LETTER
Reply to Ashton: The putative guanylyl cyclase domain of AtPepR1 and similar plant receptors We recently showed (1) that peptide ligand binding to a plant leucine-rich-repeat receptor-like kinase (LRR-RLK) (AtPepR1) initiates pathogen defense signaling cascades through elevation of cytosolic Ca2+. We provided indirect evidence consistent with AtPepR1 signaling through cytosolic guanylyl cyclase (GC) activity as follows. Receptor signaling required a cyclic nucleotide activated Ca2+ channel. Exogenous lipophilic cGMP activated channel-dependent Ca2+ elevation. Expression of the putative GC domain of the AtPepR1 receptor (“AtPepR1-GC”) in Escherichia coli increased cell cGMP levels. Affinity-purified AtPepR1-GC had a level of GC activity in vitro equal to that of other plant proteins with a similar putative GC domain (2). Ashton (3) suggests that AtPepR1-GC activity in vitro is “extraordinarily low” compared with animal GCs and that AtPepR1 and other plant proteins with a similar catalytic domain are unlikely to generate cGMP in vivo; and he questions the presence of GC activity in plants. In response to Ashton’s theoretical analysis of possible constraints to in vivo GC activity of AtPepR1 and other similar proteins, we note the following. First, we (1) specifically acknowledged Ashton’s main point in stating that although “the in vitro GC activity of. . .AtPepR1 is at least as high or higher than the activity. . .of other plant GCs. . .it is substantially lower than the GC activity of animal GC enzymes.” We provided further context about this point; when referring to the possible in vivo GC activity of AtPepR1, we explicitly stated that “this point remains speculative and is not experimentally confirmed by the work here” and that AtPepR1 “could act through cytosolic kinase activity to. . .initiate downstream. . .signaling.” Second, Ashton’s speculation that the AtPepR1-GC activity might be due to contaminating bacterial GC in the purified protein preparation ignores results (figure S3 in ref. 1) reporting increased [cGMP] of E. coli expressing AtPepR1-GC. Third, Ashton’s assertion that evidence for a regulatory role of cGMP in higher plants is “still not clear” fails to consider recent work of Isner and Maathuis (4), who used an in vivo cGMP reporter protein to show that the signal nitric oxide and hormone gibberellic acid, thought to act through cGMP
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elevation, did increase cGMP in plant cells. Ashton’s main issue is whether proteins with a GC domain akin to that found in AtPepR1 raise cGMP level in plant cells during signaling. Neither our article (1) nor Ashton’s letter (3) addresses this issue experimentally. However, we reported* that another LRR-RLK [the phytosulfokine-α (PSK-α) receptor PSKR1] with a GC domain similar to AtPepR1 and similar in vitro GC activity (not shown) does have ligand-dependent GC activity in plant cells. The unsulfated pentapeptide ligand (nPSK) does not bind to PSKR1 (5) and thus serves as an appropriate negative control for signaling induced by binding of the active sulfated pentapeptide ligand PSK-α to PSKR1. The level of cGMP was tested [using ELISA (1)] in Arabidopsis protoplasts at 5 and 15 min after application of the active PSK-α ligand that caused a statistically significant (P ≤ 0.05) elevation of in vivo cGMP compared with control protoplasts or those treated with the inactive analog (Fig. 1). Thus, these results indicate that a member of the family of plant proteins with cytosolic GC domains similar to that found in AtPepR1 is capable of generating ligand-dependent cGMP elevations in the plant cell, despite Ashton’s theoretical considerations. Gerald A. Berkowitza,1, Chris Gehringb, Helen R. Irvingc, and Lusisizwe Kwezid a Department of Plant Science, University of Connecticut, Storrs, CT 06269; bComputational Bioscience Research Centre, King Abdullah University of Science and Technology, Thuwal, Kingdom of Saudi Arabia 23955-6900; cMonash Institute of Pharmaceutical Sciences, Monash University, Parkville Campus, Parkville, Victoria 3052, Australia; and dDepartment of Biotechnology, University of the Western Cape, Bellville, South Africa 7535 1. Qi Z, et al. (2010) Ca2+ signaling by plant Arabidopsis thaliana Pep peptides depends on AtPepR1, a receptor with guanylyl cyclase activity, and cGMP-activated Ca2+ channels. Proc Natl Acad Sci USA 107:21193–21198. 2. Kwezi L, et al. (2007) The Arabidopsis thaliana brassinosteroid receptor (AtBRI1) contains a domain that functions as a guanylyl cyclase in vitro. PLoS ONE 2:e449. 3. Ashton AR (2011) Guanylyl cyclase activity in plants? Proc Natl Acad Sci USA 108: E96. 4. Isner JC, Maathuis FJ (2011) Measurement of cellular cGMP in plant cells and tissues using the endogenous fluorescent reporter FlincG. Plant J 65:329–334. 5. Matsubayashi Y, Ogawa M, Morita A, Sakagami Y (2002) An LRR receptor kinase involved in perception of a peptide plant hormone, phytosulfokine. Science 296: 1470–1472.
Author contributions: H.R.I. and L.K. performed research; and G.A.B., C.G., and H.R.I. wrote the paper. The authors declare no conflict of interest. 1
To whom correspondence should be addressed. E-mail:
[email protected].
*Kwezi L, et al. Oral Presentation and Poster Session, 15th International Workshop on Plant Membrane Biology, September 19–24, 2010, Adelaide, South Australia, abstr P2-30.
PNAS | May 10, 2011 | vol. 108 | no. 19 | E97–E98
Fig. 1. Cyclic GMP levels following treatment with PSK-α (0.1 μM) or nPSK (0.1 μM) in Arabidopsis protoplasts over 15 min; controls contain no peptide ligand. Asterisks indicate significant differences (P < 0.05; 2-way ANOVA, Bonferroni post test) from respective controls.
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Berkowitz et al.
