The maize glutamine synthetase GS1-2 gene is preferentially expressed in kernel pedicels and is developmentally-regulated.

The pedicel region of Zea mays kernels contains a unique form of maize glutamine synthetase (GS), GSp1. RNA blot analysis using GS gene-specific probes revealed that the expression of the GS1-2 gene was specific to the pedicel and that it increased in the kernels during development. This pattern of the maize GS1-2 gene expression is consistent with the tissue specificity of the GSp1 protein and suggests that it encodes the GSp1 isoform of maize GS.

Regulation of the maize ubiquitin (Ubi-1) promoter in developing maize (Zea mays L.) seeds examined using transient gene expression in kernels grown in vitro.

Kernel culture was assessed for evaluating novel gene expression in developing maize (Zea mays L.) seeds by comparing the transient expression of maize ubiquitin (Ubi-1) promoter-driven ß-glucuronidase (GUS) delivered by particle bombardment in kernels grown in culture with those grown in planta. With kernels from either source, GUS expression, as determined by histochemical staining, was widespread in young, actively growing kernels, but it diminished with kernel age and by 25 days after pollination was found only in the embryo. Transient expression of Ubi-1 in kernels grown in vitro was not affected by wounding, ethylene treatment, pathogen invasion, or heat shock. In contrast, the plant hormones indole-3-acetic acid and kinetin both stimulated transient Ubi-1 expression in the endosperm, particularly at the periphery. Transient gene expression in developing maize seeds grown in vitro should allow for facile and rapid evaluation of the tissue-specificity and environmental responses of novel gene constructs in developing maize seeds.

Immunolocalization of a Unique Form of Maize Kernel Glutamine Synthetase Using a Monoclonal Antibody.

The pedicel (basal maternal tissue) of maize (Zea mays L.) kernels contains a physically and kinetically unique form of glutamine synthetase (GSp1) that is involved in the conversion of transport forms of nitrogen into glutamine for uptake by the developing endosperm (M.J. Muhitch [1989] Plant Physiol 91: 868-875). A monoclonal antibody has been raised against this kernel-specific GS that does not cross-react either with a second GS isozyme found in the pedicel or with the GS isozymes from the embryo, roots, or leaves. When used as a probe for tissue printing, the antibody labeled the pedicel tissue uniformly and also labeled some of the pericarp surrounding the lower endosperm. Silver-enhanced immunogold staining of whole-kernel paraffin sections revealed the presence of GSp1 in both the vascular tissue that terminates in the pedicel and the pedicel parenchyma cells, which are located between the vascular tissue and the basal endosperm transfer cells. Light staining of the subaleurone was also noted. The tissue-specific localization of GSp1 within the pedicel is consistent with its role in the metabolism of nitrogenous transport compounds as they are unloaded from the phloem.

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.

Purification and Characterization of Two Forms of Glutamine Synthetase from the Pedicel Region of Maize (Zea mays L.) Kernels.

Maize (Zea mays L.) kernel pedicels, including vascular tissues, pedicel parenchyma, placento-chalazal tissue, and the surrounding pericarp, contained two forms of glutamine synthetase (EC 6.3.1.2), separable by anion exchange chromatography under mildly acidic conditions. The earlier-eluting activity (GS(p1)), but not the later-eluting activity (GS(p2)), was chromatographically distinct from the maize leaf and root glutamine synthetases. The level of GS(p1) activity changed in a developmentally dependent manner while GS(p2) activity was constitutive. GS(p1) and GS(p2) exhibited distinct ratios of transferase to hydroxylamine-dependent synthetase activities (5 and 23, respectively), which did not change with kernel age. Purified pedicel glutamine synthetases had native relative molecular masses of 340,000, while the subunit relative molecular masses differed slightly at 38,900 and 40,500 for GS(p1) and GS(p2), respectively. Both GS forms required free Mg(2+) with apparent K(m)s = 2.0 and 0.19 millimolar for GS(p1) and GS(p2), respectively. GS(p1) had an apparent K(m) for glutamate of 35 millimolar and exhibited substrate inhibition at glutamate concentrations greater than 90 millimolar. In contrast, GS(p2) exhibited simple Michaelis-Menten kinetics for glutamate with a K(m) value of 3.4 millimolar. Both isozymes exhibited positive cooperativity for ammonia, with S(0.5) values of 100 and 45 micromolar, respectively. GS(p1) appears to be a unique, kernel-specific form of plant glutamine synthetase. Possible functions for the pedicel GS isozymes in kernel nitrogen metabolism are discussed.

Acetolactate Synthase Activity in Developing Maize (Zea mays L.) Kernels.

Acetolactate synthase (EC 4.1.3.18) activity was examined in maize (Zea mays L.) endosperm and embryos as a function of kernel development. When assayed using unpurified homogenates, embryo acetolactate synthase activity appeared less sensitive to inhibition by leucine + valine and by the imidazolinone herbicide imazapyr than endosperm acetolactate synthase activity. Evidence is presented to show that pyruvate decarboxylase contributes to apparent acetolactate synthase activity in crude embryo extracts and a modification of the acetolactate synthase assay is proposed to correct for the presence of pyruvate decarboxylase in unpurified plant homogenates. Endosperm acetolactate synthase activity increased rapidly during early kernel development, reaching a maximum of 3 micromoles acetoin per hour per endosperm at 25 days after pollination. In contrast, embryo activity was low in young kernels and steadily increased throughout development to a maximum activity of 0.24 micromole per hour per embryo by 45 days after pollination. The sensitivity of both endosperm and embryo acetolactate synthase activities to feedback inhibition by leucine + valine did not change during kernel development. The results are compared to those found for other enzymes of nitrogen metabolism and discussed with respect to the potential roles of the embryo and endosperm in providing amino acids for storage protein synthesis.

Imidazolinones and acetohydroxyacid synthase from higher plants: properties of the enzyme from maize suspension culture cells and evidence for the binding of imazapyr to acetohydroxyacid synthase in vivo.

Acetohydroxyacid synthase has been purified from maize (Zea mays, var Black Mexican Sweet) suspension culture cells 49-fold by a combination of ion exchange chromatography, gel filtration, and hydroxyapatite chromatography. Use of the nondenaturing, zwitterionic detergent 3-([3-cholamidopropyl]dimethyl-ammonio)-1-propanesulfonate was necessary to dissociate the enzyme from the heterogeneous, high molecular weight aggregates in which it appears to reside in vitro. The solubilized maize acetohydroxyacid synthase had a relative molecular mass of 440,000. The purified enzyme was highly unstable. Acetohydroxyacid synthase activities in crude extracts of excised maize leaves and suspension cultured cells were reduced 85 and 58%, respectively, by incubation of the tissue with 100 micromolar (excised leaves) and 5 micromolar (suspension cultures) of the imidazolinone imazapyr prior to enzyme extraction, suggesting that the inhibitor binds tightly to the enzyme in vivo. Binding of imazapyr to maize acetohydroxyacid synthase could also be demonstrated in vitro. Evidence is presented which suggests that the interaction between imazapyr and the enzyme is reversible. Imazapyr also exhibited slow-binding properties when incubated with maize cell acetohydroxyacid synthase in extended time course experiments. Initial and final K(i) values for the inhibition were 15 and 0.9 micromolar, respectively. The results suggest that imazapyr is a slow, tight-binding inhibitor of acetohydroxyacid synthase.

Influence of culture age and spermidine treatment on the accumulation of phenolic compounds in suspension cultures.

The influence of cell age on phenol accumulation was examined by determining the quantity of individual phenols which accumulated in Paul's scarlet rose cultures of increasing age. During log-phase growth (days 7 and 11), only gallic acid and epicatechin-catechin were detected; whereas, during early and late stationary phase (days 14 and 35), several other phenols were present in addition to gallic acid and epicatechincatechin. When stationary-phase cultures were provided with a supplement of sucrose and spermidine, a treatment previously shown to arrest the senescence of rose cultures (Muhitch, Edwards, Fletcher 1983 Plant Cell Rep 2: 82-84), the cells then accumulated a higher level and a wider assortment of phenols. These results suggest that extending the lifespan of mature nondividing cell cultures offers a means of increasing the yield of secondary products by cultured cells.

Isolation and Identification of the Phenols of Paul's Scarlet Rose Stems and Stem-Derived Suspension Cultures.

The phenols of Paul's Scarlet rose stems and stem-derived cell cultures have been analyzed using C(18)-reversed-phase high performance liquid chromatography.Rose stems were found to contain gallic acid, (+)catechin, (-)epicatechin, the dimers (-)epicatechin-(+)catechin and (+)catechin-(+)catechin, a polymeric procyanidin, ferulic acid, and several gallotannins. In contrast, a cell suspension of Paul's Scarlet rose which has been maintained in culture for over 25 years contained only low levels of gallic acid and (-)epicatechin-(+)catechin. The phenol content of a second rose cell line which was started from the same initial isolate in 1957, but which was maintained in a laboratory other than our own was quantitatively and qualitatively similar to the cell line kept in our laboratory for the last 20 years. A third cell line which we started 6 months ago contained a wide variety of phenols, most of which were in common with those of rose stems.Selective subculturing of smaller cell clumps of our oldest cell line failed to enhance either the quantities or the diversity of phenols which accumulated in these cultured cells. Possible reasons for the failure of selective subculturing to enhance phenol levels in this long-established cell line are discussed.

Influence of diamines and polyamines on the senescence of plant suspension cultures.

The diamines putrescine and cadaverine and the polyamines spermine and spermidine inhibited the senescence of nonphotosynthetic cultures of Paul's Scarlet rose. Response was observed when the media of stationary phase cultures was adjusted to either 1 mM of cadaverine or putrescine; or 0.1 µM of either spermine or spermidine along with 2% sucrose in all cases. Senescence of the cultures was followed by microscopic examination of cell aliquots removed at 10 day intervals and treated with the vital stain, fluorescein diacetate.

Influence of methionine sulfoximine on the photosynthesis of isolated chloroplasts.

Methionine sulfoximine provided at a concentration which inhibits photosynthesis in intact leaves (10 mM) had no significant influence on the rate of photosynthesis of isolated pea leaf chloroplasts. In contrast, ammonium, 3-(3,4-dichlorophenyl)-1,1-dimethylurea, and D,L-glyceraldehyde all strongly inhibited the photosynthesis of isolated chloroplasts. We conclude that low concentrations of methionine sulfoximine (up to 10 mM) have no direct effect on the photosynthetic process.

Influence of Methionine sulfoximine on the photosynthesis of isolated chloroplasts.

Methionine sulfoximine provided at a concentration which inhibits photosynthesis in intact leaves (10 mM) had no significant influence on the rate of photosynthesis of isolated pea leaf chloroplasts. In contrast, ammonium, 3-(3,4-dichlorophenyl)-1,1-dimethylurea, and D,L-glyceraldehyde all strongly inhibited the photosynthesis of isolated chloroplasts. We conclude that low concentrations of methionine sulfoximine (up to 10 mM) have no direct effect on the photosynthetic process.