The role of indole-3-acetic acid and characterization of PIN transporters in complex streptophyte alga Chara braunii

Abstract

Introduction

During the transition from water to land, plants underwent a series of developmental innovations, leading to the establishment of a complex body (Harrison, 2017; Donoghue et al., 2021; Bowman, 2022). A key regulator of land plant development is the phytohormone auxin, which acts through local biosynthesis and directional transport, resulting in the formation of concentration gradients (Friml, 2022). The directional flow of auxin, facilitated by the PIN family of auxin efflux carriers, is critical for plant morphogenesis and adaptive growth responses to environmental cues (Gao et al., 2008; Luschnig & Friml, 2024). Recent research has provided significant insights into the mechanisms of auxin action (Kuhn et al., 2024), including the structural elucidation of three PIN auxin efflux carriers (Su et al., 2022; Ung et al., 2022; Yang et al., 2022). However, the question of how and when auxin became a pivotal driver of morphological changes in land plants remains largely unanswered. This question cannot be fully addressed by studying only land plants but requires a comprehensive investigation of their algal relatives (Skokan et al., 2019). Land plants (embryophytes) and streptophyte green algae, from which they emerged, together form the group Streptophyta (Becker & Marin, 2009). Streptophyte algae comprise six clades, exhibiting significant morphological diversity within these clades (Buschmann, 2020; Bierenbroodspot et al., 2024). Among these six clades, Chara spp. and Nitella spp., members of the Charophyceae family, possess the highest degree of complexity with respect to their body plan. Due to its large internodal cells, transparent gravitropic rhizoids, and rapid cytoplasmic streaming, Chara has been a model organism for decades, facilitating the study of fundamental cell biological processes (Kurtović et al., 2024).

Although genome sequencing revealed that Chara braunii lacks the auxin biosynthetic pathway involving the TAA and YUCCA genes, which converts tryptophan to an auxin, indole-3-acetic acid (IAA) (Nishiyama et al., 2018), earlier and recent studies are consistently confirming the presence of IAA in the biomass of various species of Chara and Nitella (Jahnke & Libbert, 1964; Sztein et al., 2000; Hackenberg & Pandey, 2014; Beilby et al., 2015; Schmidt et al., 2024), suggesting the presence of an alternative biosynthetic pathway. Besides IAA, other phytohormones have been identified in the biomass of C. braunii, including cytokinin N⁶-(∆²-isopentenyl)-adenine, ethylene, and jasmonic acid (Schmidt et al., 2024). However, much less is known about their effects or role in Chara, and therefore, they are not the focus of this study. On the other hand, auxin transport has been implicated in Chara in several studies. Dibb-Fuller & Morris (1992) demonstrated auxin influx and efflux in Chara cells, using radio-labeled IAA. In addition, they showed that efflux was unaffected by the PIN-dependent auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) (Abas et al., 2021). By contrast, Boot et al. (2012) found NPA-sensitive polar auxin transport in Chara corallina. Later, PIN-like proteins were detected by immunolocalization using heterologous anti-AtPIN2 antibodies in the antheridial filaments of Chara vulgaris (Żabka et al., 2016). Finally, genome sequencing confirmed that Chara indeed possesses six homologs of PIN auxin efflux carriers (Nishiyama et al., 2018), the highest number among all streptophyte algae (Hori et al., 2014; Cheng et al., 2019; Liang et al., 2020; Vosolsobě et al., 2020), at least five ATP-binding cassette B (ABCB) homologs, but no AUX/LAX influx carriers (Nishiyama et al., 2018). Furthermore, exogenously applied IAA has been shown to promote rhizoid growth in decapitated Chara thalli (Klämbt et al., 1992), affect ion transport (S. Zhang et al., 2016), induce transient depolymerization of microtubules (Jin et al., 2008), and accelerate the process of differentiation of antheridial filament cells (Godlewski, 1980). However, none of these studies further explored potential modes of IAA perception by Chara. The genome of Chara encodes certain elements of the auxin signaling pathway, in particular, an AUXIN RESPONSE FACTOR (ARF) and two Aux/IAA sequences; however, these components are not functionally equivalent to those in the canonical auxin signaling pathway of land plnts (Mutte et al., 2018). The lack of a canonical signaling pathway can be further supported by the absence of the canonical TIR1 receptor (Mutte et al., 2018; Nishiyama et al., 2018; Bowman et al., 2021). On the other hand, Chara encodes a single homolog (Carrillo-Carrasco et al., 2023) of a cell surface auxin receptor Auxin Binding Protein1 (ABP1) (Friml et al., 2022), suggesting the presence of a noncanonical signaling pathway (Kuhn et al., 2024).

Given the morphological complexity of Chara, the number of PIN homologs in the Chara genome, and the ambiguous results in literature, this study focuses on describing auxin responses and functional evaluation of Chara PIN homologs. We show that IAA treatments promoted side branching of regenerated thalli upon decapitation. To test the possible involvement of carrier-mediated IAA transport in the Chara growth responses, we cloned two of six PIN homologs and showed that in Nicotiana tabaccum, BY-2 cells CbPINa, but not CbPINc, influences the accumulation of radioactively labeled auxin. Immunolocalization using specific antibodies showed that both CbPINa and CbPINc are associated with the plasma membrane (PM) in vegetative and generative cells of Chara. However, their expressions in Arabidopsis thaliana, and bryophyte Marchantia polymorpha, did not rescue the mutant phenotypes, despite their association with the PM and polar localization of CbPINa in gametangiophore stalks of Marchantia. To analyze rapid auxin action in Chara, we integrated phosphoproteomic analysis with cytoplasmic streaming assay, revealing IAA-specific changes in the phosphoproteome. We identified MAP4K homolog as a dominant target of IAA response and also the activation of a RAF-like kinase homolog. This observation provides evidence of conserved rapid auxin signaling in streptophytes, as reported by Kuhn et al. (2024). Additionally, our study demonstrates that IAA promotes fast cytoplasmic streaming in branchlet internodal cells, suggesting a link between auxin-triggered phosphorylation events and cytoplasmic streaming dynamics.