Evolutionarily conserved switch-defective/sucrose nonfermentable (SWI/SNF) ATP-dependent chromatin remodelling complexes (CRCs) alter nucleosome positioning and chromatin states, affecting gene expression to regulate important processes such as proper development and hormonal signalling pathways. We employed transcript profiling, chromatin immunoprecipitation (ChIP), mass spectrometry, yeast two-hybrid and bimolecular fluorescence complementation protein-protein interaction studies, along with hormone and metabolite profiling and phenotype assessments, to distinguish the SWP73A and SWP73B subunit functions in Arabidopsis. We identified a novel subclass of SWI/SNF CRCs defined by the presence of the SWP73A subunit. Therefore, we propose a refined classification of SWI/SNF CRCs in Arabidopsis, introducing BRM-associated SWI/SNF (BAS)-A (containing SWP73A) and BAS-B (containing SWP73B) subclasses. The SWP73A- and SWP73B-carrying SWI/SNF CRCs exhibit differential properties, demonstrated by distinct chromatin binding patterns and divergent effects on hormone biosynthesis and metabolism. We additionally found that SWP73A plays a specific role in the regulation of auxin signalling, root development, metabolism and germination that cannot be fully compensated by SWP73B. We recognised that some atypical subclasses of SWI/SNF CRCs may be likely formed in mutant lines with inactivated SWP73 subunits. Our study reveals that the duplication of the SWP73 subunit genes contributes to unique and shared functions of SWI/SNF CRC subclasses in the regulation of various processes in Arabidopsis.

Recombineering approaches exploiting the bacteriophage λ Red recombination functions are widely used for versatile modification of eukaryotic genes carried by bacterial artificial chromosomes (BACs) in E. coli. Whereas BAC transformation provides a simple means for integration of modified genes into the genomes of animal cells to generate knock-in and knockout lines, successful application of this strategy is hampered by low frequency of homologous recombination in higher plants. However, plant cells can be transformed at a high frequency using the transferred DNA (T-DNA) of Agrobacterium, which is stably and randomly integrated into the plant genome. The function of plant genes that are modified by recombineering and transferred by Agrobacterium T-DNA vectors into plant cells can thus be suitably studied using genetic complementation of knockout mutations induced by either T-DNA insertions or genome editing with T-DNA-based Crisp/Cas9 constructs. Here we describe two recombineering protocols for modification and transfer of plant genes from BACs into Agrobacterium T-DNA plant transformation vectors. The first protocol uses a conditional suicide ccdB gene cassette to assist the genetic complementation assays by generation of point mutations, deletions, and insertions at any gene position. The second “turbo”-recombineering protocol exploits various I-SceI insertion cassettes for fusing of fluorescent protein tags to the plant gene products to facilitate the characterization of their in vivo interacting partners by affinity purification, mass spectrometry, and cellular localization studies.

The C-type hybrid-proline-rich protein (HyPRP) AtCWLP and its homolog AtPRP940 are referred as cell wall (CW)-plasma-membrane (PM) linker proteins, but little is known about their functions. Here we show that N-terminal proline-rich domains of CWLP and PRP940, containing glycosylated hydroxyproline residues, contact the CW, while their C-terminal 8CM domains function as PM-scaffolds. Both proteins are detected in PM nanodomains (PM-ND) and show co-localization and co-immunoprecipitation with aquaporins PIP2;1 and PIP2;7. Inhibition of actin polymerization by latrunculin B promotes CWLP-endosome appearance, while blocking the actomyosin-based transport by a truncated form of myosin XI-K relaxes lateral boundaries of CWLP-PIP2;1 PD-NDs. Mass spectrometry data indicate that CWLP co-purifies with dynamins implicated in fission of endocytic PD-ND invaginations. Lack of co-localization and co-immunoprecipitation with aquaporin-binding flotillin (FLOT2) indicates that CWLP and PRP940 mark a new distinct type of PM-ND. Yeast two-hybrid and co-immunoprecipitation assays demonstrate that CWLP and PRP940 interact with multiple aquaporins and several protein phosphatase PP2A-B’’ regulatory subunits. By preventing irreversible separation of CW and PM, and likely assisting PP2A-mediated dephosphorylation of aquaporins and closure of their water channels, overexpression of CWLP confers tolerance to plasmolysis, dehydration and freezing in Arabidopsis and to water shortage in potato plants. Summary Statement Arabidopsis Hybrid-Proline-Rich Proteins CWLP and PRP940 occur in association with dynamins, recruit PP2A protein phosphatases to aquaporin water channels in plasma-membrane (PM) nanodomains and elevate tolerance to cellular dehydration.

To accelerate the isolation of plant protein complexes and study cellular localization and interaction of their components, an improved recombineering protocol is described for simple and fast site-directed modification of plant genes in bacterial artificial chromosomes (BACs). Coding sequences of fluorescent and affinity tags were inserted into genes and transferred together with flanking genomic sequences of desired size by recombination into Agrobacterium plant transformation vectors using three steps of E. coli transformation with PCR-amplified DNA fragments. Application of fast-track recombineering is illustrated by the simultaneous labelling of CYCLIN-DEPENDENT KINASE D (CDKD) and CYCLIN H (CYCH) subunits of kinase module of TFIIH general transcription factor and the CDKD-activating CDKF;1 kinase with green fluorescent protein (GFP) and mCherry (green and red fluorescent protein) tags, and a PIPL (His(18)-StrepII-HA) epitope. Functionality of modified CDKF;1 gene constructs is verified by complementation of corresponding T-DNA insertion mutation. Interaction of CYCH with all three known CDKD homologues is confirmed by their co-localization and co-immunoprecipitation. Affinity purification and mass spectrometry analyses of CDKD;2, CYCH, and DNA-replication-coupled HISTONE H3.1 validate their association with conserved TFIIH subunits and components of CHROMATIN ASSEMBLY FACTOR 1, respectively. The results document that simple modification of plant gene products with suitable tags by fast-track recombineering is well suited to promote a wide range of protein interaction and proteomics studies.

The history of life consists of a series of major evolutionary transitions, including emergence and radiation of complex multicellular eukaryotes from unicellular ancestors. The cells of multicellular organisms, with few exceptions, contain the same genome, however, their organs are composed of a variety of cell types that differ in both structure and function. This variation is largely due to the transcriptional activity of different sets of genes in different cell types. This indicates that complex transcriptional regulation played a key role in the evolution of complexity in eukaryotes. In this review, we summarize how gene duplication and subsequent evolutionary innovations, including the structural evolution of nucleosomes and chromatin-related factors, contributed to the complexity of the transcriptional system and provided a basis for morphological diversity.

The evolutionary conserved family of Selenoproteins performs redox-regulatory functions in bacteria, archaea and eukaryotes. Among them, members of the SELENOPROTEIN O (SELO) subfamily are located in mammalian and yeast mitochondria, but their functions are thus far enigmatic. Screening of T-DNA knockout mutants for resistance to the proline analogue thioproline (T4C), identified mutant alleles of the plant SELO homologue in Arabidopsis thaliana. Absence of SELO resulted in a stress-induced transcriptional activation instead of silencing of mitochondrial proline dehydrogenase, and also high elevation of Δ(1)-pyrroline-5-carboxylate dehydrogenase involved in degradation of proline, thereby alleviating T4C inhibition and lessening drought-induced proline accumulation. Unlike its animal homologues, SELO was localized to chloroplasts of plants ectopically expressing SELO-GFP. The protein was co-fractionated with thylakoid membrane complexes, and co-immunoprecipitated with FNR, PGRL1 and STN7, all involved in regulating PSI and downstream electron flow. The selo mutants displayed extended survival under dehydration, accompanied by longer photosynthetic activity, compared with wild-type plants. Enhanced expression of genes encoding ROS scavenging enzymes in the unstressed selo mutant correlated with higher oxidant scavenging capacity and reduced methyl viologen damage. The study elucidates SELO as a PSI-related component involved in regulating ROS levels and stress responses.

Plants generate rhythmic metabolism during the repetitive day/night cycle. The circadian clock produces internal biological rhythms to synchronize numerous metabolic processes such that they occur at the required time of day. Metabolism conversely influences clock function by controlling circadian period and phase and the expression of core-clock genes. Here, we show that AKIN10, a catalytic subunit of the evolutionarily conserved key energy sensor sucrose non-fermenting 1 (Snf1)-related kinase 1 (SnRK1) complex, plays an important role in the circadian clock. Elevated AKIN10 expression led to delayed peak expression of the circadian clock evening-element GIGANTEA (GI) under diurnal conditions. Moreover, it lengthened clock period specifically under light conditions. Genetic analysis showed that the clock regulator TIME FOR COFFEE (TIC) is required for this effect of AKIN10. Taken together, we propose that AKIN10 conditionally works in a circadian clock input pathway to the circadian oscillator.

Plants modify organ growth and tune morphogenesis in response to various endogenous and environmental cues. At the cellular level, organ growth is often adjusted by alterations in cell growth, but the molecular mechanisms underlying this control remain poorly understood. In this study, we identify the DNA BINDING WITH ONE FINGER (DOF)-type transcription regulator OBF BINDING PROTEIN4 (OBP4) as a repressor of cell growth. Ectopic expression of OBP4 in Arabidopsis (Arabidopsis thaliana) inhibits cell growth, resulting in severe dwarfism and the repression of genes involved in the regulation of water transport, root hair development, and stress responses. Among the basic helix-loop-helix transcription factors known to control root hair growth, OBP4 binds the ROOT HAIR DEFECTIVE6-LIKE2 (RSL2) promoter to repress its expression. The accumulation of OBP4 proteins is detected in expanding root epidermal cells, and its expression level is increased by the application of abscisic acid (ABA) at concentrations sufficient to inhibit root hair growth. ABA-dependent induction of OBP4 is associated with the reduced expression of RSL2 Furthermore, ectopic expression of OBP4 or loss of RSL2 function results in ABA-insensitive root hair growth. Taken together, our results suggest that OBP4-mediated transcriptional repression of RSL2 contributes to the ABA-dependent inhibition of root hair growth in Arabidopsis.

Many bacterial pathogens deploy specific proteins, referred to as effectors, to manipulate host defense responses. Effector repertoires are shaped to target host immune signaling pathways at multiple levels. The bacteria use host cells as a source of eukaryotic cofactors that are indispensable for effector enzymatic activity or acquisition of their final conformation. They also co-opt host cellular machinery to activate pathogenic effectors via post-translational modifications and translocation to their site of action. A number of effectors possess organelle-targeting signals that control their subcellular distribution. Pseudomonas syringae pv. phaseolicola is the causative agent of halo blight in common bean. Similar to other pathogenic Gram-negative bacteria, it delivers a set of type III effectors into host cells. HopQ1 is one of these effectors. We showed previously that HopQ1 specifically interacted with plant 14-3-3s, and this association affected its subcellular localization [1]. Our current studies demonstrate that the position of HopQ1 is regulated by several other host factors involved in the plant defense response. Intracellular calcium concentration and redox status, which change in response to pathogen attack, may control HopQ1 monomer/dimer equilibrium and thereby control HopQ1 nucleocytoplasmic partitioning. Plant kinases activated in response to pathogen infection change the HopQ1 phosphorylation status, which in turn determines its localization. The resulting cellular distribution patterns of HopQ1 change during the course of microbial infection. These results suggest that bacteria hijack host defense signaling components to dynamically tune the intracellular localization of their virulence factors. We hypothesize that this mechanism would enable effectors to perform systematic searches for their corresponding virulence targets.

Increased nutrient uptake is a major hallmark of cancer and correlates with a poor survival rate. Many of the oncogenic events that drive cancer development influence metabolism by increasing glucose and glutamine uptake while other genetic events and processes also influence metabolism without increased nutrient uptake. Together these events reprogram metabolism away from catabolic metabolism and promote the anabolic pathways necessary for cell growth and proliferation. Glycolysis/gluconeogenesis is the key metabolic pathway that provides intermediates for cell growth and proliferation. We have targeted the central glycolytic enzyme aldolase which reversibly cleaves fructose-bisphosphate into triose-Ps by screening a 135 000 compound library for inhibitors of aldolase activity. One compound, UM0112176, allosterically inhibits aldolase activity at micromolar levels (IC50 ≈ 5 μM) and is cytotoxic to cancer cells (EC50 ≈ 5 μM) grown in glucose only, in glutamine only, and in complete cell culture media. The compound has good selectivity when tested against normal tissue culture cells exhibiting a 10–20-fold therapeutic window. Intracellular targeting of aldolase by UM0112176 is consistent with aldolase substrate/product accumulation depending on the direction of the glycolytic flux. The mechanism of action by UM0112176 is surprising as the compound not only targets the important metabolic role of aldolase but also impacts its moonlighting activities in F-actin polymerization, and novel activities related to ROS production and DNA repair. The elucidation of the essential role played by aldolase in metabolism and associated moonlighting activities, as revealed through the mechanism of action by UM0112176, will be the topic of the conference. L6.2

Virulence proteins VirDl and VirD2 are sub- units of a relaxosome-like protein complex that mediates con- jugational transfer of a Ti plasmid segment, the T-DNA, from Agrobacterium into higher plants. The VirDl-VirD2 complex binds to 25-bp repeats at the borders of the T-DNA and catalyzes sequence-specific nicking of the conjugative DNA strand (the T-strand) at the third base of these repeats. Nuclear localization signals present in VirD2 target the T-strand to plant cell nuclei. In addition, VirD2 probably plays a role in the high-frequency integration of the T-DNA into the plant genome by illegitimate recombination. Whereas Agrobacterium trans- formation of dicots is very efficient, T-DNA integration in most monocots can barely be detected. To develop an artificial T-DNA delivery system for monocots, a technique for efficient in vitro production of T-strand DNAs was established by using VirDl and VirD2 proteins purified from overexpressing Esch- erichia coli strains. The topoisomerase-like VirD2 enzyme was shown to mediate precise, sequence-specific cleavage of T-DNA border sequences carried by single-stranded DNA templates, even in the absence of VirDl protein. During this reaction, VirD2 remains covalently bound to the 5' end of artificial T-strand DNAs. In contrast, VirD2, alone or in complex with VirDl, fails to nick linear double-stranded DNA templates in vitro.

and hormone signaling pathways by indicating intriguing similaritiesand differencesinplantsandhumans,andsummarizeproposedmechanismsofSWI/SNF action on target loci. We postulate that, given their viability, severalplant SWI/SNF mutants may serve as an attractive model for searching forconserved functions of SWI/SNF CRCs in hormone signaling, cell cycle control,and

SWI/SNF-type ATP-dependent chromatin remodeling complexes (CRCs) are evolutionarily conserved multiprotein machineries controlling DNA accessibility by regulating chromatin structure. We summarize here recent advances highlighting the role of SWI/SNF in the regulation of hormone signaling pathways and their crosstalk in Arabidopsis thaliana. We discuss the functional interdependences of SWI/SNF complexes and key elements regulating developmental and hormone signaling pathways by indicating intriguing similarities and differences in plants and humans, and summarize proposed mechanisms of SWI/SNF action on target loci. We postulate that, given their viability, several plant SWI/SNF mutants may serve as an attractive model for searching for conserved functions of SWI/SNF CRCs in hormone signaling, cell cycle control, and other regulatory pathways.

Upon DNA damage, cyclin‐dependent kinases (CDKs) are typically inhibited to block cell division. In many organisms, however, it has been found that CDK activity is required for DNA repair, especially for homology‐dependent repair (HR), resulting in the conundrum how mitotic arrest and repair can be reconciled. Here, we show that Arabidopsis thaliana solves this dilemma by a division of labor strategy. We identify the plant‐specific B1‐type CDKs (CDKB1s) and the class of B1‐type cyclins (CYCB1s) as major regulators of HR in plants. We find that RADIATION SENSITIVE 51 (RAD51), a core mediator of HR, is a substrate of CDKB1‐CYCB1 complexes. Conversely, mutants in CDKB1 and CYCB1 fail to recruit RAD51 to damaged DNA. CYCB1;1 is specifically activated after DNA damage and we show that this activation is directly controlled by SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1), a transcription factor that acts similarly to p53 in animals. Thus, while the major mitotic cell‐cycle activity is blocked after DNA damage, CDKB1‐CYCB1 complexes are specifically activated to mediate HR.

The Arabidopsis CONSTITUTIVELY PHOTOMORPHOGENIC1/SUPPRESSOR OF PHYA-105 (COP1/SPA) complex is a key repressor of light signaling that inhibits light responses in darkness. It acts as an E3 ubiquitin ligase, which ubiquitinates positively acting light-signaling intermediates, mainly transcription factors, thereby targeting them for proteolytic degradation by the 26S proteasome. In the light, photoreceptors directly interact with the COP1/SPA complex, leading to its inactivation, which subsequently allows the target transcription factors to accumulate and initiate vast reprogramming of gene expression (Huang et al., 2014Huang X. Ouyang X. Deng X.W. Beyond repression of photomorphogenesis: role switching of COP/DET/FUS in light signaling.Curr. Opin. Plant Biol. 2014; 21C: 96-103Crossref Scopus (129) Google Scholar). Genetic and biochemical studies indicate that COP1 and SPA proteins act in concert to repress photomorphogenesis, i.e. as members of the COP1/SPA complex(es) (Laubinger et al., 2004Laubinger S. Fittinghoff K. Hoecker U. The SPA quartet: a family of WD-repeat proteins with a central role in suppression of photomorphogenesis in Arabidopsis.Plant Cell. 2004; 16: 2293-2306Crossref PubMed Scopus (177) Google Scholar, Yang and Wang, 2006Yang J. Wang H. The central coiled-coil domain and carboxyl-terminal WD-repeat domain of Arabidopsis SPA1 are responsible for mediating repression of light signaling.Plant J. 2006; 47: 564-576Crossref PubMed Scopus (43) Google Scholar, Zhu et al., 2008Zhu D. Maier A. Lee J.H. Laubinger S. Saijo Y. Wang H. Qu L.J. Hoecker U. Deng X.W. Biochemical characterization of Arabidopsis complexes containing CONSTITUTIVELY PHOTOMORPHOGENIC1 and SUPPRESSOR OF PHYA proteins in light control of plant development.Plant Cell. 2008; 20: 2307-2323Crossref PubMed Scopus (168) Google Scholar). However, a spa cop1 null mutant lacking the whole COP1/SPA complex has not been described so far. Moreover, the phenotypes of cop1 null mutants and spa quadruple mutants with mutations in all four SPA genes (SPA1-SPA4) are not identical, although this would be expected for a required co-action of COP1 and SPA proteins. cop1 null mutants arrest growth at the seedling stage, whereas a spa quadruple mutant proceeds through development and produces seed, despite being very dwarfed (McNellis et al., 1994McNellis T.W. Von Arnim A.G. Araki T. Komeda Y. Miséra S. Deng X.-W. Genetic and molecular analysis of an allelic series of cop1 mutants suggests functional roles for the multiple protein domains.Plant Cell. 1994; 6: 487-500Crossref PubMed Scopus (304) Google Scholar, Laubinger et al., 2004Laubinger S. Fittinghoff K. Hoecker U. The SPA quartet: a family of WD-repeat proteins with a central role in suppression of photomorphogenesis in Arabidopsis.Plant Cell. 2004; 16: 2293-2306Crossref PubMed Scopus (177) Google Scholar). However, the interpretation of SPA function in these spa mutants was hindered by the lack of null alleles. The spa quadruple mutant analyzed so far is not null for SPA2 since the spa2-1 allele produces and accumulates a truncated SPA2 protein lacking the C-terminal ∼100 amino acids (Laubinger et al., 2004Laubinger S. Fittinghoff K. Hoecker U. The SPA quartet: a family of WD-repeat proteins with a central role in suppression of photomorphogenesis in Arabidopsis.Plant Cell. 2004; 16: 2293-2306Crossref PubMed Scopus (177) Google Scholar, Zhu et al., 2008Zhu D. Maier A. Lee J.H. Laubinger S. Saijo Y. Wang H. Qu L.J. Hoecker U. Deng X.W. Biochemical characterization of Arabidopsis complexes containing CONSTITUTIVELY PHOTOMORPHOGENIC1 and SUPPRESSOR OF PHYA proteins in light control of plant development.Plant Cell. 2008; 20: 2307-2323Crossref PubMed Scopus (168) Google Scholar). Also, spa1-7 and spa4-1 carry T-DNA insertions at the proximity of the 3′ end of the respective coding sequence, so that there is a possibility that truncated SPA1 and SPA4 proteins are produced. Hence, it cannot be excluded that the viability of this spa quadruple mutant is due to residual production of partially functional SPA proteins. Here, we have isolated spa null mutant alleles and generated a spa quadruple null mutant and two different types of cop1 spa quintuple mutants to address the following questions with respect to the degree of COP1/SPA co-action. (1) Are Arabidopsis plants which fail to produce any SPA proteins viable, i.e. does COP1 indeed have residual activity in the absence of SPAs? (2) Are COP1 and SPAs necessary for embryogenesis, i.e. do SPA proteins have residual activity in the absence of COP1 during embryogenesis? (3) Is the C-terminal WD-repeat domain truly essential for COP1/SPA function and can the SPA WD-repeat domains partially replace the functions of the WD-repeat domain of COP1? By screening the MPIPZ T-DNA insertion collection, we identified genuine null alleles in SPA2 and SPA4 (spa2-2, spa4-3; Supplemental Figure 1). We crossed the new null alleles with the previously identified spa1-100 and spa3-1 null alleles to generate higher-order spa mutants. The null spa2-2 and spa4-3 single mutants and derived double and triple spa null mutants exhibited seedling and adult phenotypes that were indistinguishable from those of the previously characterized multiple mutant allele combinations (Supplemental Figures 2 and 3). The null spa quadruple mutant lacking all four SPA proteins, hereafter referred to as spaQn, undergoes constitutive photomorphogenesis, and is viable, fertile, and able to complete its life cycle (Figure 1A and 1B), as was reported previously for the spaQ mutant which is not null for all four SPAs (Laubinger et al., 2004Laubinger S. Fittinghoff K. Hoecker U. The SPA quartet: a family of WD-repeat proteins with a central role in suppression of photomorphogenesis in Arabidopsis.Plant Cell. 2004; 16: 2293-2306Crossref PubMed Scopus (177) Google Scholar). This result confirms that plants lacking all SPA proteins are indeed viable, which is in contrast to the seedling growth arrest observed in cop1 null mutants (McNellis et al., 1994McNellis T.W. Von Arnim A.G. Araki T. Komeda Y. Miséra S. Deng X.-W. Genetic and molecular analysis of an allelic series of cop1 mutants suggests functional roles for the multiple protein domains.Plant Cell. 1994; 6: 487-500Crossref PubMed Scopus (304) Google Scholar). Hence, we can now unambiguously conclude that COP1 alone, i.e. in the absence of SPA proteins, has residual activity that allows the plant to complete its life cycle. COP1 activity is nevertheless strongly enhanced by SPA proteins. spaQn mutants differed from spaQ mutants in that seedlings and plants appeared darker, suggesting higher anthocyanin content in spaQn than in spaQ plants (Figure 1A and 1B). Indeed, spaQn seedlings accumulated higher levels of anthocyanin than spaQ seedlings (Supplemental Figure 4A). Hence, the spaQ mutant has residual SPA activity, possibly due to the truncated SPA2-1 protein it produces. Besides controlling seedling deetiolation and leaf expansion, the COP1/SPA complex is required to suppress flowering under non-inductive short-day conditions (McNellis et al., 1994McNellis T.W. Von Arnim A.G. Araki T. Komeda Y. Miséra S. Deng X.-W. Genetic and molecular analysis of an allelic series of cop1 mutants suggests functional roles for the multiple protein domains.Plant Cell. 1994; 6: 487-500Crossref PubMed Scopus (304) Google Scholar, Laubinger et al., 2006Laubinger S. Marchal V. Gentilhomme J. Wenkel S. Adrian J. Jang S. Kulajta C. Braun H. Coupland G. Hoecker U. Arabidopsis SPA proteins regulate photoperiodic flowering and interact with the floral inducer CONSTANS to regulate its stability.Development. 2006; 133: 3213-3222Crossref PubMed Scopus (239) Google Scholar). Previous results indicated overlapping but also distinct functions of the four SPA genes in seedling growth and leaf expansion (Laubinger et al., 2004Laubinger S. Fittinghoff K. Hoecker U. The SPA quartet: a family of WD-repeat proteins with a central role in suppression of photomorphogenesis in Arabidopsis.Plant Cell. 2004; 16: 2293-2306Crossref PubMed Scopus (177) Google Scholar, Balcerowicz et al., 2011Balcerowicz M. Fittinghoff K. Wirthmueller L. Maier A. Fackendahl P. Fiene G. Koncz C. Hoecker U. Light exposure of Arabidopsis seedlings causes rapid de-stabilization as well as selective post-translational inactivation of the repressor of photomorphogenesis SPA2.Plant J. 2011; 65: 712-723Crossref PubMed Scopus (44) Google Scholar). The regulation of flowering time, however, has not yet been analyzed in this regard. Figure 1C shows that SPA1 and SPA4 are sufficient to strongly repress flowering in short days. Hence, SPA1 and SPA4 are the primary SPA genes responsible for photoperiodic flowering, while SPA2 and SPA3 provide only minor contributions in regulating the transition from vegetative to reproductive growth. spaQn mutants flowered very early in short days and long days and were, thus, fully insensitive to day length (Figure 1D). COP1 function is thought to be specific to light signal transduction. On the other hand, cop1 null mutants arrest growth at the seedling stage (McNellis et al., 1994McNellis T.W. Von Arnim A.G. Araki T. Komeda Y. Miséra S. Deng X.-W. Genetic and molecular analysis of an allelic series of cop1 mutants suggests functional roles for the multiple protein domains.Plant Cell. 1994; 6: 487-500Crossref PubMed Scopus (304) Google Scholar), suggesting fundamental defects that may not solely be related to light signaling. Consistent with this idea, cop1 mutants exhibit increased DNA damage, although this DNA damage can apparently be repaired prior to cell division (Dohmann et al., 2008Dohmann E.M. Levesque M.P. De Veylder L. Reichardt I. Jurgens G. Schmid M. Schwechheimer C. The Arabidopsis COP9 signalosome is essential for G2 phase progression and genomic stability.Development. 2008; 135: 2013-2022Crossref PubMed Scopus (78) Google Scholar). Human COP1 is also involved in DNA damage-induced cell cycle block by controlling the stability of p53 (Dornan et al., 2004Dornan D. Wertz I. Shimizu H. Arnott D. Frantz G.D. Dowd P. O'Rourke K. Koeppen H. Dixit V.M. The ubiquitin ligase COP1 is a critical negative regulator of p53.Nature. 2004; 429: 86-92Crossref PubMed Scopus (588) Google Scholar). However, Arabidopsis cop1 null mutants proceed through embryogenesis, a process with complex and well-defined cell division patterns, suggesting that cell division is not fundamentally impaired in the absence of COP1. We therefore asked whether SPA proteins are at least partially active during embryogenesis and thus allow seed formation in the absence of COP1. To this end, we aimed to generate a homozygous spaQn cop1-5 quintuple mutant which is fully devoid of both COP1 and SPAs. Indeed, homozygous spaQn cop1-5 quintuple mutant seeds were identified in progeny of a selfed spa123 (−/−) spa4-3 (+/−) cop1-5 (+/−) plant. Hence, embryogenesis clearly does not require COP1/SPA function. In conclusion, fundamental cellular processes can proceed in the absence of COP1/SPA activity. Interestingly, light is required for growth of the shoot apex and leaf organ initiation. Hence, major disturbances specifically in meristem function of cop1 and cop1 spa null mutants are likely responsible for the growth arrest at the seedling stage (Yoshida et al., 2011Yoshida S. Mandel T. Kuhlemeier C. Stem cell activation by light guides plant organogenesis.Genes Dev. 2011; 25: 1439-1450Crossref PubMed Scopus (117) Google Scholar). spaQn cop1-5 quintuple mutant seedlings had a shape very similar to that of cop1-5 single mutants in both darkness and light (Figure 1E). Both the quintuple mutant and the cop1-5 mutant failed to develop beyond the seedling stage. In total, these results show that SPA proteins have no activity in the absence of COP1. The only detectable difference between cop1-5 and the spaQn cop1-5 quintuple mutant was a higher anthocyanin content in the quintuple mutant when compared with cop1-5 or spaQn (Figure 1E and Supplemental Figure 4B). This suggests a possible COP1-independent function of SPA proteins in anthocyanin accumulation. However, since the cop1-5 and the spaQn alleles were derived from different Arabidopsis accessions, we cannot exclude the possibility that these differences are due to the mixed genetic background in the quintuple null mutant. In their C-termini, both COP1 and SPA carry a WD-repeat domain which mediates direct interactions with substrates and with DDB1 in the higher-order CUL4-DDB1COP1/SPA E3 ubiquitin ligase (Chen et al., 2010Chen H. Huang X. Gusmaroli G. Terzaghi W. Lau O.S. Yanagawa Y. Zhang Y. Li J. Lee J.H. Zhu D. et al.Arabidopsis CULLIN4-damaged DNA binding protein 1 interacts with CONSTITUTIVELY PHOTOMORPHOGENIC1-SUPPRESSOR OF PHYA complexes to regulate photomorphogenesis and flowering time.Plant Cell. 2010; 22: 108-123Crossref PubMed Scopus (168) Google Scholar, Huang et al., 2014Huang X. Ouyang X. Deng X.W. Beyond repression of photomorphogenesis: role switching of COP/DET/FUS in light signaling.Curr. Opin. Plant Biol. 2014; 21C: 96-103Crossref Scopus (129) Google Scholar). In general, mutations in the respective WD-repeat domain abolish COP1 and SPA1 function. Nevertheless, the cop1-4 mutant, which carries a premature STOP codon and therefore accumulates a truncated COP1 lacking all WD repeats, has only a partial loss-of-function phenotype (McNellis et al., 1994McNellis T.W. Von Arnim A.G. Araki T. Komeda Y. Miséra S. Deng X.-W. Genetic and molecular analysis of an allelic series of cop1 mutants suggests functional roles for the multiple protein domains.Plant Cell. 1994; 6: 487-500Crossref PubMed Scopus (304) Google Scholar). This mutant is viable and has a plant size intermediate between those of the spa quadruple mutant and the wild-type. Hence, the COP1-4 protein is partially functional despite the missing the WD-repeat domain. To investigate whether the SPA proteins are responsible for the observed residual COP1-4 activity, we generated cop1-4 spaQn quintuple mutants. Figure 1F shows that this quintuple mutant had a “fusca” phenotype that was more severe than those of the cop1-4 and spaQn mutants. The cop1-4 spaQn quintuple mutant exhibited a seedling phenotype very similar to that of the cop1-5 null mutant (Figure 1F) and, like cop1-5, failed to develop beyond the seedling stage (Supplemental Figure 5). This result indicates that the COP1-4 protein does not retain any activity in the absence of SPA proteins. We therefore conclude that the WD-repeat domains provided by the SPA proteins can at least partially substitute for the lack of the COP1 WD-repeat domain in the COP1-4 protein. The severe phenotype of the cop1-4 spaQn quintuple mutant further confirms that the WD repeats are essential for signaling activity of the COP1/SPA complex, i.e. a COP1-4 protein per se has no apparent activity. This work was supported by grants from the Deutsche Forschungsgemeinschaft DFG (SFB 635 TPC2 to U.H. and KO1438/16-1 to C.K.). X.Y. was a recipient of a graduate fellowship from the International Max Planck Research School of Molecular Plant Development which is co-funded by the Max Planck Institute for Plant Breeding Research and the University of Cologne.

The ethylene response factor VII (ERF-VII) transcription factor RELATED TO APETALA2.12 (RAP2.12) was previously identified as an activator of the ALCOHOL DEHYDROGENASE1 promoter::luciferase (ADH1-LUC) reporter gene. Here we show that overexpression of RAP2.12 and its homologues RAP2.2 and RAP2.3 sustains ABA-mediated activation of ADH1 and activates hypoxia marker genes under both anoxic and normoxic conditions. Inducible expression of all three RAP2s conferred tolerance to anoxia, oxidative and osmotic stresses, and enhanced the sensitivity to abscisic acid (ABA). Consistently, the rap2.12-2 rap2.3-1 double mutant showed hypersensitivity to both submergence and osmotic stress. These findings suggest that the three ERF-VII-type transcription factors play roles in tolerance to multiple stresses that sequentially occur during and after submergence in Arabidopsis. Oxygen-dependent degradation of RAP2.12 was previously shown to be mediated by the N-end rule pathway. During submergence the RAP2.12, RAP2.2 and RAP2.3 are stabilized and accumulates in the nucleus affecting the transcription of stress response genes. We conclude that the stabilized RAP2 transcription factors can prolong the ABA-mediated activation of a subset of osmotic responsive genes (e.g. ADH1). We also show that RAP2.12 protein level is affected by the REALLY INTERESTING GENE (RING) domain containing SEVEN IN ABSENTIA of Arabidopsis thaliana 2 (SINAT2). Silencing of SINAT1/2 genes leads to enhanced RAP2.12 abundance independently of the presence or absence of its N-terminal degron. Taken together, our results suggest that RAP2.12 and its homologues RAP2.2 and RAP2.3 act redundantly in multiple stress responses. Alternative protein degradation pathways may provide inputs to the RAP2 transcription factors for the distinct stresses.

Arabidopsis thaliana SWP73A and SWP73B are homologs of mammalian BRAHMA-associated factors (BAF60s) that tether SWITCH/SUCROSE NONFERMENTING chromatin remodeling complexes to transcription factors of genes regulating various cell differentiation pathways. Here, we show that Arabidopsis thaliana SWP73s modulate several important developmental pathways. While undergoing normal vegetative development, swp73a mutants display reduced expression of FLOWERING LOCUS C and early flowering in short days. By contrast, swp73b mutants are characterized by retarded growth, severe defects in leaf and flower development, delayed flowering, and male sterility. MNase-Seq, transcript profiling, and ChIP-Seq studies demonstrate that SWP73B binds the promoters of ASYMMETRIC LEAVES1 and 2, KANADI1 and 3, and YABBY2, 3, and 5 genes, which regulate leaf development and show coordinately altered transcription in swp73b plants. Lack of SWP73B alters the expression patterns of APETALA1, APETALA3, and the MADS box gene AGL24, whereas other floral organ identity genes show reduced expression correlating with defects in flower development. Consistently, SWP73B binds to the promoter regions of APETALA1 and 3, SEPALLATA3, LEAFY, UNUSUAL FLORAL ORGANS, TERMINAL FLOWER1, AGAMOUS-LIKE24, and SUPPRESSOR OF CONSTANS OVEREXPRESSION1 genes, and the swp73b mutation alters nucleosome occupancy on most of these loci. In conclusion, SWP73B acts as important modulator of major developmental pathways, while SWP73A functions in flowering time control.

The stem cell niche in the root meristem maintains pluripotent stem cells to ensure a constant supply of cells for root growth. Despite extensive progress, the molecular mechanisms through which root stem cell fates and stem cell niche activity are determined remain largely unknown. In Arabidopsis thaliana, the Pleiotropic Regulatory Locus 1 (PRL1) encodes a WD40-repeat protein subunit of the spliceosome-activating Nineteen Complex (NTC) that plays a role in multiple stress, hormone and developmental signaling pathways. Here, we show that PRL1 is involved in the control of root meristem size and root stem cell niche activity. PRL1 is strongly expressed in the root meristem and its loss of function mutation results in disorganization of the quiescent center (QC), premature stem cell differentiation, aberrant cell division, and reduced root meristem size. Our genetic studies indicate that PRL1 is required for confined expression of the homeodomain transcription factor WOX5 in the QC and acts upstream of the transcription factor PLETHORA (PLT) in modulating stem cell niche activity and root meristem size. These findings define a role for PRL1 as an important determinant of PLT signaling that modulates maintenance of the stem cell niche and root meristem size.