Donald R. McCarty

The Viviparous-1 developmental gene of maize encodes a novel transcriptional activator

The Viviparous-1 (Vp1) gene of maize is specifically required for expression of the maturation program in seed development. We show that Vp1 encodes a 73,335 dalton protein with no detectable homology to known proteins. An acidic transcriptional activation sequence was identified by fusion to the GAL4 DNA-binding domain. Expression of VP1 in maize protoplasts resulted in strong activation (greater than 130-fold) of a reporter gene fused to the promoter of a presumptive target gene. The acidic domain in VP1 was essential for transactivation and could be functionally replaced by the activator sequence of the herpes simplex virus VP16 protein. Our results indicate that VP1 is a novel transcription factor possibly involved in potentiation of a seed-specific hormone response.

MAX3/CCD7 Is a Carotenoid Cleavage Dioxygenase Required for the Synthesis of a Novel Plant Signaling Molecule

BACKGROUND Plant development is exquisitely environmentally sensitive, with plant hormones acting as long-range signals that integrate developmental, genetic, and environmental inputs to regulate development. A good example of this is in the control of shoot branching, where wide variation in plant form can be generated in a single genotype in response to environmental and developmental cues. RESULTS Here we present evidence for a novel plant signaling molecule involved in the regulation of shoot branching. We show that the MAX3 gene of Arabidopsis is required for the production of a graft-transmissible, highly active branch inhibitor that is distinct from any of the previously characterized branch-inhibiting hormones. Consistent with its proposed function in the synthesis of a novel signaling molecule, we show that MAX3 encodes a plastidic dioxygenase that can cleave multiple carotenoids. CONCLUSIONS We conclude that MAX3 is required for the synthesis of a novel carotenoid-derived long-range signal that regulates shoot branching.

The conserved B3 domain of VIVIPAROUS1 has a cooperative DNA binding activity.

The biochemical activities that underlie the genetically defined activator and repressor functions of the VIVIPAROUS1 (VP1) protein have resisted in vitro analysis. Here, we show that a glutathione S-transferase (GST) fusion protein, including only the highly conserved B3 domain of VP1, has a highly cooperative, sequence-specific DNA binding activity. GST fusion proteins that include larger regions of the VP1 protein have very low activity, indicating that removal of the flanking protein sequences is necessary to elicit DNA binding in vitro. DNA competition and DNase I footprinting analyses show that B3 binds specifically to the Sph element involved in VP1 activation of the C1 gene, whereas binding to the G-box-type VP1-responsive element is of low affinity and is nonspecific. Footprint analysis of the C1 promoter revealed that sequences flanking the core TCCATGCAT motif of Sph also contribute to the recognition of the Sph element in its native context. The salient features of the in vitro GST-B3 DNA interaction are in good agreement with the protein and DNA sequence requirements defined by the functional analyses of VP1 and VP1-responsive elements in maize cells.

CRINKLY4: A TNFR-Like Receptor Kinase Involved in Maize Epidermal Differentiation

The maize crinkly4 (cr4) mutation affects leaf epidermis differentiation such that cell size and morphology are altered, and surface functions are compromised, allowing graft-like fusions between organs. In the seed, loss of cr4 inhibits aleurone formation in a pattern that reflects the normal progression of differentiation over the developing endosperm surface. The cr4 gene was isolated by transposon tagging and found to encode a putative receptor kinase. The extracellular domain contains a cysteine-rich region similar to the ligand binding domain in mammalian tumor necrosis factor receptors (TNFRs) and seven copies of a previously unknown 39-amino acid repeat. The results suggest a role for cr4 in a differentiation signal.

Sugar Levels Modulate Differential Expression of Maize Sucrose Synthase Genes.

The two genes encoding sucrose synthase in maize (Sh1 and Sus1) show markedly different responses to changes in tissue carbohydrate status. This enzyme is widely regarded as pivotal to sucrose partitioning, import, and/or metabolism by developing plant organs. Excised maize root tips were incubated for varying periods in different sugars and a range of concentrations. The Sh1 mRNA was maximally expressed under conditions of limited carbohydrate supply (~0.2% glucose). In contrast, Sus1 transcript levels were low or nondetectable under sugar-depleted conditions and peaked at 10-fold greater glucose concentrations (2.0%). Responses to other metabolizable sugars were similar, but L-glucose and elevation of osmolarity with mannitol had little effect. Plentiful sugar supplies thus increased expression of Sus1, whereas reduced sugar availability enhanced Sh1. At the protein level, shifts in abundance of subunits encoded by Sh1 and Sus1 were much less pronounced but corresponded to changes in respective mRNA levels. Although total enzyme activity did not show net change, cellular localization of sucrose synthase protein was markedly altered. In intact roots, sucrose synthase was most prevalent in the stele and apex. In contrast, sugar depletion favored accumulation in peripheral cells, whereas high sugar levels resulted in elevated expression in all cell types. The differential response of the two sucrose synthase genes to sugars provides a potential mechanism for altering the pattern of enzyme distribution in response to changing carbohydrate status and also for adjusting the sucrose-metabolizing capacity of importing cells relative to levels of available photosynthetic products.

Molecular Analysis of viviparous-1: An Abscisic Acid- Insensitive Mutant of Maize

The viviparous-1 (vp1) gene in maize controls multiple developmental responses associated with the maturation phase of seed formation. Most notably, mutant embryos have reduced sensitivity to the hormone abscisic acid, resulting in precocious germination, and blocked anthocyanin synthesis in aleurone and embryo tissues. The Vp1 locus was cloned by transposon tagging, using the Robertsons Mutator element present in the vp1-mum1 mutant allele. Detection of DNA rearrangements in several spontaneous and transposable element-induced mutant vp1 alleles, including a partial deletion of the locus, confirmed the identity of the clone. The Vp1 gene encodes a 2500-nucleotide mRNA that is expressed specifically in embryo and endosperm tissues of the developing seed. This transcript is absent in seed tissues of vp1 mutant stocks. Expression of C1, a regulatory gene for the anthocyanin pathway, is selectively blocked at the mRNA level in vp1 mutant seed tissues, indicating the Vp1 may control the anthocyanin pathway by regulating C1. We suggest that the Vp1 gene product functions to potentiate multiple signal transduction pathways in specific seed tissues.

Repression of the LEAFY COTYLEDON 1/B3 Regulatory Network in Plant Embryo Development by VP1/ABSCISIC ACID INSENSITIVE 3-LIKE B3 Genes

Plant embryo development is regulated by a network of transcription factors that include LEAFY COTYLEDON 1 (LEC1), LEC1-LIKE (L1L), and B3 domain factors, LEAFY COTYLEDON 2 (LEC2), FUSCA3 (FUS3), and ABSCISIC ACID INSENSITIVE 3 (ABI3) of Arabidopsis (Arabidopsis thaliana). Interactions of these genes result in temporal progression of overlapping B3 gene expression culminating in maturation and desiccation of the seed. Three VP1/ABI3-LIKE (VAL) genes encode B3 proteins that include plant homeodomain-like and CW domains associated with chromatin factors. Whereas val monogenic mutants have phenotypes similar to wild type, val1 val2 double-mutant seedlings form no leaves and develop embryo-like proliferations in root and apical meristem regions. In a val1 background, val2 and val3 condition a dominant variegated leaf phenotype revealing a VAL function in vegetative development. Reminiscent of the pickle (pkl) mutant, inhibition of gibberellin biosynthesis during germination induces embryonic phenotypes in val1 seedlings. Consistent with the embryonic seedling phenotype, LEC1, L1L, ABI3, and FUS3 are up-regulated in val1 val2 seedlings in association with a global shift in gene expression to a profile resembling late-torpedo-stage embryogenesis. Hence, VAL factors function as global repressors of the LEC1/B3 gene system. The consensus binding site of the ABI3/FUS3/LEC2 B3 DNA-binding domain (Sph/RY) is strongly enriched in the promoters and first introns of VAL-repressed genes, including the early acting LEC1 and L1L genes. We suggest that VAL targets Sph/RY-containing genes in the network for chromatin-mediated repression in conjunction with the PKL-related CHD3 chromatin-remodeling factors.

The Carotenoid Cleavage Dioxygenase 1 Enzyme Has Broad Substrate Specificity, Cleaving Multiple Carotenoids at Two Different Bond Positions

In many organisms, various enzymes mediate site-specific carotenoid cleavage to generate biologically active apocarotenoids. These carotenoid-derived products include provitamin A, hormones, and flavor and fragrance molecules. In plants, the CCD1 enzyme cleaves carotenoids at 9,10 (9′,10′) bonds to generate multiple apocarotenoid products. Here we systematically analyzed volatile apocarotenoids generated by maize CCD1 (ZmCCD1) from multiple carotenoid substrates. ZmCCD1 did not cleave geranylgeranyl diphosphate or phytoene but did cleave other linear and cyclic carotenoids, producing volatiles derived from 9,10 (9′,10′) bond cleavage. Additionally the Arabidopsis, maize, and tomato CCD1 enzymes all cleaved lycopene to generate 6-methyl-5-hepten-2-one. 6-Methyl-5-hepten-2-one, an important flavor volatile in tomato, was produced by cleavage of the 5,6 or 5′,6′ bond positions of lycopene but not geranylgeranyl diphosphate, ζ-carotene, or phytoene. In vitro, ZmCCD1 cleaved linear and cyclic carotenoids with equal efficiency. Based on the pattern of apocarotenoid volatiles produced, we propose that CCD1 recognizes its cleavage site based on the saturation status between carbons 7 and 8 (7′ and 8′) and carbons 11 and 12 (11′ and 12′) as well as the methyl groups on carbons 5, 9, and 13 (5′, 9′, and 13′).

Viviparous1 Alters Global Gene Expression Patterns through Regulation of Abscisic Acid Signaling

Maize (Zea mays) Viviparous1 (VP1) and Arabidopsis ABI3 are orthologous transcription factors that regulate key aspects of plant seed development and ABA signaling. To understand VP1-regulated gene expression on a global scale, we have performed oligomicroarray analysis of transgenic Arabidopsis carrying 35S::VP1 in an abi3 null mutant background. We have identified 353 VP1/ABA-regulated genes by GeneChip analysis. Seventy-three percent of the genes were affected by both VP1 and ABA in vegetative tissues, indicating a tight coupling between ABA signaling and VP1 function. A large number of seed-specific genes were ectopically expressed in vegetative tissue of 35S::VP1 plants consistent with evidence that VP1 and ABI3 are key determinants of seed-specific expression. ABI5, a positive regulator of ABA signaling, was activated by VP1, indicating conservation of the feed-forward pathway mediated by ABI3. ABA induction of ABI1 and ABI2, negative regulators of ABA signaling, was strongly inhibited by VP1, revealing a second pathway of feed-forward regulation. These results indicate that VP1 strongly modifies ABA signaling through feed-forward regulation of ABI1/ABI5-related genes. Of the 32 bZIP transcription factors represented on the GeneChip, genes in the ABI5 clade were specifically coregulated by ABA and VP1. Statistical analysis of 5′ upstream sequences of the VP1/ABA-regulated genes identified consensus abscisic responsive elements as an enriched element, indicating that many of the genes could be direct targets of the ABI5-related bZIPs. The Sph element is an enriched sequence motif in promoters of genes co-activated by ABA and VP1 but not in promoters of genes activated by ABA alone. This analysis reveals that distinct combinatorial patterns of promoter elements distinguish subclasses of VP1/ABA coregulated genes.

Overlap of Viviparous1 (VP1) and abscisic acid response elements in the Em promoter: G-box elements are sufficient but not necessary for VP1 transactivation.

The relationship between promoter sequences that mediate Viviparous1 (VP1) transactivation and regulation by abscisic acid (ABA) in the wheat Em promoter was investigated using deletion analysis and directed mutagenesis. The Em1a G-box is strongly coupled to VP1 transactivation as well as to ABA regulation; however, the Em promoter includes additional components that can support VP1 transactivation without ABA responsiveness or synergism. Oligonucleotide tetramers of several G-box sequences, including Em1a, Em1b, and the dyad G-box element from the UV light-regulated parsley chalcone synthase gene, were sufficient to confer VP1 transactivation and the synergistic interaction with ABA to the -45 cauliflower mosaic virus 35S core promoter. These data suggest that VP1 can activate transcription through at least two classes of cis-acting sequences, including the G-box elements and the Sph regulatory motif found in the C1 promoter. The contrasting roles of these motifs in the Em and C1 promoters suggest a basis for the differential regulation of the corresponding genes by VP1.