Stigma receptors control intraspecies and interspecies barriers in Brassicaceae

doi:10.1038/s41586-022-05640-x

. 2023 Feb;614(7947):303-308.

doi: 10.1038/s41586-022-05640-x. Epub 2023 Jan 25.

Stigma receptors control intraspecies and interspecies barriers in Brassicaceae

Jiabao Huang^#^{1

2}, Lin Yang^#^{1

2}, Liu Yang^{1

2}, Xiaoyu Wu^{1

2}, Xiaoshuang Cui^{1

2}, Lili Zhang^{1

2}, Jiyun Hui^{1

2}, Yumei Zhao^{1

2}, Hongmin Yang^{1

2}, Shangjia Liu^{1

2}, Quanling Xu^{1

2}, Maoxuan Pang^{1

2}, Xinping Guo^{1

2}, Yunyun Cao^{1

2}, Yu Chen^{1

2}, Xinru Ren^{1

2}, Jinzhi Lv^{1

2}, Jianqiang Yu^{1

2}, Junyi Ding¹, Gang Xu¹, Nian Wang¹, Xiaochun Wei³, Qinghui Lin⁴, Yuxiang Yuan³, Xiaowei Zhang³, Chaozhi Ma⁵, Cheng Dai⁵, Pengwei Wang⁵, Yongchao Wang⁶, Fei Cheng⁷, Weiqing Zeng⁸, Ravishankar Palanivelu⁹, Hen-Ming Wu¹⁰, Xiansheng Zhang¹, Alice Y Cheung¹¹, Qiaohong Duan^{12

13}

Affiliations

¹ State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai'an, China.
² College of Horticulture Science and Engineering, Shandong Agricultural University, Tai'an, China.
³ Institute of Horticulture, Henan Academy of Agricultural Sciences, Zhengzhou, China.
⁴ Computer Network Information Centre, Chinese Academy of Sciences, Beijing, China.
⁵ National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, China.
⁶ Shandong Yiyi Agricultural Science and Technology Co., Ltd, Tai'an, China.
⁷ College of Horticulture, Qingdao Agricultural University, Qingdao, China.
⁸ International Flavors & Fragrances, Wilmington, DE, USA.
⁹ School of Plant Sciences, University of Arizona, Tucson, AZ, USA.
¹⁰ Department of Biochemistry and Molecular Biology, Molecular Cell Biology and Plant Biology Programs, University of Massachusetts, Amherst, MA, USA.
¹¹ Department of Biochemistry and Molecular Biology, Molecular Cell Biology and Plant Biology Programs, University of Massachusetts, Amherst, MA, USA. acheung@biochem.umass.edu.
¹² State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai'an, China. duanqh@sdau.edu.cn.
¹³ College of Horticulture Science and Engineering, Shandong Agricultural University, Tai'an, China. duanqh@sdau.edu.cn.

^# Contributed equally.

PMID: 36697825
PMCID: PMC9908550
DOI: 10.1038/s41586-022-05640-x

Stigma receptors control intraspecies and interspecies barriers in Brassicaceae

Jiabao Huang et al. Nature. 2023 Feb.

. 2023 Feb;614(7947):303-308.

doi: 10.1038/s41586-022-05640-x. Epub 2023 Jan 25.

Authors

Affiliations

¹ State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai'an, China.
² College of Horticulture Science and Engineering, Shandong Agricultural University, Tai'an, China.
³ Institute of Horticulture, Henan Academy of Agricultural Sciences, Zhengzhou, China.
⁴ Computer Network Information Centre, Chinese Academy of Sciences, Beijing, China.
⁵ National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, China.
⁶ Shandong Yiyi Agricultural Science and Technology Co., Ltd, Tai'an, China.
⁷ College of Horticulture, Qingdao Agricultural University, Qingdao, China.
⁸ International Flavors & Fragrances, Wilmington, DE, USA.
⁹ School of Plant Sciences, University of Arizona, Tucson, AZ, USA.
¹⁰ Department of Biochemistry and Molecular Biology, Molecular Cell Biology and Plant Biology Programs, University of Massachusetts, Amherst, MA, USA.
¹¹ Department of Biochemistry and Molecular Biology, Molecular Cell Biology and Plant Biology Programs, University of Massachusetts, Amherst, MA, USA. acheung@biochem.umass.edu.
¹² State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai'an, China. duanqh@sdau.edu.cn.
¹³ College of Horticulture Science and Engineering, Shandong Agricultural University, Tai'an, China. duanqh@sdau.edu.cn.

^# Contributed equally.

PMID: 36697825
PMCID: PMC9908550
DOI: 10.1038/s41586-022-05640-x

Erratum in

Author Correction: Stigma receptors control intraspecies and interspecies barriers in Brassicaceae.
Huang J, Yang L, Yang L, Wu X, Cui X, Zhang L, Hui J, Zhao Y, Yang H, Liu S, Xu Q, Pang M, Guo X, Cao Y, Chen Y, Ren X, Lv J, Yu J, Ding J, Xu G, Wang N, Wei X, Lin Q, Yuan Y, Zhang X, Ma C, Dai C, Wang P, Wang Y, Cheng F, Zeng W, Palanivelu R, Wu HM, Zhang X, Cheung AY, Duan Q. Huang J, et al. Nature. 2023 Mar;615(7952):E20. doi: 10.1038/s41586-023-05816-z. Nature. 2023. PMID: 36864127 Free PMC article. No abstract available.

Abstract

Flowering plants have evolved numerous intraspecific and interspecific prezygotic reproductive barriers to prevent production of unfavourable offspring¹. Within a species, self-incompatibility (SI) is a widely utilized mechanism that rejects self-pollen^2,3 to avoid inbreeding depression. Interspecific barriers restrain breeding between species and often follow the SI × self-compatible (SC) rule, that is, interspecific pollen is unilaterally incompatible (UI) on SI pistils but unilaterally compatible (UC) on SC pistils^1,4-6. The molecular mechanisms underlying SI, UI, SC and UC and their interconnections in the Brassicaceae remain unclear. Here we demonstrate that the SI pollen determinant S-locus cysteine-rich protein/S-locus protein 11 (SCR/SP11)^2,3 or a signal from UI pollen binds to the SI female determinant S-locus receptor kinase (SRK)^2,3, recruits FERONIA (FER)^7-9 and activates FER-mediated reactive oxygen species production in SI stigmas^10,11 to reject incompatible pollen. For compatible responses, diverged pollen coat protein B-class^12-14 from SC and UC pollen differentially trigger nitric oxide, nitrosate FER to suppress reactive oxygen species in SC stigmas to facilitate pollen growth in an intraspecies-preferential manner, maintaining species integrity. Our results show that SRK and FER integrate mechanisms underlying intraspecific and interspecific barriers and offer paths to achieve distant breeding in Brassicaceae crops.

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Conflict of interest statement

The authors declare no competing interests.

Figures

**Fig. 1. SRK controls stigmatic ROS in SI to reject intraspecific and interspecific pollen.**
a, Open flowers and phylogenetic tree for Brassicaceae species from maximum likelihood analysis using internal transcribed spacer sequences. Distantly related *Cochlearia danica* served as an outgroup. Scale bar, mean number of nucleotide substitutions per site. b, Aniline blue staining showing intraspecific and interspecific pollen growth in mature and bud-stage *B. rapa* stigmas. c, H₂DCFDA staining of ROS in unpollinated (UP) or pollinated *B. rapa* stigmas. d,e, Relaxed SI and UI in AS-BrSRK46-treated *B. rapa S*₄₆ stigmas (d) and stigmas from SRK-defective *B. rapa*, BrSRK^∆TM (e). f,g, Intraspecific and interspecific pollen (f) and ROS responses (g) in stigmas from SC *A. thaliana*, that is, wild type (WT), and stigmas from SI *A. thaliana* expressing *A. halleri S*₁₃ genes (*A. tha*-S₁₃). The values in the b–g images, shown as average ± s.d., indicate average number of pollen tubes in the stigma (b,d,e,f) and average ROS intensity (c,g). The same data are also presented in box plots with all data points (Extended Data Figs. 1, 2). Scale bars, 0.5 cm (a), 500 μm (b–e) and 200 μm (f,g). Each experiment was repeated at least three times with consistent results.

**Fig. 2. UI and SI converge on stigmatic ROS activation for pollen rejection via the SRK–FER interaction.**
a,b, Treating *B. rapa* stigmas with AS-BrFER1 or crossing *fer-4* into SI *A. thaliana* stigmas, S₁₃/*fer-4*, relaxed SI and UI. The values in the a,b images, shown as average ± s.d., indicate average number of pollen tubes in the stigma. The same data are also presented in box plots with all data points (Extended Data Fig. 3). c–e, Pull-down (pd) (c,d) and co-immunoprecipitation (co-IP) (e) assays showing protein extracts from SI and UI pollen. BrSCR46 augmented the BrSRK46–BrFER1 interaction. The protein samples were derived from the same experiment and the blots were processed in parallel (c–e). For gel source data, see Supplementary Fig. 1. Scale bars, 500 μm (a) and 200 μm (b). Each experiment was repeated at least three times with consistent results.

**Fig. 3. Species-preferential interaction between PCP-Bs and FER maintains interspecies barrier.**
a, In *A. thaliana* stigmas, *A. thaliana* pollen tubes were longer than those of *B. rapa*, *B. oleracea* and *B. vulgaris* at 1 HAP. b, Faster hydration and growth of *B. rapa* pollen on *A. thaliana fer-4* stigmas. The orange plots indicate relative pollen width and the blue plots indicate pollen tube length. The equatorial diameter of a pollen grain, indicated by white dashed lines, was measured in ImageJ for pollen width. c, Relaxed interspecies barrier in *fer-4* stigmas. Ratios of pollen tube length in intraspecies and interspecific crosses are used as a measure for barrier strength. In a–c, the arrows indicate pollen tube front. d,e, Species-preferential ROS reduction in *A. thaliana* stigmas by pollen (d) and PCP-Bs (e) from intraspecies and interspecies. f, Pull-down assay showing AtPCP-Bγ competed dose-dependently with GST–BrPCP-B3 for interaction with AtFER (ED)–FLAG. The protein samples were derived from the same experiment and the blots were processed in parallel (f). For gel source data, see Supplementary Fig. 1. Scale bars, 200 μm (a–e) and 20 μm (pollen in b). For box plots (a–e), the centre line indicates the median, the box limits denote the lower and upper quartiles, the dots indicate individual data points, and the whiskers denote the highest and lowest data points. P values were determined by two-tailed Student t-tests. n (in blue) indicates the number of stigmas or pollen grains. In f, for the data bar, average ± s.d. is shown; average intensities from three biological replicates of the blot are represented on the left (two-tailed t-test, n = 3). Each experiment was repeated at least three times with consistent results. Source data

**Fig. 4. Compatible pollination induces NO to suppress FER-mediated ROS production.**
a, DAF-FM DA staining of pollination-induced NO responses and pollen hydration in *A. thaliana* stigmas. The equatorial diameter of a pollen grain, indicated by white dashed lines, was measured in ImageJ for pollen width. b,c, Species-preferential elevation of NO in *A. thaliana* stigmas. d,e, FER-dependent NO elevation in *A. thaliana* stigmas induced by pollen (d) and AtPCP-Bγ (e). The values in the a–e images, shown as average ± s.d., indicate average NO intensity (a–e) and equatorial diameter of pollen grains (a). The same data are also presented in box plots with all data points (Extended Data Fig. 8). f,g, Nitrosation of FER. BrFER1 was nitrosated in vitro by the NO donor GSNO (f), and AtFER–GFP from transformed *A. thaliana* stigmas was nitrosated in vivo by SC pollination (g). h,i, Pull-down assays showing that nitrosation of FER in vitro (h) and by pollination (i) reduced its interaction with the downstream ROP2 signalling module. The protein samples were derived from the same experiment and the blots were processed in parallel (f–i). For gel source data, see Supplementary Fig. 1. Scale bars, 200 μm (a–e) and 50 μm (pollen in a). For box plots (b,c), the centre line indicates the median, the box limits denote the lower and upper quartiles, the dots indicate individual data points, and the whiskers denote the highest and lowest data points. n (in blue) indicates the number of stigmas or pollen grains. P values were determined by two-tailed Student t-tests. Each experiment was repeated at least three times with consistent results. Source data

**Fig. 5. Breaking the stigmatic barrier for distant breeding of Brassicaceae crops.**
a,b, Reducing stigmatic ROS by a ROS scavenger, increasing NO by a NO generator (a), and disrupting the BrSRK–BrFER interaction and BrFER1-to-BrRBOH signalling by AS-ODNs (b) alleviated the interspecific and intergeneric reproductive barrier. The arrowheads indicate enlarged ovules. The dashed lines denote the outline of hybrid embryos. The values in the a,b images, shown as average ± s.d., indicate average number of enlarged ovules in the pod. The same data are also presented in box plots with all data points (Extended Data Fig. 10). Scale bars, 0.5 cm (siliques) and 100 μm (emb ryos). Each experiment was repeated at least three times with consistent results. c, Model of FER-regulated ROS as a shared signalling node in SI, UI, SC and UC responses. In stigmas from SI species, SI pollen and UI pollen activate ROS via the SRK–FER–ROP2–RBOHs pathway, functioning along with the ARC1-mediated processes. Stigmas from SC species are compatible to interspecific pollen, but species-preferential interaction between PCP-Bs and FER initiates a faster compatible response via NO-mediated nitrosation of FER and RBOHs to suppress ROS production, promoting intraspecies precedence and protecting species integrity. The dashed lines and ‘?’ indicate ‘to be determined’. P, phosphate; P-some, proteosome; Ub, ubiquitin.

**Extended Data Fig. 1. ROS underlies the rejection of interspecific pollen during UI.**
a, The developmentally regulated SI and the progressive increase in the amount of SRK from bud stage to maturity. b, The number of intra– and various interspecific pollen tubes in mature or bud-stage *B. rapa* stigmas. Mature *B. rapa* stigmas rejected SI and all the interspecific pollen examined, but bud-stage *B. rapa* stigmas allowed the growth of SI pollen and interspecific *B. oleracea* pollen, and still rejected intergeneric *B. vulgaris* and *A. thaliana* pollen. See Fig. 1b. c, Imaging of H₂DCFDA-stained ROS under confocal microscope and under wide-field fluorescence microscope showed comparable changes of ROS in *B. rapa* stigmas Dotted line outlined the area for ROS quantification. Wide-field observation, with its considerably high expedition to sample entire stigma specimens, was used for the massive amount of data gathering required for this study. d, ROS intensity in unpollinated (UP) or pollinated *B. rapa* stigma. See Fig. 1c. e–g, Scavenging ROS by Na-SA suppressed stigmatic ROS induction, and promoted the growth of SI pollen and *B. oleracea* pollen in mature and *B. vulgaris* pollen in bud-stage *B. rapa* stigmas. Scale bars, 500 μm. Data bar (a): average ± SD. Average relative expression levels from three biological replicates of stigmas (two tailed t-test, n = 3). Box plots (b–g): centre line, median; box limits, lower and upper quartiles; dots, individual data points; whiskers, highest and lowest data points. n (in blue), number of stigmas. P values, two-tailed t-tests. Each experiment was repeated at least thrice with consistent results. Source data

**Extended Data Fig. 2. SRK underlies the rejection of interspecific pollen during UI.**
a, Quantitative RT-PCR showing AS-BrSRK46 treatment reduced *BrSRK46* expression in mature *B. rapa* stigmas (S₄₆) and further reduced its already low level in bud-stage stigmas (S₄₆). b, AS-BrSRK46 treatment of mature *B. rapa* stigmas (S₄₆) broke the inhibition to *B. oleracea* pollen not *B. vulgaris* pollen. AS-BrSRK46 treatment of bud-stage stigmas (S₄₆) broke inhibition to *B. vulgaris* pollen. See Fig. 1d. c, d, AS-BrSRK46 treatment reduced ROS in mature *B. rapa* stigmas (S₄₆) and further reduced its already low ROS in bud-stage stigmas (S₄₆). e–g, Sequence alignment (e), phylogenetic analysis (f) and expression of SRK in FPsc (Brara.G02663) (g). SRK from FPsc lacks the coding sequence for the transmembrane domain and we name it *BrSRK*^∆TM hereafter. h–j, Mature and bud-stage stigmas of FPsc were defective in SI and UI pollen-triggered ROS increase (h, i) and the rejection of SI and UI pollen (j). See Fig. 1e. k, *SRK13* expression from SI *A. thaliana* stigmas transformed with *Arabidopsis halleri* AhSP11/SCR13-AhSRK13-AhARC1. These three transgenes were in a single construct. l, m, The number of intra– or interspecific pollen tubes (l) and ROS changes (m) in SC *A. thaliana* stigmas and in SI *A. thaliana* stigmas. See Fig. 1f, g. n, *B. oleracea* pollen of S₃₆-haplotype was rejected in *B. rapa* stigmas of S₄₆, S₁₂, S₉, S₄₀, or S₃₈, showing the dependence on SRK but the independence of SCR-SRK interaction in the rejection of interspecific pollen. Scale bars, 500 μm (c, d, h, i, n). Data bars (a, g, k): average ± SD. Average relative expression levels from three biological replicates of stigmas (two tailed t-test, n = 3). Box plots (b–d, h–j, l–n): centre line, median; box limits, lower and upper quartiles; dots, individual data points; whiskers, highest and lowest data points. n (in blue), number of stigmas. P values, two-tailed t-tests. Each experiment was repeated at least thrice with consistent results. Source data

**Extended Data Fig. 3. FER/ANJ signalling regulates stigmatic ROS during UI.**
a, b, Quantitative RT-PCR showing AS-BrFER1 treatment suppressed the expression of *BrFER1*(a) and promoted the growth of SI pollen and *B. oleracea* pollen in mature *B. rapa* stigmas and *B. vulgaris* pollen in bud stage (b) *B. rapa* stigmas. See Fig. 2a. c, d, AS-BrFER1 treatment suppressed SI and UI-induced ROS increase in mature and bud stage *B. rapa* stigmas at 10 MAP. e, f, The expression of *FER* and *SRK* in stigmas of *A. tha* (SI)*/fer-4* (e) and loss of FER in SI *A. thaliana* promoted the growth of SI pollen, *B. oleracea* pollen and *B. vulgaris* pollen in SI *A. thaliana* stigmas (f). See Fig. 2b. g, Loss of FER in SI *A. thaliana* suppressed SI and UI-induced ROS increase in *A. tha* (SI)*/fer-4* stigmas at 10 MAP. h, Domain structures of BrANJs proteins showing only BrANJ1 have intact extracellular and intracellular domain. i, j, AS-BrANJ1 treatment suppressed the expression of *BrANJ1*(i) and promoted the growth of SI pollen and *B. oleracea* pollen in mature *B. rapa* stigmas and *B. vulgaris* pollen in bud stage *B. rapa* stigmas (j). k, l, AS-BrANJ1 treatment suppressed SI and UI-induced ROS increase in mature (k) and bud stage (l) *B. rapa* stigmas at 10 MAP. Scale bars, Scale bars, 500 μm (c, d, j, k, l), 200 μm (g). Data bars (a, e, i): average ± SD. Average relative expression levels from three biological replicates of stigmas (two tailed t-test, n = 3). Box plots (b–d, f, g, j–l): centre line, median; box limits, lower and upper quartiles; dots, individual data points; whiskers, highest and lowest data points. n (in blue), number of stigmas. P values, two-tailed t-tests. Each experiment was repeated at least thrice with consistent results. Source data

**Extended Data Fig. 4. ROS are produced by RBOHs during UI.**
a, Quantitative RT-PCR showing AS-BrRBOHF treatment suppressed the expression of *BrRBOHF*. b, c, AS-BrRBOHF treatment suppressed SI and UI-induced ROS increase in mature (b) and bud-stage (c) *B. rapa* stigmas at 10 MAP. d, The RBOH enzymatic activity in mature *B. rapa* stigmas were increased after pollination with SI and UI pollen but decreased after pollination with CP pollen. e, AS-BrRBOHF treatment promoted the growth of SI pollen and *B. oleracea* pollen in mature and *B. vulgaris* pollen in bud-stage *B. rapa* stigmas. Scale bars, 500 μm. Data bar (a): average ± SD. Average relative expression levels from three biological replicates of stigmas (two tailed t-test, n = 3). Data bars (d): Average activity of ROS producing enzymes from three technical replicates of one stigma sample containing 100 stigmas (two tailed t-test, n = 3). Box plots (b, c, e): centre line, median; box limits, lower and upper quartiles; dots, individual data points; whiskers, highest and lowest data points. n (in blue), number of stigmas. P values, two-tailed t-tests. Each experiment was repeated at least thrice with consistent results unless otherwise specified. Source data

**Extended Data Fig. 5. SRK is correlated with stigmatic ROS during UI.**
a, b, BrSRK46 kinase domain (KD) interacts with BrFER1 (KD) in yeast two-hybrid assay (a) and BiFC (b). c, d, Protein extract from SI pollen and *B. oleracea* pollen (S₃₆), but not that from CP pollen (S₁₂), increased ROS in *B. rapa* stigmas (S₄₆) (c), and enhanced interaction between BrSRK46 and BrFER1 (d) in the pull-down assay. See Fig. 2c. e–g. ROS in *B. rapa* stigmas (S₄₆) were increased (e), and the BrSRK46-BrFER1 interaction enhanced in the pull-down (f) and Co-IP assays (g), by GST-BrSCR46, not GST-BrSCR12, which is active for S₁₂ stigma. The protein samples were derived from the same experiment and the blots were processed in parallel (d, f, g). For gel source data, see Supplementary Fig. 1. See Fig. 2d, e. h, i, Nitro blue tetrazolium staining or tobacco leaves and H₂DCFDA staining of protoplasts from tobacco leaves co-expressing BrFER1, BrRBOHD2, and BrSRK46 showing the enhancement of ROS by BrSCR46, not by BrSCR12. Scale bars, 500 μm (c, e); 50 μm (i). Data bars (d, f, g): average ± SD. Average relative intensities from three biological replicates of the blots shown in Fig. 2c–e (two tailed t-test, n = 3). Box plots (c, e, h, i): centre line, median; box limits, lower and upper quartiles; dots, individual data points; whiskers, highest and lowest data points. n (in blue), number of stigmas, tobacco leaves, protoplast samples. P values, two-tailed t-tests. Each experiment was repeated at least thrice with consistent results. Source data

**Extended Data Fig. 6. The relationship of FER-ROS signaling with MLPK and ARC1 during SI and UI.**
a, b, Quantitative RT-PCR showing AS-BrMLPK and AS-BrARC1 treatment suppressed the expression of *BrMLPK* and *BrARC1*, respectively, in mature *B. rapa* stigmas. c, d, AS-BrMLPK (c) and AS-BrARC1 (d) treatment promoted the growth of SI and UI pollen tubes in mature *B. rapa* stigmas (S₄₆). e, AS-BrMLPK treatment of mature *B. rapa* stigmas (S₄₆) inhibited SI and UI pollen-induced ROS increase. f, AS-BrARC1 treatment of mature *B. rapa* stigmas (S₄₆) did not affect SI and UI pollen-induced ROS increase. Scale bars, 500 μm. Data bars (a, b): average ± SD. Average relative expression levels from three biological replicates of stigmas (two tailed t-test, n = 3). Data bar: average ± s.d. Box plots (c–f): centre line, median; box limits, lower and upper quartiles; dots, individual data points; whiskers, highest and lowest data points. n (in blue), number of stigmas. P values, two-tailed t-tests. Each experiment was repeated at least thrice with consistent results. Source data

**Extended Data Fig. 7. Species-specific match of PCP-Bs and FER.**
a–c, When observed at 1.5 HAP, the reduced reproductive barrier to *B. rapa* and *B. oleracea* pollen in *A. thaliana fer* mutant (a, *srn*) and *rbohd* mutant (b, c) stigmas was obvious. See also Fig. 3c. d, Phylogenetic analysis of PCP-B genes from *A. thaliana*, *B. oleracea*, and *B. rapa*. We name BrapaB3, an ortholog of AtPCP-Bγ, BrPCP-B3 hereafter. e, Amino acid sequence alignment of PCP-Bs from *A. thaliana*, *B. oleracea*, and *B. rapa* shows the comparison of PCP-B sequences. They all shared a common pattern of seven or eight cysteines in the mature polypeptide. Red rectangle shows the conserved cysteines (C30, C32, C33 of AtPCP-B), mutation of which render a loss in the ability for stigmatic ROS reduction and pollen hydration. f, Synthetic AtPCP-Bγ and recombinant MBP-AtPCP-Bγ show similar function in reducing ROS of UP *A. thaliana* stigmas, but GST-BrPCP-B3 shows no effect in reducing ROS at 5 or 10 MAT. g, *B. rapa* stigmas and *A. thaliana* stigmas, which express divergent FER, responded species-preferentially to BrPCP-B3 in reducing ROS. h, AtPCP-Bγ was much faster than GST-BrPCP-B3 in reducing ROS of *A. thaliana* roots, which also express FER. i, GST-BrPCP-B3 was more effective in reducing ROS of *B. rapa* roots than that of *A. thaliana* roots. j, Quantified data of pull-down assay showing the inefficiency of GST-BrPCP-B3 to compete with AtPCP-Bγ in interaction with AtFER (ED)-FLAG. The protein samples were derived from the same experiment and the blots were processed in parallel. For gel source data, see Supplementary Fig.1. See also Fig. 3f. Scale bars, 200 μm (a–c, f, g) and roots (h, i). Data bar (j): average ± SD. Average relative intensities from three biological replicates of the blot represented on the left (two tailed t-test, n = 3). Box plots (a–c, f, g–i): centre line, median; box limits, lower and upper quartiles; dots, individual data points; whiskers, highest and lowest data points. n (in blue), number of stigmas or roots. P values, two-tailed t-tests. Each experiment was repeated at least thrice with consistent results. Source data

**Extended Data Fig. 8. Stigmatic NO is required for ROS reduction in *A. thaliana* stigmas during SC and UC.**
a, b, Confocal and wide-field imaging showing the increase of stigmatic NO at 5 MAP with SC pollen (a) and quantified data of SC-induced NO and pollen hydration in *A. thaliana* stigmas, both showed NO peaking at ~5 MAP (b). See Fig. 4a. c, d, FER-dependent elevation of NO in *A. thaliana* stigmas induced by pollen and At-PCP-Bγ .See Fig. 4d, e. e, AtPCP-Bγ was much faster than GST-BrPCP-B3 in increasing NO of *A. thaliana* roots. f, GST-BrPCP-B3 was more effective in increasing NO of *B. rapa* roots than that of *A. thaliana* roots. g, Roots of *fer-4* was not responsive to AtPCP-Bγ in inducing NO. h, i, (Left to right) cPTIO scavenged SC-induced NO, suppressed SC-induced ROS reduction, and inhibited SC pollen hydration and tube growth in *A. thaliana* stigmas. j–n, Relative to WT, *noa1* stigmas showed lower NO and higher ROS levels, and slower hydration of SC (WT *A. thaliana*) pollen and pollen tube growth (j, k, l). Relative to WT, *hot5-4* stigmas showed faster NO and lower ROS levels, and faster hydration of SC (WT *A. thaliana*) pollen and pollen tube growth (j, m, n). o, p, Mutations in NOA1, *noa1* (o) and GSNOR, *hot5-4* (p), enhanced and reduced, respectively, the reproductive barrier to *B. rapa* or *B. oleracea* pollen in *A. thaliana* stigmas at 1.5 HAP. Scale bars, 200 μm (a, h, j, o, p) and roots (e–g); 50 μm for pollen (h, j). Box plots (a–p): centre line, median; box limits, lower and upper quartiles; dots, individual data points; whiskers, highest and lowest data points. n (in blue), number of stigmas, pollen grains or roots. P values, two-tailed t-tests. Each experiment was repeated at least thrice with consistent results. Source data

**Extended Data Fig. 9. The inverse relationship of NO and ROS and nitrosation of BrFER1 and RBOHs in *B. rapa* stigmas.**
a, b, Intraspecific CP pollen (2^nd row), but not SI and *B. oleracea* (UI) pollen, induced NO in *B. rapa* stigmas. c–e, Scavenging NO increased ROS (c) and RBOH enzymatic activity of UP *B.rapa* stigmas (d), and inhibited the growth of CP pollen tubes in *B. rapa* stigmas (e). f–h, GSNO treatment increased NO (f), reduced ROS (f) and RBOH enzymatic activity of UP *B. rapa* stigmas (g), and promoted the growth of SI or *B. oleracea* pollen in mature or *B. vulgaris* polen in bud-stage *B. rapa* stigmas (h). i, BrFER1(KD), not BrFER1 (ED), was nitrosated by GSNO. See also Fig. 4f. j, k, LC-MS spectrum showing the nitrosated cysteine residues in BrFER1 protein. l, FER amino acid sequence alignment showing the nitrosated cysteines, Cys730 and Cys752 (*) are evolutionarily conserved. m, Pull-down assay shows GSNO treatment induced quantitative inhibition of MBP-BrFER1(KD) with GST-BrROP2 complex. See also Fig. 4h. n, The nitrosomimetic mutation, Cys730W, reduced the amount of BrFER1 that was pulled down by GFP-BrROP2. o, Phylogenetic analysis of BrRBOHs and AtRBOHD. BrRBOHD1 and BrRBOHD2 are closer to AtRBOHD than other BrRBOHs. p–r, RBOHD was nitrosated by NO. *In vitro* nitrosation of BrRBOHD1 by GSNO (p); CP-induced nitrosation of GFP-AtRBOHD protein from *A. thaliana* stigmas (q), and LC–MS spectra showing nitrosation of Cys891 of GST-BrRBOHD1 (CT, C-terminal) (r). Nitrosated amino acid residues were labelled with TMT. Proteins were representative of more than three independent preparations. s, t, GST-BrRBOHD2 (CT) (s) and GST-BrRBOHF (CT) (t) proteins were nitrosated *in vitro*. The protein samples were derived from the same experiment and the blots were processed in parallel (i, m, n, p, q, s, t). For gel source data, see Supplementary Fig. 1. Scale bars, 500 μm. Line chart (b): average ± SD. Average relative NO intensities from three biological replicates of stigmas shown in a (two tailed t-test, n = 3). Data bars (d, g): Average activity of ROS producing enzymes from three technical replicates of one stigma sample containing 100 stigmas (two tailed t-test, n = 3). Data bar (m, n): average ± SD. Average relative intensities from three biological replicates of stigmas in (a) and blots in Fig 4h and in (n) (two tailed t-test, n = 3). Box plots (c, e, f, h): centre line, median; box limits, lower and upper quartiles; dots, individual data points; whiskers, highest and lowest data points. n (in blue), number of stigmas. P values, two-tailed t-tests. Each experiment was repeated at least thrice with consistent results unless otherwise specified. Source data

**Extended Data Fig. 10. Breaking interspecific barriers in *Brassica* crops.**
a, b, Reducing ROS by Na-SA or increasing NO by GSNO increased the number of enlarged ovules in the pod of ♀*B. rapa* × ♂*B. oleracea* or ♀*B. rapa* × ♂*B. vulgaris*. See Fig. 5a. c, Suppressing the expression of *BrSRK46*, *BrBrFER1*, or *BrRBOHF* by AS-ODNs increased the number of enlarged ovules in the pod of ♀*B. rapa* × ♂*B. oleracea* or ♀*B. rapa* × ♂*B. vulgaris*. See also Fig. 5b. d, *B. oleracea* pollen tubes or *B. vulgaris* pollen tubes in *B. rapa* pistils. The reduced number of enlarged ♀*B. rapa* × ♂*B. vulgaris* ovules is possibly due to fewer *B. vulgaris* pollen tubes had exited the transmitting tissue and targeted the *B. rapa* ovules due to additional barriers based on their evolutionary distance (Fig. 1a). Arrowheads point to bundles of pollen tubes in the transmitting tissue, stars point to ovules penetrated by a pollen tube. Scale bars, 100 μm (d). Box plots (a–c): centre line, median; box limits, lower and upper quartiles; dots, individual data points; whiskers, highest and lowest data points. n (in blue), number of pistils. P values, two-tailed t-tests. Each experiment was repeated at least thrice with consistent results. Source data

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Pollen-stigma incompatibility within and between species: Tread lightly, sedate the dogs, and don't wake the guards!
Tran TC, Lenhard M. Tran TC, et al. Dev Cell. 2023 Mar 13;58(5):335-337. doi: 10.1016/j.devcel.2023.02.010. Dev Cell. 2023. PMID: 36917929

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References

1. Broz AK, Bedinger PA. Pollen–pistil interactions as reproductive barriers. Annu. Rev. Plant Biol. 2021;72:615–639. doi: 10.1146/annurev-arplant-080620-102159. - DOI - PubMed
1. Jany E, Nelles H, Goring DR. The molecular and cellular regulation of Brassicaceae self-incompatibility and self-pollen rejection. Int. Rev. Cell Mol. Biol. 2019;343:1–35. doi: 10.1016/bs.ircmb.2018.05.011. - DOI - PubMed
1. Nasrallah JB. Self-incompatibility in the Brassicaceae: regulation and mechanism of self-recognition. Curr. Top. Dev. Biol. 2019;131:435–452. doi: 10.1016/bs.ctdb.2018.10.002. - DOI - PubMed
1. Lewis D, Crowe LK. Unilateral incompatibility in flowering plants. Heredity. 1958;12:233–256. doi: 10.1038/hdy.1958.26. - DOI
1. Kitashiba H, Nasrallah JB. Self-incompatibility in Brassicaceae crops: lessons for interspecific incompatibility. Breed Sci. 2014;64:23–37. doi: 10.1270/jsbbs.64.23. - DOI - PMC - PubMed

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[1] Broz AK, Bedinger PA. Pollen–pistil interactions as reproductive barriers. Annu. Rev. Plant Biol. 2021;72:615–639. doi: 10.1146/annurev-arplant-080620-102159. - DOI - PubMed

[2] Broz AK, Bedinger PA. Pollen–pistil interactions as reproductive barriers. Annu. Rev. Plant Biol. 2021;72:615–639. doi: 10.1146/annurev-arplant-080620-102159. - DOI - PubMed

[3] Jany E, Nelles H, Goring DR. The molecular and cellular regulation of Brassicaceae self-incompatibility and self-pollen rejection. Int. Rev. Cell Mol. Biol. 2019;343:1–35. doi: 10.1016/bs.ircmb.2018.05.011. - DOI - PubMed

[4] Jany E, Nelles H, Goring DR. The molecular and cellular regulation of Brassicaceae self-incompatibility and self-pollen rejection. Int. Rev. Cell Mol. Biol. 2019;343:1–35. doi: 10.1016/bs.ircmb.2018.05.011. - DOI - PubMed

[5] Nasrallah JB. Self-incompatibility in the Brassicaceae: regulation and mechanism of self-recognition. Curr. Top. Dev. Biol. 2019;131:435–452. doi: 10.1016/bs.ctdb.2018.10.002. - DOI - PubMed

[6] Nasrallah JB. Self-incompatibility in the Brassicaceae: regulation and mechanism of self-recognition. Curr. Top. Dev. Biol. 2019;131:435–452. doi: 10.1016/bs.ctdb.2018.10.002. - DOI - PubMed

[7] Lewis D, Crowe LK. Unilateral incompatibility in flowering plants. Heredity. 1958;12:233–256. doi: 10.1038/hdy.1958.26. - DOI

[8] Lewis D, Crowe LK. Unilateral incompatibility in flowering plants. Heredity. 1958;12:233–256. doi: 10.1038/hdy.1958.26. - DOI

[9] Kitashiba H, Nasrallah JB. Self-incompatibility in Brassicaceae crops: lessons for interspecific incompatibility. Breed Sci. 2014;64:23–37. doi: 10.1270/jsbbs.64.23. - DOI - PMC - PubMed

[10] Kitashiba H, Nasrallah JB. Self-incompatibility in Brassicaceae crops: lessons for interspecific incompatibility. Breed Sci. 2014;64:23–37. doi: 10.1270/jsbbs.64.23. - DOI - PMC - PubMed

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Stigma receptors control intraspecies and interspecies barriers in Brassicaceae

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