Noriaki Tanabe

Molecular Characterization of Organelle-Type Nudix Hydrolases in Arabidopsis

Nudix (for nucleoside diphosphates linked to some moiety X) hydrolases act to hydrolyze ribonucleoside and deoxyribonucleoside triphosphates, nucleotide sugars, coenzymes, or dinucleoside polyphosphates. Arabidopsis (Arabidopsis thaliana) contains 27 genes encoding Nudix hydrolase homologues (AtNUDX1 to -27) with a predicted distribution in the cytosol, mitochondria, and chloroplasts. Previously, cytosolic Nudix hydrolases (AtNUDX1 to -11 and -25) were characterized. Here, we conducted a characterization of organelle-type AtNUDX proteins (AtNUDX12 to -24, -26, and -27). AtNUDX14 showed pyrophosphohydrolase activity toward both ADP-ribose and ADP-glucose, although its Km value was approximately 100-fold lower for ADP-ribose (13.0 ± 0.7 μm) than for ADP-glucose (1,235 ± 65 μm). AtNUDX15 hydrolyzed not only reduced coenzyme A (118.7 ± 3.4 μm) but also a wide range of its derivatives. AtNUDX19 showed pyrophosphohydrolase activity toward both NADH (335.3 ± 5.4 μm) and NADPH (36.9 ± 3.5 μm). AtNUDX23 had flavin adenine dinucleotide pyrophosphohydrolase activity (9.1 ± 0.9 μm). Both AtNUDX26 and AtNUDX27 hydrolyzed diadenosine polyphosphates (n = 4–5). A confocal microscopic analysis using a green fluorescent protein fusion protein showed that AtNUDX15 is distributed in mitochondria and AtNUDX14 -19, -23, -26, and -27 are distributed in chloroplasts. These AtNUDX mRNAs were detected ubiquitously in various Arabidopsis tissues. The T-DNA insertion mutants of AtNUDX13, -14, -15, -19, -20, -21, -25, -26, and -27 did not exhibit any phenotypical differences under normal growth conditions. These results suggest that Nudix hydrolases in Arabidopsis control a variety of metabolites and are pertinent to a wide range of physiological processes.

Quantitative analysis of the chloroplast molecular chaperone ClpC/Hsp93 in Arabidopsis reveals new insights into its localization, interaction with the Clp proteolytic core, and functional importance.

Background: ClpC/Hsp93 is a chloroplast molecular chaperone whose function is essential for plant viability. Results: Chloroplast ClpC localized in both the stromal and envelope-membrane fractions associates with the Clp proteolytic core. Conclusion: ClpC functions primarily as the chaperone component of the chloroplast Clp protease. Significance: Learning how ClpC contributes to key functions of the Clp protease is crucial to understanding the importance of this essential enzyme for chloroplast biology. The molecular chaperone ClpC/Hsp93 is essential for chloroplast function in vascular plants. ClpC has long been held to act both independently and as the regulatory partner for the ATP-dependent Clp protease, and yet this and many other important characteristics remain unclear. In this study, we reveal that of the two near-identical ClpC paralogs (ClpC1 and ClpC2) in Arabidopsis chloroplasts, along with the closely related ClpD, it is ClpC1 that is the most abundant throughout leaf maturation. An unexpectedly large proportion of both chloroplast ClpC proteins (30% of total ClpC content) associates to envelope membranes in addition to their stromal localization. The Clp proteolytic core is also bound to envelope membranes, the amount of which is sufficient to bind to all the similarly localized ClpC. The role of such an envelope membrane Clp protease remains unclear although it appears uninvolved in preprotein processing or Tic subunit protein turnover. Within the stroma, the amount of oligomeric ClpC protein is less than that of the Clp proteolytic core, suggesting most if not all stromal ClpC functions as part of the Clp protease; a proposal supported by the near abolition of Clp degradation activity in the clpC1 knock-out mutant. Overall, ClpC appears to function primarily within the Clp protease, as the principle stromal protease responsible for maintaining homeostasis, and also on the envelope membrane where it possibly confers a novel protein quality control mechanism for chloroplast preprotein import.

Functional Analysis of the Hsp93/ClpC Chaperone at the Chloroplast Envelope

Chloroplast chaperone Hsp93 associates with the inner envelope membrane via interaction with the Tic110 translocon and likely functions in quality control but not in import propulsion of translocating preproteins. The Hsp100-type chaperone Hsp93/ClpC has crucial roles in chloroplast biogenesis. In addition to its role in proteolysis in the stroma, biochemical and genetic evidence led to the hypothesis that this chaperone collaborates with the inner envelope TIC complex to power preprotein import. Recently, it was suggested that Hsp93, working together with the Clp proteolytic core, can confer a protein quality control mechanism at the envelope. Thus, the role of envelope-localized Hsp93, and the mechanism by which it participates in protein import, remain unclear. To analyze the function of Hsp93 in protein import independently of its ClpP association, we created a mutant of Hsp93 affecting its ClpP-binding motif (PBM) (Hsp93[P-]), which is essential for the chaperone’s interaction with the Clp proteolytic core. The Hsp93[P-] construct was ineffective at complementing the pale-yellow phenotype of hsp93 Arabidopsis (Arabidopsis thaliana) mutants, indicating that the PBM is essential for Hsp93 function. As expected, the PBM mutation negatively affected the degradation activity of the stromal Clp protease. The mutation also disrupted association of Hsp93 with the Clp proteolytic core at the envelope, without affecting the envelope localization of Hsp93 itself or its association with the TIC machinery, which we demonstrate to be mediated by a direct interaction with Tic110. Nonetheless, Hsp93[P-] expression did not detectably improve the protein import efficiency of hsp93 mutant chloroplasts. Thus, our results do not support the proposed function of Hsp93 in protein import propulsion, but are more consistent with the notion of Hsp93 performing a quality control role at the point of import.

Point mutation of a plastidic invertase inhibits development of the photosynthetic apparatus and enhances nitrate assimilation in sugar-treated Arabidopsis seedlings

Because the photosynthetic apparatus contains a massive amount of nitrogen in plants, the regulation of its development by sugar signals is important to the maintenance of the carbon-nitrogen balance. In this study we isolated an Arabidopsis mutant (sicy-192) whose cotyledon greening was inhibited by treatments with sugars such as sucrose, glucose, and fructose. In the mutant, the gene encoding plastidic alkaline/neutral invertase (INV-E) was point-mutated at codon 294, with Tyr substituted for Cys (C294Y). Interestingly, the greening of cotyledons in the knock-out INV-E lines was not inhibited by treatment with the sugars. In addition, the knock-out INV-E lines expressing an INV-E:C294Y or INV-E:C294A gene had the same phenotype as sicy-192 mutants, whereas the lines expressing a wild-type INV-E gene had the same phenotype as wild-type plants. A recombinant INV-E:C294Y protein had the same enzymatic activity as a recombinant INV-E protein, suggesting that the Cys-294 residue of INV-E is important for its functions in the chloroplasts. On treatment with sucrose, the expression of photosynthesis-related genes was weaker in seedlings of mutant plants than wild-type seedlings, whereas the activity of nitrate reductase was stronger in the mutant plants than wild-type plants. These findings suggest that Cys-294 of INV-E is associated with the development of the photosynthetic apparatus and the assimilation of nitrogen in Arabidopsis seedlings to control the ratio of sucrose content to hexose content.

Plant-specific SR-related protein atSR45a interacts with spliceosomal proteins in plant nucleus

Serine/arginine-rich (SR) protein and its homologues (SR-related proteins) are important regulators of constitutive and/or alternative splicing and other aspects of mRNA metabolism. To clarify the contribution of a plant-specific and stress-responsive SR-related protein, atSR45a, to splicing events, here we analyzed the interaction of atSR45a with the other splicing factors by conducting a yeast two-hybrid assay and a bimolecular fluorescence complementation analysis. The atSR45a-1a and -2 proteins, the presumed mature forms produced by alternative splicing of atSR45a, interacted with U1-70K and U2AF35b, splicing factors for the initial definition of 5′ and 3′ splice sites, respectively, in the early stage of spliceosome assembly. Both proteins also interacted with themselves, other SR proteins (atSR45 and atSCL28), and PRP38-like protein, a homologue of the splicing factor essential for cleavage of the 5′ splice site. The mapping of deletion mutants of atSR45a proteins revealed that the C-terminal arginine/serine-rich (RS) domain of atSR45a proteins are required for the interaction with U1-70K, U2AF35b, atSR45, atSCL28, PRP38-like protein, and themselves, and the N-terminal RS domain enhances the interaction efficiency. Interestingly, the distinctive N-terminal extension in atSR45a-1a protein, but not atSR45a-2 protein, inhibited the interaction with these splicing factors. These findings suggest that the atSR45a proteins help to form the bridge between 5′ and 3′ splice sites in the spliceosome assembly and the efficiency of spliceosome formation is affected by the expression ratio of atSR45a-1a and atSR45a-2.

Identification of alternative splicing events regulated by an Arabidopsis serine/arginine-like protein, atSR45a, in response to high-light stress using a tiling array.

We have demonstrated that an Arabidopsis serine/arginine rich-like protein, atSR45a, interacts with other splicing factors and its expression is markedly induced by high-light stress, suggesting the involvement of atSR45a in the regulation of stress-responsive alternative splicing. A whole-genome tiling array identified the alternative splicing of genes regulated by atSR45a by comparing gene expression profiles in wild-type and knockout atSR45a (KO-sr45a) plants under high-light stress. The expression levels of genomic regions within 217 genes were significantly altered in the KO-sr45a plants compared with the wild-type plants. Many genes encoded factors involved in signal transduction, cell cycle and DNA processing, protein fate and transcription. A semi-quantitative reverse transcription-PCR (RT-PCR) analysis confirmed changes in the transcript levels and/or alternative splicing efficiency under high-light stress in 18 genes, suggesting that atSR45a affects directly or indirectly not only alternative splicing efficiency but also the transcription of these target genes. Changes in the expression of atSR45a in response to high-light stress temporally correlated with changes in the alternative splicing efficiency and transcript levels of three and one target genes, respectively. Sequencing of the alternatively spliced variants of three target genes showed that atSR45a suppresses the splicing efficiency of intron retention-type alternative splicing events. These findings indicated the importance of atSR45a to the diversification of the transcriptome under high-light stress.

A small RNA mediated regulation of a stress-activated retrotransposon and the tissue specific transposition during the reproductive period in Arabidopsis

Transposable elements (TEs) are key elements that facilitate genome evolution of the host organism. A number of studies have assessed the functions of TEs, which change gene expression in the host genome. Activation of TEs is controlled by epigenetic modifications such as DNA methylation and histone modifications. Several recent studies have reported that TEs can also be activated by biotic or abiotic stress in some plants. We focused on a Ty1/copia retrotransposon, ONSEN, that is activated by heat stress (HS) in Arabidopsis. We found that transcriptional activation of ONSEN was regulated by a small interfering RNA (siRNA)-related pathway, and the activation could also be induced by oxidative stress. Mutants deficient in siRNA biogenesis that were exposed to HS at the initial stages of vegetative growth showed transgenerational transposition. The transposition was also detected in the progeny, which originated from tissue that had differentiated after exposure to the HS. The results indicated that in some undifferentiated cells, transpositional activity could be maintained quite long after exposure to the HS.

Enhancements in sucrose biosynthesis capacity affect shoot branching in Arabidopsis

We previously demonstrated that transgenic tobacco plants expressing cyanobacterial fructose-1,6-/sedoheptulose-1,7-bisphosphatase in the cytosol increased the number of lateral shoots and leaves at elevated CO2 levels. These findings suggest that alterations in carbon partitioning affect the development of shoot branching. In order to elucidate the underlying mechanisms at the molecular level, we generated transgenic Arabidopsis plants overexpressing cyanobacterial fructose-1,6-bisphosphatase-II in the cytosol (AcF). At elevated CO2 levels, the number of lateral shoots was significantly increased in AcF plants. Sucrose and hexose levels were also higher in AcF plants than in wild-type plants. The expression levels of MAX1, MAX4, YUCCA8, YUCCA9, and BRC1, which are involved in auxin or strigolactone biosynthesis and responses, were lower in AcF plants than in wild-type plants. These results suggest that alterations in sugar partitioning affect hormone metabolism and responses, resulting in enhanced shoot branching.

Arabidopsis clade IV TGA transcription factors, TGA10 and TGA9, are involved in ROS-mediated responses to bacterial PAMP flg22

Reactive oxygen species (ROS) produced in chloroplasts have been proposed to act as signaling molecules for plant immunity through pathogen-associated molecular patterns (PAMPs), such as flg22. To elucidate this process, we herein conducted genetic screening of flg22-sensitive mutants from T-DNA insertion lines lacking chloroplastic H2O2-responsive genes. The results obtained showed that knockout mutants lacking a clade IV TGA transcription factor, TGA10, were more sensitive to the flg22 treatment than wild-type plants. Furthermore, although no flg22-sensitive phenotype was detected in the knockout mutant of another clade IV TGA9, double knockout tga9 tga10 mutants showed more sensitivity to flg22 than single knockout mutants. Transcripts of TGA10 and TGA9 were strongly induced by flg22 in leaves, and this was facilitated by the double knockout of stromal and thylakoid-bound ascorbate peroxidases (APX), which are major H2O2 scavengers in chloroplasts. The flg22-induced H2O2 accumulation was maintained at high level in these APXs mutants, indicating the clade IV TGAs may be induced by the ROS. Furthermore, TGA10 was required for the complete activation of the expression of several flg22-responsive genes in plants treated with this PAMP. These have provided a new insight into the relationship between the TGA transcription factors and ROS-mediated signaling in PAMPs responses.

Identification and characterization of Arabidopsis AtNUDX9 as a GDP-d-mannose pyrophosphohydrolase: its involvement in root growth inhibition in response to ammonium

Highlight AtNUDX9, a GDP-d-Man pyrophosphohydrolase in Arabidopsis, is involved in the regulation of GDP-d-Man levels affecting ammonium sensitivity via modulation of protein N-glycosylation in the roots.