Cytokinin‐dependent specification of the functional megaspore in the Arabidopsis female gametophyte

The life cycle of higher plants alternates between the diploid sporophytic and the haploid gametophytic phases. In angiosperms, male and female gametophytes develop within the sporophyte. During female gametophyte (FG) development, a single archesporial cell enlarges and differentiates into a megaspore mother cell, which then undergoes meiosis to give rise to four megaspores. In most species of higher plants, including Arabidopsis thaliana, the megaspore closest to the chalaza develops into the functional megaspore (FM), and the remaining three megaspores degenerate. Here, we examined the role of cytokinin signaling in FG development. We characterized the FG phenotype in three triple mutants harboring non‐overlapping T–DNA insertions in cytokinin AHK receptors. We demonstrate that even the strongest mutant is not a complete null for the cytokinin receptors. Only the strongest mutant displayed a near fully penetrant disruption of FG development, and the weakest triple ahk mutant had only a modest FG phenotype. This suggests that cytokinin signaling is essential for FG development, but that only a low threshold of signaling activity is required for this function. Furthermore, we demonstrate that there is elevated cytokinin signaling localized in the chalaza of the ovule, which is enhanced by the asymmetric localization of cytokinin biosynthetic machinery and receptors. We show that an FM‐specific marker is absent in the multiple ahk ovules, suggesting that disruption of cytokinin signaling elements in Arabidopsis blocks the FM specification. Together, this study reveals a chalazal‐localized sporophytic cytokinin signal that plays an important role in FM specification in FG development.     

罗汉果大孢子发生及雌配子体发育与花部形态、胚珠变化的关系

为弄清罗汉果(Siraitia grosvenorii)大孢子发生、雌配子体发育过程与花部形态特征、胚珠的关系,运用石蜡切片法对罗汉果子房进行了显微观察。结果表明,罗汉果的胚珠倒生,双珠被,厚珠心,大孢子四分体呈线型排列,合点端一个大孢子分化为功能大孢子,成熟胚囊为蓼型。花蕾形态、胚珠变化与大孢子发生、雌配子体的发育时期具有一定相关性,当子房长度为7.0 mm≤L<9.0 mm,珠心呈椭圆形时,约有45.83%的大孢子母细胞处于减数分裂时期。因此,依据罗汉果花部形态可有效确定大孢子发生与雌配子体发育的时期。     

黄瓜花性别分化与内源多胺的关系

研究了黄瓜雌、雄花几个主要发育时期和性别逆转过程中内源多胺的变化。结果表明 ,雄花在不同发育时期 ,内源腐胺含量均高于雌花 ,腐胺含量的显著升高伴随着花粉粒的形成 ,高腐胺含量是雄花发育的特征。雌花在大孢子母细胞时期以后直到雌花发育成熟 ,其内源尸胺含量均高于雄花 ,高尸胺含量可能有利于雌花的发育。高水平的内源多胺、精胺和亚精胺可能有利于雌花大孢子母细胞的形成。亚精胺和腐胺含量随着大小孢子四分体形成和大孢子核的连续分裂而分别表现下降和上升。雌性系黄瓜经硝酸银诱导雄花处理后 ,茎尖内源亚精胺含量下降 ,腐胺含量上升 ,从而诱导雄花形成 ;雄性系黄瓜经乙烯利诱导雌花处理后 ,茎尖内源亚精胺含量上升 ,腐胺含量下降 ,从而诱导雌花形成     

In vitro flowering of Boronia megastigma Nees. and the effect of 6-benzylaminopurine

Successful flower bud initiation and development was achieved on Boronia megastigma in vitro. The effect of cytokinin on flowering was investigated in environmental conditions that promote flowering as well as under conditions that stimulate vegetative growth (nonfloral promotory conditions). Flower initiation and differentiation was enhanced by cytokinin; however, many flower buds reverted when the media contained high levels of cytokinin. Anthesis occurred only on media that had no cytokinin added and under floral promotory conditions.     

Petal: a reliable explant for direct bulblet regeneration of endangered wild populations of <Emphasis Type="Italic">Fritillaria imperialis</Emphasis> L.

Wild populations of Fritillaria imperialis L. are facing extinction and need urgent conservation. This paper presents an efficient system for in vitro direct bulblet regeneration of these populations by petal culturing of flower buds. Petals at different developmental stages, green-closed flower bud (before nectar secretion) and red-closed flower bud (beginning of nectar secretion), were used as explants, and the effects of various proportions of cytokinin to auxin on direct bulblet regeneration pathway were evaluated. More explants switched on direct regeneration pathway in combination of auxins (0.6 mg l⁻¹ NAA + 0.4 mg l⁻¹ IAA) with higher level of cytokinin (1 mg l⁻¹ BAP). In contrast, auxins (0.6 mg l⁻¹ NAA + 0.4 mg l⁻¹ IAA) with lower level of cytokinin (0.1 mg l⁻¹ BAP) produced more bulblets per regenerated explant. In green-closed flower bud stage, direct bulblets regenerated from the end of petal where it was connected to the receptacle, while nectar secretion site was the place of bulblet formation in red-closed flower bud stage. In addition, genotype-dependency of direct bulblet regeneration pathway was investigated by using two different wild populations of Fritillaria imperialis. This plant regeneration procedure was applicable to different Fritillaria genotypes and regenerated bulblets were normal.     

Changes in cytokinin activity during flower development in Cosmos sulphureus Cav

Endogenous cytokinin activity was determined in the flowers of Cosmos sulphureus Cav. from bud emergence to full bloom using the soybean callus bioassay. Cytokinin activity was low early in flower development but increased prior to full bloom. In Sephadex LH-20 column chromatography of flower extracts, the cytokinins present co-eluted with zeatin, zeatin riboside and glucoside cytokinin. While the former two predominated prior to full bloom, cytokinin glucoside activity appeared to be at a maximum at full bloom. The possible relevance of these findings is discussed in relation to flower development.     

Changes in Cytokinins before and during Early Flower Bud Differentiation in Lychee (Litchi chinensis Sonn.)

Lychee (Litchi chinensis) has been analyzed for cytokinins in buds before and after flower bud differentiation, using reversephase high performance liquid chromatography in combination with Amaranthus bioassay and gas chromatography-mass spectrometry-selected ion monitoring. Four cytokinins, zeatin, zeatin riboside, N⁶-(δ²-isopentenyl)adenine, and N⁶-(δ⁶-isopentenyl) adenine riboside, were detected in buds. There was an increase of cytokinin activity in the buds during flower bud differentiation. In dormant buds, the endogenous cytokinin content was low, and the buds did not respond to exogenous cytokinin application. Application of kinetin promotes flower bud differentiation significantly after bud dormancy. These results are interpreted as an indication that the increase in endogenous cytokinin levels during flower bud differentiation may be correlative rather than the cause of flower bud initiation.     

Bushiness and cytokinin sensitivity in micropropagated Zantedeschia

Bushiness, the development of short, multiple stems, has been reported in some commercial hybrids of Zantedeschia, particularly the yellow-flowered cultivar Florex Gold derived from tissue culture. This cultivar exhibited a greater sensitivity to 6-benzylaminopurine when compared to four other cultivars using an in vitro root length bioassay, suggesting it may be pre-disposed to bushiness. The carry-over effect of cytokinin from micropropagation on subsequent tuber formation and flower development, in three selections of the cultivar Florex Gold, was also investigated. Cytokinin levels used in commercial Zantedeschia micropropagation protocols did not alter tuber or flower development. However, excessively high levels did affect development, also indicating that cytokinin may influence bushiness. Multi-eyed tubers used to initiate in vitro cultures were shown to have a significant effect on first year tuber formation and subsequent flower weight, compared to few-eyed tubers, although these plants did not necessarily develop bushy symptoms. Plant location within the greenhouse also influenced flowering although these plants did not display multiple stemmed bushy symptoms.     

Studies on male and female meiosis in Indian Allium

Frequency and position of chiasmata at diplotene were studied in pollen mother cells and megaspore mother cells of the same plants of two wild Allium species (A. consanquineum and A. kachrooi) and two cultivated species (a. cepa and A. nigrum), all diploid with 2n=16. Contrary to most reports on sex differences in recombination, chiasma frequencies were higher in male than in female meiosis in all cases. Chiasmata were non-localised except in A. kachrooi megaspore mother cells where they were proximally localised.     

Induction of early flowering in Cymbidium niveo-marginatum Mak in vitro

 Many orchids take several years to flower. We have been able to induce early flowering in the temperate orchid Cymbidium niveo-marginatum Mak in vitro. The combined treatment of cytokinin (6-benzylaminopurine), restricted nitrogen supply with phosphorus enrichment, and root excision (pruning) induced transition of the Cymbidium shoot from a vegetative to a reproductive stage. Nearly 100% of the plants flowered within 90 days only when the combined treatment was applied. When root excision and/or 6-benzylaminopurine were omitted from the combined treatments, flower induction was significantly reduced. The auxin transport inhibitor, 2,3,5-triiodobenzoic acid prevented flowering of Cymbidium in vitro, although auxin (α-naphthaleneacetic acid) itself did not induce flowering. Gibberellic acid markedly delayed flowering in C. niveo-marginatum even when the flower-promoting treatment was applied. Paclobutrazol, an anti-gibberellin agent, totally blocked the inductive effects of either cytokinin or pruning. These observations suggest that concerted actions of auxin, cytokinin, and gibberellin, as well as nutrient concentration and putative promoting/suppressing agents, determine the timing of Cymbidium orchid transition from the vegetative to reproductive stage. Received: 22 July 1998 / Revision revised: 23 November 1998 / Accepted: 2 December 1998     

The role of cytokinin in ovule development in Arabidopsis

The life cycle of higher plants alternates between the haploid gametophyte and diploid sporophyte. The female gametophyte (FG), surrounded by the sporophyte, develops within the ovule and orients along the chalazal/micropylar axis. This polarity is important in cell specification and development for both the ovule and FG. Previously, cytokinin was shown to act in the sporophytic tissue to regulate FG development.^1,2 In the highlighted study,³ we further showed that enriched cytokinin signaling in chalaza, the central domain of the ovule, is required for the specification of the functional megaspore, which usually occurs in the chalazal-most megaspore after meiosis. The restricted cytokinin signaling in the chalaza is achieved by localized cytokinin biosynthesis and perception. Here, we discuss the implications of this and other studies for the understanding of the role of two-component signaling in FG development and the genetic and cellular interactions between gametophytic and sporophytic cells. Further, we show that cytokinin-deficient mutants display distorted cell morphology in the inner integument and elevated mitotic activity in the maternal sporophyte. These results suggest that cytokinin negatively regulates cell proliferation in the sporophytic tissues surrounding the developing FG, consistent with previous results indicating that cytokinin deficiency causes an increase in the number of cells in the embryos and consequently an enlarged seed size.     

STUDIES ON FOSSIL AZOLLA: PRIMITIVE TYPES OF MEGASPORES AND MASSULAE FROM THE CRETACEOUS

Fossil Salviniaceae are described from the Claggett Shale and Judith River Formation, late Cretaceous (Campanian stage) of Montana. A new genus, Parazolla, from the Claggett Shale, has megaspores in which the swimming apparatus is composed of a number of elongate floats attached to the megaspore body and invested by coiled hairs. The floats separate at maturity. Massulae (bearing microspores) have simple hair-like glochidia, many of which are knobbed at their tips. Glochidia tend to resemble the perisporial hairs of the megaspore body. This resemblance provides fossil evidence of the homology of these two hair-like structures among living species of Azolla. In Azolla simplex from the Judith River Formation the megaspore has a single cap-like so-called columellate float. Massulae, which have anchor-shaped glochidia, are associated with these megaspores. A. simplex is the oldest species of Azolla and Parazolla the oldest member of the Salviniaceae so far found.     

A CONTRIBUTION TO THE EARLY TERTIARY FOSSIL RECORD OF THE SALVINIACEAE

Megaspores and massulae of Azolla, Azollopsis, and Salvinia are recorded from the Paleocene and Eocene of Montana and the Dakotas. Three new species of Azolla are described and two species of Azolla are redescribed. The columella of the floating apparatus of the megaspore apparatuses of Azolla appears to be a phylogenetic localization of the perispore around the megaspore. A new section of Azolla (section Kremastospora) is created for species whose megaspore apparatuses have many floats and whose massulae have hooked (anchor-shaped) glochidia. Salvinia preauriculata is based only on leaf remains from the Eocene of North America. Megaspores and massulae which probably are conspecific with the leaf remains are described. The megaspores and massulae of the fossil species are much smaller than those of the living species, S. auriculata, with which S. preauriculata has been compared. The similar size of the megaspores and massulae of the fossil species suggests a less specialized condition than in living species, where these two structures differ greatly in size. Azollopsis tomentosa, previously known from the late Cretaceous, has been found in the Eocene.     

Expression of three <Emphasis Type="Italic">Arabidopsis</Emphasis> cytokinin oxidase/dehydrogenase promoter::GUS chimeric constructs in tobacco: response to developmental and biotic factors

Expression patterns of three Arabidopsis thaliana cytokinin oxidase/dehydrogenase promoter::GUS reporter fusions were investigated in tobacco plants. While cytokinin oxidase/dehydrogenase promoter 2 showed no expression in tobacco, the cytokinin oxidase/dehydrogenase promoters 3 and 4 were active in various tissues throughout development of the tobacco. Recently, the 1452 bp promoter region of AtCKX3 was reported as almost inactive in Arabidopsis. In contrast, the 1627 bp DNA fragment preceding the AtCKX3 coding region drove expression of the reporter GUS gene in various tobacco tissues. The promoter was mainly expressed in tobacco leaves and roots during early stages of development but also later in young flower buds as well as in pollen grains. The construct was particularly active before (hypocotyl region) and during (vascular system) lateral root initiation, supporting the idea of an inhibitory role of active cytokinins in the process of root initiation. The cytokinin oxidase/dehydrogenase promoter 4::GUS fusion in tobacco was shown to share some common (but weaker) expression patterns with promoter 3, namely in the leaves and pollen, but also conferred specific expression in tobacco root cap cells and trichomes. In addition, the response of cytokinin oxidase/dehydrogenase promoter::GUS reporter fusions to infection with the leafy gall-forming bacteria Rhodococcus fascians was examined. While an avirulent strain of R. fascians did not induce expression of any of the cytokinin oxidase/dehydrogenase promoters, the cytokinin oxidase/dehydrogenase promoter 3::GUS fusion was specifically induced at the site of infection when plants were challenged with a virulent strain of R. fascians, providing a possible explanation for the lack of significantly elevated cytokinin concentrations in tissues infected with virulent strains of R. fascians.This revised version was published online in August 2005 with some black and white figures replaced by coloured figures.     

ULTRASTRUCTURAL STUDIES OF IN SITU DEVONIAN SPORES: BARINOPHYTON CITRULLIFORME

Each sporangium in the Upper Devonian taxon Barinophyton citrulliforme contains both microspores and megaspores. Microspores range up to 50 μm in diam and possess a homogeneous sporoderm characterized by an outer separable layer. The sporoderm of the megaspores (up to 900 μm) is constructed of sporopollenin units that are loosely arranged in the outer portion of the wall, and that give the megaspore wall a spongy organization. Ultrastructural evidence suggests that the small spores were not abortive megaspores, but that both spore types were functional. The spores of this plant, as well as other Devonian spores that show less dramatic size differences, are suggested as demonstrating a phase in the evolution of heterospory where sex determination was established in spores within the same sporangium prior to the evolution of micro- and megasporangia.     

BREEDING SYSTEM IN FICUS CARICA,THE COMMON FIG. I. FLORAL DIVERSITY

Coevolution in Ficus carica (Moraceae) and the fig wasp (Blastophaga psenes, Agaonidae, Chalcidoidea) has resulted in a complex breeding system involving two tree morphs (Caprifig and Edible fig), three floral forms (long-styled female, short-styled female, and male flowers) and the insect pollinator. The two female floral forms have been reported to differ only in style length and stigma shape. In the present study, we demonstrate that the two female flowers differ from inception—short-styled flower primordia are smaller and exhibit significantly greater individual variation than do those of the long-styled flower, and the relative growth rate of each flower type differs. Mature forms exhibit disparity in style length, in stigma characteristics, and in degree of fusion of stylar lobes. Female flowers of both tree morphs are unisexual from inception. Male flowers of the Caprifig tree morph are initiated as hermaphrodites and gynoecium abortion occurs before megaspore mother cell stage. A single inflorescence therefore expresses two pathways to unisexuality. Hermaphrodite flower primordia were repeatedly found in the supposedly unisexual female syconium of the Edible fig tree morph. Based on its developmental morphology, Ficus carica appears to be of gynomonoecious ancestry.     

Benzylaminopurine induces phenocopies of floral meristem and organ identity mutants in wild-typeArabidopsis plants

Arabidopsis thaliana (L.) Heynh. has been used as a model system to investigate the regulatory genes that control and coordinate the determination, differentiation and morphogenesis of the floral meristem and floral organs. We show here that benzylaminopurine (BAP), a cytokinin, influences flower development inArabidopsis and induces partial phenocopies of known floral homeotic mutants. Application of BAP to wild-type inflorescences at three developmental stages results in: (i) increase in floral organ number; (ii) formation of abnormal floral organs and (iii) induction of secondary floral buds in the axils of sepals. These abnormalities resemble the phenotypes of mutants,clv1 (increase in organ number),ap1,ap2,ap3 (abnormal floral organs) andap1 (secondary floral buds in the axils of first-whorl organs). In addition, BAP induces secondary floral buds in the axils of perianth members ofapt2-6, ap3-1 andag mutants, and accentuates the phenotype of theapt2-1 mutant to resemble theapt2-6 mutant. These observations suggest that exogenous BAP suppresses the normal functioning of the genes for floral meristem identity and thereby affects flower development and the later stages of floral organ differentiation.Abbreviations BAP N6-benzylaminopurine - CK cytokinin     

Changes in cytokinin activities before,during and after floral initiation in Polianthes tuberosa

The contents of endogenous cytokinin in tuberose corms (Polianthes tuberosa) at vegetative, early floral initiation, and flower development stages were investigated. We also determined the influence of exogenous cytokinin treatment on the corm apex at three different growth stages in relation to floral initiation and development in tuberose. The exogenous cytokinin effectively induced floral initiation and development, especially at the early floral initiation and flower development stages. Endogenous cytokinins were higher in early floral initiation and development stages in comparison to the vegetative stage. During floral initiation stage, the zeatin and dihydrozeatin increased significantly, while the cytokinins, zeatin riboside, dihydrozeatin riboside, ⁶N-(δ²-isopentenyl) adenine, and ⁶N-(δ²-isopentenyl) adenine riboside at consistently low levels. The increase of cytokinin levels in tuberose corms during floral induction suggests a role for cytokinins in tuberose apex evocation. Moreover, these results indicate that cytokinins seem to promote the development of flower buds rather than inducing flowering in tuberose.     

平基槭花性别分化的细胞形态学观察

平基槭为杂性花,雄花与两性花同株,本文对其花性别分化过程进行了细胞形态学观察。结果发现,在花性别分化的早期,雄花和两性花的花芽中雌、雄蕊原基均具备,只是在花芽发育到一定时期,雄花的雌蕊原基发生选择性败育,败育发生在大孢子母细胞减数分裂为4个大孢子时期。两性花的雌蕊可以正常膨大结实,雄蕊花药虽然可以形成二核花粉,但不能正常开裂,属于不育雄蕊。初步分析认为,两性花雄蕊花药不能正常开裂与花粉囊壁纤维层木质化程度低有关。