Tomato agamous-like6 parthenocarpy is facilitated by ovule integument reprogramming involving the growth regulator KLUH.

Fruit set is established during and soon after fertilization of the ovules inside the quiescent ovary, but the signaling pathways involved remain obscure. The tomato (Solanum lycopersicum) CRISPR loss-of-function mutant of the transcription factor gene AGAMOUS-like6 (SlAGL6; slagl6CR-sg1) is capable of fertilization-independent setting of normal, yet seedless (parthenocarpic), fruit. To gain insight into the mechanism of fleshy fruit set, in this study, we investigated how slagl6CR-sg1 uncouples fruit set from fertilization. We found that mutant ovules were enlarged due to integument over-proliferation and failed to differentiate an endothelium, the integument's innermost layer, upon maturation. A causal relationship between slagl6 loss-of-function and these abnormal phenotypes is inferred from the observation that SlAGL6 is predominantly expressed in the immature ovule integument, and upon ovule maturation, its expression shifts to the endothelium. The transcriptome of unfertilized mutant ovules profoundly differs from that of wild-type and exhibits substantial overlap with the transcriptomes of fertilized ovules sporophytic tissues. One prominent upregulated gene was the fertilization-induced cytochrome P450 cell proliferation regulator SlKLUH. Indeed, ectopic overexpression of SlKLUH stimulated both integument growth in unfertilized ovules and parthenocarpy, suggesting that its suppression by SlAGL6 is paramount for preventing fertilization-independent fruit set. Taken together, our study informs on the transcriptional programs that are regulated by SlAGL6 and demonstrates that it acts from within the ovule integument to inhibit ovary growth beyond anthesis. That by suppressing components of the fertilization-induced ovule reprogramming underlying fruit set.

Tomato facultative parthenocarpy results from SlAGAMOUS-LIKE 6 loss of function.

The extreme sensitivity of the microsporogenesis process to moderately high or low temperatures is a major hindrance for tomato (Solanum lycopersicum) sexual reproduction and hence year-round cropping. Consequently, breeding for parthenocarpy, namely, fertilization-independent fruit set, is considered a valuable goal especially for maintaining sustainable agriculture in the face of global warming. A mutant capable of setting high-quality seedless (parthenocarpic) fruit was found following a screen of EMS-mutagenized tomato population for yielding under heat stress. Next-generation sequencing followed by marker-assisted mapping and CRISPR/Cas9 gene knockout confirmed that a mutation in SlAGAMOUS-LIKE 6 (SlAGL6) was responsible for the parthenocarpic phenotype. The mutant is capable of fruit production under heat stress conditions that severely hamper fertilization-dependent fruit set. Different from other tomato recessive monogenic mutants for parthenocarpy, Slagl6 mutations impose no homeotic changes, the seedless fruits are of normal weight and shape, pollen viability is unaffected, and sexual reproduction capacity is maintained, thus making Slagl6 an attractive gene for facultative parthenocarpy. The characteristics of the analysed mutant combined with the gene's mode of expression imply SlAGL6 as a key regulator of the transition between the state of 'ovary arrest' imposed towards anthesis and the fertilization-triggered fruit set.

Interplay of MYB factors in differential cell expansion, and consequences for tomato fruit development.

We previously identified SlFSM1 as an early fruit-specific gene encoding a short protein harboring a non-canonical SANT/MYB-like domain. Here, we investigated the role of FSM1 during fruit development in tomato and its mode of action. By analyzing tomato plants ectopically expressing FSM1, we established that it negatively affects cell expansion, particularly of those cells with the highest potential to expand, such as those residing inner to the vascular bundles in the fruit pericarp. This function of FSM1 differs from that of the snapdragon FSM1-like gene, RAD, which through an antagonistic activity with DIV participates in establishing floral asymmetry. Revealing an additional component of the FSM1/RAD regulatory complex, we show here that FSM1 physically interacts with FSB1, a previously uncharacterized factor harboring an atypical MYB repeat. We also demonstrate that FSB1 physically interacts with the transcription factor MYBI, a homolog of DIV. Our results show that the formation of the FSB1-MYBI complex is competed by FSM1, which recognizes in FSB1 the same region as MYBI does. Taken together, these studies expose a function for the FSM1/FSB1/MYBI complex in controlling tomato cell expansion, while revealing a mechanism by which competing MYB-MYB interactions could participate in the control of gene expression.

Improved yielding and reduced puffiness under extreme temperatures induced by fruit-specific expression of rolB in processing tomatoes.

Tomato fruit production is severely hampered by both extremely high and low temperatures, mainly due to impaired microsporogenesis and pollination under these conditions. Even mild temperature stress, leading to partial damage to pollen viability can result in the production of under-fertilized puffy fruits of poor quality, while severe stress can abolish fruit set completely. Genetic or transgenic parthenocarpy that enables fertilization-independent fruit development offers a solution for tomato yielding under conditions unfavorable for pollen production and/or fertilization. A transgenic processing tomato UC82 line, expressing rolB specifically during early stages of fruit development was compared to the parental line with respect to yield and fruit quality under extreme temperatures. Under both high and low temperatures the transgenic line performed significantly better than the parental line. Its yield was significantly higher mainly due to a higher number of fruits that did develop, and also because of increased fruit weight. While the UC82 fruits developed under high temperatures were very puffy and severely malformed, the transgenic fruits maintained improved jelly fill and were of smooth and regular shape. Interestingly, under high temperatures the improved jelly fill in the transgenic line was accompanied by a higher number of seeds, suggesting that not only the developing seeds promote development of the placental tissue but also that proliferation of this tissue supports better seed development.

Modulated fatty acid desaturation via overexpression of two distinct omega-3 desaturases differentially alters tolerance to various abiotic stresses in transgenic tobacco cells and plants.

Changes in the degree of fatty acid (FA) desaturation are implicated in plant responses to various abiotic stresses, including heat, salt and drought. However, it is still not known whether decreased levels of linolenic acid, found in many plants subjected to salt and drought stress, reflect a mechanism of defence or damage. We addressed this question by generating tobacco cells and plants ectopically overexpressing two FA desaturases: the cytosolic FAD3 or the plastidic FAD8. A remarkable increase in the ratio of total linolenic to linoleic acids resulted from overexpression of FAD3, whereas ectopic overexpression of FAD8 induced an increased ratio mainly in the plastidic lipids. Here we present evidence that overexpressing FAD8 imposes much greater heat sensitivity than does FAD3 overexpression, in both cultured cells and whole plants. Overexpression of either FAD3 or FAD8 increases tolerance to drought in tobacco plants and to osmotic stress in cultured cells. These findings suggest that a drought-induced decreased level of linolenic acid reflects damage. Our results point to the potential of exploiting FAD overexpression as a tool to ameliorate drought tolerance.

The tomato early fruit specific gene Lefsm1 defines a novel class of plant-specific SANT/MYB domain proteins.

We describe here a novel plant-specific gene, Lefsm1 (fruit SANT/MYB-like 1) harboring a single SANT/MYB domain. The expression of Lefsm1 is specific to the very early stages of tomato (Lycopersicon esculentum) fruit development. Ectopic expression of Lefsm1 results in severe developmental alterations manifested in retarded growth, and reduced apical dominance during tomato and Arabidopsis seedling development. A promoter sequence residing 1.0 kb upstream to the translation initiation codon confers the organ-specific expression of the gene. Lefsm1 belongs to a novel small gene family consisting of five to six members in tomato, Arabidopsis and rice. The SANT/MYB domain of LeFSM1 and its orthologs in Arabidopsis and rice differs from that of all other plant or animal MYB proteins and from the SANT domains found in part of the chromatin remodeling proteins. Together, our results indicate that Lefsm1 is a founding member of a small family of proteins containing a novel MYB/SANT domain which is likely to participate in the regulation of a plant-specific developmental program.

Induction of parthenocarpy in tomato via specific expression of the rolB gene in the ovary.

The molecular signals for the development of the ovary into fruit following ovule fertilization are not clear. However, in many species, including tomato ( Lycopersicon esculentum Mill.), auxins and auxin transport inhibitors can substitute for fertilization as activators of fruit set, suggesting that this plant hormone plays a key role in this process. In agreement, transgenes for auxin biosynthesis expressed under ovary- or ovule-specific promoters were shown earlier to enable parthenocarpic (i.e. seedless) fruit development. In the present study, we tested an alternative approach for the induction of parthenocarpy that is based on ovary-specific expression of the Agrobacterium rhizogenes-derived gene rolB. This gene was chosen because rolB transgenic plants manifest several syndromes characteristic of auxin treatment. Tomato plants transformed with a chimeric construct containing the rolB gene fused to the ovary- and young-fruit-specific promoter TPRP-F1 developed parthenocarpic fruits. Fruit size and morphology, including jelly fill in the locules of the seedless fruits, were comparable to those of seeded fruits of the parental line. Although it is not known whether ROLB signals for the same cassette of genes involved in fertilization-dependent fruit development, it clearly activates a battery of genes that enable successful completion of seedless fruit development in tomato.