Arabidopsis CAP1-mediated ammonium sensing required reactive oxygen species in plant cell growth

Abstract

[Ca²⁺]_cyt-associated protein kinase (CAP) gene 1 is a receptor-like kinase that belongs to CrRLK1L (Catharanthus roseus Receptor like kinase) subfamily. CAP1 has been identified as a novel modulator of NH₄⁺ in the tonoplast, which regulates root hair growth by maintaining the cytoplasmic Ca²⁺ gradients. Different expression pattern of tonoplast intrinsic protein (TIP2;3) in the CAP1 knock out mutant and wild type on Murashige and Skoog (MS) medium suggested that CAP1 influences transport activity to regulate the compartmentalization of NH₄⁺ into vacuole. Lower expression level of Oxidative Signal-Inducible1(OXI1) in the cap1-1 root and the abnormal reactive oxygen species (ROS) gradient in root hair of cap1-1 on MS medium indicated that ROS signaling involve in CAP1-regulated root hair growth. Wild-type-like ROS distribution pattern in the cap1-1 root hair can be reestablished in seedlings grown on NH₄⁺ deficient medium, which indicated that CAP1 functions as a sensor for NH₄⁺ signaling in maintaining tip-focused ROS gradient in root hairs polar growth.

Receptor-like kinases (RLKs) in Arabidopsis constitute a major gene family; over 600 genes encode a big transmembrane kinase proteins family. The diversity extracellular domains possess the ability in binding variable ligand, which makes this family important in signal transduction of growth and development. CrRLK1L (Catharanthus roseus Receptor like kinase), a subfamily of RLK, comprises of 17 members, 6 members have been identified so far, and they all function in cell growth regulation.^1-7

The first identified of this family is THESEUS1 (THE1) through screening the suppressor for cellulose-deficient mutant procuste1-1 (prc1-1).³ THE1 has a role in cell-wall-integrity-signaling pathway, cellulose synthesis deficient activated THE1 involved signal transduction to inhibit cell elongation. FERONIA (FER), the second characterized CrRLK1L, participates in many physiology processes: triggers the cessation of pollen tube growth, regulates brassinosteroid (BR) related cell elongation, and promotes root hair development.^4,5 The opposite function of FER in inhibition pollen tube growth and promotion root hair tip growth has been suggested own to the different tissue and different signal pathway.⁴ Other two members, ANXUR1 (ANX1) and ANXUR2 (ANX2) are closely related to FER and act redundantly to maintain pollen tube growth integrity where FER functions in arresting growth of the invading pollen tube while ANXs in maintaining the tip growth.^6,8 Another member of CrRLK1L, HERCULES1 (HERK1) closely related with FER and THE1, functions in regulating cell elongation in BR signaling.⁴

We have found another member of this family, CAP1, which also showed its function in cell growth regulation.⁷ The tonoplast-localized CAP1 has been identified as a novel modulator of NH₄⁺ homeostasis signal which regulates the polar growth of root hairs by maintaining tip-focused cytoplasmic Ca²⁺ gradients.⁷ The abnormal root hair growth of cap1-1 will be rescued by removing NH₄⁺ from the MS medium. In CAP1 related root hair growth, NH₄⁺ not only served as a kind of nutrient but a kind of signaling molecule to regulate root hair growth. The cytoplasmic NH₄⁺ level must be sensed and tightly controlled to avoid NH₄⁺ toxicity. Arabidopsis use ammonium transporters (AMTs) to absorb NH₄⁺,⁹ transcriptional and post-transcriptional regulation of these AMTs can prevent excess NH₄⁺ accumulation and toxicity.^10-12 The working model of CAP1 provide an example of the cytoplast NH₄⁺ concentrations sensing and modulation for the cells to keep a relative safe level of ammonium.

In yeast, a working model of the control of normal pseudohyphal growth by the ammonium transporter Mep2 and its positive regulator, Npr1 kinase, has been proposed and tested.^13-15 During NH₄⁺ limitation, Mep2 appears to act as a specific sensor in stimulating pseudohyphal development.¹⁶ Furthermore, decoupled mutants lacking only one of the dual functions have been isolated,^17,18 suggesting that both sensing and transport of ammonium are the independent processes accomplished by one protein. Our previous work identified CAP1 could functionally complement a yeast mutant defective in high-affinity NH₄⁺ uptake by Npr1 kinase which support the receptor kinase model for cytoplasmic NH₄⁺ homeostasis.⁷ These results suggested that CAP1 as a putative NH₄⁺ sensor participates in the regulation of polar growth in root hairs depending on NH₄⁺ homeostasis .

It is indicated that the capacity of CAP1 senses the internal NH₄⁺ status leads it to control the homeostasis by regulating compartmentalization to vacuole.⁷ Arabidopsis tonoplast intrinsic protein AtTIP2;1 and AtTIP2;3 participate in vacuolar ammonium compartmentation.^19,20 We observed that expression of TIP2;3 in cap1-1 roots is higher (about 2.6-fold, P < 0.05, Student t-test; Fig. 1) than that in wild type for seedlings grown on MS medium. This suggested that cap1 mutant shown the disordered ion transport system in cells. However, after 24 h of NH₄⁺ deprivation, similar expression level was showed in cap1-1 and wild type (P > 0.05, Student t-test; Fig. 1), which coincides with the similar root hair growth for both genotypes grown on the NH₄⁺ depletion medium. The different expression pattern of TIP2;3 in wild type and cap1-1 indicated that CAP1 functions in NH₄⁺ homeostasis partly due to the decrease of the transporter gene expression to avoid NH₄⁺ toxicity.

Figure 1.AtTIP2;3 gene expression for WT and cap1-1. After grew for 7 d on MS or 6 d on MS plus 24 h on NH₄⁺-free medium (MS-NH₄⁺), RNA were extracted from the roots. Three independent biological repeats were done, bars represent means ± SE.

It is well known that ROS pathway is important for plant growth and development.^21-23 Moreover, CAP1 has been shown involved in root hair growth.⁷ It would be interesting to know whether ROS acts as the important second messenger involved in the CAP1-mediated root hair growth regulatory process. H₂O₂ can directly induce OXI1 (Oxidative Signal-Inducible1) serine/threonine protein kinase gene expression, and OXI1 functions as a positive regulator of root hair tip growth.²⁴ Our qRT-PCR data showed that OXI1 has a higher transcript activity in the wild type root than in the mutant, about 3-fold of the mutant (P < 0.01, Student t-test; Fig. 2). The lower expression of OXI1 in the mutant coincides with the shorter root hairs, suggesting that ROS signaling may be recruited in this CAP1-controlled root hair growth.

Figure 2.OXI1 gene expression in WT and cap1-1. After grew on MS medium for 7 d, RNA were extracted from these seedlings roots. Data represent the mean and bars represent the standard error of the mean. Three independent biological repeats were done.

Deficient of CAP1 results in the abnormal root hairs, and lower expression of OXI1 revealed that cap1-1 may have a disturbed ROS level in the root. ROS also affect the establishment of Ca²⁺ gradients and polarized growth,²⁵ tip-focused ROS is necessary for maintaining polar growth.²² It has been shown that cap1-1 reestablished the tip-focused Ca²⁺ gradient when NH₄⁺ removed from the MS medium.⁷ In order to know how ROS distributed in root hairs of wild type and cap1 mutants, we examined ROS levels of root hairs by using 2, 7-dichlorofluorescin diacetate (H2DCF-DA). Wild type root hairs on MS showed tip-focused H₂O₂ concentration gradient, while cap1-1 root hairs showed no gradient or higher H₂O₂ concentration at the root hair bases (Fig. 3A, top panel). For analysis of the relative ROS content, we measured the average fluorescence intensity of the same areas every 10 µm apart from the hair apex. The relative fluorescence intensity was obtained by comparing with the hair apex region fluorescence. Five root hairs of each genotype have been used for this comparison. The results clearly showed CAP1 deficient mutant lost the tip-focused ROS gradient, significant difference showed between wild type and the mutant (P < 0.01, Student t-test; Fig. 3B, top panel). While this different distribution pattern between wild type and cap1-1 cannot see when seedlings grown on the NH₄⁺-free medium, both wild type and cap1-1 showed similar tip-focused ROS gradient (Fig. 3A and B, bottom panels; P > 0.05, Student t-test). These findings suggested that ROS signaling as the downstream components participate in the CAP1-regulated root hair tip growth. CAP1 mediates NH₄⁺ homeostasis and regulates the polar growth of root hairs not only by maintaining cytoplasmic Ca²⁺ gradients but also ROS gradient. Not only CAP1, other CrRLK1Ls regulated plant cell growth also involve ROS signal pathway. In study the regulation mechanism of FER on root hair tip growth, accumulation of ROS in root hair tip has been identified, suggested ROS as an important downstream components involve in this pathway.⁵ In the the1 mutant, the activation of genes participate in protection against ROS suggested that ROS production also mediated the THE1-controlled cell growth process.^3,26 Therefore, ROS signal is required for NH₄⁺ detoxification via CAP1 sensing NH₄⁺ concentration in the cytoplasm and regulating its uptake and relocation.

Figure 3. ROS accumulation pattern in root hairs of WT and cap1-1. (A) Transmission (top) and pseudocolor fluorescent (bottom) images of intracellular ROS levels in WT and cap1–1 root hairs for grew on both MS medium or NH₄⁺-free medium (MS-NH₄⁺). Images were acquired after loading the seedlings with H2DCF-DA. (B) Fluorescence intensity were measured at 4 equivalent areas along the root hairs and relative intensity were got by comparing with the fluorescence intensity of the hair apex (n = 5 root hairs, bars represent means ± SE). Top for root hairs on MS medium; bottom for root hairs on NH₄⁺-free medium.

Under anoxic soil conditions, ammonium will be the primary nitrogen source. AMT1 also uses a trans-activation allosteric mechanism involving the conserved C-terminus, indicating that this unique regulatory system was developed early in evolution and has been maintained.¹¹ The identification of CAP1 as an NH₄⁺-homeostasis modulator raises the possibility that CAP1 could sense NH₄⁺ signaling to regulate root hair growth. Undoubtedly this is just the beginning for understanding ammonium as a signaling molecule regulating ROS and Ca²⁺ distribution and functions in the CAP1-controlled root hair tip growth. Therefore, by combining system biological analysis with functional genomics, this will enable us to gain more insight into the evolutionary link among nutrient, toxicity, and signaling of ammonium and the construction of the holistic view of ammonium signaling in plants.