P-ring: The conserved nature of phosphorus enriched cells in seedling roots of distantly related species

ABSTRACT

Plants require sunlight, carbon dioxide, water and mineral ions for their growth and development. Roots in vascular plants sequester water and ions from soil and transport them to the aboveground parts of the plant. Due to heterogeneous nature of soil, roots have evolved several regulatory barriers from molecular to organismic level that selectively allows certain ions to enter the vascular tissues for transport according to the physiological and metabolic demands of plant cell. Current literature profusely elaborates about apoplastic barriers, but the possibility of the existence of a symplastic regulation through phosphorous-enriched cells has not been mentioned. Recent investigations on native ion distribution in seedling roots of several species (Pinus pinea, Zea mays and Arachis hypogaea) identified an ionomic structure termed as “P-ring”. The P-ring is composed of a group of phosphorous-rich cells arranged in radial symmetry encircling the vascular tissues. Physiological investigations indicate that the structure is relatively inert to external temperature and ion fluctuations while anatomical studies indicates that they are less likely to be apoplastic in nature. Furthermore, their localization surrounding vascular tissues and in evolutionarily distinct plant lineages might indicate their conserved nature and involvement in ion regulation. Undoubtedly, this is an interesting and important observation that has significant merit for further investigations by the plant science community.

KEYWORDS:

• Plant roots
• ion transport
• P-ring
• symplastic regulation
• phosphorous

Introduction

The regulation of ion and water transport is critical for the growth and development of air-land (terrestrial) plants. The Casparian band in plant roots has attracted the attention of a significant number of plant biologists due to its role as an apoplastic barrier and in the regulation of ion transport^1–5. Though the distribution and size of plasmodesmata can influence symplastic transport at a cellular level and symplatic transport is tightly regulated at the molecular level by transporters and channels, the current literature does not mention any distinct anatomical feature within roots that can be identified as responsible for symplastic regulation of ion transport^6–10. The native or baseline distribution of ions in seedling roots of Pinus pinea (Gymnosperm), Zea mays (Monocot) and Arachis hypogaea (Eudicot) may indicate some possibility of such symplastic regulation^11–14.

Cryo-SEM/EDS was used to estimate the distribution of ions in root tissues. One of the most significant outcomes of these studies was identification of a group of cells arranged in radial symmetry that contained relatively high amount of phosphorous ion as compared to their surrounding cells. This ionomic structure was referred as “P-ring” because it was rich in phosphorous and had a radial symmetry. The striking similarity in the development, physiology, and anatomy of this ring across distinct evolutionary lineages of plants indicates that such a sub-anatomical/ionomic feature might be evolutionarily conserved in air-land plants (Figure 1, Figure 2 & Figure 3). The first appearance of the P-ring varied around 3 mm to 4 mm from the root tip depending on the species and were localized surrounding the vascular tissues in all root samples that were analyzed. In Pinus pinea, the P-ring was localized in the inner cortical cells that most likely developed into endodermal cells at maturity. In Zea mays and Arachis hypogaea, the P-ring was primarily found in pericycle at developmentally mature regions, while in the younger regions of the seedling root, it consisted of cells that surrounded vascular tissues but were not differentiated enough to be identified as pericycle cells. Another salient ionomic characteristic of the P-ring was its lower potassium content as compared to surrounding cells^12–14.

Figure 1. The diagram represents the cross section of a typical root with the red arrow indicating the location of P-ring. Vascular tissues (1); Pericycle (2); Endodermis (3); Inner Cortical Cells (4); Cortex (5) and Epidermis (6) are labelled to understand the spatial resolution of the P-ring in different species of plant roots..

Figure 2. An SEM image (using secondary electrons) of a cross section of Arachis hypogaea seedling root at 15 mm from the root apex.

Figure 3. An EDS (elemental mapping) showing phosphorous distribution in a cross section of Arachis hypogaea seedling root at 15 mm from the root apex. The yellow arrow indicates the location of P-ring. The red color is a false color generated by the IXRF mapping software..

Characterization of P-ring

The identification of P-ring was subsequently followed by some experiments to investigate the physiology and biochemical characters of the ring. The phosphorous concentration in the P-ring was estimated to be approximately twice the amount found in surrounding tissues. When roots were grown at different temperatures, the origin and localization of the P-ring were unaffected. However, the most striking results were obtained when the phosphorous distribution in the cells forming the P-ring remained unchanged even though exposed to high concentration of competing anions such as nitrate, phosphate, chloride, and cations such as potassium, rubidium, calcium and sodium¹². Though the initial experiments and instrumentation used are limited in their capacity to conclusively prove the symplastic nature of the ring, it cannot be ascertained that the structure is involved in ion regulation. However, it raises several questions regarding the evolutionary, developmental, and physiological significance of this ring in plant roots and its relation to ion transport. Apart from foliar absorption, roots are the main organs for sequestration and transport of water and ions¹⁵. The localization of the P-ring in root tissues that are in close proximity to vascular tissues indicates that they might be anatomically well positioned to complement the apoplastic barrier in ion transport regulation.

The striking similarity in the position of the P-ring surrounding the vascular tissues in all three species reported (Pinus pinea, Zea mays and Arachis hypogaea) might indicate significant evolutionary homology among land plants with respect to phosphorous ion regulation in root growth and development. Acquisition of phosphorous from soil and translocation of sugars is one of the fundamental foundations of root-mycorrhizal associations^16,17. Such a mutualistic association has coevolved and has helped plants to colonize the land by improving procurement of resources such as water and minerals^18,19. Roots of most land plants have such an association reflecting the successful relationship in course of evolution. It is worth investigating if the P-ring has any relation with phosphorous signaling and carbon partitioning with respect to root-mycorrhizal association. If we consider the root axis to be a reflection of the developmental timeline, P-ring was first observed closer to the root tip in the young region of the roots. In such developmentally young regions, apoplastic barrier such as the Casparian band is either absent or has not attained maturity. Thus, the development of P-ring is unlikely due to the apoplastic resistance of ion transport. Thus, the developmental mechanisms that produce them and their role in root physiology are important questions that need investigation. In a cellular environment, besides the ionic form, phosphorous can be a constituent of biomolecules, macromolecular complexes, and conjugated with proteins, lipids, or carbohydrates. In seeds, phosphorous is stored as phytates and within cell, phosphorous is found in nucleic acids, ATP, and membranes. Phosphorous serves a key role in enzyme activation and deactivation through phosphorylation and dephosphorylation, and thus, is a critical nutrient for plant growth and development^20,21.

Possible function of the P-ring

The relative inertness of the P-ring when exposed to varying temperature and ionic pressure indicated that the phosphorous contained in the P-ring is not ionic in nature but rather is of organic nature such as a high molecular weight compound that is relatively immobile to minor fluctuations of temperature or major fluctuations of ionic environment. Though the exact functionality of the P-ring has not been established, it can be hypothesized (Table 1) that such a ring would be extremely important for ion regulation in growing root tips which do not have apoplastic barrier but are the first to encounter ionic changes in soil while penetrating deeper inside soil. Thus, in absence of an apoplastic barrier such as the Casparian band, a symplastically located P-ring can serve the purpose of regulation of ion transport that will be a necessary anatomical and evolutionary innovation for maintaining ion homeostasis. However, the role of P-ring in regulating the transport of uncharged molecule such as boric acid is worth investigating as it is the primary chemical form in which the micronutrient boron passively enters the plant cell from external environment²². Soil environment is ununiform and several biotic, abiotic, and edaphic factors may become detrimental to the maturation of seedling unless ionic homeostasis can be maintained. A symplastic regulation by phosphorous-enriched cells such as the P-ring might orchestrate selective exchange of water and ions from the external environment and plant vascular tissues, thus maintaining ionic homeostasis in plants. The biochemical nature of the P-ring could be further ascertained by treating seedling roots with ATPase inhibitor and phytase and observing their effect on the P-ring development. A more direct approach will be the use of Raman microscopy for “chemical Imaging” of the P-ring; however, it is difficult to predict if the results can be analyzed to extrapolate useful data because unlike chemical compounds, biological compounds are extremely complex and has a high amount of heterogeneity; thus, Raman microscopy might yield many signals that might be difficult to interpret or might not yield conclusive data. However, Raman microscopy is increasingly used for high-resolution molecular mapping of biomolecules^23–27.

Table 1. The different characteristics of the P-ring that was observed and their plausible explanation relating to the hypothesis of “Symplastic Regulation”.

	Observations	Inferences
1	Identified in evolutionarily distinct lineages	Must be fundamental to root physiology thus conserved across species
2	Surrounds vascular tissues	Possibly involved in loading, unloading and regulation of solutes that enters or leaves from vascular tissues
3	Closely associated with or overlaps Endodermis and Pericycle	Must be involved with solute transport regulation and/or growth of lateral roots
4	Is rich in phosphorous	Must be involved with growth, energetics, structure, and enzymatic reactions
5	Has relatively lower potassium content	Lower water influx
6	Localized in absence of the Casparian band	May have barrier functions in absence of apoplastic barrier to maintain ion homeostasis
7	Resistant to temperature fluctuations	Indicates organic and bound or caged nature of the phosphorous that can resist diffusion of ions
8	Resistant to ionic pressure	Indicates organic and bound or caged nature of the phosphorous that can resist ion movement
9	Composed of cells that lacks thick cell wall	Highly likely the location of the phosphorous is in symplasm
10	Composed of cells that lacks conspicuous vacuole	Highly like that the location of the phosphorous in outside vacuoles

Beside the chemical and biochemical nature of phosphorous in the P-ring, it is also important to localize them at a sub-cellular level to understand their transport, storage, and role in cell physiology. Anatomical studies have reflected that the cells where P-ring starts developing in younger regions of the root lack conspicuous vacuoles. In these regions, vacuoles are very small in size; thus, they might not have developed the capacity for accumulation of phosphorous ions. Furthermore, the probability of a high amount of phosphorous being associated with cell wall is less likely because these cells are thin and do not have lignified thick cell walls. However, the phosphorous in the P-ring might be associated with proteins or lipids in the cell membrane or organelles. Phosphorous plays a key role in cell growth and development, actively dividing cells such as lateral root primordium, and cells near the root tips are identified as phosphorous rich. The P-ring may act as a signaling dock that regulates lateral root emergence through phosphorous signaling. Thus, it is not surprising that the phosphorous-rich pericycle cells can differentiate into founder cells in phosphorous-deficient conditions for lateral root emergence^28,29. It can be hypothetically stated that the relative central position of the P-ring in root cross section and the phosphorous-rich pericycle cells indicates that the phosphorous contained in these cells may be utilized for vascular/non-vascular tissue growth on either side of the P-ring besides supporting or signaling the lateral root formation. The P-ring, though rich in phosphorous, is found to have a lower potassium content compared to surrounding tissues. Potassium is the major osmoticum in plant cells and has a highly mobile character. A large percentage of potassium that is sequestered by root from soil solution and transported to the above-ground plant parts are recycled back to the root. Potassium decreases water potential and can direct the movement of water toward cells that have high potassium content. Several research works have indicated the role of potassium in improving water content of plant tissues and ameliorating drought stress and other abiotic and biotic stresses^30–32,33. Thus, the relatively low potassium content in the P-ring indicates that these cells might not allow movement of high volume of water. Cell-specific transcriptomics involving the investigation of aquaporin gene expression in these cells might be able to confirm the status and distribution of membrane-bound aquaporins. Such transcriptomic data may support in building a consensus about the P-ring being involved in symplastic regulation of ion transport.

The several roles that can be ascribed to the p-ring, their presence in evolutionary distinct lineages, location surrounding the vascular tissues, close association with endodermis/pericycle cells, low potassium content, relative inertness to temperature and ionic pressure, development in the absence of the Casparian band and in cells that lack conspicuous vacuoles or thick cell walls indicate that they may be involved in symplastic regulation of ion transport. For the abovementioned reasons, it is not unreasonable to state that they might complement the function of apoplastic barrier such as the Casparian band and may have a critical role in solute transport regulation which is unexplored.

Conclusions

Plant roots are key evolutionary innovations that increased the capacity of vascular plants for nutrient and water sequestration. Roots enabled plants to colonize land and increased in size, thus fundamentally changing the terrestrial ecosystem³⁴. However, roots encounter environment that is rapidly changing and heterogeneous in nature. To maintain ionic homeostasis, the root employs several barriers at organ, tissue, cellular and subcellular level which tightly controls the movement of solutes from soil to the vascular tissues of plant roots³⁵. Solute moves through apoplastic, symplastic, and trans-cellular route and involves polarized transport³⁶. The directional movement of water and ions is strictly regulated at molecular level by membrane-bound transporters and channels. Thus, solute transport in roots is strictly regulated at organ, tissue, cell, organelle, and molecular level. Current literature does not mention any phosphorous-enriched cells that can regulate solute transport at an ionomic level. Though ionomic regulations such as calcium signaling are ubiquitously involved in regulating several physiological functions in plants, their role is mostly perceived as modulators rather than regulators, and nanomolar concentrations of calcium are involved in calcium signaling.

On the contrary, the concentration of phosphorous found in P-ring is of several millimolar; thus, it might have functions beyond phosphorous signaling. The P-ring might be the first evidence of ionomic regulators that orchestrate solute transport in root through an ionomic component, thus indicating a separate level of regulation than what is already known. Improvement in technology has allowed the identification of ion-transport regulation from microscopic to molecular level; thus, it will not be surprising if ionomic level of regulation is discovered in near-future and get stacked as one more level of ion regulation. Phosphorous is essential for cell division, cell growth, cell signaling, stress response and is involved in several physiological processes such as photosynthesis and respiration. Phosphorous is involved in biosynthesis of nucleic acids, membranes and is required in several enzymatic functions. Though several phosphorous transporters have been identified and the molecular mechanism of phosphorous transport from soil solution to plant cells has been investigated²⁰, it is essential to integrate knowledge from different route of investigation and collaborate to have more complete understanding of ion regulation. Such understanding will lead to better management of crops, fertilizers, soil, environment, and human health, besides increasing the potential of growing plants beyond the soil on earth. Significant amount of genetic information on the above-ground plant traits is available^37,38; on the contrary, the biology of plant roots has not received such attention^39,40. Understanding plant roots along with innovation in technology^41,42 and better agricultural management practices^43,44 are essential to meet the goals of sustainable agriculture and environment.

Acknowledgments

All authors contributed equally.

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No potential conflict of interest was reported by the author(s).

Correction Statement

This article has been corrected with minor changes. These changes do not impact the academic content of the article.

Additional information

Funding

We deeply thank the editor of “Plant Signaling & Behavior” for being extremely generous and waving the publication cost.