Ethylene acts as a negative regulator of glucose induced lateral root emergence in Arabidopsis

Abstract

Plants, being sessile organisms, are more exposed to the hazards of constantly changing environmental conditions globally. During the lifetime of a plant, the root system encounters various challenges such as obstacles, pathogens, high salinity, water logging, nutrient scarcity etc. The developmental plasticity of the root system provides brilliant adaptability to plants to counter the changes exerted by both external as well as internal cues and achieve an optimized growth status. Phytohormones are one of the major intrinsic factors regulating all aspects of plant growth and development both independently as well as through complex signal integrations at multiple levels. We have previously shown that glucose (Glc) and brassinosteroid (BR) signalings interact extensively to regulate lateral root (LR) development in Arabidopsis.¹ Auxin efflux as well as influx and downstream signaling components are also involved in Glc-BR regulation of LR emergence. Here, we provide evidence for involvement of ethylene signaling machinery downstream to Glc and BR in regulation of LR emergence.

In plants, root system development and maintenance is under control of various factors such as light, gravity, phytohormones, soil water and nutrient content etc.^2-5 Auxin is involved at almost every step of LR initiation and development.^6-8 Apart from auxin, phytohormones such as BR, cytokinin, ethylene and ABA have also been reported to regulate LR development.^3,8-12 However, most of these endogenous and exogenous signals integrate with auxin mediated mechanisms to regulate lateral root development.^3,8 Glc can also affect various aspects of root growth and development.^1,13-15 Glc mainly employs auxin response machinery to modulate early seedling growth and development in Arabidopsis.¹³ Recently, we have shown that Glc can induce LR emergence via HXK1 mediated signaling and BRI1 mediated BR signaling works further downstream to HXK1 to regulate LR emergence.¹

Ethylene exerts an inhibitory effect on lateral root formation mainly via modulating auxin transport and signaling mediated machinery.^11,16-18 There are a number of reports describing interaction between sugar/Glc and ethylene signaling pathways in plants.^14,19-24 Similarly, BR and ethylene signaling also interact to regulate various aspects of early seedling growth and establishment such as, primary root growth, etiolated hypocotyl growth, apical hook formation, root and hypocotyl directional growth in light and dark respectively.^14,25-28 Based on previous reports and our present findings, here we provide evidence for the involvement of ethylene signaling during Glc-BR control of LR emergence.

Involvement of ethylene signaling components in regulating Glc-BR induced LR emergence

Previously, we have shown the involvement of BR signaling, auxin signaling and auxin transport components during Glc induced LR emergence.^1,13 Here, we investigated the possible involvement of ethylene during Glc-BR regulated LR emergence. Since ethylene exerts an inhibitory effect on LR emergence, we analyzed the effect of ethylene on Glc and BR induced LR emergence. Glc induced LR emergence was significantly reduced in presence of ethylene precursor (1-aminocyclopropane-carboxylic acid hydrochloride (ACC) (Fig. 1A). In our whole genome transcription profiling study, we found that the transcript levels of many ethylene response related genes was differentially regulated by exogenous Glc treatment (Table 1). Interestingly, Glc was able to cause repression in transcript abundance of majority of these genes which include; genes involved in ethylene biosynthesis such as 1-AMINOCYCLOPROPANE-1-CARBOXYLATE SYNTHASE8 (ACS8), ethylene receptor CONSTITUTIVE TRIPLE RESPONSE 1 (CTR1), downstream signaling component ETHYLENE-INSENSITIVE 2 (EIN2), EIN3, EIN3-like 1 (EIL1), and several ethylene-responsive element-binding factor family proteins such as ERF1, ERF2, ERF5, ERF6 etc. (Table 1). Altogether, these results suggested mainly an antagonistic effect of ethylene on Glc response and vice versa.

Figure 1.

Involvement of ethylene signaling components during Glc-BR control of LR emergence. (A) Ethylene can effectively reduce Glc-induced LR emergence. (B) Ethylene signaling work downstream to BR as Glc-induced LR emergence of bzr1-1D was abolished upon co-treatment with ACC. (C) Emerged LR count in WT and ethylene mutants upon treatment with increased Glc concentrations. The ethylene receptor mutant etr1-1 and signaling mutants ein2 and ein3 show enhanced LR emergence; while response of eto2 mutant was very less as compared to their respective WTs upon treatment with Glc. (D) Exogenous IAA treatment (50 nM) along with increased Glc concentrations (3% w/v) could induce LR emergence in eto2 mutant suggesting that auxin works further downstream to ethylene during LR emergence. (E) A testable model for Glc-hormone interaction during LR emergence based on previous findings and present observations. Data shown is the average of 2 biological replicate having at least 15 seedlings; error bars represent SE; (Student's t-test; P < 0.05), * control vs. treatment; ** WT vs. mutant.

Table 1.

Differential regulation of genes involved in ethylene response upon treatment with 3% Glc in light grown WT (Col-0) seedlings.

Probe Set ID	Represent-ative Public ID	FC 3% Glc vs 0% Glc	Gene Title	Gene Symbol
247543_at	AT5G61600	−16.48	Ethylene-responsive element-binding family protein
245250_at	AT4G17490	−13.21	Ethylene responsive element binding factor 6	ATERF6
260451_at	AT1G72360	−11.84	Ethylene-responsive element-binding protein, putative
245252_at	AT4G17500	−4.79	Ethylene responsive element binding factor 1	ATERF-1
266302_at	AT2G27050	−2.67	Ethylene-insensitive3-like 1	EIL1
253054_at	AT4G37580	−2.63	Hookless1	HLS1
250911_at	AT5G03730	−2.51	Constitutive triple response 1	CTR1
263653_at	AT1G04310	−2.44	Ethylene response sensor 2	ERS2
248794_at	AT5G47220	−2.12	Ethylene responsive element binding factor 2	ERF2
257981_at	AT3G20770	−2.08	Ethylene-insensitive 3	EIN3
253066_at	AT4G37770	−2.05	1-aminocyclopropane-1-carboxylate synthase8	ACS8
265194_at	AT1G05010	−2.04	Ethylene-forming enzyme	EFE
246935_at	AT5G25350	−1.97	EIN3-binding f box protein 2	EBF2
248799_at	AT5G47230	−1.9	Ethylene responsive element binding factor 5	ERF5
263467_at	AT2G31730	−1.87	Ethylene-responsive protein, putative
245098_at	AT2G40940	−1.68	Ethylene response sensor 1	ERS1
250928_at	AT5G03280	−1.45	Ethylene insensitive 2	EIN2
261874_at	AT1G50640	−1.38	Ethylene responsive element binding factor 3	ERF3
261041_at	AT1G17440	1.47	Enhanced ethylene response 4	EER4
261315_at	AT1G53170	1.56	Ethylene responsive element binding factor 8	ERF8
254434_at	AT4G20880	1.59	Ethylene-regulated nuclear protein	ERT2
264083_at	AT2G31230	1.78	Ethylene responsive element binding factor 15	ATERF15
253746_at	AT4G29100	1.82	Ethylene-responsive family protein
254926_at	AT4G11280	1.83	1-aminocyclopropane-1-carboxylate synthase6	ACS6
265577_at	AT2G20100	2.02	Ethylene-responsive family protein
261108_at	AT1G62960	2.25	1-aminocyclopropane-1-carboxylate synthase10	ACS10
247378_at	AT5G63120	2.43	Ethylene-responsive DEAD box RNA helicase, putative	RH30
246932_at	AT5G25190	3.52	Ethylene-responsive element-binding protein, putative
249042_at	AT5G44350	3.67	Ethylene-responsive nuclear protein -related

In our previous study, brassinazole resistant1-1D (bzr1-1D) mutant having endogenously high BR signaling showed significantly higher LR emergence upon Glc treatment. We then tested the effect of ACC supplementation to the Glc-induced LR emergence of bzr1-1D and it was found to be highly reduced upon ACC inclusion (Fig. 1B). This result proves that ethylene works antagonistically to- and downstream to- BR as well as Glc for controlling LR emergence.

Further, to investigate which components of ethylene signaling cascade are involved in Glc induced LR emergence, WT and ethylene perception and signaling mutant seeds were surface sterilized and imbibed at 4°C for 48h. Imbibed seeds were germinated and grown vertically on petri dishes containing ½ MS supplemented with 1% sucrose and solidified with 0.8% agar for 5 d under climate-controlled growth rooms in a long day condition, with 22°C ± 2°C temperature and 60 µmoles/m²/s light intensity. 5d old light grown seedlings were then transferred to the treatment medium without or with increasing Glc concentrations (0%, 1%, 3% w/v). Plates were kept vertically in culture room conditions and seedlings were monitored for LR emergence 4-d after treatment. The ethylene receptor mutant ethylene resistant1-1 (etr1-1), ethylene signaling mutants ethylene insensitive2 (ein2-1) and ein3-1 exhibited significantly more LR emergence upon Glc treatment, whereas the ethylene overproducer2 (eto2) mutant showed very less emerged LR count as compared to their respective WTs (Fig. 1C). All these results together suggested that ethylene receptor and downstream components of ethylene signal transduction pathway such as and EIN2, EIN3 work downstream to both Glc and BR to antagonize LR emergence in Arabidopsis.

To find out the involvement of auxin response machinery, we checked whether exogenous auxin application could rescue LR emergence defect in eto2 mutant. In Glc free medium, IAA treatment alone could not cause any significant LR emergence, but increasing Glc concentration (1%, 3% w/v) along with IAA (50 nM) treatment caused significant enhancement in LR emergence in eto2 mutant (Fig. 1D). These results proved auxin to be the downstream most factor, even to ethylene, during LR emergence. Based on previous reports and our experimental data, we propose a testable model for possible interactions and interconnections between Glc-phytohormone signals during LR emergence (Fig. 1E). Our findings have allowed us to verify the possible Glc-BR-ethylene-auxin interactions during LR emergence.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

This work was financially supported by the grant from Department of Biotechnology, Ministry of Science and Technology, Government of India (BT/PR3302/AGR/02/814/2011), NIPGR core grant and Council of Scientific and Industrial Research, India (research fellowships to A.G. and M.S.).