Ethylene Receptors Function as Components of High-Molecular-Mass Protein Complexes in Arabidopsis

Background

The gaseous plant hormone ethylene is perceived in Arabidopsis thaliana by a five-member receptor family composed of ETR1, ERS1, ETR2, ERS2, and EIN4.

Methodology/Principal Findings

Gel-filtration analysis of ethylene receptors solubilized from Arabidopsis membranes demonstrates that the receptors exist as components of high-molecular-mass protein complexes. The ERS1 protein complex exhibits an ethylene-induced change in size consistent with ligand-mediated nucleation of protein-protein interactions. Deletion analysis supports the participation of multiple domains from ETR1 in formation of the protein complex, and also demonstrates that targeting to and retention of ETR1 at the endoplasmic reticulum only requires the first 147 amino acids of the receptor. A role for disulfide bonds in stabilizing the ETR1 protein complex was demonstrated by use of reducing agents and mutation of Cys4 and Cys6 of ETR1. Expression and analysis of ETR1 in a transgenic yeast system demonstrates the importance of Cys4 and Cys6 of ETR1 in stabilizing the receptor for ethylene binding.

Conclusions/Significance

These data support the participation of ethylene receptors in obligate as well as ligand-dependent non-obligate protein interactions. These data also suggest that different protein complexes may allow for tailoring of the ethylene signal to specific cellular environments and responses.     

Regulatory Roles of Cytokinins and Cytokinin Signaling in Response to Potassium Deficiency in Arabidopsis

Potassium (K) is an important plant macronutrient that has various functions throughout the whole plant over its entire life span. Cytokinins (CKs) are known to regulate macronutrient homeostasis by controlling the expression of nitrate, phosphate and sulfate transporters. Although several studies have described how CKs signal deficiencies for some macronutrients, the roles of CKs in K signaling are poorly understood. CK content has been shown to decrease under K-starved conditions. Specifically, a CK-deficient mutant was more tolerant to low K than wild-type; however, a plant with an overaccumulation of CKs was more sensitive to low K. These results suggest that K deprivation alters CK metabolism, leading to a decrease in CK content. To investigate this phenomenon further, several Arabidopsis lines, including a CK-deficient mutant and CK receptor mutants, were analyzed in low K conditions using molecular, genetic and biochemical approaches. ROS accumulation and root hair growth in low K were also influenced by CKs. CK receptor mutants lost the responsiveness to K-deficient signaling, including ROS accumulation and root hair growth, but the CK-deficient mutant accumulated more ROS and exhibited up-regulated expression of HAK5, which is a high-affinity K uptake transporter gene that is rapidly induced by low K stress in ROS- and ethylene-dependent manner in response to low K. From these results, we conclude that a reduction in CK levels subsequently allows fast and effective stimulation of low K-induced ROS accumulation, root hair growth and HAK5 expression, leading to plant adaptation to low K conditions.     

Photoactivated UVR8-COP1 Module Determines Photomorphogenic UV-B Signaling Output in Arabidopsis

In Arabidopsis, ultraviolet (UV)-B-induced photomorphogenesis is initiated by a unique photoreceptor UV RESISTANCE LOCUS 8 (UVR8) which utilizes its tryptophan residues as internal chromophore to sense UV-B. As a result of UV-B light perception, the UVR8 homodimer shaped by its arginine residues undergoes a conformational switch of monomerization. Then UVR8 associates with the CONSTITUTIVELY PHOTOMORPHOGENIC 1-SUPPRESSOR OF PHYA (COP1-SPA) core complex(es) that is released from the CULLIN 4-DAMAGED DNA BINDING PROTEIN 1 (CUL4-DDB1) E3 apparatus. This association, in turn, causes COP1 to convert from a repressor to a promoter of photomorphogenesis. It is not fully understood, however, regarding the biological significance of light-absorbing and dimer-stabilizing residues for UVR8 activity in photomorphogenic UV-B signaling. Here, we take advantage of transgenic UVR8 variants to demonstrate that two light-absorbing tryptophans, W233 and W285, and two dimer-stabilizing arginines, R286 and R338, play pivotal roles in UV-B-induced photomorphogenesis. Mutation of each residue results in alterations in UV-B light perception, UVR8 monomerization and UVR8-COP1 association in response to photomorphogenic UV-B. We also identify and functionally characterize two constitutively active UVR8 variants, UVR8^W285A and UVR8^R338A, whose photobiological activities are enhanced by the repression of CUL4, a negative regulator in this pathway. Based on our molecular and biochemical evidence, we propose that the UVR8-COP1 affinity in plants critically determines the photomorphogenic UV-B signal transduction coupling with UVR8-mediated UV-B light perception.     

Zeaxanthin Protects Plant Photosynthesis by Modulating Chlorophyll Triplet Yield in Specific Light-harvesting Antenna Subunits

Plants are particularly prone to photo-oxidative damage caused by excess light. Photoprotection is essential for photosynthesis to proceed in oxygenic environments either by scavenging harmful reactive intermediates or preventing their accumulation to avoid photoinhibition. Carotenoids play a key role in protecting photosynthesis from the toxic effect of over-excitation; under excess light conditions, plants accumulate a specific carotenoid, zeaxanthin, that was shown to increase photoprotection. In this work we genetically dissected different components of zeaxanthin-dependent photoprotection. By using time-resolved differential spectroscopy in vivo, we identified a zeaxanthin-dependent optical signal characterized by a red shift in the carotenoid peak of the triplet-minus-singlet spectrum of leaves and pigment-binding proteins. By fractionating thylakoids into their component pigment binding complexes, the signal was found to originate from the monomeric Lhcb4–6 antenna components of Photosystem II and the Lhca1–4 subunits of Photosystem I. By analyzing mutants based on their sensitivity to excess light, the red-shifted triplet-minus-singlet signal was tightly correlated with photoprotection in the chloroplasts, suggesting the signal implies an increased efficiency of zeaxanthin in controlling chlorophyll triplet formation. Fluorescence-detected magnetic resonance analysis showed a decrease in the amplitude of signals assigned to chlorophyll triplets belonging to the monomeric antenna complexes of Photosystem II upon zeaxanthin binding; however, the amplitude of carotenoid triplet signal does not increase correspondingly. Results show that the high light-induced binding of zeaxanthin to specific proteins plays a major role in enhancing photoprotection by modulating the yield of potentially dangerous chlorophyll-excited states in vivo and preventing the production of singlet oxygen.     

Tropical Plant Extracts Modulating the Growth of Mycobacterium ulcerans

Mycobacterium ulcerans, the etiologic agent of Buruli ulcer, has been detected on aquatic plants in endemic tropical regions. Here, we tested the effect of several tropical plant extracts on the growth of M. ulcerans and the closely related Mycobacterium marinum. M. ulcerans and M. marinum were inoculated on Middlebrook 7H11 medium with and without extracts from tropical aquatic plants, including Ammannia gracilis, Crinum calamistratum, Echinodorus africanus, Vallisneria nana and Vallisneria torta. Delay of detection of the first colony and the number of colonies at day 7 (M. marinum) or day 16 (M. ulcerans) were used as endpoints. The first M. ulcerans colonies were detected at 8 ± 0 days on control Middlebrook 7H11 medium, 6.34 ± 0.75 days on A. gracilis-enriched medium (p<0.01), 6 ± 1 days on E. africanus- and V. torta-enriched media (p<0.01), 6 ± 0 days on V. nana-enriched medium (p<0.01) and 5.67 ± 0.47 days on C. calamistratum-enriched medium (p<0.01). Furthermore, the number of detected colonies was significantly increased in C. calamistratum- and E. africanus-enriched media at each time point compared to Middlebrook 7H11 (p<0.05). V. nana- and V. torta-enriched media significantly increased the number of detected colonies starting from day 6 and day 10, respectively (p<0.001). At the opposite, A. gracilis-enriched medium significantly decreased the number of detected colonies starting from day 8 PI (p<0.05). In conclusion, some aquatic plant extracts, could be added as adjuvants to the Middlebrook 7H11 medium for the culturing of M. marinum and M. ulcerans.     

LIK1, A CERK1-Interacting Kinase,Regulates Plant Immune Responses in Arabidopsis

Chitin, an integral component of the fungal cell wall, is one of the best-studied microbe-associated molecular patterns. Previous work identified a LysM receptor-like kinase (LysM-RLK1/CERK1) as the primary chitin receptor in Arabidopsis. In order to identify proteins that interact with CERK1, we conducted a yeast two-hybrid screen using the intracellular kinase domain of CERK1 as the bait. This screen identified 54 putative CERK1-interactors. Screening mutants defective in 43 of these interacting proteins identified only two, a calmodulin like protein (At3g10190) and a leucine-rich repeat receptor like kinase (At3g14840), which differed in their response to pathogen challenge. In the present work, we focused on characterizing the LRR-RLK gene where mutations altered responses to chitin elicitation. This LRR-RLK was named LysM RLK1-interacting kinase 1 (LIK1). The interaction between CERK1 and LIK1 was confirmed by co-immunoprecipitation using protoplasts and transgenic plants. In vitro experiments showed that LIK1 was directly phosphorylated by CERK1. In vivo phosphorylation assays showed that Col-0 wild-type plants have more phosphorylated LIK1 than cerk1 mutant plants, suggesting that LIK1 may be directly phosphorylated by CERK1. Lik1 mutant plants showed an enhanced response to both chitin and flagellin elicitors. In comparison to the wild-type plants, lik1 mutant plants were more resistant to the hemibiotrophic pathogen Pseudomonas syringae, but more susceptible to the necrotrophic pathogen Sclerotinia sclerotiorum. Consistent with the enhanced susceptibility to necrotrophs, lik1 mutants showed reduced expression of genes involved in jasmonic acid and ethylene signaling pathways. These data suggest that LIK1 directly interacts with CERK1 and regulates MAMP-triggered innate immunity.     

The Bacterial Effector HopX1 Targets JAZ Transcriptional Repressors to Activate Jasmonate Signaling and Promote Infection in Arabidopsis

Pathogenicity of Pseudomonas syringae is dependent on a type III secretion system, which secretes a suite of virulence effector proteins into the host cytoplasm, and the production of a number of toxins such as coronatine (COR), which is a mimic of the plant hormone jasmonate-isoleuce (JA-Ile). Inside the plant cell, effectors target host molecules to subvert the host cell physiology and disrupt defenses. However, despite the fact that elucidating effector action is essential to understanding bacterial pathogenesis, the molecular function and host targets of the vast majority of effectors remain largely unknown. Here, we found that effector HopX1 from Pseudomonas syringae pv. tabaci (Pta) 11528, a strain that does not produce COR, interacts with and promotes the degradation of JAZ proteins, a key family of JA-repressors. We show that hopX1 encodes a cysteine protease, activity that is required for degradation of JAZs by HopX1. HopX1 associates with JAZ proteins through its central ZIM domain and degradation occurs in a COI1-independent manner. Moreover, ectopic expression of HopX1 in Arabidopsis induces the expression of JA-dependent genes, represses salicylic acid (SA)-induced markers, and complements the growth of a COR-deficient P. syringae pv. tomato (Pto) DC3000 strain during natural bacterial infections. Furthermore, HopX1 promoted susceptibility when delivered by the natural type III secretion system, to a similar extent as the addition of COR, and this effect was dependent on its catalytic activity. Altogether, our results indicate that JAZ proteins are direct targets of bacterial effectors to promote activation of JA-induced defenses and susceptibility in Arabidopsis. HopX1 illustrates a paradigm of an alternative evolutionary solution to COR with similar physiological outcome.     

mNanog Possesses Dorsal Mesoderm-Inducing Ability by Modulating Both BMP and Activin/Nodal Signaling in Xenopus Ectodermal Cells

Background

In Xenopus early embryogenesis, various genes are involved with mesoderm formation. In particular, dorsal mesoderm contains the organizer region and induces neural tissues through the inhibition of bone morphogenetic protein (BMP) signaling. In our initial study to identify novel genes necessary for maintaining the undifferentiated state, we unexpectedly revealed mesoderm-inducing activity for mNanog in Xenopus.

Methodology/Principal Findings

The present series of experiments investigated the effect of mNanog gene expression on Xenopus embryo. Ectopic expression of mNanog induced dorsal mesoderm gene activity, secondary axis formation, and weakly upregulated Activin/nodal signaling. The injection of mNanog also effectively inhibited the target genes of BMP signaling, while Xvent2 injection downregulated the dorsal mesoderm gene expression induced by mNanog injection.

Conclusions/Significance

These results suggested that mNanog expression induces dorsal mesoderm by regulating both Activin/nodal signaling and BMP signaling in Xenopus. This finding highlights the possibly novel function for mNanog in stimulating the endogenous gene network in Xenopus mesoderm formation.     

Strigolactone Regulates Anthocyanin Accumulation,Acid Phosphatases Production and Plant Growth under Low Phosphate Condition in Arabidopsis

Phosphate is an essential macronutrient in plant growth and development; however, the concentration of inorganic phosphate (Pi) in soil is often suboptimal for crop performance. Accordingly, plants have developed physiological strategies to adapt to low Pi availability. Here, we report that typical Pi starvation responses in Arabidopsis are partially dependent on the strigolactone (SL) signaling pathway. SL treatment induced root hair elongation, anthocyanin accumulation, activation of acid phosphatase, and reduced plant weight, which are characteristic responses to phosphate starvation. Furthermore, the expression profile of SL-response genes correlated with the expression of genes induced by Pi starvation. These results suggest a potential overlap between SL signaling and Pi starvation signaling pathways in plants.     

Cloning of the Cryptochrome-Encoding PeCRY1 Gene from Populus euphratica and Functional Analysis in Arabidopsis

Cryptochromes are photolyase-like blue/UV-A light receptors that evolved from photolyases. In plants, cryptochromes regulate various aspects of plant growth and development. Despite of their involvement in the control of important plant traits, however, most studies on cryptochromes have focused on lower plants and herbaceous crops, and no data on cryptochrome function are available for forest trees. In this study, we isolated a cryptochrome gene, PeCRY1, from Euphrates poplar (Populus euphratica), and analyzed its structure and function in detail. The deduced PeCRY1 amino acid sequence contained a conserved N-terminal photolyase-homologous region (PHR) domain as well as a C-terminal DQXVP-acidic-STAES (DAS) domain. Secondary and tertiary structure analysis showed that PeCRY1 shares high similarity with AtCRY1 from Arabidopsis thaliana. PeCRY1 expression was upregulated at the mRNA level by light. Using heterologous expression in Arabidopsis, we showed that PeCRY1 overexpression rescued the cry1 mutant phenotype. In addition, PeCRY1 overexpression inhibited hypocotyl elongation, promoted root growth, and enhanced anthocyanin accumulation in wild-type background seedlings grown under blue light. Furthermore, we examined the interaction between PeCRY1 and AtCOP1 using a bimolecular fluorescence complementation (BiFc) assay. Our data provide evidence for the involvement of PeCRY1 in the control of photomorphogenesis in poplar.     

ABA-Mediated ROS in Mitochondria Regulate Root Meristem Activity by Controlling PLETHORA Expression in Arabidopsis

Although research has determined that reactive oxygen species (ROS) function as signaling molecules in plant development, the molecular mechanism by which ROS regulate plant growth is not well known. An aba overly sensitive mutant, abo8-1, which is defective in a pentatricopeptide repeat (PPR) protein responsible for the splicing of NAD4 intron 3 in mitochondrial complex I, accumulates more ROS in root tips than the wild type, and the ROS accumulation is further enhanced by ABA treatment. The ABO8 mutation reduces root meristem activity, which can be enhanced by ABA treatment and reversibly recovered by addition of certain concentrations of the reducing agent GSH. As indicated by low ProDR5:GUS expression, auxin accumulation/signaling was reduced in abo8-1. We also found that ABA inhibits the expression of PLETHORA1 (PLT1) and PLT2, and that root growth is more sensitive to ABA in the plt1 and plt2 mutants than in the wild type. The expression of PLT1 and PLT2 is significantly reduced in the abo8-1 mutant. Overexpression of PLT2 in an inducible system can largely rescue root apical meristem (RAM)-defective phenotype of abo8-1 with and without ABA treatment. These results suggest that ABA-promoted ROS in the mitochondria of root tips are important retrograde signals that regulate root meristem activity by controlling auxin accumulation/signaling and PLT expression in Arabidopsis.     

Innate,translation‐dependent silencing of an invasive transposon in Arabidopsis

Co‐evolution between hosts’ and parasites’ genomes shapes diverse pathways of acquired immunity based on silencing small (s)RNAs. In plants, sRNAs cause heterochromatinization, sequence degeneration, and, ultimately, loss of autonomy of most transposable elements (TEs). Recognition of newly invasive plant TEs, by contrast, involves an innate antiviral‐like silencing response. To investigate this response’s activation, we studied the single‐copy element EVADÉ (EVD), one of few representatives of the large Ty1/Copia family able to proliferate in Arabidopsis when epigenetically reactivated. In Ty1/Copia elements, a short subgenomic mRNA (shGAG) provides the necessary excess of structural GAG protein over the catalytic components encoded by the full‐length genomic flGAG‐POL. We show here that the predominant cytosolic distribution of shGAG strongly favors its translation over mostly nuclear flGAG‐POL. During this process, an unusually intense ribosomal stalling event coincides with mRNA breakage yielding unconventional 5’OH RNA fragments that evade RNA quality control. The starting point of sRNA production by RNA‐DEPENDENT‐RNA‐POLYMERASE‐6 (RDR6), exclusively on shGAG, occurs precisely at this breakage point. This hitherto‐unrecognized “translation‐dependent silencing” (TdS) is independent of codon usage or GC content and is not observed on TE remnants populating the Arabidopsis genome, consistent with their poor association, if any, with polysomes. We propose that TdS forms a primal defense against EVD de novo invasions that underlies its associated sRNA pattern.