ROS and calcium oscillations are required for polarized root hair growth

ABSTRACT

Root hairs are filamentous extensions from epidermis of plant roots with growth limited to the apical dome. Cell expansion undergoes tightly regulated processes, including the coordination between cell wall loosening and cell wall crosslinking, to form the final shape and size. Tip-focused gradients and oscillations of reactive oxygen species (ROS) together with calcium ions (Ca²⁺) as indispensable regulated mechanisms control rapid and polarized elongation of root hair cells. ROS homeostasis mediated by plasma membrane-localized NADPH oxidases, known as respiratory burst oxidase homologues (RBOHs), and class III cell wall peroxidases (PRXs), modulates cell wall properties during cell expansion. The expression levels of RBOHC, an NADPH oxidase that produces ROS, and class III PRXs are directly upregulated by ROOT HAIR DEFECTIVE SIX-LIKE 4 (RSL4), encoding a basic-helix-loop-helix (bHLH) transcription factor, to modulate root hair elongation. Cyclic nucleotide-gated channels (CNGCs), as central regulators of Ca²⁺ oscillations, also regulate root hair extension. Here, we review how the gradients and oscillations of Ca²⁺ and ROS interact to promote the expansion of root hair cells.

KEYWORDS:

• ROS
• calcium
• CNGC
• homeostasis
• RBOH
• root hair

1. Introduction

Root hairs are unicellular extension from root epidermal cells, aiding plants in nutrient acquisition and anchorage as well as interaction with microbe in soil. The expansion rate of root hairs is determined by a complex process including the driving force from vacuolar turgor pressure, the maintenance from the exocytosis of new cell wall materials and local loosening of the existing cell wall, and exceeds 1 µm/min in Arabidopsis¹. Arabidopsis root hairs can reach 1 mm or longer in length and approximately 10 µm in diameter.¹ The root maturation zone is characterized by certain epidermal cells becoming root hairs. Immature epidermal cells becoming root hair or non-hair cells can be controlled by a position-dependent mechanism.^2–5 The cortex of Arabidopsis primary root consists of a ring of eight cells (Figure 1). At an early stage, the epidermal cells at the junction of two underlying cortical cells (“H” position) will develop root hairs (trichoblasts), whereas non-hair cells (atrichoblasts) contact only one cortical cell (“N” position) (Figure 1). Therefore, eight immature root-hair cells in Arabidopsis are separated by either one or two immature non-hair cells^4,5,11 (Figure 1). Additionally, cell-cell communication is important for establishing cell identity in the root epidermis. The Myb-like protein CAPRICE (CPC) translocates from atrichoblasts to trichoblasts and represses the expression of the negative regulator GLABRA2 (GL2), thereby establishing hair cell identity.^12,13 ROOT HAIR DEFECTIVE6 (AtRHD6) encodes a basic-helix-loop-helix (bHLH) transcription factor and positively regulates the development of H cells.¹⁴ GL2, as an HD-Zip transcription factor, inhibits RHD6 expression in atrichoblasts, thereby preventing differentiation into hair cells.^15–17 The expression of AtRHD6 was not observed in the cpc mutant.^18,19 Epidermal cell differentiation can be acquired at an early stage prior to the initiation of cell elongation.⁵ The morphology of trichoblasts is distinguished from atrichoblasts prior to hair initiation.⁵ Trichoblasts in the meristematic zone display a high rate of cell division,¹⁵ reduced cell length before root hair initiation, relatively dense cytoplasm,^4,5 and less vacuolated.⁵

Figure 1. Regulation of tip growth of root hairs by ROS and Ca²⁺. This model is based on the previous works.^1,6–10Apoplastic ROS generated by PRXs and RBOHC (RHD2) control cell wall expansion or crosslinking in the apical zone of root hairs. Ca²⁺ oscillations focused in root hair tip are modulated by CNGC5, CNGC6, CNGC9 and CNGC14. The CBL1/CIPK26 complex phosphorylates and activates RBOHC to generate ROS. RSL4 directly regulates the expression of genes encoding RBOHC and PRX01 (At1g05240), PRX44 (At4g26010), PRX73 (At5g67400), and PRX69 (AT5G64100). Blue asterisks indicate cortex. Black asterisks indicate H cell position. Red asterisks indicate N cell position.

ROOT HAIR DEFECTIVE SIX-LIKE 4 (RSL4) encodes a bHLH transcription factor that promotes root hair cell elongation.¹⁹ The Rho-of-plant (ROP) guanosine triphosphatases (GTPases) are specifically localized to future sites of root hair emergence, and overexpression of ROP2 results in additional and misplaced root hairs.^20–22 Plant RAPID ALKALINIZATION FACTORS (RALFs) are secreted peptides that bind to Catharanthus roseus RECEPTOR-LIKE KINASE 1-like (CrRLK1L) family members such as FERONIA (FER), regulating plant cell size and shape.^23–26 The RALF1-FER complex promotes the translation of RSL4 and ROP2 mRNAs.^27,28 In turn, high-level accumulation of RSL4 protein suppresses RALF1 expression via directly binding to the promoter of RALF1 gene,^27,28 fine tuning root hair elongation.

2. Regulation of root hair elongation by reactive oxygen species (ROS)

The cell wall, as a dynamic structure, determines the shape of root hairs. The cell wall not only provides support for expansion at the tip but also functions in resisting against the turgor pressure.²⁹ Cell wall integrity is maintained through the coordination between cell wall loosening and restriction of growth via cell wall stiffening. In root hairs, cell walls are primarily composed of cellulose, xyloglucans, pectins, and hydroxyproline-rich glycoproteins that include extensins and arabinogalactan-proteins.^30,31 Following root hair initiation, the apical zone is characterized by apoplastic ROS and cytoplasmic calcium ion (Ca²⁺) gradients. Hair cell elongation requires a balance between cell wall loosening (ROS activity) and stiffening (crosslinking of polymers). The crosslink between calcium ions and demethylated pectins forms egg-box structures, increasing cell wall stiffness.³² ROS can be generated in the apoplast, and have different forms with different functions, such as, dioxygen (O₂), singlet oxygen (¹O₂), superoxide radical (O₂^•−), hydrogen peroxide (H₂O₂), and hydroxyl radical (OH^•)³³ An increase in apoplastic H₂O₂ concentration affects the degree of cell wall crosslinking by oxidizing cell wall compounds, thus decreasing cell expansion.^34–36 OH^• mediates scission of long-chain polysaccharides, such as pectin and xyloglucan, leading to non-enzymatic loosening of cell walls.^37,38 ROS accumulate at the apex of root hairs during elongation. In contrast, when root hairs stop growing, ROS accumulation at the apex decreases.^39,40 Thus, abnormal accumulation of ROS in root hair cells brings about exacerbated growth or root hair burst.⁶ Respiratory burst homologs (RBOHs) and peroxidases (PRXs) are the main generators of ROS at the apoplast in plants.^41–44 The ROS primarily generated by RBOH oxidase are required for normal root hair elongation.^34,40,45,46 In plants, RBOHs contain six predicted membrane-spanning domains and two Ca²⁺-binding EF-hand motifs in an N-terminal cytoplasmic region, which is absent in the homologue of mammalian NOX1-4/gp91phox.^47–49 RBOHs are activated upon Ca²⁺ binding the EF-hand and phosphorylation in the N-terminal region of RBOH.^42,50,51 In Arabidopsis, root hairs are initiated correctly but incapable of elongation in rbohc (root hair defective 2, rhd2) mutants grown on medium buffered to pH 5.0.^28,34 In maize, RHT5 (ROOTHAIRLESS 5), encoding a monocot-specific NADPH oxidase, is responsible for establishing the high levels of ROS in the tips of growing root hairs, which is required for cell elongation.⁵² Consistent with these findings, the elongation of wild-type hairs of Arabidopsis was inhibited by diphenyleneiodonium chloride (DPI), an inhibitor of NADPH oxidases.⁴⁰ In addition, OH^• partially rescued the growth of rbohc mutants, but this growth lacked spatial control.⁵³ Interestingly, supercentipedel1 (SCN1), a RhoGTPase GDP dissociation inhibitor (RhoGDI), can spatially restrict ROS to a single point on the trichoblast.³⁹ Therefore, in scn1 mutants, multiple tip-growing axes were formed from single bulges,^39,54 and ROS were produced at ectopic foci in root hair cells.³⁹ Trans-Golgi network-localized YPT-INTERACTING PROTEIN 4a and YPT-INTERACTING PROTEIN 4b (YIP4a/b) can induce activation and plasma membrane accumulation of ROP GTPases during root hair initiation.²² RHD2 accumulated at a single location in the wild type whereas it appeared at several distinct loci in the scn1 mutant.⁵¹ These results suggested that SCN1 RhoGDI controls the spatial accumulation of RHD2 to regulate the ROS distribution in root hairs.^39,51 In addition, SCN1 RhoGDI is likely to act by regulating ROP2 GTPase that is located to the hair tip, and regulates actin microfilament dynamics in root hair cells.^21,39,55 Further study indicates that such microfilaments are required for the localization of RHD2 to the growing tip.⁵¹ These findings reveal that normal, long, polarized root hair growth requires not only ROS production but also correct ROS localization.

In addition to NADPH oxidase, class III PRXs can be secreted into the apoplastic space and modulate ROS levels in apoplasts to mediate root hair growth.³⁵ Class III PRXs are involved in the consumption or release of H₂O₂ and the generation of other ROS. In the hydroxylic cycle, OH^• is generated from H₂O₂ and oxygen by Class III PRXs. In peroxidative and hydroxylic cycles, the apoplast H₂O₂ concentration is regulated by Class III PRXs.^35,56 In the oxidative cycle, together with NADPH oxidase/RBOH proteins, PRXs contribute to O₂^•− production by oxidizing ¹O_2.⁵⁷ In this regard, some PRXs appear to promote the polymerization of cell wall components, whereas others likely cause cell wall loosening via polysaccharide cleavage to promote polarized growth.^37,58,59 Mutations in two apoplastic class III peroxidases, PRX44 and PRX57, led to root hair cell wall rupture because the walls of root hair cells of the mutants were thin and mechanically weakened.⁶⁰ Moreover, PRX62 and PRX69 stimulate Arabidopsis root hair elongation at low temperatures, likely through modulating ROS homeostasis and cell wall extensin-insolubilization.⁶ PRX01, PRX44, and PRX73 function in ROS homeostasis and control Tyr-crosslinking of cell wall extensins during polar expansion of root hair cells.^7,61 RSL4 directly binds to the promoter regions of RBOHC and PRX1, 44, 73, and 69 to increase their expression, triggering apoplast oxidation and polarized root hair elongation.^6,7

3. Regulation of root hair elongation by Ca²⁺

During root hair elongation Ca²⁺ is distributed primarily at the tip apex (about 1 μM) and reduced to 0.1–0.2 μM at the base of the root hair.^46,62,63 When root hair elongation ceases, the Ca²⁺ gradient is changed.^1,46 An increased cytoplasmic Ca²⁺ concentration provokes polar secretion, rearrangement of the actin cytoskeleton, movement of organelles, and enzyme activity,⁶⁴ facilitating the elongation of tip-growing cells in roots.^65,66 In addition to the Ca²⁺ concentration gradient, Ca²⁺ oscillations play a key role in transmitting signaling events for tip growth.^46,66,67 Early studies primarily focused on the importance of the Ca²⁺ gradient and oscillations in root hair growth. Recently, Ca²⁺ channels contributing to the regulation of Ca²⁺ signatures to control polarized tip growth of root hairs have been identified.⁹ The CNGC14 nonselective cation channel mediates Ca²⁺ influx, which is required for maintaining the correct Ca²⁺ signature for the integrity of root hairs.⁶⁸ Cyclic nucleotide-gated channel 14 (cngc14) mutants exhibited very short and some branched root hairs, with altered amplitude and frequency of Ca²⁺ oscillations compared to the wild type.⁶⁸ Moreover, CNGC14 physically interacts with calmodulin 7 (CaM7) to suppress CNGC14 activity, potentially affecting Ca²⁺ oscillations during polarized growth of root hair.⁶⁹ Also, CNGC5, CNGC6, and CNGC9 maintain tip-focused Ca²⁺ oscillations, which are required for root hair growth and polarity. CNGC6, CNGC9, and CNGC14 triple mutants showed root hair burst after transition to the rapid growth phase.⁷⁰ Similarly, triple cngc5/6/9 mutants exhibit markedly attenuated cytosolic Ca²⁺ oscillations in shorter and branched root hairs.⁷¹

An increase in Ca²⁺ concentration could impact the pH at the cell surface and ROS generation.⁷² In root hairs, apoplastic ROS, apoplastic and cytoplasmic pH, and cytoplasmic Ca²⁺ oscillations occurred with a frequency of two to four peaks per minute and lag behind the growth oscillations by 8.0, 7.0, and 5.3 s, respectively.^34,73 RBOH proteins are activated by Ca²⁺ via direct binding of Ca²⁺ to the EF-hand motifs of RBOH proteins and Ca²⁺-induced phosphorylation of RBOH by kinases such as Ca²⁺-dependent protein kinase 5 (CPK5) and calcineurin B-interacting protein kinase (CIPK26).^10,74,75 ROS and Ca²⁺ form a positive feedback loop in root hair elongation.^34,51,76 ROS produced by RBOHC oxidase activate hyperpolarization-activated Ca²⁺ channels (HACCs) and stimulate Ca²⁺ influx into the cytoplasm.^40,77 In turn, a high level of Ca²⁺ triggers RBOHC oxidase activity by binding to EF-hands and promoting the phosphorylation of the residues S318/322, maintaining active growth.⁵¹ Recent studies showed that calcineurin B-like protein (CBL1)-CBL-interacting protein kinase (CIPK26) complex activates RBOHC by phosphorylation, triggering ROS generation.¹⁰

Surface pH affects cell wall viscosity by mediating the regulation of expansins, non-hydrolytic cell wall-loosening proteins, and certain cell wall-modifying enzymes.^78–81 Low pH promotes not only root hair initiation but also elongation. At root hair initiation sites, the pH in the apoplastic space is reduced from 5.0 to 4.5⁶². Low pH induces rapid extension of plant cell walls.^82,83 Membrane H⁺-ATPases (AHAs) directly regulate apoplastic pH, and AHA2 is highly expressed in growing root hairs.^84,85 FER interacts with RALF1 peptide to inhibit the activity of AHA2 possibly via Ser⁸⁹⁹ phosphorylation, thereby alkalizing the apoplast and suppressing cell expansion.^86,87 Ca²⁺ modulates extracellular pH via H⁺-ATPases, which alter cell wall structure during growth.⁸⁸ However, the root hair elongation stopped in rbohc mutants when exposed to pH 5.0, whereas rbohc mutants restored root hair elongation under pH 6.0³⁴. This indicates that extracellular pH and ROS function in a coordinated and complementary manner to regulate the expansion of the growing root hair.³⁴ However, little is known about the interaction of ROS and ions in modulating plant root hair development and growth.

4. Conclusions

Polarized root hair growth is tightly regulated by internal and external signals. ROS and Ca²⁺ are essential for the regulation of root hair elongation. Gradients and oscillations of apoplastic ROS, cytoplasmic Ca²⁺, and H⁺ are tightly linked and modulate cell wall dynamics during polar growth of root hairs. A better understanding of the mechanism underlying root hair expansion by ROS and Ca²⁺ has been achieved. However, it is unclear how ROS, Ca²⁺, and H⁺ regulate each other. Visualization of ROS and H⁺ dynamics in plant tissues and cells is challenging. Despite the identification of genes responsible for Ca²⁺ transport, further research is clearly needed to identify the Ca²⁺ transporters or channels that regulate final root hair size and to clarify how the channels are regulated by ROS. Information on the mechanism underlying root hair growth will enable the breeding of crops that can thrive under nutrient-deficient conditions and thereby increase yields.

Disclosure statement

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

Additional information

Funding

This research was supported by grants from the National Natural Science Foundation of China (31970300), the Natural Science Foundation of Jiangsu Province (BK20190025), Jiangsu Agriculture Science and Technology Innovation Fund (CX-20-2007), and the Fundamental Research Funds for the Central Universities (KYGD2022002) to Q.Z, and the National Natural Science Foundation of China (32100185) for X.Z.