Showing papers on "Endosperm published in 1997"

Fertilization-independent seed development in Arabidopsis thaliana

Abstract: We report mutants in Arabidopsis thaliana (fertilization-independent seed: fis) in which certain processes of seed development are uncoupled from the double fertilization event that occurs after pollination. These mutants were isolated as ethyl methanesulfonate-induced pseudo-revertants of the pistillata phenotype. Although the pistillata (pi) mutant has short siliques devoid of seed, the fis mutants in the pi background have long siliques containing developing seeds, even though the flowers remain free of pollen. The three fis mutations map to loci on three different chromosomes. In fis1 and fis2 seeds, the autonomous endosperm nuclei are diploid and the endosperm develops to the point of cellularization; the partially developed seeds then atrophy. In these two mutants, proembryos are formed in a low proportion of seeds and do not develop beyond the globular stage. When FIS/fis plants are pollinated by pollen from FIS/FIS plants, ≈50% of the resulting seeds contain fully developed embryos; these seeds germinate and form viable seedlings (FIS/FIS). The other 50% of seeds shrivel and do not germinate; they contain embryos arrested at the torpedo stage (FIS/fis). In normal sexual reproduction, the products of the FIS genes are likely to play important regulatory roles in the development of seed after fertilization.

A maize zinc-finger protein binds the prolamin box in zein gene promoters and interacts with the basic leucine zipper transcriptional activator Opaque2

Abstract: The prolamin box (P-box) is a highly conserved 7-bp sequence element (5′-TGTAAAG-3′) found in the promoters of many cereal seed storage protein genes. Nuclear factors from maize endosperm specifically interact with the P-box present in maize prolamin genes (zeins). The presence of the P-box in all zein gene promoters suggests that interactions between endosperm DNA binding proteins and the P-box may play an important role in the coordinate activation of zein gene expression during endosperm development. We have cloned an endosperm-specific maize cDNA, named prolamin-box binding factor (PBF), that encodes a member of the recently described Dof class of plant Cys2-Cys2 zinc-finger DNA binding proteins. When tested in gel shift assays, PBF exhibits the same sequence-specific binding to the P-box as factors present in maize endosperm nuclei. Additionally, PBF interacts in vitro with the basic leucine zipper protein Opaque2, a known transcriptional activator of zein gene expression whose target site lies 20 bp downstream of the P-box in the 22-kDa zein gene promoter. The isolation of the PBF gene provides an essential tool to further investigate the functional role of the highly conserved P-box in regulating cereal storage protein gene expression.

Transgenic rice (Oryza sativa) endosperm expressing daffodil (Narcissus pseudonarcissus) phytoene synthase accumulates phytoene, a key intermediate of provitamin A biosynthesis

Abstract: Rice (Oryza sativa L.), the major food staple for more than two billion people, contains neither beta-carotene (provitamin A) nor C40 carotenoid precursors thereof in its endosperm. To improve the nutritional value of rice, genetic engineering was chosen as a means to introduce the ability to make beta-carotene into rice endosperm tissue. Investigation of the biochemical properties of immature rice endosperm using [14C]-labelled substrates revealed the presence of geranyl geranyl diphosphate, the C20 general isoprenoid precursor necessary for C40 carotenoid biosynthesis. Phytoene synthase, which condenses two molecules of geranyl geranyl diphosphate, is the first of four specific enzymes necessary for beta-carotene biosynthesis in plants. Therefore, the Japonica rice model variety Taipei 309 was transformed by microprojectile bombardment with a cDNA coding for phytoene synthase from daffodil (Narcissus pseudonarcissus) under the control of either a constitutive or an endosperm-specific promoter. In transgenic rice plants, the daffodil enzyme is active, as measured by the in vivo accumulation of phytoene in rice endosperm. Thus, it is demonstrated for the first time that it is in principle possible to engineer a critical step in provitamin A biosynthesis in a non-photosynthetic, carotenoid-lacking plant tissue. These results have important implications for long-term prospects of overcoming worldwide vitamin A deficiency.

Ethylene-Mediated Programmed Cell Death during Maize Endosperm Development of Wild-Type and shrunken2 Genotypes.

Abstract: We characterized the progression of programmed cell death during maize (Zea mays L.) endosperm development of starchy (Su; wild-type) and shrunken2 (sh2) genotypes and tested the involve ment of ethylene in mediating this process. Histological and viability staining demonstrated that endosperm cell death was initiated earlier and progressed more rapidly in sh2 endosperm compared with Su endosperm. Internucleosomal DNA fragmentation accompanied endosperm cell death and occurred more extensively in sh2 endosperm. 1-Aminocyclopropane-1-carboxylic acid levels peaked approximately 16 d after pollination (dap) in Su endosperm and gradually decreased during subsequent development, whereas two large 1-aminocyclopropane-1-carboxylic acid peaks were observed in sh2 endosperm, the first between 16 and 20 dap and the second at 36 dap. Ethylene levels were elevated in sh2 kernels compared with Su kernels, with an initial peak 20 dap approximately 3-fold higher than in Su kernels and a second peak 36 dap approximately 5-fold higher than that in Su kernels. Ethylene treatment of Su kernels resulted in earlier and more extensive endosperm cell death and DNA fragmentation. Aminoethoxyvinylglycine treatment of sh2 kernels reduced the extent of DNA fragmentation. We conclude that ethylene is involved in triggering programmed cell death in developing maize endosperm and is responsible for the aberrant phenotype of sh2 kernels.

Cellular and molecular biology of plant seed development

Abstract: Section A: Control of Seed Development. 1. Embryogenesis in Dicotyledonous Plants R. Yadegari, R. Goldberg. 2. Development of the Suspensor: Differentiation, Communication, and Programmed Cell Death During Plant Embryogenesis B.W. Schwartz, et al. 3. Endosperm Structure and Development D.A. DeMason. 4. Hormonal Regulation of Seed Development R. Morris. Section B: The Synthesis and Accumulation of Stored Metabolites. 5. The Biochemistry and Cell Biology of Embryo Storage Proteins N.C. Nielsen, et al. 6. The Prolamin Storage Proteins of Wheat and Its Relatives G. Galili. 7. The Prolamin Proteins of Maize, Sorghum and Coix C.E. Coleman, et al. 8. The Storage Proteins of Rice and Oat D.G. Muench, T.W. Okita. 9. The Protease Inhibitors of Seeds K.A. Wilson. 10. Starch Synthesis in the Maize Seed L.C. Hannah. 11. Synthesis and Storage of Fatty Acids J. Browse. 12. Accumulation and Storage of Phosphate and Minerals V. Raboy. 13. Genetic Regulation of Carbohydrate and Protein Accumulation in Seeds M. Motto, et al. Section C: Control of Seed Maturation and Germination. 14. Lea Proteins and the Desiccation Tolerance of Seeds L. Dure. 15. Seed Maturation and Control of Dormancy J. Harada. Section D: Manipulation of Seeds Through Biotechnology. 16. Biotechnological Approaches to Altering Seed Composition E. Krebbers, et al.

The wheat transcriptional activator SPA: a seed-specific bZIP protein that recognizes the GCN4-like motif in the bifactorial endosperm box of prolamin genes.

Abstract: The conserved bifactorial endosperm box found in the promoter of wheat storage protein genes comprises two different cis elements that are thought to be involved in regulating endosperm-specific gene expression. Endosperm nuclear extracts contain binding activities. One is called ESBF-I, which binds to the endosperm motif (EM), and the other is called ESBF-II, which binds to the GCN4-like motif(GLM). Here, we present a functional analysis of the endosperm box of a low-molecular-weight glutenin gene found on the 1D1 chromosome of hexaploid wheat (LMWG-1D1) in transgenic tobacco plants. Our analysis demonstrates the necessity of the EM and GLM for endosperm-specific gene expression and suggests the presence in tobacco of functional counterparts of wheat ESBF-I and ESBF-II. Furthermore, we describe the isolation and characterization of cDNA clones encoding SPA, a seed-specific basic leucine zipper protein from wheat that can activate transcription from the GLMs of the -326-bp LMWG-1D1 promoter in both maize and tobacco leaf protoplasts. This activation is also partially dependent on the presence of functional EMs, suggesting interactions between SPA with ESBF-I-like activities.

ZmEsr, a novel endosperm‐specific gene expressed in a restricted region around the maize embryo

Abstract: A novel endosperm-specific gene named Esr (embryo surrounding region) has been isolated by differential display between early developmental stages of maize endosperms and embryos. It is expressed in a restricted region of the endosperm, surrounding the entire embryo at early stages (4 to 7 days after pollination, DAP) and ever-decreasing parts of the suspensor at subsequent stages. The expression starts at 4 DAP and is maintained until at least 28 DAP. A minimum of three Esr genes are present in the maize genome and at least two of them map to the short arm of chromosome 1 at position 56. The Esr genes contain no introns and show no significant nucleotide or amino acid sequence homologies to sequences in the databases. The open reading frames encode basic proteins of 14 kDa with presumptive signal peptides at their N-terminal followed by a hypervariable and a conserved region. The gene product may play a role in the nutrition of the developing embryo or in the establishment of a physical barrier between embryo and endosperm.

Carbohydrate Content and Enzyme Metabolism in Developing Canola Siliques

Abstract: Little biochemical information is available on carbohydrate metabolism in developing canola (Brassica napus L.) silique (pod) wall and seed tissues. This research examines the carbohydrate contents and sucrose (Suc) metabolic enzyme activities in different aged silique wall and seed tissues during oil filling. The silique wall partitioned photosynthate into Suc over starch and predominantly accumulated hexose. The silique wall hexose content and soluble acid invertase activity rapidly fell as embryos progressed from the early- to late-cotyledon developmental stages. A similar trend was not evident for alkaline invertase, Suc synthase (SuSy), and Suc-phosphate synthase. Silique wall SuSy activities were much higher than source leaves at all times and may serve to supply the substrate for secondary cell wall thickening. In young seeds starch was the predominant accumulated carbohydrate over the sampled developmental range. Seed hexose levels dropped as embryos developed from the early- to midcotyledon stage. Hexose and starch were localized to the testa or liquid endosperm, whereas Suc was evenly distributed among seed components. With the switch to oil accumulation, seed SuSy activity increased by 3.6-fold and soluble acid invertase activity decreased by 76%. These data provide valuable baseline knowledge for the genetic manipulation of canola seed carbon partitioning.

Correlation between activities of starch debranching enzyme and α-polyglucan structure in endosperms of sugary-1 mutants of rice

Abstract: Summary The biochemical lesion of the sugary-1 mutation was examined in five different mutants of rice with varying phenotypes but with mutations at the same locus. The cells in the inner part of the endosperm of all mutants tested contained phytoglycogen instead of starch, while the cells located in the outer part of the endosperm tissue from some mutants were filled with numerous starch granules. The molecular size of phytoglycogen was markedly smaller than that of amylopectin as measured by Sephacryl S-1000 chromatography. Analysis of the distribution of α-1,4 chain lengths revealed that in phytoglycogen the number of A-chains dramatically increased, while long B chains with DP ≥ 37 remarkably decreased or were almost absent, which resulted in the disappearance of the cluster structure. The results suggest that changes in the balance of enzymic activities induced by the mutations brought about a drastic alteration in polyglucan structure and the shape of the polyglucan granule. The greater the extent of phytoglycogen regions in su1 endosperm tissues became, the greater was the phytoglycogen content, and the greater the reduction in the activity of starch debranching enzyme, a type of enzyme referred to as R-enzyme (RE), limit dextrinase or pullulanase. Immunoblot analysis showed that the reduction in RE activity was due to a decrease in the amount of RE protein, and that the reduction in RE was specific since proteins of starch-branching enzymes I and IIa and ADP-glucose pyrophosphorylase were not markedly affected by su1 mutations. The proportion of starch region to the whole endosperm tissue of various su1 mutants was correlated with the RE activity in these endosperms. The results strongly suggest that the reduction in RE activity is involved in the su1 phenotype and that the enzyme plays an essential role in determining the fine structure of the amylopectin molecule

Downregulation of ovule-specific MADS box genes from petunia results in maternally controlled defects in seed development.

Abstract: A maternally determined seed defect has been obtained by downregulation of the petunia MADS box genes Floral Binding Protein 7 (FBP7) and FBP11. These genes have been previously shown to play central roles in the determination of ovule identity. Aberrant development of the seed coat and consequent degeneration of the endosperm have been observed in transgenic plants in which these two genes are downregulated by cosuppression. Analysis of the expression pattern of FBP7 and FBP11 and genetic analysis confirmed the maternal inheritance of the phenotype. The FBP7 promoter was cloned and fused to reporter genes. One of these reporter genes was the BARNASE gene for targeted cell ablation. Our results indicate that FBP7 promoter activity is restricted to the seed coat of developing seeds and that it is completely silent in the gametophytically derived tissues. The mutants used in this study provided a unique opportunity to investigate one of the poorly understood aspects of seed development: the interaction of embryo, endosperm, and maternal tissues.

Differential Expression and Properties of Starch Branching Enzyme Isoforms in Developing Wheat Endosperm

Abstract: Three forms of starch branching enzyme (BE) from developing hexaploid wheat (Triticum aestivum) endosperm have been partially purified and characterized. Immunological cross-reactivities indicate that two forms (WBE-IAD, 88 kD, and WBE-IB, 87 kD) are related to the maize BE I class and that WBE-II (88 kD) is related to maize BE II. Comparison of the N-terminal sequences from WBE-IAD and WBE-II with maize and rice BEs confirms these relationships. Evidence is presented from the analysis of nullisomic-tetrasomic wheat lines demonstrating that WBE-IB is located on chromosome 7B and that the WBE-IAD fraction contains polypeptides that are encoded on chromosomes 7A and 7D. The wheat endosperm BE classes are differentially expressed during endosperm development. WBE-II is expressed at a constant level throughout mid and late endosperm development. In contrast, WBE-IAD and WBE-IB are preferentially expressed in late endosperm development. Differences are also observed in the kinetic characteristics of the enzymes. The WBE-I isoforms have a 2- to 5-fold higher affinity for amylose than does WBE-II, and the WBE-I isoforms are activated up to 5-fold by phosphorylated intermediates and inorganic phosphate, whereas WBE-II is activated only 50%. The potential implications of this activation of BE I for starch biosynthesis are discussed.

Tissue-specific expression in transgenic maize of four endosperm promoters from maize and rice

Abstract: The tissue-specific, developmental, and genetic control of four endosperm-active genes was studied via expression of GUS reporter genes in transgenic maize plants The transgenes included promoters from the maize granule-bound starch synthase (Waxy) gene (zmGBS), a maize 27 kDa zein gene (zmZ27), a rice small subunit ADP-glucose pyrophosphorylase gene (osAGP) and the rice glutelin 1 gene (osGT1) Most plants had a transgene expression profile similar to that of the endogenous gene: expression in the pollen and endosperm for the zmGBS transgene, and endosperm only for the others Histological analysis indicated expression initiated at the periphery of the endosperm for zmGBS, zmZ27 and osGT1, while osAGP transgene activity tended to start in the lower portion of the seed Transgene expression at the RNA level was proportional to GUS activity, and did not influence endogenous gene expression Genetic analysis showed that there was a positive dosage response with most lines Activity of the zmGBS transgene was threefold higher in a low starch (shrunken 2) genetic background This effect was not seen with zmZ27 or osGTI transgenes The expression of the transgenes is discussed relative to the known behaviour of the endogenous genes, and the developmental programme of the maize endosperm

Expression of a mutant alpha -zein creates the floury2 phenotype in transgenic maize

Abstract: The maize floury2 mutation results in the formation of a soft, starchy endosperm with a reduced amount of prolamin (zein) proteins and twice the lysine content of the wild type. The mutation is semidominant and is associated with small, irregularly shaped protein bodies, elevated levels of a 70-kDa chaperone in the endoplasmic reticulum, and a novel 24-kDa polypeptide in the zein fraction. The 24-kDa polypeptide is a precursor of a 22-kDa α-zein protein that is not properly processed. The defect is due to an alanine-to-valine substitution at the C-terminal position of the signal peptide, which causes the protein to be anchored to the endoplasmic reticulum. We postulated that the phenotype associated with the floury2 mutation is caused by the accumulation of the 24-kDa α-zein protein. To test this hypothesis, we created transgenic maize plants that produce the mutant protein. We found that endosperm in seeds of these plants manifests the floury2 phenotype, thereby confirming that the mutant α-zein is the molecular basis of this mutation.

cDNA cloning and tissue specific expression of a gene for sucrose transporter from rice (Oryza sativa L.).

Abstract: We describe the cloning and expression analysis of a sucrose transporter cDNA from a monocot (the rice plant, Oryza sativa L.). The cDNA clone (OsSUT1) encoded an open reading frame of 1,611 bp (537 amino acids) and showed 76.8 to 79.7% similarity at the amino acid level to other sucrose transporters of dicot species. The predicted membrane topology of OsSUT1 protein is made up of 12 transmembrane helices which is consistent with most of the mono- and disaccharide transporters previously identified. When OsSUT1 cDNA was introduced into yeast and expressed, the cells rapidly accumulated sucrose demonstrating that OsSUT1 does, in fact, encode a sucrose transporter. From genomic Southern hybridization OsSUT1 appeared to be a single copy gene. OsSUT1 was expressed in source organs such as leaf blade, leaf sheath and germinating seed whereas little or no expression was observed in some sink organs such as the panicles before heading and the roots. Transcript was observed at high levels in panicles after heading, particularly in the portion containing endosperm and embryo. In addition, expression of OsSUT1 was high in etiolated seedlings and decreased during light-induced greening.

Independent genetic control of maize starch-branching enzymes IIa and IIb: Isolation and characterization of a Sbe2a cDNA

Abstract: In maize (Zea mays L.) three isoforms of starch-branching enzyme (SBEI, SBEIIa, and SBEIIb) are involved in the synthesis of amylopectin, the branched component of starch. To isolate a cDNA encoding SBEIIa, degenerate oligonucleotides based on domains highly conserved in Sbe2 family members were used to amplify Sbe2-family cDNA from tissues lacking SBEIIb activity. The predicted amino acid sequence of Sbe2a cDNA matches the N-terminal sequence of SBEIIa protein purified from maize endosperm. The size of the mature protein deduced from the cDNA also matches that of SBEIIa. Features of the predicted protein are most similar to members of the SBEII family; however, it differs from maize SBEIIb in having a 49-amino acid N-terminal extension and a region of substantial sequence divergence. Sbe2a mRNA levels are 10-fold higher in embryonic than in endosperm tissue, and are much lower than Sbe2b in both tissues. Unlike Sbe2b, Sbe2a-hybridizing mRNA accumulates in leaf and other vegetative tissues, consistent with the known distribution of SBEIIa and SBEIIb activities.

An Aspartic Endopeptidase is Involved in the Breakdown of Propeptides of Storage Proteins in Protein-Storage Vacuoles of Plants

Abstract: To understand the mechanism of the maturation of various proteins in protein-storage vacuoles, we purified a 48-kDa aspartic endopeptidase composed of 32-kDa and 16-kDa subunits from castor bean. Immunocytochemical and cell fractionation analyses of the endosperm of maturing castor bean seed showed that the aspartic endopeptidase was localized in the matrix of the protein-storage vacuoles, where a variety of seed storage proteins were also present. The amount of the aspartic endopeptidase increased at the mid-maturation stage of the seeds before accumulation of the storage proteins. To determine how the aspartic endopeptidase is responsible for maturation of seed proteins in concert with the vacuolar processing enzyme, we prepared 35S-labeled proproteins of seed proteins from the endoplasmic reticulum fraction of pulse-labeled maturing endosperm and used the authentic proproteins as substrates for in vitro processing experiments. The purified aspartic endopeptidase was unable to convert any of three endosperm proproteins, pro2S albumin, proglobulin, and proricin, into their mature sizes, while the purified vacuolar processing enzyme could convert all three proproteins. We further examined the activity of aspartic endopeptidase on the cleavage of an internal propeptide of Arabidopsis pro2S albumin, which is known to be removed post-translationally. The aspartic endopeptidase cleaved the propeptide at three sites under acidic conditions. These results suggest that aspartic endopeptidase cannot directly convert pro2S albumin into the mature form, but it may play a role in trimming the C-terminal propeptides from the subunits that are produced by the action of the vacuolar processing enzyme.

Influence of Gene Dosage on Carbohydrate Synthesis and Enzymatic Activities in Endosperm of Starch-Deficient Mutants of Maize

Abstract: In cereals, starch is synthesized in endosperm cells, which have a ploidy level of three. By studying the allelic dosage of mutants affecting starch formation in maize (Zea mays L.) kernels, we determined the effect of down-regulated enzyme activity on starch accumulation and the activity of associated enzymes of carbohydrate metabolism. We found a direct relationship between the amount of starch produced in the endosperm and the gene dosage of amylose extender-1, brittle-2, shrunken1, and sugary-1 mutant alleles. Changes in starch content were found to be caused by changes in the duration as well as the rate of starch synthesis, depending on the mutant. Branching enzyme, ADP-glucose pyrophosphorylase, and sucrose synthase activities were linearly reduced in endosperm containing increasing dosages of amylose extender-1, brittle-2, and shrunken-1 alleles, respectively. De-branching enzyme activity declined only in the presence of two or three copies of sugary-1. No enzyme-dosage relationship occurred with the dull1 mutant allele. All mutants except sugary-1 displayed large increases (approximately 2- to 5-fold) in activity among various enzymes unrelated to the structural gene. This occurred in homozygous recessive genotypes, as did elevated concentrations of soluble sugars, and differed in magnitude and distribution among enzymes according to the particular mutation.

Parental Effects on Seed Mass: Seed Coat but Not Embryo/Endosperm Effects

Abstract: Many biologists studying environmentally induced parental effects have indirectly suggested that the parental environment alters seed mass by altering the amount of endosperm or embryo tissue in the seed. We tested this hypothesis by measuring the effects of parental temperature on total seed mass, seed coat mass, and embryo/endosperm mass in offspring of Plantago lanceolata. Parental temperature significantly affected total seed and coat mass but not endosperm/embryo mass. Thus, larger seeds do not contain more resources in the embryo or endosperm than do small seeds. Rather they have more coat mass, which probably strongly influences germination. These results suggest caution when making assumptions about the pathways by which environmentally induced parental effects are transmitted in plant species. We also observed that controlled crosses differed significantly in their response to parental temperature, which provides evidence for genetic variation in environmentally induced parental effects, i.e., intergenerational phenotypic plasticity, in natural populations of P. lanceolata. Many studies of parental (maternal) effects have suggested that parental environment can alter offspring fitness by influencing the amount of resources that a maternal parent packs into its seeds. These resources represent capital that is used later by the offspring for seedling establishment and subsequent growth and reproduction (see reviews by Roach and Wulff, 1987; Gutterman, 1992; Wulff, 1995). This hypothesis is based on two observations: first, many studies of parental effects show that parental environment influences seed mass; second, many other studies show that seed mass can strongly influence growth, competitive ability, and other fitness traits (see references cited in above reviews). These observations suggest that seeds enlarged by the parental environment have more stored resources in the endosperm, or embryo, than do smaller seeds. To test this hypothesis, we examined the tissue(s) involved in mediating the effect of parental temperature on offspring seed mass in Plantago lanceolata L. (Plantaginaceae), a cosmopolitan temperate herbaceous plant species. The experiment that we describe here complements a study that has already documented the effects of parental temperature on a suite of life-history traits in this species (Lacey, 1996). That study showed that parental temperature influences offspring seed mass, germination, 1

Endo-[beta]-Mannanase Activity from Individual Tomato Endosperm Caps and Radicle Tips in Relation to Germination Rates

Abstract: Endo-[beta]-mannanase is hypothesized to be a rate-limiting enzyme in endosperm weakening, which is a prerequisite for radicle emergence from tomato (Lycopersicon esculentum Mill.) seeds. Using a sensitive, single-seed assay, we have measured mannanase activity diffusing from excised tomato endosperm caps following treatments that alter the rate or percentage of radicle emergence. Most striking was the 100- to more than 10,000-fold range of mannanase activity detected among individual seeds of highly inbred tomato lines, which would not be detected in pooled samples. In some cases a threshold-type relationship between mannanase activity and radicle emergence was observed. However, when radicle emergence was delayed or prevented by osmoticum or abscisic acid, the initial increase in mannanase activity was unaffected or even enhanced. Partially dormant seed lots displayed a bimodal distribution of activity, with low activity apparently associated with dormant seeds in the population. Gibberellin- and abscisic acid-deficient mutant seeds exhibited a wide range of mannanase activity, consistent with their variation in hormonal sensitivity. Although the presence of mannanase activity in the endosperm cap is consistently associated with radicle emergence, it is not the sole or limiting factor under all conditions.

Molecular Cloning, Expression and Subcellular Localization of a BiP Homolog from Rice Endosperm Tissue

Abstract: The ER luminal binding protein, BiP, has been linked to prolamine protein body formation in rice. To obtain further information on the possible role of this chaperone in protein body formation we have cloned and sequenced a BiP cDNA homolog from rice endosperm. The rice sequence is very similar to the maize BiP exhibiting 92% nucleotide identity and 96% deduced amino acid sequence identity in the coding region. Substantial amino acid sequence homology exists between rice BiP and BiP homologs from several other plant and animal species including long stretches of conservation through the amino-terminal ATPase domain. Considerable variation, however, is observed within the putative carboxy-terminal peptide-binding domain between the plant and nonplant BiP sequences. A single hand of approximately 2.4 kb was visible when RNA gel blots of total RNA purified from seed tissue were probed with radiolabeled rice BiP cDNA. This band increased in intensity during seed development up to 10 days after flowering, and then decreased gradually until seed maturity. Protein gel blots indicated that BiP polypeptide accumulation parallels that of the prolamine polypeptides throughout seed development. Immunocytochemical analysis demonstrated that BiP is localized in a non-stochastic fashion in the endoplasmic reticulum membrane complex of developing endosperm cells. It is abundant on the periphery of the protein inclusion body but not in the central portion of the protein body or in the cisternal ER membranes connecting the protein bodies. These data support a model which proposes that BiP associates with the newly synthesized prolamine polypeptide to facilitate its folding and assembly into a protein inclusion body, and is then recycled.

A Study of Maize Endosperm Hardness in Relation to Amylose Content and Susceptibility to Damage

Abstract: The relative amounts of amylose and amylopectin in maize starch were determined in samples representing hard and soft endosperm. Although differences were small, amylose content differed significantly (P < 0.001 and P < 0.05) between the two types of endosperm, with hard endosperm containing a higher percentage of amylose. Scanning electron microscopy was used to determine that the surface appearance of starch granules from hard and soft endosperm differed. Starch granules from soft endosperm had randomly distributed pores on their surfaces, which had a rough appearance. Few pores were observed on granules from hard endosperm. A fairly common occurrence with starch granules from soft endosperm was the development of wrinkles or fissures upon prolonged exposure to the beam of the electron microscope. Thus, a correlation existed between endosperm hardness, amylose content, and susceptibility to wrinkling and fissures. The granules of the soft endosperm of maize, presumably less mature than the gran...

Cloning gibberellin dioxygenase genes from pumpkin endosperm by heterologous expression of enzyme activities in Escherichia coli

Abstract: Gibberellin (GA) plant hormones are biosynthesized via complex pathways, the final steps of which are catalyzed by 2-oxoglutarate-dependent dioxygenases. Here, the cloning of two such enzymes, the GA 7-oxidase and the GA 20-oxidase, is reported using a novel approach, namely, by screening for GA dioxygenase activities expressed as T7 gene 10 fusion proteins in recombinant Escherichia coli. In vitro translation products of mRNA from endosperm of immature pumpkin seeds contained three GA dioxygenase activities, including 7-oxidase, 20-oxidase, and 3β-hydroxylase. A cDNA expression library was prepared from the endosperm mRNA in λMOSElox. An aliquot of the amplified library was converted to plasmids in vivo and used for transformation of E. coli BL21(DE3), which thereafter expressed recombinant fusion proteins containing 7-oxidase, 20-oxidase, and 3β-hydroxylase activities. By screening for specific GA dioxygenase expression, clones harboring 7-oxidase and 20-oxidase cDNA were isolated. The ORF of the 7-oxidase cDNA is 945 bp long, encoding for 314 amino acid residues with a calculated Mr of 35,712 and pI of 5.7. Recombinant GA 7-oxidase oxidizes GA12-aldehyde to GA12 and GA14-aldehyde to GA14. Evidence was obtained for further metabolism of GA12 by the 7-oxidase to four products, two of which are monohydroxylated GA12. The ORF of the 20-oxidase is—apart from seven changes, resulting in four amino acid substitutions—identical to the 20-oxidase cDNA previously cloned from pumpkin cotyledon mRNA; both 20-oxidases have the same catalytic properties.

Retrotransposon insertion into the maize waxy gene results in tissue-specific RNA processing.

Abstract: We previously reported that three alleles of the maize waxy (wx) gene were alternatively spliced as a result of the insertion of retrotransposons into intronic sequences. In addition, inefficient splicing of element sequences with the surrounding intron produced wild-type transcripts that presumably were responsible for the observed residual gene expression in the endosperm. In this study, we report that one of these alleles, wxG, has a tissue-specific phenotype with 30-fold more WX enzymatic activity in pollen than in the endosperm. Quantification of wxG-encoded transcripts in pollen and the endosperm demonstrates that this difference can be accounted for by tissue-specific differences in RNA processing. Specifically, there is approximately 30-fold more correctly spliced RNA in pollen than in the endosperm. Based on an analogy to similar examples of tissue-specific alternative splicing in animal systems, we hypothesize that the tissue-specific phenotype of the wxG allele may reflect differences in the concentration of splicing factors in these tissues.

Molecular cloning of a cDNA encoding a (1→4)-β-mannan endohydrolase from the seeds of germinated tomato (Lycopersicon esculentum)

Abstract: Mannose-containing polysaccharides are widely distributed in cell walls of higher plants. During endosperm mobilization in germinated tomato seeds (1→4)-β-mannan endohydrolases (EC 3.2.1.78) participate in the enzymic depolymerization of these cell wall polysaccharides. A cDNA encoding a (1→4)-β-mannanase from the endosperm of germinated tomato (Lycopersicon esculentum Mill.) seeds has been isolated and characterized. The amino acid sequence deduced from the 5′-region of the cDNA exactly matches the sequence of the 65 NH2-terminal amino acids determined directly from the purified enzyme. The mature enzyme consists of 346 amino acid residues, it has a calculated Mr of 38 950 and an isoelectric point of 5.3. Overall, the enzyme exhibits only 28–30% sequence identity with fungal (1→4)-β-mannanases, but more highly conserved regions, which may represent catalytic and substrate-binding domains, can be identified. Based on classification of the tomato (1→4)-β-mannanase as a member of the family 5 group of glycosyl hydrolases, Glu-148 and Glu-265 would be expected to be the catalytic acid and the catalytic nucleophile, respectively. Southern hybridization analyses indicate that the enzyme is derived from a family of about four genes. Expression of the genes, as determined by the presence of mRNA transcripts in Northern hybridization analyses, occurs in the endosperm of germinated seeds; no transcripts are detected in hypocotyls, cotyledons, roots or leaves.

Waxy Endoperm Mutants of Bread Wheat(Triticum aestivum L.) and Their Starch Properties

Abstract: Two waxy bread wheat lines, KI07Wx1 and KI07Wx2, have been developed by treating cv. Kanto 107 seeds with ethyl methanesulphonate (EMS). About 2, 000 seeds of the cultivar were soaked for 4 hours in 0.5% EMS dissolved in 7% ethanol, and then 2, 000 seeds were sown in a green-house and 10, 634 M2 seeds were obtained from 1, 872 M1 plants. Cross sections of the distal endosperms of 4, 000 M2 seeds were stained with an iodine-potassium iodide solution, and two seeds stained red-brown. Since the M3 and M4 seeds derived from the remaining proximal halves of these two seeds showed the characteristic feature of waxy endo-sperm, the waxy endosperm character was considered to be genetically fixed in these two mutant lines, which were designated as KI07Wx1 and KI07Wx2. The amylose content of endosperm starch from both mutant lines was 0.9%, and no waxy proteins were detected in the starch of either line. The agronomic characters of KI07Wx1 and KI07Wx2 were very similar to those of cv. Kanto 107 grown under field conditions, except for the heading dates of the mutant lines and the 1, 000-grain weight of KI07Wx2. These results indicate that the mutagen treatment of seeds is effective in inducing of waxy, i.e. amylose-free, mutants when a bread wheat cultivar has a single waxy protein in their endosperm starch.