Developmental morphology of the ovules of Amborella trichopoda (Amborellaceae) and Chloranthus serratus (Chloranthaceae)

The developmental morphology of the outer integument in the pendent orthotropous ovules of Amborella trichopoda (Amborellaceae) and Chloranthus serratus (Chloranthaceae) was studied. In both species the outer integument is semiannular at an early stage and becomes cup-shaped but dorsiventrally somewhat asymmetric at later stages. The outer integument, which is initiated first on the concave and lateral sides of the ovule, differs from that of the anatropous ovules of other basal families with the outer integument semiannular at an early stage or throughout development. The bilateral symmetry of the outer integument is shared by these orthotropous and anatropous ovules. The developmental pattern of the outer integument and ovule incurving characterize the ovule of the Amborellaceae and Chloranthaceae, which is not equivalent to typical orthotropous ovules of eudicots. A phylogenetic analysis of ovule characters in basal angiosperms suggests that anatropous ovules with cup-shaped outer integuments and orthotropous ovules were derived independently in several clades and that the ovules of Amborella and Chloranthus might also be derivative.     

Ovule differences between single-kernelled and double-kernelled fruits in almond (Prunus dulcis)

In order to detect differences between almond cultivars that commonly produce one or two seeds, we compared ovule sizes and developmental stages at anthesis and three days after anthesis. The results showed that the presence of only small differences in size between primary and secondary ovules during flowering seemed to be a necessary but not sufficient condition for the production of double‐kernelled fruits. In contrast, differences in the developmental stage of both ovules did not prevent the production of double kernels. Large differences in ovule size at anthesis or in the following days resulted in fruits with single seeds. The behaviour of the secondary ovules in cultivars producing fruit with single seeds was very different, even in closely related cultivars. The degeneration of the secondary ovule seems to be genetically programmed and follows a genotype‐dependent pattern.     

Ovule development at anthesis in apricot (Prunus armeniaca) varieties in a Mediterranean climate

The stage of ovule development at anthesis has been studied in 2002 and 2003 in 35 apricot varieties and 4 new apricot cultivars grown in a Mediterranean climate. Significant differences were found among varieties as well as a high heterogeneity of the developmental stage of the ovules examined in each variety. In general, a relative delay of the ovule development was observed, the presence of mature ovules at anthesis being negligible. In the 2 years of the study, differences in the developmental stage of primary ovules at anthesis were not significant and correlation to flowering time was not observed, which emphasised the previously reported genetic determination of this trait.     

组织透明技术在植物解剖学和植物发育生物学实验教学中的应用

整体组织透明和组织切片技术是研究植物组织和器官的结构及其发育过程的常用实验手段。该文针对本科生植物学课程的侧根、胚珠(特别是胚囊)以及胚胎发育内容设计了相应的实验教学课程。该实验以拟南芥幼苗、不同发育时期的花蕾和种子为材料,经固定、脱色和组织透明,利用正置显微镜观察侧根、胚珠以及胚胎的发育过程。根组织经透明后,在显微镜下可以清晰地观察到从侧根原基的建成到侧根发生的八个发育阶段。各个发育时期的胚珠经组织透明后,在显微镜下可以清晰地观察到大孢子母细胞、功能性大孢子(FG1期)以及FG2~FG7期的胚囊。各个发育时期的种子经透明后,在显微镜下可以清晰地观察到胚胎发育的1细胞期、8细胞期、球形期、心形期、鱼雷期、拐杖期和成熟期。这些实验结果可以使学生对拟南芥等双子叶植物的侧根、胚珠以及胚胎发育过程有较为系统的了解。该实验方法在其他较小的双子叶植物中也适用。     

Homeotic Transformation of Ovules into Carpel-like Structures in Arabidopsis

Ovules are specialized reproductive organs that develop within the carpels of higher plants. In Arabidopsis, mutations in two genes, BELL1 (BEL1) and APETALA2 (AP2), disrupt ovule development. In Bel1 ovules, the inner integument fails to form, the outer integument develops abnormally, and the embryo sac arrests at a late stage of megagametogenesis. During later stages of ovule development, cells of the outer integument of a Bel1 ovule sometimes develop into a carpel-like structure with stigmatic papillae and second-order ovules. The frequency of carpel-like structures was highest when plants were grown under conditions that normally induced flowering and was correlated with ectopic expression in the ovule of AGAMOUS (AG), an organ-identity gene required for carpel formation. Together, these results suggested that BEL1 negatively regulates AG late in ovule development. Likewise, mutants homozygous for the strong AP2 allele ap2-6 sometimes displayed structures with carpel-like features in place of ovules. However, such abnormal Ap2 ovules are much less ovulelike in morphology and form earlier than the Bel1 carpel-like structures. Because one role of the AP2 gene is to negatively regulate AG expression early in flower development, it is possible that AP2 works in a similar manner in the ovule. A novel ovule phenotype observed in Bel1/Ap2-6 double mutants suggested that BEL1 and AP2 genes function independently during ovule development.     

Structural aspects of ovule and seed development and nonrandom abortion inMelilotus officinalis (Fabaceae)

Summary Only one ovule matures into a seed inMelilotus officinalis. Although eight ovules form within an ovary, only the basal ovule develops into a mature seed, whereas the other ovules degenerate. The investigation of ovule and seed structure at different developmental stages and a comparison of quantitative characters of differently fated ovules within an ovary were undertaken by light, phase contrast, and fluorescence microscopy. In this species, campylotropous ovules develop simultaneously on marginal placentae in an apocarpous unilocular gynoecium. Megasporo- and megagametogenesis proceed normally and are completed in bud. The maturation of the Polygonum type embryo sac takes place after the flower opens. Shortly before fertilization, synergids show signs of degeneration in all ovules. At this stage, neither the structure nor the sizes of ovules within one ovary differ significantly. In spite of this, only the basal ovule develops into a seed. Rarely, one of the upper-situated ovules or the basal and another ovule mature into seeds. Seed enlargement is insignificant until the stage when globular embryo and nuclear endosperm are formed. At the seed-filling stage, other ovules have collapsed and the seed gradually comes to occupy the total volume of the pod. The fruit-to-seed length ratio decreases considerably during seed ripening. At fertilization, ovary length is four times greater than ovule length. In the mature state, the fruit and seed lengths are approximately equal. Seed size and weight diminish with an increase in seed number within a pod, although pod size remains constant. It is assumed that nonrandom abortion of young seeds inM. officinalis is under maternal control and is not related to structural abnormalities in ovule development or with limitation in pollen. We suppose that evolution of this species may have proceeded in the direction of a decrease in seed number and an increase in its sizes, which may play an important role in seed dispersal and seedling establishment.     

Temporal and Spatial Expression of MADS Box Genes, FBP7 and FBP11, During Initiation and Early Development of Ovules in Wild Type and Mutant Petunia hybrida

Abstract: The temporal and spatial distribution of the Petunia Floral Binding Proteins 7 and 11 (FBP7/11) were determined immunocytochemically during ovule initiation and development. In wild type plants, FBP7/11 were first detected in the placenta before ovule primordia were formed. At ovule primordium stage, FBP7/11 levels increased in the placenta and appeared in ovule primordia at the sites where integument primordia developed. At the megagametogenesis stage, FBP7/11 were present at high levels in the placenta, funicle and integument, but not in the nucellus or gametophyte. Transgenics with cosuppression of FBP7/11 formed normal ovule primordia on the placenta from which both normal ovules and carpel-like structures developed. The amount of FBP7/11 was low in the ovules and undetectable in the carpel-like structures. Plants with ectopic expression of FBP7/11 developed normal ovules on the placenta and, in addition, ovule- and carpel-like structures on sepals. Placental and sepal ovules showed the same labeling pattern as observed in wild type ovules. FBP7/11 levels were, however, low or undetectable in the carpel-like structures. The results indicate that FBP7/11 only have indirect roles in ovule primordium initiation. However, at least small quantities are needed for proper ovule differentiation. Thus, the amount of FBP7/11 is related to the type of development after primordium formation, i.e., towards the formation of real ovules or carpel-like structures.     

北京地区油松胚珠发育时期的确定

采用显微观察法对北京地区不同发育时期的油松胚珠进行解剖结构观察,以确定北京地区油松胚珠的发育时期。结果表明,北京地区油松胚珠的发育历时13个月(从2003年4月~2004年5月),具体时期为:2003年4月12日为胚珠分化期,4月21日左右为大孢子母细胞时期,5月至8月为游离核时期,2003年8月至2004年2月为胚珠休眠期,2004年4月15日左右进入雌配子体细胞化时期,4月21日左右为颈卵器原始细胞活动期,5月8日左右为中央细胞分裂期,5月14日左右为成熟颈卵器时期。本研究结果中油松胚珠经历的发育阶段与前人的研究基本一致,但在时间上存在差异,而且北京地区比辽宁兴城地区油松胚珠发育时期平均提前了大约20~30 d。     

MORPHOLOGY,VASCULAR ANATOMY AND EMBRYOLOGY OF PISTILLATE AND STAMINATE FLOWERS OF ASPARAGUS OFFICINALIS

Flowers of three pistillate (female), two heterogametic staminate (male) and two homogametic male genotypes of Asparagus officinalis L. were compared for morphology and vascular anatomy of the flower and for embryological development to the stage of mature ovules and pollen. Flowers are liliaceous, the staminate with rudimentary pistils and the pistillate with collapsed anthers. The uncomplicated vascular pattern differs between staminate and pistillate flowers only in the size and degree of maturation of bundles to stamens and carpels. Longer styles appear to be correlated with a greater extent of ovule development in ovaries of staminate flowers. Microsporogenesis in males is normal with wall development corresponding to the Monocotyledonous type. The tapetum is glandular and binucleate, cytokinesis successive, the tetrads isobilateral or occasionally decussate, and the mature pollen grain two-celled. A pair of heteromorphic, possibly sex, chromosomes was observed in heterogametic male plants. Anther development is initially the same in pistillate flowers, but the tapetum degenerates precociously followed by collapse of microspore mother cells. In pistillate flowers the ovules are hemitropous, bitegmic, and slightly crassinucellate. Megasporogenesis-megagametogenesis conforms to the Polygonum type. In staminate flowers ovule development is like that in pistillate flowers until degeneration starts in nucellar and integumentary cells at the chalazal end. Ovules in both homogametic male genotypes rarely complete meiosis, while in the heterogametic males it is normally completed with about one ovule in 20 flowers forming a mature megagametophyte. Since manipulation of sex expression in Asparagus could be important in developing inbred male and female lines for breeding purposes, those aspects of the morphological and embryological observations presented which might be useful in planning experiments to induce sex changes are discussed briefly.     

The influence of polyamines on apricot ovary development and fruit set

Polyamine (PA) concentrations of ovaries of two apricot cultivars, ‘S405‐17’ and ‘Bergeron’, collected at three stages of floral development, were analysed. The relationships between PA profiles, ovule development and fruit load between the two apricot cultivars were also determined. We show that PA levels vary with ovary development, depending on the cultivar. Higher concentrations of putrescine (PUT), spermidine and spermine were found in S405‐17 cultivar at all phenological stages. In addition, S405‐17 cultivar had more developed ovules and a higher fruit load than Bergeron. Moreover, the exogenous application of 10 mM PUT in Bergeron increased the percentage of functional ovules from 17.1% (control) to 33.3%. These results prove the effects of PAs on ovary development and indicate the possible improvement of apricot ovule development by their exogenous application. All these results define the relationship between PA content and ovule development in apricot. Further studies would confirm the use of free endogenous PA concentrations as markers of the developmental stage of the ovary, because determination of PA contents is easier and faster than serial sectioning.     

EFFECTS OF NONRANDOM SEED ABORTION ON PROGENY PERFORMANCE IN PHASEOLUS COCCINEUS L

In this study, we sought to determine if Phaseolus coccineus normally aborts potentially viable seeds, and whether seed abortion is nonrandom with respect to progeny vigor. The ovaries of Phaseolus coccineus typically have six linearly arranged ovules. The three ovule positions at the stylar end are more likely to mature seeds than the three ovules at the basal end of the ovary. When we destroyed the developing ovules at the stylar end of the fruit after fertilization but before seed abortion, there was a significant increase in the probability that the ovules in the three basal positions would produce a mature seed. The probability of seed maturation in control fruits (no ovules destroyed) ranged from 38.3 to 42.7% over the three field seasons, whereas in the experimental fruits it ranged from 64.3 to 79.7% (similar to that of ovules at the stylar end in control fruits). We did not find any significant change in the probability of seed production in the three ovule positions in the stylar end of the ovary (the positions with high probability of seed maturation) when the basal ovules were experimentally destroyed. These results indicate that potentially viable seeds are regularly aborted in P. coccineus, with seed abortion more frequent in the basal ovule positions than at the stylar positions. In two greenhouse studies and one field study, we compared the vigor of progeny produced in the control fruits with the vigor of progeny produced in fruits where three ovules (either stylar or basal end) were destroyed. We found that the performance of the progeny from the three positions at the stylar end of the control fruits did not differ from the performance of the progeny from experimental fruits in which the three basal ovule positions were destroyed. In contrast, the progeny from the basal positions of the control fruits outperformed the progeny from the experimental fruits when the seeds were produced in the three basal ovules (stylar ovules destroyed). Our findings indicate that when the experimental treatment increased the probability of seed maturation, there was a significant decrease in the average vigor of the progeny in the remaining (basal) ovule positions.     

Heterochrony and its role in sex determination of cryptically dioecious Consolea (Cactaceae) staminate flowers

Ovule development, megasporogenesis, and megagametogenesis were studied in six cryptically dioecious species of Consolea. All species showed uniform development typical for the Opuntioideae. Ovule development proceeds acropetally, but shows developmental asynchrony across floral morphs. At anthesis, female morph ovules are functional and available for fertilization, whereas staminate flower ovules are senescing and incapable of being fertilized. In occasional plants of some species, staminate flowers may reach anthesis with a few functional apical ovules capable of seed formation. Such plants are described as inconstant/leaky males. Ovule fertility differences across morphs are interpreted as resulting from heterochronic ovule development and senescence, although variation in embryo sac longevity cannot be ruled out. Significantly, ovule abortion follows a common pattern and timing in staminate flowers of both male morphs in all species. Thus, on the basis of this uniformity, a common origin for the cryptically dioecious breeding system in Consolea is hypothesized. Furthermore, staminate expression in Consolea appears to be controlled by a common, genetically determined heterochronic ovule developmental programme affecting the relative timing of ovule receptivity and flower opening. This is the first report of heterochrony as a mechanism of male sex determination.  © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society , 2008, 156 , 305–326.     

Cloning of an ovule specific promoter from Arabidopsis thaliana and expression of beta-glucuronidase

Tissue specific expression of transgenes in plant species has several advantages over constitutive expression. Identification of ovule specific promoters would be useful in genetic engineering of plants with a variety of desirable traits such as genetically engineered parthenocarpy, female sterile plants or seedless fruits. Relative inaccessibility and difficulty in harvesting adequate amounts of tissue at known developmental stages has impeded the progress in cloning of promoters involved in ovule development. In the present study an ovule specific promoter was cloned from Arabidopsis AGL11 gene and used to express GUS (beta-glucuronidase) gene in transgenic Arabidopsis. Histochemical staining of GUS appeared in the center of young ovary (ovules), but no detectable GUS activity was observed in vegetative plant tissues, sepals, petals and androecium. AGL11 gene promoter can be useful to modify the developmental path of plants by expressing either plant hormones or lethal genes for agronomic purpose.     

The D-lineage MADS-box gene OsMADS13 controls ovule identity in rice

Genes that control ovule identity were first identified in Petunia. Co-suppression of both FLORAL BINDING PROTEIN 7 (FBP7) and FBP11, two D-lineage genes, resulted in the homeotic transformation of ovules into carpelloid structures. Later in Arabidopsis it was shown that three genes, SHATTERPROOF1 (SHP1), SHP2, and SEEDSTICK (STK), redundantly control ovule identity, because in the stk shp1 shp2 triple mutant ovules lose identity and are transformed into carpel and leaf-like structures. Of these three Arabidopsis genes STK is the only D-lineage gene, and its expression, like FBP7 and FBP11, is restricted to ovules. OsMADS13 is the rice ortholog of STK, FBP7, and FBP11. Its amino acid sequence is similar to the Arabidopsis and Petunia proteins, and its expression is also restricted to ovules. We show that the osmads13 mutant is female sterile and that ovules are converted into carpelloid structures. Furthermore, making carpels inside carpels, the osmads13 flower is indeterminate, showing that OsMADS13 also has a function in floral meristem determinacy. OsMADS21 is most likely to be a paralog of OsMADS13, although its expression is not restricted to ovules. Interestingly, the osmads21 mutant did not show any obvious phenotype. Furthermore, combining the osmads13 and the osmads21 mutants did not result in any additive ovule defect, indicating that osmads21 does not control ovule identity. These results suggest that during evolution the D-lineage gene OsMADS21 has lost its ability to determine ovule identity.     

Gynoecium development inRubiaceae-Vanguerieae,with particular reference to the “stylar head”-complex and secondary pollen presentation

Gynoecium development in taxa of the tribeVanguerieae (Rubiaceae, subfam.Antirheoideae) was studied, using primarily a 2- and a 5-carpellate genus (Keetia andVangueria) as examples. All investigated taxa, characterized by ovaries with a solitary, ± apically inserted, pendulous anatropous ovule per locule/carpel, showed a very similar gynoecium development. Comparisons with other uniovulateRubiaceae (taxa of subfam.Rubioideae) revealed that notwithstanding the place of insertion and orientation of the solitary ovules (apically vs. basally inserted, pendulous vs. erect, anatropous ovules) the gynoecium development follows the same pattern; differing ovule insertion and orientation can easily be explained by the principle of variable proportions. — Since secondary pollen presentation is characteristic for the tribe, particular attention was paid to the development of the conspicuous “stylar head”-complex, defined here as structural unit comprised of pollen presenting organ, “receptaculum pollinis”, plus stigmatic (i.e., receptive) surfaces. It was found that their morphological differentiation, following the same pattern in all investigated taxa, starts at very early stages of floral development. They already had their final shape at a stage when the ovule development in the ovary had just barely started. The fully developed, actual “receptaculum pollinis” is made up of enormously enlarged, ± cylindrical epidermis cells which have peculiar ring-like thickenings in vicinity of and parallel to the outer tangential walls (a “mechanical barrier” preventing pollen tubes from entering?) and a much smaller-celled subepidermal tissue. With regard to shape, size, and exposure of the stigmatic surfaces, the investigated taxa exhibited certain morphological differences. These were found to be correlated with differences in androecium structure (and, ultimately, pollen presentation).     

Embryo sac isolation inArabidopsis thaliana: A simple and efficient technique for structure analysis and mutant selection

Arabidopsis thaliana has been widely used as a model plant in gene function analysis. However, its tiny flower and curved embryo sac make it difficult to study gene expression during megagametogenesis, fertilization, and early embryogenesis, especially in the screening of mutants from those developmental processes. The techniques currently available are sectioning and whole-mount clearing of ovules; however, sectioning is time consuming and laborious for quantitative analysis, and whole-mount clearing, makes clear cytological observation impossible. Reported here is a simple and efficient method based on enzymatic isolation of embryo sacs that enables both quantitative analysis and elaborate cytological observation for gene expression investigation and mutant screening.     

An Efficient Method for Quantitative,Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.