Amyloplast Envelope Research Articles

Mecanismos asociados con la modificación del endospermo en maíz de calidad proteínica

Protein quality maize (QPM) combines the protein quality of the opaque-2 (o2) mutant with a vitreous endosperm. These characteristics have allowed breeding programs worldwide to produce QPM genotypes that help alleviate malnutrition of people in developing countries from Africa, Asia and Latin America with a cereal-based diet. However, the development of these materials has been inefficient due to the limited knowledge about the molecular basis of the conversion of the soft o2 endosperm into a vitreous phenotype in QPM. This conversion has been associated with an increase in small protein bodies rich in 27 kDa γ-zein, the synthesis of starch with a higher proportion of amylose and short-intermediate amylopectin chain branches that favors the compaction of the starch granules, as well as alterations in the amyloplast envelope that favors the interaction between starch granules and protein bodies. Additional studies about the mechanisms involved in the modification of the endosperm in QPM will contribute to produce materials with good agronomic characteristics and protein quality.

Carotenoids modulate kernel texture in maize by influencing amyloplast envelope integrity

The mechanism that creates vitreous endosperm in the mature maize kernel is poorly understood. We identified Vitreous endosperm 1 (Ven1) as a major QTL influencing this process. Ven1 encodes β-carotene hydroxylase 3, an enzyme that modulates carotenoid composition in the amyloplast envelope. The A619 inbred contains a nonfunctional Ven1 allele, leading to a decrease in polar and an increase in non-polar carotenoids in the amyloplast. Coincidently, the stability of amyloplast membranes is increased during kernel desiccation. The lipid composition in endosperm cells in A619 is altered, giving rise to a persistent amyloplast envelope. These changes impede the gathering of protein bodies and prevent them from interacting with starch grains, creating air spaces that cause an opaque kernel phenotype. Genetic modifiers were identified that alter the effect of Ven1A619, while maintaining a high β-carotene level. These studies provide insight for breeding vitreous kernel varieties and high vitamin A content in maize.

OsBT1 encodes an ADP-glucose transporter involved in starch synthesis and compound granule formation in rice endosperm

Starch is the main storage carbohydrate in higher plants. Although several enzymes and regulators for starch biosynthesis have been characterized, a complete regulatory network for starch synthesis in cereal seeds remains elusive. Here, we report the identification and characterization of the rice Brittle1 (OsBT1) gene, which is expressed specifically in the developing endosperm. The osbt1 mutant showed a white-core endosperm and a significantly lower grain weight than the wild-type. The formation and development of compound starch granules in osbt1 was obviously defective: the amyloplast was disintegrated at early developmental stages and the starch granules were disperse and not compound in the endosperm cells in the centre region of osbt1 seeds. The total starch content and amylose content was decreased and the physicochemical properties of starch were altered. Moreover, the degree of polymerization (DP) of amylopectin in osbt1 was remarkably different from that of wild-type. Map-based cloning of OsBT1 indicated that it encodes a putatively ADP-glucose transporter. OsBT1 coded protein localizes in the amyloplast envelope membrane. Furthermore, the expression of starch synthesis related genes was also altered in the osbt1 mutant. These findings indicate that OsBT1 plays an important role in starch synthesis and the formation of compound starch granules.

Deficiency of Starch Synthase IIIa and IVb Alters Starch Granule Morphology from Polyhedral to Spherical in Rice Endosperm.

Starch granule morphology differs markedly among plant species. However, the mechanisms controlling starch granule morphology have not been elucidated. Rice (Oryza sativa) endosperm produces characteristic compound-type granules containing dozens of polyhedral starch granules within an amyloplast. Some other cereal species produce simple-type granules, in which only one starch granule is present per amyloplast. A double mutant rice deficient in the starch synthase (SS) genes SSIIIa and SSIVb (ss3a ss4b) produced spherical starch granules, whereas the parental single mutants produced polyhedral starch granules similar to the wild type. The ss3a ss4b amyloplasts contained compound-type starch granules during early developmental stages, and spherical granules were separated from each other during subsequent amyloplast development and seed dehydration. Analysis of glucan chain length distribution identified overlapping roles for SSIIIa and SSIVb in amylopectin chain synthesis, with a degree of polymerization of 42 or greater. Confocal fluorescence microscopy and immunoelectron microscopy of wild-type developing rice seeds revealed that the majority of SSIVb was localized between starch granules. Therefore, we propose that SSIIIa and SSIVb have crucial roles in determining starch granule morphology and in maintaining the amyloplast envelope structure. We present a model of spherical starch granule production.

Geometrical Formation of Compound Starch Grains in Rice Implements Voronoi Diagram.

Starch forms transparent grains, called starch grains (SGs), in amyloplasts. One of the major morphological SG forms in Poaceae, called a compound SG, is formed by assemblies of small starch granules in an amyloplast. Starch granules assemble as a well-ordered structure; however, the mechanism that regulates this organization has not been identified. In this study, we examined how starch granules grow and converge into the final SG morphology. First, we found that the number of starch granules in an amyloplast is almost constant from the early developmental stage until endosperm maturity. Next, we quantitatively evaluated the geometrical similarities between starch granules and a Voronoi diagram, which is a mathematical tessellation of space based on the distance to a specific set of points in the space. The in silico growth simulation showed that the geometrical patterns of compound SGs resembling a Voronoi diagram is determined by physical interactions among the free-growing starch granules and the amyloplast envelope membrane. The geometrical similarity between compound SGs and a Voronoi diagram is likely a result of maximum loading and storage of starch in the amyloplast. The simulation described in this study provides a greater understanding of how compound SGs are formed and also has the potential to explain morphological variations of SGs.

Multiple Subcellular Localizations of Dehydrin-like Proteins in the Embryonic Axes of Common Beech (Fagus sylvatica L.) Seeds During Maturation and Dry Storage

The accumulation and localization of dehydrin-like proteins were analyzed in the embryonic axes of beech (Fagus sylvatica L.) seeds during maturation between the 12th and 19th week after flowering and in the dry state after 2 years of storage. Three dehydrin-like proteins were reported using an antibody specific to the K-segment, and their subcellular localization was investigated using an immunocytochemistry approach. All dehydrin-like proteins were phosphorylated, and two, with molecular masses of 26 and 44 kDa, accumulated in mature seeds. During seed maturation, dehydrin-like proteins were frequently detected inside the round or segmented nuclei, along the cell plasma membrane and associated with small vesicles. Moreover, they were found to be attached to the mitochondrial and amyloplast envelopes. These proteins were also located in the cytosol and were associated with membrane structures throughout the cytoplasm. Conversely, in the embryonic axes of stored seeds, fewer specific locations were found; dehydrin-like proteins were associated mostly with amyloplasts, and were detected to a lesser extent in the nuclei, vacuoles, and other cytoplasmic structures. Bioinformatic tools were used to predict the putative beech dehydrin sites that interact with DNA, proteins, and lipid membranes to highlight the reported subcellular locations. Here, we present and discuss the possible roles of dehydrin-like proteins in seeds with respect to their subcellular localizations.

Light-Induced Degradation of Starch Granules in Turions of Spirodela polyrhiza Studied by Electron Microscopy

Spirodela polyrhiza forms turions, starch-storing perennial organs. The light-induced process of starch degradation starts with an erosion of the surface of starch grains. The grain size decreases over a period of red irradiation and the surface becomes rougher. The existence of funnel-shaped erosion structures demonstrates that starch degradation is also possible inside the grains. Neither etioplasts nor clues as to their transition into chloroplasts were found in the storage tissue by transmission electron microscopy. Juvenile chloroplasts always contained the starch grains which remained from amyloplasts. No chloroplasts were found which developed independently of starch grains. Amyloplasts are therefore the only source of chloroplasts in the cells of irradiated turions. The intactness of amyloplast envelope membranes could not be directly proved by electron microscopy. However, the light-induced transition of amyloplasts into chloroplasts provides indirect evidence for the integrity of the envelope membranes throughout the whole process. The starch grains are sequestered from the cytosolic enzymes, and only plastid-localized enzymes, which have access to the starch grains, can carry out starch degradation. In this respect the turion system resembles transitory starch degradation as known from Arabidopsis leaves. On the other hand, with α-amylase playing the dominant role, it resembles the mechanism operating in the endosperm of cereals. Thus, turions appear to possess a unique system of starch degradation in plants combining elements from both known starch-storing systems.

Physicochemical properties and development of wheat large and small starch granules during endosperm development

Wheat mature seeds have large, lenticular A-type starch granules, and small, spherical B-type and irregular C-type starch granules. During endosperm development, large amyloplasts came from proplastid, divided and increased in number through binary fission from 4 to 12 days after flowering (DAF). Large starch granules formed and developed in the large amyloplast. One large amyloplast had only one large starch granule. Small amyloplasts came from the protrusion of large amyloplast envelope, divided and increased in number through envelope protrusion after 12 DAF. B-type starch granules formed and developed in small amyloplast from 12 to 18 DAF, C-type starch granules formed and developed in small amyloplast after 18 DAF. Many B- and C-type starch granules might form and develop in one small amyloplast. The amyloplast envelopes were asynchronously degraded and starch granules released into cell matrix when amyloplasts were full of starch granules. Apparent amylose contents of large starch granules were higher than that of small starch granules, and increased with endosperm development. The swelling powers and crystallinity of large starch granule were lower than that of small starch granules, and decreased with endosperm development. Small starch granules displayed broader gelatinization temperature ranges than did large starch granules.

Development of Small Starch Granule in Barley Endosperm

The structural characteristics and physicochemical properties of barley starch differ with granule size, which are important in final product applications of barley flours. A barley ( Hordeum vulgare L.) cultivar, Yangsimai 3, was used to investigate the development of small starch granule in endosperm under electron microscope to provide references to barley breeding. Large starch granules were formed and developed at the early stage of endosperm development. Large amyloplasts with large starch granules were divided and increased in number through binary fission. One large amyloplast had only 1 large starch granule. Small starch granules could be formed and develop at the middle and late stage of endosperm development. Endosperm cells had large amyloplasts that exhibited protrusion, and some of the protrusions contained small starch granules. Small amyloplasts with small starch granules formed new small amyloplasts to produce small starch granules by the way of protruding their envelopes. Many small starch granules were formed and developed in 1 small amyloplast. The amyloplast envelope began to degrade and release starch granules into cell matrix when amyloplast was full of starch granules. These results showed that small amyloplasts came from the protrusion of amyloplast envelopes. Many small starch granules, which were compound starch granules, were formed and developed in 1 small amyloplast.

The Maize Phytoene Synthase Gene Family: Overlapping Roles for Carotenogenesis in Endosperm, Photomorphogenesis, and Thermal Stress Tolerance

Carotenoids are essential for photosynthesis and photoprotection; they also serve as precursors to signaling molecules that influence plant development and biotic/abiotic stress responses. With potential to improve plant yield and nutritional quality, carotenoids are targets for metabolic breeding/engineering, particularly in the Poaceae (grass family), which includes the major food crops. Depending on genetic background, maize (Zea mays) endosperm carotenoid content varies, and therefore breeding-enhanced carotenoid levels have been of ongoing interest. The first committed step in the plastid-localized biosynthetic pathway is mediated by the nuclear-encoded phytoene synthase (PSY). The gene family in maize and other grasses contains three paralogs with specialized roles that are not well understood. Maize endosperm carotenoid accumulation requires PSY1 expression. A maize antibody was used to localize PSY1 to amyloplast envelope membranes and to determine PSY1 accumulation in relation to carotenoid accumulation in developing endosperm. To test when and if PSY transcript levels correlated with carotenoid content, advantage was taken of a maize germplasm diversity collection that exhibits genetic and chemical diversity. Total carotenoid content showed statistically significant correlation with endosperm transcript levels at 20 d after pollination for PSY1 but not PSY2 or PSY3. Timing of PSY1 transcript abundance, previously unknown, provides critical information for choosing breeding alleles or properly controlling introduced transgenes. PSY1 was unexpectedly found to have an additional role in photosynthetic tissue, where it was required for carotenogenesis in the dark and for heat stress tolerance. Leaf carotenogenesis was shown to require phytochrome-dependent and phytochrome-independent photoregulation of PSY2 plus nonphotoregulated PSY1 expression.

Molecular and Biochemical Analysis of the Plastidic ADP-glucose Transporter (ZmBT1) from Zea mays

Physiological studies on the Brittle1 maize mutant have provided circumstantial evidence that ZmBT1 (Zea mays Brittle1 protein) is involved in the ADP-Glc transport into maize endosperm plastids, but up to now, no direct ADP-Glc transport mediated by ZmBT1 has ever been shown. The heterologous synthesis of ZmBT1 in Escherichia coli cells leads to the functional integration of ZmBT1 into the bacterial cytoplasmic membrane. ZmBT1 transports ADP-Glc in counterexchange with ADP with apparent affinities of about 850 and 465 mum, respectively. Recently, a complete ferredoxin/thioredoxin system has been identified in cereal amyloplasts and BT1 has been proposed as a potential Trx target protein (Balmer, Y., Vensel, W. H., Cai, N., Manieri, W., Schurmann, P., Hurkman, W. J., and Buchanan, B. B. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 2988-2993). Interestingly, we revealed that the transport activity of ZmBT1 is reversibly regulated by redox reagents such as diamide and dithiothreitol. The expression of ZmBT1 is restricted to endosperm tissues during starch synthesis, whereas a recently identified BT1 maize homologue, the ZmBT1-2, exhibits a ubiquitous expression pattern in hetero- and autotrophic tissues indicating different physiological roles for both maize BT1 isoforms. BT1 homologues are present in both mono- and dicotyledonous plants. Phylogenetic analyses classify the BT1 family into two phylogenetically and biochemically distinct groups. The first group comprises BT1 orthologues restricted to cereals where they mediate the ADP-Glc transport into cereal endosperm storage plastids during starch synthesis. The second group occurs in mono- and dicotyledonous plants and is most probably involved in the export of adenine nucleotides synthesized inside plastids.

Characterization of ADP-glucose transport across the cereal endosperm amyloplast envelope

Most of the carbon used for starch biosynthesis in cereal endosperms is derived from ADP-glucose (ADP-Glc) synthesized by extra-plastidial AGPase activity, and imported directly across the amyloplast envelope. The properties of the wheat endosperm amyloplast ADP-Glc transporter were analysed with respect to substrate kinetics and specificities using reconstituted amyloplast envelope proteins in a proteoliposome-based assay system, as well as with isolated intact organelles. Experiments with liposomes showed that ADP-Glc transport was dependent on counter-exchange with other adenylates. Rates of ADP-Glc transport were highest with ADP and AMP as counter-exchange substrates, and kinetic analysis revealed that the transport system has a similar affinity for ADP and AMP. Measurement of ADP and AMP efflux from intact amyloplasts showed that, under conditions of ADP-Glc-dependent starch biosynthesis, ADP is exported from the plastid at a rate equal to that of ADP-Glc utilization by starch synthases. Photo-affinity labelling of amyloplast membranes with the substrate analogue 8-azido-[alpha-32P]ADP-Glc showed that the polypeptide involved in substrate binding is an integral membrane protein of 38 kDa. This study shows that the ADP-Glc transporter in cereal endosperm amyloplasts imports ADP-Glc in exchange for ADP which is produced as a by-product of the starch synthase reaction inside the plastid.

Effect of Temperature on the Decomposition of Reserves during Germination and Early Growth of Rice Plants

Morphological changes in starch granules in endosperm cells of rice, cv. Cisadane (javanica) and cv. Koshihikari (japonica) , during germination and early growth of seedlings at 17°C, ambient temperature (field condition, 16-24°C) and 38, respectively, were examined with scanning and transmission electron microscopes. With the treatment at 17°C, a groove-like structure appeared on the surface of amyloplasts at five days after seeding (DDS) .After the amyloplast envelope disappeared and starch granules were separated from each other, small holes, which were observed at higher temperatures, did not appear, and a "rough structure" appeared on the granule surface. At 15 DDS, starch granules adjacent to scutellum were partly decomposed but those at the distal end of the seed did not decompose until 15 DAS. In the seed germinated at 38°C, starch granules in endosperm cells adjacent to scuteflum began to decompose within one day after seeding, and small holes appeared on the surface of starch granules. At further s ages of decomposition, a layer-like structures was observed inside of the starch granule that followed by the appearance of highly viscous materials on the resolvinggranules and decomposed almost completely at 15 DAS. At this time, many mitochondria that seemed to be related with decomposition of starch granules were observed by transmission electron microscopy. At an ambient temperature, the decompositionhas progressed at 15 DDS with the vanishing of amyloplast envelopes in the whole of endosperm. In cv. Koshihikari most of the starch granules had decomposed at 20 DAS.

Morphological Studies on the Mobilization of Reserves in Japanese Yam (Dioscorea japonica Thunb.) Seed Tuber and Eddo (Colocasia esculenta Schott var. antiquorum Hubbard & Rehder) Seed Corm on and after Sprouting

We examined the mechanism of reserve mobilization in Japanese yam seed tubers and eddo seed corms on and after sprouting. The decomposition of starch in pith parenchyma cells of Japanese yam tubers and eddo corms progressed from the region distant from vascular bundles to that adjacent to vascular bundles. In eddo corms, the starch also decomposed from the proximal to the distal region adjacent to the sprout or regenerate plant body. In the yam tubers, the decomposition process was similar in the proximal, middle and distal regions. The first step in the reserve mobilization in pith parenchyma cells was the decomposition of the amyloplast envelope. Subsequently, starch granules decomposed. In Japanese yam tubers, the envelope and starch granules started to decompose from the peripheral regions of the amyloplasts. The observation of soluble polysaccharides, which was the decomposition product of starch granules, was made possible by the quick freezing-vacuum freeze-drying method. By this method, we demonstrated that the soluble polysaccharides in the parenchyma cells decomposed and decreased in density. In addition, the mucilage in the mucilage duct started to decompose and decreased in density from the proximal to the distal part of the corm and also from the periphery to the center of the duct. It was shown that not only starch mobilization but also mucilage mobilization mainly supported sprouting and the growth of the regenerate plant body during about the first half of the vegetative stage.

Immunolocalization of actin in root statocytes of Lens culinaris L.

Lentil root statocytes show a strict structural polarity of their organelles with respect to the g vector. These cells are involved in the perception of gravity and are responsible for the orientation of the root. Actin filaments take part in the positioning of their organelles and could also be involved in the transduction of the gravitropic signal. A pre-embedding immunogold silver technique was carried out with a monoclonal antibody in order to study the distribution of actin cytoskeleton in the statocytes at the electron microscopic level. Some areas were never labelled (cell wall, vacuole, nucleoplasm, mitochondria, starch grains of the amyloplasts) or very slightly labelled (stroma of the amyloplasts). The labelling was scattered in the cytoplasm always close to, or on the nuclear and amyloplast envelopes and the tonoplast. Associations of 2 to 6 dots in file were observed, but these short files were not oriented in one preferential direction. They corresponded to a maximum distance of 0.9 micron. This work demonstrated that each statocyte organelle was enmeshed in an actin web of short filaments arranged in different ways. The images obtained by rhodaminephalloidin staining were in accordance with those of immunogold labelling. The diffuse fluorescence of the cytoplasm could be explained by the fact that the meshes of the web should be narrow. The vicinity of actin and of the amyloplasts envelope could account for the movement of these organelles that was observed in spatial microgravity.

Morphological Studies on the Mobilization of Reserves in Germinating Rice Seed

The first step in the mobilization of the reserves in germinating rice seed was the decomposition of amyloplast envelopes in endosperm cells adjacent to scutellum. Destruction of amyloplast envelopes caused the separation of the starch granules. Small holes appeared on the surface of the released starch granule adjacent to scutellum at 3 days after imbibition (DAI). At later stages, the number and size of the holes increased on the surface of the starch granule. Ring-like structure in the interior of starch granule was recognized in endosperm cell adjacent to scutellum at 6 DAI. Like this structure appeared in the starch granule at the central part and at the top part of endosperm at 7 and 14 DAI respectively. At 18 DAI, the remaining starch granules became smaller in size and were covered with high-viscosity materials assumed to be soluble polysaccharides. Transmission electron microscopy showed that the intact starch granules were enveloped tightly within the amyloplast membrane. When the amyloplast was degraded, the exterior of the starch granules also started to decompose. The starch granules were decomposed from the exterior rather than the interior.

Sucrose-Starch Conversion in Heterotrophic Tissues of Plants

The historical progress in recent years pertaining to the sucrose-starch conversion in heterotrophic tissues of plants has been described. Special attention has been focused on the enzymatic breakdown of sucrose to produce hexose units that are transported to the amyloplast compartment by means of specific translocator molecules and act as glucose donors for starch biosynthesis. Although the current prevailing view is that variable mechanisms operate in different plant tissues and organs, it is often argued that the following enzymic steps are essential in the overall step of sucrose to starch conversion: sucrose + UDP -> UDPGlc + Fru(sucrose synthase-SS) (UDPGlc UDPGlc + PPi -» G1P + UTPpyrophosphorylase-UGPase) (ADPGlc GlP + ATP -» ADPGlc + PPipyrophosphorylase-AGPase) (starch ADPGlc -> starchsynthase) The presence of an ADPGlc-specific translocator in the amyloplast envelope has been demonstrated in a number of plant sources, which indicates the potential role of ADPGlc-synthesizing machineries loc...

Surface Localization of Zein Storage Proteins in Starch Granules from Maize Endosperm1

Starch granules from maize (Zea mays) contain a characteristic group of polypeptides that are tightly associated with the starch matrix (C. Mu-Forster, R. Huang, J.R. Powers, R.W. Harriman, M. Knight, G.W. Singletary, P.L. Keeling, B.P. Wasserman [1996] Plant Physiol 111: 821-829). Zeins comprise about 50% of the granule-associated proteins, and in this study their spatial distribution within the starch granule was determined. Proteolysis of starch granules at subgelatinization temperatures using the thermophilic protease thermolysin led to selective removal of the zeins, whereas granule-associated proteins of 32 kD or above, including the waxy protein, starch synthase I, and starch-branching enzyme IIb, remained refractory to proteolysis. Granule-associated proteins from maize are therefore composed of two distinct classes, the surface-localized zeins of 10 to 27 kD and the granule-intrinsic proteins of 32 kD or higher. The origin of surface-localized delta-zein was probed by comparing delta-zein levels of starch granules obtained from homogenized whole endosperm with granules isolated from amyloplasts. Starch granules from amyloplasts contained markedly lower levels of delta-zein relative to granules prepared from whole endosperm, thus indicating that delta-zein adheres to granule surfaces after disruption of the amyloplast envelope. Cross-linking experiments show that the zeins are deposited on the granule surface as aggregates. In contrast, the granule-intrinsic proteins are prone to covalent modification, but do not form intermolecular cross-links. We conclude that individual granule intrinsic proteins exist as monomers and are not deposited in the form of multimeric clusters within the starch matrix.

ADP-glucose drives starch synthesis in isolated maize endosperm amyloplasts: characterization of starch synthesis and transport properties across the amyloplast envelope.

We recently developed a method of purifying amyloplasts from developing maize (Zea mays L.) endosperm tissue [Neuhaus, Thom, Batz and Scheibe (1993) Biochem. J. 296, 395-401]. In the present paper we analyse how glucose 6-phosphate (Glc6P) and other phosphorylated compounds enter the plastid compartment. Using a proteoliposome system in which the plastid envelope membrane proteins are functionally reconstituted, we demonstrate that this type of plastid is able to transport [14C]Glc6P or [32P]Pi in counter exchange with Pi, Glc6P, dihydroxyacetone phosphate and phosphoenolpyruvate. Glucose 1-phosphate, fructose 6-phosphate and ribose 5-phosphate do not act as substrates for counter exchange. Besides hexose phosphates, ADP-glucose (ADPGlc) also acts as a substrate for starch synthesis in isolated maize endosperm amyloplasts. This process exhibits saturation kinetics with increasing concentrations of exogenously supplied [14C]ADPGlc, reaching a maximum at 2mM. Ultrasonication of isolated amyloplasts greatly reduces the rate of ADPGlc-dependent starch synthesis, indicating that the process is dependent on the intactness of the organelles. The plastid ATP/ADP transporter is not responsible for ADPGlc uptake. Data are presented that indicate that ADPGlc is transported by another translocator in counter exchange with AMP. To analyse the physiology of starch synthesis in more detail, we examined how Glc6P- and ADPGlc-dependent starch synthesis in isolated maize endosperm amyloplasts interact. Glc6P-dependent starch synthesis is not inhibited by increasing concentrations of ADPGlc. In contrast, the rate of ADPGlc-dependent starch synthesis is reduced by increasing concentrations of ATP necessary for Glc6P-dependent starch synthesis. The possible modes of inhibition of ADPGlc-dependent starch synthesis by ATP are discussed with respect to the stromal generation of AMP required for ADPGlc uptake.

BT1, a possible adenylate translocator, is developmentally expressed in maize endosperm but not detected in starchy tissues from several other species

Brittle 1 protein (BT1), the major protein in amyloplast envelope membranes of wild‐type maize endosperm, is required for normal levels of starch accumulation in maize kernels. To gain information about the Bt1 locus in control of starch synthesis in plants, we examined Bt1 expression in kernels from different ages and from different genotypes of maize as well as in starchy tissues from different species. Both BT1 protein and Bt1 transcripts were detected in kernels from about 10 days post‐pollination (DPP) and increased up to 14 DPP. BT1 protein level was similar in kernels after 14 DPP. Furthermore, membranes from amyloplasts containing larger starch granules contained more BT1 protein than those containing smaller starch granules from the same age of kernels. These results indicate that Bt1 gene expression was developmentally regulated and that BT1 protein level was correlated with starch accumulation in normal maize endosperm. Unexpectedly, except for a weak band at a higher molecular mass in microsomal proteins extracted from spring barley grain which reacted with maize BT1 antibody, no BT1 protein was observed in starchy tissues from several other species, including grains of sorghum, wheat, winter barley and oats, potato tubers, pea roots and cauliflower buds. Thus either the BT1 antibody raised against a fusion protein including the 56 amino acid residues in the carboxyl terminal sequence of maize endosperm BT1 protein does not recognize BT1 homologues in amyloplasts from other tissues or species or amyloplasts from different tissues or species have alternative membrane transporters.