Showing papers on "Endosperm published in 1998"

Parent-of-origin effects on seed development in Arabidopsis thaliana

Abstract: Many flowering plants are polyploid, but crosses between individuals of different ploidies produce seeds that develop abnormally and usually abort. Often, seeds from interploidy crosses develop differently depending on whether the mother or father contributes more chromosome sets, suggesting that maternal and paternal genomes are not functionally equivalent. Here we present the first cytological investigation of seed development following interploidy crosses in Arabidopsis thaliana. We find that crosses between diploid and tetraploid plants in either direction, resulting in double the normal dose of maternal or paternal genomes in the seed, produce viable seeds containing triploid embryos. However, development of the seed and in particular the endosperm is abnormal, with maternal and paternal genomic excess producing complementary phenotypes. A double dose of maternal genomes with respect to paternal contribution inhibits endosperm development and ultimately produces a smaller embryo. In contrast, a double dose of paternal genomes promotes growth of the endosperm and embryo. Reciprocal crosses between diploids and hexaploids, resulting in a triple dose of maternal or paternal genomes, produce seeds that begin development with similar but more extreme phenotypes than those with a double dose, but these invariably abort. One explanation of our observations is that seeds with maternal or paternal excess contain different doses of maternally or paternally expressed imprinted loci affecting endosperm development.

Characterization of dull1, a Maize Gene Coding for a Novel Starch Synthase

Abstract: The maize dull1 ( du1 ) gene is a determinant of the structure of endosperm starch, and du1 – mutations affect the activity of two enzymes involved in starch biosynthesis, starch synthase II (SSII) and starch branching enzyme IIa (SBEIIa). Six novel du1 – mutations generated in Mutator -active plants were identified. A portion of the du1 locus was cloned by transposon tagging, and a nearly full-length Du1 cDNA sequence was determined. Du1 codes for a predicted 1674-residue protein, comprising one portion that is similar to SSIII of potato, as well as a large unique region. Du1 transcripts are present in the endosperm during the time of starch biosynthesis, but the mRNA was undetectable in leaf or root tissue. The predicted size of the Du1 gene product and its expression pattern are consistent with those of maize SSII. The Du1 gene product contains two repeated regions in its unique N terminus. One of these contains a sequence identical to a conserved segment of SBEs. We conclude that Du1 codes for a starch synthase, most likely SSII, and that secondary effects of du1 – mutations, such as reduction of SBEIIa, result from the primary deficiency in this starch synthase.

Genetic evidence that the two isozymes of sucrose synthase present in developing maize endosperm are critical, one for cell wall integrity and the other for starch biosynthesis.

Abstract: In maize, two paralogous genes, Sh1 and Sus1, encode two biochemically similar isozymes of sucrose synthase, SS1 and SS2, respectively. Previous studies have attributed the mild starch deficiency of the shrunken1 (sh1) endosperm to the loss of the SS1 isozyme in the mutant. Here we describe the first mutation in the sucrose synthase1 (Sus1) gene, sus1-1, and the isolation of a double recessive genotype, sh1 sus1-1. Combined data from diverse studies, including Northern and Western analyses, RT-PCR and genomic PCR, cloning and sequencing data for the 3′ region, show that the mutant sus1-1 gene has a complex pattern of expression, albeit at much reduced levels as compared to the Sus1 gene. Endosperm sucrose synthase activity in sh1 sus1-1 was barely 0.5% of the total activity in the Sh1 Sus1 genotype. Significantly, comparative analyses of Sh1 Sus1, sh1 Sus1 and sh1 sus1-1 genotypes have, for the first time, allowed us to dissect the relative contributions of each isozyme to endosperm development. Starch contents in endosperm of the three related genotypes were 100, 78 and 53%, respectively. Anatomical analyses, which confirmed the previously described early cell degeneration phenotype unique to the sh1 Sus1 endosperm, revealed no detectable difference between the two sh1 genotypes. We conclude that the SS1 isozyme plays the dominant role in providing the substrate for cellulose biosynthesis, whereas the SS2 protein is needed mainly for generating precursors for starch biosynthesis.

The titan mutants of Arabidopsis are disrupted in mitosis and cell cycle control during seed development

Abstract: We describe in this report a novel class of mutants that should facilitate the identification of genes required for progression through the mitotic cell cycle during seed development in angiosperms. Three non-allelic titan (ttn) mutants with related but distinct phenotypes are characterized. The common feature among these mutants is that endosperm nuclei become greatly enlarged and highly polyploid. The mutant embryo is composed of a few giant cells in ttn1, several small cells in ttn2, and produces a normal plant in ttn3. Condensed chromosomes arrested at prophase of mitosis are found in the free nuclear endosperm of ttn1 and ttn2 seeds. Large mitotic figures with excessive numbers of chromosomes are visible in ttn3 endosperm. The ttn1 mutation appears to disrupt cytoskeletal organization because endosperm nuclei fail to migrate to the chalazal end of the seed. How double fertilization leads to the establishment of distinct patterns of mitosis and cytokinesis in the embryo and endosperm is a central question in plant reproductive biology. Molecular isolation of TITAN genes should help to answer this question, as well as related issues concerning cell cycle regulation, chromosome movement and endosperm identity in angiosperms.

An endosperm-specific DOF protein from barley, highly conserved in wheat, binds to and activates transcription from the prolamin box of a native B-hordein promoter in barley endosperm

Abstract: A cDNA encoding a DNA-binding protein of the DOF class of transcription factors was isolated from a barley endosperm library. The deduced amino acid sequence for the corresponding protein is 94% identical through the DOF domain to the prolamin-box (P-box) binding factor PBF from maize. The gene encoding the barley PBF (BPBF) maps to chromosome 7H, and its expression is restricted to the endosperm where it precedes that of the hordein genes. The BPBF expressed in bacteria as a GST-fusion binds a P-box 5'-TGTAAAG-3' containing oligonucleotide derived from the promoter region of an Hor2 gene. Binding was prevented when the P-box motif was mutated to 5'-TGTAgAc-3'. A P-box binding activity, present in barley and wheat endosperm nuclei, interacted similarly to BPBF with this synthetic oligonucleotide, and the binding was abolished by 1,10-phenanthroline. Transient expression experiments in developing barley endosperms demonstrate that BPBF transactivates transcription from the P-box element of a native Hor2 promoter and that direct binding of BPBF to its target site is essential for transactivation since mutations in the DOF DNA-binding domain or in the P-box motif of this promoter abolished both binding and transactivation. Evidence was also obtained for the presence in wheat of a Pbf homologue having similar DNA-binding properties to that of BPBF. These results strongly implicate this endosperm-specific DOF protein from barley as an important activator of hordein gene expression and suggest the evolutionary conservation of the Pbf gene function among small grain cereals.

Brittle-1, an adenylate translocator, facilitates transfer of extraplastidial synthesized ADP--glucose into amyloplasts of maize endosperms.

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

Developmental control of H+/amino acid permease gene expression during seed development of Arabidopsis

Abstract: Summary Long distance transport of amino acids is mediated by several families of differentially expressed amino acid transporters. The two genes AAP1 and AAP2 encode broad specificity H+-amino acid co-transporters and are expressed to high levels in siliques of Arabidopsis, indicating a potential role in supplying the seeds with organic nitrogen. The expression of both genes is developmentally controlled and is strongly induced in siliques at heart stage of embryogenesis, shortly before induction of storage protein genes. Histochemical analysis of transgenic plants expressing promoter-GUS fusions shows that the genes have non-overlapping expression patterns in siliques. AAP1 is expressed in the endosperm and the cotyledons whereas AAP2 is expressed in the vascular strands of siliques and in funiculi. The endosperm expression of AAP1 during early stages of seed development indicates that the endosperm serves as a transient storage tissue for organic nitrogen. Amino acids are transported in both xylem and phloem but during seed filling are imported only via the phloem. AAP2, which is expressed in the phloem of stems and in the veins supplying seeds, may function in uptake of amino acids assimilated in the green silique tissue, in the retrieval of amino acids leaking passively out of the phloem and in xylem-to-phloem transfer along the path. The promoters provide excellent tools to study developmental, hormonal and metabolic control of nitrogen nutrition during development and may help to manipulate the timing and composition of amino acid import into seeds.

Characterization of a Low-Molecular-Weight Glutenin Subunit Gene from Bread Wheat and the Corresponding Protein That Represents a Major Subunit of the Glutenin Polymer

Abstract: Both high- and low-molecular-weight glutenin subunits (LMW-GS) play the major role in determining the viscoelastic properties of wheat (Triticum aestivum L.) flour. To date there has been no clear correspondence between the amino acid sequences of LMW-GS derived from DNA sequencing and those of actual LMW-GS present in the endosperm. We have characterized a particular LMW-GS from hexaploid bread wheat, a major component of the glutenin polymer, which we call the 42K LMW-GS, and have isolated and sequenced the putative corresponding gene. Extensive amino acid sequences obtained directly for this 42K LMW-GS indicate correspondence between this protein and the putative corresponding gene. This subunit did not show a cysteine (Cys) at position 5, in contrast to what has frequently been reported for nucleotide-based sequences of LMW-GS. This Cys has been replaced by one occurring in the repeated-sequence domain, leaving the total number of Cys residues in the molecule the same as in various other LMW-GS. On the basis of the deduced amino acid sequence and literature-based assignment of disulfide linkages, a computer-generated molecular model of the 42K subunit was constructed.

Isolation and characterization of the zSSIIa and zSSIIb starch synthase cDNA clones from maize endosperm

Abstract: Two starch synthase clones, zSSIIa and zSSIIb, were isolated from a cDNA library constructed from W64A maize endosperm. zSSIIa and zSSIIb are 3124 and 2480 bp in length, and contain open reading frames of 732 and 698 amino acid residues, respectively. The deduced amino acid sequences of the two clones share 58.1% sequence identity. Amino acid sequence identity between the zSSIIa and zSSIIb clones and the starch synthase II clones of potato and pea ranges between 45 to 51%. The predicted amino acid sequence from each SSII cDNA contains the KXGGL consensus motif at the putative ADP-Glc binding site. Both clones also contain putative transit peptides followed by the VRAA(E)A motif, the consensus cleavage site located at the C-terminus of chloroplast transit peptides. The identity of the zSSIIa and zSSIIb clones as starch synthases was confirmed by expression of enzyme activity in Escherichia coli. Genomic DNA blot analysis revealed two copies of zSSIIa and a single copy of zSSIIb. zSSIIa was expressed predominantly in the endosperm, while transcripts for zSSIIb were detected mainly in the leaf at low abundance. These findings establish that the zSSIIa and zSSIIb genes are characteristically distinct from genes encoding granule-bound starch synthase I (Waxy protein) and starch synthase I.

Expression Pattern of the Carrot EP3 Endochitinase Genes in Suspension Cultures and in Developing Seeds

Abstract: Carrot (Daucus carota) extracellular protein 3 (EP3) class IV endochitinases were previously identified based on their ability to rescue somatic embryos of the temperature-sensitive cell line ts11. Whole-mount in situ hybridization revealed that a subset of the morphologically distinguishable cell types in embryogenic and nonembryogenic suspension cultures, including ts11, express EP3 genes. No expression was found in somatic embryos. In carrot plants EP3 genes are expressed in the inner integumentary cells of young fruits and in a specific subset of cells located in the middle of the endosperm of mature seeds. No expression was found in zygotic embryos. These results support the hypothesis that the EP3 endochitinase has a “nursing” function during zygotic embryogenesis and that this function can be mimicked by suspension cells during somatic embryogenesis.

Characterization of a Granule-Bound Starch Synthase Isoform Found in the Pericarp of Wheat

Abstract: Waxy wheat (Triticum aestivum L.) lacks the waxy protein, which is also known as granule-bound starch synthase I (GBSSI). The starch granules of waxy wheat endosperm and pollen do not contain amylose and therefore stain red-brown with iodine. However, we observed that starch from pericarp tissue of waxy wheat stained blue-black and contained amylose. Significantly higher starch synthase activity was detected in pericarp starch granules than in endosperm starch granules. A granule-bound protein that differed from GBSSI in molecular mass and isoelectric point was detected in the pericarp starch granules but not in granules from endosperm. This protein was designated GBSSII. The N-terminal amino acid sequence of GBSSII, although not identical to wheat GBSSI, showed strong homology to waxy proteins or GBSSIs of cereals and potato, and contained the motif KTGGL, which is the putative substrate-binding site of GBSSI of plants and of glycogen synthase of Escherichia coli. GBSSII cross-reacted specifically with antisera raised against potato and maize GBSSI. This study indicates that GBSSI and GBSSII are expressed in a tissue-specific manner in different organs, with GBSSII having an important function in amylose synthesis in the pericarp.

Biophysical, physiological and biochemical processes regulating seed germination

Abstract: In the physiological sense, germination begins with seed water uptake and ends with the initiation of elongation by the embryonic axis, usually the radicle. The driving forces and constraints on expansion by the embryo are examined, particularly for seeds in which the embryo is surrounded by endosperm and testa tissues that restrict growth. Models have been developed to predict germination based on thermal time, hydrotime and combined hydrothermal time. These population-based models indicate that the timing of germination is closely tied to physiologically determined temperature and water potential thresholds for radicle emergence which vary among individual seeds in a population. The restraint imposed by tissues surrounding the radicle is a major determinant of the threshold water potential. Enzymatic weakening of these tissues is a key event regulating the timing of radicle emergence. Considerable evidence suggests that endo-β-mannanase is involved in this process in a number of species, although it is doubtful that it is the sole determinant of when radicle emergence occurs. Molecular and biochemical studies are revealing the complexity of events occurring in endosperm and embryo cells associated with the completion of germination. Unique permeability properties and the presence of enzymes associated with pathogen resistance suggest additional functional roles for the tissues enclosing the embryo. The insights gained from physiology and modelling are being extended by the application of molecular techniques to identify and determine the function of genes expressed in association with germination. Single-seed assay methods, in vivo reporters, specific modification of gene expression and mutagenesis will be critical technologies for advancing our understanding of germination

Characterization of SU1 Isoamylase, a Determinant of Storage Starch Structure in Maize

Abstract: Function of the maize (Zea mays) gene sugary1 (su1) is required for normal starch biosynthesis in endosperm. Homozygous su1mutant endosperms accumulate a highly branched polysaccharide, phytoglycogen, at the expense of the normal branched component of starch, amylopectin. These data suggest that both branched polysaccharides share a common precursor, and that the product of the su1 gene, designated SU1, participates in kernel starch biosynthesis. SU1 is similar in sequence to a-(136) glucan hydrolases (starch-debranching enzymes [DBEs]). Specific antibodies were produced and used to demonstrate that SU1 is a 79-kD protein that accumulates in endosperm coincident with the time of starch biosynthesis. Nearly full-length SU1 was expressed in Escherichia coli and purified to apparent homogeneity. Two biochemical assays confirmed that SU1 hydrolyzes a-(136) linkages in branched polysaccharides. Determination of the specific activity of SU1 toward various substrates enabled its classification as an isoamylase. Previous studies had shown, however, that su1- mutant endosperms are deficient in a different type of DBE, a pullulanase (or R enzyme). Immunoblot analyses revealed that both SU1 and a protein detected by antibodies specific for the rice (Oryza sativa) R enzyme are missing from su1- mutant kernels. These data support the hypothesis

Endosperm Development after Fusion of Isolated, Single Maize Sperm and Central Cells in Vitro

Abstract: We demonstrate here the possibility of endosperm development in vitro after the fusion of pairs of an isolated sperm and an isolated central cell of maize. The occurrence of karyogamy and the time course of the fusion of sperm and central cell nuclei are presented. The fusion of the sperm nucleus occurred either with one of the two polar nuclei or with the secondary nucleus and was completed within 2 hr after in vitro cell fusion. The in vitro study of early events after cell and nuclear fusion indicates that the resulting primary endosperm cell develops into a characteristic tissue capable of self-organization apart from the mother tissue. The technology presented here opens the way for new cellular and molecular studies, especially of early events after sperm and central cell fusion. These studies should lead to a better understanding of the processes of double fertilization and endosperm development.

Ethylene-responsive element binding protein (EREBP) expression and the transcriptional regulation of class I β-1,3-glucanase during tobacco seed germination

Abstract: Class I β-1,3-glucanase (βGLU I) is transcriptionally induced in the micropylar endosperm just before its rupture prior to the germination (i.e. radicle emergence) of Nicotiana tabacum L. cv. ‘Havana 425’ seeds. Ethylene is involved in endosperm rupture and high-level βGLU I expression; but, it does not affect the spatial and temporal pattern of βGLU I expression. A promoter deletion analysis of the tobacco βGLU I B gene suggests that (1) the distal −1452 to −1193 region, which contains the positively acting ethylene-responsive element (ERE), is required for high-level, ethylene-sensitive expression, (2) the regions −1452 to −1193 and −402 to 0 contribute to down-regulation by abscisic acid (ABA), and (3) the region −402 to −211 is necessary and sufficient for low-level micropylar-endosperm-specific expression. Transcripts of the ERE-binding proteins (EREBPs) showed a novel pattern of expression during seed germination: light or gibberellin was required for EREBP-3 and EREBP-4 expression; EREBP-4 expression was constitutive and unaffected by ABA or ethylene; EREBP-3 showed transient induction just before endosperm rupture, which was earlier in ethylene-treated seeds and inhibited by ABA. No expression of EREBP-1 and EREBP-2 was detected. In contrast to βGLU I, EREBP-3 and EREBP-4 were not expressed specifically in the micropylar endosperm. The results suggest that transcriptional regulation of βGLU I could depend on: activation of ethylene signalling pathways acting via EREBP-3 with the ERE as the target, and ethylene-independent signalling pathways with targets in the proximal promoter region that are likely to determine spatial and temporal patterns of expression.

Molecular cloning of starch synthase I from maize (W64) endosperm and expression in Escherichia coli

Abstract: Summary A full length cDNA clone encoding a starch synthase (zSS) from maize endosperm (inbred line W64) was isolated and characterized. The cDNA clone (Ss1) is 2907 bp in length and contains an open reading frame of 1866 bp corresponding to a polypeptide of 622 amino acid residues including a transit peptide of 39 amino acids. The Ss1 cDNA clone was identified as zSSI by its direct alignment with sequences to: (i) the N-terminus obtained from the granuleassociated form of the zSSI polypeptide, (ii) four internal peptide fragments obtained from the granule-associated form of the zSSI protein, and (iii) one internal fragment from the soluble form of the zSSI protein. The deduced amino acid sequence of Ss1 shares 75.7 % sequence identity with rice soluble Ss and contains the highly conserved KSGGLGDV putative ADP‐Glc binding site. Moreover, Ss1 exhibited significant activity when expressed in E. coli and the expressed protein is recognized by the antibody raised against the granule associated zSSI protein. Ss1 transcripts were detected in endosperm beginning at 15 days after pollination, but were not found in embryo, leaf or root. Maize contains a single copy of the Ss1 gene, which maps close to the Waxy locus of chromosome 9.

Enhanced stability of maize endosperm ADP-glucose pyrophosphorylase is gained through mutants that alter subunit interactions

Abstract: Temperature lability of ADP-glucose pyrophosphorylase (AGP; glucose-1-phosphate adenylyltransferase; ADP: α-d-glucose-1-phosphate adenylyltransferase, EC 27727), a key starch biosynthetic enzyme, may play a significant role in the heat-induced loss in maize seed weight and yield Here we report the isolation and characterization of heat-stable variants of maize endosperm AGP Escherichia coli cells expressing wild type (WT) Shrunken2 (Sh2), and Brittle2 (Bt2) exhibit a reduced capacity to produce glycogen when grown at 42°C Mutagenesis of Sh2 and coexpression with WT Bt2 led to the isolation of multiple mutants capable of synthesizing copious amounts of glycogen at this temperature An increase in AGP stability was found in each of four mutants examined Initial characterization revealed that the BT2 protein was elevated in two of these mutants Yeast two-hybrid studies were conducted to determine whether the mutant SH2 proteins more efficiently recruit the BT2 subunit into tetramer assembly These experiments showed that replacement of WT SH2 with the heat-stable SH2HS33 enhanced interaction between the SH2 and BT2 subunits In agreement, density gradient centrifugation of heated and nonheated extracts from WT and one of the mutants, Sh2hs33, identified a greater propensity for heterotetramer dissociation in WT AGP Sequencing of Sh2hs33 and several other mutants identified a His-to-Tyr mutation at amino acid position 333 Hence, a single point mutation in Sh2 can increase the stability of maize endosperm AGP through enhanced subunit interactions

The evolution of double fertilization and endosperm: an ”historical” perspective

Abstract: One hundred years ago, the developmental origin of endosperm from double fertilization was discovered independently by Navashin and Guignard. For much of the twentieth century, specific events related to the evolutionary origin of the endosperm of flowering plants remained a mystery. However, during the past 20 years, advances in phylogenetic reconstruction of seed plants, genetic theory associated with kin selection, and comparative study of the reproductive biology of the closest living relatives of angiosperms (Gnetales) have advanced our understanding of the evolutionary events associated with the origin of double fertilization and endosperm. Recent developmental analyses of Ephedra and Gnetum (members of Gnetales) indicate that these nonflowering seed plants undergo a regular process of double fertilization that yields two diploid zygotes. Use of explicit genetic and developmental criteria for analysis of evolutionary homology demonstrates congruence with the hypothesis that double fertilization processes in Gnetales and angiosperms were inherited from a common ancestor of the two lineages. In its rudimentary form, the second fertilization event in the ancestors of flowering plants yielded a supernumerary diploid embryo that was genetically identical to the normal embryo, a process most similar to what occurs in extant Ephedra. Subsequent to the divergence of the angiosperm stem lineage, the supernumerary embryo derived from double fertilization was developmentally modified into an embryo-nourishing structure, endosperm, that now characterizes angiosperms.

A cysteine endopeptidase with a C-terminal KDEL motif isolated from castor bean endosperm is a marker enzyme for the ricinosome, a putative lytic compartment

Abstract: A papain-type cysteine endopeptidase with a molecular mass of 35 kDa for the mature enzyme, was purified from germinating castor bean (Ricinus communis L.) endosperm by virtue of its capacity to process the glyoxysomal malate dehydrogenase precursor protein to the mature subunit in vitro (C. Gietl et al., 1997, Plant Physiol 113: 863-871). The cDNA clones from endosperm of germinating seedlings and from developing seeds were isolated and sequence analysis revealed that a very similar or identical peptidase is synthesised in both tissues. Sequencing established a presequence for co-translational targeting into the endoplasmic reticulum, an N-terminal propeptide and a C-terminal KDEL motif for the castor bean cysteine endopeptidase precursor. The 45-kDa pro-enzyme stably present in isolated organelles was enzymatically active. Immunocytochemistry with antibodies raised against the purified cysteine endopeptidase revealed highly specific labelling of ricinosomes, organelles which co-purify with glyoxysomes from germinating Ricinus endosperm. The cysteine endopeptidase from castor bean endosperm, which represents a senescing tissue, is homologous to cysteine endopeptidases from other senescing tissues such as the cotyledons of germinating mung bean (Vigna mungo) and vetch (Vicia sativa), the seed pods of maturing French bean (Phaseolus vulgaris) and the flowers of daylily (Hemerocallis sp.).

Barley BLZ1: a bZIP transcriptional activator that interacts with endosperm-specific gene promoters

Abstract: A cDNA encoding a bZIP transcription factor was obtained from barley endosperm and used to identify the corresponding gene from a genomic library. The gene, designated Blz1, contained six exons and five introns, plus a 442 nt-long 5'-untranslated leader sequence, and was located on chromosome 5H. The Blz1 mRNA was detected early in endosperm development and was also expressed in roots and leaves. The BLZ1 protein was a potent transcriptional activator in a yeast system; 85% of its activity was associated with the first 203 amino acid residues at the N-terminus, which included two acidic regions. Presumptive involvement of Blz1 in the regulation of gene expression in endosperm was ascertained by the DNA-binding properties of BLZ1 in electrophoretic mobility shift assays (EMSA) and by transient expression in barley developing endosperms, using, as effectors, Blz1 in both sense and anti-sense orientations. In the co-bombardment experiments, the beta-glucuronidase (GUS) reported gene responded to Blz1 if under the control of the endosperm-specific ltr1 promoter or under a synthetic promoter containing the endosperm box of gene Hor2. Sucrose synthase promoters Ss1 and Ss2 and synthetic promoters containing mutated sequences of Hor2 were unaffected in trans by Blz1.

Nucellain, a Barley Homolog of the Dicot Vacuolar-Processing Protease, Is Localized in Nucellar Cell Walls

Abstract: The nucellus is a complex maternal grain tissue that embeds and feeds the developing cereal endosperm and embryo. Differential screening of a barley (Hordeum vulgare) cDNA library from 5-d-old ovaries resulted in the isolation of two cDNA clones encoding nucellus-specific homologs of the vacuolar-processing enzyme of castor bean (Ricinus communis). Based on the sequence of these barley clones, which are called nucellains, a homolog from developing corn (Zea mays) grains was also identified. In dicots the vacuolar-processing enzyme is believed to be involved in the processing of vacuolar storage proteins. RNA-blot and in situ-hybridization analyses detected nucellain transcripts in autolysing nucellus parenchyma cells, in the nucellar projection, and in the nucellar epidermis. No nucellain transcripts were detected in the highly vacuolate endosperm or in the other maternal tissues of developing grains such as the testa or the pericarp. Using an antibody raised against castor bean vacuolar-processing protease, a single polypeptide was recognized in protein extracts from barley grains. Immunogold-labeling experiments with this antibody localized the nucellain epitope not in the vacuoles, but in the cell walls of all nucellar cell types. We propose that nucellain plays a role in processing and/or turnover of cell wall proteins in developing cereal grains.

Callose Deposition Is Responsible for Apoplastic Semipermeability of the Endosperm Envelope of Muskmelon Seeds

Abstract: Semipermeable cell walls or apoplastic "membranes" have been hypothesized to be present in various plant tissues. Although often associated with suberized or lignified walls, the wall component that confers osmotic semipermeability is not known. In muskmelon (Cucumis melo L.) seeds, a thin, membranous endosperm completely encloses the embryo, creating a semipermeable apoplastic envelope. When dead muskmelon seeds are allowed to imbibe, solutes leaking from the embryo are retained within the envelope, resulting in osmotic water uptake and swelling called osmotic distention (OD). The endosperm envelope of muskmelon seeds stained with aniline blue, which is specific for callose (beta-1,3-glucan). Outside of the aniline-blue-stained layer was a Sudan III- and IV-staining (lipid-containing) layer. In young developing seeds 25 d after anthesis (DAA) that did not exhibit OD, the lipid layer was already present but callose had not been deposited. At 35 DAA, callose was detected as distinct vesicles or globules in the endosperm envelope. A thick callose layer was evident at 40 DAA, coinciding with development of the capacity for OD. Removal of the outer lipid layer by brief chloroform treatment resulted in more rapid water uptake by both viable and nonviable (boiled) seeds, but did not affect semipermeability of the endosperm envelope. The aniline-blue-staining layer was digested by beta-1,3-glucanase, and these envelopes lost OD. Thus, apoplastic semipermeability of the muskmelon endosperm envelope is dependent on the deposition of a thick callose-containing layer outside of the endosperm cell walls.

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

Abstract: 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.

Inhibition of maize endosperm cell division and endoreduplication by exogenously applied abscisic acid

Abstract: Abscisic acid (ABA) is thought to play a role in inhibiting or aborting kernel growth during water deficit. To test the responsiveness of early endosperm development to ABA concentrations, cylinders containing (±)ABA in a buffered agar medium were applied to the apical pericarp surface of kernels on intact, well-watered maize (Zea mays L. cv. Pioneer Brand 3925) plants from 5 to 11 days after pollination (DAP). Endosperm nuclei were analyzed by flow cytometry to assess effects on cell division and endoreduplication. ABA treatments of ≥ 100 µM substantially decreased endosperm cell numbers and fresh weight accumulation, but did not affect average cell size. ABA at ≥ 300 µM decreased the proportion of nuclei in the size classes ≥ 12C, indicating that the rate of transition to endoreduplication status was inhibited, and decreased the progressive advance from 12C to 24C to 48C, indicating that the rate of S-phase cycling of endoreduplicating cells was inhibited. We conclude that cell division was more responsive to ABA concentrations than were endoreduplication or cell expansion growth.

Spatial and temporal regulation of DNA fragmentation in the aleurone of germinating barley

Abstract: During germination of barley grains, the appearance of DNA fragmentation started in aleurone cells near the embryo and extended to the distal end in a time-dependent manner. DNA fragmentation was demonstrated to occur only after the expression of α-amylase mRNA in the aleurone layer. In addition, cell wall degradation started in cells near the embryo on the sides facing the endosperm. Subsequently cell wall degradation extended to the lateral cell walls and to cells more to the distal end of the grain. A typical alteration of the nucleus was observed by electron microscopy and an almost complete degradation of DNA was found in the nucleus while the nuclear envelope remained intact. The results indicate that programmed cell death occurred in aleurone cells during germination. A model is proposed for the regulation of programmed cell death in aleurone cells during germination involving ABA levels and cell wall degradation.