Pumpkin Cotyledons Research Articles

Features of gas exchange and use of reserve substances in pumpkin seedlings in conditions of skoto- and photomorphogenesis under the influence of gibberellin and chlormequat-chloride

We investigated the effect of gibberellin and the antigibberellic agent chlormequat-chloride on gas exchange and use of reserve substances in pumpkin seedlings during germination in the light and in the dark. We established that an artificial strengthening or growth inhibition of pumpkin seedlings in conditions of skotomorphogenesis caused an increase in of respiration intensity. Gibberellin treatment increased the proportion of assimilation processes in carbon dioxide gas exchange of seedlings, and growth inhibition by retardant caused an increase in respiratory costs when the nutrition type switches in the light from heterotrophic to autotrophic. The formation by seedlings of the demand for reserve assimilates from cotyledons was largely determined by change of activity of subapical meristems, which is manifested in the acceleration of seed germination, enhancing of histogenesis for the actions of gibberellin and in the weakening of these processes under the influence of retardants. Reserve substances used both oil and nitrogen-containing compounds. The content of protein nitrogen in pumpkin cotyledons decreased more in the light than in the dark, moreover growth inhibition by the retardant slowed down and growth increase by gibberellin accelerated this process both in conditions of photomorphogenesis and skotomorphogenesis.

First Report of Seedling Blight of Watermelon Caused by Acidovorax citrulli Transmitted from Rootstock of Pumpkin in China.

Bacterial fruit blotch (BFB) is a devastating disease caused by Acidovorax citrulli, which was first observed in the United States in 1988 (3). A. citrulli can cause severe infection on a wide range of cucurbits, including watermelon, cantaloupe, and pumpkin. Cotyledon symptoms are brown, angular, necrotic spots or large necrotic lesions. The disease is seedborne, so seeds usually serve as the primary inoculum source for BFB outbreaks (2). In July 2012, seedling blight was observed by local farmers from Anhui province in China on watermelon seedlings grafted to pumpkin rootstocks; lesions were morphologically similar to those caused by A. citrulli. Presence of A. citrulli was detected in symptomatic samples by using species-specific primers BX-L1/BX-S-R2 (1). The seed company claimed seeds of watermelon (cv. Changfeng) were certified free of bacterial fruit blotch, but pumpkin seeds (cv. Kangkuxianfeng-1) had not been tested for A. citrulli. For investigating the inoculum source, the remaining seeds of watermelon (cv. Changfeng) and pumpkin (cv. Kangkuxianfeng-1) for seedling production were collected from the farmer and processed for pathogen extraction as described by Walcott and Gitaitis (2). Two microliters of seed wash was used as template for PCR using primers BX-L1/BX-S-R2 (1). The experiment was conducted three times. A 279-bp DNA fragment was consistently amplified by PCR from seed wash of pumpkin seeds, but not from the seed wash of watermelon seeds. Three Acidovorax-like strains (A1, A2, and A3) were isolated from pumpkin seed wash using TWZ semi-selective medium (0.5% peptone, 0.025% CaCl2, 1% Tween-80, 50 mg/liter berberine, 50 mg/liter cycloheximide, 50 mg/liter 2,3,5-triphenyltetrazolium chloride). PCR was performed on the 16S rDNA gene from isolate strain A1, A2, and A3 (1,492 bp; GenBank Accession Nos. JX875533, JX875534, and JX875535) with primers rp1/fd2 (4), and PCR products were sequenced. Results of sequence analysis showed the sequences of strains A1, A2, and A3 were 99% identical to that of the type strain of A. citrulli AAC00-1 (NC_008752). Pathogencity was confirmed by injection of pumpkin cotyledons with bacterial suspensions of each isolate. Collected pumpkin seeds (n = 100 seeds) and watermelon seeds (n = 100 seeds) were planted in plastic pots containing sterilized field soil at room temperature to detect A. citrulli by a wet chamber growing out test. Eight days later, brown, angular, necrotic spots or wilt developed in pumpkin seedlings, but no symptoms were noted on the watermelon seedlings. To our knowledge, this is the first report of A. citrulli causing watermelon seedling blight transmitted from pumpkin seeds by grafting in China.

Isolation of glyoxysomes from pumpkin cotyledons.

Peroxisomes are single-membrane-bound organelles found in virtually all eukaryotes. In plants, there are several classes of peroxisomes. Glyoxysomes are found in germinating seedlings and contain enzymes specific for the glyoxylate cycle, including isocitrate lyase and malate synthase. After seedlings become photosynthetic, leaf peroxisomes participate in reactions of the photorespiration pathway and contain characteristic enzymes such as glycolate oxidase and hydroxypyruvate reductase. As leaves begin to senesce, leaf peroxisomes are transformed back into glyoxysomes. Root peroxisomes in the nodules of legumes, for example, sequester enzymes such as allantoinase and uricase, which contribute to nitrogen metabolism in these tissues. Thus, peroxisomes participate in many metabolic pathways and contain specific enzyme complements, depending on the tissue source. All peroxisomes contain catalase to degrade hydrogen peroxide and enzymes to accomplish beta-oxidation of fatty acids. Glyoxysomes can be isolated from pumpkin cotyledons by standard differential centrifugation and density separation, as described in this article.

Developmental analysis of a putative ATP/ADP carrier protein localized on glyoxysomal membranes during the peroxisome transition in pumpkin cotyledons.

In order to clarify the peroxisomal membrane proteins (PMPs), we characterized one of the major PMPs, PMP38. The deduced amino acid sequence for its cDNA in Arabidopsis thaliana contained polypeptides with 331 amino acids and had high similarity with those of Homo sapiens PMP34 and Candida boidinii PMP47 known as homologues of mitochondrial ATP/ADP carrier protein. We expected PMP38 to be localized on peroxisomal membranes, because it had the membrane peroxisomal targeting signal. Cell fractionation and immunocytochemical analysis using pumpkin cotyledons revealed that PMP38 is localized on peroxisomal membranes as an integral membrane protein. The amount of PMP38 in pumpkin cotyledons increased and reached the maximum protein level after 6 d in the dark but decreased thereafter. Illumination of the seedlings caused a significant decrease in the amount of the protein. These results clearly showed that the membrane protein PMP38 in glyoxysomes changes dramatically during the transformation of glyoxysomes to leaf peroxisomes, as do the other glyoxysomal enzymes, especially enzymes of the fatty acid beta-oxidation cycle, that are localized in the matrix of glyoxysomes.

Pumpkin peroxisomal ascorbate peroxidase is localized on peroxisomal membranes and unknown membranous structures.

To investigate the roles of peroxisomal membrane proteins in the reversible conversion of glyoxysomes to leaf peroxisomes, we characterized several membrane proteins of glyoxysomes. One of them was identified as an ascorbate peroxidase (pAPX) that is localized on glyoxysomal membranes. Its cDNA was isolated by immunoscreening. The deduced amino acid sequence encoded by the cDNA insert does not have a peroxisomal targeting signal (PTS), suggesting that pAPX is imported by one or more PTS-independent pathways. Subcellular fractionation of 3- and 5-d-old cotyledons of pumpkin revealed that pAPX was localized not only in the glyoxysomal fraction, but also in the ER fraction. A magnesium shift experiment showed that the density of pAPX in the ER fraction did not increase in the presence of Mg(2+), indicating that pAPX is not localized in the rough ER. Immunocytochemical analysis using a transgenic Arabidopsis which expressed pumpkin pAPX showed that pAPX was localized on peroxisomal membranes, and also on a unknown membranous structure in green cotyledons. The overall results suggested that pAPX is transported to glyoxysomal membranes via this unknown membranous structure.

Light regulates alternative splicing of hydroxypyruvate reductase in pumpkin.

Hydroxypyruvate reductase (HPR) is a leaf peroxisomal enzyme that functions in the glycolate pathway of photorespiration in plants. We have obtained two highly similar cDNAs for pumpkin HPR (HPR1 and HPR2). It has been revealed that two HPR mRNAs might be produced by alternative splicing from a single type of pre-mRNA. The HPR1 protein, but not the HPR2 protein, was found to have a targeting sequence into leaf peroxisomes at the C-terminus, suggesting that alternative splicing controls the subcellular localization of the two HPR proteins. Immunoblot analysis and subcellular fractionation experiments showed that HPR1 and HPR2 proteins are localized in leaf peroxisomes and the cytosol, respectively. Moreover, indirect fluorescence microscopy and analyses of transgenic tobacco cultured cells and Arabidopsis thaliana expressing fusion proteins with green fluorescent protein (GFP) revealed the different subcellular localizations of the two HPR proteins. Both mRNAs were induced developmentally and by light, but with quantitative differences. Almost equal amounts of the mRNAs were detected in pumpkin cotyledons grown in darkness, but treatment with light greatly enhanced the production of HPR2 mRNA. These findings indicate that light regulates alternative splicing of HPR mRNA, suggesting the presence of a novel mechanism of mRNA maturation, namely light-regulated alternative splicing, in higher plants.

Protein Phosphorylation in Polysomes of Pumpkin Cotyledons after Coumarin Treatment

Abstract: The natural compound, coumarin, caused a change in protein pattern and influenced the phosphorylation status of some ribosome‐associated proteins of pumpkin seedlings in vivo and in vitro. Low concentrations of coumarin stimulated ribosome‐associated protein phosphorylation only in cotyledons but not in roots and stems. Two phosphoproteins whose phosphorylation state was influenced upon coumarin treatment could be isolated and characterized by their relative molecular weight of about 58 and 65 kDa and pl‐values at 5.2 and 5.7, respectively. These phosphoproteins are not major constituents of small or large subunits of ribosomes. We did not find any influence of coumarin on phosphorylation of ribosomal proteins S6, LAO and LAI–3.

Molecular Characterization of a Glyoxysomal Long Chain Acyl-CoA Oxidase That Is Synthesized as a Precursor of Higher Molecular Mass in Pumpkin

A cDNA clone for pumpkin acyl-CoA oxidase (EC 1.3.3.6; ACOX) was isolated from a lambdagt11 cDNA library constructed from poly(A)+ RNA extracted from etiolated cotyledons. The inserted cDNA clone contains 2313 nucleotides and encodes a polypeptide of 690 amino acids. Analysis of the amino-terminal sequence of the protein indicates that the pumpkin acyl-CoA oxidase protein is synthesized as a larger precursor containing a cleavable amino-terminal presequence of 45 amino acids. This presequence shows high similarity to the typical peroxisomal targeting signal (PTS2). Western blot analysis following cell fractionation in a sucrose gradient revealed that ACOX is localized in glyoxysomes. A partial purification of ACOX from etiolated pumpkin cotyledons indicated that the ACOX cDNA codes for a long chain acyl-CoA oxidase. The amount of ACOX increased and reached to the maximum activity by day 5 of germination but decreased about 4-fold on the following days during the subsequent microbody transition from glyoxysomes to leaf peroxisomes. By contrast, the amount of mRNA was already high at day 1 of germination, increased by about 30% at day 3, and faded completely by day 7. These data indicated that the expression pattern of ACOX was very similar to that of the glyoxysomal enzyme 3-ketoacyl-CoA thiolase, another marker enzyme of the beta-oxidation spiral, during germination and suggested that the expression of each enzyme of beta-oxidation is coordinately regulated.

Glyoxysomal malate dehydrogenase in pumpkin: cloning of a cDNA and functional analysis of its presequence.

Glyoxysomal malate dehydrogenase (gMDH) is an enzyme of the glyoxylate cycle that participates in degradation of storage oil. We have cloned a cDNA for gMDH from etiolated pumpkin cotyledons that encodes a polypeptide consisting of 356 amino acid residues. The nucleotide and N-terminal amino acid sequences revealed that gMDH is synthesized as a precursor with an N-terminal extrapeptide. The N-terminal presequence of 36 amino acid residues contains two regions homologous to those of other microbody proteins, which are also synthesized as large precursors. To investigate the functions of the N-terminal presequence of gMDH, we generated transgenic Arabidopsis that expressed a chimeric protein consisting of beta-glucuronidase and the N-terminal region of gMDH. Immunological and immunocytochemical studies revealed that the chimeric protein was imported into microbodies such as glyoxysomes and leaf peroxisomes and was then subsequently processed. Site-directed mutagenesis studies showed that the conserved amino acids in the N-terminal presequence, Arg-10 and His-17, function as recognition sites for the targeting to plant microbodies, and Cys-36 in the presequence is responsible for its processing. These results correspond to those from the analyses of glyoxysomal citrate synthase (gCS), which was also synthesized as a large precursor, suggesting that common mechanisms that can recognize the targeting or the processing of gMDH and gCS function in higher plant cells.

Pseudomonas syringae pv. syringae causes wart‐like eruptions on fruit of buttercup squash (Cucurbita maxima)

Fruit of buttercup squash (Cucurbita maxima D. hybrids ‘Delica’ and ‘Kurijiman') grown at Mangatawhiri, South Auckland, New Zealand, are prone to develop wart‐like eruptions during the first 2–3 weeks after harvest. This disfigurement renders the fruit unacceptable for export, and was a serious problem in the 1993–94 season. Isolations from “warts” resulted in the recovery of large numbers of bacteria (>107/g) in the spongy internal tissues. Fungi were infrequently isolated from warts. Bacterial isolates were screened for pathogenicity to pumpkin cotyledons and for the capacity to reproduce symptoms on fruit. Isolates strongly pathogenic to cotyledons and which also induced a typical waiting response were all identified as Pseudomonas syringae pv. syringae on the basis of LOPAT determinative tests, syringomycin production, and pathogenicity to lilac. The same organism was consistently re‐isolated from warts produced following artificial inoculation. One re‐isolate was tested for wart‐inducing capacity, and was found to induce typical symptoms on fruit. Other strains of P. syringae pv. syringae from other cultivars of C. maxima, obtained from the International Collection of Microorganisms from Plants (ICMP), also produced typical symptoms, whereas P. viridiflava, Xanthomonas campestris pv. cucurbitae, and P. syringae pv. syringae from melon and cucumber did not. This is the first report of P. syringae pv. syringae causing significant disease symptoms on fruit of C. maxima.

Stromal and thylakoid-bound ascorbate peroxidases are produced by alternative splicing in pumpkin

A cDNA encoding stromal ascorbate peroxidase (sAPX) was isolated using poly(A) + RNA of pumpkin cotyledons by RT-PCR. The cDNA encodes a polypeptide with 372 amino acids and shares complete sequence identity with pumpkin thylakoid-bound ascorbate peroxidase (tAPX), except for the deletion of a putative membrane spanning region located in the carboxy domain of tAPX. Southern blot hybridization and analysis of intron structure indicated that mRNAs for sAPX and tAPX, whose suborganellar localizations in chloroplasts are different, are produced by alternative splicing. Immunoblot analysis showed that the accumulation of sAPX and tAPX was differently regulated during germination and subsequent greening of pumpkin cotyledons.

Changes in the chlorophyll fluorescence characteristics of chloroplasts from intact pumpkin cotyledons, caused by organ excision and kinetin treatment

The chlorophyll (Chl) fluorescence emission as well as excitation and polarization characteristics of chloroplasts from intact cotyledons were determined in pumpkin seedlings after removal of one cotyledon (co-cotyledon) or apical bud or primary root, or after kinetin treatment of derooted seedlings. Qualitatively, the fluorescence emission and excitation spectra of chloroplasts were similar. The fluorescence emission spectra showed a maximum at 685 (F685) and a hump at 735 nm (F735), whereas the excitation spectra showed peaks at 439, 471, 485, and 676 nm. The fluorescence intensities at F685 and F735 differed in various groups of seedlings, as indicated by changes in their ratios. Similarly, the ratios of 471/439, 485/439, and 676/439 nm were also different. Variability in the Chl fluorescence intensity values and the fluorescence polarization of chloroplasts prepared from various seedling types may suggest a different degree of binding between the pigment complexes and light-harvesting Chl-protein (LHCP), resulting in different rates of photoexcitation energy loss in the form of fluorescence emission. Kinetin treatment improved the coupling of pigment complexes with reaction centre, as indicated by low polarization values in derooted and kinetin-treated seedlings, which suggests the development of a suntype chloroplast.

A rapid increase in the level of binding protein (BiP) is accompanied by synthesis and degradation of storage proteins in pumpkin cotyledons.

The binding protein (BiP) has been implicated in cotranslational folding of nascent polypeptides, and in the recognition and disposal of aberrant polypeptides. To elucidate the involvement of BiP in the biosynthesis of vacuolar proteins, we have characterized the protein in pumpkin cotyledons during seed maturation and seedling growth. Isolated microsomes from maturing pumpkin cotyledons contained a significant amount of BiP, protein-disulfide isomerase and calreticulin. We have purified a 70-kDa protein; sequences of the N-terminus and internal fragments of this protein exhibited a high identity to the sequence of soybean. Immunoblot analysis with specific antibodies raised against the purified BiP showed that the amount of BiP in a cotyledon increased markedly at the middle stages and then decreased. The increase was accompanied by the synthesis of storage proteins and the development of the endoplasmic reticulum in the cotyledons at the middle stage of seed maturation. Most of these storage proteins degraded dramatically between 2 and 5 days after seed germination, and the degradation was also accompanied by a rapid increase in the level of BiP. Subcellular fractionation of the 4-day-old cotyledons showed a high accumulation of BiP in the endoplasmic reticulum. It is possible that BiP might be involved in the synthesis of seed storage proteins during maturation and in the synthesis of hydrolytic enzymes responsible for the degradation of the storage proteins during seed germination.

A Pumpkin 72-kDa Membrane Protein of Precursor-Accumulating Vesicles Has Characteristics of a Vacuolar Sorting Receptor

Precursor-accumulating (PAC) vesicles were previously shown to mediate the transport of the precursor of a major storage protein (pro2S albumin) to protein-storage vacuoles in developing pumpkin cotyledons. In this study, we characterized two homologous proteins from PAC vesicles, a 72 kDa protein (PV72) and an 82 kDa protein (PV82). PV72 and PV82 showed an ability to bind to peptides derived from both an internal propeptide and a C-terminal peptide of pro2S albumin. PV72 was predicted to be a type I integral membrane protein with epidermal growth factor (EGF)-like motifs. These results suggest that PV72 and PV82 are potential sorting receptors for 2S albumin to protein-storage vacuoles.

CDNA cloning and expression of a gene for isocitrate lyase in pumpkin cotyledons.

A cDNA clone for isocitrate lyase (ICL) was isolated from a cDNA library prepared from the poly(A)+ RNA of etiolated pumpkin cotyledons. The cDNA encoded a polypeptide with 576 amino acids, whose sequence is more than 79% identical to those of ICL from other higher plants and contains the C-terminal tripeptide, Ser-Arg-Met, which is a putative targeting signal to microbodies. Immunogold analysis revealed that ICL protein is exclusively localized in microbodies in etiolated pumpkin cotyledons. Double labeling experiments with protein A-gold particles of different sizes showed that ICL protein coexists with a leaf-peroxisomal enzyme, glycolate oxidase, in the microbodies of greening and senescing pumpkin cotyledons, indicating that transformation between glyoxysomes and leaf peroxisomes occurs directly during greening and senescence of the cotyledons. Immunoblot analysis showed that the amount of ICL protein increased markedly during germination and decreased rapidly when seedlings were exposed to illumination, which induced the microbody transition from glyoxysomes to leaf peroxisomes. In senescing cotyledons, the level of the ICL protein and ICL mRNA increased again with the reverse transition of microbodies from leaf peroxisomes to glyoxysomes. Changes in the amount of ICL protein did not correspond to the changes in the level of ICL mRNA during greening and senescence of the cotyledons, an indication that post-transcriptional regulation plays an important role in the microbody transition.

CDNA cloning and expression of a gene for 3-ketoacyl-CoA thiolase in pumpkin cotyledons

A cDNA clone for 3-ketoacyl-CoA thiolase (EC 2.3.1.16) was isolated from a lambda gt11 cDNA library constructed from the poly(A)+ RNA of etiolated pumpkin cotyledons. The cDNA insert contained 1682 nucleotides and encoded 461 amino acid residues. A study of the expression in vitro of the cDNA and analysis of the amino-terminal sequence of the protein indicated that pumpkin thiolase is synthesized as a precursor which has a cleavable amino-terminal presequence of 33 amino acids. The amino-terminal presequence was highly homologous to typical amino-terminal signals that target proteins to microbodies. Immunoblot analysis showed that the amount of thiolase increased markedly during germination but decreased dramatically during the light-inducible transition of microbodies from glyoxysomes to leaf peroxisomes. By contrast, the amount of mRNA increased temporarily during the early stage of germination. In senescing cotyledons, the levels of the thiolase mRNA and protein increased again with the reverse transition of microbodies from leaf peroxisomes to glyoxysomes, but the pattern of accumulation of the protein was slightly different from that of malate synthase. These results indicate that expression of the thiolase is regulated in a similar manner to that of other glyoxysomal enzymes, such as malate synthase and citrate synthase, during seed germination and post-germination growth. By contrast, during senescence, expression of the thiolase is regulated in a different manner from that of other glyoxysomal enzymes.

Transcriptional regulation of hydroxypyruvate reductase gene expression by cytokinin in etiolated pumpkin cotyledons

To understand the mechanisms by which the expression of a specific gene is modulated by cytokinin, the regulation of hydroxypyruvate reductase (HPR) transcript levels by N6-benzyladenine (BA) in etiolated pumpkin (Cucurbita pepo L. cv. Halloween) cotyledons was investigated. A pumpkin HPR cDNA was generated by reverse transcriptase-polymerase chain reaction and its nucleotide sequence was determined. An antisense HPR RNA was prepared for RNase protection analysis of HPR-mRNA expression patterns in the cotyledons of dark-grown pumpkin seedlings. Treatment of the cotyledons with BA was shown to modulate HPR mRNA levels in a dose- and time-dependent manner. Similarly, nuclear run-on studies showed that the rate of transcription was also enhanced by BA treatment of the cotyledons. These results suggest that the enhancement of HPR mRNA by cytokinin is, at least in part, at the level of transcription.

Evidences of glyoxylate cycle in peroxisomes of senescent cotyledons

The metabolic pathway of the glyoxylate cycle has been investigated in peroxisomes isolated from senescent pumpkin ( Cucurbita sp.) cotyledons. β-oxidation activity, as well as activities of glyoxylate cycle enzymes isocitrate lyase (EC 4.1.3.1), malate synthase (EC 4.1.3.2), malate dehydrogenase (EC 1.1.1.37) and citrate synthase (EC 4.1.3.7) were detected. In order to establish if there is a channelling of acetyl CoA into the glyoxylate cycle, peroxisomes have been incubated with various substrates. The incubations show acetyl CoA utilization by the glyoxylate cycle. When the incubation medium for citrate formation is used, all the label from [1- 14C]acetyl CoA is recovered in citrate, whereas only 18% of the added radioactivity is recovered in malic acid (by isocitrate lyase and malate synthase) after a long incubation time (3 h). Only by feeding peroxisomes with [1,5- 14C]citric acid and exogenous aconitase (EC 4.2.1.3) can a weak formation of other organic acids (glyoxylate and succinate) be note. The requirement for exogenous aconitase to carry out the peroxisomal glyoxylate cycle points towards the isocitric acid step as a crucial factor for the operation of the global cycle.

NADP+-isocitrate dehydrogenase in germinating and senescing pumpkin cotyledons

NADP+-isocitrate dehydrogenase in germinating and senescing pumpkin cotyledons

NADP+‐isocitrate dehydrogenase in germinating and senescing pumpkin cotyledons

NADP+‐isocitrate dehydrogenase (IDH, EC 1.1.1.42) was studied during the post‐germinative growth of pumpkin (Cucurbita pepo L. cv. Alberello di Sarzana) seedlings. In cotyledons. IDH activity increased in the dark and declined after illumination. Native PAGE showed that at least two isozymes of low electrophoretic mobility are present in cotyledons and absent in other pumpkin tissues. Anion exchange chromatography performed on extracts both from 4‐day‐old etiolated cotyledons and from illuminated cotyledons confirmed the trend of the IDH isoforms. In senescing cotyledons an additional IDH isoform with higher electrophoretic mobility appears. Overall the data indicate the presence of specific NADP+‐IDH isoforms in etiolated cotyledons and senescing cotyledons, when glyoxylate cycle enzymes are active. A possible role for these IDH isoforms is proposed.