Evolutionary-conserved RLF, a cytochrome b5-like heme-binding protein, regulates organ development in Marchantia polymorpha

Introduction

Heme is a porphyrin complex with a centrally coordinated iron atom. In plants, heme shares a metabolic pathway with Chl biosynthesis up to the production of protoporphyrin IX, after which ferrochelatase coordinates Fe²⁺ to protoporphyrin IX (Roper & Smith, 1997; Hederstedt, 2012). In the Protein Data Bank (https://www.rcsb.org), which contains 222 415 proteins, 4272 protein chains have been identified as heme-binding proteins (HBPs) from entries containing heme types b and c (Li et al., 2011). The binding of these HBPs to heme is maintained by the coordination of specific amino acids, such as histidine, within the apoprotein to the iron atoms of heme. In general, in various organisms, HBPs are essential for diverse biological processes such as steroid biosynthesis, aerobic respiration, and programmed cell death because of their role in electron transfer, substrate oxidation, and metal ion storage (Reedy & Gibney, 2004; Layer et al., 2010). In plants, HBPs, such as cytochrome c, which is involved in electron transfer during photosynthesis, and SOUL4, which is implicated in lipid metabolism within chloroplast fat droplets (plastoglobule), have demonstrated diverse physiological effects (Kerfeld & Krogmann, 1998; Shanmugabalaji et al., 2020). Recently, proteomic analyses in Arabidopsis thaliana have identified a variety of HBPs, such as basic/helix–loop–helix type nuclear transcription factors and intracellular signaling factors involved in GTPase activation (Shimizu et al., 2020). These studies have revealed that HBPs function as intracellular signals regulating diverse physiological functions. However, it remains unclear how they are involved in plant organ development.

One group of HBPs in plants is a family of proteins with a cytochrome b₅-like heme-binding domain (Cytb5-HBD). In plants, the metabolic pathways mediated by most Cytb5-HBD proteins are diverse, including fatty acid desaturation, lignin biosynthesis, and nitrate reduction (Nagano et al., 2012; Gou et al., 2019). The genome of A. thaliana has 15 proteins with a Cytb5-HBD, including five members of the cytochrome b₅ family (At1g26340 (CB5A), At2g32720 (CB5B), At2g46650 (CB5C), At5g48810 (CB5D), and At5g53560 (CB5E)) (Maggio et al., 2007), one cytochrome b₅-like protein (At1g60660 (CB5LP)), four membrane-associated progesterone binding proteins (At2g24940 (MAPR2), At3g48890 (MAPR3), At4g14965 (MAPR4), and At5g52240 (MAPR5)) (Yang et al., 2005), two Δ-8 sphingolipid desaturases (At3g61580 (SLD1) and At2g46210 (SLD2)) (Sperling et al., 1998), two nitrate reductases (At1g77760 (NR1) and At1g37130 (NR2)) (Cheng et al., 1988; Wilkinson & Crawford, 1991) and At5g09680/REDUCED LATERAL ROOT FORMATION (RLF) (Ikeyama et al., 2010). In A. thaliana, cytochrome b₅ isoforms, SLD1, SLD2, NIA1, NIA2, and proteins of the MAPR family generally do not play a significant role in organ development. CB5D is involved in lignin biosynthesis, and the cb5d mutant shows a reduction in S-lignin content. However, no abnormalities in organ development have been observed in cb5d mutants (Gou et al., 2019). SLD1 and SLD2 are involved in Δ-8 sphingolipid desaturation. Their loss-of-function mutants show significantly reduced root growth under sodium dodecyl sulfate (SDS) stress, but no growth defects are observed under normal conditions (Chen et al., 2012). Similarly, NIA1 and NIA2 are involved in the regulation of nitrate metabolism and nitric oxide (NO) production; however, the null-allelic mutant nia1-3 nia2-1 is lethal, and no studies have reported effects on organ development. Additionally, single mutants of either NIA1 or NIA2 do not exhibit significant effects on plant growth or development (Wilkinson & Crawford, 1993; Tang et al., 2022). Membrane-associated progesterone binding proteins, particularly MAPR5, contribute to lipid and steroid metabolism and regulate autophagy under starvation conditions, potentially influencing energy homeostasis. However, no severe developmental abnormalities have been reported in MAPR mutants or overexpression lines (Kimura et al., 2012; Ryu et al., 2017; Wu et al., 2021).

Whereas most of the Cytb5-HBD proteins in A. thaliana regulate a variety of metabolic reactions, At5g09680/RLF is a unique Cytb5-HBD protein that regulates organ development, including lateral root (LR) formation (Ikeyama et al., 2010). In general, LR formation is essential for root system architecture in most vascular plants and is regulated by auxin. In A. thaliana, LR formation is regulated by several auxin signaling modules including the SOLITARY-ROOT/IAA14, an auxin/indole-3-acetic acid (Aux/IAA) protein – AUXIN RESPONSE FACTOR (ARF) 7 and ARF19 module (Fukaki et al., 2005; Okushima et al., 2007). The rlf loss-of-function mutants of RLF show a marked reduction in the number of LRs while their ARF7/19-mediated auxin signaling is not affected, suggesting that RLF regulates LR formation independently of auxin response (Ikeyama et al., 2010). In addition, the rlf mutations cause aberrant organ development, including reduced primary root growth and decreased leaf size (Ikeyama et al., 2010). While the study of RLF in A. thaliana sheds light on the significance of Cytb5-HBD proteins for plant growth and development, a crucial gap remains in our understanding of how Cytb5-HBD proteins orchestrate growth and development of plant organs. First, it is not known whether RLF binds to heme and works as HBP in planta. Second, there is limited understanding of whether Cytb5-HBD proteins share a common role in regulating organ development in land plants. To address these issues, we extend our investigation of RLF to the model bryophyte species, Marchantia polymorpha (Ishizaki et al., 2016; Fernandez-Pozo et al., 2022) in addition to A. thaliana. M. polymorpha forms distinct organs specialized for vegetative or reproductive functions, such as the thallus, which serves as the main photosynthetic structure, gemma cups that produce gemmae, and reproductive organs like antheridiophores and archegoniophores. Its genetic tractability and evolutionary position as a nonvascular plant make it an ideal model for exploring these questions.

In this study, we demonstrate that the heme-binding ability of RLF is necessary for its biological function. In addition, we unveil the role of MpRLF, an ortholog of RLF in M. polymorpha, in orchestrating proper vegetative and reproductive development. Our findings highlight the crucial role of the MpRLF gene in plant organ development. Notably, the similarities between MpRLF and A. thaliana RLF suggest the existence of a conserved mechanism, governed by RLF that intricately regulates organ development across diverse plant lineages.