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Receptor-like kinases (RLKs) play important roles in plant immunity signaling; thus, many are hijacked by pathogen effectors to promote successful pathogenesis. Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of rice leaf blight disease. The strain PXO99A has 18 non-TAL (transcription activation-like) effectors; however, their mechanisms of action and host target proteins remain largely unknown. Although the effector XopR from the Xoo strain MAFF311018 was shown to suppress PAMP-triggered immune responses in Arabidopsis, its target has not yet been identified. Here, we show that PXO99A XopR interacts with BIK1 at the plasma membrane. BIK1 is a receptor-like cytoplasmic kinase (RLCK) belonging to the RLK family of proteins and mediates PAMP-triggered stomatal immunity. In turn, BIK1 phosphorylates XopR. Furthermore, XopR suppresses PAMP-triggered stomatal closure in transgenic Arabidopsis expressing XopR. In addition, XopR is able to associate with RLCKs other than BIK1. These results suggest that XopR likely suppresses plant immunity by targeting BIK1 and other RLCKs.

Natural variation can be exploited to identify allelic variants of proteins. In this study, patch clamp was used to determine transport properties of two AtTPK1 alleles from Landsberg and Kas-2 ecotypes. No difference in conductance or ion selectivity was observed but the Kas version of TPK1 showed different Ca(2+) dependence in its open probability compared to Ler. Leaves from Kas showed lower rates of water loss than those of Ler, in either the absence or presence of ABA, an observation that is consistent with higher TPK1 channel activity at comparable cytoplasmic Ca(2+) concentrations. A model that explains the results is presented., (© 2015 Federation of European Biochemical Societies.)

Land plants possess myosin classes VIII and XI. Although some information is available on the molecular properties of class XI myosins, class VIII myosins are not characterized. Here, we report the first analysis of the enzymatic properties of class VIII myosin. The motor domain of Arabidopsis class VIII myosin, ATM1 (ATM1-MD), and the motor domain plus one IQ motif (ATM1-1IQ) were expressed in a baculovirus system and characterized. ATM1-MD and ATM1-1IQ had low actin-activated Mg(2+)-ATPase activity (Vmax = 4 s(-1)), although their affinities for actin were high (Kactin = 4 μM). The actin-sliding velocities of ATM1-MD and ATM1-1IQ were 0.02 and 0.089 μm/s, respectively, from which the value for full-length ATM1 is calculated to be ∼0.2 μm/s. The results of actin co-sedimentation assay showed that the duty ratio of ATM1 was ∼90%. ADP dissociation from the actin·ATM1 complex (acto-ATM1) was extremely slow, which accounts for the low actin-sliding velocity, low actin-activated ATPase activity, and high duty ratio. The rate of ADP dissociation from acto-ATM1 was markedly biphasic with fast and slow phase rates (5.1 and 0.41 s(-1), respectively). Physiological concentrations of free Mg(2+) modulated actin-sliding velocity and actin-activated ATPase activity by changing the rate of ADP dissociation from acto-ATM1. GFP-fused full-length ATM1 expressed in Arabidopsis was localized to plasmodesmata, plastids, newly formed cell walls, and actin filaments at the cell cortex. Our results suggest that ATM1 functions as a tension sensor/generator at the cell cortex and other structures in Arabidopsis.

The target of rapamycin (TOR) kinase pathway regulates various biological processes, including translation, synthesis of ribosomal proteins, and transcription of rRNA. The ribosomal protein S6 (RPS6) is one of the well known downstream components of the TOR pathway. Ribosomal proteins have been known to have diverse functions in regulating cellular metabolism as well as protein synthesis. So far, however, little is known about other possible role(s) of RPS6 in plants, besides being a component of the 40 S ribosomal subunit and acting as a target of TOR. Here, we report that RPS6 may have a novel function via interaction with histone deacetylase 2B (AtHD2B) that belongs to the plant-specific histone deacetylase HD2 family. RPS6 and AtHD2B were localized to the nucleolus. Co-expression of RPS6 and AtHD2B caused a change in the location of both RPS6 and AtHD2B to one or several nucleolar spots. ChIP analysis suggests that RPS6 directly interacts with the rRNA gene promoter. Protoplasts overexpressing both AtHD2B and RPS6 exhibited down-regulation of pre-18 S rRNA synthesis with a concomitant decrease in transcription of some of the ribosomal proteins, suggesting their direct role in ribosome biogenesis and plant development. This is consistent with the mutation in rps6b that results in reduction in 18 S rRNA transcription and decreased root growth. We propose that the interaction between RPS6 and AtHD2B brings about a change in the chromatin structure of rDNA and thus plays an important role in linking TOR signaling to rDNA transcription and ribosome biogenesis in plants.

The molecular mechanisms underlying plant cell totipotency are largely unknown. Here, we present a protocol for the efficient regeneration of plants from Arabidopsis thaliana protoplasts. The specific liquid medium used in our study leads to a high rate of reentry into the cell cycle of most cell types, providing a powerful system to study dedifferentiation/regeneration processes in independent somatic cells. To identify the early events in the establishment of totipotency, we monitored the genome-wide transcript profiles of plantlets and protoplast-derived cells (PdCs) during the first week of culture. Plant cells rapidly dedifferentiated. Then, we observed the reinitiation and reorientation of protein synthesis, accompanied by the reinitiation of cell division and de novo cell wall synthesis. Marked changes in the expression of chromatin-associated genes, especially of those in the histone variant family, were observed during protoplast culture. Surprisingly, the epigenetic status of PdCs and well-established cell cultures differed, with PdCs exhibiting rare reactivated transposons and epigenetic changes. The differentially expressed genes identified in this study are interesting candidates for investigating the molecular mechanisms underlying plant cell plasticity and totipotency. One of these genes, the plant-specific transcription factor ABERRANT LATERAL ROOT FORMATION4, is required for the initiation of protoplast division.

Background: Quantitative information on gene activity at single cell-type resolution is essential for the understanding of how cells work and interact. Root hairs, or trichoblasts, tubular-shaped outgrowths of specialized cells in the epidermis, represent an ideal model for cell fate acquisition and differentiation in plants., Results: Here, we provide an atlas of gene and protein expression in Arabidopsis root hair cells, generated by paired-end RNA sequencing and LC/MS-MS analysis of protoplasts from plants containing a pEXP7-GFP reporter construct. In total, transcripts of 23,034 genes were detected in root hairs. High-resolution proteome analysis led to the reliable identification of 2,447 proteins, 129 of which were differentially expressed between root hairs and non-root hair tissue. Dissection of pre-mRNA splicing patterns showed that all types of alternative splicing were cell type-dependent, and less complex in EXP7-expressing cells when compared to non-root hair cells. Intron retention was repressed in several transcripts functionally related to root hair morphogenesis, indicative of a cell type-specific control of gene expression by alternative splicing of pre-mRNA. Concordance between mRNA and protein expression was generally high, but in many cases mRNA expression was not predictive for protein abundance., Conclusions: The integrated analysis shows that gene activity in root hairs is dictated by orchestrated, multilayered regulatory mechanisms that allow for a cell type-specific composition of functional components.

Hardly anything is known about translational control of plant mitochondrial gene expression. Here, we provide evidence for differential translation of mitochondrial transcripts in Arabidopsis thaliana. We found that silencing of the nuclear RPS10 gene encoding mitochondrial ribosomal protein S10 disturbs the ratio between the small and large subunits of mitoribosomes, with an excess of the latter. Moreover, a portion of the small subunits are incomplete, lacking at least the S10 protein. rps10 cells also have an increased mitochondrial DNA copy number per cell, causing an upregulation of all mitochondrial transcripts. Mitochondrial translation is also altered so that it largely overrides the hyperaccumulation of transcripts, and as a consequence, only ribosomal proteins are oversynthesized, whereas oxidative phosphorylation subunits are downregulated. Expression of nuclear-encoded components of mitoribosomes and oxidative phosphorylation system (OXPHOS) complexes seems to be less affected. The ultimate coordination of expression of the nuclear and mitochondrial genomes occurs at the complex assembly level. These findings indicate that mitoribosomes can regulate gene expression by varying the efficiency of translation of mRNAs for OXPHOS and ribosomal proteins.

Phototropins (phot1 and phot2), blue light-receptor protein kinases in plants, mediate stomatal opening by activating the plasma membrane H(+)-ATPase in guard cells, but the signaling from phototropins to the H(+)-ATPase remains unknown. A recent study concluded that ROOT PHOTOTROPISM2 (RPT2) is involved in the primary step of this process. However, this conclusion is based solely on the determination of stomatal apertures in the epidermis. We investigated the role of RPT2 in blue light-dependent stomatal opening in more detail. We generated double mutants of rpt2 and phototropins (phot1 or phot2) in the Col ecotype background and obtained the typical phenotypes of rpt2 mutants, including the impairment in phototropism. In contrast, neither blue light-dependent H(+) pumping nor blue light-dependent H(+)-ATPase activation in guard cells was affected in the rpt2 mutants of rpt2, phot1 rpt2, and phot2 rpt2. Stomata in these rpt2 mutants opened widely by blue light in both epidermal peels and intact leaves, and no difference in the responses was found between the wild type and the mutants. From these results, we concluded that RPT2 gene disruption does not affect blue light-dependent stomatal opening., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

ADP-ribosylation factor1 (Arf1), a member of the small GTP-binding proteins, plays a pivotal role in protein trafficking to multiple organelles. In its GDP-bound form, Arf1 is recruited from the cytosol to organelle membranes, where it functions in vesicle-mediated protein trafficking. However, the mechanism of Arf1-GDP recruitment remains unknown. Here, we provide evidence that two Glo3p-type Arf GTPase-activating proteins (ArfGAPs), ArfGAP domain8 (AGD8) and AGD9, are involved in the recruitment of Arf1-GDP to the Golgi apparatus in Arabidopsis (Arabidopsis thaliana). RNA interference plants expressing low levels of AGD8 and AGD9 exhibited abnormal Golgi morphology, inhibition of protein trafficking, and arrest of plant growth and development. In RNA interference plants, Arf1 was poorly recruited to the Golgi apparatus. Conversely, high levels of AGD8 and AGD9 induced Arf1 accumulation at the Golgi and suppressed Golgi disruption and inhibition of vacuolar trafficking that was caused by overexpression of AGD7. Based on these results, we propose that the Glo3p-type ArfGAPs AGD8 and AGD9 recruit Arf1-GDP from the cytosol to the Golgi for Arf1-mediated protein trafficking, which is essential for plant development and growth.

In eukaryotic cells, protein trafficking plays an essential role in biogenesis of proteins that belong to the endomembrane compartments. In this process, an important step is the sorting of organellar proteins depending on their final destinations. For vacuolar proteins, vacuolar sorting receptors (VSRs) and receptor homology-transmembrane-RING H2 domain proteins (RMRs) are thought to be responsible. Arabidopsis (Arabidopsis thaliana) contains seven VSRs. Among them, VSR1, VSR3, and VSR4 are involved in sorting storage proteins targeted to the protein storage vacuole (PSV) in seeds. However, the identity of VSRs for soluble proteins of the lytic vacuole in vegetative cells remains controversial. Here, we provide evidence that VSR1, VSR3, and VSR4 are involved in sorting soluble lytic vacuolar and PSV proteins in vegetative cells. In protoplasts from leaf tissues of vsr1vsr3 and vsr1vsr4 but not vsr5vsr6, and rmr1rmr2 and rmr3rmr4 double mutants, soluble lytic vacuolar (Arabidopsis aleurain-like protein:green fluorescent protein [GFP] and carboxypeptidase Y:GFP and PSV (phaseolin) proteins, but not the vacuolar membrane protein Arabidopsis βFructosidase4:GFP, exhibited defects in their trafficking; they accumulated to the endoplasmic reticulum with an increased secretion into medium. The trafficking defects in vsr1vsr4 protoplasts were rescued by VSR1 or VSR4 but not VSR5 or AtRMR1. Furthermore, of the luminal domain swapping mutants between VSR1 and VSR5, the mutant with the luminal domain of VSR1, but not that of VSR5, rescued the trafficking defects of Arabidopsis aleurain-like protein:GFP and phaseolin in vsr1vsr4 protoplasts. Based on these results, we propose that VSR1, VSR3, and VSR4, but not other VSRs, are involved in sorting soluble lytic vacuolar and PSV proteins for their trafficking to the vacuoles in vegetative cells.

Di- and tripeptide transporters of the PTR/NRT1 (peptide transporter/nitrate transporter1)-family are localized either at the tonoplast (TP) or plasma membrane (PM). As limited information is available on structural determinants required for targeting of plant membrane proteins, we performed gene shuffling and domain swapping experiments of Arabidopsis PTRs. A 7 amino acid fragment of the hydrophilic N-terminal region of PTR2, PTR4 and PTR6 was required for TP localization and sufficient to redirect not only PM-localized PTR1 or PTR5, but also sucrose transporter SUC2 to the TP. Alanine scanning mutagenesis identified L(11) and I(12) of PTR2 to be essential for TP targeting, while only one acidic amino acid at position 5, 6 or 7 was required, revealing a dileucine (LL or LI) motif with at least one upstream acidic residue. Similar dileucine motifs could be identified in other plant TP transporters, indicating a broader role of this targeting motif in plants. Targeting to the PM required the loop between transmembrane domain 6 and 7 of PTR1 or PTR5. Deletion of either PM or TP targeting signals resulted in retention in internal membranes, indicating that PTR trafficking to these destination membranes requires distinct signals and is in both cases not by default., (© 2012 John Wiley & Sons A/S.)

Cytokinesis is the process of partitioning the cytoplasm of a dividing cell, thereby completing mitosis. Cytokinesis in the plant cell is achieved by the formation of a new cell wall between daughter nuclei using components carried in Golgi-derived vesicles that accumulate at the midplane of the phragmoplast and fuse to form the cell plate. Proteins that play major roles in the development of the cell plate in plant cells are not well defined. Here, we report that an AP180 amino-terminal homology/epsin amino-terminal homology domain-containing protein from Arabidopsis (Arabidopsis thaliana) is involved in clathrin-coated vesicle formation from the cell plate. Arabidopsis Epsin-like Clathrin Adaptor1 (AtECA1; At2g01600) and its homologous proteins AtECA2 and AtECA4 localize to the growing cell plate in cells undergoing cytokinesis and also to the plasma membrane and endosomes in nondividing cells. AtECA1 (At2g01600) does not localize to nascent cell plates but localizes at higher levels to expanding cell plates even after the cell plate fuses with the parental plasma membrane. The temporal and spatial localization patterns of AtECA1 overlap most closely with those of the clathrin light chain. In vitro protein interaction assays revealed that AtECA1 binds to the clathrin H chain via its carboxyl-terminal domain. These results suggest that these AP180 amino-terminal homology/epsin amino-terminal homology domain-containing proteins, AtECA1, AtECA2, and AtECA4, may function as adaptors of clathrin-coated vesicles budding from the cell plate.

In plants, the ERF/EREBP family of transcriptional regulators plays a key role in adaptation to various biotic and abiotic stresses. These proteins contain a conserved AP2 DNA-binding domain and several uncharacterized motifs. Here, we describe a short motif, termed 'EDLL', that is present in AtERF98/TDR1 and other clade members from the same AP2 sub-family. We show that the EDLL motif, which has a unique arrangement of acidic amino acids and hydrophobic leucines, functions as a strong activation domain. The motif is transferable to other proteins, and is active at both proximal and distal positions of target promoters. As such, the EDLL motif is able to partly overcome the repression conferred by the AtHB2 transcription factor, which contains an ERF-associated amphiphilic repression (EAR) motif. We further examined the activation potential of EDLL by analysis of the regulation of flowering time by NF-Y (nuclear factor Y) proteins. Genetic evidence indicates that NF-Y protein complexes potentiate the action of CONSTANS in regulation of flowering in Arabidopsis; we show that the transcriptional activation function of CONSTANS can be substituted by direct fusion of the EDLL activation motif to NF-YB subunits. The EDLL motif represents a potent plant activation domain that can be used as a tool to confer transcriptional activation potential to heterologous DNA-binding proteins., (© 2012 Mendel Biotechnology Inc. The Plant Journal © 2012 Blackwell Publishing Ltd.)

The expression of auxin-responsive genes is regulated by the TIR1/AFB auxin receptor-dependent degradation of Aux/IAA transcriptional repressors, which interact with auxin-responsive factors (ARFs). Most of the 29 Aux/IAA genes present in Arabidopsis have not been functionally characterized to date. IAA8 appears to have a distinct function from the other Aux/IAA genes, due to its unique transcriptional response to auxin and the stability of its encoded protein. In this study, we characterized the function of Arabidopsis IAA8 in various developmental processes governed by auxin and in the transcriptional regulation of the auxin response. Transgenic plants expressing estrogen-inducible IAA8 (XVE::IAA8) exhibited significantly fewer lateral roots than the wild type, and an IAA8 loss-of-function mutant exhibited significantly more. Ectopic overexpression of IAA8 resulted in abnormal gravitropism. The strong induction of early auxin-responsive marker genes by auxin treatment was delayed by IAA8 overexpression. GFP-fusion analysis revealed that IAA8 localized not only to the nucleus, but, in contrast to other Aux/IAAs, also to the cytosol. Furthermore, we demonstrated that IAA8 interacts with TIR1, in an auxin-dependent fashion, and with ARF proteins, both in yeast and in planta. Taken together, our results show that IAA8 is involved in lateral root formation, and that this process is regulated through the interaction with the TIR1 auxin receptor and ARF transcription factors in the nucleus.

Many plant host factors are known to interact with viral proteins during pathogenesis, but how a plant virus induces a specific disease symptom still needs further research. A lily strain of Cucumber mosaic virus (CMV-HL) can induce discrete necrotic spots on infected Arabidopsis (Arabidopsis thaliana) plants; other CMV strains can induce similar spots, but they are not as distinct as those induced by CMV-HL. The CMV 2b protein (2b), a known RNA-silencing suppressor, is involved in viral movement and symptom induction. Using in situ proximity ligation assay immunostaining and the protoplast assays, we report here that CMV 2b interacts directly with Catalase3 (CAT3) in infected tissues, a key enzyme in the breakdown of toxic hydrogen peroxide. Interestingly, CAT3, normally localized in the cytoplasm (glyoxysome), was recruited to the nucleus by an interaction between 2b and CAT3. Although overexpression of CAT3 in transgenic plants decreased the accumulation of CMV and delayed viral symptom development to some extent, 2b seems to neutralize the cellular catalase contributing to the host defense response, thus favoring viral infection. Our results thus provide evidence that, in addition to altering the type of symptom by disturbing microRNA pathways, 2b can directly bind to a host factor that is important in scavenging cellular hydrogen peroxide and thus interfere specifically with that host factor, leading to the induction of a specific necrosis.

Higher plants contain biologically active proteins that are recognized by antibodies against human atrial natriuretic peptide (ANP). We identified and isolated two Arabidopsis thaliana immunoreactive plant natriuretic peptide (PNP)-encoding genes, AtPNP-A and AtPNP-B, which are distantly related members of the expansin superfamily and have a role in the regulation of homeostasis in abiotic and biotic stresses, and have shown that AtPNP-A modulates the effects of ABA on stomata. Arabidopsis PNP (PNP-A) is mainly expressed in leaf mesophyll cells, and in protoplast assays we demonstrate that it is secreted using AtPNP-A:green fluorescent protein (GFP) reporter constructs and flow cytometry. Transient reporter assays provide evidence that AtPNP-A expression is enhanced by heat, osmotica and salt, and that AtPNP-A itself can enhance its own expression, thereby generating a response signature diagnostic for paracrine action and potentially also autocrine effects. Expression of native AtPNP-A is enhanced by osmotica and transiently by salt. Although AtPNP-A expression is induced by salt and osmotica, ABA does not significantly modulate AtPNP-A levels nor does recombinant AtPNP-A affect reporter expression of the ABA-responsive RD29A gene. Together, these results provide experimental evidence that AtPNP-A is stress responsive, secreted into the apoplastic space and can enhance its own expression. Furthermore, our findings support the idea that AtPNP-A, together with ABA, is an important component in complex plant stress responses and that, much like in animals, peptide signaling molecules can create diverse and modular signals essential for growth, development and defense under rapidly changing environmental conditions.

Plant architecture is a predictable but flexible trait. The timing and position of organ initiation from the shoot apical meristem (SAM) contribute to the final plant form. While much progress has been made recently in understanding how the site of leaf initiation is determined, the mechanism underlying the temporal interval between leaf primordia is still largely unknown. The Arabidopsis ZRIZI (ZRZ) gene belongs to a large gene family encoding multidrug and toxic compound extrusion (MATE) transporters. Unique among plant MATE transporters identified so far, ZRZ is localized to the membrane of a small organelle, possibly the mitochondria. Plants overexpressing ZRZ in initiating leaves are short, produce leaves much faster than wild-type plants and show enhanced growth of axillary buds. These results suggest that ZRZ is involved in communicating a leaf-borne signal that determines the rate of organ initiation., (© The Author 2011. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.)

Arabidopsis thaliana AtNUDT7 Nudix pyrophosphatase hydrolyzes NADH and ADP-ribose in vitro and is an important factor in the cellular response to diverse biotic and abiotic stresses. Several studies have shown that loss-of-function Atnudt7 mutant plants display many profound phenotypes. However the molecular mechanism of AtNUDT7 function remains elusive. To gain a better understanding of this hydrolase cellular role, proteins interacting with AtNUDT7 were identified. Using AtNUDT7 as a bait in an in vitro binding assay of proteins derived from cultured Arabidopsis cell extracts we identified the regulatory protein RACK1A as an AtNUDT7-interactor. RACK1A-AtNUDT7 interaction was confirmed in a yeast two-hybrid assay and in a pull-down assay and in Bimolecular Fluorescence Complementation (BiFC) analysis of the proteins transiently expressed in Arabidopsis protoplasts. However, no influence of RACK1A on AtNUDT7 hydrolase catalytic activity was observed. In vitro interaction between RACK1A and the AGG1 and AGG2 gamma subunits of the signal transducing heterotrimeric G protein was also detected and confirmed in BiFC assays. Moreover, association between AtNUDT7 and both AGG1 and AGG2 subunits was observed in Arabidopsis protoplasts, although binding of these proteins could not be detected in vitro. Based on the observed interactions we conclude that the AtNUDT7 Nudix hydrolase forms complexes in vitro and in vivo with regulatory proteins involved in signal transduction. Moreover, we provide the initial evidence that both signal transducing gamma subunits bind the regulatory RACK1A protein.

Protein-protein interactions are an important aspect of the gene regulation process. The expression of a gene in response to certain stimuli, within a specific cell type or at a particular developmental stage, involves a complex network of interactions between different regulatory proteins and the cis-regulatory elements present in the promoter of the gene. A number of methods have been developed to study protein-protein interactions in vitro and in vivo in plant cells, one of which is bimolecular fluorescence complementation (BiFC). BiFC is a relatively simple technique based upon the reconstitution of a fluorescent protein. The interacting protein complex can be visualized directly in a living plant cell when two non-fluorescent fragments, of an otherwise fluorescent protein, are fused to proteins found within that complex. Interaction of tagged proteins brings the two non-fluorescent fragments into close proximity and reconstitutes the fluorescent protein. In addition, the subcellular location of an interacting protein complex in the cell can be simultaneously determined. Using this approach, we have successfully demonstrated a protein-protein interaction between a R2R3 MYB and a basic helix-loop-helix MYC transcription factor related to flavonoid biosynthetic pathway in tobacco protoplasts.

In Arabidopsis, CORYNE (CRN), a new member of the receptor kinase family, was recently isolated as a key player involved in the CLAVATA3 (CLV3) signaling pathway, thereby playing an important role in regulating the development of shoot and root apical meristems. However, the precise relationships among CLAVATA1 (CLV1), CLAVATA2 (CLV2), and CRN receptors remain unclear. Here, we demonstrate the subcellular localization of CRN and analyze the interactions among CLV1, CLV2, and CRN using firefly luciferase complementation imaging (LCI) assays in both Arabidopsis mesophyll protoplasts and Nicotiana benthamiana leaves. Fluorescence targeting showed that CRN was localized to the plasma membrane. The LCI assays coupled with co-immunoprecipitation assays demonstrated that CLV2 can directly interact with CRN in the absence of CLV3. Additional LCI assays showed that CLV1 did not interact with CLV2, but can interact weakly with CRN. We also found that CLV1 can interact with CLV2-CRN heterodimers, implying that these three proteins may form a complex. Moreover, CRN, rather than CLV1 and CLV2, was able to form homodimers without CLV3 stimulation. Taken together, our results add direct evidence to the newly proposed two-parallel receptor pathways model and therefore provide new insights into the CLV3 signaling pathway.

The last steps of the Leu biosynthetic pathway and the Met chain elongation cycle for glucosinolate formation share identical reaction types suggesting a close evolutionary relationship of these pathways. Both pathways involve the condensation of acetyl-CoA and a 2-oxo acid, isomerization of the resulting 2-malate derivative to form a 3-malate derivative, the oxidation-decarboxylation of the 3-malate derivative to give an elongated 2-oxo acid, and transamination to generate the corresponding amino acid. We have now analyzed the genes encoding the isomerization reaction, the second step of this sequence, in Arabidopsis thaliana. One gene encodes the large subunit and three encode small subunits of this enzyme, referred to as isopropylmalate isomerase (IPMI) with respect to the Leu pathway. Metabolic profiling of large subunit mutants revealed accumulation of intermediates of both Leu biosynthesis and Met chain elongation, and an altered composition of aliphatic glucosinolates demonstrating the function of this gene in both pathways. In contrast, the small subunits appear to be specialized to either Leu biosynthesis or Met chain elongation. Green fluorescent protein tagging experiments confirms the import of one of the IPMI small subunits into the chloroplast, the localization of the Met chain elongation pathway in these organelles. These results suggest the presence of different heterodimeric IPMIs in Arabidopsis chloroplasts with distinct substrate specificities for Leu or glucosinolate metabolism determined by the nature of the different small subunit.

The Anaphase Promoting Complex (APC) controls CDK activity by targeting the ubiquitin-dependent proteolysis of S-phase and mitosis-promoting cyclins. Here, we report that the ectopic expression of the Arabidopsis CDC27a, an APC subunit, accelerates plant growth and results in plants with increased biomass production. CDC27a overexpression was associated to apical meristem restructuration, protoplasts with higher (3)H-thimidine incorporation and altered cell-cycle marker expression. Total protein extracts immunoprecipitated with a CDC27a antibody showed ubiquitin ligase activity, indicating that the Arabidopsis CDC27a gets incorporated into APC complexes. These results indicate a role of AtCDC27a in regulation of plant growth and raise the possibility that the activity of the APC and the rates of plant cell division could be regulated by the concentration of the CDC27a subunit.

Nitrate, one of the major nitrogen sources for plants, is stored in the vacuole. Nitrate accumulation within the vacuole is primarily mediated by the NO(3)(-)/H(+) exchanger AtCLCa, which belongs to the chloride channel (CLC) family. Crystallography analysis of hCLC5 suggested that the C-terminal domain, composed by two cystathionine beta-synthetase motifs in all eukaryotic members of the CLC family is able to interact with ATP. However, interaction of nucleotides with a functional CLC protein has not been unambiguously demonstrated. Here we show that ATP reversibly inhibits AtCLCa by interacting with the C-terminal domain. Applying the patch clamp technique to isolated Arabidopsis thaliana vacuoles, we demonstrate that ATP reduces AtCLCa activity with a maximum inhibition of 60%. ATP inhibition of nitrate influx into the vacuole at cytosolic physiological nitrate concentrations suggests that ATP modulation is physiologically relevant. ADP and AMP do not decrease the AtCLCa transport activity; nonetheless, AMP (but not ADP) competes with ATP, preventing inhibition. A molecular model of the C terminus of AtCLCa was built by homology to hCLC5 C terminus. The model predicted the effects of mutations of the ATP binding site on the interaction energy between ATP and AtCLCa that were further confirmed by functional expression of site-directed mutated AtCLCa.

In Arabidopsis, bric-a-brac, tramtrack and broad (BTB) domain scaffold proteins form a family of 80 proteins that have involvement in various signaling pathways. The five members of the subfamily of BTB AND TAZ DOMAIN proteins (BT1-BT5) have a typical domain structure that is only observed in land plants. Here, we present a functional analysis of the BT family, of which at least four members are encoded by auxin-responsive genes. BT1 is a short-lived protein that is characteristically targeted for degradation by the 26S proteasome. Expression pattern, gene structure and sequence analyses indicate that BT1 and BT2 are closely related. They both localize to the nucleus and the cytosol, whereas the remaining BT proteins were determined as cytosolic proteins. Detailed molecular and phenotypic analysis of plants segregating for null mutations in the BT family revealed substantial redundancy among the BT members, and highlighted that BT proteins perform crucial roles in both male and female gametophyte development. BT2 seems to be the predominant gene in this process, in which it is functionally replaced by BT3 and BT1 through reciprocal transcription regulation. Compensational expression alters the steady-state mRNA levels among the remaining BT family members when other BT members are lost, and this contributes towards functional redundancy. Our data provide a surprising example of functional redundancy among genes required during gametophyte development, something that could not be detected in the current screens for gametophyte mutants., (© 2009 The Authors. Journal compilation © 2009 Blackwell Publishing Ltd.)

Sorting nexins are conserved proteins that function in vesicular trafficking and contain a characteristic phox homology (PX) domain. Here, we characterize the ubiquitously expressed Arabidopsis thaliana sorting nexin AtSNX2b. Sub-cellular fractionation studies indicate that AtSNX2b is peripherally associated with membranes. The AtSNX2b PX domain binds to phosphatidylinositol 3-phosphate in vitro and this association is required for the localization of GFP-AtSNX2b to punctate structures in vivo, identified as the trans-Golgi network, prevacuolar compartment and endosomes. Overexpression of GFP-tagged AtSNX2b produces enlarged GFP-labeled compartments that can also be labeled by the endocytic tracer FM4-64. Endocytic trafficking of FM4-64 to the vacuole is arrested in these GFP-AtSNX2b compartments, and similar FM4-64-accumulating compartments are seen upon overexpression of untagged AtSNX2b. This suggests that exit of membrane components from these enlarged or aggregated endosomes is inhibited. Vacuolar proteins containing an N-terminal propeptide, but not those with a C-terminal propeptide, are also present in these enlarged compartments. We hypothesize that AtSNX2b is involved in vesicular trafficking from endosomes to the vacuole.

SR proteins are multidomain splicing factors which are important for spliceosome assembly and for regulation of alternative splicing. In mammalian nuclei these proteins localise to speckles from where they are recruited to transcription sites. By using fluorescent protein fusion technology and different experimental approaches it has been shown that Arabidopsis SR proteins, in addition to diffuse nucleoplasmic staining, localise into an irregular nucleoplasmic network resembling speckles in mammalian cells. As Arabidopsis SR proteins fall into seven conserved sub-families we investigated co-localisation of members of the different sub-families in transiently transformed tobacco protoplast. Here we demonstrate the new finding that members of different SR protein sub-families localise into distinct populations of nuclear speckles with no, partial or complete co-localisation. This is particularly interesting as we also show that these proteins do interact in a yeast two-hybrid assay as well as in pull-down and in co-immunopreciptiation assays. Our data raise the interesting possibility that SR proteins are partitioned into distinct populations of nuclear speckles to allow a more specific recruitment to the transcription/pre-mRNA processing sites of particular genes depending on cell type and developmental stage.

In plant cells, the vacuole functions as a major calcium store. Although a calmodulin-regulated Ca2+-ATPase (ACA4) is known to be present in prevacuolar compartments, the presence of an ACA-type Ca2+-ATPase in the mature vacuole of a plant cell has not been verified. Here we provide evidence that ACA11 localizes to the vacuole membrane. ACA11 tagged with GFP was expressed in stable transgenic plants, and visualized in root cells and protoplasts by confocal microscopy. A Ca2+-ATPase function for ACA11 was confirmed by complementation of yeast mutants. A calmodulin binding domain was identified within the first 37 residues of the N-terminal autoinhibitory region.

In plants one bona fide Galpha subunit has been identified, as well as a single Gbeta and two Ggamma subunits. To study the roles of lipidation motifs in the regulation of subcellular location and heterotrimer formation in living plant cells, GFP-tagged versions of the Arabidopsis thaliana heterotrimeric G protein subunits were constructed. Mutational analysis showed that the Arabidopsis Galpha subunit, GPalpha1, contains two lipidation motifs that were essential for plasma membrane localization. The Arabidopsis Gbeta subunit, AGbeta1, and the Ggamma subunit, AGG1, were dependent upon each other for tethering to the plasma membrane. The second Ggamma subunit, AGG2, did not require AGbeta1 for localization to the plasma membrane. Like AGG1, AGG2 contains two putative lipidation motifs, both of which were necessary for membrane localization. Interaction between the subunits was studied using fluorescence resonance energy transfer (FRET) imaging by means of fluorescence lifetime imaging microscopy (FLIM). The results suggest that AGbeta1 and AGG1 or AGbeta1 and AGG2 can form heterodimers independent of lipidation. In addition, FLIM-FRET revealed the existence of GPalpha1-AGbeta1-AGG1 heterotrimers at the plasma membrane. Importantly, rendering GPalpha1 constitutively active did not cause a FRET decrease in the heterotrimer, suggesting no dissociation upon GPalpha1 activation.

The tonoplast monosaccharide transporter (TMT) family comprises three isoforms in Arabidopsis thaliana, and TMT-green fluorescent protein fusion proteins are targeted to the vacuolar membrane. TMT promoter-beta-glucuronidase plants revealed that the TONOPLAST MONOSACCHARIDE TRANSPORTER1 (TMT1) and TMT2 genes exhibit a tissue- and cell type-specific expression pattern, whereas TMT3 is only weakly expressed. TMT1 and TMT2 expression is induced by drought, salt, and cold treatments and by sugar. During cold adaptation, tmt knockout lines accumulated less glucose and fructose compared with wild-type plants, whereas no differences were observed for sucrose. Cold adaptation of wild-type plants substantially promoted glucose uptake into isolated leaf mesophyll vacuoles. Glucose uptake into isolated vacuoles was inhibited by NH(4)(+), fructose, and phlorizin, indicating that transport is energy-dependent and that both glucose and fructose were taken up by the same carrier. Glucose import into vacuoles from two cold-induced tmt1 knockout lines or from triple knockout plants was substantially lower than into corresponding wild-type vacuoles. Monosaccharide feeding into leaf discs revealed the strongest response to sugar in tmt1 knockout lines compared with wild-type plants, suggesting that TMT1 is required for cytosolic glucose homeostasis. Our results indicate that TMT1 is involved in vacuolar monosaccharide transport and plays a major role during stress responses.

In plants, the chromatin-associated high mobility group (HMG) proteins occur in two subfamilies termed HMGA and HMGB. The HMGA proteins are characterized by the presence of four AT-hook DNA binding motifs, and the HMGB proteins contain an HMG box DNA binding domain. As architectural factors, the HMG proteins appear to be involved in the regulation of transcription and other DNA-dependent processes. We have examined the subcellular localization of Arabidopsis thaliana HMGA, HMGB1, and HMGB5, revealing that they localize to the cell nucleus. They display a speckled distribution pattern throughout the chromatin of interphase nuclei, whereas none of the proteins associate with condensed mitotic chromosomes. HMGA is targeted to the nucleus by a monopartite nuclear localization signal, while efficient nuclear accumulation of HMGB1/5 requires large portions of the basic N-terminal part of the proteins. The acidic C-terminal domain interferes with nucleolar targeting of HMGB1. Fluorescence recovery after photobleaching experiments revealed that HMGA and HMGB proteins are extremely dynamic in the nucleus, indicating that they bind chromatin only transiently before moving on to the next site, thereby continuously scanning the genome for targets. By contrast, the majority of histone H2B is basically immobile within the nucleus, while linker histone H1.2 is relatively mobile.

S-Adenosylmethionine (SAM) is formed exclusively in the cytosol but plays a major role in plastids; SAM can either act as a methyl donor for the biogenesis of small molecules such as prenyllipids and macromolecules or as a regulator of the synthesis of aspartate-derived amino acids. Because the biosynthesis of SAM is restricted to the cytosol, plastids require a SAM importer. However, this transporter has not yet been identified. Here, we report the molecular and functional characterization of an Arabidopsis thaliana gene designated SAM TRANSPORTER1 (SAMT1), which encodes a plastid metabolite transporter required for the import of SAM from the cytosol. Recombinant SAMT1 produced in yeast cells, when reconstituted into liposomes, mediated the counter-exchange of SAM with SAM and with S-adenosylhomocysteine, the by-product and inhibitor of transmethylation reactions using SAM. Insertional mutation in SAMT1 and virus-induced gene silencing of SAMT1 in Nicotiana benthamiana caused severe growth retardation in mutant plants. Impaired function of SAMT1 led to decreased accumulation of prenyllipids and mainly affected the chlorophyll pathway. Biochemical analysis suggests that the latter effect represents one prominent example of the multiple events triggered by undermethylation, when there is decreased SAM flux into plastids.

Fluorescent cell division cycle (CDC)48 proteins were studied in living plant protoplasts. CDC48A and somatic embryogenesis receptor like kinase 1 (SERK1) were found to co-localize in the endoplasmatic reticulum (ER) and at the plasma membrane (PM), but not in endosomal compartments. Fluorescent lifetime imaging microscopy (FLIM) was used to detect Förster resonance energy transfer (FRET) between CrFP/YFP-tagged CDC48A and SERK1. FRET is indicative of direct protein-protein interaction. CDC48A was found to interact only with SERK1 in small areas at the PM, but not in endosomes. These findings confirm and extend our previous findings that CDC48A in plants directly interacts with SERK1.

For the full activation of cyclin-dependent kinases (CDKs), not only cyclin binding but also phosphorylation of a threonine (Thr) residue within the T-loop is required. This phosphorylation is catalyzed by CDK-activating kinases (CAKs). In Arabidopsis three D-type CDK genes (CDKD;1-CDKD;3) encode vertebrate-type CAK orthologues, of which CDKD;2 exhibits high phosphorylation activity towards the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II. Here, we show that CDKD;2 forms a stable complex with cyclin H and is downregulated by the phosphorylation of the ATP-binding site by WEE1 kinase. A knockout mutant of CDKD;3, which has a higher CDK kinase activity, displayed no defect in plant development. Instead, another type of CAK - CDKF;1 - exhibited significant activity towards CDKA;1 in Arabidopsis root protoplasts, and the activity was dependent on the T-loop phosphorylation of CDKF;1. We propose that two distinct types of CAK, namely CDKF;1 and CDKD;2, play a major role in CDK and CTD phosphorylation, respectively, in Arabidopsis.

In plants, excess reactive oxygen species are toxic molecules induced under environmental stresses, including pathogen invasions and abiotic stresses. Many anti-oxidant defense systems have been reported to require NADPH as an important reducing energy equivalent. However, the sources of NADPH and the molecular mechanisms of maintaining cytoplasmic redox balance are unclear. Here, we report the biological function of a putative cytoplasmic NADH kinase (NADK3) in several abiotic stress responses in Arabidopsis. We found that cytoplasmic NADPH is provided mostly by the product of the NADK3 gene in Arabidopsis. Expression of he NADK3 gene is responsive to abscisic acid (ABA) and abiotic stress conditions, including methyl violgen (MV), high salinity and osmotic shock. An NADK3 null mutant showed hypersensitivity to oxidative stress in both seed germination and seedling growth. Seed germination of the mutant plants also showed increased sensitivity to ABA, salt and mannitol. Furthermore, stress-related target genes were identified as upregulated in the mutant by mannitol and MV. Our study indicates that this cytoplasmic NADH kinase, a key source of the cellular reductant NADPH, is required for various abiotic stress responses.

Boron (B) is essential in plants but often present at low concentrations in the environment. To investigate how plants survive under conditions of B limitation, we conducted a transcriptome analysis and identified NIP5;1, a member of the major intrinsic protein family, as a gene upregulated in B-deficient roots of Arabidopsis thaliana. Promoter-beta-glucuronidase fusions indicated that NIP5;1 is strongly upregulated in the root elongation zone and the root hair zone under B limitation, and green fluorescent protein-tagged NIP5;1 proteins localized to the plasma membrane. Expression in Xenopus laevis oocytes demonstrated that NIP5;1 facilitated the transport of boric acid in addition to water. Importantly, two T-DNA insertion lines of NIP5;1 displayed lower boric acid uptake into roots, lower biomass production, and increased sensitivity of root and shoot development to B deficiency. These results identify NIP5;1 as a major plasma membrane boric acid channel crucial for the B uptake required for plant growth and development under B limitation.

Mutants that are defective in brassinosteroid (BR) biosynthesis or signaling display severely retarded growth patterns due to absence of growth-promoting effects by BRs. Arabidopsis (Arabidopsis thaliana) DWARF4 (DWF4) catalyzes a flux-determining step in the BR biosynthetic pathways. Thus, it is hypothesized that the tissues of DWF4 expression may represent the sites of BR biosynthesis in Arabidopsis. Here we show that DWF4 transcripts accumulate in the actively growing tissues, such as root, shoot apices with floral clusters, joint tissues of root and shoot, and dark-grown seedlings. Conforming to the RNA gel-blot analysis, DWF4:beta-glucuronidase (GUS) histochemical analyses more precisely define the tissues that express the DWF4 gene. Examination of the endogenous levels of BRs in six and seven different tissues of wild type and brassinosteroid insensitive1-5 mutant, respectively, revealed that BRs are significantly enriched in roots, shoot tips, and joint tissues of roots and shoots. In addition, DWF4:GUS expression was negatively regulated by BRs. DWF4:GUS activity was increased by treatment with brassinazole, a BR biosynthetic inhibitor, and decreased by exogenous application of bioactive BRs. When DWF4:GUS was expressed in a different genetic background, its level was down-regulated in brassinazole resistant1-D, confirming that BRASSINAZOLE RESISTANT1 acts as a negative regulator of DWF4. Interestingly, in the brassinosteroid insensitive2/dwf12-1D background, DWF4:GUS expression was intensified and delocalized to elongating zones of root, suggesting that BRASSINOSTEROID INSENSITIVE2 is an important factor that limits DWF4 expression. Thus, it is likely that the DWF4 promoter serves as a focal point in maintaining homeostasis of endogenous bioactive BR pools in specific tissues of Arabidopsis.

The transit peptides of nuclear-encoded chloroplast proteins are necessary and sufficient for targeting and import of proteins into chloroplasts. However, the sequence information encoded by transit peptides is not fully understood. In this study, we investigated sequence motifs in the transit peptide of the small subunit of the Rubisco complex by examining the ability of various mutant transit peptides to target green fluorescent protein reporter proteins to chloroplasts in Arabidopsis (Arabidopsis thaliana) leaf protoplasts. We divided the transit peptide into eight blocks (T1 through T8), each consisting of eight or 10 amino acids, and generated mutants that had alanine (Ala) substitutions or deletions, of one or two T blocks in the transit peptide. In addition, we generated mutants that had the original sequence partially restored in single- or double-T-block Ala (A) substitution mutants. Analysis of chloroplast import of these mutants revealed several interesting observations. Single-T-block mutations did not noticeably affect targeting efficiency, except in T1 and T4 mutations. However, double-T mutants, T2A/T4A, T3A/T6A, T3A/T7A, T4A/T6A, and T4A/T7A, caused a 50% to 100% loss in targeting ability. T3A/T6A and T4A/T6A mutants produced only precursor proteins, whereas T2A/T4A and T4A/T7A mutants produced only a 37-kD protein. Detailed analyses revealed that sequence motifs ML in T1, LKSSA in T3, FP and RK in T4, CMQVW in T6, and KKFET in T7 play important roles in chloroplast targeting. In T1, the hydrophobicity of ML is important for targeting. LKSSA in T3 is functionally equivalent to CMQVW in T6 and KKFET in T7. Furthermore, subcellular fractionation revealed that Ala substitution in T1, T3, and T6 produced soluble precursors, whereas Ala substitution in T4 and T7 produced intermediates that were tightly associated with membranes. These results demonstrate that the transit peptide contains multiple motifs and that some of them act in concert or synergistically.

Cellular and physiological evidence suggests the presence of a novel class of systemically mobile plant molecules that are recognized by antibodies against vertebrate atrial natriuretic peptides (ANPs). In order to characterize the function of these immunoanalogues we have expressed the full-length recombinant (AtPNP-A[1-126]) and demonstrate that this molecule induces osmoticum-dependent H(2)O uptake into protoplasts at nanomolar concentrations and thus affects cell volume. A similar response is also seen with a recombinant that does not contain the signal peptide (AtPNP-A[26-126]) as well as a short domain (AtPNP-A[33-66]) that shows homology to the vertebrate peptide. Taken together, these findings suggest that AtPNP-A has an important and systemic role in plant growth and homeostasis.

Null-mutations of the Arabidopsis FKBP-like immunophilin TWISTED DWARF1 (TWD1) gene cause a pleiotropic phenotype characterized by reduction of cell elongation and disorientated growth of all plant organs. Heterologously expressed TWD1 does not exhibit cis-trans-peptidylprolyl isomerase (PPIase) activity and does not complement yeast FKBP12 mutants, suggesting that TWD1 acts indirectly via protein-protein interaction. Yeast two-hybrid protein interaction screens with TWD1 identified cDNA sequences that encode the C-terminal domain of Arabidopsis multidrug-resistance-like ABC transporter AtPGP1. This interaction was verified in vitro. Mapping of protein interaction domains shows that AtPGP1 surprisingly binds to the N-terminus of TWD1 harboring the cis-trans peptidyl-prolyl isomerase-like domain and not to the tetratrico-peptide repeat domain, which has been shown to mediate protein-protein interaction. Unlike all other FKBPs, TWD1 is shown to be an integral membrane protein that colocalizes with its interacting partner AtPGP1 on the plasma membrane. TWD1 also interacts with AtPGP19 (AtMDR1), the closest homologue of AtPGP1. The single gene mutation twd1-1 and double atpgp1-1/atpgp19-1 (atmdr1-1) mutants exhibit similar phenotypes including epinastic growth, reduced inflorescence size, and reduced polar auxin transport, suggesting that a functional TWD1-AtPGP1/AtPGP19 complex is required for proper plant development.

Compared to mammals, little is known about the unfolded protein response (UPR) in plants. Using an oligonucleotide array comprising approximately 8200 Arabidopsis genes we investigated the effect of endoplasmic reticulum (ER) stress on gene expression. Expression of 26 genes increased, including at least nine whose products act in the ER, while their transcriptional activations were confirmed by promoter analyses. Among them, BiP-L, a novel BiP, whose expression appeared to be regulated by two promoter sequences perfectly matching mammalian ERSE. Cloning and sequencing of full-length BiP-L cDNA showed it contained a signal peptide sequence and the ER retention signal (HDEL). Interestingly, BiP-L was substantially different from the other two Arabidopsis BiP genes in genomic organization and sequence homology. Furthermore, phylogenetic analysis showed that the BiP-L protein is the most distal form among the reported plant BiP proteins. RNA levels of BiP-L were very low in various mature Arabidopsis plant organs, while significant levels of BiP-L only observed in stressed seedlings. Transcription of BiP-L during ER stress was shown to be regulated by a feedback loop.

In eukaryotes, transcription factors of the E2F family, in addition to having a role in cell proliferation, participate in regulating apoptosis, differentiation and development. In Arabidopsis thaliana, eight gene sequences have been identified as encoding E2F or DP homologues. DP proteins form heterodimers with E2Fs. The aim of this work was to characterize the functions of three of these factors: AtE2F-a, AtE2F-b and AtDP-a. Here we report that AtE2F-a and AtE2F-b transactivate a reporter gene via an E2F consensus cis-acting element in Arabidopsis protoplasts. AtE2F-a is a more potent activator than AtE2F-b. Furthermore, co-expression of the E2F partner AtDP-a, or the DNA binding protein AtPur alpha, modulates the activation of AtE2F-a. Taken together, these results suggest that AtE2F-a, AtE2F-b and AtDP-a share features characteristic of members of the E2F family of transcription factors. Moreover, over-expression of AtE2F-a and AtDP-a can induce differentiated, non-dividing, leaf cells to re-enter S-phase. We conclude that the transcription factor AtE2F-a plays an important role in progression into S phase, which probably correlates with its capacity to stimulate transcription.