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.

The genomic route to tomato breeding: Past, present, and future.

Over the past 10,000 years, tomato species have undergone both unintentional and intentional selection to enhance their favorable traits for human consumption and manufacturing. These selection processes have significantly influenced the genomes of tomato species and have played a critical role in improving tomato varieties. In this review, we summarize recent advances in tomato genome sequencing, explore the impact of human-driven selection, and recapitulate key genes associated with important agronomic traits in tomato breeding. We provide several examples of genomics-guided tomato breeding to highlight the potential of genome resources in facilitating tomato improvement. Furthermore, we elaborate the progress and strategies of tomato breeding through genome design and present how such efforts can help future enhancements of tomato to align with the demands of sustainability and evolving human societies.

Exploring the influence of a single-nucleotide mutation in EIN4 on tomato fruit firmness diversity through fruit pericarp microstructure.

Tomato (Solanum lycopersicum) stands as one of the most valuable vegetable crops globally, and fruit firmness significantly impacts storage and transportation. To identify genes governing tomato firmness, we scrutinized the firmness of 266 accessions from core collections. Our study pinpointed an ethylene receptor gene, SlEIN4, located on chromosome 4 through a genome-wide association study (GWAS) of fruit firmness in the 266 tomato core accessions. A single-nucleotide polymorphism (SNP) (A → G) of SlEIN4 distinguished lower (AA) and higher (GG) fruit firmness genotypes. Through experiments, we observed that overexpression of SlEIN4AA significantly delayed tomato fruit ripening and dramatically reduced fruit firmness at the red ripe stage compared with the control. Conversely, gene editing of SlEIN4AA with CRISPR/Cas9 notably accelerated fruit ripening and significantly increased fruit firmness at the red ripe stage compared with the control. Further investigations revealed that fruit firmness is associated with alterations in the microstructure of the fruit pericarp. Additionally, SlEIN4AA positively regulates pectinase activity. The transient transformation assay verified that the SNP (A → G) on SlEIN4 caused different genetic effects, as overexpression of SlEIN4GG increased fruit firmness. Moreover, SlEIN4 exerts a negative regulatory role in tomato ripening by impacting ethylene evolution through the abundant expression of ethylene pathway regulatory genes. This study presents the first evidence of the role of ethylene receptor genes in regulating fruit firmness. These significant findings will facilitate the effective utilization of firmness and ripening traits in tomato improvement, offering promising opportunities for enhancing tomato storage and transportation capabilities.

Defective mutations in STAY-GREEN 1, PHYTOENE SYNTHASE 1, and MYB12 genes lead to formation of green ripe fruit in tomato.

Modern tomatoes produce colorful mature fruits, but many wild tomato ancestors form green or gray green ripe fruits. Here, tomato cultivar 'Lvbaoshi' (LBS) that produces green ripe fruits was found to contain three recessive loci responsible for fruit development. The colorless peel of LBS fruits was caused by a 603 bp deletion in the promoter of SlMYB12. The candidate genes of the remaining two loci were identified as STAY-GREEN 1 (SlSGR1) and PHYTOENE SYNTHASE 1 (SlPSY1). SGR1 and PSY1 co-suppression by RNAi converted the pink fruits into green ripe fruits in transgenic plants. An amino acid change in PSY1 and a deletion in the promoter of SGR1 were also identified in several wild tomatoes bearing green or gray ripe fruits. Overexpression of PSY1 from green ripe fruit wild tomatoes in LBS plants could only partially rescue the green ripe fruit phenotype of LBS, and transgenic lines expressing ProSGR1::SGR1 from Solanum pennellii also failed to convert purple-flesh into red-flesh fruits. This work uncovers a novel regulatory mechanism by which SlMYB12, SlPSY1, and SlSGR1 control fruit color in cultivated and some wild tomato species.

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.

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.

SlTCP24 and SlTCP29 synergistically regulate compound leaf development through interacting with SlAS2 and activating transcription of SlCKX2 in tomato.

The complexity of compound leaves results primarily from the leaflet initiation and arrangement during leaf development. However, the molecular mechanism underlying compound leaf development remains a central research question. SlTCP24 and SlTCP29, two plant-specific transcription factors with the conserved TCP motif, are shown here to synergistically regulate compound leaf development in tomato. When both of them were knocked out simultaneously, the number of leaflets significantly increased, and the shape of the leaves became more complex. SlTCP24 and SlTCP29 could form both homodimers and heterodimers, and such dimerization was impeded by the leaf polarity regulator SlAS2, which interacted with SlTCP24 and SlTCP29. SlTCP24 and SlTCP29 could bind to the TCP-binding cis-element of the SlCKX2 promoter and activate its transcription. Transgenic plants with SlTCP24 and SlTCP29 double-gene knockout had a lowered transcript level of SlCKX2 and an elevated level of cytokinin. This work led to the identification of two key regulators of tomato compound leaf development and their targeted genes involved in cytokinin metabolic pathway. A model of regulation of compound leaf development was proposed based on observations of this study.

SlZF3 regulates tomato plant height by directly repressing SlGA20ox4 in the gibberellic acid biosynthesis pathway.

Plant height is an important target trait for crop genetic improvement. Our previous work has identified a salt-tolerant C2H2 zinc finger, SlZF3, and its overexpression lines also showed a semi-dwarf phenotype, but the molecular mechanism remains to be elucidated. Here, we characterized the dwarf phenotype in detail. The dwarfism is caused by a decrease in stem internode cell elongation and deficiency of bioactive gibberellic acids (GAs), and can be rescued by exogenous GA3 treatment. Gene expression assays detected reduced expression of genes in the GA biosynthesis pathway of the overexpression lines, including SlGA20ox4. Several protein-DNA interaction methods confirmed that SlZF3 can directly bind to the SlGA20ox4 promoter and inhibit its expression, and the interaction can also occur for SlKS and SlKO. Overexpression of SlGA20ox4 in the SlZF3-overexpressing line can recover the dwarf phenotype. Therefore, SlZF3 regulates plant height by directly repressing genes in the tomato GA biosynthesis pathway.

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 STP1 Deletion 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 STP1 Insertion to enhance STP1 expression, but not to the promoter of STP1 Deletion , 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.

SlBBX20 attenuates JA signalling and regulates resistance to Botrytis cinerea by inhibiting SlMED25 in tomato.

Jasmonic acid (JA) plays an important role in regulating plant growth and defence responses. Here, we show that a transcription factor that belongs to the B-box (BBX) family named SlBBX20 regulates resistance to Botrytis cinerea in tomato by modulating JA signalling. The response to JA was significantly suppressed when SlBBX20 was overexpressed in tomato. By contrast, the JA response was enhanced in SlBBX20 knockout lines. RNA sequencing analysis provided more evidence that SlBBX20 modulates the expression of genes that are involved in JA signalling. We found that SlBBX20 interacts with SlMED25, a subunit of the Mediator transcriptional co-activator complex, and prevents the accumulation of the SlMED25 protein and transcription of JA-responsive genes. JA contributes to the defence response against necrotrophic pathogens. Knocking out SlBBX20 or overexpressing SlMED25 enhanced tomato resistance to B. cinerea. The resistance was impaired when SlBBX20 was overexpressed in plants that also overexpressed SlMED25. These data show that SlBBX20 attenuates JA signalling by regulating SlMED25. Interestingly, in addition to developing enhanced resistance to B. cinerea, SlBBX20-KO plants also produced higher fruit yields. SlBBX20 is a potential target gene for efforts that aim to develop elite crop varieties using gene editing technologies.