Comparative in situ analyses of cell wall matrix polysaccharide dynamics in developing rice and wheat grain

Cell wall polysaccharides of wheat and rice endosperm are an important source of dietary fibre. Monoclonal antibodies specific to cell wall polysaccharides were used to determine polysaccharide dynamics during the development of both wheat and rice grain. Wheat and rice grain present near synchronous developmental processes and significantly different endosperm cell wall compositions, allowing the localisation of these polysaccharides to be related to developmental changes. Arabinoxylan (AX) and mixed-linkage glucan (MLG) have analogous cellular locations in both species, with deposition of AX and MLG coinciding with the start of grain filling. A glucuronoxylan (GUX) epitope was detected in rice, but not wheat endosperm cell walls. Callose has been reported to be associated with the formation of cell wall outgrowths during endosperm cellularisation and xyloglucan is here shown to be a component of these anticlinal extensions, occurring transiently in both species. Pectic homogalacturonan (HG) was abundant in cell walls of maternal tissues of wheat and rice grain, but only detected in endosperm cell walls of rice in an unesterified HG form. A rhamnogalacturonan-I (RG-I) backbone epitope was observed to be temporally regulated in both species, detected in endosperm cell walls from 12 DAA in rice and 20 DAA in wheat grain. Detection of the LM5 galactan epitope showed a clear distinction between wheat and rice, being detected at the earliest stages of development in rice endosperm cell walls, but not detected in wheat endosperm cell walls, only in maternal tissues. In contrast, the LM6 arabinan epitope was detected in both species around 8 DAA and was transient in wheat grain, but persisted in rice until maturity.

Wheat seed embryo excision enables the creation of axenic seedlings and Koch’s postulates testing of putative bacterial endophytes

Early establishment of endophytes can play a role in pathogen suppression and improve seedling development. One route for establishment of endophytes in seedlings is transmission of bacteria from the parent plant to the seedling via the seed. In wheat seeds, it is not clear whether this transmission route exists, and the identities and location of bacteria within wheat seeds are unknown. We identified bacteria in the wheat (Triticum aestivum) cv. Hereward seed environment using embryo excision to determine the location of the bacterial load. Axenic wheat seedlings obtained with this method were subsequently used to screen a putative endophyte bacterial isolate library for endophytic competency. This absence of bacteria recovered from seeds indicated low bacterial abundance and/or the presence of inhibitors. Diversity of readily culturable bacteria in seeds was low with 8 genera identified, dominated by Erwinia and Paenibacillus. We propose that anatomical restrictions in wheat limit embryo associated vertical transmission, and that bacterial load is carried in the seed coat, crease tissue and endosperm. This finding facilitates the creation of axenic wheat plants to test competency of putative endophytes and also provides a platform for endophyte competition, plant growth, and gene expression studies without an indigenous bacterial background.

Effects of abscisic acid, ethylene and sugars on the mobilization of storage proteins and carbohydrates in seeds of the tropical tree Sesbania virgata (Leguminosae)

Endospermic legumes are abundant in tropical forests and their establishment is closely related to the mobilization of cell-wall storage polysaccharides. Endosperm cells also store large numbers of protein bodies that play an important role as a nitrogen reserve in this seed. In this work, a systems approach was adopted to evaluate some of the changes in carbohydrates and hormones during the development of seedlings of the rain forest tree Sesbania virgata during the period of establishment. Seeds imbibed abscisic acid (ABA), glucose and sucrose in an atmosphere of ethylene, and the effects of these compounds on the protein contents, alpha-galactosidase activity and endogenous production of ABA and ethylene by the seeds were observed. The presence of exogenous ABA retarded the degradation of storage protein in the endosperm and decreased alpha-galactosidase activity in the same tissue during galactomannan degradation, suggesting that ABA represses enzyme action. On the other hand, exogenous ethylene increased alpha-galactosidase activity in both the endosperm and testa during galactomannan degradation, suggesting an inducing effect of this hormone on the hydrolytic enzymes. Furthermore, the detection of endogenous ABA and ethylene production during the period of storage mobilization and the changes observed in the production of these endogenous hormones in the presence of glucose and sucrose, suggested a correlation between the signalling pathway of these hormones and the sugars. These findings suggest that ABA, ethylene and sugars play a role in the control of the hydrolytic enzyme activities in seeds of S. virgata, controlling the process of storage degradation. This is thought to ensure a balanced flow of the carbon and nitrogen for seedling development.

Storage proteins and cell wall mobilisation in seeds of Sesbania virgata (Cav.) Pers. (Leguminosae)

The endosperm of seeds of Sesbania virgata (Cav.) Pers. accumulates galactomannan as a cell wall storage polysaccharide. It is hydrolysed by three enzymes, one of them being alpha-galactosidase. A great amount of protein bodies is found in the cytoplasm of endospermic cells, which are thought to play the major role as a nitrogen reserve in this seed. The present work aimed at understanding how the production of enzymes that degrade storage compounds is controlled. We performed experiments with addition of inhibitors of transcription (actinomycin-d and alpha-amanitin) and translation (cycloheximide) during and after germination. In order to follow the performance of storage mobilisation, we measured fresh mass, protein contents and alpha-galactosidase activity. All the inhibitors tested had little effect on seed germination and seedling development. Actinomycin-d and cycloheximide provoked a slight inhibition of the storage protein degradation and concomitantly lead to an elevation of the alpha-galactosidase activity. Although alpha-amanitin showed some effect on seedling development at latter stages, it presented the former effect and did not change galactomannan degradation performance. Our data suggest that some of the proteases may be synthesised de novo, whereas alpha-galactosidase seems to be present in the endosperm cells probably as an inactive polypeptide in the protein bodies, being probably activated by proteolysis when the latter organelle is disassembled. These evidences suggest the existence of a connection between storage proteins and carbohydrates mobilisation in seeds of S. virgata, which would play a role by assuring a balanced afflux of the carbon and nitrogen to the seedling development.

The role of carbohydrates in seed germination and seedling establishment of Himatanthus sucuuba, an Amazonian tree with populations adapted to flooded and non-flooded conditions

Background and Aims In the Amazonian floodplains plants withstand annual periods of flooding which can last 7 months. Under these conditions seedlings remain submerged in the dark for long periods since light penetration in the water is limited. Himatanthus sucuuba is a tree species found in the `varzea` (VZ) floodplains and adjacent non-flooded `terra-firme` (TF) forests. Biochemical traits which enhance flood tolerance and colonization success of H. sucuuba in periodically flooded environments were investigated. Methods Storage carbohydrates of seeds of VZ and TF populations were extracted and analysed by HPAEC/PAD. Starch was analysed by enzyme (glucoamylase) degradation followed by quantification of glucose oxidase. Carbohydrate composition of roots of VZ and TF seedlings was studied after experimental exposure to a 15-d period of submersion in light versus darkness. Key Results The endosperm contains a large proportion of the seed reserves, raffinose being the main nonstructural carbohydrate. Around 93% of the cell wall storage polysaccharides (percentage dry weight basis) in the endosperm of VZ seeds was composed of mannose, while soluble sugars accounted for 2.5%. In contrast, 74% of the endosperm in TF seeds was composed of galactomannans, while 22% of the endosperm was soluble sugars. This suggested a larger carbohydrate allocation to germination in TF populations whereas VZ populations allocate comparatively more to carbohydrates mobilized during seedling development. The concentration of root non-structural carbohydrates in non-flooded seedlings strongly decreased after a 15-d period of darkness, whereas flooded seedlings were less affected. These effects were more pronounced in TF seedlings, which showed significantly lower root non-structural carbohydrate concentrations. Conclusions There seem to be metabolic adjustments in VZ but not TF seedlings that lead to adaptation to the combined stresses of darkness and flooding. This seems to be important for the survival of the species in these contrasting environments, leading these populations to different directions during evolution.

Structural aspects of the zygotic embryogenesis of Acca sellowiana (O. Berg) Burret (Myrtaceae)

(Structural aspects of the zygotic embryogenesis of Acca sellowiana (O. Berg) Burret (Myrtaceae)). Acca sellowiana has anatropous, bitegmic and crassinucellate ovules. The outer and inner integuments are double-layered except in the micropyle, where they are composed of more layers; the micropyle is zig-zag shaped. The egg apparatus lies at the micropylar pole, and the zynergids present a conspicuous filiform apparatus. The antipodal cells are present in the chalazal region, persisting before the occurrence of double fertilization. The zygote is visible 21 days after pollination; nuclear endosperm is already present. The first mitotic division of the zygote occurs at 24(th) day. The globular, cordiform and torpedo embryo stages can be seen at 30, 45 and 60 days after pollination, respectively. The mature embryo characterized by the presence of a well-developed hypocotyl-radicular axis with two fleshy and folded cotyledons was observed 120 days after pollination. Endosperm is absent in the seeds, and the embryo has spiral form, characteristic of Myrtinae. The zygotic embryology studies of A. sellowiana indicate that this species has embryological characteristics which are in agreement with those reported for Myrtaceae (Myrteae, Myrtinae), and also broaden the knowledge about the sexual reproduction of this native species, whose commercial cultivation has been growing.

Identification of non-zein proteins in BR473 maize protein bodies by LC-nanoESI-MS/MS

The nutritional value of maize seed is limited due to its high content of storage proteins (zeins), which are deficient in essential amino acids such as lysine and tryptophan. In a previous paper, we showed that protein bodies obtained from BR473 maize variety, developed by Embrapa (Brazilian Agricultural Research Corporation), were mainly constituted by Z27 and a smaller quantity of Z50 gamma-zeins. Besides zein proteins, other not identified protein band in the SDS/PAGE was also observed, which could indicate the presence of non-zein proteins additionally to gamma-zeins. In the present paper, we have demonstrated the presence of non-zein proteins in BR473 maize protein bodies by LC-nanoESI-MS/MS and database searching. This fact could be related to the excellent energetic value and higher protein quality of BR473 maize grains, since high lysine concentration in some maize varieties has been related to the presence of cytoskeleton proteins that are non-zeins. We have identified the following proteins: Brittle-1 protein (chloroplast precursor), Legumin-1, glyceroldehyde-3-phosphate dehydrogenase, and elongation factor 1-alpha.

Morfologia e avaliação do crescimento inicial de plântulas de Bactris gasipaes Kunth. (arecaceae) em diferentes substratos

Este trabalho teve por objetivos descrever a morfologia dos diásporos, as fases da germinação e determinar o substrato mais adequado para o crescimento inicial de plântulas de pupunha. Periodicamente, unidades representativas de cada fase de germinação foram retiradas para a descrição da seqüência dos eventos morfológicos. Os substratos usados para o crescimento inicial foram Plantmax HT, areia, terra (latossolo roxo) e outro com proporções iguais de terra, areia e esterco (TAE). Avaliou-se o crescimento inicial das plântulas aos 101 dias após o transplante, com base na sua altura, número de folhas, comprimento e largura das folhas. Observou-se que as sementes são albuminosas, com endosperma oleaginoso e de consistência relativamente dura. O embrião é lateral, periférico e relativamente indiferenciado, de forma cônica. A germinação inicia-se com o desenvolvimento de uma massa de células indiferenciadas na depressão micropilar. Posteriormente, esta massa de células torna-se cilíndrica, com a diferenciação dos primórdios caulinar e radicular. O primórdio caulinar é constituído por três bainhas envolvendo a primeira folha. Estas se abrem sucessivamente, permitindo a emergência da folha primária. Entre os substratos testados, de acordo com os parâmetros avaliados, os mais adequados para crescimento inicial de mudas de Bactris gasipaes Kunth foram o TAE e o Plantmax.

Aspectos morfológicos, germinação e desenvolvimento inicial de plântulas de Archontophoenix alexandrae (F. Mueller) H. Wendl. e Drude (Arecaceae) em diferentes substratos

Este trabalho teve por objetivo descrever a morfologia dos diásporos e as suas fases de germinação, bem como determinar o substrato mais adequado para a germinação das sementes e o crescimento das plântulas de palmeira-da-rainha (Archontophoenix alexandrae (F. Mueller) H. Wendl. e Drude). Periodicamente, unidades representativas de cada fase de germinação foram retiradas para as descrições morfológicas. Os substratos utilizados foram Plantmax®, areia, terra (latossolo roxo) e outro com proporções iguais de terra, areia e esterco (TAE). Foi instalado um experimento em delineamento inteiramente casualizado. Avaliaram-se a porcentagem, o tempo médio de germinação e o crescimento inicial das plântulas aos 135 dias após a emergência, com base na altura, diâmetro do colo e número, comprimento e largura das folhas. As sementes são albuminosas, com endosperma ruminado e oleaginoso, e o embrião é lateral, periférico e relativamente indiferenciado. A germinação é do tipo criptocotiledonar hipógea, iniciando-se com a formação de uma massa de células indiferenciadas na depressão micropilar. Essa massa de células torna-se cilíndrica, com a diferenciação dos primórdios caulinares e radiculares. Concomitantemente, ocorre o desenvolvimento de raízes adventícias no eixo embrionário. O substrato mais indicado para a germinação de sementes de palmeira-da-rainha é o Plantmax®, porém, para o crescimento inicial, indica-se Plantmax® e TAE.

Morfologia de frutos e sementes de espécies arbóreas do gênero Solanum L.

O objetivo do trabalho foi caracterizar a morfologia do fruto e da semente de três espécies florestais nativas do Brasil, Solanum granuloso-leprosum Dunal, S. lycocarpum A.St.-Hil. e S. pseudoquina A.St.-Hil., recomendadas para plantações destinadas à recuperação de áreas degradadas. Os frutos são indeiscentes, carnosos, do tipo baga, globosos, polispérmicos e constituídos por dois ou mais lóculos. As sementes são estenospérmicas, campilótropas, elipsóides, comprimidas, apresentando seção longitudinal largo-ovalada ou achatado-ovalada e seção transversal elíptica. Hilo mediano-marginal localizado em uma depressão e micrópila arredondada. Sementes albuminosas, com endosperma abundante, periférico, carnoso-firme, semitransparente e de coloração esbranquiçada. Embrião axial, linear, contínuo e curvado. em Solanum lycocarpum e S. pseudoquina o embrião é circinado e em S. granuloso-leprosum é espiralado.

Morphological Aspects and Storage of Dypsis leptocheilos (Hodel) Beentje & J. Dransf. (Arecaceae) Seeds

Dypsis leptocheilos (Hodel) Beentje & J. Dransf. palms are widely used in landscaping, however there is a lack of studies about its biology, especially on the morphology of its seedling disseminules and the ideal seed storage time without loss of viability. The objectives of this work were to describe the morphological aspects and to study the storage effect on the germination of D. leptocheilos seeds. The experiments were performed at FCAV/UNESP, Jaboticabal Campus, São Paulo State, Brazil. In order to obtain a morphology description 100 disseminules were sowed in vermiculite. The external and internal sides of the disseminules as well as the representative phases of the germination process were sketched with the help of a light chamber attached to a stereomicroscope. Five treatments were arranged in order to study the storage effects on the seeds (four months of storage and straight after harvest), with four replicates with 25 disseminules each (seeds with attached endocarp) in a completely randomized design. The germination test was conducted over a 140 days period and noted every two days. In order to calculate the germination percentage (% G) and the Germination Speed Index (GSI), the seeds were considered germinated when the germinative button appeared. The data were submitted to a polynomial regression analysis to verify the behavior of the variables over the storage period. The D. leptocheilos seeds have rounded shape, ruminate endosperm and hard consistency. The embryo is lateral, peripheral and relatively undifferentiated, with approximately 3 mm long and conical shape. The germination percentage increased from 17% for seeds sowed straight after harvest to 73% for those stored over a three-month period. For the GSI the increase was about 0.85%. It was concluded that the germination of D. leptocheilos is of an adjacent ligulate type. Furthermore, the seeds stored for 3 months at 14 degrees C showed both higher percentage and germination speed.

Seed anatomy and germination of Phoenix roebelenii O'Brien (Arecaceae)

O objetivo desse trabalho foi estudar a morfologia, anatomia e o processo germinativo de sementes de Phoenix roebelenii. Para o levantamento dos dados biométricos foram utilizadas 100 sementes de frutos recém-colhidos, deixados secar ao ar por um dia. Para a germinação, quatro repetições de 50 sementes tratadas com Vitavax-Thiran foram semeadas em bandejas de plástico, contendo Sphagnum sp. como substrato e mantidas sob condições ambientais de laboratório. Detalhes da morfologia da semente foram documentados com o uso de microscopia eletrônica de varredura e esquematizados com auxílio de câmara clara, acoplada ao estereomicroscópio. Foram confeccionadas lâminas permanentes com cortes do embrião, para o estudo de sua anatomia. As dimensões médias das sementes foram: 10,32mm comprimento, 5,21mm largura e 3,91mm espessura. O peso de 1000 sementes foi de 151,1g e 1kg continha 6600 unidades. O início da germinação variou entre 27 e 58 dias. As sementes são albuminosas, com endosperma duro e o embrião é pouco diferenciado, lateral e periférico. A germinação inicia-se pela abertura de um opérculo, através do qual é emitido o pecíolo cotiledonar com o eixo embrionário na extremidade. O pecíolo funciona, internamente, como um haustório, digerindo gradativamente o endosperma. Na sua parte posterior, desenvolve-se a plúmula, que emerge através de uma fenda. Nota-se o aparecimento de raízes secundárias na porção anterior da raiz primária.

Sucrose and nitrogen supplies regulate growth of maize kernels

The growth of maize (Zea mays L.) kernels depends on the availability of carbon (C) and nitrogen (N) assimilates supplied by the mother plant and the capacity of the kernel to use them. Our objectives were to study the effects of N and sucrose supply levels on growth and metabolism of maize kernels. Kernel explants of Pioneer 34RO6 were cultured in vitro with varying combinations of N (5 to 30 mM) and sucrose (117 to 467 mM). Maximum kernel growth was obtained with 10 mM N and 292 mM sucrose in the medium, and a deficiency of one assimilate could not be overcome by a sufficiency of the other. Increasing the N supply led to increases in the kernel sink capacity (number of cells and starch granules in the endosperm), activity of certain enzymes (soluble and bound invertases, sucrose synthase, and aspartate aminotransaminase), starch, and the levels of N compounds (total-N, soluble protein, and free amino acids), and decreased the levels of C metabolites (sucrose and reducing sugars). Conversely, increasing the sucrose supply increased the level of endosperm C metabolites, free amino acids, and ADPG-PPase and alanine transaminase activities, but decreased the activity of soluble invertase and concentrations of soluble protein and total-N. Thus, while C and N are interdependent and essential for accumulation of maximum kernel weight, they appear to regulate growth by different means. Nitrogen supply aids the establishment of kernel sink capacity, and promotes activity of enzymes relating to sucrose and nitrogen uptake, while sucrose regulates the activities df invertase and ADPG-PPase. (C) 1999 Annals of Botany Company.

Mechanism and control of Genipa americana seed germination

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Método de preparo das sementes de mamoneira (Ricinus communis L.) para o teste de tetrazólio

O teste de tetrazólio é um método rápido e eficaz para avaliar a viabilidade e o vigor de sementes. O presente trabalho teve por objetivo padronizar o método de preparo das sementes de mamoneira (Ricinus communis L.) para a avaliação do potencial fisiológico pelo teste de tetrazólio. Foram testados os seguintes métodos de preparo das sementes: corte longitudinal mediano através do tegumento, endosperma e embrião; corte longitudinal diagonal sem atingir o eixo embrionário; remoção do tegumento; remoção do tegumento com corte longitudinal mediano através do endosperma e embrião; e remoção do tegumento com corte longitudinal mediano, paralelo aos cotilédones, através do endosperma e embrião. Antes dos preparos, as sementes foram pré-condicionadas entre papel toalha umedecido por 18 horas a 30ºC, e após os preparos, as sementes foram imersas na solução de tetrazólio na concentração de 0,5% e mantidas em câmara escura a 35ºC para o desenvolvimento da coloração. Avaliou-se a uniformidade da coloração das sementes após cada preparo, por meio da comparação entre eles. Para a avaliação do potencial fisiológico das sementes de mamoneira pelo teste de tetrazólio o método indicado de preparo é a remoção do tegumento, com posterior corte longitudinal e mediano, no sentido do comprimento, através do endosperma e embrião.