A 21-bp InDel in the promoter of STP1 selected during tomato improvement accounts for soluble solid content in fruits

Domestication and improvement are important processes that generate the variation in genome and phonotypes underlying crop improvement.Unfortunately,during selection for certain attributes,other valuable traits may be inadvertently discarded.One example is the decline in fruit soluble solids content(SSC)during tomato breeding.Several genetic loci for SSC have been identified,but few reports on the underlying mechanisms are available.In this study we performed a genome-wide association study(GWAS)for SSC of the red-ripe fruits in a population consisting of 481 tomato accessions with large natural variations and found a new quantitative trait locus,STP1,encoding a sugar transporter protein.The causal variation of STP1,a 21-bp InDel located in the promoter region 1124 bp upstream of the start codon,alters its expression.STP1 Insertion accessions with an 21-bp insertion have higher SSC than STP1Deletion accessions with the 21-bp deletion.Knockout of STP1 in TS-23 with high SSC using CRISPR/Cas9 greatly decreased SSC in fruits.In vivo and in vitro assays demonstrated that ZAT10-LIKE,a zinc finger protein transcription factor(ZFP TF),can specifically bind to the promoter of STP1Insertion to enhance STP1 expression,but not to the promoter of STP1Deletion,leading to lower fruit SSC in modern tomatoes.Diversity analysis revealed that STP1 was selected during tomato improvement.Taking these results together,we identified a naturally occurring causal variation underlying SSC in tomato,and a new role for ZFP TFs in regulating sugar transporters.The findings enrich our understanding of tomato evolution and domestication,and provide a genetic basis for genome design for improving fruit taste.

A NAC transcription factor, NOR-like1, is a new positive regulator of tomato fruit ripening

Ripening of the model fruit tomato(Solanum lycopersicum)is controlled by a transcription factor network including NAC(NAM,ATAF1/2,and CUC2)domain proteins such as No-ripening(NOR),SlNAC1,and SlNAC4,but very little is known about the NAC targets or how they regulate ripening.Here,we conducted a systematic search of fruit-expressed NAC genes and showed that silencing NOR-like1(Solyc07g063420)using virus-induced gene silencing(VIGS)inhibited specific aspects of ripening.Ripening initiation was delayed by 14 days when NOR-like1 function was inactivated by CRISPR/Cas9 and fruits showed obviously reduced ethylene production,retarded softening and chlorophyll loss,and reduced lycopene accumulation.RNA-sequencing profiling and gene promoter analysis suggested that genes involved in ethylene biosynthesis(SlACS2,SlACS4),color formation(SlGgpps2,SlSGR1),and cell wall metabolism(SlPG2a,SlPL,SlCEL2,and SlEXP1)are direct targets of NOR-like1.Electrophoretic mobility shift assays(EMSA),chromatin immunoprecipitation-quantitative PCR(ChIP-qPCR),and dual-luciferase reporter assay(DLR)confirmed that NOR-like1 bound to the promoters of these genes both in vitro and in vivo,and activated their expression.Our findings demonstrate that NOR-like1 is a new positive regulator of tomato fruit ripening,with an important role in the transcriptional regulatory network.

The Influence of Co-Suppressing Tomato 1-Aminocyclopropane-1-Carboxylic Acid OxidaseⅠon the Expression of Fruit Ripening-Related and Pathogenesis-Related Protein Genes

The purpose of this study is to explore the influence of co-suppressing tomato ACC oxidase Ⅰ on the expression of fruit ripening-related and pathogenesis-related protein genes, and on the biosynthesis of endogenous ethylene and storage ability of fruits. Specific fragments of several fruit ripening-related and pathogenesis-related protein genes from tomato （Lycopersicon esculentum） were cloned, such as the l-aminocyclopropane-1-carboxylic acid oxidase 1 gene （LeAC01）, 1- aminocyclopropane-l-carboxylic acid oxidase 3 gene （LeAC03）, EIN3-binding F-box 1 gene （LeEBF1）, pathogenesis-related protein 1 gene （LePR1）, pathogenesis-related protein 5 gene （LePR5）, and pathogenesis-related protein osmotin precursor gene （LeNP24） by PCR or RT-PCR. Then these specific DNA fragments were used as probes to hybridize with the total RNAs extracted from the wild type tomato Ailsa Craig （AC＋＋） and the LeAC01 co-suppression tomatoes （V1187 and T4B）, respectively. At the same time, ethylene production measurement and storage experiment of tomato fruits were carded out. The hybridization results indicated that the expression of fruit ripening-related genes such as LeACO3 and LeEBF1, and pathogenesis-related protein genes such as LePR1, LePR5, and LeNP24, were reduced sharply, and the ethylene production in the fruits, wounded leaves decreased and the storage time of ripening fruits was prolonged, when the expression of LeACO1 gene in the transgenic tomato was suppressed. In the co-suppression tomatoes, the expression of fruit ripening-related and pathogenesis-related protein genes were restrained at different degrees, the biosynthesis of endogenous ethylene decreased and the storage ability of tomato fruits increased.

Mutagenesis of SlNAC4 by CRISPR/Cas9 alters gene expression and softening of ripening tomato fruit

Softening is one of the key fruit quality traits,which results from the selective expression of cell wall metabolism genes during ripening.The identification of transcription factors(TFs)that regulate fruit softening is an important field in order to understand and control fruit softening.In tomato,NAC(NAM,ATAF,and CUC)TFs members have been demonstrated to be involved in fruit ripening regulation,including NAC-NOR(nonripening),NOR-like1,SlNAC4,SlNAC1.Here,we generated slnac4 mutant knockout(CR-SlNAC4)tomato plant by a clustered regularly interspaced short palindromic repeats genomic targeting system(CRISPR/Cas9)and SlNAC4 overexpressing(OE-SlNAC4)plant.In addition to confirming the previously reported results that SlNAC4 positively regulates fruit ripening,we found that SlNAC4 has a strong effect on tomato fruit softening.Compared with the control fruit,fruit softening was inhibited in slnac4 fruit and conversely was accelerated in OE-SlNAC4 tomato fruit.Through RNA-sequencing(RNA-seq)analysis,we found that expression levels of SlEXP1(expansin)and SlCEL2(endo-β-1,4 glucanase)genes involved in cell wall metabolism were significantly different in WT(wild type)/slnac4 and WT/OE-SlNAC4 fruit.Further study showed that these genes contained a NAC TF binding domain in their promoter regions.In vitro electrophoretic mobility shift assays(EMSA)and dual-luciferase reporter assays(DLR)demonstrated that these two genes were the direct targets of SlNAC4 binding and transactivation.The results enriched the function of SlNAC4 and provided a new dimension in understanding the regulation of tomato fruit softening.

协同抑制番茄ACO1对果实成熟及病程相关蛋白基因表达的影响

目的探讨协同抑制番茄ACO1基因对果实成熟和病程相关蛋白基因表达、内源乙烯生物合成及果实耐贮性的影响。方法采用PCR或RT-PCR方法克隆了番茄ACC氧化酶1、ACC氧化酶3、EBF1、PR1、PR5以及NP24基因片段,并以此制备探针,以协同抑制ACO1的转基因番茄和野生型番茄为研究对象,进行Northern杂交,同时测定了伤害叶片和果实的乙烯释放量,并进行了果实贮藏试验等。结果Northern杂交结果表明,番茄ACO1基因表达被抑制后,与果实成熟相关基因LeACO3和LeEBF1,以及病程相关蛋白基因LePR1、LePR5和LeNP24的表达量急剧降低。乙烯释放量测定试验和果实贮藏试验结果表明,协同抑制LeACO1番茄完整和受伤叶片以及完整果实内源乙烯释放量相对于野生型番茄大大减少,成熟果实贮藏时间延长。结论协同抑制番茄ACO1基因表达的同时,与果实成熟相关基因和病程相关蛋白基因的表达也不同程度地受到抑制,而且其内源乙烯生物合成减少,果实耐贮性增强。

Symbiosome-like intracellular colonization of cereals and other crop plants by nitrogen-fixing bacteria for reduced inputs of synthetic nitrogen fertilizers

It has been forecast that the challenge of meeting increased food demand and protecting environmental quality will be won or lost in maize, rice and wheat cropping systems, and that the problem of environmental nitrogen enrichment is most likely to be solved by substituting synthetic nitrogen fertilizers by the creation of cereal crops that are able to fix nitrogen symbiotically as legumes do. In legumes, rhizobia present intracellularly in membrane-bound vesicular compartments in the cytoplasm of nodule cells fix nitrogen endosymbiotically. Within these symbiosomes, membrane-bound vesicular compartments, rhizobia are supplied with energy derived from plant photosynthates and in return supply the plant with biologically fixed nitrogen, usually as ammonia. This minimizes or eliminates the need for inputs of synthetic nitrogen fertilizers. Recently we have demonstrated, using novel inoculation conditions with very low numbers of bacteria, that cells of root meristems of maize, rice, wheat and other major non-legume crops, such as oilseed rape and tomato, can be intracellularly colonized by the non-rhizobial, non-nodulating, nitrogen fixing bacterium, Gluconacetobacter diazotrophicus that naturally occurs in sugarcane. G. diazotrophicus expressing nitrogen fixing (nifH) genes is present in symbiosome-like compartments in the cytoplasm of cells of the root meristems of the target cereals and non-legume crop species, somewhat similar to the intracellular symbiosome colonization of legume nodule cells by rhizobia. To obtain an indication of the likelihood of adequate growth and yield, of maize for example, with reduced inputs of synthetic nitrogen fertilizers, we are currently determining the extent to which nitrogen fixation, as assessed using various methods, is correlated with the extent of systemic intracellular colonization by G. diazotrophicus, with minimal or zero inputs.

Establishment of Withania somnifera Hairy Root Cultures for the Production of Withanolide A

Withania sominifera （Indian ginseng） was transformed by Agrobacterium rhizogenes. Explants from seedling roots, stems, hypocotyls, cotyledonary nodal segments, cotyledons and young leaves were inoculated with A. rhizogenes strain R1601. Hairy （transformed） roots were induced from cotyledons and leaf explants. The transgenic status of hairy roots was confirmed by polymerase chain reaction using nptll and rolB specific primers and, subsequently, by Southern analysis for the presence of nptll and rolB genes in the genomes of transformed roots. Four clones of hairy roots were established; these differed in their morphology. The doubling time of faster growing cultures was 8-14 d with a fivefold increase in biomass after 28 d compared with cultured, non-transformed seedling roots. MS-based liquid medium was superior for the growth of transformed roots compared with other culture media evaluated （SH, LS and N6）, with MS-based medium supplemented with 40 g/L sucrose being optimal for biomass production. Cultured hairy roots synthesized withanolide A, a steroidal lactone of medicinal and therapeutic value. The concentration of withanolide A in transformed roots （157.4 μg/g dry weight） was 2.7-fold more than in non-transformed cultured roots （57.9 μg/g dry weight）.

Stamen specification and anther development in rice

Male reproductive development is a complex biological process which includes the formation of the stamen with differentiated anther tissues, in which microspores/pollens are generated, then anther dehiscence and subsequently pollination. Stamen specification and anther development involve a number of extraordinary events such as meristem transition, cell division and differentiation, cell to cell communication, etc., which need the cooperative interaction of sporophytic and gametophytic genes. The advent of various tools for rice functional gene identification, such as complete genome sequence, genome-wide microarrays, collections of mutants, has greatly facilitated our understanding of mechanisms of rice stamen specification and anther development. Male sterile lines are critical for hybrid rice breeding, therefore understanding these processes will not only contribute greatly to the basic knowledge of crop developmental biology, but also to the development of new varieties for hybrid rice breeding in the future.