A Kelch Domain-Containing F-Box Coding Gene Negatively Regulates Flavonoid Accumulation in Muskmelon.

The flavonoids are phenylpropanoid-derived metabolites that are ubiquitous in plants, playing many roles in growth and development. Recently, we observed that fruit rinds of yellow casaba muskmelons (Cucumis melo 'Inodorous Group') accumulate naringenin chalcone, a yellow flavonoid pigment. With RNA-sequencing analysis of bulked segregants representing the tails of a population segregating for naringenin chalcone accumulation followed by fine mapping and genetic transformation, we identified a Kelch domain-containing F-box protein coding (CmKFB) gene that, when expressed, negatively regulates naringenin chalcone accumulation. Additional metabolite analysis indicated that downstream flavonoids are accumulated together with naringenin chalcone, whereas CmKFB expression diverts the biochemical flux toward coumarins and general phenylpropanoids. These results show that CmKFB functions as a posttranscriptional regulator that diverts flavonoid metabolic flux.

A model for transport of a viral membrane protein through the early secretory pathway: minimal sequence and endoplasmic reticulum lateral mobility requirements.

Viral movement proteins exploit host endomembranes and the cytoskeleton to move within the cell via routes that, in some cases, are dependent on the secretory pathway. For example, melon necrotic spot virus p7B, a type II transmembrane protein, leaves the endoplasmic reticulum (ER) through the COPII-dependent Golgi pathway to reach the plasmodesmata. Here we investigated the sequence requirements and putative mechanisms governing p7B transport through the early secretory pathway. Deletion of either the cytoplasmic N-terminal region (CR) or the luminal C-terminal region (LR) led to ER retention, suggesting that they are both essential for ER export. Through alanine-scanning mutagenesis, we identified residues in the CR and LR that are critical for both ER export and for viral cell-to-cell movement. Within the CR, alanine substitution of aspartic and proline residues in the DSSP ß-turn motif (D7 AP10 A) led to movement of discrete structures along the cortical ER in an actin-dependent manner. In contrast, alanine substitution of a lysine residue in the LR (K49 A) resulted in a homogenous ER distribution of the movement protein and inhibition of ER-Golgi traffic. Moreover, the ability of p7B to recruit Sar1 to the ER membrane is lost in the D7 AP10 A mutant, but enhanced in the K49 A mutant. In addition, fluorescence recovery after photobleaching revealed that K49 A but not D7 AP10 A dramatically diminished protein lateral mobility. From these data, we propose a model whereby the LR directs actin-dependent mobility toward the cortical ER, where the cytoplasmic DSSP ß-turn favors assembly of COPII vesicles for export of p7B from the ER.

Histological study of organogenesis in Cucumis melo L. after genetic transformation: why is it difficult to obtain transgenic plants?

Melon (Cucumis melo L.) is widely considered as a recalcitrant species for genetic transformation. In this study, we developed different regeneration and transformation protocols and we examined the regeneration process at different steps by histological studies. The highest regeneration rate (1.13 ± 0.02 plants per explant) was obtained using cotyledon explants of the 'Védrantais' genotype on Murashige and Skoog (MS) medium supplemented with 0.2 mg/l 6-benzylaminopurine (BAP) and 0.2 mg/l dimethylallylaminopurine (2-iP). Agrobacterium tumefaciens-mediated transformations with the uidA reporter gene were realized on cotyledon explants cultivated in these conditions: 70-90% of explants expressed a transient GUS activity during the early stages of regeneration, however, only few transgenic plants were obtained (1.8-4.5% of stable transformation with the GV2260pBI101 strain). These results revealed a low capacity of melon GUS-positive cells to regenerate transgenic plants. To evaluate the influence of the Agrobacterium infection on plant regeneration, histological analyses were conducted on explants 2, 7, 15, and 28 days after co-culture with the GV2260pBI101 strain. Genetic transformation occurred in epidermal and sub-epidermal cells and reached the meristematic structures expressing a high level of GUS activity during 14 days of culture; but after this period, most of the meristematic structures showed premature cell vacuolization and disorganization. This disruption of the GUS-positive meristematic areas could be responsible of the difficulties encountered to regenerate melon plants after genetic transformation.

Overexpression of the downward leaf curling (DLC) gene from melon changes leaf morphology by controlling cell size and shape in Arabidopsis leaves.

A plant-specific gene was cloned from melon fruit. This gene was named downward leaf curling (CmDLC) based on the phenotype of transgenic Arabidopsis plants overexpressing the gene. This expression level of this gene was especially upregulated during melon fruit enlargement. Overexpression of CmDLC in Arabidopsis resulted in dwarfism and narrow, epinastically curled leaves. These phenotypes were found to be caused by a reduction in cell number and cell size on the adaxial and abaxial sides of the epidermis, with a greater reduction on the abaxial side of the leaves. These phenotypic characteristics, combined with the more wavy morphology of epidermal cells in overexpression lines, indicate that CmDLC overexpression affects cell elongation and cell morphology. To investigate intracellular protein localization, a CmDLC-GFP fusion protein was made and expressed in onion epidermal cells. This protein was observed to be preferentially localized close to the cell membrane. Thus, we report here a new plant-specific gene that is localized to the cell membrane and that controls leaf cell number, size and morphology.

The perisperm-endosperm envelope in Cucumis: structure, proton diffusion and cell wall hydrolysing activity.

BACKGROUND: and Aims The envelope surrounding the embryo in cucurbit seed, which consists of a single layer of live endosperm cells covered by lipid- and callose-rich layers, is reported to show semi-permeability and also to act as the primary barrier to radicle emergence. Structure, development and permeability of the envelope and activity of cell wall hydrolases during germination of cucumber and muskmelon seeds were investigated. METHODS: Sections of seeds were stained with aniline blue and Sudan III. Proton diffusion and endo-beta-mannanase activity were detected by tissue printing. A gel-diffusion assay was performed to quantify endo-beta-mannanase activity, while the activity of beta-glucanase was determined with laminarin as the substrate and glucose formation measured using the GOD-POD method. KEY RESULTS: The lipid layer differentiated during seed development in cucumber in the epidermis of a multilayered nucellus, whereas the callose layer appeared to develop outside the endosperm cell layer. Accordingly, the envelope has been called the perisperm-endosperm (PE) envelope. Chloroform treatment of seeds, which resulted in a substantial reduction in Sudan staining of the lipid layer, also enhanced the permeability of the PE envelope to 2,3,5-triphenyltetrazolium chloride. Proton diffusion occurred when the PE envelopes from seeds had their inner surface in contact with bromocresol purple-containing agarose gels, but not when their outer surface was in contact. Substantial endo-beta-mannanase activity was present in the caps of the PE envelopes, whereas a marked increase in beta-glucanase activity was observed in radicles prior to germination. CONCLUSIONS: The lipid layer seems to contribute to the semi-permeability of the PE envelope. The diffusion of protons might create an acidic environment conducive to the activity of cell wall hydrolases, namely endo-beta-mannanase (EC 3.2.1.78) and beta-glucanase [beta(1-->3)glucanohydrolase; EC 3.2.1.6], which, in turn, may play a role in the weakening of the PE envelope necessary for the protrusion of the radicle in cucumber and muskmelon seeds.

Powdery mildew (Sphaerotheca fuliginea) resistance in melon is selectable at the haploid level.

The major cause of powdery mildew in melons (Cucumis melo L.) is the fungus Sphaerotheca fuliginea. There are several cultivar- and season-specific races of this fungus. In order to control powdery mildew, it is important to introduce resistance to fungal infection into new cultivars during melon breeding. Haploid breeding is a powerful tool for the production of pure lines. In this study, it was investigated whether powdery mildew resistance could be manifested at the haploid level from two disease-resistant melon lines, PMR 45 and WMR 29. the effects of various races of S. fuliginea on diploid and haploid plants of PMR 45 and WMR 29 and of a disease-susceptible line, Fuyu 3 were measured. The responses of haploid and diploid plants to powdery mildew were identical. In addition, haploids that were generated from hybrids between Fuyu 3 and disease-resistant lines were examined. Seven out of 13 haploids from a Fuyu 3xPMR 45 cross and 10 out of 12 haploids from a Fuyu 3xWMR 29 cross were classified as resistant plants because they showed the same responses as their disease-resistant diploid parents to the various fungal races. These results indicate that resistance in PMR 45 and WMR 29 is selectable at the haploid level. All of the plant responses were observed by microscopy. A possible mechanism for generating powdery mildew resistance in two different melon lines is discussed.