Nitrogen depletion enhances endodermal suberization without restricting transporter-mediated root influx
Introduction
Radial movement of water and nutrients from the soil through roots involves passing across concentric layers of epidermis, endodermis and cortical cells before reaching the vasculature system for long-distance translocation to the shoot (Sattelmacher, 2001). Nutrient ions and water can move across plasma membranes, via membrane-bound transporters and channels (transcellular route), or plasmodesmata, from one protoplast to another (symplastic route). Alternatively, they can move within the extracellular space outside the plasma membrane (apoplastic route). The composite transport model explains that the pathways are arranged in parallel allowing nutrient ions to switch between them depending on the resistance for solute flow (Steudle and Frensch, 1996; Steudle and Peterson, 1998). This enables a form of regulation to meet shoot nutrient demand (Plett et al., 2020).
The presence of endodermal barriers is dependent on the stage of development. Early in the development of fully elongated endodermal cells, aligned patches of lignin are seen that later fuse to form Casparian strips. In mature cells, Casparian strips terminate the apoplastic pathway, forcing water and nutrients to move via the symplastic/transcellular route. In later stages of root development, suberin, a biopolyester, deposits in the inner surface of primary endodermal cell wall forming a hydrophobic, waxy layer and deposition increases with root age (Meyer and Peterson, 2013). Suberin and casparian bands are both root barriers but whilst the latter only restricts apoplastic passage of nutrient ions, suberin, based on its deposition between the plasma membrane and the plasmalemma, may also restrict access of nutrient ions to their transporters (the transcellular route) but leaving the symplastic route open to the passage of ions (Barberon et al., 2016; Geldner, 2013).
Suberization appears to be an adaptive response to nutrient stress. This concept was supported by the ionomic studies of Barberon et al. (2016) using transgenic line over-expressing the Cuticle Destructing Factor 1 (AtCDEF1). The ELTP::CDEF1 and CASP1::CDEF1 lines have a functional Casparian band but no detectable suberin (Naseer et al., 2012), and a lower potassium (K) concentration in the shoot. Additionally, deficiencies in the macro-nutrients sulphur (S), K, and phosphorous (P) resulted in enhanced suberization of wild type Arabidopsis plants (Barberon et al., 2016; Li et al., 2020). Early development of suberin lamellae was also observed in the K-deficient skor mutant and the S-deficient sultr1;1 sultr1;2 mutant (Barberon et al., 2016).
In contrast, deficiencies in the micro-nutrients zinc, iron (Fe) and Mn resulted in delayed deposition of suberin lamellae (Barberon et al., 2016; Chen et al., 2019; Sijmons et al., 1985). Discontinuous (patchy) suberization was observed in the Fe deficient mutant, irt1, and in the Fe and Mn deficient, nramp1 mutant (Barberon et al., 2016). These results may indicate that radial transport of micronutrients predominantly occurs across the endodermis via the apoplastic route, although the cation exchange capacity of the apoplast may result in root accumulation of these aforementioned nutrient ions. Barberon (2017) also suggests that enhanced endodermal suberization prevents leakage of some macronutrients (i.e. S, K, and P) from the stele to the apoplast of cortical cells.
Under conditions of prolonged drought or deficiencies of certain nutrients, root cortical senescence results in increased deposition of aliphatic suberin (Schneider et al., 2017), and, the endodermis may in some conditions become the outermost protective layer of the root (Meyer and Peterson, 2013). Aliphatic suberin, which is composed of primary alcohols, fatty acids, α–ω dicarboxylic acids (diacids), and ω-hydroxy acids (ω−OH acids), is thought to prevent movement of water and gases due to its high hydrophobicity (Schreiber et al., 1999) whilst aromatic suberin, whose most abundant constituents are ferulic and coumaric acids, has a greater protective role against pathogens as well as preventing solute movement (Kreszies et al., 2018).
The extracellular space around the cell wall is water-filled, enabling ions such as nitrate () to move along with water apoplastically, through cell-wall interstitial spaces and characterized by very rapid turn-over kinetics for ions such as (Kronzucker et al., 1995a, 1995b; Siddiqi et al., 1991). However, is also able to cross the plasma membrane via both low-affinity nitrate transporter systems (LATS) and high-affinity nitrate transporter systems (HATS). LATS function significantly only when external concentrations are high (> 1 mM) and often display linear transport kinetics (Kronzucker et al., 1995a, 1995b; Siddiqi et al., 1990). Nitrate transporters of the NPF/NRT1, the nitrate transporter 1/peptide transporter family (Léran et al., 2014), function as LATS but the dual-affinity transporter AtNPF6.3/NRT1.1 can switch its mode of action dependent on its phosphorylation state which varies as a function of external (Liu and Tsay, 2003). The high-affinity nitrate pathway is considered to dominate over the low-affinity pathway in oilseed rape (Malagoli et al., 2004), dwarf maize (Garnett et al., 2013) and wheat (Melino et al., 2015) at lower levels of external N supply. HATS function is confined to lower external concentrations (0.01−1 mM) and displays saturable transport activity and both constitutive and inducible components (Siddiqi et al., 1990).
Armand et al. (2019) recently demonstrated that low N caused enhanced and developmentally early deposition of suberin lamellae in barley roots with a concomitant decrease in root hydraulic conductivity when compared to the control supplied with both NH4+ and . Suberization is considered to create a barrier to reduce back-flow of water and thereby losses from the stele to the apoplast of the cortex (Kreszies et al., 2018). On the other hand, in roots of rice, low ammonium (NH4+) supply, when ammonium was the sole N source, resulted in reduced suberin and lignin content as compared to the control, whereas high NH4+ supply enhanced suberin and lignin content (Ranathunge et al., 2016).
Broadly, we aim here to understand the effect of N nutrition on transport pathways of from the root to the shoot. Specifically, the aim of this project was to assess whether N-depletion induced suberin composition changes in early suberized barley roots using supply to the control as the sole source of N to avoid complications with differences in the effect of NH4+ doses seen by Ranathunge et al. (2016). Furthermore, in roots with early and enhanced suberization, we assessed whether suberin deposition could reduce transporter mediated influx (transcellular route).
Three malt barley cultivars that are currently grown and used as parents in breeding programs were selected to assess whether the root architecture and biochemical responses to N-depletion were consistent or whether genetic variation for this trait exists. One of these cultivars, Scarlett, was previously used in a study demonstrating enhanced cell wall suberization under osmotic stress (Kreszies et al., 2019). The results of this present study showed that reduced N translocation to the shoot under N-deplete conditions was correlated with early and enhanced cell wall suberization and induction of the high-affinity transport system. The evidence suggests that early and enhanced suberization under N-deplete conditions does not affect transcellular routes for transport in barley.
Section snippets
Plant material and growth conditions
Three barley (Hordeum vulgare L. spp. vulgare) cultivars were selected for this experiment, Australian cultivars Bass and La Trobe and the German cultivar Scarlett. Bass and La Trobe are Australian malt varieties that attract a higher premium than other varieties on the export market (Robertson, D. Senior Barley Trader). Cultivar La Trobe has a higher predicted yield than cv. Bass across multiple environments and years in Australia (app.nvtonline.com.au, 2019). Scarlett is a German variety with
Plant growth responses to N depletion
Barley plants were grown with a steady-state supply of nitrate (), provided at 1.5 mM for 14 d before a treatment group was moved to N starvation for seven days (0 mM, Switch), while the control group was maintained at a steady state of 1.5 mM . Plants grown under N-deplete condition had a reduced number of tillers, above-ground biomass (Table 1), and chlorophyll content (Fig. 2). Additionally, root dry weight, but not fresh weight, was increased (Table 1) as reflected in the root
Discussion
Plants must continuously acclimate to keep pace with spatiotemporal fluctuations in N availability. Conditions of water deficit, causing reduced transpirational water fluxes, can impair the acquisition of nitrate and ammonium (Plett et al., 2020). Plants use a myriad of strategies to overcome localised N deficiencies which, aside from adjustments in the expression of genes encoding N transporters, their abundance and regulation, includes changes in root system architecture, the organization of
Conclusion
Reduced root to shoot translocation of under N-deplete conditions occurs despite induction of the high-affinity transport system. We speculate that this is due to blocking of the apoplastic transport route, however, whether this was due to reduced transpiration or enhanced endodermal suberization, or a combination of both is inconclusive. Maintenance of shoot N in plants grown under optimal N nutrition must therefore rely on both the apoplastic and symplastic/transcellular transport
CRediT authorship contribution statement
V.J. Melino: Conceptualization, Methodology, Software, Validation, Investigation, Formal analysis, Visualization, Writing - original draft, Writing - review & editing. D.C. Plett: Conceptualization, Methodology, Writing - review & editing, Supervision, Funding acquisition. P. Bendre: Investigation, Validation, Formal analysis. H.C. Thomsen: Validation, Formal analysis, Investigation. V.V. Zeisler-Diehl: Validation, Formal analysis, Investigation. L. Schreiber: Conceptualization, Methodology,
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgements
The authors would like to acknowledge the University of Melbourne, School of Agriculture and Food research investment fund, Australia. We acknowledge the Melbourne Histology and Histopathology platform and expert advice of Laura Leone and the University of Melbourne Biological Optical Microscopy (BOMP) platform. We would like to acknowledge Melbourne TrACEES Platform for the service and expert advice by Michael Hall.
References (61)
- et al.
Adaptation of root function by nutrient-induced plasticity of endodermal differentiation
Cell
(2016) - et al.
CYP86A1 from Arabidopsis thalianaEncodes a cytochrome P450-Dependent fatty acid omega-hydroxylase
Biochem. Biophys. Res. Commun.
(1998) - et al.
Suberized transport barriers in Arabidopsis, barley and rice roots: from the model plant to crop species
J. Plant Physiol.
(2018) - et al.
A unified nomenclature of nitrate transporter 1/peptide transporter family members in plants
Trends Plant Sci.
(2014) - et al.
Molecular cloning of higher plant homologues of the high-affinity nitrate transporters of Chlamydomonas reinhardtii and Aspergillus nidulans
Gene
(1996) - et al.
Regulation of root nitrate uptake at the NRT2.1 protein level in Arabidopsis thaliana
J. Biol. Chem.
(2007) - et al.
Cortex cell hydraulic conductivity, endodermal apoplastic barriers and root hydraulics change in barley (Hordeum vulgare L.) in response to a low supply of N and P
Ann. Bot.
(2019) The endodermis as a checkpoint for nutrients
New Phytol.
(2017)- et al.
Root suberin forms an extracellular barrier that affects water relations and mineral nutrition in Arabidopsis
PLoS Genet.
(2009) - et al.
A berberine-aniline blue fluorescent staining procedure for suberin, lignin, and callose in plant tissue
Protoplasma
(1988)
Efficient lipid staining in plant material with Sudan red 7B or fluoral yellow 088 in polyethylene glycol-glycerol
Biotech. Histochem.
The CesA gene family of barley. Quantitative analysis of transcripts reveals two groups of co-expressed genes
Plant Physiol.
RootGraph: a graphic optimization tool for automated image analysis of plant roots
J. Exp. Bot.
Rapid colorimetric determination of nitrate in plant-tissue by nitration of salicylic-acid
Commun. Soil Sci. Plant Anal.
The intensity of manganese deficiency strongly affects root endodermal suberization and ion homeostasis
Plant Physiol.
CYP86B1 is required for very long chain ω-Hydroxyacid and α, ω-Dicarboxylic acid synthesis in root and seed suberin polyester
Plant Physiol.
Barley breeding history, progress, objectives, and technology
The response of the maize nitrate transport system to nitrogen demand and supply across the lifecycle
New Phytol.
Variation for N uptake system in maize: genotypic response to N supply
Front. Plant Sci.
The endodermis
Annu. Rev. Plant Biol.
Identification of nitrogen use efficiency genes in barley: searching for QTLs controlling complex physiological traits
Front. Plant Sci.
The Arabidopsis cytochrome P450 CYP86A1 encodes a fatty acid ω-hydroxylase involved in suberin monomer biosynthesis
J. Exp. Bot.
Signaling pathways underlying nitrogen-dependent changes in root system architecture: from model to crop species
J. Exp. Bot.
The Arabidopsis nitrate transporter NRT2.4 plays a double role in roots and shoots of nitrogen-starved plants
Plant Cell
Osmotic stress enhances suberization of apoplastic barriers in barley seminal roots: analysis of chemical, transcriptomic and physiological responses
New Phytol.
Compartmentation and flux characteristics of nitrate in spruce
Planta
Kinetics of influx in spruce
Plant Physiol.
The Arabidopsis nitrate transporter NRT2.5 plays a role in nitrate acquisition and remobilization in nitrogen-starved plants
Plant J.
Apoplastic barriers, aquaporin gene expression and root and cell hydraulic conductivity in phosphate-limited sheepgrass plants
Physiol. Plant.
Root characteristics of vigorous wheat improve early nitrogen uptake
Aust. J. Agric. Res.
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2021, Current Opinion in Plant BiologyCitation Excerpt :More surprisingly, suberin plasticity was also observed in response to mineral deficiencies especially in Arabidopsis where potassium or sulfur deficiencies were shown to induce suberization while manganese, iron or zinc deficiencies were shown to reduce suberization and induce its degradation through abscisic acid (ABA) and ethylene signaling, respectively [24,26]. Suberin plasticity in response to mineral deficiencies has been also described in barley roots in response to manganese or nitrogen deficiencies [36–38]. In addition, suberin is also highly plastic in response to biotic interactions including nematodes, pathogenic and beneficial microbes [∗∗39–∗∗42].
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These authors should be considered joint first author