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

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 granule-associated 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.

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

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.

Physical association of starch biosynthetic enzymes with starch granules of maize endosperm. Granule-associated forms of starch synthase I and starch branching enzyme II.

Antibodies were used to probe the degree of association of starch biosynthetic enzymes with starch granules isolated from maize (Zea mays) endosperm. Graded washings of the starch granule, followed by release of polypeptides by gelatinization in 2% sodium dodecyl sulfate, enables distinction between strongly and loosely adherent proteins. Mild aqueous washing of granules resulted in near-complete solubilization of ADP-glucose pyrophosphorylase, indicating that little, if any, ADP-glucose pyrophosphorylase is granule associated. In contrast, all of the waxy protein plus significant levels of starch synthase I and starch branching enzyme II (BEII) remained granule associated. Stringent washings using protease and detergent demonstrated that the waxy protein, more than 85% total endosperm starch synthase I protein, and more than 45% of BEII protein were strongly associated with starch granules. Rates of polypeptide accumulation within starch granules remained constant during endosperm development. Soluble and granule-derived forms of BEII yielded identical peptide maps and overlapping tryptic fragments closely aligned with deduced amino acid sequences from BEII cDNA clones. These observations provide direct evidence that BEII exits as both soluble and granule-associated entities. We conclude that each of the known starch biosynthetic enzymes in maize endosperm exhibits a differential propensity to associate with, or to become irreversibly entrapped within, the starch granule.

Response of enzymes and storage proteins of maize endosperm to nitrogen supply.

To examine the effects of N nutrition upon endosperm development, maize (Zea mays) kernels were grown in vitro with either 0, 3.6, 7.1, 14.3, or 35.7 millimolar N. Kernels were harvested at 20 days after pollination for determination of enzyme activities and again at maturity for quantification of storage products and electrophoretic separation of zeins. Endosperm dry weight, starch, zein-N, and nonzein-N all increased in mature kernels as N supply increased from zero to 14.3 millimolar. The activities of sucrose synthase, aldolase, phosphoglucomutase, glutamate-pyruvate transaminase, glutamate-oxaloacetate transaminase, and acetolactate synthase increased from 1- to 2.5-fold with increasing N supply. Adenosine diphosphate-glucose pyrophosphorylase and both ATP- and PPi-dependent phosphofructokinases increased to lesser extents, while no significant response was detected for hexose kinases and glutamine synthetase. Nitrogen-induced changes in enzyme activities were often highly correlated with changes in final starch and/or zein-N contents. Separation of zeins indicated that these peptides were proportionately enhanced by N supply, with the exception of C-zein, which was relatively insensitive to N. These data indicate that at least a portion of the yield increase in maize produced by N fertilization is induced by a modification of kernel metabolism in response to N supply.

Nitrogen-induced changes in the growth and metabolism of developing maize kernels grown in vitro.

Cereal kernel growth and grain yield are functions of endosperm starch accumulation. The objective of this study was to examine how various metabolic factors in developing maize (Zea mays L.) endosperm influence starch deposition. Kernels were grown in vitro on medium with: (a) zero N (-N), (b) optimum N (+N), or (c) -N from 3 to 20 days after pollination followed by +N until maturity (+/-N) to produce different degrees of endosperm growth and to promote an enhancement of starch synthesis midway through development. At intervals, kernels were harvested and levels of enzyme activities and carbohydrate and N constituents examined. Endosperm starch and protein accumulation were decreased in -N compared to +N kernels, but relief of N starvation increased both constituents. With greater movement of N into +/-N kernels, endosperm sugar concentrations declined suggesting an inverse relationship between C and N transport. Unusually high concentrations of sugar in N stressed kernels did not appear to limit or enhance starch production. Rather, increased accumulation of starch in +/-N endosperm was correlated with significant increases in the enzymatic activities of sucrose synthase and PPi-linked phosphofructokinase, and to a lessor extent hexokinase. In addition, the occurrence of specific proteins of the albumin/globulin fraction either increased, decreased, or remained unchanged in relation to starch synthesis. These data suggest that lack of N limits starch deposition in maize endosperm primarily through an influence on synthesis of key proteins.

Roles of carbohydrate supply and phytohormones in maize kernel abortion.

Kernels at the ear tip of field grown maize (Zea mays L.) often abort during flowering, resulting in significant yield loss. The objective of this study was to determine if abortion is initiated by an inadequate supply of carbohydrates for growth of ear tip kernels, and/or by a hormonal signal. Field grown maize plants were either unshaded or shaded during flowering to increase kernel abortion. Nonstructural carbohydrates, indoleacetic acid (IAA), abscisic acid (ABA), and cytokinins were measured in aborting and nonaborting kernels, before and after abortion occurred. Kernel abortion was initiated 8 days after anthesis (DAA) and was complete by 12 DAA, when kernels ceased dry weight accumulation. Concentrations of reducing sugars, sucrose, and starch in aborting kernels were not significantly different from those in nonaborting kernels up to 12 DAA. Also, total carbohydrate concentrations were higher in the cob of aborting than of nonaborting kernels from 8 to 26 DAA. These data suggest that kernel abortion is not initiated by an inadequate supply of carbohydrates. However, accumulation of reducing sugars in the cob of aborting kernels suggests that transfer of sugars from cob to kernels is impaired early in the abortion process. Differences in IAA, ABA, and cytokinin concentrations between aborting and nonaborting kernels were only observed after abortion was complete. Kernel abortion is apparently not initiated by a signal of these hormones. After completion of abortion, aborted kernels contained higher concentrations of ABA and lower concentrations of IAA than nonaborted kernels. There was also a trend toward higher concentrations of zeatin riboside in nonaborted kernels. Abscisic acid may promote kernel abortion after the process has been initiated. A mechanism for kernel abortion is discussed.

Growth and composition of maize kernels cultured in vitro with varying supplies of carbon and nitrogen.

This study employed in vitro seed culture to determine how C and N supply influence the growth (i.e. starch accumulation) and protein composition of maize (Zea mays L.) endosperm. Immature kernels were grown to maturity on liquid medium containing various concentrations of C (sucrose at 234 millimolar [low] and 468 millimolar [high]) and N (amino acid mixture ranging in N from 0 to 144 millimolar). Low C supply limited starch, but not N, accumulation in the endosperm. With high C, endosperm starch and protein content increased concomitantly as N supply increased from 0 to 13.4 millimolar. Endosperm growth was unaffected by additional N until concentrations exceeding approximately 72 millimolar reduced starch accumulation. A similar inhibition of starch deposition occurred with lower N concentrations when kernels were grown with low C. Endosperm total N content reached a point of saturation with approximately 36 millimolar N in the medium, regardless of C supply. Zein synthesis in the endosperm responded positively across all N levels, while glutelin content remained static and albumin/globulin proteins were reduced in amount when N supply was greater than 36 millimolar. A reciprocal, inverse relationship was observed in mature endosperm tissue between the concentrations of free amino acids and soluble sugars. Our data suggest that under N stress starch and protein accumulation in the endosperm are interdependent, at least in appearance, but are independent otherwise.

Increased induction of regenerable callus cultures from cultured kernels of the maize inbred FR27rhm.

Kernels of the maize inbred FR27rhm were cultured on various media to determine if the treatments would alter the frequency of formation of regenerable callus (induction frequency) by embryos excised from the kernels when they were placed on callus induction medium. The addition of 60 µM dicamba (3,6-dichloro-o-anisic acid) to the kernel culture medium resulted in an induction frequency of 27-38% compared to 0% for controls on standard kernel culture medium. Embryos excised from dicamba-treated kernels also showed in-ovule callus-like tissue proliferation. The increased induction frequency and the callus-like tissue proliferation could also be produced by injecting the ears of field grown FR27rhm plants, 3-d post pollination, with 1.08 µmoles of dicamba. The results indicate that treatment of the developing ear with dicamba, in vivo or the developing kernel in vitro, may be an effective means to increase the frequency of regenerable callus induction from recalcitrant maize genotypes, such as the B73 derivative FR27rhm.

Gramine Accumulation in Leaves of Barley Grown under High-Temperature Stress.

The indole alkaloid gramine is toxic to animals and may play a defensive role in plants. Under certain conditions, shoots of barley cultivars such as ;Arimar' and CI 12020 accumulate gramine (N,N-dimethyl-3-aminomethylindole) and lesser amounts of its precursors 3-aminomethylindole (AMI) and N-methyl-3-aminomethylindole (MAMI); other cultivars such as ;Proctor' do not. When grown at optimal temperatures (21 degrees C/16 degrees C, day/night), Arimar contained a high level of gramine in the first leaf (approximately 6 milligrams per gram dry weight), but progressively less accumulated in successive leaves so that the gramine level in the shoot as a whole fell sharply with age. In Arimar and CI 12020 plants transferred at the two- to three-leaf stage from 21 degrees C/16 degrees C to supra-optimal temperatures (>/=30 degrees C/25 degrees C), there was massive gramine accumulation in leaves which developed at high temperature, so that gramine level in the whole shoot remained high (about 3-8 milligrams per gram dry weight).Proctor lacked both constitutive gramine accumulation in the first leaf and heat-induced gramine accumulation in later leaves. The following evidence indicates that this results from a lesion in the pathway of synthesis (tryptophan -->--> AMI --> MAMI --> gramine) between tryptophan and AMI. (a) Proctor and Arimar leaves readily absorbed [(14)C]gramine, but neither cultivar degraded it extensively. (b) Arimar leaf tissue incorporated [(14)C]formate label into the N-methyl groups of gramine and MAMI, and converted [methylene-(14)C]tryptophan to AMI, MAMI, and gramine; Proctor leaf tissue did not, even when a trapping pool of unlabeled gramine was supplied. (c) Proctor converted [(14)C]MAMI to gramine as actively as Arimar. (d) Proctor incorporated [(14)C]formate label into gramine and MAMI when supplied with AMI; the ratio [(14)C]gramine/[(14)C]MAMI fell with leaf age, suggesting that the two N-methylations involve different enzymes. Inasmuch as Proctor leaf tissue did not methylate added tryptamine or tyramine, the N-methyltransferase(s) of gramine synthesis may be substrate specific.In sterile culture at optimal temperatures, 10 millimolar gramine did not affect autotrophic growth of Arimar or Proctor plantlets or heterotrophic growth of callus. At supra-optimal temperature, plantlet growth was reduced by gramine although callus growth was not. We speculate that gramine-accumulating cultivars may suffer autotoxic effects at high leaf temperatures.