Activation tagging using T-DNA vectors that contain multimerized transcriptional enhancers from the cauliflower mosaic virus (CaMV) 35S gene has been applied to Arabidopsis plants. New activation-tagging vectors that confer resistance to the antibiotic kanamycin or the herbicide glufosinate have been used to generate several tens of thousands of transformed plants. From these, over 30 dominant mutants with various phenotypes have been isolated. Analysis of a subset of mutants has shown that overexpressed genes are almost always found immediately adjacent to the inserted CaMV 35S enhancers, at distances ranging from 380 bp to 3.6 kb. In at least one case, the CaMV 35S enhancers led primarily to an enhancement of the endogenous expression pattern rather than to constitutive ectopic expression, suggesting that the CaMV 35S enhancers used here act differently than the complete CaMV 35Spromoter. This has important implications for the spectrum of genes that will be discovered by this method.

red light, although cry1 acted as a phyA/phyB-dependent modulator of chlorophyll accumulation under these conditions. All three photoreceptors contributed to most blue light deetiolation responses, either redundantly or additively; however, phyB acted as a modulator of cotyledon expansion dependent on the presence of cry1. As reported previously, flowering time in long days was promoted by phyA and inhibited by phyB, with each suppressing the other's effect. In addition to the effector/modulator relationships described above, measurements of hypocotyls from blue-light-grown seedlings demonstrated phytochrome activity in blue light and cry1 activity in a phyAphyB mutant background.

SummaryPCR-based detection of single nucleotide polymorphisms is a powerful tool for the plant geneticist. Cleaved amplified polymorphic sequence analysis is the most widely used approach for the detection of single nucleotide polymorphisms. However, this technique is limited to mutations which create or disrupt a restriction enzyme recognition site. This paper presents a modification of this technique where mismatches in a PCR primer are used to create a polymorphism based on the target mutation. This technique is useful for following known mutations in segregating populations and genetic mapping of isolated DNAs used for positional based cloning of new genes. In addition, a computer program has been developed that facilitates the design of these PCR primers.

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The Arabidopsis bas1-D mutation suppresses the long hypocotyl phenotype caused by mutations in the photoreceptor phytochrome B (phyB). The adult phenotype of bas1-D phyB-4 double mutants mimics that of brassinosteroid biosynthetic and response mutants. bas1-D phyB-4 has reduced levels of brassinosteroids and accumulates 26-hydroxybrassinolide in feeding experiments. The basis for the mutant phenotype is the enhanced expression of a cytochrome P450 (CYP72B1). bas1-D suppresses a phyB-null allele, but not a phyA-null mutation, and partially suppresses a cryptochrome-null mutation. Seedlings with reduced BAS1 expression are hyperresponsive to brassinosteroids in a light-dependent manner and display reduced sensitivity to light under a variety of conditions. Thus, BAS1 represents one of the control points between multiple photoreceptor systems and brassinosteroid signal transduction.

Calmodulin is a small Ca(2+)-binding protein proposed to act as the intracellular Ca2+ receptor that translates Ca2+ signals into cellular responses. We have constructed mutant yeast calmodulins in which the Ca(2+)-binding loops have been altered by site-directed mutagenesis. Each of the mutant proteins has a dramatically reduced affinity for Ca2+; one does not bind detectable levels of 45Ca2+ either during gel filtration or when bound to a solid support. Furthermore, none of the mutant proteins change conformation even in the presence of high Ca2+ concentrations. Surprisingly, yeast strains relying on any of the mutant calmodulins not only survive but grow well. In contrast, yeast strains deleted for the calmodulin gene are not viable. Thus, calmodulin is required for growth, but it can perform its essential function without the apparent ability to bind Ca2+.

The Arabidopsis thaliana genome encodes 29 AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED (AHL) genes, which evolved into two phylogenic clades. The AHL proteins contain one or two AT-hook motif(s) and one plant and prokaryote conserved (PPC)/domain of unknown function #296 (DUF296) domain. Seedlings lacking both SOB3/AHL29 and ESC/AHL27 confer a subtle long-hypocotyl phenotype compared with the WT or either single-null mutant. In contrast, the missense allele sob3-6 confers a dramatic long-hypocotyl phenotype in the light. In this study, we examined the dominant-negative feature of sob3-6 and found that it encodes a protein with a disrupted AT-hook motif that abolishes binding to AT-rich DNA. A loss-of-function approach demonstrated different, yet redundant, contributions of additional AHL genes in suppressing hypocotyl elongation in the light. We showed that AHL proteins interact with each other and themselves via the PPC/DUF296 domain. AHLs also share interactions with other nuclear proteins, such as transcription factors, suggesting that these interactions also contribute to the functional redundancy within this gene family. The coordinated action of AHLs requires an AT-hook motif capable of binding AT-rich DNA, as well as a PPC/DUF296 domain containing a conserved Gly-Arg-Phe-Glu-Ile-Leu region. Alteration of this region abolished SOB3/AHL29's physical interaction with transcription factors and resulted in a dominant-negative allele in planta that was phenotypically similar to sob3-6. We propose a molecular model where AHLs interact with each other and themselves, as well as other nuclear proteins, to form complexes which modulate plant growth and development.enhanceosome | seedling establishment

Seed germination is regulated by endogenous hormonal cues and external environmental stimuli such as water, low temperature, and light. After germination, the young seedling must rapidly establish its root system and the photoautotrophic capability appropriate to its surrounding environment. Light and the phytohormone abscisic acid (ABA) both regulate seed germination and seedling development, although how light and ABA signals are integrated at the molecular level is not understood. Here, we found that the previously described light-signaling component HY5 also mediates ABA response in seed germination, early seedling growth, and root development in Arabidopsis. HY5 binds to the promoter of the transcription factor ABI5 gene with high affinity and is required for the expression of ABI5 and ABI5-targeted late embryogenesis-abundant genes in seeds. Chromatin immunoprecipitation also indicated that the binding of HY5 to the ABI5 promoter is significantly enhanced by ABA. Overexpression of ABI5 restores ABA sensitivity in hy5 and results in enhanced light responses and shorter hypocotyls in the wild type. Our studies identified an unexpected mode of light and ABA signal integration that may help young seedlings better adapt to environmental stresses.light response ͉ signal transduction T he plant hormone abscisic acid (ABA) plays essential roles in several aspects of plant growth and development, including seed maturation, seed dormancy, and adaptation to environmental stresses such as drought and high salinity (1-4). Genetic studies of ABA regulation of seed germination and gene expression have identified a number of Arabidopsis mutants with altered ABA sensitivities. One of the ABA insensitive mutants, abi5, was isolated for its ability to germinate in the presence of higher concentrations of exogenous ABA (5). ABI5 encodes a basic leucine zipper (bZIP) transcription factor whose accumulation inhibits seed germination and early seedling establishment (6-8). ABI5 regulates the expression of ABA induced, mostly seed-specific, AtEM genes that encode class I late embryogenesis-abundant (LEA) proteins important for seed maturation (6, 9, 10). Interestingly, the ABI5 gene was also expressed in young seedlings, suggesting that ABI5 may have additional functions beyond seed development and germination (11). It is known that seed germination and early seedling development are also regulated by light (12). Yet little is known how light and ABA signals are integrated in the regulation of this common developmental transition from seeds to seedlings.Whereas the role of ABA in seedling photomorphogenesis is unclear, ABA is well recognized for its critical roles in plant adaptation to drought stress. In addition to inducing stomatal closure and the activation of stress-responsive genes, ABA was recently found to be involved in lateral root development (13-15). Genetic studies indicated that the inhibition of lateral root elongation and potential promotion of primary root growth by drought stress and ABA are adaptive responses o...