The interaction between glucose and cytokinin signaling in controlling Arabidopsis thaliana seedling root growth and development

ABSTRACT

Cytokinin (CK) and glucose (GLC) control several common responses in plants. There is an extensive overlap between CK and GLC signal transduction pathways in Arabidopsis. Physiologically, both GLC and CK could regulate root length in light. CK interacts with GLC via HXK1 dependent pathway for root length control. Wild-type (WT) roots cannot elongate in the GLC free medium while CK-receptor mutant ARABIDOPSIS HISTIDINE KINASE4 (ahk4) and type B ARR triple mutant ARABIDOPSIS RESPONSE REGULATOR1, 10,11 (arr1, 10,11) roots could elongate even in the absence of GLC as compared with the WT. The root hair initiation was also found defective in CK signaling mutants ahk4, arr1,10,11 and arr3,4,5,6,8,9 on increasing GLC concentration (up to 3%); and lesser number of root hairs were visible even at 5% GLC as compared with the WT. Out of 941 BAP regulated genes, 103 (11%) genes were involved in root growth and development. Out of these 103 genes, 60 (58%) genes were also regulated by GLC. GLC could regulate 5736 genes, which include 327 (6%) genes involved in root growth and development. Out of these 327 genes, 60 (18%) genes were also regulated by BAP. Both GLC and CK signaling cannot alter root length in light in auxin signaling mutant AUXIN RESPONSE3/INDOLE-3-ACETIC ACID17 (axr3/iaa17) suggesting that they may involve auxin signaling component as a nodal point. Therefore CK- and GLC- signaling are involved in controlling different aspects of root growth and development such as root length, with auxin signaling components working as downstream target.

KEYWORDS:

• Arabidopsis
• cytokinin
• glucose
• lateral roots
• root hairs
• root length
• signaling

Introduction

The root system architecture mainly consists of primary root (PR), lateral roots (LR) and root hairs. In nature, root is constantly exposed to a variety of adverse environmental conditions such as temperature fluctuations, flood, drought, soil compaction, lack of essential nutrients, exposure to toxic minerals and pathogens, etc.Root is a highly dynamic structure and can adjust their growth as per changing environmental conditions by modulating its length; thickness; tropic movement; lateral root and root hair formation.^1,2,3,4,5,6 The optimal root architecture is essential to provide fitness to the plant in nature since it provides mechanical support to the aerial parts of the plant body, anchorage to the soil, helps in water and nutrients uptake from the soil, synthesize metabolites, can serve as energy storage organ, and interacts with symbiotic organisms.^3,4 Therefore, a detailed understanding of all the pathways and their cross-talk involved in root growth and development is an important prerequisite in plant biology.

Phytohormones play significant roles as a rapid endogenous signaling molecules to determine optimal root architecture in response to these cues.^7,8,9 The core of hormone regulatory network involved in shaping root growth pattern comprises of 2 well-known classical plant hormones, cytokinin (CK) and auxin, interacting at the levels of metabolism, transport, signaling and gene expression.^{8,10,11,12,13,14} CKs are essential molecules playing key roles during different phases of plant growth and development.¹⁵ CK signaling involves multistep phosphorelay which is the more advanced and complex version of bacterial 2 component system (TCS).^13,15,16 ARABIDOPSIS HISTIDINE KINASE2 (AHK2), AHK3 and AHK4 are membrane spanning proteins that serve as CK receptors and are primarily localized at the ER membrane.^17,18,19 Among CK receptors, AHK4 uniquely possesses dual activity since it leads to phosphorylation of AHPs in the presence of CK, while it causes dephosphorylation of AHPs in the absence of CK.²⁰ Five His phosphotransfer proteins (AHP1-AHP5) function as redundant positive regulators, whereas AHP6 (pseudo-HPt) serves as a negative regulator of CK signaling cascade.²¹ AHPs continuously shuttle between the cytoplasm and nucleus independent of their phosphorylation status or of CK level. Only nuclear phosphorylated AHPs can phosphorylate ARABIDOPSIS RESPONSE REGULATORS/ARRs.²² The ARRs are fall into 2 categories, type A ARRs (ARR3-ARR9, ARR15-ARR17) and type B ARRs (ARR1, ARR2, ARR10-ARR14, ARR18-ARR21). Phosphorylated type B response regulators can induce the transcription of type A ARRs, CYTOKININ RESPONSE FACTORS (CRFs) and some other CK early-responsive genes. Type A response regulators act as negative regulators and attenuates CK signaling whereas type B response regulators function as positive regulators of CK signaling cascade.²³

In literature, there are reports which suggest that sugar (Glucose/GLC) can also affect root growth and development.^{3,24,25,26,27} GLC signaling pathways existing in Arabidopsis are mainly of 2 types; i) AtHXK1 (HEXOKINASE1) -dependent pathway (AtHXK1-signaling function dependent and AtHXK1-catalytic activity dependent) and, ii) AtHXK1-independent pathway which involves G-protein mediated signaling.²⁸ In AtHXK1 signaling function dependent pathway, HXK1 acts as an intracellular GLC sensor. HXK1 loss-of-function mutant (S177A mutation) glucose insensitive2.1 (gin2.1) retained the signaling function (GLC sensing) but not the catalytic activity (GLC phosphorylation).²⁹ In AtHXK1-independent pathway, plasma membrane bound protein regulator of G-protein signaling (RGS1) acts as an external glucose sensor.³⁰

Sugar and CK are key molecules in plants and there are several reports indicating that both these molecules regulate a myriad of similar processes. CK and GLC can act agonistically,^31,32,33,34 antagonistically,^29,35 and independent of each other.³⁶ Earlier our group reported a novel root directional response termed as “CK-induced root growth response” which is significantly enhanced when GLC is supplemented in the medium.³ In addition, our group also reported that at whole genome level, large number of genes (89%) were agonistically regulated by GLC and CK.³⁷ GLC could also affect genes involved in CK biosynthesis, degradation and signaling.³⁷ Physiologically, both GLC and CK could control root growth in light, hypocotyl length in dark, chlorophyll and anthocyanin content.³⁷ Mishra et al. (2009) reported that increasing GLC concentrations affect primary root length, lateral root numbers, root hairs and gravitropic curvature of the roots of Arabidopsis seedlings.²⁵ Increasing GLC concentrations can also influence root length, lateral roots, root deviation and root hair elongation in auxin signaling mutants indicating that auxin signaling is involved in controlling GLC-regulated root phenotypes.²⁵ GLC acts through G-protein complex to attenuate auxin-mediated bimodality in regulating lateral root formation.²⁶ High GLC concentrations reduced root meristem size by repressing the accumulation of PIN1 and thus reducing auxin level.²⁷ In summary, CK, auxin and GLC control several root-related responses. Here, we are showing how CK- and GLC-signaling interact in controlling root growth and development in Arabidopsis.

Results

Involvement of GLC signaling components in controlling root growth

To find out the effect of CK and GLC on root length; light-grown 5-d-old Columbia (Col) seedlings were transferred to different concentrations of GLC and 6-benzylaminopurine (BAP) containing half strength MS medium in the light for 3-d as mentioned by Kushwah and Laxmi (2014).³⁷ BAP decreased the root length in Col at all the concentrations tested. GLC supplementation in the medium increased the root length. The increase in root length was found to be more at 1% GLC and 3% GLC, whereas found less at 5% GLC when compared with the untreated control (Fig. 1).³⁷ Thus for regulating primary root length, GLC and CK work antagonistically at lower GLC concentrations (1% and 3%) and act agonistically at higher GLC concentration (5%) (Fig. 1). CK-sensitivity of GLC signaling mutants was assayed to find out whether GLC-CK interaction controls root length via HXK1-dependent or HXK1-independent pathway. HXK1-dependent pathway mutant gin2 was found resistant/less sensitive at all the GLC concentrations tested (Fig. 2) whereas HXK1-independent pathway mutants rgs1, G-PROTEIN ALPHA SUBUNIT1 (gpa1) and THYLAKOID FORMATION1 (thf1) were found to have similar sensitivity (Fig. 3A and B), toward CK for root length control as compared with WT. These results altogether indicate that CK interacts with GLC via HXK1 dependent pathway to control root length.

Figure 1. GLC-CK signaling interaction in WT for root length control: BAP decreased the root length in Col at all the concentrations tested. GLC supplementation in the medium increased the root length. The increase in root length was found to be more at 1% GLC and 3% GLC, whereas found less at 5% GLC when compared with the untreated control. For controlling root length, GLC and CK work antagonistically at lower GLC concentrations but act agonistically at higher GLC concentration. Data shown is the average root length and error bars represent SD. Means accompanied by the different letters in a group at mentioned GLC concentration are statistically different (All Pairs, Tukey HSD post ANOVA test at P≤ 5%).

Figure 2. HXK1-dependence of CK signaling for root length control: Root growth of L. erecta and HXK1-dependent pathway mutant gin2.1. The gin2.1 mutant was found less sensitive at all the GLC concentrations tested to CK for root length control as compared with WT. Data shown is the average root length and error bars represent SD. Means accompanied by the different letters in a group at mentioned GLC concentration are statistically different (All Pairs, Tukey HSD post ANOVA test at P ≤ 5%).

Figure 3. Root length of HXK1-independent pathway mutants: (A and B) The HXK1-independent pathway mutants rgs1.1, rgs1.2, thf1.1 and gpa1.1 were found to have similar sensitivity to CK for root length control as compared with WT. Data shown is the average root length and error bars represent SD. Means accompanied by the different letters in a group at mentioned GLC concentration are statistically different (All Pairs, Tukey HSD post ANOVA test at P ≤ 5%).

Involvement of CK signaling components in controlling root growth

GLC-sensitivity of CK signaling mutants was assayed to find out the role of CK signaling elements in controlling root growth. In CK-perception mutant ahk4 and type B ARR mutant arr1,10,11; root length was found more even in the absence of GLC as compared with the WT (Fig. 4A).

Figure 4. CK signaling dependence of GLC response for root length, lateral root numbers and root hairs: (A) In CK-receptor mutant ahk4 and type B ARR triple mutant arr1,10,11, root length was found to be more even in the absence of GLC as compared with the WT. (B) CK perception mutant ahk4, type B ARR triple mutant arr1,10,11 and type A ARR mutant arr3,4,5,6,8,9 displayed similar lateral root numbers as compared with the WT. (C) The root hair initiation was defective in ahk4, arr1,10,11 and arr3,4,5,6,8,9 on increasing GLC concentration and lesser number of root hairs were visible even on 5% GLC as compared with WT. Data shown is the average root length/lateral root numbers and error bars represent SD. Student's t test, P < 0.05, *control vs treatment.

According to Mishra et al. (2009), GLC supplementation in the medium increases lateral roots in WT.²⁵ CK perception mutant ahk4, type B ARR triple mutant arr1,10,11 and type A ARR sextuple mutant arr3,4,5,6,8,9 displayed similar lateral root numbers as compared with the WT (Fig. 4B).

The increasing GLC concentration also increases root hair initiation and elongation.²⁵ In auxin signaling mutants, GLC at higher concentrations could cause root hair initiation, however root hair elongation was severely compromised.²⁵ The root hair initiation was found defective in CK signaling mutants ahk4, arr1,10,11 and arr3,4,5,6,8,9 on increasing GLC concentration (up to 3%) and lesser number of root hairs were visible even at 5% GLC as compared with the WT (Fig. 4C).

In microarray data published by Kushwah and Laxmi (2014), out of 941 genes regulated by BAP (0% GLC + 0 M BAP versus 0% GLC + 1 µM BAP), 103 (11%) genes were involved in root growth and development (Fig. S1A).³⁷ Out of these 103 genes, 60 (58%) genes were also regulated by GLC (Fig. S1A). Out of these 103 BAP-regulated genes which were involved in root growth and development, 6 genes were also involved in auxin biosynthesis and signaling (Fig. S1B). Out of these 6 genes, 4 genes were also regulated by GLC (Fig. S1B). Among the genes involved in auxin biosynthesis and signaling, one of the important member is SHORT HYPOCOTYL2 (SHY2)/ INDOLE-3-ACETIC ACID3 (IAA3) (Fig. S1B). The size of root meristem is established by a fine balance between the antagonistic interaction of auxin and CK which converges on SHY2.³⁸

GLC could regulate 5736 genes (0% GLC vs. 3% GLC), which include 327 (6%) genes involved in root growth and development (Fig. S2A). Out of these 327 genes, 60 (18%) genes were also regulated by BAP (Fig. S2A). Out of these 327 GLC-regulated genes which are involved in root growth and development, 12 genes are also involved in auxin biosynthesis and signaling (Fig. S2B). Out of these 12 genes, only one gene was also regulated by BAP (Fig. S2B). The genes involved in auxin biosynthesis and signaling are, LIKE AUXIN RESISTANT 1 (LAX1), ROOTY 1 (RTY1), PIN-FORMED 1 (PIN1), AUXIN-INDUCED IN ROOT CULTURES 12 (AIR12), AUXIN RESISTANT 4 (AXR4), HOMEOBOX 53 (HB53), P-GLYCOPROTEIN 1 (PGP1), MILDEW RESISTANCE LOCUS O 11 (MLO11), NAC DOMAIN CONTAINING PROTEIN 1 (NAC1), TRANSPARENT TESTA GLABRA 1 (TTG1) (Fig. S2B). These results altogether indicate that several genes involved in auxin signaling and homeostasis are also commonly regulated by CK and GLC.

Involvement of auxin signaling components in controlling root growth

CK and GLC sensitivity of auxin signaling mutants was assayed to find out if both these pathways culminate at auxin signaling or not. The auxin signaling mutants TRANSPORT INHIBITOR RESPONSE1 (tir1), AUXIN RESISTANT1.3 (axr1.3) and Aux/IAA gain of function mutant SOLITARY ROOT1 (slr1)/ INDOLE-3-ACETIC ACID14 (iaa14) were found to have similar sensitivity, whereas Aux/IAA gain of function mutant AUXIN RESPONSE3 (axr3)/INDOLE-3-ACETIC ACID17 (iaa17) was found highly resistant to different concentrations of CK and GLC for root length control as compared with the WT (Fig. 5).

Figure 5. Involvement of auxin signaling downstream to both GLC and CK in controlling root growth: The auxin signaling mutants tir1, axr1.3 and slr1 were found to have similar sensitivity whereas, axr3 mutant was found highly resistant for different concentrations of GLC and CK for root length control as compared with the WT. Data shown is the average root length and error bars represent SD. Means accompanied by the different letters in a group at mentioned GLC concentration are statistically different (All Pairs, Tukey HSD post ANOVA test at P≤ 5%).

Discussion

There are various reports in literature which indicates that for many plant responses, GLC and CK act antagonistically whereas, for others they act agonistically and for some responses both these molecules function independently of each other.^{3,29,31,32,33,34,35,36,37}

HXK-dependent pathway mutant gin2 was found less sensitive to all the GLC concentrations tested toward CK in terms of root length inhibition, whereas HXK-independent pathway mutants rgs1, gpa1 and thf1 were found to have similar sensitivity toward CK for root length regulation as compared with the WT. Therefore, similar to hypocotyl length regulation in dark, for root length control also CK interacts with GLC via AtHXK1-dependent pathway.³⁷

In CK-perception mutant ahk4 and type B ARR triple mutant arr1,10,11, root length was found to be more even in the absence of GLC as compared with the WT. This suggests that the positive elements of CK signaling might contribute toward non-elongation of root in the absence of GLC. The root length changes were significantly compromised in several auxin signaling mutants on increasing GLC concentration.²⁵ This suggests that CK and GLC signaling may also work via affecting auxin signaling as a downstream component.

CK perception mutant ahk4, type B ARR triple mutant arr1,10,11 and type A ARR sextuple mutant arr3,4,5,6,8,9 displayed similar lateral root numbers as compared with the WT. Lateral root induction was significantly compromised in auxin perception mutant tir1 and signaling mutants slr1/iaa14, axr2/iaa7 and axr3/iaa17 on increasing GLC concentration.²⁵ This suggests that GLC mediated regulation of lateral root numbers involve auxin signaling rather than CK signaling.

The root hair initiation was found defective in CK signaling mutants ahk4, arr1,10,11 and arr3,4,5,6,8,9 and only few root hairs were visible at high GLC concentration (5%). The root hair density and length significantly affect the root-soil interface.⁶ According to Mishra et al. (2009), increasing GLC concentrations promote root hair initiation but the root hair elongation is compromised in auxin mutants indicating significant role of auxin signaling in GLC mediated root hair elongation.²⁵ High GLC is able to overcome the repression of root hair initiation in AUX/IAA gain-of-function mutants (root hairless mutants) slr1, axr2 and axr3.²⁵ This suggests that for GLC mediated root hair initiation CK signaling is involved whereas, for GLC mediated root hair elongation auxin signaling is involved.

Altogether, these results suggest that CK receptor, type B and type A RRs are involved in various GLC induced root growth and developmental changes. There are previous reports showing that type A ARRs integrate CK signaling with circadian rhythm, light, pathogen and other hormone signaling cascades.³⁸ The phosphorylated ARR4 interacts with phyB and therefore serves as a converging node for the integration of CK and light signaling.^39,40 For shoot meristem development and root stem-cell niche establishment, auxin and CK signaling converges on ARR7 and ARR15 (type A RRs).⁴¹ Type A ARRs are involved in GLC mediated control of hypocotyl length in dark.³⁷ Here, we are reporting that for GLC-CK interaction, CK receptor as well as type A and type B RRs act as downstream components.

It is now evident from several studies that physiologic phenomenon are regulated in a complex web like manner by the interaction of several hormones.⁴² CK is known to interact either in synergistic or antagonistic manner with other hormones, especially ethylene and auxin.^38,43,44 There are several reports indicating the cross-talk between auxin and CK in controlling root and lateral roots growth and development.^{14,15,38,41,45,46} There are also various reports confirming that interaction between sugar and auxin controls root and lateral roots growth and development.^25,26,47 According to Mishra et al. (2009), increasing GLC concentrations can affect primary root length, gravitropism, lateral roots and root hairs in auxin perception and signaling mutants (TIR1, AXR2/IAA7, AXR3/IAA17 and SLR1/IAA14).²⁵ GLC acts via G-protein signaling to attenuate the auxin-mediated bimodality during lateral root formation.²⁶ In Arabidopsis, N-MYC DOWNREGULATED-LIKE1 (NDL1) protein interacts with Gβγ dimers, establish local auxin maxima and thus regulate lateral root initiation and emergence.⁴⁷ ABA INSENSITIVE 5 (ABI5) by repressing PIN1 accumulation functions in the GLC-mediated inhibition of the root meristem zone.²⁷ These reports in literature suggest that GLC modulates root growth via affecting auxin signaling and/or transport. The resistance of AUX/IAA gain-of-function mutant axr3 toward GLC and CK for root length control suggests that both these pathways may converge at AXR3 protein for root length regulation. Therefore, auxin signaling might act as a nodal point for GLC-CK interaction in controlling root length. There are also reports about the functional importance of cross-talk between auxin, CK and GLC signaling in regulating various developmental processes.^3,37 Resistance of axr3 toward GLC and CK for hypocotyl length control also suggests that GLC and CK signaling may converge at AUX/IAA proteins for controlling hypocotyl length in dark.³⁷ All these results together indicate that in addition to hypocotyl growth, CK- and GLC- signaling are also involved in controlling different aspects of root growth and development such as primary root length and root hairs with auxin signaling components as a downstream target. Understanding of key genetic components involved in the determination of optimal root system architecture can help us to engineer better performing crops under adverse environmental conditions.

Materials and methods

Plant materials

Following seed stocks were obtained from Arabidopsis Biological Resource Center (ABRC): ahk4 (CS6563, At2g01830); arr1,10,11 (CS6993, At3g16857/At4g31920/At1g67710); arr3,4,5,6,8,9 (CS25279, At1g59940/At1g10470/At3g48100/At5g62920/At2g41310/At3g57040); tir1.1 (CS3798, At3g62980); axr1.3 (CS3075, At1g05180); axr3.1 (CS57504, At1g04250); gin2.1 (CS6383, At4g29130). The rgs1.1 and rgs1.2 (At3g26090);⁴⁸ gpa1.1 (At2g26300);⁴⁹ thf1.1 (At2g20890)⁵⁰ lines were procured from the original published source. The slr1 (At4g14550) mutant line was provided by Dr. Hidehiro Fukaki (Nara Institute of Science and Technology, Nara, Japan).⁵¹ The ahk4; arr1,10,11; gpa1.1 were in the Ws background, gin2.1 mutant was derived from Ler background whereas all other mutant lines used were in the Col background. All chemicals were purchased from Sigma (St. Louis, MO, USA). The BAP stock was prepared in DMSO (dimethyl sulfoxide).

Seedling growth

Seeds were sterilized, imbibed at 4˚C for 2-d and were sown on plates (120 × 120 mm) containing half-strength MS medium (pH 5.7) supplemented with 1% sucrose and 0.8% agar. Seed germination was performed in growth room under long day conditions (16 h light/8 h dark, 80 µmol m⁻² s⁻¹ light intensity and 22˚C ± 2˚C temperature). Afterwards, 5-d-old seedlings were transferred on plates (120 × 120 mm) containing half-strength MS medium (pH 5.7) supplemented with GLC (0%, 1%, 3%, 5%), BAP (0M, 10⁻⁷M, 5 × 10⁻⁷M, 10⁻⁶M) and agar (0.8%) and root tips were marked. Thereafter, seedlings were grown vertically for next 3-d in climate controlled long day growth chambers.

Measurement of root length and lateral roots

For root length, digital images were captured using Nikon Coolpix digital camera on the 3^rd-d of seedling transfer and lengths were quantified using ImageJ. For root hairs digital images were captured with Nikon Coolpix digital camera connected with a Nikon SMZ1500 Stereo-Zoom microscope. Lateral roots were quantified directly under Nikon SMZ1500 Stereo-Zoom microscope on 3^rd-d of seedling transfer. Data shown is the average of 10–15 seedlings and error bars represent SD. Student's t test with paired 2-tailed distribution and all pairs Tukey HSD post ANOVA test at P ≤ 5% were used as mentioned specifically for statistical analysis.

Disclosure of potential conflicts of interest

No potential conflict of interest were disclosed.

Funding

This work was financially supported by the National Institute of Plant Genome Research (NIPGR) core grant, Department of Biotechnology, Government of India (Grant No. BT/PR14398/BRB/843/2010) and Department of Biotechnology, Government of India research fellowship to SK.