Gibberellin and miRNA156-targeted SlSBP genes synergistically regulate tomato floral meristem determinacy and ovary patterning.

Many developmental processes associated with fruit development occur at the floral meristem (FM). Age-regulated microRNA156 (miR156) and gibberellins (GAs) interact to control flowering time, but their interplay in subsequent stages of reproductive development is poorly understood. Here, in tomato (Solanum lycopersicum), we show that GA and miR156-targeted SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL or SBP) genes interact in the tomato FM and ovary patterning. High GA responses or overexpression of miR156 (156OE), which leads to low expression levels of miR156-silenced SBP genes, resulted in enlarged FMs, ovary indeterminacy and fruits with increased locule number. Conversely, low GA responses reduced indeterminacy and locule number, and overexpression of a S. lycopersicum (Sl)SBP15 allele that is miR156 resistant (rSBP15) reduced FM size and locule number. GA responses were partially required for the defects observed in 156OE and rSBP15 fruits. Transcriptome analysis and genetic interactions revealed shared and divergent functions of miR156-targeted SlSBP genes, PROCERA/DELLA and the classical WUSCHEL/CLAVATA pathway, which has been previously associated with meristem size and determinacy. Our findings reveal that the miR156/SlSBP/GA regulatory module is deployed differently depending on developmental stage and create novel opportunities to fine-tune aspects of fruit development that have been important for tomato domestication.

The peach HECATE3-like gene FLESHY plays a double role during fruit development.

Tight control of cell/tissue identity is essential for a correct and functional organ patterning, an important component of overall fruit development and eventual maturation and ripening. Despite many investigations regarding the molecular determinants of cell identity in fruits of different species, a useful model able to depict the regulatory networks governing this relevant part of fruit development is still missing. Here we described the peach fruit as a system to link the phenotype of a slow ripening (SR) selection to an altered transcriptional regulation of genes involved in determination of mesocarp cell identity providing insight toward molecular regulation of fruit tissue formation. Morpho-anatomical observations and metabolomics analyses performed during fruit development on the reference cultivar Fantasia, compared to SR, revealed that the mesocarp of SR maintained typical immaturity traits (e.g. small cell size, high amino acid contents and reduced sucrose) throughout development, along with a strong alteration of phenylpropanoid contents, resulting in accumulation of phenylalanine and lignin. These findings suggest that the SR mesocarp is phenotypically similar to a lignifying endocarp. To test this hypothesis, the expression of genes putatively involved in determination of drupe tissues identity was assessed. Among these, the peach HEC3-like gene FLESHY showed a strongly altered expression profile consistent with pit hardening and fruit ripening, generated at a post-transcriptional level. A double function for FLESHY in channelling the phenylpropanoid pathway to either lignin or flavour/aroma is suggested, along with its possible role in triggering auxin-ethylene cross talk at the start of ripening.

microRNA156-targeted SPL/SBP box transcription factors regulate tomato ovary and fruit development.

Fruit ripening in tomato (Solanum lycopersicum L.) is well understood at the molecular level. However, information regarding genetic pathways associated with tomato ovary and early fruit development is still lacking. Here, we investigate the possible role(s) of the microRNA156/SQUAMOSA promoter-binding protein-like (SPL or SBP box) module (miR156 node) in tomato ovary development. miR156-targeted S. lycopersicum SBP genes were dynamically expressed in developing flowers and ovaries, and miR156 was mainly expressed in meristematic tissues of the ovary, including placenta and ovules. Transgenic tomato cv. Micro-Tom plants over-expressing the AtMIR156b precursor exhibited abnormal flower and fruit morphology, with fruits characterized by growth of extra carpels and ectopic structures. Scanning electron microscopy and histological analyses showed the presence of meristem-like structures inside the ovaries, which are probably responsible for the ectopic organs. Interestingly, expression of genes associated with meristem maintenance and formation of new organs, such as LeT6/TKN2 (a KNOX-like class I gene) and GOBLET (a NAM/CUC-like gene), was induced in developing ovaries of transgenic plants as well as in the ovaries of the natural mutant Mouse ear (Me), which also displays fruits with extra carpels. Conversely, expression of the MADS box genes MACROCALYX (MC) and FUL1/TDR4, and the LEAFY ortholog FALSIFLORA, was repressed in the developing ovaries of miR156 over-expressors, suggesting similarities with Arabidopsis at this point of the miR156/SPL pathway but with distinct functional consequences in reproductive development. Altogether, these observations suggest that the miR156 node is involved in maintenance of the meristematic state of ovary tissues, thereby controlling initial steps of fleshy fruit development and determinacy.

The NTT transcription factor promotes replum development in Arabidopsis fruits.

Fruits are complex plant structures that nurture seeds and facilitate their dispersal. The Arabidopsis fruit is termed silique. It develops from the gynoecium, which has a stigma, a style, an ovary containing the ovules, and a gynophore. Externally, the ovary consists of two valves, and their margins lay adjacent to the replum, which is connected to the septum that internally divides the ovary. In this work we describe the role for the zinc-finger transcription factor NO TRANSMITTING TRACT (NTT) in replum development. NTT loss of function leads to reduced replum width and cell number, whereas increased expression promotes replum enlargement. NTT activates the homeobox gene BP, which, together with RPL, is important for replum development. In addition, the NTT protein is able to bind the BP promoter in yeast, and when this binding region is not present, NTT fails to activate BP in the replum. Furthermore, NTT interacts with itself and different proteins involved in fruit development: RPL, STM, FUL, SHP1 and SHP2 in yeast and in planta. Moreover, its genetic interactions provide further evidence about its biological relevance in replum development.

The WOX13 homeobox gene promotes replum formation in the Arabidopsis thaliana fruit.

The Arabidopsis fruit forms a seedpod that develops from the fertilized gynoecium. It is mainly comprised of an ovary in which three distinct tissues can be differentiated: the valves, the valve margins and the replum. Separation of cells at the valve margin allows for the valves to detach from the replum and thus dispersal of the seeds. Valves and valve margins are located in lateral positions whereas the replum is positioned medially and retains meristematic properties resembling the shoot apical meristem (SAM). Members of the WUSCHEL-related homeobox family have been involved in stem cell maintenance in the SAM, and within this family, we found that WOX13 is expressed mainly in meristematic tissues including the replum. We also show that wox13 loss-of-function mutations reduce replum size and enhance the phenotypes of mutants affected in the replum identity gene RPL. Conversely, misexpression of WOX13 produces, independently from BP and RPL, an oversized replum and valve defects that closely resemble those of mutants in JAG/FIL activity genes. Our results suggest that WOX13 promotes replum development by likely preventing the activity of the JAG/FIL genes in medial tissues. This regulation seems to play a role in establishing the gradient of JAG/FIL activity along the medio-lateral axis of the fruit critical for proper patterning. Our data have allowed us to incorporate the role of WOX13 into the regulatory network that orchestrates fruit patterning.

Redundant CArG Box Cis-motif Activity Mediates SHATTERPROOF2 Transcriptional Regulation during Arabidopsis thaliana Gynoecium Development.

In the Arabidopsis thaliana seed pod, pod shatter and seed dispersal properties are in part determined by the development of a longitudinally orientated dehiscence zone (DZ) that derives from cells of the gynoecial valve margin (VM). Transcriptional regulation of the MADS protein encoding transcription factors genes SHATTERPROOF1 (SHP1) and SHATTERPROOF2 (SHP2) are critical for proper VM identity specification and later on for DZ development. Current models of SHP1 and SHP2 regulation indicate that the transcription factors FRUITFULL (FUL) and REPLUMLESS (RPL) repress these SHP genes in the developing valve and replum domains, respectively. Thus the expression of the SHP genes is restricted to the VM. FUL encodes a MADS-box containing transcription factor that is predicted to act through CArG-box containing cis-regulatory motifs. Here we delimit functional modules within the SHP2 cis-regulatory region and examine the functional importance of CArG box motifs within these regulatory regions. We have characterized a 2.2kb region upstream of the SHP2 translation start site that drives early and late medial domain expression in the gynoecium, as well as expression within the VM and DZ. We identified two separable, independent cis-regulatory modules, a 1kb promoter region and a 700bp enhancer region, that are capable of giving VM and DZ expression. Our results argue for multiple independent cis-regulatory modules that support SHP2 expression during VM development and may contribute to the robustness of SHP2 expression in this tissue. Additionally, three closely positioned CArG box motifs located in the SHP2 upstream regulatory region were mutated in the context of the 2.2kb reporter construct. Mutating simultaneously all three CArG boxes caused a moderate de-repression of the SHP2 reporter that was detected within the valve domain, suggesting that these CArG boxes are involved in SHP2 repression in the valve.

Rosaceae Fruit Development, Ripening and Post-harvest: An Epigenetic Perspective.

Rosaceae is a family with an extraordinary spectrum of fruit types, including fleshy peach, apple, and strawberry that provide unique contributions to a healthy diet for consumers, and represent an excellent model for studying fruit patterning and development. In recent years, many efforts have been made to unravel regulatory mechanism underlying the hormonal, transcriptomic, proteomic and metabolomic changes occurring during Rosaceae fruit development. More recently, several studies on fleshy (tomato) and dry (Arabidopsis) fruit model have contributed to a better understanding of epigenetic mechanisms underlying important heritable crop traits, such as ripening and stress response. In this context and summing up the results obtained so far, this review aims to collect the available information on epigenetic mechanisms that may provide an additional level in gene transcription regulation, thus influencing and driving the entire Rosaceae fruit developmental process. The whole body of information suggests that Rosaceae fruit could become also a model for studying the epigenetic basis of economically important phenotypes, allowing for their more efficient exploitation in plant breeding.

Beyond the Divide: Boundaries for Patterning and Stem Cell Regulation in Plants.

The initiation of plant lateral organs from the shoot apical meristem (SAM) is closely associated with the formation of specialized domains of restricted growth known as the boundaries. These zones are required in separating the meristem from the growing primordia or adjacent organs but play a much broader role in regulating stem cell activity and shoot patterning. Studies have revealed a network of genes and hormone pathways that establish and maintain boundaries between the SAM and leaves. Recruitment of these pathways is shown to underlie a variety of processes during the reproductive phase including axillary meristems production, flower patterning, fruit development, and organ abscission. This review summarizes the role of conserved gene modules in patterning boundaries throughout the life cycle.