2024 Volume 36 Issue 1 Pages 21-26
Gametophore shoot formation from protonemal tissues in Physcomitrium patens is used as a model to investigate the evolution of shoot architecture. The establishment of cellular polarity facilitates this morphological innovation by regulating oriented cell division. Despite the discovery of molecular mechanisms relevant to the cellular polarization, it remains largely unclear how the cellular physiological conditions are adjusted to accommodate the developmental and growth regimes. Arginine metabolism has been identified as a metabolic signature that is altered during gametophore shoot formation. The functional characterization of arginine metabolism should be investigated to determine whether and how arginine metabolism contributes to gametophore shoot formation. Here, I summarize the current knowledge on the molecular mechanism regulating gametophore shoot formation in P. patens. I describe also how arginine metabolism is modified during gametophore shoot formation. Based on this information, I finally discuss the future directions to explore the role of arginine metabolism in gametophore shoot formation by delineating the dynamics of the metabolic network using an isotope labeling experiment.
The evolution of land plants accompanied the innovative transition of their body plan from a two-dimensional to three-dimensional architecture. Oriented cell division facilitates this architectural transition and helps generate diverse plant forms (Donoghue et al. 2021, Harrison and Morris 2017). Cellular polarity plays a role in determining the angle of the division plane that forms, representing a key initial cue underlying these developmental processes (Bowman et al. 2002, Tomescu et al. 2014). Many molecular machines that explain the establishment of cellular polarity have been identified (Harrison 2017, Ramalho et al. 2022). However, how cellular physiology is modified during polarity establishment and subsequent tissue formation remains largely unexplored. Given that metabolic demand seems to have changed with the developmental and growth regimes, the current challenge is to unravel the relevant physiology.
The primary metabolic network is generally sustained under environmental perturbation to maintain cellular homeostasis. Feedback regulates metabolic activity to ensure network robustness (Okamura and Hirai 2017, Watanabe et al. 2021). This tight maintenance is important because disruption of the primary metabolic network has deleterious effects on plant growth and can be lethal (Engel et al. 2007, Gipson et al. 2017). Despite the robustness of the primary metabolic network, it can adjust to the developmental and growth regimes to modify the network dynamics (Miyazawa and Aulehla 2018, Teleman 2016). However, it is unclear how the conflict between robustness and flexibility is resolved in the primary metabolic network without one interfering with the other.
This review focuses on the establishment of cellular polarity during gametophore shoot formation in the moss Physcomitrium patens. This process might be a good experimental model for investigating how the robust primary metabolic network is modified because arginine metabolism is significantly altered to facilitate gametophore shoot formation (Kawade et al. 2020).
Leafy gametophore shoots arise from filamentous protonemal tissues in P. patens. This developmental change resembles the architectural transition from two- to three-dimensional tissues during the evolution of land plants. While protonemal tissues exhibit cell division perpendicular to the growth direction, gametophore shoots generate different cell types by oriented cell division with an oblique plane to sculpt the tissue architecture. Successive rounds of oriented cell division follow a stereotypic pattern, which helps form leafy gametophore shoots in a reproducible manner (Kosetsu et al. 2017, Harrison et al. 2009).
Molecular genetic studies have shown that the calpain protease family protein DEFECTIVE KERNEL (DEK) 1 and ubiquitin-associated protein NO GAMETOPHORES (NOG) 1 regulate oriented cell division in P. patens gametophores (Demko et al. 2014, Moody et al. 2018, Olsen et al. 2015, Perroud et al. 2014). The CLAVATA signaling pathway, which regulates stem cell functions in Arabidopsis thaliana (Brand et al. 2000, Clark et al. 1997, Reddy and Meyerowitz 2005), is also involved in regulating oriented cell division in gametophores (Whitewoods et al. 2018). NOG2 encodes the enzyme shikimate O-hydroxycinnamoyltransferase, which is located downstream of the NOG1 pathway and upstream of the CLAVATA signaling pathway (Moody et al. 2021). An orthologous A. thaliana gene encoding shikimate O-hydroxycinnamoyltransferase contributes to phenylpropanoid biosynthesis, which is linked to lignin formation in vascular plants (Hoffmann et al. 2004).
PIN-mediated auxin transport might be involved in gametophore cellular polarization, highlighting the conserved crucial role of polar auxin transport during evolution of the plant body plan (Bennett et al. 2014, Viaene et al. 2014). A more recent study showed that two genes encoding GRAS family transcription factors, P. patens SHORTROOT and SCARECROW, facilitate the oriented cell division required for leaf vein formation (Ishikawa et al. 2023).
The transcriptional co-activator ANGUSTIFOLIA3 (AN3)/GROWTH REGULATING FACTOR (GRF)-INTERACTING FACTOR1 (GIF1) was first shown to promote cell proliferation in A. thaliana leaves (Horiguchi et al. 2005, Kim and Kende 2004). The molecular mechanisms of AN3/GIF1 action have been explored using transcriptome, protein–protein interaction, and chromatin immunoprecipitation sequencing analyses (Horiguchi et al. 2011, Nelissen et al. 2015, Vercruyssen et al. 2014) and are reviewed in detail elsewhere (Kim and Tsukaya 2015). The significant contribution of AN3/GIF1 to shoot formation is observed also in P. patens. Loss-of-function mutants of P. patens AN3/GIF1 family proteins exhibit severely dwarfed gametophore shoots but relatively normal protonemal tissue growth. This phenotype is at least partly attributable to defects in cell proliferation and expansion in the leaves. These morphological abnormalities are restored when A. thaliana AN3/GIF1 is ectopically expressed in P. patens with disruptions of the counterpart genes (Kawade et al. 2020), suggesting conservation of the molecular functions in these two plant species.
In addition to the growth and developmental roles of the AN3/GIF1-mediated molecular machinery, some transcriptional signatures suggest that AN3/GIF1 is involved in regulating metabolism (Horiguchi et al. 2011, Vercruyssen et al. 2014). Furthermore, a recent study showed that AN3/GIF1 physically interacts with FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR, which is a core regulator of iron uptake and homeostasis (Zheng et al. 2023). Perhaps AN3/GIF1 adjusts the metabolic or physiological status while engaging in shoot formation.
Loss-of-function of AN3/GIF1 in P. patens causes a significant increase in the arginine level, specifically in the gametophore shoots showing stunted growth. The levels of other metabolites closely related to arginine metabolism are also affected in the mutants. However, there is little change in the glycolytic pathway, tricarboxylic acid (TCA) cycle, or energy status represented by the adenosine triphosphate (ATP) and adenosine diphosphate (ADP) contents. These metabolic profiles suggest that arginine metabolism is a downstream target of AN3/GIF1 activity. This idea is supported by the observation that arginine treatment of wild-type P. patens recapitulates the stunted shoot growth and transcriptional changes detected in the mutants (Kawade et al. 2020). Future work is needed to delineate how arginine metabolism is modified and the role of this metabolic change, to understand novel aspects of arginine metabolism in plant growth and development.
In addition to its role as a proteinogenic amino acid, arginine plays an important role as a network hub in the primary metabolic network connecting the urea cycle, the polyamine biosynthetic pathway, and nitric oxide production (Kawade et al. 2023). Arginine serves also as a nitrogen reservoir for nitrogen allocation and re-utilization (Slocum 2005, Winter et al. 2015). This begins with arginine hydrolyzation to ornithine and urea by arginase (Siddappa and Marathe 2020). A loss-of-function mutation in arginase compromises this catabolic pathway, causing excessive arginine accumulation in panicles and impaired grain development in rice (Ma et al. 2013). Because this arginase-deficient rice mutant shows relatively normal shoot growth during the vegetative phase, the arginine catabolic pathway likely has a major role in the reproductive phase of rice. Ornithine is produced by arginine hydrolyzation and, after a subsequent catabolic reaction via ornithine-δ-aminotransferase, serves as a substrate of proline metabolism. Interference of this catabolic pathway via a loss-of-function mutation in ornithine-δ-aminotransferase results in ornithine overaccumulation, which in turn stimulates feedback inhibition of the arginase enzymatic activity (Liu et al. 2018). In strong agreement with this metabolic perturbation, the ornithine-δ-aminotransferase mutants exhibit reproductive growth and developmental abnormalities, similar to the arginase mutants (Liu et al. 2018). Exogenous nitrogen supplements ameliorate these defects in arginase- or ornithine-δ-aminotransferase-deficient rice (Liu et al. 2018, Ma et al. 2013), suggesting a link between arginine metabolism and nitrogen status. Arginine biosynthesis from argininosuccinate is mediated by argininosuccinate lyase. This metabolic reaction was recently shown to be involved in regulating root elongation in response to surrounding ammonium levels via auxin-mediated machinery (Xie et al. 2023).
Metabolomics or metabolome analysis is an approach used to identify broad metabolite levels at a specific time point, giving a snapshot of the metabolic network, which is instrumental for estimating the physiological cellular status. However, it is difficult to infer the dynamics of the metabolic network, including the activity of each metabolic pathway (metabolic flow). Given that metabolism operates as a dynamic network adjusting pathway influx and efflux in a context-dependent manner, information related to network dynamics would help delineate the associated physiological implications (Allen and Young 2020, Antoniewicz 2018, Jang et al. 2018, Okahashi et al. 2016). Labeling with radioactive or stable isotope tracers can directly quantify the metabolic dynamics in vivo. Although there are some technical limitations to be resolved, such as analysis throughput and tracer incorporation, studies have succeeded in identifying the metabolic dynamics in plants using this technique (António et al. 2016, Hasunuma et al. 2010, Joshi et al. 2019, Okamura et al. 2021).
When stable isotope 13C- or 15N-labelled arginine is used, the isotope labeling pattern during arginine metabolism can be quantified (Figure 1). Such an experiment unraveled a novel co-catabolic pathway of arginine and succinate that facilitates symbiotic nitrogen fixation (Flores-Tinoco et al. 2020). Our group is currently installing this experimental technique to compare the isotope-labeling patterns between protonemal and gametophore tissues in P. patens.
Figure 1. A schematic diagram of an arginine metabolism isotope-labeling experiment. The metabolic reaction from arginine to agmatine, ornithine, or citrulline is depicted after cellular incorporation of stable isotope 13C-labelled arginine (A) or 15N-labelled arginine (B).
Whereas arginine plays a vital role in nitrogen allocation and re-utilization (Liu et al. 2018, Ma et al. 2013, Xie et al. 2023), exogenous supplementation of nitrogen fails to rescue the stunted shoot growth in the P. patens AN3/GIF1 mutants (Kawade et al. 2020). This suggests that nitrogen metabolism is not relevant to the altered arginine metabolism during gametophore shoot formation in P. patens. However, there is a potential link between arginine metabolism and the metabolic processes related to the cell wall because the expression of genes encoding cell wall-modifying enzymes (expansins A1 and A9) and a cell wall reconstruction enzyme (xyloglucan endotransglucosylase/hydrolase 9) was decreased significantly in the gametophores of the P. patens AN3/GIF1 mutants (Kawade et al. 2020). This is consistent with the enzymatic role of PpNOG2 orthologs in lignin formation in vascular plants (Hoffmann et al. 2004, Moody et al. 2021). Given that cell wall modification by some expansin family proteins is involved in the stem cell formation in chloronemata (Sakakibara et al. 2014), future studies should investigate whether and how altered arginine metabolism affects stem cell formation via cell-wall modification in the gametophores of the P. patens AN3/GIF1 mutants.
A loss-of-function mutation in the P. patens NOG2 gene might compromise the pathway of flavonoid biosynthesis from p-coumaroyl-CoA because this metabolite is a precursor of both lignin and flavonoid biosynthesis. Because flavonoids inhibit auxin transport (Besseau et al. 2007), auxin-mediated cellular polarization might be affected in the P. patens NOG2 mutants. To specify whether either or both metabolic pathways are linked to arginine metabolism during gametophore shoot formation, isotope tracing of 13C- or 15N-labelled arginine would be helpful.
Oriented cell division is a visible phenotype that is instructed by cellular polarity. This has been investigated by identifying molecules exhibiting spatially biased distribution (Adamowski and Friml 2015, Naramoto et al. 2021). However, metabolic network has been studied less in the context of cellular polarity. Here, I suggest that the metabolic network is polarized in a broad sense when metabolic flow is biased at the intersection between different metabolic pathways (Figure 1 and 2). Such network polarization in metabolism might have physiological impacts, which in turn affect plant growth and development. Uncovering the unrecognized dynamics of a metabolic network during cellular polarization would help determine how this visible phenotype is established via invisible metabolic changes. Given that arginine connects various metabolic pathways as a network hub to facilitate many roles (Kawade et al. 2023, Winter et al. 2015), arginine metabolism would be an interesting target to delineate metabolic network polarization. Visualization of the network dynamics in arginine metabolism during gametophore shoot formation in P. patens helps determine how this is regulated in a robust but flexible manner in the primary and developmental metabolism. Future studies should also examine whether altered arginine metabolism contributes to the establishment of polarity in gametophore shoots in P. patens.
Figure 2. A schematic diagram of an arginine metabolism isotope-labeling experiment. The metabolic reaction from arginine to agmatine, ornithine, or citrulline is depicted after cellular incorporation of stable isotope 13C-labelled arginine (A) or 15N-labelled arginine (B).
I apologize to all colleagues whose work has not been cited due to space limitations. This work was supported by Japan Society for the Promotion of Science (JSPS) (KAKENHI Grant Number JP22K06289).
