Nuclear factor Y-A3b binds to the SINGLE FLOWER TRUSS promoter and regulates flowering time in tomato.

The control of flowering time is essential for reproductive success and has a major effect on seed and fruit yield and other important agricultural traits in crops. Nuclear factors Y (NF-Ys) are transcription factors that form heterotrimeric protein complexes to regulate gene expression required for diverse biological processes, including flowering time control in plants. However, to our knowledge, there has been no report on mutants of individual NF-YA subunits that promote early flowering phenotype in plants. In this study, we identified SlNF-YA3b, encoding a member of the NF-Y transcription factor family, as a key gene regulating flowering time in tomato. Knockout of NF-YA3b resulted in an early flowering phenotype in tomato, whereas overexpression of NF-YA3b delayed flowering in transgenic tomato plants. NF-YA3b was demonstrated to form heterotrimeric protein complexes with multiple NF-YB/NF-YC heterodimers in yeast three-hybrid assays. Biochemical evidence indicated that NF-YA3b directly binds to the CCAAT cis-elements of the SINGLE FLOWER TRUSS (SFT) promoter to suppress its gene expression. These findings uncovered a critical role of NF-YA3b in regulating flowering time in tomato and could be applied to the management of flowering time in crops.

EARLY FLOWERING is a dominant gain-of-function allele of FANTASTIC FOUR 1/2c that promotes early flowering in tomato.

Flowering time, an important factor in plant adaptability and genetic improvement, is regulated by various genes in tomato (Solanum lycopersicum). In this study, we characterized a tomato mutant, EARLY FLOWERING (EF), that developed flowers much earlier than its parental control. EF is a dominant gain-of-function allele with a T-DNA inserted 139 bp downstream of the stop codon of FANTASTIC FOUR 1/2c (FAF1/2c). The transcript of SlFAF1/2c was at elevated levels in the EF mutant. Overexpressing SlFAF1/2c in tomato plants phenocopied the early flowering trait of the EF mutant. Knocking out SlFAF1/2c in the EF mutant reverted the early flowering phenotype of the mutant to the normal flowering time of the wild-type tomato plants. SlFAF1/2c promoted the floral transition by shortening the vegetative phase rather than by reducing the number of leaves produced before the emergence of the first inflorescence. The COP9 signalosome subunit 5B (CSN5B) was shown to interact with FAF1/2c, and knocking out CSN5B led to an early flowering phenotype in tomato. Interestingly, FAF1/2c was found to reduce the accumulation of the CSN5B protein by reducing its protein stability. These findings imply that FAF1/2c regulates flowering time in tomato by reducing the accumulation and stability of CSN5B, which influences the expression of SINGLE FLOWER TRUSS (SFT), JOINTLESS (J) and UNIFLORA (UF). Thus, a new allele of SlFAF1/2c was discovered and found to regulate flowering time in tomato.

Developing a highly efficient CGBE base editor in watermelon.

Cytosine and adenosine base editors (CBEs and ABEs) are novel genome-editing tools that have been widely utilized in molecular breeding to precisely modify single-nucleotide polymorphisms (SNPs) critical for plant agronomic traits and species evolution. However, conventional BE editors are limited to achieve C-to-T and A-to-G substitutions, respectively. To enhance the applicability of base editing technology in watermelon, we developed an efficient CGBE editor (SCGBE2.0) by removing the uracil glycosylase inhibitor (UGI) unit from the commonly used hA3A-CBE and incorporating the uracil-DNA glycosylase (UNG) component. Seven specific guide RNAs (sgRNAs) targeting five watermelon genes were designed to assess the editing efficiency of SCGBE. The results obtained from stably transformed watermelon plants demonstrated that SCGBE2.0 could efficiently induce C-to-G mutations at positions C5-C9 in 43.2% transgenic plants (with a maximum base conversion efficiency of 46.1%) and C-to-A mutation at position C4 in 23.5% transgenic plants (with a maximum base conversion efficiency of 45.9%). These findings highlight the capability of our integrated SCGBE2.0 editor to achieve C-to-G/A mutations in a site-preferred manner, thus providing an efficient base editing tool for precise base modification and site-directed saturated mutagenesis in watermelon.

Creation of a watermelon haploid inducer line via ClDMP3-mediated single fertilization of the central cell.

The use of doubled haploids is one of the most efficient breeding methods in modern agriculture. Irradiation of pollen grains has been shown to induce haploids in cucurbit crops, possibly because it causes preferential fertilization of the central cell over the egg cell. Disruption of the DMP gene is known to induce single fertilization of the central cell, which can lead to the formation of haploids. In the present study, a detailed method of creating a watermelon haploid inducer line via ClDMP3 mutation is described. The cldmp3 mutant induced haploids in multiple watermelon genotypes at rates of up to 1.12%. These haploids were confirmed via fluorescent markers, flow cytometry, molecular markers, and immuno-staining. The haploid inducer created by this method has the potential to greatly advance watermelon breeding in the future.

The tomato CONSTANS-LIKE protein SlCOL1 regulates fruit yield by repressing SFT gene expression.

BACKGROUND: CONSTANS (CO) and CONSTANS-LIKE (COL) transcription factors have been known to regulate a series of cellular processes including the transition from the vegetative growth to flower development in plants. However, their role in regulating fruit yield in tomato is poorly understood. RESULT: In this study, the tomato ortholog of Arabidopsis CONSTANS, SlCOL1, was shown to play key roles in the control of flower development and fruit yield. Suppression of SlCOL1 expression in tomato was found to lead to promotion of flower and fruit development, resulting in increased tomato fruit yield. On the contrary, overexpression of SlCOL1 disturbed flower and fruit development, and significantly reduced tomato fruit yield. Genetic and biochemical evidence indicated that SlCOL1 controls inflorescence development by directly binding to the promoter region of tomato inflorescence-associated gene SINGLE-FLOWER TRUSS (SFT) and negatively regulating its expression. Additionally, we found that SlCOL1 can also negatively regulate fruit size in tomato. CONCLUSIONS: Tomato SlCOL1 binds to the promoter of the SFT gene, down-regulates its expression, and plays a key role in reducing the fruit size.

Highly efficient, genotype-independent transformation and gene editing in watermelon (Citrullus lanatus) using a chimeric ClGRF4-GIF1 gene.

Efficient genetic transformation has the potential to advance research and breeding in watermelon (Citrullus lanatus), but regeneration from tissue culture remains challenging. Previous work showed that expressing a fusion of two interacting transcription factors, GROWTH-REGULATING FACTOR4 (GRF4) and GRF-INTERACTING FACTOR1 (GIF1), improved regeneration in wheat (Triticum aestivum). By overexpressing a chimeric fusion of ClGRF4 and ClGIF1, we achieved highly efficient transformation in watermelon. Mutating the mi396 microRNA target site in ClGRF further boosted the transformation efficiency up to 67.27% in a genotype-independent manner. ClGRF4-GIF1 can also be combined with clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing tools to achieve highly efficient gene editing in watermelon, which we used to successfully create diploid seedless watermelon. This research thus puts forward a powerful transformation tool for future watermelon research and breeding.

L2, a chloroplast metalloproteinase, regulates fruit ripening by participating in ethylene autocatalysis under the control of ethylene response factors.

Although autocatalytic ethylene biosynthesis plays an important role in the ripening of climacteric fruits, our knowledge of the network that promotes it remains limited. We identified white fruit (wf), a tomato mutant that produces immature fruit that are white and that ripen slowly. We found that an inversion on chromosome 10 disrupts the LUTESCENT2 (L2) gene, and that white fruit is allelic to lutescent2. Using CRISPR/Cas9 technology we knocked out L2 in wild type tomato and found that the l2-cr mutants produced phenotypes that were very similar to white fruit (lutescent2). In the l2-cr fruit, chloroplast development was impaired and the accumulation of carotenoids and lycopene occurred more slowly than in wild type. During fruit ripening in l2-cr mutants, the peak of ethylene release was delayed, less ethylene was produced, and the expression of ACO genes was significantly suppressed. We also found that exogenous ethylene induces the expression of L2 and that ERF.B3, an ethylene response factor, binds to the promoter of the L2 gene and activates its transcription. Thus, the expression of L2 is regulated by exogenous ethylene. Taken together, our results indicate that ethylene may affect the expression of L2 gene and that L2 participates in autocatalytic ethylene biosynthesis during tomato fruit ripening.

NF-Y plays essential roles in flavonoid biosynthesis by modulating histone modifications in tomato.

NF-Y transcription factors are reported to play diverse roles in a wide range of biological processes in plants. However, only a few active NF-Y complexes are known in plants and the precise functions of NF-Y complexes in flavonoid biosynthesis have not been determined. Using various molecular, genetic and biochemical approaches, we found that NF-YB8a, NF-YB8b and NF-YB8c - a NF-YB subgroup - can interact with a specific subgroup of NF-YC and then recruit either of two distinct NF-YAs to form NF-Y complexes that bind the CCAAT element in the CHS1 promoter. Furthermore, suppressing the expression of particular NF-YB genes increased the levels of H3K27me3 at the CHS1 locus and significantly suppressed the expression of CHS1 during tomato fruit ripening, which led to the development of pink-coloured fruit with colourless peels. Altogether, by demonstrating that NF-Y transcription factors play essential roles in flavonoid biosynthesis and by providing significant molecular insight into the regulatory mechanisms that drive the development of pink-coloured tomato fruit, we provide a major advance to our fundamental knowledge and information that has considerable practical value for horticulture.

UF, a WOX gene, regulates a novel phenotype of un-fused flower in tomato.

Flower formation is a basic condition for fruit set in all flowering plants. The normal stamen of tomato flower fused together to form a yellow cylinder surrounding the carpels. In this study, we identified an un-fused flower (uf) tomato mutant that is defective in petal, carpal and stamen fusion and lateral outgrowth. After RNA-seq-based BSA (BSR), the candidate region location was identified in the long arm of chromosome 3. Using map-based cloning with InDel and CAPS markers, the UF candidate gene was mapped in a 104 kb region. In this region, a WOX (WUSCHEL-related homeobox) transcription factor SlWOX1 was considered as a candidate of UF as there is a 72bp deletion in its second exon in uf mutant. The mutations of SlWOX1 generated by CRISPR/CAS9 approach under wild-type background reproduced the phenotypes of uf mutant, indicating that the SlWOX1 gene is indeed UF. Interestingly, expression analysis of organ lateral polarity determinant genes showed that abaxial genes (SlYABBY5 and SlARF4) and adaxial genes (AS and HD-ZIPIII) were significantly down-regulated in the uf mutant, which is different to that in Arabidopsis and petunia. In conclusion, this work revealed a novel function of SlWOX1 in the regulation of flower development in tomato.

miR156a-targeted SBP-Box transcription factor SlSPL13 regulates inflorescence morphogenesis by directly activating SFT in tomato.

The inflorescences and lateral branches of higher plants are generated by lateral meristems. The structure of the inflorescence has a direct effect on fruit yield in tomato (Solanum lycopersicum). We previously demonstrated that miR156a plays important roles in determining the structures of the inflorescences and lateral branches in tomato by suppressing the expression of the SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) transcription factor gene family. However, information on regulatory pathways associated with inflorescence morphogenesis is still lacking. In this study, we demonstrate that SPL13 is the major SPL involved in miR156a-regulated tomato inflorescence structure determination and lateral branch production. Suppressing the expression of SPL13 in tomato increases the number of inflorescences on vegetative branches and lateral branches, decreases the number of flowers and fruit, and reduces fruit size and yield. Genetic and biochemical evidence indicate that SPL13 controls inflorescence development by positively regulating the expression of the tomato inflorescence-associated gene SINGLE FLOWER TRUSS (SFT) by directly binding to its promoter region. Thus, our findings provide a major advance to our understanding of the miR156a-SlSPL-based mechanism that regulates plant architecture and yield in tomato.

Association analysis between feed efficiency and expression of key genes of the avTOR signaling pathway in meat-type ducks.

Genes involved in the target of rapamycin (TOR) signaling pathway are implicated in nutrient translation, cell proliferation and differentiation, and anabolism, which can affect both growth and feed intake. However, the role of TOR signaling in the regulation of feed intake and feed efficiency in poultry is not clear. In the present study, a total of 1000 ducks, of similar initial weight, were chosen and transferred to individual cages to determine their residual feed intake (RFI) from the age of 21 to 42 days. Subsequently, 60 ducks, which were divided into high (HRFI) and low (LRFI) groups according to their RFI, were chosen to analyze the TOR signaling activities in the liver. The differential expression level of genes involved in the TOR signaling pathway was assayed by the real-time polymerase chain reaction. In the liver, the expression of AKT, avTOR, avLST8, and S6K1 was significantly higher in LRFI ducks than in HRFI ducks; avTOR and AKT were negatively associated with the feed conversion ratio and RFI. Furthermore, PI3K was moderately positively associated with AKT; AKT was strongly positively associated with PI3K, avTOR, avLST8, and S6K1; and avTOR was strongly positively associated with S6K1. In conclusion, the activation of avTOR signaling in the liver of LRFI ducks might be ascribed to higher energy state or more active nutrient transport (amino acids), or both, than those in the liver of HRFI ducks. The results of the present study indicate that AKT and avTOR of TOR signaling might be used as candidate genes to assess molecular regulation of feed efficiency.

Expression and diversification analysis reveals transposable elements play important roles in the origin of Lycopersicon-specific lncRNAs in tomato.

Long noncoding RNAs (lncRNAs) regulate gene expression and biological processes. With the development of high-throughput RNA sequencing technology, lncRNAs have been extensively studied in recent years. Nevertheless, the expression and evolution of lncRNAs in plants remain poorly understood. Here, we identified 413 and 709 multi-exon noncoding transcripts from 353 and 595 loci of the cultivar tomato Heinz1706 and its wild relative LA1589, respectively. Systematic comparison of the sequence and expression of lncRNAs showed that they are poorly conserved in Solanaceae, with only < 0.4% lncRNAs present in all sequenced genomes of tomato and potato. Sequence analysis of Lycopersicon-specific lncRNA loci in Solanum lycopersicum and S. pennellii showed that the origins of these molecules are associated with transposable elements (TEs). LncRNA-314, a fruit-specific lncRNA expressed in S. lycopersicum and S. pimpinellifolium, but not in S. pennellii, originated through two evolutionary events: speciation of S. pennellii resulted in insertion of a long terminal repeat (LTR) retrotransposon into chromosome 10 and contributed to most of the transcribed region of lncRNA-314; and a large deletion in Lycopersicon generated the promoter region and part of the transcribed region of lncRNA-314. These results provide novel insights into the evolution of lncRNAs in plants.