Diurnal Rhythms in the Red Seaweed Gracilariopsis chorda are Characterized by Unique Regulatory Networks of Carbon Metabolism

Abstract

Cellular and physiological cycles are driven by endogenous pacemakers, the diurnal and circadian rhythms. Key functions such as cell cycle progression and cellular metabolism are under rhythmic regulation, thereby maintaining physiological homeostasis. The photoreceptors phytochrome and cryptochrome, in response to light cues, are central input pathways for physiological cycles in most photosynthetic organisms. However, among Archaeplastida, red algae are the only taxa that lack phytochromes. Current knowledge about oscillatory rhythms is primarily derived from model species such as Arabidopsis thaliana and Chlamydomonas reinhardtii in the Viridiplantae, whereas little is known about these processes in other clades of the Archaeplastida, such as the red algae (Rhodophyta). We used genome-wide expression profiling of the red seaweed Gracilariopsis chorda and identified 3,098 rhythmic genes. Here, we characterized possible cryptochrome-based regulation and photosynthetic/cytosolic carbon metabolism in this species. We found a large family of cryptochrome genes in G. chorda that display rhythmic expression over the diurnal cycle and may compensate for the lack of phytochromes in this species. The input pathway gates regulatory networks of carbon metabolism which results in a compact and efficient energy metabolism during daylight hours. The system in G. chorda is distinct from energy metabolism in most plants, which activates in the dark. The green lineage, in particular, land plants, balance water loss and CO2 capture in terrestrial environments. In contrast, red seaweeds maintain a reduced set of photoreceptors and a compact cytosolic carbon metabolism to thrive in the harsh abiotic conditions typical of intertidal zones.

Keywords: Gracilariopsis chorda; cryptochrome; cytosolic carbon metabolism; horizontal gene transfers; rhythmic gene.

Fig. 1.

Rhythmic gene expression patterns in the red seaweed Gracilariopsis chorda. The dark areas in the plots are nighttime hours. The major peak patterns are labeled as pattern-a to pattern-l, grouped into morning-phased (a-b-c), dusk-phased (d-e-f), evening-phased (g-h-i), and dawn-phased (j-k-l) genes. The two fluctuating rhythmic gene expression patterns are considered minor peak patterns (m and n). The relative gene expression patterns are marked with the “+” and “–” symbols, indicating “increase” and “decrease” in gene expression when compared to the previous time point, respectively (see Materials and Methods).

Fig. 2.

Gene expression of cryptochromes (CRYs) in G. chorda. The horizontal axis indicates each sampling point (hours), and the vertical axis indicates the z-score of the target gene expression. The red- and black-colored gene names indicate rhythmic and nonrhythmic genes, respectively (asterisk: significant correlation in rhythmic pattern). The in-frame GFP fusion of the Gc-pCRY1 (PXF43553.1) gene was not available (N/A) in this study (see Materials and Methods). The tree, resulting from a ML phylogenetic analysis of CRYs, is shown in a simplified format (supplementary fig. S1, Supplementary Material online). The images on the right side are results of subcellular localization of CRYs in G. chorda when expressed in the heterologous system of A. thaliana. Bioinformatic predictions of subcellular localization using CRYs in G. chorda are provided (See Methods).

Fig. 3.

Model for the DN cycle of gene expression involving metabolism of cytosolic C₃ and C₄ organic acids in G. chorda (asterisk: significant correlation in rhythmic pattern).

Fig. 4.

Comparison of diurnal gene regulation and cellular metabolism in plants and red algae. a) Comparison of major DN regulatory systems in terrestrial and aquatic habitats. b) Comparison of carbon metabolism in plants and G. chorda. The simplified model of chloroplast carbon metabolism in C₃, C₄, and CAM plants is based on previous studies (Drincovich et al. 2001; Lai et al. 2002; Wheeler et al. 2005; Tronconi et al. 2008, 2018; Aubry et al. 2011; Maier et al. 2011; Zones et al. 2015; Rao and Dixon 2016; Shen et al. 2017; Chen et al. 2019; Khoshravesh et al. 2020; Tay et al. 2021; Winter and Smith 2022), whereas the model of cytosolic carbon metabolism in G. chorda is based on gene expression analysis (this study). Abbreviations: PHY, phytochrome; CRY, cryptochrome; CA, carbonic anhydrase; PEP, phosphoenolpyruvate; OAA, oxaloacetate; PEPC, PEP carboxylase; PEPCK, PEP carboxykinase; MDH, malate dehydrogenase; PPDK, pyruvate orthophosphate dikinase; NADP-ME, NADP-malic enzyme; NAD-ME, NAD-malic enzyme; Calvin C₃ cycle, Calvin–Benson–Bassham cycle; TCA cycle, tricarboxylic acid cycle.