BR and RALFs
The findings in this study suggest that the downregulation of AtRALF23 may be part of the mechanism by which BR stimulates plant growth and cell expansion. Others have proposed that BR promotes cell elongation, in part, by altering the mechanical properties of the cell wall (Zurek et al., 1994). It is thought that BR may be acting much like auxin, or perhaps through the auxin signaling pathway, by inducing cell-wall acidification (Hardtke, 2007; Hardtke et al., 2007). Cerana et al. (1983) showed that BR stimulated growth and proton extrusion in Azuki bean epicotyls (Vigna angularis). The effects of BR and auxin (indole-3-acetic acid, IAA) in that system appeared to be additive, suggesting that the hormones may be acting independently. BL also stimulated root growth and proton extrusion in maize root segments (Romani et al., 2006).
AtRALF23 is an inhibitor of plant growth, and is downregulated by BR. Pearce et al. (2001b) demonstrated that a chemically synthesized tomato RALF inhibited root growth in tomato and Arabidopsis seedlings. Wu et al. (2007) showed that silencing of the single RALF gene in Nicotiana attenuata (NaRALF) using an inverted repeat (ir) construct resulted in seedlings with roots that grew more rapidly, but produced abnormal root hairs. The irRALF lines tended not to acidify the medium around the tips of the root hairs, and the authors argued that the abnormal root hairs resulted from a higher apoplastic pH. They proposed that the irRALF phenotype was probably a result of pH effects, because when they grew seedlings on medium buffered to pH 5.5, they could restore the wild-type phenotype in the irRALF lines. The authors did point out that the effects of irRALF were contrary to expectations, as RALF induces alkalinization, and not acidification, of the medium in tobacco cell suspension cultures (Pearce et al., 2001b).
Our findings are quite different from Wu et al. (2007), but are consistent with the observations that RALF induces rhizosphere alkalinization. In our experiments, the AtRALF23-myc overexpressers prevented the acidification of the medium surrounding the roots. On the other hand, the roots of wild-type and s1p-3 seedlings clearly acidified the rhizosphere, based on pH indicator color changes. The s1p-3 mutant appeared even more effective than the wild type in acidifying the medium, consistent with the expectation that the mutant fails to process AtRALF23 and produce an active alkalinization factor.
RALFs tend to counteract cell-wall acidification, presumably by blocking membrane-associated proton pumps leading to rapid media alkalization (Moura et al., 2006). It is not known how RALFs elicit an alkalization response. A RALF receptor has not yet been identified, although Scheer et al. (2005) used a labeled form of a tomato RALF ([125I]azido-LeRALF), and found two membrane proteins that would bind the peptide. The authors reported that the alkalization response caused by LeRALF was effectively blocked by suramin, a heterocyclic, polysulfonated inhibitor of ligand–receptor interactions, suggesting that the alkalization response caused by LeRALF was, indeed, produced through ligand–receptor interaction (Scheer et al., 2005). However, the proteins that bind LeRALF have not yet been isolated and identified; therefore, the LeRALF receptor is not known.
Other evidence, nonetheless, supports the proposition that RALFs are ligands for receptor-mediated responses. Alkalization in response to a tobacco RALF, NtRALF, is very fast, peaking at less than 5 min after adding the peptide to tobacco cell-suspension cultures (Pearce et al., 2001b). In addition, NtRALF elicits a rapid MAP kinase response that is coincident with the alkalization response (Pearce et al., 2001b). However, it is not known whether RALFs affect proton pumping as a final effector in a signaling pathway, or whether RALFs interact directly with a membrane-associated proton pump. Such a pump mediating the BR-induced acidification response has not been firmly identified. On the other hand, RALF signaling may be very indirect, affecting other signaling processes that in turn impact on the operation of membrane-associated proton pumps. In this regard, it is interesting that Haruta et al. (2008) found that AtRALF1 elevates cytoplasmic Ca2+ levels in Arabidopsis seedlings, as indicated by aequorin luminescence. It is possible that changes in cytoplasmic Ca2+ levels might trigger other ion pumps or gates, leading to the alkalization response associated with RALFs.
In any case, BRs regulate RALF23 by downregulating its expression. BRs are well known for their ability to regulate gene expression, and the BR signaling pathway controls transcription factors that up- or downregulate BR-responsive genes (see recent reviews: Belkhadir and Chory, 2006; Gendron and Wang, 2007; Li and Jin, 2007). The BR signaling pathway affects the phosphorylation and activity of BES1, one of two transcription factors that activate or repress BR-responsive genes (Yin et al., 2005, 2002). We observed that AtRALF23 was downregulated in a constitutively activated bes1-D mutant, indicating that AtRALF23 is regulated by the BR signaling pathway. We do not know yet whether the AtRALF23 is a direct target of BES1; however, the presence of BES1 binding sites in the AtRALF23 gene promoter imply that BES1 may directly regulate AtRALF23 expression (RS, J-XL, HG, YY and SHH, unpublished data).
The repression of AtRALF23 by BR signaling may have several implications for BR-induced cell elongation. Most obviously, downregulation of a negative regulator (AtRALF23) for cell elongation may be required for optimal BR-induced growth. The AtRALF23 overexpression phenotypes strongly support this possibility. AtRALF23 overexpression is characterized by a reduction in vegetative growth, particularly in root elongation and leaf expansion in seedlings, as well as BR-induced hypocotyls elongation. AtRALF23 overexpressing plants also show a reduction in inflorescence stem elongation, and a loss in apical dominance. An AtRALF23 loss-of-function phenotype has not yet been characterized because a knock-out in the gene has not been identified, and RNAi constructs specific for individual RALFs are challenging to produce, because some of the sequences of the 34 members of the RALF gene family are similar. Some of the other members of the RALF gene family are also negative growth regulators. Like AtRALF23, AtRALF1 overexpression results in semidwarf plants with reduced leaf and root growth (Matos et al., 2008). Unlike AtRALF23, AtRALF1 expression is not downregulated by BL treatment; however, AtRALF1 is normally expressed at much lower levels than AtRALF23 (Arabidopsis eFP browser, http://www.bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi).
In their study of AtRALF1, Matos et al. (2008) observed that a canonical subtilase site (dibasic AA) upstream from the presumed start of the mature peptide was important for the processing of the precursor. They mutated the site and demonstrated that the precursor was not processed, and the construct did not produce the overexpression phenotype. They showed that the AtRALF1 precursor can be processed in extracts by microsomal fractions, but were unable to identify the enzyme responsible for the proteolysis. Nonetheless, their findings are consistent with the possibility that a protease associated with the secretory pathway might be responsible for the processing of AtRALF1.
An important finding in our study was the discovery that AtS1P is involved in AtRALF23 processing. The first function reported for AtS1P in plants was the processing of membrane-associated transcription factors (Liu et al., 2007b). AtS1P participates in processing AtbZIP17, a factor involved in endoplasmic reticulum (ER) stress responses. AtS1P is largely located in the Golgi apparatus (Liu et al., 2007b), and it is thought that in response to ER stress, these transcription factors are released from the ER and translocated to the Golgi, where they undergo proteolytic processing.
RALFs are thought to be secreted peptides, and since we showed that AtRALF23 requires AtS1P for processing, it is likely that the AtRALF23 propeptide is processed in the Golgi on its way through the secretory pathway. Escobar et al. (2003) tracked RALF after transfecting Nicotiana benthamiana leaves with NtRALF-GFP via a viral vector. They first found NtRALF-GFP in the ER, and later in the apoplast. It would seem possible that some AtS1P is secreted, and that AtRALF23 propeptide is proteolytically processed in the apoplast. AtS1P has a C-terminal transmembrane domain that is thought to anchor the enzyme to the membranes of the secretory pathway. In mammalian cells it was found that a small quantity of S1P undergoes autocatalytic cleavage to release the truncated enzyme, which is then secreted into the media (Cheng et al., 1999; Espenshade et al., 1999). It is unlikely that proAtRALF23 is cleaved by AtS1P in the apoplast, because the Arabidopsis enzyme has a pH optimum of 7.5 (RS, unpublished data), and little or no activity at the acidic pH of the apoplast. However, if a small quantity of proAtRALF23 is processed, and begins to promote the alkalization of the apoplast, then that might generate an apoplastic environment conducive to the processing of AtRALF23 by a released form of AtS1P.
In the case of Arabidopsis PHYTOSULFOKINE 4 (AtPSK4), the expression of both the gene and the processing activity are upregulated by wounding. When the AtPSK4 transgene is constitutively expressed, processing regulates the appearance of the mature AtPSK4 peptide (Srivastava et al., 2008). Although processing is required for AtRALF23 activity, it is unlikely that processing regulates AtRALF23 in response to BL. The microarray data of Nemhauser et al. (2004) indicate that AtRALF23 expression is regulated by BL, but that the expression of AtS1P is not.
AtS1P processing cuts the AtRALF23 propeptide cleanly at the N terminus of the predicted mature peptide. Hence, AtS1P cleavage is probably the only proteolytic step needed to activate AtRALF23. This is in contrast to the processing of AtPSK4 by AtSBT1.1, which releases the peptide from the larger precursor by cutting three amino acid residues from the N terminus of the predicted mature peptide (Srivastava et al., 2008). The activation of AtPSK4 is likely to require the trimming of three residues from the N terminus and five residues from the C terminus of the released peptide, by more than one protease. However, results from this study demonstrate that after the removal of the signal peptide, AtRALF23 maturation appears to be dependent only on one protease, AtS1P.