[Show abstract][Hide abstract] ABSTRACT: Rhizosphere acidification is essential for iron (Fe) uptake into plant roots. Plasma membrane (PM) H(+)-ATPases play key roles in rhizosphere acidification. However, it is not fully understood how PM H(+)-ATPase activity is regulated to enhance root Fe uptake under Fe-deficient conditions. Here, we present evidence that cytochrome b5 reductase 1 (CBR1) increases unsaturated fatty acid levels, which stimulates PM H(+)-ATPase activity and thereby leads to rhizosphere acidification. CBR1-overexpressing (CBR1-OX) Arabidopsis thaliana plants had higher levels of unsaturated fatty acids (18:2 and 18:3), higher PM H(+)-ATPase activity, and lower rhizosphere pH than wild-type plants. By contrast, cbr1-2 loss-of-function mutant plants had lower unsaturated fatty acid levels, lower PM H(+)-ATPase activity, and higher rhizosphere pH than controls. Reduced PM H(+)-ATPase activity in cbr1-2 was restored in vitro by addition of unsaturated fatty acids. Transcript levels of CBR1, fatty acids desaturase2 (FAD2), and fatty acids desaturase3 (FAD3) increased under Fe-deficient conditions. We propose that CBR1 has a crucial role to increase unsaturated fatty acid levels, which activates the PM H(+)-ATPase and thereby reduces rhizosphere pH. This reaction cascade ultimately promotes root Fe uptake.
[Show abstract][Hide abstract] ABSTRACT: COPI vesicles are essential to the retrograde transport of proteins in the early secretory pathway. The COPI coatomer complex consists of seven subunits, termed α-, β-, β'-, γ-, δ-, ε-, and ζ-COP, in yeast and mammals. Plant genomes have homologs of these subunits, but the essentiality of their cellular functions has hampered the functional characterization of the subunit genes in plants. Here we have employed virus-induced gene silencing (VIGS) and dexamethasone (DEX)-inducible RNAi of the COPI subunit genes to study the in vivo functions of the COPI coatomer complex in plants. The β'-, γ-, and δ-COP subunits localized to the Golgi as GFP-fusion proteins and interacted with each other in the Golgi. Silencing of β'-, γ-, and δ-COP by VIGS resulted in growth arrest and acute plant death in Nicotiana benthamiana, with the affected leaf cells exhibiting morphological markers of programmed cell death. Depletion of the COPI subunits resulted in disruption of the Golgi structure and accumulation of autolysosome-like structures in earlier stages of gene silencing. In tobacco BY-2 cells, DEX-inducible RNAi of β'-COP caused aberrant cell plate formation during cytokinesis. Collectively, these results suggest that COPI vesicles are essential to plant growth and survival by maintaining the Golgi apparatus and modulating cell plate formation.
[Show abstract][Hide abstract] ABSTRACT: The phytohormone abscisic acid (ABA) plays crucial roles in numerous physiological processes during plant growth and abiotic stress responses. The endogenous ABA level is controlled by complex regulatory mechanisms involving biosynthesis, catabolism, transport and signal transduction pathways. This complex regulatory network may target multiple levels, including transcription, translation and post-translational regulation of genes involved in ABA responses. Most of the genes involved in ABA biosynthesis, catabolism and transport have been characterized. The local ABA concentration is critical for initiating ABA-mediated signalling during plant development and in response to environmental changes. In this chapter we discuss the mechanisms that regulate ABA biosynthesis, catabolism, transport and homoeostasis. We also present the findings of recent research on ABA perception by cellular receptors, and ABA signalling in response to cellular and environmental conditions.
No preview · Article · Sep 2015 · Essays in Biochemistry
[Show abstract][Hide abstract] ABSTRACT: In eukaryotic cells, organellar proteome biogenesis is pivotal for cellular function. Chloroplasts contain a complex proteome, the biogenesis of which includes post-translational import of nuclear-encoded proteins. However, the mechanisms determining when and how nascent chloroplast-targeted proteins are sorted in the cytosol are unknown. Here, we establish the timing and mode of interaction between ankyrin repeat-containing protein 2 (AKR2A), the cytosolic targeting factor of chloroplast outer membrane (COM) proteins, and its interacting partners during translation at the single-molecule level. The targeting signal of a nascent AKR2A client protein residing in the ribosomal exit tunnel induces AKR2A binding to ribosomal RPL23A. Subsequently, RPL23A-bound AKR2A binds to the targeting signal when it becomes exposed from ribosomes. Failure of AKR2A binding to RPL23A in planta severely disrupts protein targeting to the COM; thus, AKR2A-mediated targeting of COM proteins is coupled to their translation, which in turn is crucial for biogenesis of the entire chloroplast proteome.
No preview · Article · Apr 2015 · Nature Communications
[Show abstract][Hide abstract] ABSTRACT: Engineered nanomaterials (ENMs) enable the control and exploration of intermolecular interactions inside microscopic systems, but the potential environmental impacts of their inevitable release remain largely unknown. Plants exposed to ENMs display effects, such as increase in biomass and chlorophyll, distinct from those induced by exposure to their bulk counterparts, but few studies have addressed the mechanisms underlying such physiological results. The current investigation found that exposure of Arabidopsis thaliana to nano zero-valent iron (nZVI) triggered high plasma membrane H+-ATPase activity. The increase in activity caused a decrease in apoplastic pH, an increase in leaf area and also wider stomatal aperture. Analysis of gene expression indicated that the levels of the H+-ATPase isoform responsible for stomatal opening, AHA2, were 5-fold higher in plants exposed to nZVI than in unexposed control plants. This is the first study to show that nZVI enhances stomatal opening by inducing the activation of plasma membrane H+-ATPase, leading to the possibility of increased CO2 uptake.
Full-text · Article · Dec 2014 · Environmental Science and Technology
[Show abstract][Hide abstract] ABSTRACT: Chloroplasts produce carbohydrates, hormones, vitamins, amino acids, pigments, nucleotides, ATP, and secondary metabolites. Channels and transporters are required for the movement of molecules across the two chloroplast envelope membranes. These transporters and channel proteins are grouped into two different types, including β-barrel proteins and transmembrane-domain (TMD) containing proteins. Most β-barrel proteins are localized at the outer chloroplast membrane, and TMD-containing proteins are localized at the inner chloroplast membrane. Many of these transporters and channels are encoded by nuclear genes; therefore, they have to be imported into chloroplasts after translation on cytosolic ribosomes. These proteins should have specific targeting signals for their final destination in the chloroplast membrane and for assembly into specific complexes. In this review, we summarize recent progress in the identification, functional characterization, and biogenesis of transporters and channels at the chloroplast envelope membranes, and discuss outstanding questions regarding transporter and channel protein biogenesis.
[Show abstract][Hide abstract] ABSTRACT: Clathrin is a highly conserved coat protein that plays a critical role in lipid vesicle-mediated trafficking at multiple routes in various post-Golgi compartments. It consists of large and small subunits, and exists in the cytosol as triskelions composed of three pairs of small and large subunits. For vesicle formation, the triskelions are recruited to the membrane of specific compartments where they undergo self-polymerization to produce coats for lipid vesicles. However, clathrin has no ability to bind directly to lipid membranes. Therefore, accessory proteins are necessary for its recruitment to the donor compartment where vesicles are formed. A large number of accessory proteins, called adaptor proteins, have been identified and characterized extensively at the molecular and cellular levels in animal cells and yeast. Recently, the roles of many adaptor proteins have been elucidated in plant cells. As expected from the conserved nature of lipidmediated trafficking in eukaryotic cells, these plant adaptor proteins for clathrin show a high degree of functional conservation with those found in animal cells and yeast. At the same time, they are also involved in plant-specific processes such as the transition from the PSV to the lytic vacuole and cell-plate formation. Here, we summarize recent advances in the physiological roles of adaptor proteins in plant cells.
No preview · Article · Oct 2014 · Journal of Plant Biology
[Show abstract][Hide abstract] ABSTRACT: In organellogenesis of the chloroplast from endosymbiotic cyanobacteria, the establishment of protein-targeting mechanisms to the chloroplast should have been pivotal. However, it is still mysterious how these mechanisms were established and how they work in plant cells. Here we show that AKR2A, the cytosolic targeting factor for chloroplast outer membrane (COM) proteins, evolved from the ankyrin repeat domain (ARD) of the host cell by stepwise extensions of its N-terminal domain and that two lipids, monogalactosyldiacylglycerol (MGDG) and phosphatidylglycerol (PG), of the endosymbiont were selected to function as the AKR2A receptor. Structural analysis, molecular modeling, and mutational analysis of the ARD identified two adjacent sites for coincidental and synergistic binding of MGDG and PG. Based on these findings, we propose that the targeting mechanism of COM proteins was established using components from both the endosymbiont and host cell through a modification of the protein-protein-interacting ARD into a lipid binding domain.
[Show abstract][Hide abstract] ABSTRACT: Pandemics in poultry caused by the highly pathogenic avian influenza (HPAI) A virus occur too frequently globally, and there is growing concern about the HPAI A virus due to the possibility of a pandemic among humans. Thus, it is important to develop a vaccine against HPAI suitable for both humans and animals. Various approaches are underway to develop such vaccines. In particular, an edible vaccine would be a convenient way to vaccinate poultry because of the behaviour of the animals. However, an edible vaccine is still not available. In this study, we developed a strategy of effective vaccination of mice by the oral administration of transgenic Arabidopsis plants (HA-TG) expressing haemagglutinin (HA) in the endoplasmic reticulum (ER). Expression of HA in the ER resulted in its high-level accumulation, N-glycosylation, protection from proteolytic degradation and long-term stability. Oral administration of HA-TG with saponin elicited high levels of HA-specific systemic IgG and mucosal IgA responses in mice, which resulted in protection against a lethal influenza virus infection with attenuated inflammatory symptoms. Based on these results, we propose that oral administration of freeze-dried leaf powders from transgenic plants expressing HA in the ER together with saponin is an attractive strategy for vaccination against influenza A virus.
[Show abstract][Hide abstract] ABSTRACT: The phytohormone abscisic acid (ABA) plays a crucial role in numerous aspects of plant growth and environmental stress responses. Endogenous ABA levels are regulated by a balance between its biosynthetic and catabolic activities. This balance may occur at multiple levels and includes the expression of genes involved in these processes. ABA UDP-glucosyltransferase (UGT), the major player in the ABA conjugation pathway, has been shown to have a marginal effect on free ABA levels. However, recent studies provide new insight into the importance of the ABA conjugation pathway in contributing to the control of ABA homeostasis. Gain-of-function and loss-of-function mutant analyses have revealed that UGT71B6, an ABA UGT, and its 2 closely related homologs, UGT71B7 and UGT71B8, play a crucial role in ABA homeostasis and in adaptation to various abiotic stresses.
[Show abstract][Hide abstract] ABSTRACT: Chloroplasts and mitochondria are endosymbiotic organelles thought to be derived from endosymbiotic bacteria. In present-day eukaryotic cells, these two organelles play pivotal roles in photosynthesis and ATP production. In addition to these major activities, numerous reactions, and cellular processes that are crucial for normal cellular functions occur in chloroplasts and mitochondria. To function properly, these organelles constantly communicate with the surrounding cellular compartments. This communication includes the import of proteins, the exchange of metabolites and ions, and interactions with other organelles, all of which heavily depend on membrane proteins localized to the outer envelope membranes. Therefore, correct and efficient targeting of these membrane proteins, which are encoded by the nuclear genome and translated in the cytosol, is critically important for organellar function. In this review, we summarize the current knowledge of the mechanisms of protein targeting to the outer membranes of mitochondria and chloroplasts in two different directions, as well as targeting signals and cytosolic factors.
[Show abstract][Hide abstract] ABSTRACT: The phytohormone abscisic acid (ABA) is crucial for plant growth and adaptive responses to various stress conditions. Plants continuously adjust the ABA level to meet physiological needs, but how ABA homeostasis occurs is not fully understood. This study provides evidence that UGT71B6, an ABA uridine diphosphate glucosyltransferase (UGT), and its two closely related homologs, UGT71B7 and UGT71B8, play crucial roles in ABA homeostasis and in adaptation to dehydration, osmotic stress and high-salinity stresses. UGT RNAi plants that had low levels of these three UGT transcripts displayed hypersensitivity to exogenous ABA and high-salt conditions during germination, and exhibited a defect in plant growth. However, the ectopic expression of UGT71B6 in the atbg1 mutant background aggravated the ABA-deficient phenotype of atbg1 mutant plants. In addition, modulation of the expression of the three UGTs affects the expression of CYP707A1 to CYP707A4, which encode ABA 8'-hydroxylases; four CYP707As were expressed at higher levels in the UGT RNAi plants but at lower levels in the UGT71B6:GFP-overexpressing plants. Based on these data, this study proposes that UGT71B6 and its two homologs play a critical role in ABA homeostasis that depends on intrinsic cellular and environmental conditions in plants.
[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.
Preview · Article · Dec 2013 · Journal of Plant Biology
[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: Key message:
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.
No preview · Article · Oct 2013 · Plant Cell Reports
[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.
Preview · Article · Sep 2013 · Nucleic Acids Research
[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.