COP1 plays a prominent role in drought stress tolerance in Arabidopsis and Pea

Accepted Manuscript COP1 plays a prominent role in drought stress tolerance in Arabidopsis and pea Maryam Moazzam Jazi, Samaneh Ghasemi, Seyed Mahdi Seyedi, Vahid Niknam PII:

S0981-9428(18)30353-X

DOI:

10.1016/j.plaphy.2018.08.015

Reference:

PLAPHY 5376

To appear in:

Plant Physiology and Biochemistry

Received Date: 5 July 2018 Accepted Date: 8 August 2018

Please cite this article as: M.M. Jazi, S. Ghasemi, S.M. Seyedi, V. Niknam, COP1 plays a prominent role in drought stress tolerance in Arabidopsis and pea, Plant Physiology et Biochemistry (2018), doi: 10.1016/j.plaphy.2018.08.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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COP1 plays a prominent role in drought stress tolerance in Arabidopsis and Pea

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Maryam Moazzam Jazi1, Samaneh Ghasemi2, Seyed Mahdi Seyedi1*, Vahid Niknam2

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Biotechnology, Tehran, Iran

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Tehran, Iran

Plant

Biotechnology

Department,

National

Correspondence: Seyed Mahdi Seyedi ([email protected])

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Genetic

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Department of Plant Biology, School of Biology, College of Sciences, Tehran University,

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Abstract

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Constitutively photomorphogenic 1(COP1) is an E3 ubiquitin ligase that has been studied extensively in

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the photomorphogenesis- and light-related processes in Arabidopsis. However, the possible role of COP1

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in plant drought stress response remains unknown. Hence, in the present study, the stomatal behavior as

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one of the key elements in plant dehydration response was investigated in Arabidopsis cop1-4 and pea

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light-independent photomorphogenesis (lip1) mutants. We observed that water loss rate in the cop1-4 and

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lip1 detached leaves were significantly much faster than wild-type, resulting from failing to reduce the

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stomatal aperture by the mutants. But, interestingly, the cop1-4 and lip1 isolated leaves treated with

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abscisic acid (ABA) as well as cop1-4 soil-grown under drought stress could close their stomata as wild-

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type. Hence, COP1 plays a fundamental role in the regulation of stomatal movements in response to

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dehydration and its function was conserved during evolution in both Arabidopsis and pea. Further

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evaluations showed the cop1-4 mutant was not significantly damaged from the oxidative stress derived

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from soil water limiting conditions when compared to wild-type. Similarly, the up-regulation level of

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several key stress-responsive genes was relatively lower in cop1-4 than wild-type. Therefore, COP1

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might be considered as a potential key regulator of both short-and long-term dehydration response.

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Multiple stress-related cis-elements were also detected in the COP1 promoter region, which supported its

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up-regulation in response to drought, salt, and cold stresses. Besides, we figured out the constitutively

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open stomata of cop1-4 in darkness can be as a result of the reduced AtMYB61 expression.

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Key words: Arabidopsis, COP1, Drought stress, Pea, Stomata, Water loss.

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Abbreviations

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ABA, abscisic acid; ABRE, abscisic acid-responsive elements; CAT, catalase; COP1, constitutively

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photomorphogenic 1; COR15a, cold-regulated protein 15a; CRT, cryptochromes; CTAB, cetyltrimethyl

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ammonium bromide; DRE, dehydration response element; ERD1, early response to dehydration 1; FC,

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field capacity; HSF, heat shock transcription factor; HY5, long hypocotyl 5; LIP, light-independent

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photomorphogenesis; MDA, Malondialdehyde; PHOT, phototropins; PHYA, phytochrome A; PHYB,

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phytochrome B; PVP, Polyvinylpyrrolidone; ROS, reactive oxygen species; RWC, relative water content;

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SOD, superoxide dismutase; SUMO, Small ubiquitin-related modifier; TBA, thiobarbituric acid

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Introduction

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Water deficiency, as a result of climate change, is a major threat to supply global food resources for the

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increasing population worldwide. Drought stress is considered as one of the most important limiting

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factor for sustainable agriculture, particularly in arid and semi-arid areas, resulting in reduced crop yield

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by more than 50% every year (Atkinson and Urwin, 2012; Singh et al., 2015). It is adversely affects the

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plant growth, development, survival and productivity, hence the investigation of plant drought stress

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responses has gained widespread attention over recent years. Plants have evolved specific defence

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mechanisms in response to environmental stresses, including drought, which is characterized with a series

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of morphological, physiological, biochemical, and molecular changes in stressed plants (Krishnan and

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Pereira, 2008; Wang, 2014). Osmotic adjustment through the accumulation of compatible solutes, such as

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proline is a prevalent physiological response in plants exposed to various abiotic stresses, such as drought,

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salinity and cold (Krasensky and Jonak, 2012). Proline acts as an osmoprotectant, a molecular chaperone

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stabilizing macromolecules, and reactive oxygen species (ROS) scavenger (Szabados and Savouré, 2010).

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The glutamate-originated pathway appears to be the predominant proline biosynthesis pathway, especially

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under stress conditions (Hayat et al., 2012).

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Oxidative stress resulted from the extensive generation of ROS is an unavoidable aspect of different

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stresses (Noctor et al., 2014). Under drought conditions, stress-induced stomatal closure and the restricted

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CO2 uptake leads to the over-reduction of photosynthetic electron transport chain, favouring the electron

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transfer to oxygen and producing various kinds of ROS in the chloroplasts (Harb et al., 2010; Noctor et

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al., 2014). Although ROSs plays a key role in the signal transduction pathways at their steady-state level,

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they can initiate destructive oxidative processes such as lipid peroxidation and chlorophyll bleaching in

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the stressed plants (Miller et al., 2008; Sharma et al., 2012). Therefore, to scavenge ROS and alleviate the

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oxidative damages, plants have developed a wide range of enzymatic and non-enzymatic antioxidants,

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which superoxide dismutase (SOD) and catalase (CAT) comprised the primary enzymatic antioxidants.

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Similarly, flavonoids, cartenoids and anthocyanin are the main non-enzymatic antioxidants, accumulating

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in response to oxidative stress in most plant species (Sharma et al., 2012; You and Chan, 2015).

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Principally, plant drought responses are modulated by multiple stress-responsive genes, like RD22 and

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P5CS. The gene expression alternation ultimately promotes the cellular homeostasis, toxins detoxification

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and growth recovery of stressed plants (Nuruzzaman et al., 2013). At least six independent systems

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regulate gene expression in response to water deprivation in Arabidopsis, three of which are abscisic acid

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(ABA)-independent and the other three are ABA-dependent (Yamaguchi-Shinozaki and Shinozaki, 2006;

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Yoshida et al., 2014). Under water stress, the ABA-induced stomatal closure followed by reduced water

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loss is one of the essential aspects of drought tolerance in plants, and it is considered as a drought

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avoidance mechanism (Basu et al., 2016; Buckley, 2005). Two R2R3-MYB transcription factors,

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AtMYB60 and AtMYB61, are the main regulators of stomatal movements. The AtMYB60 expression is

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induced by white and blue light and repressed by darkness and drought stress; hence the stomatal aperture

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size is correlated with the AtMYB60 transcript level (Daszkowska-Golec and Szarejko, 2013). Contrary to

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AtMYB60, darkness promotes the expression of AtMYB61, leading to decreased stomatal aperture while

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other stomatal closuring signals, like ABA, salt and drought do not altered the AtMYB61 expression

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pattern (Liang et al., 2005).

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Among environmental signals, light is a major factor in the reflection of various plant cell movements and

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developmental processes including, stomatal movements, de-etiolation, and cotyledon opening (Ma et al.,

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2002). It has been revealed that phototropins (PHOT) and cryptochromes (CRY) are involved in blue

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light-induced stomatal opening and that COP1, a negative regulator of photomorphogenesis, likely acts

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downstream of CRY and PHOT signaling pathway to repress stomatal opening (Smirnova et al., 2012).

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COP1 is an E3 ubiquitin-ligase contributed to the degradation of photomorphogenic-related factors,

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including long hypocotyl 5 (HY5), phytochrome A (PHYA), phytochrome B (PHYB) and cryptochrome

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(CRY), resulting in photomorphogenesis repression (Jia et al., 2014; Osterlund et al., 2000).

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It has been proved that the occurrence of partial duplication within the pea homolog of COP1 give rise to

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create lip1 mutant (Sullivan and Gray, 2000). The lip1 mutants showed light-grown development when

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grown in darkness as they displayed short stems along with open and expanded shoots, like cop1-4

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(Seyyedi et al., 1999; Stoop‐Myer et al., 1999). COP1 function has been well documented during

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Arabidopsis developmental modifications in the presence and absence of light (Chang et al., 2011; Chen

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et al., 2015; Subramanian et al., 2004). However, to the best of knowledge, there is no report elucidating

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the involvement of COP1 in response to drought stress in Arabidopsis and pea. It was very recently

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demonstrated that Small ubiquitin-related modifier (SUMO) conjugation mediated by AtSIZ1 negatively

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regulated through the E3 ubiquitin-ligase activity of COP1 under abiotic stresses, thereby COP1 controls

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Arabidopsis abiotic stresses response via the modulation of the AtSIZ1 level (Kim et al., 2016). However,

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the impact of COP1 on the plant physiological and molecular responses under abiotic treatments was not

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reported.

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In the present study, wild-type (Col-0) and cop1-4 mutant of Arabidopsis (Arabidopsis thaliana) were

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subjected to dehydration treatment at two levels, short-term and long term by detaching leaves and

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stopping irrigation, respectively. In order to uncover the probable COP1 role in Arabidopsis drought

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response, some of major physiological and molecular parameters were evaluated. We also used abi1-1

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mutant, which is the drought-sensitive and ABA-insensitive plant as the control for the experiment.

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Furthermore, wild-type pea (Pisum sativum cv Alaska) and lip1 mutant were utilized to evaluate their

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stomata response to short-term and long-term dehydration.

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Materials and Methods

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Plant growth conditions and abiotic treatments

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Arabidopsis (A. thaliana), ecotype Columbia (Col-0), cop1-4, and abi1-1 mutants as well as pea (P.

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sativum cv Alaska) and lip1 mutant were used in this study. Surface-sterilized seeds were directly sown

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on pots containing a mixture of perlite, vermiculite and peat and stratified at 4°C in dark for 2 days. The

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seedlings were grown under controlled conditions at 22˚C and 70% relative humidity under long-day

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conditions (16h/8h, light/dark cycle). Short-term dehydration was imposed by excising leaf and placing

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on the laboratory bench at 23˚C and 27% relative humidity.

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Long-term drought treatment was applied to four-week-old plants (Arabidopsis and pea) by water

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withholding. The level of drought stress was gravimetrically checked (Ramegowda et al., 2013); briefly,

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pots along with plants were weighed daily until the end of experiment. Control plants were maintained at

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100% soil field capacity (FC). Upon the arrival of 75% FC (after 2 days) and 50% FC (after 4 days),

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leaves were sampled. For salt treatment, Arabidopsis plants were hydroponically grown in Hoagland’s

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nutrient solution with aeration, and the nutrient solution was renewed every three days. Four-week-old

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plants were exposed to salt stress (200 mM) by adding NaCl to the solution and leaves were collected at

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1, 3, and 5 days of salt application. For cold treatment, four-week-old soil-grown Arabidopsis plants were

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transferred to a growth chamber with 4°C for 3 days (cold acclimation), then subjected to -7°C for 6

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hours, followed by transferring to 4°C for one day. The leaves were collected after each step. For dark

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treatment, four-week-old plants grown in soil were kept at darkness for 2 days, and then leaves sampled

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under faint green light. In all cases, sampled leaves were frozen immediately in liquid nitrogen and stored

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at -80°C until use. All experiments were conducted in three biological replicates.

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Leaf relative water content

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Relative water content (RWC) was calculated by the following formula: RWC = [(fresh mass - dry mass)

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/ (saturated mass - dry mass)] × 100.

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Water loss assay

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For transpirational water loss assay, leaves from several four-week-old plants were detached, so that the

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leaves area of cop1-4 and lip1 mutants was similar to the corresponding wild-type. The leaves were

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placed in the filter paper on a laboratory bench at 23°C and 27% relative humidity and weighted at

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designated time intervals. The loss in fresh weight presented as the percentage of initial fresh weight at

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each time point (Li et al., 2008).

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Stomatal aperture measurement

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The abaxial epidermis was peeled from the leaves of multiple four-week-old plants under drought

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treatment as well as normal growth conditions and the stomata were observed by light microscopy (Zeiss

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) connected to a fitted camera. The stomatal pore width was measured using ProgRes Capture Pro 2.1

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software. For ABA-induced stomatal closure assay, the leaves were floated in the opening buffer

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containing 10 mM KCl, 10 mM CaCl2, and 10 mM MES, 0.01% Tween 20, pH 6.5 at 22°C for 2 hours to

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open the stomata. Then, the leaves were transferred to the same buffer supplemented with 10 µmol ABA,

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maintained in these conditions for 2 hours and the pore size was calculated (Tanaka et al., 2005).

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Stomatal density (i.e. the number of stomata per unit area) in both adaxial and abaxial leaf surface was

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determined in the four-week-old plants. Ten random parts (each part consisted of 0.1 mm2) from four

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leaves were counted for determination of stomatal density.

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Hydrogen peroxide and lipid peroxidation level measurement

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The H2O2 concentration was determined using the standard curve generated with known concentrations of

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H2O2 as described by (Loreto and Velikova, 2001). Malondialdehyde (MDA) level as the main index of

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lipid peroxidation was determined by the thiobarbituric acid (TBA) reaction according to Du and

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Bramlage (Du and Bramlage, 1992). The MDA concentration was calculated using extinction coefficient

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of absorbance 155 mM-1 cm-1.

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Proline content measurement

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Proline content was determined via reaction with ninhydrin reagent (Magné and Larher, 1992). Briefly,

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100 mg of fresh leaves was homogenized in 1.2 ml of 3% (w/v) aqueous sulphosalycylic acid and the

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homogenate filtered. After adding 2 ml ninhydrin (1%, w/v) to the supernatant and incubating at 98°C for

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one hour, the micro tubes were cooled and treated with 2 ml toluene. The absorbance of the upper phase

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was spectrophotometrically assessed at 518 nm after. The proline concentration was calculated according

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to a calibration curve.

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Enzymatic and non-enzymatic antioxidant assay

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For enzymatic antioxidant activity assay, 200 mg of fresh leaves was homogenized in 0.1 M Trise-HCl

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buffer (pH 7.8) containing 1 mM EDTA and 4% Polyvinylpyrrolidone (PVP) at 4°C. The resulting

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supernatant was used for enzyme assays. Proteins were quantified according to Bradford (Bradford, 1976)

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using albumin bovine serum as a standard. The per unit SOD activity was estimated by adding 40 µl of

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the enzymatic extract to a solution containing 13 mM methionine, 75 µM p-nitroblue tetrazolium chloride

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(NBT), 100 µM EDTA and 2 µM riboflavin in a 50 mm potassium phosphate buffer pH 7.5. The reaction

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started with 10 min illumination under a 30 W fluorescent lamp and stopped by turning light off. Non-

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illuminated and illuminated reactions without supernatant served as calibration standards, and the final

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reaction products were measured at 560 nm (Dhindsa et al., 1981).

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The CAT activity was assessed following the modified method by measuring the decrease in absorbance

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at 240 nm of the reaction mixture with a final volume of 2 ml containing 15 mM H2O2 in 50 mM K-

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phosphate buffer (pH 7.0) and 10 µL extract (Aebi, 1984).

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As the non-enzymatic antioxidant, the anthocyanin level was measured by homogenising leaves (50 mg)

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in acidic methanol (99:1, v/v) and recording the absorbance of the supernatant at 530 and 657 nm (Laby et

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al., 2000). The anthocyanin content expressed as follows:

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Relative units of anthocyanin/g fresh weight of tissue = OD530 - (0.25×OD657) × extraction volume (ml)

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× 1 / weight of tissue sample (g).

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Total RNA isolation and gene expression analysis

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Total RNA was extracted from 200 mg of leaves using a modified CTAB (cetyltrimethyl ammonium

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bromide) method (Moazzam-Jazi et al., 2015). After the treatment of extracted RNA with RNase free

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DNase I (Fermentas), single-stranded cDNA was synthesized from total RNA according to the

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manufacturer’s instructions (RevertAid First Strand cDNA Synthesis Kit, Fermentas). Real-time PCR was

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performed using the SYBR Green Real-time PCR master mix (Ampliqon, Denmark). The reaction

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mixture contained 1 µL of cDNA sample, 0.5 µL of each the forward and reverse primers (10 µM) and 10

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µL real-time master mix with a final volume of 20 µL. The cycling conditions were set as follows, initial

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activation of DNA polymerase at 95°C for 15 min, followed by 40 cycles 94°C for 30s, 58-60°C for 30s

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and 72°C for 30s for all genes except for P5CS1 that was 90s. The relative gene expressions of KIN2,

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P5CS1, RD22, NCED3, ABI1, MYB60, and COP1 were analysed by Pfaffl formula (Pfaffl, 2001) within

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REST 2009 software. Also, APK2a was used as the internal control. All samples were amplified in

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triplicate. The primer sequences are available in Table 1.

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Table1. Primer pairs were used for gene expression analysis Gene name

Gene Identifier

Forward primer sequence 5´-3´

APK2a

NM_101304.4

GGAGAGATACTTTCATCGCCTAA CC

GACCTGCTTTTGCCAATCCG

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KIN2

NM_121602.4

CAAAACACACATCAAAAACG

AACATTGCTCTTCTCCTCAG

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RD22

NM_122472.4

CACAAGGGAAGACCGATTTAC

TTCTCCTCTCCACTAACTTTTC TG

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P5CS1

NM_001202785.1

GTCATTTTGGTGTCATCTGGTGC

CATCAAGCTGGTCAAACATAG TC

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NCED3

NM_112304.3

TCCAGCTCTTCATTTCCCTAA

CGGCCATTGAAATAGACCAA

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ABI1

NM_118741.3

GATATCTCCGCCGGAGATGAGAT C

CATTCCACTGAATCACTTTTC CTC

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MYB60

NM_100755.3

GGACCATGGACTCCTGAAGA

CCACGTTTAATTCCAGGTCT

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COP1

NM_128855.4

TTGTTCCAGCGGTGAAACCT

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Tm (ºC)

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Reverse primer sequence 5´-3´

CAGATCCGGTGCTCCAATCT

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Promoter analysis

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The 1 kb upstream sequence from the translational initiation codon of COP1 in A. thaliana was obtained.

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In order to assess the transcription factor binding sites within the promoter region, the sequence was

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analysed using the web-based tool, “The Plant Promoter Analysis Navigator (PlantPAN 2.0;

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http://PlantPAN2.itps.ncku.edu.tw).” This tool provides resources for detecting cis-regulatory elements

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and the corresponding transcription factors in a gene promoter (Chow et al., 2016). The biological

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function

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(http://bioinfo.cau.edu.cn/agriGO/) via the singular enrichment analysis tool. The analysis was conducted

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with Fisher’s exact test and FDR cutoff of 0.05 was considered for significance.

all

transcription

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AgriGO

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Statistical analysis

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A completely randomized design with three biological replicates was considered and all physiologic data

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analyzed using SPSS software (version of 22). The mean comparison was performed by Duncan's

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Multiple Range Test (DMRT) at the 0.05 level of confidence.

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Results

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The contrasting response of cop1-4 stomata to dehydration

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One of the earliest plant responses to drought stress is the restriction of transpirational water loss through

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stomatal closure. Previous studies revealed that the COP1 possess the regulatory role in the stomatal

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movements (Mao et al., 2005). With this information in mind, the sensitivity of cop1-4 mutant leaves to

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short-term dehydration was examined by leaf detachment. We measured the water loss rate of wild-type

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and cop1-4 leaves after incubation at 22°C in the light. Based on our results, the weight of detached

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leaves reached to the half of initial weight in the wild type and cop1-4 mutant after 180 and 30 minutes of

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leaf detachment, respectively (Fig. 1), implying that the water loss rate in the mutant leaves was much

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faster than the wild type and responsible for the appearance of wilt symptoms in the mutant.

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Fig.1. Rate of leaf water loss in cop1-4 mutant and wild-type during short-term dehydration. Water loss

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rate is expressed as the percentage of initial fresh weight. Each data point is presented as the mean ± SD

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(n=3).

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Considering the water loss assay and the central role of stomata in the modulation of transpiration rate, we

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estimated the stomatal aperture of leaves under control conditions and 10, 20, and 30 minutes after leaf

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detachment. We observed that the stomatal aperture of cop1-4 leaves was much larger than wild-type

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under normal growth conditions; the average stomatal aperture of 200 stomata was 2.8 µm in the wild-

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type while it was 6.3 µm in cop1-4 (Fig. 2). The wild-type stomata started to close after 20 minutes of leaf

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detachment and their average aperture reached to 1.5 µm, but, interestingly, the cop1-4 stomata remained

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open in the same conditions (Fig. 2A).

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Considering the key role of ABA in the coordination of stomatal conductance with the plant water supply

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(Aasamaa and Sõber, 2011), we incubated leaves with exogenous ABA (10 µmol) for 40 and 120 minutes

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to explore whether ABA treatment induces a change on the stomatal aperture of cop1-4. Following the

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assessment of stomata status, we found that the cop1-4 stomata were sensitive to ABA and their stomatal

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pores reduced in response to exogenous ABA as the wild-type plants only after 120 minutes. As

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illustrated in Fig. 2B, the average stomatal aperture of wild-type and cop1-4 leaves from 2.6 and 5.4 µm

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before ABA application, respectively, were decreased to 1.2 and 2.5 µm in response to ABA treatment for

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120 minutes (Fig. 2B), suggesting that the cop1-4 stomata is not defective in ABA-mediated stomatal

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closure, but they are less sensitive to ABA than wild-type.

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As our results demonstrated, the cop1-4 mutant is defective to sense the leaf detachment-imposed

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dehydration and reduces their stomatal aperture. Consequently, we speculated that it may result in the

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enhanced drought sensitivity at the whole plant, too. To address this possibility, wild-type and cop1-4

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mutant were planted in the soil and four-week-old plants subjected to drought treatment by withholding

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water. After the stomata status evaluation, surprisingly, we found out that the stomatal behaviour was

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similar in both with-type and mutant in response to soil water deficiency. As shown in Fig. 2C, the

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stomatal aperture average significantly decreased in the stressed plants, it was reached to 1.7 µm and 2.54

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µm from 3 µm and 6.5 µm in the wild type and cop1-4 leaves, respectively, after four days of drought

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stress (Fig. 2C).

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Fig.2. Stomatal apertures of cop1-4

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mutant and wild-type leave. (A) The

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conditions and short-term dehydration imposed by leaf detachment. (B) The cop1-4 mutant and wild-type stomata response to the leaf treatment with 10

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stomatal aperture under normal growth

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µmol ABA. (C) The cop1-4 mutant and wild-type stomatal aperture in response

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soil

water

shortage

conditions. Data are the average stomatal aperture of 200 stomata. Means followed by the same letter(s) are not significantly different at 0.05 level of probability (DMRT).

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A representative illustration of open and closed stomata of cop1-4 and wild-type was shown in Fig. 3.

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Fig.3. Representative image of open

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and closed stomata of cop-1-4 and wild-type. (A) Wild-type, open. (B)

Wild-type, closed. (C) cop1-4, open.

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(4) cop1-4, closed.

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Furthermore, we investigated the adaxial and abaxial stomatal density of wild-type and cop1-4 mutant

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under control growth conditions (Fig. 4 A). As Fig. 4 showed, there is no significant difference in the

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number of stomata per unit of leaf area between wild-type and cop1-4 under normal growth conditions.

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Surveying the abaxial stomatal density in plants under soil drought stress revealed that this parameter has

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remained constant after 2 and 4 days of drought stress in both wild-type and cop1-4 (Fig. 4B).

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Fig.4 Adexial and abexial stomatal density of wild-type and cop1-4 mutant under normal growth

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conditions (A) and the abexial stomatal density of wild-type and cop1-4 mutant under soil drought stress

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(B). Data are the average stomatal density of 120 stomata.

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Interestingly, we observed that cop1-4 mutants soil-grown were more tolerant to drought stress than wild-

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type under our experimental conditions (Fig.5).

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Fig.5. Phenotypic picture of plants

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under normal growth conditions (A)

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treatment (B), imposed by withholding

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water.

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and after 4 days of soil drought

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The lip1 stomata contrasting response to dehydration and ABA treatment

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As LIP1 in pea is ortholog for COP1 and the lip1 mutant exhibits the similar characteristics to cop1-4

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(Sullivan and Gray, 2000), we were curious to assess its stomatal behaviour in response to dehydration as

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well as exogenous ABA treatment. Interestingly, we found a resemblance in stomatal response between

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cop1-4 and lip1 mutants under our experimental conditions. Water loss assay showed that the lip1

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detached leaves weight attained to the half of initial weight after 40 minutes while wild-type isolated

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leaves required 10.5 hours to reach the same weight, implying that lip1 fails to close its stomata in

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response to short-term dehydration as cop1-4 (Fig. 6A).

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The lip1 stomata are significantly wider than wild-type under normal growth conditions similar to those

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of cop1-4. Based on our microscopic measurements, the average stomatal aperture is 4.2 and 6.5 µm in

369

wild-type and lip1 mutant, respectively (Fig. 6B). But, the treatment of detached leaves with 10 µmol

370

ABA for 120 minutes resulted in a reduced stomatal pores in both wild-type and lip1 mutant, so that the

371

stomatal aperture of wild-type and lip1 decreased to 2.9 and 3.5 µm in response to ABA treatment.

372

Therefore, neither cop1-4 nor lip1 appear impaired in the ABA-induced stomatal closure (Fig. 6B). Our

373

findings suggested that COP1 can play an essential role in regulating the stomatal movements in both

374

Arabidopsis and pea.

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Fig.6. lip1 mutant and wild-type

376

stomatal behaviour. (A) lip1 mutant

377

and wild-type stomatal behaviour in

378

response to short-term dehydration as

379

evidenced by leaf water loss assay.

380

Water loss rate is expressed as the

381

percentage of initial fresh weight.

382

Each data point is presented as the

383

mean ± SD (n=3). (B) The lip1 mutant

384

and wild-type stomata response to the

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385

leaf treatment with 10 µmol ABA. (C)

386

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The lip1 mutant and wild-type stomata

387

after 2 and 4 days of drought stress.

388

Data are the average stomatal aperture

389

of 200 stomata. Means followed by

390

the same letter(s) are not significantly

391

different at 0.05 level of probability

392

(DMRT).

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393 394 395 396

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Furthermore, drought treatment was applied on the soil-grown pea lip1 mutant and wild-type by water

399

withholding and the stomatal behaviour surveyed after 2 and 4 days of soil drought stress. Interestingly,

400

we observed that lip1 stomatal aperture decreased in response to soil water shortage as wild-type (Fig.

401

6C). As Fig. 6C showed, the average of stomatal aperture reduced to 4.3 µm and 2.8 µm from 6.1 µm and

402

4.3 µm in lip1 mutant and wild-type, respectively, after 4 days of drought stress.

403 404

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Evaluation of main physiological parameters related to drought stress in cop1-4

405 406

RWC content

407

RWC is an important index of plant water status and reflects the balance between the water supply and

408

transpiration rate. Hence, in order to estimate the whole plant transpiration under drought stress, we

13

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examined the RWC level of wild-type and cop1-4 plants after two and four days of the cessation of

410

irrigation. Based on the results, there was no difference between wild-type and cop1-4 mutant in the RWC

411

content during drought treatment, the RWC of both wild-type and cop1-4 plants significantly diminished

412

after two days of drought stress (Fig. 7A). The similarity in the level of RWC under soil water shortage

413

conditions further support that in contrast to cop1-4 detached leaves, the cop1-4 whole plant could limit

414

the transpirational water loss via the stomatal closure and maintain the RWC as wild-type plants.

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415 Proline content

417

It is well documented that proline has an important role in protecting plants under drought stress. To

418

evaluate this hypothesis and to determine the effects of cop1 mutation on drought-induced proline

419

accumulation, proline level was measured in wild-type, cop1-4, and abi1-1 mutants under normal and

420

long-term drought stress conditions. Interestingly, we found that proline content in cop1-4 mutant leaves

421

was about 4.5 fold higher than that of the wild-type under normal growth conditions (Fig. 7B). As shown

422

in Fig. 7B, soil water deficiency resulted in proline accumulation in wild-type and abi1-1 mutant leaves

423

while the level of this compatible solute remained relatively constant until the fourth day of drought

424

treatment, when significantly increased when compared to well-watered conditions. However, the cop-1-4

425

leaves exhibited the lowest proline content while abi1-1 and col had the highest proline level at the end of

426

drought treatment.

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H2O2 and MDA content

429

Since the increased ROS production is one of the major effects of a drought stress on plants and can

430

induce lipid peroxidation, the levels of H2O2 (the main ROS in plants) and MDA (the key by-product of

431

lipid peroxidation) were measured to survey the oxidative damage level during drought stress. As

432

illustrated in Figs. 7C and 7D, the H2O2 and MDA content showed no significant difference between

433

wild-type and mutant plants under normal growth conditions. However, H2O2 level was increased 2- and

434

6-fold in wild-type and abi1-1 mutant leaves, respectively, after four days of water withholding as

435

compared with the control conditions. Interestingly, no significant change in the H2O2 content was

436

observed in the cop1-4 leaves in these conditions (Fig. 7C). Regarding MDA content, in contrast to wild-

437

type and abi1-1 mutant, the MDA content was not significantly altered in the cop1-4 leaves during soil

438

water deficient treatment. However, the highest MDA content was obtained from wild-type and abi1-1

439

mutant plants after four days of stopping irrigation (Fig. 7D). Based on the results, cop1-4 mutant

440

suffered from the lower level of drought-induced oxidative damage than wild-type under our

441

experimental conditions.

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442 443

Fig.7. RWC (A), Proline (B), H2O2 (C) and MDA (D) levels in cop1-4, abi1-1 mutants as well as wild-

444

type leave in response to soil water shortage conditions. Data represent the mean ± SD (n = 3). Means

445

followed by the same letter(s) are not significantly different at 0.05 level of probability (DMRT).

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446 Antioxidant activities level

448

ROS level is modulated by a well-defined detoxification system, involving a number of antioxidants that

449

reduce the reactive oxygen species content and minimize their deleterious effects. Here, the activity of

450

two main antioxidant enzymes, SOD and CAT were surveyed to monitor enzymatic ROS detoxification

451

in the plants under drought stress. Based on our results, there was no significant difference in SOD and

452

CAT activities among wild-type and the two mutants, cop1-4 and abi1-1 leaves under normal growth

453

conditions (Fig. 8A). While the CAT activity tends to be increased during drought treatment in wild-type

454

and reached to its highest activity (2.4 fold) at the end of experiment, cop1-4 exhibited no significant

455

change in the enzyme activity. The CAT activity was enhanced after two days of drought stress in abi1-1

456

leaves, and then significantly deceased after four days of water shortage in this mutant (Fig. 8A). In the

457

case of SOD activity, wild-type and abi1-1 leaves elevated SOD activity by 1.6 and 2.2 fold after four

458

days of drought treatment as compared to the control growth conditions whereas no alternation in the

459

enzyme activity was observed in drought-stressed cop1-4 mutant leaves (Fig. 8B).

460

Furthermore, anthocyanins are a large class of water-soluble pigments in the flavonoid group found in all

461

plant tissues, which can decrease leaf osmotic potential and operate as antioxidant in plants under abiotic

462

stresses (Rossel et al., 2007). Hence, to examine the effects of water deficiency on this non-enzymatic

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antioxidant level, especially in the cop1-4, anthocyanin content in wild-type and two mutants, cop1-4 and

464

abi1-1 leaves was measured. As shown in Fig. 8C, there was no significant difference in anthocyanin

465

content among wild-type, cop1-4, and abi1-1 plants under normal growth conditions, while soil water

466

deficiency gave rise to an excess production of anthocyanin in all plants. The anthocyanin level was

467

elevated by about 2.5 times in wild-type and cop1-4 leaves and 4 times in abi1-1 at the end of experiment

468

(fourth day of treatment). The more anthocyanin accumulation in abi1-1 leaves may implicate the severe

469

dehydration stress in the mutant.

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470 471

Fig.8. Enzymatic antioxidant activities

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472

level of CAT (A) and SOD (B) as well

473

as the non-enzymatic antioxidant level,

474

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anthocyanin (C) content, in cop1-4,

475

abi1-1 mutants, and wild-type leaves in

476

response to soil water stress. Data

477

represent the mean ± SD (n = 3). Means

478

followed by the same letter(s) are not

479

significantly different at 0.05 level of

480

484 485 486 487 488 489 490 491

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probability (DMRT).

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492

Gene expression analysis in cop1-4

493

Plant drought response is a complex biological process that needs to be surveyed at both physiological

494

and molecular levels. Here, to explore the effects of cop1 mutation on drought tolerance at the molecular

495

level, the expression profiling of several key stress-responsive genes, including MYB60, P5CS1, RD22,

496

KIN2, NCED3, and ABI1 was evaluated by qRT-PCR analysis in the wild-type and the two mutants’

16

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leaves, cop1-4 and abi1-1 under controlled and drought treatment conditions. Also, the COP1 expression

498

pattern was assessed in the plants under control, drought, salt and cold treatments as well as in the

499

presence and the absence of light. Furthermore, considering the fact that the cop1-4 stomata are

500

constitutively open in darkness (Mao et al., 2005) and AtMYB61 plays a major role in dark-induced

501

stomatal closure (Liang et al., 2005), the expression pattern of AtMYB61 was monitored in wild-type and

502

cop1-4 under light and darkness.

503

Based on the expression analysis of AtMYB60 gene, the transcript level of AtMYB60 was significantly

504

decreased after two days of drought treatment in the wild-type and cop1-4 leaves. The reduced gene

505

expression pattern was retained until the fourth day of treatment, when wild-type and cop1-4 leaves

506

accumulated the lowest level of AtMYB60 transcript, so that there was about 4 folds reduction in gene

507

expression at this time. As compared with wild-type and cop1-4 mutant, the gene expression was

508

diminished with a delay of two days, after four days of desiccation in abi1-1 mutant (Fig. 9A). Given that

509

the AtMYB60 function in the drought-induced stomatal closure, our results are consistent with the

510

stomatal behaviour at the stressed plants and illustrated that cop1-4 whole plant can lessen their stomatal

511

aperture via the reduced AtMYB60 transcript level in response to soil water limiting conditions.

512

In regards to stress-responsive genes, the wild-type as well as the mutants, cop1-4 and abi1-1 exhibited

513

elevated levels of KIN2, RD22, and P5CS1 under dehydration treatment compared with normal growth

514

conditions. But, interestingly, the up-regulation level of the genes was significantly lower in cop1-4

515

leaves than wild-type under drought treatment compared to normal growth conditions as illustrated in

516

Figs. 9B-9D

517

As ABA plays a fundamental role in the stress signalling pathway, leading to plant adaptation to adverse

518

growth conditions, the mRNA level of NCED3 and ABI1 genes involved in ABA biosynthesis and signal

519

transduction pathways, respectively, were assessed during drought treatment. As presented in Figs.9E and

520

9F, the transcripts abundance had no difference between wild-type and cop1-4 mutant under normal

521

growth conditions. However, the enhanced transcript level of NCED3 and ABI1 were observed in wild-

522

type, cop1-4 and abi1-1 after two days of desiccation. The induction of these genes in cop1-4 mutant can

523

suggest that this mutant is not deficient in ABA-related pathways.

525 526

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Fig. 9. Relative expression level of MYB60, KIN2, RD22, P5C1, NCED3 and ABI1 (A-F) in cop1-4, abi1-

529

1 mutants and wild-type leaves in response to soil water limiting conditions. Data represent the mean ±

530

SD (n = 3). Means followed by the same letter(s) are not significantly different at 0.05 level of probability

531

(DMRT).

532

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To elucidate the potential involvement of COP1 gene in plant abiotic stresses response, we evaluated its

534

expression under drought, salinity, and cold stresses. Our results, surprisingly demonstrated that COP1

535

expression was strongly induced by the applied stresses in wild-type and abi1-1 leaves, but not

536

significantly in the cop1-4 (Fig. 10). As illustrated in Fig. 10, in contrast to cop1-4 mutant, the COP1

537

transcript was accumulated in wild-type and abi1-1 leaves after two days of drought treatment until the

538

end of experiment (Fig. 10). Similarly, the same trend was detected in response to salt and cold stresses

539

(Fig. 10). As Fig. 10 displays, wild-type and abi1-1 mutant exhibited the elevated COP1 transcript level

540

after one day salinity treatment until five days of treatment while cop1-4 mutant accumulated this

541

transcript after three days of salt stress, however, the transcript abundance in cop1-4 leaves was

542

significantly lower than wild-type (Fig. 10). In the case of cold stress, while the COP1 transcript level

543

was notably increased in the wild-type and abi1-1 mutant at 4°C and -7°C, the transcript abundance was

544

approximately remained constant in cop1-4 leaves (Fig. 10).

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545

546 547

Fig. 10. Relative expression level of COP1 in cop1-4, abi1-1 mutants and wild-type leaves in response to

548

drought, salt, and cold stresses. Data represent the mean ± SD (n = 3). Means followed by the same

549

letter(s) are not significantly different at 0.05 level of probability (DMRT).

550

Earlier studies revealed that the stomatal aperture of cop1-4 mutant was much larger than wild-type and

552

our findings determined that the mutant was extremely sensitive to leaf detachment-imposed dehydration

553

(Fig. 1). With this information in mind, we assessed the expression level of NCED3 and COP1 genes in

554

detached leaves of wild-type and cop1-4 mutant at 10, 20, and 30 minutes after leaf detachment. As

555

shown in Figs. 11A and 11B, NCED3 and COP1 transcript accumulation were significantly induced in

556

both wild-type and cop1-4 leaves within 20 and 30 min after leaf detachment, respectively, implicating

557

that similar to NCED3, the COP1 could be an early-responsive gene. This broad responsiveness of COP1

558

to various types of abiotic stresses suggests it may be a component of plant abiotic stress response.

560 561 562 563

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564 565

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566 567

Fig. 11. Relative expression levels of

568

NCED3 (A) and (B) COP1 (B) in

569

cop1-4

570

response to short-term dehydration

571

imposed by leaf detachment. Data

572

represent the mean ± SD (n = 3).

573

Means followed by the same letter(s)

574

are not significantly different at 0.05

575

level of probability (DMRT).

wild-type

leaves

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and

576

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577 578 579 580 581 582

We also investigated the probable effect of cop1 mutation on the steady-state mRNA level of AtMYB61 in

584

the presence and absence of light. Surprisingly, wild-type and cop1-4 plants showed the opposite

585

expression pattern. As Fig. 12A illustrates, in contrast to cop1-4, the transcript abundance of AtMYB61 is

586

significantly higher in the darkness than the light presence in wild-type plants. Similarly, the expression

587

analysis of COP1 demonstrated that both wild-type and cop1-4 displayed the decreased transcript

588

abundance in darkness when compared to the presence of light, but the reduction level was much higher

589

(11 folds) in cop1-4 than wild-type in darkness (Fig. 12B). Our results can infer that COP1 expression in

590

darkness is required for the expression of AtMYB61 that is essential for dark-induced stomatal closure.

592 593 594 595

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597 Fig.12. Relative expression levels of

599

MYB61 (A) and COP1 (B) in cop1-4

600

and wild-type leaves in the presence

601

and absence of light. Data represent

602

the mean ± SD (n = 3). Means

603

followed by the same letter(s) are not

604

significantly different at 0.05 level of

605

probability (DMRT).

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606 607

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608 609 610 611 612 613 614 Promoter analysis

616

The gene expression induction or suppression under various developmental and environmental conditions

617

is mainly regulated via gene promoters and their cis-acting elements (Hernandez-Garcia and Finer, 2014).

618

In the current study, the altered expression pattern of COP1 gene in response to various abiotic stresses

619

prompted us to survey its promoter region for the probable stress-related cis-acting elements and the

620

corresponding transcription factors. The survey of 1 kb upstream sequences from translational initiation

621

codon of COP1 revealed the presence of multiple stress-associated cis-acting elements, including 3

622

abscisic acid-responsive elements (ABRE), 16 ABRE-like sequence required for etiolation-induced

623

expression of ERD1 (early response to dehydration 1) and 2 dehydration response element (DRE) in the

624

promoter region (Supplementary file S1). In addition, one salt-tolerance zinc finger protein binding site,

625

12 MYB recognition sites, one binding site for cold-regulated protein 15a (COR15a), and 20 heat shock

626

transcription factor (HSF) binding sites were detected in the COP1 promoter. The results implied that

627

COP1 is an abiotic stress-responsive gene that whose expression regulated in both ABA-dependent and

628

ABA-independent pathways. It is consistent with the altered COP1 expression level in response to

629

drought, salt, and cold stresses in both wild-type and abi1 mutant. Furthermore, in order to get insight into

630

the function of associated transcription factors (TFs) to COP1 promoter, all identified TFs were subjected

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to singular enrichment analysis embedded in AgriGO. Based on the analysis, several gene ontology (GO)

632

terms related to abiotic stresses including, response to stress (GO:0006950), response to abiotic stimulus

633

(GO:0009628), response to water deprivation (GO:0009414), response to abscisic acid stimulus

634

(GO:0009737), abscisic acid mediated signaling pathway (GO:0009738), response to salt stress

635

(GO:0009651), response to cold (GO:0009409), cellular response to heat (GO:0034605), response to

636

osmotic stress (GO:0006970), response to hydrogen peroxide (GO:0042542), and hyperosmotic salinity

637

response (GO:0042538) were significantly enriched (FDR < 0.01), which further support the COP1

638

function under various stresses. The complete list of significantly enriched GO term is presented in

639

Supplementary file S2.

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631

SC

640 641 Discussion

643

Understanding the drought signal transduction pathway and the consequent plant response to reduce the

644

detrimental effects of water scarcity has continued to be one of the main objectives for scientists

645

(Obidiegwu et al., 2015; Pinheiro and Chaves, 2010). Drought stress response was extensively studied

646

in various plants, especially in Arabidopsis (Oh et al., 2005; Todaka et al., 2015; Wang et al., 2008). The

647

current study as the first attempt to uncover the different attributes of cop1-4 as well as lip1 mutants in

648

response to drought stress added the novel and main player, COP1, to drought signalling pathway. The

649

COP1 importance in plant drought response is related to its pivotal roles in developing stomata and

650

regulating stomatal movements (Mao et al., 2005). Stomatal closure is an early and rapid plant reaction to

651

water deficiency that is regulated through a complex network of signalling pathways (Merilo et al., 2015).

652

Here, we found that the lip1 stomatal apertures, like cop1-4, are significantly larger than wild-type under

653

normal growth conditions. It suggests that the COP1 function in the regulation of stomatal pore size is

654

conserved during evolution, in both Arabidopsis and pea. Interestingly, we discovered that contrary to

655

wild-type, the cop1-4 and lip1 detached leaves failed to decrease their stomatal aperture in response to

656

leaf detachment imposed dehydration, accounting for up to 50% of water loss during only 30 and 90

657

minutes after leaf excising in cop1-4 and lip1, respectively. It suggested that the COP1 plays a prominent

658

role in the sense and the rapid stomata response to short-term dehydration in the detached leaves in both

659

Arabidopsis and pea.

660

Guard cell ABA signaling is important for regulating basal stomatal openness and rapid stomatal

661

responses to environmental stimuli (Merilo et al., 2015). However, the appropriate hormone functioning

662

relies on the proper level of biologically active ABA, correlating with several processes including, ABA

663

biosynthesis, catabolism, conjugation, deconjugation, transport, and perception (Daszkowska-Golec and

664

Szarejko, 2013). Here, the sensitivity of cop1-4 detached leaves to exogenous ABA treatment after 120

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22

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minutes, not after 40 minutes, implied that cop1-4 stomata is less sensitive to ABA than wild-type.

666

Similarly, Khanna et al (2014) reported that the stomata of GFP–TUA6,cop1-4 line resulted from

667

crossing GFP–TUA6 line with the cop1-4 mutant did not respond to 40 minutes treatment with 10 µM

668

ABA (Khanna et al., 2014). Although, ABA-mediated stomatal closure is not defective in the cop1-4

669

mutant, the delay in stomatal response to ABA may be long enough to induce rapid wilting in detached

670

leaves.

671

In Arabidopsis, ABA was synthesized mainly in leaves via the induction of NCED3 expression (Merilo et

672

al., 2015). NCED3 is considered to be the main regulatory enzyme, which its transcript up-regulated

673

within 15–30 min after leaf detachment or dehydration (Behnam et al., 2013; Xiong and Zhu, 2003).

674

Although, we could clearly detect the enhanced level of NCED3 transcript level within 20 minutes in both

675

wild-type and cop1-4 detached leaves, the cop1-4 stomata remained open, unlike wild-type. It can

676

propose that the accumulation of NCED3 transcript and the subsequent ABA biosynthesis appear to occur

677

normally in cop1-4 mutant; however, it is not sufficient for rapid stomatal closure in response to short-

678

term dehydration.

679

Unexpectedly, cop1-4 and lip1 whole plants were not as sensitive as the detached leaves to drought

680

treatment as they exhibited the decreased stomatal apertures accompanied by the similar level of RWC

681

between the mutant and wild-type during soil water deficient. This discrepancy highlights the root-shoot

682

signalling role in plant stress response. Contrary to detached leaves, as stress was imposed gradually on

683

the intact plants and the root-shoot signalling precede the ABA-induced stomatal closure, the ABA

684

concentration and time is enough to induce stomatal closure in the mutants as wild-type. Christmann et al

685

(2007) reported soil water stress generates a hydraulic signal in the shoot, which acts upstream of ABA

686

signalling and stomatal closure; however, both hydraulic signal and ABA is required for stomatal closure

687

in response to water stress. Another study by Pantin et al (2013) showed that ABA has two effects on

688

stomata: a direct biochemical effect on the guard cells and an indirect hydraulic impact by reducing leaf

689

water permeability triggered within vascular tissues. Under drought stress, ABA concentration rises in the

690

vascular parenchyma as a result of importing root-derived ABA and locally synthesized ABA. ABA

691

signalling in the bundle sheath by inactivating bundle sheath aquaporins (PIP) resulted in the reduced leaf

692

hydraulic conductance. Vascular ABA then transfers to the guard cells and promotes stomatal closure

693

(Pantin et al., 2013). Overall, our findings can suggest that the COP1 plays an important but a transient

694

role in initiating the rapid stomatal response to short-term dehydration and this role was evolutionary

695

conserved in both Arabidopsis and Pea.

696

Moreover, surveying the adaxial and abaxial stomatal density of wild-type and cop1-4 mutant under

697

control growth conditions and soil drought treatment revealed that there is no significant difference in

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stomatal density between wild-type and mutant. The similar results reported by previous studies

699

(Balcerowicz et al., 2014; Kang et al., 2009).

700

Although the stomatal aperture of cop1-4 was much wider than wild-type, both wild-type and cop1-4

701

displayed the similar level of RWC under normal growth conditions. As RWC implies the balance

702

between transpiration rate and water reservation, it indicated that cop1-4 mutant is able to conserve leaf

703

water probably via an efficient osmo-regulation system. It has been shown that proline accumulation

704

resulted in lowered cell osmotic potential and facilitated the cell turgor pressure maintenance (Hayat et

705

al., 2012). In line with this report, we found that cop1-4 leaves significantly contained more proline level

706

than wild-type under well-watered conditions. Therefore, the proline accumulation may be the essential

707

factor for compensating the increased water loss via the wide stomatal aperture and maintaining relative

708

leaf water content in cop1-4 mutants as wild-type plants.

709

The enhanced proline content as a common phenomenon in the stressed plants has also been widely

710

described; it is important not only for osmoregulation, but also for protecting cells by enhancing the

711

protein and cell membrane stability (Bhaskara et al., 2015; Estrada-Melo et al., 2015; Szabados and

712

Savouré, 2010). In the present study, while the increased content of proline was detected in wild-type and

713

abi1-1 after two days of drought stress, cop1-4 significantly accumulated this osmolyte after four days of

714

desiccation. It appears that the high constitutive levels of proline in cop1-4 can protect cell against the

715

detrimental effects of stress without additional proline enhancement until mutant exposed to longer

716

treatment periods or more severe stress, leading to further proline accumulation. The similar situation has

717

been also described in a drought-tolerant citrus genotype (Zandalinas et al., 2016).

718

Plants suffering from various stresses, including water deficiency often exhibit oxidative stress symptoms

719

as evidenced by the elevated reactive oxygen species and MDA levels (Das and Roychoudhury, 2014).

720

Compared with other ROS, H2O2 is a relatively long-lived molecule that is able to diffuse across cell

721

membranes. This characteristic is compatible with its role as a signalling molecule during growth and

722

stress plant response (Sharma et al., 2012). However, the high concentration of H2O2 can be a sign of

723

undesirable situation and extremely harmful for organism survival. Here, H2O2 and MDA content were

724

presented the positive association as also described by previous studies (Murshed et al., 2013; Ren et al.,

725

2016). In contrast to cop1-4 leaves, both H2O2 and MDA level were significantly incremented in wild-

726

type and abi1-1 under drought treatment. It implicated that cop1-4 mutant did not undergo the detectable

727

oxidative damage resulting from applied drought stress.

728

Enzymatic and non-enzymatic antioxidants are considered as an efficient defence system to cope with

729

excessive ROS and ameliorate the oxidative damage level. Among the enzymatic antioxidants, SOD acts

730

as one of the central and preliminary antioxidants by converting O2.− into H2O2 and maintaining the

731

intracellular O2.− within normal range (Das and Roychoudhury, 2014). In consistent with previous studies

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in Arabidopsis (Jung, 2004) and rubber tree (Wang, 2014), SOD activity was enhanced in wild-type and

733

abi1-1 leaves during drought stress while no significant change was detected in cop1-4 mutants, it is

734

probably due to either superoxide radical levels was not as high as for SOD activation in the mutant or the

735

non-enzymatic antioxidants such as anthocyanin play a main role in these conditions. Similarly, the lack

736

of significant CAT activity enhancement in cop1-4 can refer to the low level of H2O2 in the mutant as

737

reported that catalase breaks down high concentration of H2O2 because of its low affinity (high Km) for

738

this substrate (Mhamdi et al., 2010). It has been demonstrated that environmental stresses cause either

739

enhancement or depletion of CAT activity, depending on the intensity, duration, and type of the stress

740

(Janiak et al., 2015). Therefore, the drought-induced reductions in CAT activities in abi1-1 leaves may be

741

as a result of sever oxidative stress as also evidenced by the increased H2O2 and MDA levels. Overall, the

742

antioxidants activity was in accordance to H2O2 and MDA content during drought stress and may confirm

743

that oxidative damage level in cop1-4 and abi1-1 leaves is lower and higher than wild-type leaves,

744

respectively.

745

Many drought-inducible genes with various functions respond to water deficiency at the transcriptional

746

level. K1N2, RD22, and COR15a are of key stress-responsive genes encode the variety of LEA and like-

747

LEA proteins that are able to act as chaperones to stabilize proteins and membrane structure in the

748

stressed plants to confer cellular tolerance to dehydration (Goyal et al., 2005; Janiak et al., 2015). Based

749

on our results, the expression of these genes were induced in leaves of wild-type and two mutants, cop1-4

750

and abi1-1 in response to water deficiency; however, their expression levels in cop1-4 leaves were not as

751

high as wild-type, hence the improved drought tolerance of cop1-4 whole plant might not be correlated

752

with these genes. The expression of stress-inducible genes is regulated by the ABA-dependent and ABA-

753

independent pathways (Joshi et al., 2016). A similar expression pattern of the above-mentioned genes in

754

abi1-1 mutant in our study indicated that their induction is ABA-independent, as previous studies have

755

been also reported (Jensen et al., 1996).

756

There are two closely related P5CS genes in Arabidopsis that mainly P5CS1 expression is promoted by

757

drought and salt stresses (Ben Rejeb et al., 2015; Székely et al., 2008). In agreement with previous

758

researches, the increased steady state level of P5CS1 mRNA was detected in the drought stressed plants.

759

While the proline content of cop1-4 leaves was significantly higher than wild-type under normal growth

760

conditions, the P5CS1 transcript accumulation had not been followed the same pattern. It can refer to the

761

proline feedback inhibition effect as previous studies was also reported that proline biosynthesis in plants

762

primarily regulated at the transcriptional level by end product (Porcel et al., 2004; Sharma and Verslues,

763

2010). Although the role of ABA in regulating proline accumulation requires further examination due to

764

contradictory data obtained in various species by different researchers (Guan et al., 2014; Verslues and

765

Bray, 2005), we could clearly observed the enhanced P5CS1 transcript and proline levels in abi1-1

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mutant leaves, suggesting that no ABA involvement is required for proline accumulation or drought-

767

induced P5CS1 is regulated by both ABA-dependent and ABA-independent pathways.

768

In Arabidopsis genome, at least nine genes are identified to code NCED that among them NCED3 showed

769

higher expression in both roots and shoots in response to drought stress (Rasheed et al., 2016). Similarly,

770

we found the elevated NCED3 expression in wild-type and two mutants, cop1-4 and abi1-1 leaves under

771

water limiting conditions. Hence, the mutation in COP1 gene does not interfere with ABA biosynthesis

772

pathway at least at the transcription level. Further, Barrero et al revealed that there is a positive

773

correlation between the enhanced NCED3 transcript level and ABA content (Barrero et al., 2006). Thus,

774

the cop1-4 mutant can produce enough ABA, which facilitates the stomatal closure as evidenced by the

775

reduced stomatal pores in cop1-4 whole plant in response to soil water deficiency. Besides the

776

environmental signals, Arabidopsis stomatal aperture was modulated by several transcription factors,

777

including AtMYB60 and AtMYB61. Here, we found that the steady-state level of AtMYB60 was reduced in

778

wild-type, cop1-4, and abi1-1 leaves. The AtMYB60 response in abi1-1 refers to the presence of cis-acting

779

elements responsible for down-regulating gene expression in response to dehydration in an ABA-

780

independent manner (Rusconi et al., 2013). However, abi1-1 could not able to close their stomata in

781

response to water shortage conditions, it may propose that the ABA-independent pathway mediated

782

AtMYB60 down-regulation is not enough for drought-induced stomatal closure. The down-regulated

783

AtMYB60 was in line with decreased stomatal pore in wild-type as reported by previous report (Cominelli

784

et al., 2005) as well as cop1-4 leaves in response to soil water deficiency. Consequently, the diminished

785

stomatal aperture in the stressed cop1-4 whole plant could be resulted from the reduced AtMYB60

786

transcript level.

787

AtMYB61 is a pleiotropic regulator of plant stomatal aperture, lateral root formation, and xylem cell

788

differentiation. It has suggested that this gene has an active role for stomatal opening inhibition in

789

darkness (Baldoni et al., 2015). Here, we figured out cop1-4 leaves accumulated AtMYB61 and COP1 in

790

much lower than wild-type in darkness. Considering that stomatal closure was impaired in the cop1-4

791

leaves in darkness and COP1 expression can complement the constitutive stomatal opening phenotype in

792

this mutant (Mao et al., 2005), our findings can uncover that the expression of both COP1 and AtMYB61

793

may essential for the reduced stomatal pores in darkness and AtMYB61 might play a key role on the

794

downstream of COP1 for promoting darkness-induced stomatal closure.

795

Additionally, we deciphered that COP1 expression pattern can undergo by various abiotic stresses

796

(drought, salinity, and cold), offering the COP1 could be a stress-responsive gene. While COP1 transcript

797

abundance remained relatively constant in cop1-4 mutant, it was significantly up-regulated in wild-type

798

and abi1-1 mutant during abiotic stresses. The COP1 expression in abi1-1 mutant revealed that its

799

expression is regulated through both ABA-dependent and ABA-independent pathways in response to

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unfavourable conditions as also evidenced by the presence of both ABRE and DRE cis-acting elements in

801

the COP1 promoter region. Similarly, the existence of several enriched ABA- and stress-related GO terms

802

among the biological functions of transcription factors associated to the COP1 promoter could further

803

confirm the altered gene expression pattern in response to applied various abiotic treatments.

804

Taken together, COP1 can act as a central regulator of abiotic stresses response pathway with diverse

805

roles in detached leaves and whole plant levels. The conserved function of COP1 in both Arabidopsis and

806

pea during evolution can emphasis its crucial importance. Our study adds a new level of complexity to the

807

overall understanding of main effectors required for stomatal closure as well as the root-shoot signalling

808

pathway under drought stress. Further efforts are needed to resolve exactly how COP1 mediates plant

809

response to both short-term and long-term dehydration.

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Acknowledgements

813

We are grateful to Prof. Xing-Wang Deng for providing cop1-4 seeds as well as Dr. William Thompson

814

and Dr. Shannon Frances for providing lip1 seeds.

815 816 References

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1012 1013 1014

Supporting information

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Supplementary file 2. List of significantly enriched GO terms associated with all transcriptions factors

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bind to COP1 promoter region.

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Supplementary file 1. List of stress-related cis-acting elements in COP1 promoter region.

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Highlights

2. COP1 is an abiotic stress-responsive gene in Arabidopsis.

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1. COP1 plays a fundamental role in the regulation of stomatal movements in response to dehydration in both Arabidopsis and Pea.

3. cop1-4 mutant stomata respond to short-term and long-term dehydration in a different manner in

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Arabidopsis.

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Author contributions M.M.J, S.GH, and S.M.S designed the research. M.M.J and S.GH carried out the experiment and analyzed the data. M.M.J wrote the manuscript. S.M.S and V.N supervised the project. All authors read

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and approved the manuscript.