Functional Characterization Of Tomato Curl3 Gene In Metabolism And Development

Brassinosteroids(BRs)play a critical role in plant growth and development;however,genetic evidence for the BR-signaling mediated regulation of plant growth remains elusive in fleshy crop species.These physiological responses are mediated through a network of molecular interactions between the BR signalosome and other plant hormones.The interactions between hormones,environmental signals,and developmental programs are so complex that the description and modeling of the whole system is very challenging.The research is of great significance in understanding the role and mechanisms of BR signaling in controlling the vast area of plant physiology and biochemistry and to highlight the valuable potential of BR signaling in agricultural production.In this study,to elucidate the possible effects of BR signaling in the growth and development of tomato plant we developed a CRISPR-mediated BR-insensitive curl3 mutant but due to its highly pleiotropic phenotype,we further obtained a weak(intermediate)recessive allele mutant(abs1)at the same cu3 locus,with less exaggerated phenotype as compared to the curl3 mutant.Here,we clarified the function of Sl Curl3,the key BR signaling gene in tomato,in the regulation of plant growth and architecture,primary metabolite regulation,photosynthesis,phytohormone homeostasis,floral organ and fruit development.The main results are summarized as follows.1)In this study,a BRI1(BRASSINOSTEROID INSENSITIVE 1)homologous gene,Curl3 was isolated from Solanum lycopersicum and CRISPR-mediated knockdown mutants were created and functionally characterized.The loss-of-function of the BR signaling mutant showed significant alterations in plant physiology and biochemistry.curl3 mutant exhibited extremely abridged plant height,curled leaf structure,and altered biochemical and metabolic pathway activities.Moreover,phytohormones such as abscisic acid contents were upregulated significantly while a significant decrease in cytokinin active form zeatin was observed in BRinsensitive plants.As for primary metabolites including sugars and acids.Sucrose,fructose and glucose decreased significantly in BR-insensitive mutant.In contrast,there was a considerable increase in citric acid content in the young leaves of the curl3 mutant.Surprisingly,BRinsensitivity increased the concentration of free amino acids i.e.valine,phenylalanine,glutamine,and asparagine as a mechanism to maintain plant growth balance.In conclusion,these discoveries provide a new and clearer understanding of metabolic changes in response to BRinsensitivity.2)Brassinolide(BR)enhances the efficiency of photosynthetic machinery through the activation of metabolic pathways,photochemistry,and ribulose activity.However,the potential mechanism mediating these processes through BR signaling still needed to be discussed.In this study,we found the concealed BR-signaling mediated effects on tomato leaf morphophysiological and biochemical traits including chlorophyll accumulation,and photosynthetic efficiency.Under controlled conditions,the altered brassinolide sensitivity1(abs1)mutant showed decreased leaf area and biomass as a result of substantially decreased vascularization and epidermal cell size.abs1 mutant displayed significantly decreased chlorophyll accumulation and suppression in photosynthetic machinery components like photochemical quenching,electron transport rate,the maximal quantum yield of PSII photochemistry,and net photosynthetic rate.Whereas an increase in minimal fluorescence yield and non-photochemical quenching,suggests that abs1 mutant leaf has weakened abilities to harvest and transfer light energy.Moreover,the transcriptome analysis revealed differentially expressed genes involved in the chlorophyll biosynthesis and photosystem(PSI and PSII)reaction center.The abs1 mutant depicted the decreased expression level of genes encoding light-harvesting chlorophyll a/b binding proteins and photosystem II binding protein A required for the reaction center of the PSII complex.Besides,hormonal profiling of the abs1 mutant indicates the complexity of the BR and other phytohormones interactions.Our findings concluded that the BR signaling reduction transcriptionally impaired chlorophyll synthesis,harvesting quantum photon and transferring light energy to PSI and PSII,leading to a decrease in overall photosynthetic capacity.3)The expression pattern of BR-signaling components at different floral developmental stages revealed that BR signaling actively participates during various floral stages,indicating its possible role in flower development.In this study,altered brassinosteroid sensitivity1(abs1)mutant was used to explore the regulatory roles of BR signaling in flower development.Moreover,the abs1 mutant displayed several phenotypic defects,including an altered floral architecture and reduced male fertility.In addition,the tomato floral development and pollenassociated genes were suppressed in the abs1 mutant.Furthermore,the abs1 mutant exhibited reduced expression of BZR and GA related genes suggesting an important role of BR and GA crosstalk during flower development.Our data imply that Curl3 may affect floral organ and pollen development by modulating BR and GA cross-talk in tomato.Overall,these findings provide a deeper understanding of morphological and transcriptional changes during flower development in response to BR-insensitivity.4)In this study,the expression pattern of BRI1 homologous BR receptor gene Curl3 in fruit tissues at different developmental stages revealed that BR signaling actively participates throughout the fruit development,indicating its possible role in fruit development.The altered brassinolide sensitivity1 mutant was used to explore the regulatory roles of BR signaling during fruit development.The BR signaling mutant exhibited a typical dwarf phenotype along with reduced vegetative growth,fruit size,and fruit weight.Microscopic and transcriptional evaluation of the abs1 mutant fruits implies that reduced cell size and cell number is responsible for the phenotypic variations.Additionally,we also found that the altered content of phytohormones such as auxin,gibberellin,cytokinin,and ethylene level contributed to altered fruit development in abs1 mutant.Moreover,expression levels of fruit growth and cell development-specific genes were downregulated in BR-insensitive plants;culminating in reduced cell size,cell number,and cell layers.Altogether,these findings provide a profound understanding of physio-chemical changes during fruit development in response to BR-insensitivity.These insights may enhance efforts to improve the productivity of fruit crops through genetic engineering of Curl3 via precise control of BR signaling.