RALFs

Abstract

Peptide signaling regulates a variety of developmental processes and environmental responses in plants.^1–6 For example, the peptide systemin induces the systemic defense response in tomato⁷ and defensins are small cysteine-rich proteins that are involved in the innate immune system of plants.^8,9 The CLAVATA3 peptide regulates meristem size¹⁰ and the SCR peptide is the pollen self-incompatibility recognition factor in the Brassicaceae.^11,12 LURE peptides produced by synergid cells attract pollen tubes to the embryo sac.⁹ RALFs are a recently discovered family of plant peptides that play a role in plant cell growth.

The Discovery of RALF

During a search for systemins in tobacco, an unrelated peptide factor was discovered that alkalinizes suspension culture medium even more rapidly than systemins.¹³ This factor was named RALF, for Rapid ALkalinization Factor. RALF peptide was first purified from tobacco leaves using the suspension cell alkalinization assay, and an N-terminal peptide sequence was obtained. This sequence allowed the identification of a full length tobacco cDNA indicating that the RALF gene encodes a 115 aa secreted preproprotein that is processed to release a 49 aa active peptide from the C-terminus. Highly conserved active RALF peptides were also isolated from tomato and alfalfa cells, and a synthetic peptide based on the tomato sequence was produced. Both native and oxidized synthetic peptides were able to produce alkalinization responses at nanomolar concentrations, but alkylation of the reduced form of the peptide (which prevents formation of disulfide bridges) inactivated the synthetic peptide. A role for RALFs in defense was considered, since both systemins and RALFs induce alkalinization of medium and MAP kinase activation. However, application of RALF peptide to tobacco leaves did not induce a defense response as detectable by the induction of tobacco trypsin inhibitor. Studies using poplar-derived RALFs further supported the notion that these peptides are not involved in defense.¹⁴ In this case, RALFs were isolated from poplar leaves and shown to induce alkalinization of poplar suspension cell culture medium. Using the amino acid sequence of the peptides, two cDNAs encoding poplar RALF peptides, ptdRALF1 and ptdRALF2, were isolated. Fungal elicitors did not induce expression of either RALF gene, and RALF peptide treatment of cell cultures did not induce the expression of the phenylalanine ammonia lyase (PAL) gene, a defense marker. Together, these two initial studies provided convincing evidence that the primary role of RALF peptides is not in a defense response.

To investigate other possible roles for RALF peptides, Pearce et al.¹³ germinated Arabidopsis and tomato seeds in the presence of 10 µM synthetic RALF. A striking inhibition of root growth and root hair initiation was observed, and was shown to be reversible by transfer of seedlings to RALF-free medium with subsequent recovery of normal growth. These results suggested a growth regulation function for RALFs.

RALF Genes and Peptide Structure

Searches of EST databases by Pearce et al.¹³ revealed that highly conserved RALF genes exist in a wide array of plant families including dicots, monocots and gymnosperms. There are now more than 100 RALF or RALF-Like gene sequences from numerous plant species in GenBank with at least 76% identity to tomato RALF. An alignment of a diverse set of RALF peptides is shown in Figure 1. Most, but not all, RALF genes encode a prepropeptide; all are predicted to be targeted to the endomembrane system (TargetP scores for probability of secretion for RALFs in Fig. 1 vary from 0.95–0.99) and then proteolytically processed near a conserved dibasic site. The predicted active mature peptide of about 50 aa at the C-terminus is very highly conserved, and includes four conserved cysteines in the active peptide region that are likely to be involved in disulfide bridges.¹³ The predicted mature peptide also possesses other well-conserved sequences including a YISY motif near the mature N-terminus, a GASYY motif between the first and second conserved cysteines, and a PYXRGCS motif that contains the third conserved cysteine residue.

Figure 1.

Amino acid alignment of RALFs used in functional studies. SlRALF Solanum lycopersicum vegetative SGN-U316452; PtdRALF1 Hybrid Populus AY172330; NaRALF Nicotiana attenuata root AY456269.1; AtRALF1 Arabidopsis thaliana NM_100171; AtRALF23 Arabidopsis thaliana NM_112530; MtRALF Medicago truncatula MtC90970; SacRALF1 Saccharum spp CA182793.1; SlPRALF Solanum lycopersicum pollen SGN-U324197. SGN unigene sequences can be found at http://solgenomics.net/index.pl, and Medicago unigene sequences can be found at http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=medicago. All other gene annotations refer to GenBank accession numbers. Signal peptides shown are as predicted by TargetP (www.cbs.dtu.dk/services/TargetP/). Pro Peptides begin after the predicted signal cleavage site and end with dibasic processing site (arrow). Identical residues are boxed in black, and similar residues are shaded in gray. Consensus sequence of the predicted mature peptide indicates conservation in at least 6 out of 8 sequences. Asterisks indicate conserved cysteine residues.

In cases where a genome sequence is available, it has been shown that each plant species contains a family of RALF genes and that these genes are expressed in a wide array of plant tissues. for example, about 40 RALF or RALF-like (RALFL) genes are encoded in the Arabidopsis genome, and these have variable expression patterns.^2,15,16 In some cases, a subset of RALF genes is expressed in a highly tissue-specific manner, for example ovule-specific expression of a subset of RALF genes has been observed in Solanum chacoense and Arabidopsis,^15,16 and pollen-specific RALFs have been identified in broccoli and tomato.^17,18

RALF Processing and Localization

Golgi-localized subtilisin-related proteinases called proprotein convertases are typically involved in the processing of prohormone proteins in animals at dibasic sites.^19–21 RALF precursors possess a conserved dibasic site upstream to the active peptide, and recent studies have shown that this site is required for pro-peptide processing and activity of both AtRALF1 and AtRALF23.^22,23 In the case of tomato pollen SlRALF, mature peptide was only detected in the medium in an in vitro pollen germination system.¹⁸ These results are also consistent with localization of a Nicotiana benthamiana RALF fused to GFP, which localized first to the ER and later to the cell wall in N. benthamiana leaf cells.²⁴ These results together indicate an extracellular localization for processed mature RALF peptides.

RALF Receptor(s)

The extracellular RALF peptide is likely to exert its effects through binding to a specific receptor(s), as is the case for other known peptides, including those in plants.^7,25–28 Consistent with this, suramin, an inhibitor of receptor-ligand interactions, strongly inhibited alkalinization of suspension cell medium by tomato RALF.²⁹ A synthetic biologically active ¹²⁵I-azido-RALF bound to suspension culture cells in a saturable manner (saturation reached by 2 nM) with unlabeled peptide competing for binding of the labeled peptide. When the photoaffinity cross linker was activated, two proteins of 25 kDa and 120 kDa were labeled. Fractionation and solubilization studies indicated that these RALF-binding proteins are intrinsic membrane proteins. Acid washes removed the labeled peptide, suggesting that the peptide was not internalized.²⁹ The amino acid sequences of the 25 and 120 kDa RALF-binding proteins have not been elucidated as yet. Another possible class of RALF-binding proteins is the cell wall localized Leucine-rich Repeat eXtensin chimera (LRX) proteins.^30–33 When a tomato pollen-specific LRX recognition domain was used as a bait in a Yeast 2 Hybrid screen, a pollen specific RALF was identified.¹⁸ While it is intriguing to note that several plant peptide receptors bind to their ligands via Leucine-rich Repeats (LRRs), the relationship of the RALF-binding proteins identified by Scheer et al.²⁹ and the cell wall localized LRX proteins is not yet known.

RALF Function

A consistent view of RALF peptides as negative regulators of plant growth is emerging from functional analysis based on peptide assays and gene regulation studies (Table 1).

Table 1.

Functional studies of RALF peptides and genes

Species	Tissue	Gene	Activity	Reference
Solanum lycopersicum	Leaves (at least)	SlRALF	Exogenous peptide causes alkalinization of growth medium and inhibition of tomato and Arabidopsis root growth	Pearce, et al. 200113
Hybrid Populus	Most tissues	PtdRALF1, PtdRALF2	Exogenous peptide causes alkalinization of cell culture growth medium	Haruta and Constabel, 200314
Nicotiana attenuata	Roots, petioles	NaRALF	RNAi downregulation causes long roots, abnormal root hairs	Wu, et al. 200734
Arabidopsis thaliana	Roots, stems	AtRALF1	Overexpression causes semi-dwarfism, exogenous peptide causes cytoplasmic Ca++ spike and inhibition of hypocotyl elongation	Matos, et al. 2008;22 Haruta, et al. 2008,37 Mingossi, et al. 201036
Medicago trunculata	Roots (at least)	MtRALFL1	Overexpression causes reduced number and abnormal nodule development, regulated by bacterial Nod factors	Combier, et al. 200835
Arabidopsis thaliana	Seedlings, regulated by BR	AtRALF23	Overexpression impairs brassinolide-induced hypocotyl elongation and causes semi-dwarfism	Srivastava, et al. 200923
Saccharum spp	Roots, expanding leaves	SacRALF1	Exogenous peptide causes inhibition of microcalli development	Mingossi, et al. 201036
Solanum lycopersicum	Pollen	SlPRALF	Exogenous peptide causes inhibition of pollen tube growth	Covey, et al. 201018

RALFs were discovered as bioactive peptides that caused the alkalinization of suspension cell medium,¹³ and a similar alkalinization response was observed with RALFs isolated from poplar.¹⁴ The tomato RALF peptide also inhibited root growth in tomato and Arabidopsis seedlings,¹³ suggesting a role in growth regulation.

Several gene regulation studies have also been conducted to probe RALF function. Downregulation of a root-expressed NaRALF gene in Nicotiana attenuata resulted in increased root growth and abnormal root hair growth, further supporting the notion that RALFs regulate plant growth.³⁴ In this study, root trichoblasts expanded in a bulbous fashion and eventually burst, rather than establishing normal tip growth. Levels of reactive oxygen species were reduced in the root hair initiation zone. These studies indicated that RALF effects on growth and extracellular pH are closely tied, since normal root hair growth was partially restored at low pH, pH oscillations at the trichoblast surface were slowed, and pH increased at trichoblast tips in NaRALF downregulated plants. Further, these plants grew poorly in competition with normal plants in alkaline soil.

Overexpression of RALF genes can inhibit overall plant growth or specific aspects of plant growth, depending on the targeted RALF gene. Transgenic studies in Arabidopsis showed that overexpression of either AtRALF1 or AtRALF23 resulted in semi-dwarf plants.^22,23 In Medicago trunculata, a root-expressed RALF gene (MtRALFL1) was discovered as being upregulated by nodulation factors.³⁵ Overexpression of the MtRALFL1 gene in transgenic plants resulted in a reduction in nodules and an increase in aborted infection threads.

Peptide-based studies have also been carried out to test RALF function. Recombinant AtRALF1 peptide was shown to inhibit Arabidopsis hypocotyl elongation, and both AtRALF1 and sugar cane RALF (SacRALF1) peptides inhibited cell elongation in microcalli derived from sugar cane suspension culture cells.³⁶ Synthetic tomato pollen SlPRALF inhibits in vitro pollen tube growth but the peptide is effective only during a specific developmental window.¹⁸ When SlPRALF was added to pollen grains in germination medium, pollen tube germination was initiated, resulting in a very small (<10 micron) structure called a “glebula,” but normal tip growth was inhibited. When added to actively growing pollen tubes that were less than 50 microns in length, SlPRALF inhibited further growth. However, once tubes were longer than 50 microns, they became resistant to the peptide.

All of these studies, in a variety of plant families and in a variety of tissues, support a role for RALF peptides in negatively regulating cell growth, in particular by inhibiting cell expansion.

Possible Mechanisms of RALF Action

RALFs affect a number of cellular processes including proton flux and MAP kinase activation, as well as levels of reactive oxygen species and cytoplasmic Ca⁺⁺.^13,23,34,37 How direct the connection of proton flux is to the regulation of cell expansion by RALFs is an open question at this point. Certainly changes in proton flux can have direct effects on the flow of other ions, metabolites, water and hormones into or out of cells. For example, extracellular pH strongly affects the entry of auxin into cells. Further, changes in pH can have drastic effects on intracellular and extracellular enzyme activity. For example, RALFs may modulate the cell wall landscape by influencing the activity of pH-sensitive cell wall modification proteins, including pectin methylesterases, exo-β-glucanases and/or expansins.^38–44

However, changes in proton flux may be downstream from the primary event(s) triggered by RALFs. The observation that both downregulation³⁴ and overexpression²³ of RALFs results in transgenic plants being unable to acidify growth medium does not support a simple proton pump activation/inhibition model. Both the activation of MAP kinases and the alkalinization of cell medium occur within minutes of exposure to peptide.¹³ However, even more rapid responses to RALFs have been reported. In Arabidopsis, screens for bioactive peptides using Arabidopsis seedlings expressing aequorin (a bioluminescent Ca⁺⁺ reporter) identified AtRALF1 as a factor that induced a transient internal Ca⁺⁺ spike within 40 s.³⁷ This suggests that the initiation of a Ca⁺⁺ signaling cascade may be the primary RALF response, and that changes in proton flux and MAP kinase activation may be downstream results of a calcium signaling pathway. Calcium is known to regulate many aspects of plant cell growth.⁴⁵ For example, a steep Ca⁺⁺ gradient at the pollen tube tip has been shown to be a critical regulator of pollen tube growth.^46–49 Further physiological, biochemical and genetic studies will be required to finally resolve the mechanisms by which this peptide hormone regulates plant cell growth. Another interesting area of RALF biology is the modulation of RALF action by other plant hormones. For example, PtdRALF2 transcripts are downregulated by methyl jasmonate in poplar cell suspension cultures¹⁴ and AtRALF23 is significantly downregulated by brassinolide.²³

Conclusions

RALF peptides represent a class of plant-specific growth regulators. Compared to what is known about other plant hormones, work on RALFs is just beginning, with the identification of the receptor(s) and signal transduction pathways yet to be elucidated. RALFs appear to act in many plant species and in many tissues during growth and development, making it of special interest for plant developmental biologists.

Abbreviations

RALF

rapid alkalinization factor

MAP

mitogen-activated protein

EST

expressed sequence tag