Roles and mechanisms of Ca²⁺ in regulating primary root growth of plants

ABSTRACT

Calcium (Ca²⁺) as a universal signal molecule plays pivotal roles in plant growth and development. It regulates root morphogenesis mainly through mediating phytohormone and stress signalings or affecting these signalings. In recent years, much progress has been made in understanding the roles of Ca²⁺ in primary root development. Here, we summarize recent advances in the functions and mechanisms of Ca²⁺ in modulating primary root growth in plants under normal and stressful conditions.

KEYWORDS:

• Ca²⁺
• primary root
• auxin
• hormone
• reactive oxygen species
• stress

Text

Root system including the primary root (PR) plays key roles in sustaining the aerial parts and absorbing nutrient and water in soil, and is vital for plant survival. PR growth is largely established by the division of meristem cells at the meristematic zone, and subsequent elongation of the cells at the elongation zone and differentiation of the cells at the maturation zone in the root tip.^1–3 It is coordinately regulated by multiple hormones such as auxin, cytokinin (CTK), gibberellins, ethylene (ET), brassinosteroid (BR) and abscisic acid (ABA), and by diverse environmental cues like water and nutrient availability, and light in plants.^1–4 Among these, auxin is extremely important. The biosynthesis, degradation, transport and distribution of auxin significantly impact PR growth.^1,2,4 Besides hormones and environmental factors, secondary messengers such as reactive oxygen species (ROS) (H₂O₂ and superoxide anion), nitric oxide, inositol trisphosphate (InsP3) and calcium (Ca²⁺) are crucial for the regulation of PR development.^5–8

Ca²⁺ as a nutrient and a signal molecule plays pivotal roles in regulating stomatal movement, responses to various biotic and abiotic stresses, and growth and development including root morphogenesis in plants.^6,9-11 It acts alone or through activating specific calcium sensors, for example, calmodulins, calcineurin B-like proteins (CBLs), calcium-dependent protein kinases (CPKs) and other Ca²⁺ binding proteins.^9,12 Accumulating evidence reveals that Ca²⁺ is essential for PR growth.^13–17 Previous studies have shown that Ca²⁺ participates in auxin-controlled maintenance of the quiescent center, and inositol trisphosphate-stimulated Ca²⁺ signaling affects auxin transport and Pin-FORMED (PIN) polarity during root growth of plants.^5,6,18 In this review, we discuss recent advances in the roles and regulatory mechanisms of Ca²⁺ in plant PR development under normal and stressful conditions.

Ca²⁺ regulates PR growth through affecting or mediating auxin signaling

Auxin is the central regulator of PR development. Its local accumulation and gradients in the growing root tip (with a maximum at the quiescent center) determine the maintenance and development of root apical meristem (RAM), from which root cells are produced. Auxin stimulates cell proliferation in the RAM.^1,2 Its accumulation and gradient formation depend on the auxin efflux transporters PINs and influx transporters AUX1/Like AUX1. Auxin at a high concentration induces SCF^TIR1-mediated degradation of Aux/IAA transcriptional repressors. The Aux/IAA degradation releases its binding proteins auxin response factors (ARFs), further activating downstream auxin-responsive genes to initiate diverse cellular responses.^19,20

There exist complex interactions between Ca²⁺ and auxin during root growth. Ca²⁺ impacts PR development through modulating auxin accumulation, transport and signaling, as well as the expression of many auxin-related genes. Ca²⁺ also functions as a transducer of auxin signaling during PR elongation. It has been demonstrated that nuclear calcium signatures influence auxin signaling, altering PR development. Leitão et al found that nuclear Ca²⁺ signals generated in meristematic cell regulate PR growth in Arabidopsis.¹⁶ Ca²⁺ signals (Ca²⁺ signatures) are modulated by nuclear membrane-localized Ca²⁺ channels DOES NOT MAKE INFECTIONS 1 (AtDMI1). Increases in the expression of AtDMI1 decrease the frequency of Ca²⁺ spikes, resulting in shorter PRs. By contrast, disruption of AtDMI1 increases the duration of Ca²⁺ spikes, causing longer PRs. Further study showed that AtDMI1 is required for PR growth. AtDMI1 negatively affects auxin accumulation and signaling, and the transcription of genes required for auxin biosynthesis, transport, and signaling in the RAM. These genes include Gretchen Hagen3 (GH3) gene GH3.8 (encoding the enzyme catalyzing the synthesis of IAA amide conjugates), PURIN7 (encoding 5ʹ-phosphoribosyl-4- (N-succinocarboxamide)-5-aminoimidazole synthetase), PINOID (encoding AGCVIII kinase) and PIN1. Auxin also modulates nuclear Ca²⁺ signals.¹⁶ Thus, nuclear Ca²⁺ signals integrate growth-related and auxin-dependent signals to regulate PR growth in Arabidopsis (Figure 1).

Figure 1. Mechanisms for nuclear Ca²⁺ regulating PR growth. Overexpression of AtDMI1 decreases Ca²⁺ frequency, inhibiting auxin signaling, decreasing the expression of PLETHORA1 and PLETHORA2, and retarding PR growth. Impairment of AtDMI1 increases Ca²⁺ duration, stimulating auxin signaling and the expression of PLETHORA1 and PLETHORA2, and PR growth. Thick up and down arrows, respectively, represent increases and decreases in their levels; thin arrows indicate positive regulation, and bars indicate negative regulation. The black cross means impairment of AtDMI1.

Ca²⁺-activated CBL-interacting protein kinase 25 (CIPK25) also positively regulates root meristem development by affecting auxin signalings.¹⁷ CIPKs and CBLs are crucial signaling modules that relay calcium signals, and phosphorylate downstream targets to modulate plant adaptations to diverse environmental stresses, and growth and development including root growth.^9,12,21 It has been reported that Arabidopsis mutant cipk25 exhibits short PR phenotype and has a slower root growth rate with fewer meristem cells. Overexpression of CIPK25 causes the opposite root phenotype to cipk25. The cipk25 lines have reduced acropetal and basipetal auxin transport, decreased responsiveness to auxin-evoked root growth inhibition, and arrested auxin-responsive promoter activity. Moreover, mutation of CIPK25 leads to clear reduction of the expression of auxin-responsive genes including PIN1, PIN2, PIN3 and PIN7, and ARF5, ARF6 and ARF8, but causes the increase in the expression of short hypocotyl 2 (SHY2), one of key member of Aux/IAA family (IAA3).¹⁷ Further studies revealed that disruption in SHY2 effectively rescues the short root phenotype of cipk25. Moreover, CIPK25 interacts with CBL4 and CBL5, suggesting that Ca²⁺ may activate CBL4 and CBL5, further motivates CIPK25 and modulates PR growth in Arabidopsis (Figure 2). Additionally, chickpea CIPK25 (CaCIPK25) has shown to facilitate PR development after being overexpressed in tobacco,²² reflecting the conservative function of CIPK25 in the regulation of root growth in plants.

Figure 2. Mechansims for cytosolic Ca²⁺ modulating PR growth. Cytosolic Ca²⁺ signalings regulate and mediate the signalings of auxin, CTK, BR, ABA, ET, and Cd and salt stress to affect PR development. Ca²⁺ regulates CBL4/5 and CIPK25, influencing auxin transport and response, and promoting PR growth. CTK inhibits CIPK25, dampening PR elongation. Ca²⁺ elevations regulated by CNGC2 and AtrbohD/F suppress PR growth. ABA activates CPK4/6, further promotes ET biosynthesis, inhibiting PR growth. ABA activates MPK6, leading to the reduction of ROS and Ca²⁺ levels and shortened roots. CMI1- and AtGLR3.6-impacted increases of Ca²⁺ facilitate PR growth. FER-modulated Ca²⁺ inhibits salt-caused damage of cell-wall integrity, and positively impacts root growth. Ca²⁺-activated CML38 antagonizes AtRALF1, which interferes with BR signaling, modulating PR elongation. Ca²⁺ attenuates Cd stress-induced disorder of auxin signaling, facilitating PR growth. Different auxinic compounds like IAA, IBA, NAA and 2,4-D elicit distinct Ca²⁺ signalings, favoring PR development. Arrows show positive regulation, and bars indicate negative regulation. The dashed line means uncharacterized regulation. The circle with a cross in it reveals damage of cell-wall integrity.

Recently, a study revealed that Ca²⁺-binding protein CMI1/KRP1 [Ca²⁺-dependent modulator of ICR1 (Interactor of constitutively active ROP1)/KIC-related protein 1] functions in the root meristem growth in Arabidopsis. It modulates auxin response and conveys auxin-triggered cytoplasmic Ca²⁺ signaling, positively affecting PR growth (Figure 2).¹⁵ Auxin has been observed to upregulate the expression of gene CMI1 through auxin receptors TIR1/AFB (Transport inhibitor1/Auxin-signaling F-Box) proteins. CMI1 defect causes the increased auxin response, altered expression of PIN2 protein, arrested increase of auxin-induced Ca²⁺, reduced root meristem size and shortened PRs. Moreover, CMI1 regulates auxin-elicited Ca²⁺ signaling in a cell type/tissue-specific manner. It acts as a monomer, and is able to bind Ca²⁺ at low concentrations (10⁻⁹–10⁻⁸ M), which leads to the change of its secondary structure.¹⁵

In addition, CNGC2 (Cyclic nucleotide gated ion channel 2), also named DND1 (Defense, no death1), translocates Ca²⁺ signaling and mediates auxin responses, thereby positively impacting auxin-caused inhibition of PR growth in Arabidopsis.¹⁴ CNGCs are nonselective cation channels that have been addressed to act as Ca²⁺ channels in plants. Loss-of-function mutant cngc2/dnd1 displays defective auxin responses and less sensitivity to auxin-repressed PR growth. The effects of the mutant can be rescued by mutation of the gene repressor of defense, no death1 (RDD1), which encodes the auxin biosynthesis gene YUCCA6. Moreover, cngc2/dnd1 exhibits clear alterations of auxin-triggered enhancement of cytosolic Ca^2+.14 These results support the notion that CNGC2 inhibits PR development via influencing auxin-mediated Ca²⁺ signaling in Arabidopsis (Figure 2).

Ca²⁺ has also been observed to alleviate cadmium (Cd) caused PR growth inhibition by attenuating Cd-evoked disorder of auxin homeostasis in roots of Arabidopsis.²³ Cd treatment clearly inhibits PR elongation, and modifies the level and distribution of auxin and the expression of some genes involved in auxin biosynthetic, catabolic and transport, for example, PAT1 (PR-anthranilate transferase1), CYP79B2 (a cytochrome P450 gene), CYP79B3, NIT2 (Nitrilase2), TAA1 (Tryptophan aminotransferase1), AUX1, PIN2, PIN4, GH3.9 and GH3.17 in roots. Ca²⁺ application markedly alleviates or changes these effects of Cd, promoting PR growth (Figure 2).²³ Another study indicated that Cd- or Ca²⁺ deficiency-induced transcription factor MYB59 negatively regulates PR growth by affecting Ca²⁺ homeostasis and signaling in Arabidopsis.²⁴ Yet, whether MYB59 modulates auxin homeostasis need to be further investigated.

Additionally, different cytosolic calcium signatures have been found to be induced by diverse auxin and auxinic compounds, and modulate PR growth of Arabidopsis.²⁵ Treatments with indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), 1-naphthalene acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D) or mecoprop [2-(4-chloro-2-methylphenoxy) propanoic acid] lead to marked increases in cytosolic Ca2+ levels in roots and PR length (Figure 2). Moreover, each of the compounds triggers a specific and unique cytosolic Ca2+ signature, implying that different Ca²⁺ signalings evoked by distinct auxin types may have distinguishing effects on PR growth in plants.²⁵

Ca²⁺ modulates PR development by impacting CTK signaling

CTK is a crucial modulator of PR growth. It suppresses RAM growth by promoting cell differentiation.²⁶ In Arabidopsis, CTK signaling is mainly mediated by histidine kinases, which activate Arabidopsis response regulators (ARRs), further stimulating the expression of CTK target genes and initiating downstream responses.²⁶ CTK regulates root growth through interacting with auxin. CTK activates ARR1, which upregulates the expression of SHY2, resulting in the inhibition of ARF activity, downregulation of PINs and repression of root cell division. Conversely, auxin facilitates SHY2 degradation, activates ARFs, and stimulates expression of PINs and cell division.^26,27 CTK-dependent auxin degradation also contributes to root growth regulation in plants.²⁸ In Arabidopsis, CIPK25 has shown to have positive roles in PR growth.¹⁷ Exogenous application of CTK decreases the expression of CIPK25, which is dependent upon ARR1 and ARR12, two important components of CTK signaling in controlling root meristem size. Moreover, loss-of-function of CIPK25 promotes the expression of SHY2, and SHY2 mutation completely rescues the short root phenotype of cipk25 (Figure 2). Treatment with CTK also results in further reduction of PIN1 expression level in cipk25 relative to wild type (WT), in agreement with CTK-caused additional decrease of meristematic cell number in cipk25 compared with WT plants.¹⁷ These results indicate that CIPK25 promotes root growth through integrating CTK and auxin signalings (Figure 2).

Ca²⁺ positive impacts PR elongation through interfering with BR signaling

BR is an important hormone for PR development in plants.²⁹ Ca²⁺ promotes PR growth by influencing BR signaling.^30,31 Ca²⁺-regulated calmodulin-like protein 38 (CML38), a secreted protein, has been addressed to physically interact with rapid alkalinization factor 1 (AtRALF1), a small secreted peptide hormone in Arabidopsis. CML38 promotes but AtRALF1 suppresses PR elongation.^30–32 CML38 antagonizes AtRALF1-mediated root inhibition in Ca²⁺-dependent but not pH-dependent manner.³¹ The transgenic plants overexpressing AtRALF1 exhibit reduced root cell size, and AtRALF1 silencing leads to the opposite effects. BR reagent brassinolide (BL) promotes PR growth at concentrations of 0.01, 0.1, and 1 nM in WT plants of Arabidopsis. However, root growth of AtRALF1 overexpressors is less sensitive to these doses of BL whereas that of AtRALF1-silenced plants is sensitive to BL. Moreover, AtRALF1 stimulates the expression of two BR-downregulated genes implicated in BR biosynthetic pathway: the cytochrome P450 monooxygenases constitutive photomorphism and dwarfism and dwarf4. BL treatment reduces the transcriptional levels of AtRALF1-inducible genes like proline rich protein 1 (AtPRP1), AtPRP3, hydroxyproline-rich glycoprotein 2 (AtHRPG2) and xyloglucan endotransglucosylase4 (TCH4).³⁰ Hence, Ca²⁺ affects the activity of AtRALF1 through activation of CML38, further modulating BR signaling and PR growth (Figure 2).

Ca²⁺ serves roles in ABA-inhibited PR growth via transducing ROS signals or affecting ethylene biosynthesis

ROS has been addressed to play critical roles in the regulation of stomatal closure, adaptations to diverse biotic and abiotic stress, and plant growth and development.^33–36 They also mediate hormone and stress signalings, and regulate RAM and PR development.^37–39 ROS are generated through both enzymic and non-enzymic reactions in plants. Plasma membrane NADPH oxidases, also known as respiratory burst oxidase homologues (Rbohs), have been documented to be important producers of ROS.⁴⁰ Rbohs are involved in the regulation of numerous biological processes including stomatal movement, responses to pathogen attack and various abiotic stresses, and growth and development in plants.^41–44 They are also key regulators of root growth.^13,37,44,45 Rbohs possess two Ca²⁺-binding EF-hand motifs in the cytoplasmic N-terminal region that regulate their activity by Ca²⁺. Rbohs also promote the increase in cytoplasmic Ca²⁺ levels in guard cells and root cells in ABA signaling.^46,47 Ca²⁺ mediates ROS signaling to positively influence ABA-inhibited PR growth.¹³ In Arabidopsis, ABA-repressed PR elongation is impaired in the double mutants of NADPH oxidases AtrbohD and AtrbohF.⁴⁸ Moreover, disruptions of AtrbohD and AtrbohF clearly abolish ABA-promoted ROS generation, cytosolic Ca²⁺ increases, and the activation of plasma membrane Ca²⁺-permeable channels. Exogenous H₂O₂ effectively activates the Ca²⁺ currents in PRs of AtrbohD and AtrbohF mutant seedlings, demonstrating that Ca²⁺ functions downstream of ROS to regulate ABA-inhibited PR growth.¹³ Differently, Han et al found that Arabidopsis mitogen-activated protein kinase 6 (MPK6) negatively affects ABA- or moderate H₂O₂-induced increases in cytosolic Ca²⁺, further facilitating the inhibition of cell expansion of PRs by ABA or H₂O₂.⁴⁹ That is, Ca²⁺ is negatively correlated with PR elongation suppressed by ABA (Figure 2). The reason and underlying mechanism for the discrepancy of the role of Ca²⁺ in the two studies need to be further investigated.

Evidence revealed that the activities of AtrbohD and AtrbohF are modulated by Ca²⁺ and Ca²⁺ sensors.⁵⁰ In Arabidopsis, AtrbohD is phosphorylated and activated by CPK5 during innate immune responses, and AtrbohF is subject to regulation by CBL1, CBL9, CIPK26 and CIPK11.^50–52 Both CIPK26 and CIPK11 interact and phosphorylate AtrbohF. Co-expression of CIPK26 with CBL1 or CBL9 and co-expression of CIPK11 with CBL1 or CBL9 clearly increase ROS generation by AtrbohF in a human embryo kidney cell line HEK293T. Moreover, direct Ca²⁺ binding to AtrbohF serves a role in the activation of the NADPH oxidase by phosphorylation of CBL1/CIPK26 or CBL1/CIPK11 complexes in HEK293T cells.⁵⁰ Yet, whether CPK5, CBL1, CBL9, CIPK26 and CIPK11 contribute to ABA-inhibited PR growth through activating AtrbohD and AtrbohF needs to be addressed in future studies.

Besides, ET signaling and level exert effects on ABA inhibition of PR growth.⁵³ ABA is able to activate CPK4 and CPK11, which phosphorylate and stabilize ACC (1–aminocyclopropane-1–carboxylate synthase) synthase 6, one of the key enzymes of ET biosynthesis, further increasing ET production in Arabidopsis.⁵⁴ Therefore, Ca²⁺ may positively modulate ABA-suppressed PR elongation by enhancing the activities of CPK4 and CPK11 and biosynthesis of ET in plants.

Ca²⁺ facilitates PR growth by regulating cell wall reformation

In plants, the expansion of cells including PR cells has to undergo the relaxation of cell walls without the loss of cell integrity.⁵⁵ Therefore, the maintenance of cell-wall integrity is essential for root growth under normal especially stress conditions. Ca²⁺ signal has been demonstrated to exert effects in maintaining cell-wall integrity and modulates PR elongation under salt stress.⁵⁶ In the presence of high concentration of salt, PR growth of Arabidopsis WT plants is inhibited; however, PRs from the mutant of gene FERONIA (FER), which encodes a receptor kinase localized in plasma-membrane, are defective. Further studies suggest that FER is responsible for the maintenance of cell-wall integrity under salinity stress. FER interacts directly with pectin, which protects cell walls against saline stress-damage through cross-linking. Importantly, FER triggers cell-specific cytosolic Ca²⁺ transients that are necessary to maintain cell-wall integrity under salt stress (Figure 2).⁵⁶ Accordingly, Ca²⁺ signal promotes PR growth through facilitating pectin cross-linking under saline condition.

In previous studies, CML38 has shown to interplay with and counteract AtRALF1-inhibited PR growth in Arabidopsis. Moreover, AtRALF1 is able to induce the expression of genes involved in cell-wall rearrangement including AtPRP1, AtPRP3, AtHRPG2 and TCH4,^30,31 highlighting the important role of the CML38-relayed Ca²⁺ signal in regulating cell wall reformation and PR development.

Other Ca²⁺-dependent mechanisms involved in PR growth

Experimental data suggest that Arabidopsis Ca²⁺-ATPase 8 (ACA8) mediates Ca²⁺ signaling, coordinating PR development in the absence of sucrose.⁵⁷ ACA8 is localized in the plasma membrane, and plays a role in the establishment of the root meristem when sucrose is unavailable. ACA8 downregulates the expression of cell cycle regulator genes, for instance, CYCB (Cyclin B), CYCD and CDKB in roots under sucrose deprivation.⁵⁷

Ca²⁺ signal may affect PR growth by modulating PLETHORA1 and PLETHORA2, two essential regulators of root development. It is because that AtDMI1, the regulator of nuclear Ca²⁺ signature, downregulates the expression of PLETHORA1 and PLETHORA2 during PR growth in Arabidopsis (Figure 1).¹⁶

Ca²⁺ may also function in PR development controlled by glutamate receptor-like protein AtGLR3.6 in Arabidopsis.⁵⁸ AtGLR3.6 positively modulates root meristem size and PR growth by altering cell division and negatively impacting the level of cyclin-dependent kinase inhibitor Kip-related protein 4 (KRP4) in an auxin-dependent manner. Mutation in AtGLR3.6 causes retarded PR growth and upregulated expression of KRP4. Furthermore, disruption of AtGLR3.6 results in significant reduction of cytosolic Ca²⁺ elevation in roots. Ca²⁺ application decreases the abundances of KRP4 and induces PR elongation, indicating that Ca²⁺ signal plays a role in AtGLR3.6-stimulated PR growth in Arabidopsis.⁵⁸ However, the relationship of Ca²⁺ and auxin signaling during root growth was not investigated.

In summary, Ca²⁺ mediates the signalings of multiple hormones particularly auxin, environmental cues and plant development to regulate PR growth. Changes in Ca²⁺ levels and Ca²⁺ signature in both the nucleus and cytoplasm have great contributions to the development of PRs. Numerous proteins include Ca²⁺-permeable channels, CBLs, CIPKs, CPKs, CMLs, Ca²⁺-binding proteins and other proteins are involved in the modulation of Ca²⁺ signalings and root growth (Figures 1 and 2). Despite these progresses, many open questions about Ca²⁺ affecting PR development of plants remain. For instance, how does nuclear Ca²⁺ coordinate cytosolic Ca²⁺ during root morphogenesis? What are the main sources of cytosolic Ca²⁺ (apoplast or diverse organelles)? What is the detailed relationship between Ca²⁺ concentrations, Ca²⁺ signal property and PR development? Moreover, how does Ca²⁺ simultaneously coordinate multiple different internal and external cues to regulate PR growth? What are the underlying mechanisms? These questions need to be thoroughly investigated in the coming days.

Disclosure of potential conflicts of interest

The authors declare that they have no conflicts of interest in the study.

Additional information

Funding

This work was supported by the National Natural Science Foundation of China [31870248].