O. sativa L. ssp. japonica. cv. Zhonghua11, ZH11). 101 Diffuse GFP signals were detected in the cytoplasm, and the signals did not overlap with 102 mitochondria, chloroplasts, or nuclear signals (Fig. 1c). To validate these results, we 103 extracted mitochondrial and chloroplast proteins and carried out western blot experiments 104 with anti-OsPPR2-1 and different organelle-specific commercial antibodies. The 105 immunoblots showed a cytosolic localization for OsPPR2-1 (Fig. 1d). Together, these 106 observations support the conclusion that OsPPR2-1 is a PPR protein localized to the cytosol. 107 To examine the evolutionary relationships among OsPPR2-1 and its homologs, we 108 performed phylogenetic analysis via the maximum likelihood (ML) method using MEGA 109 software version X. OsPPR2-1 shares high sequence similarity with its homologs in other 110 plants, including the monocot maize (Zea mays) and the dicots Arabidopsis and soybean 111 (Glycine max). We detected a low level of similarity (~30%) between OsPPR2-1 and other 112 PPR proteins in rice (Extended Data Fig. 1a and 1b). These results suggest that OsPPR2-1 113 is a unique PPR gene in rice and that PPR genes are conserved among the species examined. 114 115 Knockdown of OsPPR2-1 Expression Leads to Reduced Fertility 116 To explore the role of OsPPR2-1 in rice development, we downregulated OsPPR2-1 117 expression using RNA interference (RNAi, driven by the maize Ubiquitin promoter) and 118 made prr2-1 mutants using clustered regularly interspaced short palindromic repeats 119 (CRISPR)-associated RNA-guided endonuclease Cas9 (CRISPR/Cas9) gene editing. Eight 120 independent transgenic lines were obtained, including three RNAi lines and five 121 CRISPR/Cas9 lines. For each method, we selected two stable lines that exhibited the lowest 122 OsPPR2-1 transcript levels for further study (Extended Data Fig. 2f). The RNAi progeny 123 plants were named ppr2R-1 and ppr2R-2 and the CRISPR/Cas9 plants were named 124 Casppr2-1 and Casppr2-2. (Fig. 2a and Extended Data Fig. 2a, Extended Data Fig. 3a–3b). 125 Although these four RNAi and CRISPR/Cas9 lines exhibited normal vegetative 126 growth, their reproductive growth was abnormal. In these lines, pale-yellow and even white 127 anthers were observed (Fig. 2a and Extended Data Fig. 2a). We examined the pollen 128 viability in the lines by iodine-potassium iodide (I2-KI) staining, which stains viable pollen. 129 The pollen grains of plants with downregulated OsPPR2-1 expression appeared lighter than 130 wild-type (WT) pollen. The pollen staining rates were 23.43 ± 1.8%, 31.2 ± 2.1%, 21.1 ± 131 1.3%, and 40.5 ± 2.1% for ppr2R-1, ppr2R-2, Casppr2-1, and Casppr2-2, respectively, 132 which were much lower than that of the WT (95.36 ± 2.4%) (Fig. 2b and 2d, Extended 133 Data Fig. 2b and 2d). Furthermore, the seed-setting rates of ppr2R-1, ppr2R-2, Casppr2-1, 134 and Casppr2-2 were 31.2 ± 2.1%, 35.2 ± 1.5%, 18.5 ± 1.1%, and 21.3 ± 1.3%, respectively, 135 which were greatly reduced compared with the rate of 93.1 ± 1.2% in WT plants (Fig. 2c, 136 Extended Data Fig. 2c and 2e). These results indicate that the knockdown of OsPPR2-1 137 led to abnormal anther development, decreased pollen fertility, and reduced seed-setting 138 rates. 139 140 Downregulating OsPPR2-1 Causes Abnormal Plastid Development in the Tapetum 141 To further investigate the defects in the anthers of OsPPR2-1-knockdown plants, we 142 generated semi-thin sections of anthers during development and observed them by light 143 microscopy. There were no detectable morphological differences between OsPPR2-1- 144 knockdown and WT anthers before stage 7. At stage 8, the tapetum of WT anthers began 145 to degrade and appeared (Fig. 2e). However, in the anthers of plants with 146 knocked-down expression of OsPPR2-1, the tapetal cells exhibited increased vacuolization 147 (Fig. 2j and 2o, Extended Data Fig. 4f and 4k). In WT pollen from stage 9 to stage 11, the 148 tapetum became thinner, the cytoplasm became condensed, and the tapetum cells continued 149 to degrade. The were then released and bicellular pollen was generated (Fig. 150 2f–2h). 151 Compared to normal WT anther development, in OsPPR2-1-knockdown plants from 152 stages 9 to 11, the tapetal cells exhibited irregular swelling and the cytoplasm began to 153 expand into the anther locule. After being released from the callose, the microspores were 154 compressed into irregular shapes. (Fig. 2k–2m and 2p–2r, Extended Data Fig. 4g–4i and 155 4l–4n). In WT anthers at stage 12, the tapetum disappeared completely. The microspores 156 progressed through the second , and mature pollen grains containing two 157 reproductive nuclei and one nutrient nucleus were generated (Fig. 2i). However, in 158 OsPPR2-1-knockdown anthers during the same stage, the tapetum was not completely 159 degraded and the microspores collapsed after being released and were not filled with starch 160 grains (Fig. 2n and 2s, Extended Data Fig. 4j and 4o). These results indicate that the normal 161 degradation of tapetal cells was blocked in ppr2R-1, ppr2R-2, Casppr2-1, and Casppr2-2 162 anthers, thus affecting pollen development and pollen fertility. 163 To further characterize the abnormalities in the tapetal cells of ppr2R-1, ppr2R-2, 164 Casppr2-1, and Casppr2-2 plants at stages 10–12, we observed the ultrastructural features 165 of the tapetum by transmission electron microscopy (TEM). Consistent with the 166 observations we made of the semi-thin sections, the WT tapetum continued to degrade at 167 stage 10. In addition, fewer organellar structures, such as plastids and endoplasmic 168 reticulum, were observed (Fig. 3a). However, in ppr2R-1, ppr2R-2, Casppr2-1, and 169 Casppr2-2 plants at this stage, the tapetum swelled abnormally and expanded to the center 170 of the anther locule. Numerous proplastids of different shapes (spindle-shaped and round) 171 were present in the cytoplasm (Fig. 3d and 3g, Extended Data Fig. 5b–5e, 5s and 5v). At 172 stage 11 in WT plants, the tapetum was degraded into fragments and elaioplasts were 173 detected (Fig. 3b), whereas in OsPPR2-1-knockdown plants, the tapetum cells continued 174 to expand inward, exhibiting a loose structure, and vacuolated proplastids did not 175 differentiate into elaioplasts (Fig. 3e and 3h, Extended Data Fig. 5g–5j, 5t and 5w). Notably, 176 there were significantly more tapetal plastids in OsPPR2-1-knockdown anthers compared 177 to WT anthers (Extended Data Fig. 8a–8h). At stage 12, unlike the WT tapetum (Fig. 3c), 178 the tapetum in OsPPR2-1-knockdown anthers was not completely degraded, and 179 vacuolated elaioplasts and other proplastids were still observed (Fig. 3f and 3i, Extended 180 Data Fig. 5l–5o, 5u and 5x). These results suggest that downregulating OsPPR2-1 affects 181 the normal development of elaioplasts in the anther tapetum. 182 183 OsPPR2-1-Knockdown Plants Exhibit Sustained Programmed Cell Death in the 184 Tapetum 185 Tapetum degradation is considered to result from programmed cell death (PCD) of the 186 tapetum cells. Our TEM analysis indicated that the tapetum degradation process was 187 abnormal from stage 9 to stage 12 in OsPPR2-1-knockdown anthers. To determine whether 188 the altered OsPPR2-1 expression in these anthers affects tapetal PCD, we performed a 189 terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay to examine 190 DNA fragmentation, a hallmark of PCD. In WT tapetal cells, PCD signals from the tapetum 191 were first observed at the beginning of meiosis. Strong TUNEL signals were observed from 192 stage 8 to stage 9, with the strongest signals detected at stage 9 (Fig. 4a–4b). PCD signals 193 gradually weakened from stage 10 to stage 11 and disappeared at stage 12 (Fig. 4c–4e). By 194 contrast, in ppr2R-1, ppr2R-2, Casppr2-1, and Casppr2-2 tapetal cells, much stronger 195 TUNEL signals were observed at stage 8 (Fig. 4f and 4k, Extended Data Fig. 6f and 6k), 196 and strong signals were still detected at stage 9, appearing in the epidermis, endothecium, 197 and microspores (Fig. 4g and 4l, Extended Data Fig. 6g and 6l). The signals persisted 198 during stages 10 to 11 and became weaker at stage 12 (Fig. 4h–4j, 4m–4o, Extended Data 199 Fig. 6h–6j, 6m–6o). These observations are consistent with the results of the cytological 200 observation mentioned above. Therefore, the TUNEL assay indicated that downregulating 201 OsPPR2-1 led to abnormal, prolonged PCD in the tapetum. 202 203 Transcriptome Analysis of Anthers in OsPPR2-1-Knockdown Lines to Identify 204 OsPPR2-1 Target Genes 205 The cytological analysis showed abnormal tapetal plastid development, and a 206 significant increase in the number of plastids (Fig. 3, Extended Data Fig. 8). To identify 207 how OsPPR2-1 regulates plastid development, we analyzed the transcriptome data of 208 anthers from WT and OsPPR2-1-knockdown plants at stages 8, 9, and 10, and screened for 209 target genes that OsPPR2-1 might regulate during plastid development. According to the 210 transcriptome analysis, the transcript levels of 214 genes differed between WT and 211 OsPPR2-1-knockdown plants in the three stages, among which the transcripts of 107 genes 212 increased (log2 >= 1, probability > 0.8) (Supplementary Table 4) and the transcripts of 107 213 genes decreased (log2 <= - 1, probability > 0.8) compared with the WT (Supplementary 214 Table 5). 215 Among the differentially expressed genes, we identified 18 transcription factor genes, 216 11 that were upregulated, and 7 that were downregulated (Supplementary Table 6), but 217 their annotations suggested that most of these transcription factors are not related to the 218 regulation of plastid development. Intriguingly, the expression of the OsGLK1 219 transcription factor gene increased in the OsPPR2-1-knockdown plants and OsGLK1 220 (Os06g0348800) plays key roles in plastid development and maintenance and regulation 221 of the accumulation of chloroplast proteins encoded by genes in the nucleus 20-22. 222 To identify whether knockdown of OsPPR2-1 affects potential targets of OsGLK1, we 223 compared our data to a previous study of the transcriptome effects of overexpressing 224 OsGLK1 under the control of the CaMV 35S promoter in rice calli; this study identified 75 225 nucleus-encoded chloroplast-related genes that increased in expression in these calli20. Our 226 transcriptome data showed that the expression of 53 of the 75 OsGLK1-regulated genes 227 was higher in OsPPR2-1-knockdown plants than in the WT (Supplementary Table 7). 228 Therefore, our data show that knockdown of OsPPR2-1 affects the expression of many 229 genes that are likely direct or indirect targets of OsGLK1. 230 We also examined the expression of OsGLK1 in WT and OsPPR2-1-knockdown 231 anthers by qRT-PCR analysis. In WT anthers, OsGLK1 expression fluctuated during 232 different stages of development. OsGLK1 was expressed at high levels during stages 5, 6, 233 and 10 but at low levels during stages 7, 8, 9, and 11. Almost no expression was detected 234 at stage 12. However, in the ppr2R-1 and Casppr2-1 plants, OsGLK1 was expressed at 235 significantly higher levels than in the WT during all stages of anther development. Even at 236 stage 12, the expression level of OsGLK1 remained high (Fig. 5a). By contrast, the 237 expression of OsGLK2, a homolog of OsGLK1, was almost unchanged in both WT and 238 OsPPR2-1-knockdown plants (Extended Data Fig. 7j). 239 To validate the transcriptome data, we measured the transcript levels of most of the 240 GLK1 target genes21 by qRT-PCR and observed that the expression of the GLK1 target 241 genes were upregulated in OsPPR2-1-knockdown anthers (Extended Data Fig. 9a–9k). 242 Based on the transcriptome data, we identified OsGLK1 as a candidate target gene of 243 OsPPR2-1. 244 245 Overexpressing OsGLK1 Causes Abnormal Tapetum Development 246 To determine whether increasing the expression of OsGLK1 influences the 247 development of tapetal plastids, thereby affecting tapetum and pollen development, we 248 constructed an OsGLK1-overexpression vector driven by the maize Ubiquitin promoter and 249 transferred it into WT plants. Of the four transgenic lines generated, we selected two 250 independent transgenic lines with strong OsGLK1 expression, and we named these lines 251 OEGLK1-1 and OEGLK1-2. 252 Compared to WT plants, OEGLK1-1 and OEGLK1-2 lines showed pale-white anthers 253 and reduced seed-setting rates (Fig. 5b–5d). We also examined the pollen viability of these 254 plants by I2-KI staining. The pollen viability of the OEGLK1-1 and OEGLK1-2 lines was 255 more than 30% lower than that of WT plants (Fig. 5b and 5e). These results indicate that 256 overexpressing OsGLK1 led to reduced pollen fertility. 257 To investigate the cytological features responsible for the reduced pollen fertility 258 caused by OsGLK1 overexpression, we examined semi-thin sections of WT, OEGLK1-1, 259 and OEGLK1-2 anthers at different developmental stages. Compared to normal WT anther 260 development (Fig. 5f–5j), in OEGLK1-1 and OEGLK1-2 plants, the tapetum abnormally 261 expanded inward at stage 9, the tapetum degradation process was prolonged, and defective 262 development occurred during later stages of anther development (Fig. 5k–5t). 263 Further observation by TEM showed that plastids in the tapetum did not develop properly 264 and the plastids did not differentiate into normal elaioplasts in the tapetum of OEGLK1-1 265 and OEGLK1-2 anthers (Fig. 6a–6i, Extended Data Fig. 7a–7i). We also detected the target 266 genes regulated by OsGLK1, and the results showed that the expression levels of these 267 genes increased in the OEGLK1-1 and OEGLK1-2 anthers (Extended Data Fig. 9a–9k). 268 To determine the effect of OsGLK1 overexpression on PCD in the tapetum, we 269 performed a TUNEL assay (Fig. 6j–6x). Strong positive TUNEL signals persisted in 270 OEGLK1-1 and OEGLK1-2 tapetum cells and did not decrease from stages 8–12. Therefore, 271 we obtained consistent results from the observations of semi-thin sections, TEM, and 272 TUNEL assays of OsPPR2-1-knockdown and OsGLK1-overexpression plants. These 273 results indicate that overexpressing OsGLK1 led to prolonged tapetum degeneration and 274 PCD. 275 276 OsPPR2-1 Regulates the Expression of OsGLK1 277 PPR proteins have been generally shown to function in RNA metabolism, RNA 278 cleavage, and RNA editing8, suggesting that the OsGLK1 mRNA could be a target of 279 OsPPR2-1. To further verify the regulatory effect of OsPPR2-1 on OsGLK1 expression, 280 we measured OsGLK1 expression in the anthers of plants overexpressing OsPPR2-1 281 (OEPPR2-1 and OEPPR2-2). OsGLK1 expression was significantly reduced in these 282 anthers compared to the control (Extended Data Fig. 7k). 283 Because the phenotypes of OEGLK1-1 plants were similar to those of OsPPR2-1 284 knockdown plants we reasoned that OsGLK1 is directly regulated by OsPPR2-1. To 285 investigate this hypothesis, we knocked down the expression level of OsGLK1 in pprR2-1 286 plants, and the progeny (#1 and #2) showed partially rescued pollen fertility with decreased 287 OsGLK1 expression compared to the WT (Extended Data Fig. 11). These results suggest 288 that OsPPR2-1 and OsGLK1 interact and that OsGLK1 is regulated by OsPPR2-1. 289 To investigate the binding of OsPPR2-1 with OsGLK1 mRNA, we performed RNA 290 immunoprecipitation (RIP) assays and an RNA-electrophoretic mobility shift assay (RNA- 291 EMSA). For RIP, after fixing anther tissue to stabilize the RNA–protein complexes and 292 grinding the tissue to release the nucleic acids, we immunoprecipitated the samples with 293 anti-OsPPR2-1 antibody. We purified the immunoprecipitated RNA samples from these 294 complexes and subjected them to qRT-PCR to detect the enrichment of different fragments 295 of OsGLK1 mRNA. Three fragments of OsGLK1 mRNA (G1, G2, and G4) were enriched 296 by OsPPR2-1, suggesting that OsPPR2-1 binds to OsGLK1 mRNA. 297 OsGLK2 is a homolog of OsGLK1. These genes share a highly conserved region, 298 which is designated T123. The nucleotide sequence alignment of OsGLK1 and OsGLK2 299 highlighting the conserved T1 region is shown in Extended Data Fig. 10. We then used 300 RIP to test whether OsPPR2-1 could bind to the T1 regions of the OsGLK1 mRNA, using 301 OsGLK2 as a negative control. The enrichment of the T1 regions was relatively low (Fig. 302 7a). RNA-EMSA confirmed this binding, showing that OsPPR2-1 could bind to the 303 OsGLK1 mRNA and did not bind to the OsGLK2 mRNA (Fig. 7b). 304 Since OsGLK1 transcripts were significantly higher in plants with downregulated 305 OsPPR2-1 expression, and when OsPPR2-1 was highly expressed OsGLK1 expression 306 was downregulated, this indicated that OsPPR2-1 negatively regulated the expression of 307 OsGLK1. To further analyze the effect of OsPPR2-1 on OsGLK1, we performed in vitro 308 cleavage assays. We used in vitro transcription of the full-length OsGLK1 coding region 309 and several short fragments that could bind to the RIP assay. Using the OsACTIN full- 310 length coding region or the homologous T1 fragment, Glk2-2 fragment of the OsGLK2 311 mRNA as negative control RNA, we expressed the full-length and incomplete OsPPR2-1 312 recombinant protein for the in vitro RNA cleavage reaction at the same time. We repeated 313 the experiments several times, and the result showed that the recombinant OsPPR2-1 could 314 cleave the OsGLK1 RNA directly, but not the OsACTIN RNA or the homologous T1 315 fragment and Glk2-2 fragment of the OsGLK2 mRNA (Fig. 7c and 7d). These results 316 demonstrate that OsPPR2-1 specifically binds to OsGLK1 RNA and degrades OsGLK1 317 mRNA directly. 318 The above results suggest that OsPPR2-1 interacts with and degrades OsGLK1 319 mRNA, thus affecting plastid development in the tapetum and regulating tapetal 320 degradation and PCD, thereby affecting pollen development (Fig. 7e). 321 322 DISCUSSION 323 Plastids Play an Important Role in Tapetum Degradation 324 Normal tapetum development and degradation are crucial for successful male 325 reproductive development in rice. PCD must begin on time to ensure successful tapetum 326 degradation, since this process supplies the nutrients needed for microspore release and 327 provides proteins, , and polysaccharides for pollen maturation. Advancing or 328 delaying the PCD process in the tapetum will lead to pollen abortion24. The regulation of 329 tapetum degradation and PCD has been widely studied25,26. Two kinds of factors regulate 330 tapetum degradation and PCD: reactive oxygen species (ROS)27-29 and transcription 331 factors30. 332 Plastids are unique and have specific patterns of development in tapetal 333 cells. A few reports have shown that plastid development-related genes regulate tapetum 334 PCD31. For example, Brassica napus BnaC.Tic40 is essential for tapetal development and 335 PCD32. In addition, OsCER1, a protein that affects plastid differentiation, causes delayed 336 tapetum PCD and altered pollen fertility33. These results highlight a link between plastid 337 development, tapetum development, and PCD. 338 In a crucial step for tapetum degradation, tapetum plastids are converted into 339 elaioplasts. Notably, aberrant tapetum plastid development in plants led to tapetum altered 340 nutrient accumulation33-34. In the current study, plants with downregulated OsPPR2-1 341 expression exhibited vacuolated proplastids and plastids that failed to differentiate into 342 normal elaioplasts in the tapetum (Fig. 3). In these plants, the supply of nutrients to the 343 microspores was disturbed and tapetal degradation was prolonged. OsGLK1 is a key 344 regulator of plastid development. Plants overexpressing OsGLK1 exhibited abnormal 345 swelling of the tapetum, and the tapetum degradation process continued to stage 12, which 346 in turn led to prolonged tapetal PCD. Our results show that normal development of the 347 elaioplasts correlates closely with OsPPR2-1 expression and plays an important role in the 348 degradation of the tapetum, which is essential for anther development in rice. 349 350 OsPPR2-1 Regulates OsGLK1 Expression to Mediate Tapetum Development 351 To date, more than 400 PPR proteins have been identified in rice, Arabidopsis, and 352 other model plants2,35. Most functional PPR proteins are localized to mitochondria and 353 chloroplasts, and a few are present in the nucleus36. As trans-acting factors, PPRs are 354 mainly involved in the post-transcriptional modification of RNA, which influences the 355 expression of genes related to plant growth and development by RNA processing, splicing, 356 and editing37-39. PPR proteins play important roles in the regulation of seed development40, 357 restoring male fertility13, the formation of chloroplasts and leaf growth41, and root growth42. 358 In the current study, we characterized the cytosol-localized PPR protein OsPPR2-1, which 359 belongs to the P subfamily of PPR proteins (Fig. 1c). Our results demonstrate that OsPPR2- 360 1 binds to OsGLK1 mRNA and negatively regulates OsGLK1 expression to control plastid 361 development and tapetal PCD, thereby affecting pollen development in rice. 362 OsGLK1 expression was significantly upregulated in the tapetum of OsPPR2-1- 363 knockdown plants compared to the WT (Fig. 5a). By contrast, the expression level of 364 OsGLK2, a homolog of OsGLK1, was almost unchanged in these plants compared to the 365 WT (Extended Data Fig. 7j). RIP analysis and RNA-EMSA indicated that OsPPR2-1 366 directly binds to OsGLK1 but not to OsGLK2, suggesting that OsPPR2-1 directly regulates 367 OsGLK1 expression. The in vitro cleavage assay demonstrated that OsGLK1 mRNA could 368 be cleaved by OsPPR2-1 directly (Fig. 7c). Based on these results, we hypothesize that 369 OsPPR2-1 functions as the RNase to regulate the expression of OsGLK1. 370 OsGLK1 controls plastid development in non-photosynthetic tissues20,21,43,44. In 371 Arabidopsis, GLK1 not only controls plastid development, but it also plays key roles in 372 plastid maintenance. The upregulation of GLK1 delays leaf senescence45. In the current 373 study, OsGLK1 expression fluctuated during anther development in WT plants, with higher 374 mRNA levels in stage 5, 6, and 10 anthers and lower levels in stage 12 anthers. By contrast, 375 the mRNA level of OsGLK1 was significantly higher in OsPPR2-1-knockdown plants 376 during almost all stages of anther development, with no significant differences among the 377 stages (Fig. 5a). Overexpressing OsGLK1 in WT plants also led to abnormal plastid 378 development and tapetum degradation. Plastids differentiate into elaioplasts during 379 tapetum development, a process required for the phase transition46,47. Our findings suggest 380 that the fluctuating expression of OsGLK1 is required for the differentiation of plastids into 381 elaioplasts. In addition, the data suggest that OsPPR2-1 is responsible for removing 382 OsGLK1 mRNA during a specific stage of anther development to ensure the normal 383 development of elaioplasts, thus regulating pollen development in rice. 384 In conclusion, we report that normal expression of OsPPR2-1 is necessary for tapetal 385 PCD and pollen development. Furthermore, our results demonstrate that OsGLK1 386 expression regulated by OsPPR2-1 mediates tapetum development. Our characterization 387 of OsPPR2-1 expression and confirmation of the relationship between OsPPR2-1 and 388 OsGLK1 provides valuable insight into tapetal PCD and pollen development and 389 emphasizes the importance of plastid development in various processes within plants. 390 391 MATERIALS AND METHODS 392 393 Plant Materials and Growth Conditions 394 All transgenic and WT rice plants (Oryza sativa ssp. japonica cv. Zhonghua11, ZH11) 395 used in this study were grown in the paddy field at South China Agricultural University, 396 Guangzhou, China. The transgenic lines included ppr2R-1, ppr2R-2, Casppr2-1, Casppr2- 397 2, OEGLK1-1, OEGLK1-2, OEPPR2-1, and OEPPR2-2. 398 399 Phenotypic Characterization of Transgenic Plants 400 For phenotypic characterization, the plants were photographed with a digital camera 401 (Canon 750D). Anthers from different developmental stages were collected and confirmed 402 by examining semi-thin sections. Transmission electron microscopy (TEM) was performed 403 as described as by Li et al48. Plastid number was analyzed by counting all plastids in each 404 photograph obtained by TEM. Statistical analysis of each sample was performed by the 405 Student’s t-test. Anthers from different developmental stages, as defined by Zhang et al, 406 were collected based on spikelet length and lemma/palea morphology19. Flowers and 407 anthers were photographed under a stereoscopic microscope (Olympus SZX10) to examine 408 their morphology. Pollen viability was analyzed by 1% I2-KI staining, followed by 409 photography under a Leica DNRXA microscope. 410 411 Vector Construction and Plant Transformation 412 The coding sequence of OsPPR2-1 was amplified from full-length cDNA from rice cv. 413 ZH11 by reverse-transcription PCR (RT-PCR). To construct the RNAi vector for OsPPR2- 414 1, an ~300-bp fragment of the cDNA sequence driven by the Ubiquitin promoter was 415 inserted into the pYLRNAi vector49. To generate the CRISPR/Cas9 vector, the OsPPR2-1 416 and OsGLK1 target sequences were constructed using pYLgRNA-OsU3 and pYLgRNA- 417 OsU6, as previously described50. To construct the OsGLK1-overexpression vector, a 1368 418 bp fragment of the OsGLK1 cDNA sequence driven by the maize Ubiquitin promoter was 419 inserted into the pYLox vector51. The WT cDNA sequence was used as a template for PCR 420 amplification. To generate the p35S::-OsPPR2-1-GFP vectors, full-length OsPPR2-1 421 cDNA was amplified and fused with the N-terminus of GFP in the pUC18 vector under the 422 control of the Cauliflower mosaic virus (CaMV) 35S promoter52. For the Ubi::FLAG- 423 OsPPR2-1 construct, full-length OsPPR2-1 cDNA was amplified and fused with the C- 424 terminus of FLAG in the pYLox vector driven by the Ubiquitin promoter51. For knockdown 425 of OsGLK1 expression in the pprR2-1 background, we knockdown the OsGLK1 in the 426 pprR2-1 RNAi line and screened the progeny lines for decreased expression of OsPPR2-1 427 and OsGLK1. We selected two lines, named #1 and #2, for further analysis. 428 All constructs were confirmed by sequencing, introduced into Agrobacterium 429 tumefaciens EHA105 cells, and transformed into ZH11 by Agrobacterium-mediated 430 transformation53. All primers used for vector construction are listed in Supplemental Table 431 1. 432 433 Phylogenetic Analysis and Sequence Alignment 434 Phylogenetic analysis of PPR2-1 and the other PPR proteins from various eukaryotes 435 was performed using the maximum likelihood (ML) method. The best-fit models of 436 evolution for the amino acid were selected following the Akaike Information Criterion 437 (AIC) with Prot Test server 2.454. Phylogenetic analyses of rice, maize (Zea mays), soybean 438 (Glycine max), and Arabidopsis PPR proteins were subsequently performed using the ML 439 method with RaxML 8.1.5 with 1000 bootstrap replicates with the WAG+I+G+F model55. 440 The phylogenetic tree was drawn using MEGA version X software. The accession numbers 441 of the proteins used for phylogenetic analysis are listed in Supplementary Table 2 and 442 Supplementary Table 3. 443 The nucleotide sequence alignment of OsGLK1 and OsGLK2 was analyzed by 444 MAFFT version 7 (Multiple alignment program for amino acid or nucleotide sequences, 445 http://mafft.cbrc.jp/alignment/software/). 446 447 Expression Analysis via qRT-PCR 448 Total RNA was isolated from rice anthers using TRIzol reagent (Invitrogen, USA). 449 DNase I-treated total RNA (1.0 µg) was used for reverse transcription with HiScript II Q 450 RT SuperMix for qPCR reagent (Vazyme, Nanjing). Quantitative PCR was performed as 451 previously described56. qRT-PCR analyses were performed with three repeats per sample 452 using RealStar Green Fast Mixture (GenStar, Beijing) with a qTOWER3G Real-Time PCR 453 Detection System (Analytikjena, Germany). Data were normalized to the expression of the 454 rice ACTIN1 gene (OsACTIN1). Primers used for qRT-PCR analysis are listed in 455 Supplementary Table 1. 456 457 In situ Hybridization 458 Specific regions of OsPPR2-1 were amplified using the corresponding primers 459 (Supplementary Table 1) and transcribed in vitro as probes using a DIG RNA Labeling kit 460 (Roche, Switzerland). Anthers at different developmental stages were isolated from fresh 461 young panicles, immediately fixed, embedded in paraffin (Sigma-Aldrich, USA), and 462 sectioned to a thickness of 5–7 µm. Hybridization and immunological detection were 463 performed as previously described57. Briefly, the sections were placed on slides in 464 hybridization buffer (40 µL per slide) containing the probes, covered with coverslips, and 465 incubated overnight at 45°C. Immunological detection of the hybridized probes was 466 performed using a DIG Nucleic Acid Detection kit (Roche) according to the 467 manufacturer’s protocol. 468 469 Subcellular Localization 470 For transient expression, protoplasts were isolated from the leaf sheaths of rice plants 471 as described previously58. Briefly, for protoplast transformation, 10 µL of plasmid carrying 472 OsPPR2-1 with GFP fragments, 100 µL of protoplasts, and 110 µL of PEG solution (40% 473 PEG4000, 0.3 M mannitol, and 0.1 M CaCl2) were mixed gently and incubated for 15 min. 474 Following transformation, the cells were washed with W5 solution and resuspended in WI 475 solution (4 mM MES, pH 5.7, 0.5 M mannitol, and 20 mM KCl). The cells were incubated 476 for 12–16 h after transformation. GFP signals were observed under a Carl Zeiss LSM510 477 Meta confocal microscope28. The excitation and emission wavelengths for chloroplasts 478 were 488 and 545 nm and the excitation and emission wavelengths for mitochondria were 479 579 and 599 nm, the excitation and emission wavelengths for the nucleus were 561 and 480 580 nm, respectively29. 481 The immunoblot membrane was successively incubated with OsPPR2-1-specific 482 antibody (OsPPR2-1-specific rabbit polyclonal antibody generated by GenScript, Nanjing, 483 China, 1:2,000 dilution) and secondary antibody goat anti-rabbit IgG HRP (Boster 484 Biological Technology Co. Ltd., BA1054, 1:10,000 dilution). The nuclear proteins and 485 cytosolic proteins were isolated by the Bestbio plant nuclear and cytosolic proteins 486 extraction kit (Bestbio, Beijing, #BB3169). The mitochondrial and chloroplast proteins 487 were isolated by the plant mitochondrial and chloroplast proteins extraction kit (Biorab, 488 Beijing, #HR0145, #HR0159), respectively. 489 The quality of the subcellular fractionations was verified by western blot experiments 490 with different organelle-specific commercial antibodies: Anti-Histone H3 Antibody 491 (Merck Millipore) was selected as the nuclear protein control, TIC40 antibody (PhytoAB 492 Inc. PHY1248S, 1:2,000 dilution) was selected as the chloroplast protein control, the COX 493 IV (Bioss, Beijing, China, bsm-33037M, 1:2,000 dilution) was selected as mitochondrial 494 protein control. 495 496 TUNEL Assay 497 The TUNEL assay was performed using a TUNEL kit (DeadEnd Fluorometric 498 TUNEL system; Promega) according to the manufacturer’s instructions. The anther 499 samples were collected at different developmental stages. The samples were analyzed 500 under a confocal laser-scanning microscope (LSM510, Zeiss). The overlays of fluorescein 501 and propidium iodide signals were considered to be TUNEL-positive signals. All 502 photographs were taken using the same settings. 503 504 Transcriptome Analysis 505 RNA samples used for transcriptome analysis were prepared from stage 8, stage 9, and 506 stage 10 anthers from WT (ZH11) and ppr2R-1 (OsPPR2-1 RNAi) plants grown under 507 normal field conditions. RNA sequencing was performed by Beijing Genomics Institution 508 (BGI). The RNAseq data were analyzed using Gene Chip Operating software (GCOS 1.4). 509 Following cluster generation and library preparation, reads were generated on the 510 BGIseq500 platform (BGI-Shenzhen, China). Differential expression analysis between 511 WT and ppr2R-1 plants was performed using the output from Significant Analysis of 512 EBseq59. A false discovery rate of < 0.05 and fold change R1.7 were used as the thresholds 513 for significantly differential expression. Gene functions were annotated using the NCBI 514 (ncbi.nlm.nih.gov), Pfam (https://pfam.xfam.org/), and Swiss-Prot (https://www. 515 uniprot.org/contact) databases. The differentially expressed genes in ppr2R-1 versus WT 516 plants are listed in Supplementary Table 4 to Supplementary Table 7. 517 518 RNA IP-qPCR Analysis 519 RNA IP (RIP) assays were performed as described previously60 with minor 520 modifications. Briefly, approximately 2 g of stage 11 anther tissue from OEPPR2-1 plants 521 was fixed in 1% formaldehyde under a vacuum. The anther samples were washed with cold 522 sterile water and ground to a powder in liquid nitrogen. The RNA–protein complexes were 523 immunoprecipitated using an anti-PPR2-1-specific rabbit polyclonal antibody generated 524 by GenScript (Nanjing, China). Normal rabbit immunoglobulin G (IgG, #2729, Cell 525 Signaling Technology) antibody was used as a negative control. Unrelated RNA sequences, 526 including OsACTIN1 and the G1 to G4 region of the OsGLK1 coding region, were used as 527 internal controls. The T1 region of OsGLK2 is the region with the strongest sequence 528 similarity to OsGLK1. Following RIP, the RNA was extracted from the precipitated 529 complexes, and qRT-PCR was performed with gene-specific primers listed in 530 Supplementary Table 1. 531 532 RNA-EMSA 533 A full-length PPR2-1 protein containing the PPR motif fused with the His-tag was 534 obtained by the baculovirus expression system using the method described in He et 535 al61(2020). The recombinant protein was purified using Nuvia IMAC Ni-Charged resin 536 (Bio-Rad, Cat # 780-0800). A biotinylated probe and a non-biotinylated probe were 537 generated by amplifying the G2 and G4 regions of the OsGLK1 mRNA, which were used 538 in the RIP assay, and the T1 region of the OsGLK1 mRNA, which has the strongest 539 similarity to OsGLK2. The Glk2-2 region of the OsGLK2 mRNA was used as the negative 540 control. These regions were labeled using synthetic biotinylated primers and non-labeled 541 primers (Thermo Fisher Scientific, 20160). Reactions were carried out in accordance with 542 the manufacturer’s protocol for LightShift Chemiluminescent RNA EMSA (REMSA) kit 543 (Thermo Fisher Scientific, 20158). Samples were run on 6% native polyacrylamide gel 544 electrophoresis (PAGE) and transferred onto 0.45 µm nylon membranes (GE Healthcare) 545 for chemiluminescence imaging detection with X-ray film. To determine the specificity of 546 binding, several experiments were conducted with unlabeled sequence in excess. The 547 primers are listed in Supplementary Table 1. 548 549 RNA in vitro cleavage 550 The full-length OsPPR2-1 and its incomplete coding sequences were constructed into 551 prokaryotic expression vector pET30a. The recombinant proteins were purified using 552 Nuvia IMAC Ni-Charged resin (Bio-Rad, Cat # 780-0800) to gain the full-length PPR2-1 553 protein (OsPPR2-1) and its incomplete PPR2-1 protein (OsPPR2-1∆). The purity and 554 concentration of purified proteins were determined by SDS-PAGE. The OsGLK1, the 555 OsACTIN mRNA, and T1 RNA transcription templates were amplified by PCR with the 556 primer containing the T7 promoter and purified for in vitro transcription. The reaction 557 system was configured with the T7 RNA Polymerase (Promega, USA, P2075), in vitro 558 transcription was conducted at 37°C for 1–2 h, and the template DNA was removed by 559 DNase I (TAKARA, Japan, Cat # 2270A) at 37°C for 15 min. The products were purified 560 and recovered with the RNA probe purification kit (OMEGA, R6249), and the RNA probes 561 were tested by Urea-PAGE. The reaction system of the in vitro cleavage experiment was 562 performed with a mixture containing 1 µL 10 X cyto buffer (400 mM Tris-HCl, pH 8.4, 20 563 mM MgCl2, 20 mM DTT, 20 µM KCl), and 1 µL RiboLock RNase Inhibitor (40 U/µL, 564 Thermo Fisher Scientific, EO0381), 500 ng ~1 ug in vitro transcribed RNA, and 1 µg full- 565 length PPR2-1 protein or incomplete PPR2-1 protein in a total volume of 10 µL. The 566 enzyme digestions were incubated at 30°C for 15–20 minutes, then tested by denaturing 567 PAGE at 150V for 60 min. The gel was stained by the SYBR Gold Nucleic Acid Gel Stain 568 (Thermo Fisher Scientific, S11494) and photographed in the Gel Image Detection System 569 (Analytikjena, Germany). The primers are listed in Supplementary Table 1. 570 571 572 AUTHOR CONTRIBUTIONS 573 C.Z. directed the project. S.Z. performed the experiments with the help of J.D., J.L., and 574 J.L. All authors discussed and interpreted the results. S.Z. and C.Z. wrote the manuscript. 575 576 577 ACKNOWLEDGEMENTS 578 We thank the Instrumental Analysis & Research Center of South China Agricultural 579 University for TEM analysis; Prof. Jingchen Sun, College of Animal Science, South China 580 Agricultural University for the assistance to express the OsPPR2-1 protein via an insect 581 virus vector; and Science Corporation of Gene, Guangzhou for advice on transcriptome 582 analysis. This research was supported by grants from the National Natural Science 583 Foundation of China (Grants 31921004, 31871231, 31371225), and the China Postdoctoral 584 Science Foundation (2019M662941). The authors declare no competing financial interests. 585 586 REFERENCES 587 588 1. Pusnik, M. Small, I. Read, L. K. Fabbro, T. & Schneider, A. Pentatricopeptide repeat 589 proteins in Trypanosoma brucei function in mitochondrial . Mol Cell Biol. 590 27, 6876-6888(2007). 591 2. Lurin, C. et al. 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776 777 Fig. 1 Expression analysis of OsPPR2-1. 778 a, qRT-PCR analysis of OsPPR2-1 expression. BStage 6, stage 1 to stage 6. Data 779 are shown as means ± SD (n=3). 780 b, RNA in situ hybridization analysis of OsPPR2-1 in WT anthers from stage 8 to 781 stage 11. Ta, tapetum. Bars = 25 µm. 782 c, Subcellular localization of OsPPR2-1 in rice leaf sheath protoplasts. 783 p35S::Tic40-eGFP, p35S::COX11-mcherry, and p35S::NLS-mKATE were used as 784 the chloroplast, mitochondrial, and nuclear markers, respectively. Bar = 10 µm. 785 d, OsPPR2-1 was detected in different cell compartment protein extracts using 786 western blot analysis. The COX IV, Tic 40, and H3 antibodies were used as the 787 indicators of chloroplast, mitochondria, and nuclear protein controls, respectively. 788 CP, cytosolic protein; Chl, chloroplast protein; Mit, mitochondria protein; NP, 789 nuclear protein; Total, Total protein. 790 791 Figure 2. Phenotypic comparison and anther development of WT, ppr2R-1, 792 and Casppr2-1 plants. 793 a, Comparison of WT, ppr2R-1, and Casppr2-1 spikelets after removing the lemma 794 and palea. b, Staining of WT, ppr2R-1, and Casppr2-1 pollen grains with I2-KI dye. 795 c, Comparison of the seed-setting rates of WT, ppr2R-1, and Casppr2-1 plants. 796 Data are means ± SD (n= 30); **, P < 0.01 according to Student’s t-test. 797 d, Comparison of pollen viability in WT, ppr2R-1, and Casppr2-1 plants. The data 798 represent the mean of ten independent experiments ± SE; **, P < 0.01 according 799 to Student’s t-test. e to s, Bright-field photomicrographs of transverse sections 800 showing anther and microspore development in WT (e–i), ppr2R-1 (j–n), and 801 Casppr2-1 (o–s) plants from stage 8 to stage 12. Ta, tapetum. Bars = 2 mm in (a), 802 50 µm in (b), 25 μm in (e) to (s). 803 804 805 Fig. 3. High magnification views of TEM images of the tapetum. 806 a to i, High magnification views of TEM images showing tapetal cells and plastids 807 in WT (a–c), ppr2R-1 (d–f), and Casppr2-1 (g–i) plants from stage 10 to stage 12. 808 Red arrowheads indicate plastids. Ela, elaioplast; Msp, microspore parietal cell; Pl, 809 proplastid; Pe, ; Ub, Ubisch body; V, ; T, tapetum. Bars = 1 µm 810 in (a) to (i). 811 812 813 814 Fig. 4. TUNEL assays of anthers to detect DNA fragmentation due to PCD. 815 816 a to o, DNA fragment signals in WT (a–e), ppr2R-1 (f–j), and Casppr2-1 (k–o) 817 anthers from stage 8 to stage 12. The red fluorescence is from anthers stained 818 with propidium iodide (PI), as viewed under a confocal laser-scanning microscope; 819 images are overlays of green fluorescence from the TUNEL assay with PI staining. 820 Mp, mature pollen; Ta, tapetal cell; Bars = 20 μm. 821 822 823 Fig. 5. Phenotypic characterization and transverse sections of OEGLK1-1 824 and OEGLK1-2 anthers. 825 a, qRT-PCR analysis of OsGLK1 transcript levels in WT, ppr2R-1, and Casppr2-1 826 anthers from stage 9 to 12. Error bars indicate SD. Two biological replicates were 827 used; each reaction represents three technical repeats. Asterisks represent 828 significant differences determined by Student’s t-test at P < 0.001 (***), P < 0.01 829 (**), P < 0.05 (*). 830 b, Spikelets (top) and pollen grains (bottom) from WT, OEGLK1-1, and OEGLK1- 831 2 plants. Pollen grains were stained with I2-KI. Bars = 2 mm for spikelets and 50 832 μm for pollen grains. 833 c, Expression analysis of OsGLK1 in WT, OEGLK1-1, and OEGLK1-2 anthers at 834 stage 12. Error bars indicate SD. Each reaction represents three technical repeats. 835 ***, P < 0.001 according to Student’s t-test. 836 d, Comparison of the seed-setting rates of WT, OEGLK1-1, and OEGLK1-2 plants. 837 Data are means ± SD (n= 30), **, P < 0.01 according to Student’s t-test. 838 e, Comparison of the viability of mature WT, OEGLK1-1, and OEGLK1-2 pollen. 839 Data are means ± SD (n= 20), **, P < 0.01 according to Student’s t-test. 840 f to t, Comparison of transverse sections of anthers from WT (f–j), OEGLK1-1 (k– 841 o), and OEGLK1-2 plants (p–t). Ta, tapetal cell; Bars = 20 μm. 842 843 Fig. 6. Transmission electron microscopy and TUNEL assay of WT, OEGLK1- 844 1, and OEGLK1-2 anthers. 845 a to i, Higher magnification views of TEM images showing tapetal cells and plastids 846 of WT (a–c), OEGLK1-1 (d–f), and OEGLK1-2 (g–i) anthers from stage 10 to stage 847 12. Red arrowheads indicate plastids. E, epidermis; Ela, elaioplast; Mt, 848 ; Pl, plastid; Pe, peroxisome; Ub, Ubisch body; V, vacuole. Bars = 1 849 µm in (a) to (i). 850 j to x, DNA fragment signals in WT (j–n), OEGLK1-1 (o–s), and OEGLK1-2 (t–x) 851 anthers. The red fluorescence is from anthers stained with PI under a confocal 852 laser-scanning microscope; images are overlays of green fluorescence from the 853 TUNEL assay and PI staining. Ta, tapetal cell; Bars = 20 μm. 854 855 Fig. 7. OsPPR2-1 directly binds to OsGLK1 mRNA and promotes its 856 degradation; a proposed model of the role of OsPPR2-1-OsGLK1 in 857 regulating elaioplast development in anthers and PCD in the tapetum in rice. 858 a, The binding of OsPPR2-1 to OsGLK1 mRNA, as measured by RNA 859 immunoprecipitation (RIP) followed by qRT-PCR. The fold-enrichment values of 860 OsGLK1 mRNA determined by RIP-qPCR using stage 11 anthers from transgenic 861 plants overexpressing PR2-1 (OEPPR2-1) are shown. Negative control, anti-IgG 862 (normal rabbit IgG antibody). G1–G4, four different regions of OsGLK1 mRNA. T1, 863 The most similar sequence in the coding regions between OsGLK1 and OsGLK2. 864 Error bars indicate SD. Each reaction represents three technical replicates. 865 b, RNA-EMSA analysis for the binding of OsPPR2-1 to the OsGLK1 mRNA 866 fragments. The RNA fragments (G2, G4, T1) from the RIP-PCR analysis (Fig. 7b) 867 were end-labeled with biotin, and the unlabeled RNA fragments were used as a 868 cold competitor. The 20× and 50× contain 20– and 50–fold more unlabeled probes 869 than labeled probes, respectively. The Glk2-2 probe was used as a negative 870 control RNA fragment from the OsGLK2 mRNA. 871 c, Image of the OsPPR2-1 cleavage assay. The OsGLK1 RNA and OsACTIN RNA 872 were incubated with either recombinant OsPPR2-1 or the incomplete OsPPR2-1 873 (OsPPR2-1∆). OsACTIN RNA was used as a negative control. 874 d. The homologous T1 RNA fragment and the Glk2-2 fragment of the OsGLK2 875 mRNA from the RIP-PCR analysis and RNA-EMSA were incubated with either 876 recombinant OsPPR2-1 or the mutant OsPPR2-1 (OsPPR2-1∆). After the 877 reactions, the reaction mixtures were analyzed on a denaturing polyacrylamide gel. 878 Molecular weight marker sizes (M) are indicated in bases. 879 e, Proposed model of the role of OsPPR2-1–OsGLK1 in regulating plastid 880 development in the tapetum of rice anthers. According to this model, the 881 appropriate plastid differentiates into an elaioplast in the tapetum. OsGLK1 882 ensures that the tapetum is degraded at the proper time, which is directly regulated 883 by OsPPR2-1. E, epidermis; En, endothecium; T, tapetum. 884 Supplemental Information: a

Extended Data Fig. 1. Phylogenetic analysis of PPR proteins. a, Phylogenetic analysis of PPR proteins in various organisms. Os: Oryza sativa L; Gm: Glycine max; At: Arabidopsis thaliana; Zm: Zea mays. The protein accession numbers are listed in Supplementary Table 2. b, Phylogenetic analysis of most of the PPR proteins in rice. The phylogenetic trees were drawn using MEGA X software. The protein accession numbers are listed in Supplementary Table 3.

Extended Data Fig. 2. Phenotypic comparison of WT, ppr2R-2, and Casppr2- 2 plants. a, Comparison of WT, ppr2R-2, and Casppr2-2 spikelets after removing the lemma and palea. b, Staining of WT, ppr2R-2, and Casppr2-2 pollen grains with I2-KI dye. c, Comparison of the seed-setting rates of WT, ppr2R-2, and Casppr2-2 plants. Data are means ± SD (n= 30); Asterisks represent significant differences determined by Student’s t-test at **, P < 0.01. d, Comparison of pollen viability in WT, ppr2R-2, and Casppr2-2 plants. Data are means ± SD (n= 20); Asterisks represent significant differences determined by Student’s t-test at **, P < 0.01. e, WT, ppr2R-2, and Casppr2-2 panicles showing the seed-setting rates. f, qPCR analysis of OsPPR2-1 transcript levels in WT, ppr2R-1, ppr2R-2, Casppr2-1, and Casppr2-2 plants. Error bars indicate SD. Each reaction represents three technical repeats. ***, P < 0.001; **, P < 0.01 according to Student’s t-test. Bars = 2 mm in a, 50 µm in b, 200 μm in e.

885 Extended Data Fig. 3. Analysis of CRISPR/Cas9-generated T0 transgenic Casppr2-1 plants. a, Schematic of the OsPPR2-1 gene indicating the positions of the deletions in the Casppr2-1 mutants. b, The mutated sites in CRISPR/Cas9 T0 transgenic Casppr2-1 plants. The WT sequence is shown at the top with the protospacer adjacent motif (PAM) sequence and the target sequence highlighted in red. “Sub” indicates the substitute-mutation bases; “–” and “Del” indicate the deleted bases.

Extended Data Fig. 4. Anther development in WT, ppr2R-2, and Casppr2-2 plants. a to o, Bright-field photomicrographs of transverse sections showing anther and microspore development in WT (a–e), ppr2R-2 (f–j), and Casppr2-2 (k–o) anthers from stage 8 to stage 12. Ta, tapetum. Bars = 25 μm in (a) to (o).

Extended Data Fig. 5. TEM observation of tapetal cells in WT, ppr2R-2, and Casppr2-2 anthers. a to o, TEM images showing tapetal cells of WT (a, f, k), ppr2R-1 (b, g, l), ppr2R- 2 (c, h, m), Casppr2-1 (d, i, n), and Casppr2-2 (e, j, o) anthers from stage 10 to stage 12. p to x, High magnification views of TEM images showing tapetal cells and plastids of WT (p–r), ppr2R-2 (s–u), and Casppr2-2 (v–x) anthers from stage 10 to stage 12. Red arrows indicate plastids. Amy, ; Bp, bicellular pollen; Ela, elaioplast; En, endothecium; Mt, mitochondrion; Mp, mature pollen; Msp, microspore parietal cell; Pl, proplastid; Pe, peroxisome; Ub, Ubisch body; T, tapetum. Bars = 1 µm in (a) to (x).

Extended Data Fig. 6. Detection of DNA fragmentation in anthers. a to o, TUNEL assay showing DNA fragment signals in WT (a–e), ppr2R-2 (f–j), and Casppr2-2 (k–o) anthers from stage 8 to stage 12. The red fluorescence is from anthers stained with propidium iodide (PI) under a confocal laser-scanning microscope; images are overlays of green fluorescence from the TUNEL assay with PI staining. Ta, tapetal cell; Bars = 20 μm.

Extended Data Fig. 7. TEM showing tapetal cells of WT, OEGLK1-1, and OEGLK1-2 anthers, and expression analysis of OsGLK1 and OsGLK2 in WT, OEPPR2-1-, and OsPPR2-1-knockdown plants. a to i, TEM images showing tapetal cells of WT (a–c), OEGLK1-1 (d–f), and OEGLK1-2 (g–i) anthers from stage 10 to stage 12. Bp, bicellular pollen; En, endothecium; Mp, mature pollen; Msp, microspore parietal cell; T, tapetum. Bars = 1 µm in (a) to (i). j, qRT-PCR analysis of OsGLK2 expression in WT, ppr2R-1, and Casppr2-1 anthers at stage 9 to stage 12. Error bars indicate SD. Each reaction represents three technical repeats. k, qRT-PCR analysis of OsGLK1 expression in WT, OEPPR2-1, and OEPPR2-2 anthers at stage 11. Error bars indicate SD. Each reaction represents three technical repeats. Asterisks represent significant differences determined by Student’s t-test at ***, P < 0.001.

Extended Data Fig. 8. Higher magnification views and statistical analysis of the tapetal plastids of anthers at stage 11. a to g, High magnification views of TEM images showing tapetal cells and plastids of WT (a), ppr2R-1 (b), ppr2R-2 (c), Casppr2-1 (d), Casppr2-2 (e), OEGLK1-1 (f), and OEGLK1-2 (g) anthers at stage 11. Red arrows indicate plastids. Ela, elaioplast; Pl, plastid. Bars = 0.5 µm. h, Number of tapetal plastids in anthers at stage 11. Data are shown as means ± SD, (n = 20). Asterisks represent significant differences determined by Student’s t-test at **, P < 0.01.

Extended Data Fig. 9. Expression of the target genes of OsGLK1 during anther development in WT, OsPPR2-1-knockdown, and OEGLK1 plants. a to k, qRT-PCR analysis of several target genes, Os04g0690800 (a), Os05g0291700 (b), Os08g0104600 (c), Os04g0459500 (d), Os07g0562700 (e), Os09g0346500 (f), Os04g0678700 (g), Os02g0197600 (h), Os10g0567400 (I), Os05g0162800 (j), and Os08g0157600 (k), of OsGLK1 in WT, ppr2R-1, ppr2R-2, Casppr2-1, Casppr2-2, OEGLK1-1, and OEGLK1-2 anthers at stage 8 and stage 9. Error bars indicate SD. Each reaction represents three technical repeats. Asterisks represent significant differences determined by Student’s t-test at ***, P < 0.001; **, P < 0.01; *, P < 0.05.

Extended Data Fig. 10. Nucleotide sequence alignment of the OsGLK1 and OsGLK2 coding regions. The nucleotide sequence alignment between OsGLK1 and OsGLK2 cDNAs were drawn with MAFFT version 7. The G1 (between the brown arrows), G2 (black arrows), G3 (orange arrows), G4 (blue arrows), and T1 (red arrows) fragments were used in the RNA immunoprecipitation and RNA-EMSA from Fig. 7a and Fig. 7b.

Extended Data Fig. 11. Phenotypic comparison of WT and OsGLK1-PPR2-1- knockdown plants. a, Comparison of WT, #1, and #2 (plants with knockdown of OsGLK1 in the pprR2- 1 background) spikelets after removing the lemma and palea. b, Staining of WT, #1, and #2 pollen grains with I2-KI dye. c, Comparison of pollen viability in WT, #1, and #2 plants. The data represent the mean of ten independent experiments ± SE. d, Comparison of the seed-setting rates in WT, #1, and #2 plants. Data are means ± SD (n = 10), *, P < 0.05 according to Student’s t-test. e, qPCR analysis of OsPPR2-1 transcript levels in WT, #1, and #2 anthers. Error bars indicate SD. Each reaction represents three technical repeats. ***, P < 0.001 according to Student’s t-test. f, qPCR analysis of OsGLK1 transcript levels in WT, #1, and #2 anthers. Error bars indicate SD. Each reaction represents three technical repeats. *, P < 0.05 according to Student’s t-test. Bars = 2 mm in (a), 50 µm in (b). g, The OsGLK1 mutated sites in #1 and #2 plants. The WT sequence is shown at the top with the PAM sequence and the target sequence highlighted in red. “–” indicates deleted bases.

886 Supplementary Table 1. Primers used in this study Primer name Forward primer Reverse primer Objective

PPR2i AAAAAAGCTTAGGAGGAGATGCAG AAAAGGATCCTCATGTAGAGGATGG Transgene ATCATG TCTCG Construction

PPR2cas9-U3 GGCAGGTGGTTCCGGCGTGTCGA AAACTCGACACGCCGGAACCACC Transgene Construction PPR2cas9-U6b GGCGCTGAAGCCGGCGCGACGGT AAACACCGTCGCGCCGGCTTCAG Transgene Construction GLK1cas9-U3 GGCAGCACACCGACGTCGTCCGC AAACGCGGACGACGTCGGTGTGC Transgene Construction

GLK1cas9-U6b GCCGCGTGGACCGTGCCAACCAT AAACATGGTTGGCACGGTCCACG Transgene Construction GLK1-OE GATGATAAAAAGCTTACTATGCTTG GGATCCATAACGCGTACTTCCACACG Transgene CCGTGTCGCCGGC CTGGAGGAACGT Construction

PPR2-OE AAAAAAGCTTCTCCTCCACGCCGC AAAAACTAGTTTGAATGGATAAACC Transgene CGCAGCC GCTACGC Construction

GFP-PPR2 AAAAACTAGTAATCGAAACGTCGTT AAAAACTAGTGCTGTCGTCAGATGA Subcellular CTGATCG AGAAC Localization

PPR2Cas9test GCCGCCGCAGCCATGTCGCG GGCGACGGTGGCCTCCTCGG Target sequencing

qPPR2 AGGGTTGTGGAGGCCCGGAA CGCTCGGCATAGCGTCGTCA qRT-PCR

qGLK1 AAGAATCAGCAGGACGCACA AGGAACGTTTGCTACGCCTT qRT-PCR

Actin CACATTCCAGCAGATGTGGA GCGATAACAGCTCCTCTTGG qRT-PCR

qGLK2 TGCTCTGGGAGCACAAGCC CTCGCTGTCCCTCACCATG qRT-PCR

Os04g0459500 AAGGGTGACATCCCCACCTA CGAGGATCTTCACGAAGGGG qRT-PCR

Os02g0197600 TCAGGCTCTTCTCTCTGGGA TAGCTCAGGGACTGCTTGGA qRT-PCR

Os10g0567400 ACTGTGGCATCGCTGTCTTT CGGGTCATCGACATGGAACA qRT-PCR Os07g0562700 CCGAGCTCAAGGTCAAGGAG TGAACTTGGTGGCGTAGACC qRT-PCR

Os04g0690800 GCGTTCAAGAGCAGAACCAA GTTCTCCTTGGTGAACCCGAT qRT-PCR

Os05g0291700 ATGGGAGAAGCTCAGCACAC GGGCTTCACAACAGCTCTCT qRT-PCR

Os08g0104600 AGCAACAAGCTGGGAGACAG GAGTAAGGCAGGTCGATCCC qRT-PCR

Os09g0346500 CACCATGGCGCTCTCCTCCC GTGAGGTAGCTCGGCGGCTC qRT-PCR

Os05g0162800 CCTTGACGCCCTGATTCTGT TGGATTCTGAAGCCGCCATT qRT-PCR

Os08g0157600 CCAATGGTGCAGGGAAGGAT TGCAGCAGACCAATCCACAT qRT-PCR

Os04g0678700 AAGGCGTACAAGGACAGCAA GTCTCCTCGTGGAATCTCCG qRT-PCR

PPR2-Sense AATTAATACGACTCACTATAGGGCTT AAGCTGTCCATCAGGGCATC in situ hybridization GCCATTATGACACGCCG

PPR2antiSense CTTGCCATTATGACACGCCG AATTAATACGACTCACTATAGGGAAG in situ hybridization CTGTCCATCAGGGCATC

G1 GGAGTTGTCCCGTGTCCAAT TGTGCGTCCTGCTGATTCTT RIP-qPCR/EMSA

G2 AGAGCATCGACGCAGCTATC CTGTCCACTGAAGGTGGCTT RIP-qPCR/EMSA

G3 GGTTGAGGCCAAGTCTTCGT TCCAGTCCACCTTCGCTTTC RIP-qPCR/EMSA

G4 GCTTTCTGGCACCACCCTTA ATTGGACACGGGACAACTCC RIP-qPCR/EMSA

T1 GATAGACAAGGCCGTGCCGT GGCGTCCTTCCTTGGCCCGC RIP-qPCR/EMSA

OsGLK1 ATGCTTGCCGTGTCGCCGGC AATTAATACGACTCACTATAGGGTCC In vitro cleaveage ACACGCTGGAGGAACGT OsActin ATGGCTGACGCAGAGGACAT AATTAATACGACTCACTATAGGGGAA In vitro cleaveage GCACTTCCTGTGGACG

Supplementary Table 2. Protein accession numbers used in the phylogenetic analysis in Extended Data Fig. 1a Arabidopsis Protein Oryza sativa L thaliana Glycine max Zea mays Als3 XP_015615836.1 NP_201558.3 XP_003539000.1 XP_008656745.1 MPR25 XP_015635686.1 NP_188854.1 KRH14042.1 ONM14274.1 OGR1 XP_015619829.1 NP_174678.2 KRH23904.1 NP_001169361.1 OTP51 XP_015626611.1 NP_565382.4 XP_003528385.1 NP_001343984.1 PPR1 XP_015611720.1 NP_177599.1 XP_003545972.2 NP_001168043.1 PGL12 XP_015620120.1 NP_177302.1 XP_003534476.1 NP_001141997.1 PGL1 XP_015618645.1 NP_193307.2 XP_003529581.1 NP_001146425.2 PPR2-1 XP_015626415.1 NP_181260.1 XP_003532699.1 AQK67735.1 PPR6 XP_015639788.1 NP_173004.1 XP_003529817.2 XP_008650050.1 PPS1 ABA99524.2 NP_193101.2 XP_006574752.1 ONM17712.1 SLA4 BAC83621.1 NP_196000.2 XP_003520676.1 XP_008670964.1 SMK1 XP_015616471.1 NP_173449.1 XP_003523930.1 XP_020404227.1 SPED1-D XP_015643803.1 NP_174428.1 XP_003520007.1 AQK82374.1 TCD10 XP_015614726.1 NP_194913.1 XP_003527773.1 XP_008660079.1 V4 XP_015634085.1 CAC05458.1 XP_003533047.1 XP_008669276.2 WSL4 XP_015627373.1 NP_172461.1 XP_003523769.1 NP_001141010.1 WSL5 XP_015635904.1 OAO94867.1 XP_003556878.1 AQK47407.1

Supplementary Table 3. Protein accession numbers used in the phylogenetic analysis in Extended Data Fig. 1b Gene ID Protein Accession ID Os01g0263400 XP_015621386.1 Os01g0355100 XP_015627306.1 Os01g0506100 XP_015621124.1 Os01g0589900 BAS72941.1 Os01g0611900 XP_015622180.1 Os01g0783100 XP_015620999.1 Os01g0785700 XP_015622164.1 Os01g0793200 XP_015635951.1 Os01g0839400 XP_015641633.1 Os01g0852900 XP_015616080.1 Os02g0167200 XP_015625552.1 Os02g0170000 XP_015623551.1 Os02g0191200 XP_015625578.1 Os02g0226900 BAD25660.1 Os02g0266200 XP_015626980.1 Os02g0470000 XP_015624639.1 Os02g0582300 XP_015626384.1 Os02g0644600 XP_015625390.1 Os02g0759500 XP_015623291.1 Os02g0769900 XP_015625798.1 Os03g0165100 BAS82461.1 Os03g0201400 XP_015632829.1 Os03g0314400 XP_015628579.1 Os03g0317100 XP_015628601.1 Os03g0363700 XP_015632338.1 Os03g0441400 XP_015628135.1 Os03g0746400 BAF13163.1 Os03g0807400 EEC76380.1 Os03g0844000 AAS07350.1 Os03g0852700 XP_015632922.1 Os04g0118700 XP_015634033.1 Os04g0218100 BAF14173.1 Os04g0221300 BAS88174.1 Os04g0349600 BAS88716.1 Os04g0463800 CAE76014.1 Os04g0477200 XP_015634992.1 Os04g0488200 XP_015633384.1 Os04g0488500 BAF15066.1 Os04g0514500 XP_015636795.1 Os04g0643700 XP_015635889.1 Os05g0112900 XP_015640061.1 Os05g0275000 XP_015639397.1 Os05g0275100 EEC78894.1 Os05g0294600 XP_015637547.1 Os05g0313600 EEE63208.1 Os05g0370000 XP_015640363.1 Os05g0439300 XP_015638217.1 Os05g0534900 XP_015640809.1 Os05g0548600 XP_015637688.1 Os05g0572900 XP_015638111.1 Os06g0112000 XP_015643851.1 Os06g0114366 XP_015641390.1 Os06g0125300 XP_015644126.1 Os06g0228900 BAS96892.1 Os06g0231400 XP_015640992.1 Os06g0249500 BAD45723.1 Os06g0499301 BAD45366.1 Os06g0690900 XP_015643390.1 Os06g0694300 BAS99267.1 Os06g0710800 XP_015644397.1 Os07g0101200 BAD31827.1 Os07g0179000 XP_015647522.1 Os07g0213300 BAT00611.1 Os07g0249100 XP_015647722.1 Os07g0300200 XP_015647503.1 Os07g0513200 XP_015646847.1 Os07g0578800 BAT02318.1 Os07g0621100 XP_015646617.1 Os07g0670000 BAT03146.1 Os07g0671200 XP_015647917.1 Os08g0107800 BAD09394.1 Os08g0131000 XP_015648489.1 Os08g0153600 BAT03863.1 Os08g0191900 XP_015648115.1 Os08g0248400 XP_015651079.1 Os08g0300700 XP_015650984.1 Os08g0340900 XP_015648262.1 Os08g0402600 XP_015648665.1 Os08g0481000 XP_015649802.1 Os08g0494350 EEC83802.1 Os09g0110200 XP_015612107.1 Os09g0251500 XP_015610800.1 Os09g0327200 XP_015651189.1 Os09g0407800 BAD36246.1 Os09g0411600 XP_015651329.1 Os09g0412900 XP_015610904.1 Os09g0417500 XP_015610781.1 Os09g0423300 XP_015651316.1 Os09g0542800 XP_015651356.1 Os09g0555400 XP_015611699.1 Os10g0116000 BAT09660.1 Os10g0358700 XP_015614766.1 Os10g0368902 BAT10547.1 Os10g0476900 BAT11301.1 Os10g0477200 XP_015614114.1 Os10g0484300 XP_015614400.1 Os10g0488900 EAY79002.1 Os10g0497300 BAD08215.1 Os10g0540100 XP_015613796.1 Os10g0558600 AAK55452.1 Os11g0103000 EAY81947.1 Os11g0114800 XP_015617192.1 Os11g0275400 XP_015617492.1 Os11g0357094 XP_015629550.1 Os11g0433101 XP_015615676.1 Os11g0482400 XP_015615477.1 Os11g0587100 XP_015615245.1 Os11g0607100 XP_015617145.1 Os11g0661000 XP_015617498.1 Os11g0680200 BAT15245.1 Os12g0109300 ABA96198.2 Os12g0114400 ABA96280.1 Os12g0130900 BAF29091.1 Os12g0152600 XP_015619721.1 Os12g0156900 XP_015618146.1 Os12g0181900 XP_015618676.1 Os12g0407900 XP_015620458.1 Os12g0490100 XP_015618777.1 Os12g0557800 BAF30026.1 Os12g0638900 XP_015620082.1

Supplementary Table 4. The upregulated genes in the transcriptome analysis of ppr2R-1 anthers Stage 8 Stage 9 Stage 10 Gene ID log2 Fold Change log2 Fold Change log2 Fold Change Os08g0157600 2.5812793 3.9816889 2.9396895 Os05g0279900 3.660117 3.714665 3.219169 Os04g0683700 1.377188 1.187543 1.016886 Os01g0363300 1.74026 1.298879 1.275056 Os03g0760800 2.539395 1.530233 2.26551 Os07g0115200 6.716991 8.26872 6.640491 Os07g0499300 9.391244 1.299781 9.203348 Os01g0303800 2.66464 1.79271 3.467855 Os02g0626600 2.085183 2.115733 1.854225 Os04g0116800 8.363292 1.503522 4.396605 Os09g0314500 2.678222 1.220469 1.808743 Os02g0187700 1.130521 1.309865 1.339599 Os03g0243750 2.384717 1.005085 1.806201 Os02g0755000 1.485427 1.227459 1.401167 Os07g0252400 1.456079 1.048667 1.792492 Os05g0102000 3.079241 2.644456 3.741467 Os06g0136600 2.349066 1.524076 1.484111 Os03g0293500 1.717269 1.50763 1.208278 Os08g0512700 2.644305 2.734514 1.55092 Os07g0162450 3.474259 3.537726 4.858276 Os06g0643500 2.585438 1.238791 1.670534 Os09g0528500 1.957078 1.52594 1.469357 Os05g0563600 3.039237 1.012951 1.525857 Os03g0850700 2.491312 1.395215 1.600855 Os03g0299700 3.715644 4.561388 4.87599 Os05g0196200 1.08823 1.107269 1.090866 Os01g0318202 1.525353 2.102553 2.744297 Os02g0687900 3.976002 1.261976 2.153499 Os01g0219500 3.655844 1.537544 1.121118 Os05g0397300 2.112081 1.099736 1.406341 Os04g0352400 5.549134 4.091258 1.186251 Os02g0712512 9.866506 2.512403 2.234558 Os12g0576100 2.904117 10.5493 3.352531 Os10g0430200 3.724893 2.053151 2.069581 Os06g0318533 3.189486 4.775086 5.194909 Os07g0694700 2.053039 1.384103 1.31645 Os03g0387900 1.129416 2.713077 2.16476 Os03g0788500 1.510912 1.080756 1.245643 Os01g0537250 4.333138 6.523999 5.855875 Os02g0744851 4.500928 1.547638 3.357472 Os12g0111600 1.760107 1.457776 2.344964 Os03g0832200 4.679627 1.040133 2.023745 Os03g0243700 3.285519 1.083096 1.412907 Os03g0687400 6.614765 1.900857 2.095706 Os01g0636500 3.884334 1.101554 3.731449 Os10g0567900 4.631957 3.819073 4.624738 Os03g0437600 3.820814 1.474163 3.699695 Os05g0438500 2.233729 1.276212 2.872376 Os11g0384789 3.820814 1.474163 3.699695 Os05g0427400 2.820047 1.269568 1.463967 Os01g0787000 1.927041 1.237081 1.799503 Os09g0416900 2.353867 1.245781 2.001385 Os03g0127500 3.237113 1.886136 1.740487 Os06g0581300 1.531787 1.399102 1.620124 Os08g0448000 1.537241 1.234569 2.043082 Os03g0132000 2.106862 1.113772 2.010683 Os04g0529700 6.835248 1.863916 3.225281 Os04g0635100 6.061792 1.505785 2.375225 Os03g0770851 1.86504 1.590047 1.595046 Os08g0415600 2.1516 1.210843 1.412614 Os09g0334500 2.006726 1.694803 2.192463 Os04g0413500 2.285109 2.133006 1.983874 Os05g0162800 1.5782601 1.5146642 1.6432247 Os08g0545850 6.376537 1.630726 2.304728 Os01g0585300 2.989453 2.434175 2.54667 Os02g0786900 3.313788 1.226139 1.645732 Os08g0160600 1.484985 1.014275 1.960158 Os05g0512301 1.454058 1.377079 1.504098 Os02g0732200 1.35069 1.250699 1.340491 Os04g0693300 1.531967 1.178615 1.925313 Os07g0287400 3.558364 2.220806 6.452485 Os01g0743200 2.176624 1.997368 1.235263 Os03g0676400 3.091448 1.288811 3.857367 Os06g0142200 1.605179 1.899601 3.969293 Os08g0448050 1.609439 1.688434 1.984814 Os04g0528200 7.570252 1.498371 1.853653 Os12g0575300 1.072252 1.080522 1.544866 Os02g0629800 6.135125 1.601914 5.242589 Os02g0491400 2.273258 1.33195 1.697242 Os05g0592300 1.973033 2.106365 1.715703 Os01g0883900 1.629736 1.186342 1.24435 Os05g0279850 3.478661 2.569021 3.16624 Os03g0788550 1.409807 1.271884 1.469562 Os01g0210500 1.016422 1.304249 1.073313 Os06g0142400 4.612829 4.201882 5.053411 Os06g0662200 2.112396 1.842086 2.291301 Os02g0712500 1.480206 1.40175 1.474199 Os01g0580500 2.047644 1.930258 2.622132 Os06g0696400 1.244125 1.276736 2.00086 Os02g0697550 4.816419 1.934655 6.31691 Os08g0545800 5.871069 1.649762 1.995298 Os11g0521500 1.839609 1.927803 2.550705 Os04g0578400 3.704655 3.582246 2.908885 Os06g0472000 3.027885 2.266417 3.773521 Os07g0412100 2.380378 2.175239 1.42552 Os03g0308800 2.048817 3.130161 2.353865 Os01g0919900 3.290739 1.845064 1.975785 Os02g0729400 3.884005 2.57511 2.669784 Os05g0291700 3.258097 1.354151 1.723688 Os02g0744900 3.8477279 1.6948331 3.0644925 Os04g0459500 5.7774393 1.4460234 4.232237 Os04g0690800 2.4142785 1.2251911 1.3014818 Os04g0678700 5.1860695 1.3352453 3.8729433 Os08g0104600 2.170783 1.654825 1.903221 Os10g0567400 2.4841995 1.1156028 2.199015 Os09g0346500 4.7787796 1.5214703 3.537965 Os06g0348800 2.6027071 1.3830415 2.0324755 The differentially expressed genes listed were upregulated in WT versus ppr2R- 1. The genes in red are related to chloroplast development.

Supplementary Table 5. The downregulated genes in the transcriptome analysis of ppr2R-1 anthers

stage 8 stage 9 stage 10 Gene ID log2 Fold Change log2 Fold Change log2 Fold Change Os03g0265900 −3.020883583 −1.711368144 −1.349901415 Os01g0567533 −5.090386236 −5.847181757 −3.171290226 Os04g0511200 −5.893301531 −3.936070154 −2.571434972 Os01g0225550 −2.565722144 −4.315917516 −1.703018262 Os01g0247600 −1.060261535 −2.026624382 −1.119762478 Os02g0236900 −1.822031499 −2.276485124 −2.817587689 Os03g0703300 −3.909523252 −4.808865123 −2.332298236 Os08g0110600 −1.98495612 −1.077711045 −1.093463178 Os06g0728000 −1.112255929 −1.101330362 −1.046033675 Os07g0461900 −1.475862692 −2.828410021 −1.403628441 Os06g0698300 −2.613174097 −3.572574838 −1.310206179 Os01g0962100 −1.378484585 −2.908996705 −1.464130044 Os06g0339800 −1.089403252 −1.508689259 −1.739445517 Os08g0529901 −1.691109905 −2.446158099 −1.423601992 Os10g0142100 −1.042854161 −2.107888235 −1.255263246 Os03g0291200 −1.588744592 −2.780112252 −1.360719746 Os02g0774100 −1.716310113 −1.732455542 −2.306606063 Os04g0675400 −6.114609953 −3.797098753 −2.49131568 Os01g0793800 −2.398707466 −2.88718764 −4.284945453 Os07g0150700 −1.893851503 −1.597660865 −1.053077838 Os06g0166400 −1.132025103 −1.040711689 −1.142140052 Os03g0723400 −1.994493524 −2.593488372 −2.434609819 Os10g0389500 −3.852855576 −4.830743466 −3.360256214 Os07g0209100 −1.567682368 −3.00746545 −2.222070008 Os06g0237400 −4.095897861 −4.137246723 −3.718174053 Os12g0128700 −1.008405705 −2.007494537 −1.024373834 Os09g0275200 −2.490354192 −2.31835774 −1.260941655 Os01g0306400 −5.163386392 −3.938774657 −3.237535908 Os03g0244466 −3.076429282 −1.641546029 −1.421371433 Os04g0571300 −1.1556902 −2.589942468 −1.761418638 Os03g0184100 −3.880195729 −2.431521685 −2.561681677 Os02g0586900 −6.163475226 −7.66503366 −4.253467798 Os03g0416400 −1.344466917 −3.304829905 −2.054348922 Os11g0532200 −1.521119925 −2.400072297 −2.330621122 Os05g0355400 −2.268596752 −3.387940228 −2.221533419 Os11g0547000 −1.621565607 −1.1678124 −1.097527922 Os08g0417000 −2.108659345 −1.87658065 −1.142722054 Os06g0705400 −9.009828617 −1.588756074 −2.162638647 Os06g0298200 −1.557481764 −1.976007144 −1.542891105 Os12g0428000 −3.980157545 −4.073152426 −3.14797303 Os11g0120300 −1.285964161 −2.564839219 −1.242701404 Os09g0551600 −1.212994632 −1.220075794 −1.024012349 Os06g0132550 −1.366061144 −1.875523238 −1.632364855 Os07g0209201 −1.601619108 −2.342524384 −1.987751646 Os11g0454200 −2.402342694 −1.535019187 −1.465269083 Os05t0478000 −3.616999977 −3.225420114 −2.018813272 Os05g0421100 −8.62935662 −3.264962187 −2.707403437 Os03g0586500 −3.843348519 −1.502793959 −1.768100628 Os08g0335200 −2.004865755 −2.722574502 −2.321928095 Os06g0594400 −1.187130267 −1.428266045 −1.291307581 Os02g0724000 −1.398688428 −2.98862521 −1.271392727 Os09g0361200 −2.36186684 −3.492933362 −1.469230656 Os06g0559400 −2.119512731 −2.792734971 −3.03212732 Os06g0673500 −1.249523168 −2.383918192 −1.463220836 Os06g0594300 −1.388008354 −1.444399177 −1.56965565 Os09g0505900 −4.451379346 −4.557464783 −1.209767047 Os09g0483200 −3.296122494 −3.722117095 −1.563356273 Os11g0454300 −3.164531743 −1.712460631 −2.191560752 Os03g0749100 −2.788843365 −3.644639604 −4.774742245 Os01g0860500 −3.655429817 −2.649802104 −3.172010902 Os01g0153600 −3.20004499 −3.842643672 −3.519664684 Os06g0553200 −1.027914943 −1.033676118 −1.970333775 Os01g0785300 −2.811927652 −4.429382411 −2.840119353 Os02g0161900 −1.240701427 −1.067344227 −1.055116872 Os03g0749200 −3.77807713 −3.831151024 −4.931076305 Os01g0313600 −6.12462164 −4.604723849 −4.041531223 Os01g0342800 −1.034104471 −1.334698967 −1.92812478 Os09g0322100 −2.605034199 −1.012197638 −1.605959687 Os04g0486950 −1.110640126 −3.205765179 −1.382559876 Os01g0733200 −4.939285356 −1.720468736 −2.857817264 Os01g0606900 −2.328301867 −1.294425222 −2.069677888 Os06g0589700 −1.90779938 −3.701085811 −2.35995762 Os03g0826800 −6.37435985 −4.700625286 −4.12740367 Os07g0113300 −1.530715021 −2.176172482 −1.362936851 Os08g0537900 −2.527172146 −3.036871446 −3.245707588 Os10g0395200 −1.25252292 −2.225999295 −1.853831929 Os06g0147100 −2.352103209 −2.718439537 −2.447858043 Os06g0725000 −1.829004353 −1.766688754 −1.232272152 Os11g0444700 −1.459234972 −1.952980016 −1.525697814 Os01g0225600 −2.019439881 −3.782739623 −1.810508019 Os01g0860450 −2.999589151 −3.627496545 −5.004220466 Os06g0115300 −1.797706496 −2.480736332 −1.148404265 Os01g0153625 −4.296682144 −3.858977146 −3.455846331 Os03g0820500 −1.264027655 −2.977666136 −2.330179534 Os01g0605700 −2.907427812 −4.89479628 −2.034105033 Os08g0472800 −2.231360885 −3.756842883 −2.187540624 Os03g0826900 −6.172784656 −5.113027126 −5.579385783 Os05g0453500 −1.889640304 −1.30230531 −1.169429812 BGI_novel_G001486 −1.11135555 −1.909186335 −1.170867128 Os01g0314800 −2.013370055 −2.62177142 −1.305554331 Os08g0389700 −2.162636018 −4.470185179 −3.319508142 Os09g0437500 −3.169469318 −3.153520051 −2.262495817 Os03g0413100 −9.499845887 −3.054506286 −3.495048435 Os07g0663800 −2.850083466 −5.183221824 −4.821671713 Os08g0137400 −1.162610994 −1.782537178 −1.889125888 Os01g0567600 −4.641086721 −6.508452779 −1.97648155 Os02g0690000 −1.198440569 −1.676641962 −1.188674766 Os08g0241800 −2.402890741 −3.784255723 −1.681370456 Os12g0586100 −3.581852573 −3.545755239 −2.512432976 Os06g0606401 −1.187019936 −1.95557843 −1.79542578 Os02g0671100 −3.081838617 −3.343107298 −2.344101736 Os05g0468500 −4.6794801 −5.187542779 −1.100793172 Os10g0103700 −1.487370962 −1.336280297 −1.755923424 Os03g0666600 −2.917625871 −2.250793938 −1.035340229 Os01g0822800 −3.208967909 −2.687555926 −1.818687721 Os05g0560200 −1.149417234 −2.043164321 −1.960004044 Os07g0663750 −3.076621282 −9.857980995 −6.038135129

Supplementary Table 6. The up- and downregulated transcription factors in the transcriptome analysis of ppr2R-1 anthers

Upregulated transcription factors Os06g0348800 OsGLK1 Os08g0157600 LHY, homolog of At5g37260 Os05g0162800 REVEILLE 8, homolog of At5g17300 Os03g0387900 LNK1 Os12g0180800 transcription factor bHLH110 Os02g0187700 transcription factor MYB44 Os03g0127500 bZIP transcription factor Os09g0334500 WRKY family transcription factor-like, WRKY transcription factor 30 Os01g0318400 Sequence-specific DNA binding transcription factor Os06g0662200 bZIP transcription factor RISBZ5; Rice seed bZIP5; Full=bZIP transcription factor 52; OsbZIP52: negative regulator of cold and drought stress response Os01g0636500 zinc finger MYM-type protein 1-like, transposase

Downregulated transcription factors

Os10g0103700 homeobox-leucine zipper protein HOX15 Os06g0298200 zinc finger protein CONSTANS-LIKE 9 Os02g0724000 zinc finger protein CONSTANS-LIKE 9 Os06g0166400 ethylene-responsive transcription factor RAP2-9 Os01g0306400 protein DOG1-like 4 Os05g0560200 transcription factor TGA9 Os01g0733200 heat stress transcription factor C-1b

Supplementary Table 7. Comparison of upregulated genes in the transcriptome analysis of ppr2R-1 anthers with the transcriptome of Nakamura et al., 2009

Stage 8 Stage 9 Stage 10 Gene ID log2 Fold Change log2 Fold Change log2 Fold Change PSI subunits Os08g0560900 0.209567118 3.095095547 2.689573624 Os07g0435300 0.100965574 0.821824404 1.287305069 Os03g0778100 0.386001612 2.49908095 2.048037459 Os09g0481200 0.694474925 3.25030042 2.417356316 Os05g0560000 0.107872114 2.22684234 1.547573915 Os07g0148900 0.381096375 2.413121454 2.080489918 Os12g0420400 −0.485599436 1.394522116 1.488082958 Os12g0189400 0.528853733 2.658262996 3.014849687 Os04g0414700 0.929574873 3.519484215 2.548477167 Os06g0320500 0.345678114 2.989492342 2.277754657 Os07g0577600 0.605889538 2.082336714 1.708856985 Os02g0197600 0.789953226 3.004118459 2.100001293 Os08g0435900 0.059980697 3.238866503 2.447351389 Os02g0764500 0.664433903 5.454739923 3.032914622 Os09g0439500 −1.249594149 −0.179071337 0.565439942 PSII subunits Os01g0501800 0.441188861 1.477609291 1.346516566 Os07g0141400 0.218273503 2.167589525 2.350938251 Os02g0578400 −4.665072676 1.076621282 7.918863237 Os08g0200300 0.244703384 1.857059117 1.636531755 Os01g0869800 1.99382977 0.030381746 0.04419474 Os01g0773700 0.233709718 0.930997556 0.89533985 Os03g0343900 0.125050253 3.28916574 2.941695025 Os08g0119800 −4.889177992 0 0 Os01g0600900 −0.022892855 3.872838238 2.853643732 Os09g0346500 1.521470341 4.778779564 3.537965021 Os01g0720500 0.578053833 3.34017733 2.888179703 Os03g0592500 0.972156946 3.719440153 2.765874917 Os07g0562700 0.720714689 4.001916652 3.030779593 Os07g0558400 0.250065097 3.619079317 3.149126386 Os11g0242800 0.808597593 3.858606622 3.423596822 Os04g0457000 0.326687175 1.692791189 1.063912052 Intersystemic electron transport Os07g0556200 0.697491738 2.065828257 1.944471509 Os03g0805600 2.171357668 0.850365802 0.847480136 Os02g0792800 −0.21109402 −0.10168414 −0.658330182 Os11g0242400 0.054295977 −0.0736671 0.902121351 Os06g0101600 0.485831035 2.730805819 2.769858809 Os08g0104600 1.654824815 2.170782669 1.903221121 Os04g0412200 0.553670195 1.148784587 0.317633443 Os03g0685000 −0.844623307 −0.451307449 −1.874854814 Os06g0107700 −0.848595659 5.512724275 0.948113258 Os02g0103800 0.125791029 3.254639965 2.527814137 ATPase Os07g0513000 0.536827021 0.890319246 0.309328058 Os02g0750100 −0.360402243 0.379812467 0.490894926 Os03g0278900 0.46406247 1.384021684 1.771905219

CO2 assimilation Os12g0292400 −0.154722595 4.691279719 1.954432527 Os12g0274700 −0.644308833 0.563052253 0.728829794 Os12g0291100 0.083636077 0.761234343 0.879427186 Os12g0291400 −0.968563341 0.907303325 1.540472861 Os11g0707000 0.615415008 1.418198655 1.360907175 Os05g0496200 0.124351047 1.777576432 1.763177622 Os03g0129300 0.12679445 2.056078673 1.668148152 Os04g0459500 1.446023422 5.777439344 4.232236982 Os01g0303000 −2.636372522 1.174497731 0.58599632 Os03g0306800 0.136255406 1.375866902 0.496498863 Os11g0171300 0.290407822 0.42196886 0.36159824 Os01g0866400 0.402585758 −3.787143978 −1.493920302 Os03g0267300 −0.693205209 1.047305715 −0.639410285 Os04g0234600 0.804470409 2.844251729 3.999507192 Os03g0169100 0.08723101 0.150236167 0.007295224 Os02g0698000 0.32762908 2.612294483 2.270240176 Plastid genome expression Os08g0163400 0.65708239 0.030659636 1.360523872 Os03g0271100 −0.083453136 −0.56617225 0.196416832 Os05g0586600 5.661198087 5.927354698 4.385912551 Os08g0242800 0.392772316 −1.253003012 0.535173476 Chl biosynthesis Os03g0337600 0.609407677 0.879953673 1.666725862 Os03g0563300 −0.491762386 2.221863865 0.534113359 Os03g0811100 −0.430211022 0.579965284 0.755549534 Os03g0323200 0.822679255 1.717200708 1.756401008 Os06g0132400 0.268146231 0.512700758 8.856425529 Os01g0279100 −0.014597376 3.943235116 3.984369348 Os04g0678700 1.335245265 5.186069548 3.872943264 Os10g0496900 0.028816572 2.775390083 2.493032075 Os02g0744900 1.69483311 3.847727923 3.06449252 Os05g0349700 −0.379740238 0.568578406 0.749840167 Os10g0567400 1.115602751 2.484199524 2.199015039 The 53 genes in red were also identified as upregulated genes in the transcriptome analysis of ppr2R-1 anthers compared to the previous transcriptome study of OsGLK1-overexpressing rice calli (Nakamura et al., 2009).

Figures

Figure 1

Expression analysis of OsPPR2-1. a, qRT-PCR analysis of OsPPR2-1 expression. BStage 6, stage 1 to stage 6. Data are shown as means ± SD (n=3). b, RNA in situ hybridization analysis of OsPPR2-1 in WT anthers from stage 8 to stage 11. Ta, tapetum. Bars = 25 μm. c, Subcellular localization of OsPPR2-1 in rice leaf sheath protoplasts. p35S::Tic40-eGFP, p35S::COX11-mcherry, and p35S::NLS-mKATE were used as the chloroplast, mitochondrial, and nuclear markers, respectively. Bar = 10 μm. d, OsPPR2-1 was detected in different cell compartment protein extracts using western blot analysis. The COX IV, Tic 40, and H3 antibodies were used as the indicators of chloroplast, mitochondria, and nuclear protein controls, respectively. CP, cytosolic protein; Chl, chloroplast protein; Mit, mitochondria protein; NP, nuclear protein; Total, Total protein.

Figure 2 Phenotypic comparison and anther development of WT, ppr2R-1, and Casppr2-1 plants. a, Comparison of WT, ppr2R-1, and Casppr2-1 spikelets after removing the lemma and palea. b, Staining of WT, ppr2R-1, and Casppr2-1 pollen grains with I2-KI dye. c, Comparison of the seed-setting rates of WT, ppr2R-1, and Casppr2-1 plants. Data are means ± SD (n= 30); **, P < 0.01 according to Student’s t-test. d, Comparison of pollen viability in WT, ppr2R-1, and Casppr2-1 plants. The data represent the mean of ten independent experiments ± SE; **, P < 0.01 according to Student’s t-test. e to s, Bright-eld photomicrographs of transverse sections showing anther and microspore development in WT (e–i), ppr2R-1 (j–n), and Casppr2-1 (o–s) plants from stage 8 to stage 12. Ta, tapetum. Bars = 2 mm in (a), 50 μm in (b), 25 μm in (e) to (s). Figure 3

High magnication views of TEM images of the tapetum. a to i, High magnication views of TEM images showing tapetal cells and plastids in WT (a–c), ppr2R-1 (d–f), and Casppr2-1 (g–i) plants from stage 10 to stage 12. Red arrowheads indicate plastids. Ela, elaioplast; Msp, microspore parietal cell; Pl, proplastid; Pe, peroxisome; Ub, Ubisch body; V, vacuole; T, tapetum. Bars = 1 μm in (a) to (i).

Figure 4

TUNEL assays of anthers to detect DNA fragmentation due to PCD. a to o, DNA fragment signals in WT (a–e), ppr2R-1 (f–j), and Casppr2-1 (k–o) anthers from stage 8 to stage 12. The red uorescence is from anthers stained with propidium iodide (PI), as viewed under a confocal laser-scanning microscope; images are overlays of green uorescence from the TUNEL assay with PI staining. Mp, mature pollen; Ta, tapetal cell; Bars = 20 μm. Figure 5

Phenotypic characterization and transverse sections of OEGLK1-1 and OEGLK1-2 anthers. a, qRT-PCR analysis of OsGLK1 transcript levels in WT, ppr2R-1, and Casppr2-1 anthers from stage 9 to 12. Error bars indicate SD. Two biological replicates were used; each reaction represents three technical repeats. Asterisks represent signicant differences determined by Student’s t-test at P < 0.001 (***), P < 0.01 (**), P < 0.05 (*). b, Spikelets (top) and pollen grains (bottom) from WT, OEGLK1-1, and OEGLK1- 2 plants. Pollen grains were stained with I2-KI. Bars = 2 mm for spikelets and 50 μm for pollen grains. c, Expression analysis of OsGLK1 in WT, OEGLK1-1, and OEGLK1-2 anthers at stage 12. Error bars indicate SD. Each reaction represents three technical repeats. ***, P < 0.001 according to Student’s t-test. d, Comparison of the seed-setting rates of WT, OEGLK1-1, and OEGLK1-2 plants. Data are means ± SD (n= 30), **, P < 0.01 according to Student’s t-test. e, Comparison of the viability of mature WT, OEGLK1-1, and OEGLK1-2 pollen. Data are means ± SD (n= 20), **, P < 0.01 according to Student’s t-test. f to t, Comparison of transverse sections of anthers from WT (f–j), OEGLK1-1 (k– o), and OEGLK1-2 plants (p–t). Ta, tapetal cell; Bars = 20 μm. Figure 6

Transmission electron microscopy and TUNEL assay of WT, OEGLK1-1, and OEGLK1-2 anthers. a to i, Higher magnication views of TEM images showing tapetal cells and plastids of WT (a–c), OEGLK1-1 (d–f), and OEGLK1-2 (g–i) anthers from stage 10 to stage 12. Red arrowheads indicate plastids. E, epidermis; Ela, elaioplast; Mt, mitochondrion; Pl, plastid; Pe, peroxisome; Ub, Ubisch body; V, vacuole. Bars = 1 μm in (a) to (i). j to x, DNA fragment signals in WT (j–n), OEGLK1-1 (o–s), and OEGLK1-2 (t– x) anthers. The red uorescence is from anthers stained with PI under a confocal laser-scanning microscope; images are overlays of green uorescence from the TUNEL assay and PI staining. Ta, tapetal cell; Bars = 20 μm.

Figure 7

OsPPR2-1 directly binds to OsGLK1 mRNA and promotes its degradation; a proposed model of the role of OsPPR2-1-OsGLK1 in regulating elaioplast development in anthers and PCD in the tapetum in rice a, The binding of OsPPR2-1 to OsGLK1 mRNA, as measured by RNA immunoprecipitation (RIP) followed by qRT- PCR. The fold-enrichment values of OsGLK1 mRNA determined by RIP-qPCR using stage 11 anthers from transgenic plants overexpressing PR2-1 (OEPPR2-1) are shown. Negative control, anti-IgG (normal rabbit IgG antibody). G1–G4, four different regions of OsGLK1 mRNA. T1, The most similar sequence in the coding regions between OsGLK1 and OsGLK2. Error bars indicate SD. Each reaction represents three technical replicates. b, RNA-EMSA analysis for the binding of OsPPR2-1 to the OsGLK1 mRNA fragments. The RNA fragments (G2, G4, T1) from the RIP-PCR analysis (Fig. 7b) were end-labeled with biotin, and the unlabeled RNA fragments were used as a cold competitor. The 20× and 50× contain 20– and 50–fold 868 more unlabeled probes than labeled probes, respectively. The Glk2-2 probe was used as a negative control RNA fragment from the OsGLK2 mRNA. c, Image of the OsPPR2-1 cleavage assay. The OsGLK1 RNA and OsACTIN RNA were incubated with either recombinant OsPPR2-1 or the incomplete OsPPR2-1 (OsPPR2-1Δ). OsACTIN RNA was used as a negative control. d. The homologous T1 RNA fragment and the Glk2-2 fragment of the OsGLK2 mRNA from the RIP-PCR analysis and RNA-EMSA were incubated with either recombinant OsPPR2-1 or the mutant OsPPR2-1 (OsPPR2-1Δ). After the reactions, the reaction mixtures were analyzed on a denaturing polyacrylamide gel. Molecular weight marker sizes (M) are indicated in bases. e, Proposed model of the role of OsPPR2-1–OsGLK1 in regulating plastid development in the tapetum of rice anthers. According to this model, the appropriate plastid differentiates into an elaioplast in the tapetum. OsGLK1 ensures that the tapetum is degraded at the proper time, which is directly regulated by OsPPR2-1. E, epidermis; En, endothecium; T, tapetum.