Identification of a homolog of Arabidopsis DSP4 (SEX4) in chestnut: its induction and accumulation in stem amyloplasts during winter or in response to the cold_

Oligosaccharide synthesis is an important cryoprotection strategy used by woody plants during winter dormancy. At the onset of autumn, starch stored in the stem and buds is broken down in response to the shorter days and lower temperatures resulting in the buildup of oligosaccharides. Given that the enzyme DSP4 is necessary for diurnal starch degradation in Arabidopsis leaves, this study was designed to address the role of DSP4 in this seasonal process in Castanea sativa Mill. The expression pattern of the CsDSP4 gene in cells of the chestnut stem was found to parallel starch catabolism. In this organ, DSP4 protein levels started to rise at the start of autumn and elevated levels persisted until the onset of spring. In addition, exposure of chestnut plantlets to 4 °C induced the expression of the CsDSP4 gene. In dormant trees or cold-stressed plantlets, the CsDSP4 protein was immunolocalized both in the amyloplast stroma and nucleus of stem cells, whereas in the conditions of vegetative growth, immunofluorescence was only detected in the nucleus. The studies indicate a potential role for DSP4 in starch degradation and cold acclimation following low temperature exposure during activity–dormancy transition.

Brittle-1, an Adenylate Translocator, Facilitates Transfer of Extraplastidial Synthesized ADP-Glucose into Amyloplasts of Maize Endosperms1

Amyloplasts of starchy tissues such as those of maize (Zea mays L.) function in the synthesis and accumulation of starch during kernel development. ADP-glucose pyrophosphorylase (AGPase) is known to be located in chloroplasts, and for many years it was generally accepted that AGPase was also localized in amyloplasts of starchy tissues. Recent aqueous fractionation of young maize endosperm led to the conclusion that 95% of the cellular AGPase was extraplastidial, but immunolocalization studies at the electron- and light-microscopic levels supported the conclusion that maize endosperm AGPase was localized in the amyloplasts. We report the results of two nonaqueous procedures that provide evidence that in maize endosperms in the linear phase of starch accumulation, 90% or more of the cellular AGPase is extraplastidial. We also provide evidence that the brittle-1 protein (BT1), an adenylate translocator with a KTGGL motif common to the ADP-glucose-binding site of starch synthases and bacterial glycogen synthases, functions in the transfer of ADP-glucose into the amyloplast stroma. The importance of the BT1 translocator in starch accumulation in maize endosperms is demonstrated by the severely reduced starch content in bt1 mutant kernels.

Regulation of carotenoid biosynthesis in banana fruit

This thesis investigated the basis for massive differences in provitamin A carotenoid content in banana fruits. Rather than gene expression levels, carotenoid storage capacity and product degradation explained much of the differences. Such information should provide important insights for future developments in the biofortification of banana. A high carotenoid-containing cultivar, 'Asupina' and a popular commercial but low carotenoid-containing cultivar, 'Cavendish' were used in the investigations.

The quest for golden bananas: Investigating carotenoid regulation in a Fe’i group Musa cultivar

The regulation of carotenoid biosynthesis in a high-carotenoid-accumulating Fe’i group Musa cultivar, “Asupina”, has been examined and compared to that of a low-carotenoid-accumulating cultivar, “Cavendish”, to understand the molecular basis underlying carotenogenesis during banana fruit development. Comparisons in the accumulation of carotenoid species, expression of isoprenoid genes, and product sequestration are reported. Key differences between the cultivars include greater carotenoid cleavage dioxygenase 4 (CCD4) expression in “Cavendish” and the conversion of amyloplasts to chromoplasts during fruit ripening in “Asupina”. Chromoplast development coincided with a reduction in dry matter content and fruit firmness. Chromoplasts were not observed in “Cavendish” fruits. Such information should provide important insights for future developments in the biofortification and breeding of banana.

青藏高原青稞淀粉资源的评价

青稞（Hordeum vulgare L.var．nudum Hook．f.），即裸大麦，是兼食用、饲用和酿造于一体的作物，有着重要的利用价值。淀粉是青稞籽粒中含量最多、最重要的碳水化合物，淀粉含量、直支淀粉比将会直接影响淀粉的功能特性，进而影响淀粉的应用领域。我国青藏高原青稞的栽培和食用历史悠久，特色青稞资源极其丰富。目前关于青藏高原青稞淀粉特性的报道还不多见，筛选和培育特色淀粉青稞利于拓展青稞的应用领域, 从而提高其经济价值。 本研究以114份青藏高原青稞品种(系)为实验材料，通过SDS-PAGE对材料的胚乳淀粉颗粒结合蛋白（SGAPs）进行分离，确定各蛋白的分子量大小、组合类型和多态性等。然后按照国标法测试材料的籽粒总淀粉含量和直链淀粉含量，通过微型糊化粘度仪分析相应的淀粉糊化特性，最后使用显微镜观察比较了青稞的淀粉颗粒形态特征。主要结果如下： 1、114种青稞中共分离出20种不同的SGAP条带，条带分子量为35.00～112.39 KDa，分布频率为12.28～97.37%。材料含有的SGAPs条带数从10到14不等，超过一半的材料含11种SGAP条带。20种条带形成16种组合类型，其中西藏地区青稞包含所有16个组合类型，四川地区青稞包含其中12个组合类型。青藏高原青稞籽粒淀粉颗粒结合蛋白的差异很大，遗传多样性丰富。 2、114份青稞的总淀粉含量、直链淀粉含量、直支淀粉比、峰值粘度、糊化温度和峰值温度的变幅分别为51.26～66.70%、14.64～29.74%、0.17～0.42、194～1135BU、58.8～65.2℃和81.4～92.4℃，相应的平均值分别为59.82%、23.60%、0.31、722.30BU、62.1℃和88.8℃。群体在总淀粉含量、直链淀粉含量、直支淀粉比、峰值粘度、糊化温度和峰值温度上的分布具有明显的正态性；所有胚乳淀粉体的淀粉粒都呈复粒结构。对西藏和四川的材料进行了分组比较， 两地区的青稞在直链淀粉含量和直支淀粉比上的差异达到显著水平。 3、筛选出18份具有特殊淀粉特性的青稞品种，其中5份材料的总淀粉含量超过65%，包括NB63-1、NB67、甘孜白六棱、98221-1和NB63；3份材料的直链淀粉含量大于29%，包括藏青85、藏青3号和喜马拉6号；8份材料的直支淀粉比小于0.25，包括99033-6、春青稞、阿坝330、Jan-03、米麦114、396、NB63-1和92013；7份材料的糊化温度低于60℃，同时材料的峰值粘度大于1000BU,并且峰值温度低于90℃，包括足捉春、Jan-03、阿坝330、米麦114、春青稞、20003和阿青5号。 4、各淀粉特性间存在高度相关性。直链淀粉含量和直支淀粉比与糊化温度成极显著正相关，与峰值粘度成极显著负相关，与A型淀粉粒数量和大小呈负相关。不同SGAPs组合的品种之间，淀粉含量和淀粉糊化特性间差异均达显著水平。SGAP2、SGAP5、SGAP6和SGAP7可能对籽粒直链淀粉含量、直支淀粉比和糊化温度有正向效应；SGAP3、SGAP9∼SGAP20可能对峰值粘度有正向效应。 本研究对青藏高原青稞淀粉资源进行了较为全面的评价，对该区青稞淀粉特性有了系统的认识。研究筛选出的特殊青稞品种可作为青稞育种和青稞淀粉工业应用的潜在资源，淀粉特性差异巨大的众多青稞品种也为拓宽青稞应用领域提供了丰富的资源保障。本研究对部分SGAPs在性质上的鉴定和功能上的初步推断为青稞材料的筛选提供了指导，也为品质育种提供了理论参考。 Hulless barley (naked barley, Hordeum vulgare L.) is a short- season, early maturing crop with a wide range of adaptation. It has been attracting more and more attention due to its superior nutrition and extensive industrial applications. Starch is the main ingredient in hulless barley seeds which makes up 65 percent of hulless barley’s dry weight. The ratio of the amylose/amylopectin and the size, shape, distribution of starch granules can affect the physico-chemical and functional properties of starch, which may turn affect its utilizations. The Qinghai-Tibet Plateau, which is located in southwestern China, is a typical area of vertical agricultural ecosystem and one of the barley origin centers with abundant hulless barley resources. There are little reports about hulless barley in Qinghai-Tibet Plateau at present. To screen and cultivate some characteristic hulless barley can improve its value. An improved SDS-PAGE was used to identify SGAPs combination of 114 hulless barley varieties. Starch content (total starch and amylose starch) was determined according to the standard methods GB5006-85 and GB/T 15683 using PerkinElmer M341 Precision Automatic Polarimeter and UV spectrophotometer 755B respectively. The pasting properties were measured by BRABENDER Micrio Visco-Amylo- Graph 803201. The morphology of starch granules were observed and compared with Axioplan 2 Imaging light microscopy. The following were the results obtained: 1. There were 20 major SGAPs presented in 114 varieties, with the molecular weight ranged from 35.00 to 112.39 KDa, and the frequencies ranged from 12.28% to 97.37%. The number of SGAP bands in each accession varied from 10 to 14, more than half of the population had 11 bands. There were 16 distinct SGAP patterns in the 114 varieties, the Tibet hulless barley had all of the 16 types and the Sichuan hulless barley had 12 types. The results indicated the Qinghai-Tibet Plateau hulless barley had a polymorphism of the SGAPs. 2. The ranges of the total starch content, amylose content, Am/Ap, peak viscosity, pasting temperature and peak temperature of the 114 hulless barley were 51.26～66.70%,14.64～29.74%,0.17～0.42,194～1135BU,58.8～65.2 and 81.4℃～92.4, with an average of ℃59.82%, 23.60%, 0.31, 722.30BU, 62.1 and 88.8,℃℃ respectively. The distributions of the total starch content, amylose content, Am/Ap, peak viscosity, pasting temperature and peak temperature were visibly normal school. All of the amyloplasts in endosperm of varieties showed bimodal size distributions.The main starch properties of hulless barley from Tibet and Sichuan were separated and compared, the differences on amylose content and Am/Ap were obvious. 3. Eighteen accessions which had special starch properties were screened out. Five accessions with total starch content beyond 65%, including NB63-1, NB67, Ganzibailiuleng, 98221-1 and NB63; three accessions, Zangqing85, Zangqing3 and Ximala6, with the highest amylose content (>29%); five accessions with Am/Ap less than 0.25, including 99033-6, Chun Qingke, A Ba 330, Jan-03, Mi Mai114, 396, NB63-1 and 92013; seven accessions had a pasting temperature under 60, ℃meanwhile their peak viscosity beyond 1000BU and their peak temperature under 90℃，including Zu Cuochun, Jan-03, A Ba 330, Mi Mai 114, Chun Qingke, 20003 and A Qing 5. 4. There were high correlations between starch properties. Amylose content and Am/Ap were positively correlated to pasting temperature, negatively correlated to peak viscosity, negatively correlated to the number and granule size of A-type granule. Different SGAP combinations caused significant diversities in starch content and pasting properties. SGAP2, SGAP5, SGAP6 and SGAP7 may have positive effect on amylose content, Am/Ap and pasting temperature; SGAP3, SGAP9∼SGAP20 may have positive effect on peak viscosity. Our research made a comprehensive evaluation on the hulless barley starch from the Qinghai-Tibet Plateau, we can get a systemic understanding. Some special accessions were screened out can be used on the hulless barley breeding lines and industries utilization.The combination of the SGAPs may become a criterion to evaluate the hulless barley endosperm starch quality. Consequently, the results will be good information for further studies on the hulless barley.

Nuclear magnetic resonance water relaxation time changes in bananas during ripening: a new mechanism

BACKGROUND: Nuclear magnetic resonance studies of banana fragments during ripening show an increase on the water transverse relaxation time (T(2)) and a decrease in water self-diffusion coefficient (D). As T(2) and D are normally directly correlated, we studied these two properties in intact bananas during ripening, in an attempt to rule out the effect of injury on the apparent discrepancies in the behavior of T(2) and D. RESULTS: The results show that injury in bananas causes a decrease in T(2) of the water in vacuoles (T(2vac)). They also show that T(2vac) increased and D decreased during ripening, ruling out the injury effect. To explain the apparent discrepancies, we propose a new hypothesis for the increase in T(2) values, based on the reduction of Fe(3+) ions to Fe(2+) by galacturonic acid, produced by the hydrolysis of pectin and a decrease in internal oxygen concentration during ripening. CONCLUSION: As injury alters T(2) values it is necessary to use intact bananas to study relaxation times during ripening. The novel interpretation for the increase in T(2vac) based on reduction of Fe(+3) and O(2) concentration is an alternative mechanism to that based on the hydrolysis of starch in amyloplasts. (C) 2010 Society of Chemical Industry

Detection of brassinosteroids in pollen of Lolium perenne L. by immunocytochemistry

Bioactive brassinosteroids have been localized in developing and mature pollen of anhydrously fixed rye-grass (Lolium perenne) by immunocytochemistry using polyclonal antibodies to castasterone generated in rabbits. Tricellular pollen fixed by freeze-substitution was also labelled in the starch granules. Study of the developmental sequence of the pollen through the microsporocyte, microspore, bicellular and tricellular stages showed that the brassinosteroids were increasingly sequestered in starch granules as the amyloplasts matured, supporting the view that these are storage organelles for these potent plant growth promoters. In bicellular pollen, heavy labelling was seen in the zone within 0.5 μm of the starch granule, where stromal tissue remains. Thus, the stroma may be the site of synthesis of these compounds. During aqueous fixation, the brassinosteroids leached from the starch granules of tricellular pollen, indicating that they would be quickly available after imbibition to influence the physiology of germinating pollen. The results from high-performance liquid chromatography of dansylaminophenylboronates from partially purified extracts of freshly dehisced tricellular pollen of rye-grass showed 25-methylcastasterone may be a minor component, together with two unknown peaks. No specific binding of brassinolide to any soluble proteins extracted from tricellular rye-grass pollen was observed using the antibodies in gel electrophoresis or enzyme-linked immunosorbent assays.

Cellular localization of water soluble, allergenic proteins in rye-grass (Lolium perenne) pollen using monoclonal and specific IgE antibodies with immunogold probes

A postembedding method has been developed for localizing water soluble allergens in rye-grass pollen. This uses dry fixation in glutaraldehyde vapour, followed by 2,2-dimethoxypropane, prior to a 100% ethanol series leading into embedment in LR Gold. This has allowed the attachment of specific monoclonal antibodies to the allergen, which are themselves probed with specific immunogold labels to the antibodies. Wall and cytoplasmic sites have been identified, representing an improvement of fixation and localization of allergens over previous studies employing polyclonal, broad spectrum antibodies.

Rye-grass allergens are labelled in mature pollen grains in the exine (tectum, nexine and central chamber), and in the electron opaque areas of the cytoplasm, especially mitochondria. The allergens are absent from the intine, polysaccharide (P) particles, amyloplasts, Golgi bodies and endoplasmic reticulum. IgE antibodies derived from humans allergic to rye-grass pollen, bind to similar sites in the cytoplasm but only to the outer surface of the pollen grain wall. This method now provides a valuable tool for further developmental studies on the pollen grains, in order to establish the site/s of synthesis of the allergens.

Isolation of cDNA encoding a newly identified major allergenic protein of rye-grass pollen : intracellular targeting to the amyloplast

We have identified a major allergenic protein from rye-grass pollen, tentatively designated Lol pIb of 31kDa and with pI 9.0. A cDNA clone encoding Lol pIb has been isolated, sequenced, and characterized. Lol pIb is located mainly in the starch granules. This is a distinct allergen from Lol pI, which is located in the cytosol. Lol pIb is synthesized in pollen as a pre-allergen with a transit peptide targeting the allergen to amyloplasts. Epitope mapping of the fusion protein localized the IgE binding determinant in the C-terminal domain.

Development of plastids in pollen and tapetum of rye-grass, Lolium perenne L

Proplastids of both tapetal cells and microsporocytes were present early in anther development. Tapetal proplastids differentiated—probably into elaioplasts—at late microspore stage. The tapetal cytoplasm was completely resorbed by early tricellular pollen stage. Microspore proplastids differentiated into amyloplasts at early bicellular stage, and were present in both vegetative and generative cells. In the generative cell, the amyloplasts were ephemeral and apparently degenerated within autophagic vacuoles. Plastids were absent from sperm cells. Vegetative cell amyloplasts increased in number apparently by fission such that one amyloplast produced one amyloplast and one proplastid per division. Mature pollen grains were estimated to contain between 550 and 820 amyloplasts with only one starch granule per plastid.

The floral nectary of Hymenaea stigonocarpa (Fabaceae, caesalpinioideae): Structural aspects during floral development

Background and Aims Considering that few studies on nectary anatomy and ultrastructure are available for chiropterophilous flowers and the importance of Hymenaea stigonocarpa in natural 'cerrado' communities, the present study sought to analyse the structure and cellular modifications that take place within its nectaries during the different stages of floral development, with special emphasis on plastid dynamics.Methods For the structural and ultrastructural studies the nectary was processed as per usual techniques and studied under light, scanning and transmission electron microscopy. Histochemical tests were employed to identify the main metabolites on nectary tissue and secretion samples.Key Results The floral nectary consists of the inner epidermis of the hypanthium and vascularized parenchyma. Some evidence indicates that the nectar release occurs via the stomata. The high populations of mitochondria, and their juxtaposition with amyloplasts, seem to be related to energy needs for starch hydrolysis. Among the alterations observed during the secretory phase, the reduction in the plastid stromatic density and starch grain size are highlighted. When the secretory stage begins, the plastid envelope disappears and a new membrane is formed, enclosing this region and giving rise to new vacuoles. After the secretory stage, cellular structures named 'extrastomatic bodies' were observed and seem to be related to the nectar resorption.Conclusions Starch hydrolysis contributes to nectar formation, in addition to the photosynthates derived directly from the phloem. In these nectaries, the secretion is an energy-requiring process. During the secretion stage, some plastids show starch grain hydrolysis and membrane rupture, and it was observed that the region previously occupied by this organelle continued to be reasonably well defined, and gave rise to new vacuoles. The extrastomatic bodies appear to be related to the resorption of uncollected nectar.

Flowering phenology, nectary structure and breeding system in Corymborkis flava (Spiranthoideae : Tropidieae), a terrestrial orchid from a Neotropical forest

Flowering phenology, breeding system and nectary structure of Corymborkis flava (Sw.) Kuntze were studied in a fragment of the Atlantic Forest in south-eastern Brazil. The flowering period extended from March (end of rainy season) to early June and seed dispersal occurred from June to September (dry season). Flowering peak occurred mainly in April, with up to 34 open flowers per plant being observed. The yellow, odourless and tubular flowers lasted similar to 7.8 days. The flowers present a perigonal nectary located in the basal lateral parts of the labellum; this is the first report on the nectary location and characterisation in the Tropidieae tribe. At the pre-anthesis stage, cells of both secretory parenchyma and epidermis of the nectary are filled with compound amyloplasts. However, starch grains were not observed in these tissues in senescent flowers, indicating that these starch grains are hydrolysed and used as source of sugars for nectar production. The nectar accumulates between the cuticle and the outer periclinal wall of the epidermal cells before. owing out into the nectar chamber. C. flava is a self-compatible species and spontaneous self-pollination does not occur because of hercogamy. The high pollinia removal (0.80) and insertion (0.82) per flower, as well as the high natural fruit-set indicate an efficient natural pollination system. The present study contributes for the knowledge of the diversity of reproductive strategies and nectary structures in Orchidaceae.

Ontogenia do óvulo e da Antera de Cybistax antisyphilitica (Mart.) Mart. (Bignoniaceae)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Floral nectar production and nectary structure of a bee-pollinated shrub from Neotropical savanna

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Recuperação de amidos remanescentes em resíduos de extração da fécula de mandioca

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)