Plants protect themselves against herbivory with a diverse array of repellent or toxic secondary metabolites. However, many herbivorous insects have developed counteradaptations that enable them to feed on chemically defended plants without apparent negative effects. Here, we present evidence that larvae of the specialist insect, Pieris rapae (cabbage white butterfly, Lepidoptera: Pieridae), are biochemically adapted to the glucosinolatemyrosinase system, the major chemical defense of their host plants. The defensive function of the glucosinolate-myrosinase system results from the toxic isothiocyanates that are released when glucosinolates are hydrolyzed by myrosinases on tissue disruption. We show that the hydrolysis reaction is redirected toward the formation of nitriles instead of isothiocyanates if plant material is ingested by P. rapae larvae, and that the nitriles are excreted with the feces. The ability to form nitriles is due to a larval gut protein, designated nitrile-specifier protein, that by itself has no hydrolytic activity on glucosinolates and that is unrelated to any functionally characterized protein. Nitrile-specifier protein appears to be the key biochemical counteradaptation that allows P. rapae to feed with impunity on plants containing glucosinolates and myrosinases. This finding sheds light on the ecology and evolution of plant-insect interactions and suggests novel highly selective pest management strategies. O ne of the best-studied groups of plant defense compounds are the glucosinolates (Fig. 1), amino acid-derived thioglycosides found in several plant families (1), including the agriculturally important crops of the Brassicaceae such as oilseed rape, cabbage, and broccoli and the model plant Arabidopsis thaliana (2). Glucosinolates cooccur with myrosinases (thioglucoside glucohydrolases, EC 3.2.3.1), and together these two components constitute an activated plant defense system known as the ''mustard oil bomb'' (3). On tissue damage, the nontoxic glucosinolates are hydrolyzed by myrosinases into biologically active derivatives (Fig. 1 A). The outcome of the hydrolysis reaction depends on the structure of the glucosinolate side chain and the reaction conditions (4). The most common class of hydrolysis products, isothiocyanates (mustard oils), has frequently been shown to be highly toxic to both generalist and specialist insect herbivores (5, 6).Despite the toxicity of isothiocyanates, several lepidopteran insect species use glucosinolate-and myrosinase-containing plants as hosts. Although the neurophysiological bases of host plant choice in these species have been studied extensively (7), relatively little is known about how they overcome the toxicity of their host plants. Among the well known specialist insect herbivores on glucosinolate-containing plants, Pieris rapae is one of the most abundant butterflies in Northern and Central Europe, and it has recently also become a pest in North America. P. rapae has been a model insect for studying herbivore host selection (7,8), but the bioc...

Functional genomics approaches, which use combined computational and expression-based analyses of large amounts of sequence information, are emerging as powerful tools to accelerate the comprehensive understanding of cellular metabolism in specialized tissues and whole organisms. As part of an ongoing effort to identify genes of essential oil (monoterpene) biosynthesis, we have obtained sequence information from 1,316 randomly selected cDNA clones, or expressed sequence tags (ESTs), from a peppermint (Mentha x piperita) oil gland secretory cell cDNA library. After bioinformatic selection, candidate genes putatively involved in essential oil biosynthesis and secretion have been subcloned into suitable expression vectors for functional evaluation in Escherichia coli. On the basis of published and preliminary data on the functional properties of these clones, it is estimated that the ESTs involved in essential oil metabolism represent about 25% of the described sequences. An additional 7% of the recognized genes code for proteins involved in transport processes, and a subset of these is likely involved in the secretion of essential oil terpenes from the site of synthesis to the storage cavity of the oil glands. The integrated approaches reported here represent an essential step toward the development of a metabolic map of oil glands and provide a valuable resource for defining molecular targets for the genetic engineering of essential oil formation.functional genomics ͉ isoprenoid biosynthesis ͉ monoterpene biosynthesis E ssential oil plants have been valued historically for their medicinal, culinary, and fragrance properties and include members of the genus Mentha (mint), which produce some of the most widely used essential oils (1). The essential oil of peppermint is biosynthesized and stored in specialized anatomical structures, termed peltate glandular trichomes, on leaf surfaces (2, 3). Given the commercial value of these oils, the processes involved in their biosynthesis and secretion are attractive targets for genetic engineering. The characteristic flavor and aroma components of mint oils are monoterpenes (4). However, because of the complexity of the monoterpene biosynthetic pathway and the difficulties in purifying the responsible enzymes (5), obtaining gene probes from the corresponding target proteins by classical biochemical approaches has presented a considerable challenge.The partial sequencing of anonymous cDNA clones [expressed sequence tags (ESTs)] has become a rapid and cost-effective means of gaining information about gene expression and coding capacity of Arabidopsis thaliana (6) and rice (7). In addition, a few groups have analyzed ESTs expressed in specialized tissues and organs to assist in the identification of new genes involved in specialized pathways, such as those of developing rice endosperm (8), isolated guard cells of Brassica campestris (9), wood-forming tissues of poplar (10), and immature xylem of Loblolly pine (11).Here we report on a functional genomics approach (Fig. 1A) direct...

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With the recent development of techniques for analyzing transmembrane thylakoid proteins by two-dimensional gel electrophoresis, systematic approaches for proteomic analyses of membrane proteins became feasible. In this study, we established detailed two-dimensional protein maps of Chlamydomonas reinhardtii light-harvesting proteins (Lhca and Lhcb) by extensive tandem mass spectrometric analysis. We predicted eight distinct Lhcb proteins. Although the major Lhcb proteins were highly similar, we identified peptides which were unique for specific lhcbm gene products. Interestingly, lhcbm6 gene products were resolved as multiple spots with different masses and isoelectric points. Gene tagging experiments confirmed the presence of differentially N-terminally processed Lhcbm6 proteins. The mass spectrometric data also revealed differentially N-terminally processed forms of Lhcbm3 and phosphorylation of a threonine residue in the N terminus. The N-terminal processing of Lhcbm3 leads to the removal of the phosphorylation site, indicating a potential novel regulatory mechanism. At least nine different lhca-related gene products were predicted by comparison of the mass spectrometric data against Chlamydomonas expressed sequence tag and genomic databases, demonstrating the extensive variability of the C. reinhardtii Lhca antenna system. Out of these nine, three were identified for the first time at the protein level. This proteomic study demonstrates the complexity of the light-harvesting proteins at the protein level in C. reinhardtii and will be an important basis of future functional studies addressing this diversity.In all eukaryotic oxygenic photosynthetic organisms, lightharvesting chlorophyll a-or b-binding proteins (LHC proteins) function in the collection and transfer of light energy to the reaction centers of photosystem II (PSII) (Lhcb proteins) and photosystem I (PSI) (Lhca proteins). Additionally these proteins are also involved in light dissipation and energy quenching. Therefore, light-harvesting proteins are important components of the photosynthetic machinery that optimize photosynthetic function and minimize photooxidative damage in response to light quantity and quality. It has been known for several years that light-harvesting proteins are products of many genes. This concept is illustrated by a recent analysis of the Arabidopsis genome which revealed that the lhc gene family is composed of more than 20 genes (24). Besides the large number of lhc gene products, posttranslational modifications, such as phophorylation, contribute to even more complexity at the protein level (31, 45). Phosphorylation of the major Lhcb proteins of PSII is important in the process of state transitions. This process leads to a redistribution of excitation energy between PSII and PSI by reorganization of the antennae and thereby regulates energy flow between the photosystems. The importance of phosphorylation for state transitions is shown by the phenotype of the Chlamydomonas reinhardtii Stt7 mutant. This mutant is markedly ...

Iron deficiency induces a remodeling of the photosynthetic apparatus in Chlamydomonas reinhardtii. In this study we showed that a key mechanistic event in the remodeling process of photosystem I (PSI) and its associated light-harvesting proteins (LHCI) is the N-terminal processing of Lhca3. N-terminal processing of Lhca3 is documented independently by two-dimensional gel electrophoresis and tandem mass spectrometric (MS/ MS) analysis as well as by quantitative comparative MS/MS peptide profiling using isotopic labeling of proteins. Dynamic remodeling of the LHCI complex under iron deficiency is further exemplified by depletion of Lhca5 and up-regulation of Lhca4 and Lhca9 polypeptides in respect to photosystem I. Most importantly, the induction of N-terminal processing of Lhca3 by progression of iron deficiency correlates with the functional drop in excitation energy transfer efficiency between LHCI and PSI as assessed by low temperature fluorescence emission spectroscopy. Using an RNA interference (RNAi) strategy, we showed that the truncated form of Lhca3 is essential for the structural stability of LHCI. Depletion of Lhca3 by RNAi strongly impacted the efficiency of excitation energy transfer between PSI and LHCI, as is the case for iron deficiency. However, in contrast to iron deficiency, comparative MS/MS peptide profiling using isotopic labeling of proteins demonstrated that RNAi depletion of Lhca3 caused strong reduction of almost all Lhca proteins in isolated PSI particles.