Endosperm Tissue Research Articles

Endosperm Morphogenesis in Wheat: Transfer of Nutrients from the Antipodals to the Lower Endosperm1

The morphogenesis of early cell formation in the endosperm of wheat (Triticum aestivum L.) was studied by light microscopy. The objective of this study was to provide a description of endosperm cell formation and corresponding changes in the surrounding diploid and haploid tissues during the first 4 days of wheat endosperm growth. Entire spikelets of large‐kerneled, soft white winter and small‐kerneled hard red winter wheat cultivars were harvested daily, embedded in paraffin, and sectioned longitudinally. The microscopic sections provided a series of photographs documenting the continuous development of the endosperm, embryo sac, and maternal tissues during their early growth. During the first 4 days of development, dense endosperm cells were formed in the lower one‐fourth of the embryo sac, immediately above the embryo. The cytoplasm that made up those cells came from endosperm vesicles formed higher in the embryo sac. These vesicles were formed in the free nuclear endosperm bordering the antipodals. As the vesicles enlarged, they became spherical, were freed, and fell to the gravitational bottom of the embryo sac. There they coalesced, in an appropriate position to be used as nourishment for the embryo. During the period of endosperm vesicle formation, there was concurrent degeneration of the antipodals. By the end of the fourth day postanthesis (PA), the antipodals had been largely consumed and vesicle formation had stopped. Also by that time, walls had grown in from the nucellus and had partitioned the mass of nucleate cytoplasm, above the embryo, into endosperm cells. By 15 days PA, these cells had been consumed entirely by the developing embryo. Cytoplasmic vesicles were found to be the developmental mechanism by which nutrients are transferred from the antipodals to the lower endosperm and ultimately to the embryo.

Characterization and distribution of invertase activity in developing maize (Zea mays) kernels

Invertase (β‐fructofuranoside fructohydrolase, EC 3.2.1.26) activity in developing maize (Zea mays L. inbred W64A) was separated into soluble and particulate forms. The particulate form was solubilized by treatment with 1 M NaCl or with other salts. However, CaCl2 inhibited invertase activity, and neither detergents nor 0.5 M methyl mannoside were effective in solubilizing the invertase activity. The soluble and particulate invertases were both glycoproteins, both had pH optima of 5.0 and Km values for sucrose of 2.83 and 1.84 mM, respectively. The apparent molecular weight of salt‐solubilized invertase was 40 kDa. Gel filtration of the soluble invertase showed multiple peaks with apparent molecular weights ranging from 750 kDa to over 9 000 kDa. Histochemical staining of cell wall preparations for invertase activity suggested that the particulate invertase is associated with the cell wall. Also, nearly all the invertase activity was localized in the basal endosperm and pedicel tissues, which are sites of sugar transport. No invertase activity was found in the upper endosperm, the embryo or in the placento‐chalazal tissue. In contrast, sucrose synthase (EC 2.4.1.13) activity was found primarily in the embryo and the upper endosperm, which are areas of active biosynthesis of storage compounds.

Effects of Growth Regulators and Glutamine on In Vitro Development of Zygotic Embryos of Taro (Colocasia esculenta var. antiquorum)

Zygotic embryos of taro, Colocasia esculenta var. antiquorum cultured on Linsmaier-Skoog (LS) medium without the addition of hormones develop into mature plants only in the presence of endosperm tissue. Growth is usually evident within the first week of culture when embryos swell and become green. Embryos excised from endosperm and cultured on LS containing 0-01 mg 1−1 naphthaleneacetic acid (NAA), and 0–01 mg 1−1 6-dimethylaminopurine (6-DMAP) grow at a rate comparable with controls for the first week of culture. During the second week, growth rates are higher than controls primarily because embryos form elongated hypocotyl regions which often produce swollen tissues and/or callus. In the presence of 200 mg 1−1 glutamine and a range of concentrations of 6-dimethylaminopurine, benzyladenine, or NAA, elongation of the hypocotyl axis is inhibited, and a compact callus may develop. Embryos grown on LS containing 200 mg 1−1 glutamine and 2.0 mg 1−1 2, 4, 5-trichlorophenoxyacetic acid form friable callus which was used to generate short-lived suspension cultures.

Structural and transcriptional analysis of DNA sequences flanking genes that encode 19 kilodalton zeins.

The nucleotide sequence of gz19ab11, a clone that corresponds to the coding and flanking sequences of an Mr 19,000 alpha zein, was determined. Comparison of the DNA sequences flanking this gene with those of other members of the gene subfamily showed that sequence conservation extends 820 nucleotides into the 5' flanking region and 130 nucleotides into the 3' flanking region. Southern blot analysis of maize DNA indicated that highly repetitive sequences are located within 950 bp 5' and 300 bp 3' to the protein coding region of these genes. The coding region of gz19ab11 is similar to but not identical with cDNA clones corresponding to Mr 19,000 zeins, and analysis of zein transcripts indicated that this gene is expressed exclusively in endosperm tissue. RNAs which correspond to transcripts originating 60 nucleotides, and more than 800 nucleotides, upstream of the initiation codon were detected for this and a related gene. However, the concentration of the large RNA species was several orders of magnitude less than that of the shorter RNAs. The functional significance of these large RNA transcripts in zein gene expression is unclear.

Starch characteristics in cultures of normal and mutant maize endosperm

Vigorously growing suspension cultures of 'normal', amylose-extender (ae) and waxy (wx) maize endosperm were established from near isogenic lines of maize inbred A636. The recovery of the ability to produce vigorous cultures of ae and wx endosperm by backcrossing demonstrate the genetic control of endosperm growth in vitro. Phenotypic expression of the endosperm mutants in culture was studied by examining the properties of starch accumulated in endosperm cultures and starch from developing and mature kernels of the same genotype. After 9 months in culture, the amylose contents of the starch in normal callus tissue and normal endosperm tissue were not significantly different, 28.2% and 31.7%, respectively. Starch granules from normal cultures and endosperm stained blue-black with iodine and were round to polygonal in shape. The starches of wx endosperm and callus cultures contained no amylose, and wx starch granules stained brown-orange with iodine. Although, wx starch granules were primarily round, a few granules with "jagged edges" were observed in starch samples isolated from cultures and kernels. The percent amylose in starch from ae callus was significantly lower than the amylose content of starch from ae endosperm tissue, 39.9% and 67.7%, respectively. Starch granules from ae endosperm and cultures were smaller than normal and wx starch granules. Irregular starch granules which are typical of ae endosperm were present in ae callus tissue, but were less frequently observed. We conclude that specific endosperm mutant phenotypes are expressed in vitro.

Effect of gibberellin on protein and non-structural carbohydrate in dormant Avena fatua caryopses

Application of gibberellic acid (GA 3) to dormant Avena fatua L. caryopses resulted in the termination of dormancy within 24 h as indicated by germination between 24 and 48 h. During the period of imbibition from 0 to 24 and 24 to 48 h changes occurred in protein and carbohydrate metabolism in GA-treated and untreated caryopses. Germination did not occur in untreated caryopses, therefore physiological changes in these caryopses were not associated with the termination of dormancy. GA-treatment increased the concentration of soluble and SDS-extractable protein in the endosperm tissue by 4 and 5%, respectively, over the 24 h untreated material; no changes were apparent when the protein profiles of GA-treated and untreated tissues were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 0, 24 and 48 h after imbibition. The concentration of hexose and sucrose in the GA-treated endosperm tissue increased 189 and 151 μmol, respectively, over the untreated material at 24 h. Gibberellic acid had no effect on starch metabolism in the endosperm tissue in the first 24 h, the period associated with the termination of dormancy. The concentration of hexose increased by 57 μmol and starch decreased by 80 μmol in the GA-treated embryo tissue within 24 h. Our results demonstrate that exogenously applied GA influences sucrose and hexose metabolism in the endosperm tissue. The specific effect of GA on starch and hexose metabolism in the dormant A. fatua caryopsis embryo tissue may be associated with the termination of dormancy.

Purification and Properties of Nonproteolytic Degraded ADPglucose Pyrophosphorylase from Maize Endosperm

ADPglucose pyrophosphorylase from developing endosperm tissue of starchy maize (Zea mays) was purified 88-fold to a specific activity of 34 micromoles alpha-glucose-1-P produced per minute per milligram protein. Rabbit antiserum to purified spinach leaf ADPglucose pyrophosphorylase was able to inhibit pyrophosphorolysis activity of the purified enzyme by up to 90%. The final preparation yielded four major protein staining bands following sodium dodecyl sulfate polyacrylamide gel electrophoresis. When analyzed by Western blot hybridization only the fastest migrating, 54 kilodaltons, protein staining band cross-reacted with affinity purified rabbit antispinach leaf ADPglucose pyrophosphorylase immunoglobulin. The molecular mass of the native enzyme was estimated to be 230 kilodaltons. Thus, maize endosperm ADPglucose pyrophosphorylase appears to be comprised of four subunits. This is in contrast to the respective subunit and native molecular masses of 96 and 400 kilodaltons reported for a preparation of maize endosperm ADPglucose pyrophosphorylase (Fuchs RL and JO Smith 1979 Biochim Biophys Acta 556: 40-48). Proteolytic degradation of maize endosperm ADPglucose pyrophosphorylase appears to occur during incubation of crude extracts at 30 degrees C or during the partial purification of the enzyme according to a previously reported procedure (DB Dickinson, J Preiss 1969 Arch Biochem Biophys 130: 119-128). The progressive appearance of a 53 kilodalton antigenic peptide suggested the loss of a 1 kilodalton proteolytic fragment from the 54 kilodalton subunit. The complete conservation of the 54 kilodalton subunit structure following extraction of the enzyme in the presence of phenylmethylsulfonyl fluoride and/or chymostain was observed. The allosteric and catalytic properties of the partially purified proteolytic degraded versus nondegraded enzyme were compared. The major effect of proteolysis was to enhance enzyme activity in the absence of added activator while greatly decreasing its sensitivity to the allosteric effectors 3-P-glycerate and inorganic phosphate.

Water Relations of Tomato Seed Germination

Water uptake during the germination of UC 82B tomato seeds was triphasic. Seed Ψ measurements indicated that phase I imbibition occurred because of the large Ψ gradient between the seed and the imbibition solution (water). During phase II the seed Ψ was in equilibrium with the water. Phase III water uptake recommenced with the onset of radicle emergence without changes in Ψ or Ψπ. Changes in embryo water content were also triphasic. During phase II the embryo Ψ remained at - 1.5 MPa, not in equilibrium with the imbibing solution. At radicle emergence it was - 0.8 MPa and rose to -0.3 MPa as the radicle elongated. There was no evidence of a lowering of embryo Ψπ nor of a build up of Ψp prior to radicle emergence. Water uptake studies with excised embryos indicated that, within the seed, the enclosing tissues prevented the embryo from taking up water. It is suggested that embryo water content is restricted by the constraint on embryo expansion caused by the enclosing endosperm tissue. Lowering of embryo Ψπ to build up Ψp is not necessary for radicle emergence. The control of germination may lie in the mechanism which leads to weakening of this mechanical restraint of the endosperm.

Role of Placental Sac in Endosperm Carbohydrate Import in Sorghum Caryopses1

An anatomical study of tissues involved with carbohydrate import into developing caryopsis endosperm in three cultivars of sorghum [Sorghum bicolor (L.) Moench] was complemented by determination of the assimilate concentration of placental sac fluids to investigate the role of the placental sac. Individual panicles were tagged daily at anthesis. Caryopses were harvested from anthesis to maturity and from 10 to 20 days postanthesis at 5‐day intervals for microscopic examination and placental sac fluid carbohydrate analysis. Formation of endosperm tissue by 5 days postanthesis coincided with differentiation of transfer cells within the outer surface of endosperm tissue adjacent to the placental region. All caryopsis tissues thought to be associated with carbohydrate movement to the endosperm (vascular tissue, chalazal tissue, remnant nucellar tissue, the placental sac, and aleurone transfer cells) were present in the cultivars by 10 days postanthesis. The role of the placental sac as an apoplastic sink for hexose import into the endosperm was supported by anatomical location and high glucose and fructose concentrations relative to sucrose concentrations in placental sac fluids of all cultivars at 10,15, and 20 days postanthesis. A model for hexose import into the endosperm following vascular unloading in sorghum was developed based on prior findings for maize (Zea mays L.). Results indicate that the placental sac provides an intermediate apoplastic sink for assimilate accumulation from placental tissues and subsequent hexose import by transfer cells of the endosperm.

Excision of Ds produces waxy proteins with a range of enzymatic activities.

The waxy (wx) locus of maize encodes an enzyme responsible for the synthesis of amylose in endosperm tissue. The phenotype of the Dissociation (Ds) insertion mutant wx-m1 is characterized by endosperm sectors that contain different levels of amylose. We have cloned the Wx gene from this allele and from two germinal derivatives, S5 and S9, that produce intermediate levels of amylose. The Ds insertion in wx-m1 is in exon sequences, is 409 bp in length and represents an example of a class of Ds elements that are not deletion derivatives of the Activator (Ac) controlling element. The two germinal derivatives, S5 and S9, lack the Ds element but contain an additional 9 and 6 bp, respectively, at the site of Ds insertion. The level of Wx mRNA and Wx protein in S5 and S9 is essentially the same as in normal endosperm tissue but Wx enzymatic activity is reduced. Thus, the lesions in S5 and S9 lead to the addition of amino acids in the Wx protein, resulting in Wx enzymes with altered specific activities. This work supports the notion that the maize transposable elements may serve a function in natural populations to generate genetic diversity, in this case, proteins with new enzymatic properties.

GENETIC POLYMORPHISM IN THE MORPHOLOGICALLY REDUCED DWARF MISTLETOES (ARCEUTHOBIUM, VISCACEAE): AN ELECTROPHORETIC STUDY

Arceuthobium is a well defined genus of obligate parasites of conifers. Assessment of taxonomic and phylogenetic relationships in this group is difficult owing to morphological reductions and complex host relationships. In the present study, genetic relationships within and among 19 taxa were examined using starch gel electrophoresis of triploid seed endosperm tissue. Allelic frequency data for eleven polymorphic loci were derived from analysis of 40 natural populations collected from the U.S. and Mexico. The genus showed remarkably high levels of genetic diversity: averaged across the 19 taxa, 66.7% of the loci were polymorphic with an average of 2.23 alleles per locus. This level of polymorphism is approximately double the average value reported for many dicotyledons and stands in contrast to the overall uniform morphology of these parasites.Unweighted pair group cluster analysis (UPGMA) of genetic similarity measures was conducted for all sampled populations and the results compared to a phenetic system of classification for the genus. Similarities between the two studies include the recognition of two subgenera based upon the segregation of the verticillately branched Arceuthobium americanum from the remainder of the taxa examined. Analysis of isozyme data supported a grouping of six taxa: A. vaginatum ssp. cryptopodum, A. vaginatum ssp. durangense, A. gillii, A. rubrum, A. divaricatum, and A. douglasii. The placement of the latter three taxa in other groups by phenetic criteria provides evidence for molecular divergence not seen using morphological features. The Campylopodum group of taxa comprised eleven members that were linked at similarity values of 80% or greater. Populations were not unambiguously grouped according to species as defined by the phenetic study or at similarity levels comparable to other well defined species in this study. This group is either not reproductively isolated or molecular differentiation is cryptic due to rapid adaptive radiation onto numerous host tree species.

Free and bound indole-acetic acid is low in the endosperm of the maize mutantdefective endosperm-B18

The maize mutant defective endosperm-B18 (de (*)-B18), which is recessive to its wildtype, accumulates substantially less dry matter in the endosperm than its normal counterpart. Both free and bound indole-acetic acid (IAA) content has been measured at 5 different developmental stages. In endosperm tissue, the level of IAA is at least 15 times lower in the mutantde (*) -B18 than in the wildtype. The situation found in the diploid tissues is somewhat different: in the mature embryo the level of total IAA is lower in the mutant than in the wildtype, while in 4-day old seedlings the level of total IAA is, to some degree, similar in both genotypes. Naphthalene-acetic acid (NAA), a stable synthetic auxin which mimics IAA in its biochemical effects, is able to normalize the seed weight of the mutant when applied to developing grains. The results favor the conclusion that in maize endosperm the mutationde (*) -B18 is involved in IAA metabolism.

Evidence for the existence of endosperm balance number in the true clovers (Trifolium spp.)

The endosperm balance number (EBN) hypothesis was first advanced to explain results from interspecific crosses in Solanum and later in Impatiens. According to the EBN hypothesis, normal endosperm development following intra- or inter-specific crosses depends on having a ratio of two EBNs from the female to one EBN from the male in the endosperm tissue. EBNs may differ among related species. Successful hybrids can be obtained between species with the same EBN. The ploidy level of an individual species can be varied to modify its EBN, making it cross compatible with a species sharing its modified EBN. Interspecific crosses within Trifolium have been limited and difficult. Crosses reported in the literature, including evidence from our own study, suggest that EBN is operating in Trifolium and have been used to assign EBN numbers to some clover species. The use of 2n eggs enabled two species, differing in EBN, to be crossed. An understanding of the EBN mechanism that operates in Trifolium should make successful interspecific hybrids easier to obtain in the future.Key words: endosperm balance number, hybrids (interspecific), 2n gametes, Trifolium.

Tissue-specific and light-dependent changes of chromatin organization in barley (Hordeum vulgare).

The DNase I sensitivity of the nuclear genes encoding the NADPH-protochlorophyllide oxidoreductase, the light-harvesting chlorophyll a/b protein (LHCP), the hordeins and a 15-kDa protein of unknown function was assayed in chromatin of etiolated and green leaves and endosperm tissue of barley (Hordeum vulgare L.). A tissue-specific differentiation of chromatin structure was found for the LHCP, hordein and 15-kDa protein genes. The genes for the LHCP and the 15-kDa protein, which are expressed in leaf tissue, display DNase I sensitivity in leaves but not in endosperm. Hordein genes which are expressed solely in endosperm, were insensitive to low levels of digestion with DNase I in leaves but sensitive in endosperm. The effect of light on chromatin structure was determined by comparing leaves of etiolated plants and plants which had been grown under a day/night cycle. Only in the case of the 15-kDa protein is there a remarkable change from a DNAse-I-sensitive configuration in etiolated leaves to a more resistant one in leaves from illuminated plants. The gene for the NADPH-protochlorophyllide oxidoreductase was found to be equally sensitive to DNase I in leaves and endosperm.

Accumulation and subcellular localization of glutelin-2 transcripts during maturation of maize endosperm

The expression of glutelin-2 genes has been studied during maize endosperm maturation and compared to the expression of two different zein genes. Glutelin-2 mRNA in endosperm accumulates in parallel to zein mRNAs, nevertheless, the opaque-2 mutation does not affect glutelin-2 transcription. Subcellular fractionation of endosperm tissue shows that glutelin-2 mRNA is accumulated in the protein body fraction indicating that these organelles are the place of translation of glutelin-2 mRNA. The results indicate that the levels of glutelin-2 transcripts in maize endosperm increase following a temporal pattern similar to that displayed by zein transcripts, the typical maize storage protein.

ADPglucose Pyrophosphorylase Is Encoded by Different mRNA Transcripts in Leaf and Endosperm of Cereals

Western blots of soluble protein from wheat, rice, and corn showed that ADPglucose pyrophosphorylase subunits have a size of 50 kilodaltons from endosperm tissue and 43 and 46 kilodaltons from leaf. Antisera to ADPglucose pyrophosphorylase precipitated in vitro translation products of 73 and 76 kilodaltons when leaf poly(A)(+) RNA was used, whereas endosperm mRNA directed the synthesis of 50 and 56 kilodalton polypeptides. To further study the nature of these mRNA species, an ADPglucose pyrophosphorylase cDNA clone from rice endosperm polyadenylated RNA was obtained and used as a hybridization probe. Northern blots showed that ADPglucose pyrophosphorylase mRNA was slightly larger in leaf (2100 bases) than in endosperm tissue (1900 bases). These studies indicated that in cereals there are at least two tissue specific forms of ADPglucose pyrophosphorylase that are encoded by distinct mRNA transcripts. Analysis of genomic DNA by Southern blotting suggested that ADPglucose pyrophosphorylase is encoded by a small gene family.

Differential Synthesis in Vitro of Barley Aleurone and Starchy Endosperm Proteins

To widen the selection of proteins for gene expression studies in barley seeds, experiments were performed to identify proteins whose synthesis is differentially regulated in developing and germinating seed tissues. The in vitro synthesis of nine distinct barley proteins was compared using mRNAs from isolated endosperm and aleurone tissues (developing and mature grain) and from cultured (germinating) aleurone layers treated with abscisic acid (ABA) and GA(3). B and C hordein polypeptides and the salt-soluble proteins beta-amylase, protein Z, protein C, the chymotrypsin inhibitors (CI-1 and 2), the alpha-amylase/subtilisin inhibitor (ASI) and the inhibitor of animal cell-free protein synthesis systems (PSI) were synthesized with mRNA from developing starchy endosperm tissue. Of these proteins, beta-amylase, protein Z, and CI- 1 and 2 were also synthesized with mRNA from developing aleurone cells, but ASI, PSI, and protein C were not. CI-1 and also a probable amylase/protease inhibitor (PAPI) were synthesized at high levels with mRNAs from late developing and mature aleurone. These results show that mRNAs encoding PAPI and CI-1 survive seed dessication and are long-lived in aleurone cells. Thus, expression of genes encoding ASI, PSI, protein C, and PAPI is tissue and stage-specific during seed development. Only ASI, CI-1, and PAPI were synthesized in significant amounts with mRNA from cultured aleurone layers. The levels of synthesis of PAPI and CI-1 were independent of hormone treatment. In contrast, synthesis of alpha-amylase (included as control) and of ASI showed antagonistic hormonal control: while GA promotes and ABA reduces accumulation of mRNA for alpha-amylase, these hormones have the opposite effect on ASI mRNA levels.

Biosynthesis of the Macrocyclic Diterpene Casbene in Castor Bean (Ricinus communis L.) Seedlings

Casbene is a macrocyclic diterpene hydrocarbon that is produced in young castor bean (Ricinus communis L.) seedlings after they are exposed to Rhizopus stolonifer or other fungi. The activities of enzymes that participate in casbene biosynthesis were measured in cell-free extracts of 67-hour castor bean seedlings (a) that had been exposed to R. stolonifer spores 18 hours prior to the preparation of extracts, and (b) that were maintained under aseptic conditions throughout. Activity for the conversion of mevalonate to isopentenyl pyrophosphate does not change significantly after infection. On the other hand, the activities of farnesyl pyrophosphate synthetase (geranyl transferase), geranylgeranyl pyrophosphate synthetase (farnesyl transferase), and casbene synthetase are all substantially greater in infected tissues in comparison with control seedlings maintained under sterile conditions. The subcellular localization of these enzymes of casbene biosynthesis was investigated in preparations of microsomes, mitochondria, glyoxysomes, and proplastids that were resolved by centrifugation in linear and step sucrose density gradients of homogenates of castor bean endosperm tissue from both infected and sterile castor bean seedlings. Isopentenyl pyrophosphate isomerase and geranyl transferase activities are associated with proplastids from both infected and sterile seedlings. Significant levels of farnesyl transferase and casbene synthetase are found only in association with the proplastids of infected tissues and not in the proplastids of sterile tissues. From these results, it appears that at least the last two steps of casbene biosynthesis, geranylgeranyl pyrophosphate synthetase and casbene synthetase, are induced during the process of infection, and that the enzymes responsible for the conversion of isopentenyl pyrophosphate to casbene are localized in proplastids.

DEVELOPMENT AND EVALUATION OF A FREEZE‐ETCH, FREEZE‐FRACTURE TECHNIQUE APPLIED TO DEVELOPING WHEAT ENDOSPERM

A technique was developed and evaluated whereby differentiating wheat endosperm tissue could be processed for the freeze‐etch, freeze‐fracture technique without the use of chemical fixatives. Field grown, developing hard red winter wheat (cv. Newton) caryopses were infiltrated with glycerol prior to freezing in liquid nitrogen‐cooled Freon‐22. Frozen samples were placed in cryogenic vials and stored in liquid nitrogen until needed. Freeze‐fracture was carried out under standard conditions. Evaluation of replicas from glycerol‐imbibed endosperm was made by comparing them to replicas obtained from freshly frozen untreated endosperm, and endosperm that had been prefixed in glutaraldehyde and paraformaldehyde. Other evaluations were made by comparing replicas of glycerol‐treated endosperm to thin sections obtained from wheat that had been imbibed with glycerol, fixed, dehydrated, and infiltrated and embedded in epoxy resin for routine electron microscopy. The results indicate that if the unfixed glycerol‐treated wheat endosperm is handled carefully, replicas can be obtained which show few artifacts due to glycerol and freezing. Typical artifacts such as vesiculation of RER, ice crystal damage, production of fusion intermediates, and membrane particle segregation can be nearly eliminated when this technique is applied to developing wheat endosperm.

Heat Inactivation of Starch Synthase in Wheat Endosperm Tissue

The effect of temperature on accumulation of starch was studied in grain slices of wheat (Triticum aestivum cv SUN9E), taken 15 days after anthesis. As compared with pretreatment of such slices at 25 degrees C, pretreatment at 30 or 35 degrees C reduced the subsequent conversion of sucrose to starch. In contrast to rice (Oryza sativa cv Calrose), pretreatment of wheat soluble starch synthase in vitro at 30 degrees C or higher temperatures reduced its activity. In zymograms using nondenaturing polyacrylamide gel electrophoresis followed by activity staining, the slowest migrating band represented the most temperature sensitive isozyme. Although preincubation of a soluble enzyme sample in vitro at 25 degrees C did not result in loss of starch synthase activity, it did result in a gradual shift of zymogram banding pattern toward faster migrating species. Pretreatment of isolated starch granules at 40 degrees C increased their bound starch synthase activity. Both soluble and bound enzymes in the grains of whole wheat plants lost activity when the plants were held above 30 degrees C for 30 minutes or longer. Both activities lost from the grains after a 1 hour treatment at 37 degrees C were restored in 1 to 2 days by a return to 21 degrees C. In slices, inactivation of the soluble starch synthase was increased by incubation with 2,4-dinitrophenol. It is tentatively suggested that in vivo heat inactivation of soluble starch synthase may be a direct effect of heat on the enzyme protein and that of bound enzyme an indirect effect involving metabolic factors.