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Physiology and metabolism
Physiology and metabolism The second coming of plant biochemistry and physiology Editorial overview Eran Pichersky and Krishna Niyogi Current Opinion in Plant Biology 2006, 9:217–219
1369-5266/$ – see front matter Published by Elsevier Ltd. DOI 10.1016/j.pbi.2006.03.017
Eran Pichersky Department of Molecular, Cellular, and Developmental Biology (MCDB), University of Michigan, 830 N University Street, Ann Arbor, Michigan 48109, USA e-mail: [email protected]
Eran’s research focuses on the mechanisms by which plants acquire the ability to synthesize new compounds, with emphasis on volatiles that are involved in defense and the attraction of pollinators. Specific projects examine the evolution of the substrate/product specificities of terpene synthases, methyltransferases, acyltransferases, esterases, and dehydrogenases. Krishna Niyogi Department of Plant and Microbial Biology, 111 Koshland Hall, University of California, Berkeley, California 94720-3102, USA e-mail: [email protected]
Kris’ lab studies photoprotection, photosynthesis, and photo-oxidative stress responses. By isolating and characterizing algal and plant mutants, they aim to assess the physiological significance of different protective mechanisms with the hope of manipulating plant productivity and the ability of plants to grow in different, often adverse, environments.
www.sciencedirect.com
The availability of huge amounts of gene sequence information has had a major effect on the study of plant metabolism and physiology, as it has on most other areas of plant biology. It could be argued that plant metabolism is the area that has been affected the most by the genomics boom, first negatively and then positively. After World War II, the availability of radioactive isotopes and new instruments, together with the large influx of money and investigators into universities and research institutes everywhere and particularly in the USA, resulted in a ‘golden age’ of plant biochemistry. It is still amazing to read papers from the period between late 1940s and early 1980s and discover the range and depth of investigations into a multitude of plant metabolites, the enzymes responsible for their biosynthesis, and the physiological roles of such metabolites. It must be noted, however, that even then and certainly now, the elucidation of specific reactions and biochemical pathways lagged far behind the identification of the compounds produced by such pathways and the demonstration of the physiological effects of the compounds. Furthermore, in those days, the identification of the enzymes responsible for the synthesis of plant metabolites was a long and laborious process. In cases where an enzyme was purified to ‘near homogeneity’, as the phrase went, its biochemical characterization could be highly sophisticated, but the structural analysis was often rather limited. When the era of gene isolation and characterization began in earnest in the early 1980s, it seems that activity in the area of plant biochemistry was strongly curtailed. Initially, some biochemists continued with their enzyme identification and characterization work, with the goal of obtaining enough of a structural ‘handle’ (protein sequence or antibodies) to enable them to ‘clone’ the gene. Soon, methods that made it possible to isolate specific genes without having any information about the protein were developed, culminating, of course, in whole-genome sequencing. Genetic methods such as the generation of mutants, the identification of such mutants for further study (mostly by selection or screening regimes that rely on visual inspection), and the identification of the genes responsible for the observed phenotype became predominant approaches in the general field of plant physiology and development. It was not only the methods that changed, but the particular questions as well. Even when a gene clearly encoded a protein that had a metabolic function, the questions most commonly asked were: in which tissues is the gene expressed, under what conditions, how much, and so on. Beyond the basic protein statistics (size, similarity to other proteins, and predicted subcellular localization), the biochemical function of the protein was rarely examined. Typically, the function of a protein was investigated (indeed, defined) by examining what happened when its gene was mutated or its expression altered. Current Opinion in Plant Biology 2006, 9:217–219
218 Biochemistry and physiology
While genomics and its related ‘omics’ are still going strong, and continue to make important contributions to our understanding of plants, ‘classical’ biochemistry is clearly making a comeback. Arabidopsis has close to 30 000 genes, and some other plant species have even more, and we would all like to know how together these genes make a plant a plant. There is no question that the majority of plant genes encode proteins that have some sort of activity involving the chemical transformation of either small or large molecules — in other words, they are enzymes. Other proteins interact with metabolites as receptors or transporters (sometimes with attendant enzymatic activities as well). Our task is now to identify these functions. As Markus Lange points out in the opening review of this issue, in the post-genomic era this task usually begins by trying to integrate and correlate the vast amount of gene sequence data, gene expression data, protein abundance data and metabolic data. New algorithms are available that can point to a putative biochemical function of a given protein. However, a biochemical demonstration of activity is still necessary to establish a proof. Other articles in this issue report on recent progress in several areas of metabolism and physiology that have greatly benefited from the availability of genomic sequences and expressed sequence tag (EST) databases. The availability of such sequence information can be applied in two basic ways. In one, the investigators attempt to understand a whole process in one or a few well-studied organisms. For example, Linda Walling describes the fascinating gamut of amino-terminal processing that proteins undergo after they are synthesized on the ribosomes. In Arabidopsis, at least 28 different proteases have been found to be involved in this process. Adam et al. detail the various functions of the complement of proteases that are found in plastids, with an emphasis on Clp and FtsH, two major proteases that are of prokaryotic origin. Benning et al. describe genetic and biochemical approaches, mainly in Arabidopsis, to understand the mechanisms of lipid trafficking to, from, and within plastids. Tanaka and Tanaka review recent advances in understanding chlorophyll metabolism (including its biosynthesis, interconversions, and degradation) and the regulation of this complex pathway, which has important consequences for leaf senescence, programmed cell death, and plastid signaling. Pilon et al. describe transporters and metallochaperones that are necessary for the regulated delivery of copper to various compartments. They go on to discuss the assembly of copper into ethylene receptors and several critical enzymes, as well as a newly recognized role for copper in the synthesis of molybdenum cofactor. DNA sequence information is also accumulating for emerging model organisms that are less well studied. Allen et al. describe new insights into nitrogen metabolism that have been gleaned from the growing genomic resources for Current Opinion in Plant Biology 2006, 9:217–219
diatoms, photosynthetic eukaryotes that play a key role in global ecology and biogeochemistry. On the other hand, Kenji Matsui in his review of green leaf volatiles, Christiane Nawarth in her review of the cuticle, and Mary Wildermuth in her review of benzoic acid and its derivatives all describe areas of plant metabolism that are only beginning to benefit from the use of genomic approaches to elucidate the pathways that lead to the synthesis of physiologically important compounds. A second approach to identifying biochemical function is to take a group of related sequences and compare them, then to investigate the enzymes that they encode. This approach is particularly useful in studying ‘secondary’, or specialized, metabolites that account for the majority of compounds made by plants. Such specialized metabolites number in the hundred of thousands. Of course each plant species synthesizes only a fraction of those, and although there is some overlap in the range of compounds synthesized by various species, most compounds are unique to a given lineage. But diversification occurs also within the genome. Thus, the Arabidopsis genome contains at least 30 genes that encode terpene synthases (TPSs), which are responsible for the variety of monoterpenes, sesquiterpenes, and diterpenes observed in this species (see article by Dorothea Tholl). Other species also have related TPSs but some of these produce terpenes that Arabidopsis cannot make. In addition, plants have a family of genes that encode triterpene synthases, which are structurally unrelated to TPSs. The products of the triterpene synthases serve both structural and defense roles, and the triterpene synthase family exhibits intra- and interspecific diversification similar to that seen in the TPS family (Phillips et al.). As described by Auldridge et al., studies of carotenoid cleavage dioxygenases in several plants have revealed the roles of these enzymes in producing a diverse array of apocarotenoid molecules, including aromatic volatiles, abscisic acid, and a novel regulator of lateral shoot branching. The comparative approach has also been very useful in understanding the biochemical functions of several large families of metal transporters in plants, as reviewed by Colangelo and Guerinot. The acyltransferase family named BAHD (John D’Auria) constitutes another such example. In the past, metabolites were usually discovered first and the goal was to find the enzymes that make them. To be sure, many metabolites are still being discovered, particularly in large-scale metabolic profiling projects, whose biosynthetic enzymes (or receptors, or transporters) are unknown and need to be identified. But the large-scale sequencing projects are raising a new type of challenge: enzymes without known products (and substrates). Fortunately, the frequent observation that a newly discovered protein is homologous to previously characterized proteins allows us to make an educated guess as to the www.sciencedirect.com
Editorial overview Pichersky and Niyogi 219
type of substrate used by the new enzyme and the type of product formed by it. Occasionally, in vitro biochemical assays with a new enzyme result in the discovery that the enzyme catalyzes the formation of a compound not previously seen (see, for example, Phillips et al.). Thus, we are now at a point where ‘reverse biochemistry’ has become a useful tool to complement ‘forward’ biochem-
www.sciencedirect.com
istry. When employed together, and in conjunction with all the other tools now available and discussed in this issue, both biochemical approaches — from the metabolite to the enzyme (the classical approach), or from the enzyme to the metabolite — are likely to accelerate our rate of discovery in plant metabolism, and thus have a profound effect on our understanding of plant physiology.
Current Opinion in Plant Biology 2006, 9:217–219
1369-5266/$ – see front matter Published by Elsevier Ltd. DOI 10.1016/j.pbi.2006.03.017
Eran Pichersky Department of Molecular, Cellular, and Developmental Biology (MCDB), University of Michigan, 830 N University Street, Ann Arbor, Michigan 48109, USA e-mail: [email protected]
Eran’s research focuses on the mechanisms by which plants acquire the ability to synthesize new compounds, with emphasis on volatiles that are involved in defense and the attraction of pollinators. Specific projects examine the evolution of the substrate/product specificities of terpene synthases, methyltransferases, acyltransferases, esterases, and dehydrogenases. Krishna Niyogi Department of Plant and Microbial Biology, 111 Koshland Hall, University of California, Berkeley, California 94720-3102, USA e-mail: [email protected]
Kris’ lab studies photoprotection, photosynthesis, and photo-oxidative stress responses. By isolating and characterizing algal and plant mutants, they aim to assess the physiological significance of different protective mechanisms with the hope of manipulating plant productivity and the ability of plants to grow in different, often adverse, environments.
www.sciencedirect.com
The availability of huge amounts of gene sequence information has had a major effect on the study of plant metabolism and physiology, as it has on most other areas of plant biology. It could be argued that plant metabolism is the area that has been affected the most by the genomics boom, first negatively and then positively. After World War II, the availability of radioactive isotopes and new instruments, together with the large influx of money and investigators into universities and research institutes everywhere and particularly in the USA, resulted in a ‘golden age’ of plant biochemistry. It is still amazing to read papers from the period between late 1940s and early 1980s and discover the range and depth of investigations into a multitude of plant metabolites, the enzymes responsible for their biosynthesis, and the physiological roles of such metabolites. It must be noted, however, that even then and certainly now, the elucidation of specific reactions and biochemical pathways lagged far behind the identification of the compounds produced by such pathways and the demonstration of the physiological effects of the compounds. Furthermore, in those days, the identification of the enzymes responsible for the synthesis of plant metabolites was a long and laborious process. In cases where an enzyme was purified to ‘near homogeneity’, as the phrase went, its biochemical characterization could be highly sophisticated, but the structural analysis was often rather limited. When the era of gene isolation and characterization began in earnest in the early 1980s, it seems that activity in the area of plant biochemistry was strongly curtailed. Initially, some biochemists continued with their enzyme identification and characterization work, with the goal of obtaining enough of a structural ‘handle’ (protein sequence or antibodies) to enable them to ‘clone’ the gene. Soon, methods that made it possible to isolate specific genes without having any information about the protein were developed, culminating, of course, in whole-genome sequencing. Genetic methods such as the generation of mutants, the identification of such mutants for further study (mostly by selection or screening regimes that rely on visual inspection), and the identification of the genes responsible for the observed phenotype became predominant approaches in the general field of plant physiology and development. It was not only the methods that changed, but the particular questions as well. Even when a gene clearly encoded a protein that had a metabolic function, the questions most commonly asked were: in which tissues is the gene expressed, under what conditions, how much, and so on. Beyond the basic protein statistics (size, similarity to other proteins, and predicted subcellular localization), the biochemical function of the protein was rarely examined. Typically, the function of a protein was investigated (indeed, defined) by examining what happened when its gene was mutated or its expression altered. Current Opinion in Plant Biology 2006, 9:217–219
218 Biochemistry and physiology
While genomics and its related ‘omics’ are still going strong, and continue to make important contributions to our understanding of plants, ‘classical’ biochemistry is clearly making a comeback. Arabidopsis has close to 30 000 genes, and some other plant species have even more, and we would all like to know how together these genes make a plant a plant. There is no question that the majority of plant genes encode proteins that have some sort of activity involving the chemical transformation of either small or large molecules — in other words, they are enzymes. Other proteins interact with metabolites as receptors or transporters (sometimes with attendant enzymatic activities as well). Our task is now to identify these functions. As Markus Lange points out in the opening review of this issue, in the post-genomic era this task usually begins by trying to integrate and correlate the vast amount of gene sequence data, gene expression data, protein abundance data and metabolic data. New algorithms are available that can point to a putative biochemical function of a given protein. However, a biochemical demonstration of activity is still necessary to establish a proof. Other articles in this issue report on recent progress in several areas of metabolism and physiology that have greatly benefited from the availability of genomic sequences and expressed sequence tag (EST) databases. The availability of such sequence information can be applied in two basic ways. In one, the investigators attempt to understand a whole process in one or a few well-studied organisms. For example, Linda Walling describes the fascinating gamut of amino-terminal processing that proteins undergo after they are synthesized on the ribosomes. In Arabidopsis, at least 28 different proteases have been found to be involved in this process. Adam et al. detail the various functions of the complement of proteases that are found in plastids, with an emphasis on Clp and FtsH, two major proteases that are of prokaryotic origin. Benning et al. describe genetic and biochemical approaches, mainly in Arabidopsis, to understand the mechanisms of lipid trafficking to, from, and within plastids. Tanaka and Tanaka review recent advances in understanding chlorophyll metabolism (including its biosynthesis, interconversions, and degradation) and the regulation of this complex pathway, which has important consequences for leaf senescence, programmed cell death, and plastid signaling. Pilon et al. describe transporters and metallochaperones that are necessary for the regulated delivery of copper to various compartments. They go on to discuss the assembly of copper into ethylene receptors and several critical enzymes, as well as a newly recognized role for copper in the synthesis of molybdenum cofactor. DNA sequence information is also accumulating for emerging model organisms that are less well studied. Allen et al. describe new insights into nitrogen metabolism that have been gleaned from the growing genomic resources for Current Opinion in Plant Biology 2006, 9:217–219
diatoms, photosynthetic eukaryotes that play a key role in global ecology and biogeochemistry. On the other hand, Kenji Matsui in his review of green leaf volatiles, Christiane Nawarth in her review of the cuticle, and Mary Wildermuth in her review of benzoic acid and its derivatives all describe areas of plant metabolism that are only beginning to benefit from the use of genomic approaches to elucidate the pathways that lead to the synthesis of physiologically important compounds. A second approach to identifying biochemical function is to take a group of related sequences and compare them, then to investigate the enzymes that they encode. This approach is particularly useful in studying ‘secondary’, or specialized, metabolites that account for the majority of compounds made by plants. Such specialized metabolites number in the hundred of thousands. Of course each plant species synthesizes only a fraction of those, and although there is some overlap in the range of compounds synthesized by various species, most compounds are unique to a given lineage. But diversification occurs also within the genome. Thus, the Arabidopsis genome contains at least 30 genes that encode terpene synthases (TPSs), which are responsible for the variety of monoterpenes, sesquiterpenes, and diterpenes observed in this species (see article by Dorothea Tholl). Other species also have related TPSs but some of these produce terpenes that Arabidopsis cannot make. In addition, plants have a family of genes that encode triterpene synthases, which are structurally unrelated to TPSs. The products of the triterpene synthases serve both structural and defense roles, and the triterpene synthase family exhibits intra- and interspecific diversification similar to that seen in the TPS family (Phillips et al.). As described by Auldridge et al., studies of carotenoid cleavage dioxygenases in several plants have revealed the roles of these enzymes in producing a diverse array of apocarotenoid molecules, including aromatic volatiles, abscisic acid, and a novel regulator of lateral shoot branching. The comparative approach has also been very useful in understanding the biochemical functions of several large families of metal transporters in plants, as reviewed by Colangelo and Guerinot. The acyltransferase family named BAHD (John D’Auria) constitutes another such example. In the past, metabolites were usually discovered first and the goal was to find the enzymes that make them. To be sure, many metabolites are still being discovered, particularly in large-scale metabolic profiling projects, whose biosynthetic enzymes (or receptors, or transporters) are unknown and need to be identified. But the large-scale sequencing projects are raising a new type of challenge: enzymes without known products (and substrates). Fortunately, the frequent observation that a newly discovered protein is homologous to previously characterized proteins allows us to make an educated guess as to the www.sciencedirect.com
Editorial overview Pichersky and Niyogi 219
type of substrate used by the new enzyme and the type of product formed by it. Occasionally, in vitro biochemical assays with a new enzyme result in the discovery that the enzyme catalyzes the formation of a compound not previously seen (see, for example, Phillips et al.). Thus, we are now at a point where ‘reverse biochemistry’ has become a useful tool to complement ‘forward’ biochem-
www.sciencedirect.com
istry. When employed together, and in conjunction with all the other tools now available and discussed in this issue, both biochemical approaches — from the metabolite to the enzyme (the classical approach), or from the enzyme to the metabolite — are likely to accelerate our rate of discovery in plant metabolism, and thus have a profound effect on our understanding of plant physiology.
Current Opinion in Plant Biology 2006, 9:217–219