The effects of illumination and trophic strategy on gene expression in Chlamydomonas reinhardtii

Highlights

• Impact of illumination and trophic strategy on C.reinhardtii growth & gene expression

• Identification of novel housekeeping genes for reliable comparisons across conditions

• Analysis of the response of central carbon pathways under a variety of conditions

• Expression profiles of commonly used promoters for recombinant protein synthesis

Abstract

Microalgae are of substantial biotechnological interest due their polyphyletic nature which grants them access to a wide array of high-value metabolites. The inherent genetic diversity of microalgae combined with their metabolic plasticity when grown using different trophic and illumination strategies necessitate the establishment of a reference knowledge base. In the present study we present a detailed characterisation of the combined effects of wavelength selection and trophic strategy on the growth kinetics and gene expression profile of the model microalgae Chlamydomonas reinhardtii grown under moderate to high light intensity (400 μmol_ph m⁻²·s⁻¹). The aim is twofold: (a) to establish a list of reliable housekeeping genes valid for quantitative comparisons across several combinations of different wavelengths and trophic strategies and (b) to enable the investigation of the response of central carbon metabolic pathways under these process conditions. White, blue and red light emitting diodes (LEDs) were used to grow pH controlled photo- and mixo-trophic cultures over a period of 136 h in batch mode. A panel of 10 candidate genes, along with biomass growth rate and pigment content were dynamically monitored across all conditions. Statistical analysis identified genes (acetyl-CoA carboxylase subunit α and photosystem I reaction centre subunit II) with less variability observed in their expression levels across the entirety of conditions evaluated compared to housekeeping genes often referred to in literature (receptor of activated protein kinase C and ribosomal protein (large subunit) 19). Further analysis of gene expression profiles revealed substantial differences in response to changes in both wavelength selection (upregulation of ribulose bisphosphate carboxylase small subunit under red phototrophic growth) and trophic strategy (upregulation of malate synthase from phototrophic to mixotrophic conditions). The systematic approach used to establish reliable reference genes presented herein enables robust comparisons of cellular responses across different conditions to better understand algal metabolism and improve process performance.

Introduction

Freshwater microalgae, like Chlamydomonas reinhardtii, have evolved an extensively versatile metabolism that increases their chances of survival in diverse environmental conditions in the presence (mixotrophy) or absence (autotrophy) of a readily available source of nutrients [1]. Due to this metabolic plasticity, and despite significant research efforts, our knowledge of algal metabolism remains nascent, relative to other crops, and our control over biomass composition remains rudimentary [2]. One of the main reasons that fully understanding algal metabolism and how it responds to varying environmental and bioprocessing conditions is far from trivial is the uniqueness of incident light as a critical process parameter. The effects of light on microalgal physiology are mediated by a variety of specialised, light harvesting and light sensing molecules. Chlorophylls, carotenoids, rhodopsins, phototropins and cryptochromes each have a unique absorption spectrum and are involved in different cellular processes like photosynthesis, phototaxis [3] or cell cycle control [4,5].

In addition, light in and of itself is complex in nature as it can vary in terms of intensity, photoperiod and spectral composition. Light intensity has a well documented and characterised positive effect on biomass growth both under phototrophic and mixotrophic conditions [6]. However, excessively high light intensities have been shown to trigger stress responses, like carotenoid production, in a number of algal species [7,8] or even completely inhibit growth (photoinhibition) [9]. Phototrophic studies on biomass growth of C. reinhardtii have shown that below saturating light levels (25–100 μmol_ph m⁻² s⁻¹), illumination with red and red-blue light can achieve a higher biomass yield on photons compared to white light [10]. However, at or above saturating light intensities (>1500 μmol_ph m⁻² s⁻¹) the yield of biomass on photons was found to be inversely proportional to the specific photon absorption rate. Consequently, illumination at wavelengths considered to be sub-optimal due to their relatively low specific absorption rate (e.g. illumination with yellow and warm white light) has been found to yield higher biomass yields than red or blue light at high light intensities [11].

The advent of light emitting diodes (LEDs) has enhanced our ability to accurately control several properties of incident light, beyond intensity, and investigate their effects on biomass growth and biochemical composition [12]. This has enabled in depth investigations of the effects of the frequency and duration of light/dark cycles, spanning timescales from milliseconds to hours (diel-cycle) [[13], [14], [15]]. Studies on the impact of light quality on biomass composition, primarily focused on pigment and/or lipid content in specialised producer species like Haematoccocus. pluvialis and Phaeodactylum tricornutum [16], have shown a variety of possible responses across species. Therefore, there is a need to understand the diverse and complex effects of the spectral composition of incident light on microalgal physiology and metabolism.

Associating gene expression profiles with changes in microalgal physiology under narrow band illumination can help unravel the specific effects of discrete regions of the photosynthetically active radiation (PAR) region of the visible light spectrum (400–750 nm). However, relative quantification of reverse transcription-quantitative real time polymerase chain reaction (RT-qPCR) measurements requires the existence of a set of reference genes with stable expression profiles across a variety of culture conditions. A number of studies have previously proposed candidate reference genes for C. reinhardtii with no clear consensus available in the literature due to the limited number of experimental conditions examined in each study [[17], [18], [19]]. A comprehensive study of the stability of four candidate reference genes under nitrogen deprivation both in photoautotrophic and mixotrophic conditions was recently published [20], however is limited to only white light illumination. An extensive transcriptomic analysis of C. reinhardtii gene expression under a variety of nutrient depletion conditions has been recently published [21].

Given the biotechnological potential of triggering desired responses in a microalgal culture by utilising a tailored spectral composition, this study investigates the effects of wavelength selection and trophic strategy on gene expression in C. reinhardtii. The genetic responses elicited by monochromatic LEDs with narrow peak intensities at the two extremes of the PAR range (Blue, 440-480 nm and Red, 640-670 nm) under phototrophic and mixotrophic conditions were compared against white light control cultures. A set of 10 candidate reference genes was compiled from literature and their stability was assessed across all 6 experimental conditions. The novel reference genes identified herein, coupled with dynamic biomass and pigment content measurements, were used to evaluate the effect of wavelength and trophic strategy induced changes at key metabolic nodes. Finally, the expression levels of promoter genes and/or 5′−/3′-untranslated regions (5′−/3′-UTR) previously targeted for the expression of recombinant proteins in C. reinhardtii were characterised to discern favourable illumination conditions and trophic strategies.