Cytokinin-modulated gene expression in excised pumpkin cotyledons

Comparison of two-dimensional polyacrylamide gel electrophoretic maps of proteins isolated from benzyladenine-treated and untreated pumpkin (Cucurbita pepo L. cv Halloween) cotyledons showed that the expression of certain proteins is enhanced, induced, or suppressed by the cytokinin treatment. The amount of poly(A)⁺ mRNA isolated from cotyledons incubated with 10⁻⁴ molar benzyladenine for five days was about four-fold over the water-incubated control. The activity of hydroxypyruvate reductase prepared from purified cotyledonous microbodies and analyzed by native gel electrophoresis is proportionally enhanced by sequentially higher concentrations (10⁻⁹ to 10⁻⁴ molar) of benzyladenine. Ethidium bromide (1 microgram per milliliter) did not inhibit hydroxypyruvate reductase activity; thus, the enzyme synthesis does not appear to be controlled by organelle genes. Hydroxypyruvate reductase synthesis is inhibited by cycloheximide, cordycepin, and to a certain degree by actinomycin D. These data support the view of a close association between cytokinin action and gene expression.     

Pumpkin hydroxypyruvate reductases with and without a putative C-terminal signal for targeting to microbodies may be produced by alternative splicing

Two full-length cDNAs encoding hydroxypyruvate reductase were isolated from a cDNA library constructed with poly(A)⁺ RNA from pumpkin green cotyledons. One of the cDNAs, designated HPR1, encodes a polypeptide of 386 amino acids, while the other cDNA, HPR2 encodes a polypeptide of 381 amino acids. Although the nucleotide and deduced amino acid sequences of these cDNAs are almost identical, the deduced HPR1 protein contains Ser-Lys-Leu at its carboxy-terminal end, which is known as a microbody-targeting signal, while the deduced HPR2 protein does not. Analysis of genomic DNA strongly suggests that HPR1 and HPR2 are produced by alternative splicing.     

Import of peroxisomal hydroxypyruvate reductase into glyoxysomes

A new procedure was used to purify the peroxisomal matrix enzyme hydroxypyruvate reductase (HPR) from green leaves of pumpkin (Cucurbita pepo L.) and spinach (Spinacia oleracea L.). Monospecific antibodies were prepared against this enzyme in rabbits. Immunoprecipitation of HPR from watermelon (Citrullus vulgaris Schrad.) yielded a single protein with a subunit molecular weight of 45 kDa. Immunohistochemical labeling of HPR was found exclusively in watermelon microbodies. Isolated polyadenylated mRNA from light-grown watermelon cotyledons was injected into Xenopus laevis oocytes. The heterologous in-vivo translation product of HPR exhibited the same molecular weight as the immunoprecipitate from watermelon cotyledons, indicating the lack of a cleavable extra sequence. The watermelon HPR translated in oocytes was imported into isolated glyoxysomes from castor bean (Ricinus communis L.) endosperm and remained resistant to proteolysis after the addition of proteinase K. The HPR did not change its apparent molecular weight during sequestration; however, it may have changed its conformation.Abbreviations HPR hydroxypyruvate reductase - PMSF phenylmethylsulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

cDNA cloning and differential gene expression of three catalases in pumpkin

Three cDNA clones (cat1, cat2, cat3) for catalase (EC 1.11.1.6) were isolated from a cDNA library of pumpkin (Cucurbita sp.) cotyledons. In northern blotting using the cDNA-specific probe, the cat1 mRNA levels were high in seeds and early seedlings of pumpkin. The expression pattern of cat1 was similar to that of malate synthase, a characteristic enzyme of glyoxysomes. These data suggest that cat1 might encode a catalase associated with glyoxysomal functions. Furthermore, immunocytochemical analysis using cat1-specific anti-peptide antibody directly showed that cat1 encoding catalase is located in glyoxysomes. The cat2 mRNA was present at high levels in green cotyledons, mature leaf, stem and green hypocotyl of light-grown pumpkin plant, and correlated with chlorophyll content in the tissues. The tissue-specific expression of cat2 had a strong resemblance to that of glycolate oxidase, a characteristic enzyme of leaf peroxisomes. During germination of pumpkin seeds, cat2 mRNA levels increased in response to light, although the increase in cat2 mRNA by light was less than that of glycolate oxidase. cat3 mRNA was abundant in green cotyledons, etiolated cotyledons, green hypocotyl and root, but not in young leaf. cat3 mRNA expression was not dependent on light, but was constitutive in mature tissues. Interestingly, cat1 mRNA levels increased during senescence of pumpkin cotyledons, whereas cat2 and cat3 mRNAs disappeared during senescence, suggesting that cat1 encoding catalase may be involved in the senescence process. Thus, in pumpkin, three catalase genes are differentially regulated and may exhibit different functions.     

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.     

Phytohormonal changes in greening and senescing intact cotyledons of oilseed rape and pumpkin: influence of the growth retardant BAS 111 ˙˙ W

The triazole growth retardant BAS 111‥W delayed senescence in cotyledons of pumpkin ( Cucurbita maxima L. cv. Gelbe genetzte Riesenmelone) and stimulated chlorophyll synthesis in greening cotyledons of oilseed rape ( Brassica napus L. cv. Petranova) seedlings. In both cases, changes of phytohormone-like substances in the cotyledons were analyzed on a fresh weight basis by immunoassay.
After soil treatment with increasing retardant concentrations, a close correlation was observed in senescing cotyledons of pumpkin between a reduced loss in total chlorophyll and increasing levels of dihydrozeatin riboside (DZR) and trans -zeatin riboside (ZR)-type cytokinins. In contrast, the levels of isopentenyladenosine (IPA)-type cytokinins, 3-indoleacetic acid (IAA) and gibberellin (GA) did not change significantly. The levels of abscisic acid (ABA) were slightly elevated at low retardant concentrations but dropped considerably below those of controls at higher doses. Consequently, the molar ratio of total cytokinin to ABA content changed from approximately 1:40 in controls (50% of initial chlorophyll) to 1:3 in cotyledons treated with 3 mg BAS 111‥W plant⁻¹ (85% of initial chlorophyll). These changes, together with the known reduction of ethylene production by plants treated with nitrogen-heterocyclic retardants, can explain the delayed senescence in pumpkin cotyledons. Likewise, when etiolated, BAS 111‥W-treated seedlings of oilseed rape were exposed to light, the stimulation of chlorophyll synthesis in the cotyledons was accompanied by an accumulation of DZR- and, particularly, ZR-type cytokinins and IAA. In contrast, GA and ABA contents decreased slightly. We conclude that the influence of BAS 111‥W on cytokinin levels might be involved in the stimulation of greening.     

Role of polyamines in the cytokinin-dependent physiological processes II. Modulation of polyamine levels during cytokinin-stimulated expansion of cucumber cotyledons

The cucumber cotyledon expansion test was used as a model system to study a possible relationship between cytokinin and polyamines. When kinetin was applied to excised cotyledons incubated in the dark it caused a marked increase in the activity of arginine decarboxylase. As a result of ADC action, putrescine content also rose markedly, whereas the level of spermidine and spermine decreased. However, inhibition of putrescine biosynthesis with D-arginine did not affect cytokinin promotion growth. Applied alone, putrescine had no significant effect on growth. These results indicate that the large increase in putrescine content that derives from cytokinin treatment cotyledons is not essential for cytokinin-induced expansion of cotyledons. Addition of K⁺ and Ca²⁺ ions to the cotyledons incubated with cytokinin caused a marked reduction in the putrescine level and ADC activity. The higher level of putrescine (35 %) and spermine (62 %) bound to chromatin and the large increase (174 %) in spermidine content bound to ribosomes which derive from cytokinintreated cotyledons in relation to literature data can indicate that these polyamines may play an important role in gene expression during cytokinin-stimulated expansion of cucumber cotyledons. The inhibition of cytokinin effect, viz. enlargement of the cotyledons by inhibitors of spermidine biosynthesis, additionally suggessted a possible involvement of polyamines in cytokinin action.     

Uptake and effects of N-benzyladenine in excised watermelon cotyledons: influence of cotyledon age

Some characteristics of uptake of [8-¹⁴C]N⁶-benzyladenine (BA) by watermelon (Citrullus vulgaris Schrad., cv. Fairfax) cotyledons that were either excised immediately after 24 hours inhibition (day 0) or cultured in the dark for 48 hours on moist filter paper (day 2) have been compared.

The uptake of BA seems to be passive in cotyledons of both kinds. The initial rate of uptake is, however, much slower in day 2 cotyledons. This is probably due to a higher resistance of cell membranes to BA influx. When the day 2 cotyledons are frozen and thawed, so that the membrane barrier is abolished, the amount of BA taken up is the same as in day 0 cotyledons.

In spite of the lower rate of uptake, the physiological effects of BA in day 2 cotyledons are as strong as in day 0 cotyledons and occur with a shorter lag time. Sensitivity to BA seems indeed to start 24 to 48 hours after excision.