Cloning of gibberellin 3 beta-hydroxylase cDNA and analysis of endogenous gibberellins in the developing seeds in watermelon

We have isolated Cv3h, a cDNA clone from the developing seeds of watermelon, and have demonstrated significant amino acid homology with gibberellin (GA) 3 beta-hydroxylases. This cDNA clone was expressed in Escherichia coli as a fusion protein that oxidized GA(9) and GA(12) to GA(4) and GA(14), respectively. The Cv3h protein had the highest similarity with pumpkin GA 2 beta,3 beta-hydroxylase, but did not possess 2 beta-hydroxylation function. RNA blot analysis showed that the gene was expressed primarily in the inner parts of developing seeds, up to 10 d after pollination (DAP). In the parthenocarpic fruits induced by treatment with 1-(2-chloro-4-pyridyl)-3-phenylurea (CPPU), the embryo and endosperm of the seeds were undeveloped, whereas the integumental tissues, of maternal origin, showed nearly normal development. Cv3h mRNA was undetectable in the seeds of CPPU-treated fruits, indicating that the GA 3 beta-hydroxylase gene was expressed in zygotic cells. In our analysis of endogenous GAs from developing seeds, GA(9) and GA(4) were detected at high levels but those of GA(20) and GA(1) were very low. This demonstrates that GA biosynthesis in seeds prefers a non-13-hydroxylation pathway over an early 13-hydroxylation pathway. We also analyzed endogenous GAs from seeds of the parthenocarpic fruits. The level of bioactive GA(4 )was much lower there than in normal seeds, indicating that bioactive GAs, unconnected with Cv3h, exist in integumental tissues during early seed development.     

Cloning and Molecular Analyses of a Gibberellin 20-Oxidase Gene Expressed Specifically in Developing Seeds of Watermelon

To understand the biosynthesis and functional role of gibberellins (GAs) in developing seeds, we isolated Cv20ox, a cDNA clone from watermelon (Citrullus lanatus) that shows significant amino acid homology with GA 20-oxidases. The complementary DNA clone was expressed in Escherichia coli as a fusion protein, which oxidized GA(12) at C-20 to the C(19) compound GA(9), a precursor of bioactive GAs. RNA-blot analysis showed that the Cv20ox gene was expressed specifically in developing seeds. The gene was strongly expressed in the integument tissues, and it was also expressed weakly in inner seed tissues. In parthenocarpic fruits induced by 1-(2-chloro-4-pyridyl)-3-phenylurea treatment, the expression pattern of Cv20ox did not change, indicating that the GA 20-oxidase gene is expressed primarily in the maternal cells of developing seeds. The promoter of Cv20ox was isolated and fused to the beta-glucuronidase (GUS) gene. In a transient expression system, beta-glucuronidase staining was detectable only in the integument tissues of developing watermelon seeds.     

Early Fruit Development in the Watermelon: Anatomical Comparison of Pollinated, Auxin-induced Parthenocarpic and Unpollinated Fruits

The anatomy of pollinated, auxin-induced parthenocarpic andunpollinated watermelon fruits was observed for nine days afterflowering. Parthenocarpic fruits were larger and had higherfresh weight and percentage water than pollinated fruits atday 1 but the positions were reversed by day 9. Unpollinatedfruits did not increase in size after day 3. Pericarp cells were small, of regular shape and showed no obviouschange with either time or treatment. Cell number increasedin the pollinated and parthenocarpic but not in the unpollinatedfruits. Cells divided in the flesh of the parthenocarpic but not ofthe pollinated fruits which increased in size by cell enlargementonly. Starch, present in the cells of the flesh and placentaat day 0 was absent from the unpollinated fruits at day 6. Ovules grew in both pollinated and parthenocarpic fruits largelydue to cell division in the nucellus and integuments; the pollinatedovules were larger than the parthenocarpic throughout. Embryoand endosperm development occurred in the pollinated but notin the parthenocarpic ovules. Starch was present throughoutthe nine-day period in the integuments of the pollinated andparthenocarpic ovules but was lost from the integuments of theunpollinated ovules by day 6. Pollinated and parthenocarpicovules contributed increasingly to fruit dry weight over thenine-day period. It is suggested that the ovule tissues, in particular the nucellusand integument may exert control over early development in bothpollinated and parthenocarpic fruits.     

Light Alters Cytosolic and Plastidic Phosphorylase Distribution in Pearl Millet Leaves

Treatment of pollinated pea (Pisum sativum L. cv Alaska, line V1) ovaries with 3,5-dioxo-4-butyryl-cyclohexane carboxylic acid ethyl ester (LAB), an acylcyclohexanedione derivative that competitively inhibits 2-oxoglutarate-dependent gibberellin (GA) dioxygenases, caused a reduction of pod elongation proportional to the amount of inhibitor applied. The effect of LAB was counteracted by GA1 and GA3, and partially by GA20. The inhibitor decreased the contents of GA1 and GA3 (the purported active GAs) and GA8, increased those of GA19 and GA20, and did not affect that of GA29 in both the pod and the developing seeds. These results provide evidence that GA1 and/or GA3 control pod development in pea and show that GA20 is not active per se. In contrast to its effect on pollinated ovaries, LAB promoted parthenocarpic development of unpollinated ovaries, which is associated with an increase of GA1 and GA8 content. The inhibitor enhanced the response of unpollinated ovaries to GA1 and GA20, but it did not alter the response to GA3. LAB is proposed to promote parthenocarpic development and enhance the response to exogenous GAs by blocking the 2[beta]-hydroxylation of GA1 more efficiently than 3[beta]-hydroxylation of GA20.     

p-CPA increases the endogenous IAA content of parthenocarpic muskmelon fruit

We examined the effects of seed formation andpara-chlorophenoxyacetic acid (p-CPA)treatment on the growth and endogenous indole acetic acid (IAA) content ofmuskmelon fruit. The growth of parthenocarpic muskmelon fruit induced by 1-(2-chloro-4-pyridyl)-3-phenylurea (CPPU) declined 15 days after anthesis (DAA),resulting in smaller fruit than those pollinated at harvest.p-CPA improved the growth of parthenocarpic fruit thatweretreated between 10 and 25 DAA. Endogenous IAA levels in the seedsof artificially pollinated fruit were at their highest at 10 DAA,then decreased, and increased again after 30 to 45 DAA, whereas,the levels in the empty seeds of parthenocarpic fruit were significantly lowerthroughout development. Although endogenous IAA levels in the placenta ofpollinated fruit were lower than those in the seeds, the changing patterns werevery similar to those in the seeds. Endogenous IAA levels in the mesocarp ofpollinated fruit remained lower than those in the placenta throughout fruitgrowth, and the pattern of change was similar to that of the placenta. Levelsinthe seed, placenta and mesocarp of p-CPA-nontreatedparthenocarpic fruit stayed lower than those in pollinated fruit.p-CPA increased the levels of IAA in the seeds, placenta,and mesocarp of parthenocarpic fruit after the first treatment (10DAA) to 15 DAA, while those in the mesocarp increasedsignificantly after the second treatment (25 DAA), but did notincrease in empty seed and placenta.     

Genetic engineering of parthenocarpic fruit development in tomato

Parthenocarpy was engineered in two genotypes of Lycopersicon esculentum Mill. by using the DefH9-iaaM chimeric gene. The parthenocarpic trait consists of fruit set and growth in the absence of fertilization. Seedless parthenocarpic fruits were obtained from emasculated flowers, and fruits with seeds from pollinated flowers. All parthenocarpic tomato plants analysed expressed the DefH9-iaaM gene during flower development. The fruit set percentage of emasculated transgenic flowers was similar to that of control plants. In 7 out of 8 independent transgenic plants, the fresh weight of fruits derived from pollinated or emasculated flowers did not significantly differ from that of fruits obtained by pollination of the control plants. The pH of the parthenocarpic fruit was generally unaffected and the soluble solid concentration was either unchanged or increased. Thus, the DefH9-iaaM gene is a genetic tool that might be used to improve tomato productivity.     

Fruit characteristics, seed production and pollen tube growth in the wild chilli pepper Capsicum flexuosum

Fruits formed after different pollination regimes (flowers hand pollinated, unpollinated, and open pollinated) and the seeds obtained were characterized in the wild chilli pepper Capsicum flexuosum Sendtn. Pollen tube development in vivo and ovary growth were also analyzed. Seedless fruits and empty seeds were abundant among the fruits from hand pollinated and open pollinated flowers, while no more than one seed with embryo was found in a low percentage of fruits from such pollination treatments. Parthenocarpic fruits were formed from unpollinated flowers. Pollen tube growth was arrested in the upper third of the style for almost all pollen tubes except for a single one that may continue elongating occasionally. The ovary size increased continuously after pollination, even without fertilization. The sum of the evidence registered may help to explain the low number of seeds with embryo harvested, the abundance of seedless fruits formed from pollinated flowers (possibly parthenocarpic), and the high rate of parthenocarpic fruits formed from unpollinated flowers.     

Class I chitinase and beta-1,3-glucanase are differentially regulated by wounding,methyl jasmonate,ethylene, and gibberellin in tomato seeds and leaves

Class I chitinase (Chi9) and beta-1,3-glucanase (GluB) genes are expressed in the micropylar endosperm cap of tomato (Lycopersicon esculentum) seeds just before radicle emergence through this tissue to complete germination. In gibberellin (GA)-deficient mutant (gib-1) seeds, expression of Chi9 and GluB mRNA and protein is dependent upon GA. However, as expression occurs relatively late in the germination process, we investigated whether the genes are induced indirectly in response to tissue wounding associated with endosperm cap weakening and radicle protrusion. Wounding and methyl jasmonate (MeJA) induced Chi9 expression, whereas ethylene, abscisic acid, sodium salicylate, fusicoccin, or beta-aminobutyric acid were without effect. Chi9 expression occurred only in the micropylar tissues when seeds were exposed to MeJA or were wounded at the chalazal end of the seed. Expression of Chi9, but not GluB, mRNA was reduced in germinating seeds of the jasmonate-deficient defenseless1 tomato mutant and could be restored by MeJA treatment. Chi9 expression during germination may be associated with "wounding" from cell wall hydrolysis and weakening in the endosperm cap leading to radicle protrusion, and jasmonate is involved in the signaling pathway for this response. Among these treatments and chemicals (other than GA), only MeJA and wounding induced a low level of GluB expression in gib-1 seeds. However, MeJA, wounding, and particularly ethylene induced both genes in leaves, whereas GA induced only Chi9 in leaves. Although normally expressed simultaneously during tomato seed germination, Chi9 and GluB genes are regulated distinctly and tissue specifically by hormones and wounding.     

The promoter of the asi gene directs expression in the maternal tissues of the seed in transgenic barley

The bifunctional alpha-amylase/subtilisin inhibitor (BASI) is an abundant protein in barley seeds, proposed to play multiple and apparently diverse roles in regulation of starch hydrolysis and in seed defence against pathogens. In the Triticeae, the protein has evolved the ability to specifically inhibit the main group of alpha-amylases expressed during germination of barley and encoded by the amyl gene family found only in the Triticeae. The expression of the asi gene that encodes BASI has been reported to be controlled by the hormones abscisic acid (ABA) and gibberellic acid (GA). Despite many studies at the gene and protein level, the function of this gene in the plant remains unclear. In this study, the 5'-flanking region (1033 bp, 1033-asi promoter) and the 3'-flanking region (655 bp) of the asi gene were isolated and characterised. The 1033-asi promoter sequence showed homology to a number of ciselements that play a role in ABA and GA regulated expression of other genes. With a green fluorescent protein gene (gfp) as reporter, the 1033-asi promoter was studied for spatial, temporal and hormonal control of gene expression. The 1033-asi promoter and its deletions direct transient gfp expression in the pericarp and at low levels in mature aleurone cells, and this expression is not regulated by ABA or GA. In transgenic barley plants, the 1033-asi promoter directed tissue-specific expression of the gfp gene in developing grain and germinating grain but not in roots or leaves. In developing grain, expression of gfp was observed specifically in the pericarp, the vascular tissue, the nucellar projection cells and the endosperm transfer cells and the hormones ABA or GA did not regulate this expression. In mature germinating grain gfp expression was observed in the embryo but not in aleurone or starchy endosperm. However, GA induced gfp expression in the aleurone of mature imbibed seeds from which the embryo had been removed. Expression in maternal rather than endosperm tissues of the grain suggests that earlier widespread assumptions that the protein is expressed largely in the endosperm may have been largely based on analysis of mixed grain tissues. This novel pattern of expression suggests that both activities of the protein may be primarily involved in seed defence in the peripheral tissues of the seed.     

The role of endogenous gibberellin in seed and fruit development of tomato: Studies with a gibberellin-deficient mutant

The role of endogenous gibberellin (GA) in seed and fruit development was studied with the use of the GA-deficient ga-1 mutant of tomato ( Lycopersicon esculentum Mill. cv. Moneymaker). Flowers of the ga-1 mutant were abnormal and sterile, but parthenocarpic fruit development was observed occasionally on the dwarf plants. A single application of GA₄₊₇ restored the fertility of the mutant flowers and resulted in seed set. Development of GA-producing and GA-deficient seeds in GA-deficient fruits was compared by pollination of ga-1/ga-1 flowers with wild-type or ga-1 pollen, respectively. In ga-1/ga-1 seed dehydration started about 1 week earlier than in Ga-1/ga-1 seeds. Ultimate fresh and dry weights of mature Ga-1/ga-1 seeds were higher than those of ga-1/ga-1 seeds and showed negative correlations with the total number of seeds per fruit. Total content and composition of seed proteins were not influenced by the GA-deficiency. Germination of the mature seeds depended on embryonal GA synthesis and was not influenced by maternal GA production. Final fresh weight of the ga-1/ga-1 fruits was positively correlated with the number of seeds per fruit. In these fruits, the minimum number of seeds for growth above the parthenocarpic level was about 10 or 35 in the presence of Ga-1/ga-1 or ga-1/ga-1 seeds, respectively. Fruits containing GA-producing seeds reached a higher fresh weight than those containing GA-deficient seeds, and their ripening was delayed by one week. It is concluded that gibberellin is indispensable for the development of fertile flowers and for seed germination, but only promoting in later stages of fruit and seed development.     

Distribution of gibberellins and expressional analysis of GA 20-oxidase genes of morning glory during fruit maturation

Gibberellins A1/3 (GA1/3) and GA20 appeared earlier in surrounding tissues (pericarps/carpel/placenta) than in developing seeds of morning glory. The content of GA1/3 became higher in seeds than in the surrounding tissues at 9 days after anthesis (DAA), while that of GA20 stayed lower in seeds even at 12 DAA, suggesting the possibility that GA20 was translocated into seeds from the surrounding tissues and converted to GA1/3. The site of biosynthesis of GA20 in the fruits was determined by RNA-blotting and in situ hybridization of GA 20-oxidase genes (InGA20ox1, InGA20ox2). InGA20ox1 was not expressed in the surrounding tissues but in seeds, while no signal due to InGA20ox2 was detected in neither tissue. The expression of InGA20ox1 started in the seed coat near the hilum and spread in the seed coat like those of GA 3-oxidase and GA-inducible alpha-amylase genes. These observations suggest that GA biosynthesis is tissue-specifically and time-dependently regulated in the fruit of morning glory.     

Phytochrome regulation and differential expression of gibberellin 3beta-hydroxylase genes in germinating Arabidopsis seeds.

Despite extensive studies on the roles of phytochrome in photostimulated seed germination, the mechanisms downstream of the photoreceptor that promote germination are largely unknown. Previous studies have indicated that light-induced germination of Arabidopsis seeds is mediated by the hormone gibberellin (GA). Using RNA gel blot analyses, we studied the regulation of two Arabidopsis genes, GA4 and GA4H (for GA4 homolog), both of which encode GA 3beta-hydroxylases that catalyze the final biosynthetic step to produce bioactive GAs. The newly isolated GA4H gene was expressed predominantly during seed germination. We show that expression of both GA4 and GA4H genes in imbibed seeds was induced within 1 hr after a brief red (R) light treatment. In the phytochrome B-deficient phyB-1 mutant, GA4H expression was not induced by R light, but GA4 expression still was, indicating that R light-induced GA4 and GA4H expression is mediated by different phytochromes. In contrast to the GA4 gene, the GA4H gene was not regulated by the feedback inhibition mechanism in germinating seeds. Our data demonstrate that expression of GA 3beta-hydroxylase genes is elevated by R light, which may result in an increase in biosynthesis of active GAs to promote seed germination. Furthermore, our results suggest that each GA 3beta-hydroxylase gene plays a unique physiological role during light-induced seed germination.     

Comprehensive hormone profiling in developing Arabidopsis seeds: examination of the site of ABA biosynthesis, ABA transport and hormone interactions

ABA plays important roles in many aspects of seed development, including accumulation of storage compounds, acquisition of desiccation tolerance, induction of seed dormancy and suppression of precocious germination. Quantification of ABA in the F(1) and F(2) populations originated from crosses between the wild type and an ABA-deficient mutant aba2-2 demonstrated that ABA was synthesized in both maternal and zygotic tissues during seed development. In the absence of zygotic ABA, ABA synthesized in maternal tissues was translocated into the embryos and partially induced seed dormancy. We also analyzed the levels of ABA metabolites, gibberellins, IAA, cytokinins, jasmonates and salicylic acid (SA) in the developing seeds of the wild type and aba2-2. ABA metabolites accumulated differentially in the silique and seed tissues during development. Endogenous levels of SA were elevated in aba2-2 in the later developmental stages, whereas that of IAA was reduced compared with the wild type. These data suggest that ABA metabolism depends on developmental stages and tissues, and that ABA interacts with other hormones to regulate seed developmental processes.     

Expression of the le Mutation in Young Ovaries of Pisum sativum and Its Effect on Fruit Development

The effect of the le mutation on the growth and gibberellin (GA) content of developing fruits was investigated using the near-isogenic lines of Pisum sativum L. 205+ (LeLe) and 205- (lele). Although stem elongation is known to be reduced in 205- plants by approximately 65%, the growth of pods and seeds was unaffected by the le mutation. GA1, GA3, and GA20 stimulated parthenocarpic development of unpollinated ovaries on both 205+ and 205- plants. GA20 was less active on 205- ovaries than on 205+, whereas GA1 had similar, high activity in both lines. The activity of GA3 was even higher than that of GA1 in both lines. Decapitation of 205+ plants induced parthenocarpic development of unpollinated ovaries, but this treatment was much less effective on 205- plants. The contents of GA1 and GA8 in entire ovaries 6 d after anthesis, as well as in the pod and fertilized ovules, were substantially lower in 205- than in 205+ plants, whereas the reverse was true for the levels of GA20 and GA29. These results suggest that 3[beta]-hydroxylation of GA20 to GA1 is reduced in ovaries as well as in vegetative tissues. Thus, the le mutation appears to be expressed in young reproductive organs of the 205- line, even though it does not affect the fruit phenotype. Because the content of GA3 in the ovary was similar in the two lines, one explanation for the normal fruit size in the 205- line is that GA3 is the native regulator of pod growth. Alternatively, sufficient GA1 may still be produced in 205- fruits to maintain normal pod growth.     

Tissue-specific organic acid metabolism in reproductive and non-reproductive parts of the fig fruit is partially induced by pollination

Organic acids are important components of overall fruit quality through flavor, taste, nutritional and medicinal values. Pollinated fig (Ficus carica L.) fruit quality is enhanced by increased acidity. We quantified the major organic acids and characterized the expression pattern of organic acid metabolic pathway-related genes in the reproductive part – inflorescence and non-reproductive part – receptacle of parthenocarpic and pollinated fig fruit during ripening. Essentially, pollinated fruit contains seeds in the inflorescence, as opposed to no seeds in the parthenocarpic inflorescence. The major organic acids – citrate and malate – were found in relatively high quantities in the inflorescence compared to the receptacle of both parthenocarpic and pollinated fig fruit. Notably, pollination increased citric acid content significantly in both inflorescence and receptacle. Genes related to the phosphoenolpyruvate carboxylase (PEPC) cycle, tricarboxylic acid cycle, citrate catabolism and glyoxylate cycle were identified in fig fruit. Expression levels of most of these genes were higher in inflorescences than in receptacles. In particular, FcPEPC and FcFUM (encoding fumarase) had significantly higher expression in the inflorescence of pollinated fruit. Most importantly, expression of the glyoxylate cycle genes FcMLS and FcICL (encoding malate synthase and isocitrate lyase, respectively) was induced to strikingly high levels in the inflorescence by pollination, and their expression level was highly positively correlated with the contents of all organic acids. Therefore, the glyoxylate cycle may be responsible for altering the accumulation of organic acids to upgrade the fruit taste during ripening, especially in the pollinated, seeded inflorescence.     

Immunohistochemistry of active gibberellins and gibberellin-inducible alpha-amylase in developing seeds of morning glory

Gibberellins (GAs) in developing seeds of morning glory (Pharbitis nil) were quantified and localized by immunostaining. The starch grains began to be digested after the GA contents had increased and reached a plateau. Immunohistochemical staining with the antigibberellin A(1)-methyl ester-antiserum, which has high affinity to biologically active GAs, showed that GA(1) and/or GA(3) were localized around starch grains in the integument of developing young seeds, suggesting the participation of GA-inducible alpha-amylase in this digestion. We isolated an alpha-amylase cDNA (PnAmy1) that was expressed in the immature seeds, and using an antibody raised against recombinant protein, it was shown that PnAmy1 was expressed in the immature seeds. GA responsiveness of PnAmy1 was shown by treating the young fruits 9 d after anthesis with GA(3). RNA-blot and immunoblot analyses showed that PnAmy1 emerged soon after the rapid increase of GA(1/3). An immunohistochemical analysis of PnAmy1 showed that it, like the seed GA(1/3), was also localized around starch grains in the integument of developing young seeds. The localization of GA(1/3) in the integument coincident with the expression of PnAmy1 suggests that both function as part of a process to release sugars for translocation or for the further development of the seeds.