A maize zinc-finger protein binds the prolamin box in zein gene promoters and interacts with the basic leucine zipper transcriptional activator Opaque2

The prolamin box (P-box) is a highly conserved 7-bp sequence element (5′-TGTAAAG-3′) found in the promoters of many cereal seed storage protein genes. Nuclear factors from maize endosperm specifically interact with the P-box present in maize prolamin genes (zeins). The presence of the P-box in all zein gene promoters suggests that interactions between endosperm DNA binding proteins and the P-box may play an important role in the coordinate activation of zein gene expression during endosperm development. We have cloned an endosperm-specific maize cDNA, named prolamin-box binding factor (PBF), that encodes a member of the recently described Dof class of plant Cys2-Cys2 zinc-finger DNA binding proteins. When tested in gel shift assays, PBF exhibits the same sequence-specific binding to the P-box as factors present in maize endosperm nuclei. Additionally, PBF interacts in vitro with the basic leucine zipper protein Opaque2, a known transcriptional activator of zein gene expression whose target site lies 20 bp downstream of the P-box in the 22-kDa zein gene promoter. The isolation of the PBF gene provides an essential tool to further investigate the functional role of the highly conserved P-box in regulating cereal storage protein gene expression.

A deletion in an indole synthase gene is responsible for the DIMBOA-deficient phenotype of bxbx maize

The biosynthesis of DIMBOA, a pesticidal secondary metabolite of maize, branches off the tryptophan pathway. We have previously demonstrated that indole is the last intermediate common to both the tryptophan and hydroxamic acid pathways. The earliest discovered mutant in the DIMBOA pathway, bxbx (benzoxazineless), is deficient in the production of DIMBOA and related compounds. This paper presents evidence that a gene identified by Kramer and Koziel [Kramer, V. C. & Koziel, M. G. (1995) Plant Mol. Biol. 27, 1183–1188] as maize tryptophan synthase α (TSA) is the site of the genetic lesion in the DIMBOA-deficient mutant maize line bxbx. We demonstrate that the TSA gene has sustained a 924-bp deletion in bxbx compared with its counterpart in wild-type maize. We report that the TSA gene maps to the same location as the bxbx mutation, on the short arm of chromosome 4. We present evidence that the very early and very high level of expression of TSA corresponds to the timing and level of DIMBOA biosynthesis but is strikingly different from the expression of the maize tryptophan synthase β (TSB) genes. We show that feeding indole to bxbx seedlings restores their ability to synthesize DIMBOA. We conclude that the maize enzyme initially named tryptophan synthase α in fact is a DIMBOA biosynthetic enzyme, and we propose that it be renamed indole synthase. This work confirms and enlarges upon the findings of Frey et al. [Frey, M. Chomet, P., Glawischniq, E., Stettner, C., Grün, S., Winklmair, A., Eisenreich, W., Bacher, A., Meeley, R. B., Briggs, S. P., Simcox, K. & Gierl, A. (1997) Science 277, 696–699], which appeared while the present paper was in review.

Overexpression of thioredoxin h leads to enhanced activity of starch debranching enzyme (pullulanase) in barley grain

Biochemically active wheat thioredoxin h has been overexpressed in the endosperm of transgenic barley grain. Two DNA constructs containing the wheat thioredoxin h gene (wtrxh) were used for transformation; each contained wtrxh fused to an endosperm-specific B1-hordein promoter either with or without a signal peptide sequence for targeting to the protein body. Twenty-two stable, independently transformed regenerable lines were obtained by selecting with the herbicide bialaphos to test for the presence of the bar herbicide resistance gene on a cotransformed plasmid; all were positive for this gene. The presence of wtrxh was confirmed in 20 lines by PCR analysis, and the identity and level of expression of wheat thioredoxin h was assessed by immunoblots. Although levels varied among the different transgenic events, wheat thioredoxin h was consistently highly expressed (up to 30-fold) in the transgenic grain. Transgenic lines transformed with the B1-hordein promoter with a signal peptide sequence produced a higher level of wheat thioredoxin h on average than those without a signal sequence. The overexpression of thioredoxin h in the endosperm of germinated grain effected up to a 4-fold increase in the activity of the starch debranching enzyme, pullulanase (limit dextrinase), the enzyme that specifically cleaves α-1,6 linkages in starch. These results raise the question of how thioredoxin h enhances the activity of pullulanase because it was found that the inhibitor had become inactive before the enzyme showed appreciable activity.

Major recent and independent changes in levels and patterns of expression have occurred at the b gene, a regulatory locus in maize

The b locus encodes a transcription factor that regulates the expression of genes that produce purple anthocyanin pigment. Different b alleles are expressed in distinct tissues, causing tissue-specific anthocyanin production. Understanding how phenotypic diversity is produced and maintained at the b locus should provide models for how other regulatory genes, including those that influence morphological traits and development, evolve. We have investigated how different levels and patterns of pigmentation have evolved by determining the phenotypic and evolutionary relationships between 18 alleles that represent the diversity of b alleles in Zea mays. Although most of these alleles have few phenotypic differences, five alleles have very distinct tissue-specific patterns of pigmentation. Superimposing the phenotypes on the molecular phylogeny reveals that the alleles with strong and distinctive patterns of expression are closely related to alleles with weak expression, implying that the distinctive patterns have arisen recently. We have identified apparent insertions in three of the five phenotypically distinct alleles, and the fourth has unique upstream restriction fragment length polymorphisms relative to closely related alleles. The insertion in B-Peru has been shown to be responsible for its unique expression and, in the other two alleles, the presence of the insertion correlates with the phenotype. These results suggest that major changes in gene expression are probably the result of large-scale changes in DNA sequence and/or structure most likely mediated by transposable elements.

Comparative mapping of Andropogoneae: Saccharum L. (sugarcane) and its relation to sorghum and maize

Comparative genetic maps of Papuan Saccharum officinarum L. (2n = 80) and S. robustum (2n = 80) were constructed by using single-dose DNA markers (SDMs). SDM-framework maps of S. officinarum and S. robustum were compared with genetic maps of sorghum and maize by way of anchor restriction fragment length polymorphism probes. The resulting comparisons showed striking colinearity between the sorghum and Saccharum genomes. There were no differences in marker order between S. officinarum and sorghum. Furthermore, there were no alterations in SDM order between S. officinarum and S. robustum. The S. officinarum and S. robustum maps also were compared with the map of the polysomic octoploid S. spontaneum ‘SES 208’ (2n = 64, x = 8), thus permitting relations to homology groups (“chromosomes”) of S. spontaneum to be studied. Investigation of transmission genetics in S. officinarum and S. robustum confirmed preliminary results that showed incomplete polysomy in these species. Because of incomplete polysomy, multiple-dose markers could not be mapped for lack of a genetic model for their segregation. To coalesce S. officinarum and S. robustum linkage groups into homology groups (composed of homologous pairing partners), they were compared with sorghum (2n = 20), which functioned as a synthetic diploid. Groupings suggested by comparative mapping were found to be highly concordant with groupings based on highly polymorphic restriction fragment length polymorphism probes detecting multiple SDMs. The resulting comparative maps serve as bridges to allow information from one Andropogoneae to be used by another, for breeding, ecology, evolution, and molecular biology.

Adenosine diphosphate glucose pyrophosphatase: A plastidial phosphodiesterase that prevents starch biosynthesis

A distinct phosphodiesterasic activity (EC 3.1.4) was found in both mono- and dicotyledonous plants that catalyzes the hydrolytic breakdown of ADPglucose (ADPG) to produce equimolar amounts of glucose-1-phosphate and AMP. The enzyme responsible for this activity, referred to as ADPG pyrophosphatase (AGPPase), was purified over 1,100-fold from barley leaves and subjected to biochemical characterization. The calculated Keq′ (modified equilibrium constant) value for the ADPG hydrolytic reaction at pH 7.0 and 25°C is 110, and its standard-state free-energy change value (ΔG′) is −2.9 kcal/mol (1 kcal = 4.18 kJ). Kinetic analyses showed that, although AGPPase can hydrolyze several low-molecular weight phosphodiester bond-containing compounds, ADPG proved to be the best substrate (Km = 0.5 mM). Pi and phosphorylated compounds such as 3-phosphoglycerate, PPi, ATP, ADP, NADP+, and AMP are inhibitors of AGPPase. Subcellular localization studies revealed that AGPPase is localized exclusively in the plastidial compartment of cultured cells of sycamore (Acer pseudoplatanus L.), whereas it occurs both inside and outside the plastid in barley endosperm. In this paper, evidence is presented that shows that AGPPase, whose activity declines concomitantly with the accumulation of starch during development of sink organs, competes with starch synthase (ADPG:1,4-α-d-glucan 4-α-d-glucosyltransferase; EC 2.4.1.21) for ADPG, thus markedly blocking the starch biosynthesis.

Phase identity of the maize leaf is determined after leaf initiation

The vegetative development of the maize shoot can be divided into juvenile and adult phases based on the types of leaves produced at different times in shoot development. Models for the regulation of phase change make explicit predictions about when the identity of these types of leaves is determined. To test these models, we examined the timing of leaf type determination in maize. Clones induced in transition leaf primordia demonstrated that the juvenile and adult regions of these leaves do not become clonally distinct until after the primordium is 700 μm in length, implying that these cell fates were undetermined at this stage of leaf development. Adult shoot apices were cultured in vitro to induce rejuvenation. We found that leaf primordia as large as 3 mm in length can be at least partially rejuvenated by this treatment, and the location of rejuvenated tissue is correlated with the maturation pattern of the leaf. The amount and distribution of juvenile tissue in rejuvenated leaves suggests that rejuvenation occurs nearly simultaneously in all leaf primordia. In vitro culture rejuvenated existing leaf primordia and the P0 primordium, but did not change the identity of subsequent primordia or the total number of leaves produced by the shoot. This result suggests that leaf identity can be regulated independently of the identity of the shoot apical meristem, and it implies that vegetative phase change is not initiated by a change in the identity of the shoot apical meristem.

The MITE family Heartbreaker (Hbr): Molecular markers in maize

Transposable elements are ubiquitous in plant genomes, where they frequently comprise the majority of genomic DNA. The maize genome, which is believed to be structurally representative of large plant genomes, contains single genes or small gene islands interspersed with much longer blocks of retrotransposons. Given this organization, it would be desirable to identify molecular markers preferentially located in genic regions. In this report, the features of a newly described family of miniature inverted repeat transposable elements (MITEs) (called Heartbreaker), including high copy number and polymorphism, stability, and preference for genic regions, have been exploited in the development of a class of molecular markers for maize. To this end, a modification of the AFLP procedure called transposon display was used to generate and display hundreds of genomic fragments anchored in Hbr elements. An average of 52 markers were amplified for each primer combination tested. In all, 213 polymorphic fragments were reliably scored and mapped in 100 recombinant inbred lines derived from a cross between the maize inbreds B73 × Mo17. In this mapping population, Hbr markers are distributed evenly across the 10 maize chromosomes. This procedure should be of general use in the development of markers for other MITE families in maize and in other plant and animal species where MITEs have been identified.

A knob-associated tandem repeat in maize capable of forming fold-back DNA segments: Are chromosome knobs megatransposons?

A class of tandemly repeated DNA sequences (TR-1) of 350-bp unit length was isolated from the knob DNA of chromosome 9 of Zea mays L. Comparative fluorescence in situ hybridization revealed that TR-1 elements are also present in cytologically detectable knobs on other maize chromosomes in different proportions relative to the previously described 180-bp repeats. At least one knob on chromosome 4 is composed predominantly of the TR-1 repeat. In addition, several small clusters of the TR-1 and 180-bp repeats have been found in different chromosomes, some not located in obvious knob heterochromatin. Variation in restriction fragment fingerprints and copy number of the TR-1 elements was found among maize lines and among maize chromosomes. TR-1 tandem arrays up to 70 kilobases in length can be interspersed with stretches of 180-bp tandem repeat arrays. DNA sequence analysis and restriction mapping of one particular stretch of tandemly arranged TR-1 units indicate that these elements may be organized in the form of fold-back DNA segments. The TR-1 repeat shares two short segments of homology with the 180-bp repeat. The longest of these segments (31 bp; 64% identity) corresponds to the conserved region among 180-bp repeats. The polymorphism and complex structure of knob DNA suggest that, similar to the fold-back DNA-containing giant transposons in Drosophila, maize knob DNA may have some properties of transposable elements.

The earliest archaeological maize (Zea mays L.) from highland Mexico: New accelerator mass spectrometry dates and their implications

Accelerator mass spectrometry age determinations of maize cobs (Zea mays L.) from Guilá Naquitz Cave in Oaxaca, Mexico, produced dates of 5,400 carbon-14 years before the present (about 6,250 calendar years ago), making those cobs the oldest in the Americas. Macrofossils and phytoliths characteristic of wild and domesticated Zea fruits are absent from older strata from the site, although Zea pollen has previously been identified from those levels. These results, together with the modern geographical distribution of wild Zea mays, suggest that the cultural practices that led to Zea domestication probably occurred elsewhere in Mexico. Guilá Naquitz Cave has now yielded the earliest macrofossil evidence for the domestication of two major American crop plants, squash (Cucurbita pepo) and maize.

TRM1, a YY1-like suppressor of rbcS-m3 expression in maize mesophyll cells

The genes rbcS and rbcL encode, respectively, the small and large subunits of the photosynthetic carbon dioxide fixation enzyme ribulose bisphosphate carboxylase/oxygenase. There is a single rbcL gene in each chloroplast chromosome; a family of rbcS genes is located in the nuclear genome. These two genes are not expressed in mesophyll cells but are in adjacent bundle-sheath cells of leaves of the C4 plant Zea mays. Two regions of the maize gene rbcS-m3 are required for suppressing expression in mesophyll cells. One region is just beyond the translation termination site in the 3′ region, and the other is several hundred base pairs upstream of the transcription start site. A binding site for a protein with limited homology to the viral, yeast, and mammalian transcription repressor-activator YY1 (Yin-Yang I), has now been identified in the 3′ region. A maize gene for a protein with zinc fingers homologous to those of YY1 has been isolated, characterized, and expressed in Escherichia coli. The gene is designated trm1 (transcription repressor-maize 1). The protein TRM1 binds to the YY1-like site and, in addition, TRM1 binds to two sequence regions in the 5′ region of the gene that have no homology to the YY1 site. Mutagenesis or deletion of any of these three sequences eliminates repression of rbcS-m3 reporter genes in mesophyll cells.

Proline Accumulation in Maize (Zea mays L.) Primary Roots at Low Water Potentials. II. Metabolic Source of Increased Proline Deposition in the Elongation Zone1

The proline (Pro) concentration increases greatly in the growing region of maize (Zea mays L.) primary roots at low water potentials (ψw), largely as a result of an increased net rate of Pro deposition. Labeled glutamate (Glu), ornithine (Orn), or Pro was supplied specifically to the root tip of intact seedlings in solution culture at high and low ψw to assess the relative importance of Pro synthesis, catabolism, utilization, and transport in root-tip Pro deposition. Labeling with [3H]Glu indicated that Pro synthesis from Glu did not increase substantially at low ψw and accounted for only a small fraction of the Pro deposition. Labeling with [14C]Orn showed that Pro synthesis from Orn also could not be a substantial contributor to Pro deposition. Labeling with [3H]Pro indicated that neither Pro catabolism nor utilization in the root tip was decreased at low ψw. Pro catabolism occurred at least as rapidly as Pro synthesis from Glu. There was, however, an increase in Pro uptake at low ψw, which suggests increased Pro transport. Taken together, the data indicate that increased transport of Pro to the root tip serves as the source of low-ψw-induced Pro accumulation. The possible significance of Pro catabolism in sustaining root growth at low ψw is also discussed.

Impacts of Aluminum on the Cytoskeleton of the Maize Root Apex. Short-Term Effects on the Distal Part of the Transition Zone1

Using monoclonal tubulin and actin antibodies, Al-mediated alterations to microtubules (MTs) and actin microfilaments (MFs) were shown to be most prominent in cells of the distal part of the transition zone (DTZ) of an Al-sensitive maize (Zea mays L.) cultivar. An early response to Al (1 h, 90 μm) was the depletion of MTs in cells of the DTZ, specifically in the outermost cortical cell file. However, no prominent changes to the MT cytoskeleton were found in elongating cells treated with Al for 1 h in spite of severe inhibition of root elongation. Al-induced early alterations to actin MFs were less dramatic and consisted of increased actin fluorescence of partially disintegrated MF arrays in cells of the DTZ. These tissue- and development-specific alterations to the cytoskeleton were preceded by and/or coincided with Al-induced depolarization of the plasma membrane and with callose formation, particularly in the outer cortex cells of the DTZ. Longer Al supplies (>6 h) led to progressive enhancements of lesions to the MT cytoskeleton in the epidermis and two to three outer cortex cell files. Our data show that the cytoskeleton in the cells of the DTZ is especially sensitive to Al, consistent with the recently proposed specific Al sensitivity of this unique, apical maize root zone.

Change in Apoplastic Aluminum during the Initial Growth Response to Aluminum by Roots of a Tolerant Maize Variety1

Root elongation, hematoxylin staining, and changes in the ultrastructure of root-tip cells of an Al-tolerant maize variety (Zea mays L. C 525 M) exposed to nutrient solutions with 20 μm Al (2.1 μm Al3+ activity) for 0, 4, and 24 h were investigated in relation to the subcellular distribution of Al using scanning transmission electron microscopy and energy-dispersive x-ray microanalysis on samples fixed by different methods. Inhibition of root-elongation rates, hematoxylin staining, cell wall thickening, and disturbance of the distribution of pyroantimoniate-stainable cations, mainly Ca, was observed only after 4 and not after 24 h of exposure to Al. The occurrence of these transient, toxic Al effects on root elongation and in cell walls was accompanied by the presence of solid Al-P deposits in the walls. Whereas no Al was detectable in cell walls after 24 h, an increase of vacuolar Al was observed after 4 h of exposure. After 24 h, a higher amount of electron-dense deposits containing Al and P or Si was observed in the vacuoles. These results indicate that in this tropical maize variety, tolerance mechanisms that cause a change in apoplastic Al must be active. Our data support the hypothesis that in Al-tolerant plants, Al can rapidly cross the plasma membrane; these data clearly contradict the former conclusions that Al mainly accumulates in the apoplast and enters the symplast only after severe cell damage has occurred.

In Vitro Biosynthesis of Phosphorylated Starch in Intact Potato Amyloplasts1

Intact amyloplasts from potato (Solanum tuberosum L.) were used to study starch biosynthesis and phosphorylation. Assessed by the degree of intactness and by the level of cytosolic and vacuolar contamination, the best preparations were selected by searching for amyloplasts containing small starch grains. The isolated, small amyloplasts were 80% intact and were free from cytosolic and vacuolar contamination. Biosynthetic studies of the amyloplasts showed that [1-14C]glucose-6-phosphate (Glc-6-P) was an efficient precursor for starch synthesis in a manner highly dependent on amyloplast integrity. Starch biosynthesis from [1-14C]Glc-1-P in small, intact amyloplasts was 5-fold lower and largely independent of amyloplast intactness. When [33P]Glc-6-P was administered to the amyloplasts, radiophosphorylated starch was produced. Isoamylase treatment of the starch followed by high-performance anion-exchange chromatography with pulsed amperometric detection revealed the separated phosphorylated α-glucans. Acid hydrolysis of the phosphorylated α-glucans and high-performance anion-exchange chromatography analyses showed that the incorporated phosphate was preferentially positioned at C-6 of the Glc moiety. The incorporation of radiolabel from Glc-1-P into starch in preparations of amyloplasts containing large grains was independent of intactness and most likely catalyzed by starch phosphorylase bound to naked starch grains.