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.     

Gibberellins in developing fruits of Pisum sativum cv. Alaska: Studies on their role in pod growth and seed development

Gibberellins A₁, A₈, A₂₀ and A₂₉ were identified by capillary gas chromatography-mass spectrometry in the pods and seeds from 5-d-old pollinated ovaries of pea (Pisum sativum cv. Alaska). These gibberellins were also identified in 4-d-old non-developing, parthenocarpic and pollinated ovaries. The level of gibberellin A₁ within these ovary types was correlated with pod size. Gibberellin A₁, applied to emasculated ovaries cultured in vitro, was three to five times more active than gibberellin A₂₀. Using pollinated ovary explants cultured in vitro, the effects of inhibitors of gibberellin biosynthesis on pod growth and seed development were examined. The inhibitors retarded pod growth during the first 7 d after anthesis, and this inhibition was reversed by simultaneous application of gibberellin A₃. In contrast, the inhibitors, when supplied to 4-d-old pollinated ovaries for 16 d, had little effect on seed fresh weight although they reduced the levels of endogenous gibberellins A₂₀ and A₂₉ in the enlarging seeds to almost zero. Paclobutrazol, which was one of the inhibitors used, is xylem-mobile and it efficiently reduced the level of seed gibberellins without being taken up into the seed. In intact fruits the pod may therefore be a source of precursors for gibberellin biosynthesis in the seed. Overall, the results indicate that gibberellin A₁, present in parthenocarpic and pollinated fruits early in development, regulates pod growth. In contrast the high levels of gibberellins A₂₀ and A₂₉, which accumulate during seed enlargement, appear to be unnecessary for normal seed development or for subsequent germination.Abbreviations GA_(a) gibberellin A_n - GC-MS combined gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - PFK perfluorokerosene - PVP polyvinylpyrrolidone     

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.     

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.     

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.     

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.     

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.     

Multiple paternal genotypes in embryogenic tissue derived from individual immature loblolly pine seeds

Pine embryogenic tissue derived from immature zygotic embryos may consist of multiple genotypes due to simple polyembryony. To test this hypothesis, megagametophytes with intact zygotic embryos were cultured from immature loblolly pine (Pinus taeda L.) seeds of clone WV42 control pollinated with a 1:1:1 pollen mix of clones WV44, WV47, and WV48. Each pollen parent contained a marker allele at one or more of the following loci: aconitase, malic dehydrogenase, 6-phosphogluconate dehydrogenase, and shikimate dehydrogenase, allowing determination of the paternal parent. After two to four weeks in culture, embryogenic tissue derived from zygotic embryos extruded from megagametophytes was separated into individual embryos and sectors of embryogenic tissue. The paternal genotype of each resulting cell line was determined by starch gel electrophoresis. Three of thirty-six explants produced multiple cell lines with genotypic differences among the cell lines within each explant. Our results unequivocally show that it is possible to initiate embryogenic tissue from more than one zygotic embryo of a loblolly pine seed and that the resulting cell lines may be genetically different.Abbreviations ACO aconitase - MDH malic dehydrogenase - SKDH shikimate dehydrogenase - 6PGD 6-phosphogluconate dehydrogenase     

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.     

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.     

Parthenocarpy and seed predation by insects in Bursera morelensis

Background and Aims

While parthenocarpy (meaning the production of fruits without seeds) may limit fecundity in many plants, its function is not clear; it has been proposed, however, that it might be associated with a strategy to avoid seed predation. Bursera morelensis is a dioecious endemic plant that produces fruits with and without seeds, and its fruits are parasitized by insects. Its reproductive system is not well described and no published evidence of parthenocarpy exists for the species. The purpose of this work was to describe the breeding system of B. morelensis and its relationship to seed predation by insects.

Methods

The breeding system was described using pollination experiments, verifying the presence of parthenocarpic fruits and apomictic seeds. Reproductive structures from flower buds to mature fruits were quantified. For fruits, an anatomical and histological characterization was made. The number of fruits in which seeds had been predated by insects was correlated with parthenocarpic fruit production.

Key Results

The major abortion of reproductive structures occurred during fruit set. The results discard the formation of apomictic seeds. Flowers that were not pollinated formed parthenocarpic fruits and these could be distinguished during early developmental stages. In parthenocarpic fruits in the first stages of development, an unusual spread of internal walls of the ovary occurred invading the locule and preventing ovule development. Unlike fruits with seeds, parthenocarpic fruits do not have calcium oxalate crystals in the ovary wall. Both fruit types can be separated in the field at fruit maturity by the presence of dehiscence, complete in seeded and partial in parthenocarpic fruits. Trees with more parthenocarpic fruits had more parasitized fruits.

Conclusions

This is the first time the anatomy of parthenocarpic fruits in Burseraceae has been described. Parthenocarpic fruits in B. morelensis might function as a deceit strategy for insect seed predators as they are unprotected both chemically and mechanically by the absence of calcium oxalate crystals.Key words: Parthenocarpy, Bursera morelensis, predation, seeds, insects, breeding system, calcium oxalate crystals     

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.     

Cytoplasmic HSP70 homologues of pea: differential expression in vegetative and embryonic organs

Eukaryotes express several cytoplasmic HSP70 genes, and their encoded proteins participate in diverse cellular processes. Three cDNAs encoding highly expressed cytoplasmic HSP70 homologues from Pisum sativum were cloned and characterized. They were designated PsHSP71.2, PsHSC71.0, and PsHSP70b. These HSP70 genes have different expression profiles in leaves: PsHSP71.2 is observed only in response to heat stress, PsHSC71.0 is present constitutively, and PsHSP70b is weakly constitutively expressed, but induced strongly in response to heat stress. In addition to being heat induced, the PsHSP71.2 mRNA is also expressed in zygotic, but not maternal organs of developing pea seeds, while PsHSC71.0 and PsHSP70b mRNAs are present in maternal and zygotic organs throughout seed development. Immunoblot analysis of parallel protein samples detects a 70 kDa polypeptide in all samples, and a 72 kDa polypeptide that corresponds to the PsHSP71.2 gene product is observed in cotyledons beginning at mid-maturation and in axes beginning between late maturation and desiccation. This polypeptide is not detected in the seed coat. The 72 kDa polypeptide remains abundant in both cotyledons and axes through germination, but declines substantially between 48 and 72 h after the onset of imbibition. Differential control of HSP70 expression during heat stress, seed maturation, and germination is consistent with the hypothesis that there are functional distinctions between cytoplasmic HSP70s.     

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.