Showing papers on "Endosperm published in 1993"

Activities of Hydrogen Peroxide-Scavenging Enzymes in Germinating Wheat Seeds

Abstract: During imbibition and germination of wheat (Triticum aestivum) in the dark over 72 h, activities of the enzymes of the ascorbate (AsA)-dependent H 2 O 2 -scavenging pathway, AsA peroxidase, monodehydroascorbate (MDAsA) reductase, dehydroascorbate (DHAsA) reductase and glutathione (GSSG) reductase as well as superoxide dismutase (SOD), catalase and guaiacol peroxidase were determined both in whole grains and in isolated embryos and endosperm. With the exception of DHAsA reductase, activities of the other enzymes assayed increased in germinating seeds, especially during radicle emergence (between 24-48 h of imbibition)

Endosperm Origin, Development, and Function

Abstract: Endosperm has been studied from a variety of vantage points: evolution, role in seed development and germination, genetics, physiology, and biochemistry. This tissue represents a renewable, biodegradable source of materials; much effort has been directed to improve its use in feed and food making as well as its refinement to secondary products such as oils and plastics. Although there is a vast literature dealing with each of these topics, we still have a remarkably superficial understanding of most of them. There has been revitalized interest in understanding the endosperm in relation to seed-specific developmental processes. lnformation from these studies could provide a basis for developing more efficient approaches for plant improvement and use. Recent advances in molecular biology have created the possibility for detailed study of many of the genetic and molecular mechanisms involved in endosperm development. This research could conceivably lead to answers to many basic questions in developmental biology as well as to new tools that enhance practical uses of endosperm. In this review, it is not our intention to present a comprehensive overview of what is known about endosperm. Rather, we have chosen to summarize some of the research that has been done, making note of comprehensive reviews and pointing out important questions that remain open to scientific inquiry.

Elevated temperature reduces starch deposition in wheat endosperm by reducing the activity of soluble starch synthase

Abstract: Temperatures of more than 25° C adversely affect the activity of soluble starch synthase (SSS), an amyloplastic enzyme, in endosperm of wheat (Triticum aestivum L cv Mardler) Enzyme rate was found to have a temperature optimum between 20 and 25°C This effect was apparently reversible after a short period of exposure to elevated temperature We also found that with a prolonged period of exposure to elevated temperature there was another temperature-related phenomenon which caused a loss of enzyme activity that appeared to be much slower to reverse We have termed this effect of temperature on SSS activity “knockdown” The knockdown in SSS activity also occurred in-vivo However, elevated temperature did not affect the activities of several other enzymes in the pathway of starch synthesis (ADP-glucose pyrophosphorylase, UDP-glucose pyrophosphorylase, sucrose synthase, phosphoglucomutase, phosphoglucose isomerase, bound starch synthase or hexokinase) Because the knockdown effect appeared to be specific to the enzyme SSS, we quantified the effect of knockdown on flux of carbon into starch and used these data to calculate the flux-control coefficient for SSS Using data at 10–20°C the flux-control coefficient was CStarch10–20C = 050, whereas at 20–30° C the flux-control coefficient was CStarch20–30C = 138, and between 30–40°C the flux-control coefficient was CStarch30–40C = 069 Using data at 10–30°C the flux-control coefficient was CStarch10–30C = 115, and at 10–40°C the flux-control coefficient was CStarch10–40C = 082 In conclusion, we suggest that SSS is a major site of regulation of starch synthesis in developing wheat grain During periods of high temperature the control point in the pathway of starch synthesis is apparently not associated exclusively with ADP-glucose pyrophosphorylase In field conditions, in which temperatures are fluctuating, there will likely be periods of control of starch synthesis being exerted predominantly by SSS During periods at lower temperature, control of flux may be exerted by SSS, perhaps in combination with other flux-controlling enzymes in the pathway Our data point-out a crucial new aspect of quantifying control strengths of enzymes in plants: the determination of enzyme control strengths should be done in carefully regulated temperature conditions Thus, since temperature is a major determinant of real flux through a pathway and the individual enzymes can respond differently to changing temperature conditions, the control strengths of individual steps in a pathway may vary with changing environmental conditions This is particularly pronounced in starch deposition, because of the temperature instability of SSS

Novel plants and processes for obtaining them

Abstract: The invention relates to a transgenic or mutated plant having genomic material which alters the normal starch synthesis pathway within the plant. More specifically, the present invention relates to a plant having a genotype which creates new forms of starch in significant quantity. Particularly, the invention relates to grain having an embryo with a genotype heterozygous for two or more wild type genes (for example, Aa/Bb) and an endosperm having a genotype heterozygous for such genes (for example, AAa/BBb or AAa/bbB or aaA/BBb or aaA/bbB) and the starch produced therefrom.

Characterization of Starch Structures of 17 Maize Endosperm Mutant Genotypes with Oh43 Inbred Line Background.

Abstract: The objectives were to characterise the structure and morphology of starch from 17 maize endosperm mutant genotypes in a common Oh43 inbred background to help in understanding the influences of recessive mutant genes on the maize starches

The nitrogen response of a barley C‐hordein promoter is controlled by positive and negative regulation of the GCN4 and endosperm box

Abstract: The 431 bp C-hordein promoter of lambda-1-17 exhibits a specific response to amino acids and NH4NO3 in developing barley (Hordeum vulgare L.) endosperms. With the aid of particle bombardment it is shown that the GCN4 motif ATGA(C/G)TCAT is the dominating cis-acting element in this response. But synergistic interaction with the neighbouring endosperm motif TGTAAAGT within the bifactorial prolamin element and cooperation with upstream sequences including a second prolamin-like element is an absolute requirement for a strong, positive regulation by an optimal nitrogen regime. Low nitrogen levels convert the GCN4 box into a negative motif. In contrast the endosperm box on its own exerted a silencing activity, independent of nitrogen nutrition. Sequence comparisons revealed that GCN4- and endosperm-like motifs are widely distributed among plant promoters. Their putative role in nitrogen regulation is discussed.

Role of Auxin in Maize Endosperm Development (Timing of Nuclear DNA Endoreduplication, Zein Expression, and Cytokinin).

Abstract: The timing of developmental events and regulatory roles of auxin were examined in maize (Zea mays L.) endosperms. Zeatin, zeatin riboside, and indole-3-acetic acid (IAA) levels were determined by enzyme-linked immunosorbent (ELISA). Zeatin and zeatin riboside increased to maximal concentrations at an early stage (9 d after pollination [DAP]), corresponding to the stage when cell division rate was maximal. In contrast, IAA concentration was low at 9 DAP and abruptly increased from 9 to 11 DAP, thus creating a sharp decline in the cytokinin to auxin ratio. Coincident with the increase in IAA was an increase in DNA content per nucleus, attributed to postmitotic DNA replication via endoreduplication. Exogenous application of 2,4-dichlorophenoxyacetic acid (2,4-D) at 5 or 7 DAP hastened the time course of DNA accumulation per nucleus and increased the average nuclear diameter, whereas 2-(para-chlorophenoxy)isobutyric acid delayed such development. Exogenously applied 2,4-D hastened the accumulation of the zein polypeptides of apparent molecular masses of 12, 14, and 16 kD and the expression of mRNA hybridizing with a zein DNA probe. We conclude that an abrupt increase in auxin induces cellular differentiation events in endosperm, including endoredupliction and expression of particular zein storage proteins.

Zein composition in hard and soft endosperm of maize

Abstract: Maize protein composition and distribution may directly influence endosperm texture and physical properties. To test this hypothesis, we compared compositions of alcohol-soluble proteins in maize endosperm from the hard and soft fractions of eight normal genotypes. Kernels were hand-dissected to obtain fractions differing in texture. Endosperm fractions were extracted with a solution containing alcohol, reducing agent, and sodium acetate and were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by reversed-phase high-performance liquid chromatography. We found more (an average of 3.3 times more) alpha-zeins (19 and 22 kDa) in hard endosperm fractions than in soft endosperm fractions, In contrast, soft endosperm fractions contained nearly twice as much 27-kDa gamma-zein (based on percent) than did hard endosperm fractions. Thus, distribution of the various types of zeins was not uniform throughout the maize endosperm. Our results suggest that the zein composition of protein bodies in normal maize kernels may be correlated with texture of the endosperm from which the sample was obtained.

Developmental and pathogen‐induced expression of three barley genes encoding lipid transfer proteins

Abstract: Clones for three barley non-specific lipid transfer proteins (LTP2, LTP3, and LTP4; formerly Cw18, Cw20 and Cw21, respectively) which had been previously shown to inhibit growth of plant pathogens, were selected and characterized from a cDNA library derived from young etiolated leaves. Genes Ltp2 and Ltp4 were located in chromosome 3H and gene Ltp3 was assigned to chromosome 7H by Southern blot analysis of wheat-barley disomic addition lines, using gene-specific probes (3'-ends of cDNAs). These assignments were confirmed by the polymerase chain reaction, using specific primers. The three genes were expressed in stem, shoot apex, leaves and roots (at low levels) throughout development. Genes Ltp3 and Ltp4 were expressed at high levels, and Lpt2 at low levels, in the spike (rachis, lemma plus palea and grain coats). Neither of the mRNAs was detected in endosperm. The proteins were localized by tissue-printing with polyclonal antibodies in the outer cell layer of the exposed surfaces of the plant, throughout the embryo, and in vascular tissues. Expression levels in leaves were moderately increased by 0.34 M NaCl and by 0.1 mM abscisic acid and were not affected by cold, drought, salicylate, 2,6-dichloro-isonicotinic acid, ethylene or ethephon. Methyl Jasmonate (10 microM) switched off all three genes. Inoculation with Av6 or vir6 isolates of the fungal pathogen Erysiphe graminis increased the three mRNAs, especially that of LTP4, which reached a maximum nine-fold increase 12-16 h after infection.

Germination and Dormancy of Abscisic Acid- and Gibberellin-Deficient Mutant Tomato (Lycopersicon esculentum) Seeds (Sensitivity of Germination to Abscisic Acid, Gibberellin, and Water Potential)

Abstract: Germination responses of wild-type (MM), abscisic acid (ABA)-deficient (sitw), and gibberellin (GA)-deficient (gib-1) mutant tomato (Lycopersicon esculentum Mill. cv Moneymaker) seeds to ABA, GA4+7, reduced water potential ([psi]), and their combinations were analyzed using a population-based threshold model (B.R. Ni and K.J. Bradford [1992] Plant Physiol 98: 1057–)1068). Among the three genotypes, sitw seeds germinated rapidly and completely in water, MM seeds germinated more slowly and were partially dormant, and gib-1 seeds did not germinate without exogenous GA4+7. Times to germination were inversely proportional to the differences between the external osmoticum, ABA, or GA4+7 concentrations and the corresponding threshold levels that would either prevent ([psi]b, log[ABAb]) or promote (log[GAb]) germination. The sensitivity of germination to ABA, GA4+7, and [psi] varied widely among individual seeds in the population, resulting in a distribution of germination times. The rapid germination rate of sitw seeds was attributable to their low mean [psi]b (-1.17 MPa). Postharvest dormancy in MM seeds was due to a high mean [psi]b (-0.35 MPa) and a distribution of [psi]b among seeds such that some seeds were unable to germinate even on water. GA4+7 (100 [mu]M) stimulated germination of MM and gib-1 seeds by lowering the mean [psi]b to -0.75 MPa, whereas ABA inhibited germination of MM and sitw seeds by increasing the mean [psi]b. The changes in [psi]b were not due to changes in embryo osmotic potential. Rather, hormonal effects on endosperm weakening opposite the radicle tip apparently determine the threshold [psi] for germination. The analysis demonstrates that ABA- and GA-dependent changes in seed dormancy and germination rates, whether due to endogenous or exogenous growth regulators, are based primarily upon corresponding shifts in the [psi] thresholds for radicle emergence. The [psi] thresholds, in turn, determine both the rate and final extent of germination within the seed population.

Insensitivity of Barley Endosperm ADP-Glucose Pyrophosphorylase to 3-Phosphoglycerate and Orthophosphate Regulation

Abstract: Crude extracts of starchy endosperm from barley (Hordeum vulgare cv Bomi) contained high pyrophosphorolytic activity (up to 0.5 [mu]mol of glucose-1-P formed min-1 mg-1 of protein) of ADP-glucose pyrophosphorylase (AGP) when assayed in the absence of 3-phosphoglycerate (3-PGA). This high activity was observed regardless of whether AGP had been extracted in the presence or absence of various protease inhibitors or other protectants. Western blot analysis using antibodies specific for either the small or large subunit of the enzyme demonstrated that the large, 60-kD subunit was prone to proteolysis in crude extracts, with a half-time of degradation at 4[deg]C (from 60 to 53 to 51 kD) on the order of minutes. The presence of high concentrations of protease inhibitors decreased, but did not prevent this proteolysis. The small, 51-kD subunit of barley endosperm AGP was relatively resistant to proteolysis, both in the presence or absence of protease inhibitors. For the crude, nonproteolyzed enzyme, 3-PGA acted as a weak activator of the ADP-glucose synthetic reaction (about 25% activation), whereas in the reverse reaction (pyrophosphorolysis) it served as an inhibitor rather than an activator. For both the synthetic and pyrophosphorolytic reactions, inorganic phosphate (Pi) acted as a weak competitive or mixed inhibitor of AGP. The relative insensitivity to 3-PGA/Pi regulation has been observed with both the nonproteolyzed crude enzyme and partially purified (over 60-fold) AGP, the latter characterized by two bands for the large subunit (molecular masses of 53 and 51 kD) and one band for the small subunit (51 kD). Addition of 3-PGA to assays of the partially purified, proteolyzed enzyme had little or no effect on the Km values of all substrates of AGP, but it reduced the Hill coefficient for ATP (from 2.1 to 1.0). These findings are discussed with respect to previous reports on the structure and regulation of higher plant AGP.

Starch branching enzyme II from maize endosperm.

Abstract: ADP-Glc pyrophosphorylase (EC 2.7.7.27), starch synthases (EC 2.4.1.21), and SBEs (EC 2.4.1.18) are the key enzymes in the pathway of plant starch biosynthesis. ADPGlc pyrophosphorylase and starch synthase catalyze the formation of ADP-Glc, the substrate for starch synthesis, and the polymerization of Glc into the amylose and amylopectin fractions of the starch granule, respectively. SBEs catalyze branch point formation by the cleavage and reattachment of a-1,4-linked Glc chains to a-1,6 branch points in the growing starch molecule (Borovsky et al., 1979; Boyer, 1985). Branching enzymes are proposed to interact with starch synthases in formation of amylopectin (Boyer and Preiss, 1981). Multiple forms of SBE have been identified in maize (Zea mays L.) endosperm (Boyer and Preiss, 1978, 1981). Three forms of SBE, I, Ila, and Ilb, from developing maize endosperm have been characterized by their leve1 of branching activity on amylose and amylopectin and by kinetic and immunological properties (Boyer and Preiss, 1978; Fisher and Boyer, 1983; Singh and Preiss, 1985). Distinct differences have been shown between SBE I and IIa or IIb by the above parameters, but only small differences exist between SBE IIa and IIb. Genetic evidence suggests that IIa and IIb are products of separate genes (Boyer and Preiss, 1981; Hedman and Boyer, 1982). The precise role of each isoform in starch formation has yet to be determined. To further understand these enzymes, efforts to clone the genes have been undertaken. We report here the cloning of a SBE I1 cDNA from maize (Table I). Three XgtlO cDNA libraries were constructed from endosperm. poly(A)+ RNA 14, 22, and 29 DAP. A heterologous nucleic acid probe, clone pJSBE5, the cDNA for pea SBE I, was used to screen the 14-DAP library (Bhattacharyya et al., 1990). After purifying and subcloning into plasmid pBluescript I1 SK(Stratagene), a full-length cDNA of 2725 bp was isolated. Northern blots of total maize RNA isolated from endosperm tissue 12 DAP and probed with the cloned maize cDNA revealed a single transcript of approximately 2.7 kb. Deduced amino acid sequence was compared with the pea SBE I (Bhattacharyya et al., 1990), maize SBE I (Baba et al., 1991), and rice SBE I (Nakamura et al., 1992) translated cDNA sequences using Intelligenetics software. Levels of residue identity were 71, 52, and 52%, respectively. From these results, we conclude that we have cloned a second

In vivo footprinting of a low molecular weight glutenin gene (LMWG-1D1) in wheat endosperm

Abstract: The quality of the wheat grain is determined by the quantity and composition of storage proteins (prolamins) which are synthesized exclusively in endosperm tissue. We are investigating the mechanisms underlying the regulation of expression of a prolamin gene, the low molecular weight glutenin gene LMWG-1D1. The LMWG-1D1 promoter contains the endosperm box, a sequence motif highly conserved in the promoter region of a large number of storage protein genes, which is thought to confer endosperm-specific expression of prolamin genes. Here we show by in vivo DMS footprinting of wheat endosperm tissue that the endosperm box becomes occupied by putative trans-acting factors during grain ripening. During early stages of development the endosperm motif within the 5' half of the endosperm box becomes occupied first, followed by binding of a second activity to a GCN4/jun-like motif in the 3' half just prior to the stage of maximum gene expression. Occupancy of the endosperm box is highly tissue-specific: no protection was observed in husk and leaf tissues. Several binding activities were identified in vitro from nuclear protein extracts of wheat endosperm which bind specifically to the endosperm and GCN4/jun motifs identified by in vivo footprinting.

The origin of lysine-containing proteins in opaque-2 maize endosperm

Abstract: The reduction of zein synthesis in the maize (Zea mays L.) opaque-2 mutant is associated with an increased percentage of lysine in the endosperm protein. When expressed on an endosperm basis, we found that W64A opaque-2 contains 490 μg of lysine compared with 350 μg in W64A normal. SDS-PAGE analysis of endosperm proteins indicated that several non-zein proteins are more abundant in the mutant than in normal genotype. To determine the subcellular origin of these proteins, we separated an endosperm homogenate from developing kernels by sucrose density gradient centrifugation and used marker enzyme assays and immunoblot analyses to identify cellular components. Amino acid analysis of proteins in the gradient fractions showed that the majority of the lysine occurs in soluble proteins at the top of the gradient. To identify these proteins, we prepared a complex antiserum against the entire soluble protein fraction and used it to immunoscreen an endosperm cDNA expression library. Sequence analysis of clones identified mRNAs involved in carbohydrate metabolism, amino acid biosynthesis, and protein synthesis. RNA dot blot hybridization analysis with these clones revealed significant variation in the levels of transcripts between normal and opaque-2 endosperm, but we identified several mRNAs that are elevated in opaque-2 and that may encode proteins responsible for the enhanced lysine content.

Partial Purification of a cis-trans-Isomerase of Zeatin from Immature Seed of Phaseolus vulgaris L

Abstract: Investigation of the conversion of exogenous cis-zeatin to trans-zeatin in immature seeds of Phaseolus vulgaris L. led to the isolation of a cis-trans-isomerase from the endosperm. The enzyme was purified more than 2000-fold by chromatography on a series of fast protein liquid chromatography (anion exchange, gel filtration, and hydrophobic interaction) and concanavalin A columns. The enzymic reaction favors conversion from the cis to the trans form and requires flavin, light, and dithiothreitol. cis-Zeatin riboside is also a substrate for the enzyme. Retention on the concanavalin A column indicated that the enzyme is a glycoprotein. The enzyme was stable for at least 8 weeks when stored at -80[deg] C. The occurrence of cis-trans-isomerization suggests that cis-zeatin and cis-zeatin riboside formed by tRNA degradation could be precursors of biologically active cytokinins.

Cytokinins in Plant Pathogenic Bacteria and Developing Cereal Grains

Abstract: Cytokinin analysis by immunoaffinity chromatography (IAC), high-performance liquid chromatography (HPLC) and radioimmunoassay (RIA) or enzyme-linked immunosorption assay (ELISA) has been used to study two separate topics: the role of tRNA in bacterial cytokinin biosynthesis and the changes in cytokinin concentration which occur during cereal grain development. Transfer RNA isopentenylation in the gall-forming plant pathogen Agrobacterium tumefaciens is encoded by the chromosomal miaA locus. Mutation of miaA reduces tRNA isopentenylation significantly and preliminary data suggest that turnover of isopentenylated tRNA is responsible for low level secretion of free N6-isopentenyladenine (iP) by the bacteria. However, the major route of cytokinin biosynthesis by gall-forming plant patho- genic bacteria is not via tRNA turnover but by direct biosynthesis mediated by dimethylallylpyro- phosphate: 5'-AMP transferase (DMAPP :AMP transferase) encoded by such genes as ipt, tzs (from A, tumefaciens) or ptz (from Pseudomonas savastanoi). Analysis of cytokinin levels in developing wheat and rice grains in the period immediately following pollination showed large transient increases in zeatin (Z) and zeatin riboside (ZR) which coincided with the period of maximum endosperm cell division reported by others. Detailed analyses of maize kernels, where development can be staged readily, showed that Z and ZR concentrations peaked 9 days after pollination (DAP). During the period 8-10 DAP, cytokinin oxidase underwent a significant increase in specific activity, indicating that cytokinin catabolism was enhanced as endosperm cell division ended.

Endosperm starch properties in several wheat cultivars preferred for Japanese noodles

Abstract: To characterize superior genotypes for the white Japanese noodle, endosperm starch properties including amylose content, flour peak viscosity and starch-granule bound Waxy (Wx) proteins were compared using several cultivars preferred for noodle manufacture. Amylose contents from three seasons trials and flour peak viscosity from two seasons trials varied among cultivars. Low amylose content was a common property in the noodle cultivars, whereas a high peak viscosity was not always the case. When the Wx proteins were analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis, a clear reduction in the amount of low molecular weight protein or a lack of the high molecular weight protein occurred in the noodle cultivars. Segregation of Wx proteins was detectable in a B1F1 population, indicating that the Wx protein analysis has a potential as a surrogate of selecting low amylose genotypes in early generations.

Effects of osmotic preconditioning on nuclear replication activity in seeds of pepper (Capsicum annuum)

Abstract: Flow cytometric determination of nuclear DNA contents in embryos of dry, fully matured pepper seeds revealed only 2C signals. Therefore, pepper belongs to those species in which the quiescent embryo arrests nuclear division in the presynthetic G 1 phase. In whole seeds, two other peaks were observed which corresponded with the 3C and 6C DNA content and originated from the triploid endosperm. After 36 to 48 h of imbibition in water (48 to 60 h before visible germination), a rise in the 4C signal was observed in the root tip of the embryo. Osmotic preconditioning (priming) of pepper seeds for 7, 14 or 21 days in polyethylene glycol (PEG) or KNO 3 +K 3 PO 4 considerably reduced the time to 50% germination (t 50 ) and the mean germination time, and the effect was proportional to the duration of the priming treatments

Lack of aconitase in glyoxysomes and peroxisomes

Abstract: The aim of this work was to find out whether aconitase [citrate (isocitrate) hydro-lyase, EC 4.2.1.3] which is rapidly inactivated by H2O2, is present in the microbodies from plant cells. The separation of intact organelles from castor-bean (Ricinus communis) endosperm and potato (Solanum tuberosum) tuber indicated that aconitase activity is essentially limited to the mitochondria and cytosol fraction, but was not detected in highly purified castor-bean endosperm and potato tuber peroxisomes. An isotropic e.p.r. signal of the type expected for the 3Fe cluster of oxidized aconitase was not detected in microbodies. In immunoblot analyses, antibodies raised against potato tuber mitochondrial aconitase did not cross-react with any glyoxysomal or peroxisomal protein. Positive reactions were found for cytosol fraction and mitochondria of castor-bean endosperm. The operation of the full glyoxylate cycle in isolated glyoxysomes requires the presence of aconitase in the incubation medium. It is concluded that glyoxysomes are probably devoid of aconitase and that the glyoxylate cycle requires a detour via the cytosol, which contains a powerful aconitase activity.

Dominant Negative Mutants of Opaque2 Suppress Transactivation of a 22-kD Zein Promoter by Opaque2 in Maize Endosperm Cells.

Abstract: In maize endosperm, genes encoding the 22-kD zein class of storage proteins are regulated by the OPAQUE2 locus. The Opaque2 (O2) protein shares homology with the basic domain/leucine zipper class of transcriptional activators. Using microprojectile bombardment, we have shown that O2 is capable of transactivating a 22-kD zein promoter in maize endosperm suspension cultures and in longitudinal sections of intact endosperm. Two mutant forms of the O2 gene were constructed by deleting regions that encode either the basic domain or the first 175 N-terminal residues of the O2 protein. When either of these mutant O2 genes was coexpressed with wild-type O2 in a maize endosperm expression system, O2-mediated transactivation of the 22-kD zein promoter was inhibited specifically and in a dose-dependent manner. Electrophoretic mobility shift assays and immunoprecipitation studies indicated that the mutant O2 proteins form heterodimers with wild-type O2 in vitro. The mutant lacking the basic domain forms heterodimers with wild-type O2, which can no longer bind DNA. In contrast, the product of the N-terminal truncation allele forms homodimers and heterodimers with wild-type O2, both of which can still bind DNA. Because the N-terminal region contains an activation domain, it is likely that these latter complexes are deficient in transactivation. Dominant negative inhibitors of gene expression, such as those constructed here, provide an alternative to antisense RNA approaches for inactivation of gene function in plants.