CRISPR/Cas9-mediated mutations of FANTASTIC FOUR gene family for creating early flowering mutants in tomato.

Flowering time is of great agricultural importance and the timing and extent of flowering usually determines yield and availability of flowers, fruits and seeds. Identification of genes determining flowering has important practical applications for tomato breeding. Here we demonstrate the roles of the FANTASTIC FOUR (FAF) gene family in regulating tomato flowering time. In this plant-specific gene family, SlFAF1/2a shows a constitutive expression pattern during the transition of the shoot apical meristem (SAM) from vegetative to reproductive growth and significantly influences flowering time. Overexpressing SlFAF1/2a causes earlier flowering compared with the transformations of other genes in the FAF family. SlFAF1/2c also positively regulates tomato flowering, although to a lesser extent. The other members of the SlFAF gene family, SlFAF1/2b, SlFAF3/4a and SlFAF3/4b, are negative regulators of tomato flowering and faf1/2b, faf3/4a and faf3/4b single mutants all display early flowering. We generated a series of early flowering mutants using the CRISPR/Cas9 editing system, and the faf1/2b faf3/4a faf3/4b triple mutant flowering earliest compared with other mutants. More importantly, these mutants show no adverse effect on yield. Our results have uncovered the role of the FAF gene family in regulating tomato flowering time and generated early flowering germplasms for molecular breeding.

Multiple-model GWAS identifies optimal allelic combinations of quantitative trait loci for malic acid in tomato.

Malic acid (MA) is an important flavor acid in fruits and acts as a mediator in a series of metabolic pathways. It is important to understand the factors affecting MA metabolism for fruit flavor improvement and to understand MA-mediated biological processes. However, the metabolic accumulation of MA is controlled by complex heredity and environmental factors, making it difficult to predict and regulate the metabolism of MA. In this study, we carried out a genome-wide association study (GWAS) on MA using eight milestone models with two-environment repeats. A series of associated SNP variations were identified from the GWAS, and 15 high-confidence annotated genes were further predicted based on linkage disequilibrium and lead SNPs. The transcriptome data of candidate genes were explored within different tomato organs as well as various fruit tissues, and suggested specific expression patterns in fruit pericarp. Based on the genetic parameters of population differentiation and SNP distribution, tomato MA content has been more influenced by domestication sweeps and less affected by improvement sweeps in the long-term history of tomato breeding. In addition, genotype × environment interaction might contribute to the difference in domestication phenotypic data under different environments. This study provides new genetic insights into how tomato changed its MA content during breeding and makes available function-based markers for breeding by marker-assisted selection.

Variation in the fruit development gene POINTED TIP regulates protuberance of tomato fruit tip.

The domestication of tomato has led to striking variations in fruit morphology. Here, we show a genome-wide association study (GWAS) to understand the development of the fruit tip and describe a POINTED TIP (PT) gene that encodes a C2H2-type zinc finger transcription factor. A single nucleotide polymorphism is found to change a histidine (H) to an arginine (R) in the C2H2 domain of PT and the two alleles are referred to as PTH and PTR. Knocking out PTH leads to development of pointed tip fruit. PTH functions to suppress pointed tip formation by downregulating the transcription of FRUTFULL 2 (FUL2), which alters the auxin transport. Our evolutionary analysis and previous studies by others suggest that the PTR allele likely hitch-hiked along with other selected loci during the domestication process. This study uncovers variation in PT and molecular mechanism underlying fruit tip development in tomato.

A mutation in a C2H2-type zinc finger transcription factor contributed to the transition toward self-pollination in cultivated tomato.

The degree of stigma exsertion has a major influence on self-pollination efficiency in tomato, and its improvement is essential for raising productivity and for fixing advantageous traits in cultivated tomato. To study the evolution of stigma exsertion degree in tomato, we searched for genes associated with this trait and other aspects of flower morphology, including the lengths of anthers, styles, and ovaries. We performed a genome-wide association on 277 tomato accessions and discovered a novel stigma exsertion gene (SE3.1). We reannotated the structure of the gene, which encodes a C2H2-type zinc finger transcription factor. A mutation of the lead single nucleotide polymorphism creates a premature termination codon in SE3.1 and an inserted stigma in cultivated tomatoes. SE3.1 is essential for the conversion of flush stigmas to inserted stigmas. This conversion has a major impact on the rate of self-fertilization. Intriguingly, we found that both SE3.1 and Style2.1 contribute to the transition from stigma exsertion to insertion during the domestication and improvement of tomato. Style2.1 controls the first step of exserted stigmas to flush stigmas, and SE3.1 controls the second step of flush stigmas to inserted stigmas. We provide molecular details for the two-step process that controls the transition from stigma exsertion to insertion, which is of great agronomic importance in tomato.

Interactions between ShPP2-1, an F-box family gene, and ACR11A regulate cold tolerance of tomato.

There is a critical need to identify germplasm resources and genes that promote cold tolerance of tomato because global tomato production is threatened by cold stress. We found that the expression of an F-box gene family member named ShPP2-1 from Solanum habrochaites is cold inducible and studied its contribution to cold tolerance. Overexpression of ShPP2-1 in cultivated tomato (AC) reduced cold tolerance by intensifying damage to cell membranes. To explore the underlying molecular mechanism, we conducted a yeast two-hybrid library screen and found that a protein containing ACT domain repeats named ACR11A interacts with PP2-1. Overexpression of SlACR11A in AC enhanced the cold tolerance of seedlings and germinating seeds. Cold tolerance decreased in tomato plants that overexpressed both of these genes. Additionally, we performed seed germination experiments in the cold with 177 tomato accessions and identified two alleles of SlACR11A that differ in one single-nucleotide polymorphism. We found that one of these alleles, SlACR11AG, is significantly enriched in cold-tolerant tomato plants. Taken together, our findings indicate that the combination of low expression levels of PP2-1 and high expression levels of ACR11A can promote cold tolerance. These genes may therefore serve as direct targets for both genetic engineering and improvement projects that aim to enhance the cold tolerance of tomato.

MAPK11 regulates seed germination and ABA signaling in tomato by phosphorylating SnRKs.

Seed germination is a critical stage in the plant life cycle and it plays an important role in the efficiency of agricultural production. However, our knowledge of the mechanisms that regulate seed germination remains limited. In this study, we identified a novel gene, MAPK11, that encodes mitogen-activated protein kinase 11; its expression was significantly higher in seeds of tomato varieties with a low optimum germination temperature than in those with a high optimum germination temperature. In tests at 25 °C, overexpression of MAPK11 in an accession with optimum germination at 25 °C resulted in a decrease in germination, whereas RNAi of MAPK11 in an accession with optimum germination at 15 °C resulted in increased germination. Furthermore, we found that lines overexpressing MAPK11 exhibited hypersensitivity to ABA during germination. These observations were at least partially explained by the fact that MAPK11 up-regulated both NCED1 expression and ABA biosynthesis, and that it also affected ABA signaling and negatively regulated germination by influencing the phosphorylation of SnRK2.2 in vivo. In addition, we found that MAPK11 interacts with and phosphorylates SnRK1 in vivo, thereby potentially inhibiting its activation. SnRK1 interacted with ABI5 and suppressed the transcription of ABI5, thereby affecting ABA signaling and the regulation of germination. Our results demonstrate that ABA signaling in tomato is affected by a mechanism that depends on MAPK11 phosphorylating SnRKs, and this ultimately influences seed germination.

ShCIGT, a Trihelix family gene, mediates cold and drought tolerance by interacting with SnRK1 in tomato.

Abiotic stress, such as drought and cold stress, have a major impact on plant growth and development. The trihelix transcription factor family plays important roles in plant morphological development and adaptation to abiotic stresses. In this study, we isolated a cold-induced gene named ShCIGT from the wild tomato species Solanum habrochaites and found that it contributes to abiotic stress tolerance. ShCIGT belongs to the GT-1 subfamily of the trihelix transcription factors. It was constitutively expressed in various tissues. Its expression was induced by multiple abiotic stresses and abscisic acid (ABA). Overexpression of ShCIGT in cultivated tomato enhanced cold and drought stress tolerance. In addition, the transgenic plants displayed a reduced sensitivity to ABA during post-germination growth. We found that ShCIGT interacts with SnRK1, an energy sensor in the metabolic signaling network, which controls plant metabolism, growth and development, and stress tolerance. Based on these data, we conclude ShCIGT may improve abiotic-stress tolerance in tomato by interacting with SnRK1.

[Use of limited sampling strategy for estimating area under concentration-versus-time curve of mycophenolate sodium in renal allograft recipients].

OBJECTIVE: To establish a formula for estimating area under the concentration-versus-time curve (AUC) of mycophenolate sodium in Chinese renal allograft recipients with a limited sampling model. METHODS: A total of 35 renal allograft recipients were recruited from 2010 to 2013 to receive enteric-coated mycophenolate sodium (EC-MPS), calcineurin (CNI) and prednisone as immunosuppressive triple therapy. The serum concentration of mycophenolic acid (MPA) was assayed by enzyme multiplied immunoassay technique (EMIT) at pre-dose (C0), 0.5 (C0.5), 1.0 (C1), 1.5 (C1.5), 2.0 (C2), 3.0 (C3), 4.0 (C4), 6.0 (C6), 8.0 (C8) and 12.0 (C12) h post-dose respectively. Pharmacokinetic parameters of MPA (C0, C12, Cmax, Tmax, AUC0-12 h) were calculated by software WINNOLIN. Simplified formulae for estimation of MPA-AUC in tacrolimus (Tac) group or cyclosporin A (CsA) group were established by multiple stepwise regression analysis. RESULTS: There were variable MPA AUC0-12 h levels between 14 and 67 mg×h/L (mean: 37 ± 14). The MPA trough level (C0) had no correlations with MPA AUC0-12 h (r(2) = 0.090) . The simplified MPA AUC formula for Tac group was AUC = 5.678+1.718×C4+2.853×C6+1.812×C8+3.413×C12 with four sampling points (C4, C6, C8, C12). Estimated AUC with the formula had correlations with AUC0-12 h (r(2) = 0.890). The mean absolute predict error (APE) was 3.45% (0.41%-24.71%) and the proportion of APE above 15% stood at 11.1% (2/18) . In CsA group, the simplified MPA AUC formula was AUC = 7.072+1.525×C3+1.558×C4+ 1.573×C6+2.285×C8. The correlation was r(2) = 0.952, mean APE was 6.50% (0.02%-12.91%) and proportion of APE above 15% stood at 0. The above formulae were observed to have agreement with AUC0-12 h by Bland-Altman analysis. CONCLUSION: The simplified MPA AUC formulae with 4-point sampling provide an effective approach for estimating full MPA AUC0-12 h in Chinese renal recipients on EC-MPS plus tacrolimus or cyclosporin A.