Fertility Restoration Of TGMS Lines By Delayed Callose Degradation During Pollen Development

The increasing global warming and the natural disaster including heat-waves,droughts,floods,cold-waves and other weather extremes which occurs frequently across the globe have bring a negatively inestimable impact on the environment and the development of human society and economy.Among them,the development of agricultural production and food security are deeply influenced by the global warming.An increasing occurrence of extreme weather events makes the improvement of agricultural production by increasing productivity and reducing crop losses due to environmental factors of significant importance for feeding a growing global population.Accelerated modern agriculture improvement must leverage naturally evolved elite traits and transformative engineering to establish the sustainable and resilient agricultural production systems for the caloric needs of an increasing global population.Thus,key to addressing the challenge is a better understanding of the molecular pathways by which plants dynamically respond to environment,while maintaining efficiency of growth and nutrient use.In our country,the application of temperature-sensitive genic male sterility（TGMS）in two-line hybrid breeding system enables a drastic increase in the yields of crops.Frequent occurrence of extreme weather events like the sudden temperature dropping in summer has bring serious negative impacts on two-line hybrid breeding.Thus,a further understanding of the molecular mechanism by which TGMS lines respond to different temperatures and transition their fertility is of paramount importance for improving their elite traits at nucleotide-scale precision to cope with the potential food crisis resulting from global warming.Despite the fact that several TGMS genes have been reported and analyzed,the general mechanism of how low temperature could recover the fertility of TGMS lines has long been a mystery.It has been reported that low ambient temperature recover the fertility of various TGMS lines on account of slowing of development.In this study,a genetic screening for restorer of rvms-2（res）was carried out to explore the mechanism of the fertility restoration of TGMS lines.The res3 rvms-2 line exhibits increased fertility as indicated by longer siliques with seeds compared to rvms-2 under 24 ℃.One of the res candidates,res3,was identified at At1g33430,previously named as UNEVEN PATTERRN OF EXINE1/KAONASHI4（UPEX1/KNS4）,which encodes an arabinogalactanβ-（1,3）-galactosyltransferase.A wild-type coding sequences of RES3downstream of its own promoter were fused to VENUS（Pro RES3:VENUS-RES3）to determine the subcellular localization of RES3.The reporter construct was introduced into wild-type Arabidopsis plants,and imaging showed the VENUS fluorescence was specially detected in the tapetal cells ranging from stage 7 to stage 10.Cytological data showed that the mutation in RES3/UPEX1 leads to dramatically swollen tapetal cells and a delay in tetrad callose degradation.Studies have shown that ANTHER-SPECIFIC PROTEIN6（A6）is involved in the degradation of tetrad callose wall.Furthermore,the secretion of A6 proteins was blocked in res3,which resulting in the delayed degradation of tetrad callose.These data suggested that the secretory function of the tapetum was partly affected resulting from the mutation in RES3/UPEX1.A construct containing the coding DNA sequence of A6 driven by the A9 promoter（proA9::A6）which would advance the expression ofβ-（1,3）-glucanase was transferred into wild-type and res3 rvms-2 plants.The fertility of T₁ transformants in wild-type background was unaffected,whereas those in res3 rvms-2 mutants were back to sterile.In addition,the premature expression of A6 could not eliminate the effect of res3 on tapetum morphology.Further genetic data showed that a delay in tetrad callose degradation resulting from a6s also could restore the fertility of rvms-2.Thus,we proposed that the delay in tetrad callose degradation resulting from res3 is responsible for the fertility restoration of rvms-2.Semi-thin sections and TEM observation showed that the delay in tetrad callose degradation provides valuable time to accumulate sporopollenin for rvms-2 microspores,the relatively intact nexine structure allows microspores to reach maturity.In addition to rvms-2,the fertility of multiple known TGMS lines including acos5,cyp703a2,abcg26 and cals5-2,could also by restored by delayed callose degradation.The exine layer of these TGMS lines are defective,and the ruptured microspore fails to form the intine layer under normal condition.The delayed tetrad callose degradation leads to the formation of a intact nexine layer,which is vital for intine formation,before the release of microspores.These microspores could reach maturity with the protection of the intine layer.The core regulatory pathway of tapetal development and function including the key regulators of DYSFUNCTIONAL TAPETUM1（DYT1）,DEFECTIVE IN TAPETAL DEVELOPMENT AND FUNCTION1（TDF1）,ABORTED MICROSPORES（AMS）,MALE STERILE188（MS188）and MALE STERILE1（MS1）has been well established.qRT-PCR analysis showed that the expression of RES3 was significantly decreased in dyt1,tdf1 and ams.The Electrophoretic mobility shift assay（EMSA）and Chromatin immunoprecipitation（ChIP）suggested that AMS could directly bind to the E-box region（CANNTG）in the RES3 promoter.We further found that the 35S promoter-driven AMS expression significantly induced the activity of RES3 promoter in Arabidopsis mesophyll protoplasts.These results suggest that AMS directly regulates the expression of RES3.We proposed that RES3/UPEX1 may catalyze the glycosylation of A6s to facilitate its secretion from tapetum to locule for rapid tetrad callose degradation after exine establishment.The accurate synthesis and rapid secretion of callase（A6s）are crucial for tetrad callose degradation,so that the preliminary exine could develop into intact pollen wall after the microspore release.Collectively,our study reveal that the delayed degeneration of callose wall may broadly compensate the defects of pollen wall formation to prevent pollen from breakdown in various TGMS mutants,and provide further understanding of the general mechanism underlying the sterility-fertility conversion of TGMS lines.In addition,we proposed that AMS is also required for the establishment of tapetum secretion function which is of importance to demonstrate the roles of transcriptional regulatory pathway（DYT1-TDF1-AMS-MS188-MS1）in pollen development.