[show abstract][hide abstract] ABSTRACT: In Arabidopsis thaliana, the R2R3 MYB-like transcription factor MYB30 is a positive regulator of the pathogen-induced hypersensitive response and of brassinosteroid and abscisic acid signaling. Here, we show that MYB30 expressed under the control of the strong phloem-specific SUC2 promoter accelerates flowering both in long and short days. Early flowering is mediated by elevated expression of FLOWERING LOCUS T (FT), which can be observed in the absence and presence of CONSTANS (CO), the main activator of FT. CO-independent activation by high MYB30 expression results in FT levels that remain below those observed in the wild-type plants, which show an additive CO-dependent activation. In contrast, TWIN SISTER OF FT (TSF) is repressed in plants expressing high levels of MYB30 in the phloem. In transient assays, MYB30 and CO additively increase the activity of a reporter construct driven by a 1 kb FT promoter. Acceleration of flowering by MYB30 does not require the presence of salicylic acid and is independent of FLC. Taken together, increased levels of MYB30, which was reported to be induced in response to the perception of pathogens, can accelerate flowering and MYB30 may thus be a candidate to mediate cross-talk between gene networks involved in biotic stress perception and flowering time.
PLoS ONE 01/2014; 9(2):e89799. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: In mammals, the Voltage-dependent anion channels (VDACs) are predominant proteins of the outer mitochondrial membrane (OMM) where they contribute to the exchange of small metabolites essential for respiration. They were shown to be as well associated with the plasma membrane (PM) and act as redox enzyme or are involved in ATP release for example. In Arabidopsis, we show that four out of six genomic sequences encode AtVDAC proteins. All four AtVDACs are ubiquitously expressed in the plant but each of them displays a specific expression pattern in root cell types. Using two complementary approaches, we demonstrate conclusively that the four expressed AtVDACs are targeted to both mitochondria and plasma membrane but in differential abundance, AtVDAC3 being the most abundant in PM, and conversely, AtVDAC4 almost exclusively associated with mitochondria. These are the first plant proteins to be shown to reside in both these two membranes. To investigate a putative function of AtVDACs, we analyzed T-DNA insertion lines in each of the corresponding genes. Knock-out mutants for AtVDAC1, AtVDAC2 and AtVDAC4 present slow growth, reduced fertility and yellow spots in leaves when atvdac3 does not show any visible difference compared to wildtype plants. Analyses of atvdac1 and atvdac4 reveal that yellow areas correspond to necrosis and the mitochondria are swollen in these two mutants. All these results suggest that, in spite of a localization in plasma membrane for three of them, AtVDAC1, AtVDAC2 and AtVDAC4 have a main function in mitochondria.
[show abstract][hide abstract] ABSTRACT: Acyl chain length is thought to be crucial for biophysical properties of the membrane, in particular during cell division, when active vesicular fusion is necessary. In higher plants, the process of cytokinesis is unique, because the separation of the two daughter cells is carried out by de novo vesicular fusion to generate a laterally expanding cell plate. In Arabidopsis thaliana, very-long-chain fatty acid (VLCFA) depletion caused by a mutation in the microsomal elongase gene PASTICCINO2 (PAS2) or by application of the selective elongase inhibitor flufenacet altered cytokinesis. Cell plate expansion was delayed and the formation of the endomembrane tubular network altered. These defects were associated with specific aggregation of the cell plate markers YFP-Rab-A2a and KNOLLE during cytokinesis. Changes in levels of VLCFA also resulted in modification of endocytosis and sensitivity to brefeldin A. Finally, the cytokinesis impairment in pas2 cells was associated with reduced levels of very long fatty acyl chains in phospholipids. Together, our findings demonstrate that VLCFA-containing lipids are essential for endomembrane dynamics during cytokinesis.
[show abstract][hide abstract] ABSTRACT: Sphingolipids are a class of structural membrane lipids involved in membrane trafficking and cell polarity. Functional analysis of the ceramide synthase family in Arabidopsis thaliana demonstrates the existence of two activities selective for the length of the acyl chains. Very-long-acyl-chain (C > 18 carbons) but not long-chain sphingolipids are essential for plant development. Reduction of very-long-chain fatty acid sphingolipid levels leads in particular to auxin-dependent inhibition of lateral root emergence that is associated with selective aggregation of the plasma membrane auxin carriers AUX1 and PIN1 in the cytosol. Defective targeting of polar auxin carriers is characterized by specific aggregation of Rab-A2(a)- and Rab-A1(e)-labeled early endosomes along the secretory pathway. These aggregates correlate with the accumulation of membrane structures and vesicle fragmentation in the cytosol. In conclusion, sphingolipids with very long acyl chains define a trafficking pathway with specific endomembrane compartments and polar auxin transport protein cargoes.
The Plant Cell 06/2011; 23(6):2362-78. · 9.25 Impact Factor
[show abstract][hide abstract] ABSTRACT: Very-long-chain fatty acids (VLCFAs) are essential for many aspects of plant development and necessary for the synthesis of seed storage triacylglycerols, epicuticular waxes, and sphingolipids. Identification of the acetyl-CoA carboxylase PASTICCINO3 and the 3-hydroxy acyl-CoA dehydratase PASTICCINO2 revealed that VLCFAs are important for cell proliferation and tissue patterning. Here, we show that the immunophilin PASTICCINO1 (PAS1) is also required for VLCFA synthesis. Impairment of PAS1 function results in reduction of VLCFA levels that particularly affects the composition of sphingolipids, known to be important for cell polarity in animals. Moreover, PAS1 associates with several enzymes of the VLCFA elongase complex in the endoplasmic reticulum. The pas1 mutants are deficient in lateral root formation and are characterized by an abnormal patterning of the embryo apex, which leads to defective cotyledon organogenesis. Our data indicate that in both tissues, defective organogenesis is associated with the mistargeting of the auxin efflux carrier PIN FORMED1 in specific cells, resulting in local alteration of polar auxin distribution. Furthermore, we show that exogenous VLCFAs rescue lateral root organogenesis and polar auxin distribution, indicating their direct involvement in these processes. Based on these data, we propose that PAS1 acts as a molecular scaffold for the fatty acid elongase complex in the endoplasmic reticulum and that the resulting VLCFAs are required for polar auxin transport and tissue patterning during plant development.
The Plant Cell 02/2010; 22(2):364-75. · 9.25 Impact Factor
[show abstract][hide abstract] ABSTRACT: Flowering of Arabidopsis is induced by long summer days (LDs). The transcriptional regulator CONSTANS (CO) promotes flowering, and its transcription is increased under LDs. We systematically misexpressed transcription factors in companion cells and identified several DOF proteins that delay flowering by repressing CO transcription. Combining mutations in four of these, including CYCLING DOF FACTOR 2 (CDF2), caused photoperiod-insensitive early flowering by increasing CO mRNA levels. CO transcription is promoted to differing extents by GIGANTEA (GI) and the F-box protein FKF1. We show that GI stabilizes FKF1, thereby reducing CDF2 abundance and allowing transcription of CO. Despite the crucial function of GI in wild-type plants, introducing mutations in the four DOF-encoding genes into gi mutants restored the diurnal rhythm and light inducibility of CO. Thus, antagonism between GI and DOF transcription factors contributes to photoperiodic flowering by modulating an underlying diurnal rhythm in CO transcript levels.
[show abstract][hide abstract] ABSTRACT: The putative RNA-binding protein SUPPRESSOR OF GENE SILENCING 3 (SGS3) protects RNA from degradation before transformation into dsRNA by the RNA-dependent RNA polymerase RDR6 during plant post-transcriptional gene silencing and trans-acting small interfering (siRNA) pathways. In this study, we show that SGS3 acts as a homodimer, and that the point mutation sgs3-3 impairs post-transcriptional gene silencing in a dominant-negative manner through the formation of SGS3/sgs3-3 heterodimers. Unlike complete-loss-of-function sgs3 mutants, which are impaired in the accumulation of both micro RNA-directed TAS cleavage products and mature trans-acting siRNAs, the sgs3-3 mutant overaccumulates TAS cleavage products and exhibits slightly reduced trans-acting siRNA accumulation. Together, these results suggest that sgs3-3 is a neomorphic allele that shows increased RNA protective activity, resulting in decreased RNA processing by downstream post-transcriptional gene silencing and trans-acting siRNA pathway components.
[show abstract][hide abstract] ABSTRACT: The SNF1/AMPK/SnRK1 kinases are evolutionary conserved kinases involved in yeast, mammals, and plants in the control of energy balance. These heterotrimeric enzymes are composed of one alpha-type catalytic subunit and two gamma- and beta-type regulatory subunits. In yeast it has been proposed that the beta-type subunits regulate both the localization of the kinase complexes within the cell and the interaction of the kinases with their targets. In this work, we demonstrate that the three beta-type subunits of Arabidopsis (Arabidopsis thaliana; AKINbeta1, AKINbeta2, and AKINbeta3) restore the growth phenotype of the yeast sip1Deltasip2Deltagal83Delta triple mutant, thus suggesting the conservation of an ancestral function. Expression analyses, using AKINbeta promoterbeta-glucuronidase transgenic lines, reveal different and specific patterns of expression for each subunit according to organs, developmental stages, and environmental conditions. Finally, our results show that the beta-type subunits are involved in the specificity of interaction of the kinase with the cytosolic nitrate reductase. Together with previous cell-free phosphorylation data, they strongly support the proposal that nitrate reductase is a real target of SnRK1 in the physiological context. Altogether our data suggest the conservation of ancestral basic function(s) together with specialized functions for each beta-type subunit in plants.
[show abstract][hide abstract] ABSTRACT: The functional genomics approach requires systematic analysis of protein subcellular distribution and interaction networks, preferably by optimizing experimental simplicity and physiological significance. Here, we present an efficient in planta transient transformation system that allows single or multiple expression of constructs containing various fluorescent protein tags in Arabidopsis cotyledons. The optimized protocol is based on vacuum infiltration of agrobacteria directly into young Arabidopsis seedlings. We demonstrate that Arabidopsis epidermal cells show a subcellular distribution of reference markers similar to that in tobacco epidermal cells, and can be used for co-localization or bi-molecular fluorescent complementation studies. We then used this new system to investigate the subcellular distribution of enzymes involved in sphingolipid metabolism. In contrast to transformation systems using tobacco epidermal cells or cultured Arabidopsis cells, our system provides the opportunity to take advantage of the extensive collections of mutant and transgenic lines available in Arabidopsis. The fact that this assay uses conventional binary vectors and a conventional Agrobacterium strain, and is compatible with a large variety of fluorescent tags, makes it a versatile tool for construct screening and characterization before stable transformation. Transient expression in Arabidopsis seedlings is thus a fast and simple method that requires minimum handling and potentially allows medium- to high-throughput analyses of fusion proteins harboring fluorescent tags in a whole-plant cellular context.
The Plant Journal 10/2008; 56(1):169-79. · 6.58 Impact Factor
[show abstract][hide abstract] ABSTRACT: Very-long-chain fatty acids (VLCFAs) are synthesized as acyl-CoAs by the endoplasmic reticulum-localized elongase multiprotein complex. Two Arabidopsis genes are putative homologues of the recently identified yeast 3-hydroxy-acyl-CoA dehydratase (PHS1), the third enzyme of the elongase complex. We showed that Arabidopsis PASTICCINO2 (PAS2) was able to restore phs1 cytokinesis defects and sphingolipid long chain base overaccumulation. Conversely, the expression of PHS1 was able to complement the developmental defects and the accumulation of long chain bases of the pas2-1 mutant. The pas2-1 mutant was characterized by a general reduction of VLCFA pools in seed storage triacylglycerols, cuticular waxes, and complex sphingolipids. Most strikingly, the defective elongation cycle resulted in the accumulation of 3-hydroxy-acyl-CoA intermediates, indicating premature termination of fatty acid elongation and confirming the role of PAS2 in this process. We demonstrated by in vivo bimolecular fluorescence complementation that PAS2 was specifically associated in the endoplasmic reticulum with the enoyl-CoA reductase CER10, the fourth enzyme of the elongase complex. Finally, complete loss of PAS2 function is embryo lethal, and the ectopic expression of PHS1 led to enhanced levels of VLCFAs associated with severe developmental defects. Altogether these results demonstrate that the plant 3-hydroxy-acyl-CoA dehydratase PASTICCINO2 is an essential and limiting enzyme in VLCFA synthesis but also that PAS2-derived VLCFA homeostasis is required for specific developmental processes.
Proceedings of the National Academy of Sciences 10/2008; 105(38):14727-31. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: In plants, seasonal changes in day length are perceived in leaves, which initiate long-distance signaling that induces flowering at the shoot apex. The identity of the long-distance signal has yet to be determined. In Arabidopsis, activation of FLOWERING LOCUS T (FT) transcription in leaf vascular tissue (phloem) induces flowering. We found that FT messenger RNA is required only transiently in the leaf. In addition, FT fusion proteins expressed specifically in phloem cells move to the apex and move long distances between grafted plants. Finally, we provide evidence that FT does not activate an intermediate messenger in leaves. We conclude that FT protein acts as a long-distance signal that induces Arabidopsis flowering.
[show abstract][hide abstract] ABSTRACT: The CCT (for CONSTANS, CONSTANS-LIKE, TOC1) domain is found in 45 Arabidopsis thaliana proteins involved in processes such as photoperiodic flowering, light signaling, and regulation of circadian rhythms. We show that this domain exhibits similarities to yeast HEME ACTIVATOR PROTEIN2 (HAP2), which is a subunit of the HAP2/HAP3/HAP5 trimeric complex that binds to CCAAT boxes in eukaryotic promoters. Moreover, we demonstrate that CONSTANS (CO), which promotes Arabidopsis flowering, interacts with At HAP3 and At HAP5 in yeast, in vitro, and in planta. Mutations in CO that delay flowering affect residues highly conserved between CCT and the DNA binding domain of HAP2. Taken together, these data suggest that CO might replace At HAP2 in the HAP complex to form a trimeric CO/At HAP3/At HAP5 complex. Flowering was delayed by overexpression of At HAP2 or At HAP3 throughout the plant or in phloem companion cells, where CO is expressed. This phenotype was correlated with reduced abundance of FLOWERING LOCUS T (FT) mRNA and no change in CO mRNA levels. At HAP2 or At HAP3 overexpression may therefore impair formation of a CO/At HAP3/At HAP5 complex leading to reduced expression of FT. During plant evolution, the number of genes encoding HAP proteins was greatly amplified, and these proteins may have acquired novel functions, such as mediating the effect of CCT domain proteins on gene expression.
The Plant Cell 12/2006; 18(11):2971-84. · 9.25 Impact Factor
[show abstract][hide abstract] ABSTRACT: The sucrose nonfermenting-1 protein kinase (SNF1)/AMP-activated protein kinase subfamily plays a central role in metabolic responses to nutritional and environmental stresses. In yeast (Saccharomyces cerevisiae) and mammals, the beta- and gamma-noncatalytic subunits are implicated in substrate specificity and subcellular localization, respectively, and regulation of the kinase activity. The atypical betagamma-subunit has been previously described in maize (Zea mays), presenting at its N-terminal end a sequence related to the KIS (kinase interacting sequence) domain specific to the beta-subunits (Lumbreras et al., 2001). The existence of two components, SNF1-related protein kinase (SnRK1) complexes containing the betagamma-subunit and one SnRK1 kinase, had been proposed. In this work, we show that, despite its unusual features, the Arabidopsis (Arabidopsis thaliana) homolog AKINbetagamma clearly interacts with AKINbeta-subunits in vitro and in vivo, suggesting its involvement in heterotrimeric complexes located in both cytoplasm and nucleus. Unexpectedly, a transcriptional analysis of AKINbetagamma gene expression highlighted the implication of alternative splicing mechanisms in the regulation of AKINbetagamma expression. A two-hybrid screen performed with AKINbetagamma as bait, together with in planta bimolecular fluorescence complementation experiments, suggests the existence of interactions in the cytosol between AKINbetagamma and two leucine-rich repeats related to pathogen resistance proteins. Interestingly, this interaction occurs through the truncated KIS domain that corresponds exactly to a GBD (glycogen-binding domain) recently described in mammals and yeast. A phylogenetic study suggests that AKINbetagamma-related proteins are restricted to the plant kingdom. Altogether, these data suggest the existence of plant-specific SnRK1 trimeric complexes putatively involved in a plant-specific function such as plant-pathogen interactions.
[show abstract][hide abstract] ABSTRACT: PASTICCINO1 (PAS1) is a high molecular weight FK506-binding protein (FKBP) involved in the control of cell proliferation and differentiation during plant development. Mutations in the C-terminal region of PAS1 result in severe developmental defects. We show here that the C-terminal domain of PAS1 controls the subcellular distribution of this protein. We also demonstrated in vitro and in vivo, by Forster resonance energy transfer, that this C-terminal region is required for interaction with FAN (FKBP-associated NAC), a new member of the plant-specific family of NAC transcription factors. PAS1 and FAN are translocated into the nucleus upon auxin treatment in plant seedlings. The nuclear translocation of PAS1 is dependent on the presence of the C terminus of the protein. Finally, we showed that FAN is involved in PAS1-regulated processes because FAN overproduction partly complemented the pas1 phenotype. We suggest that PAS1 regulates the function of this NAC-like transcription factor by controlling its targeting to the nucleus upon plant cell division.
Journal of Biological Chemistry 10/2006; 281(35):25475-84. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: The SNF1/AMPK/SnRK1 heterotrimeric kinase complex is involved in the adaptation of cellular metabolism in response to diverse stresses in yeast, mammals and plants. Following a model proposed in yeast, the kinase targets are likely to bind the complex via the non-catalytic beta-subunits. These proteins currently identified in yeast, mammals and plants present a common structure with two conserved interacting domains named Kinase Interacting Sequence (KIS) and Association with SNF1 Complex (ASC), and a highly variable N-terminal domain. In this paper we describe the characterisation of AKINbeta3, a novel protein related to AKINbeta subunits of Arabidopsis thaliana, containing a truncated KIS domain and no N-terminal extension. Interestingly the missing region of the KIS domain corresponds to the glycogen-binding domain (beta-GBD) identified in the mammalian AMPKbeta1. In spite of its unusual features, AKINbeta3 complements the yeast sip1Deltasip2Deltagal83Delta mutant. Moreover, interactions between AKINbeta3 and other AKIN complex subunits from A. thaliana were detected by two-hybrid experiments and in vitro binding assays. Taken together these data demonstrate that AKINbeta3 is a beta-type subunit. A search for beta-type subunits revealed the existence of beta3-type proteins in other plant species. Furthermore, we suggest that the AKINbeta3-type subunits could be plant specific since no related sequences have been found in any of the other completely sequenced genomes. These data suggest the existence of novel SnRK1 complexes including AKINbeta3-type subunits, involved in several functions among which some could be plant specific.
[show abstract][hide abstract] ABSTRACT: The SnRK1 complex, a metabolic regulator of nutrient deficit in plants, consists of a catalytic -subunit and two regulatory subunits, β and that exist as several isoforms. To obtain insight into the developmental and stress conditions that regulate the expression of these regulatory subunits, four β subunits MtAKINβ1-β4, and three subunits MtAKINβ, MtSNF4b and MtAKIN were identified and characterized in seeds of M. truncatula. Their transcripts were found to accumulate differentially in vegetative and seed tissues and appear to be differentially modulated during germination and the imposition of stress. MtAKIN and MtAKINβ3 showed identical patterns of expression upon osmotic shock, whereas transcripts of MtAKIN and MtAKINβ1 were strongly up-regulated upon starvation of the radicles. Addition of glucose during the starvation process reversed this effect. MtAKINβ2 and MtSNF4b were specifically induced upon the re-induction of desiccation tolerance in germinated, desiccation-sensitive radicles, whereas only MtSNF4b expression was repressed by an inhibitor of abscisic acid synthesis. A second subunit, MtAKINβ, was transiently expressed early during the induction of desiccation tolerance, and its expression could be modulated by blocking the respiratory ATP production by cyanide. The transcriptional regulation of the subunit isoforms showing identical expression profiles in germinating seeds appears to be under the control of different factors.
[show abstract][hide abstract] ABSTRACT: AKINalpha1, a Ser/Thr kinase from Arabidopsis thaliana belongs to the highly conserved SNF1 family of protein kinases in eukaryotes. Recent data suggest that the plant SNF1-related kinases (SnRK1 family) are key enzymes implicated in the regulation of carbohydrate and lipid metabolism. In Saccharomyces cerevisiae and mammals, the SNF1 and AMPKalpha protein kinases interact with two other families of proteins, namely SNF4/AMPKgamma and SIP1/SIP2/GAL83/AMPKbeta, to form active heterotrimeric complexes. In this paper, we describe the characterisation of three novel cDNAs. AKINbeta1 and AKINbeta2 encode proteins similar to SIP1, SIP2 and GAL83 and AKINgamma codes for a protein showing similarity with SNF4. Using the two-hybrid system, specific interactions have been shown between A. thaliana AKINbeta1/beta2, AKINgamma and AKINgamma as well as between the A. thaliana and S. cerevisiae subunits. Interestingly, AKINbeta1, AKINbeta2 and AKINgamma mRNAs accumulate differentially in A. thaliana tissues and are modulated during development and under different growth conditions. These data suggest the presence in higher plants of a conserved heterotrimeric complex. Moreover, the differential transcription of different non-catalytic subunits can constitute a first level of regulation of the SNF1-like complex in plants.
The Plant Journal 07/1999; 18(5):541-50. · 6.58 Impact Factor
[show abstract][hide abstract] ABSTRACT: The SNF1/AMPK/SnRK1 kinases are evolutionary conserved kinases involved in yeast, mammals, and plants in the control of energy balance. These heterotrimeric enzymes are composed of one a-type catalytic subunit and two g- and b-type regulatory subunits. In yeast it has been proposed that the b-type subunits regulate both the localization of the kinase complexes within the cell and the interaction of the kinases with their targets. In this work, we demonstrate that the three b-type subunits of Arabidopsis (Arabidopsis thaliana; AKINb1, AKINb2, and AKINb3) restore the growth phenotype of the yeast sip1Dsip2Dgal83D triple mutant, thus suggesting the conservation of an ancestral function. Expression analyses, using AKINb promoter:: b-glucuronidase transgenic lines, reveal different and specific patterns of expression for each subunit according to organs, developmental stages, and environmental conditions. Finally, our results show that the b-type subunits are involved in the specificity of interaction of the kinase with the cytosolic nitrate reductase. Together with previous cell-free phosphorylation data, they strongly support the proposal that nitrate reductase is a real target of SnRK1 in the physiological context. Altogether our data suggest the conservation of ancestral basic function(s) together with specialized functions for each b-type subunit in plants.