Rapid sequence and functional diversification of a miRNA superfamily targeting calcium signaling components in seed plants.

MicroRNA (miRNA)-directed posttranscriptional gene silencing (miR-PTGS) is an integral component of gene regulatory networks governing plant development and responses to the environment. The sequence homology between Sly-miR4376, a miRNA common to Solanaceae and reported to target autoinhibited Ca -ATPase 10 (ACA10) messenger RNA (mRNA) in tomato, and Arabidopsis miR391 (Ath-miR391), previously annotated as a nonconserved member of the deeply conserved miR390 family, has prompted us to revisit the function of Ath-miR391, as well as its regulatory conservation. A combination of genetic, molecular, and bioinformatic analyses revealed a hidden conservation for miR-PTGS of ACA10 homologs in spermatophytes.

Biological activity and dimerization state of modified phytochrome A proteins.

To assess potential physical interactions of type I phyA with the type II phyB-phyE phytochromes in vivo, transgenes expressing fusion gene forms of phyA were introduced into the Arabidopsis phyA mutant background. When a single c-Myc (myc) epitope is added to either the N- or C-terminus of phyA, the constructs completely complement phyA mutant phenotypes. However, addition of larger tags, such as six consecutive myc epitopes or the yellow fluorescent protein sequence, result in fusion proteins that show reduced activity.

Comparative functional analysis of full-length and N-terminal fragments of phytochrome C, D and E in red light-induced signaling.

Phytochromes (phy) C, D and E are involved in the regulation of red/far-red light-induced photomorphogenesis of Arabidopsis thaliana, but only limited data are available on the mode of action and biological function of these lesser studied phytochrome species. We fused N-terminal fragments or full-length PHYC, D and E to YELLOW FLUORESCENT PROTEIN (YFP), and analyzed the function, stability and intracellular distribution of these fusion proteins in planta. The activity of the constitutively nuclear-localized homodimers of N-terminal fragments was comparable with that of full-length PHYC, D, E-YFP, and resulted in the regulation of various red light-induced photomorphogenic responses in the studied genetic backgrounds.

Directed dimerization: an in vivo expression system for functional studies of type II phytochromes.

Type II phytochromes (phy) in Arabidopsis form homodimers and heterodimers, resulting in a diverse collection of light-stable red/far-red (R/FR) sensing photoreceptors. We describe an in vivo protein engineering system and its use in characterizing the activities of these molecules. Using a phyB null mutant background, singly and doubly transgenic plants were generated that express fusion proteins containing the phyB-phyE N-terminal photosensory regions (NB-NE PSRs), a nuclear localization sequence, and small yeast protein domains that mediate either homodimerization or heterodimerization.

Unanticipated regulatory roles for Arabidopsis phytochromes revealed by null mutant analysis.

In view of the extensive literature on phytochrome mutants in the Ler accession of Arabidopsis, we sought to secure a phytochrome-null line in the same genetic background for comparative studies. Here we report the isolation and phenotypic characterization of phyABCDE quintuple and phyABDE quadruple mutants in the Ler background. Unlike earlier studies, these lines possess a functional allele of FT permitting measurements of photoperiod-dependent flowering behavior.

Obligate heterodimerization of Arabidopsis phytochromes C and E and interaction with the PIF3 basic helix-loop-helix transcription factor.

Phytochromes are dimeric chromoproteins that regulate plant responses to red (R) and far-red (FR) light. The Arabidopsis thaliana genome encodes five phytochrome apoproteins: type I phyA mediates responses to FR, and type II phyB-phyE mediate shade avoidance and classical R/FR-reversible responses. In this study, we describe the complete in vivo complement of homodimeric and heterodimeric type II phytochromes.

The phytochrome red/far-red photoreceptor superfamily.

Proteins of the phytochrome superfamily of red/far-red light receptors have a variety of biological roles in plants, algae, bacteria and fungi and demonstrate a diversity of spectral sensitivities and output signaling mechanisms. Over the past few years the first three-dimensional structures of phytochrome light-sensing domains from bacteria have been determined.

The ACA10 Ca2+-ATPase regulates adult vegetative development and inflorescence architecture in Arabidopsis.

The Arabidopsis (Arabidopsis thaliana) compact inflorescence (cif) genotype causes altered adult vegetative development and a reduction in elongation of inflorescence internodes resulting in formation of floral clusters. The cif trait requires both a recessive mutation, cif1, and the activity of a naturally occurring dominant allele of an unlinked gene, CIF2(D). We show here that the pseudoverticillata mutation is allelic with cif1 and that the product of the CIF1 gene is ACA10, a member of the large family of P-type Ca(2+)-ATPases found in higher plants.

Diversification of phytochrome contributions to germination as a function of seed-maturation environment.

Environmental conditions during seed maturation influence germination, but the genetic basis of maternal environmental effects on germination is virtually unknown. Using single and multiple mutants of phytochromes, it is shown here that different phytochromes contributed to germination differently, depending on seed-maturation conditions. Arabidopsis thaliana wild-type seeds that were matured under cool temperatures were intensely dormant compared with seeds matured at warmer temperature, and this dormancy was broken only after warm seed-stratification followed by cold seed-stratification.

Distinct light and clock modulation of cytosolic free Ca2+ oscillations and rhythmic CHLOROPHYLL A/B BINDING PROTEIN2 promoter activity in Arabidopsis.

Plants have circadian oscillations in the concentration of cytosolic free calcium ([Ca(2+)](cyt)). To dissect the circadian Ca(2+)-signaling network, we monitored circadian [Ca(2+)](cyt) oscillations under various light/dark conditions (including different spectra) in Arabidopsis thaliana wild type and photoreceptor and circadian clock mutants. Both red and blue light regulate circadian oscillations of [Ca(2+)](cyt).

A new role for phytochromes in temperature-dependent germination.

Germination timing is a fundamental life-history trait, as seedling establishment predicates realized fitness in the wild. Light and temperature are two important cues by which seeds sense the proper season of germination. Using Arabidopsis thaliana, we provide evidence that phytochrome-mediated germination pathways simultaneously respond to light and temperature cues in ways that affect germination.

Heterodimerization of type II phytochromes in Arabidopsis.

Coimmunoprecipitation of members of the phytochrome red/farred photoreceptor family from plant extracts has been used to analyze their heteromeric binding interactions. Phytochrome (phy)B or phyD apoproteins with six myc epitopes fused to their N termini are biologically active when expressed in Arabidopsis. Immunoprecipitation of either of these tagged proteins from seedling extracts coprecipitates additional type II phytochromes: six myc (myc6)-phyB coprecipitates phyC-phyE; and myc6-phyD coprecipitates phyB and phyE.

Differential activities of the Arabidopsis phyB/D/E phytochromes in complementing phyB mutant phenotypes.

The Arabidopsis phyB, phyD, and phyE phytochromes regulate plant developmental and growth responses to continuous red light and to the ratio of red to far-red light. They are also more highly related in sequence to each other and more recently derived evolutionarily than phyA and phyC. In order to directly compare the signaling activities of these three photoreceptor apoproteins, an assay was developed based upon complementation of the phyB-1 null mutant phenotype with transgenes consisting of the PHYB promoter (PB) driving expression of the PHYB, PHYD, or PHYE coding sequences.

Signaling activities among the Arabidopsis phyB/D/E-type phytochromes: a major role for the central region of the apoprotein.

The Arabidopsis phyB, phyD, and phyE phytochromes regulate plant developmental and growth responses to continuous red light (R) and to the ratio of R to far-red (FR) light. The activities of these three photoreceptors in the control of seedling growth have been compared using a transgenic assay based upon induction of R-hypersensitivity of hypocotyl elongation by overexpression of the apoproteins from the 35S promoter. 35S-phyB, 35S-phyD, and 35S-phyE lines expressing similar levels of the respective phytochromes were isolated.

Patterns of expression and normalized levels of the five Arabidopsis phytochromes.

Using monoclonal antibodies specific for each apoprotein and full-length purified apoprotein standards, the levels of the five Arabidopsis phytochromes and their patterns of expression in seedlings and mature plants and under different light conditions have been characterized. Phytochrome levels are normalized to the DNA content of the various tissue extracts to approximate normalization to the number of cells in the tissue. One phytochrome, phytochrome A, is highly light labile.

The out of phase 1 mutant defines a role for PHYB in circadian phase control in Arabidopsis.

Arabidopsis displays circadian rhythms in stomatal aperture, stomatal conductance, and CO(2) assimilation, each of which peaks around the middle of the day. The rhythmic opening and closing of stomata confers a rhythm in sensitivity and resistance, respectively, to the toxic gas sulfur dioxide. Using this physiological assay as a basis for a mutant screen, we isolated mutants with defects in circadian timing.