Plastidial phosphorylase is required for normal starch synthesis in Chlamydomonas reinhardtii

Among the three distinct starch phosphorylase activities detected in Chlamydomonas reinhardtii, two distinct plastidial enzymes (PhoA and PhoB) are documented while a single extraplastidial form (PhoC) displays a higher affinity for glycogen as in vascular plants. The two plastidial phosphorylases are shown to function as homodimers containing two 91-kDa (PhoA) subunits and two 110-kDa (PhoB) subunits. Both lack the typical 80-amino-acid insertion found in the higher plant plastidial forms. PhoB is exquisitely sensitive to inhibition by ADP-glucose and has a low affinity for malto-oligosaccharides. PhoA is more similar to the higher plant plastidial phosphorylases: it is moderately sensitive to ADP-glucose inhibition and has a high affinity for unbranched malto-oligosaccharides. Molecular analysis establishes that STA4 encodes PhoB. Chlamydomonas reinhardtii strains carrying mutations at the STA4 locus display a significant decrease in amounts of starch during storage that correlates with the accumulation of abnormally shaped granules containing a modified amylopectin structure and a high amylose content. The wild-type phenotype could be rescued by reintroduction of the cloned wild-type genomic DNA, thereby demonstrating the involvement of phosphorylase in storage starch synthesis.     

Inheritance of parthenocarpy in summer squash (Cucurbita pepo L.)

The inheritance of the tendency to set parthenocarpic fruit in the summer squash (Cucurbita pepo L.) line Whitaker was studied. Two parental lines, Whitaker (parthenocarpic) and Caserta (non-parthenocarpic), and the F1 and F2 generations and backcrosses to both parents were tested. The parthenocarpic tendency of individual plants was scored on a scale from 1 (non-parthenocarpic fruit) to 5 (parthenocarpic fruit). The Whitaker line produced parthenocarpic fruit and had a mean score of 4.2, whereas Caserta did not set parthenocarpic fruit and had a score of 1.55. The heritability estimates indicated that genetic gains from selection were feasible. The additive-dominant model showed a good fit, with epistasis being negligible or nonexistent. The hypothesis of monogenic inheritance with incomplete dominance was not rejected within the degree of dominance range from 0.2 to 0.5. These results indicate that parthenocarpy is controlled by a single locus, with incomplete dominance in the direction of parthenocarpic expression.     

Evaluation of haploidization efficiency in winter squash (Cucurbita maxima Duch.) and pumpkin (Cucurbita moschata Duch.) through anther culture

The production of large numbers of haploids is the crucial point of the dihaploidisation process. Although in vitro haploid plants were successfully produced by irradiated pollen technique in winter squash (Cucurbita maxima Duch.) and pumpkin (Cucurbita moschata Duch.), the frequency is still insufficient for using in a large-scale breeding programme. Thus, the present study was conducted to determine the efficacy of the anther culture technique on the production of in vitro haploids in the aforementioned species for which there have been no successful reports concerning by androgenesis. The anthers at uninucleate microspore stage were collected at different florescence times and then cultured on a solid MS medium supplemented by different combinations of 2,4-D (2,4-dichlorophenoxyacetic acid), BAP (6-benzylaminopurine), KN (kinetin) with the constant addition of NAA (naphthalene acetic acid) to induce callogenesis, embryogenesis and plantlet initiation. The combination of PGR, genotype and anther collection time played an important role in the androgenic response. The highest response was obtained from 57S?21 and G9 lines with the combination of 2.0 or 4.0 mg/l BAP?+?0.05 mg/l NAA (E6 medium) at the first anther collection time. Plantlets were regenerated and rooted on MS medium supplemented by 0.01 mg/l IAA. In total, 74 plants were recovered and propagated with micro-cuttings. The ploidy analyses revealed that 35 plants (47.3?%) were haploid (n?=?20), and the others (52.7?%) were diploid (2n?=?40).     

Plastidial starch phosphorylase in sweet potato roots is proteolytically modified by protein-protein interaction with the 20S proteasome

Post-translational regulation plays an important role in cellular metabolism. Earlier studies showed that the activity of plastidial starch phosphorylase (Pho1) may be regulated by proteolytic modification. During the purification of Pho1 from sweet potato roots, we observed an unknown high molecular weight complex (HX) showing Pho1 activity. The two-dimensional gel electrophoresis, mass spectrometry, and reverse immunoprecipitation analyses showed that HX is composed of Pho1 and the 20S proteasome. Incubating sweet potato roots at 45°C triggers a stepwise degradation of Pho1; however, the degradation process can be partially inhibited by specific proteasome inhibitor MG132. The proteolytically modified Pho1 displays a lower binding affinity toward glucose 1-phosphate and a reduced starch-synthesizing activity. This study suggests that the 20S proteasome interacts with Pho1 and is involved in the regulation of the catalytic activity of Pho1 in sweet potato roots under heat stress conditions.     

Genome size variation in pumpkin (Cucurbita sp.)

Pumpkins, a subgroup of the domesticated Cucurbita species, have been reported to range in fruit type (related to size) from miniature (<100 g) to jumbo (>273 kg). In order to obtain a wide range of fruit types it is hypothesised that all potential factors affecting fruit type must be used. One factor that is often overlooked in plant studies is genome size. In various plant species, genome size variation has been associated with characteristics such as cell size, plant size and flowering time. Such characteristics are referred to as nucleotypic parameters. In order to determine if nucleotypic selection is occurring in pumpkin, 17 varieties were analysed for genome size variation in two separate experiments. The species selected encompass the total range of fruit types reported in pumpkin. Significant nuclear DNA content variation was observed in pumpkin. There was no significant correlation between genome size and fruit type. In fact, the miniature pumpkin types were found to have the same genome size as the jumbo pumpkin types. In addition, a positive correlation between genome size and stomata length (an estimate of cell size in plants) was observed. Both the miniature and jumbo types were observed to have the smallest genome size and the smallest cell size. Thus, nucleotypic selection does appear to occur in pumpkin and appears to be involved in determining fruit type, although it may not be the only factor involved.     

Plastidial alpha-glucan phosphorylase is not required for starch degradation in Arabidopsis leaves but has a role in the tolerance of abiotic stress

To study the role of the plastidial alpha-glucan phosphorylase in starch metabolism in the leaves of Arabidopsis, two independent mutant lines containing T-DNA insertions within the phosphorylase gene were identified. Both insertions eliminate the activity of the plastidial alpha-glucan phosphorylase. Measurement of other enzymes of starch metabolism reveals only minor changes compared with the wild type. The loss of plastidial alpha-glucan phosphorylase does not cause a significant change in the total accumulation of starch during the day or its remobilization at night. Starch structure and composition are unaltered. However, mutant plants display lesions on their leaves that are not seen on wild-type plants, and mesophyll cells bordering the lesions accumulate high levels of starch. Lesion formation is abolished by growing plants under 100% humidity in still air, but subsequent transfer to circulating air with lower humidity causes extensive wilting in the mutant leaves. Wilted sectors die, causing large lesions that are bordered by starch-accumulating cells. Similar lesions are caused by the application of acute salt stress to mature plants. We conclude that plastidial phosphorylase is not required for the degradation of starch, but that it plays a role in the capacity of the leaf lamina to endure a transient water deficit.     

Pattern of dry matter accumulation in developing fruit parts of early- and late-maturing pigeon peas (Cajanus cajan (L.)Millsp.)

Pigeon pea cultivars AL 15 (early-) and T₂₁ (late-maturing) were compared for dry matter accumulation in fruit parts pod wall (PW), seed coats (SC) and seed at various seed developmental stages. Significant water loss and dry matter accumulation in the fruit parts commenced much earlier in cv. AL 15 as compared with cv. T₂₁. The pod wall accumulated starch, reducing sugars and N-substances up to 21 and 28 days after anthesis (DAA) in cultivars AL 15 and T₂₁, respectively, which was later distributed to the seed. Growth of pod wall and seed was sequential, not concurrent, as the pod wall lost significant dry matter when the seeds within them reached their maximum dry matter. The fruit parts of cv. AL 15 accumulated more photosynthates than cv. T₂₁ at all comparable stages.     

Genetics of resistance to red pumpkin beetle (A ulacophora foveicollis) in summer squash (Cucurbita pepo L.)

Summary Resistance to red pumpkin beetle in summer squash was found to be controlled by polygenes. Diallel and Triple test cross analysis revealed the preponderance of non-additive and additive gene effects for resistance respectively. Absence of epistasis for resistance was indicated by both tests.     

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.     

Pumpkin (Cucurbita sp.) seed globulin VI. Proteolytic activities appearing in germinating cotyledons

N--benzoyl D,L-arginine p-nitroanilide (BAPA), leucine p-nitroanilide(LPA) and casein hydrolytic activities were assayed in germinatingcotyledons. BAPA hydrolytic activity was not detected in dryseeds, but increased rapidly from 1 to 4 days of germinationand then decreased. LPA and casein hydrolytic activities weredetected in dry seeds and increased from 2 to 4 days. Caseinhydrolytic activity decreased faster than the other two activities. BAPA hydrolytic enzyme was partially purified. It was inhibitedby p-chloromercuribenzoate and activated by ß-mercaptoethanoland dithiothreitol, but was not affected by EDTA, phenylmethylsulfonylfluoride, pumpkin trypsin inhibitor and several divalent cations.It had no ability to hydrolyze globulin or the chain to produce F_ß or smaller polypeptides,respectively, which was referred to as proteolytic activityI in the preceding paper (14), but released small peptides andamino acids from the chain and F_ß. However, it wasdifferent from proteolytic enzyme II which was present in dryseeds and inhibited by EDTA (14). Pumpkin trypsin inhibitor was purified. Its molecular weightwas estimated to be 10,500 by gel filtration. It did not inhibitthe BAPA hydrolytic enzyme. Both proteolytic activities I andII were also not reduced by the inhibitor (14). The inhibitoryactivity decreased gradually during germination. (Received November 9, 1979; )     

Aluminium-induced growth inhibition is associated with impaired efflux and influx of H+ across the plasma membrane in root apices of squash (Cucurbita pepo)

It is generally understood that the inhibition of growth of root apices is the initial effect caused by aluminium (Al) toxicity. The correlation between impaired H+-fluxes across the plasma membrane (PM) and Al-induced growth inhibition, Al accumulation and callose formation in root apices of squash (Cucurbita pepo L. cv. Tetsukabuto) is reported here. The root inhibition was dependent on Al concentration, and the duration of exposure, with the damage occurring preferentially in regions with high Al accumulation and callose formation. Using the fluorescent Al indicator (Morin), Al was localized in the cell walls of the root-tip cells after 3 h and in the whole root-tip cells after 6 h of the Al treatment (50 micro M). The inhibition of H+-pumping rate in the highly purified PM vesicles obtained from the Al-treated apical root portions (1 cm) coincided with the inhibition of root growth under Al stress. Furthermore, H+-ATPase activity of PM vesicles prepared from the control root apices was strongly inhibited by Al in vitro in a dose-dependent manner. Approximately 50% inhibition was observed when PM vesicles were preincubated at Al concentration as low as 10 micro M followed by the enzyme assay in the medium without Al. Using the pH indicator (bromocresol purple), it is shown that surface pH of the control (0 Al) root apices was strongly alkalized from the starting pH of 4.5 in a time-dependent manner. By contrast, the surface pH changed only slightly in the Al-treated root apices. The changes in surface pH mediated by altered dynamics of H+ efflux and influx across the root tip PM play an important role in root growth as affected by Al.     

Glyoxylate Cycle Enzymes in Peroxisomes Isolated from Petals of Pumpkin (Cucurbita sp.) during Senescence

The activities of the two unique enzymes of the glyoxylate cycle,isocitrate lyase (EC 4.1.3.1 [EC] ) and malate synthase (EC 4.1.3.2 [EC] ),were undetectable in petals of pumpkin (Cucurbita sp. AmakuriNankin) until the end of blooming, but they appeared duringsenescence. The activity of catalase (EC 1.11.1.6 [EC] ) increased,glycolate oxidase (EC 1.1.3.1 [EC] ) activity did not change, whilehydroxypyruvate reductase (EC 1.1.1.81 [EC] ) activity peaked at fullblooming stage and declined thereafter. After fractionationof cellular organelles on a sucrose density gradient, we detectedisocitrate lyase and malate synthase activities in peroxisomalfractions only from petals at the senescing stage. Northernblot analysis revealed that malate synthase mRNA increased duringpetal senescence. Citrate synthase (EC 4.1.3.7 [EC] ) and malate dehydrogenase(EC 1.1.1.37 [EC] ) activities were also present, while aconitase(EC 4.2.1.3 [EC] ) was not detectable in peroxisomal fractions. Moreoverthe presence of 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35 [EC] )and urate oxidase (EC 1.7.3.3 [EC] ) in the peroxisomal fractionsfrom senescing petals indicates that peroxisomes could be involvedboth in the ß-oxidation pathway and in the purinecatabolism during petal senescence. (Received May 25, 1991; Accepted September 25, 1991)     

Identification of high seed oil yield and high oleic acid content in Brazilian germplasm of winter squash (Cucurbita moschata D.)

Cucurbita moschata D. seed oil contains approximately 75% unsaturated fatty acids, with high levels of monounsaturated fatty acids and antioxidant compounds such as vitamin E and carotenoids, constituting a promising food in nutritional terms. In addition, the Brazilian germplasm of C. moschata exhibits remarkable variability, representing an important source for the genetic breeding of this vegetable and other cucurbits. The present study evaluated the productivity and profile of the seed oil of 91C. moschata accessions from different regions of Brazil maintained in the Vegetable Germplasm Bank of the Federal University of Viçosa (BGH-UFV). A field experiment was conducted between January and July 2016. The accessions showed high genetic variability in terms of characteristics related to seed oil productivity (SOP), such as the weight of seeds per fruit and productivity of seeds, providing predicted selection gains of 29.39 g and 0.26 t ha^?1, respectively. Based on the phenotypic and genotypic correlations, a greater SOP can be achieved while maintaining a high oleic acid concentration and low linoleic acid concentration, providing oil of better nutritional and chemical quality. In the variability analysis, the accessions were clustered into five groups, which had different averages for SOP and fatty acid concentration of seed oil, an approach that will guide the use of appropriate germplasm in programs aimed at genetic breeding for SOP and seed oil profile. Per se analysis identified BGH-4610, BGH-5485A, BGH-6590, BGH-5556A, BGH-5472A, and BGH-5544A as the most promising accessions in terms of SOP, with an average (μ + g) of approximately 0.20 t ha^?1. The most promising accessions for a higher oleic acid concentration of seed oil were BGH-5456A, BGH-3333A, BGH-5361A, BGH-5472A, BGH-5544A, BGH-5453A, and BGH-1749, with an average (μ + g) of approximately 30%, almost all of which were also the most promising in terms of a lower linoleic acid concentration of the seed oil, with an average (μ + g) of approximately 45%. Part of the C. moschata accessions evaluated in the present study can serve as a promising resource in genetic breeding programs for SOP and fatty acid profile, aiming at the production of oil with better nutritional and physicochemical quality.     

Effect of deicing chloride salts on ion accumulation in spruce (Picea abies (L.) sp.)

Among the inorganic chloride salts, NaCl, CaCl₂ and in a minor proportion KCl and MgCl₂ are used as deicing agents. Mixturs of these salts were merely applied with respect to their physico-chemical properties, but their effect on roadside vegetation has never been studied so far. From a screening of different salt mixtures on ion accumulation in needles and twigs of spruce tress (Picea abies sp.) it was shown that the presence of a small amount of calcium in the salt treatments had some beneficial effects on ion regulation. In the presence of calcium, sodium accumulation could be reduced. But more straightforward was its effect on the selectivity between sodium and potasium in favour of the latter. Chloride concentrations did not alter very much; their role in the presence of monovalent cations is nevertheless obvious and is discussed. The study also confirms the presence of potassium retranslocation in conifer trees. The ion characteristics are briefly discussed with respect to the ecological effects of chloride salts on tress.     

Pumpkin (Cucurbita sp.) seed globulin V. Proteolytic activities involved in globulin degradation in ungerminated seeds

Two proteolytic activities I and II involved in the globulindegradation were detected in pumpkin seeds. Activity I, hydrolyzing and ß subunits of the globulin to form F_ß,was found in both dry seeds and cycloheximide-treated cotyledons,and decreased during germination. Activity II, hydrolyzing F_ßto produce small peptides and amino acids, was not observedin dry seeds but found in cycloheximide-treated cotyledons,increased up to 4 days, and gradually decreased during germination. Activity I gave limited hydrolytic products from the globulinand the chain, but not from F_ß, the chain and some animal proteins. It was inhibitedby EDTA. On the other hand, activity II hydrolyzed F_ßand the chain faster than the globulin, the chain and some animal proteins. It was inhibitedby EDTA and p-chloromer-curibenzoate, and activated by ß-mercaptoethanol,dithiothreitol and CoCl₂. Optimum pH's were at about 6.8 andat 6.0 to 6.8 for activities I and II, respectively. The degradation process of the globulin can be divided intotwo steps: the first step is the conversion of globulin to F_ßand the second step, F_ß to small peptides and aminoacids. (Received November 9, 1979; )     

Hormonal control of floral abscission in zucchini squash (<Emphasis Type="Italic">Cucurbita pepo</Emphasis>)

In the zucchini squash, Cucurbita pepo, a well coordinated abscission of the female flower during fruit set is essential to obtain a fruit of commercial value. In Spain zucchini is mainly produced in greenhouses in Almería, where high temperatures during the spring-summer period provoke a cultivar-dependent defect in fruits known as the “sticky flower” syndrome. This disorder is characterised by an arrest in growth and maturation of floral organs, and a lack of female floral abscission, thus diminishing fruit shelf-life, commercial quality and value. The aim of the present work was to improve knowledge of the abscission process in C. pepo to better understand the fundamental causes of this disorder. The anatomical analysis of abscission shows a well defined male floral abscission zone (AZ), few hours after anthesis, which differs from the female zone which is not differentiated from the adjacent tissue until the abscission process has begun, and which occurs as a consequence of AZ cell enlargement and the dissolution of their cell walls. To evaluate the role of ethylene and auxins in the regulation of floral abscission in zucchini we performed several treatments, with: ethylene, added as 0.25% ethrel solution; AVG, the inhibitor of ethylene synthesis, at 100 μM; indol-3-acetic acid, 100 μM; and TIBA, the inhibitor of auxin polar transport, at 10 mM. These treatments show that ethylene is an accelerator of zucchini floral abscission, and also promotes abscission in isolated AZs of sticky flowers. On the other hand, IAA delays abscission of the female flowers, whilst the inhibitor of auxin polar transport promotes it. The activity of the cell wall hydrolytic enzymes, polygalacturonase and cellulase, sharply increased just before the shedding of zucchini floral organs (72 h after anthesis). Moreover, both enzyme activities were induced by ethylene, which partly explains the ethylene promoting effect.     

Phenology of flowering and starch accumulation in grape (Vitis vinifera L.) cuttings and vines

BACKGROUND AND AIMS: A reliable protocol for flowering and fruiting in cuttings was developed with the aim of (a) studying inflorescence and flower development in grapevine cuttings and field plants, and (b) assisting haploid plant production. METHODS: Inflorescence and flower development was studied in 'Gewurztraminer' (GW) and 'Pinot Noir' (PN) grape vines and cuttings grown in a glasshouse, along with variations in starch in the flowers. As there is a strong relationship between flower development and starch, the starch content of reproductive structures was estimated. KEY RESULTS: Inflorescence and flower development were similar in the vines and cuttings with consistent differences between the two cultivars. Indeed, the ontogenesis of male and female organs is not synchronous in GW and PN, with both female and male meiosis occurring earlier in PN than in GW. Moreover, changes of starch reserves were similar in the two plant types. CONCLUSIONS: Cuttings have a similar reproductive physiology to vines, and can be used to study grape physiology and to develop haploid plants.     

Pumpkin (Cucurbita sp.) seed globulin VII. Immunofluorescent study on protein bodies in ungerminated and germinating cotyledon cells

Protein bodies of pumpkin cotyledon cells were oval (about 10?7µm), and each was composed of a crystalloid, a globoidand proteinaceous matrix. They started to swell and fuse with1 day of imbibition. The proteinaceous matrix region expandedat the expense of crystalloids, and its electron density decreased.Finally, the protein bodies became central vacuoles includingmany small protein particles in about 8 days of germination. Fluorescent microscopy using antibodies raised against pumpkinseed globulin showed that fluorescence could not be observedin either protein bodies of ungerminated seeds or crystalloidsof germinating cotyledons, and only the proteinaceous matrixof germinating cotyledons became fluorescent. Probable causesof no fluorescence on crystalloids of seed globulin depositionwere considered. (Received November 9, 1979; )