BcMF21 is important for pollen development and germination in Brassica campestris ssp. chinensis

Brassica campestris Male Fertility 21 (BcMF21) was previously isolated from the flower buds of Chinese cabbage (Brassica campestris L. ssp. chinensis Makino, syn. B. rapa ssp. chinensis) and expressed specifically in tapetum and microspores during the meiosis stage and the uninucleate stage of microspore development. Here, we used antisense RNA technology to knock down the expression level of BcMF21 in B. campestris and analyzed the phenotype of the transgenic plants. Alexander staining and scanning electron microscope revealed sterility and exine deformities in the mature pollen grains of BcMF21 antisense RNA transgenic plants. The germination furrow of the BcMF21 antisense RNA transgenic pollen was covered by lipid like materials. The pollen tubes burst and could not grow normally in vitro. Therefore, we presented here BcMF21 might be an important gene for pollen development and germination.     

Morphological and functional characterization of BcMF13 in the antisense-silenced plants of Brassica campestris ssp. chinensis var. parachinensis

The gene Brassica campestris male fertility 13 (BcMF13, GenBank accession number EF158459) was isolated as a reproductive organ-specific gene from Chinese cabbage (Brassica campestris L. ssp. chinensis Makino, syn. B. rapa ssp. chinensis). It is exclusively expressed in stage four and five flower buds of fertile lines and is most strongly expressed in stamens. Here, we report a functional characterization of this BcMF13 gene in the antisense-silenced plants. The inflorescence of the BcMF13 mutant was compacted with anthers curved outside. The fertility of this mutant was greatly reduced with less than 5 seeds per silique. Under scanning electron microscopy, the mutant demonstrated numerous shriveled pollen grains with deep invaginations. The frequency of normal pollen grains was just 45.34%. The pollen mother cell, the tetrad, and the mature pollen of the BcMF13 mutant were abnormal resulting in the poor pollen vitality. Germination test in vivo suggested BcMF13 delayed the pollen tubes’ extension in the style. All these indicated BcMF13 had a vital role in pollen development of Chinese cabbage.     

In vitro culture of young floral buds of tomato (Lycopersicon esculentum mill.)

Young floral buds of tomato (Lycopersicon esculentum Mill.), with sepal primordia only, were cultured successfully in a liquid medium containing Murashige and Skoog's salts, White's vitamins and glycine, 3% (w/v) sucrose and benzylaminopurine (BAP). In 5 weeks, 60–70% of the cultured buds showed normal flower development through to anthesis, and a full complement of floral organs was produced. In the anthers, microsporogenesis occured and microspores and pollen grains were formed. The gynoecium was differentiated into an ovary, style and a stigma, and the ovary contained many ovules, some of which had developing embryo sacs. The floral organs grown in vitro were, however, smaller in size than those produced in vivo. In the control medium, i.e. without BAP, the floral buds produced petal and stamen primordia only and they too showed limited growth. The data presented shows the importance of cytokinins in the initiation and development of organs in the excised floral buds.     

Functional analysis of a novel male fertility CYP86MF gene in Chinese cabbage (Brassica campestris L. ssp. chinensis makino)

In our earlier work, a cytochrome P450 CYP86MF gene was isolated from floral bud of Chinese cabbage (Brassica campestris L. ssp. chinensis Makino, syn. B. rapa L.) by mRNA differential display PCR (DD-PCR) and rapid amplification of cDNA ends (RACE). To unravel the biological function of CYP86MF gene, the antisense fragment from the CYP86MF gene was transferred into Chinese cabbage pak-choi (B. campestris ssp. chinensis var. communis Tsen et Lee). Out of 22 plants transformed with the antisense gene constructed from the CYP86MF, 20 reached to flowering stage. Morphological investigations showed that the transgenic plants developed the normal floral organ. However, they remained self-infertile, even when artificial self-pollination was performed in the bud stage. Pollen germination test indicated that the pollen from the transgenic line TB-2 could not germinate normally. Further physiological, biochemical and cytological analyses showed that only significant difference was detectable in contents of the endogenous hormones, and a layer of unknown material adhered to the surface of microspore. The present studies thus provided valuable clues for understanding the biological function of the CYP86C subfamily genes. Furthermore, our studies also demonstrate a novel method for obtaining artificial male sterility line of Chinese cabbage.     

Cytochemical investigation of genic male-sterility in Chinese cabbage

A genic male sterile Chinese cabbage, Brassica campestris L. ssp. chinensis Makino, was examined using cytological and cytochemical methods to characterize the process of pollen abortion in this plant. Thick sections of both fertile and sterile anthers at different developmental stages were stained using Toluidine Blue O, Periodic Acid-Schiff’s (PAS) reaction and Sudan Black B to detect cytochemical changes that may occur in the distribution of insoluble polysaccharide and lipid storage bodies. Pollen abortion in sterile anthers occurs at an early stage of microspore development. During early microspore development, reductions in the number of starch grains in the connective tissue of fertile anthers coincide with the accumulation of starch grains in cells of the anther wall. In the late microspore stage, a large vacuole forms in the microspore, and tapetal cells synthesize and accumulate lipid droplets. The cellular organization of tapetal cells in sterile anthers appears similar to that in fertile anthers, except for the absence of lipid droplets in cells of sterile anthers and diffusely labeled tapetal polysaccharides, suggesting defects in nutrient storage. Supported by National Natural Science Foundation of CHINA (30170060)     

Floral morphogenesis in Sinofranchetia (Lardizabalaceae) and its systematic significance

The floral organs of Sinofranchetia chinensis Hemsl. (Lardizabalaceae) are all spiral in initiation. Stamen and petal (nectar‐leaf) primordia initiate independently and are different in shape. The petals and three stamens in the first whorl are retarded in the early developmental stages. The carpel primordia are conduplicate; the stigma is formed around the upper part of the ventral slit and the style is not differentiated. The functionally unisexual flowers are bisexual in organization in the early developmental stages. The development of the flowers on the inflorescence is spiral and centripetal. Some floral characteristics of Sinofranchetia appear to be plesiomorphic in Lardizabalaceae. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160 , 82–92.     

大白菜新型细胞质雄性不育系6w-9605A的育性鉴定和花药败育的细胞学观察

采用石蜡切片技术,研究了大白菜(Brassica campestris L.ssp.pekinensis)细胞质雄性不育系6w-9605A及其保持系6w-9605B的花药发育过程的细胞形态学特征,确定不育系花药败育时期及方式,并对不育系6w-9605A进行花器官观察和育性鉴定.结果表明:保持系6w-9605B花药发育正常;不育系6w-9605A花药发育受阻于孢原分化时期,占总败育花药的66.7％,不形成花粉囊和花粉粒,属于无花粉囊型败育;另外33.3％的败育花药可形成花粉囊,小孢子均受阻于单核靠边期或者二胞期,败育特点为绒毡层细胞异常肥大,挤压小孢子,导致小孢子和绒毡层解体;6w-9605A的不育性稳定、彻底,不育株率和不育度均为100％.     

Effects of nutrient contents and defense compounds on herbivory in reproductive organs and leaves of Iris gracilipes

Optimal defense theory (ODT) states that the plant tissue with the highest value to fitness will receive the most protection compared with other plant parts. ODT can be applied to the differences in defenses among floral organs, although most studies have concentrated on the comparison between leaves and flowers. Using Iris gracilipes, we investigated whether ODT is supported when primary and accessory floral organs and leaves are distinguished. We found that anthers and perianths tended to be attacked more severely than ovaries and leaves in the bud and flower stages and that anthers contained the highest nitrogen and phosphorus concentrations. Although ovaries were also found to contain high nitrogen and phosphorus concentrations, they were less severely attacked by herbivores than anthers, perhaps because ovaries contained the highest condensed tannins concentrations among the floral organs except for perianths in the flower stage. Thus, noting that the number of ovules is very much smaller than that of pollen grains, we concluded that ovaries are the most intensively protected, consistent with the prediction of ODT as applied to floral organs. ODT is applicable to the difference in defense allocation among floral organs.     

驴蹄草属和金莲花属(毛茛科)花器官的形态发生及系统学意义

利用扫描电镜观察了驴蹄草Caltha palustris L.和川陕金莲花Trollius buddae Schipcz.花器官的发生和发育过程。结果显示:驴蹄草和川陕金莲花的所有花器官均螺旋状向心式发生、向心式发育,花器官的螺旋状发生方式在毛茛科Ranunculaceae可能是一种基本式样;苞片、萼片与其他花器官原基的形状明显不同,显示苞片、萼片与其他花器官在系统发生上有所不同;川陕金莲花的花瓣在早期延迟发育且基部具囊,花瓣的延迟发育在毛茛科具花瓣的属中非常普遍,而花瓣基部的囊类似于耧斗菜属Aquilegia一些植物;两个属雄蕊群一纵列雄蕊中的小孢子均向心式发育,这种发育方式在毛茛科可能为基本类型。两个属植物的心皮原基均为对折式,在发育过程中,驴蹄草心皮顶端沿腹缝线形成下延的柱头组织,川陕金莲花不形成明显的柱头组织。根据花形态发生和发育特点,并结合其他研究成果,认为这两个属不应当属于同一个族。     

A SCANNING ELECTRON MICROSCOPIC STUDY ON THE INITIATION AND DEVELOPMENT OF FLORAL ORGANS OF BRASSICA NAPUS (CV. WESTAR)

The initiation and development of the floral organs of Brassica napus L. (cv. Westar) were examined using the scanning electron microscope. After transition of the vegetative apex into an inflorescence apex, flower primordia were initiated in a helical phyllotactic pattern. The sequence of initiation of the floral organs in a flower bud was that of sepals, stamens, petals and gynoecium. Of the four sepal primordia, the abaxial was initiated first, followed by the two lateral and finally the adaxial primordium. The four long stamens were initiated simultaneously in positions alternating with the sepals. The two short stamens were initiated basipetal to and outside the long stamens, and opposite the lateral sepals. The petals arose on either side of the two short stamens and the gynoecium was produced from the remainder of the apex. During development, the sepal primordia curved sharply at the tips and tightly enclosed the other organs. Stamen primordia developed tetralobed anthers at an early stage while filament elongation occurred just prior to anthesis. A unique pattern of bulbous cells was present on the abaxial surface of the anther. Growth of petal primordia lagged relative to the other floral organs but expansion was rapid prior to anthesis. The gynoecium primordium was characterized by an invagination early in development. At maturity, there was differentiation of a papillate stigma, an elongated style and a long ovary marked externally by sutures and divided internally by a septum. Distinct patterns of cuticular thickenings were observed on the abaxial and adaxial surfaces of the petals and stamens and on the surface of the style. The patterns were less obvious on the sepals and ovary. Stomata were present on both surfaces of the mature sepals, on the style and restricted areas on the abaxial surface of the anthers and nectaries but were absent from the petals, the adaxial surface of the stamens and the ovary. No hairs were present on any of the floral organs.     

Floral morphogenesis of Caltha and Trollius (Ranunculaceae) and its systematic significance

The floral morphogenesis of Caltha palustris L. and Trollius buddae Schipcz. was observed with a scanning electron microscope (SEM). The primordia of all floral organs initiate spirally and centripetally and develop centripetally. The spiral initiation sequence may be a basic pattern in Ranunculaceae. The primordia of bracts, sepals, and other floral organs are different in shape: the bract primordia are triangle, the sepal primordia crescent, and the petal (in Trollius), stamen, and carpel primordia hemispheric. This may indicate that the bracts, the sepals and other floral organs are different in origin. The petals are retarded in early developmental stages in Trollius buddae Schipcz, and have purses at the base. The retarded petals are very common in Ranunculaceae and the purse of the petal is similar to that of some Aquilegia species. The microspores in a longitudinal series of stamens develop centripetally in Caltha and Trollius; this may be a basic pattern in Ranunculaceae. The carpel primordia are plicate. In the developmental process of the carpels, the stigmatic tissue appears from the apex of the style and is decurrent along the ventral suture in Caltha, but there is no obvious stigmatic tissue in Trollius. Based on floral morphogenesis characteristics as well as the results from molecular systematics, comparative morphology and palynology studies, we consider that Caltha is not closely related to Trollius and that these two genera should not be treated in the same tribe.     

Heat stress differentially modifies ethylene biosynthesis and signaling in pea floral and fruit tissues

Key message

We identified and cloned the two precursors of miR158 and its target gene in Brassica campestris ssp. chinensis, which both had high relative expression in the inflorescences. Further study revealed that over-expression of miR158 caused reduced pollen varbility, which was caused by the degradation of pollen contents from the binucleate microspore stage. These results first suggest the role of miR158 in pollen development of Brassica campestris ssp. chinensis.

Abstract

MicroRNAs (miRNAs) play crucial roles in many important growth and development processes both in plants and animals by regulating the expression of their target genes via mRNA cleavage or translational repression. In this study, miR158, a Brassicaceae specific miRNA, was functionally characterized with regard to its role in pollen development of non-heading Chinese cabbage (Brassica campestris ssp. chinensis). Two family members of miR158 in B. campestris, namely bra-miR158a1 and bra-miR158a2, and their target gene bra027656, which encodes a pentatricopeptide repeat (PPR) containing protein, were identified. Then, qRT-PCR analysis and GUS-reporter system revealed that both bra-miR158 and its target gene had relatively high expression levels in the inflorescences. Further study revealed that over-expression of miR158 caused reduced pollen varbility and pollen germination ratio, and the degradation of pollen contents from the binucleate microspore stage was also found in those deformed pollen grains, which led to pollen shrinking and collapse in later pollen development stage. These results first shed light on the importance of miR158 in pollen development of Brassica campestris ssp. chinensis.

     

Inflorescence and floral development in Astragalus lagopoides Lam. (Leguminosae: Papilionoideae: Galegeae)

Inflorescence and floral organogenesis and development of the bushy perennial legume Astragalus lagopoides of the section Hymenostegis were studied by means of epi-illumination light microscopy. Based on our observations, the primordia of lanceolate racemose inflorescences are born in the axils of leaves. Each inflorescence apex initiates acropetally bracts and floral apices for some time and then eventually ceases meristematic activity and forms an oblong-shaped terminal structure. The formation of such atypical terminal protrusion on the inflorescence meristem is judged to be a diagnostic feature for well-organized cessation of meristem morphogenesis. Pentamerous perfect flowers of the plant show strong zygomorphy and marked overlap in time of initiation among different organ primordia. Unexpectedly, sepal initiation is bidirectional starting from the lateral sides of the floral apex. Other significant developmental feature includes the existence of two types of common primordia, which are formed successively. From the primary common primordia there are produced antesepalous stamens and secondary common primordia. In comparison, the five secondary common primordia subdivide into a petal and an antepetalous stamen primordia. Initiation of two different types of common primordia is possibly the result of rising overlap in time of initiation of organs and demonstrates an advanced developmental style in the genus Astragalus.     

白菜OguCMS相关MYB家族新基因BcMYBogu的克隆与特征分析

为研究CMS核质互作的分子机理, 将甘蓝型油菜(Brassica napus L.)和白菜(B. campestris L. ssp. chinensis Makino)杂交并连续回交6代获得白菜OguCMS, 在与保持系花药细胞学比较的基础上, 运用cDNA-AFLP筛选得到白菜OguCMS早、中期花蕾提早表达的MYB-like差异片断, 利用RACE克隆得到该片断的cDNA全长, 命名为BcMYBogu(GenBank 登录号: EF127861), 对其氨基酸序列和表达特征进行研究。结果表明, 白菜OguCMS绒毡层在四分体后增生, 高度液泡化, 导致小孢子花粉外壁异常, 细胞质同外壁分离并降解; 花药变白; BcMYBogu具有典型的MYB DNA结合域—W残基和SH[AL]QKY[RF]基序; 系统进化分析显示BcMYBogu与AtMYB26, AtMYB32和AtMYB4等聚类在同一分枝; RT-PCR分析表明BcMYBogu在莲座叶、花茎和花蕾中均有表达, 但在OguCMS花蕾中表达量显著上升。由此推测BcMYBogu是一个新的与白菜OguCMS相关的MYB家族新成员。     

Evolution of the self-pollinating flower in Clarkia xantiana (Onagraceae). I. Size and development of floral organs

Clarkia xantiana has two subspecies that differ in breeding system: ssp. xantiana, which is outcrossing, and ssp. parviflora, which is self-fertilizing. Outcrossing is the ancestral breeding system for the genus Clarkia. Flowers of ssp. parviflora have characteristics commonly associated with selfing taxa: they are smaller and have little temporal and spatial separation between mature anthers and stigma (dichogamy and herkogamy, respectively). Flower morphology and development were studied in four populations of each subspecies to establish the developmental changes that occurred in the evolution of selfing. In particular, we sought to evaluate the hypothesis that the selfing flower may have arisen as a byproduct of selection for rapid maturation in the arid environment occupied by ssp. parviflora. This hypothesis predicts that development time should be reduced in spp. parviflora relative to ssp. xantiana. We also sought to compare the pattern of covariation of flower morphology and development between subspecies to that within subspecies. Similar within vs. between patterns of covariation could be indicative of developmental or functional constraints on the independent evolution of floral parts. In spite of significant variation among populations within subspecies, the subspecies clearly differ in flower morphology and development. All floral organs, except ovaries, are smaller in ssp. parviflora than in ssp. xantiana. The flower plastochron, the duration of flower development from bud initiation to anthesis, and the duration of protandry are all shorter in ssp. parviflora than in ssp. xantiana. Maximum relative growth rates are higher for all organs in ssp. parviflora than in ssp. xantiana. Thus, progenesis (i.e., via a reduction in development time) is combined with growth acceleration in the evolution of the selfing flower. Since reduced development time and growth acceleration both allow selfing flowers to mature earlier than outcrossing ones, selection for early maturation may have contributed to the evolution of the selfing flower form. The pattern of trait covariation differs within spp. parviflora relative to the patterns within spp. xantiana and between the two subspecies, suggesting that floral parts can and have evolved independently of one another.     

Floral development in Astragalus caspicus Bieb. (Leguminosae: Papilionoideae: Galegeae)

Floral organogenesis and development of the bushy perennial legume Astragalus caspicus were studied using epi-illumination light microscopy techniques. Based on our observations, flowers are in axillary two-flowered racemes, initiate all 21 floral organs and show precocious appearance of zygomorphy. The order of floral organ initiation is unidirectional in whorls starting from the abaxial position of the flower with a high degree of overlap. Another important ontogenetic feature is the existence of two successive common primordial stages categorized as primary and secondary. The primary common primordia produce antesepalous stamens and secondary common primordia. In contrast, the five secondary common primordia subdivide into a petal and an antepetalous stamen primordia. Our findings on floral ontogeny of A. caspicus provide new evidence for the complex and variable floral initiation and development in legumes. The floral apex with strong overlapping initiation of different organs illustrates a paradox in which different capabilities must be presumed to exist simultaneously. Moreover, two extraordinary types of common primordia represent possibly an advanced evolutionary trend where time intervals between the initiations of different floral organs in Papilionoideae are shortened.