Non-invasive Assay for Chlorophyll Biosynthesis Kinetics Determination during Early Stages of <em>Arabidopsis</em> De-etiolation

Chlorophyll biosynthesis is a hallmark of de-etiolation, one of the most dramatic stages in the plant life cycle. The tightly controlled and highly ...

Masaryk University

Labdeers s.r.o

Tightly controlled spatiotemporal specificity of gene expression is intrinsic to developmental and adaptation responses of living systems throughout the kingdoms. Forward genetic screens employing well-characterized reporter lines can be used to identify as yet unknown genetic factors driving specific aspects of individual regulatory pathways. However, such screens are demanding with respect to data acquisition and analysis from thousands of mutant lines. Here, we describe a method that allows screening of a mutagenized GUS reporter line in Arabidopsis using an automated microscopy imaging system as a tool for rapid and efficient identification of mutants with modified expression profile for a gene of interest.

Automated microscopy in forward genetic screening of Arabidopsis.

Integrating important environmental signals with intrinsic developmental programmes is a crucial adaptive requirement for plant growth, survival, and reproduction. Key environmental cues include changes in several light variables, while important intrinsic (and highly interactive) regulators of many developmental processes include the phytohormones cytokinins (CKs) and ethylene. Here, we discuss the latest discoveries regarding the molecular mechanisms mediating CK/ethylene crosstalk at diverse levels of biosynthetic and metabolic pathways and their complex interactions with light. Furthermore, we summarize evidence indicating that multiple hormonal and light signals are integrated in the multistep phosphorelay (MSP) pathway, a backbone signalling pathway in plants. Inter alia, there are strong overlaps in subcellular localizations and functional similarities in components of these pathways, including receptors and various downstream agents. We highlight recent research demonstrating the importance of CK/ethylene/light crosstalk in selected aspects of plant development, particularly seed germination and early seedling development. The findings clearly demonstrate the crucial integration of plant responses to phytohormones and adaptive responses to environmental cues. Finally, we tentatively identify key future challenges to refine our understanding of the molecular mechanisms mediating crosstalk between light and hormonal signals, and their integration during plant life cycles.

Illuminating light, cytokinin, and ethylene signalling crosstalk in plant development.

In plants, the multistep phosphorelay (MSP) pathway mediates a range of regulatory processes, including those activated by cytokinins. The cross talk between cytokinin response and light has been known for a long time. However, the molecular mechanism underlying the interaction between light and cytokinin signaling remains elusive. In the screen for upstream regulators we identified a  () gene whose activity is indispensable for spatiotemporally correct expression of  (), encoding the constitutively active sensor His kinase that activates MSP signaling.  is a new allele of  () that encodes the key protein in the biosynthesis of phytochromobilin, a cofactor of photoconvertible phytochromes. Our analysis confirmed the light-dependent regulation of the  expression pattern. We show that  expression is under the control of phytochrome A (phyA), functioning as a dual (both positive and negative) regulator of  expression, presumably via the phyA-regulated transcription factors (TF) PHYTOCHROME INTERACTING FACTOR3 and CIRCADIAN CLOCK ASSOCIATED1. Changes in  expression observed in /- and  mutants correlate with misregulation of MSP signaling, changed cytokinin sensitivity, and developmental aberrations that were previously shown to be associated with cytokinin and/or CKI1 action. Besides that, we demonstrate a novel role of phyA-dependent  expression in the hypocotyl elongation and hook development during skotomorphogenesis. Based on these results, we propose that the light-dependent regulation of  provides a plausible mechanistic link underlying the well-known interaction between light- and cytokinin-controlled plant development.

Light Controls Cytokinin Signaling via Transcriptional Regulation of Constitutively Active Sensor Histidine Kinase CKI1.

Seedling de-etiolation is one of the key stages of the plant life cycle, characterized by a strong rearrangement of the plant development and metabolism. The conversion of dark accumulated protochlorophyllide to chlorophyll in etioplasts of de-etiolating plants is taking place in order of ns to µs after seedlings illumination, leading to detectable increase of chlorophyll levels in order of minutes after de-etiolation initiation. The highly complex chlorophyll biosynthesis integrates number of regulatory events including light and hormonal signaling, thus making de-etiolation an ideal model to study the underlying molecular mechanisms. Here we introduce the iReenCAM, a novel tool designed for non-invasive fluorescence-based quantitation of early stages of chlorophyll biosynthesis during de-etiolation with high spatial and temporal resolution. iReenCAM comprises customized HW configuration and optimized SW packages, allowing synchronized automated measurement and analysis of the acquired fluorescence image data. Using the system and carefully optimized protocol, we show tight correlation between the iReenCAM monitored fluorescence and HPLC measured chlorophyll accumulation during first 4h of seedling de-etiolation in wild type  and mutants with disturbed chlorophyll biosynthesis. Using the approach, we demonstrate negative effect of exogenously applied cytokinins and ethylene on chlorophyll biosynthesis during early de-etiolation. Accordingly, we identify type-B response regulators, the cytokinin-responsive transcriptional activators ARR1 and ARR12 as negative regulators of early chlorophyll biosynthesis, while contrasting response was observed in case of EIN2 and EIN3, the components of canonical ethylene signaling cascade. Knowing that, we propose the use of iReenCAM as a new phenotyping tool, suitable for quantitative and robust characterization of the highly dynamic response of seedling de-etiolation.

iReenCAM: automated imaging system for kinetic analysis of photosynthetic pigment biosynthesis at high spatiotemporal resolution during early deetiolation.

Tereza Dobisova

CEITEC - Central European Institute of Technology,
National Centre for Biomolecular Research,
Faculty of Science,
National Centre for Biomolecular Research, Faculty of Science

Masaryk University

Non-invasive Assay for Chlorophyll Biosynthesis Kinetics Determination during Early Stages of Arabidopsis De-etiolation

Tereza Dobisova

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}CEITEC - Central European Institute of Technology,National Centre for Biomolecular Research,Faculty of Science,National Centre for Biomolecular Research, Faculty of Science

Masaryk University

Non-invasive Assay for Chlorophyll Biosynthesis Kinetics Determination during Early Stages of Arabidopsis De-etiolation

CEITEC - Central European Institute of Technology,
National Centre for Biomolecular Research,
Faculty of Science,
National Centre for Biomolecular Research, Faculty of Science