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Dispatch R741

Plant peptide hormones: The long and the short of it Keith Lindsey

Figure 1 A polypeptide from tobacco has been found to be processed into multiple functional peptides, each with independent hormone-like activities. This adds to a growing set of small peptides known to function as Systemic activation signal molecules in plants. of defence

Address: School of Biological and Biomedical Sciences, University of Durham, South Road, Durham DH1 3LE, UK. E-mail: [email protected] MAP kinase Current Biology 2001, 11:R741–R743 pathway 0960-9822/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. Preproprotein: 165 amino acids Mechanically Peptide hormones of animals and yeasts have been known wounded about for many years. Few examples have been character- leaf ized in plants, however, and not without some controversy. Signal Tob Sys I Tob Sys II sequence 18 amino 18 amino Nevertheless, experimental evidence is accumulating for acids acids the existence of biologically active peptides that function Current Biology as signalling molecules in a currently small but diverse range of processes in plants, notably defence responses, A model for the systemic wound reponse in tobacco. Following cell division control and reproductive mechanisms. Now wounding, a secreted prohormone may release smaller peptides, Tob Sys I and Tob Sys II, which activate defence transcription via a the first evidence has been presented that single polypep- MAP kinase signalling pathway in non-wounded leaves. tides can be processed to generate more than one indepen- dently active peptide, in this case each with systemin-like activity [1]. Tomato systemin was first identified in a biological assay of extract fractions from tomato leaf. Functional The first plant signalling peptide to be described in plants analysis of purified HPLC fractions that induce proteinase was tomato systemin [2]. This was identified in the context inhibitor I and II gene expression led to the identification of the systemic wound response that a number of plant of the 18 amino acid systemin polypeptide [2]. Subsequent species exhibit following attack by insect pests. On pest studies demonstrated that systemin is translated as a larger attack, or after mechanical damage, wounded leaves exhibit precursor protein, prosystemin [5], and the presumption is transcriptional activation of genes encoding a range of that prosystemin is enzymically processed to generate sys- defence . In solanaceous species, such as tomato or temin. Systemin was shown by tracer studies to be mobile, potato, serine protease inhibitor proteins accumulate as and found to be active at very low (femtomolar) concentra- part of the ‘local’ reaction to damage. These proteins have tions. Evidence for functionality thus comes both from been shown to inhibit insect feeding in transgenic plants exogenous application experiments, in which systemin has [3]. The most interesting feature of the defence response, been found to move through the phloem [6] and be however is that the transcriptional activation of the protease capable of activating an array of defence-related genes [7], inhibitors is not only local, but is also systemic (Figure 1). and from studies with transgenic plants, in which tomato In other words, leaves at a distance from the wound site, plants overexpressing prosystemin were found to express but which are not themselves mechanically damaged, also proteinase inhibitors constitutively [8]. Systemin appears accumulate the protease inhibitors. This phenomenon sug- to act via the octadecanoid signalling pathway, in which gests the transmission of a signal or signals from the wound linolenic acid, a fatty acid, is released from membranes by site to other parts of the plant — a molecular ‘rapid reac- systemin and converted to jasmonic acid, a key signal in tion force’ that protects susceptible leaves. Originally known defence gene activation [9]. as ‘proteinase inhibitor inducing factor’ or PIIF, the fore- most candidate for the transmissible signal was for some The cloning of the cognate tomato cDNA revealed that it time considered to be small pectic fragments derived from encodes a polypeptide of 200 amino acids residues. This plant cell walls, which are able to elicit proteinase inhibitors represents prosystemin, the predicted precursor of an [4]. But the search for the signal led also to the tomato active 18 amino acids oligopeptide which is the presumed peptide systemin. mature systemin signal molecule [5]. Interestingly, recent R742 Current Biology Vol 11 No 18

work suggests that tomato leaf extracts do not contain the other protein, although screening of tobacco cDNA libraries enzyme activity to cleave prosystemin to the 18 amino revealed the presence of a second, very closely related acids systemin, but rather generate larger products [10]. cDNA. This encodes a second tobacco systemin precursor Both prosystemin and systemin are functional, and it seems of 164 amino acids, which contains a further tobacco sys- that it is the systemin-containing domain of prosystemin temin, Tob Sys Ia, which is identical to Tob Sys I except that is essential for activity and may be generated in vivo as for having alanine instead of threonine at position 3. a larger fragment, as well as the 18 amino acids molecule. Despite their structural divergence the various tobacco Use of the reverse transcriptase form of the polymerase systemins and tomato systemin exhibit functional similari- chain reaction (RT-PCR) has revealed that other solana- ties. The precursor mRNAs encoding the tobacco systemins ceous species, such as potato, black nightshade and bell increased in abundance rapidly following treatment of pepper, have prosystemin orthologues, with sequence iden- tobacco plants with methyl jasmonate, indicating that, like tities of 73–88% [11]. But although tobacco is a close rela- tomato prosystemin, the gene is regulated via the octade- tive of these species, and exhibits a systemic wound canoid pathway. Furthermore, treatment of plants with response, no systemin-like peptides had been identified in Tob Sys I and Tob Sys II caused the rapid activation of a this species until recently. Pearce et al. [1] developed a 48 kDa mitogen activated protein (MAP) kinase and bioassay that was based on the observed ability of tomato tobacco trypsin inhibitors, analagous to the effects of systemin to induce an alkalinization of the medium of tomato systemin. tomato cell suspension cultures. Assaying crude and HPLC- purified fractions from tobacco leaves led to the identifica- What general conclusions can be drawn from this work? tion of three polypeptides — designated polypeptides I, II The evidence for a plant prohormone, from which can be and III — that had systemin-like alkalinization effects on generated multiple polypeptides with independent sig- cultured tobacco cells. Polypeptides I and II showed signifi- nalling activities, is novel, though there are examples in cantly higher activities than tomato systemin in this system. both animals and yeast. For many years it was not recog- Polypeptide III was of much lower abundance than either nized that peptides or polypeptides are used as signalling of the other two, and sequence analysis showed that it was molecules in plants, though there are now a number (albeit virtually identical to polypeptide II, differing only by an a small number) of good candidates implicated in different additional leucine at the carboxyl terminus. processes. For some there is biochemical evidence in support of their suggested signalling activity. Apart from The two major polypeptides, designated tobacco systemin I the systemins, there are the phytosulfokines, which are (Tob Sys I) and tobacco systemin II (Tob Sys II), exhibit a small (four or five amino acids long) disulphated peptides number of unique features. Although each is of a similar originally identified in the medium of asparagus cell cul- size to the tomato systemin, being 18 amino acids residues tures [12] and generated by the processing of preprophyto- in length, there is very little homology with tomato sys- sulfokine [13]. These are probably the factors responsible temin. MALDI mass spectrometry indicated that Tob for ‘conditioning’ of the culture medium and exhibit mito- Sys I and Tob Sys II are linked to pentose sugars, which genic activity, not only in asparagus, but also in rice [14]. were released by mild hydrolysis; tomato systemin has no There is also biochemical evidence that natriuretic peptide- carbohydrate modifications. As synthetic Tob Sys I and Tob like molecules, immunologically related to those found in Sys II are about 10,000-fold less active than the glycosy- animals, may exist in plants, though to date there are no lated versions, it would appear that the carbohydrate moi- molecular genetic data to support this proposal [15]. eties are important for activity, though not essential. Tob Sys I and Tob Sys II also differ from tomato systemin in Other predicted bioactive peptides or oligopeptides containing hydroxylated prolines. have been identified by genetic, rather than biochemical, methods. The CLAVATA1 (CLV1) and CLAVATA3 (CLV3) The most novel feature of Tob Sys I and Tob Sys II was genes of Arabidopsis are both required for correct organiza- revealed when a cDNA was synthesized using a Tob Sys tion of the shoot apical meristem. Mutation of either gene I-specific primer for RT-PCR. The resulting cDNA was leads to uncontrolled proliferation of cells and consequent found to encode a predicted preproprotein of 165 amino enlargement of both vegetative and floral meristems. The acids, containing the sequences of both Tob Sys I and Tob similarity of the mutant phenotypes, together with the Sys II, towards its amino-terminal and carboxy-terminal results of epistasis experiments, suggests the two genes ends, respectively. This protein also has a secretion signal function in the same biochemical pathway, a view sup- at its amino terminus, upstream of Tob Sys I, a strong indi- ported by molecular data. CLV3 encodes a 96 amino acid cation that it is the first example of a plant ‘prohormone’ polypeptide, with a predicted signal peptide at the amino that is processed to produce multiple signalling molecules. terminus, suggesting that it is secreted [16]. CLV1, on the The preproprotein has no significant homologies with any other hand encodes a predicted receptor kinase, and in situ Dispatch R743

hybridization studies revealed spatial and temporal co- as Arabidopsis, we can expect new classes of small polypep- expression of CLV1 with CLV3 in the shoot meristem. It is tides with signalling activities to emerge. tempting to suggest that CLV3 is the ligand for CLV1, but this is still to be demonstrated unequivocally. Simi- References larly, the likely pollen-localized stigmatic ligand for the 1. Pearce G, Moura DS, Stratmann J, Ryan CA: Production of multiple plant hormones from a single polyprotein precursor. Nature 2001, Brassica receptor kinase required for self-incompatibility is 411:817-820. a secreted small (<8 kDa) polypeptide, part of the S locus 2. Pearce G, Strydom D, Johnson S, Ryan CA: A polypeptide from tomato leaves induces wound-inducible proteinase inhibitor cysteine-rich (SCR) protein family [17]. proteins. Science 1991, 253:895-898. 3. Hilder VA, Gatehouse AMR, Sheerman SE, Barker RF, Boulter D: Another example is enod40, a gene activated early in the A novel mechanism of insect resistance engineered in tobacco. Nature 1987, 330:160-163. root nodulation process following Rhizobium infection of 4. Bowles DJ: Signal transduction in the wound response of tomato legumes [18]. The enod40 gene is expressed in the root plants. Phil Trans. R Soc Lond [Biol] 1998, 353:1495-1510. pericycle, before cortical cells divide during the establish- 5. McGurl B, Pearce G, Orozco-Cardenas M, Ryan CA: Structure, expression and antisense inhibition of the systemin precursor ment of the nodule. Of relevance here is the fact that the gene. Science 1992, 255:1570-1573. 0.7 kilobase enod40 transcript from legume or non-legume 6. Narvaez-Vasquez J, Pearce G, Orozco-Cardenas M, Franceschi VR, Ryan CA: Autoradiograpic and biochemical evidence for the species has no long open reading frame, but two short systemic translocation of systemin in tomato plants. Planta 1995, open reading frames, encoding peptides of 13 and 27 195:593-600. amino acids. These are required for the activity of the 7. Bergey DR, Howe GA, Ryan CA: Polypeptide signaling for plant defensive genes exhibits analogies to defense signaling in enod40 gene of Medicago, expression of which can induce animals. Proc Natl Acad Sci USA 1996, 93:12053-12058. cortical cell division [19]. The gene enod40 may therefore 8. McGurl B, Orozco-Cardenas M, Pearce G, Ryan CA: Overexpression of the prosystemin gene in transgenic tomato plants generates a encode a polycistronic transcript that undergoes transla- systemic signal that constitutively induces proteinase inhibitor tional reinitiation. A requirement for non-translated parts synthesis. Proc Natl Acad Sci USA 1995, 91:9799-9802. of the enod40 sequence suggests that enod40 might encode 9. Ryan CA, Pearce G: Systemin: A polypeptide signal for plant defensive genes. Annu Rev Cell Dev Biol 1998, 14:1-17. a biologically active RNA molecule that requires the peptide 10. Dombrowski JE, Pearce G, Ryan CA: Proteinase inhibitor-inducing products for its full activity, possibly in RNA stabilization, activity of the prohormone systemin resides exclusively in the transport or function. To date, the enod40 peptides have C-terminal systemin domain. Proc Natl Acad Sci USA 1999, 96:12947-12952. not been detected biochemically, though genetic studies, 11. Constabel CP, Yip L, Ryan CA: Prosystemin from potato, black in which point mutations ablated their activity, indicate nightshade, and bell pepper: primary structure and biological activity of predicted systemin polypeptides. Plant Mol Biol 1998, that they are produced and are functional [19]. 36:55-62. 12. Matsubayashi Y, Sakagami Y: Phytosulfokine, sulfated polypeptides In our own laboratory, we have identified a gene, POLARIS, that induce the proliferation of single mesophyll cells of Asparagus officinalis L. Proc Natl Acad Sci USA 1996, which is expressed in the embryonic and seedling root tip 93:7623-7627. of Arabidopsis in response to positional information, and is 13. Yang HP, Matsubayashi Y, Hanai H, Sakagami Y: Phytosulfokine- rapidly transcriptionally activated in response to auxin alpha, a peptide growth factor found in higher plants: Its structure, functions, precursor and receptors. Plant Cell Physiol 2000, [20]. POLARIS encodes a mRNA of about 450 nucleotides, 41:825-830. which has an open reading frame potentially encoding a 36 14. Matsubayashi Y, Takagi L, Sakagami Y: Phytosulfokine-a, a sulfated pentapeptide, stimulates the proliferation of rice cells by means of amino acid peptide; mutation of the open reading frame specific high- and low-affinity binding sites. Proc Natl Acad Sci leads to defects in root growth. The open-reading-frame- USA 1997, 94:13357-13362. containing region of the cDNA is sufficient to comple- 15. Gehring CA: Natriuretic peptides - A new class of plant hormone? Ann Bot 1999, 83:329-334. ment the mutant phenotype, indicating that the predicted 16. Fletcher JC, Brand U, Running MP, Simon R, Meyerowitz EM: POLARIS polypeptide is indeed functional. Physiological Signaling of cell fate decisions by CLAVATA3 in Arabidopsis shoot meristems. Science 1999, 283:1911-1914. studies suggest POLARIS is required for correct cytokinin 17. Yang WC, Katinakis P, Hendriks P, Smolders A, de Vries F, Spee J, and ethylene signalling, which modulates root growth (our Van Kammen A, Bisseling T, Franssen H: Characterization of unpublished data). GmENOD40, a gene showing novel patterns of cell-specific expression during nodule development. Plant J 1993, 3:573-585. The field of plant peptide signalling is set to expand. The 18. Schopfer CR, Nasrallah ME, Nasrallah JB: The male determinant of fully sequenced Arabidopsis genome includes a large self-incompatibility in Brassica. Science 1999, 286:1697-1700. 19. Sousa C, Johansson C, Charon C, Manyani H, Sautter C, number of short open reading frames for possible peptide Kondorosi A, Crespi M: Translational and structural requirements signal molecules, as well as longer ones for predicted of the early nodulin gene enod40, a short-open reading frame- containing RNA, for elicitation of a cell-specific growth response receptor-like proteins with no known ligands. Plant in the alfalfa root cortex. Mol Cell Biol 2001, 21:354-366. carboxypeptidases are known that have peptide-generat- 20. Topping JF, Lindsey K: Promoter trap markers differentiate ing activities, but again, not many substrates of such structural and positional components of polar development in Arabidopsis. Plant Cell 1997, 9:1713-1725. enzymes have yet been identified. As the tools of genomics and proteomics are applied to the functional analysis of plant open reading frames, initially in model species such