Molecular Mechanism Of Receptor Kinase FERONIA-regulated Self-incompatibility In Brassica Rapa

The heterosis of Brassica rapa is obvious and F1 hybrids were mainly produced from self-incompatibility（SI）lines.Parents with strong SI are beneficial to improve the hybridization rate but self-pollination breeding is very difficult.This results in that strong SI lines with excellent traits cannot be used as parents greatly limiting parental selection and matching.Therefore,it is urgent to systematically analyze the SI regulation mechanism of Brassica rapa to improve the breeding efficiency of SI lines and expand the selection range of parents.The study found that the SI reaction of Brassica rapa is regulated by receptor kinase FERONIA-mediated reactive oxygen species（ROS）.Stigmatic ROS increased after self-pollination to inhibit the growth of self-pollination,but decreased after cross-pollination to promote the growth of cross-pollination.The main findings were as follows:1.Stigmatic ROS increase after self-pollination and decrease after cross-pollinationStigmas stained at different times after self-pollination and cross-pollination with H₂DCFDA.It was found that stigmatic ROS increased rapidly at 5 min after self-pollination and reached the highest level at 30 min which was three times that of unpollinated stigmas.And the stigmatic ROS continued to decrease from 5 to 30 min after cross-pollination.At the same time,it was found that the time points of the increase and decrease of stigmatic ROS were highly consistent with the time points of self-pollen inhibition and cross-pollen hydration,germination,and entry into the stigmas.These results suggest that the increase and decrease of stigmatic ROS is a specific response induced by SI and compatible pollen（CP）respectively.AS-ODNs were added to the stigmas in vitro culture system to inhibit the expression of SRK which is a pistil determinant recognized by self-pollen.The results showed that AS-Br SRK46 treatment specifically decreased the expression of Br SRK46 in the stigmas of S₄₆haplotype.It also effectively broke down the SI and promoted the growth of self-pollen.These results indicate that AS-ODN is an effective method to inhibit the expression of specific genes in the stigmas of Brassica rapa.More importantly,AS-Br SRK46 treatment effectively prevented the increase of stigmatic ROS caused by self-pollination,indicating that the increase of stigmatic ROS after self-pollination is dependent on self-pollen recognition.2.High levels of stigmatic ROS inhibit germination and growth of self-pollenTo study the relationship between stigmatic ROS and SI,different kinds of ROS scavengers were added to change the content of stigmatic ROS in vitro culture system.It was found that superoxide anion scavengers Cu Cl₂and Tiron,hydrogen peroxide scavenger KI,and hydroxyl radical scavenger sodium benzoate significantly reduced stigmatic ROS and effectively broke down SI in a concentration-dependent manner indicating that stigmatic ROS is necessary for maintaining SI.On the other hand,when superoxide anion producer X/XO or hydrogen peroxide producer Fe SO₄were applied to stigmas both of them increased stigmatic ROS and inhibited the growth of compatible pollen suggesting that stigmatic ROS was a sufficient condition for inhibiting pollen growth.These results suggest that stigmatic ROS is a necessary and sufficient factor for inhibiting self-pollen.To further investigate the effect of high levels of stigmatic ROS on self-pollen,the study invented the stigma transfer experiment.In the stigma transfer experiment,self-pollinated stigmas at 0 min,15 min,30 min,60 min were respectively transferred to the medium containing ROS scavenger Cu Cl₂and the growth state of the self-pollinated pollen was observed.The results showed that the growth of self-pollen tubes was less inhibited in the stigmas that were transferred immediately after self-pollination,while the growth of self-pollen tube was severely inhibited in the stigmas that were transferred from 15 to 30 min after self-pollination.Self-pollens all lost the ability of regermination that were transferred at 60min after self-pollination.The results indicate that ROS induced by self-pollen causes irreversible damage to itself.The study also examined the change of ROS in dual self-and cross-pollination on a single stigma,mimicking a natural pollination.Dual self-and cross-pollinations at the same time（0 min）or 15 min apart both showed the absence of self-pollen tubes at the SI half and bundles of cross-pollen tubes penetrating the CP half of each stigma.Moreover,both self-and cross-pollination induced changes in stigmatic ROS resulting in higher levels of ROS at the SI half and lower levels of ROS at the CP half.Consistently,significantly higher or lower levels of ROS were also observed at the SI half or the CP half,respectively than the unpollinated half of each stigma in these assays.These results indicate that when self-and cross-pollen landed on the same stigma at the same time,self-pollen did not affect the germination and growth of cross-pollen.3.The FERONIA receptor kinase-Rac/Rops-Rbohs signaling pathway regulates the SI responseTreating stigmas with DPI that is a specific inhibitor of NADPH oxidases effectively reduced stigmatic ROS and broke down SI,indicating that NADPH oxidases are involved in regulating the synthesis of stigmatic ROS in SI.NADPH oxidase-encoding genes BrRbohD1,BrRbohD2,Br Rboh F,and Br Rboh I in Brassica rapa were the most abundantly expressed in the stigmas.Inhibition of these four Br Rbohs with corresponding AS-ONDs effectively reduced stigmatic ROS,broke down SI and promoted self-pollen growth.Furthermore,treating stigmas of other cruciferous plants such as radish（Raphanus sativus）and ornamental kale（B.oleracea var.acephala）with DPI also reduced stigmatic ROS and broke down SI,indicating that NADPH oxidases-regulated ROS production could be a conservative and common mechanism adopted by the Brassicaceae to inhibit self-pollen.Self-and cross-pollination caused increase and decrease of stigmatic ROS at 1 min and 3min.NADPH-oxidases activity of stigma was significantly increased or decreased at 5 min after self-or cross-pollination,suggesting that NADPH-oxidase activity is tightly controlled.Adding GDP,an inhibitor of Rac/Rop GTPase,to the stigma in vitro culture system could effectively reduce stigmatic ROS and break down SI,indicating that Rac/Rop GTPase is involved in regulating SI.Br ROP2 was highly expressed Br ROP in the stigma of Brassica rapa.AS-Br ROP2 treatment could reduce stigmatic ROS and break down SI.Yeast two-hybrid and protein pull-down assay showed that Br ROP2 interacted with Br Rboh F,indicating that Br ROP2 is the major regulator of NADPH oxidase in the process of SI.Protein pull-down assay revealed that Br ROP2 interacted with Br FER1.Treating stigmas with AS-Br FER1 in Brassica rapa reduced stigmatic ROS and broke down SI,suggesting that FERONIA receptor kinase regulates stigmatic ROS production by Rac/Rop-NADPH oxidase.The above experiments show that stigmatic ROS is generated through the FERONIA receptor kinase-Rac/Rops-Rbohs signaling pathway.4.FERONIA receptor kinase-Rac/Rops-Rbohs signaling pathway regulates compatible pollination responsesWhen stigmas were treated with AS-Br FER1,AS-Br ROP2,and AS-Br Rboh F in Brassica rapa respectively,it was found that reducing the expression of three genes also reduced ROS in unpollinated stigmas.It was further found that treatment of stigmas in Brassica rapa with AS-Br FER1,AS-Br ROP2,AS-Br Rboh F after cross-pollination or direct self-pollination in the stigmas in fer and rboh D mutants of Arabidopsis significantly promoted the germination and growth of compatible pollen.Combined with the decrease of stigmatic ROS after cross-pollination,the above results indicate that production of ROS by FERONIA receptor kinase-Rac/Rops-Rbohs signaling pathway also has a certain inhibitory effect on compatible pollen.In this study,the molecular mechanism of ROS regulating SI was analyzed through the FERONIA receptor kinase-Rac/Rops-Rbohs signaling module,which provided new insights for further analysis of the molecular mechanism of SI.And it also has important theoretical implications for the improvement of SI breeding technology.