Blue Light Regulates an Auxin-Induced K -Channel Gene in the Maize

Blue light regulates an auxin-induced K؉-channel gene in the maize coleoptile I. Fuchs*, K. Philippar*, K. Ljung†, G. Sandberg†, and R. Hedrich*‡ *Lehrstuhl fu¨r Molekulare Pflanzenphysiologie und Biophysik, Universita¨t Wu¨ rzburg, Julius-von-Sachs-Platz 2, D-97082 Wu¨rzburg, Germany; and †Department of Forest Genetics and Plant Physiology, University of Umeå, SLU, SE-901 83, Sweden Edited by Anthony R. Cashmore, University of Pennsylvania, Philadelphia, PA, and approved August 7, 2003 (received for review May 14, 2003) Auxin redistribution along gravistimulated maize coleoptiles blue-light stimulus (16) and evoke blue-light induced cytoplas- -causes differential expression of the auxin-induced K؉-channel mic Ca2ϩ signals (17), most likely via calcium-permeable chan gene ZMK1 (Zea mays K؉ channel 1) and precedes the curvature nels (18). An ortholog to phototropin 1 (PHOT1) has already response. To evaluate the role of ZMK1 during phototropism, we been isolated from maize (GenBank accession no. AF033263; for here investigated blue light-stimulated coleoptiles. Four hours of a review, see ref. 19). Interestingly, the blue light-responsive blue light stimulation resulted in phototropic bending (23°). Rota- protein in maize has been shown to be most abundant in the tion on a clinostat, at nominally ‘‘zero’’ gravity, and simultaneous apical 5-mm-tip zone of coleoptiles (20), a localization in line stimulation with unidirectional blue light, however, resulted in up with the photoperceptivity of this organ (21). to 51° bending toward the light. Differential ZMK1 transcription Gravity sensing most likely results from the translocation of reached a maximum after 90 min of blue light stimulation under statholiths (22). The molecular mechanism of graviperception, gravity, whereas ZMK1 expression remained asymmetric for at however, remains still unknown. In A. thaliana, the isolation of least 180 min in photostimulated coleoptiles on a clinostat. We auxin-sensitivity mutants led to the identification of components therefore conclude that the stronger phototropic bending under of the auxin influx (AUX family; ref. 23) and efflux carrier (PIN nominally ‘‘zero’’ gravity results from prolonged differential ex- family; refs. 24 and 25). The agravitropic nature of these pression of ZMK1. Under both conditions, asymmetric expression mutants, the polar localization of the respective protein (26), and of ZMK1 could be superimposed on the lateral auxin gradient its redistribution on gravistimulation (27) point to their role in across the coleoptile tip, whereas the gene for the blue light polar and lateral auxin transport (for a review, see ref. 28). receptor phototropin 1 (PHOT1), expressed in the tip only, was not Moreover, a putative auxin receptor, ABP1, has been crystallized differentially regulated in response to blue light. The activation of with 1-naphthylacetic acid (29) and mediates auxin-induced cell the two different receptors eliciting the photo- and gravitropic expansion (30). ABP1 deletion results in embryo lethality (31). response of the coleoptile thus feeds into a common signaling Thus, two physically distinct stimuli, blue light and gravity, pathway, resulting in auxin redistribution in the coleoptile tip and merge into one common signaling pathway, leading to the finally in differential transcription of ZMK1. In the process of signal redistribution of endogenous auxin and differential growth in integration, gravity transduction restricts the magnitude of the blue light-inducible ZMK1 gradient. The spatial and temporal the two flanks of the maize coleoptile. In a search for the osmotic ؉ motor involved in auxin-induced growth, potassium-dependent distribution of ZMK1 transcripts and thus differential K uptake in ϩ both flanks of the coleoptile seem to limit the stimulus-induced steps were identified and control of the osmotic motor by K bending of this sensory organ. channels was predicted (32). In a previous study, we isolated the voltage-dependent inwardly rectifying Kϩ channel ZMK1 (Zea mays Kϩ channel 1) from maize coleoptiles (8). ZMK1 tran- tudying the tropistic curvature of plant organs, Charles scription is induced by active auxins, and gravistimulation results SDarwin (1) postulated the existence of a signal molecule in a differential expression in the two flanks of the gravire- being synthesized in the tip of photostimulated grass coleoptiles sponding coleoptile. Because coleoptile elongation and bending and enhancing growth in one flank of the organ. Went (2) and involve transcriptional activation of ZMK1 and in turn increase Cholodny (3) could show that this substance, called auxin, is ϩ ϩ in K -channel protein density (33), this K channel seems to laterally translocated in photostimulated coleoptiles, resulting in represent an essential element of the osmotic motor required for a curvature of the plant toward the light source. Using radio- gravitropic curvature. actively labeled auxin, Parker and Briggs (4) and Iino (5) ZMK1 confirmed the Cholodny–Went hypothesis (6). Whereas lateral To explore whether differential expression is involved auxin translocation in photostimulated maize seedlings is re- in the phototropic curvature of maize coleoptiles as well, we stricted to the tip (7), auxin redistribution in gravistimulated followed the redistribution of ZMK1 transcripts in photostimu- seedlings occurs along the length of the entire coleoptile (8). lated plants in the presence of gravity or at nominally ‘‘zero’’ Tropistic curvature is preceded by the asymmetric distribution of gravity. We demonstrated that differential expression of ZMK1 apoplastic protons (9), the drop in extracellular pH being caused during phototropism is coupled to the redistribution of auxin in by increased indole-3-acetic acid (IAA) levels in the faster- the coleoptile tip. Furthermore, the extent of the curvature growing flank of the organ (for the Acid Growth Theory, see ref. response of maize seedlings and the degree of differential ZMK1 10). Increased proton extrusion results from auxin activation of expression were closely correlated. Thus, we conclude, that the plasma membrane Hϩ ATPase and in the long-term rise in the number of Hϩ pumps (refs. 11–13; for a review, see ref. 14). This paper was submitted directly (Track II) to the PNAS office. Acidification of the apoplast activates expansins mediating cell Abbreviations: BLC, Blue Light Cycler; fw, fresh weight; IAA, indole-3-acetic acid; PHOT1, wall loosening, a prerequisite for cell expansion (for a review, see phototropin 1; ZMK1, Zea mays Kϩ channel 1. ref. 15). Data deposition: The sequences reported in this paper have been deposited in the GenBank Although the incoming signal is transduced with the help of database [accession nos. Y07632 (ZMK1); AF033263 (PHOT1); AAB40106 (ZmACT81); and one single phytohormone, signal perception is different in gravi- AAB40105 (ZmACT83)]. and photostimulated plants. The light signal is perceived by ‡To whom correspondence should be addressed. E-mail: [email protected] photoreceptor proteins (phototropins). In Arabidopsis thaliana, wuerzburg.de. PLANT BIOLOGY two phototropins, phot1 and -2, act together in perceiving the © 2003 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.2032704100 PNAS ͉ September 30, 2003 ͉ vol. 100 ͉ no. 20 ͉ 11795–11800 Downloaded by guest on September 30, 2021 Fig. 1. Differential expression of ZMK1 and coleoptile bending angles during gravi- and phototropism of the maize coleoptile. (a) Relative transcript level (RU, relative units) of ZMK1 in light-exposed (open bars) and shaded (solid bars) coleoptile halves after 0, 30, 60, and 90 min of stimulation with unilateral blue light. The mRNA content (n ϭ 3 Ϯ SE) was quantified as described in Materials and Methods. The level of ZMK1 mRNA in the light-exposed half at t ϭ 0 min was set to 1.0. (b) Curvature angle of coleoptiles after 4 hr of gravistimulation (g, black bar) and stimulation with unilateral blue light (bl, gray bar) and of photostimulated seedlings rotating on the BLC (bl-g, open bar). Bending angles (mean of n Ն 30 Ϯ SE) were measured as described in Materials and Methods. (c) Relative transcript level (RU) of ZMK1 in light-exposed (open bars) and shaded (closed bars) coleoptile halves of seedlings rotating on the BLC after 0, 30, 60, and 90 min of simultaneous stimulation with unilateral blue light. Blue light illumination started 30 min after the onset of clinostat rotation (Ϫ30). The mRNA content (n ϭ 3 Ϯ SE) was quantified as described in Materials and Methods. The level of ZMK1 mRNA in the light-exposed half at t ϭ 0 min was set to 1.0. ZMK1 is involved in both gravitropic and phototropic bending of Europe Bv., Amsterdam, NLPB 500, 450 nm). Light intensity was maize coleoptiles. adjusted to 10 ␮mol⅐mϪ2⅐sϪ1. Materials and Methods Quantitative Real-Time RT-PCR. Isolation of RNA from maize Maize Strains and Growth Conditions. Quantitative real-time RT- coleoptiles, quantitative real-time RT-PCR (LightCycler, Roche PCR experiments were performed on tissues from Z. mays Diagnostics), and quantification of transcripts were performed seedlings (hybrid corn cv. ‘‘Oural’’). Maize caryopses were as described in ref. 35. The following ZMK1- and PHOT1- soaked in water, germinated on moistened filter paper, and specific primers were used: ZMK1 fw (5Ј-ataacaatgggcatacag- grown under red light (0.2 ␮mol⅐mϪ2⅐sϪ1)at25°C, because red 3Ј), ZMK1 rev (5Ј-ttccgtctttcattgag-3Ј), PHOT1 fw (5Ј- Ј Ј light treatment is necessary for maize coleoptiles to acquire ccacggtcattgtcag-3 ), and PHOT1 rev (5 -ctt-cccttggttttagaatg- Ј responsiveness for time-dependent phototropism (34). After 3 ). All quantifications were normalized to the signal of actin ͞ 72 hr, the seedlings were transferred into pots filled with 1% cDNA fragments generated by the primers ZmAct 81 83 fw Ј Ј ͞ Ј agar. For phototropic stimulation, the apical 5 mm of the 4- to (5 -acacagtgccaatct-3 ) and ZmAct 81 83 rev (5 -actgagcacaat- Ј 5-day-old and 2- to 2.5-cm-long seedlings were illuminated with gttac-3 ). unilateral blue light (450 nm, 10 ␮mol⅐mϪ2⅐sϪ1). The plane passing through the two vascular bundles of the coleoptile was Analysis of Endogenous IAA Concentrations.

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