Exogenous salicylic acid improves freezing tolerance of spinach (Spinacia oleracea L.) leaves

Accepted Manuscript Exogenous salicylic acid improves freezing tolerance of spinach (Spinacia oleracea L.) leaves Hyunsuk Shin, Kyungwon Min, Rajeev Arora PII:

S0011-2240(17)30336-X

DOI:

10.1016/j.cryobiol.2017.10.006

Reference:

YCRYO 3894

To appear in:

Cryobiology

Received Date: 8 September 2017 Revised Date:

16 October 2017

Accepted Date: 17 October 2017

Please cite this article as: H. Shin, K. Min, R. Arora, Exogenous salicylic acid improves freezing tolerance of spinach (Spinacia oleracea L.) leaves, Cryobiology (2017), doi: 10.1016/ j.cryobiol.2017.10.006. 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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Exogenous salicylic acid improves freezing tolerance of spinach (Spinacia oleracea L.) leaves

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Hyunsuk Shin1, 2†, Kyungwon Min1†, Rajeev Arora1*

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Department of Horticulture, Gyeongnam National University of Science and Technology,

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Jinju 52725, S. Korea

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Both authors contributed equally to this manuscript

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Department of Horticulture, Iowa State University, Ames, IA 50011; 2 present address:

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*Corresponding author. Tel.:+1-515-294-0031; fax: +1-515-294-0730

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*E-mail address: [email protected]

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Abstract

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Salicylic acid (SA)-treatment has been reported to improve plant tolerance to various abiotic stresses. However, its

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effect on freezing tolerance has not been well investigated. We investigated the effect of exogenous SA on freezing

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tolerance of spinach (Spinacia oleracea L.) leaves. We also explored if nitric oxide (NO) and/or hydrogen peroxide

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(H2O2)-mediation was involved in this response, since these are known as primary signaling molecules involved in

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many physiological processes. A micro-centrifuge tube-based system used to apply SA to petiolate spinach leaves

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(0.5 mM over 4-d) was effective, as evident by SA content of leaf tissues. SA-treatment did not hamper leaf growth

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(fresh and dry weight; equatorial and longitudinal length) and was also not significantly different from 25%

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Hoagland controls vis-à-vis growth. SA application significantly improved freezing tolerance as evidenced by

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reduced ion-leakage and alleviated oxidative stress (lower accumulation of O2·- and H2O2) following freeze-thaw

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stress treatments (-6.5, -7.5, and -8.5 °C). Improved freezing tolerance of SA-treated leaves was paralleled by

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increased proline and ascorbic acid (AsA) accumulation. A 9-d cold acclimation (CA) treatment also improved leaf

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freezing tolerance (compared to non-acclimated control) and was accompanied by accumulation of SA and proline.

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Our results indicate that increased FST may be associated with accumulation of compatible solutes (proline) and

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antioxidants (AsA). Notably, the beneficial effect of SA on freezing tolerance was abolished when either H2O2- or

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NO-scavenger (1 µM N-acetylneuraminic acid, NANA or 100 µM hemoglobin, HB, respectively) was added to SA

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as pretreatment. Our data suggest that SA-induced freezing tolerance in spinach may be mediated by NO and H2O2

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signaling.

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Key Words

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Freezing stress; cold acclimation; oxidative stress; proline; ascorbic acid; salicylic acid; NO signaling;

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H2O2 signaling

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1. Introduction

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50 Freeze-thaw is one of the major environmental stressors impacting crop yield and distribution of plant species. Long

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enough exposure to temperatures cooler than the freezing tolerance threshold of plant tissues can result in

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irreversible injuries. By far, the primary cause of injury due to an equilibrium freezing episode is cellular

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dysfunction resulting from dehydration and contraction (during freezing) followed by rehydration and expansion

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(during thaw) [4]. Thus far, two cellular loci have been predominantly implicated to explain the mechanism of

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freeze-thaw injury: 1) structural and functional perturbations in cell membranes, evident in solute leakage [1, 64],

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and 2) excessive production of reactive oxygen species (ROS) [3, 30], which, if not adequately removed through

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antioxidants, damage cellular components including membranes, proteins, and nucleic acids [43].

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Salicylic acid (SA), a hormone-like plant phenolic, has been widely implicated as signal molecule mediating defense mechanisms against pathogens, such as hypersensitive response and development of systemic

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acquired resistance [8]. Evidence is also accumulating for SA-application enhancing tolerance against abiotic

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stresses including heat, chilling, drought, and salt stress [31, 42, 55]. However, the effect of exogenous SA on

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freezing tolerance in plant tissues has not been well investigated.

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Nitric oxide (NO) and hydrogen peroxide (H2O2) are well known key signaling molecules in plants, regulating biological processes and mediating diverse protective mechanisms against abiotic stresses [51].

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Accordingly, to gain mechanistic insights into SA’s protective role, several laboratories have explored potential

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cross-talk among SA, H2O2 and NO [34, 46, 52, 54]. Research also suggests that improved abiotic stress tolerance or

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stress-alleviation in the presence of exogenous SA may be mediated via: 1) bolstered pool and/or activity of

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enzymatic / non-enzymatic antioxidants, and/or 2) accumulation of compatible solute, proline, both of which have

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been widely correlated with acquisition of stress tolerance [2, 43, 59, 61, 67]. Indeed, pretreatment of SA improves

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chilling tolerance of maize seedlings via increase in H2O2 concentration which in turn induced an increase in

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antioxidant enzymes activities [25]. Moreover, combined application of NO and SA to wheat seedlings represses

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more efficiently the accumulation of malondialdehyde (MDA) and ROS and enhances activities of antioxidant

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enzymes than NO and SA alone [9]. However, a potential cross-talk among SA, NO, and H2O2 remains unknown in

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relation to freezing tolerance.

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Spinach (Spinacia oleracea L.), an important horticultural crop, is vulnerable to sudden and unseasonal spring frost often resulting in frost-damage and economic losses. Frequency of such occurrences is predicted to

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increase in future due to vagaries of climate change. Development of strategies or cultural practices to improve

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freezing tolerance would, therefore, be beneficial to horticulture industry. The major objectives of the present study

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were to determine whether exogenous SA-application could induce freezing tolerance in spinach leaves, and

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whether NO and / or H2O2 are involved in SA-induced response. In order to gain insight into the mode of action for

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SA-treatment, experiments were also conducted to monitor leaf growth and tissue content for SA, proline and a non-

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enzymatic antioxidant, ascorbic acid (AsA). A micro-centrifuge tube-based system (detailed under ‘Materials and

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Methods’) was used for administering exogenous SA to spinach leaves.

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2. Materials and Methods

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2.1. Plant Material and Growing Conditions

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Seeds of Spinacia oleracea L. ‘Reflect’, an F1 hybrid cultivar (Johnny’s selected seeds, Inc., Winslow, ME) were

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sown in plug flats using Sunshine LC-1 mix (Seba Beach, Alberta, Canada) and placed in growth chamber at

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15/15 °C (D/N) under average photosynthetically active radiation (PAR) of ~300 µmol m-2 s-1 and 12-h photoperiod

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provided by incandescent and fluorescent lights. Seedlings were watered as needed (~ 4-d interval). Two weeks

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later, the growth temperature was elevated to 20/18 °C (D/N) and 300 ppm EXCEL nutrition solution (Scotts Sierra

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Horticultural Products Company, Marysville, OH) was fed to seedlings through sub-irrigation. About 17-day-old

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seedlings were harvested and used for a SA and other treatments in a micro-centrifuge tube system.

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2.2. Micro-Centrifuge Tube System and Treatments

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First two true petiolate leaves were excised in deionized water to prevent embolism of xylem and inserted in a 1.5 4

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mL micro-centrifuge tubes (containing treatment solutions) through an adequate sized hole drilled in the lids of the

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tubes; these tubes are hereafter referred to as ‘micro-tubes’ (Fig. 1 inset). Control (25% Hoagland nutrition solution;

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[20]) and all the treatments, also made with 25% Hoagland as solvent, (all at ~ pH 6.5± 0.4) are as follows: (1) HG-

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control [25% Hoagland nutrition solution (HG)], (2) 0.5 mM or 1 mM SA, (3) 1 µM N-acetylneuraminic acid

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(NANA, a H2O2 scavenger), (4) 0.5 mM SA + 1 µM NANA, (5) 100 µM hemoglobin (HB, a NO scavenger), (6) 0.5

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mM SA + 100 µM HB, and (7) 0.5 mM SA+ 1 µM NANA+ 100 µM HB. Tubes held in micro-tube racks were

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placed in a growth chamber at 20/18 °C (D/N), ~300 µmol m-2 s-1, and 12-h photoperiod. To avoid any dehydration,

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especially in the early stages of sample transfer to the growth chamber, micro-tube racks were covered with a

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perforated transparent plastic dome with moist paper towels placed under it. The tubes were examined every 24 h

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and refilled with the spent solution as needed. After a 4-d treatment (precisely 96 h), samples were used for freeze-

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tolerance assays and other experimentation described below.

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2.3. Leaf Growth Measurement

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Leaf-growth was estimated by measuring length (longitudinal/equatorial), fresh weight, and dry weight. Leaf length

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and fresh weight were measured before and after the 4-d treatment in each solution. Dry weight was measured only

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after the 4-d treatment by oven-drying leaves at 75 ± 1 °C for 72 h. Data were obtained from 2 independent

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experiments with total of 32 technical replicates (16 technical replicate/experiment sampled from 8 individual

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plants; one leaf/technical replicate).

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2.4. Freezing Tolerance Measurement

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Leaf freezing tolerance was determined using the ion-leakage-based laboratory freeze-thaw protocol, as detailed by

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Chen and Arora [3]; Min et al. [41].Essentially, a pair of petiolate leaves (dip-rinsed four times with deionized

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water and blotted) was placed in a 2.5 × 20 cm test tube with petioles standing in 150 µL deionized water and slowly

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cooled (–0.5 °C/30 min) in a glycol bath (Isotemp 3028; Fisher Scientific, Pittsburgh, PA) to various freezing

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treatment temperatures following ice-nucleation at –1 °C. Tissues were held for 30 min at each temperature and 5

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thawed on ice overnight. Unfrozen control (UFC) leaves were maintained at 0 °C throughout the freeze-thaw cycle. For selecting the freezing treatment temperatures, first a LT50 curve was generated for micro-tube control (HG-control) samples by freezing the leaves at –3 to –12 °C. Tubes were removed from the glycol bath at –1 °C

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intervals. This experiment was repeated thrice, each with 5 technical replicates/temperature. Percent injury at

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individual treatment temperatures from these experiments were pooled and used to generate a sigmoid curve fitting

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the Gompertz function [39]; LT50 (°C), mid-point between the minimum and maximum injury, was defined as the

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leaf freezing tolerance. Guided by this sigmoid curve, 3 treatment temperatures, –6.5, –7.5, and –8.5 °C, were

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selected for comparing freezing tolerance of HG-control and 0.5 mM or 1.0 mM SA-treated leaves. For freezing

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tolerance experiments involving all 7 treatment solutions (i.e., HG, SA, NANA, HB, SA+NANA, SA + HB, and

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SA+NANA+HB), leaves were exposed to only two freezing treatments, -6.5 and -7.5 °C.

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Samples were removed from the glycol bath following freezing at selected temperatures and thawed as explained above. Freezing tolerance tests were independently repeated five times for HG-control, 0.5 mM and 1.0

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mM SA comparison, and thrice for the 7 treatments experiment, respectively, each including 4 to 8 technical

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replications per treatment temperature (two leaves/technical replicate). Percent injury data from these independent

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experiments were pooled to obtain treatment means.

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2.5. Staining of ROS (Superoxide and Hydrogen Peroxide)

Histochemical detection of superoxide (O2•–) and hydrogen peroxide (H2O2) was performed using the procedures

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previously used in our laboratory for spinach leaves [3, 41]. Staining intensities were visually evaluated for HG-

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control and 0.5 mM SA-treated leaves following exposure to freeze-thaw at -6.5 and -7.5 °C. Staining experiments

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were independently repeated twice, each with two replications (two leaves/replicate) per temperature per treatment

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solution. A representative picture of that data is presented here.

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2.6. Non- (NA) and Cold-Acclimation (CA) Treatments

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These treatments were administered on whole seedlings grown in the greenhouse media (not the micro-tube-treated 6

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ones). Plants grown for ~21-d in growth chamber (same conditions as described above) were considered as non-

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acclimated (NA). Nineteen-day old seedlings, grown same as NA plants, were transferred to a cold room (4 °C and

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12-h photoperiod at ~75 µmol m-2 s-1) for 9-d, and used as CA plants. LT50 of NA and CA plants was evaluated as

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described under freezing tolerance Measurements’. Experiments consisted five biological replications, each

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including 5-8 (for NA) and 4-5 (for CA) technical replicates.

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2.7. Quantification of SA, Pro, and AsA in Leaf Tissues

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SA, proline, and AsA were quantified in HG-control, 0.5mM SA, NA, and CA-tissues, using gas chromatography-

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mass spectrometry (GC-MS). Protocols used to extract SA, proline, and AsA were as described by Noutsos et al.

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[49], with some modifications. Two-hundred mg of liquid N2-ground leaf powder was mixed with internal standard

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(25 µg of ribitol) and homogenized with 0.35 mL hot methanol (60°C) for 10 min followed by 10 min sonication.

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Chloroform (0.35 mL) and 0.3 mL of HPLC grade water were then added to the mixture and vortexed for 1 min

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followed by centrifugation (10,000g) for 10 min. Upper, polar phase (200 µl), was removed and transferred into GC-

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MS vial (2 mL) and dried in a SpeedVac for 10-h. Extracts were methoximated using methoxyamine hydrochloride

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(50 µl of 20 mg/mL) and incubated for 90 min at 30 °C. Samples were silylated [by adding 70 µl of bis-trimethyl

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silyl trifluoroacetamide (BSTFA) plus 1% trimethylchlorosilane (TMCS)] for 30 min at 37 °C, and subjected to GC-

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MS (7890A GC, 5975C MSD, and separation column HP5MS; Agilent Technologies). Three target molecular

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species were identified using the method of Sumner et al. [60] by comparing the mass spectral to NIST08 Library

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and using Retention Indices followed by quantification based on internal standards.

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Measurements consisted of three biological replications, each with two technical replicates (each technical

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replicate was a 200 mg powder sub-sampled from the bulk generated by grinding 4-5 leaves).

2.8. Statistical Analysis

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Statistical differences were assessed using analysis of variance (ANOVA) employing R (version 3.2.2, The R

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Foundation for Statistical Computing, ISBN 3-900051-07-0). Mean differences were analyzed by Duncan’s multiple

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3. Results

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3.1. Growth Measurements in SA-Treated and HG-Control Leaves

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Leaves treated with 0.5 mM SA in micro-tubes for 4-d and their corresponding HG-controls continued to expand, as

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evident by increase in longitudinal and equatorial dimensions. While no significant difference for the two sets in

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equatorial growth was observed (relative to respective 0-d dimensions), SA-treated leaves grew relatively less

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longitudinally, though only by ~3% (Table 1). SA-treated leaves showed slightly lower gain in FW over 4-d

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treatment compared to HG-control but FW/DW ratios for the two sets were essentially same (Table 2).

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3.2. LT50 of Spinach Leaves Maintained in Micro-Tube System

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Freeze-thaw response curve (-3 to -12 °C) for ‘Reflect’ spinach leaves treated in micro-tubes with 25% HG for 4-d is

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presented in Fig. 1. Minimum and maximum injury percentages were 0.2 % and 81.9 %, respectively, with -6.9 °C

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corresponding to the mid-point of percent injury (41.1%), hence was considered as LT50 or freezing tolerance. A

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picture of the micro-tube system is presented as an inset.

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3.3. Effect of Exogenous SA on Freezing Tolerance and SA Content

Three test temperatures, -6.5, -7.5 and -8.5 °C, were selected to compare freezing tolerance of HG-control and SA-

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treated leaves. In preliminary experiments, four SA concentrations, i.e. 0.25, 0.5, 1.0 and 2.0 mM, were tested as leaf

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treatments. No significant difference was observed for freezing tolerance at 0.25 mM SA compared to HG control,

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while 2 mM SA treatment resulted in somewhat stunted growth and wilted appearance of leaves during the 4-d

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exposure (data not shown). Hence these two SA concentrations were dropped for subsequent experiments.

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Leaves treated with 0.5 mM SA were significantly more freeze-tolerant than HG-controls as evidenced by a reduction in freeze-thaw injury by 64.9, 43.9, and 14.2 % at -6.5, -7.5, and -8.5 °C, respectively (Fig. 2A). 8

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Likewise, percent injury for 1.0 mM SA treated tissues frozen at the same three temperatures was significantly

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reduced by 60.8, 33.1, and 12.2 %, respectively. Since no significant difference in percent injury was detected

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between 0.5 mM- and 1.0 mM SA-treatment, only 0.5 mM SA was selected for subsequent freezing tolerance and

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other experiments. Also, since the improvement in freezing tolerance by SA-treatment was more pronounced at -6.5

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and -7.5 °C stress level, the third treatment temperature (-8.5°C) was not included in subsequent experiments.

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SA content of leaves treated with 0.5 mM and 1.0 mM SA was ~6.7 and 270-fold of HG-Control (Fig. 2B), indicating that micro-tube system was effective in SA-uptake by petiolate spinach leaves.

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Distribution of superoxide (O2·-) (blue stain) and hydrogen peroxide (H2O2) (brown stain) showed similar pattern

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across two independent experiments; Fig. 3 presents a representative set of images. Although stain intensity was not

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digitally quantified, a higher accumulation of O2•– and H2O2 was apparent in injured tissues compared to unfrozen

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controls (UFCs) wherein the two ROS were essentially undetectable. Moreover, ROS accumulation increased with

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severity in freezing stress (-6.5 versus -7.5 °C). Data also indicate that ROS staining intensity in SA-treated leaves

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was lower than HG-control at both stress levels, indicating reduced ROS accumulation (compare Fig. 3A, C with

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Fig. 3B, D).

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3.5. Freezing Tolerance and SA Content of NA and CA Leaf Tissues

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Freezing tolerance (LT50) of NA and CA leaves were -5.8 and -9.9°C, respectively (Fig. 4A). SA content of NA and

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CA tissues was 0.14 and 0.65 pmol/mg FW, respectively, indicating ~5-fold accumulation during CA (Fig. 4B).

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3.6. Proline and AsA Content of SA-Treated, NA and CA Leaf Tissues

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Proline and AsA contents of 0.5 mM SA-treated tissues were significantly higher (~1.5- and 1.75-fold, respectively)

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than those of HG-Control (Fig. 5A and B). A 9-d CA treatment (4°C) of spinach seedlings also resulted in significant

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increase in proline content (~28-fold) compared to NA tissues (Fig. 5C) while no marked change in AsA content was

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observed after CA (Fig. 5D).

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To explore if SA-induced freeze-tolerance potentially involves H2O2 or NO mediation, experiments were conducted

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to evaluate freezing tolerance of leaves (at -6.5 and -7.5 °C stress treatments) that were pre-treated with ‘SA-alone’

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or in the presence of H2O2- or NO-scavenger (NANA and HB, respectively); NANA-alone, HB-alone,

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SA+NANA+HB, and HG-control were included as controls. Freezing tolerance experiment was repeated thrice,

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each with 4 to 5 technical replicates / temperature / treatment (two leaves/replicate). Means with standard errors

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from the pooled data across these experiments are presented in Fig. 6.

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Leaves treated with 0.5 mM SA suffered ~59% and 40% less injury at -6.5 and -7.5 °C, respectively,

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compared to HG-control, and thus provided the highest protection from freezing stress among six treatments.

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Notably, the beneficial effect of SA on freezing tolerance at both stress temperatures was completely offset when

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H2O2 scavenger (NANA) was added to SA as pretreatment; this treatment essentially resulted in similar freezing

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tolerance as HG-control. ‘NANA-alone’ pretreatment too was ineffective at eliciting any significant increase in

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freezing tolerance compared to HG-control but also did not adversely affect freezing tolerance. ‘SA+NO-scavenger’

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(SA+HB) appeared slightly effective at improving freezing tolerance but only at -7.5 °C. Finally, ‘HB-alone’ and

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‘SA+HB+NANA’ were not only ineffective but the only treatments that appeared somewhat deleterious, both

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causing ~ 30% increase in freeze-injury at -6.5 °C compared to controls while >2.5-fold injury compared to ‘SA-

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alone’.

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4. Discussion

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Evidence is accumulating for the beneficial effects of SA application on plant tolerance to abiotic as well as biotic

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stresses [24, 31]. However, research on SA’s effect on freezing tolerance remains scarce. Investigations also show 10

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that SA’s effect might involve NO and/or H2O2 signaling and that improved performance by tissues may result, in

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part, from increased tolerance to oxidative stress. Accordingly, we set out to explore a hypothesis that SA

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application increases freezing tolerance of Spinacia oleracea L. ‘Reflect’ leaves and that this effect involves NO

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and/or H2O2 mediation.

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To treat leaves with SA without potential confounding effect on the whole plant, we devised a micro-tube system. Our data on the SA contents of 4-d treated leaves indicates that this system allowed SA uptake through leaf

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petioles. Solution uptake was also evident by the need to having to refill the micro-tubes over time to replace spent

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solutions ensuring the submergence of petiole bases under liquids throughout. Leaf growth parameters and visual

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observations (leaf turgor or any indication of chlorosis) recorded over 4-d treatment period indicate that SA

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treatment used in this study was not detrimental. Based on the literature survey of SA concentrations used across

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diverse species for effecting tolerance against drought, chilling, salt, heat, or cold [29, 45, 47, 55], we first tested 4

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levels of SA application, 0.25, 0.5, 1.0, and 2.0 mM, of which, only 0.5 mM was selected for most of the work for

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reasons discussed below and elsewhere.

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4.1. SA Treatment Increases Freezing Tolerance

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Based on the freeze-response curve of HG-control leaves from micro-tube system (Fig. 1), we selected three stress

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temperatures, -6.5, -7.5, and -8.5 °C, to compare freezing tolerance of SA-treated and control tissues; these

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represented physiologically relevant injury levels, namely, substantial yet relatively mild (~25%), approx. LT50 level

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(~50%), and a level more severe than LT50 (~70%) but milder than maximum injury.

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Leaves treated with 0.5 mM and 1.0 mM SA were significantly less injured (ion-leakage assay) compared

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to controls at all stress levels, and both treatments were statistically equally effective at improving freezing tolerance

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(Fig. 2A). Since the leaves treated with 1.0 mM SA showed slight wilting, though only during the initial 24-h of

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treatment (data not shown), and accumulated intriguingly/abnormally high SA (~270-fold of control; Fig. 2B), this

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treatment too was dropped from subsequent physiological/biochemical investigations. Our selection of 0.5 mM SA

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for this study is in accordance with Hara et al. [15] who noted that optimal SA treatment for most plants’

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enhancement of abiotic stress tolerance ranged between 0.1 – 0.5 mM. 11

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Though several studies have explored SA’s role in conferring chilling tolerance (references in [53]), we are aware of only three reports to have shown increase in freezing tolerance by SA application [45, 62, 66]; one dealt

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with in vitro cultured potato plantlets while others employed SA ‘spray’ on winter-wheat leaves and grape leaves

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(unlike petiole-uptake in our study); moreover, neither study provided any data on SA uptake by tissues. Another

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important distinction of our study from these three is that we evaluated freezing tolerance using a temperature-

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controlled freeze-thaw protocol, regulating gradual and realistic cooling and thawing, and ensuring extracellular ice

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formation (ice-nucleation). In a recent study with WT and mutant Arabidopsis plants carrying the sid2-1 mutation, a

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loss of function of ICS1 with primary role in SA biosynthesis, Kim et al. [33] concluded that SA biosynthesis did not

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contribute to freeze-tolerance, an observation seemingly in contrast to our finding of SA-induced freezing tolerance

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in spinach leaves. Interestingly, however, a close examination of freeze-thaw response curves for WT and sid2-1

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mutant genotypes in their study reveals that despite similar LT50 (approximately -4°C) for both genotypes, sid2-1

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mutants were significantly more injured (by ~20%) compared to WT control at ~-3°C, a stress slightly milder than

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LT50, supporting our results with spinach leaves (Fig. 2A). This revelation is significant and seems to caution that

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comparing freezing tolerances across treatments based solely on LT50 may not always reveal real and significant

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freezing tolerance differences particularly in the sub-lethal range which may be more meaningful, physiologically,

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than injuries too severe to be remedied.

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Excess accumulation of ROS (O2·- and H2O2) during freeze-thaw and resultant oxidative damage by ROS

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have been widely proposed to explain freezing injury [2, 30]. Our data are consistent with this notion since freeze-

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thaw stressed leaves accumulated greater amount of O2·- and H2O2 (visual intensity) compared to unfrozen controls

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(Fig. 3); similar observations were made in our previous studies with ‘Bloomsdale’ spinach [3, 41]. ROS

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accumulation, however, was relatively higher in control than SA-treated leaves at both stress levels, supporting the

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SA-induced freezing tolerance evident from the ion-leakage assay (Fig. 2A). Several studies have shown that

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exogenous SA application protected plants against abiotic stresses by alleviating the accumulation of O2·- and/or

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H2O2 (see references [15, 31, 44]).

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4.2. Freezing Tolerance vis-à-vis SA content of NA and CA leaf tissues

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A 9-d CA of whole plants resulted in gain of 4.1°C in freezing tolerance (Fig. 4A). NA and CA LT50 values obtained

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in our study are in accordance with those previously reported in [3, 14, 41]. Notably, the freezing tolerance (LT50) of

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micro-tube-treated leaves (-6.9°C) was 1.1 °C colder (more negative) than for those sampled from whole-plants (see

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NA- LT50 ), despite the use of identically controlled freeze-thaw protocol (compare Fig. 1 and Fig. 4A). This small

320

yet significant discrepancy in freezing tolerance from the two scenarios highlights the rationale for first determining

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freeze-response curve for micro-tube treated leaves so as to select physiologically relevant freeze-stress treatments

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for this study.

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CA leaves had higher SA content than NA controls; ~5-fold accumulation during CA (9-d at 4°C) in our study is supported by a similar 4.1 to 6.3-fold accumulation in cold-exposed (12-d at 5 °C) Arabidopsis leaves [53]

325

and SA accumulation in cold acclimating wheat [35]. Increased freezing tolerance paralleled by SA accumulation

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during CA lends support to a similar outcome for leaves fed with SA in micro-tubes. However, gain in freezing

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tolerance vis-a-vis SA accumulation in CA tissues was substantially higher than that by SA-feeding (compare Fig.

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2A, B with Fig. 4A, B). This is not surprising since CA is a multi-factorial, complex response involving myriad of

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cellular, molecular and whole plant level adjustments, and SA accumulation maybe one of the contributing factor to

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this additive response.

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4.3. Why are SA-Treated Leaves More Freezing Tolerant?

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In order to explore probable physiological/biochemical explanation for SA-induced freezing tolerance, we studied

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the leaf-growth dynamic and tissue levels of two molecules (proline and AsA) known to be associated with

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acquisition of freezing tolerance.

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4.3.1. Leaf growth and increased Freezing Tolerance

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Data showed only a slight reduction (~3%) in longitudinal growth (none equatorially) in SA-fed leaves over the

340

treatment duration; these leaves also gained slightly less FW. Though unable to discount completely, we are inclined

341

to assume a negligible effect of leaf growth differences on freezing tolerance. Others have also noted a growth13

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342

retardation effect of exogenously applied SA (0.1 mM), e.g. in stem of potato microplants [40] or tobacco seedlings

343

[7].

344

4.3.2. Proline accumulation and increased Freezing Tolerance

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Proline, a compatible solute, has been widely reported to accumulate at higher level in CA tissues compared to NA

347

ones [22, 36, 50, 67] and is recognized as one of the components of abiotic stress tolerance mechanism [61]. Due to

348

its hydrophilic nature, proline is believed to protect macromolecules and membranes against freeze-induced

349

desiccation [21]. Proline accumulation under stress is also implicated in detoxification of ROS and cellular osmotic

350

adjustment [18]. In the present study, more freeze-tolerant SA-treated spinach leaves also accumulated ~45% higher

351

proline compared to HG-controls. Similarly, CA spinach leaves accumulated higher proline content compared to

352

NA. Moreover, much greater proline accumulation in CA leaves relative to SA-fed ones is also paralleled by a

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substantially greater gain in freezing tolerance by the former (compare Fig 5A vs. Fig. 5C with Fig. 2A vs. Fig. 4A).

354

In support of our results, others have also noted increased proline accumulation and concomitant amelioration of

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abiotic stresses (heat or salt) in 0.5 mM SA-treated wheat [32], Torreya grandis [37], and lentil [42]. No experiments

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were conducted in our study to investigate why SA-treatment results in proline accumulation. However, Misra and

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Saxena [42] and Khan et al. [32] noted increased activities of proline biosynthetic enzyme (γ-glutamyl kinase, a

358

component of 1-pyrroline-5-carboxylic acid (P5C) synthetase complex) in SA-treated tissues; both groups also noted

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decreased activity of proline catabolizing enzyme (proline oxidase) in SA-treated tissues, an outcome also expected

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to favor proline accumulation.

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4.3.3. AsA accumulation and increased Freezing Tolerance

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Ascorbic acid (AsA) or ascorbate is one of the important non-enzymatic antioxidants, widely implicated in

364

protecting plants against oxidative stress emanating from various abiotic stresses including freeze-thaw [2, 57] and

365

references therein). AsA essentially serves as a substrate in glutathione-ascorbate (GSH-AsA) cycle, catalyzed by

366

ascorbate peroxidase (APX) to detoxify H2O2 [59]. In our study, SA treatment markedly improved freezing

367

tolerance as well as caused lower O2•– and H2O2 accumulation following freeze-thaw, suggesting improved ROS

368

scavenging by SA-fed tissues. These tissues also accumulated higher level of AsA (~1.75-fold) compared to HG-

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control (Fig. 5B), which could be one of the components of bolstered ROS scavenging. Others have also noted AsA

370

accumulation by exogenous SA application [38, 46].

371

Understanding why SA-feeding increases AsA accumulation is beyond the scope of this study. However, literature survey leads us to offer an explanation for potential ‘indirect inducement’. Research shows that exogenous

373

SA reduces activity of catalase [5, 23, 46], another antioxidant enzyme responsible for detoxifying H2O2. Therefore,

374

it may be argued that, if not scavenged, this could result in excess H2O2 accumulation under stress conditions (such

375

as freeze-thaw). And, upregulation of APX activity will, conceivably, be needed to compensate for diminished

376

catalase activity, thereby requiring even greater amount of AsA (substrate). Indeed, increased APX activity or

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transcripts has been reported in response to SA-application [7, 29, 62].

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Upregulation of enzymatic and non-enzymatic antioxidants, including AsA, during CA has been reported

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[6, 26, 28]. To determine if CA-induced freezing tolerance was also accompanied by AsA accumulation, we

380

compared NA and CA tissues but were intrigued to find no significant change (Fig. 5D). Speculatively, AsA may not

381

constitute a major component of scaled-up antioxidant capacity in cold acclimated ‘Reflect’ spinach leaves, a

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proposal warranting further investigation.

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4.4. Does SA-Induced Freezing Tolerance Require H2O2 or NO Mediation?

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SA, NO, and H2O2 are regarded as key signaling molecules regulating various biological processes and mediating

387

diverse stress protective mechanisms [44, 48, 51]. A ‘self-amplifying feedback loop’ concept suggests a ‘duplex

388

signal transduction’ whereby SA modulates H2O2 accumulation and vice-versa [13, 16, 27, 54]. Evidence also exists

389

for SA-induced NO production [69] or enhancement of NO-regulated antioxidant enzymes [34], and for NO

390

modulating SA and H2O2 levels [13, 52]. Using an NO scavenger (hemoglobin, HB), Mostofa et al. [46]

391

demonstrated that NO mediates SA-induced salt tolerance in rice. Despite investigations of potential ‘cross-talk’

392

among SA, NO, and/or H2O2 in mediating abiotic stress tolerances, no such information exists for freezing

393

tolerance.

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Our results showed while 0.5 mM SA-treatment induced significant freezing tolerance in spinach leaves,

395

adding 1µM NANA, a H2O2-scavenger, to SA (SA+NANA) essentially abolished the beneficial effect of SA (Fig. 6).

396

This suggests that SA exerts its effect in H2O2-dependent manner, a notion reinforced by reports that H2O2 signaling 15

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acts downstream of SA [68]. Accumulation of H2O2 at exceedingly low levels has been implicated as a second

398

messenger for activating various stress responses [43, 48]. Harfouche et al. [16] noted that exogenous application of

399

SA, in a range of 0.1 – 0.5 mM, gave rise to low ROS accumulation and activation of antioxidant enzymes [19].

400

Research also shows that exogenous H2O2 enhances tolerance to salt [12], heat [65], and chilling stresses [17], and

401

in most cases, the protective mechanism included enhanced antioxidant enzymes activity. Although antioxidant

402

enzymes activities were not measured in the present study, our data on reduced ROS accumulation in freeze-stressed

403

leaves pretreated with SA, coupled with their higher AsA accumulation compared with HG-control, lend support to

404

such notion. A similar response for ‘SA+NANA’, ‘NANA only’ and ‘HG-control’ treatments suggests that 1µM

405

NANA used in our study was sufficient to remove most of H2O2, endogenous as well as any putatively induced by

406

SA, and was also not detrimental to unstressed or freeze-stressed spinach leaves.

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Our data indicates that scavenging of NO using ‘SA+HB’ pretreatment negated the beneficial effect of ‘SA alone’ on freezing tolerance, especially at -6.5°C stress. Moreover, ‘HB-alone’ treatment resulted in slightly higher

409

injury than HG-control at -6.5°C, a treatment which evoked similar response as ‘SA+NANA+HB’ pretreatment.

410

This suggests a role for NO signaling and for its mediation in SA-induced freezing tolerance in spinach leaves.

411

However, explanation for slightly lower injury at -7.5°C in ‘SA+HB’ pretreated leaves compared to HG-control,

412

‘HB alone’ or ‘SA+NANA+HB’ is intriguing. Simaei et al. [58] noted that ‘SA+NO-donor’ pretreatment improved

413

salt stress tolerance of soybean seedlings via increased antioxidant enzymes activity. Reports also exist for improved

414

tolerance by NO-donor-application against chilling [10], salt/heat [63] and drought [11]. Also, NO has been shown

415

to protect plants from oxidative stress by promoting detoxification of O2- via enhancing H2O2-scavenging enzymes

416

[56]. Fan et al. [10] reported a reduced level of electrolyte leakage in NO-donor-treated plants after chilling stress,

417

paralleled by enhanced antioxidant enzymes activity in response. Mostofa et al. [46] provided evidence for cross-

418

talk between SA, H2O2 and NO in enhancing salt stress tolerance in rice seedlings.

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In conclusion, the present study provides evidence that a 4-d treatment of 0.5 mM exogenous SA

420

improved freezing tolerance (reduced membrane leakage and lower ROS accumulation) of spinach leaves without

421

causing any detrimental effect on leaf health. The micro-tube system used here allowed efficient absorption of SA by

422

the leaves. Our results indicate that SA-induced increased freezing tolerance may be associated with greater

423

accumulation of compatible solute, proline and antioxidant AsA, and may involve NO and H2O2 mediation (Fig. 7). 16

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Future studies should test the effect of SA-feeding on freezing tolerance at whole plant level. Moreover, further

425

mechanistic elucidation of SA-induced freezing tolerance of spinach leaves is warranted, potentially involving the

426

investigation of antioxidant enzyme activity, gene expression associated with proline biosynthesis, and monitoring

427

of other factors contributing to freeze-tolerance.

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Acknowledgments

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This journal paper of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, Project no. 3601

431

was supported by Hatch Act and State of Iowa funds. Technical assistance by Mr. Peter Lawlor (Manager,

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Horticulture Greenhouses) and Drs. Ann Perera and Lucas Showman (W.M. Keck Metabolomics Laboratory), Iowa

433

State University is gratefully acknowledged.

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Fig. 1

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Freeze-thaw injury response of micro-tube-based leaves of 3-week old spinach (Spinacia oleracea L. cv. Reflect)

590

seedlings; spinach seedlings were grown in green house media for 17-d (see methods) and petiolate leaves were

591

transferred to micro-centrifuge tubes containing 25% Hoagland solution for 4-d before administering the freezing

592

test. The sigmoid curve was generated from percent injury at individual treatment temperatures using Gompertz

593

function; LT50 (°C), the mid-point (41.1% corresponding to -6.9°C) between the minimum (0.2%) and maximum

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(81.9%) injury was defined as the temperature causing 50 % injury. Inset: Illustration of micro-centrifuge tube

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system used in this study (see Methods).

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Fig. 2

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(A) Effect of exogenous SA on leaf freezing tolerance of 3-week old spinach (Spinacia oleracea L. cv. Reflect)

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seedlings; plants were grown in green house media for 17-d and petiolate leaves were incubated for 4-d in three

600

different treatments (as follows) before exposure to freezing treatments: (1) 25% Hoagland solution (HG-Control),

601

(2) 0.5mM SA, and (3) 1.0 mM SA using micro-tube system as shown in Fig. 1 inset. Unfrozen leaves

602

corresponding to each treatment were used as control (UFC). Different letters indicate significant differences

603

between treatments at p ≤0.05, according to Duncan’s multiple range test. (B) SA concentration in leaf tissues

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incubated with three solutions as explained above

605

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Fig. 3

607

Distribution of superoxide (O2· -) (A and B) and hydrogen peroxide (H2O2) (C and D) in unfrozen controls (UFC)

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and freeze-thaw injured spinach (Spinacia oleracea L. cv. Reflect) leaves, that were pretreated with either 0.5 mM

609

SA or with 25% Hoagland solution (HG-Control) before exposure to two freezing treatments, -6.5 and -7.5 °C.

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Superoxide (O2· -) and hydrogen peroxide (H2O2) were visualized by histochemical staining

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Fig. 4

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(A) Freeze-thaw injury response in non-acclimated (NA) and cold-acclimated (CA) spinach (Spinacia oleracea L.

614

cv. Reflect) leaves. The sigmoid curves were generated from percent freeze-injury using Gompertz function; LT50

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(°C) of NA leaves (-5.8°C) corresponded to mid-point injury of 47.7%, between the minimum (0.2%) and maximum

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(95.0%) injury, and that of CA leaves (-9.9 °C) corresponded to mid-point injury of 45% between the minimum

617

(0.2%) and maximum (88.7%) injury. (B) SA content in NA- and CA-spinach leaves. *, p ≤ 0.05, analyzed by

618

Student’s t-test

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619

Fig. 5

621

Proline and ascorbic acid (AsA) content in Hoagland solution (HG-control)- vs. 0.5 mM SA-treated spinach

622

(Spinacia oleracea L. cv. Reflect) leaves (A and B), and non-acclimated (NA) vs. cold-acclimated (CA) leaves (C

623

and D). Plants used for A and B were grown in green house media for 17-d and petiolate leaves were incubated for

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4-d in HG-control or SA. See methods for details on NA and CA treatments. NS, no significant difference; *, p ≤

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0.05; **, p ≤ 0.01, analyzed by Student’s t-test

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Fig. 6

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Effects of SA with or without H2O2- and NO-scavengers (NANA and HB, respectively) on freezing tolerance of

629

(Spinacia oleracea L. cv. Reflect) leaves; plants were grown in green house media for 17-d and petiolate leaves

630

were incubated in seven different treatments (as follows) for 4-d using micro-tube system before exposure to two

631

freezing treatments (i.e., -6.5 and -7.5 °C): (1) 25% Hoagland solution (HG-Control), (2) 0.5mM SA (SA), (3) 0.5

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mM SA + 1µM NANA (SA+NANA), (4) 0.5 mM SA + 100 µM HB (SA+HB), (5) 0.5 mM SA+ 1µM NANA + 100

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µM HB (SA+NANA+HB), (6) 1µM N-acetylneuraminic acid (NANA), and (7) 100 µM hemoglobin (HB). Different

634

letters indicate significant differences between treatments at p ≤0.05, according to Duncan’s multiple range test

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Schematic illustration of the effect of SA-pretreatment on freezing tolerance of spinach (Spinacia oleracea L. cv.

638

Reflect) leaves. SA-accumulating leaves suffer lower oxidative stress and reduced membrane injury (ion-leakage)

639

following a freeze-thaw cycle. Proposed mechanism involves accumulation of proline and ascorbic acid in SA-fed

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leaves in a H2O2- and NO-dependent manner. SA, salicylic acid; NO, nitric oxide; H2O2, hydrogen peroxide.

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Table 1

644

Leaf length and ratio of leaf length of 25% Hoagland solution (HG-Control)- vs. 0.5 mM SA-treated spinach

645

(Spinacia oleracea L. cv. Reflect) leaves

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Leaf length (cm) 0-d

Treatment

Ratio of leaf length (4-d/0-d)

4-d

Equatorial

HG-Control

3.39 ± 0.04

2.09 ± 0.04

0.5 mM SA

3.37 ± 0.04

2.06 ± 0.03

Longitudinal

Equatorial

Longitudinal

Equatorial

3.55 ± 0.05

2.14 ± 0.04

1.05

1.02

3.47 ± 0.05

2.10 ± 0.02

1.02y

1.02z

TE D

Longitudinal

Data are the means of 32 technical replicates from two independent experiments (16 technical replicates each) with

648

standard errors analyzed with Student’s t-test

649

y

p ≤ 0.01

650

z

non-significant

AC C

651

EP

646 647

652

Table 2

653

Leaf weight and ratio of leaf weight of Hoagland’s solution (HG-Control)- vs. 0.5 mM SA-treated spinach (Spinacia

654

oleracea L. cv. Reflect) leaves

Leaf weight (g) Ratio of leaf weight Treatment

0-d Fresh weight

4-d Fresh weight

Dry weight

25

FW (4-d)/FW (0-d)

FW (4-d)/DW

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HG-control

0.135 ± 0.005

0.156 ± 0.007

0.025 ± 0.001

1.15

6.37

0.5 mM SA

0.131 ± 0.004

0.143 ± 0.004

0.023 ± 0.001

1.10y

6.38z

655 Data are the means of 32 technical replicates from two independent experiments (16 technical replicates each) with

657

standard errors analyzed with Student’s t-test

658

y

p ≤ 0.01

659

z

non-significant

AC C

EP

TE D

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656

26

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