A mutation in the brassinosteroid biosynthesis gene CpDWF5 disrupts vegetative and reproductive development and the salt stress response in squash (Cucurbita pepo).

A Cucurbita pepo mutant with multiple defects in growth and development has been identified and characterized. The mutant dwfcp displayed a dwarf phenotype with dark green and shrinking leaves, shortened internodes and petioles, shorter but thicker roots and greater root biomass, and reduced fertility. The causal mutation of the phenotype was found to disrupt gene Cp4.1LG17g04540, the squash orthologue of the Arabidopsis brassinosteroid (BR) biosynthesis gene DWF5, encoding for 7-dehydrocholesterol reductase. A single nucleotide transition (G > A) causes a splicing defect in intron 6 that leads to a premature stop codon and a truncated CpDWF5 protein. The mutation co-segregated with the dwarf phenotype in a large BC1S1 segregating population. The reduced expression of CpDWF5 and brassinolide (BL) content in most mutant organs, and partial rescue of the mutant phenotype by exogenous application of BL, showed that the primary cause of the dwarfism in dwfcp is a BR deficiency. The results showed that in C. pepo, CpDWF5 is not only a positive growth regulator of different plant organs but also a negative regulator of salt tolerance. During germination and the early stages of seedling development, the dwarf mutant was less affected by salt stress than the wild type, concomitantly with a greater upregulation of genes associated with salt tolerance, including those involved in abscisic acid (ABA) biosynthesis, ABA and Ca2+ signaling, and those coding for cation exchangers and transporters.

Structural and functional characterization of genes PYL-PP2C-SnRK2s in the ABA signalling pathway of Cucurbita pepo.

BACKGROUND: The core regulation of the abscisic acid (ABA) signalling pathway comprises the multigenic families PYL, PP2C, and SnRK2. In this work, we conducted a genome-wide study of the components of these families in Cucurbita pepo. RESULTS: The bioinformatic analysis of the C. pepo genome resulted in the identification of 19 CpPYL, 102 CpPP2C and 10 CpSnRK2 genes. The investigation of gene structure and protein motifs allowed to define 4 PYL, 13 PP2C and 3 SnRK2 subfamilies. RNA-seq analysis was used to determine the expression of these gene families in different plant organs, as well as to detect their differential gene expression during germination, and in response to ABA and cold stress in leaves. The specific tissue expression of some gene members indicated the relevant role of some ABA signalling genes in plant development. Moreover, their differential expression under ABA treatment or cold stress revealed those ABA signalling genes that responded to ABA, and those that were up- or down-regulated in response to cold stress. A reduced number of genes responded to both treatments. Specific PYL-PP2C-SnRK2 genes that had potential roles in germination were also detected, including those regulated early during the imbibition phase, those regulated later during the embryo extension and radicle emergence phase, and those induced or repressed during the whole germination process. CONCLUSIONS: The outcomes of this research open new research lines for agriculture and for assessing gene function in future studies.

An miR164-resistant mutation in the transcription factor gene CpCUC2B enhances carpel arrest and ectopic boundary specification in Cucurbita pepo flower development.

The sex determination process in cucurbits involves the control of stamen or carpel development during the specification of male or female flowers from a bisexual floral meristem, a function coordinated by ethylene. A gain-of-function mutation in the miR164-binding site of CpCUC2B, ortholog of the Arabidopsis transcription factor gene CUC2, not only produced ectopic floral meristems and organs, but also suppressed the development of carpels and promoted the development of stamens. The cuc2b mutation induced the transcription of CpCUC2B in the apical shoots of plants after female flowering but repressed other CUC genes regulated by miR164, suggesting a conserved functional redundancy of these genes in the development of squash flowers. The synergistic androecious phenotype of the double mutant between cuc2b and etr2b, an ethylene-insensitive mutation that enhances the production of male flowers, demonstrated that CpCUC2B arrests the development of carpels independently of ethylene and CpWIP1B. The transcriptional regulation of CpCUC1, CpCUC2, and ethylene genes in cuc2b and ethylene mutants also confirms this conclusion. However, the epistasis of cuc2b over aco1a, a mutation that suppresses stamen arrest in female flowers, and the down-regulation of CpACS27A in cuc2b female apical shoots, indicated that CpCUC2B promotes stamen development by suppressing the late ethylene production.

Physiological and metabolomic responses of the ethylene insensitive squash mutant etr2b to drought.

The squash gain-of-function mutant etr2b disrupts the ethylene-binding domain of ethylene receptor CpETR2B, conferring partial ethylene insensitivity, changes in flower and fruit development, and enhanced salt tolerance. In this paper, we found that etr2b also confers a growth advantage as well as a physiological and metabolomic response that make the mutant better adapted to drought. Mutant plants had a higher root and leaf biomass than WT under both well-watered and drought conditions, but the reduction in growth parameters in response to drought was similar in WT and etr2b. Water deficit reduced all gas-exchange parameters in both WT and etr2b, but under moderate drought the mutant increased photosynthesis rate in comparison with control conditions, and showed a higher leaf CO2 concentration, transpiration rate, and stomata conductance than WT. The response of etr2b to drought indicates that ethylene is a negative regulator of plant growth under both control and drought. Since etr2b increased ABA content in well-watered plant, but prevented the induction of ABA production in response to drought, it is likely that the etr2b response under drought is not mediated by ABA. A 1H NMR metabolomic analysis revealed that etr2b enhances the accumulation of osmolytes (soluble sugars and trigonelline), unsaturated and polyunsaturated fatty acids, and phenolic compounds under drought, concomitantly with a reduction of malic- and fumaric-acid. The role of CpETR2B and ethylene in the regulation of these drought-protective metabolites is discussed.

The ethylene receptor mutation etr2b reveals crosstalk between ethylene and ABA in the control of Cucurbita pepo germination.

The enhanced salt tolerance of squash ethylene-insensitive mutants during germination and early stages of seedling development suggested that abscisic acid (ABA) could mediate this tolerance. To gain insight into the crosstalk between ethylene and ABA in seed germination, the germination rate and early seedling growth of wild type (WT) and ethylene-insensitive etr2b mutant were compared in seeds germinated under water and exogenous ABA treatment. The etr2b seeds germinated earlier than WT under both water and ABA, and the effect of ABA on radicle length and seedling growth of etr2b was lower than in WT, indicating that etr2b is also insensitive to ABA. The comparison of ABA and ethylene contents and ABA and ethylene gene expression profiles in WT and etr2b dry and imbibed seeds in either water, NaCl or ABA demonstrated a clear crosstalk between ethylene and ABA in germination. The expression profiles of ethylene genes in WT and etr2b indicated that the role of ethylene in seed germination does not appear to follow the canonical ethylene signaling pathway. Instead, etr2b reduces ABA content during formation of the seeds (dry seeds) and in response to seed imbibition and germination, which means diminished dormancy in the ethylene mutant. The etr2b mutation downregulated the expression of ABA biosynthesis and signaling genes during germination, demonstrating the positive role of ethylene receptor gene CpETR2B on seed germination and early seedling growth in squash is mediated by ABA. The reduced effect of exogenous ABA on ethylene production and ethylene gene expression in etr2b seeds suggests that this regulation is also dependent on ethylene.

Jasmonate-deficient mutant lox3a reveals crosstalk between jasmonate and ethylene in the differential regulation of male and female flower opening and early fruit development in Cucurbita pepo.

Jasmonate (JA) has been found to be a relevant hormone in floral development in numerous species, but its function in cucurbit floral development and sex determination is unknown. Crosstalk between JA and ethylene (ET) in the differential regulation of male and female floral development was investigated by using the novel JA-deficient mutant lox3a, and the ET-deficient and -insensitive mutants, aco1a and etr2b, respectively, of Cucurbita pepo. The lox3a mutation suppresses male and female flower opening and induces the development of parthenocarpic fruit. A bulked-segregant analysis coupled with whole genome sequencing and fine mapping approach allowed the identification of lox3a mutation in CpLOX3A, a LIPOXYGENASE gene involved in JA biosynthesis. The reduced JA content and expression of JA-signalling genes in male and female flowers of lox3a, and the rescue of lox3a phenotype by external application of methyl jasmonate (MeJA), demonstrated that JA controls petal elongation and flower opening, as well as fruit abortion in the absence of fertilization. JA also rescued the phenotype of ET mutants aco1a and etr2b, which are both specifically defective in female flower opening and fruit abortion. ET, the sex determining hormone of cucurbits, is induced in female flowers towards anthesis, activating JA production and promoting the aperture of the female flower, and the abortion of the unfertilized ovary. Given the close association between flower closure and parthenocarpic fruit development, we propose that flower opening can act as a switch that triggers fruit set and development in fertilized ovaries, but may alternatively induce the abortion of the unfertilized ovary. Both ET and JA from mature and senescent petals can serve as remote signals that determine the alternative development of the ovary and fruit.

Involvement of ethylene receptors in the salt tolerance response of Cucurbita pepo.

Abiotic stresses have a negative effect on crop production, affecting both vegetative and reproductive development. Ethylene plays a relevant role in plant response to environmental stresses, but the specific contribution of ethylene biosynthesis and signalling components in the salt stress response differs between Arabidopsis and rice, the two most studied model plants. In this paper, we study the effect of three gain-of-function mutations affecting the ethylene receptors CpETR1B, CpETR1A, and CpETR2B of Cucurbita pepo on salt stress response during germination, seedling establishment, and subsequent vegetative growth of plants. The mutations all reduced ethylene sensitivity, but enhanced salt tolerance, during both germination and vegetative growth, demonstrating that the three ethylene receptors play a positive role in salt tolerance. Under salt stress, etr1b, etr1a, and etr2b germinate earlier than WT, and the root and shoot growth rates of both seedlings and plants were less affected in mutant than in WT. The enhanced salt tolerance response of the etr2b plants was associated with a reduced accumulation of Na+ in shoots and leaves, as well as with a higher accumulation of compatible solutes, including proline and total carbohydrates, and antioxidant compounds, such as anthocyanin. Many membrane monovalent cation transporters, including Na+/H+ and K+/H+ exchangers (NHXs), K+ efflux antiporters (KEAs), high-affinity K+ transporters (HKTs), and K+ uptake transporters (KUPs) were also highly upregulated by salt in etr2b in comparison with WT. In aggregate, these data indicate that the enhanced salt tolerance of the mutant is led by the induction of genes that exclude Na+ in photosynthetic organs, while maintaining K+/Na+ homoeostasis and osmotic adjustment. If the salt response of etr mutants occurs via the ethylene signalling pathway, our data show that ethylene is a negative regulator of salt tolerance during germination and vegetative growth. Nevertheless, the higher upregulation of genes involved in Ca2+ signalling (CpCRCK2A and CpCRCK2B) and ABA biosynthesis (CpNCED3A and CpNCED3B) in etr2b leaves under salt stress likely indicates that the function of ethylene receptors in salt stress response in C. pepo can be mediated by Ca2+ and ABA signalling pathways.

The Ethylene Biosynthesis Gene CpACO1A: A New Player in the Regulation of Sex Determination and Female Flower Development in Cucurbita pepo.

A methanesulfonate-generated mutant has been identified in Cucurbita pepo that alters sex determination. The mutation converts female into hermaphrodite flowers and disrupts the growth rate and maturation of petals and carpels, delaying female flower opening, and promoting the growth rate of ovaries and the parthenocarpic development of the fruit. Whole-genome resequencing allowed identification of the causal mutation of the phenotypes as a missense mutation in the coding region of CpACO1A, which encodes for a type I ACO enzyme that shares a high identity with Cucumis sativus CsACO3 and Cucumis melo CmACO1. The so-called aco1a reduced ACO1 activity and ethylene production in the different organs where the gene is expressed, and reduced ethylene sensitivity in flowers. Other sex-determining genes, such as CpACO2B, CpACS11A, and CpACS27A, were differentially expressed in the mutant, indicating that ethylene provided by CpACO1A but also the transcriptional regulation of CpACO1A, CpACO2B, CpACS11A, and CpACS27A are responsible for determining the fate of the floral meristem toward a female flower, promoting the development of carpels and arresting the development of stamens. The positive regulation of ethylene on petal maturation and flower opening can be mediated by inducing the biosynthesis of JA, while its negative control on ovary growth and fruit set could be mediated by its repressive effect on IAA biosynthesis.

Mapping a Partial Andromonoecy Locus in Citrullus lanatus Using BSA-Seq and GWAS Approaches.

The sexual expression of watermelon plants is the result of the distribution and occurrence of male, female, bisexual and hermaphrodite flowers on the main and secondary stems. Plants can be monoecious (producing male and female flowers), andromonoecious (producing male and hermaphrodite flowers), or partially andromonoecious (producing male, female, bisexual, and hermaphrodite flowers) within the same plant. Sex determination of individual floral buds and the distribution of the different flower types on the plant, are both controlled by ethylene. A single missense mutation in the ethylene biosynthesis gene CitACS4, is able to promote the conversion of female into hermaphrodite flowers, and therefore of monoecy (genotype MM) into partial andromonoecy (genotype Mm) or andromonoecy (genotype mm). We phenotyped and genotyped, for the M/m locus, a panel of 207 C. lanatus accessions, including five inbreds and hybrids, and found several accessions that were repeatedly phenotyped as PA (partially andromonoecious) in several locations and different years, despite being MM. A cosegregation analysis between a SNV in CitACS4 and the PA phenotype, demonstrated that the occurrence of bisexual and hermaphrodite flowers in a PA line is not dependent on CitACS4, but conferred by an unlinked recessive gene which we called pa. Two different approaches were performed to map the pa gene in the genome of C. lanatus: bulk segregant analysis sequencing (BSA-seq) and genome wide association analysis studies (GWAS). The BSA-seq study was performed using two contrasting bulks, the monoecious M-bulk and the partially andromonoecious PA-bulk, each one generated by pooling DNA from 20 F2 plants. For GWAS, 122 accessions from USDA gene bank, already re-sequenced by genotyping by sequencing (GBS), were used. The combination of the two approaches indicates that pa maps onto a genomic region expanding across 32.24-36.44 Mb in chromosome 1 of watermelon. Fine mapping narrowed down the pa locus to a 867 Kb genomic region containing 101 genes. A number of candidate genes were selected, not only for their function in ethylene biosynthesis and signalling as well as their role in flower development and sex determination, but also by the impact of the SNPs and indels differentially detected in the two sequenced bulks.