In a review article published in the February 2002 issue of Current Trends in Plant Science (7: 78-83), Keith Lindsey, Stuart Casson and Paul Chilley (School of Biological and Biomedical Sciences, University of Durham, UK) review recent research on small peptides that play a variety of functional roles in plant growth and development. Peptides, long thought not to serve as important signaling molecules in plants (in contrast to the situation in mammalian systems), have recently been recognized as critical factors in cell division, cell expansion, meristem organization, root nodulation, defense against pathogens and self-incompatibility responses.
In their introductory remarks, the authors point out that for many years intercellular signaling in plants focused almost entirely upon the five classical plant hormones: abscisic acid, auxin, cytokinin, ethylene and gibberellin. Later, other signaling molecules such as brassinosteroids, jasmonates and nitric oxide were recognized to play important roles in plants. Perhaps more controversially, peptides may also be added to this list, as there have been reports of at least six peptide signaling molecules in plants. The majority of these peptides are formed by the cleavage of larger molecule precursor units, called proproteins, into biologically active forms.
Systemin, the first signaling peptide to be reported, was isolated from the wounded tomato leaves. Mechanical damage to potato and tomato leaves, as occurs during insect damage, causes expression of an array of genes encoding systematic wound response proteins (SWRPs). These genes encode protective proteins that inhibit insect feeding, such as protease inhibitors. SWRPs are induced not only in leaves that were directly damaged, but also in other unwounded leaves. Systemin appears to be the signal that triggers production of SWRPs. Systemin is 18 amino acids in length and is translocated from the wounded to undamaged leaves.
Systemin is derived from a proprotein of 200 amino acids called prosystemin. In addition to tomato and potato, prosystemin orthologues with 73-88% sequence identities have been reported in other solanaceous species such as Solunum nigrum and Capsicum annuum. It has been shown that both prosystemin and systemin are biologically active as long as the C-terminal region is present. Recently, two kinds of systemin called Tob Sys I and Tob Sys II have been identified in Tobacco. Both tobacco systemin forms consist of 18 amino acids, the same number found in tomato systemin. The biological activity of the tobacco systemins is also similar to tomato systemin. However, in spite of their functional similarity, structural differences occur, one major difference being the presence of attached sugar moieties to Tob Sys I and Tob Sys II. The glycosylated forms of Tob Sys I and Tob Sys II are about 10,000 times more active than non-glycosylated forms. Both tobacco systemins are derived from a single preproprotein that contains both Tob Sys I and Tob Sys II. Two preproteins have been identified; one contains Tob Sys I and Tob Sys II with a secretion signal upstream of Tob Sys I, while the other contains two copies of Tob Sys I (with a Thr to Ala change in the second copy) and one copy of Tob Sys II. The authors suggest that the tobacco preproproteins are secreted as ‘prohormones’ and that these are processed to give rise to distinct signal peptides in the systemic wound response.
Another example of a proteinaceous signaling molecule occurs in the process of root nodulation. Legume species are capable of forming symbiotic relationships with nitrogen-fixing Rhizobium bacterial species. This process results in the formation of root nodules that arise from root cortical cell divisions. Peptide-encoding RNAs encoded by the ENOD40 seem to act as a signal. ENOD40 may act to suppress the effects of ethylene during initiation of nodulation, thereby promoting cortical cell divisions. The ENOD40 genes from legumes and non-legumes correspond to approximately 700 bp transcripts that encode two conserved regions corresponding to peptides of 13- and 27-amino acids. Some controversy remains regarding whether the transcripts themselves or the encoded peptides serve as signaling molecules, but recent studies in alfalfa have indicated that translatability of the peptide-encoding region of the transcript is required for biological activity. Synthetic peptides did not show biological activity, though this could indicate a requirement for post-translational modification or could be indicative of instability. Unlike systemin, ENOD40 peptides are not encoded larger protein precursors. The presence of peptides corresponding to the ENOD40 open reading frames has not yet been detected, though the authors point out that this could be due to rapid turnover or incorporation into a larger macromolecular complex.
Another example of a peptide signaling molecule occurs during pattern development i the shoot apical meristem. The CLAVATA (CLV) genes of Arabidopsis are required for proper organization of the shoot apical meristem, which is the source of cells for new leaf development. One CLV gene, CLV3, encodes a 96-amino acid peptide. This protein, together with CLV1, regulates cell division in the apical meristematic regions. Mutation of CLV1 or CLV3 interferes with the normal meristem organization, resulting in meristem enlargement. CLV1 encodes a predicted receptor kinase, and it has been suggested that CLV3 acts as a ligand for CLV1, possibly to allow formation of a large macromolecular complex (see also the Newsworthy article entitled “SHEPHERD is the Arabidopsis GRP94 Molecular Chaperone Responsible for the Formation of Functional CLAVATA Proteins” in this edition of Arabidopsis.com.) The peptide contains an 18-amino acid secretion signal at the N-terminus. The authors point out that further investigation is necessary to gain insight to the nature of the interaction between CLV1 and CLV3 or specific domains of CLV3 and whether the 96-amino acid form of CLV3 undergoes processing to a smaller form.
Self-incompatibility interactions control outcrossing in the Brassica family. A single polymorphic locus in most Brassicas determines whether or not pollen will germinate on the stigma of the same plant. This mechanism of reproductive isolation is called sporophytic incompatibility (SI) and it serves to prevent self-fertilization. Two proteins, SLG (S-locus glycoprotein) and SRK (S-locus receptor kinase), are expressed in stigmatic papillae. SRK is thought to act as a receptor for a pollen-borne protein that determines whether or not pollen will be rejected. These pollen-borne SI determinants have recently been identified as a family of small polypeptides called S-locus cysteine-rich (SCR) proteins. The SCR genes show much structural polymorphism and encode small polypeptides with bipartite structures. Recent studies have shown that SCR binds the ectodomain of SRK in an allele-specific manner.
Plant peptides with mitogenic activity called phytosulfokines have recently been identified. It has long been known that “conditioned” media, previously used for growing cells, can facilitate establishment of new cell lines better than unused media. The active components responsible for this phenomenon have been identified from conditioned media used to culture suspension cells of asparagus. Analysis of the conditioned media led to identificaiton of pronase-sensitive factors called phytosulfokine-alpha and -beta (PSK-alpha and PSK-beta). These are small disulfated peptides that are four or five amino acids long. PSK-beta may be derived from PSK-alpha, which itself is derived from processing of a larger propeptide. Recently, four genes encoding precursors of PSK-alpha have been identified in Arabidopsis. Cognate genes for PSK-alpha have also been identified in rice seedlings, suggesting that PSK-alpha may be important for in planta cell proliferation as well as in cultured cells.
The authors conclude by describing two other small polypeptides: rapid alkanization factor (RALF) isolated from tomato, tobacco and alfalfa, and POLARIS (PLS), which was identified by the authors. The function of RALF is not known, but it does induce alkalinization of suspension-cultured cells. PLS is required for root elongation in Arabidopsis (unpublished results.)
The authors explain that the small size of peptides and their cDNAs (which are often not represented in cDNA libraries) impeded the discovery of functional peptides in plants. They indicate the future work will focus on identification of new peptide-encoding genes and their products, as well as study of “orphan” receptor kinases in Arabidopsis with no known ligands. With the help of yeast two-hybrid and proteomic technologies, the authors feel that it may be possible to identify peptides, receptors, and components of the precursor protein processing systems.