Heterotrimeric G-proteins in green algae

Abstract

Heterotrimeric G-proteins (G-proteins, hereafter) are important signaling components in all eukaryotes. The absence of these proteins in the sequenced genomes of Chlorophycean green algae has raised questions about their evolutionary origin and prevalence in the plant lineage. The existence of G-proteins has often been correlated with the acquisition of embryophytic life-cycle and/or terrestrial habitats of plants which occurred around 450 million years ago. Our discovery of functional G-proteins in Chara braunii, a representative of the Charophycean green algae, establishes the existence of this conserved signaling pathway in the most basal plants and dates it even further back to 1–1.5 billion years ago. We have now identified the sequence homologs of G-proteins in additional algal families and propose that green algae represent a model system for one of the most basal forms of G-protein signaling known to exist to date. Given the possible differences that exist between plant and metazoan G-protein signaling mechanisms, such basal organisms will serve as important resources to trace the evolutionary origin of proposed mechanistic differences between the systems as well as their plant-specific functions.

Keywords: :

• Heterotrimeric G-proteins
• Green algae
• Evolution
• Chara braunii
• Charophytes

Introduction

Heterotrimeric G-proteins are key signaling molecules involved in regulation of a wide spectrum of signal transduction pathways established in different eukaryotic organisms.¹ The basic molecular mechanism entails a switch-like signaling process with an inactive state comprised of a trimer consisting of the three subunits Gα, Gβ and Gγ, and an active state characterized by the dissociation of Gα subunit from the Gβ/Gγ dimer. The interchange between both these states is dependent on the exchange of GDP against GTP for activation and the subsequent GTP hydrolysis for inactivation on the Gα subunit.^2,3 The G-protein signaling mechanism in plants is not yet understood in the exquisite details that are available for metazoan systems where activation is exclusively mediated by the guanine nucleotide exchange (GEF) activity of a cognate G-protein coupled receptor (GPCR). In metazoans, it has been established that ligand binding to a GPCR leads to the conformational change in Gα which allows for GDP to GTP exchange. Canonical GPCRs do not seem to exist in plants and Arabidopsis Gα has been proposed to be self-activated.⁴ The origin of such differences and its prevalence across species remains to be ascertained. Additionally, almost every heterotrophic species exhibits a lineage-specific expansion of G-proteins and their regulators, and multiple genes exist for each of the G-protein subunits. In contrast, plants have a relatively simple repertoire of G-protein genes which ranges from multiple copies in plants that have gone through recent genome duplication events, e.g., soybean (4 Gα, 4 Gβ and 10 Gγ)⁵ to the bryophyte Physcomitrella patens (moss) that contains Gβ and Gγ subunits, but no Gα.⁴ The most commonly-explored plants such as Arabidopsis and rice encode for one Gα, one Gβ and a few Gγ proteins.⁶ Despite their limited quantities, plant G-proteins have been shown to be involved in regulation of multiple growth and developmental pathways, especially in modulating plant hormone responses.^7-9

We have recently discovered that basal, aquatic plants such as the green alga Chara braunii possess each of the G-protein subunits as well as its regulatory protein, RGS (Regulator of G-protein Signaling). The basic biochemical properties of Chara G-protein genes are similar to what has been reported for land plants and the Gα and RGS proteins from Chara and Arabidopsis exhibit cross-species biochemical functionality.¹⁰ We now report that G-proteins exist in additional charophyte species besides Chara braunii and their absence from the sequenced genomes of chlorophyte algae possibly reflects their loss in one specific branch of the algal lineage. These findings reaffirm that the existence of G-protein signaling in plants is not correlated with the acquisition of terrestrial habitat or predominance of embryophytic life-cycle.

Material and Methods

In silico-EST data analysis

To optimize the search for potential candidate genes and to account for different codon usage bias in different species, we used amino acid sequence query with the TBLASTN¹¹ algorithm. This allowed for screening of all six possible reading frames of target nucleotide sequences. Arabidopsis GPA1 (Accession: AEC07820), AGB1 (accession: AEE86382), AGG1 (accession: AEE80480), AGG2 (accession: AEE76694) and AGG3 (accession: Q6AWT8) were used as query for TBLASTN search using GenBank+EMBL+DDBJ database EST sequences and the nucleotide collection hosted by NCBI.

Phytohormone detection

Chara braunii were cultivated in distilled water on a sand/soil/peat mixture at room temperature and 14/10 h light/dark cycle as described previously.¹² Aquatic tissue was harvested and lyophilized for 16 h and subsequently ground in liquid N₂. Fifty mg algal dry mass was used for acidic hormone quantification. Salicylic acid (SA), abscisic acid (ABA), jasmonic acid (JA), indole 3 acetic acid (IAA), jasmonate-isoleucine conjugate (JA-Ile), indole-3-acetyl-aspartate (IAA-Asp) and JA precursor cis-(+)-12-oxo-phytodienoic acid (cis-OPDA) were assayed using an LC-MS/MS method. Hormone extraction was performed in the presence of a mixture of deuterium labeled standards (D5SA, D6ABA, D2JA, D5IAA) at 2.5 μM each which was spiked at the beginning of the extraction. Samples were homogenized twice in 900 µL MeOH/ACN (1:1 v/v) and one time in 200 μL of 30% methanol followed by analysis using Shimadzu LC system interfaced with an AB Sciex 4000 QTRAP mass spectrometer and TurboIonSpray (TIS) electrospray ion source. For LC separation, a monolithic C18 column (Onyx, 4.6 mm x 100 mm, Phenomenex) with a guard cartridge was used flowing at 1 ml.min^–1. The gradient was from 60% solvent A (0.1% [v/v] acetic acid in Milli-Q water) to 100% solvent B (90% acetonitrile [v/v] with 0.1% acetic acid [v/v]). Hormones were detected in three technical replicates using MRM transitions and quantified with standard samples.

Results and Discussion

The absence of G-protein genes in chlorophytes and their presence in Chara braunii prompted us to uncover their distribution in additional algal genomes. Our analysis shows that genes encoding G-protein subunits exist in multiple charophyte algae. In general, green algae can be divided in two major divisions: Chlorophyta and Charophyta, which represent two polyphyletic sister branches.^13-15 Charophyta, together with land plants form the monophyletic branch of Streptophyta and comprise six different taxa (Fig. 0.1): Mesostigmatophyceae, Chlorokybophyceae, Klebsormidiophyceae, Zygnematophyceae, Coleochaetophyceae and Charophyceae.^16-18 Interestingly, both monotypic taxa, Mesostigmatophyceae and Chlorokybophyceae, with Mesostigma viride and Chlorokybus atmophyticus as their only known representatives, do not show the presence of candidate genes encoding G-protein subunits in our ESTs data analysis. Mesostigma is unicellular and its phylogenetic rank under the green algae is still controversial. It is also unclear if it belongs to the plant lineage or represents a unique sister to all green algae.^19-22 Conversely, Chlorokybus belongs to the charophyte green algae and forms conglomerates of several cells possessing a non-motile vegetative phase, an evolutionary innovation which seems to be absent in Mesostigma.¹⁷

The earliest charophyte species possessing ESTs with a high similarity to the Arabidopsis thaliana G-proteins identified in our studies was Klebsormidium flaccidum (Klebsormidiophyceae), which contains orthologous sequences to AtGPA1 and AtAGB1. In addition, Coleochaete scutata, Chaetosphaeridium globosum (Coleochaetophyceae) and Nitella hyalina (Charophyceae) also exhibit one orthologous gene for the Gα subunit (Fig. 1). No promising candidates for G-protein genes could be identified in Zygnematophyceae so far. Whole genome sequencing data, which are not available for any of the charophyte green alga to date, will certainly help complete the picture of heterotrimeric G-proteins in green algae and therefore also in land plant evolution. This could be particularly true for Zygnematophyceae where G-protein genes seem to be absent but are expected to exist due to their phylogeny as sister to Coleochaetophyceae and/or Charophyceae. Zygnematophyceae are considered to be more advanced than Klebsormidiophyceae in the charophyte lineage¹⁶ and exhibit enormous diversity, with more than 4000 species and morphologies which range from unicellular to filamentous.^17,20

Figure 1. Schematic overview of the phylogeny of green algae and evolutionary innovations (modified from).^16,20 Potential genes encoding Gα, Gβ and Gγ subunits of the heterotrimeric G protein complex based on EST data analysis are labeled with orange, blue or green and selected morphological or molecular characters are shown. G-protein genes can be found in the monophyletic branch of Streptophyta such as in several charophytes (Klebsormidiophyceae, Coleochaetophyceae and Charophyceae) and embryophytes, but not in chlorophytes.

No representatives of the chlorophyte algae showed evidence for the presence of heterotrimeric G protein genes so far, which is especially remarkable because the genomes of several chlorophytes (Volvox carteri, Chlamydomonas reinhardtii, Cocomyxa subellipsoidea C-169, Micromonas pusilla CCMP1545, Micromonas pusilla RCC299 and Ostreococcus lucimarinus) are completely sequenced.²³ In fact, no G-protein homologs were identified among the glaucophytes or rodophytes (algal lineages more primitive than chlorophytes), suggesting Klebsormidiophyceae are the most primitive green plants that contain G-proteins. In general, due to the limited number of fully sequenced genomes, these findings should be considered with care. However, it is worth mentioning that the limiting factor for the identification of G-protein genes is the completeness of transcriptome datasets rather than the sequence homology of the conserved protein domains in the search. Sequence-based homology searches have successfully identified G-protein genes in a variety of plants.²⁴ Nonetheless, it can be stated without doubt that G-protein signaling of photo-autotrophic organisms evolved early in the plant lineage, long before embryophytes conquered terrestrial habitats. Furthermore, the origin of heterotrimeric G-protein signaling can be dated before major innovations like the appearance of phragmoplasts, but after or concurrent with the appearance of multicellular, filamentous morphology. This is based on the fact that Klebsormidium flaccidum, which contains G-protein genes, is a multicellular, non-branching green alga whose ancestor separated before the innovation of phragmoplasts, plasmodesmata, branching, apical cell growth, desiccation-resistant zygospores or cellulose-synthesizing rosettes.^16,17

Besides charophyte green algae, we identified homologs of Arabidopsis Gα and Gβ proteins in the sequenced genomes of two other basal photosynthetic organisms, the diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum.^25,26 Diatoms represent mostly unicellular algae and are commonly described as part of phytoplankton. Diatoms are not monophyletic with green algae and belong instead to the “supergroup” of Chromalveolates which include a plethora of different algae possessing plastids derived from a red algal endosymbiont.¹⁶ A large number of green algal genes can be found in Chromalveolates genomes²⁷ and although its phylogeny is not yet completely understood, it is supposed that these genes were incorporated in the Chromalveolates genomes by endosymbiosis of green algal ancestors.²⁸ These data further corroborate that G-protein genes have been lost only in the specific green algae lineages.

Since we are only beginning to uncover the existence of heterotrimeric G-proteins in basal plant species, it is expected that huge knowledge gaps exist concerning their evolution and function in these lineages. One of the major roles of higher plant G-proteins comprises the regulation of growth, development and stress responses by modulation of plant hormone signal transduction pathways. As a first step to evaluate the possible roles of G-proteins in green algae, we elucidated the presence of plant hormones in Chara braunii. Our data show the unambiguous presence of SA, ABA and IAA, whereas JA as well as JA derivatives and precursors were undetectable (Table 1). Since charophytes possess multi-cellular morphology including differentiation of certain cell types, cell-to-cell communication and signaling might be necessary and G-proteins could potentially be involved in regulation of phytohormone-regulated pathways in green algae, similar to higher plants. Recent reports demonstrate the role of plant hormone-regulated processes in green algae, for example polar auxin transport in Chara corallina²⁹ and gibberellic acid and brassinosteroid responses in Klebsormidium flaccidum.³⁰ Heterotrimeric G-proteins might also be involved in some of these processes since charophytes and land plants share several physiological characteristics which are absent in chlorophytes such as globular cellulose synthesizing terminal complexes,³¹ the GapA/B gene duplication³² and specific enzymes like glycolate oxidase³³ or class I aldolases.³⁴ Further analysis of additional genome sequences and characterization of G-proteins in regulating key signaling pathways in algae will shed light on their evolution, expanse, function and possible divergence from established regulatory mechanisms.

Table 1. Quantification of acidic phytohormones in Chara braunii using LC-MS/MS

Phytohormone	Amount [ng analyte /g dry weight tissue]	S.D.
SA	2440.9	309.3
ABA	3.2	0.4
JA	ND	–
JA-ILE	ND	–
OPDA	ND	–
IAA	51.4	5.9
IAA-ASP	ND	–

Amounts of salicylic acid (SA), abscisic acid (ABA), jasmonic acid (JA), isoleucine conjugated JA (JA-ILE), 12-oxo phytodienoic acid (OPDA) a JA precursor, indole-3-acteic acid (IAA), aspartate conjugated IAA (IAA-ASP) and corresponding standard deviation (S.D.) of three technical replicates are indicated (ND = not detectable or outside the limits of detection).

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

This work is supported by National Science Foundation grant (MCB-1157944) to SP. We are thankful to Dr. Ursula Goodenough, Biology Department, Washington University, St. Louis and Dr. Jae-Hyeok Lee, Department of Botany, University of British Columbia for insightful discussions.