The E3 ubiquitin ligase HOS1 is involved in ethylene regulation of leaf expansion in Arabidopsis

Abstract

Ethylene regulates a variety of physiological processes, such as flowering, senescence, abscission, and fruit ripening. In particular, leaf expansion is also controlled by ethylene in Arabidopsis. Exogenous treatment with ethylene inhibits leaf expansion, and consistently, ethylene insensitive mutants show increased leaf area. Here, we report that the RING finger-containing E3 ubiquitin ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 1 (HOS1) regulates leaf expansion in an ethylene signaling pathway. The HOS1-deficient mutant showed reduced leaf area and was insensitive to ethylene perception inhibitor, silver thiosulfate (STS). Accordingly, genes encoding ethylene signaling components were significantly up-regulated in hos1-3. This study demonstrates that the HOS1 protein is involved in ethylene signal transduction for the proper regulation of leaf expansion possibly under environmentally stressful conditions.

Keywords:

• Arabidopsis
• ethylene
• HOS1
• leaf expansion

Abbreviations

CTR1	=	CONSTITUTIVE TRIPLE RESPONSE 1
EIN	=	ETHYLENE INSENSITIVE
ERF	=	ETHYLENE-RESPONSE FACTOR
ERS	=	ETHYLENE RESPONSE SENSOR
ESE1	=	ETHYLENE AND SALT INDUCIBLE 1
ETR	=	ETHYLENE RECEPTOR
HOS1	=	HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 1
STS	=	silver thiosulfate

Leaf expansion is a crucial process that not only determines the shape and size of mature leaves but also ensures its photosynthetic capacity for plant growth and development.^1,2 It is influenced by multiplicity of environmental factors, such as water availability and day length.^3,4 In addition, endogenous hormone signaling is also intensively involved in the control of leaf expansion. Promotive roles of auxin, brassinosteroid, and gibberellin in leaf expansion have been demonstrated.^5-7

The simple hydrocarbon ethylene is a gaseous plant hormone that regulates a variety of developmental processes, such as seed germination, cell elongation, abscission, senescence, sex determination, and fruit ripening.^8-13 It has been also proposed that ethylene negatively regulates leaf expansion process. Exogenous ethylene treatment results in reduced leaf area.¹⁴ Furthermore, ethylene insensitive mutants, such as ethylene receptor 1 (etr1) and ethylene response sensor 1 (ers1), show increased leaf size.^15,16

Five receptor isoforms are responsible for ethylene perception: ETR1, ETR2, ERS1, ERS2, and ETHYLENE INSENSITIVE 4 (EIN4).¹⁷ Their ethylene perception is integrated into a negative regulator of ethylene signaling, CONSTITUTIVE TRIPLE RESPONSE 1 (CTR1) Ser/Thr protein kinase,¹⁸ which in turn regulates a series of transcriptional cascades in order to control ethylene-dependent physiological processes. EIN2 is a representative positive signaling regulator and subsequently activates genes encoding EIN3 and related EIN3-LIKE (EIL), which further regulate expression of ETHYLENE-RESPONSE FACTORs (ERFs).^19,20 In the presence of ethylene, ethylene receptors inactivate kinase activity of CTR1 so that subsequent transcriptional cascades are activated.²¹ Although ethylene signaling pathways have been intensively investigated, its intricate networks with other signaling pathways remain to be unraveled.

The RING-type E3 ligase HOS1 was originally reported as cold signaling attenuator.²² The cold-activated HOS1 protein executes protein turnover of the INDUCER OF CBF EXPRESSION1 (ICE1) MYC-like basic helix-loop-helix (bHLH) transcription factor,^23,24 which activates the C-REPEAT BINDING FACTOR (CBF)-COLD-REGULATED (COR) pathway,^23,25,26 indicating the homeostatic role in plant adaptation to long-term cold stress. In addition to its role in cold response, the HOS1 protein also mediates plant hormone signaling. For instance, HOS1 negatively regulates auxin biosynthesis in the control of hypocotyl elongation.²⁷

It has been previously reported that hos1 mutants show reduced leaf area,²⁸ although underlying molecular mechanism is elusive. Thus, we wanted to know how HOS1 regulates leaf expansion. We hypothesized that HOS1 is associated with ethylene signaling, because the hos1-3 mutant showed similar phenotypes in leaf expansion to the ctr1 mutant that exhibits constitutive ethylene responses.¹⁴

To investigate whether alterations in ethylene signaling underlie reduced leaf expansion of hos1-3, we employed a chemical inhibitor of ethylene receptors, STS. The ethylene receptors require the copper ion for high-affinity ethylene binding.²⁹ Silver can substitute for copper as a cofactor and thereby inhibit ethylene responses.³⁰ In the presence of STS, wild-type plants showed increased leaf expansion as previously reported.³¹ However, the hos1-3 mutant was insensitive to STS and showed constitutively reduced leaf area (Fig. 1A and B). Quantitative analysis revealed that wild-type leaves exhibited approximately 4-fold increase in the leaf area upon the treatment with STS, whereas the hos1-3 mutant only showed 1.5-fold increase in its leave size (Fig. 1B).

Figure 1. The reduced leaf expansion of hos1-3 is insensitive to STS. The effects of 10 μM STS on leaf expansion were examined using Arabidopsis wild-type (Columbia-0; Col-0) and hos1-3 seedlings. Biological triplicates were averaged and statistically analyzed by Student's t-test (*P < 0.05). Bars indicate standard error of the mean.

To convince the constitutive ethylene response in hos1-3, we analyzed expression of ethylene signaling genes, such as EIN3 and ETHYLENE AND SALT INDUCIBLE 1 (ESE1), in wild-type and hos1-3 seedlings. Consistent with its leaf expansion, ethylene-responsive genes were significantly upregulated in hos1-3 (Fig. 2), indicating that HOS1 negatively regulates ethylene signaling.

Figure 2. Ethylene signaling genes are up-regulated in hos1-3 mutant. Ten-day-old seedlings grown under long day conditions were used to analyze expression of genes encoding ethylene signaling components. Transcript accumulation was analyzed by quantitative RT-PCR (RT-qPCR). The EUKARYOTIC TRANSLATION INITIATION FACTOR 4A1 (eIF4a) gene (At3g13920) was used as an internal control. Biological triplicates were averaged and statistically analyzed by Student's t-test (*P<0.05). Bars indicate standard error of the mean.

Ethylene regulates leaf expansion largely through cell expansion.¹⁵ Ethylene-insensitive ers1 mutant shows increased cell expansion in leaf epidermal cells,¹⁵ whereas the ctr1 mutation leads to reduced cell expansion without substantial alterations in cell division.¹⁸ Consistently, HOS1 also regulates cell expansion in the control of hypocotyl elongation,²⁷ supporting the probable role of HOS1 in ethylene-controlled cell elongation for leaf expansion process.

HOS1 is a key signaling component that regulates multiple hormone signaling pathways. We previously reported that HOS1 regulates auxin biosynthesis in the control of hypocotyl elongation.²⁷ In addition, this study provides evidence that HOS1 also plays a role in ethylene regulation of leaf expansion. Given that auxin and ethylene signaling is elaborately intertwined,^32-34 HOS1 might establish an important crosstalk. Auxin biosynthesis and ethylene signaling would be coordinated to fine-tune HOS1-mediated developmental processes.

HOS1 was originally identified as cold signaling attenuator. Considering that HOS1 regulates ethylene signaling, we postulated that cold stress may stimulate ethylene signaling to control leaf expansion. Indeed, ethylene signaling was activated at low temperature (Fig. 3). Moreover, leaf area is also significantly reduced with lower photosynthetic activity under long-term cold conditions.³⁵ Collectively, HOS1 may underlie the processes that balance growth and environmental tolerance, ensuring plant fitness.

Figure 3. Ethylene signaling genes are induced at low temperature. Ten-day-old seedlings grown under long day conditions were exposed to cold (4°C) for 1h. Transcript accumulation was analyzed by RT-qPCR. The eIF4a gene was used as an internal control. Biological triplicates were averaged. Bars indicate standard error of the mean.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interests were disclosed.

Funding

This work was supported by the Basic Science Research (NRF-2013R1A1A1004831) and Global Research Network (NRF-2014S1A2A2028392) programs provided by the National Research Foundation of Korea and by the Next-Generation BioGreen 21 Program (PJ011192042015) provided by the Rural Development Administration. K.L. was supported by the BK21 PLUS program in the Department of Bioactive Material Sciences.