Self‐pollination and parthenocarpic ability in developing ovaries of self‐incompatible Clementine mandarins (Citrus clementina)

This study aimed to determine if self‐pollination is needed to trigger facultative parthenocarpy in self‐incompatible Clementine mandarins (Citrus clementina Hort. ex Tan.). ‘Marisol’ and ‘Clemenules’ mandarins were selected, and self‐pollinated and un‐pollinated flowers from both cultivars were used for comparison. These mandarins are always seedless after self‐pollination and show high and low ability to develop substantial parthenocarpic fruits, respectively. The time‐course for pollen grain germination, tube growth and ovule abortion was analyzed as well as that for carbohydrates, active gibberellins (GA₁ and GA₄), auxin (IAA) and abscisic acid (ABA) content in the ovary. ‘Clemenules’ showed higher pollen grain germination, but pollen tube development was arrested in the upper style 9 days after pollination in both cultivars. Self‐pollination did not stimulate parthenocarpy, whereas both un‐pollinated and self‐pollinated ovaries set fruit regardless of the cultivar. On the other hand, ‘Marisol’ un‐pollinated flowers showed greater parthenocarpic ovary growth than ‘Clemenules’ un‐pollinated flowers, i.e. higher ovule abortion rate (+21%), higher fruit set (+44%) and higher fruit weight (+50%). Further, the greater parthenocarpic ability of ‘Marisol’ paralleled higher levels of GA₁ in the ovary (+34% at anthesis). ‘Marisol’ ovary also showed higher hexoses and starch mobilization, but lower ABA levels (?64% at anthesis). Self‐pollination did not modify carbohydrates or GA content in the ovary compared to un‐pollination. Results indicate that parthenocarpy in the Clementine mandarin is pollination‐independent with its ability to set depending on the ovary hormone levels. These findings suggest that parthenocarpy in fertile self‐incompatible mandarins is constitutively regulated.     

Hormonal changes associated with fruit set and development in mandarins differing in their parthenocarpic ability

Satsuma [Citrus unshiu (Mak) Marc.] and Clementine [Citrus reticulata (Hort.) Ex. Tanaka, cv. Oroval] are two related species of seedless mandarins which differ in their tendency to set parthenocarpic fruits. Satsuma fruits naturally set parthenocarpically whereas Clementine mandarins show very low ability to set fruit in the absence of cross-pollination. The endogenous levels of gibberellins (GAs) and free and conjugated indole-acetic acid (IAA) and abscisic acid (ABA) throughout early stages of fruit development were investigated in seedless cultivars of both species. Analyses performed by full-scan combined gas chromatography-mass spectrometry (GC-MS) of extracts from ovaries at anthesis demonstrated the presence of GA₁₉, GA₂₀, GA₂₉, GA₁, GA₈, GA₃ and iso-GA₃ in Satsuma mandarin, whereas only GA₂₉, GA₃ and trace levels of GA₈ were detected in Clementine. At this developmental stage GA-like substances, as estimated by bioassay, reached their highest levels in Satsuma, while Clementine mandarins contained relatively lower levels. In both species the highest levels of free IAA were found at petal-fall stage at which time free ABA levels also peaked. Developing fruits of Clementine had higher amounts of both free IAA and ABA. In Satsuma, levels of conjugated IAA remained low throughout reproductive development whereas in Clementine they increased as the free form declined. In contrast, conjugated ABA was at low levels in Clementine but reached higher concentrations in Satsuma. These results suggest that in these mandarins the potential for setting parthenocarpic fruits is mainly influenced by the hormonal status of the fruit during the later stages of cell division and early stages of cell enlargement. Thus, the condition of low ability to set parthenocarpic fruits appears to be associated with lower levels of active GAs, lower capability to catabolize ABA to conjugated ABA and higher ability to conjugate IAA during this period.     

Abscisic acid increases in non-growing and paclobutrazol-treated fruits of seedless mandarins

Clementine ( Citrus reticulata [Hort.] Ex. Tanaka cv. Oroval) is a self-incompatible mandarin with a slow rate of fruit growth and high percentage of fruit abscission. Seedless Satsuma ( Citrus unshiu [Mak] Marc. cv. Clausellina) shows natural parthenocarpy and higher fruit set. Application of 25 μg fruit⁻¹ of paclobutrazol (PP333), an inhibitor of gibberellin biosynthesis, reduced the rate of growth and accelerated fruit abscission in both varieties. In contrast, gibberellin A₃ (GA₃) stimulated fruit growth only in the self-incompatible mandarin. Clementine fruits, in the absence of pollination, showed an approximately 2-fold transient increase in the free abscisic acid (ABA) content shortly after petal fall. In Satsuma, a very small accumulation of ABA was detected. Paclobutrazol treatment induced a 3-fold increase in ABA in Satsuma fruits but did not substantially affect the pattern of ABA accumulation in Clementine. In this variety, GA₃ suppressed the ABA increase observed in untreated fruits. These effects were observed 24 h after treatment. However, in Satsuma fruits, the effect of GA₃ on the ABA content was negligible. In addition, a comparative analysis of growing and non-growing fruits of Clementine showed that ABA, on a per unit weight basis, was always higher in the non-growing fruits. Treatment with 85 μM fluridone, an inhibitor of carotenoid biosynthesis and thus indirectly of ABA, delayed fruit abscission in Clementine, but also decreased fruit growth. Collectively, these observations indicate a relationship between high ABA content and a reduced rate of fruit growth and an acceleration of fruit abscission.     

Gibberellins inCitrus sinensis: A comparison between seeded and seedless varieties

The gibberellin (GA) content of the reproductive organs ofCitrus sinensis (L.) Osb., cv. Bianca Comuna and the seedless variety, Salustiana, were examined by combined gas chromatography-mass spectrometry (GC/MS) at different stages of development. Gibberellins A₁, A₂₀, and A₂₉ were identified in the reproductive buds of both cultivars 21 days prior to anthesis and in fruits 35 days after anthesis by comparison of their mass spectra and Kovats retention indices with those of standards. In addition, three uncharacterized isomers of GA₁ were detected, one in buds and two in fruits. The presence of GA₄ in both tissues, and of GA₈ in the reproductive buds, was indicated by the occurrence of characteristic ions at the expected retention times, although their spectra were too weak for full identification. Vegetative shoots of cv. Salustiana contained gibberellins A₁, A₁₉, A₂₀, and A₂₉, and the unidentified isomer of GA₁ present in reproductive buds. The presence of trace amounts of gibberellins A₈ and A₁₇ was also indicated. Although the two varieties did not differ qualitatively in the GAs present during flower and fruit development, the seedless variety contained slightly greater amounts. The concentrations of gibberellins A₁, A₄, and A₂₀ were determined by gas chromatography-selected ion monitoring (GC/SIM) throughout ovary development and early fruit growth. In both varieties, the maximum GA₁ concentration occurred at anthesis. Highest concentrations of gibberellins A₂₀ and A₄ were found in fruit 35 days after anthesis, although the GA₁ concentration at this stage remained low.     

Gibberellins inCitrus sinensis: A comparison between seeded and seedless varieties

The gibberellin (GA) content of the reproductive organs ofCitrus sinensis (L.) Osb., cv. Bianca Comuna and the seedless variety, Salustiana, were examined by combined gas chromatography-mass spectrometry (GC/MS) at different stages of development. Gibberellins A₁, A₂₀, and A₂₉ were identified in the reproductive buds of both cultivars 21 days prior to anthesis and in fruits 35 days after anthesis by comparison of their mass spectra and Kovats retention indices with those of standards. In addition, three uncharacterized isomers of GA₁ were detected, one in buds and two in fruits. The presence of GA₄ in both tissues, and of GA₈ in the reproductive buds, was indicated by the occurrence of characteristic ions at the expected retention times, although their spectra were too weak for full identification. Vegetative shoots of cv. Salustiana contained gibberellins A₁, A₁₉, A₂₀, and A₂₉, and the unidentified isomer of GA₁ present in reproductive buds. The presence of trace amounts of gibberellins A₈ and A₁₇ was also indicated. Although the two varieties did not differ qualitatively in the GAs present during flower and fruit development, the seedless variety contained slightly greater amounts. The concentrations of gibberellins A₁, A₄, and A₂₀ were determined by gas chromatography-selected ion monitoring (GC/SIM) throughout ovary development and early fruit growth. In both varieties, the maximum GA₁ concentration occurred at anthesis. Highest concentrations of gibberellins A₂₀ and A₄ were found in fruit 35 days after anthesis, although the GA₁ concentration at this stage remained low.     

The source of gibberellins in the parthenocarpic development of ovaries on topped pea plants

The role and source of gibberellins (GAs) involved in the development of parthenocarpic fruits of Pisum sativum L. has been investigated. Gibberellins applied to the leaf adjacent to an emasculated ovary induced parthenocarpic fruit development on intact plants. The application of gibberellic acid (GA₃) had to be done within 1 d of anthesis to be fully effective and the response was concentration-dependent. Gibberellin A₁ and GA₃ worked equally well and GA₂₀ was less efficient. [³H]Gibberellin A₁ applied to the leaf accumulated in the ovary and the accumulation was related to the growth response. These experiments show that GA applied to the leaf in high enough concentration is translocated to the ovary. Emasculated ovaries on decapitated pea plants develop without application of growth hormones. When [³H] GA₁ was applied to the leaf adjacent to the ovary a substantial amount of radioactivity accumulated in the growing shoot of intact plants. In decapitated plants, however, this radioactivity was mainly found in the ovary. There it caused growth proportional to the accumulation of CA₁. Application of LAB 150978, an inhibitor of GA biosynthesis, to decapitated plants inhibited parthenocarpic fruit development and this inhibition was counteracted by the application of GA₃ (either to the fruit, or the leaf adjacent to the ovary, or through the lower cut end of the stem). All evidence taken together supports the view that parthenocarpic pea fruit development on topped plants depends on the import of gibberellins or their precursors, probably from the vegetative aerial parts of the plant.Abbreviations FW flesh weight - GA_n gibberellin A_n - HPLC high-performance liquid chromatography     

Gibberellins in shoots and developing capsules of Populus species

Extracts of stems of growing shoots of Populus deltoides and P. trichocarpa, and developing capsules of P. deltoides were analysed for gibberellins (GAs) by gas chromatography-mass spectrometry. The following known GAs were identified by comparison of their Kovats retention indices (KRIs) and mass spectra with those of standards: GA1, GA8, GA9, GA19, GA20, 16 beta,17-dihydro-17-hydroxy GA20, GA23, GA28, GA29, GA34, GA44, and GA97. Several of these have not been previously reported from Populus. In addition, two new GAs were identified as 12 beta-hydroxy GA53 (GA127) and 16 beta,17-dihydro-17-hydroxy GA53 and their structures were confirmed by partial synthesis. Evidence was found of 16,17-dihydro-16,17-dihydroxy GA9, 16,17-dihydro-16,17-dihydroxy GA12, 12-hydroxy GA14, and GA34-catabolite by comparison of mass spectra and KRIs with published data. Several putative GAs (hydroxy- and dihydroxy-GA12-like) were also found. The catabolites of active GAs or of key precursors, hydroxylated at C-2 in stems and either C-2, C-12, C-17, or C-16,17 in capsules, were the major proportion of the GAs.     

Ecdysteroids in developing ovaries and eggs of the tobacco hornworm

Ecdysteroids of ovaries and newly-laid eggs (0- to 1-hour-old) of the tobacco hornworm are present mainly as conjugates (greater than 95%). Newly-laid eggs contain ecdysteroid conjugates equivalent to 21 micrograms of 26-hydroxyecdysone and 0.73 micrograms of ecdysone per gram of eggs. These levels are similar in ovaries of 93-hour-old adult females. In 1- to 18-hour-old eggs more than 63% of the ecdysteroids exist in the free form and the proportion is similar in 48- to 64-hour-old eggs. The ratio of 26-hydroxyecdysone to ecdysone in the conjugated form remains constant during oocyte maturation and embryogenesis. Though 26-hydroxyecdysone is without molting hormone activity in the house fly assay, the exceptionally high concentration of 26-hydroxyecdysone conjugate(s) in ovaries and newly-laid eggs, together with the fact that it is being released during embryogenesis, indicate some physiological role for 26-hydroxyecdysone.     

Endogenous free polyamines and their role in fruit set of low and high parthenocarpic ability citrus cultivars

Endogenous free polyamines (PAs), putrescine, spermidine and spermine, from developing fruitlets of Citrus species (Citrus unshiu Marc. and Citrus clementina Hort ex Tanaka) which differ in their parthenocarpic ability, and from uniflowered leafy and leafless inflorescences differing in their ability to set, have been determined by dansylation and separation of dansyl derivatives by HPLC. No significant differences in PAs content were observed between species or between leafy and leafless inflorescences which, nevertheless, significantly differed in fruit set. However, significant differences in their content were found in developing fruitlets, depending on the preceding flowering intensity of the tree and on the fruitlet load. These results suggest that, in Citrus, PAs may act as a nitrogen source rather than a regulator of fruit set.     

Masculinizing effect of testes on developing rat ovaries in organ culture

Masculinizing effect of testes on developing rat ovaries was shown in vitro by culturing testes from 17.5-day-old fetuses in contact with female genital tracts from 14.5-day-old rat fetuses. The testes induced the differentiation of epithelial cells staining for cytokeratin in the ovarian blastema. These cells formed seminiferous cord-like structures delineated by a basement membrane, in a way that resembles early stages of testicular organogenesis. In addition to the morphological masculinization, functional masculinization was obtained since the ovaries produced the anti-Müllerian hormone as shown by bioassay and immunohistochemical procedures. Across a distance, testes from 17.5-day-old fetuses failed to induce masculinization. These results suggest that testes from 17.5-day-old fetuses produce a locally diffusible factor interfering with the development and the differentiation of the fetal ovaries. The possibility that the anti-Müllerian hormone secreted by the testes may be the factor involved is discussed comparing these results with those obtained with testes from different stages and with bibliographic data.     

Imaging and quantifying carbohydrate transport to the developing ovaries of maize

BACKGROUND AND AIMS: Shade or inadequate water can inhibit photosynthesis and limit the development of maize (Zea mays) ovaries around the time of pollination, potentially reducing the number of kernels at harvest. This study investigated whether the decreased photosynthesis diminished only the sugar supply or also altered the transport path to the ovaries. METHODS: Photosynthesis and water potentials (Psiw) were measured in the leaves while dry matter delivery was monitored in the ovaries. Ovary glucose, starch and acid invertase activities were measured in situ. Stems were fed xylem-mobile safranin or phloem-mobile carboxyfluorescein (CF), and the dye transport to the ovaries was determined. KEY RESULTS: Under normal conditions, the ovaries gained in dry mass, and starch accumulated in the pedicel and ovary wall. Glucose accumulated in the pedicel, apparently in the apoplast where insoluble (cell-wall-bound) acid invertase acted on the arriving sucrose. A glucose gradient developed from pedicel to nucellus. Safranin moved in the xylem and did not reach the ovary, but CF moved in the phloem and arrived at the ovary. CF also spread into the pedicel but unlike glucose it did not enter the nucellus. Low Psiw or shade decreased leaf photosynthesis, ovary dry mass accumulation, invertase activities, pedicel glucose, starch accumulation and CF delivery. Removal of these treatments reversed the effects. CONCLUSIONS: The success of CF in tracing the general path and rate of carbohydrate transport gave visual evidence that phloem transport to the ovary decreased at low Psiw or in the shade but otherwise remained functional. The decreases indicated that losses in carbohydrate delivery are central features of failed ovary development under these conditions. The selectivity of transport into the nucellus resembled the situation later when embryo and endosperm are present and selective uptake occurs from the apoplast.     

Neonatal steroid exposure increases in vitro aromatization ability of peripubertal hamster ovaries

Neonatal female hamsters were exposed to doses of testosterone propionate or estradiol benzoate that would lead to behavioral masculinization and defeminization at adulthood. At Days 20, 30, or 40 of life, ovaries were removed and incubated in Kreb's Ringer bicarbonate for 4 h with or without the addition of 1 X 10(-9) M testosterone as a substrate. Incubation medium was assayed by radioimmunoassay (RIA) for the accumulation of estradiol. Ovaries from steroid-exposed animals aromatized testosterone to estradiol at a greatly increased rate compared to ovaries from oil-injected control animals. Serum from treated animals contained androgen, which could act as substrate for aromatization in vivo. Neonatally estrogenized females exhibited elevated circulating estradiol levels. Increased aromatizing ability could be due to early antral follicle formation as a result of elevated luteinizing hormone (LH) and/or follicle-stimulating hormone (FSH). Implications of increased aromatization ability of ovaries in the process of behavioral sexual differentiation are discussed.     

Pollen-pistil interactions and early fruiting in parthenocarpic citrus

Background and Aims

An intense pollen–pistil interaction precedes fertilization. This interaction is of particular relevance in agronomically important species where seeds or fruits are the edible part. Over time some agronomically species have been selected for the ability to produce fruit without seeds. While this phenomenon is critical for commercial production in some species, very little is known about the events behind the production of seedless fruit. In this work, the relationship between pollen–pistil interaction and the onset of fruiting was investigated in citrus mandarin.

Methods

Pistils were sequentially examined in hand-pollinated flowers paying attention to pollen-tube behaviour, and to cytochemical changes along the pollen-tube pathway. To evaluate which of these changes were induced by pollination/fertilization and which were developmentally regulated, pollinated and unpollinated pistils were compared. Also the onset of fruiting was timed and changes in the ovary examined.

Key Results

Conspicuous changes occurred in the pistil along the pollen-tube pathway, which took place in a basipetal way encompassing the timing of pollen-tube growth. However, these changes appear to be developmentally regulated as they happened in the same way and at the same time in unpollinated flowers. Moreover, the onset of fruiting occurred prior to fertilization and the very same changes could be observed in unpollinated flowers.

Conclusions

Pollen–pistil interaction in citrus showed similarities with unrelated species and families belonging to other taxa. The uncoupling of the reproductive and fruiting processes accounts for the parthenocarpic ability of unpollinated flowers to produce fruit in citrus. However, the maintenance of a functional reproductive process reflects the potential to produce seeded fruits, providing a basis for the understanding of the production of seeded or unseeded fruits and further understanding of the process of parthenocarpy in other species.     

Expression of connexin 43 mRNA and protein in developing follicles of prepubertal porcine ovaries

A major form of cell-cell communication is mediated by gap junctions, aggregations of intercellular channels composed of connexins (Cxs), which are responsible for exchange of low molecular weight (<1200 Da) cytosolic materials. These channels are a growing family of related proteins. This study was designed to determine the ontogeny of connexin 43 (Cx43) during early stages of follicular development in prepubertal porcine ovaries. A partial-length (412 base) cDNA clone was obtained from mature porcine ovaries and determined to have 98% identity with published porcine Cx43. Northern blot analysis demonstrated a 4.3-kb mRNA in total RNA isolated from prepubertal and adult porcine ovaries. In-situ hybridization revealed that Cx43 mRNA was detectable in granulosa cells of primary follicles but undetectable in dormant primordial follicles. The intensity of the signal increased with follicular growth and was greatest in the large antral follicles. Immunohistochemical evaluation indicated that Cx43 protein expression correlated with the presence of Cx43 mRNA. These results indicate that substantial amounts of Cx43 are first expressed in granulosa cells following activation of follicular development and that this expression increases throughout follicular growth and maturation. These findings suggest an association between the enhancement of intercellular gap-junctional communication and onset of follicular growth.     

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.     

l-Ascorbic acid metabolism in parthenocarpic and seeded cherry tomatoes

The auxin treatment in tomato plants during anthesis has been extensively used for setting fruits in adverse climatic conditions (e.g., low temperatures and inadequate light), which is well known that reduces pollen availability and fertility. Since auxin application may affect fruit composition and quality, we examined l-ascorbic acid metabolism in seeded fruit (set by natural pollination) and parthenocarpic fruit (set by auxin) in cherry tomato cv. Conchita. Specifically, we studied the oxidized and total ascorbic acid contents, the expression of all characterized genes of l-ascorbic acid metabolism, the activity of ascorbate peroxidase and dehydroascorbate reductase and the immunolocalization of ascorbate peroxidase. Differences were detected between seeded and parthenocarpic fruits, in the expression of some of the genes of ascorbic acid metabolism. However, strong presence of l-ascorbic acid peroxidase protein was detected on the developing seeds. Our data indicate that induced parthenocarpy in auxin treated plants has a significant influence in ascorbic acid metabolism comparing to seeded tomato fruits.     

Differential regulation of a fruit-specific 62 kDa protein in developing parthenocarpic (pat-2/pat-2) and seeded tomato fruits

The denatured protein profiles of developing tomato ( Lycopersicon esculentum Mill.) fruits, from the anthesis stage up to fruits at 30% of their final diameter, were examined in a pai-2l pat-2 parthenocarpic line and in its near isogenic non-partheno-carpic line. At anthesis no differences were detected between the protein patterns of ovaries developed on parthenocarpic and non-parthenocarpic plants. In subsequent stages the seeded and seedless fruits differed in the pattern of manifestation of several abundant proteins, none of which seem to be included in seeds The most prominent difference was found in an insoluble protein of 62 kDa; in developing seeded fruits of either the parthenocarpic or the non-parthenocarpic line, its rate of decline was much faster than in seedless fruits. In seeded fruits larger than 4-6 mm in diameter it was scarcely detected, whereas in parthenocarpic seedless 8–10 mm fruits it was still abundant. This protein is fruit specific; it is also enhanced in chemically (n-n-tolyl phthalamic acid) – induced parthenocarpic fruits of the non-parthenocarpic line. The prolonged manifestation in the parthenocarpic fruits results from de novo synthesis of this protein. There are indications that it is not a stress-related protein. This is the first demonstration of an association between the pattern of modulation of a protein and the phenotypic expression of genetically controlled parthenocarpy.