KINESIN MOTOR PROTEINS ARE ESSENTIAL FOR MALE GAMETOPHYTE DEVELOPMENT IN MARSILEA VESTITA

The male gametophyte of the semi-aquatic fern, Marsilea vestita, produces multiciliated spermatozoids in a rapid developmental sequence that is controlled post-transcriptionally when dry microspores are placed in water. Development can be divided into two phases, mitosis and differentiation. During the mitotic phase, a series of nine successive division cycles produce 7 sterile cells and 32 spermatids in 4.5-5 hours. During the next 5-6 hours, each spermatid differentiates into a corkscrew-shaped motile spermatozoid with ~140 cilia. This document focuses on the role of motor proteins in the regulation of male gametophyte development and during ciliogenesis. In order to study the mechanisms that regulate spermatogenesis, RNAseq was used to generate a reference transcriptome that allowed us to assess the abundance of transcripts at different stages of development. Over 120 kinesin-like sequences were identified in the transcriptome that represent 56 unique kinesin transcripts. Members of the kinesin-2, -4, -5, -7, -8, -9, -12, -13, and -14 families, in addition to several plant specific and ‘orphan’ kinesins are present. Most (91%) of these kinesin transcripts change in abundance throughout gametophyte development, with 52% of kinesin mRNAs enriched during the mitotic phase and 39% enriched during differentiation. Functional analyses show that the temporal regulation of kinesin transcripts during gametogenesis directly correlates with kinesin protein function. Specifically, Marsilea makes one kinesin-2 (MvKinesin-2) and two kinesin-9 (MvKinesin-9A and MvKinesin-9B) transcripts, which are present during spermatid differentiation and ciliogenesis. Silencing experiments showed that MvKinesin-2 and MvKinesin-9A are required for ciliogenesis and motility in the Marsilea male gametophyte; however, these kinesins display atypical roles during these processes. In contrast, spermatozoids produced after the silencing of MvKinesin-9B exhibit normal morphology. MvKinesin-2 is necessary for cytokinesis as well as for regulating ciliary length and MvKinesin-9A is needed for the correct orientation of basal bodies, events not typically associated with these proteins. In addition, Marsilea makes motile, ciliated gametophytes without the help of IFT dynein, outer arm dynein, or the BBsome. These results are the first to investigate the kinesin-linked mechanisms that regulate ciliogenesis in a land plant.

Somatic meiosis and development of the juvenile gametophyte in members of the Batrachospermales sensu lato (Rhodophyta)

Seven populations (six in culture and one sampled directly from nature) of the freshwater red algal families Batrachospermaceae, Lemaneaceae and Thoreaceae were examined, involving three species of Batrachospermum, two of Paralemanea and one of Thorea. All 'Chantransia' stages ultimately produced juvenile gametophytes. The production of juvenile gametophytes in the three populations of Batrachospermum was generally most abundant at 15°C and low irradiances (47-68 μmol photons m-2 s-1). The most abundant gametophyte development in the Paralemanea species was observed at 10°C and low or high irradiances (47-142 μmol photons m-2 s-1). Gametophyte production in Thoreaceae occurred at higher temperatures (20°C) and also at low irradiances. In species of the Batrachospermaceae and Lemaneaceae, the 'elimination cells' can be situated on the basal or suprabasal cell of the juvenile gametophyte, but the position is usually fixed in individual species. The presence and position of the elimination cells remain to be established in Thoreaceae. Our results corroborate a previous study suggesting that the position of elimination cells is such a constant feature that it is of potential diagnostic value at the generic or infrageneric (sectional or specific) level. The characteristics observed in the development of the juvenile gametophytes in species of Batrachospermaceae and Lemaneaceae essentially agreed with general descriptions in the previous studies. The characteristics of the Thoreaceae, with a distinctive developmental pattern of the juvenile gametophyte and the occurrence of two morphological types in the 'Chantransia' stage, support the proposal to elevate it to the ordinal level. Two remarkable observations in Batrachospermum species were the production of numerous juvenile gametophytes from filaments of the same plant of the 'Chantransia' stage and the formation of a system of rhizoidal filaments or cell agglomeration of the juvenile gametophytes, which produced new gametophytes. These two characteristics potentially increase the formation of additional gametophytes under favourable conditions.

蕨类植物配子体发育及化感作用的研究

我国的蕨类植物资源十分丰富，约有2600多种，占全世界12000种的1/5强，其中许多种具有很高的观赏价值。在欧美的一些国家，蕨类植物已经成为花卉行业的一个重要组成部分，早已进入家庭居室、宾馆、商场、办公楼和其它公共场所，绿化和美化人们的生活环境。到目前为止，全世界共已栽培成功观赏蕨类植物700多种（品种），而我国仅有十多种投入花卉市场作为盆景和切花配叶，尚未形成一定的规模。繁殖技术不过关是造成蕨类植物在我国花卉市场难以迅速发展的障碍之一。目前国内对蕨类植物的需求主要靠野外采挖得以满足，对野生蕨类植物资源造成严重的破坏，再加上自然生态环境的恶化，致使许多物种濒临灭绝。因此，加强相关的基础研究、繁殖技术和外界环境对蕨类植物影响的研究，对蕨类植物资源的利用和保护具有重要的研究价值和现实意义。 作者在近五年的研究过程中，建立了蕨类植物孢子无菌培养技术，观察记录了蕨类植物配子体的形态发育，为建立蕨类植物快速有效的繁殖技术体系打下了良好的基础。在此研究基础上，探讨了对蕨类植物生长发育构成威胁的外界环境之一—外来入侵植物紫茎泽兰对蕨类植物的生长发育的化感作用。紫茎泽兰为目前国际上研究的热点入侵植物之一，对我国农业、林业等已造成很大破坏，并对蕨类植物资源构成一定威胁。蕨类植物配子体发育过程是蕨类植物生活史中相对脆弱的环节，最容易受化感作用的影响。但迄今为止，国际上尚未开展任何紫茎泽兰对蕨类植物（包括孢子体阶段和配子体阶段）的化感作用的研究。为保护蕨类植物自然资源，填补这一研究领域的空白，开展了紫茎泽兰对蕨类植物配子体发育过程的化感作用的研究。实验结果如下： （1）建立了金毛狗（已被列入《濒危野生动植物种国际贸易公约》（CITES）附录II 名单）、凤尾蕨属植物如傅氏凤尾蕨、剑叶凤尾蕨、蜈蚣草、银脉凤尾蕨、溪边凤尾蕨、疏裂凤尾蕨、西南凤尾蕨等多种蕨类植物的孢子无菌培养技术体系。 （2）采用无机培养基培养、土壤培养和滤纸培养三种培养方法，显微镜下观察傅氏凤尾蕨、剑叶凤尾蕨、蜈蚣草、银脉凤尾蕨、溪边凤尾蕨、疏裂凤尾蕨、西南凤尾蕨、三角鳞毛蕨、乌毛蕨、普通针毛蕨、金毛狗的孢子萌发及配子体形态发育的整个过程，研究了上述蕨类植物孢子萌发的类型、配子体各发育阶段的形态特征以及到达每个发育阶段所需的时间，为蕨类植物的繁殖技术提供了科学的依据，同时为蕨类植物系统进化和形态分类学研究提供了重要的基础资料。 （3）以金毛狗、普通针毛蕨、疏裂凤尾蕨为受体植物，研究了紫茎泽兰对这三种蕨类植物的配子体发育的化感作用。选择蕨类植物孢子萌发、假根生长、配子体形态、配子体发育各阶段及所占比例为测定指标，结果表明：紫茎泽兰根、茎、叶提取液可以对这三种蕨类植物的配子体发育产生不同程度的化感作用。表现为：延迟这三种蕨类植物的孢子萌发，降低其孢子萌发率，抑制假根的伸长，并且上述抑制作用随紫茎泽兰提取液浓度的升高而增强。随着紫茎泽兰叶水提液浓度的升高，金毛狗配子体发育阶段会滞后。在紫茎泽兰根提取液处理下，普通针毛蕨配子体上的假根发生畸变。随着时间的推移，这种现象削弱或基本消失。随着根和叶提取液的浓度升高，普通针毛蕨配子体发育阶段将会滞后。高浓度的紫茎泽兰根提取液能改变疏裂凤尾蕨的配子体的形态，并使疏裂凤尾蕨的配子体发育阶段滞后，浓度越高效果越显著。这三种蕨类植物在受到紫茎泽兰化感作用的影响后表现不同，推测与它们对紫茎泽兰所含有的化感物质的不同敏感度有关。

Caractérisation d'une famille de récepteurs kinases impliqués dans le développement gamétophytique chez Arabidopsis thaliana

Au cours du développement des végétaux, de l’établissement de l’identité cellulaire des premiers organes au guidage du tube pollinique, la communication cellule à cellule est d’une importance capitale. En réponse, les voies de signalisation moléculaires sont élaborées pour la perception d’un signal extérieur et la transduction en une réponse génique via une cascade intracellulaire. Les récepteurs kinases font partie des protéines perceptrices des stimuli et constituent chez les plantes une catégorie de protéines avec une occurrence considérable, mais dont très peu d’informations détaillées sont disponibles à ce jour. Une famille de récepteurs kinases chez Arabidopsis thaliana, AtORK11 (Arabidopsis thaliana Ovule Receptor Kinase 11), a été identifiée par orthologie à un récepteur spécifique aux ovaires chez une solanacéee sauvage, Solanum chacoense. La fonction présumée de cette famille de récepteurs kinases de type leucine-rich repeat, suggérée par son patron d’expression, implique les événements relatifs au développement des gamétophytes et à la reproduction. Afin de caractériser la fonction des quatre gènes de la famille (AtORK11a, AtORK11b, AtORK11c et AtORK11d) une stratégie d’analyse de mutants d’insertion de l’ADN-T et d’évaluation du mode d’action par complémentation bimoléculaire par fluorescence (BiFC) a été entreprise. Aucune fonction précise n’a pu être attribuée aux doubles mutants d’insertion, par contre la surexpression d’une construction dominante négative indique un rôle dans le développement gamétophytique. Il a aussi été démontré que les quatre récepteurs peuvent interagir par homodimérisation aussi bien que par hétérodimérisation. Une hypothèse de redondance fonctionnelle est ainsi mise à jour parmi la famille des gènes AtORK11.

Germline-Specific MATH-BTB Substrate Adaptor MAB1 Regulates Spindle Length and Nuclei Identity in Maize

Germline and early embryo development constitute ideal model systems to study the establishment of polarity, cell identity, and asymmetric cell divisions (ACDs) in plants. We describe here the function of the MATH-BTB domain protein MAB1 that is exclusively expressed in the germ lineages and the zygote of maize (Zea mays). mab1 (RNA interference [RNAi]) mutant plants display chromosome segregation defects and short spindles during meiosis that cause insufficient separation and migration of nuclei. After the meiosis-to-mitosis transition, two attached nuclei of similar identity are formed in mab1 (RNAi) mutants leading to an arrest of further germline development. Transient expression studies of MAB1 in tobacco (Nicotiana tabacum) Bright Yellow-2 cells revealed a cell cycle-dependent nuclear localization pattern but no direct colocalization with the spindle apparatus. MAB1 is able to form homodimers and interacts with the E3 ubiquitin ligase component Cullin 3a (CUL3a) in the cytoplasm, likely as a substrate-specific adapter protein. The microtubule-severing subunit p60 of katanin was identified as a candidate substrate for MAB1, suggesting that MAB1 resembles the animal key ACD regulator Maternal Effect Lethal 26 (MEL-26). In summary, our findings provide further evidence for the importance of posttranslational regulation for asymmetric divisions and germline progression in plants and identified an unstable key protein that seems to be involved in regulating the stability of a spindle apparatus regulator(s).

The Arabidopsis male-sterile mutant, opr3, lacks the 12-oxophytodienoic acid reductase required for jasmonate synthesis

Jasmonic acid (JA) and its precursor 12-oxophytodienoic acid (OPDA) act as plant growth regulators and mediate responses to environmental cues. To investigate the role of these oxylipins in anther and pollen development, we characterized a T-DNA-tagged, male-sterile mutant of Arabidopsis, opr3. The opr3 mutant plants are sterile but can be rendered fertile by exogenous JA but not by OPDA. Cloning of the mutant locus indicates that it encodes an isozyme of 12-oxophytodienoate reductase, designated OPR3. All of the defects in opr3 are alleviated by transformation of the mutant with an OPR3 cDNA. Our results indicate that JA and not OPDA is the signaling molecule that induces and coordinates the elongation of the anther filament, the opening of the stomium at anthesis, and the production of viable pollen. Just as importantly, our data demonstrate that OPR3 is the only isoform of OPR capable of reducing the correct stereoisomer of OPDA to produce JA required for male gametophyte development.

Desenvolvimento floral e do óvulo e aspectos da reprodução em Aechmea sp. e Vriesea sp. (Bromeliaceae)

A utilização de Bromélias tem sido crescente no mercado de plantas onamentais, por outro lado, muitas espécies encontram-se ameaçadas, grande parte pelos impactos humanos no ambiente. Aechmea correia-araujoi E. Pereira & Moutinho, Aechmea gamossepala Wittm, Vriesea ensiformis (Vell.) Beer e Vriesea saundersii (Carrière) E. Morren ex Mez, espécies nativas da Mata Atlântica brasileira, têm sido alvo de extrativismo. Informações básicas sobre a espécie são essenciais para subsidiar a condução de programas de conservação e melhoramento genético, que aliados a ferramentas biotecnológicas permitem a incorporação de estratégias inovadoras aos métodos de melhoramento. Neste sentido, o objetivo do presente trabalho foi descrever essas espécies, quanto à micromorfologia floral, aspectos reprodutivos envolvidos no processo de polinização, desenvolvimento floral e deesenvolvimento gametofítico, como mecanismo de preservação e produção comercial. A caracterização morfológica e anatômica das flores das espécies de Aechmea e Vriesea contribuiu para a compreensão do processo reprodutivo. As espécies apresentam grãos de pólen com alta capacidade reprodutiva, viabilidade polínica superior a 93%, germinação in vitro maior que 80% e o estigma apresenta-se receptivo da antese ao final do dia. A ontogênese floral de A. correia-araujoi é centrípeta, os primórdios desenvolvem-se na ordem, sépala, pétala, androceu e gineceu. O apêndice petalar é formado na fase final do desenvolvimento. O primórdio de óvulo tem origem placentária e caráter trizonal, o óvulo é anátropo, bitegumentado e crassinucelado. O meristema floral de A. gamosepala se desenvolve de forma centrípeta, de forma unidirecional reversa. O estigma diferencia-se na fase inicial do desenvolvimento e os apêndices petalares, na fase final. O óvulo é anátropo, crassinucelado, bitegumentado, tétrade linear, megásporo calazal funcional, desenvolvimento tipo monospórico e Polygonum. As anteras são bitecas, tetraesporangiadas, com tapete secretor. Botões florais de 8,7 - 13,0 mm são indicados no estudo de embriogênese a partir de micrósporo. As alterações celulares e o padrão de distribuição de pectinas e AGPs foram caracterizadas por análise citoquímica com azul de toluidina, KI e DAPI e imunocitoquímica por imunofluorescência com os anticorpos para RNA, pectinas esterificadas (JIM7), não esterificadas (JIM5) e AGPs (LM2, LM6, MAC207, JIM13, JIM14) e analisadas por microscopia de fluorescência. Foram caracterizados padrões de distribuição espaço-temporal de pectinas e AGP que podem ser utilizados como marcadores de desenvolvimento gametofítico masculino. As observações feitas nesse trabalho fornecem dados sobre aspectos reprodutivos das espécies que podem ser utilizados em programas de melhoramento genético, conservação e desenvolvimento de haploides

Formation and early development of tetraspores of Polysiphonia urceolata (Rhodomelaceae, Rhodophyta)

Polysiphonia urceolata is one type of potential commercial red seaweeds used for breeding and cultivation, because of its significant biochemical and biomedical application. However, the information of breeding and seedling incubation for cultivation is limited, especially the early development. In this study, tetrasporohyte and gametophyte of P. urceolata were taken as the study materials in Huiquan Bay, Qingdao, China. The cleaned and sterilized tetrasporophytes and gametophytes were pre-cultured in sterilized seawater, then nurtured at 18A degrees C, 25 mu mol photons m(-2) s(-1) in 12:12 h (light:dark) photoperiod. Continuous observation under microscope showed that the early development consists of bipolar division stage and seedling stage. In the division stage, tetraspores germinate into bipolar sporelings that further differentiate into a colorless rhizoidal portion and a lightly pigmented upright shoot. The lightly pigmented rhizoidal cell develops to a rhizoid and the larger pigmented cell transforms to an erect axis. In the seedling stage, several quasi-protuberances appear on the erect axis and form juvenile seedlings. The results demonstrate the culture of P. urceolata from tetraspores under laboratory conditions.

Study on early-stage development of conchospore in Porphyra yezoensis Ueda

Porphyra yezoensis Ueda (Rhodophyta) is a seaweed of economic importance with a typical dimorphic life cycle consisting of a leafy gametophyte and a filamentous sporophyte. Recently, it has been recognized as a model system for fundamental and applied studies in marine biological sciences. Conchospore, a major spore linking the two distinct multicellular phases in the life cycle, is most widely used in the breeding of P. yezoensis. In this paper, the early-stage development of conchospore, including the attachment and the cell wall formation, was studied with fluorescent reagents staining and Scanning Electron Microscopy detection. Results displayed: (I) the cell wall began to be generated after culturing for 4 h in the attached conchospores; (2) the initially released conchospores were plastids with some filmy, amorphous substance on the surface, and they attached to the fibers firmly via the actively secreted mucilaginous substances after their touch to the fibers; (3) cellulase and pectolase prohibited the attachment of conchospores in the different ways; and (4) only attached conchospores generated cell walls and developed normally, while the suspending ones could not. It indicated that the cellulose played crucial roles in the permanent attachment as the pectin did in the initial attachment. The conchospore attachment seemed to trigger the cell wall formation and the further development. Affects of light on the development of conchospores were also discussed. The results showed that high intensity (200 mu mol.m(-2).s(-1)) and long-wave (>= 580 nm) light facilitated the division rate of conchospores. (C) 2008 Elsevier B.V. All rights reserved.

Early development of Costaria costata (C. Agardh) Saunders and cultivation trials

Costaria costata (C. Agardh) Saunders is one of common kelps distributed in many coastal areas worldwide; however, in China, no reports have been made on cultivation of the genus. To investigate potential cultivation of the species in the northern part of China, trials on isolation and preservation of the gametophytes were conducted using C. costata from Korea; growth and development of the gametophytes were observed. We showed that at 10 +/- 1A degrees C, 60 mu mol m(-2)s(-1) and 12:12 h (L:D), freshly released zoospores settled down within 1 hour, and then developed into the primary cell during the following 2 days. After a vegetative growth phase lasting 6-8 days, female gametophytes became 3-4 times larger in diameter than that of the primary cell, but still remained at a unicellular stage, while male gametophytes divided into 4-10 cells with only a slight change in size. Fertilization occurred within 10 days after the zoospores were released from the sporangia, and the apical and basal tissues of the juvenile sporophyte divided and differentiated into the blade and stipe. Temperature and irradiance influenced gametophytic vegetative growth and developmental patterns. Generally, low irradiance (15 mu mol m(-2)s(-1) and 30 mu mol m(-2)s(-1)) was unfavorable to the induction of fertility, but it enhanced female gametophyte division. The optimal conditions for vegetative growth were 15A degrees C and 30 mu mol m(-2)s(-1). After transplantation of the juvenile seedlings and after eight months cultivation, the harvested mature blade reached 194 cm in length and 32.7 cm in width. Our study proves that it is feasible to implement propagation and large scale cultivation of C. costata in northern China.

Formation and early development of tetraspores of Gracilaria lemaneiformis (Gracilaria, Gracilariaceae) under laboratory conditions

Morphological and culture studies of tetraspores of Gracilaria lemaneiformis were carried out under laboratory conditions. Relationships of germination rate, diameter and survival rate of tetraspores from 1st generation branches with grads of temperature and irradiance were determined, respectively. The result showed that 1st generation branches is in the majority of the tetraspores shedding and tetraspores from which had highest survival rates than other parts of the sporophytic plant. The time tetraspores used developing from giant unicells to diads, which both existed on the epidermis, then to tetraspores off the matrix, was only approximately 3 weeks all through. However, tetraspores spent more than two months developing into germlings of gametophytes. It was shown that temperature variation (10, 15, 25, 30 degrees C) with the light of 30 mu mol m(-2) s(-1) had significant effects on the germination rate and diameter, but had no apparent effect on survival rate (ANOVA, P < 0.01). Germination rates of tetraspores reached the maximum at 20 degrees C, which was significantly higher than those at other temperature levels (P < 0.01), whereas 15 degrees C seemed to be optimal temperature for the diameter. All the three growth parameters (germination rate, diameter and survival rate) yield highly significant variations with irradiance treatments at room temperature (ANOVA, P < 0.01). The optimal germination rate was detected at the irradiance of 30 mu mol m(-2) s(-1) (P < 0.01). The photon flux density which exceeds 480 nnol m(-2) s(-1) have apparently negative effect on diameter and survival rate. (c) 2005 Elsevier B.V. All rights reserved.