James J. Giovannoni

Fruit weight is a key determinant of yield in high-value vegetable crops such as tomato. Despite extensive research, the molecular mechanisms underlying this complex trait remain largely elusive, with only a few genes cloned to date based on quantitative trait loci (QTL). Here, we analysed two populations and reanalysed three previously published populations and identified 945 QTL associated with agro-morphological traits, including both previously reported and unidentified loci. We focused on SlGRF10 (GROWTH-REGULATING FACTOR 10) underlying a fruit weight QTL, fw1.2. Loss of SlGRF10 reduced fruit weight by decreasing cell size, without affecting cell number. Analysis of natural variation in SlGRF10 in over 1,000 tomato accessions revealed that increased single-nucleotide polymorphism diversity in SlGRF10 is associated with lower fruit weight. This suggests that putative impaired activity contributes to reduced fruit weight, while breeding-induced reduction of genetic variation may have promoted increased fruit weight. Transcriptome profiling of SlGRF10 knockout lines 7- and 20-days post anthesis identified several differentially expressed genes involved in cell cycle progression. Our findings not only confirm the role of SlGRF10 in regulating tomato fruit weight but also highlight a set of candidate genes associated with key morpho-physiological traits. With fw11.3, named as CELL SIZE REGULATOR (CSR), being the only QTL related to cell size determination in tomato fruits so far, SlGRF10 offers a valuable target for precision breeding and enhances our understanding of fruit weight in tomato and related fruit-bearing species.

Summary: Fruit development begins with cell division and expansion, which affects the final size and shape of the fruit. We previously showed tomato UV‐DAMAGED DNA BINDING PROTEIN 1 (SlDDB1) negatively regulates cell proliferation and organ size in an epigenetic manner, but the underlying molecular mechanism remains elusive. We identify two SlDDB1‐interacting proteins, SlMSI2 and SlSDG2, the orthologs of Arabidopsis MULTICOPY SUPPRESSOR OF IRA1 2 and SET DOMAIN GROUP 2, respectively, antagonistically controlling cell proliferation via the ubiquitin‐proteasome pathway in tomato. SlMSI2 encodes a WD40‐repeat protein and negatively involves cell proliferation and interacts with SlDDB1 and SlCUL4 to form the CRL4 ^SlMSI2 ligase complex, through which SlSDG2 is ubiquitinated for degradation. SlSDG2 codes for an H3K9 methyltransferase positively regulating cell proliferation and coordinates with a DNA methyltransferase SlCMT3a to negatively regulate expression of target genes, a subset of negative regulators involving cell division or cell cycle progression through concurrent methylation of H3K9 and DNA at the regulatory chromatin regions. The study reveals the mechanistic basis by which the CRL4 E3 ligase epigenetically regulates cell proliferation and fruit size and structure via ubiquitination‐mediated manipulation of the H3K9 methyltransferase. This work also presents novel targets for manipulating plant structure and size toward optimized agricultural outcomes.

Summary: Carotenoids are vital photosynthetic pigments for plants. Golden2‐like transcription factors (GLKs) are widely recognized as major regulators of Chl biosynthesis and chloroplast development. However, despite GLKs being subjected to intensive investigations, whether GLKs directly regulate carotenoid biosynthesis and the molecular mechanisms by which GLKs transcriptionally activate their target genes remain unclear. Here, we report that GLKs directly regulate carotenoid biosynthesis and activate their target genes in a G‐box binding factor (GBF)‐dependent manner in Arabidopsis. Both in vitro and in vivo studies reveal that GLKs physically interact with GBFs to activate transcription of phytoene synthase (PSY), the gene encoding a rate‐limiting enzyme for carotenoid biosynthesis. While GLKs possess transactivation activity, they depend on GBFs to directly bind to the G‐box motif to modulate PSY expression. Loss of GBFs impairs GLK function in regulating carotenoid and Chl biosynthesis. Since the G‐box motif is an enriched motif in the promoters of GLK‐regulated genes, the GLK–GBF regulatory module likely serves as a common mechanism underlying GLK‐regulated photosynthetic pigment biosynthesis and chloroplast development. Our findings uncover a novel regulatory machinery of carotenoid biosynthesis, discover a molecular mechanism of transcriptional regulation by GLKs, and divulge GLKs as important regulators to coordinate photosynthetic pigment synthesis in plants.

Ripening is crucial for the development of fleshy fruits that release their seeds following consumption by frugivores and are important contributors to human health and nutritional security. Many genetic ripening regulators have been identified, especially in the model system tomato, yet more remain to be discovered and integrated into comprehensive regulatory models. Most tomato ripening genes have been studied in pericarp tissue, though recent evidence indicates that locule tissue is a site of early ripening-gene activities. Here, we identified and functionally characterized an Ethylene Response Factor (ERF) gene, SlERF.D6 , by investigating tomato transcriptome data throughout plant development, emphasizing genes elevated in the locule during fruit development and ripening. SlERF.D6 loss-of-function mutants resulting from CRISPR/Cas9 gene editing delayed ripening initiation and carotenoid accumulation in both pericarp and locule tissues. Transcriptome analysis of lines altered in SlERF.D6 expression revealed multiple classes of altered genes including ripening regulators, in addition to carotenoid, cell wall, and ethylene pathway genes, suggesting comprehensive ripening control. Distinct regulatory patterns in pericarp versus locule tissues were observed, indicating tissue-specific activity of this transcription factor (TF). Analysis of SlERF.D6 interaction with target promoters revealed an APETALA 2/ETHYLENE RESPONSE FACTOR (AP2/ERF) TF ( SlDEAR2 ) as a target of SlERF.D6. Furthermore, we show that a third TF gene, SlTCP12 , is a target of SlDEAR2, presenting a tricomponent module of ripening control residing in the larger SlERF.D6 regulatory network.

β-carotene promotes pollen germination and tube growth via a mechanism that regulates reactive oxygen species homeostasis in tomato.

It is well known that if a fruit is harvested extremely early its development and function are interrupted, and it may never attain full maturity and optimal quality. Reports revealing insights regarding the alterations of maturation, ripening and postharvest quality in very early picked fruits are rare. We examined the effects of early harvesting on tomatoes by characterizing different accessions at the molecular, physiological, and biochemical levels. We found that even very early harvested fruits could achieve postharvest maturation and ripening though with some defects in pigment and cuticle formation, and seeds from very early harvested fruits could still germinate and develop as normal and healthy plants. One critical regulator of tomato cuticle integrity, SlCER1–2, was shown to contribute to cuticle defects in very early harvested fruits. Very early fruit harvest still allowing ripening and seed development indicates that the genetic and physiological programs of later maturation and ripening are set into motion early in fruit development and are not dependent on complete fruit expansion nor attachment to the plant.

Ripening is crucial for the development of fleshy fruits that release their seeds following consumption by frugivores and are important contributors to human health and nutritional security. Many genetic ripening regulators have been identified, especially in the model system tomato, yet more remain to be discovered and integrated into comprehensive regulatory models. Most tomato ripening genes have been studied in pericarp tissue, though recent evidence indicates that locule tissue is a site of early ripening-gene activities. Here we identified and functionally characterized an Ethylene Response Factor gene, SlERF.D6, by investigating tomato transcriptome data throughout plant development, emphasizing genes elevated in the locule during fruit development and ripening. SlERF.D6loss-of-function mutants resulting from CRISPR/Cas9 gene editing delayed ripening initiation and carotenoid accumulation in both pericarp and locule tissues. Transcriptome analysis of lines altered in SlERF.D6 expression revealed multiple classes of altered genes including ripening regulators, in addition to carotenoid, cell wall and ethylene pathway genes, suggesting comprehensive ripening control. Distinct regulatory patterns in pericarp versus locule tissues were observed indicating tissue-specific activity of this transcription factor. Analysis of SlERF.D6 interaction with target promoters revealed an AP2/ERF transcription factor (SlDEAR2) as a target of SlERF.D6. Furthermore, we show that a third transcription factor gene, SlTCP12, is a target of SlDEAR2, presenting a tri-component module of ripening control.

Fruit softening, an irreversible process that occurs during fruit ripening, can lead to losses and waste during postharvest transportation and storage. Cell wall disassembly is the main factor leading to loss of fruit firmness, and several ripening-associated cell wall genes have been targeted for genetic modification, particularly pectin modifiers. However, individual knockdown of most cell wall–related genes has had minimal influence on cell wall integrity and fruit firmness, with the notable exception of pectate lyase. Compared to pectin disassembly, studies of the cell wall matrix, the xyloglucan–cellulose framework, and underlying mechanisms during fruit softening are limited. Here, a tomato (Solanum lycopersicum) fruit ripening–associated α-expansin (SlExpansin1/SlExp1) and an endoglucanase (SlCellulase2/SlCel2), which function in the cell wall matrix, were knocked out individually and together using clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9-mediated genome editing. Simultaneous knockout of SlExp1 and SlCel2 enhanced fruit firmness, reduced depolymerization of homogalacturonan-type pectin and xyloglucan, and increased cell adhesion. In contrast, single knockouts of either SlExp1 or SlCel2 did not substantially change fruit firmness, while simultaneous overexpression of SlExp1 and SlCel2 promoted early fruit softening. Collectively, our results demonstrate that SlExp1 and SlCel2 synergistically regulate cell wall disassembly and fruit softening in tomato.

SUMMARY: A level of redundancy and interplay among the transcriptional regulators of floral development safeguards a plant's reproductive success and ensures crop production. In the present study, an additional layer of complexity in the regulation of floral meristem (FM) identity and flower development is elucidated linking carotenoid biosynthesis and metabolism to the regulation of determinate flowering. The accumulation and subsequent cleavage of a diverse array of ζ‐carotenes in the chloroplast biogenesis 5 (clb5) mutant of Arabidopsis results in the reprogramming of meristematic gene regulatory networks establishing FM identity mirroring that of the FM identity master regulator, APETALA1 (AP1). The immediate transition to floral development in clb5 requires long photoperiods in a GIGANTEA‐independent manner, whereas AP1 is essential for the floral organ development of clb5. The elucidation of this link between carotenoid metabolism and floral development translates to tomato exposing a regulation of FM identity redundant to and initiated by AP1 and proposed to be dependent on the E class floral initiation and organ identity regulator, SEPALLATA3 (SEP3).

Grapevine (Vitis vinifera L.) is one of the most widely cultivated fruit crops because the winemaking industry has huge economic relevance worldwide. Uncovering the molecular mechanisms controlling the developmental progression of plant organs will prove essential for maintaining high-quality grapes, expressly in the context of climate change, which impairs the ripening process. Through a deep inspection of transcriptomic data, we identified VviNAC60, a member of the NAC transcription factor family, as a putative regulator of grapevine organ maturation. We explored VviNAC60 binding landscapes through DNA affinity purification followed by sequencing and compared bound genes with transcriptomics datasets from grapevine plants stably and transiently overexpressing VviNAC60 to define a set of high-confidence targets. Among these, we identified key molecular markers associated with organ senescence and fruit ripening. Physiological, metabolic, and promoter activation analyses showed that VviNAC60 induces chlorophyll degradation and anthocyanin accumulation through the upregulation of STAY-GREEN PROTEIN 1 (VviSGR1) and VviMYBA1, respectively, with the latter being upregulated through a VviNAC60–VviNAC03 regulatory complex. Despite sharing a closer phylogenetic relationship with senescence-related homologs to the NAC transcription factor AtNAP, VviNAC60 complemented the nonripening(nor) mutant phenotype in tomato (Solanum lycopersicum), suggesting a dual role as an orchestrator of both ripening- and senescence-related processes. Our data support VviNAC60 as a regulator of processes initiated in the grapevine vegetative- to mature-phase organ transition and therefore as a potential target for enhancing the environmental resilience of grapevine by fine-tuning the duration of the vegetative phase.