Parthenocarpic fruit development in tomato

Abstract: Parthenocarpic fruit development is a very attractive trait for growers and consumers. In tomato, three main sources of facultative parthenocarpy, pat, pat-2, pat-3/pat-4, are known to have potential applications in agriculture. The parthenocarpic fruit development in these lines is triggered by a deregulation of the hormonal balance in some specific tissues. Auxins and gibberellins are considered as the key elements in parthenocarpic fruit development of those lines. An increased level of these hormones in the ovary can substitute for pollination and trigger fruit development. This has opened up genetic engineering approaches for parthenocarpy that have given promising results, both in quality and quantity of seedless fruit production.     

Self‐pollination and parthenocarpic ability in developing ovaries of self‐incompatible Clementine mandarins (Citrus clementina)

This study aimed to determine if self‐pollination is needed to trigger facultative parthenocarpy in self‐incompatible Clementine mandarins (Citrus clementina Hort. ex Tan.). ‘Marisol’ and ‘Clemenules’ mandarins were selected, and self‐pollinated and un‐pollinated flowers from both cultivars were used for comparison. These mandarins are always seedless after self‐pollination and show high and low ability to develop substantial parthenocarpic fruits, respectively. The time‐course for pollen grain germination, tube growth and ovule abortion was analyzed as well as that for carbohydrates, active gibberellins (GA₁ and GA₄), auxin (IAA) and abscisic acid (ABA) content in the ovary. ‘Clemenules’ showed higher pollen grain germination, but pollen tube development was arrested in the upper style 9 days after pollination in both cultivars. Self‐pollination did not stimulate parthenocarpy, whereas both un‐pollinated and self‐pollinated ovaries set fruit regardless of the cultivar. On the other hand, ‘Marisol’ un‐pollinated flowers showed greater parthenocarpic ovary growth than ‘Clemenules’ un‐pollinated flowers, i.e. higher ovule abortion rate (+21%), higher fruit set (+44%) and higher fruit weight (+50%). Further, the greater parthenocarpic ability of ‘Marisol’ paralleled higher levels of GA₁ in the ovary (+34% at anthesis). ‘Marisol’ ovary also showed higher hexoses and starch mobilization, but lower ABA levels (?64% at anthesis). Self‐pollination did not modify carbohydrates or GA content in the ovary compared to un‐pollination. Results indicate that parthenocarpy in the Clementine mandarin is pollination‐independent with its ability to set depending on the ovary hormone levels. These findings suggest that parthenocarpy in fertile self‐incompatible mandarins is constitutively regulated.     

Role of gibberellins in parthenocarpic fruit development induced by the genetic system pat-3/pat-4 in tomato

The role of gibberellins (GAs) in the induction of parthenocarpic fruit-set and growth by the pat-3/pat-4 genetic system in tomato ( Lycopersicon esculentum Mill.) was investigated using wild type (WT; Cuarenteno) and a near-isogenic line derived from the German line RP75/59 (the source of pat-3/pat-4 parthenocarpy). Unpollinated WT ovaries degenerated but GA₃ application induced parthenocarpic fruit growth. On the contrary, parthenocarpic growth of pat-3/pat-4 fruits, which occurs in the absence of pollination and hormone treatment, was not affected by applied GA₃. Unpollinated pat-3/pat-4 fruit growth was negated by paclobutrazol, an inhibitor of ent -kaurene oxidase, and this inhibitory effect was negated by GA₃. The quantification of the main GAs of the early 13-hydroxylation pathway (GA₁, GA₈, GA₁₉, GA₂₀, GA₂₉ and GA₄₄) in unpollinated ovaries at 3 developmental stages (flower bud, FB; pre-anthesis, PR; and anthesis, AN), by gas chromatography-selected ion monitoring, showed that the concentration of most of them was higher in pat-3/pat-4 than in WT ovaries at PR and AN stages. The concentration of GA₁, suggested previously to be the active GA in tomate, was 2–4 times higher. Unpollinated pat-3/pat-4 ovaries at FB, PR and AN stages also contained relatively high amounts (5–12 ng g⁻¹) of GA₃, a GA found at less than 0.5 ng g⁻¹ in WT ovaries. It is concluded that the mutations pat-3/pat-4 may induce natural facultative parthenocarpy capacity in tomato by increasing the concentration of GA₁ and GA₃ in the ovaries before pollination.     

Mapping and characterization of novel parthenocarpy QTLs in tomato

Parthenocarpy is the development of the fruit in absence of pollination and/or fertilization. In tomato, parthenocarpy is considered as an attractive trait to solve the problems of fruit setting under unfavorable conditions. We studied the genetics of parthenocarpy in two different lines, IL5-1 and IVT-line 1, both carrying Solanum habrochaites chromosome segments. Parthenocarpy in IL5-1 is under the control of two QTLs, one on chromosome 4 (pat4.1) and one on chromosome 5 (pat5.1). IVT-line 1 also contains two parthenocarpy QTLs, one on chromosome 4 (pat4.2) and one on chromosome 9 (pat9.1). In addition, we identified one stigma exsertion locus in IL5-1, located on the long arm of chromosome 5 (se5.1). It is likely that pat4.1, from IL5-1 and pat4.2, from IVT-line 1, both located near the centromere of chromosome 4 are allelic. By making use of the microsynteny between tomato and Arabidopsis in this genetic region, we identified ARF8 as a potential candidate gene for these two QTLs. ARF8 is known to act as an inhibitor for further carpel development in Arabidopsis, in absence of pollination/fertilization. Expression of an aberrant form of the Arabidopsis ARF8 gene, in tomato, has been found to cause parthenocarpy. This candidate gene approach may lead to the first isolation of a parthenocarpy gene in tomato and will allow further use in several crop species.     

Molecular expression of genes involved in parthenocarpic fruit set in tomato

To characterize the phenomenon of natural parthenocarpy in tomato ( Lycopersicon esculentum Mill.) two different approaches have been followed. At a developmental level, the ovary weights of three non-parthenocarpic lines and three near-isogenic parthenocarpic ( pat-2 ) lines were compared. Four developmental stages were considered: flower bud, preanthesis, anthesis and 4 days after anthesis. The parthenocarpic lines displayed ovary weights higher than their respective non-parthenocarpic lines from preanthesis to 4 days after anthesis. A molecular approach involved comparison of in vitro translation products from flower RNAs taken from the same developmental stages of non-parthenocarpic and near-isogenic parthenocarpic ( pat-2 and pat-3/pat-4 ) lines. Analysis by two-dimensional polyacrylamide gel electrophoresis showed the differential expression of a 30-kDa product in parthenocarpic materials from preanthesis to anthesis. These results suggest that the physiological and molecular events responsible for parthenocarpy begin at the preanthesis stage, before the flower is completely mature and receptive to pollination. The differential expression of this in vitro translation product in pat-2 and pat-3/pat-4 genotypes also suggests a common or confluent molecular basis in genetically controlled parthenocarpy.     

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.     

Genetic Analysis of Growth-Regulator-Induced Parthenocarpy in Arabidopsis

In Arabidopsis, seedless silique development or parthenocarpy can be induced by the application of various plant growth regulators (PGRs) to unfertilized pistils. Ecotype-specific responses were observed in the Arabidopsis ecotypes Columbia and Landsberg relative to the type of PGR and level applied. The parthenocarpic response was greatest in ecotype Landsberg, and comparisons of fruit growth and morphology were studied primarily in this ecotype. Gibberellic acid application (10 micromol pistil(-1)) caused development similar to that in pollinated pistils, while benzyladenine (1 micromol pistil(-1)) and naphthylacetic acid (10 micromol pistil(-1)) treatment produced shorter siliques. Naphthylacetic acid primarily modified mesocarp cell expansion. Arabidopsis mutants were employed to examine potential dependencies on gibberellin biosynthesis (ga1-3, ga4-1, and ga5-1) and perception (spy-4 and gai) during parthenocarpic silique development. Emasculated spy-4 pistils were neither obviously parthenocarpic nor deficient in PGR perception. By contrast, emasculated gai mutants did not produce parthenocarpic siliques following gibberellic acid application, but silique development occurred following pollination or application of auxin and cytokinin. Pollinated gai siliques had decreased cell numbers and morphologically resembled auxin-induced parthenocarpic siliques. This shows that a number of independent and possibly redundant pathways can direct hormone-induced parthenocarpy, and that endogenous gibberellins play a role in regulating cell expansion and promoting cell division in carpels.     

Cytokinin-Induced Parthenocarpic Fruit Development in Tomato Is Partly Dependent on Enhanced Gibberellin and Auxin Biosynthesis

Fruit set of plants largely depends on the biosynthesis and crosstalk of phytohormones. To date the role of cytokinins (CKs) in the fruit development is less understood. Here, we showed that parthenocarpic fruit could be induced by 1-(2-chloro-4-pyridyl)-3-phenylurea (CPPU, an active CK) in tomato ( Solanum lycopersicum cv. Micro-Tom). The fresh weight of CPPU-induced parthenocarpic fruits was comparable with that induced by GA₃. Importantly, CPPU-induced parthenocarpy was found to be compromised by simultaneous application of paclobutrazol (a GA biosynthesis inhibitor), and this effect could be restored by exogenous GA₃. Like pollination, CPPU-induced fruit showed enhanced accumulation of GA₁₊₃ and indole-3-acetic (IAA), which were accompanied by elevated expression of GA biosynthesis genes like SlGPS, SlGA20ox1, SlGA20ox2 and SlGA3ox1, and IAA biosynthesis gene ToFZY. Elevated GAs level in CPPU-induced fruits was also associated with down-regulation of GA inactivation genes, namely SlGA2ox1,2,3,4,5 in comparison with untreated control. These results suggested that CKs may induce parthenocarpy in tomato partially through modulation of GA and IAA metabolisms.     

l-Ascorbic acid metabolism in parthenocarpic and seeded cherry tomatoes

The auxin treatment in tomato plants during anthesis has been extensively used for setting fruits in adverse climatic conditions (e.g., low temperatures and inadequate light), which is well known that reduces pollen availability and fertility. Since auxin application may affect fruit composition and quality, we examined l-ascorbic acid metabolism in seeded fruit (set by natural pollination) and parthenocarpic fruit (set by auxin) in cherry tomato cv. Conchita. Specifically, we studied the oxidized and total ascorbic acid contents, the expression of all characterized genes of l-ascorbic acid metabolism, the activity of ascorbate peroxidase and dehydroascorbate reductase and the immunolocalization of ascorbate peroxidase. Differences were detected between seeded and parthenocarpic fruits, in the expression of some of the genes of ascorbic acid metabolism. However, strong presence of l-ascorbic acid peroxidase protein was detected on the developing seeds. Our data indicate that induced parthenocarpy in auxin treated plants has a significant influence in ascorbic acid metabolism comparing to seeded tomato fruits.     

Induction of parthenocarpy in tomato via specific expression of the<Emphasis Type="Italic"> rolB</Emphasis> 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.     

Cell division and cell enlargement in fruit of Lagenaria leucantha as influenced by pollination and plant growth substances

The effects of NAA (naphthaleneacetic acid), GA₃ (gibberellic acid), CPPU (N-(2-chloro-4-pyridyl)-N'-phenylurea) and pollination on fruit set, cell division and enlargement were studied in Lagenaria leucantha, an important vegetable. NAA and GA₃ were ineffective in inducing parthenocarpy, whereas CPPU induced parthenocarpic fruit significantly larger than fruit that resulted from pollination. Cell division, which occurred during the first 4 days after pollination was not reactivated by NAA or GA₃, but was effectively reactivated by CPPU. The cell number of the total cross-section of CPPU-treated fruit was 117.4% of that of pollinated fruit and 154.4% of that of unpollinated at 12 DAA (days after anthesis) respectively. The CPPU-induced parthenocarpic fruit had the largest cell cross-sectional area followed, successively, by pollinated fruit, NAA-treated fruit, GA₃-treated fruit and unpollinated fruit. These results indicate that CPPU induced parthenocarpic fruit growth by directly reactivating cell division and expansion.     

microRNA159‐targeted SlGAMYB transcription factors are required for fruit set in tomato

The transition from flowering to fruit production, namely fruit set, is crucial to ensure successful sexual plant reproduction. Although studies have described the importance of hormones (i.e. auxin and gibberellins) in controlling fruit set after pollination and fertilization, the role of microRNA‐based regulation during ovary development and fruit set is still poorly understood. Here we show that the microRNA159/GAMYB1 and ‐2 pathway (the miR159/GAMYB1/2 module) is crucial for tomato ovule development and fruit set. MiR159 and SlGAMYBs were expressed in preanthesis ovaries, mainly in meristematic tissues, including developing ovules. SlMIR159‐overexpressing tomato cv. Micro‐Tom plants exhibited precocious fruit initiation and obligatory parthenocarpy, without modifying fruit shape. Histological analysis showed abnormal ovule development in such plants, which led to the formation of seedless fruits. SlGAMYB1/2 silencing in SlMIR159‐overexpressing plants resulted in misregulation of pathways associated with ovule and female gametophyte development and auxin signalling, including AINTEGUMENTA‐like genes and the miR167/SlARF8a module. Similarly to SlMIR159‐overexpressing plants, SlGAMYB1 was downregulated in ovaries of parthenocarpic mutants with altered responses to gibberellins and auxin. SlGAMYBs likely contribute to fruit initiation by modulating auxin and gibberellin responses, rather than their levels, during ovule and ovary development. Altogether, our results unveil a novel function for the miR159‐targeted SlGAMYBs in regulating an agronomically important trait, namely fruit set.