In order to study the regulation of photosynthetic carbon flow in higher plants, many researchers have created and analyzed transgenic plants that had reduced or increased respective enzyme activity involved in the Calvin cycle, sucrose synthesis and starch metabolism. We have succeeded in enhancing the photosynthetic carbon fixation capacity by introducing certain enzymes, fructose-1,6-bisphosphatase and/or sedoheptulose-1,7-bisphosphatase, involved in the Calvin cycle. In this review, we discuss that the contribution of some enzymes and processes to controlling the metabolic flux and storage of carbohydrates and plant growth using transgenic plants. These results lead to a reassessment of ideas about the regulation of carbon metabolism and have consequences for design of bioengineering strategies to increase crop productivity in plants.

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Ferredoxin (Fd)-dependent cyclic electron transport through photosystem I (PSI) was first discovered to be cyclic photophosphorylation coupled with ATP synthesis in the chloroplast. Although pioneer studies provided important information, the physiological significance of this electron transport has been underestimated in C₃ plants. The discovery that the Arabidopsis pgr5 (proton gradient regulation) mutant shows impaired Fd-dependent cyclic electron transport was the first piece of evidence of molecular-scale information on this electron transport pathway and its physiological function. The recent advance of techniques for measuring the activity of cyclic electron transport in vivo has allowed a re-evaluation of its activity using wild type and pgr5. In this review, we discuss the major role played by Fd-dependent cyclic electron transport, especially in 1) induction of thermal dissipation, 2) contribution to ATP synthesis and 3) photoprotection of PSI.

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Expression of a variety of sink-related genes for the synthesis of storage compounds are up-regulated in response to increased levels of sugars, while expression of many genes involved in photosynthesis and the breakdown of storage compounds is repressed by sugars. Despite of extensive studies on sugar signaling in plants, only few transcription factors involved in the expression of sugar-inducible genes have been identified so far. A transgenic Arabidopsis thaliana line carrying luciferase gene under the control of a short sugar-inducible promoter derived from a sweet potato sporamin gene was used to screen for loss-of-function type as well as gain-of-function type mutants. These genetic screens resulted in the identification of novel transcription factors that are involved in the expression of at least a subset of sugar-inducible genes in Arabidopsis.

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Methionine biosynthesis in plants provides the primary source of this essential amino acid. Early pioneering works characterised many of the steps involved in the methionine biosynthetic pathway and properties of the enzymes involved. They also showed that methionine biosynthesis was strictly controlled in higher plants and part of a larger, complex pathway that involves the biosynthesis of lysine, threonine and isoleucine. The adoption of the model plant Arabidopsis thaliana has enabled us to isolate mto mutants that over-accumulate soluble methionine. Through the analysis of these mto mutants, valuable insight has been gained into the regulation and in vivo dynamics of the methionine biosynthetic pathway in higher plants. Each of the three mutant mto loci identified to date (mto1, mto2, mto3) disrupt different processes within the pathway and display distinct phenotypic profiles. They have also revealed that, in addition to the common feedback controls, the pathway is subject to changing temporal and spatial regulation over the course of the plant life cycle.

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Nicotine is most familiar to us as a principal pharmacologically active component of cigarettes. This alkaloid is synthesized in the root in response to insect damage and then transported to the aerial parts of tobacco plants. In this short review, we summarize enzymes and genes involved in nicotine biosynthesis, regulatory mechanisms of gene expression involving the NIC regulatory loci and jasmonic acid, and finally metabolic engineering of nicotine formation.

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In higher plants, lateral root (LR) formation is a major contributor to root architecture in the soil. Although it is well known that auxin promotes LR formation, the molecular mechanisms that regulate auxin-mediated LR formation are still largely unknown. Recent molecular genetic studies using Arabidopsis mutants have shown that normal auxin signaling mediated by two families of transcriptional regulators, Aux/IAAs (Auxin/Indole-3-Acetic Acid; repressors of auxin-responsive transcription) and ARFs (Auxin Response Factor; activators or repressors of auxin-responsive transcription), is necessary for LR formation. Auxin-responsive transcription mediated by SLR/IAA14 and ARF7/ARF19 in the root pericycle is particularly important for LR initiation. This review summarizes what is known about the auxin signaling that regulates LR formation in Arabidopsis, and discusses the role of Aux/IAAs and ARFs for LR initiation and subsequent primordial development.

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In plants, growth and development are modulated in response to a number of signaling molecules. In past decades, many physiological studies suggested various antagonistic or synergistic effects by multiple signaling molecules in growth regulation of plants. Recent studies have highlighted the machinery for integration of signaling pathways. Here we summarize the findings on molecular mechanisms for crosstalk, focusing on crosstalk between ethylene and other signaling pathways in Arabidopsis.

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A broad group of open tetrapyrroles derived from heme are collectively called bilins. Phycocyanobilin and phycoerythrobilin are utilized as accessory chromophore for light harvesting by phycobiliproteins that comprise the photosynthetic apparatus in cyanobacteria and algae. Phytochromes, known as a major photoreceptor in plants, contain a phytochromobilin chromophore as an essential prosthetic group for photo-sensing. The possible ancestral proteins of phytochromes, as found in cyanobacteria and eubacteria, possess phycocyanobilin and/or biliverdin as a prosthetic group. In this mini-review, the diverse functions of bilin chromophores and their biosynthesis in photosynthetic organisms are summarized and the potential applications to plant biotechnology will be discussed.

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Single plant cells have hundreds of mitochondria that move around and change their shape through the processes of fission and fusion. The Arabidopsis genome has genes for two dynamin-related proteins, DRP3A and DRP3B, that are similar to genes involved in mitochondrial fission in yeasts. DRP3A and DRP3B were localized to mitochondrial constricted sites and ends. Over-expression of DRP3A or DRP3B with point mutations caused severe elongation of mitochondria. These results show that DRP3A and DRP3B are functional homologues of the dynamin-related protein for mitochondrial fission in yeasts. On the other hand, Arabidopsis appears to have no genes similar to those involved in yeast mitochondrial fusion. Little has been written about mitochondrial fusion in plants. Using a novel photoconvertible fluorescent protein, we have recently shown that mitochondria in live plant cells rapidly undergo fusion and fission on a time scale of seconds. In this mini-review, we focus on plant mitochondrial fission and fusion, and compare them with mitochondrial fission and fusion in yeasts and animals, about which much has been learned in recent years.

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Classical yeast genetic approaches have been successfully applied for identification of genes related to the suppression of cell death. Isolated genes included several reactive oxygen species (ROS)-related genes such as SOD (superoxide dismutase), peroxidase, and GST (glutathione S-transferase). The AtBI-1 (Arabidopsis Bax Inhibitor-1), which is a plant homolog of mammalian antiapoptotic gene BI-1, was also isolated as a suppressor of Bax-mediated lethality in yeast. Overexpression of BI-1 suppresses Bax-, H₂O₂₋, salicylic acid-, and elicitor- induced cell death in plant cells. These data indicate conserved overlapping pathways that regulate ROS-mediated cell death in plants and animals.

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Trienoic fatty acids (TAs) are the most abundant fatty acid species in the membrane lipids of plant cells. While serving as the major structural organizers of biomembranes on the one hand, TAs play essential roles in mediating stress signaling on the other hand. This review focuses on recent studies proposing biochemical roles for TAs in abiotic and biotic stress tolerances, and on the emerging picture of transcriptional and post-transcriptional mechanisms that act in coordination to optimize membrane TA levels under adverse environmental conditions.

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Various genome projects have recently provided abundant data on the genomes of different organisms, from simple microbes to higher plants and animals. More tools are needed to characterize the function of these many genes. RNAi offers many advantages regarding the down-regulation of gene expression. To increase the effectiveness of RNAi, novel RNAi methods (transient RNAi, differential RNAi (dRNAi), comprehensive RNAi with simple construction, quantitatively regulated RNAi, etc.) have been developed for functional genomics in plant. These methods have been used to reveal the functions of many genes and functional networks in genomes of higher plants. The further development of a gene-substitution method based on dRNAi could provide a new tool for the characterization of plant gene functions using a more rational approach. RNAi has also been shown to be useful in metabolic engineering. Detailed characterization of the mechanism of RNAi could also provide the molecular basis for the next generation of gene engineering.

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The RNA silencing technique is an effective tool to examine the biological function of the target mRNA in plants. The recent development of versatile-type RNAi vectors, which are driven by constitutive promoters, and GATEWAY™ cloning technology makes it easy to construct the RNAi vectors with trigger sequences and to analyze the function of a target gene. Although these vectors are highly useful, constitutive defects of the target mRNA expression sometimes result in lethality or seed abortion. Here, we summarize recent approaches to RNA silencing research designed to overcome these difficulties and to dissect gene expression.

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RNA silencing is a sequence-specific gene-inactivation mechanism conserved among eukaryotes that functions as an antiviral defense in plants and animals. To counteract this defense, viruses encode RNA silencing suppressors. Over 30 RNA silencing suppressors have been identified, but the mechanisms by which they suppress RNA silencing is unclear for most of them. The best-characterized suppressor is P19, encoded by viruses of the genus Tombusvirus, which belongs to the family Tombusviridae. Three suppressors have also been identified in the genera Carmovirus, Aureusvirus, and Dianthovirus in the family Tombusviridae. In this review, we summarize recent findings regarding these four RNA silencing suppressors, focusing on their modes of action in RNA silencing suppression and their roles in virus infection.

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Understanding of molecular mechanisms of pathogenicity of Agrobacterium and plant viruses has allowed us to utilize these pathogens as efficient vectors for transgene expression in plants. These vectors are now employed for high-throughput overexpression screening of plant and pathogen genes in planta. Virus-induced gene silencing (VIGS) has become one of indispensable tools for functional analysis of genes. VIGS is also suitable for high-throughput screening of plant genes. In this review, we describe: (1) vector-mediated transient expression techniques; (2) high-throughput overexpression screening of pathogen genes, of which products elicit hypersensitive response (HR) on host plants; (3) high-throughput overexpression screening of plant genes that are related to HR-like cell death; and (4) high-throughput VIGS screening of plant genes necessary for HR.

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Among 12,000 alkaloids which are produced in plants, caffeine (1,3,7-trimethylxanthine) is one of the best known due to it uses as an ingredient of pharmaceuticals and beverages. In coffee plants, it is synthesized from xanthosine through three successive methylation and ribose removal steps. We have isolated all genes encoding the corresponding N-methyltransferases; xanthosine methyltransferase (XMT), 7-methylxanthine methyltransferase (MXMT) and 3,7-dimethylxanthine methyltransferase (DXMT), as well as for the 7-methylxanthosine nucleosidase. Using these genes, we have engineered caffeine production in two ways. The first is to decrease the caffeine content in coffee plants to cope with occasional health problems caused by caffeine uptake, and the other is to produce caffeine as an insect repellant in crop plants, originally not synthesizing caffeine. The first approach was performed using an RNAi for MXMT, yielding a 70% suppression of the caffeine level in leaves of transgenic coffee plants. The other approach was carried out by simultaneous introduction of three genes, XMT, MXMT and DXMT, into tobacco plants, which produced up to 5 μg caffeine per g fresh weight of leaves. This amount of caffeine was enough to repel tobacco cutworms (Spodoptera litura), suggesting the method to be practically efficient for construction of herbivore tolerant crops. The significance of the present study is discussed with reference to four topics: practical metabolic engineering; development of a genetic transformation system for tropical trees; generation of genetically modified (GM) plants with a minimal load on the environment; and providing GM foods that bring direct merits to consumers.

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Ethylene controls many physiological and developmental processes in plants, including fruit and flower development, reproductive physiology, and responses to environmental stimuli. Ethylene exerts its effects through the ethylene receptor of plants. Ethylene receptor genes have been isolated in a variety of plant species, and in early studies, these genes were used to genetically engineer fruit ripening in tomato and flower senescence in petunia and carnation. Recently, we demonstrated that the over-expression of mutated melon ethylene receptor genes affected pollen development and induced a male sterile phenotype in transgenic plants. One major concern regarding genetically modified plants is the transgene flow through pollen dispersal, which may pose a potential impact to the environment, especially on genetic diversity. Therefore, the inducible male sterility system using mutated ethylene receptor genes could be a possible strategy for preventing pollen dispersal from these plants, thereby reducing the potential impact associated with transgenic plants. This review summarizes the studies on the inducible male sterility system that uses ethylene receptor genes.

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Salt stress in plant cells is mainly caused by a combination of hyperosmotic stress resulting from a high concentration of Na⁺ in the environment and ionic stress resulting from the toxicity of cytosolic Na⁺. Thus, salt tolerance in plants can be improved by expressing genes involved in compatible-solute biosynthesis to increase hyperosmotic tolerance, and/or by expressing vacuolar and plasma membrane ion transporters to re-establish intracellular Na⁺ homeostasis under salt stress. To increase the salt tolerance of plants, we have identified and characterized genes that can confer increased hyperosmotic- and ionic-stress tolerance to plant cells.
We identified three paralogues of the HAL3 gene in tobacco (NtHAL3a, NtHAL3b, and NtHAL3c) that encode putative 4′-phosphopantothenoylcysteine decarboxylases. We found that overexpression of NtHAL3a in tobacco BY2 cells increased the level of proline, a major compatible solute in plants, and improved the salt tolerance of these cells. We also found that tolerance to ionic stress can be improved in plants by the expression of the yeast ENA1 gene, which encodes a sodium efflux pump that is present in fungi but not in plants. Furthermore, to understand the molecular mechanisms underlying Na⁺ and K⁺ homeostasis in rice (Oryza sativa L.), we identified two rice HKT transporters, OsHKT1 and OsHKT2, with different properties of Na⁺ and K⁺ transport. Finally, we investigated the role of the conserved glycine filter residue in the K⁺ selectivity of the two OsHKTs.

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For comprehensive understanding of gene flow consisting of pollen flow and seed flow and the consequences, one of the promising approaches is the integration of ecological and genetic studies with a model plant species or related species group to disentangle complicated interactions of the ecological and genetic processes. We present a brief summary of research aiming to disentangle the factors affecting gene flow in wild populations of Japanese Primula species as a model of bumblebee-pollinated clonal herbs.

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To establish a guideline for assessing the environmental effects of genetically modified (GM) crops, flows of genes from GM crops to their wild relatives should be understood. We investigated the gene flow between cultivated and wild varieties of carrot, Daucus carota, as a model plant system despite of the fact that no GM carrot is currently cultivated in Japan. Carrot is one of most widely cultivated vegetables and shows high genetic diversity because of its cross- and insect-mediated-pollination characteristics. Since wild carrot grows in many countries including Japan, gene flow in carrot would be one of the worst cases of dispersing unnecessary modified genes to the wild. In this study, we investigated the population density of wild carrot in Hokkaido, Japan, and the behaviors of its pollinators. There, wild carrot grows in wasteland and along roads near the coast where cultivated carrot cannot grow. We made hybrids between wild and cultivated carrots, observed their growth, and found that the hybrid could vigorously grow at the site. We also found gene markers that can distinguish wild carrot from its cultivated relative although the wild carrot population was more genetically diverse than the cultivated one. These makers will enable gene introgression from cultivated carrot to its wild relatives to be detected. This study will be a reference for assessing gene flows in GM crops.

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From a phylogenetic perspective, most plant biodiversity lie in the algae, which comprise nine divisions distinct in cell architecture. In the past decade or so, molecular phylogenies have revealed that many algal divisions are only distantly related, and belong to five different supergroups of eukaryotes. The scattered and distant distributions of algae are interpreted as the result of separate endosymbioses that occurred in various lineages of eukaryotes. Endosymbiosis is a major driving force of algal evolution and diversification, and therefore, is a key process in understanding plant evolution. In this paper, the process of plastid acquisition via endosymbiosis is considered, focusing on the evolution of protein transport machinery, which is indispensable for establishing new lineages of algae, and simultaneous lateral gene transfer from the symbiont to the host nucleus. The current understanding of protein transport diversity is reviewed in relation to the membrane topologies of plastids. The endosymbioses found in various algae and protists are natural occurrences in plant evolution and diversification, and exhibit intermediate stages of plastid acquisition. The conditions necessary for plastid establishment are also considered, using several examples of ongoing endosymbioses.

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Current issues on transgenic crops were discussed with respect to political (including legal and regulatory issues), economic, social and technological factors. Present concern on the transgenic crops was examined particularly on these components by focusing the biosafety issues and public perception. Japanese controversy on the GM crops was raised as specific cases for assessments and critiques. Regional collaboration on the uses of GM crops was also indicated in the close future needs for the sustainable uses of products from plant genetic engineering.

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