MicroProtein-mediated recruitment of CONSTANS into a TOPLESS trimeric complex represses flowering in Arabidopsis

MicroProteins are short, single domain proteins that act by sequestering larger, multi-domain proteins into non-functional complexes. MicroProteins have been identified in plants and animals, where they are mostly involved in the regulation of developmental processes. Here we show that two Arabidopsis thaliana microProteins, miP1a and miP1b, physically interact with CONSTANS (CO) a potent regulator of flowering time. The miP1a/b-type microProteins evolved in dicotyledonous plants and have an additional carboxy-terminal PF(V/L)FL motif. This motif enables miP1a/b microProteins to interact with TOPLESS/TOPLESS-RELATED (TPL/TPR) proteins. Interaction of CO with miP1a/b/TPL causes late flowering due to a failure in the induction of FLOWERING LOCUS T (FT) expression under inductive long day conditions. Both miP1a and miP1b are expressed in vascular tissue, where CO and FT are active. Genetically, miP1a/b act upstream of CO thus our findings unravel a novel layer of flowering time regulation via microProtein-inhibition

Early Signaling in plant immunity /

During pathogen infection, plants recognize microbial molecules known as pathogen associated molecular patterns (PAMPS) by surface localized pattern recognition receptors (PRRs), these results in physiological changes that limit pathogen growth in a process known as PAMP-triggered immunity (PTI). Recognition of PAMPs triggers the production of the phytohormone salicylic acid (SA), which is important for defense against biotrophic pathogens and requires redox regulated NONEXPRESSER OF PR GENES1 (NPR1), a master regulator of SA- mediated defense. Although there is significant research on the transcriptional changes related to defense signaling, there is limited information on the early defense related protein changes that occurs before major transcriptional changes. Here we present the mass spectrometry result of early changes in protein abundance, and phosphorylation of Arabidopsis thaliana plants treated with defense elicitor BTH. We observed that 48 proteins changed in abundance, and 43 in phosphorylation following BTH treatment. We also analyzed the changes in abundance in NPR1 mutant (npr1-1) and observed that 43 proteins changed in abundance. We characterized the roles of 9 of the observed proteins in defense against three pathogens Hyaloperonospora arabidopsidis (Hpa), Pseudomonas syringae pv. (Pto) DC3000, and Botrytis cinerea. Our results reveal the novel role of 2 proteins in defense against PtoDC3000, 5 proteins in defense against Hpa and 3 proteins in defense against botrytis. We further analyzed the BTH induced proteome changes observed in our mass spectrometry results of the total proteome, phosphoproteome and npr1-1 proteome with STRING, an online database of known and predicted protein interactions. The predicted networks created from STRING were used in combination with our infection bioassay results to predict the roles of our proteins in known and novel defense networks. Our study emphasizes the strength of mass spectrometry as a tool to discover proteins with observable phenotypes and to predict novel protein network

The microProteins miP1a/b act by engaging CO in a TOPLESS/TOPLESS-like co-repressor complex.

<p>(<b><i>A</i></b>) Representative image series of co-localization studies of GFP-CO and RFP-TPL co transformed with either miP1a (n = 15), the B-Box-dead version miP1a* (n = 16) or miP1aΔPFVFL (n = 9) that is lacking the TPL-interaction motif. (<b><i>B</i></b>) Yeast-three-hybrid demonstrating the formation of a CO-TPL-miP1a trimeric complex. Growth of serial dilutions on non-selective SD-medium lacking leucine, tryptophan and uracil (-L/-W/-U) show normal yeast growth. Only positive interactions were able to grow on restrictive growth medium supplemented with 10mM 3-Aminotriazole (3-AT) and lacking histidine. (<b><i>C</i></b>) <i>In vitro</i> pull-down experiments. Recombinant MBP-CO, GST-miP1a, GST-miP1aΔPFVFL, GST-ZPR3 and HIS-TPL proteins were produced in <i>E</i>. <i>coli</i>. After cell lysis, cell extracts of MBP-CO and HIS-TPL were mixed with GST-miP1a, GST-miP1aΔPFVFL or GST-ZPR3 and incubated with magnetic anti-GST coupled magnetic beads (Promega). GST-miP1a, GST-miP1aΔPFVFL and GST-ZPR3 complexes were precipitated and washed using a magnetic stand, eluted by boiling in SDS-loading buffer and separated by SDS-PAGE. HIS-TPL and MBP-CO Proteins were detected by immunoblotting. (<b><i>D</i></b>) <i>In vitro</i> pull-down experiment of the trimeric TPL-miP1a-CO complex. MBP-CO and HIS-TPL were mixed with either miP1a or miP1a* proteins. After immunoprecipitation of MBP-CO with an amylose resin proteins were detected by immunoblotting.</p

Diurnal expression profiles of <i>CO</i>, <i>FT</i>, <i>miP1a</i> and <i>miP1b</i>.

<p>Quantitative RT-PCR analysis of <i>CO</i> and <i>FT</i> (<b><i>A-F</i></b>), <i>miP1a</i> (<b><i>G</i>,<i>H</i></b>) and <i>miP1b</i> (<b>I,J</b>). Plants were grown in 16-hour long days (<b><i>A</i>, <i>C</i>, <i>E</i>, <i>G</i>, <i>H</i></b>) or 8-hour short day conditions (<b><i>B</i>, <i>D</i>, <i>F</i>, <i>H</i>, <i>J</i></b>). Samples were harvested every 3 h over a time period of 24 h. Expression levels are relative to <i>GAPDH</i> and the error bars represent the standard deviation of four technical replicates. (<b><i>A</i>,<i>B</i></b>) <i>CO</i> and <i>FT</i> expression in Col-0 wild type plants. (<b><i>C</i>,<i>D</i></b>) <i>CO</i> and <i>FT</i> expression in transgenic <i>35S</i>::<i>FLAG-miP1a</i> plants. (<b><i>E</i>,<i>F</i></b>) <i>CO</i> and <i>FT</i> expression in transgenic <i>35S</i>::<i>FLAG-miP1b</i> plants. (<b><i>G</i>,<i>H</i></b>) Expression profile of <i>miP1a</i> in LD and SD. (<b><i>I</i>,<i>J</i></b>) Expression profile of <i>miP1b</i> in LD and SD.</p

Model depicting the role of microProteins in flowering time regulation.

<p>The circadian clock is entrained by day/night cycles. In response to long days, <i>CO</i> is activated by GI/FKF1. Increasing levels of CO cause induction of <i>FT</i>, which triggers the transition from vegetative to reproductive growth. MiP1a/b act by controlling CO activity. If miP1a/b levels are ectopically high, CO activity is low and flowering is delayed.</p

Multi-level analysis of the interactions between <i>REVOLUTA</i> and <i>MORE AXILLARY BRANCHES 2</i> in controlling plant development reveals parallel, independent and antagonistic functions

Class III homeodomain leucine zipper (HD-ZIPIII) transcription factors play fundamental roles in controlling plant development. The known HD-ZIPIII target genes encode proteins involved in the production and dissipation of the auxin signal, HD-ZIPII transcription factors and components that feedback to regulate HD-ZIPIII expression or protein activity. Here, we have investigated the regulatory hierarchies of the control of MORE AXILLARY BRANCHES2 (MAX2) by the HD-ZIPIII protein REVOLUTA (REV). We found that REV can interact with the promoter of MAX2. In agreement, rev10D gain-of-function mutants had increased levels of MAX2 expression, while rev loss-of-function mutants showed lower levels of MAX2 in some tissues. Like REV, MAX2 plays known roles in the control of plant architecture, photobiology and senescence, which prompted us to initiate a multi-level analysis of growth phenotypes of hd-zipIII, max2 and respective higher order mutants thereof. Our data suggest a complex relationship of synergistic and antagonistic activities between REV and MAX2; these interactions appear to depend on the developmental context and do not all involve the direct regulation of MAX2 by REV