Calmodulin, calmodulin-related proteins and plant responses to the environment

trends in plant science reviews

Calmodulin, calmodulin-related proteins and plant responses to the environment Wayne A. Snedden and Hillel Fromm Plant cells encounter a variety of environmental stimuli. They often respond by a rapid and transient increase in the concentration of cytosolic calcium (Ca2+). These Ca2+ signals modulate cellular processes via high-affinity, Ca2+-binding proteins, such as calmodulin, which in turn regulate the activity of downstream target proteins. In recent years a number of calmodulin-related proteins have been identified that are unique to plants. In addition, plant calmodulins have novel functions not previously described in other organisms, and dynamic expression patterns of calmodulin-related genes modulated by environmental signals.

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n any organism, one of the fundamental properties of defense mechanisms is that they must be inducible in response to the external threat. This ensures that resources are not wasted. Thus, plant cells have evolved to perceive different signals from their surroundings, to integrate them and to respond by modulating various functions via signal transduction pathways. Recently, tremendous progress has been made in understanding the role of Ca2+ as a second messenger in plants. This has become possible in several ways: by employing innovative types of technology to measure, in real time, changes in the concentration of Ca2+ in cells1,2; by using novel approaches to introduce signaling components and reporter genes into single plant cells3; and by using methods for the isolation of downstream cellular targets of calmodulin4. These studies suggest that Ca2+ has a vital role in mediating plant responses to external stimuli of both abiotic origin

Fig. 1. Ca2+-bound-calmodulin-mediated signal transduction in plants. Biotic and abiotic signals are perceived by receptors, resulting, in some cases, in transient changes in Ca2+ concentrations in the cytosol and/or organelles (e.g. nucleus). Increases in free Ca2+ concentrations originating from either extracellular pools or intracellular stores are capable of binding to Ca2+-modulated proteins including calmodulin and calmodulin-related proteins. Structural modulations of these proteins enable them to interact with numerous cellular targets that control a multitude of cellular functions, such as metabolism, ion balance, the cytoskeleton and protein modifications. In addition, Ca2+ and calmodulin might also regulate the expression of genes by complex signalling cascades or by direct binding to transcription factors. Rapid changes in cellular functions result from direct interactions of calmodulin and calmodulin-related proteins with their targets (within seconds to minutes). Slower responses require gene transcription, RNA processing and protein synthesis (variable times from minutes to days). These calmodulin-mediated processes, together with cellular changes triggered by other signaling pathways, constitute the response of the plant to the external signals. Broken arrows denote Ca2+ fluxes from extracellular or intracellular stores, and question marks signify unknown signal transduction intermediates.

(e.g. light, cold, heat, movement, hypoxia and drought) and biotic origin (e.g. phytohormones, pathogens and interactions with symbionts). Thus, Ca2+ triggers a myriad of cellular processes that influence growth, development and physiology, which allow plants to adapt to the changing environment. Ca2+-dependent modulation of cellular processes occurs via intracellular Ca2+-binding proteins, of which calmodulin is one of the best characterized. It has no catalytic activity of its own, but Ca2+bound calmodulin (Ca2+–calmodulin) activates numerous target proteins involved in a variety of cellular processes (Fig. 1). Recent studies have revealed that plants possess unique calmodulinrelated proteins, the functions of which are still unknown. This review focuses on recent developments in studying calmodulin and calmodulin-related proteins, their downstream effectors and their role in regulating responses to external signals.

Cytosol Nucleus

Indirect calmodulin-mediated cellular responses (slow)

Genes

Transcription factors

Cytoskeleton ?

Metabolism

Ionic balance Protein modifications

Calmodulin Calmodulin targets Ca2+

Ca2+

Calmodulin Direct calmodulin-mediated cellular responses (rapid)

Extracellular biotic and abiotic signals

Intracellular Ca2+ store

Receptor ?

Receptor

Copyright © 1998 Elsevier Science Ltd. All rights reserved. 1360 - 1385/98/$19.00 PII: S1360-1385(98)01284-9

Other Ca2+-modulated proteins and interaction with other signalling pathways regulated by different second messengers (e.g. cGMP, inositol triphosphate and cADP-ribose)

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Fig. 2. Three-dimensional structure of mammalian calmodulin. (a) Crystal structure of Ca2+bound calmodulin (Ca2+–calmodulin). (b) Solution structure of Ca2+–calmodulin–peptide complex. The structural images show only the backbones of calmodulin and the peptide (i.e. no side chains). ␣-Helices are shown as cylinders (violet in calmodulin, light blue in the target peptide); ␤-sheets are indicated as deep-purple arrows; and Ca2+ ions are indicated as spheres in light brown. The images were created with Insight II software (BIOSYM Technology, San Diego, USA) using the Brookhaven database structure codes 3CLN and 2BBM for (a) and (b), respectively.

Structural basis of Ca2+-dependent functions of calmodulin

Most proteins that function as intracellular transducers of Ca2+ signals contain a common structural motif, the ‘EF hand’5, which is a helix–loop–helix structure that binds a single Ca2+ ion. These motifs typically occur in closely linked pairs, interacting through antiparallel ␤-sheets5. This arrangement is the basis for cooperativity in Ca2+ binding. The superfamily of EF-hand proteins is divided into several classes based on differences in number and organization of EF-hand pairs, amino acid sequences within or outside the motifs, affinity to Ca2+ and/or selectivity and affinity to target proteins5. Calmodulin is an acidic EF-hand protein present in all eukaryotes. The calmodulin prototype is composed of 148 amino acids arranged in two globular domains connected with a long flexible helix. Each globular domain contains a pair of intimately linked EF hands (Fig. 2a). One of the intriguing properties of calmodulin is that it can bind and activate numerous target proteins that share very little amino acid sequence similarity in their binding sites. Thus, the positioning of these binding domains must be empirically determined. However, the majority of known target sites for calmodulin are composed of a stretch of 12–30 contiguous amino acids with positively charged amphiphilic characteristics and a propensity to form an ␣-helix upon binding to calmodulin (Fig. 2b). This affords a tremendous potential for variability in primary sequence (i.e. target diversity). In addition, calmodulin-binding domains, or closely juxtaposed regions, often also function as autoinhibitory or pseudosubstrate domains, maintaining the target in an inactive state in the absence of a Ca2+ signal6. Recent X-ray diffraction and 300

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NMR studies of calmodulin have provided a model for the structural basis of calmodulin– target interactions7. The binding of Ca2+ to calmodulin (affinity in the range of 10⫺5 to 10⫺6 M) exposes two hydrophobic surfaces surrounded by negative charges, one in each globular domain. Ca2+–calmodulin can then bind to its targets (affinity in the nanomolar range), mainly by hydrophobic interactions with long hydrophobic side chains in the target sites. Electrostatic interactions contribute to the stability of the calmodulin–target complex. In solution, the two globular domains wrap around the target, forming an almost globular structure (Fig. 2b). However, functions of calmodulin in the Ca2+-free state have also been described7,8. For example, a mutant yeast calmodulin lacking the ability to bind calcium can still complement a mutant yeast strain lacking its calmodulin gene. Subsequent to this finding, calcium-independent calmodulin binding to the yeast spindle-pole body, required for chromosome segregation, was characterized8. Some target proteins bind calmodulin with higher affinity in the absence of Ca2+ than in its presence (e.g. neuromodulin). Other proteins, such as phosphorylase kinase, can form stable complexes with calmodulin such that the calmodulin does not dissociate even at low Ca2+ concentrations, but activation of the target protein is still Ca2+ dependent7. These findings raise the possibility that Ca2+-free calmodulin functions also occur in plants.

Unique calmodulin-related proteins: a possible basis for distinct functions

All plants seem to possess types of calmodulins that are very similar (close to 90% amino acid sequence identity) to mammalian calmodulin (Fig. 3). However, an intriguing finding in recent years is that plants possess different types of calmodulin-related proteins that are distinct from those in other organisms. The terms calmodulinrelated proteins and calmodulin isoforms are not clearly defined, and thus here we have considered as calmodulin-related proteins only those proteins that have at least 40% amino acid sequence identity to the phylogenetically conserved calmodulins. The family of plant calmodulin-related proteins includes members with varying numbers of predicted EF-hands (from three to six), and some extend beyond the normal length of calmodulin. For example, the calmodulinrelated TCH3 protein from Arabidopsis contains a duplication of a pair of EF-hands at the N-terminus (Fig. 4); and in the wheat CaM-III protein (clone TaCaM2-1; Fig. 4), the first EF-hand domain is absent, and instead there is a unique stretch of relatively hydrophobic amino acids not present in any other reported calmodulin. The C-terminal extensions of petunia CaM53 and rice CaM61 (35 and 38 amino acid extensions, respectively) have a net positive charge and each contains a C-terminal Caax-box prenylation site (CTIL and CVIL, respectively; Fig. 4) that might be involved in facilitating their association with membranes. The positive charges might also play a role in their subcellular localization, such as encouraging association with membranes and/or targeting to the nucleus. Recent studies of mammalian cells indicate that translocation of calmodulin into the nucleus is a key mechanism for regulating transcription9.

trends in plant science reviews Although the physiological significance * * * * * * of the multiple types of calmodulin-related MADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSL 40 Human CaM proteins in plants is unclear, recent studies Human CLP ---------VT---------------C---R--------Arabidopsis CaM-2 ------DD--S---------------C------------have revealed that plant calmodulin-related --E-----------------------C------------Petunia CaM72 proteins differ in their ability to activate ------DD--S---------------C------------Petunia CaM81 known calmodulin-regulated enzymes in ------DD------------------C------------Wheat CaM1-1 vitro. Soybean SCaM-4, which differs at 32 Soybean SCaM-1 ------D---S---------------C------------amino acid residues from SCaM-1 (Fig. 3), Soybean SCaM-4 ---I-S----VD-----G--------C--VE--A--I--was unable to activate pea NAD kinase, but * * * * * * was able, like SCaM-1, to activate the Human CaM GQNPTEAELQDMINEVDADGNGTIDFPEFLTMMARK 76 mammalian enzyme 3′,5′-cyclic nucleotide ---------R--MS-I-R-----V------G----Human CLP phosphodiesterase10. Similarly, differential ------------------------------NL---Arabidopsis CaM-2 activation of NAD kinase by different cal-------------S-----Q----------NL---Petunia CaM72 modulins from Arabidopsis has also been ------------------------------NL---Petunia CaM81 11 ------------------------------NL---Wheat CaM1-1 reported . Thus, there might be target ------------------------------NL---Soybean SCaM-1 specificity for different members of the calD-----E------S----------E-D---SL--KSoybean SCaM-4 modulin superfamily. In addition, amino * * * * * * acid differences within or adjacent to the Human CaM MKDTDSEEEIREAFRVFDKDGNGYISAAELRHVMTNL 113 Ca2+-binding loops might result in different -----N-----------------FV----------RHuman CLP affinities for Ca2+. For example, the human ---------LK---------Q--F------------Arabidopsis CaM-2 calmodulin-like protein (CLP; Fig. 3), which ---------LK---K-----Q-------DV------Petunia CaM72 shares 85% amino acid identity with human ---------LK---------Q--F------------Petunia CaM81 2+ calmodulin, was found to bind Ca with a ---------LK---------Q--F------------Wheat CaM1-1 ---------LK---------Q--F------------Soybean SCaM-1 mean affinity about tenfold lower than that V----A---LK---K-----Q------S------I-Soybean SCaM-4 of calmodulin12. It is therefore conceivable 2+ that Ca sensitivity is an additional factor * * * * * * in defining the in vivo roles for the different Human CaM GEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK 149 plant calmodulin-related proteins. This is ----S---------A--T------------RVLVSHuman CLP -------------K---V-----I------KV-M-Arabidopsis CaM-2 particularly relevant when one considers 2+ -----------------M------------R--L-Petunia CaM72 that Ca fluxes differ temporally, spatially -----------------V-----I------KV-M-Petunia CaM81 and in magnitude, depending upon the type -----------------V-----I------KV-M-Wheat CaM1-1 2 2+ of stimulus a cell perceives . These Ca -----------------V-----I------KV-M-Soybean SCaM-1 ---------EQ--K---L------------K--MTVR Soybean SCaM-4 patterns (Ca2+ signatures) are probably an important component in assigning the Fig. 3. Comparison of amino acid sequences of selected plant calmodulins with human physiological response needed for a particucalmodulin (CLP, ‘calmodulin-like protein’). The amino acid sequences of some phylolar stimulus. It is unclear why plants have genetically conserved calmodulins are aligned with respect to the four Ca2+ binding sites (asterisks). Amino acids are numbered (right) from the start methionine codon of the human evolved such a multiplicity of calmodulincalmodulin cDNA. GenBank accession numbers: human calmodulin, M19311; human related proteins, but the emerging picture CLP, X13461; Arabidopsis CaM-2, M38380; Petunia CaM72, M80832; Petunia CaM81, is one of tremendous flexibility in calM80836; wheat CaM1-1, U48242; soybean SCaM-1, L01430; soybean SCaM-4, L01433. modulin–target and calmodulin–Ca2+ interactions. Several questions arise from these observations: • Do all calmodulin-related proteins function as Ca2+ transducers genes (CaM-2, CaM-3 and CaM-5; Ref. 11) encode the same and, if so, what are their cellular targets? calmodulin, whereas other genes encode either very similar pro• How do the unique structural properties of calmodulin-related teins (CaM-1, CaM-4 and CaM-6; Ref. 11) or more distant proteins affect their responsiveness to Ca2+ and their affinity calmodulin-related proteins such as TCH2, TCH3 (Ref. 14) and and selectivity to target proteins? CaBP-22 (Ref. 15). A similar picture occurs in other plants. • How is the spatial and temporal expression of calmodulin- Therefore, if there is no functional redundancy in calmodulinrelated proteins in plants regulated? related genes, their expression patterns and/or downstream • How do these factors collectively relate to the physiological targets must be distinct. A striking example is the induced expresroles of calmodulin-related proteins? sion of a group of calmodulin-related genes in response to physiAddressing these questions is an exciting challenge that will help cal (e.g. touch, dark, heat and light) and chemical (e.g. auxin and unravel the complexities of Ca2+ signaling in plants. NaCl) stimuli14,16. The induced expression of at least some of these calmodulin-related genes is mediated by a rise in cytoDynamic expression of calmodulin-related genes solic Ca2+ in response to the external stimulus17. The dynamic Many plant species have been shown to possess calmodulin multi- modulation of calmodulin gene expression in plants is also regene families composed of several genes encoding an identical flected in gene-specific, developmentally regulated, organ-, tissueprotein as well as other genes encoding various calmodulin- and cell-specific expression patterns18,19. These observations related proteins. For example, in wheat, at least seven genes imply that the intracellular Ca2+-sensing machinery is continuencode the conserved calmodulin TaCaM-I; at least two other ously changing, probably reflecting a more general phenomenon genes encode TaCaM-II, which differs from TaCaM-I in just of tuning of the plant’s signal transduction network20; the sigtwo conserved amino acid substitutions; and at least one other nificance of this might be sensitization or desensitization to gene encodes TaCaM-III, a novel calmodulin-related protein external signals, preparation for a longer-term response, or other lacking the first Ca2+-binding site13. In Arabidopsis, at least three functions2. August 1998, Vol. 3, No. 8

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trends in plant science reviews light-mediated gene activation and chloroplast deHuman calmodulin velopment3. Genes involved in anthocyanin biosynthesis Plant calmodulin (e.g. Petunia CaM81) (e.g. the gene encoding chalcone synthase) can be inPetunia CaM53 CTIL duced in the aurea mutant by microinjection of cGMP, Rice CaM61 CVIL whereas Ca2+–calmodulin microinjection leads to the Wheat TaCaM2-1 induction of genes associated with the development Arabidopsis CaBP-22 of the photosynthetic complexes (e.g. CAB genes). Arabidopsis TCH3 Moreover, these signaling molecules operate in a recipArabidopsis TCH2 rocally repressive manner; Ca2+–calmodulin microChlamydomonas calmodulin injection inhibits anthocyanin production, whereas Yeast calmodulin cGMP microinjection inhibits CAB expression3. In Fig. 4. Schematic structural presentation of selected plant calmodulin-related proteins. Loops denote high2+ addition, UV-B light, but affinity Ca -binding sites. C-terminal extensions, denoted by filled boxes, of Petunia CaM53 and rice not UV-A light, appears to CaM61, contain a large number of positively charged amino acids. Each of these two proteins ends with a modulate chalcone synthase Caax-box motif (C, cysteine; a, an aliphatic amino acid; x, usually serine, methionine, cysteine, alanine, glugene expression through a tamine or leucine; CTIL and CVIL in Petunia CaM53 and rice CaM61, respectively), which is a site for protein prenylation in eukaryotes. Regions of sequence similarity between Arabidopsis CaBP-22 and TCH3 are calmodulin-mediated pathshown as shaded boxes. Open boxes represent non-homologous sequences. The degrees of similarity of the way25. Similarly, microincalmodulin and calmodulin-related proteins to human calmodulin in the aligned regions are: Petunia jection of calmodulin into CaM81, 95.3%; Petunia CaM53, 95.3%; rice CaM61, 89.3%; wheat CaM2-1, 95.2%; Arabidopsis CaBPthe zygotes of fucoid algae 22, 72.6%; Arabidopsis TCH3, 68.5%; Arabidopsis TCH2, 51.5%; Chlamydomonas calmodulin, 95.2%; enhanced photopolarization and yeast calmodulin, 73.3%. The respective GenBank accession numbers are: M19311, M80836, M80831, of the zygote axis26. In genU37936, U48690, Z12136, L34546, AF026473, M207293 and M14760. eral, however, the targets calmodulin that mediate these responses remain unknown. The diversity of plant calmodulin-binding proteins Ectopic expression of specific calmodulin isoforms, calmoduIn animals, over 25 calmodulin targets have been identified, in- lin-target proteins, or mutated proteins in transgenic plants, cluding kinases, receptors, ion channels and G-proteins21. In plants, suggests the involvement of calmodulin, directly or indirectly, relatively few calmodulin-regulated proteins have been isolated, in a large number of different developmental processes. Transeven though calmodulin has been implicated in a range of cellular genic potato plants with increased expression levels of a specific processes as diverse as responses to pathogens22, gravitropism23,24, calmodulin isoform27 exhibited developmental abnormalities, light3 and cold17. However, much of the evidence for the involve- including enhanced apical dominance, elongated tubers and aerial ment of calmodulin in plants is based on the use of pharmacologi- tubers. Nevertheless, overexpression of a calmodulin isoform cal agents that function as calmodulin antagonists in vivo. Such or its target does not always lead to phenotypic changes, sugcircumstantial data must be treated with caution, particularly given gesting that specific isoforms, threshold concentrations, or postthe presence of Ca2+-dependent protein kinases in plants with cal- translational mechanisms might be important factors that affect modulin-like domains. Consequently, in order to dissect the vari- developmental processes. For example, transgenic tobacco plants ous pathways under calmodulin control, it is necessary to identify expressing a mutated calmodulin that is incapable of being the protein targets of calmodulin. A number of plant calmodulin methylated at a specific amino acid residue display decreased targets have been cloned and these are presented in Table 1. This ap- stem internode growth, reduced seed production and reduced seed pears to be an interesting and diverse group of proteins. Moreover, and pollen viability28. These plants have significantly elevated plants seem to have evolved a unique repertoire of calmodulin tar- levels of NADP and H2O2. Dramatic phenotypic changes are also gets whose homologs in animals do not appear to be modulated by observed in transgenic tobacco plants expressing a petunia calmodulin (Table 1). The multiplicity of calmodulin-related pro- glutamate decarboxylase that is lacking its calmodulin-binding teins and the diversity of calmodulin targets imply that a broad domain29. In these transgenic plants, the stem cortex parenchyma spectrum of processes is probably modulated by these proteins in cells fail to elongate, which results in stunted plants. This phenoplants. Indeed, manipulation of calmodulin and calmodulin targets, type correlates with an increase in the levels of ␥-aminobutyric by microinjection studies and transgenic analysis, can significantly acid and reduced levels of glutamate. Cell morphology is also afalter the developmental profile of plant organelles, cells and tissues. fected in trichomes of Arabidopsis plants that have mutations A particularly interesting example of the involvement of calmodu- in AtKCBP, the gene encoding a calmodulin-binding kinesin-like lin in plant development comes from studies of photomorpho- protein30. Other cells in these plants exhibit a normal phenogenesis. Microinjection studies of calmodulin into a phytochrome- type, which suggests that different members of the kinesin family deficient aurea mutant of tomato have allowed the identification might be able to compensate for AtKCBP mutations in most of Ca2+–calmodulin-dependent and -independent pathways for cells. 302

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Table 1. Calmodulin-binding proteins in plantsa Functional group

Protein

Features

Refs

Catalyzes conversion of L-glutamate to ␥-aminobutyric acid; activated by Ca2+-bound calmodulin (Ca2+–calmodulin) and many stresses; forms a multisubunit complex, tightly associated with calmodulin. NAD kinase (gene not cloned) Catalyzes conversion of NAD to NADP; activated by Ca2+-bound calmodulin; might be involved in oxidative burst. Apyrase (nucleoside Subcellular localization is light regulated; ectopic expression triphosphatase) affects metabolism and growth in Arabidopsis. Phosphorylation Ca2+-binding, Ca2+– Has a visinin-like (three ‘EF hands’) Ca2+-binding domain calmodulin-dependent kinaseb and a separate Ca2+–calmodulin-binding domain, which is (CCaMK) autoinhibitory. Kinase homologue (MCK1) Homologous to known Ca2+–calmodulin-regulated kinases from other eukaryotes; isolated from maize roots; might be involved in gravitropism. Ion transport Ca2+-ATPases (BCA1 and Endomembrane Ca2+ pumps; calmodulin-binding domain ACA2) autoinhibitory; localized near the N-terminus; endomembrane localization is unique to plants. Transporter-like (HvCBT1) Probable ion transporter with putative cyclic nucleotide binding domain. Cytoskeleton function Kinesin-likeb (KCBP) Motor domain protein; calmodulin modulates association with tubulin; highly expressed during mitosis; might be involved in acentriolar spindle and phragmoplast formation; involved in trichome development. Elongation factor-1␣ (EF-1␣) Calmodulin modulates interaction of EF-1␣ with microtubules in vitro. Myosin (MYA1) Similar to class V myosins; contains IQ repeats with putative calmodulin-binding characteristics. DNA binding Basic leucine zipper protein Transcription factor; interacts in vitro with a DNA element in (TGA3) the promoter of the Cam-3 Arabidopsis calmodulin gene. Metabolism

Glutamate decarboxylaseb (GAD)

29, 31 and 32

28 33; and S.J. Roux, pers. commun. 34 24 35 and 36 37 30 and 38

39 40 41

a

Note that other unidentified plant calmodulin-binding proteins have been cloned. Calmodulin-regulated forms of these proteins are unique to plants.

b

Future perspectives

References

The existence of multigene families of calmodulin and calmodulin-related proteins in plants and the growing list of known targets of calmodulin suggest a complex Ca2+-mediated regulatory network controlling development and responses to the environment. Future studies should aim to dissect the specific roles of members of the calmodulin superfamily, their subcellular distribution and the physiological relevance of their interaction with target proteins. Different approaches include affinity tests, mapping of calmodulin-binding domains, assessment of protein–protein interactions in vivo, functional analysis using reverse genetics and microinjection into plant cells. In certain developmental processes, an involvement of calmodulin has clearly been shown (e.g. photomorphogenesis), but the mechanisms and calmodulin targets involved in these processes remain uncharacterized. Thus, future characterization of calmodulin target proteins is essential. Continued integrated studies in model systems, such as Arabidopsis, should help accelerate our understanding of Ca2+- and calmodulinmediated signal transduction pathways.

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Acknowledgements

We thank V. Sobolev for assistance in preparing three-dimensional structure computer images. We also thank M. Apse, T. Arazi, A. Danon, Y. Eyal and P. McCourt for critical reading of the manuscript. W.A.S. is the recipient of a Canadian National Science and Engineering Research Council postdoctoral fellowship.

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Wayne A. Snedden is at the Dept of Botany, University of Toronto, 25 Willcocks St, Toronto, Ontario, Canada M5S 2B2; Hillel Fromm* is at the Dept of Plant Sciences, Weizmann Institute of Science, Rehovot, 76100, Israel. *Author for correspondence (tel +972 8934 2102; fax +972 8934 4181; e-mail [email protected]).

Articles in next month’s issue of Trends in Plant Science Early signal transduction pathways in plant–pathogen interactions, E. Blumwald, G.S. Aharon and B.C-H. Lam Promoters that respond to chemical inducers, C. Gatz and I. Lenk The evolution of mating strategies in flowering plants, S.C.H. Barrett Regulation of nitrogen fixation in cyanobacteria, H. Böhme Phytomining, R.R. Brooks, M.F. Chambers, L.J. Nicks and B.H. Robinson Chemical ecology in the molecular era, T. Mitchell-Olds, J. Gershenzon, I.T. Baldwin and W. Boland

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