A Comparison of Stem Peroxidases in Bush and Vine Forms of Squash (Cucurbita maxima Duch.and C. pepo L.)

Total peroxidase activities and peroxidase isozymes were comparedamong 10 bush- and vine-squash cultivars of Cucurbita maximaDuch. and C. pepo (L.) Several qualitative variations of isozymeswere detected between C. maxima and C. pepo, and some differenceswere evident among varieties of C. pepo. There were no qualitativedifferences between genetically similar bush and vine strains.Quantitative differences were difficult to resolve, althoughit did appear that in varieties of C. maxima exhibiting lowlevels of peroxidase activity, isozymes C₃, C₄, and C₆ wereless intense. Depending upon the cultivars compared, total peroxidase activityin bush forms was either higher or lower than, or the same astheir vine counterparts. Since the major bush genes in the varietiesstudied are believed to be allelic, the results indicate thatthe over-all genotype of squash varieties can influence therelative expression of peroxidase activity between bush andvine forms.     

Phytochrome-mediated light regulation of nitrate reductase expression in squash cotyledons

In etiolated squash (Cucurbita maxima L.) cotyledons, nitrate-inducible NADH:nitrate reductase activity and protein were increased in darkness by red light pulses with red/far-red photoreversibility. Continuous far-red light also led to increased levels of nitrate reductase activity and protein. Poly(A)+RNA, which hybridizes to squash nitrate reductase cDNA, was also increased by light treatments. Thus, we found that after nitrate triggering, nitrate reductase expression appears to be regulated by light via phytochrome.     

Increase of cytokinin activity in detached etiolated cotyledons of squash after illumination

Cytokinins nullified the lag period of the formation of chlorophyllin detached etiolated cotyledons of squash (Cucurbita moschataDuch. var. melonaeformis Makino cv. Tokyo). One hour after illumination,cytokinin activity in detached cotyledons rapidly increasedand maintained a certain level for another hour. ¹ Present address: Department of Enviromental Studies, Collegeof Integrated Arts and Sciences, Hiroshima University, Hiroshima730, Japan. (Received November 25, 1976; )     

Cucurbit Powdery Mildew: First Insights for the Identification of the Causal Agent and Screening for Resistance of Squash Genotypes (Cucurbita moschata (Duchesne ex Lam.) Duchesne ex Poir.) in Mendoza,Argentina

The cucurbit powdery mildew (CPM) caused by different fungal species is a major concern for cucurbit crops around the world. In Argentina CPM constitutes the most common and damaging disease for cucurbits, especially for squash crops (Cucurbita moschata). The present study displays initial insights into the knowledge of the disease in western Argentina, including the determination of the prevalent species causing CPM, as well as the evaluation of the resistance of squash cultivars and breeding lines. Fungal colonies were isolated from samples collected in Mendoza province, Argentina. A field trial was also performed to assess the resistance of five squash accessions, including commercial cultivars and breeding lines. The severity of CPM was analyzed and epidemiological models were built based on empirical data. The morphological determinations and analysis with specific molecular markers confirmed Podosphaera xanthi as the prevalent causal agent of CPM in Mendoza. The results od the field trial showed differences in the resistance trait among the squash accessions. The advanced breeding line BL717/1 showed promising results as source of CPM resistance for the future development of open pollinated resistant cultivars, a crucial tool for an integrative control of the disease.     

Changes in Levels of Auxin and Abscisic Acid and the Evolution of Ethylene in Squash Hypocotyls after Treatment with Brassinolide

Brassinolide stimulated an increase in fresh weight of segmentsof hypocotyls of etiolated squash (Cucurbita maxima Duch. cv.Houkou-Aokawaamaguri). Brassinolide-treated segments containedhigher levels of IAA than water-treated segments and also showeda tendency towards decreased levels of ABA. However, treatmentwith brassinolide did not have much effect on the productionof ethylene. (Received May 30, 1988; Accepted May 22, 1989)     

De novo synthesis and hormonal regulation of a dipeptidase in Cucurbita maxima

The following evidence was obtained for the de novo synthesis of dipeptidase in squash (Cucurbita maxima Duch. var. Hubbard) cotyledons during germination: (i) the amount of [¹⁴C]leucine incorporated into the dipeptidase was greater than that found in other proteins; (ii) the enzyme coincided with a peak of radioactivity in DEAE column chromatography; and (iii) the specific radioactivity of the enzyme increased with purification. There was also a positive correlation between the rate of [¹⁴C]leucine incorporation into dipeptidase and the rate of dipeptidase development. Four plant growth regulators, gibberellic acid (GA) benzyladenine (BA), indol-3-acetic acid (IAA), and abscisic acid (ABA) were examined for their effect on the development of dipeptidase activity at 5 × 10^?6 and 5 × 10^?5 M. None of these regulators affected the activity of the isolated dipeptidase per se. In intact see ds, BA and IAA inhibited the development of dipeptidase activity at the higher concentration, ABA reduced the activity at both concentrations; however, GA enhanced its development at the higher concentration. In distal-half cotyledons, BA and GA stimulated enzyme development but they showed no synergistic effect. IAA suppressed the development of enzyme activity at the higher concentration and ABA inhibited development at both levels.     

Immunochemical Difference of Wound-Induced 1-Aminocyclopropane-1-carboxylate Synthase from the Auxin-Induced Enzyme

Immunochemical cross-reactivity of wound- and auxin-induced1-aminocyclopropane-1-carboxylate (ACC) synthase was examinedwith the antibody against wound-induced ACC synthase purifiedfrom mesocarp of winter squash (Cucurbita maxima Duch.). Theantibody recognized ACC synthase from wounded hypocotyls ofwinter squash and from wounded pericarp of tomato fruits, butnot the enzyme from IAA-treated hypocotyls of winter squash,tomato and mung bean. These results indicate that the primarystructure of the wound-induced enzyme is different from thatof the auxin-induced enzyme in the same species, and impliesthat there are two different genes for ACC synthase, one forwound induction and the other for auxin induction. (Received June 14, 1988; Accepted July 20, 1988)     

Changes in Levels of Cytokinins in Etiolated Squash Seedlings after Illumination

When etiolated seedlings of squash (Cucurbita maxima Duch. cv.Houkou-aokawaamaguri) were exposed to light, their cotyledonsbegan to accumulate chlorophyll at a low rate for the firsthour but at a high rate therafter. After 0, 1 and 2 h of illumination,cytokinin levels in cotyledons, hypocotyls and roots were analyzedby ELISA (enzyme-linked immunosorbent assay) and GC-SIM (gaschromatography-selected ion monitoring). Cytokinin levels measuredby ELISA were about 10 times higher than those measured by GC-SIM,suggesting that ELISA was not suitable for the measurementsof cytokinin levels in the extracts from these tissues. Cytokininsfound by GC-SIM were t-RZ (t-ribosyl zeatin), c-RZ, t-Z (t-zeatin),c-Z, RiP (ribosyl isopentenyladenine) and iP (isopoentenyladenine).Levels of biologically active cytokinins (t-RZ and t-Z) didnot show marked changes after illumination. Levels of c-RZ andc-Z did, however, decrease in cotyledons and increase in hypocotylsafter illumination. Promotion of the accumulation of the chlorophyllin detached squash cotyledons by exogenously applied t-Z waspartially inhibited by the addition of c-Z, suggesting thatthe decrease in the endogenous level of c-Z in cotyledons ofintact seedlings after illumination permits the light-inducedformation of chlorophyll. (Received August 8, 1990; Accepted April 4, 1991)     

Intact plastids are required for nitrate- and light-induced accumulation of nitrate reductase activity and mRNA in squash cotyledons

Induction of nitrate reductase activity and mRNA by nitrate and light is prevented if chloroplasts are destroyed by photooxidation in norflurazon-treated squash (Cucurbita maxima L.) cotyledons. The enzyme activity and mRNA can be induced if norflurazon-treated squash seedlings are kept in low-intensity red light, which minimizes photodamage to the plastids. It is concluded that induction of nitrate reductase activity and nitrate reductase mRNA requires intact plastids. If squash seedlings grown in low-intensity red light are transferred to photooxidative white light, nitrate reductase activity accumulates during the first 12 hours after the shift and declines thereafter. Thus photodamage to the plastids and the disappearance of nitrate reductase activity and mRNA are events separable in time, and disappearance of the enzyme activity is a consequence of the damage to the plastids.     

Purification of Squash NADH:Nitrate Reductase by Zinc Chelate Affinity Chromatography

NADH:nitrate reductase (EC 1.6.6.1) was isolated and purified from the green cotyledons of 5-day-old squash seedlings (Cucurbita maxima L.). The 10-hour purification procedure consisted of two steps: direct application of crude enzyme to blue Sepharose and specific elution with NADH followed by direct application of this effluent to a Zn²⁺ column with elution by decreasing the pH of the phosphate buffer from 7.0 to 6.2. The high specific activity (100 micromoles per minute per milligram protein) and high recovery (15-25%) of electrophoretically homogeneous nitrate reductase show that the enzyme was not damaged by exposure to the bound zinc. With this procedure, homogeneous nitrate reductase can be obtained in yields of 0.5 milligram per kilogram cotyledons.     

Dynamic Regulation of Novel and Conserved miRNAs Across Various Tissues of Diverse Cucurbit Species

MicroRNA genes (miRNAs) encoding small non-coding RNAs are abundant in plant genomes and play a key role in regulating several biological mechanisms. Five conserved miRNAs, miR156, miR168-1, miR168-2, miR164, and miR166 were selected for analysis from the 21 known plant miRNA families that were recovered from deep sequencing data of small RNA libraries of pumpkin and squash. A total of six novel miRNAs that were not reported before were found to have precursors with reliable fold-back structures and hence considered novel and were designated as cuc_nov_miRNAs. A set of five conserved, six novel miRNAs, and five uncharacterized small RNAs from the deep sequencing data were profiled for their dynamic regulation using qPCR. The miRNAs were evaluated for differential regulation across the tissues among four diverse cucurbit species, including pumpkin and squash (Cucurbita moschata Duch. Ex Poir. and Cucurbita pepo L.), bitter melon (Momordica charantia L.), and Luffa (Loofah) (Luffa acutangula Roxb.). Expression analysis revealed differential regulation of various miRNAs in leaf, stem, and fruit tissues. Importantly, differences in the expression levels were also found in the leaves and fruits of closely related C. moschata and C. pepo. Comparative miRNA profiling and expression analysis in four cucurbits led to identification of conserved miRNAs in cucurbits. Predicted targets for two of the conserved miRNAs suggested miRNAs are involved in regulating similar biological mechanisms in various species of cucurbits.     

Prehistoric cultivated cucurbits from the Viru Valley,Peru

A sample of 535 specimens of cucurbits were retrieved from archaeological deposits in the Viru Valley, Peru, from contexts dated between ca. 1800 B.C.– 1100 A.D. by Carbon-14 analysis and cultural associations. Cultivars ofCucurbita maxima Duch. and C.moschata (Duch.) Duch. ex Poir. are found to be restricted, respectively, to early and late chronological periods, with no overlap. Cultural and ecological factors are suggested to account for the observed temporal and spatial variations in species occurrence. Comparison of materials from other coastal valleys indicat es significant, but presently unexplainable, spatial and temporal differences in the distribution of these species. Supportive evidence for South America as an independent center of domestication for C.maxima tis offered.     

The relationship between transpiration and chlorophyll synthesis in etiolated squash cotyledons

Etiolated cotyledons of squash (Cucurbita moschata Duch. var.melonaeformis Makino cv. Tokyo) treated with kinetin, KCl orfusicoccin in the dark increased both transpiration in the darkand chlorophyll synthesis after exposure to light. The minimumperiod of kinetin treatment to stimulate transpiration was similarto that to accelerate chlorophyll synthesis. On the other hand,treatment of cotyledons with vaseline or abscisic acid markedlyinhibited both transpiration and chlorophyll synthesis, suggestingthat cytokinin-induced stimulation of chlorophyll synthesiscould be mediated through the effect on the stomatal opening. (Received December 7, 1977; )     

A re-examination of the reputed control of cotyledonary metabolism by the axis

Summary A re-examination has been made of the supposed regulation by the axis of cotyledonary isocitrate lyase (EC 4.1.3.1) activity in Cucumis sativas L. and Cucurbita maxima Duch. and greening in cucumis. When precautions are taken to ensure that isolated cotyledons and cotyledons attached to the axis have equal access to oxygen and/or water no axis effects can be observed. The results underline the importance of maintaining excised plant parts in comparable physical conditions if meaningful physiological measurements are to be made. Previous reports describing a controlling role for the axis are therefore most probably incorrect, although alternative regulatory mechanisms mediated by the axis are still possible.     

Inheritance of Resistance to Crown Gall in Pisum sativum

We screened a total of 1365 pea (Pisum sativum) lines for response to inoculation with Agrobacterium tumefaciens, strain B6, and characterized resistance in one cultivar, Sweet Snap. Sweet Snap seedlings were highly resistant to tumorigenesis under most conditions. Resistance was overcome at inoculum concentrations of greater than 10⁹ bacteria per milliliter. At such high concentrations, very small tumors developed on Sweet Snap in response to four wide-host-range Agrobacterium strains, but tumors on other cultivars were two-to sevenfold larger than those that formed on Sweet Snap. The hypervirulent strain A281 induced larger tumors on Sweet Snap than did other Agrobacterium strains, but tumors on other genotypes were more than 100% larger than those on Sweet Snap. Physiological experiments suggested that tumorigenesis in Sweet Snap is not blocked in early stages of infection, and genetic analysis indicated that inheritance of resistance to crown gall is a quantitative trait. In addition to the observed resistance in Sweet Snap, three `supersusceptible' genotypes, which developed very large tumors, also were identified.     

The isolation and some properties of a lectin (Haemagglutinin) from Cucurbita phloem exudate

The occurrence of high haemagglutinating (lectin) activity in phloem exudate from three cucurbit species is reported. The protein responsible for this lectin activity in Cucurbita maxima Duch. has been isolated by cation exchange chromatography on Sepharose and identified by gel electrophoresis. The lectin showed agglutinating activity at concentrations as low as 0.1 g/ml. No sugar, including those transported in the phloem of these species, interacted with agglutination. The lectin could not be extracted from cucurbit seed, but appeared in 5-day old seedlings. The possible role of a lectin in the sieve element is discussed.     

Chloroplast Degradation in Agcing Cotyledons of Pumpkin

The cotyledons of Cucurbita maxima provide a system which permitsthe isolation of chloro-plasts in various states of structuraldisintegration. These states are defined and corrlated withthe morphological appearance of the cotyledon and its electronmicroscopic and biochemical parameters. Removal of growth abovethe cotyledons results in cotyledons whose chloroplasts do notchange in pigment composition. The connection between light-harvestingapparatus and electron transport components remains intact inthese plastids while the electron transport chain undergoesage-induced alterations. The implications of these observationson studies of structure-function relationships in photosynthesisare discussed.     

Vacuum infiltration enhances the Agrobacterium-mediated genetic transformation in Indian soybean cultivars

For the first time we have developed a reliable and efficient vacuum infiltration-assisted Agrobacterium-mediated genetic transformation (VIAAT) protocol for Indian soybean cultivars and recovered fertile transgenic soybean plants through somatic embryogenesis. Immature cotyledons were used as an explant and three Agrobacterium tumefaciens strains (EHA 101, EHA 105, and KYRT 1) harbouring the binary vector pCAMBIA1301 were experimented in the co-cultivation. The immature cotyledons were pre-cultured in liquid somatic embryo induction medium prior to vacuum infiltration with the Agrobacterium suspension and co-cultivated for 3 days on co-cultivation medium containing 50 mg l^?1 citric acid, 100 µM acetosyringone, and 100 mg l^?1 l-cysteine. The transformed somatic embryos were selected in liquid somatic embryo induction medium containing 10 mg l^?1 hygromycin and the embryos were germinated in basal medium containing 20 mg l^?1 hygromycin. The presence and integration of the hpt II and gus genes into the soybean genome were confirmed by GUS histochemical assay, polymerase chain reaction, and Southern hybridization. Among the different combinations tested, high transformation efficiency (9.45 %) was achieved when immature cotyledons of cv. Pusa 16 were pre-cultured for 18 h and vacuum infiltrated with Agrobacterium tumefaciens KYRT 1 for 2 min at 750 mm of Hg. Among six Indian soybean cultivars tested, Pusa 16 showed highest transformation efficiency of 9.45 %. The transformation efficiency of this method (VIAAT) was higher than previously reported sonication-assisted Agrobacterium-mediated transformation. These results suggest that an efficient Agrobacterium-mediated transformation protocol for stable integration of foreign genes into soybean has been developed.     

The localization of starch in root tips

As an extension of earlier studies performed in our laboratory on enzyme localization on tissue level, the distribution of starch was examined in seedling root tips of peaPisum sativum L., broad beanFaba vulgaris Moench., maizeZea mays L., lupinLupinus albus L., and pumpkinCucurbita maxima Duch., and in tips of adventive roots of onionAllium cepa L. by means of the alcian blue /JJK procedure applied to paraffin sections. In pea, some genetic (various cultivars) and eco-physiological (different harvests, various localities, diverse way of germination, uneven seedling age, unequal stage of cell growth) factors were studied concerning the present problem. In all these cases the starch localization pattern remains constant though the amounts of starch vary in particular cell complexes. From the evaluation of the starch localization at the level of the beginning cell growth in particular species and from comparison of the findings in different species it follows that starch localization and histogenesis patterns are different. This supports the view that the two phenomena are caused or controlled by uneven factors.     

Effects of Abscisic Acid on the Synthesis of Cell-Wall Polysaccharides in Segments of Etiolated Squash Hypocotyl II. Levels of UDP-Neutral Sugars

The effects of abscisic acid (ABA) on the formation of UDP-sugarsin segments of squash (Cucurbita maxima Duch.) hypocotyls wereexamined with [³H]glucose. After a 3-h incubation, 74% of totalradioactivity incorporated into neutral sugar residues of UDP-sugarswas found in glucose residues, 19% in galactose residues andless than 7% in other sugar residues. Exogenously applied ABAincreased the extent of incorporation of [³H]glucose into UDP-glucose,UDP-galactose and UDP-xylose by 30%, 30%, and 20%, respectively.However, ABA neither affected the total incorporation of radioactivityinto segments nor the incorporation into free glucose and sucrosein the segments. Since ABA inhibits the synthesis of celluloseand hemicellulosic polysaccharides in cell walls of segmentsof squash hypocotyls prior to the suppression of elongationof segments (Wakabayashi et al. 1989a, b), the present resultsindicate that ABA inhibits cell-wall synthesis by affectingsome step(s) beyond the formation of UDP-sugars and not by decreasingthe synthesis of UDP-sugars in the hypocotyl segments. ¹Present address: Biological Laboratory, Faculty of Education,Kagawa University, Saiwai-cho 1-1, Takamatsu, 760 Japan (Received May 30, 1990; Accepted February 5, 1991)