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Higher plants produce a large variety of low-molecular weight secondary compounds. Among them, nitrogen-containing alkaloids are the most biologically active and are often used pharmaceutically. Whereas alkaloid chemistry has been intensively investigated, alkaloid biosynthesis, including the relevant biosynthetic enzymes, genes and their regulation, and especially transcription factors, is largely unknown, as only a limited number of plant species produce certain types of alkaloids and they are difficult to study. Recently, however, several groups have succeeded in isolating the transcription factors that are involved in the biosynthesis of several types of alkaloids, including bHLH, ERF, and WRKY. Most of them show Jasmonate (JA) responsiveness, which suggests that the JA signaling cascade plays an important role in alkaloid biosynthesis. Here, we summarize the types and functions of transcription factors that have been isolated in alkaloid biosynthesis, and characterize their similarities and differences compared to those in other secondary metabolite pathways, such as phenylpropanoid and terpenoid biosyntheses. The evolution of this biosynthetic pathway and regulatory network, as well as the application of these transcription factors to metabolic engineering, is discussed., (© 2013, Elsevier Inc. All Rights Reserved.)

Plants produce a variety of secondary metabolites that possess strong physiological activities. Unfortunately, however, their production can suffer from a variety of serious problems, including low levels of productivity and heterogeneous quality, as well as difficulty in raw material supply. In contrast, microorganisms can be used to produce their primary and some of their secondary metabolites in a controlled environment, thus assuring high levels of efficiency and uniform quality. In an attempt to overcome the problems associated with secondary metabolite production in plants, we developed a microbial platform for the production of plant isoquinoline alkaloids involving the unification of the microbial and plant metabolic pathways into a single system. The potential applications of this system have also been discussed.

The plant secondary metabolites benzylisoquinoline alkaloids (BIAs) have diverse pharmaceutical activities, and some are used medicinally (e.g., morphine, codeine, berberine). Recently, we constructed a platform to produce BIAs using bioengineered Escherichia coli, which could be useful for bulk production. The E. coli strain used in this system produces the important intermediate (S)-reticuline from glucose or glycerol. Although the amount produced (40 mg/L) exceeded the amount that can be purified from plants, the conversion efficiency from glycerol was only 0.15%; thus, there was much room for improvement. Our production system was developed in a jar fermenter but it is difficult to work with multiple samples using this system. In contrast, many samples can be cultured in parallel using shake flask cultures, allowing optimization of production conditions. Here, we describe bench-top production of (S)-reticuline and optimization of culture conditions using shake flask cultures. The production of (S)-reticuline reached 33.9 mg/L.

Plant cytochromes P450 (P450s) participate in a variety of biochemical pathways to produce a vast diversity of plant natural products. The number of P450 genes in plant genomes is estimated to be up to 1% of the total gene annotations of each plant species, implying that plants are huge sources for various P450-dependent reactions. Plant P450s catalyze a wide variety of monooxygenation/hydroxylation reactions in secondary metabolism, and some of them are involved in unusual reactions such as methylenedioxy-bridge formation, phenol coupling reactions, oxidative rearrangement of carbon skeletons, and oxidative C-C bond cleavage. Here, we summarize unusual P450 reactions in various plant secondary metabolisms, and describe their proposed reaction mechanisms., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The PsbP is a thylakoid lumenal subunit of photosystem II (PSII), which has developed specifically in higher plants and green algae. In higher plants, the molecular function of PsbP has been intensively investigated by release-reconstitution experiments in vitro. Recently, solution of a high-resolution structure of PsbP has enabled investigation of structure-function relationships, and efficient gene-silencing techniques have demonstrated the crucial role of PsbP in PSII activity in vivo. Furthermore, genomic and proteomic studies have shown that PsbP belongs to the divergent PsbP protein family, which consists of about 10 members in model plants such as Arabidopsis and rice. Characterization of the molecular function of PsbP homologs using Arabidopsis mutants suggests that each plays a distinct and important function in maintaining photosynthetic electron transfer. In this review, recent findings regarding the molecular functions of PsbP and other PsbP homologs in higher plants are summarized, and the molecular evolution of these proteins is discussed.

Transcription factors control many processes in plants and have high potentials to manipulate specialized metabolic pathways. Transcriptional regulation of the biosynthesis of monoterpenoid indole alkaloids (MIAs), nicotine alkaloids, and benzylisoquinoline alkaloids (BIAs) has been characterized using Catharanthus roseus, Nicotiana and Coptis plants. However, metabolic engineering in which specific transcription factors are used in alkaloid biosynthesis is limited. In this study, we characterized the effects of ectopic expression of CjWRKY1, which is a transcriptional activator with many targets in BIA biosynthesis in Coptis japonica (Ranunculaceae) and Eschscholzia californica (California poppy, Papaveraceae). Heterologous expression of CjWRKY1 in cultured California poppy cells induced increases in transcripts of several genes encoding BIA biosynthetic enzymes. Metabolite analyses indicated that the overexpression of the CjWRKY1 gene also induced increases in the accumulation of BIAs such as sanguinarine, chelerythrine, chelirubine, protopine, allocryptopine, and 10-hydroxychelerythrine in the culture medium. Previous characterization of EcbHLH1 and current results indicated that both transcription factors, WRKY1 and bHLH1, are substantially involved in the regulation of BIA biosynthesis. We discuss the function of CjWRKY1 in E. californica cells and its potential for metabolic engineering in BIA biosynthesis. [ABSTRACT FROM AUTHOR]

The PsbS protein plays an important role in dissipating excess light energy as heat in photosystem II (PSII). However, the physiological importance of PsbS under naturally fluctuating light has not been quantitatively estimated. Here we investigated energy allocation in PSII in PsbS-suppressed rice transformants (ΔpsbS) under both naturally fluctuating and constant light conditions. Under constant light, PsbS was essential for inducing the rapid formation of light-inducible thermal dissipation (ΦNPQ), which consequently suppressed the rapid formation of basal intrinsic decay (Φf,D), while the quantum yield of electron transport (ΦII) did not change. In the steady state phase, the difference between the wild type (WT) and ΔpsbS was minimized. Under regularly fluctuating light, the reduced PsbS resulted in higher ΦII upon the transition from high light to low light and in lower ΦII upon the transition from low light to high light, indicating that ΦII was, to some extent, controlled by PsbS. Under naturally fluctuating light in a greenhouse, rapid changes in ΦII were compensated by ΦNPQ in the WT, but by Φf,D in ΔpsbS. As a consequence, a significantly lower ΣNPQ integrated ΦNPQ over a whole day) and higher Σf,D were found in ΔpsbS. Furthermore, thermal dissipation associated with photoinhibtion was enhanced in ΔpsbS. These results suggest that PsbS plays an important role in photoprotective process at the induction phase of photosynthesis as well as under field conditions. The physiological relevance of PsbS as a photoprotection mechanism and the identities of ΦNPQ and Φf,D are discussed. [ABSTRACT FROM PUBLISHER]

The allocation of absorbed light energy in PSII to electron transport and heat dissipation processes in rice grown under waterlogged conditions was estimated with the lake model of energy transfer. With regard to diurnal changes in energy allocation, the peak of the energy flux to electron transport, JPSII, occurred in the morning and the peak of the energy flux to heat dissipation associated with non-photochemical quenching of Chl fluorescence, JNPQ, occurred in the afternoon. With regard to seasonal changes in energy allocation, JPSII in the rapidly growing phase was greater than that in the ripening phase, even though the leaves of rice receive less light in the growing phase than in the ripening period in Japan. This seasonal decrease in JPSII was accompanied by an increase in JNPQ. One of the reasons for the lower JPSII in the ripening phase might be a more sever afternoon suppression of JPSII. To estimate energy dissipation due to photoinhibition of PSII, JNPQ was divided into Jfast, which is associated with fast-recovering NPQ mainly due to qE, and Jslow, which is mainly due to photoinhibition. The integrated daily energy loss by photoinhibiton was calculated to be about 3–8% of light energy absorption in PSII. Strategies for the utilization of light energy adopted by rice are discussed. For example, very efficient photosynthesis under non-saturating light in the rapidly growing phase is proposed. [ABSTRACT FROM PUBLISHER]

Electron transport in photosystem II (PSII) and photosystem I (PSI) was estimated in terms of chlorophyll fluorescence and changes in P700 redox, respectively, in the unicellular green alga Dunaliella salina in the presence or absence of a nitrogen source in the culture medium. In a nitrogen-containing medium, the quantum yield of PSII (Φ) and that in PSI (Φ) were at the same level in low light, but cyclic electron transport around photosystem I (CET-PSI) was induced under high light as estimated from an increase in Φ/Φ. High light might further enhance the rate of electron transport in PSI by inducing the state 2 transition, in which the distribution of light energy is shifted to PSI at the expense of PSII. Nitrogen deficiency resulted in a decrease in Φ and an increase in Φ. As a consequence, the rate of CET-PSI was expected to increase. The high CET-PSI under N deficiency was probably associated with a high level of energy quenching ( qE) formation in PSII. [ABSTRACT FROM AUTHOR]

PsbP and PsbQ proteins are extrinsic subunits of photosystem II (PSII) and participate in the normal function of photosynthetic water oxidation. Both proteins exist in a broad range of the oxygenic photosynthetic organisms; however, their physiological roles in vivo have not been well defined in higher plants. In this study, we established and analyzed transgenic tobacco (Nicotiana tabacum) plants in which the levels of PsbP or PsbQ were severely down-regulated by the RNA interference technique. A plant that lacked PsbQ showed no specific phenotype compared to a wild-type plant. This suggests that PsbQ in higher plants is dispensable under the normal growth condition. On the other hand, a plant that lacked PsbP showed prominent phenotypes: drastic retardation of growth, pale-green-colored leaves, and a marked decrease in the quantum yield of PSII evaluated by chlorophyll fluorescence. In PsbP-deficient plant, most PSII core subunits were accumulated in thylakoids, whereas PsbQ, which requires PsbP to bind PSII in vitro, was dramatically decreased. PSII without PsbP was hypersensitive to light and rapidly inactivated when the repair process of the damaged PSII was inhibited by chloramphenicol. Furthermore, thermoluminescence studies showed that the catalytic manganese cluster in PsbP-deficient leaves was markedly unstable and readily disassembled in the dark. The present results demonstrated that PsbP, but not PsbQ, is indispensable for the normal PSH function in higher plants in vivo. [ABSTRACT FROM AUTHOR]

The oxidation level of P700 induced by far-red light (ΔAFR) in briefly dark-treated leaves of some sun plants decreased during the daytime and recovered at night. The dark recovery of decreased ΔAFR proceeded slowly, with a half-time of about 5 h. We propose that stromal over-reduction induced by sunlight was the direct cause of the depression of ΔAFR. The depression of ΔAFR found during the daytime was reproduced by controlled illumination with saturating light of fully dark-treated leaves. Simultaneous measurement of P700 redox and chlorophyll fluorescence showed that the depression of ΔAFR was associated with dark reduction of the plastoquinone pool, which represented cyclic electron transport activity. The decrease of ΔAFR in the light-stressed chloroplasts was partly reversed by treatment with 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, an inhibitor of electron transport at the cytochrome b6/f complex, and the subsequent addition of methyl viologen, an efficient electron acceptor from photosystem I (PSI), stimulated further recovery, showing that both cyclic electron flow around PSI and the charge recombination within PSI were responsible for the light-induced depression of ΔAFR. The dark level of blue-green fluorescence, an indicator of NAD(P)H concentration, from intact chloroplasts was increased by high-light stress, suggesting that NADPH accumulated in stroma as a result of the high-light treatment. Possible effects on photosynthetic activity of over-reduction and its physiological relevance are discussed. [ABSTRACT FROM AUTHOR]

Cyclic electron transport around PSI through the NAD(P)H dehydrogenase complex (NDH) in tobacco leaf disks, measured as an increase in the dark level of Chl fluorescence after the onset of darkness, was inhibited by antimycin A, an inhibitor of ferredoxin quinone reductase (FQR), suggesting that antimycin A inhibits not only the FQR-mediated cyclic flow but also the NDH-dependent flow. This electron flow was inhibited also by amytal, an inhibitor of mitochondrial NDH and by nigericin. The reduction of plastoquinone was detected when NADPH and ferredoxin were added to the suspension of the osmotically ruptured chloroplasts of the wild type and NDH-defective mutant. Because the addition of NADPH alone did not induce the reduction, membrane-bound ferredoxin NADP+reductase (FNR) was supposed to reduce ferredoxin, which may be a more direct electron donor for the plastoquinone reduction. The presence of two types of reducing enzymes was suggested from the bi-phasic inhibition of plastoquinone reduction by antimycin A in the wild type. It is proposed that the reducing activity inhibited by antimycin A at a low concentration is attributed to FQR and the less sensitive activity to NDH. [ABSTRACT FROM AUTHOR]