Elizabeth C. McKinney

Repression of Flowering in Arabidopsis Requires Activation of FLOWERING LOCUS C Expression by the Histone Variant H2A.Z

The histone variant H2A.Z has been implicated in numerous chromatin-mediated processes, including transcriptional activation, euchromatin maintenance, and heterochromatin formation. In yeast and humans, H2A.Z is deposited into chromatin by a conserved protein complex known as SWR1 and SRCAP, respectively. Here, we show that mutations in the Arabidopsis thaliana homologs of two components of this complex, ACTIN-RELATED PROTEIN6 (ARP6) and PHOTOPERIOD-INDEPENDENT EARLY FLOWERING1 (PIE1), produce similar developmental phenotypes and result in the misregulation of a common set of genes. Using H2A.Z-specific antibodies, we demonstrate that ARP6 and PIE1 are required for the deposition of H2A.Z at multiple loci, including the FLOWERING LOCUS C (FLC) gene, a central repressor of the transition to flowering. Loss of H2A.Z from chromatin in arp6 and pie1 mutants results in reduced FLC expression and premature flowering, indicating that this histone variant is required for high-level expression of FLC. In addition to defining a novel mechanism for the regulation of FLC expression, these results support the existence of a SWR1-like complex in Arabidopsis and show that H2A.Z can potentiate transcriptional activation in plants. The finding that H2A.Z remains associated with chromatin throughout mitosis suggests that it may serve an epigenetic memory function by marking active genes and poising silenced genes for reactivation.

The Arabidopsis ACT7 Actin Gene Is Expressed in Rapidly Developing Tissues and Responds to Several External Stimuli

ACT7 encodes one of the six distinct and ancient subclasses of actin protein in the complex Arabidopsis actin gene family. We determined the sequence and structure of the Arabidopsis thaliana ACT7 actin gene and investigated its tissue-specific expression and regulation. The ACT7 mRNA levels varied by 128-fold among several different tissues and organs. The highest levels of ACT7 mRNA were found in rapidly expanding vegetative organs, the lowest in pollen. A translational fusion with the 5[prime] end of ACT7 (1.9 kb) joined to the [beta]-glucoronidase reporter gene was strongly and preferentially expressed in all young, developing vegetative tissues of transgenic Arabidopsis plants. ACT7 was the only Arabidopsis actin gene strongly expressed in the hypocotyl and seed coat. Although no [beta]-glucoronidase expression was seen in developing ovules or immature seeds, strong expression was seen in dry seeds and immediately after imbibition in the entire seedling. ACT7 was the only Arabidopsis actin gene to respond strongly to auxin, other hormone treatments, light regime, and wounding, and may be the primary actin gene responding to external stimuli. The ACT7 promoter sequence contains a remarkable number of motifs with sequence similarity to putative phytohormone response elements.

The Nuclear Actin-Related Protein ARP6 Is a Pleiotropic Developmental Regulator Required for the Maintenance of FLOWERING LOCUS C Expression and Repression of Flowering in Arabidopsis

Actin-related proteins (ARPs) are found in the nuclei of all eukaryotic cells, but their functions are generally understood only in the context of their presence in various yeast and animal chromatin-modifying complexes. Arabidopsis thaliana ARP6 is a clear homolog of other eukaryotic ARP6s, including Saccharomyces cerevisiae ARP6, which was identified as a component of the SWR1 chromatin remodeling complex. We examined the subcellular localization, expression patterns, and loss-of-function phenotypes for this protein and found that Arabidopsis ARP6 is localized to the nucleus during interphase but dispersed away from the chromosomes during cell division. ARP6 expression was observed in all vegetative tissues as well as in a subset of reproductive tissues. Null mutations in ARP6 caused numerous defects, including altered development of the leaf, inflorescence, and flower as well as reduced female fertility and early flowering in both long- and short-day photoperiods. The early flowering of arp6 mutants was associated with reduced expression of the central floral repressor gene FLOWERING LOCUS C (FLC) as well as MADS AFFECTING FLOWERING 4 (MAF4) and MAF5. In addition, arp6 mutations suppress the FLC-mediated late flowering of a FRIGIDA-expressing line, indicating that ARP6 is required for the activation of FLC expression to levels that inhibit flowering. These results indicate that ARP6 acts in the nucleus to regulate plant development, and we propose that it does so through modulation of chromatin structure and the control of gene expression.

Isovariant dynamics expand and buffer the responses of complex systems: the diverse plant actin gene family.

Most plant and animal genes are members of gene families that are differentially expressed and may encode diverse protein isovariants. With the recent explosion of information in plant genomics, researchers have become acutely aware that the gene families in plants are at least as diverse as their

The evolution of new structures: clues from plant cytoskeletal genes.

How large numbers of genes were recruited simultaneously to build new organ structures is one of the greatest puzzles in evolutionary biology. Here, we present data suggesting that the vegetative and reproductive classes of actins and other cytoskeletal proteins arose concurrently with the macroevolutionary divergence of leaves and reproductive structures in the earliest land plants. That the cytoskeleton is essential for physically programming the development of organs and tissues is well established. Thus, we propose that this regulatory dichotomy represents an ancient landmark event in the global regulation of hundreds of higher-plant genes, an event that is linked to the macroevolution of plant vegetative and reproductive organs. The recent availability of sequence and expression data for large numbers of plant genes should make it possible to dissect this and other major macroevolutionary events.

One Plant Actin Isovariant, ACT7, Is Induced by Auxin and Required for Normal Callus Formation

During plant growth and development, the phytohormone auxin induces a wide array of changes that include cell division, cell expansion, cell differentiation, and organ initiation. It has been suggested that the actin cytoskeleton plays an active role in the elaboration of these responses by directing specific changes in cell morphology and cytoarchitecture. Here we demonstrate that the promoter and the protein product of one of the Arabidopsis vegetative actin genes, ACT7, are rapidly and strongly induced in response to exogenous auxin in the cultured tissues of Arabidopsis. Homozygous act7-1 mutant plants were slow to produce callus tissue in response to hormones, and the mutant callus contained at least two to three times lower levels of ACT7 protein than did the wild-type callus. On the other hand, a null mutation in ACT2, another vegetative actin gene, did not significantly affect callus formation from leaf or root tissue. Complementation of the act7-1 mutants with the ACT7 genomic sequence restored their ability to produce callus at rates similar to those of wild-type plants, confirming that the ACT7 gene is required for callus formation. Immunolabeling of callus tissue with actin subclass-specific antibodies revealed that the predominant ACT7 is coexpressed with the other actin proteins. We suggest that the coexpression, and probably the copolymerization, of the abundant ACT7 with the other actin isovariants in cultured cells may facilitate isovariant dynamics well suited for cellular responses to external stimuli such as hormones.

The Arabidopsis ACT11 actin gene is strongly expressed in tissues of the emerging inflorescence, pollen, and developing ovules.

ACT11 represents a unique and ancient actin subclass in the complex Arabidopsis actin gene family. We have isolated and characterized the Arabidopsis ACT11 actin gene and examined its expression. Southern blotting with a 5′ gene-specific probe showed that ACT11 was a single-copy gene in the genome. Northern analysis with a 3′ gene-specific probe and reverse transcriptase-mediated PCR (RT-PCR) using gene-specific primers detected ACT11 mRNA at low levels in seedling, root, leaf, and silique tissue; at moderate levels in the inflorescence stem and flower; and at very high levels in pollen. The 5′ region of the ACT11 gene, including the promoter region, the 5′-untranslated leader, the intron within the leader, and the first 19 actin codons, was fused to a β-glucuronidase (GUS) reporter gene. The expression of the ACT11/GUS fusion was examined histochemically in numerous independent transgenic Arabidopsis plants. Strong ACT11/GUS activity was detected in rapidly elongating tissues and organs (e.g., etiolated hypocotyls, expanding leaves, stems) and in floral organ primordia. As the floral buds developed into mature flowers, strong GUS activity was gradually restricted to mature pollen and developing ovules. ACT11 appears to be the only Arabidopsis actin gene expressed at significant levels in ovule, embryo, and endosperm. The unique expression patterns in reproductive organs and the sequence divergence of the ACT11 actin gene suggest that the ACT11 isovariant plays distinct and required roles during Arabidopsis development.

Class-specific interaction of profilin and ADF isovariants with actin in the regulation of plant development.

Two ancient and highly divergent actin-based cytoskeletal systems have evolved in angiosperms. Plant genomes encode complex actin and actin binding protein (ABP) gene families, most of which are phylogenetically grouped into gene classes with distinct vegetative or constitutive and reproductive expression patterns. In Arabidopsis thaliana, ectopic expression of high levels of a reproductive class actin, ACT1, in vegetative tissues causes severe dwarfing of plants with aberrant organization of most plant organs and cell types due to a severely altered actin cytoskeletal architecture. Overexpression of the vegetative class actin ACT2 to similar levels, however, produces insignificant phenotypic changes. We proposed that the misexpression of the pollen-specific ACT1 in vegetative cell types affects the dynamics of actin due to its inappropriate interaction with endogenous vegetative ABPs. To examine the functionally distinct interactions among the major classes of actins and ABPs, we ectopically coexpressed reproductive profilin (PRF4) or actin-depolymerizing factor (ADF) isovariants (e.g., ADF7) with ACT1. Our results demonstrated that the coexpression of these reproductive, but not vegetative, ABP isovariants suppressed the ectopic ACT1 expression phenotypes and restored wild-type stature and normal actin cytoskeletal architecture to the double transgenic plants. Thus, the actins and ABPs appear to have evolved class-specific, protein–protein interactions that are essential to the normal regulation of plant growth and development.

A Single Vegetative Actin Isovariant Overexpressed under the Control of Multiple Regulatory Sequences Is Sufficient for Normal Arabidopsis Development

The relative significance of gene regulation and protein isovariant differences remains unexplored for most gene families, particularly those participating in multicellular development. Arabidopsis thaliana encodes three vegetative actins, ACT2, ACT7, and ACT8, in two ancient and highly divergent subclasses. Mutations in any of these differentially expressed actins revealed only mild phenotypes. However, double mutants were extremely dwarfed, with altered cell and organ morphology and an aberrant F-actin cytoskeleton (e.g., act2-1 act7-4 and act8-2 act7-4) or totally root-hairless (e.g., act2-1 act8-2). Our studies suggest that the three vegetative actin genes and protein isovariants play distinct subclass-specific roles during plant morphogenesis. For example, during root development, ACT7 was involved in root growth, epidermal cell specification, cell division, and root architecture, and ACT2 and ACT8 were essential for root hair tip growth. Also, genetic complementation revealed that the ACT2 and ACT8 isovariants, but not ACT7, fully rescued the root hair growth defects of single and double mutants. Moreover, we synthesized fully normal plants overexpressing the ACT8 isovariant from multiple actin regulatory sequences as the only vegetative actin in the act2-1 act7-4 background. In summary, it is evident that differences in vegetative actin gene regulation and the diversity in actin isovariant sequences are essential for normal plant development.

Small Changes in the Regulation of One Arabidopsis Profilin Isovariant, PRF1, Alter Seedling Development

Profilin (PRF) is a low-molecular-weight actin binding protein encoded by a diverse gene family in plants. Arabidopsis PRF1 transcripts are moderately well expressed in all vegetative organs. A regulatory mutant in PRF1, prf1-1, was isolated from a library of T-DNA insertions. The insertion disrupted the promoter region of PRF1 100 bp upstream from the transcriptional start site. Although steady state levels of PRF1 transcripts appeared normal in mature prf1-1 plants, the levels in young seedlings were only one-half those observed in wild type. Reactions with a PRF1 isovariant–specific monoclonal antiserum and general anti-profilin antisera demonstrated that PRF1 protein levels also were one-half those found in wild-type seedlings, although total profilin levels were unaffected. Mutant seedlings no longer could downregulate PRF1 levels in the light, as did wild type. Consistent with their molecular phenotypes, young mutant seedlings displayed several morphological phenotypes but developed into apparently normal adult plants. Their initial germination rate and development were slow, and they produced excessive numbers of root hairs. Mutant seedlings had abnormally raised cotyledons, elongated hypocotyls, and elongated cells in the hypocotyl, typical of phenotypes associated with some defects in light and circadian responses. A wild-type PRF1 transgene fully complements the hypocotyl phenotypes in the prf1-1 mutant. The ability of profilin to regulate actin polymerization and participate directly in signal transduction pathways is discussed in light of the prf1-1 phenotypes.