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The albino lemma 1 (alm1) mutants of barley (Hordeum vulgare L.) exhibit obvious chlorophyll-deficient hulls. Hulls are seed-enclosing tissues on the spike, consisting of the lemma and palea. The alm1 phenotype is also expressed in the pericarp, culm nodes and basal leaf sheaths, but leaf blades and awns are normal green. A single recessive nuclear gene controls tissue-specific alm1 phenotypic expression. Positional cloning revealed that the ALM1 gene encodes a Golden 2-like (GLK) transcription factor, HvGLK2, belonging to the GARP subfamily of Myb transcription factors. This finding was validated by genetic evidence indicating that all 10 alm1 mutants studied had a lesion in functionally important regions of HvGLK2, including the three alpha-helix domains, an AREAEAA motif and the GCT box. Transmission electron microscopy revealed that, in lemmas of the alm1.g mutant, the chloroplasts lacked thylakoid membranes, instead of stacked thylakoid grana in wild-type chloroplasts. Compared with wild type, alm1.g plants showed similar levels of leaf photosynthesis but reduced spike photosynthesis by 34%. The alm1.g mutant and the alm1.a mutant showed a reduction in 100-grain weight by 15.8% and 23.1%, respectively. As in other plants, barley has HvGLK2 and a paralog, HvGLK1. In flag leaves and awns, HvGLK2 and HvGLK1 are expressed at moderate levels, but in hulls, HvGLK1 expression was barely detectable compared with HvGLK2. Barley alm1/Hvglk2 mutants exhibit more severe phenotypes than glk2 mutants of other plant species reported to date. The severe alm1 phenotypic expression in multiple tissues indicates that HvGLK2 plays some roles that are nonredundant with HvGLK1.

DOI： 10.1093/pcp/pcab001

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Bundle Sheath Defective 2, BSD2, is a stroma-targeted protein initially identified as a factor required for the biogenesis of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) in maize. Plants and algae universally have a homologous gene for BSD2 and its deficiency causes a RuBisCO-less phenotype. As RuBisCO can be the rate-limiting step in CO2 assimilation, the overexpression of BSD2 might improve photosynthesis and productivity through the accumulation of RuBisCO. To examine this hypothesis, we produced BSD2 overexpression lines in Arabidopsis. Compared with wild type, the BSD2 overexpression lines BSD2ox-2 and BSD2ox-3 expressed 4.8-fold and 8.8-fold higher BSD2 mRNA, respectively, whereas the empty-vector (EV) harbouring plants had a comparable expression level. The overexpression lines showed a significantly higher CO2 assimilation rate per available CO2 and productivity than EV plants. The maximum carboxylation rate per total catalytic site was accelerated in the overexpression lines, while the number of total catalytic sites and RuBisCO content were unaffected. We then isolated recombinant BSD2 (rBSD2) from E. coli and found that rBSD2 reduces disulfide bonds using reductants present in vivo, for example glutathione, and that rBSD2 has the ability to reactivate RuBisCO that has been inactivated by oxidants. Furthermore, 15% of RuBisCO freshly isolated from leaves of EV was oxidatively inactivated, as compared with 0% in BSD2-overexpression lines, suggesting that the overexpression of BSD2 maintains RuBisCO to be in the reduced active form in vivo. Our results demonstrated that the overexpression of BSD2 improves photosynthetic efficiency in Arabidopsis and we conclude that it is involved in mediating RuBisCO activation.

DOI： 10.1111/tpj.14617

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Mitochondria and chloroplasts (plastids) both harbour extranuclear DNA that originates from the ancestral endosymbiotic bacteria. These organelle DNAs (orgDNAs) encode limited genetic information but are highly abundant, with multiple copies in vegetative tissues, such as mature leaves. Abundant orgDNA constitutes a substantial pool of organic phosphate along with RNA in chloroplasts, which could potentially contribute to phosphate recycling when it is degraded and relocated. However, whether orgDNA is degraded nucleolytically in leaves remains unclear. In this study, we revealed the prevailing mechanism in which organelle exonuclease DPD1 degrades abundant orgDNA during leaf senescence. The DPD1 degradation system is conserved in seed plants and, more remarkably, we found that it was correlated with the efficient use of phosphate when plants were exposed to nutrient-deficient conditions. The loss of DPD1 compromised both the relocation of phosphorus to upper tissues and the response to phosphate starvation, resulting in reduced plant fitness. Our findings highlighted that DNA is also an internal phosphate-rich reservoir retained in organelles since their endosymbiotic origin.

DOI： 10.1038/s41477-018-0291-x

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Language：English   Publisher：Oxford University Press  

Endosymbiotically originated chloroplast DNA (cpDNA) encodes part of the genetic information needed to fulfill chloroplast function, including fundamental processes such as photosynthesis. In the last two decades, advances in genome analysis led to the identification of a considerable number of cpDNA sequences from various species. While these data provided the consensus features of cpDNA organization and chloroplast evolution in plants, how cpDNA is maintained through development and is inherited remains to be fully understood. In particular, the fact that cpDNA exists as multiple copies despite its limited genetic capacity raises the important question of how copy number is maintained or whether cpDNA is subjected to quantitative fluctuation or even developmental degradation. For example, cpDNA is abundant in leaves, where it forms punctate structures called nucleoids, which seemingly alter their morphologies and numbers depending on the developmental status of the chloroplast. In this review, we summarize our current understanding of 'cpDNA dynamics', focusing on the changes in DNA abundance. A special focus is given to the cpDNA degradation mechanism, which appears to be mediated by Defective in Pollen organelle DNA degradation 1 (DPD1), a recently discovered organelle exonuclease. The physiological significance of cpDNA degradation in flowering plants is also discussed.

DOI： 10.1093/pcp/pcy084

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BioMed Central Ltd.  

Background: Chlorophyll breakdown is the most obvious sign of leaf senescence. The chlorophyll catabolism pathway and the associated proteins/genes have been identified in considerable detail by genetic approaches combined with stay-green phenotyping. Arabidopsis CYO1 (AtCYO1), a protein disulfide reductase/isomerase localized in the thylakoid membrane, is hypothesized to assemble the photosystem by interacting with cysteine residues of the subunits. Results: In this study, we report that ectopic overexpression of AtCYO1 in leaves induces a stay-green phenotype during darkness, where oxidative conditions favor catabolism. In AtCYO1ox leaves, Fv/Fm and both chlorophyll a and chlorophyll b content remained high during dark-induced senescence. The thylakoid ultrastructure was preserved for a longer time in AtCYO1ox leaves than in wild type leaves. AtCYO1ox leaves maintained thylakoid chlorophyll-binding proteins associated with both PSII (D1, D2, CP43, CP47, LHCB2, and Cyt f) and PSI (PSA-A/B), as well as stromal proteins (Rubisco and ferredoxin-NADP+ reductase). AtCYO1ox did not affect senescence-inducible gene expression for chlorophyll catabolism or accumulation of chlorophyll catabolites. Conclusions: Our results suggest that ectopic overexpression of AtCYO1 had a negative impact on the initiation of chlorophyll degradation and proteolysis within chloroplasts. Our findings cast new light on the redox regulation of protein disulfide bonds for the maintenance of functional chloroplasts.

DOI： 10.1186/s12870-018-1294-5

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press  

Chlorophyll is an essential molecule for acquiring light energy during photosynthesis. Mutations that result in chlorophyll retention during leaf senescence are called - € stay-green' mutants. One of the several types of stay-green mutants, Type E, accumulates high levels of chlorophyll in the pre-senescent leaves, resulting in delayed yellowing. We isolated delayed yellowing1-1 (dye1-1), a rice mutant whose yellowing is delayed in the field. dye1-1 accumulated more chlorophyll than the wild-type in the pre-senescent and senescent leaves, but did not retain leaf functionality in the - €- senescent green leaves', suggesting that dye1-1 is a Type E stay-green mutant. Positional cloning revealed that DYE1 encodes Lhca4, a subunit of the light-harvesting complex I (LHCI). In dye1-1, amino acid substitution occurs at the location of a highly conserved amino acid residue involved in pigment binding
indeed, a severely impaired structure of the PSI-LHCI super-complex in dye1-1 was observed in a blue native PAGE analysis. Nevertheless, the biomass and carbon assimilation rate of dye1-1 were comparable to those in the wild-type. Interestingly, Lhcb1, a trimeric LHCII protein, was highly accumulated in dye1-1, in the chlorophyll-protein complexes. The high accumulation of LHCII in the LHCI mutant dye1 suggests a novel functional interaction between LHCI and LHCII.

DOI： 10.1093/jxb/erx468

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER GMBH, URBAN & FISCHER VERLAG  

In the dicotyledonous plant Arabidopsis thaliana, the cotyledon chloroplast biogenesis factor AtCYO1 is crucial for the biogenesis of cotyledon chloroplasts. Arabidopsis mutants lacking AtCYO1 have pale cotyledons but develop normal mature leaves. In the monocotyledonous plant Oryza sativa, the gene OsCYO1 has high sequence identity to AtCYO1, but its function is unknown. We examined the role of OsCYO1 in O. sativa. We first confirmed that transformation with OsCYO1 could recover the phenotype of the Arabidopsis cyo1 mutant. Similar to AtCYO1, recombinant OsCYO1 has protein disulfide reductase (PDR) activity, which increased as a function of dieosin glutathione disulfide concentration with an apparent K-m of 3.2 mu M and K-cat of 0.53 min(-1). The PDR activity was reduced when NADPH or NADH was used as an electron donor; however, PDR activity was observed with OsCYO1 and glutathione, suggesting that glutathione may serve as a reducing agent for OsCYO1 in vivo. In O. sativa, the OsCYO1 transcript level was higher in leaves compared with the coleoptile, which is the first leaf-like organ that forms during rice embryogenesis. Many OsCYO1 mutant lines defective in RNA interference had green leaves, however, three mutant lines had not only albino coleoptile but also albino leaves. Those having green leaves reduced photosynthetic performance in leaves. Our results demonstrate that OsCYO1 is enzymatically equivalent to AtCYO1 but that the physiological role of OsCYO1 in monocotyledonous plants may differ from that of AtCYO1 in dicotyledonous plants. (C) 2016 Elsevier GmbH. All rights reserved.

DOI： 10.1016/j.jplph.2016.10.005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Chloroplasts are not generated de novo but proliferate from a pre-existing population of plastids present in meristematic cells. Chloroplast division is executed by the co-ordinated action of at least two molecular machineries: internal machinery located on the stromal side of the inner envelope membrane and external machinery located on the cytosolic side of the outer envelope membrane. To date, molecular studies of chloroplast division in higher plants have been limited to several species such as Arabidopsis. To elucidate chloroplast division in rice, we performed forward genetics and isolated a mutant displaying large chloroplasts among an ethyl methanesulfonate (EMS)-mutagenized Oryza sativa spp japonica Nipponbare population. Using a map-based approach, this mutation, termed giant chloroplast (gic), was allocated in a gene that encodes a protein that is homologous to Paralog of ARC6 (PARC6), which is known to play a role in chloroplast division. GIC is unique in that it has a long C-terminal extension that is not present in other PARC6 homologs. Characterization of gic phenotypes in a rice field showed that gic exhibited defective growth in seed setting, suggesting that the gic mutant negatively affects the reproductive stage. This report is the first describing a chloroplast division mutant in monocotyledons and its effect on plant development.

DOI： 10.1093/pcp/pcv024

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Blue light (BL) induces stomatal opening through the activation of H+-ATPases with subsequent ion accumulation in guard cells. In most plant species, red light (RL) enhances BL-dependent stomatal opening. This RL effect is attributable to the chloroplasts of guard cell, the only cells in the epidermis possessing this organelle. To clarify the role of chloroplasts in stomatal regulation, we investigated the effects of RL on BL-dependent stomatal opening in isolated epidermis, guard cell protoplasts, and intact leaves of Arabidopsis thaliana. In isolated epidermal tissues and intact leaves, weak BL superimposed on RL enhanced stomatal opening while BL alone was less effective. In guard cell protoplasts, RL enhanced BL-dependent H+-pumping and DCMU, a photosynthetic electron transport inhibitor, eliminated this effect. RL enhanced phosphorylation levels of the H+-ATPase in response to BL, but this RL effect was not suppressed by DCMU. Furthermore, DCMU inhibited both RL-induced and BL-dependent stomatal opening in intact leaves. The photosynthetic rate in leaves correlated positively with BL-dependent stomatal opening in the presence of DCMU. We conclude that guard cell chloroplasts provide ATP and/or reducing equivalents that fuel BL-dependent stomatal opening, and that they indirectly monitor photosynthetic CO2 fixation in mesophyll chloroplasts by absorbing PAR in the epidermis.

DOI： 10.1371/journal.pone.0108374

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Language：English   Publisher：OXFORD UNIV PRESS  

During leaf senescence, macromolecules such as proteins and lipids are known to be degraded for redistribution into upper tissues. Similarly, nucleic acids appear to undergo fragmentation or degradation during senescence, but the physiological role of nucleic acid degradation, particularly of genomic DNA degradation, remains unclear. To date, more than a dozen of plant deoxyribonucleases have been reported, whereas it remains to be verified whether any of them degrade DNA during leaf senescence. This review summarizes current knowledge related to the plant nucleases that are induced developmentally or in a tissue-specific manner and are known to degrade DNA biochemically. Of these, several endonucleases (BFN1, CAN1, and CAN2) and an exonuclease (DPD1) in Arabidopsis seem to act in leaf senescence because they were shown to be inducible at the transcript level. This review specifically examines DPD1, which is dual-targeted to chloroplasts and mitochondria. Results show that, among the exonuclease family to which DPD1 belongs, DPD1 expression is extraordinary when estimated using a microarray database. DPD1 is the only example among the nucleases in which DNA degradation has been confirmed in vivo in pollen by mutant analysis. These data imply a significant role of organelle DNA degradation during leaf senescence and implicate DPD1 as a potential target for deciphering nucleotide salvage in plants.

DOI： 10.1093/jxb/eru091

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© Zhejiang University Press, Hangzhou and Springer-Verlag Berlin Heidelberg 2013. The Arabidopsis thaliana kas3 mutant was isolated based on its hypersensitivity of photosystem (PS) II to low temperature using a chlorophyll (Chl) fluorescence imaging system. Chl content was lower in kas3 seedlings cultured at 23 °C than in the wild type, but maximum PSII activity was only mildly affected. We also clarified that the activity and levels of photosynthetic electron transport machinery were reduced after chilling treatment. The kas3 mutation causes an amino acid alteration in 3-ketoacyl-ACP synthase III (KasIII) which catalyzes the first decarboxy condensation step in de novo fatty biosynthesis in plastids. The defect in KasIII led to the partial loss of the de novo synthesis pathway for fatty acids in plastids. Consequently, the total fatty acid level was reduced to 75% of the wild-type level in kas3 at 23 °C and was further reduced to 60% at 4 °C. The full activity of KasIII is required for the biogenesis of intact photosynthetic machinery in thylakoid membranes and is especially important for the process responding to low temperature.

DOI： 10.1007/978-3-642-32034-7_136

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Sigma factor is a subunit of plastid-encoded RNA polymerase that regulates the transcription of plastid-encoded genes by recognizing a set of promoters. Sigma factors have increased in copy number and have diversified during the evolution of land plants, but details of this process remain unknown. Liverworts represent the basal group of embryophytes and are expected to retain the ancestral features of land plants. In liverwort (Marchantia polymorpha L.), we isolated and characterized a T-DNA-tagged mutant (Mpsig1) of sigma factor 1 (MpSIG1). The mutant did not show any visible phenotypes, implying that MpSIG1 function is redundant with that of other sigma factors. However, quantitative reverse-transcription polymerase chain reaction and RNA gel blot analysis revealed that genes related to photosynthesis were downregulated, resulting in the minor reduction of some protein complexes. The transcript levels of genes clustered in the petL, psaA, psbB, psbK, and psbE operons of liverwort were lower than those in the wild type, a result similar to that in the SIG1 defective mutant in rice (Oryza sativa). Overexpression analysis revealed primitive functional divergence between the SIG1 and SIG2 proteins in bryophytes, whereas these proteins still retain functional redundancy. We also discovered that the predominant sigma factor for ndhF mRNA expression has been diversified in liverwort, Arabidopsis (Arabidopsis thaliana), and rice. Our study shows the ancestral function of SIG1 and the process of functional partitioning (subfunctionalization) of sigma factors during the evolution of land plants.

DOI： 10.1093/gbe/evt137

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

The Arabidopsis thaliana kas3 mutant was isolated based on the hypersensitivity of PSII to low temperature using a Chl fluorescence imaging technique. Chl content was lower in kas3 seedlings cultured at 23 degrees C than in the wild type, but PSII activity was only mildly affected. However, after the chilling treatment at 4 degrees C for 7 d, PSII activity was severely impaired in kas3. PSII was more sensitive to light at 4 degrees C in the presence of lincomycin, suggesting that the kas3 mutation accelerates at least the PSII photodamage. The kas3 mutation causes an amino acid alteration in 3-ketoacyl-ACP synthase III (KasIII), leading to the partial loss of the de novo synthesis pathway for fatty acids in plastids. Consequently, the total fatty acid level was reduced to 75% of the wild-type level in kas3 at 23 degrees C and was further reduced to 60% at 4 degrees C. The composition of fatty acids was also slightly affected in kas3 at both 4 and 23 degrees C. Consistent with the results of the electron transport analysis, the chilling treatment also destabilized PsaA and cytochrome (Cyt) f and D1 in kas3. An analysis of double mutants with pgr1 conditionally defective in Cyt b(6)f activity and with var2 defective in FtsH protease suggested that the kas3 mutation has pleiotropic effects on chloroplast function, probably impacting both the Cyt b(6)f activity and translation in chloroplasts at 23 degrees C. The full activity of KasIII is required for the biogenesis of the intact electron transport machinery in thylakoid membranes and is especially important for the process of responding to low temperature.

DOI： 10.1093/pcp/pcq085

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC PLANT CELL & MOLECULAR BIOL  

The Arabidopsis thaliana cfd (cytochrome f deficient) mutant was isolated by the sensitivity of its photosystem II to low temperature using a chlorophyll fluorescence imaging technique. The cfd mutant is defective in intersystem electron transport even at 23 degrees C, secondarily leading to photodamage of PSII at 4 degrees C. Map-based cloning revealed that the cfd phenotype is due to a mutation in At1g49380, which encodes a putative plastid-targeting protein with high similarity to Ccs1 in Chlamydomonas reinhardtii. Ccs1 is required for c-type cytochrome (Cyt) assembly in chloroplasts. Consistent with the high sequence similarity of At1g49380 and Ccs1, the levels of Cyt f heme and Cyt f were low in the cfd mutant. We conclude that CFD is an ortholog of Chlamydomonas Ccs1. In vitro ferredoxin-dependent plastoquinone reduction activity was not affected in cfd, suggesting that system II c-type Cyt biogenesis is required for the machinery of photosystem I cyclic electron transport.

DOI： 10.5511/plantbiotechnology.10.0614a

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PLANT BIOLOGISTS  

In higher plants, the chloroplast NAD(P)H dehydrogenase (NDH) complex mediates photosystem I (PSI) cyclic and chlororespiratory electron transport. We reported previously that NDH interacts with the PSI complex to form a super-complex (NDH-PSI). In this study, NDH18 and FKBP16-2 (FK506 Binding Protein 16-2), detected in the NDH-PSI super-complex by mass spectrometry, were shown to be NDH subunits by the analysis of their knockdown lines. On the basis of extensive mutant characterization, we propose a structural model for chloroplast NDH, whereby NDH is divided into four subcomplexes. The subcomplex A and membrane subcomplex are conserved in cyanobacteria, but the subcomplex B and lumen subcomplex are specific to chloroplasts. Two minor light-harvesting complex I proteins, Lhca5 and Lhca6, were required for the full-size NDH-PSI supercomplex formation. Similar to crr pgr5 double mutants that completely lack cyclic electron flow activity around PSI, the lhca6 pgr5 double mutant exhibited a severe defect in growth. Consistent with the impaired NDH activity, photosynthesis was also severely affected in mature leaves of lhca6 pgr5. We conclude that chloroplast NDH became equipped with the novel subcomplexes and became associated with PSI during the evolution of land plants, and this process may have facilitated the efficient operation of NDH.

DOI： 10.1105/tpc.109.068791

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PLANT BIOLOGISTS  

The cytochrome b(6)f (Cyt b6f) complex in flowering plants contains nine conserved subunits, of which three, PetG, PetL, and PetN, are bitopic plastid-encoded low-molecular-weight proteins of largely unknown function. Homoplastomic knockout lines of the three genes have been generated in tobacco (Nicotiana tabacum 'Petit Havana') to analyze and compare their roles in assembly and stability of the complex. Deletion of petG or petN caused a bleached phenotype and loss of photosynthetic electron transport and photoautotrophy. Levels of all subunits that constitute the Cyt b6f complex were faintly detectable, indicating that both proteins are essential for the stability of the membrane complex. In contrast, Delta petL plants accumulate about 50% of other Cyt b6f subunits, appear green, and grow photoautotrophically. However, Delta petL plants show increased light sensitivity as compared to wild type. Assembly studies revealed that PetL is primarily required for proper conformation of the Rieske protein, leading to stability and formation of dimeric Cyt b(6)f complexes. Unlike wild type, phosphorylation levels of the outer antenna of photosystem II (PSII) are significantly decreased under state II conditions, although the plastoquinone pool is largely reduced in Delta petL, as revealed by measurements of PSI and PSII redox states. This confirms the sensory role of the Cyt b(6)f complex in activation of the corresponding kinase. The reduced light-harvesting complex II phosphorylation did not affect state transition and association of light-harvesting complex II to PSI under state II conditions. Ferredoxin-dependent plastoquinone reduction, which functions in cyclic electron transport around PSI in vivo, was not impaired in Delta petL.

DOI： 10.1104/pp.107.100131

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The rice mutants M249 and M134 accumulate chlorophyllides a and b which are esterified with incompletely reduced alcohols such as geranylgeraniol, dihydrogeranylgeraniol, and tetrahydrogeranylgeraniol. Quantities of α-tocopherol, phylloquinone, and menaquinones in leaves of these mutants were determined by high performance liquid chromatography (HPLC) with a fluorescence detector after post-column chemical reduction to convert quinones to fluorescent quinols. Methylnaphthoquinones, varying in the reduction state of the side chain (menaquinones), were detected in leaf segments of the rice mutants on HPLC analyses with both high selectivity and sensitivity to plant quinones. Mutant M249 preferentially accumulated menaquinone, which contains tetrahydrogeranylgeraniol as its side chain. However, mutant M134 exhibited preferential accumulation of menaquinone with a geranylgeraniol side chain. In both mutants, the accumulation patterns of menaquinones with different prenyl side chains were similar to those of chlorophyll with the corresponding prenyl side chains. The content of P700, the photosystem I primary electron donor, in the wild type was greater than that of either mutant, on both a chlorophyll and a fresh weight basis. However, the ratios of total methylnaphthoquinones to P700 were similar in both the wild type and the mutants. Since no comparative large differences in photosynthetic activity exist between the wild type and the mutants, these results suggest that the hydrogenation of the methylnaphthoquinone side chain to phytol is not an essential requirement for it to function as an electron acceptor in photosystem I. On the other hand, α-tocopherol was detected in fully developed leaves of the wild type, but not in those of the mutants. Accumulation of menaquinones and the loss of α-tocopherol in mutant leaves suggest that the reduction of chlorophyll-geranylgeraniol to phytol and that of geranylgeranyl pyrophosphate to phytyl pyrophosphate are catalysed by the same enzyme.

DOI： 10.1093/jxb/erh218

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Language：English   Publisher：Japanese Society of Plant Physiologists  

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Other Link： https://projects.repo.nii.ac.jp/?action=repository_uri&item_id=184967

Language：English   Publisher：Japanese Society of Plant Physiologists  

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Other Link： https://projects.repo.nii.ac.jp/?action=repository_uri&item_id=184965

Publisher：長岡工業高等専門学校  

Measurements of naphthoquinones and plastoquinones are carried out by high performance liquid chromatography (HPLC) with a spectrophotometric detector. However, this method lack both selectivity and sensitivity, and quinones measurements require concentrating extract and removing substances strongly absorbing ultraviolet light as chlorophylls and carotenoids from quinone extract. Therefore, HPLC method with fluorescence detection after post-column chemical reduction was developed for measurements of phylloquinone (PK), menaquinone (MK), plastoquinone A-45 (PQ)and tocopheryl quinone (TQ) in leaf segments without the pretreatment as removing chlorophylls from quinone extracts. The quinone derivatives extracted from leaves were separated by reversed-phase C18 column at 35℃ with a mixture of equivalent volume of methanol and ethanol as a mobile phase at a flow rate of 1.0ml/min. Separated naphthoquinone derivatives were detected by monitoring fluorescence intensity at 430 nm of quinoles excited at 320 nm with a fluorescence detector after reaction with ethanolic sodium borohydride of 0.045% (W/V) at a flow rate of 1.0 ml/min in a reaction coil (0.5mm i.d. x 200cm) kept at 35℃ connected online chromatographic column. Tocophery quinone and plastoquinone were detected by measuring fluorescence(Ex=290nm, Em=320nm)of the quinone derivatives, the other separation and reduction conditions except for fluorescence detection is the same as in naphthoquinone analysis. The calibration curves constructed by plotting the peak area against the amount of standard quinone/-ol were linear over the tested range from 7.5 to 2000 pmol. The proposed method is simpler, more specific snd sensitive than other conventional methods.

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