Overexpression of Arabidopsis aspartic protease APA1 gene confers drought tolerance

Journal Pre-proof Overexpression of Arabidopsis aspartic protease APA1 gene confers drought tolerance ´ ´ Fiol Diego Fernando, Daleo Gustavo Raul, ´ D’Ippolito Sebastian, Guevara Mar´ıa Gabriela

PII:

S0168-9452(20)30008-X

DOI:

https://doi.org/10.1016/j.plantsci.2020.110406

Reference:

PSL 110406

To appear in:

Plant Science

Received Date:

4 September 2019

Revised Date:

26 December 2019

Accepted Date:

31 December 2019

´ D, Fernando FD, Raul ´ DG, Gabriela GM, Overexpression Please cite this article as: Sebastian of Arabidopsis aspartic protease APA1 gene confers drought tolerance, Plant Science (2020), doi: https://doi.org/10.1016/j.plantsci.2020.110406

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier.

Overexpression of Arabidopsis aspartic protease APA1 gene confers drought tolerance. Authors: D´Ippólito Sebastián, Fiol Diego Fernando, Daleo Gustavo Raúl, Guevara María Gabriela*. Biological Research Institute, National Council of Scientific and Technique Research (CONICET), University of Mar del Plata, Mar del Plata (UNMDP), Argentina.

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* corresponding author. Biological Research Institute, National Council of Scientific and Technique Research (CONICET), University of Mar del Plata, Mar del Plata (UNMDP), Argentina. E-mail address: [email protected] (M.G. Guevara).

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The expression level of APA1 was induced by drought and ABA. Overexpression of aspartic protease APA1 gene increased Arabidopsis tolerance to drought. Leaves of OE-APA1 plants have a reduced stomatal index and water loss. Overexpression of APA1 up-regulates ABA signaling pathway genes.

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Highlights

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Abstract

Drought is an environmental stress that severely affects plant growth and crop production. Different

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studies have focused on drought responses but the molecular bases that regulate these mechanisms are still unclear. We report the participation of Aspartic Protease (APA1) in drought tolerance. Overexpressing APA1 Arabidopsis plants (OE-APA1), showed a phenotype more tolerant to drought compared with WT. On the contrary, apa1 insertional lines were more sensitive to this stress

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compared to WT plants. Morphological and physiological differences related with the water loss were observed between leaves of OE- APA1 and WT plants. OE-APA1 leaves showed lower stomata index and stomata density as well as a smaller of the stomatic aperture compared to WT plants. qPCR analysis in OE-APA1 leaves, showed higher expression levels of genes related to ABA signaling and synthesis. Analysis of plant lines expressing APA1 promoter fused to GUS showed that APA1 is

expressed in epidermal and stomata cells. In summary, this work suggests that APA1 is involved in ABA-dependent response that its overexpression confers drought tolerance in Arabidopsis. Keywords: aspartic protease, drought tolerance, ABA signaling, guard cell, Arabidopsis

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1. Introduction Plants are constantly exposed to biotic and abiotic stresses that significantly affect their growth and

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development [1]. In addition, global climate change amplify the stress generated by the decrease in water availability by an overall increase in temperature, concentration of gases and an intensification of the

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hydrogeological cycle [2]. Drought is one of the environmental factors that have a greater impact on the production and distribution of crops [3]. In the past four decades, it was estimated a loss of 1820 million Mg

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of maize, rice and wheat caused by drought [4] and according to predictions associated with climate change, losses in potato production will exceed 30% in the next 10 years [5]. Due to these losses, it is necessary to

face of drought stress conditions.

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direct greater effort towards the understanding of the defense mechanisms developed by the plant in the

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Plants have evolved complex physiological and biochemical adaptations to cope with water stress. Water use efficiency is an important selection trait and plants have evolved different molecular mechanisms to reduce their consumption and adjust their growth to adverse environmental conditions [6,7]. The response is controlled by complex regulatory events mediated by abscisic acid (ABA) signaling [8,9], ion transport [10],

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and the activities of transcription factors (TFs) involved in the regulation of stomatal responses [11], all of which are integrated into orchestrated molecular networks, enabling plants to adapt and survive [5]. Protein breakdown and recycling are an essential part of the plant response to stress. Proteases can

be involved in different events including: removal of inactivated, denatured or abnormal proteins, signal propagation, reutilization of amino acids, and modification of protein content during stress conditions that require changes in metabolic status [12].

Aspartic proteases (APs; EC 3.4.23) comprise one of the four superfamily’s of proteolytic enzymes that are widely distributed among vertebrates, plants, yeasts, nematodes, parasites, fungi, bacteria and viruses [13,14]. Most APs plant have common features, they are active at acidic pH, inhibited by pepstatin A and they contain two aspartic acid residues responsible for the catalytic activity [14,15]. APs have been implicated in the processing of proteins in different been involved in the processing of proteins in different plant organs and implicated in defense responses against

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pathogens [16–18] , senescence [14] and programmed cell death and reproduction [15].

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AP increase in both, gene expression levels and activity were reported in Phaseolus vulgaris L. and Vigna unguiculata L. plants subjected to water stress [19]. In addition it was reported that ASPG1,

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an A. thaliana guard cell-expressed AP, confers tolerance to drought stress [20]. Although it is widely

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accepted that plant proteases play a key role in responses to various environmental stimuli, the physiological importance of APs in responses to drought stress has not yet been studied in depth.

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In this work, we demonstrated that overexpression of the aspartic protease APA1 confers

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drought tolerance in Arabidopsis, probably via ABA-dependent signaling. 2. Material and methods

2.1 Plant material and growth conditions. A. thaliana ecotype Columbia were used to generate overexpressing plants. apa1 insertional lines

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(SALK_110749, Fig. 1B) were from the Arabidopsis Biological Resource Center (ABRC; http://www.arabidopsis.org/abrc/). The homozygosis of this line was checked by genotyping. Before germination, all seeds were sterilized with 50% ethanol in 10 % SDS and vernalized for two days at 4°C. Seeds were sown on 1/2 MS medium (Sigma-Aldrich, St. Louis, MO, USA) and 2% (w/v)

sucrose. Plants were grown in a growth chamber under fluorescent lights (150 mmol/m2s) at 22 ± 3 °C with 60 % relative humidity under 16-h light/8-h dark cycle. 2.2 Constructs designs and plasmid propagation. 2.2.1 Generations of vector for overexpressing APA1 (OE-APA1). PCR to amplify APA1 cDNA open reading frame (1521bp) was carried out with specific primers (Table

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S1) for 40 cycles of 95 °C for 30 s, 50 °C for 40 s on the basis of annealing temperatures of different primer pairs and 72 °C for 2 min. APA1 cDNA was cloned into pENTR-D/TOPO (Invitrogen, USA)

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followed by their mobilization into destination vector containing the CaMV 35S promoter, pMDC83

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2.2.2 Generation of plasmid for ProAPA1:GUS.

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[21].

APA1 DNA promotor fragment (1941 bp upstream from ATG) was amplified by PCR with specific

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primers (Table S1) for 40 cycles of 95 °C for 30 s, 55 °C for 40 s on the basis of annealing temperatures of different primer pairs and 72 °C for 2 min. PCR product was cloned into pENTR-D/TOPO

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(Invitrogen, USA) followed by their mobilization into destination vector containing the βglucuronidase (GUS) gene, pMDC162 [34].

2.2.3 Plasmid propagation.

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Plasmids generated both in 2.2.1 and 2.2.2 were propagated in Escherichia coli strain Top10F (Invitrogen, Carlsbad, USA). The Luria-Bertani medium (1% bacto-peptone, 0.5% yeast extract, and 1% NaCl) in liquid or solid (1.5% agar) was used to culture Top10F0 cells at 37 °C with 10 mg/mL of kanamycin (pENTR-D/TOPO-cDNA-APA1

and pENTR-D/TOPO-Pro-APA1) or

spectinomycin

(pMDC83-cDNA-APA1 or pMDC162-Pro-APA1) for selection of transformants. Then, both

destinations vectors were introduced into Agrobacterium tumefaciens GV3101 (pMP90) by electroporation using a GENE PULSER II electroporation system (Bio-Rad Laboratories, Hercules, CA, USA).

2.3 Generation of A. thaliana plants OE-APA1. A. thaliana WT plants were transformed by the floral dip method as described previously [22]

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Transformant plants were selected on MS plates with 50 mg/ml Hygromycin, transplanted to soil,

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and APA1 overexpression was confirmed by qRT-PCR. The primers used are listed in Table S1.

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2.4 Water stress

Seedlings grown in plates were transplanted (see 2.3)in a pot, containing a pre-weighed amount of

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a vermiculite-perlite mix. Different plant lines (WT, apa1 and OE-APA1) were kept under a 16- h

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light/8-h dark cycle at 22 °C with 60% relative humidity in growth chambers. Plants were grown in normal water condition for 28 days and mild water deficit was applied as previously described [23]. Briefly, soil water content was maintained at 0.35 g water g-1 dry soil in every pot until day 28 and

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then, soil water content was raised at 0.35 (Well-Watered=WW) or 0.2 (mild watered deficit=MWD) g water g-1 dry soil. Subsequently, the stress was maintained by watering each two days to maintain the same weight (WW or MWD) in all the pots. During two weeks, plant phenotype was followed

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and photographs (Nikon Coolpix P80, Nikon Corp., Tokyo, Japan) and different samples were taken at times as is indicated in legends.

2.5 Physiological measurements 2.5.1 Chlorophyll content.

Chlorophyll content was estimated as previously described [24]. Measurements of SPAD values were determined using a SPAD-502 (Konica-Minolta). SPAD values were taken in four different leaves from each genotype at 10 days of WW or MWD. In addition, total chlorophyll concentration was determined by absorbance as previously described [25] with some modifications. A total of 100 mg from rosette leave of each genotype in control or MWD were pulverized with liquid nitrogen. Samples were incubated with 1.5 mL of 100 % acetone in the dark for 4 h and centrifuged for 15 min

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at 3000 rpm. Absorbance was measured in the supernatant at 645 and 663 nm. Total chlorophyll

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content was determined using the next formula: 20.2*A645 + 8.02*A663 (µg/ml).

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2.5.2. Root length.

To determined root length, four roots from each genotype were excised and washed 10 days after

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WW or MWD. Pictures were taken and length was quantify using ImageJ (Image J,

2.5.3 Total area leaf.

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http://imagej.nih.gov/ij/).

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Leaf area measurements were performed as described previously [26]. Photographs were taken with a Nikon Coolpix P80 (Nikon Corp., Tokyo, Japan) on successive days after WW or MWD. Total rosette surface area was measured and analyzed using Fiji ImageJ1, as described previously [27].

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Data were processed, and statistical analysis was performed using GraphPad Prism software (GraphPad Software Inc., San Diego, CA).

2.5.4 Determination of cell size, stomata index and stomata density.

The density of stomata and pavement cells was determined by counting number of cells in mm 2 in a microscopy Nikon Eclipse E200 fluorescence microscope (Nikon Corp., Tokyo, Japan). Stomatal index was calculated according to Royer [41] [Stomata * 100/(Stomata + pavement cells)].

2.5.5 Stomatal aperture and ABA treatments. Stomata pore area was analyzed as described previously [28]. Epidermal strips excised from the

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abaxial side of expanded Arabidopsis leaves of each genotype at 10 days after WW or MWD was

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used. Images of stomata were digitalized using a Nikon DS-Fi 1 camera coupled to a Nikon Eclipse E200 fluorescence microscope (Nikon Corp., Tokyo, Japan) and the stomatal aperture width was

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measured using ImageJ analysis software.

In ABA treatment, before ABA addition (10 µM), peels were submerged in opening buffer (10 mM

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K-MES, pH 6.1, 10 mM KCl) for 3 h. The pore aperture quantification was analyzed as described

2.6 Water measurements

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2.6.1 Water consumption

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above.

Water consumption was calculated as previously described [23]. Briefly, each two days, from 0 to 14 of MWD the quantity of water added in each pot was registered by weight and used to calculate

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water uptake.

2.6.2 Water Content Water content in 10 days plants of WW or MWD was determined as previously described [29]. Fresh weight (FW) was scored in four rosettes form each genotype immediately after separation of root

and rosette. Dry weight was scored after 24 h at 80°C.Water content was then determined as the ratio: (fresh weight2 dry weight)/fresh weight [23].

2.6.3. Water loss Four leaves for three different plants from each genotype were cut 10 days after MWD and weighted every 20 minutes during 100 min. The percentage (%) of lost water was expressed as the ratio:

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2.7. RNA isolation and expression analyses by real time RT-PCR.

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[initial weight – weight]/[initial weight] × 100 [23].

Total RNA from Arabidopsis mature leaves were extracted using TRIZOL Reagent (Invitrogen,

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Shanghai, China) according to the manufacturer's instructions. Total RNA (500 ng) was reverse

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transcribed using MMLV reverse transcriptase (PROMEGA, USA) and Random primers (Invitrogen, USA). Quantitative PCRs was conducted in a 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) with SYBR Premix PCR Master Mix (Applied Biosystems, UK). Fluorescence was

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measured at 78–80◦C during 40 cycles. The designed sequences used in each quantification is described in Table S1.

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2.8 GUS assay.

2.8.1. Histochemical analysis. GUS activity was detected as described previously [30]. Tissues from transgenic plants transformed with ProAPA1:GUS were sampled and incubated in the GUS assay solution in a dark at 37 °C for 12 h, followed by treatment with ethanol series, fixation and examination or GUS activity.

2.8.2. ABA and osmotic stress assay. One-week old seedlings were grown on MS plates and incubated with ABA 5 µM or 30% PEG for 0, 2, 4 and 8 hs. Images of seedlings were digitalized using a Cannon EOS Rebel T5i camera (Cannon, Japan) coupled to a Nikon stereoscopic microscope SMZ745 (Nikon Corp., Tokyo, Japan).

2.8.3. Stomata quantification.

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Leaves from ProAPA1:GUS subjected to 10 days of WW or MWD were excised and blue staining was

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analyzed using a Cannon EOS Rebel T5i camera (Cannon, Japan) coupled to a Nikon stereoscopic microscope SMZ745 (Nikon Corp., Tokyo, Japan). Staining in epidermal and stomata cells was

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analyzed using a Nikon DS-Fi 1 camera coupled to a Nikon Eclipse E200 fluorescence microscope

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(Nikon Corp., Tokyo, Japan)

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2.9. Statistical analysis.

The data were presented as the mean ±SE with at least three biological replicates. Student’s t-tests

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(two-tailed analysis) at P < 0.01 or P <0.05 were used to analyze the significant differences.

3. Results

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3.1 APA1 is induced by drought

To analyze the expression of APA1 under drought conditions, total RNA was isolated from

A. thaliana leaves in control conditions, defined as well-watered (WW), or in mild water deficit (MWD). Transcript level of APA1 was determined by quantitative real time PCR (qRT-PCR) at different times after WW and MWD treatments. Results obtained showed that APA1 was up regulated after 10 days of water stress (Fig. 1A).

3.2 OE-APA1 plants display enhanced tolerance to drought stress A. thaliana plants were transformed with 35S:APA1 construct and subjected to MWD. We followed plants in a time-course experiment and observed phenotypical differences between genotypes after 10 days of stress (Fig. S1). Independent overexpressing lines, designated as OE-APA1 and OE-APA1.2, were selected and seeds were collected. Augmented transcript levels of APA1 in

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overexpressing plants and null expression in insertional lines (Fig. 1B) were confirmed (Fig. S2). Under WW conditions, the phenotype of OE-APA1 plants did not differ from those of WT or apa1

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plants. However, after 10 days of MWD, OE-APA1 plants improved tolerance to drought stress (Fig. 2A). Overexpressing plants under MWD behaved as non-stressed plants. These results were

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observed in the phenotype as well in the chlorophyll content and in the shortening of principal root

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length (Fig. 2 B and C). On the other hand, apa1 under MWD showed changes in phenotype, chlorophyll content and in root length comparable with WT under the same condition (Fig. 2 A-C,

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Fig. S3). In addition, WT and apa1 plants grown under MWD showed a reduction the total leaf area. This reduction was not observed in OE-APA1 lines grown under the same conditions. Cell density

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was analyzed to determine if the reduction in total leaf area was a consequence of a difference in the cell size. Results showed that cell density did not change upon MWD in OE-APA1 lines (Fig. 3 A and C).

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3.3 Overexpressing OE-APA1 affects leaf water loss Hydric parameters were measured in order to determine if drought tolerance of OE-APA1 plants is related with the ability of these plants to lose and consume water. We determined a lower water loss of OE-APA1 plants compared to WT and apa1 plants, calculated in detached leaves under MWD (Fig. 4B). We also measure water content and observed that OE-APA1 plants under MWD showed a

higher content of water (Fig. 4 C). On the other hand, there were not significant differences in the water consumption in plant genotypes tested under both, control and stress conditions (Fig. 4A). 3.4 APA1 overexpression affects stomata behavior. Results obtained with the different genotypes on water dynamics drove us to investigate stomatal behavior, a main factor affecting water use properties in plant leaves. We observed

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aperture, density and index of stomata in the lower epidermis of the rosette leaves in WT, OE-APA1

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and apa1 plants under WW or MWD.

The OE-APA1 plants showed reduced stomatal pore aperture compared with WT and apa1

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plants. This difference was present in WW and was maintained under MWD (Fig. 5A-B). The stomatal index was significantly lower in leaves of OE-APA1 plants than in WT and apa1 plants, both in WW

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as in plants under MWD (Fig. 5C). In addition, as well the differences did not result significatively,

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we observed a tendency in a reduction of the number of stomata per area in OE-APA1 plants respectively compared with WT under WW or MWD. On the other hand, apa1 showed a major

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stomata density than others genotypes in WW conditions (Fig. 5D). Due that ABA regulate stomatal closure, we determined the aperture pore size of WT, OEAPA1 and apa1 plants in response to it hormone. We observed that OE-APA1 plants already have the stomatic pore closed before ABA treatment and that the addition of hormone does not intensify

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the pore closure (Fig. 5 E).

3.5 Overexpression of APA1 affect expression of ABA signaling-related genes To determine if the function of APA1 in the stomata behavior is mediated by ABA signaling, we quantified the expression of this gene and related ABA genes in WT, OE- APA1 and apa1 plants in WW conditions. We measure APA1 expression in WT plants with or without ABA addition and

observed that external ABA application induce 4-fold APA1 gene expression (Fig. 6A). Then, we quantified the expression of two ABA responsive genes (RD19 and RAB18) and one gene that is implicated in ABA synthesis (NCED2). As is shown in Fig. 6 B, C and D, compared to WT, all genes resulted down regulated in apa1 and induced in OE-AP1 plants. In addition, two homeodomainleucine zipper class I genes; HB7 and HB12, both strongly induced by water-deficit and abscisic acid (ABA) [31,32] were modified in the same sense, down regulated in apa1 and induced in OE-APA1

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plants (Fig. 6 E and F). These findings suggest that the observed increase in drought tolerance

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conferred by overexpression of APA1 could be mediated via ABA and ABA-responsive gene expression.

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3.6 Expression profile of APA1

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To characterize the expression patterns of APA1, a 1941 bp upstream from ATG fragment upstream of the initiation codon ATG was fused to the β-glucuronidase (GUS) reporter gene in the pMDC162

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vector [21] (Fig. 7, upper panel). APA1 expression was detected in leaves in WW or MWD conditions. Specifically, we observed expression in vascular tissues (Fig. 7 A, B, E, F), in epidermal cells (Fig. 7 C

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and G) and stomata cells (Fig. 7 D and H). Both in epidermal and guard cells, APA1 expression seems to be restricted to chloroplasts (white arrows). In ABA treatment we observed that APA1 have a basal expression in hypocotyls (0 hours) and this expression was induced in a time course and detected strongly in vascular tissues. In PEG treatments, we observed a basal expression in

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hypocotyls and an induction at 2 hours of treatment, appearing in vascular tissues and maintained at the similar levels until 8 hours of treatment (Fig. 8). 4. Discussion Aspartic proteases (APs) genes have been identified in A. thaliana, B. oleracea, L. esculentum, M. trunculata, N. benthamiana, N. tabacum, O. sativa, P. abies, Z. mays [12]. Different

studies have related AP function in physiological processes during plant development as leaf senescence [33], seed ripening and germination [34], the immunity response [35,36], cell death [37] and reproduction [38]. Even though, little is known about AP and its participation in the response to abiotic stresses. Here we show that APA1 overexpression enhances ABA sensitivity promoting adaptive drought tolerance in Arabidopsis.

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4.1 APA1 overexpression promotes drought tolerance. APs are involved in the response to different pathogens infection in tomato and tobacco

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[39] in potato [16,40] in Cucurbita maxima [41], in rice [42] and in A. thaliana [43]. APs has been

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described as intermediates in the response to different abiotic stresses. Induction of a tomato AP in the response to wounding treatment [44], induction of a wheat AP in response to dark, wounding

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and UV-B light [45] have been reported. In addition, a common been AP is induced under drought stress [46]. Transcriptomic analyses demonstrated that an A. thaliana gene encoding an AP, named

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APA1, is induced in stomata cells in response to salt [47] cold and drought [20]. However, the roles

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of APA1 in the mentioned stresses were not studied in detail. In this work, we characterize APA1 gene functions associated with drought stress response for the first time. We cloned the APA1 cDNA sequence in a vector under the Cauliflower Mosaic virus 35S enhancer and performed MWD assays for WT, overexpressing and apa1 insertional lines

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in Arabidopsis plants. We observed that OE-APA1 lines resulted more tolerant to MWD than WT and, inversely, apa1 was more susceptible to this stress. Specifically, in plants under MWD, overexpressing lines showed more total area leaf, a less chlorophyll content and a less reduction in the shortened of principal root length compared to WT and apa1. We observed that OE-APA1 plants responded to MWD treatments showing an increment in the number of pavemented cells and a decrease in the stomata index and density of stomata cells

compared with WT. This rearrangement in the stomata cells could explain the tolerance of overexpressing plants to drought. To validate that, we analyzed different hydric parameters and we observed that even though there was no difference in water consumption between different genotypes, overexpressing leaves submitted to MWD losses less water and have a slight major content of water than WT plants. In contrast, apa1 lines showed elevated loss of water in leaves. This result could be explained by a lower transpiration rates of OE-APA1 plants and consequently a

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possible reason for the enhanced drought tolerance of the OE-APA1 plants. It was reported that the

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overexpression of another guard cell expressed AP (ASPG1) in Arabidopsis confer tolerance to water stress and that this aptitude was related with a lower water loss in leaves [20]. In addition, in this

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work, also is observed that overexpressing lines showed an increment in the stomatal closure. We therefore looked in detail the stomata aperture and its response to ABA in the different plants both

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in control conditions and in MWD. We observed that the pore in overexpressing plants were smaller

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than those of WT and apa1 plants. In this work, we conclude that the effect of overexpressing APA1 in water tolerance is due its influence in ABA-mediated stomatal behavior, both from a decrease in

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stomatal density and index, as well from a reduced aperture pore size. 4.2 The possible function of APA1

We reported that APA1 was induced in response to drought stress and ABA treatment. This induction was associated with an increment in the ABA signaling response. Two well-known

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different ABA responsive genes (RD29 and RAB18) and one gene that is implicated in the ABA synthesis (NCED2) resulted induced in APA1 overexpressing lines and repressed in apa1 plants. RAB18 and RD29 encode for a late embryogenesis abundant protein (LEA). In plants, the LEA function is associated with tolerance against drought, low temperature, and salinity via ABA signaling modifications [48]. NCED2 encodes for a 9-cis-epoxycarotenoid dioxygenase, an enzyme

which cleaves 9-cisxanthophylls to xanthoxin, a precursor of ABA. It gene was reported to be induced in response to drought in barley [49] and to osmotic stress in Vitis vinifera [50]. In addition, we observed that overexpressing lines, showed an induction in the expression of HB7 and HB12 genes. These two genes encode for transcription factors (TFs) belongs to the Homeodomain leucineZipper family and were reported to be induced by drought and ABA treatments [31,32]. It was described [31] that both TFs modulate the expression of ABA receptors PYL/PYRs and their

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interactors PP2C and SnRK2s, affecting different development stages in response to ABA as well the

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adaptative response to drought. In other study [32] it was described that HB7 overexpressing plants consumed less water than WT and lost less water by leaf transpiration. In addition, they report that

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HB7 seems to be the responsible for stomata closure. Interestingly, it was related HB7 and HB12 expression with an increment in the rosette leaves development in plants under water stress

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conditions [51], an event similar to what was observed in this work. Taken together, ours results,

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suggest that in addition to the reduced stomata index, APA1 overexpression confer drought tolerance via regulation of the ABA signaling pathway to consequently modify the opening of the

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stomatal pore.

The role of chaperones and proteases under stress conditions was recently discussed [52]. The authors provide evidences that in chloroplasts, chaperone systems refold proteins after stress, while proteases degrade misfolded and aggregated proteins that cannot be refolded. In order to

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identify potential substrates of APA1, in silico analysis was performed using several interactome analysis tools (Fig. S4; Table S2). Data obtained shown that APA1 could interact with gene products of two families, the BEL1 gene family and the KNOTTED1-like homeobox gene family [54]. Both genes families are regulated in response to drought, however their roles in abiotic stress response are still known [55]. Additionally, the results suggest a potential interaction between APA1 and an extracellular myrosinase binding protein like (MBP). This protein has been identified a novel

component in ABA signaling in guard cells [10,56,57]. Further work aimed to confirm these APA1 protein interactions should be done in order to establish the network of action of APA1 during drought stress. 5. Concluding remarks. We demonstrated that APA1 confers tolerance to mild water deficit when is overexpressed

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in Arabidopsis. This is, at least in part, due its role as an intermediate in the ABA-induced stomatal closure. Specifically, ABA-induced APA1 expression, could promote, directly or indirectly, the

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expression of ABA related genes and/or interact with different drought related proteins, in

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particular those associated with stomata closure and density. (Fig. 9). Even though, further analysis as endogenous ABA determination would be necessary to completely determine the precise

Conflict of interest

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mechanistic basis of this response.

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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Acknowledgments

This work was supported in part by National Scientific and Technical Research Council grant (CONICET) to M.G.G.; Scientific Research Commission of the Province of Buenos Aires (CIC) grants to M.G.G. and G. R. D. and University of Mar del Plata grant to M.G.G. S.D, D.F.F. and M.G.G. are established researchers of CONICET. G.R. D. is established researcher of CIC.

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Fig. 1. APA1 expression in A. thaliana plants under WW and MWD and apa1 insertional line representation. (A) Total RNA was isolated from leaf samples collected 10 days after MWD or in WW. Expression levels in qRT-PCR assays were calculated relative to expression of UBIQUITIN mRNA. Data are means of three replicates with ± SD. Asterisk means significative difference. (B) Graphical representation of the APA1 locus (At1G11910) in chromosome 1 of Arabidopsis. Insertion points of apa1 line is shown.

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Fig. 2. Changes in phenotype and in chlorophyll content in OE-APA1 Arabidopsis under drought stress. (A) WT, apa1 and OE plants were planted in pots and cultured for two weeks under WW conditions. Then, MWD was applied and at day 10 plants were photographed. (B) Spad Values were taken for WT, OE and apa1 plants in control or 10 days of MWD. (C) Principal root length of WT, OE and apa1 plants in control or under 10 days of MWD. Asterisk means significant differences in relation to WT in WW. Results were analysed by Student´s t test (P≤ 0.05). Data are means of three replicates with ± SD

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Fig. 3. Total area leaf and pavement cell density in OE-APA1 and apa1 plants. (A) Total area leaf of WT, OE and apa1 plants WW or under 1o days of MWD were quantify by ImageJ. Asterisk means significant differences in relation to WT in MWD (B) Pavement cell density of plants under 10 days of MWD was determined from lower epidermis cell strips. This assay did not show significative differences. Data are means of three replicates with ± SD (C) Representative picture of epidermis strips of plants under MWD. Results were calculated from at least three plants.

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Fig. 4. Water use in OE-APA1 and apa1 plants. (A). Water consumption was evaluated in WT; OE and apa1 plants by weighting the pots in WW or during the MWD (until day 14th). (B) Water loss from detached leaves of WT; OE and apa1 plants at the indicated time points. Water loss rates are indicated as the percentage of the initial fresh weight (% FW). Results are shown as the mean from three replicated samples from three different plants of each genotype analysed. Asterisk means significant differences in relation to WT in same condition. (C) Water content rates were calculated as the ratio between relating dry weight (W1) and fresh weight (W2). Asterisk means significant differences in relation to WT in WW. Results are shown as the mean from three replicated samples from three different plants of each genotype with ± SD.

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Fig. 5. Stomatal behaviour of WT, OE and apa1 plants in control or under MWD. (A) Stomatal guard cells were observed in WW or 10 days after MWD was applied. Photographs are representative for each case. Aperture stomata quantification is represented in (B). Stomatal index (C) and density (D) in lower epidermis of full-size rosette leaves of WT, OE and apa1 in WW or under 10 days of MWD. (E) Stomatal pore aperture in epidermis strips from of WT, OE and apa1 plants under 10 days of MWD and treated with 10 µM ABA. Data are means of three replicates with ± SD. Different letters indicate significant differences.

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Fig. 6. Analysis of expression of ABA-signalling-related genes in WT, apa1 and OE-APA1 plants under well-watered conditions. Total RNA was isolated from leaf samples collected in 4th- week plants and expression gene levels were calculated relative to expression of UBQ. Data are means of three replicates with ± SD. Asterisks means significative differences respect to WT.

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Fig. 7. Detection of tissue-specific localization of the APA1 protein in leaves. Plants transformed with proAPA1:GUS (upper panel) were irrigated with water (A-D) or summited to MWD for 10 days Bars=1mm (A,B, E,F), 0.01mm (C,D,G,H).White arrows indicate expression in chloroplasts.

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Fig 8. APA1 expression in seedling treated with ABA or PEG. One-week-old seedlings expressing APA1:promoter fussed to GUS reported gene were treated with ABA 5 µM or 30% PEG and incubated for different hours. Seedlings were incubated with X-GAL substrate during 24 hours, distained with ethanol 70 % and photographed. Bars=1mm.

Fig. 9. Model of Interactions between APA1 and ABA signalling mediators in the response to drought. ABA generated by drought induce APA1 expression. APA1 protein induce the expression of different genes related with the ABA response to drought as HB7, HB12, RD29, RAB18. Expression

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of this genes conduce to a different response as aperture and density stomata modifications. Red arrows: results reported in this work. (1) [48] (2) [50] (3) [32,51].