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.     

A predicted plastid rhomboid protease affects phosphatidic acid metabolism in Arabidopsis thaliana

The thylakoid membranes of the chloroplast harbor the photosynthetic machinery that converts light into chemical energy. Chloroplast membranes are unique in their lipid makeup, which is dominated by the galactolipids mono‐ and digalactosyldiacylglycerol (MGDG and DGDG). The most abundant galactolipid, MGDG, is assembled through both plastid and endoplasmic reticulum (ER) pathways in Arabidopsis, resulting in distinguishable molecular lipid species. Phosphatidic acid (PA) is the first glycerolipid formed by the plastid galactolipid biosynthetic pathway. It is converted to substrate diacylglycerol (DAG) for MGDG Synthase (MGD1) which adds to it a galactose from UDP‐Gal. The enzymatic reactions yielding these galactolipids have been well established. However, auxiliary or regulatory factors are largely unknown. We identified a predicted rhomboid‐like protease 10 (RBL10), located in plastids of Arabidopsis thaliana, that affects galactolipid biosynthesis likely through intramembrane proteolysis. Plants with T‐DNA disruptions in RBL10 have greatly decreased 16:3 (acyl carbons:double bonds) and increased 18:3 acyl chain abundance in MGDG of leaves. Additionally, rbl10‐1 mutants show reduced [¹⁴C]–acetate incorporation into MGDG during pulse?chase labeling, indicating a reduced flux through the plastid galactolipid biosynthesis pathway. While plastid MGDG biosynthesis is blocked in rbl10‐1 mutants, they are capable of synthesizing PA, as well as producing normal amounts of MGDG by compensating with ER‐derived lipid precursors. These findings link this predicted protease to the utilization of PA for plastid galactolipid biosynthesis potentially revealing a regulatory mechanism in chloroplasts.     

Rice debranching enzyme isoamylase3 facilitates starch metabolism and affects plastid morphogenesis

Debranching enzymes, which hydrolyze α-1 and 6-glucosidic linkages in α-polyglucans, play a dual role in the synthesis and degradation of starch in plants. A transposon-inserted rice mutant of isoamylase3 (isa3) contained an increased amount of starch in the leaf blade at the end of the night, indicating that ISA3 plays a role in the degradation of transitory starch during the night. An epitope-tagged ISA3 expressed in Escherichia coli exhibited hydrolytic activity on β-limit dextrin and amylopectin. We investigated whether ISA3 plays a role in amyloplast development and starch metabolism in the developing endosperm. ISA3-green fluorescent protein (GFP) fusion protein expressed under the control of the rice ISA3 promoter was targeted to the amyloplast stroma in the endosperm. Overexpression of ISA3 in the sugary1 mutant, which is deficient in ISA1 activity, did not convert water-soluble phytoglycogen to starch granules, indicating that ISA1 and ISA3 are not functionally redundant. Both overexpression and loss of function of ISA3 in the endosperm generated pleomorphic amyloplasts and starch granules. Furthermore, chloroplasts in the leaf blade of isa3 seedlings were large and pleomorphic. These results suggest that ISA3 facilitates starch metabolism and affects morphological characteristics of plastids in rice.     

Measurement of Metabolites Associated with Nonaqueously Isolated Starch Granules from Immature Zea mays L. Endosperm

Starch granules with associated metabolites were isolated from immature Zea mays L. endosperm by a nonaqueous procedure using glycerol and 3-chloro-1,2-propanediol. The soluble extract of the granule preparation contained varying amounts of neutral sugars, inorganic phosphate, hexose and triose phosphates, organic acids, adenosine and uridine nucleotides, sugar nucleotides, and amino acids. Based on the metabolites present and on information about translocators in chloroplast membranes, which function in transferring metabolites from the chloroplast stroma into the cytoplasm, it is suggested that sucrose is degraded in the cytoplasm, via glycolysis, to triose phosphates which cross the amyloplast membrane by means of a phosphate translocator. It is further postulated that hexose phosphates and sugars are produced from the triose phosphates in the amyloplast stroma by gluconeogenesis with starch being formed from glucose 1-phosphate via pyrophosphorylase and starch synthase enzymes. The glucose 1-phosphate to inorganic phosphate ratio in the granule preparation was such that starch synthesis by phosphorylase is highly unlikely in maize endosperm.     

Roles of isoamylase and ADP-glucose pyrophosphorylase in starch granule synthesis in rice endosperm

Amyloplast-targeted green fluorescent protein (GFP) was used to monitor amyloplast division and starch granule synthesis in the developing endosperm of transgenic rice. Two classical starch mutants, sugary and shrunken, contain reduced activities of isoamylase1 (ISA1) and cytosolic ADP-glucose pyrophosphorylase, respectively. Dividing amyloplasts in the wild-type and shrunken endosperms contained starch granules, whereas those in sugary endosperm did not contain detectable granules, suggesting that ISA1 plays a role in granule synthesis at the initiation step. The transition from phytoglycogen to sugary-amylopectin was gradual in the boundary region between the inner and outer endosperms of sugary. These results suggest that the synthesis of sugary-amylopectin and phytoglycogen involved a stochastic process and that ISA1 activity plays a critical role in the stochastic process in starch synthesis in rice endosperm. The reduction of cytosolic ADP-glucose pyrophosphorylase activity in shrunken endosperm did not inhibit granule initiation but severely restrained the subsequent enlargement of granules. The shrunken endosperm often developed pleomorphic amyloplasts containing a large number of underdeveloped granules or a large cluster of small grains of amyloplasts, each containing a simple-type starch granule. Although constriction-type divisions of amyloplasts were much more frequent, budding-type divisions were also found in the shrunken endosperm. We show that monitoring GFP in developing amyloplasts was an effective means of evaluating the roles of enzymes involved in starch granule synthesis in the rice endosperm.     

Isolation and characterization of an amyloplast envelope-enriched fraction from immature maize endosperm

Gardner, H. W., Miernyk, J. A., Christianson, D. D. and Khoo, U. 1987. Isolation and characterization of an amyloplast envelope-enriched fraction from immature maize endosperm.
A 10000–100000 g pellet obtained by centrifugation of homogenates from immature (25 days after pollination) de-embryonated maize ( Zea mays L., cv. W64A-normal and a typical hybrid) kernels was further fractionated by sedimentation on discontinuous sucrose density gradients. Particles with the highest carotenoid content (0.68% by weight carotenoids based upon total lipid) sedimented at densities of 1.083-1.106 g ml^-1, coincident with the plastid envelope marker enzyme, galacto-syltransferase (EC 2.4.1.46). Lipids extracted from the carotenoid-rich fraction were mainly digalactosyldiacylglycerols, monogalactosyldiacylglycerols, phosphatidylcholines, phosphatidylinositols and phosphatidylglycerols, in order of molar abundance. With increasing particle density (>1.106 g ml¹) the phospholipid and neutral lipid content increased, and the proportion of carotenoids and galactolipids decreased. Electron micrographs of the carotenoid-rich fraction revealed vesicles ranging in size from < 0.1 to 0.5 um, as well as smaller granular membranes. The carotenoid-rich membrane fraction was progressively more difficult to isolate as the endosperm matured, and freezing the immature endosperm prevented subsequent isolation. The lipid and enzyme composition and ultrastructural characteristics of the isolated fraction suggest that it is composed of amyloplast envelope vesicles.     

Structural insights and membrane binding properties of MGD1, the major galactolipid synthase in plants

Monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the major lipid components of photosynthetic membranes, and hence the most abundant lipids in the biosphere. They are essential for assembly and function of the photosynthetic apparatus. In Arabidopsis, the first step of galactolipid synthesis is catalyzed by MGDG synthase 1 (MGD1), which transfers a galactosyl residue from UDP‐galactose to diacylglycerol (DAG). MGD1 is a monotopic protein that is embedded in the inner envelope membrane of chloroplasts. Once produced, MGDG is transferred to the outer envelope membrane, where DGDG synthesis occurs, and to thylakoids. Here we present two crystal structures of MGD1: one unliganded and one complexed with UDP. MGD1 has a long and flexible region (approximately 50 amino acids) that is required for DAG binding. The structures reveal critical features of the MGD1 catalytic mechanism and its membrane binding mode, tested on biomimetic Langmuir monolayers, giving insights into chloroplast membrane biogenesis. The structural plasticity of MGD1, ensuring very rapid capture and utilization of DAG, and its interaction with anionic lipids, possibly driving the construction of lipoproteic clusters, are consistent with the role of this enzyme, not only in expansion of the inner envelope membrane, but also in supplying MGDG to the outer envelope and nascent thylakoid membranes.     

Green light for galactolipid trafficking

Galactolipids not only play a crucial role in photosynthesis but are also important for the adaptation of membrane-lipid composition in plants to phosphate-limiting conditions. The enzymes of galactolipid assembly have been localised to the envelope membranes of chloroplasts. Lipid trafficking is essential for galactolipid synthesis and redistribution because lipid precursors originate from two compartments, the endoplasmic reticulum (ER) and the plastid, and because galactolipids have to be transported to extraplastidial membranes during phosphate deprivation. Analysis of Arabidopsis mutants that are impaired in galactolipid synthesis (i.e. dgd1 and dgd2) or in ER-to-plastid lipid transport (i.e. tgd1) has resulted in the identification of a processive galactosyltransferase whose function is still enigmatic.     

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.     

Ultrastructural characterization of maize (Zea mays L.) kernels exposed to high temperature during endosperm cell division

This study reports the ultrastructural changes in maize endosperm that result from exposure to high temperature during cell division. Kernels were grown in vitro at 25 ºC continuously (control) and at 5 d after pollination (DAP) subsamples were transferred to continuous 35 ºC for either 4 or 6 d. The 4 d treatment reduced kernel mass by 40% and increased kernel abortion three-fold. The 6-d high-temperature treatment resulted in a 77% reduction in kernel mass and a 12-fold increase in kernel abortion. Evaluation of the kernels at 11 DAP using scanning and transmission electron microscopy revealed that the reduced kernel mass and/or abortion was associated with the disruption of cell division and amyloplast biogenesis in the periphery of the endosperm. This was further confirmed by the presence of an irregular-shaped nucleus, altered size of the nucleolus, highly dense nucleoplasm, and a decrease in the number of proplastids and amyloplasts. Thus, the endosperm cavity was not filled, the total number of endosperm cells was reduced by 35 and 70%, and the number of starch granules was decreased by 45 and 80% after exposure to 4 and 6 d of high-temperature treatments, respectively. This also resulted in a 35–70% reduction in total starch accumulation. KI/I₂ staining and light microscopy revealed that starch accumulation in the peripheral endosperm cells was reduced more severely than in the central zones. However, the scanning electron micrographs of cells from the central endosperm showed that the number and the size of apparently viable amyloplasts were reduced and isolated granules were smaller and/or showed enhanced pitting. These ultrastructural data support the hypothesis that high temperature during endosperm cell division reduces kernel sink potential and subsequently mature kernel mass, mainly by disrupting cell division and amyloplast biogenesis in the peripheral and central endosperm.     

Btl, a structural gene for the major 39–44 kDa amyloplast membrane polypeptides

The aim of the present work was to investigate the relationship between the Btl gene (Btl) and the major 39–44 kDa amyloplast membrane polypeptides which were deficient in amyloplast membranes of brittlel (btl) kernels of maize (Zea mays L.). A rapid yet gentle procedure for the isolation of amyloplasts from immature kernels is described. These amyloplasts were relatively free of contamination by other cellular components, and immunological studies showed that they contained polypeptides which reacted with antibodies to maize starch branching enzyme and ADP-Gle pyrophosphorylase. Purified membranes isolated from the amyloplast contained a poly-peptide which reacted with antibodies to the P_i-translocator from spinach chloroplasts. However, a cluster of 39–44 kDa polypeptides accounted for about 40% of the total amyloplast membrane protein from W64A kernels. These polypeptides were specifically recognized by antibodies raised against a fusion protein consisting of 56 amino acids of the carboxyl terminus of the BTI protein and glutathione S-transferase. The BT1 antibodies also reacted with the abundant polypeptides in amyloplast membranes from hybrid kernels (Doebler 66XP and Pioneer 3780), and the shrunkenl and shrunken2 mutant genotypes, but no BTl reacting polypeptides were present in amyloplast membranes from btl mutant kernels. We were unable to detect BTl by the immunoblot procedure in microsomal membranes from embryo and pericarp tissues from the kernel, from seedling roots and shoots, or in membranes from mitochondria and chloroplasts. The same BTl immunoblot pattern was obtained for proteins extracted from microsomal membranes from developing endosperm and from purified amyloplast membranes. A linear relationship between the number of copies of Btl alleles and the levels of BTl in endosperm microsomal membranes was demonstrated in a gene dosage series. BTl was not extracted from amyloplast membranes by chloroform/methanol or by alkaline buffer at pH 11.5, but was partially extracted by 0.1 M NaOH. These lines of evidence support the conclusion that Btl is the structural gene for the major 39–44 kDa amyloplast membrane polypeptides and that these polypeptides are integral proteins specific to amyloplast membranes from the endosperm.     

Polypeptides of the Maize Amyloplast Stroma : Stromal Localization of Starch-Biosynthetic Enzymes and Identification of an 81-Kilodalton Amyloplast Stromal Heat-Shock Cognate

In the developing endosperm of monocotyledonous plants, starch granules are synthesized and deposited within the amyloplast. A soluble stromal fraction was isolated from amyloplasts of immature maize (Zea mays L.) endosperm and analyzed for enzyme activities and polypeptide content. Specific activities of starch synthase and starch-branching enzyme (SBE), but not the cytosolic marker alcohol dehydrogenase, were strongly enhanced in soluble amyloplast stromal fractions relative to soluble extracts obtained from homogenized kernels or endosperms. Immunoblot analysis demonstrated that starch synthase I, SBEIIb, and sugary1, the putative starch-debranching enzyme, were each highly enriched in the amyloplast stroma, providing direct evidence for the localization of starch-biosynthetic enzymes within this compartment. Analysis of maize mutants shows the deficiency of the 85-kD SBEIIb polypeptide in the stroma of amylose extender cultivars and that the dull mutant lacks a >220-kD stromal polypeptide. The stromal fraction is distinguished by differential enrichment of a characteristic group of previously undocumented polypeptides. N-terminal sequence analysis revealed that an abundant 81-kD stromal polypeptide is a member of the Hsp70 family of stress-related proteins. Moreover, the 81-kD stromal polypeptide is strongly recognized by antibodies specific for an Hsp70 of the chloroplast stroma. These findings are discussed in light of implications for the correct folding and assembly of soluble, partially soluble, and granule-bound starch-biosynthetic enzymes during import into the amyloplast.     

Critical regulation of galactolipid synthesis controls membrane differentiation and remodeling in distinct plant organs and following environmental changes

The plant galactolipids, monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), are the most abundant lipids in chloroplast membranes, and they constitute the majority of total membrane lipids in plants. MGDG is synthesized by two types of MGDG synthase, type-A (MGD1) and type-B (MGD2, MGD3). These MGDG synthases have distinct roles in Arabidopsis. In photosynthetic organs, Type A MGD is responsible for the bulk of MGDG synthesis, whereas Type B MGD is expressed in non-photosynthetic organs such as roots and flowers and mainly contributes to DGDG accumulation under phosphate deficiency. Similar to MGDG synthesis, DGDG is synthesized by two synthases, DGD1 and DGD2; DGD1 is responsible for the majority of DGDG synthesis, whereas DGD2 makes its main contribution under phosphate deficiency. These galactolipid synthases are regulated by light, plant hormones, redox state, phosphatidic acid levels, and various stress conditions such as drought and nutrient limitation. Maintaining the appropriate ratio of these two galactolipids in chloroplasts is important for stabilizing thylakoid membranes and maximizing the efficiency of photosynthesis. Here we review progress made in the last decade towards a better understanding of the pathways regulating plant galactolipid biosynthesis.     

Identification of the maize amyloplast stromal 112-kD protein as a plastidic starch phosphorylase

Amyloplast is the site of starch synthesis in the storage tissue of maize (Zea mays). The amyloplast stroma contains an enriched group of proteins when compared with the whole endosperm. Proteins with molecular masses of 76 and 85 kD have been identified as starch synthase I and starch branching enzyme IIb, respectively. A 112-kD protein was isolated from the stromal fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to tryptic digestion and amino acid sequence analysis. Three peptide sequences showed high identity to plastidic forms of starch phosphorylase (SP) from sweet potato, potato, and spinach. SP activity was identified in the amyloplast stromal fraction and was enriched 4-fold when compared with the activity in the whole endosperm fraction. Native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses showed that SP activity was associated with the amyloplast stromal 112-kD protein. In addition, antibodies raised against the potato plastidic SP recognized the amyloplast stromal 112-kD protein. The amyloplast stromal 112-kD SP was expressed in whole endosperm isolated from maize harvested 9 to 24 d after pollination. Results of affinity electrophoresis and enzyme kinetic analyses showed that the amyloplast stromal 112-kD SP preferred amylopectin over glycogen as a substrate in the synthetic reaction. The maize shrunken-4 mutant had reduced SP activity due to a decrease of the amyloplast stromal 112-kD enzyme.     

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.Starch is the most important carbohydrate storage material and contains the Glc polymers amylose and amylopectin. At least four classes of enzymes, ADP-Glc pyrophosphorylase (AGPase), starch synthase (SS), starch branching enzyme (BE), and starch debranching enzyme (DBE), are necessary for efficient starch biosynthesis in storage tissues.SSs (EC 2.4.1.21) play a central role in starch synthesis during α-glucan elongation by adding Glc residues from ADP-Glc to the nonreducing ends via α-1,4-glucosidic linkages. Rice (Oryza sativa) contains 11 SS genes that are grouped into six classes, SSI to SSV and granule-bound starch synthase (GBSS; Supplemental Fig. S1; Hirose and Terao, 2004; Ohdan et al., 2005). Every class contains multiple isozymes, except for SSI and SSV; SSI, SSIIa, SSIIIa, and GBSSI are highly expressed in developing rice endosperm (Hirose and Terao, 2004; Ohdan et al., 2005). SSI elongates short amylopectin chains with degree of polymerization (DP) from 6 or 7 to DP 8 to 12 (Fujita et al., 2006). SSIIa elongates amylopectin from DP 6 to 12 to DP 13 to 24 (Umemoto et al., 2002; Nakamura et al., 2005), and SSIIIa elongates long amylopectin chains with DP 33 or greater (Fujita et al., 2007). GBSSI synthesizes amylose and extra-long amylopectin chains (Sano, 1984; Takeda et al., 1987; Hizukuri, 1995). The functions of other SS isozymes, such as SSIIb, SSIIc, SSIIIb, SSIVa, SSIVb, SSV, and GBSSII, remain largely unknown due to the lack of respective mutant lines. It is not clear how SS isozymes contribute to starch granule formation.Rice endosperm amyloplasts produce characteristic compound-type starch granules, which consist of dozens of polyhedral, sharp-edged granules (Matsushima et al., 2010). Compound-type starch granules are the most common type in endosperm of Poaceae species (Tateoka, 1962; Grass Phylogeny Working Group, 2001; Prasad et al., 2011; Matsushima et al., 2013). Simple-type starch granules (one starch granule per amyloplast) are produced in some species of the Bambusoideae, Pooideae, Micrairoideae, Chloridoideae, and Panicoideae subfamilies. The taxonomic relationships in the Poaceae do not enable an accurate prediction of granule morphology (Tateoka 1962; Shapter et al., 2008; Matsushima et al., 2013).Two studies that changed starch granule shape from simple type to compound type have been reported (Suh et al., 2004; Myers et al., 2011). A hull-less cultivar of cv Betzes barley (Hordeum vulgare), cv Nubet, contains simple-type and bimodal starch granules, which are typical of wild-type barley. Chemical mutagenesis of cv Nubet produced a mutant called franubet, which contains compound-type starch granules (Suh et al., 2004). In the maize monogalactosyldiacylglycerol synthase-deficient mutant opaque5, simple-type granules are replaced by compound-type granules separated by a membranous structure (Myers et al., 2011). The molecular mechanisms that control starch granule morphology in cereal endosperm are largely unknown, although an alteration in membrane lipid synthesis may be involved (Myers et al., 2011).A structural model for the compound-type amyloplast is shown Figure 1. The amyloplast envelope contains an outer envelope membrane (OEM), inner envelope membrane (IEM), and intermembrane space (IMS). Each starch granule is enclosed by an IEM, and granules are separated by a septum-like structure (SLS; Yun and Kawagoe, 2010). In this model, the IMS and SLS are directly connected, and fluorescent proteins such as GFP and Cherry can move freely between the two (Fig. 1; Kawagoe, 2013). The chloroplast envelope membrane contains little protein compared with the thylakoid membrane (Heber and Heldt, 1981). The endosperm amyloplast envelope membrane contains even less protein. Low protein content could be a major reason why the amyloplast envelope in rice endosperm is difficult to observe using high-resolution electron microscopy. In transgenic rice, a fluorescent protein fused to an IEM protein, the ADP-Glc transporter BRITTLE1, visualized the amyloplast IEM (Yun and Kawagoe, 2010). Fluorescent proteins fused to the chloroplast OEM protein OEP7 visualized the amyloplast OEM in endosperm (Kawagoe, 2013). These studies revealed that the outermost membranes of rice amyloplasts are OEM and contain intraamyloplast compartments. Starch is synthesized within the amyloplast compartments and is ultimately formed as compound-type granules that are individually wrapped in IEM (Yun and Kawagoe, 2010; Kawagoe, 2013).Open in a separate windowFigure 1.Structural model of the wild-type amyloplast in developing rice endosperm. The OEM is in black, the IEM is in magenta, the IMS is in green, and the SLS is in blue. G, Starch granules.Confocal microscopy analyses of the rice IEM protein, BRITTLE1, revealed that an SLS, or cross wall, divides starch granules in the amyloplast (Yun and Kawagoe, 2010). A model for the synthesis of compound-type starch granules consisting of polyhedral, sharp-edged granules proposed that the SLS functions as a mold that casts growing granules into a characteristic shape (Yun and Kawagoe, 2010; Kawagoe, 2013). The model postulates a central role for the SLS in producing characteristic compound-type granules, although neither the SLS components nor the enzymes affecting its properties have been characterized.Arabidopsis (Arabidopsis thaliana) SS genes are grouped into six classes. Leaf transitory starch biosynthesis has been investigated in single mutants of SSI, SSII, SSIII, and SSIV and in various double and triple SS mutants (Ral et al., 2004; Delvallé et al., 2005; Zhang et al., 2005, 2008; Szydlowski et al., 2009, 2011). Starch granules in leaf chloroplasts are reduced in number but enlarged in the ssIV mutant (Roldán et al., 2007; Crumpton-Taylor et al., 2013) and in the ssIV double and triple mutants (Szydlowski et al., 2009). Immature ssIV leaves have no starch granules but accumulate the starch synthase substrate ADP-Glc at high concentrations. Starch granules are flattened and discoid in wild-type leaves but are rounded in mature leaves of ssIV, suggesting that SSIV is essential for coordinating granule formation with chloroplast division during leaf expansion (Crumpton-Taylor et al., 2013). The ssIII ssIV double mutant does not accumulate measurable amounts of starch in the leaves, despite the presence of SSI and SSII activity (Szydlowski et al., 2009), implying that Arabidopsis SSIII and SSIV are involved in the initiation of starch granule formation and that either SSIII or SSIV is sufficient. Overexpression of AtSSIV increases the starch level in Arabidopsis leaves and potato (Solanum tuberosum) tubers (Gámez-Arjona et al., 2011). In transgenic plants, the AtSSIV-GFP fusion protein is enriched in specific regions at the edge of granules in Arabidopsis chloroplasts and potato tuber amyloplasts. In rice, SSIVa and SSIVb are expressed in the endosperm and other organs at an early developmental stage (Hirose and Terao, 2004; Ohdan et al., 2005).In this study, two rice allelic SSIVb-deficient mutant lines (ss4b) were generated by insertion of the retrotransposon Tos17 and crossed with the SSIIIa null mutant (ss3a). Surprisingly, the ss3a ss4b endosperm produced spherical starch granules that were separated from each other within amyloplasts, whereas the single mutants produced compound-type polyhedral starch granules. The SSIVb and GBSSI enzymes were localized to distinct compartments in developing amyloplasts. We discuss the changes in rice starch structure due to the deficiency of both SSIIIa and SSIVb, the alteration in starch granule morphology, and possible unconventional functions of SSIIIa and SSIVb. We also present a model of how spherical granules are produced in ss3a ss4b rice endosperm.     

Surface Localization of Zein Storage Proteins in Starch Granules from Maize Endosperm : Proteolytic Removal by Thermolysin and in Vitro Cross-Linking of Granule-Associated Polypeptides

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 δ-zein was probed by comparing δ-zein levels of starch granules obtained from homogenized whole endosperm with granules isolated from amyloplasts. Starch granules from amyloplasts contained markedly lower levels of δ-zein relative to granules prepared from whole endosperm, thus indicating that δ-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.It has long been known that starch granules contain bound polypeptides, with protein levels of isolated starch granules from maize (Zea mays) ranging from 0.3 to 1.0% based upon measurement of N₂ (May, 1987). A recent study by our laboratory demonstrates that isolated starch granules from maize contain several dozen strongly bound polypeptides (Mu-Forster et al., 1996). The granule-associated proteins include starch-biosynthetic enzymes such as the waxy protein, SSI, and SBEIIb. These polypeptides are not removed from intact starch granules by protease treatment or detergent washing; therefore, they are believed to bind to the starch and to become irreversibly entrapped within the starch matrix.Based upon staining intensities of polypeptides extracted from the starch granule (Mu-Forster et al., 1996), approximately one-half of the granule-associated proteins in maize consist of low-molecular-mass polypeptides ranging between 10 and 27 kD. These bands fall within the size range displayed by the zein storage proteins, however, the spatial distribution of these polypeptides within the starch granule is unknown. Zeins have been defined as alcohol-soluble proteins that occur principally in protein bodies of maize endosperm and that may or may not require reduction before extraction (Wilson, 1991). The association of zeins with starch granules during endosperm development would not be expected because zein genes do not contain transit peptides that would target these proteins through the amyloplast envelope into the amyloplast stroma.The objective of this study was to establish the topology of granule-associated zeins in starch granules from maize endosperm. To accomplish this, it was necessary to distinguish between surface-localized and internalized polypeptides. Our working hypothesis defines polypeptides localized at the starch granule surface as those that are susceptible to hydrolysis upon treatment of intact granules with exogenous proteases. Conversely, internal granule proteins are defined as those that (a) become susceptible to proteolysis only following thermal disruption of the starch matrix, and (b) resist extraction by 2% SDS at room temperatures (Denyer et al., 1993; Rahman et al., 1995; Mu-Forster et al., 1996).In this study we were able to distinguish between surface-localized and internalized granule-associated polypeptides in starch granules from maize endosperm by use of the thermophilic protease thermolysin. Thermolysin is well suited for this purpose because it is highly active at starch-gelatinization temperatures, and has also been shown to effectively hydrolyze hydrophobic proteins located at the surfaces of chloroplasts and other subcellular organelles (Cline et al., 1984; Xu and Chitnis, 1995). Upon extended incubation of intact starch granules with thermolysin at subgelatinization temperatures, we found that zeins were selectively removed from the starch granule surface. All other granule-associated polypeptides remained inaccessible to proteolytic attack or to extraction by 2% SDS, unless the starch matrix was first disrupted by gelatinization. Our results distinguish between the surface-localized and granule-intrinsic proteins of maize endosperm, and establish that zeins are localized at the starch-granule surface. In addition, cross-linking experiments were conducted to determine nearest-neighbor relationships among zein subunits localized at the granule surface and granule intrinsic polypeptides localized within the starch matrix.     

STARCH GRANULE (AMYLOPLAST) DEVELOPMENT IN ENDOSPERM OF SEVERAL ZEA MAYS L. GENOTYPES AFFECTING KERNEL POLYSACCHARIDES

Endosperm cell and starch granule (amyloplast) development of six maize (Zea mays L.) genotypes, normal, amylose-extender (ae), sugary (su), waxy (wx), amylose-extender sugary (ae su), and amylose-extender waxy (ae wx), was compared. Endosperms of all genotypes were indistinguishable at 14 days after pollination. Cells were highly vacuolated and those in the central crown area of the kernel contained small starch granules in close association with the nucleus. Cellular and nuclear enlargement occurred during endosperm development in all genotypes, and major and minor gradients in physiological age of endosperm cells were observed in all kernels. Amyloplast development varied with genotype. Plastid development in normal and wx cells was characterized by an initial starch granule formation followed by granule enlargement to cell maturity. Endosperms homozygous for ae (ae, ae su, and ae wx) developed abnormal plastid-granules. Secondary granule formations preceded development of abnormality in ae and ae su, but not in ae wx endosperms. In contrast to ae and ae su starch granules, ae wx granules were highly birefringent indicating a high degree of crystallinity. In all three ae genotypes, abnormality increased as a function of kernel and physiological cell age. The su mutant had two distinct effects on amyloplast development. First, a mobilization of the initially formed starch, and second a synthesis and accumulation of phytoglycogen and the formation of large rounded plastids. In ae su plastid development, there was a mobilization of the starch initially formed (resulting in irregularly shaped, nonbirefringent granules) but only small amounts of phytoglycogen were produced.