Plants Embrace a Stepchild: the Discovery of Peptide Growth Regulators Henk J Franssen

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Plants embrace a stepchild: the discovery of peptide growth regulators Henk J Franssen

Over the past decade, peptides have been added to the signalling cascades in plants do exist; avirulence genes of collection of signalling molecules in plants. As the impact of several pathogens encode peptides and the corresponding peptide hormones in non-plants is enormous, a comparison of plant resistence genes encode putative receptor proteins, plant and non-plant peptide signal molecules at this stage containing leucine-rich repeats (LRRs) [4•]. Indeed, in deserves our attention—not only to reveal common and unique animals several LRR receptors recognise peptide hor- features, but also to point to new avenues of future research mones [5]. Strikingly, LRR encoding genes have also been on plant hormones. shown to be crucial in controlling shoot and flower meristem size [6] and embryogenesis [7]. Although the Addresses (peptide) ligands for these LRR containing receptors have Department of Molecular Biology, Agricultural University, Dreijenlaan 3, not been identified as yet, the above data support the role 6703HA Wageningen, The Netherlands; e-mail: of peptides in plant development. [email protected]

Current Opinion in Plant Biology 1998, 1:384–387 This review will describe the state-of-art of plant peptides within the context of the broader knowledge of non-plant http://biomednet.com/elecref/1369526600100384 peptide signals. © Current Biology Ltd ISSN 1369-5266

Abbreviations The imperium of non-plant signalling peptides GFP green fluorescent protein Research on non-plant peptides has resolved the succes- LRR leucine rich repeat sive steps in peptide signalling: peptide production, ORF open reading frame transport and perception and, finally, integration at the cel- lular level [3]. The generation of active peptides is Introduction controlled at the transcriptional- and post-translational Intrinsic to multicellular organisms is the continuous flow level. At the latter, this is regulated by proteolytic process- of information between cells to co-ordinate the role and ing, usually by proteases belonging to the family of serine functioning of individual cells within the organism. proteases which recognise a dibasic motif [3,8] and/or by post-translational modification of the primary translation Intercellular communication in plants has long been products [3]. Translocation out of the cell involves either thought to be the exclusive playground of the classic plant the secretory pathway or a membrane targeting secretory hormones [1]. In recent years, however, this group of signal mechanism [3,9,10]. The presence of specific amino acids molecules has been enriched with oligosaccharides, brassi- within the prohormones determines by which of these two nosteroids, polyamines and jasmonates [2]. Although at first secretory pathways the active hormone is secreted, in glance these signal molecules seem to be specific to plants, numerous cases only after external stimuli. Perception of several structurally related molecules are operational in non- the peptide signals requires the presence of specific cell plants as well; for example, serotonin resembles auxin, nitric surface receptors on receiving cells that provide the first oxide ethylene, steroids brassinosteroids and prostaglandins link in the signal perception- transduction cascade. Four jasmonic acid. Apparently, the nature of signal molecules major classes of peptide hormone receptors can be distin- has been conserved throughout plant and animal kingdoms. guished; G-protein linked receptors, ligand gated It is amazing, therefore, that a prominent group of signal ion-channel receptors, receptor kinases and receptors with molecules in non-plant eukaryotes, peptide hormones, had intrinsic enzymatic activity [3]. to await its discovery in plants for so long. Stepchild spotted In non-plant eukaryotes, hundreds of peptide hormones To date, four peptide signal molecules have been discov- have been discovered that play a key role in embryogene- ered in plants; systemin, enod40, cyi1a and sulfokins sis, cell division and proliferation and neural transmission (Table 1). [3]. Indeed, several of these processes are also intrinsic to plant development but, strikingly, non-peptide signal mol- Systemin was identified as the active factor that is trans- ecules have traditionally been ascribed the major roles in ported out of the wounds of wounded tomato plants to most of these processes [1]. In other words, there was felt distal tissues to induce the expression of two well charac- to be no need to look for a potential role for peptide sig- terised wound-inducible proteinase inhibitor encoding nals in these processes. genes [11,12]. These genes are systemically induced in leaves of pathogen-attacked tomato and potato plants and, The unraveling of the genetic basis of plant–pathogen therefore, are part of the inducible defense repertoire of interactions, however, has revealed that peptide-activated the plant. In the systemin perceiving cells the induction of Plants embrace a stepchild Franssen 385

Table 1 assay, the mutant plant cyi shares several phenotypes con- sistent with an altered cytokinin metabolism or response. Amino acid sequences of the four plant signalling peptides Furthermore, it is striking that in this T-DNA tagging, a Systemin [11,37] gene was tagged that is transcribed into an mRNA of 1000 Tomato A V Q S K P P S K R D P P K M Q T D bases and that in this mRNA the first ATG is the start Potato-1 A V H S T P P S K R D P P K M Q T D codon of the longest ORF, yet it codes for a protein of just Potato-2 A A H S T P P S K R D P P K M Q T D Nightshade A V R S T P P P K R D P P K M Q T D 22 amino acids. Pepper A V H S T P P S K R P P P K M Q T D Sulfokins were recognised as a group of mitogenic factors enod40 [16,21] that induce proliferation of plant cells in low-density sus- M Q W D E A I H G S Tobacco pension cell cultures. The activity of sulfokins are Soybean M E L C W Q T S I H G S Pea M K L L C W Q K S I H G S indispensable by auxin or cytokinin [20]. cyi1a [19·] Strikingly, for three out of the four peptides isolated so far Tobacco M A S S R H Q M Q C T K Y N K S L H T H G T — enod40, sulfokins and cyi1a — a role in cell division and proliferation is plausible. Herein, enod40 and cyi1a seem Sulfokin [20,22·] Asparagus/rice Y I Y T Q to interplay with the activity of the classical hormones Y I Y T auxin and cytokinin, whereas sulfokins are working inde- pendently of these hormones. Thus, a picture emerges indicating that plant peptides are involved in fine tuning the proteinase inhibitor encoding genes is accomplished the activity of other (hormone) signals in plants. through the induction of jasmonic acid biosynthesis [13]. Are we relatives? Enod40 was originally isolated as a gene that becomes acti- As the research field covering plant peptide signalling vated during root nodule formation on legumes as a result comes of age, it is impelling to address the question as to of the interaction of these plants with soil-borne Rhizobium what extent plant peptides and non-plant peptides share bacteria [14,15]. properties. This is not only useful for the sake of comparison but also important to shed light on the gaps in our From transient expression studies of a transgene construct knowledge. consisting of a translational fusion of the enod40 open reading frame (ORF) and green fluorescent protein (GFP) in Plant peptides are active in the nanomolar to picomolar protoplasts, it can be inferred that the enod40 ORF is trans- range [11,16,20]. lated in plant cells and the existence of the resulting peptide in nodules has been shown by ELISA [16]. Clones have been isolated for systemin, enod40 and cyi1a Ballistic targeting into Medicago truncatula (Mt) of a [11,14,15,19•]. Systemin is synthesised as part of a propep- Mtenod40 cDNA clone comprising the peptide encoding tide of 200 amino acids from which the active systemin, region leads to division of root cortical cells [17•]. Alfalfa consisting of 18 amino acids (Table 1), is released — most transformed with antisense enod40 leads to impaired regen- likely through proteolyses [12]. In contrast, both enod40 eration of calli, while overexpression of enod40 gives rise to and cyi1a are encoded by a short open reading frame com- embryogenic tumors [18], both indicating that the function prising 10–13 [16] and 22 amino acids [19•] (Table 1), of enod40 is not restricted to nodule formation. The identi- respectively. These peptides seem to be synthesised fication of an enod40 homolog in tobacco is in line with this directly as active entities. Thus, the question arises as to hypothesis [16]. Furthermore, from these observations it what are the mechanisms underlying the control of activi- can be inferred that enod40 plays a role in cell prolifera- ty of these peptides. tion, which in most cases is controled by an auxin–cytokinin balance. Strikingly, both synthetic tobac- It has been put forward that a second region that is highly co and soybean enod40 peptides allow tobacco protoplasts conserved among all enod40 mRNAs and is located in the to divide at supra optimal auxin concentrations [16]. 3′UTR and non-coding [16,17•,18,21] might have a regula- Although doubts now exist about the value of the tobacco tory role in the translation of the peptide encoding part. protoplast assay used, tomato cell suspension cultures This hypothesis is formed on the observation that the exposed to tomato enod40 are altered in their response to responses after transient expression of DNA constructs auxin as well, reflecting the significance of enod40 like containing either the peptide spanning region or the peptides in non legumes (H Franssen, unpublished data). 3′UTR are identical [16,17•]. This can be explained by assuming that the endogenous enod40 is expressed at low The gene encoding cyi1a was identified through activation levels, but not translatable as a result of the binding of a T-DNA tagging of tobacco and screening for tagged lines of protein in the 3′UTR. As a consequence of transfection of which protoplasts division occurs independent of cytokinin cells with 3′UTR containing constructs the inhibitory pro- and auxin [19•]. Again, despite the current dispute over this tein is diluted and thus released from the endogenous 386 Cell signalling and gene regulation

enod40 mRNA, which is now translatable. Such a mecha- Purposeful research can now be initiated to unravel exist- nism is reminiscent of, for instance, the translational ing peptide hormone involving cascades. Genetic control of bicoid mRNA by the caudal gene product in approaches will be crucial in identifying the participants Drosophila [16]. Sulfokins have been isolated from the involved, and physiological and biochemical approaches dicot Asparagus officinalis and the monocot rice [20,22•]. will be needed to answer questions related to the mecha- Sulfokins consist of 4–5 amino acids: among them two nisms by which they work. tyrosines, the sulfation of which is required for biological activity (Table 1) [20]. The plant sulfokins share this post- To get an impression of how long it might take to fill in the translational modification with cholecystokinin and gaps in our knowledge, the history of insulin, the first pep- leucosulfakinin, two sulfated peptides isolated from ani- tide hormone discovered in mammals, provides an mals and insects [23,24], respectively. The non-plant illustrious and also somewhat frightening example. sulfokins are released from larger primary translation products. Genes encoding the plant peptides, however, have not yet been isolated. In 1922 Banting and Best [33] identified and partially purified insulin. Forty five years later it was found that insulin Only for systemin has translocation out of the cell been is proteolytically cleaved from a larger precursor, proinsulin demonstrated [11], but as prosystemin lacks all the charac- [34]. In the mid 80’s the receptor for insulin was isolated teristic features of a secretory polypeptide, the mechanism [35] and even 75 years after the discovery of insulin it involved is unknown. Sulfokins have been purified from turned out that its story is not completed — recently, it was conditioned medium implying they have been translocated. found that also the C-peptide that is released from the pro- There is no evidence as yet for secretion of enod40 or cyi1a. hormone by proteolytical events displays biological activity [36].

In the search for receptors, sytemin- and sulfokin binding It is clear that we are standing at the beginning of the proteins have been identified. Systemin binds to a 50kD exploration of a new field in plant biology and the excite- protein that has kex2p-protease like activity (i.e. belongs ment about plant peptide signal molecules will certainly to the family of serine proteases recognising a dibasic last for a great part of the 21st century. motif) [25], and the nature of the sulfokin binding proteins awaits elucidation [22•]. References and recommended reading Papers of particular interest, published within the annual period of review, Despite the patchy knowledge of peptide signalling, it have been highlighted as: seems plausible to conclude that plant peptides are part of • of special interest •• of outstanding interest signal transduction cascades that resemble those resolved for peptide hormones in non-plants. At the same time, 1. Raven PH, Evert RF, Eichhorn SE: Biology of plants, edn 5. New York: Worth Publishers Inc.; 1992. many questions concerning the constitution of these cascades arise that await further research to be resolved. 2. Karssen CM, van Loon LC, Vreugdenhil D: Progress in plant growth regulation. Dordecht: Kluwer Academic Publishers; 1992. Perspectives: a bright future for Cinderella 3. Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD: Molecular biology of the cell, edn3. New York: Garland Publishing Inc.; 1994. Now the paradigm of no peptide signals in plants has been abandoned, it can be anticipated that more signalling pep- 4. Baker B, Zambryski P, Staskawicz B, Dinesh-Kumar SP: Signaling in · plant–microbe interactions. Science 1997, 276:726-732. tides will be discovered. The progress made in plant An overview is presented of LRR containing receptors involved in genome- and expressed sequence tag (EST) sequencing plant–microbe interactions, and homology to non-plant resistance-related LRR receptors is discussed (see [6,7,27]). projects will certainly contribute to this. Furthermore, the number of genes encoding (putative) receptors is increas- 5. Kobe B, Deisenhofer J: The leucine-rich repeat: a versatile binding motif. Trends Biochem Sci 1994, 19:415-420. ing, among them LRR containing proteins [4•,6,7,26,27], crinkly4, a tumor necrosis factor-like receptor [28] and a 6. Clark SE, Williams RW, Meyerowitz EM: The CLAVATA1 gene encodes a putative receptor kinase that controls shoot and floral putative G-protein-coupled receptor [29]. It can be expect- meristem size in Arabidopsis. Cell 1997, 89:575-585. ed that, like in non-plants, peptides serve as ligands for 7. Schmidt EDL, Guzzo F, Toonen MAJ, de Vries SC: A leucine-rich these receptors in plants as well. repeat containing receptor-like kinase marks somatic plant cells competent to form embryos. Development 1997, 124:2049-2062.

In addition, so-called non-translated RNAs should be rein- 8. Harris RB: Processing of pro-hormone precursor proteins. Arch vestigated for the presence of short ORFs[30,31,32]. A Biochem Biophys 1989, 275:315-333. surprising discovery in the course of identifying plant sig- 9. Kuchler K, Sterne RE, Thorner, J: Saccharomyces cerevisiae STE6 nalling peptides, is the identification of mRNAs encoding gene product: a novel pathway for protein export in eukaryotic cells. EMBO J 1989, 8:3973-3984. short peptides. Studies on transgenes consisting of a translational fusion of the putative ORF and a marker gene, like 10. Massagué J, Pandiella A: Membrane-anchored growth factors. Annu Rev Biochem 1993, 62:515-541. GFP, either transiently expressed or after stable integration, will be valuable in determining whether small 11. Pearce G, Strydom D, Johnson S, Ryan CA: A polypeptide from tomato leaves induces wound-inducible proteinase inhibitor peptides are made in planta. proteins. Science 1991, 253:895-898. Plants embrace a stepchild Franssen 387

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