Physiological roles of the light, oxygen, or voltage domains of phototropin 1 and phototropin 2 in Arabidopsis

Abstract

Phototropins (phot1 and phot2) are plant blue-light receptors that mediate phototropism, chloroplast movement, stomatal opening, rapid inhibition of growth of etiolated seedlings, and leaf expansion in Arabidopsis (Arabidopsis thaliana). Their N-terminal region contains two light, oxygen, or voltage (LOV) domains, which bind flavin mononucleotide and form a covalent adduct between a conserved cysteine and the flavin mononucleotide chromophore upon photoexcitation. The C-terminal region contains a serine/threonine kinase domain that catalyzes blue-light-activated autophosphorylation. Here, we have transformed the phot1 phot2 (phot1-5 phot2-1) double mutant with PHOT expression constructs driven by the cauliflower mosaic virus 35S promoter. These constructs encode either wild-type phototropin or phototropin with one or both LOV-domain cysteines mutated to block their photochemistry. We selected multiple lines in each of the eight resulting categories of transformants for further physiological analyses. Specifically, we investigated whether LOV1 and LOV2 serve the same or different functions for phototropism and leaf expansion. Our results show that the LOV2 domain of phot1 plays a major role in phototropism and leaf expansion, as does the LOV2 domain of phot2. No complementation of phototropism or leaf expansion was observed for the LOV1 domain of phot1. However, phot2 LOV1 was unexpectedly found to complement phototropism to a considerable level. Similarly, transformants carrying a PHOT transgene with both LOV domains inactivated developed strong curvatures toward high fluence rate blue light. However, we found that the phot2-1 mutant is leaky and produces a small level of full-length phot2 protein. In vitro experiments indicate that cross phosphorylation can occur between functional phot2 and inactivated phot1 molecules. Such a mechanism may occur in vivo and therefore account for the functional activities observed in the PHOT transgenics with both lov domains inactivated. The implications of this mechanism with respect to phototropin function are discussed.

Figure 1.

Constructs and expression levels of phot1 or phot2 in the several transformants. A, Diagram of the various PHOT transgenes used to create transgenic lines in the phot1 phot2 double-mutant background. PHOT transgenes containing a wild-type or mutagenized version of PHOT1 or PHOT2 with LOV1 and/or LOV2 C39A mutation (series 1–8). B, Western blots of membrane fractions prepared from wild-type (gl1), phot1 phot2 double mutant, or independent transgenic Arabidopsis seedlings expressing the series of constructs illustrated in A. Membrane protein extracts were probed with anti-phot1 antibody. Fifty micrograms of protein were loaded in each lane. Arrow indicates the molecular mass of phot1 (120 kD). C, Expression levels in control and transgenic seedlings. Total RNA was extracted from the seedling lines as in part B, above, grown under continuous white light at 22°C for 4 weeks. PHOT1, PHOT2, and UBQ10 cDNA were amplified by RT-PCR under semiquantitative conditions, resolved on agarose gels, and detected by hybridization. The signal intensity was determined by PhosphorImager and standardized to that of UBQ-10. The ratio of the phototropin signal to that of UBQ-10 was then normalized to that of the wild-type control. The experiment was done three times with the PHOT2 transgenes with similar results and once with the PHOT1 transgenes as results were consistent with those from the western analysis.

Figure 2.

Hypocotyl phototropism in etiolated wild-type (gl1), phot1 phot2 double mutant, and the transformant seedling lines of Arabidopsis. Hypocotyl curvatures of dark-grown seedlings 2.5-d-old at the onset of irradiation were measured after an 8-h exposure at 1 μmol m⁻² s⁻¹ (transformed with PHOT1 constructs [A]) or 20 μmol m⁻² s⁻¹ (transformed with PHOT2 constructs [B]) fluence rate of blue light. The hypocotyl curvatures of 20 seedlings were measured in each case and average curvatures are shown. Error bars represent the mean ± se. The experiments in each box were done three times with similar results. Representative results from one experiment are shown.

Figure 3.

Time course for the development of phototropic curvature in etiolated wild-type (gl1), untransformed phot1 phot2 double mutant, and representative double-mutant seedlings transformed with the PHOT1 expression constructs (A) and PHOT2 expression constructs (B) shown in Figure 1A (the data for line 3-a are almost hidden beneath those for line 4-a and phot1 phot2). The magnitude of the phototropic responses was determined against irradiation time. For the phot1 transformants, the fluence rate was 1 μmol m⁻² s⁻¹ and for the phot2 transformants, it was 20 μmol m⁻² s⁻¹ of blue light. Hypocotyl curvatures of 20 seedlings were measured in each case and average curvatures are shown. Error bars represent the mean ± se. Each experiment was done three times and representative results are shown. Responses of phot1-5 single and phot2-1 single mutants are also shown.

Figure 4.

Phototropism fluence rate response curves in the etiolated wild-type (gl1), the untransformed phot1 phot2 double mutant, and representative double-mutant seedlings transformed with PHOT1 expression constructs (A) and PHOT2 (B) expression constructs shown in Figure 1A. Curvatures were measured after an 8-h exposure to unilateral blue light at the indicated fluence rate. Hypocotyl curvatures of 20 seedlings were measured in each case with similar results and average curvatures are shown. Error bars represent the mean ± se. Each experiment was performed three times with similar results and representative results are shown. Hypocotyl curvatures for all of the phot1 + (series 4) transgenic lines (C) and the phot2 + (series 8) transgenic lines (D) are shown. Responses of wild-type (gl1), phot1 phot2 double mutant, phot1-5 single, and phot2-1 single mutant are also shown.

Figure 5.

Expression levels of phot2 protein in the wild-type (gl1), phot1 phot2 double mutant, and phot1-5. Western blots of membrane fractions prepared from wild-type, phot1 phot2 double mutant, and phot1 single mutant (phot1-5) were probed with anti-phot2 antibody. Fifty micrograms of protein was loaded in each lane. Protein was extracted from leaves of 2-week-old light-grown seedlings (white light, fluence rate 45 μmol m⁻² s⁻¹).

Figure 6.

Effect of mutating either the conserved Cys (C39A) within each LOV domain in LOV1 and LOV2 or the conserved Asps in the kinase domain, required for kinase activity (D806N in phot1, D720N in phot2) on light-activated in vitro autophosphorylation of phot1 or phot2 expressed in the insect cells. Lanes 1 and 2: both LOV domains of phot1 mutated (phot1); lanes 3 and 4: phot1 kinase domain mutated (phot1); lanes 5 and 6: wild-type phot1; lanes 7 and 8: phot2 kinase domain mutated (phot2); lanes 9 and 10: wild-type phot2. D, Dark; L, light (saturating white light); , LOV-domain mutated; , kinase-domain mutated. Western-blot analysis of phot1 and phot2 protein levels is shown below. Soluble protein extracts prepared from insect cells were probed with anti-phot1 antibody (left) or anti-phot2 antibody (right).

Figure 7.

Light-activated cross phosphorylation of inactivated phot1 by functional phot2. Proteins were produced by coexpression of the two photoreceptor genes in insect cells. Lanes 1 and 2: functional phot2 plus phot1 with its kinase domain inactivated (phot1); lanes 3 and 4: phot2 with its kinase domain inactivated (phot2) plus phot1 with its kinase domain inactivated (phot1); lanes 5 and 6: functional phot2 plus phot1 with both LOV domains mutated (phot1 = phot1); lanes 7 and 8: phot2 with its kinase domain inactivated (phot2) plus phot1 both LOV domains mutated (phot1 = phot2). Inactivation of the proteins is as described in Figure 6. Western-blot analysis of phot1 and phot2 protein levels is shown below. Soluble protein extracts prepared from insect cells were probed with anti-phot1 antibody (left) or anti-phot2 antibody (right).

Figure 8.

Leaf-expansion phenotypes in wild-type (gl1), untransformed phot1 phot2 double mutant, and representative double-mutant seedlings transformed with PHOT expression constructs (series 1–8) shown in Figure 1A. Responses of phot1 single and phot2 single mutant are also shown. A, Plants were grown in soil under continuous white light (45 μmol m⁻² s⁻¹) in the growth chamber (22°C) for 4 weeks (bar = 1 cm). B, The leaf-expansion index of the fifth rosette leaves was expressed as the ratio of the projection of the leaf width before and after artificial uncurling. Each value is the mean ± se of 12 leaves.