Identification Of The G??s Mutants And Functional Studies Of RGB1 In Rice Seedlings'Growth And Development

It is well known that the heterotrimeric G proteins are important signal mediators in response to numerous external signals in eukaryotic cells.These heterotrimeric G proteins usually refer to the G proteins.So far,more than 20 genes for a subunit,5 genes for ? subunit,and 12 genes for y subunit have been identified in mammalian genomes,which can be combined to form more than one thousand G proteins.In clearly contrast to the G proteins in animal cells,there are usually only one to five plant G proteins.For example,Arabidopsis G proteins contain an a subunit(AtGPA1),a ? subunit(AGB1)and three y subunits(AGG1,AGG2 and AGG3).Function analysis of Arabidopsis Ga(GPA1)and G?(AGB1)distinctly indicates that G proteins mediate the entire developmental process.There are an a subunit(RGA1),a ? subunit(RGB 1)and five y subunits(RGG1,RGG2,OsGGC2,GS3,qPE9-1)of G proteins in rice.The loss-of-function allele of RGA1 results in dwarfing,wider stems,short and wide leaves,erect ears,small grains and non-elongation between the second segments caused by inhibition and gibberellin signaling.GS3 and qPE9-1/DEP1 are the major QTLs for grain yield in rice.GS3 which is highly expressed in young ears mainly regulates grain size.qPE9-1/DEP1 which is expressed in most tissues and highly expressed in inflorescence meristem of rice mainly regulates ear length.qPE9-1/DEP1 and OsGGC2 are positively regulating seed size.The y subunit role is dependent on the G protein a subunit and the? subunit.However,the loss-of-function mutations in RGB1 have not been reported.Moreover,the research on the mechanism of G-protein-regulated plant growth and development is mostly concentrated on Arabidopsis,there are few studies on how rice G protein regulates rice growth and development.In this study,all members of the rice G protein ? subunit and y subunit were knocked out by the CRISPR/Cas9 system.In order to ensure the consistency of the research background,the variety Zhonghua 11 was used for rice transgenic plants.The obtained mutants were used to analyze their functions.The main results are as follows:Part 1 Identification and phenotypic analysis of a loss-of-function mutation in RGB11.The binary expression vector p35S::Cas9-RGB1 was constructed and transformed into the japonica rice 'Zhonghua 11'(ZH11)via the Agrobacterium-mediated transformation method.The sequencing results of the RGB1 T0 generation showed that 6 plants were edited in the 17 transgenic lines.One of the edited plant was a homozygous mutant(-3 bp)and the rest were RGB1 heterozygous mutants.Besides the wild type and heterozygous mutants,the mutants of '-3bp'and '+3bp' in RGBI target loci were found in Ti generations.The RGB1 heterozygous individual plants had normal pollen fertility and the normal germ and radicle in the embryo.2.To identify the genotype,seeds and grains(de-hulled)from the different RGB1 heterozygous mutant lines in T2 generations were germinated and grown for a week.Germination percentage of the de-hulled grains and seeds was from 58.33%to 65.0%and from 72.57%to 75.69%,respectively.Genomic DNA was extracted from individual non-germinated seeds,and the DNA fragment including the target sequence was amplified and sequenced.Three types of RGB1 loss-of-function mutant lines named rgbl-1(+1 bp),rgbl-2(-1 bp)and rgbl-3(-11 bp)were identified.All three mutants were unable to produce roots and shoots even in 5-day-old seedlings(after germination),s?ggesting that loss-of-function mutants in RGB1 are seedling-lethal.3.To identify the cause of seedling lethality in the rice rgbl mutants,we analyzed the phenotype of the wild type and rgbl seedlings at different stages during early post-embryogenic development.Seedlings of the three rgbl mutant lines stopped growing on the first day after germination.From 48 h to 96 h,the color of the rgbl-1 mutant radicles changed from yellowish to brown and then to dark brown,while the wild type radicles were always white.Callus induction experiments showed that embryo-induced callus of the rgbl mutant was inhibited and the elongation of the shoots was significantly inhibited compared to the control.The embryos in the rgbl mutant were longitudinally cut and found to be brown in the radicle,which was different from the wild type.This result indicates that RGB1 is a key gene for rice seedlings' development and callus induction.4.The promoter activity and mRNA expression of RGB 1 was examined during germination and postgermination.The results showed that RGB1 expression was weak at the germination stage and increased with the development of seedlings.It was specifically expressed in the vascular tissues,root epidermis and hypocotyls of the embryos.During the development of the root,RGB1 mRNA was detected in the metaxylem of the primary root,the adventitious root,the beginning of the lateral root,the meristem of the lateral root,and the epidermis and vascular tissue of the mature part of the primary root.It appears that the primary root stopped growing one day after germination due to the arrested cell development.5.The development of the shoot-root axis in the rgb1-1 mutant was suppressed in 2-day-old and 3-day-old seedlings.The length of the shoot-root axis in the rgb1-1 mutant was significantly shorter than the wild type.Microscopic observation showed that cell elongation was significantly reduced in the longitudinal plane in the mutant shoot-root axis.In addition to the limited nubmer of shoots and roots,the development of the embryo shoot-root axis was suppressed in the rgbl mutants.Part 2.RGB1 invovles in auxin,cytokinin,and brassinsteroid signaling pathway to promote seedling growth and development1.To investigate the molecular function of the RGB 1 protein in seedling development,we used RNA sequencing(RNA-seq)to analyze the transcriptome differences in the embryos of the wild type and the rgb1-1 mutants in 3-day-old seedlings(Figure 7e).Compared to the control,a total of 9,743 differentially expressed genes were detected in rgb1-1,of which 52.54%(5,119 genes)were down-regulated and 47.46%(4,624 genes)showed up-regulated expression.KEGG enrichment analysis showed that these genes were significantly enriched in phenylpropanoid biosynthesis,DNA replication,diterpenoid biosynthesis,ribosome,and plant hormone signal transduction.We examined the expression of genes specifically expressed in the SAM,the vascular tissues,and the root in the rgb1-1 mutant and the wild type via heatmapping.The heatmaps showed that most(81.8%)of the genes were significantly downregulated in the rgb1-1 mutants compared to the wild type.In the rgb1-1 vascular tissues,the auxin transport genes PIN1A,PIN1B,PIN1C,PIN2,and PIN3A,the auxin responsive genes OsARF11,OsARF16,and OsARF19,and the cytokinin responsive A-type genes RR1,RR2,RR3,RR4,RR5,RR6,and RR7 were largely downregulated,while expression of the cytokinin degradation genes CKX4 and CKX9 were upregulated.In rgb1-1 roots,the GSK2,BAK1,and BRI1 genes in the BR pathway and the auxin responsive genes IAA11,IAA13,IAA23,and IAA24 were significantly downregulated.2.Pearson's correlation test showed a strong correlation between the qRT-PCR and RNA-seq data(r=0.85;p<0.05).This confirmed the reliablity of the qRT-PCR and RNA-seq results obtained in this study.In addition to verifying the RNA-seq results,we further investigated the expression of genes related to auxin signaling and auxin content in 2-day-old and 3-day-old seedlings.OsARF4,OsARF9,and OsARF13 were up regulated more than 3-fold in the rgbl mutants compared to the wild type in 2-day-old seedlings,and OsARF11 and OsARF19 were downregulated more than 3-fold in 3-day-old seedlings(Figure 7g).The expression of OsIAA2,OsIAA7,OsIAA10,OsIAA13,OsIAA16,OsIAA17,OsIAA24,OsIAA26,OsIAA27 was significantly reduced in the rgbl mutant versus the wild type more than 5-fold on day 3 after germination.In the rgbl mutants,OsPINId,OsPIN2,and OsPIN5C were downregulated both in 2-day-old and 3-day-old seedlings and more than 30-fold in the 3-day-old seedlings3.IAA concentration in the rgbl mutants were significantly less than in WT in 2-day-old seedlings but significantly more than in WT in 3-day-old seedlings.In addition to IAA,we also measured the cytokinin and BR concentration in the rgbl mutants and the control.The IPA content was undetectable in the rgbl mutants but was present in the wild type,and the content of BL in the rgb1 mutant was>10-fold that of the control in the embryos of 3-day-old seedlings.These results indicate that RGB 1 integrates the auxin,cytokinin,and brassinsteroid pathways to regulate seedling growth in rice.Part 3.Identification and phenotypic analysis of the mutants of Gy in rice.1.To study the function of the G protein gamma subunits RGG1,RGG2,GS3,qPE9-1,OsGGC2,the binary vectors such as p2×35S::Cas9-RGG1,p2×35S::Cas9-RGG2,p2×35S::Cas9-RGG1&RGG2,p2 x35S::Cas9-GS3,p2×35S::Cas9-qPE9-1,p2×35S::Cas9-OsGGC2 were constructed via the CRISPR/Cas9 system.The vectors were transformed into the Zhonghua11 by Agrobacterium-mediated method and the positive transgenic lines were obtained in the To generations.2.After multi-generation sequencing analysis,the homozygous mutants of rggl,gs3,qpe9-1 and ggc2 were isolated.The rggl mutants were named rggl-1(-7bp),rggl-2(-2bp),rggl-3(1SNP,-16 bp),rggl-4(+1 bp);the gs3 mutants were named gs3-1(-1 bp),gs3-2(-2 bp),gs3-3(-34 bp),gs3-4(+1bp);the qpe9-1 mutants were namedqpe9-1-1(-lbp),qpe9-1-2(-2bp),qpe9-1-3(-5bp),qpe9-1-4(-7 bp),qpe9-1-5(+1 bp)and the ggc2 mutant was named ggc2-1(-41 bp).3.We investigate the agronomic traits of rggl,gs3,qpe9-1 and ggc2 mutant lines and the control.The results showed that the plant height,ear length,flag leaf length,flag leaf width and tiller number of different types of the rgg1 mutants were not significantly different from the control,indicating that the loss of function of RGG1 did not affect rice vegetative growth under natural conditions.The qpe9-1 showed depressed plant height,the flag leaves and the panicles.It indicated that qPE9-1 not only affected the reproductive growth but also the vegetative growth.The plant height of the ggc2 mutant was not significantly different from the control,indicating that ggc2 did not affect the rice vegetative growth.4.The sequencing results of the RGG2,RGG1RGG2 T0 generatioin strains showed that 3 of the 8 RGG2 transgenic lines and 6 of 34 RGG1RGG2 transgenic lines were edited.The genotypes of all the surviving seedlings of the RGG2,RGG1RGG2 Ti generations were heterozygous mutants and the wide type.The rgg2 single and the rgg1rgg2 double mutants may be lethal.5.Combined with the phenotypic characteristics of the RGB 1 mutants,the seeds and grains(de-hulled)from the different RGG1,RGG1RGG2 heterozygous mutant lines in T2 generations were germinated and grown for a week.There were a clear phenotypic separation in RGG2 and RGG1RGG2 heterozygous mutant seedlings.About 25%of the seedlings showed growth inhibition in RGG2 heterozygous mutant lines,browning at the leaf pillow and reduced root mass.About 25%of the seedlings of the RGG1RGG2 heterotrimeric mutants could not produce the roots and the leaf growth stopped at the 4th day after germination.Genomic DNA was extracted from the individual suppressed seedlings and the DNA fragment including the RGG1 and RGG2 target loci were amplified and sequenced.One type of RGG2 loss-of-function mutant line named rgg2-1(-2bp)and three types of RGG1RGG2 loss-of-function mutant lines named rgglrgg2-1(-7bp,+1bp),rgg1rgg2-2(1bp SNIP-16bp,-1bp),rgg1rgg2-3(+1bp,-lbp).The rgg2-1 mutants were lethal in 30-day-old seedlings.And all three rgglrgg2 mutants were unable to grow after 4 days after germination,suggesting that loss-of-function mutants in RGG1RGG2 are seedling-lethal.