[show abstract][hide abstract] ABSTRACT: In eukaryotic cells, a major proportion of the cellular proteins localize to various subcellular organelles where they are involved in organelle-specific cellular processes. Thus, the localization of a particular protein in the cell is an important part of understanding the physiological role of the protein in the cell. Various approaches such as subcellular fractionation, immunolocalization and live imaging have been used to define the localization of organellar proteins. Of these various approaches, the most powerful one is the live imaging because it can show in vivo dynamics of protein localization depending on cellular and environmental conditions without disturbing cellular structures. However, the live imaging requires the ability to detect the organelles in live cells. In this study, we report generation of a new set of transgenic Arabidopsis plants using various organelle marker proteins fused to a fluorescence protein, monomeric Cherry (mCherry). All these markers representing different subcellular organelles such as chloroplasts, mitochondria, peroxisomes, endoplasmic reticulum (ER) and lytic vacuole showed clear and specific signals regardless of the cell types and tissues. These marker lines can be used to determine localization of organellar proteins by colocalization and also to study the dynamics of organelles under various developmental and environmental conditions.
Journal of Plant Biology 12/2013; 56(6). · 0.99 Impact Factor
[show abstract][hide abstract] ABSTRACT: Multiple transcription factors (TFs) play essential roles in plants under abiotic stress, but how these multiple TFs cooperate in abiotic stress responses remains largely unknown. In this study, we provide evidence that the NAC (for NAM, ATAF1/2, and CUC2) TF ANAC096 cooperates with the bZIP-type TFs ABRE binding factor and ABRE binding protein (ABF/AREB) to help plants survive under dehydration and osmotic stress conditions. ANAC096 directly interacts with ABF2 and ABF4, but not with ABF3, both in vitro and in vivo. ANAC096 and ABF2 synergistically activate RD29A transcription. Our genome-wide gene expression analysis revealed that a major proportion of abscisic acid (ABA)-responsive genes are under the transcriptional regulation of ANAC096. We found that the Arabidopsis thaliana anac096 mutant is hyposensitive to exogenous ABA and shows impaired ABA-induced stomatal closure and increased water loss under dehydration stress conditions. Furthermore, we found the anac096 abf2 abf4 triple mutant is much more sensitive to dehydration and osmotic stresses than the anac096 single mutant or the abf2 abf4 double mutant. Based on these results, we propose that ANAC096 is involved in a synergistic relationship with a subset of ABFs for the transcriptional activation of ABA-inducible genes in response to dehydration and osmotic stresses.
[show abstract][hide abstract] ABSTRACT: Transgenic Arabidopsis and lettuce plants overexpressing AtHSP17.8 showed ABA-hypersensitive but abiotic stress-resistant phenotypes. ABA treatment caused a dramatic induction of early ABA-responsive genes in AtHSP17.8 -overexpressing transgenic lettuce. Plant small heat shock proteins function as chaperones in protein folding. In addition, they are involved in responses to various abiotic stresses, such as dehydration, heat and high salinity in Arabidopsis. However, it remains elusive how they play a role in the abiotic stress responses at the molecular level. In this study, we provide evidence that Arabidopsis HSP17.8 (AtHSP17.8) positively regulates the abiotic stress responses by modulating abscisic acid (ABA) signaling in Arabidopsis, and also in lettuce, a heterologous plant when ectopically expressed. Overexpression of AtHSP17.8 in both Arabidopsis and lettuce leads to hypersensitivity to ABA and enhanced resistance to dehydration and high salinity stresses. Moreover, early ABA-responsive genes, ABI1, ABI5, NCED3, SNF4 and AREB2, were rapidly induced in AtHSP17.8-overexpressing transgenic Arabidopsis and lettuce. Based on these data, we propose that AtHSP17.8 plays a crucial role in abiotic stress responses by positively modulating ABA-mediated signaling in both Arabidopsis and lettuce. Moreover, our results suggest that stress-tolerant lettuce can be engineered using the genetic and molecular resources of Arabidopsis.
[show abstract][hide abstract] ABSTRACT: The nucleotide sequence around the translational initiation site is an important cis-acting element for post-transcriptional regulation. However, it has not been fully understood how the sequence context at the 5'-untranslated region (5'-UTR) affects the translational efficiency of individual mRNAs. In this study, we provide evidence that the 5'-UTRs of Arabidopsis genes showing a great difference in the nucleotide sequence vary greatly in translational efficiency with more than a 200-fold difference. Of the four types of nucleotides, the A residue was the most favourable nucleotide from positions -1 to -21 of the 5'-UTRs in Arabidopsis genes. In particular, the A residue in the 5'-UTR from positions -1 to -5 was required for a high-level translational efficiency. In contrast, the T residue in the 5'-UTR from positions -1 to -5 was the least favourable nucleotide in translational efficiency. Furthermore, the effect of the sequence context in the -1 to -21 region of the 5'-UTR was conserved in different plant species. Based on these observations, we propose that the sequence context immediately upstream of the AUG initiation codon plays a crucial role in determining the translational efficiency of plant genes.
Nucleic Acids Research 09/2013; · 8.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: Clathrin-mediated endocytosis (CME) regulates many aspects of plant development, including hormone signaling and responses to environmental stresses. Despite the importance of this process, the machinery that regulates CME in plants is largely unknown. In mammals, the heterotetrameric ADAPTOR PROTEIN COMPLEX-2 (AP-2) is required for the formation of clathrin-coated vesicles at the plasma membrane (PM). Although the existence of AP-2 has been predicted in Arabidopsis thaliana, the biochemistry and functionality of the complex is still uncharacterized. Here, we identified all the subunits of the Arabidopsis AP-2 by tandem affinity purification and found that one of the large AP-2 subunits, AP2A1, localized at the PM and interacted with clathrin. Furthermore, endocytosis of the leucine-rich repeat receptor kinase, BRASSINOSTEROID INSENSITIVE1 (BRI1), was shown to depend on AP-2. Knockdown of the two Arabidopsis AP2A genes or overexpression of a dominant-negative version of the medium AP-2 subunit, AP2M, impaired BRI1 endocytosis and enhanced the brassinosteroid signaling. Our data reveal that the CME machinery in Arabidopsis is evolutionarily conserved and that AP-2 functions in receptor-mediated endocytosis.
[show abstract][hide abstract] ABSTRACT: Fertilization in flowering plants requires the temporal and spatial coordination of many developmental processes, including pollen production, anther dehiscence, ovule production, and pollen tube elongation. However, it remains elusive as to how this coordination occurs during reproduction. Here, we present evidence that endocytosis, involving heterotetrameric adaptor protein complex 2 (AP-2), plays a crucial role in fertilization. An Arabidopsis thaliana mutant ap2m displays multiple defects in pollen production and viability, as well as elongation of staminal filaments and pollen tubes, all of which are pivotal processes needed for fertilization. Of these abnormalities, the defects in elongation of staminal filaments and pollen tubes were partially rescued by exogenous auxin. Moreover, DR5rev:GFP (for green fluorescent protein) expression was greatly reduced in filaments and anthers in ap2m mutant plants. At the cellular level, ap2m mutants displayed defects in both endocytosis of N-(3-triethylammonium-propyl)-4-(4-diethylaminophenylhexatrienyl) pyridinium dibromide, a lypophilic dye used as an endocytosis marker, and polar localization of auxin-efflux carrier PIN FORMED2 (PIN2) in the stamen filaments. Moreover, these defects were phenocopied by treatment with Tyrphostin A23, an inhibitor of endocytosis. Based on these results, we propose that AP-2-dependent endocytosis plays a crucial role in coordinating the multiple developmental aspects of male reproductive organs by modulating cellular auxin level through the regulation of the amount and polarity of PINs.
[show abstract][hide abstract] ABSTRACT: Adaptor protein (AP) complexes are the predominant coat proteins of membrane vesicles in post-Golgi trafficking of mammalian cells. Each AP complex contains a specific medium subunit, μ-adaptin, that selects cargo proteins bearing sequence-specific sorting motifs. Much less is known about the AP complexes and their μ subunits in plants. Because of uncertain homology, the μ-adaptins of Arabidopsis have been designated muA through muD [Happel et al. (2004) Plant J 37(5):678-693]. Furthermore, only muD has been assigned to a specific AP complex, AP-3, involved in Golgi-vacuolar trafficking [Niihama et al. (2009) Plant Cell Physiol 50(12):2057-2068, Zwiewka et al. (2011) Cell Res 21(12):1711-1722, and Wolfenstetter et al. (2012) Plant Cell 24(1):215-232]. In contrast, the μ subunit of neither the post-Golgi trafficking AP-1 complex nor the endocytic AP-2 complex has been identified. Here, we report the functional analysis of redundant AP-1 μ-adaptins AP1M1 (also known as muB1) and AP1M2 (also known as muB2). Coimmunoprecipitation revealed that both AP1M2 and its less strongly expressed isoform AP1M1 are complexed with the large subunit γ-adaptin of AP-1. In addition, AP1M2 was localized at or near the trans-Golgi network. Knockout mutations of AP1M2 impaired pollen function and arrested plant growth whereas the ap1m1 ap1m2 double mutant was nearly pollen-lethal. At the cellular level, the absence of AP1M2 entailed inhibition of multiple trafficking pathways from the trans-Golgi network to the vacuole and to the plasma membrane in interphase and to the plane of cell division in cytokinesis. Thus, AP-1 is crucial in post-Golgi trafficking in plant cells and required for cell division and plant growth.
Proceedings of the National Academy of Sciences 06/2013; · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: BACKGROUND: Neuroblastoma Amplified Gene (NAG) was identified as a gene co-amplified with the N-myc gene, whose genomic amplification correlates with poor prognosis of neuroblastoma. Later it was found that NAG is localized in endoplasmic reticulum (ER) and is a component of the syntaxin 18 complex that is involved in Golgi-to-ER retrograde transport in human cells. Homologous sequences of NAG are found in plant databases, but its function in plant cells remains unknown. RESULTS: Nicotiana benthamania Neuroblastoma-Amplified Gene (NbNAG) encodes a protein of 2,409 amino acids that contains the secretory pathway Sec39 domain and is mainly localized in the ER. Silencing of NbNAG by virus-induced gene silencing resulted in growth arrest and acute plant death with morphological markers of programmed cell death (PCD), which include chromatin fragmentation and modification of mitochondrial membrane potential. NbNAG deficiency caused induction of ER stress genes, disruption of the ER network, and relocation of bZIP28 transcription factor from the ER membrane to the nucleus, similar to the phenotypes of tunicamycin-induced ER stress in a plant cell. NbNAG silencing caused defects in intracellular transport of diverse cargo proteins, suggesting that a blocked secretion pathway by NbNAG deficiency causes ER stress and programmed cell death. CONCLUSIONS: These results suggest that NAG, a conserved protein from yeast to mammals, plays an essential role in plant growth and development by modulating protein transport pathway, ER stress response and PCD.
[show abstract][hide abstract] ABSTRACT: The plant hormone abscisic acid (ABA) plays pivotal roles in many important physiological processes including stomatal closure, seed dormancy, growth and various environmental stresses. In these responses, ABA action is under the control of complex regulatory mechanisms involving homeostasis, perception and signaling. Recent studies provide new insights into these processes, which are of great importance in understanding the mechanisms underlying the evolutionary principle of how plants can survive as a sessile organism under ever-changing environmental conditions. They also form the basis for designing plants that have an enhanced resistance to various stresses in particular abiotic stress.
[show abstract][hide abstract] ABSTRACT: In eukaryotic cells consisting of many different types of organelles, targeting of organellar proteins is one of the most fundamental cellular processes. Proteins belonging to the ER, chloroplasts and mitochondria are targeted individually from the cytosol to their cognate organelles. Since the targeting to these organelles occurs in the cytosol during or after translation, the most crucial aspect is how specific targeting to these three organelles can be achieved without interfering with other targeting pathways. For these organelles, multiple mechanisms are used for targeting proteins, but the exact mechanism used depends on the type of protein and organelle, the location of targeting signals in the protein and the location of the protein in the organelle. In this review, we discuss the various mechanisms involved in protein targeting to the ER, chloroplasts and mitochondria and how the targeting specificity is determined for these organelles in plant cells.
[show abstract][hide abstract] ABSTRACT: In eukaryotic cells, a large number of proteins are transported to their final destination after translation by a process called intracellular trafficking. Transient gene expression, either in plant protoplasts or in specific plant tissues, is a fast, flexible, and reproducible approach to study the cellular function of proteins, protein subcellular localizations, and protein-protein interactions. Here we describe the general method of protoplast isolation, polyethylene glycol-mediated protoplast transformation and immunostaining of protoplast or intact root tissues for studying the localization of protein in Arabidopsis.
Methods in molecular biology (Clifton, N.J.) 01/2013; 1043:113-20.
[show abstract][hide abstract] ABSTRACT: ADP-ribosylation factor 1 (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 domain 8 (AGD8) and AGD9, are involved in the recruitment of Arf1-GDP to the Golgi apparatus in Arabidopsis. RNAi plants expressing low levels of AGD8 and AGD9 exhibited abnormal Golgi morphology, inhibition of protein trafficking, and arrest of plant growth and development. In RNAi 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 recruits Arf1-GDP from the cytosol to the Golgi for Arf1-mediated protein trafficking, which is essential for plant development and growth.
[show abstract][hide abstract] ABSTRACT: The retromer is involved in recycling lysosomal sorting receptors in mammals. A component of the retromer complex in Arabidopsis thaliana, vacuolar protein sorting 29 (VPS29), plays a crucial role in trafficking storage proteins to protein storage vacuoles. However, it is not known whether or how vacuolar sorting receptors (VSRs) are recycled from the prevacuolar compartment (PVC) to the trans-Golgi network (TGN) during trafficking to the lytic vacuole (LV). Here, we report that VPS29 plays an essential role in the trafficking of soluble proteins to the LV from the TGN to the PVC. maigo1-1 (mag1-1) mutants, which harbor a knockdown mutation in VPS29, were defective in trafficking of two soluble proteins, Arabidopsis aleurain-like protein (AALP):green fluorescent protein (GFP) and sporamin:GFP, to the LV but not in trafficking membrane proteins to the LV or plasma membrane or via the secretory pathway. AALP:GFP and sporamin:GFP in mag1-1 protoplasts accumulated in the TGN but were also secreted into the medium. In mag1-1 mutants, VSR1 failed to recycle from the PVC to the TGN; rather, a significant proportion was transported to the LV; VSR1 overexpression rescued this defect. Moreover, endogenous VSRs were expressed at higher levels in mag1-1 plants. Based on these results, we propose that VPS29 plays a crucial role in recycling VSRs from the PVC to the TGN during the trafficking of soluble proteins to the LV.
[show abstract][hide abstract] ABSTRACT: The majority of mitochondrial proteins are encoded in the nuclear genome and imported into mitochondria posttranslationally from the cytosol. An N-terminal presequence functions as the signal for the import of mitochondrial proteins. However, the functional information in the presequence remains elusive. This study reports the identification of critical sequence motifs from the presequence of Arabidopsis thaliana F1-ATPase γ-subunit (pFAγ). pFAγ was divided into six 10-amino acid segments, designated P1 to P6 from the N to the C terminus, each of which was further divided into two 5-amino acid subdivisions. These P segments and their subdivisions were substituted with Ala residues and fused to green fluorescent protein (GFP). Protoplast targeting experiments using these GFP constructs revealed that pFAγ contains several functional sequence motifs that are dispersed throughout the presequence. The sequence motifs DQEEG (P4a) and VVRNR (P5b) were involved in translocation across the mitochondrial membranes. The sequence motifs IAARP (P2b) and IAAIR (P3a) participated in binding to mitochondria. The sequence motifs RLLPS (P2a) and SISTQ (P5a) assisted in pulling proteins into the matrix, and the sequence motif IAARP (P2b) functioned in Tom20-dependent import. In addition, these sequence motifs exhibit complex relationships, including synergistic functions. Thus, multiple sequence motifs dispersed throughout the presequence are proposed to function cooperatively during protein import into mitochondria.
[show abstract][hide abstract] ABSTRACT: 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 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, it remains controversial about the identity of VSRs for soluble proteins of the lytic vacuole in vegetative cells. 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, rmr1rmr2 and rmr3rmr4 double mutants, soluble lytic vacuolar (AALP:GFP and CPY:GFP) and PSV (phaseolin) proteins, but not a vacuolar membrane protein At[beta]Fruct4:GFP, exhibited defects in their trafficking; they accumulated to the endoplasmic reticulum (ER) 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 AALP: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.
[show abstract][hide abstract] ABSTRACT: Cuticular waxes are synthesized by the extensive export of intracellular lipids from epidermal cells. However, it is still not known how hydrophobic cuticular lipids are exported to the plant surface through the hydrophilic cell wall. The LTPG2 gene was isolated based on Arabidopsis microarray analysis; this gene is predominantly expressed in stem epidermal peels as compared with in stems. The expression of LTPG2 transcripts was observed in various organs, including stem epidermis and silique walls. The composition of the cuticular wax was significantly altered in the stems and siliques of the ltpg2 mutant and ltpg1 ltpg2 double mutant. In particular, the reduced level of the C29 alkane, which is the major component of cuticular waxes in ltpg1 ltpg2 stems and siliques, was similar to the sum of reduced values of either parent. The total cuticular wax load was reduced by approximately 13% and 20% in both ltpg2 and ltpg1 ltpg2 siliques, respectively, and by approximately 14% in ltpg1 ltpg2 stems when compared with the wild-type. Similarly, severe alterations in the cuticular layer structure of epidermal cells of ltpg2 and ltpg1 ltpg2 stems and silique walls were observed. In tobacco epidermal cells, intracellular trafficking of the fluorescent LTPG/LTPG1 and LTPG2 to the plasma membrane was prevented by a dominant-negative mutant form of ADP-ribosylation factor 1, ARF1(T31N). Taken together, these results indicate that LTPG2 is functionally overlapped with LTPG/LTPG1 during cuticular wax export or accumulation and LTPG/LTPG1 and LTPG2 are targeted to the plasma membrane via the vesicular trafficking system.
Plant and Cell Physiology 08/2012; 53(8):1391-403. · 4.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: The phytohormone abscisic acid (ABA) plays a critical role in various physiological processes, including adaptation to abiotic stresses. In Arabidopsis thaliana, ABA levels are increased both through de novo biosynthesis and via β-glucosidase homolog1 (BG1)-mediated hydrolysis of Glc-conjugated ABA (ABA-GE). However, it is not known how many different β-glucosidase proteins produce ABA from ABA-GE and how the multiple ABA production pathways are coordinated to increase ABA levels. Here, we report that a previously undiscovered β-glucosidase homolog, BG2, produced ABA by hydrolyzing ABA-GE and plays a role in osmotic stress response. BG2 localized to the vacuole as a high molecular weight complex and accumulated to high levels under dehydration stress. BG2 hydrolyzed ABA-GE to ABA in vitro. In addition, BG2 increased ABA levels in protoplasts upon application of exogenous ABA-GE. Overexpression of BG2 rescued the bg1 mutant phenotype, as observed for the overexpression of NCED3 in bg1 mutants. Multiple Arabidopsis bg2 alleles with a T-DNA insertion in BG2 were more sensitive to dehydration and NaCl stress, whereas BG2 overexpression resulted in enhanced resistance to dehydration and NaCl stress. Based on these observations, we propose that, in addition to the de novo biosynthesis, ABA is produced in multiple organelles by organelle-specific β-glucosidases in response to abiotic stresses.
The Plant Cell 05/2012; 24(5):2184-99. · 9.25 Impact Factor
[show abstract][hide abstract] ABSTRACT: Transient gene expression, in plant protoplasts or specific plant tissues, is a key technique in plant molecular cell biology, aimed at exploring gene products and their modifications to examine functional subdomains, their interactions with other biomolecules, and their subcellular localization. Here, we highlight some of the major advantages and potential pitfalls of the most commonly used transient gene expression models and illustrate how ectopic expression and the use of dominant mutants can provide insights into protein function.
The Plant Cell 04/2012; 24(4):1316-26. · 9.25 Impact Factor