Light on perenniality: Para-dormancy is based on ABA-GA antagonism and endo-dormancy on the shutdown of GA biosynthesis.

Perennial para- and endo-dormancy are seasonally separate phenomena. Whereas para-dormancy is the suppression of axillary buds (AXBs) by a growing shoot, endo-dormancy is the short-day elicited arrest of terminal and AXBs. In hybrid aspen (Populus tremula x P. tremuloides) compromising the apex releases para-dormancy, whereas endo-dormancy requires chilling. ABA and GA are implicated in both phenomena. To untangle their roles, we blocked ABA biosynthesis with fluridone (FD), which significantly reduced ABA levels, downregulated GA-deactivation genes, upregulated the major GA3ox-biosynthetic genes, and initiated branching. Comprehensive GA-metabolite analyses suggested that FD treatment shifted GA production to the non-13-hydroxylation pathway, enhancing GA4 function. Applied ABA counteracted FD effects on GA metabolism and downregulated several GA3/4 -inducible α- and γ-clade 1,3-ß-glucanases that hydrolyze callose at plasmodesmata (PD), thereby enhancing PD-callose accumulation. Remarkably, ABA-deficient plants repressed GA4 biosynthesis and established endo-dormancy like controls but showed increased stress sensitivity. Repression of GA4 biosynthesis involved short-day induced DNA methylation events within the GA3ox2 promoter. In conclusion, the results cast new light on the roles of ABA and GA in dormancy cycling. In para-dormancy, PD-callose turnover is antagonized by ABA, whereas in short-day conditions, lack of GA4 biosynthesis promotes callose deposition that is structurally persistent throughout endo-dormancy.

Lipid Body Dynamics in Shoot Meristems: Production, Enlargement, and Putative Organellar Interactions and Plasmodesmal Targeting.

Post-embryonic cells contain minute lipid bodies (LBs) that are transient, mobile, engage in organellar interactions, and target plasmodesmata (PD). While LBs can deliver γ-clade 1,3-ß-glucanases to PD, the nature of other cargo is elusive. To gain insight into the poorly understood role of LBs in meristems, we investigated their dynamics by microscopy, gene expression analyzes, and proteomics. In developing buds, meristems accumulated LBs, upregulated several LB-specific OLEOSIN genes and produced OLEOSINs. During bud maturation, the major gene OLE6 was strongly downregulated, OLEOSINs disappeared from bud extracts, whereas lipid biosynthesis genes were upregulated, and LBs were enlarged. Proteomic analyses of the LB fraction of dormant buds confirmed that OLEOSINs were no longer present. Instead, we identified the LB-associated proteins CALEOSIN (CLO1), Oil Body Lipase 1 (OBL1), Lipid Droplet Interacting Protein (LDIP), Lipid Droplet Associated Protein1a/b (LDAP1a/b) and LDAP3a/b, and crucial components of the OLEOSIN-deubiquitinating and degradation machinery, such as PUX10 and CDC48A. All mRFP-tagged LDAPs localized to LBs when transiently expressed in Nicotiana benthamiana. Together with gene expression analyzes, this suggests that during bud maturation, OLEOSINs were replaced by LDIP/LDAPs at enlarging LBs. The LB fraction contained the meristem-related actin7 (ACT7), "myosin XI tail-binding" RAB GTPase C2A, an LB/PD-associated γ-clade 1,3-ß-glucanase, and various organelle- and/or PD-localized proteins. The results are congruent with a model in which LBs, motorized by myosin XI-k/1/2, traffic on F-actin, transiently interact with other organelles, and deliver a diverse cargo to PD.

Plant Lipid Bodies Traffic on Actin to Plasmodesmata Motorized by Myosin XIs.

Late 19th-century cytologists observed tiny oil drops in shoot parenchyma and seeds, but it was discovered only in 1972 that they were bound by a half unit-membrane. Later, it was found that lipid bodies (LBs) arise from the endoplasmic reticulum. Seeds are known to be packed with static LBs, coated with the LB-specific protein OLEOSIN. As shown here, apices of Populus tremula x P. tremuloides also express OLEOSIN genes and produce potentially mobile LBs. In developing buds, PtOLEOSIN (PtOLE) genes were upregulated, especially PtOLE6, concomitant with LB accumulation. To investigate LB mobility and destinations, we transformed Arabidopsis with PtOLE6-eGFP. We found that PtOLE6-eGFP fusion protein co-localized with Nile Red-stained LBs in all cell types. Moreover, PtOLE6-eGFP-tagged LBs targeted plasmodesmata, identified by the callose marker aniline blue. Pharmacological experiments with brefeldin, cytochalasin D, and oryzalin showed that LB-trafficking requires F-actin, implying involvement of myosin motors. In a triple myosin-XI knockout (xi-k/1/2), transformed with PtOLE6-eGFP, trafficking of PtOLE6-eGFP-tagged LBs was severely impaired, confirming that they move on F-actin, motorized by myosin XIs. The data reveal that LBs and OLEOSINs both function in proliferating apices and buds, and that directional trafficking of LBs to plasmodesmata requires the actomyosin system.

ARR22 overexpression can suppress plant Two-Component Regulatory Systems.

In plants, several developmental processes are co-coordinated by cytokinins via phosphorylation dependent processes of the Two-Component System (TCS). An outstanding challenge is to track phosphorelay flow from cytokinin perception to its molecular outputs, of which gene activation plays a major role. To address this issue, a kinetic-based reporter system was expounded to track TCS phosphorelay activity in vivo that can distinguish between basal and cytokinin dependent effects of overexpressed TCS members. The TCS phosphorelay can be positively activated by cytokinin and inhibited by pharmaceuticals or naturally interfering components. In this case we took advantage of the phosphohistidine-phosphatase Arabidopsis Response Regulator (ARR) 22 and investigated its phosphocompetition with other TCS members in regulating promoters of ARR5 and WUS in Arabidopsis thaliana cell culture protoplasts. In congruency with the proposed function of ARR22, overexpression of ARR22 blocked the activation of all B-type ARRs in this study in a TCS dependent manner. Furthermore, this effect could not be mimicked by A-type response regulator overexpression or compensated by AHP overexpression. Compared to other reporter assays, ours mimicked effects previously observed only in transgenic plants for all of the TCS proteins studied, suggesting that it is possible to expose phosphocompetition. Thus, our approach can be used to investigate gene signaling networks involving the TCS by leveraging ARR22 as a TCS inhibitor along with B-type ARR overexpression.

The plant cell wall integrity maintenance and immune signaling systems cooperate to control stress responses in Arabidopsis thaliana.

Cell walls surround all plant cells, and their composition and structure are modified in a tightly controlled, adaptive manner to meet sometimes opposing functional requirements during growth and development. The plant cell wall integrity (CWI) maintenance mechanism controls these functional modifications, as well as responses to cell wall damage (CWD). We investigated how the CWI system mediates responses to CWD in Arabidopsis thaliana CWD induced by cell wall-degrading enzymes or an inhibitor of cellulose biosynthesis elicited similar, turgor-sensitive stress responses. Phenotypic clustering with 27 genotypes identified a core group of receptor-like kinases (RLKs) and ion channels required for the activation of CWD responses. A genetic analysis showed that the RLK FEI2 and the plasma membrane-localized mechanosensitive Ca2+ channel MCA1 functioned downstream of the RLK THE1 in CWD perception. In contrast, pattern-triggered immunity (PTI) signaling components, including the receptors for plant elicitor peptides (AtPeps) PEPR1 and PEPR2, repressed responses to CWD. CWD induced the expression of PROPEP1 and PROPEP3, which encode the precursors of AtPep1 and AtPep3, and the release of PROPEP3 into the growth medium. Application of AtPep1 and AtPep3 repressed CWD-induced phytohormone accumulation in a concentration-dependent manner. These results suggest that AtPep-mediated signaling suppresses CWD-induced defense responses controlled by the CWI mechanism. This suppression was alleviated when PTI signaling downstream of PEPR1 and PEPR2 was impaired. Defense responses controlled by the CWI maintenance mechanism might thus compensate to some extent for the loss of PTI signaling elements.

The Arabidopsis leucine-rich repeat receptor kinase MIK2/LRR-KISS connects cell wall integrity sensing, root growth and response to abiotic and biotic stresses.

Plants actively perceive and respond to perturbations in their cell walls which arise during growth, biotic and abiotic stresses. However, few components involved in plant cell wall integrity sensing have been described to date. Using a reverse-genetic approach, we identified the Arabidopsis thaliana leucine-rich repeat receptor kinase MIK2 as an important regulator of cell wall damage responses triggered upon cellulose biosynthesis inhibition. Indeed, loss-of-function mik2 alleles are strongly affected in immune marker gene expression, jasmonic acid production and lignin deposition. MIK2 has both overlapping and distinct functions with THE1, a malectin-like receptor kinase previously proposed as cell wall integrity sensor. In addition, mik2 mutant plants exhibit enhanced leftward root skewing when grown on vertical plates. Notably, natural variation in MIK2 (also named LRR-KISS) has been correlated recently to mild salt stress tolerance, which we could confirm using our insertional alleles. Strikingly, both the increased root skewing and salt stress sensitivity phenotypes observed in the mik2 mutant are dependent on THE1. Finally, we found that MIK2 is required for resistance to the fungal root pathogen Fusarium oxysporum. Together, our data identify MIK2 as a novel component in cell wall integrity sensing and suggest that MIK2 is a nexus linking cell wall integrity sensing to growth and environmental cues.

Chimeric Autofluorescent Proteins as Photophysical Model System for Multicolor Bimolecular Fluorescence Complementation.

The yellow fluorescent protein (YFP) is frequently used in a protein complementation assay called bimolecular fluorescence complementation (BiFC), and is employed to visualize protein-protein interactions. In this analysis, two different, nonfluorescent fragments of YFP are genetically attached to proteins of interest. Upon interaction of these proteins, the YFP fragments are brought into proximity close enough to reconstitute their original structure, enabling fluorescence. BiFC allows for a straightforward readout of protein-protein interactions and furthermore facilitates their functional investigation by in vivo imaging. Furthermore, it has been observed that the available color range in BiFC can be extended upon complementing fragments of different proteins that are, like YFP, derived from the Aequorea victoria green fluorescent protein, thereby allowing for a multiplexed investigation of protein-protein interactions. Some spectral characteristics of "multicolor" BiFC (mcBiFC) complexes have been reported before; however, no in-depth analysis has been performed yet. Therefore, little is known about the photophysical characteristics of these mcBiFC complexes because a proper characterization essentially relies on in vitro data. This is particularly difficult for fragments of autofluorescent proteins (AFPs) because they show a very strong tendency to form supramolecular aggregates which precipitate ex vivo. In this study, this intrinsic difficulty is overcome by directly fusing the coding DNA of different AFP fragments. Translation of the genetic sequence in Escherichia coli leads to fully functional, highly soluble fluorescent proteins with distinct properties. On the basis of their construction, they are designated chimeric AFPs, or BiFC chimeras, here. Comparison of their spectral characteristics with experimental in vivo BiFC data confirmed the utility of the chimeric proteins as a BiFC model system. In this study, nine different chimeras were thoroughly analyzed at both the ensemble and the single-molecular level. The data indicates that mutations believed to be photophysically silent significantly alter the properties of AFPs.

The interaction of the Arabidopsis response regulator ARR18 with bZIP63 mediates the regulation of PROLINE DEHYDROGENASE expression.

As the first and rate-limiting enzyme of proline degradation, PROLINE DEHYDROGENASE1 (PDH1) is tightly regulated during plant stress responses, including induction under hypoosmolarity and repression under water deficit. The plant receptor histidine kinases AHKs, elements of the two-component system (TCS) in Arabidopsis thaliana, are proposed to function in water stress responses by regulating different stress-responsive genes. However, little information is available concerning AHK phosphorelay-mediated downstream signaling. Here we show that the Arabidopsis type-B response regulator 18 (ARR18) functions as a positive osmotic stress response regulator in Arabidopsis seeds and affects the activity of the PDH1 promoter, known to be controlled by C-group bZIP transcription factors. Moreover, direct physical interaction of ARR18 with bZIP63 was identified and shown to be dependent on phosphorylation of the conserved aspartate residue in the ARR18 receiver domain. We further show that bZIP63 itself functions as a negative regulator of seed germination upon osmotic stress. Using reporter gene assays in protoplasts, we demonstrated that ARR18 interaction negatively interferes with the transcriptional activity of bZIP63 on the PDH1 promoter. Our findings provide new insight into the function of ARR18 and bZIP63 as antagonistic regulators of gene expression in Arabidopsis.

Structure-function analysis of Arabidopsis thaliana histidine kinase AHK5 bound to its cognate phosphotransfer protein AHP1.

The multi-step phosphorelay (MSP) system defines a key signal transduction pathway in plants and many eukaryotes. In this system, external stimuli first lead to the activation of a histidine kinase, followed by transfer of a phosphoryl group from the receiver domain of the kinase (HK(RD)) to downstream, cytosolic phosphotransfer proteins (HPs). In order to establish the determinants of specificity for this signaling relay system, we have solved the first crystal structure of a plant HK(RD), AHK5(RD), in complex with one of its cognate HPs, AHP1. AHP1 binds AHK5(RD) via a prominent hydrogen bond docking ridge and a hydrophobic patch. These features are conserved among all AHP proteins, but differ significantly from other structurally characterized prokaryotic and eukaryotic HPs. Surface plasmon resonance experiments show that AHK5(RD) binds to AHP1-3 with similar, micromolar affinity, consistent with the transient nature of this signaling complex. Our correlation of structural and functional data provide the first insight, at the atomic level as well as with quantitative affinity data, into the molecular recognition events governing the MSP in plants.

Identification of two-component system elements downstream of AHK5 in the stomatal closure response of Arabidopsis thaliana.

To optimize water use efficiency, plants regulate stomatal closure through a complex signaling process. Hydrogen peroxide (H2O2) is produced in response to several environmental stimuli, and has been identified as a key second messenger involved in the regulation of stomatal aperture. The Arabidopsis histidine kinase 5 (AHK5) has been shown to regulate stomatal closure in response to H2O2 and other stimuli that depend on H2O2. AHK5 is a member of the two-component system (TCS) in Arabidopsis. The plant TCS comprises three different protein types: the hybrid histidine kinases (HKs), the phosphotransfer proteins (HPs) and the response regulators (RRs). Here we determined TCS elements involved in H2O2- and ethylene-dependent stomatal closure downstream of AHK5. By yeast and in planta interaction assays and functional studies, AHP1, 2 and 5 as well as the response regulators ARR4 and ARR7 were identified acting downstream of AHK5 in the ethylene and H2O2 response pathways of guard cells. Furthermore, we demonstrate that aspartate phosphorylation of ARR4 is only required for the H2O2- but not for the ethylene-induced stomatal closure response. Our data suggest the presence of a complex TCS signaling network comprising of at least AHK5, several AHPs and response regulators, which modulate stomatal closure in response to H2O2 and ethylene.

The Arabidopsis B-type response regulator 18 homomerizes and positively regulates cytokinin responses.

In higher plants, the two-component system (TCS) is a signaling mechanism based on a His-to-Asp phosphorelay. The Arabidopsis TCS involves three different types of proteins, namely the histidine kinases (AHKs), the histidine phosphotransfer proteins (AHPs) and the response regulators (ARRs). The ARRs comprise three different families, namely A, B and C types, according to their protein structure. While some members of the B-type family of ARRs have been studied extensively and reported to act as DNA-binding transcriptional regulators, very limited information is available for other B-type ARRs such as ARR18. In this study, we characterize in detail the molecular and functional properties of ARR18. ARR18 acts as a transcriptional regulator in plant cells and forms homodimers in planta as shown by FRET-FLIM studies. As demonstrated by mutational analysis, the aspartate at position 70 (D70) in the receiver domain of ARR18 acts as crucial phosphorylation site. The modification of D70 affects the response regulator's ability to homodimerize and to activate its target genes. Furthermore, physiological investigations of Arabidopsis lines ectopically expressing ARR18 introduce ARR18 as a new member within the cytokinin-regulated response pathway regulating root elongation.