Susana Manzano

Plant sex chromosomes: molecular structure and function.

Recent molecular and genomic studies carried out in a number of model dioecious plant species, including Asparagus officinalis, Carica papaya, Silene latifolia, Rumex acetosa and Marchantia polymorpha, have shed light on the molecular structure of both homomorphic and heteromorphic sex chromosomes, and also on the gene functions they have maintained since their evolution from a pair of autosomes. The molecular structure of sex chromosomes in species from different plant families represents the evolutionary pathway followed by sex chromosomes during their evolution. The degree of Y chromosome degeneration that accompanies the suppression of recombination between the Xs and Ys differs among species. The primitive Ys of A. officinalis and C. papaya have only diverged from their homomorphic Xs in a short male-specific and non-recombining region (MSY), while the heteromorphic Ys of S. latifolia, R. acetosa and M. polymorpha have diverged from their respective Xs. As in the Y chromosomes of mammals and Drosophila, the accumulation of repetitive DNA, including both transposable elements and satellite DNA, has played an important role in the divergence and size enlargement of plant Ys, and consequently in reducing gene density. Nevertheless, the degeneration process in plants does not appear to have reached the Y-linked genes. Although a low gene density has been found in the sequenced Y chromosome of M. polymorpha, most of its genes are essential and are expressed in the vegetative and reproductive organs in both male and females. Similarly, most of the Y-linked genes that have been isolated and characterized up to now in S. latifolia are housekeeping genes that have X-linked homologues, and are therefore expressed in both males and females. Only one of them seems to be degenerate with respect to its homologous region in the X. Sequence analysis of larger regions in the homomorphic X and Y chromosomes of papaya and asparagus, and also in the heteromorphic sex chromosomes of S. latifolia and R. acetosa, will reveal the degenerative changes that the Y-linked gene functions have experienced during sex chromosome evolution.

Expansion of Microsatellites on Evolutionary Young Y Chromosome

Sex chromosomes are an ideal system to study processes connected with suppressed recombination. We found evidence of microsatellite expansion, on the relatively young Y chromosome of the dioecious plant sorrel (Rumex acetosa, XY1Y2 system), but no such expansion on the more ancient Y chromosomes of liverwort (Marchantia polymorpha) and human. The most expanding motifs were AC and AAC, which also showed periodicity of array length, indicating the importance of beginnings and ends of arrays. Our data indicate that abundance of microsatellites in genomes depends on the inherent expansion potential of specific motifs, which could be related to their stability and ability to adopt unusual DNA conformations. We also found that the abundance of microsatellites is higher in the neighborhood of transposable elements (TEs) suggesting that microsatellites are probably targets for TE insertions. This evidence suggests that microsatellite expansion is an early event shaping the Y chromosome where this process is not opposed by recombination, while accumulation of TEs and chromosome shrinkage predominate later.

Involvement of ethylene biosynthesis and signalling in fruit set and early fruit development in zucchini squash ( Cucurbita pepo L.)

BackgroundWe have identified a kind of parthenocarpy in zucchini squash which is associated with an incomplete andromonoecy, i.e. a partial conversion of female into bisexual flowers. Given that andromonoecy in this and other cucurbit species is caused by a reduction of ethylene production in the female flower, the associated parthenocarpic development of the fruit suggested the involvement of ethylene in fruit set and early fruit development.ResultsWe have compared the production of ethylene as well as the expression of 13 ethylene biosynthesis and signalling genes in pollinated and unpollinated ovaries/fruits of two cultivars, one of which is parthenocarpic (Cavili), while the other is non-parthenocarpic (Tosca). In the latter, unpollinated ovaries show an induction of ethylene biosynthesis and ethylene signal transduction pathway genes three days after anthesis, which is concomitant with the initiation of fruit abortion and senescence. Fruit set and early fruit development in pollinated flowers of both cultivars and unpollinated flowers of Cavili is coupled with low ethylene biosynthesis and signalling, which would also explain the partial andromonoecy in the parthenocarpic genotype. The reduction of ethylene production in the ovary cosegregates with parthenocarpy and partial andromonoecy in the selfing progeny of Cavili. Moreover, the induction of ethylene in anthesis (by ethephon treatments) reduced the percentage of bisexual parthenocarpic flowers in Cavili, while the inhibition of ethylene biosynthesis or response (by AVG and STS treatments) induces not only andromonoecy but also the parthenocarpic development of the fruit in both cultivars.ConclusionsResults demonstrate that a reduction of ethylene production or signalling in the zucchini flower is able to induce fruit set and early fruit development, and therefore that ethylene is actively involved in fruit set and early fruit development. Auxin and TIBA treatments, inducing fruit set and early fruit development in this species, also inhibit ethylene production and the expression of ethylene biosynthesis and response genes. A model is presented that discusses the crosstalk between ethylene and auxin in the control of fruit set and early fruit development in zucchini squash.

The role of ethylene and brassinosteroids in the control of sex expression and flower development in Cucurbita pepo

In this paper we compare the sensitivity of different squash genotypes to ethylene and brassinosteroids by studying the effects of different ethylene and brassinosteroid treatments on the sexual expression and flower development of different C. pepo genotypes: Bolognese (Bog) and Vegetable Spaghetti (Veg), two contrasting lines for ethylene production and sensitivity, as well as Cora, a standard commercial hybrid. Results have demonstrated that ethylene has a much greater effect on sexual expression and flower development in C. pepo than brassinosteroids. Ethephon increases the number of female flowers per plant and reduces the first male phase of development, while treatments with the ethylene inhibitors AVG and STS reduce the number of female flowers per plant and expand the first male phase of development. The differential response observed between genotypes appears to be related to ethylene production and sensitivity. Bog, which produces more ethylene and is more sensitive to this hormone, responded much better to AVG and STS, reducing the number of female flowers per plant, while Veg, which is characterised by lower production of and sensitivity to ethylene, responded better to ethephon by reducing the first male phase of development and increasing the number of female flowers per plant. The differential abortion of female and male flowers in ethephon, AVG and STS treatments, as well as the occurrence of bisexual flowers in the AVG and STS treated plants of the more ethylene sensitive genotypes, demonstrate that ethylene is also involved in the development of female flowers. Female flower buds require a minimal level of ethylene not only to complete their development and maturation without a premature abortion, but also to arrest the development of stamens in the third whorl and to promote the appropriate growth of the carpels. On the contrary, the role of brassinosteroids in the sexual expression of C. pepo was not so evident. The application of brassinazole, an inhibitor of brassinosteroid biosynthesis slightly changes the production of ethylene in the three analysed genotypes, but those changes have little effect on their sexual phenotypes, and they do not alter the development of the unisexual flowers.

Involvement of Ethylene Biosynthesis and Signalling in the Transition from Male to Female Flowering in the Monoecious Cucurbita pepo

It is well established that ethylene is the main hormonal regulator of sexual expression in the Cucurbitaceae family, controlling not only the sexual fate of individual floral buds, but also the female flower transition, that is, the time at which the first female flower appears and therefore the number of female flowers per plant. Although sex determination of individual flower buds is known to be controlled by specific ethylene biosynthesis ACS genes in melon and cucumber, the role of ethylene genes in the control of the transition to female flowering is still unknown. We have identified two contrasting monoecious inbred lines of Cucurbita pepo, Bolognese (Bog) and Vegetable spaghetti (Veg), which differ in female flower transition but not in flower development. In Bog, which is very sensitive to ethylene, the transition to female flowering is very early, whereas in Veg, which is much less sensitive to ethylene, the transition occurs much later. In this article we compare the production of ethylene and the expression profiles of seven genes involved in the biosynthesis, perception, and signalling of ethylene in the two contrasting lines. Bog, with earlier female flower transition, showed higher ethylene production and CpACO1 expression in the apex at an earlier stage of plant development, when Bog is already producing female flowers, but Veg has not transitioned to female flowering yet. Moreover, the expression of the ethylene receptor and CTR-like genes in the apex of Veg and Bog plants indicates that these genes negatively regulate female flower transition during the earlier stages of plant development. The earlier transition to female flowering in Bog is not only associated with a higher production of ethylene in the apex but also with a premature decline of ethylene negative regulators (receptors and CTR-like) in the apex of the plant. These results provide the basis for a model that explains the regulation of female flowering transition in monoecious cucurbits.

A Major Gene Conferring Reduced Ethylene Sensitivity and Maleness in Cucurbita pepo

External treatment with ethylene had indicated earlier that this hormone is the main factor controlling sex determination in Cucurbita pepo. Up to now, however, there was no genetic evidence that supported the relationship between ethylene production, or perception, and sexual expression in this species. Here we demonstrate that the extreme male phenotype of the Vegetable Spaghetti (Veg) inbred line of C. pepo subspecies pepo is determined by a major gene that confers reduced ethylene sensitivity in plants. The production of female flowers in the Veg line is very delayed and reduced with respect to the contrasting Bolognese (Bog) line, ranging between 5 and 35% of female flowers per plant. This enhanced maleness trait segregates as a single gene in the F2 and backcross (BC) generations, and co-segregates with a weak ethylene-insensitive phenotype in the F2 population, suggesting that the gene responsible for the Veg phenotype could be the result of a mutation in a receptor or response gene for ethylene. Although the etiolated seedlings of the Veg line, and the most androecious plants in the F2 generation, produce more ethylene than those of the contrasting line, they are less sensitive to this hormone, as indicated by a weaker triple response and a delayed abscission of ethylene-treated male flowers. Given that the sexual phenotype of F2 plants is correlated with ethylene sensitivity, with the more sensitive plants producing the higher number of female flowers, our results demonstrate that the ethylene response is directly involved in the control of sex determination in C. pepo. It regulates the induction of female flower production, and therefore the extension of the initial phase of development in which the plant produces only male flowers, as well as the number of female flowers per plant.

Cloning and characterisation of two CTR1-like genes in Cucurbita pepo: regulation of their expression during male and female flower development.

Ethylene is an essential regulator of flower development in Cucurbita pepo, controlling the sexual expression, and the differentiation and maturation of floral organs. To study the action mechanism of ethylene during the male and female flower development, we have identified two CTR1 homologues from C. pepo, CpCTR1 and CpCTR2, and analysed their expressions during female and male flower development and in response to external treatments with ethylene. CpCTR1 and CpCTR2 share a high homology with plant CTR1-like kinases, but differ from other related kinases such as the Arabidopsis EDR1 and the tomato LeCTR2. The C-terminal ends of both CpCTR1 and CpCTR2 have all the conserved motifs of Ser/Thr kinase domains, including the ATP-binding signature and the protein kinase active site consensus sequence, which suggests that CpCTR1 and CpCTR2 could have the same function as CTR1 in ethylene signalling. The transcripts of both genes were detected in different organs of the plant, including roots, leaves and shoots, but were mostly accumulated in mature flowers. During the development of male and female flowers, CpCTR1 and CpCTR2 expressions were concomitant with ethylene production, which indicates that both genes could be upregulated by ethylene, at least in flowers. Moreover, external treatments with ethylene, although did not alter the expression of these two genes in seedlings and leaves, were able to upregulate their expression in flowers. In the earlier stages of flower development, when ethylene production is very low, the expression of CpCTR1 and CpCTR2 is higher in male floral organs, which agrees with the role of these genes as negative regulators of ethylene signalling, and explain the lower ethylene sensitivity of male flowers in comparison with female flowers. The function of the upregulation of these two genes in later stages of female flower development, when the production of ethylene is also increased, is discussed.

Molecular and functional characterization of CpACS27A gene reveals its involvement in monoecy instability and other associated traits in squash (Cucurbita pepo L.)

A number of Cucurbita pepo genotypes showing instable monoecy or partial andromonoecy, i.e. an incomplete conversion of female into bisexual flowers, have been detected. Given that in melon and cucumber andromonoecy is the result of reduction of ethylene production in female floral buds, caused by mutations in the ethylene biosynthesis genes CmACS7 and CsACS2; we have cloned and characterized two related C. pepo genes, CpACS27A and CpACS27B. The molecular structure of CpACS27A and its specific expression in the carpels of female flowers during earlier stages of flower development suggests that this gene is the Cucurbita ortholog of CmACS7 and CsACS2. CpACS27B is likely to be a paralogous pseudogene since it has not been found to be expressed in any of the analyzed tissues. CpACS27A was sequenced in Bolognese (Bog) and Vegetable Spaghetti (Veg), two monoecious inbred lines whose F2 was segregating for partial andromonoecy. The Bog allele of CpACS27A carried a missense mutation that resulted in a substitution of the conserved serine residue in position 176 by an alanine. Segregation analysis indicated that this mutant variant is necessary but not sufficient to confer the andromonoecious phenotype in squash. In concordance with its involvement in stamen arrest, a reduction in CpACS27A expression has been found in bisexual flower buds at earlier stages of development. This reduction in CpACS27A expression was concomitant with a downregulation of other ethylene biosynthesis and signaling genes during earlier and later stages of ovary development. The role of CpACS27A is discussed regarding the regulation of ethylene biosynthesis and signaling genes in the control of andromonoecy-associated traits, such as the delayed maturation of corolla and stigma as well as the parthenocarpic development of the fruit.

Sources of parthenocarpy for Zucchini breeding: relationship with ethylene production and sensitivity

Parthenocarpy is becoming an essential trait for off-season greenhouse production of Zucchini squash. Given that winter conditions promote a reduction in the number of male flowers and in the activity of pollinators, the application of synthetic auxins is currently the most widespread method to induce fruit set. We have evaluated the parthenocarpic tendency of 48 long-fruited accessions of Cucurbita pepo spp. pepo, from morphotypes Zucchini, Vegetable marrow and Cocozelle, including 45 traditional cultivars and 3 commercial hybrids, with the goal of identifying new sources of parthenocarpy for breeding programs. After the first screening, 20 selected accessions were evaluated for the growth rate of unpollinated fruit. Twelve of the selected accessions identified as either strongly parthenocarpic or non-parthenocarpic, were compared for fruit rate growth, ethylene production and ethylene sensitivity. Apart from the three control hybrids, the fastest parthenocarpic fruit growth was observed in ‘CpCAL112’, ‘CM-37’, ‘E-27’, ‘PI261610’, and ‘V-185’. The source of the parthenocarpy of some of these accessions differs from that of the hybrids as it was not associated with the conversion of female into bisexual flowers or with the so-called “fruits with attached flowers” syndrome, which is an undesirable trait in current parthenocarpic hybrids. The alternative sources of parthenocarpy may be of great importance in current Zucchini breeding programs. We also demonstrate that the parthenocarpy of these accessions is associated with downregulation of ethylene production in unpollinated fruits during the first days post anthesis (DPA). In non-parthenocarpic accessions, unpollinated fruits boosted ethylene production at 3 DPA, concomitantly with fruit abortion and senescence, while in parthenocarpic accessions, fruits produced little ethylene at 3 DPA. Therefore, ethylene production in ovaries/fruits at 3 DPA can be used as a marker to identify and select parthenocarpy in Zucchini squash. However, in the cultivars tested here, ethylene production and sensitivity in vegetative organs and in male flowers earlier than 3 PDA do not appear well associated with parthenocarpy.

Individual Shrink Wrapping of Zucchini Fruit Improves Postharvest Chilling Tolerance Associated with a Reduction in Ethylene Production and Oxidative Stress Metabolites

We have studied the effect of individual shrink wrapping (ISW) on the postharvest performance of refrigerated fruit from two zucchini cultivars that differ in their sensitivity to cold storage: Sinatra (more sensitive) and Natura (more tolerant). The fruit was individually shrink wrapped before storing at 4°C for 0, 7 and 14 days. Quality parameters, ethylene and CO2 productions, ethylene gene expression, and oxidative stress metabolites were assessed in shrink wrapped and non-wrapped fruit after conditioning the fruit for 6 hours at 20°C. ISW decreased significantly the postharvest deterioration of chilled zucchini in both cultivars. Weight loss was reduced to less than 1%, pitting symptoms were completely absent in ISW fruit at 7 days, and were less than 25% those of control fruits at 14 days of cold storage, and firmness loss was significantly reduced in the cultivar Sinatra. These enhancements in quality of ISW fruit were associated with a significant reduction in cold-induced ethylene production, in the respiration rate, and in the level of oxidative stress metabolites such as hydrogen peroxide and malonyldialdehyde (MDA). A detailed expression analysis of ethylene biosynthesis, perception and signaling genes demonstrated a downregulation of CpACS1 and CpACO1 genes in response to ISW, two genes that are upregulated by cold storage. However, the expression patterns of six other ethylene biosynthesis genes (CpACS2 to CpACS7) and five ethylene signal transduction pathway genes (CpCTR1, CpETR1, CpERS1, CpEIN3.1 and CpEN3.2), suggest that they do not play a major role in response to cold storage and ISW packaging. In conclusion, ISW zucchini packaging resulted in improved tolerance to chilling concomitantly with a reduction in oxidative stress, respiration rate and ethylene production, as well as in the expression of ethylene biosynthesis genes, but not of those involved in ethylene perception and sensitivity.