A plant-specific protein essential for blue-light-induced chloroplast movements

Abstract

In Arabidopsis (Arabidopsis thaliana), light-dependent chloroplast movements are induced by blue light. When exposed to low fluence rates of light, chloroplasts accumulate in periclinal layers perpendicular to the direction of light, presumably to optimize light absorption by exposing more chloroplast area to the light. Under high light conditions, chloroplasts become positioned parallel to the incoming light in a response that can reduce exposure to light intensities that may damage the photosynthetic machinery. To identify components of the pathway downstream of the photoreceptors that mediate chloroplast movements (i.e. phototropins), we conducted a mutant screen that has led to the isolation of several Arabidopsis mutants displaying altered chloroplast movements. The plastid movement impaired1 (pmi1) mutant exhibits severely attenuated chloroplast movements under all tested fluence rates of light, suggesting that it is a necessary component for both the low- and high-light-dependant chloroplast movement responses. Analysis of pmi1 leaf cross sections revealed that regardless of the light condition, chloroplasts are more evenly distributed in leaf mesophyll cells than in the wild type. The pmi1-1 mutant was found to contain a single nonsense mutation within the open reading frame of At1g42550. This gene encodes a plant-specific protein of unknown function that appears to be conserved among angiosperms. Sequence analysis of the protein suggests that it may be involved in calcium-mediated signal transduction, possibly through protein-protein interactions.

Figure 1.

Schematic of device used for chloroplast movement mutant screen. Modified petri dishes containing up to eight leaves can be placed on the turntable. The turntable rotates to eight precise positions so that each leaf can be located between the red measuring beam and the photodetector. The design allows for rapid removal and replacement of petri dishes while ensuring that each measurement of light transmittance is made through the same area of each leaf enabling fast and accurate measurements from a large number of leaves. LEDs, light-emitting diodes.

Figure 2.

BL-induced chloroplast movements in wild-type and pmi mutants. The plots show the average change (± se) in the percentage of RL transmittance of leaves relative to the average transmittance measured prior to the first BL treatment. RL transmittance was measured in dark-acclimated leaves for 90 min before sequential treatments of 0.3, 20, and 60 μmol·m⁻²·s⁻¹ of BL (450 nm ± 25 nm) indicated by the arrowheads at 90, 150, and 210 min. Number of leaves (n) is shown for each treatment. Inset is an autofluorescence micrograph of the giant chloroplasts in pmi4 palisade cells. WT, Wild type. Bar = 10 μm.

Figure 3.

BL-induced chloroplast migration and positioning in wild-type and pmi1 leaf cells. Dark-acclimated wild-type (A–C) and pmi1-1 (D–F) palisade cells were exposed to sequential 1-h treatments of low- and high-fluence-rate white light from below. Micrographs from time points 0 (A and D), 60 (B and E), and 120 (C and F) min are shown. Bar = 10 μm. Cross sections of wild-type and pmi1-1 rosette leaves exposed to 1 h of either darkness (G and J), low BL (H and K), or high BL (I and L) show chloroplast positioning in all cell layers. Bar = 30 μm. Bar graphs depict the percentage (± se) of chloroplasts located along the anticlinal and periclinal cells walls in fixed wild-type and pmi1-1 leaf cross sections and represent averages from 200 to 400 cells per BL treatment.

Figure 4.

Organization of the actin cytoskeleton in wild-type and pmi1 leaf cells. Confocal images of wild-type (A) and pmi1 (B) palisade cells expressing GFP-mtalin. GFP fluorescence is shown in green and chloroplast autofluorescence in red. Images represent projections of 36 optical 0.2-μm sections. Bar = 10 μm.

Figure 5.

Mapping of the PMI1 locus and organization of the corresponding PMI1 gene. A, Genetic interval located south of the centromere on chromosome 1 is shown with corresponding SSLP and CAPS markers used to fine map the pmi1-1 EMS mutation. Arrows indicate the positions of the markers and the number of recombinants per number of chromosomes tested. B, The pmi1-1 chloroplast movement defect was rescued by the 8-kb overlapping region of BAC T8D8 and F8D11. The positions of the two genes located in this genetic interval are shown. C, Diagram of the PMI1 gene (T8D8.2). Exons are depicted as thick bars and introns as thin lines. Positions of the initiation and termination codons as well as the pmi1-1 point mutation and pmi1-2 T-DNA insertion are shown. The position of the original annotated and third putative start codons are indicated by an arrowhead and an asterisk, respectively.

Figure 6.

BL-induced chloroplast movements of rescued pmi1-1 transgenic and pmi1-2 plants. RL transmittance was measured in dark-acclimated leaves for 45 min before exposure to either 40 (A and C) or 0.3 (B and D) μmol·m⁻²·s⁻¹ of BL (450 nm ± 25 nm) indicated by the arrowheads. The plots show the average change (± se) in the percentage of RL transmittance of leaves relative to the average transmittance measured for the leaves prior to the BL treatment. Pro:PMI1 is the wild-type PMI1 gene driven by its own promoter. Number of leaves (n) is shown for each treatment.

Figure 7.

Diagram of PMI1 protein. A, The deduced amino acid sequence of PMI1 based on GenBank cDNA sequence and RT-PCR results. The putative cleavage signal is underlined. The raised boxes indicate the coil-coiled sequences and the dotted box the putative bombesin-like domain. The two conserved domains revealed by sequence alignment with orthologous sequences are in boldface. B, Diagram of the PMI1 protein. The positions of the original annotated and third putative start codons are indicated by an arrowhead and an asterisk, respectively. Arrows indicate the position of the pmi1-1 and pmi1-2 mutations. a.a., amino acids.

Figure 8.

Alignment of conserved central (A) and C-terminal (B) domains of PMI1 from Arabidopsis with putative orthologous sequences from O. sativa (Os), Z. mays (Zm), G. max (Gm), and M. trunculata (Mt). Orthologous sequences were obtained from a BLAST search of the National Center for Biotechnology Information and The Institute for Genomic Research gene indices and maize database using AtPMI1 as a reference. Sequences were aligned using ClustalW (http://www.ebi.ac.uk/clustalw/) and the output produced by BoxShade (http://www/ch.embnet.org/software/BOX_form.html). Black boxes indicate identity, and gray represents similarity between residues. Alignment of At5g20610 and At5g26160, putative members of the PMI1 gene family, are shown as well as the protein accession numbers for the orthologous sequences.

Figure 9.

Expression of PMI1 in Arabidopsis tissues. Total RNA was extracted from various wild-type (Col) plant tissues and PMI1 transcript measured by RT-PCR using gene-specific primers spanning the 70-bp intron of At1g42550. The product from the fully spliced mRNA is shown. Primers spanning the two introns of profilin1 were used as a control. RNA was combined from the rosette leaves (RL), stems (S), cauline leaves (CL), flowers (F), and roots (R) of three different 9-week-old plants.