G protein-coupled receptors (GPCRs) at the cell surface activate heterotrimeric G proteins by inducing the G protein alpha (Galpha) subunit to exchange guanosine diphosphate for guanosine triphosphate. Regulators of G protein signaling (RGS) proteins accelerate the deactivation of Galpha subunits to reduce GPCR signaling. Here we identified an RGS protein (AtRGS1) in Arabidopsis that has a predicted structure similar to a GPCR as well as an RGS box with GTPase accelerating activity. Expression of AtRGS1 complemented the pheromone supersensitivity phenotype of a yeast RGS mutant, sst2Delta. Loss of AtRGS1 increased the activity of the Arabidopsis Galpha subunit, resulting in increased cell elongation in hypocotyls in darkness and increased cell production in roots grown in light. These findings suggest that AtRGS1 is a critical modulator of plant cell proliferation.

The transition from the juvenile to adult phase in plants is controlled by diverse exogenous and endogenous cues such as age, day length, light, nutrients, and temperature. Previous studies have shown that the gradual decline in microRNA156 (miR156) with age promotes the expression of adult traits. However, how age temporally regulates the abundance of miR156 is poorly understood. We show here that the expression of miR156 responds to sugar. Sugar represses miR156 expression at both the transcriptional level and post-transcriptional level through the degradation of miR156 primary transcripts. Defoliation and photosynthetic mutant assays further demonstrate that sugar from the pre-existing leaves acts as a mobile signal to repress miR156, and subsequently triggers the juvenile-to-adult phase transition in young leaf primordia. We propose that the gradual increase in sugar after seed germination serves as an endogenous cue for developmental timing in plants.DOI: http://dx.doi.org/10.7554/eLife.00269.001

In Arabidopsis thaliana, the cryptochrome (CRY) blue light photoreceptors and the phytochrome (phy) red/far-red light photoreceptors mediate a variety of light responses. COP1, a RING motif-containing E3 ubiquitin ligase, acts as a key repressor of photomorphogenesis. Production of stomata, which mediate gas and water vapor exchange between plants and their environment, is regulated by light and involves phyB and COP1. Here, we show that, in the loss-of-function mutants of CRY and phyB, stomatal development is inhibited under blue and red light, respectively. In the loss-of-function mutant of phyA, stomata are barely developed under far-red light. Strikingly, in the loss-of-function mutant of either COP1 or YDA, a mitogen-activated protein kinase kinase kinase, mature stomata are developed constitutively and produced in clusters in both light and darkness. CRY, phyA, and phyB act additively to promote stomatal development. COP1 acts genetically downstream of CRY, phyA, and phyB and in parallel with the leucine-rich repeat receptor-like protein TOO MANY MOUTHS but upstream of YDA and the three basic helix-loop-helix proteins SPEECHLESS, MUTE, and FAMA, respectively. These findings suggest that light-controlled stomatal development is likely mediated through a crosstalk between the cryptochrome-phytochrome-COP1 signaling system and the mitogen-activated protein kinase signaling pathway.

Signal transduction typically begins by ligand-dependent activation of a concomitant partner which is otherwise in its resting state. However, in cases where signal activation is constitutive by default, the mechanism of regulation is unknown. The Arabidopsis thaliana heterotrimeric Gα protein self-activates without accessory proteins, and is kept in its resting state by the negative regulator, AtRGS1 (Regulator of G protein Signaling 1), which is the prototype of a seven transmembrane receptor fused with an RGS domain. Endocytosis of AtRGS1 by ligand-dependent endocytosis physically uncouples the GTPase accelerating activity of AtRGS1 from the Gα protein, permitting sustained activation. Phosphorylation of AtRGS1 by AtWNK8 kinase causes AtRGS1 endocytosis, required both for G protein-mediated sugar signaling and cell proliferation. In animals, receptor endocytosis results in signal desensitization, whereas in plants, endocytosis results in signal activation. These findings reveal how different organisms rearrange a regulatory system to result in opposite outcomes using similar phosphorylation-dependent endocytosis.

Anthocyanins are synthesized in the cytosolic surface of the endoplasmic reticulum (ER) but dominantly accumulate in the vacuole. Little is known about how anthocyanins are transported from the ER to the vacuole. Here, we provide evidence supporting that Transparent Testa 19 (TT19), a glutathione S-transferase (GST), functions as a carrier to transport cyanidin and/or anthocyanins to the tonoplast. We identified a novel tt19 mutant (tt19-7), which barely accumulates anthocyanins but produces a 36% higher level of flavonol than the wild-type (WT), from ethyl methanesulfonate mutagenized seeds. Expressing TT19-fused green fluorescence protein (GFP) in tt19-7 rescues the mutant phenotype in defective anthocyanin biosynthesis, indicating that TT19-GFP is functional. We further showed that TT19-GFP is localized not only in the cytoplasm and nuclei, but also on the tonoplast. The membrane localization of TT19-GFP was further ascertained by immunoblot analysis. In vitro assay showed that the purified recombinant TT19 increases water solubility of cyanidin (Cya) and cyanidin-3-O-glycoside (C3G). Compared with C3G, Cya can dramatically quench the intrinsic tryptophan fluorescence of TT19 to much lower levels, indicating a higher affinity of TT19 to Cya than to C3G. Isothermal titration calorimetry analysis also confirmed physical interaction between TT19 and C3G. Taken together, our data reveal molecular mechanism underlying TT19-mediated anthocyanin transportation.

Mutations in genes encoding components of the heterotrimeric G-protein complex were previously shown to confer altered sensitivity to increased levels of D-glucose. This suggests that G-protein coupling may be a novel sugar-signaling mechanism in Arabidopsis thaliana. THYLAKOID FORMATION1 (THF1) is here demonstrated in vivo as a Ga interaction partner that functions downstream of the plasma membrane-delimited heterotrimeric G-protein (GPA1) in a D-glucose signaling pathway. THF1 is a plastid protein localized to both the outer plastid membrane and the stroma. Contact between root plastidic THF1 and GPA1 at the plasma membrane occurs at sites where the plastid membrane abuts the plasma membrane, as demonstrated by Fö rster resonance energy transfer (FRET). A probable role for THF1 in sugar signaling is demonstrated by both biochemical and genetic evidence. Root growth in the thf1-1 null mutant is hypersensitive to exogenous D-glucose, and THF1-overexpressing roots are resistant to inhibition of growth rate by high D-glucose. Additionally, THF1 levels are rapidly degraded by D-glucose but not L-glucose. The interaction between THF1 and GPA1 has been confirmed by in vitro and in vivo coimmunoprecipitation, FRET analysis, and genetic epistasis and provides evidence of a sugar-signaling mechanism between plastids and the plasma membrane.

SummaryVirescence, a phenotype in which leaves green more slowly than usual, is recognized to play a role in protection from photo-oxidative damage before healthy chloroplasts are developed. The elucidation of the molecular mechanisms underlying virescence will provide insights into how the development of chloroplasts is controlled. In this study, we find that knockout alleles of Yellow Seedlings 1 (YS1) in Arabidopsis lead to a virescent phenotype, which disappears by 3 weeks after germination. The ys1 mutation resulted in marked decreases in photosynthetic capacity and photosynthetic pigment complexes, and disturbed ultrastructure of thylakoid membranes in 8-day-old seedlings. However, cotyledons of ys1 seedlings pre-treated in the dark for 5 days turn green almost as fast as the wild type in light, revealing that the developmental defects in ys1 are limited to the first few days after germination. Inspection of all known plastid RNA editing and splicing events revealed that YS1 is absolutely required for editing of site 25992 in rpoB transcripts encoding the beta subunit of the plastid-encoded RNA polymerase (PEP). YS1 is a nuclear-encoded chloroplast-localized pentatricopeptide repeat protein differing from previously described editing factors in that it has a C-terminal DYW motif. A defect in PEP activity is consistent with the changes in plastid transcript patterns observed in ys1 seedlings. We conclude that the activity of PEP containing RpoB translated from unedited transcripts is insufficient to support rapid chloroplast differentiation.

Development of thylakoid membranes depends upon the transport of membrane vesicles from the chloroplast inner envelope and subsequent fusion of vesicles within the interior of the plastid. The Arabidopsis (Arabidopsis thaliana) Thylakoid formation1 (Thf1) gene product is shown here to control an important step required for the normal organization of these vesicles into mature thylakoid stacks and ultimately for leaf development. The Arabidopsis Thf1 gene encodes an imported chloroplast protein, as shown by in vitro import and localization of a Thf1-green fluorescent protein fusion product in transgenic plants. This gene is conserved in oxygenic photoautotrophs ranging from cyanobacteria to flowering land plants. Transcript levels for Thf1 are induced in the light and decrease under dark conditions, paralleling profiles of light-regulated nuclear genes involved in chloroplast function. Disruption of the Thf1 gene via T-DNA insertion results in plants that are severely stunted with variegated leaf patterns. Nongreen sectors of variegated leaves lacking Thf1 expression contain plastids that accumulate membrane vesicles on the interior and lack organized thylakoid structures. Green sectors of Thf1-disrupted leaves contain some chloroplasts that form organized thylakoid membranes, indicating that an inefficient compensatory mechanism supports thylakoid formation in the absence of Thf1. Genetic complementation of a Thf1 knockout line confirms the role of this gene in chloroplast and leaf development. Transgenic plants expressing the Thf1 gene in antisense orientation are stunted with altered thylakoid organization, especially in young seedlings. The data indicate that the Thf1 gene product plays a crucial role in a dynamic process of vesicle-mediated thylakoid membrane biogenesis.The development of normal chloroplasts is a crucial step in the survival of photosynthetic eukaryotes. Maturation and activity of the chloroplast is highly dependent on its relationship with the nucleus, as the bulk of proteins that function in the chloroplast are encoded in the nucleus and are posttranslationally imported from the cytoplasm (Keegstra and Cline, 1999). Lesions in genes encoding plastid-localized proteins can have severe effects on the many metabolic processes that occur there and ultimately can prevent normal plant growth and development. Demonstrating the overriding importance of chloroplast proteins, the majority of mutations in a screen of mutagenized Arabidopsis (Arabidopsis thaliana), designed to identify genes important for seedling viability, appeared to affect chloroplast formation or function (Budziszewski et al., 2001). Environmental conditions, especially light, also play important roles in triggering chloroplast development and orchestrating gene expression required for plastid function (Anderson, 1986). In spite of the dependence of plants on the photosynthesis occurring on chloroplast thylakoid membranes, there is much to be learned regarding the mechanisms by which these internal membranes originate and how th...