Blue-light induced biosynthesis of ROS contributes to the signaling mechanism of Arabidopsis cryptochrome

Abstract

Cryptochromes are evolutionarily conserved blue light receptors with many roles throughout plant growth and development. They undergo conformational changes in response to light enabling interaction with multiple downstream signaling partners. Recently, it has been shown that cryptochromes also synthesize reactive oxygen species (ROS) in response to light, suggesting the possibility of an alternate signaling mechanism. Here we show by fluorescence imaging and microscopy that H₂0₂ and ROS accumulate in the plant nucleus after cryptochrome activation. They induce ROS-regulated transcripts including for genes implicated in pathogen defense, biotic and abiotic stress. Mutant cryptochrome alleles that are non-functional in photomorphogenesis retain the capacity to induce ROS-responsive phenotypes. We conclude that nuclear biosynthesis of ROS by cryptochromes represents a new signaling paradigm that complements currently known mechanisms. This may lead to novel applications using blue light induced oxidative bursts to prime crop plants against the deleterious effects of environmental stresses and toxins.

Figure 1

Domain structure and photochemical reactions of Arabidopsis cryptochrome 1. Upper panel light sensing (flavin binding) domain and C-terminal domain are indicated showing position of point mutants used in this study. Lower panel: flavin photoreduction and reoxidation reactions which have been linked to biological signaling. Flavin (FAD) is oxidized in the dark-adapted state of the receptor and becomes reduced in the presence of light, forming the neutral radical (FADH°) flavin redox state which is correlated with protein conformational change linked to biological activation. Subsequent to flavin reduction, flavin reoxidation occurs in a light-independent reaction leading to the production of Reactive Oxygen Species (ROS). Currently known paradigms for plant cryptochrome signaling are discussed in ref..

Figure 2

Production and subcellular localization of ROS in Cryptochrome-1 overexpressing (WS01) and double mutant cry1cry2 (C1C2) seedlings exposed to blue light. Primary root tips from 8-day old etiolated Arabidopsis seedling (see methods) roots either lacking both cry1 and cry2 (C1C2 mutant) or over- expressing cryptochrome 1 (WS01) were treated with HOECHST for nuclear staining and SOSG for ROS staining for 30 minutes, exposed to blue light for 10 minutes, and then immediately viewed by a Zeiss AxioImager.Z1/ApoTome microscope. (a) Images show single z section that cross the nucleus. Colocalized pixels appear in white on the merged images and on the colocalized pixels maps. Scale bars are 25 μm. (b) using the segmentation and ROI manager tool on imageJ, fluorescence intensity of white and red pixels were quantified from these images for each nucleus and exported to LibreOffice calculator. The mean fluorescence intensity of >50 individual nuclei is shown.

Figure 3

Effect of cryptochrome on transcription of representative ROS-regulated genes. Quantitative real-time PCR indicating the relative expression of twelve ROS-responsive genes investigated in WS (wild type), cry1,  cry2, and cry1 cry2 mutant seedlings. All seedlings were null mutants (absence of protein) . Four-day-old dark-grown seedlings were transferred to blue light (60 μmol m⁻² s⁻¹) for 3 h. prior to RNA extraction and qPCR analysis. Three replicates (biological) were used to compute bar errors (representing the S.D.).

Figure 4

C-terminal point mutant of Arabidopsis cry1 retains light response and ROS biosynthetic capacity. Wild type (a) and E531K (b) mutant proteins were expressed and purified from baculovirus expression system as previously described. ‘Dark’ shows spectra of isolated proteins prior to illumination. ‘Light’ indicates spectra after photoreduction for 30 seconds at 500 μmol m⁻² s⁻¹ blue light in the presence of 5 mM DTT . Samples were subsequently returned to dark and spectra taken at the indicated times (in min). (c). 100micromolar concentration of proteins were illuminated at 500 μmol m⁻² s⁻¹ blue light and aliquots taken at the indicated time for detection of ROS by AMPLEX RED fluorescence detection method as described. Aliquots were taken at the indicated times. Error bars represent SD of three independent measurements.

Figure 5

Phenotypes of cryptochrome C-terminal point mutants. Wild type and mutant alleles were grown for seven days on petri plates at the indicated light fluence (upper panel) and hypocotyl length of seedlings was measured. Error bars represent SD of measurement from 10 seedlings. Anthocyanin accumulation was determined as described, error represents SD of three independent measurements.

Figure 6

(a) C-terminal point mutants retain ability to induce ROS regulated gene expression. qPCR showing the relative expression of 12 ROS-responsive genes investigated in WS (wild type), Cry1 (null allele), and the indicated C-terminal point mutant seedlings. Four-day-old dark-grown seedlings were transferred to blue light (60 μmol m⁻² s⁻¹) for 3 h. prior to RNA extraction and qPCR analysis. Data are from three biological replicates n = 3; error bars indicate ± SD. No differences in expression were noted in dark grown seedlings without blue light treatment (supplementary Fig. 1). (b) Expression of ROS regulated genes in Trp triad W400F mutant Arabidopsis seedlings. qPCR shows expression of 12 ROS regulated genes in wild type (Ws), cry1 mutant, and expressing W400F Trp triad mutants (lines A and B - see Supplementary Fig. 3 for levels of mutant protein expression). No effect on gene expression is observed for the control cry mutant alleles. Data are from 3 biological replicates (n = 3), error bars indicate SD.