Sugar Transporter Protein 1 (STP1) contributes to regulation of the genes involved in shoot branching via carbon partitioning in Arabidopsis.

We previously demonstrated that alterations in sugar partitioning affect the expression of genes involved in hormone biosynthesis and responses, including BRANCHED1 (BRC1), resulting in enhanced shoot branching in transgenic Arabidopsis plants overexpressing cyanobacterial fructose-1,6-bisphosphatase-II in the cytosol (AcF). The exogenous treatment of wild-type Arabidopsis plants with sugars showed the same transcript characteristics, indicating that sugars act as a signal for branching. We also found that the reductions induced in BRC1 expression levels in wild-type plants by the sugar treatments were suppressed in the knockout mutant of sugar transporter 1 (stp1-1). Intracellular sugar contents were similar in stp1-1 and wild-type plants following the sugar treatments, suggesting that STP1 acts as a factor for the regulation of shoot branching depending on extracellular sugar contents. Abbreviations: BRC1: BRABCHED1; FBP/SBPase: fructose-1,6-/sedoheptulose-1,7-bisphosphatase; Glc: glucose; HXK: hexokinase; SnRK1.1/AKIN10: SNF1-RELATED PROTEIN KINASE 1.1; Suc: sucrose; SnRK1: sucrose non-fermenting 1-related protein kinase; STP: sugar transporter protein.

The basic helix-loop-helix transcription factor, bHLH11 functions in the iron-uptake system in Arabidopsis thaliana.

Iron (Fe) is a micronutrient that is essential for plant development and growth. Basic helix-loop-helix (bHLH) transcription factors are a superfamily of transcription factors that are important regulatory components in transcriptional networks in plants. bHLH transcription factors have been divided into subclasses based on their amino acid sequences and domain structures. Among the members of clade IVb (PYE, bHLH121, and bHLH11), the functions of bHLH11 remain unclear. In the present study, we characterized bHLH11 as a negative regulator of Fe homeostasis. bHLH11 expression levels were high in the roots and up-regulated after plants were transferred to Fe sufficient conditions. Although T-DNA knockout mutants of bHLH11 were lethal, dominant negative (DN-) and overexpression (OX-) of bHLH11 plants exhibited sensitivity to Fe deficiency. Furthermore, the expression of FIT, a master regulator of Fe deficiency responses, was suppressed in the transgenic plants. These results suggest that the transcriptional repressor bHLH11 functions as a negative regulator of FIT-dependent Fe uptake and modulates Fe levels in Arabidopsis plants. Salicylic acid (SA) modulates the expression of genes involved in Fe-deficient responses. We found that SA levels were elevated in DN- and OX-bHLH11 plants. The T-DNA insertion mutant sid2-1, which was defective for the production of SA, did not exhibit sensitivity to Fe deficiency; however, the crossed plants of OX-bHLH11 and sid2-1 relieved sensitivity to the Fe deficiency observed in OX-bHLH11 plants. These results suggest that the accumulation of SA is closely related to iron homeostasis.

Suppression of Chloroplastic Alkenal/One Oxidoreductase Represses the Carbon Catabolic Pathway in Arabidopsis Leaves during Night.

Lipid-derived reactive carbonyl species (RCS) possess electrophilic moieties and cause oxidative stress by reacting with cellular components. Arabidopsis (Arabidopsis thaliana) has a chloroplast-localized alkenal/one oxidoreductase (AtAOR) for the detoxification of lipid-derived RCS, especially α,ß-unsaturated carbonyls. In this study, we aimed to evaluate the physiological importance of AtAOR and analyzed AtAOR (aor) mutants, including a transfer DNA knockout, aor (T-DNA), and RNA interference knockdown, aor (RNAi), lines. We found that both aor mutants showed smaller plant sizes than wild-type plants when they were grown under day/night cycle conditions. To elucidate the cause of the aor mutant phenotype, we analyzed the photosynthetic rate and the respiration rate by gas-exchange analysis. Subsequently, we found that both wild-type and aor (RNAi) plants showed similar CO2 assimilation rates; however, the respiration rate was lower in aor (RNAi) than in wild-type plants. Furthermore, we revealed that phosphoenolpyruvate carboxylase activity decreased and starch degradation during the night was suppressed in aor (RNAi). In contrast, the phenotype of aor (RNAi) was rescued when aor (RNAi) plants were grown under constant light conditions. These results indicate that the smaller plant sizes observed in aor mutants grown under day/night cycle conditions were attributable to the decrease in carbon utilization during the night. Here, we propose that the detoxification of lipid-derived RCS by AtAOR in chloroplasts contributes to the protection of dark respiration and supports plant growth during the night.

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 dehydroascorbate reductase 1 and 2 modulate redox states of ascorbate-glutathione cycle in the cytosol in response to photooxidative stress.

Ascorbate and glutathione are indispensable cellular redox buffers and allow plants to acclimate stressful conditions. Arabidopsis contains three functional dehydroascorbate reductases (DHAR1-3), which catalyzes the conversion of dehydroascorbate into its reduced form using glutathione as a reductant. We herein attempted to elucidate the physiological role in DHAR1 and DHAR2 in stress responses. The total DHAR activities in DHAR knockout Arabidopsis plants, dhar1 and dhar2, were 22 and 92%, respectively, that in wild-type leaves. Under high light (HL), the levels of total ascorbate and dehydroascorbate were only reduced and increased, respectively, in dhar1. The oxidation of glutathione under HL was significantly inhibited in both dhar1 and dhar2, while glutathione contents were only enhanced in dhar1. The dhar1 showed stronger visible symptoms than the dhar2 under photooxidative stress conditions. Our results demonstrated a pivotal role of DHAR1 in the modulation of cellular redox states under photooxidative stress.

Enhanced photosynthetic capacity increases nitrogen metabolism through the coordinated regulation of carbon and nitrogen assimilation in Arabidopsis thaliana.

Plant growth and productivity depend on interactions between the metabolism of carbon and nitrogen. The sensing ability of internal carbon and nitrogen metabolites (the C/N balance) enables plants to regulate metabolism and development. In order to investigate the effects of an enhanced photosynthetic capacity on the metabolism of carbon and nitrogen in photosynthetically active tissus (source leaves), we herein generated transgenic Arabidopsis thaliana plants (ApFS) that expressed cyanobacterial fructose-1,6-/sedoheptulose-1,7-bisphosphatase in their chloroplasts. The phenotype of ApFS plants was indistinguishable from that of wild-type plants at the immature stage. However, as plants matured, the growth of ApFS plants was superior to that of wild-type plants. Starch levels were higher in ApFS plants than in wild-type plants at 2 and 5 weeks. Sucrose levels were also higher in ApFS plants than in wild-type plants, but only at 5 weeks. On the other hand, the contents of various free amino acids were lower in ApFS plants than in wild-type plants at 2 weeks, but were similar at 5 weeks. The total C/N ratio was the same in ApFS plants and wild-type plants, whereas nitrite levels increased in parallel with elevations in nitrate reductase activity at 5 weeks in ApFS plants. These results suggest that increases in the contents of photosynthetic intermediates at the early growth stage caused a temporary imbalance in the free-C/free-N ratio and, thus, the feedback inhibition of the expression of genes involved in the Calvin cycle and induction of the expression of those involved in nitrogen metabolism due to supply deficient free amino acids for maintenance of the C/N balance in source leaves of ApFS plants.

Wax Ester Fermentation and Its Application for Biofuel Production.

In Euglena cells under anaerobic conditions, paramylon, the storage polysaccharide, is promptly degraded and converted to wax esters. The wax esters synthesized are composed of saturated fatty acids and alcohols with chain lengths of 10-18, and the major constituents are myristic acid and myristyl alcohol. Since the anaerobic cells gain ATP through the conversion of paramylon to wax esters, the phenomenon is named "wax ester fermentation". The wax ester fermentation is quite unique in that the end products, i.e. wax esters, have relatively high molecular weights, are insoluble in water, and accumulate in the cells, in contrast to the common fermentation end products such as lactic acid and ethanol.A unique metabolic pathway involved in the wax ester fermentation is the mitochondrial fatty acid synthetic system. In this system, fatty acid are synthesized by the reversal of ß-oxidation with an exception that trans-2-enoyl-CoA reductase functions instead of acyl-CoA dehydrogenase. Therefore, acetyl-CoA is directly used as a C2 donor in this fatty acid synthesis, and the conversion of acetyl-CoA to malonyl-CoA, which requires ATP, is not necessary. Consequently, the mitochondrial fatty acid synthetic system makes possible the net gain of ATP through the synthesis of wax esters from paramylon. In addition, acetyl-CoA is provided in the anaerobic cells from pyruvate by the action of a unique enzyme, oxygen sensitive pyruvate:NADP+ oxidoreductase, instead of the common pyruvate dehydrogenase multienzyme complex.Wax esters produced by anaerobic Euglena are promising biofuels because myristic acid (C14:0) in contrast to other algal produced fatty acids, such as palmitic acid (C16:0) and stearic acid (C18:0), has a low freezing point making it suitable as a drop-in jet fuel. To improve wax ester production, the molecular mechanisms by which wax ester fermentation is regulated in response to aerobic and anaerobic conditions have been gradually elucidated by identifying individual genes related to the wax ester fermentation metabolic pathway and by comprehensive gene/protein expression analysis. In addition, expression of the cyanobacterial Calvin cycle fructose-1,6-bisphosphatase/sedohepturose-1,7-bisphosphatase, in Euglena provided photosynthesis resulting in increased paramylon accumulation enhancing wax ester production. This chapter will discuss the biochemistry of the wax ester fermentation, recent advances in our understanding of the regulation of the wax ester fermentation and genetic engineering approaches to increase production of wax esters for biofuels.

A gain-of-function mutation of plastidic invertase alters nuclear gene expression with sucrose treatment partially via GENOMES UNCOUPLED1-mediated signaling.

Plastid gene expression (PGE) is one of the signals that regulate the expression of photosynthesis-associated nuclear genes (PhANGs) via GENOMES UNCOUPLED1 (GUN1)-dependent retrograde signaling. We recently isolated Arabidopsis sugar-inducible cotyledon yellow-192 (sicy-192), a gain-of-function mutant of plastidic invertase, and showed that following the treatment of this mutant with sucrose, the expression of PhANGs as well as PGE decreased, suggesting that the sicy-192 mutation activates a PGE-evoked and GUN1-mediated retrograde pathway. To clarify the relationship between the sicy-192 mutation, PGE, and GUN1-mediated pathway, plastid and nuclear gene expression in a double mutant of sicy-192 and gun1-101, a null mutant of GUN1 was studied. Plastid-encoded RNA polymerase (PEP)-dependent PGE was markedly suppressed in the sicy-192 mutant by the sucrose treatment, but the suppression as well as cotyledon yellow phenotype was not mitigated by GUN1 disruption. Microarray analysis revealed that the altered expression of nuclear genes such as PhANG in the sucrose-treated sicy-192 mutant was largely dependent on GUN1. The present findings demonstrated that the sicy-192 mutation alters nuclear gene expression with sucrose treatment via GUN1, which is possibly followed by inhibiting PEP-dependent PGE, providing a new insight into the role of plastid sugar metabolism in nuclear gene expression.

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

GDP-d-mannose (GDP-d-Man) is an important intermediate in ascorbic acid (AsA) synthesis, cell wall synthesis, protein N-glycosylation, and glycosylphosphatidylinositol-anchoring in plants. Thus, the modulation of intracellular levels of GDP-d-Man could be important for maintaining various cellular processes. Here an Arabidopsis GDP-d-Man pyrophosphohydrolase, AtNUDX9 (AtNUDT9; At3g46200), which hydrolysed GDP-d-Man to GMP and mannose 1-phosphate, was identified. The K m and V max values for GDP-d-Man of AtNUDX9 were 376±24 µM and 1.61±0.15 µmol min(-1) mg(-1) protein, respectively. Among various tissues, the expression levels of AtNUDX9 and the total activity of GDP-d-Man pyrophosphohydrolase were the highest in the roots. The GDP-d-Man pyrophosphohydrolase activity was increased in the root of plants grown in the presence of ammonium. No difference was observed in the levels of AsA in the leaf and root tissues of the wild-type and knockout-nudx9 (KO-nudx9) plants, whereas a marked increase in N-glycoprotein levels and enhanced growth were detected in the roots of KO-nudx9 plants in the presence of ammonium. These results suggest that AtNUDX9 is involved in the regulation of GDP-d-Man levels affecting ammonium sensitivity via modulation of protein N-glycosylation in the roots.

Characterization and physiological role of two types of chloroplastic fructose-1,6-bisphosphatases in Euglena gracilis.

The chloroplastic fructose-1,6-bisphosphatase (FBPase) is a late-limiting enzyme in the Calvin cycle. In the present study, we isolated and characterized the cDNAs encoding two types of chloroplastic FBPase isoforms (EgFBPaseI and II) from Euglena gracilis. The Km values of recombinant EgFBPaseI and EgFBPaseII for fructose 1,6-bisphosphate (Fru 1,6-P2) were 165 ± 17 and 2200 ± 200 µM, respectively. The activity of EgFBPaseI was inhibited by 1mM H2O2 and recovered when incubated with DTT. The activity of EgFBPaseII was resistant to concentrations of H2O2 up to 1mM, which was distinct from those of EgFBPaseI and spinach chloroplastic FBPase. The suppression of EgFBPaseI gene expression by gene silencing markedly decreased photosynthetic activity and inhibited cell growth. The results of the present study clearly demonstrated that EgFBPaseI played a critical role in photosynthesis in Euglena chloroplasts.

Diversity of regulatory mechanisms of photosynthetic carbon metabolism in plants and algae.

To clarify the regulatory mechanisms of the Calvin cycle in algae, we analyzed the molecular properties of the enzymes involved in this cycle. We demonstrated that these enzymes were not regulated by redox modulation through the ferredoxin/thioredoxin system under light/dark conditions and were not sensitive to treatments with hydrogen peroxide in vitro, unlike the chloroplastic thiol-modulated enzymes of plants. On the other hand, we found that cyanobacteria possessed a unique enzyme involved in the Calvin cycle. The CP12 protein played an important role in regulating carbon metabolism in the Calvin cycle in cyanobacteria and eukaryotic algae. This review described the regulatory mechanisms of the Calvin cycle in algae and also the effects of alterations to photosynthetic carbon metabolism on plant productivity, carbon partitioning, and the carbon/nitrogen balance using transgenic plants expressing algal genes.

Identification and characterization of cytosolic fructose-1,6-bisphosphatase in Euglena gracilis.

Euglena gracilis has the ability to accumulate a storage polysaccharide, a ß-1,3-glucan known as paramylon, under aerobic conditions. Under anaerobic conditions, E. gracilis cells degrade paramylon and synthesize wax esters. Cytosolic fructose-1,6-bisphosphatase (FBPase) appears to be a key enzyme in gluconeogenesis and position branch point of carbon partitioning between paramylon and wax ester biosynthesis. We herein identified and characterized cytosolic FBPase from E. gracilis. The Km and Vmax values of EgFBPaseIII were 16.5 ± 1.6 µM and 30.4 ± 7.2 µmol min(-1) mg protein(-1), respectively. The activity of EgFBPaseIII was not regulated by AMP or reversible redox modulation. No significant differences were observed in the production of paramylon in transiently suppressed EgFBPaseIII gene expression cells by RNAi (KD-EgFBPaseIII); nevertheless, FBPase activity was markedly decreased in KD-EgFBPaseIII cells. On the other hand, the growth of KD-EgFBPaseIII cells was slightly higher than that of control cells.

O2-dependent large electron flow functioned as an electron sink, replacing the steady-state electron flux in photosynthesis in the cyanobacterium Synechocystis sp. PCC 6803, but not in the cyanobacterium Synechococcus sp. PCC 7942.

To determine whether alternative electron flow (AEF) can replace the photosynthetic electron flow in cyanobacteria, we used an open O2-electrode system to monitor O2-exchange over a long period. In air-grown Synechocystis sp. PCC 6803 (S. 6803(WT)), the quantum yield of PSII, Y(II), held even after photosynthesis was suppressed by CO2 shortage. The S. 6803 mutant, deficient in flavodiiron (FLV) proteins 1 and 3, showed the same phenotype as S. 6803(WT). In contrast, Y(II) decreased in Synechococcus sp. PCC 7942 (S. 7942). These results suggest that AEF functioned as the Y(II) in S. 6803 and replaced the photosynthetic electron flux. In contrast, the activity of AEF in S. 7942 was lower. The affinity of AEF for O2 in S. 6803 did not correspond to those of FLVs in bacteria or terminal oxidases in respiration. AEF might be driven by photorespiration.

H2O2-triggered retrograde signaling from chloroplasts to nucleus plays specific role in response to stress.

Recent findings have suggested that reactive oxygen species (ROS) are important signaling molecules for regulating plant responses to abiotic and biotic stress and that there exist source- and kind-specific pathways for ROS signaling. In plant cells, a major source of ROS is chloroplasts, in which thylakoid membrane-bound ascorbate peroxidase (tAPX) plays a role in the regulation of H(2)O(2) levels. Here, to clarify the signaling function of H(2)O(2) derived from the chloroplast, we created a conditional system for producing H(2)O(2) in the organelle by chemical-dependent tAPX silencing using estrogen-inducible RNAi. When the expression of tAPX was silenced in leaves, levels of oxidized protein in chloroplasts increased in the absence of stress. Microarray analysis revealed that tAPX silencing affects the expression of a large set of genes, some of which are involved in the response to chilling and pathogens. In response to tAPX silencing, the transcript levels of C-repeat/DRE binding factor (CBF1), a central regulator for cold acclimation, was suppressed, resulting in a high sensitivity of tAPX-silenced plants to cold. Furthermore, tAPX silencing enhanced the levels of salicylic acid (SA) and the response to SA. Interestingly, we found that tAPX silencing-responsive genes were up- or down-regulated by high light (HL) and that tAPX silencing had a negative effect on expression of ROS-responsive genes under HL, suggesting synergistic and antagonistic roles of chloroplastic H(2)O(2) in HL response. These findings provide a new insight into the role of H(2)O(2)-triggered retrograde signaling from chloroplasts in the response to stress in planta.

Cytosolic ascorbate peroxidase 1 protects organelles against oxidative stress by wounding- and jasmonate-induced H(2)O(2) in Arabidopsis plants.

BACKGROUND: Reactive oxygen species (ROS) are not only cytotoxic compounds leading to oxidative damage, but also signaling molecules for regulating plant responses to stress and hormones. Arabidopsis cytosolic ascorbate peroxidase 1 (APX1) is thought to be a central regulator for cellular ROS levels. However, it remains unclear whether APX1 is involved in plant tolerance to wounding and methyl jasmonate (MeJA) treatment, which are known to enhance ROS production. METHODS: We studied the effect of wounding and MeJA treatment on the levels of H(2)O(2) and oxidative damage in the Arabidopsis wild-type plants and knockout mutants lacking APX1 (KO-APX1). RESULTS: The KO-APX1 plants showed high sensitivity to wounding and MeJA treatment. In the leaves of wild-type plants, H(2)O(2) accumulated only in the vicinity of the wound, while in the leaves of the KO-APX1 plants it accumulated extensively from damaged to undamaged regions. During MeJA treatment, the levels of H(2)O(2) were much higher in the leaves of KO-APX1 plants. Oxidative damage in the chloroplasts and nucleus was also enhanced in the leaves of KO-APX1 plants. These findings suggest that APX1 protects organelles against oxidative stress by wounding and MeJA treatment. GENERAL SIGNIFICANCE: This is the first report demonstrating that H(2)O(2)-scavenging in the cytosol is essential for plant tolerance to wounding and MeJA treatment.

Improvement of vitamin E quality and quantity in tobacco and lettuce by chloroplast genetic engineering.

Vitamin E (tocopherol: Toc) is an important lipid-soluble antioxidant synthesized in chloroplasts. Among the 8 isoforms of vitamin E, α-Toc has the highest activity in humans. To generate transgenic plants with enhanced vitamin E activity, we applied a chloroplast transformation technique. Three types of the transplastomic tobacco plants (pTTC, pTTMT and pTTC-TMT) carrying the Toc cyclase (TC) or γ-Toc methyltransferase (γ-TMT) gene and the TC plus γ-TMT genes as an operon in the plastid genome, respectively, were generated. There was a significant increase in total levels of Toc due to an increase in γ-Toc in the pTTC plants. Compared to the wild-type plants, Toc composition was altered in the pTTMT plants. In the pTTC-TMT plants, total Toc levels increased and α-Toc was a major Toc isoform. Furthermore, to use chloroplast transformation to produce α-Toc-rich vegetable, TC-overexpressing transplastomic lettuce plants (pLTC) were generated. Total Toc levels and vitamin E activity increased in the pLTC plants compared with the wild-type lettuce plants. These findings indicated that chloroplast genetic engineering is useful to improve vitamin E quality and quantity in plants.

Activation of γ-aminobutyrate production by chloroplastic H(2)O(2) is associated with the oxidative stress response.

We isolated an Arabidopsis knockout line lacking glutamate decarboxylase 1 (GAD1), one that produced γ-aminobutyrate (GABA), as an oxidative stress-insensitive mutant, and found that chloroplastic H(2)O(2) enhances GAD1 expression and GABA levels. This suggests a possible relationship between GABA metabolism and the chloroplastic H(2)O(2)-mediated stress response.

The involvement of Arabidopsis glutathione peroxidase 8 in the suppression of oxidative damage in the nucleus and cytosol.

A family of eight genes with homology to mammalian glutathione peroxidase (GPX) isoenzymes, designated AtGPX1-AtGPX8, has been identified in Arabidopsis thaliana. In this study we demonstrated the functional analysis of Arabidopsis AtGPX8 with peroxidase activity toward H(2)O(2) and lipid hydroperoxides using thioredoxin as an electron donor. The transcript and protein levels of AtGPX8 in Arabidopsis were up-regulated coordinately in response to oxidative damage caused by high-light (HL) stress or treatment with paraquat (PQ). Furthermore, the knockout Arabidopsis mutants of AtGPX8 (KO-gpx8) exhibited increased sensitivity to oxidative damage caused by PQ treatment in root elongation compared with the wild-type plants. In contrast, transgenic lines overexpressing AtGPX8 (Ox-AtGPX8) were less sensitive to oxidative damage than the wild-type plants. The levels of oxidized proteins in the KO-gpx8 and Ox-AtGPX8 lines were enhanced and suppressed, respectively, compared with the wild-type plants under HL stress or PQ treatment. The fusion protein of AtGPX8 tagged with green fluorescent protein was localized in the cytosol and nucleus of onion epidermal cells. In addition, the AtGPX8 protein was detected in the cytosolic and nuclear fractions prepared from leaves of Arabidopsis plants using the AtGPX8 antibody. Oxidative DNA damage under treatment with PQ increased in the wild-type and KO-gpx8 plants, while it decreased in the OX-AtGPX8 plants. These results suggest that AtGPX8 plays an important role in the protection of cellular components including nuclear DNA against oxidative stress.

An Arabidopsis FAD pyrophosphohydrolase, AtNUDX23, is involved in flavin homeostasis.

Although flavins, riboflavin (RF), FMN and FAD, are essential for primary and secondary metabolism in plants, the metabolic regulation of flavins is still largely unknown. Recently, we found that an Arabidopsis Nudix hydrolase, AtNUDX23, has FAD pyrophosphohydrolase activity and is distributed in plastids. Levels of RF and FAD but not FMN in Arabidopsis leaves significantly increased under continuous light and decreased in the dark. The transcript levels of AtNUDX23 as well as genes involved in flavin metabolism (AtFADS, AtRibF1, AtRibF2, AtFMN/FHy, LS and AtRibA) significantly increased under continuous light. The pyrophosphohydrolase activity toward FAD was enhanced in AtNUDX23-overexpressing (OX-NUDX23) plants and reduced in AtNUDX23-suppressed (KD-nudx23) plants, compared with the control plants. Interestingly intracellular levels of RF, FMN and FAD significantly decreased in not only OX-NUDX23 but also KD-nudx23 plants. The transcript levels of the flavin metabolic genes also decreased in both plants. Similarly, the increase in intracellular levels on treatment with flavins caused a reduction in the transcript levels of genes involved in flavin metabolism. These results suggest that negative feedback regulation of the metabolism of flavins through the hydrolysis of FAD by AtNUDX23 in plastids is involved in flavin homeostasis in plant cells.

Enzymatic and molecular characterization of Arabidopsis ppGpp pyrophosphohydrolase, AtNUDX26.

Not only in bacteria but also in plant cells, guanosine-3',5'-tetraphosphate (ppGpp) is an important signaling molecule, that affects various cellular processes. In this study, we identified nucleoside diphosphates linked to some moiety X (Nudix) hydrolases, AtNUDX11, 15, 25, and 26, having ppGpp pyrophosphohydrolase activity from Arabidopsis plants. Among these, AtNUDX26 localized in chloroplasts had the highest Vmax and kcat values, leading to high catalytic efficiency, kcat/Km. The activity of AtNUDX26 required Mg2+ or Mn2+ ions as cofactor and was optimal at pH 9.0 and 50 °C. The expression of AtNUDX26 and of ppGpp metabolism-associated genes was regulated by various types of stress, suggesting that AtNUDX26 regulates cellular ppGpp levels in response to stress and impacts gene expression in chloroplasts. This is the first report on the molecular properties of ppGpp pyrophosphohydrolases in plants.