Induction of Direct or Priming Resistance against Botrytis cinerea in

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Induction of direct or priming resistance against Botrytis cinerea in strawberries by #-aminobutyric acid and their effects on sucrose metabolism Kaituo Wang, Yunxia Liao, Qi Xiong, Jianquan Kan, Shifeng Cao, and Yonghua Zheng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00947 • Publication Date (Web): 01 Jul 2016 Downloaded from http://pubs.acs.org on July 2, 2016

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Journal of Agricultural and Food Chemistry

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Induction of direct or priming resistance against Botrytis cinerea in

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strawberries by β-aminobutyric acid and their effects on sucrose

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metabolism

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Kaituo Wang,†,‡,§ Yunxia Liao,† Qi Xiong,† Jianquan Kan,‡ Shifeng Cao,ǁ, * and Yonghua

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Zheng§

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†

College of Life Science and Engineering, Chongqing Three Gorges University, Chongqing,

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404000, People’s Republic of China

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‡

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China

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§

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Jiangsu 210095, People’s Republic of China

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ǁ

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Nanjing 210014, People’s Republic of China

College of Food Science, Southwest University, Chongqing, 400715, People’s Republic of

College of Food Science and Technology, Nanjing Agricultural University, Nanjing,

Nanjing Research Institute for Agricultural Mechanization, Ministry of Agriculture,

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Running title: β-aminobutyric acid on Different forms of Defense Response

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and Sucrose Metabolism in Strawberries

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* To whom correspondence should be addressed. Tel.: +86-25-84346256. Fax:

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+86-25-84346256. E-mail: [email protected].

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ABSTRACT

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The specific forms of disease resistance induced by β-aminobutyric acid (BABA) and

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their impacts on sucrose metabolism of postharvest strawberries were determined in the

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present research. Treatment with 10 to 500 mmol L-1 BABA inhibited the Botrytis cinerea

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infection, possibly directly by suppressing the fungus growth and indirectly by triggering

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disease resistance. Moreover, BABA-induced resistance against B. cinerea infection in

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strawberries was associated with either one of two mechanisms, depending on the

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concentration used: BABA at concentrations higher than 100 mmol L-1 directly induced the

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defense response including a H2O2 burst, modulation of the expression of PR genes

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including FaPR1, FaChi3, Faβglu and FaPAL and increased activities of chitinase,

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β-1,3-glucanase and PAL, whereas BABA at 10 mmol L-1 activated a priming response

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because the BABA-treated fruits exhibited an increased capacity to express molecular

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defense only when the fruits were inoculated with B. cinerea. Activation of the priming

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defense appeared almost as effective against B. cinerea as inducing direct defense.

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However, the primed strawberries maintained higher activities of SS-synthesis, SPS and

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SPP enzymes) and lower level of SS-cleavage during the incubation; these activities

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contributed to higher sucrose, fructose and glucose contents, sweetness index and sensory

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scores compared with fruits exhibiting the direct defense. Thus, it is plausible that the

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priming defense, which can be activated by BABA at relatively low concentrations,

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represents an optimal strategy for combining the advantages of enhanced disease protection

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and soluble sugar accumulation.

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KEYWORDS: strawberry, β-aminobutyric acid, induced resistance, direct, priming,

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sucrose metabolism

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INTRODUCTION

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Strawberry (Fragaria × ananassa) is a large-scale horticultural crop grown worldwide

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and is a rich natural source of anthocyanin antioxidants.1 Nevertheless, this typical

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non-climacteric fruit appears greatly perishable and susceptible to infection by fungal

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pathogens. Botrytis cinerea Pers.: Fr (gray mold) is the main cause of decay in strawberry

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but has been effectively controlled by some synthetic fungicides.2 Application of synthetic

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fungicides on harvested strawberry is being curtailed due to their potentially negative

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influences on the human health and environment; thus, alternative approaches to disease

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control are needed. Among recent new approaches, inducing disease resistance by various

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environment-friendly chemicals against fungal infection in postharvest fruits is attracting

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increasing interest.3

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In plants, induced resistance has been deemed to a state of enhanced defensive

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capacity by a stimulation of biotic or abiotic elicitors that can fuel appropriate cellular

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defense responses against pathogen invasion. Moreover, these elicitors can enhance the

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plant’s basal level of resistance by one specific form of defense responses, namely direct

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activation or priming mechanism for a faster and stronger response in plant against

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pathogen challenge.4 It has been reported that the direct expression of diastase resistance in

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plants can incur allocation costs that arise from the diversion of metabolites and energy

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from growth and development towards defense.5 In contrast, priming-related defense

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usually offers broad-spectrum protection without significant fitness costs from plant

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metabolism and growth because the priming defense processes remain dormant until

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pathogen infection such that the plant can avoid expending energy under low disease

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pressure. Therefore, priming for enhanced defense reflects a kind of cost-effective behavior

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in evolutional disease management of plants.6,7 Several chemical elicitors could potentially ACS Paragon Plus Environment


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induce the priming response against pathogens, e.g., salicylic acid (SA) and its photostable

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functional analog benzothiadiazole (BTH), methyl jasmonate (MeJA), dichloroisonicotinic

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acid (INA), saccharin and vitamins such as thiamine and riboflavin.8 In addition to these

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well-studied chemical inducers, β-aminobutyric acid (BABA) belongs to a non-protein

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amino acid and functions as an effective elicitor for inducing resistance in model plants

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such as Arabidopsis9 and tobacco.10 Experimental evidence has also shown that exogenous

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BABA effectively activated defensive genes, contributing to the establishment of resistance

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against various microbial pathogens in citrus,11 apple,12 mango13 and grape berries.14 In

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Arabidopsis, BABA-induced resistance was based on the potentiation of natural defense

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against biotic stresses by means of either the priming or the direct inducible defense, most

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likely depending on the dose of the specific inducer used.15 However, the specific forms

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and molecular mechanism underlying BABA-induced resistance in harvested fruits remain

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unknown.

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Soluble sugars are not only important attributes of sensory quality in fruits but sever as

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signaling molecules that regulate expressions of a variety of genes implicated in defense

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processes and metabolic pathway, resultantly determining fruit ripening and the

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accumulation of secondary metabolites.16 Glucose is the main sugar in strawberry fruit at

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all developmental stages and is normally stored in cellular vacuoles. Strawberries also

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contain sucrose and fructose at lower levels.17 Increased concentrations of soluble sugars

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during grape ripening were accompanied by an enhanced expression of defense-related

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genes, showing a positive association between sugar metabolism and disease resistance.18

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However, the direct induction of defense response by high MeJA concentrations negatively

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affected the fruit quality parameters of grapes, including lower soluble sugar concentrations

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and antioxidant capacities.19 However, the changes in soluble sugar components in

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harvested strawberries that accompany the expressions of defense responses after treatment ACS Paragon Plus Environment

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with resistance-inducing elicitors is still unclear.

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The aims of this study were to investigate (a) the specific characteristics of disease

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resistance that were induced by BABA elicitations under different concentrations and (b)

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the effects of BABA treatments on sucrose metabolism to obtain a further understanding of

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whether direct or priming resistance inhibited the accumulation of soluble sugars and

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caused sweetness and sensory loss in harvested strawberries.

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MATERIALS AND METHODS

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Fruit and pathogen. Strawberry (Fragaria × ananassa Duch. cv. Fengxiang) fruits,

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without synthetic fungicide application, were hand-harvested randomly at the fully ripe

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stage from an organic orchard in Wanzhou, Chongqing city, China, and then transferred to

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our laboratory within 2 h. The fruits of uniform color and size were sorted against any

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visual defects and randomly divided into two groups of 800 fruits each, which were

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subdivided into five subgroups of 160 fruits each.

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A B. cinerea strain was separated from infected strawberries and maintained on potato

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dextrose agar (PDA) at 25 °C for 14 d. Then spore suspensions were prepared by rinsing

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the spores from the fungal cultures with a sterilized bacteriological loop and removing

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adhering mycelial fragments by filtration of four layers of sterile cheesecloth. Finally, the

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concentration of the spore suspensions was modified to 5 × 104 spores mL-1 using a

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hemocytometer counting chamber.

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BABA (purity ≥ 99%, Sigma Co., USA) were dissolved in sterile distilled water containing 0.01 % (v/v) Tween-20 as required by application.

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Effects of BABA treatments on different forms of induced disease resistance in ACS Paragon Plus Environment


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strawberries. For the first group, each subgroup of 160 fruits was immersed in a BABA

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solution at one of the following concentrations: 0 (control), 1, 10, 100 and 500 mmol L-1 at

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20 °C; the immersed fruits remained for 15 min and were then air-dried for 30 min

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followed by surface sterilization and draining on one small sheet of filter paper at 20 °C.

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The fruits were then uniformly wounded with dissecting needles at the equatorial region to

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form two symmetrical holes of diameter 1.5 mm and depth 2 mm. Afterwards, each wound

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was inoculated with 20 µL of the prepared spore suspensions of B. cinerea and allowed to

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slowly air-dry for 1 h. For the second group, the strawberries in each subgroup were

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subjected to BABA treatment and wounded as described above. Then, a mock-inoculation

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was made by pipetting 20 µL of sterile distilled water into each wound. Finally, the fruits in

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both groups were placed in polyethylene boxes, and then incubated at 20 °C and 90 % RH

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for 5 d. Tissue samples of healthy pulp from 40 fruits in each treatment of each replicate at

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each time point were collected before the incubation (time 0) and daily throughout the

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entire incubation period. The samples of the fruits from each treatment were mixed and

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frozen in liquid nitrogen, and finally stored at -80 °C until analysis of H2O2 generation,

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enzyme activities and gene expressions. Each treatment contained three replicates, and the

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entire experiment was conducted twice with similar results.

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Disease assessment. A strawberry can be considered as an infected fruit when its

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visible fungal lesion around the wound area was more than 2 mm wide. Disease incidence

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(the percentage of infected fruits) and the lesion diameter were calculated on day 1, 3 and 5

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of the incubation.

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H2O2 concentration measurement. The concentration of endogenous H2O2 was

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determined according to the method of the titanium (IV) method as described by Patterson

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et al.20

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Defense-related enzyme assays. Activities of chitinase (EC 3.2.1.14), β-1,3-glucanase ACS Paragon Plus Environment

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(EC 3.2.1.6) and phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) were determined

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according to the method of Abeles et al.21 and Assis et al.22, respectively. All of the

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activities were expressed as units per mg protein. Protein content in the supernatants was

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measured by the bovine serum albumin (BSA) method of Bradford.23

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Pathogenesis-related (PR) gene expression analysis. Frozen sample (5 g) was gently

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ground into a powder form under the protection of liquid nitrogen. Afterwards, total RNA

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was extracted from 0.4 g of the powder according to the protocol of Yu et al.24 Reverse

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transcription (RT) was conducted using 2 µg of total RNA and the SuperRT First Strand

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cDNA Synthesis Kit (Invitrogen, Carlsbad, CA, USA) following the instructions. The

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synthesized cDNA was used as a template to amplify PCR.

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Real-time quantitative PCR (qRT-PCR) were conducted using a StepOnePlusTM

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Real-Time PCR System (Applied Biosystems, USA) in triplicates with specific primers of

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the defensive genes (Table 1). The thermal cycling conditions included an initial

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denaturation at 95 °C for 7 min followed by 40 cycles of amplification: denaturation at

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95 °C for 15 s, primer annealing at a primer-specific temperature ranging from 50 °C to

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60 °C for 30 s, achieving by a melting curve analysis program. All qRT-PCR reactions were

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normalized by the threshold cycle value (CT) comparing with a housekeeping gene

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Fa18S-rRNA (GenBank ID: AF163494.1) according to the 2-∆∆C(T) method.25

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Effects of BABA treatments on sucrose metabolism. Another second group of freshly

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harvested strawberries without pathogenic inoculation was treated with BABA at the

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concentrations of 0 (control), 1, 10, 100 and 500 mmol L-1, and then stored at 20 °C for 5 d.

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Tissue samples from approximately 50 fruits in each treatment of each replicate at each

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time point were collected before treatment (time 0) and daily during storage before

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measurement of parameters relevant to sucrose metabolism. The mixed samples from each

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treatment were frozen until analysis. Three replicates of 300 fruits were used per treatment, ACS Paragon Plus Environment


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and the entire experiment was conducted twice with similar results.

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Soluble sugar and fruit sweetness determinations. 5 g of the frozen samples was

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extracted in triplicate with 10 mL of pre-cooled 95 % ethanol, and the homogenates were

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then centrifuged for 10 min (10,000 × g). Afterwards, the combined supernatants were

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dried in a vacuum rotary evaporator (40 °C). The dry residual were redissolved in 10 mL of

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deionized water, and then filtered through a miniature reversed-phase C18 cartridge and a

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0.45-µm cellulose acetate membrane (Supelco Co., Bellefonte, PA, USA) for sugar

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assessments. Each sample (20 µL) was injected into an Agilent HPLC system equipped

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with a Zorbax carbohydrate analytical column (150 × 4.6 mm, 5 µm); deionized water was

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used as the mobile phase within 45 min at 0.8 mL min-1. The absorbance was scanned

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between 190 and 575 nm. External standardization was applied to quantify the individual

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sugars with calibration curves of authentic standards.26

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The parameter of sweetness index of strawberry was used to assess the sweetness

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perception and calculated using the formula: (2.30 [fructose]) + (1.35 [sucrose]) + (1.00

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[glucose]).27

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Enzyme assay. Activities of SPS (sucrose phosphate synthase, EC 2.4.1.14), SS

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(sucrose synthase, EC 2.4.1.13) and SPP (sucrose-6-phosphate phosphatase, EC 3.1.3.24)

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were measured using the methods of Hubbard et al.28, Miron and Schaffer29 and Chen et

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al.30, respectively.

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Sensory evaluation. After storage at 20 °C for 5 d, the sensory scores of the

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strawberries were evaluated according to our previous method.31 Ten trained panelists were

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required to score each fruit from five aspects such as firmness, visual appearance, juiciness,

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flavor and taste (including sweetness and acidity) on a nine-point scale according to our

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previous standard.31


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Effects of BABA on the growth of B. cinerea in vitro. The spore germination and germ

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tube elongation of the pathogen were estimated in potato dextrose broth (PDB) by our

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previous method32. Each treatment comprised three replicates of 10 PDB tubes, and the

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entire experiment was repeated twice with similar results.

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Cellular leakage. Cytoplasmic contents leaked from B. cinerea mycelium were

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measured as described by Qin et al.33 and expressed as gram per kilogram of dry mycelium

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weight.

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Statistical Analysis. All values were shown as the mean ± standard errors of three

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replicates from one independent experiment. Statistical analysis was conducted using the

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SPSS package program version 13.0 (SPSS Inc., Chicago, IL). Duncan’s multiple range test

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was chosen to compare the means at P < 0.05.

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RESULTS

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Effects of BABA treatments and B. cinerea inoculation on inhibiting postharvest

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disease in strawberries. The efficacy of BABA for controlling gray mold disease in

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strawberries was affected by the BABA concentration (Figure 1). In mock-inoculated

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strawberries, treatment with BABA concentrations from 10 to 100 mmol L-1 decreased

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disease incidence during the incubation at 20 °C; BABA at 1 mmol L-1 was found to

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provide little positive effect on the control of disease development (Figure 1A). Although

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inoculation with B. cinerea accelerated the decay development compared with the

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non-inoculated strawberries, both of the disease incidence and lesion diameter in

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BABA-treated fruits exhibited constitutively lower than the B. cinerea-inoculated controls

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except on the BABA concentration at 1 mmol L-1, where no beneficial effect can be found

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during the whole incubation (Figure 1B and C). ACS Paragon Plus Environment


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Effects of BABA treatment and B. cinerea inoculation on H2O2 concentration in

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strawberries. In mock-inoculated strawberries, no significant change in H2O2

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concentration was found in the controls and in the 1 mmol L-1 BABA-treated fruits

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throughout the incubation period. However, the H2O2 concentration increased with the

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similar time kinetics responded to BABA concentrations at 100 or 500 mmol L-1 on the first

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day of incubation; at this time, the H2O2 concentration reached a maximal value that was

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1.96-fold higher than that in the controls. Thereafter, H2O2 concentration gradually declined

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to background levels. Moreover, the mock-inoculated strawberries that were treated with 10

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mmol L-1 BABA displayed a gradual increase in H2O2 generation during the last 2 d of

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incubation (Figure 2A). H2O2 generation was activated in B. cinerea-inoculated

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strawberries to a greater degree, and concentrations reached a maximal level on the first

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day (approximately 3.5-fold higher than the value in mock-inoculated fruits), which was

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followed by a rapid decrease during the remaining 4 d. Pretreatment with 10, 100 or 500

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mmol L-1 of BABA resulted in higher level of H2O2 than the 1 mmol L-1 BABA-treated or

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control fruits throughout the incubation (Figure 2B).

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Effects of BABA treatment and B. cinerea-inoculation on the activities of

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defense-related enzyme in strawberries. The activities of chitinase and β-1,3-glucanase

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in controls rose slowly during the incubation for 5 d; meanwhile, PAL activity decreased

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gradually until the end of the incubation. Treatment of the fruits with 1 or 10 mmol L-1

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BABA had little influence on the defensive enzyme activities. In contrast, BABA

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treatments at 100 or 500 mmol L-1 directly promoted activities of chitinase, β-1,3-glucanase

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and PAL, and the activities were all significant higher than the corresponding control

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activities over the incubation (Figure 3A, C and E). Inoculation with B. cinerea resulted in ACS Paragon Plus Environment

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an evident inducible enhancement of the activities of chitinase, β-1,3-glucanase and PAL in

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strawberries. 1 mmol L-1 BABA did not affect the activities of the enzymes in comparison

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with the only B. cinerea-inoculated fruits. Treatment with 10 mmol L-1 BABA primed the

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fruits for disease resistance as shown by the promoted activities of the defensive enzymes

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in the BABA-treated and subsequently B. cinerea-inoculated fruits (Figure 3B, D and F).

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Effects of BABA treatment and B. cinerea-inoculation on defense-related gene

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expression in strawberries. To assess the defense responses involved in BABA-induced

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resistance, qRT-PCR was applied for quantifying the transcript levels of PR genes in mock-

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or B. cinerea-inoculated strawberries. In mock-inoculated fruits, the expression levels of

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FaPR1, FaChi3, Faβglu and FaPAL in the controls were low and stable throughout 5 days

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of incubation at 20 °C. BABA at 1 or 10 mmol L-1 showed no inducible effect on the

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transcript levels of all four defense genes, whereas treatment with 50 or 100 mmol L-1

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BABA directly enhanced the expression levels of the PR genes throughout the incubation

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(Figure 4A, C, E and G). After inoculation with B. cinerea, higher expression levels of

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FaPR1, FaChi3, Faβglu and FaPAL were recorded in pathogen-inoculated fruits compared

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with those in mock-inoculated fruits over the period. Treatment with 1 mmol L-1 BABA did

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not affect the pathogen-induced activation of all defense-related genes. In the strawberries

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that were pretreated with 100 or 500 mmol L-1 BABA and then inoculated with B. cinerea,

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the PR genes maintained higher transcript levels than only B. cinerea-inoculated fruits at

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each time point. Furthermore, the expression levels of FaChi3, Faβglu and FaPAL in the B.

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cinerea-inoculated strawberries were more strongly induced by 10 mmol L-1 BABA

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treatment and remained at constantly higher levels in comparison with the

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pathogen-inoculated fruits treated with 50 or 100 mmol L-1 BABA (Figure 4B, D, F and

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H). ACS Paragon Plus Environment


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Effects of BABA treatments on sucrose metabolism in strawberries. SS-synthesis

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activity increased within the first 2 d and thereafter gradually decreased until the 5th day in

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the control strawberries (Figure 5A). The trend of the change in SPS and SPP activities

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was similar, showing that the activities gradually rose with incubation time (Figure 5C and

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D). SS-cleavage activity in the controls maintained very stable during the storage (Figure

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5B). Treatment with 1 mmol L-1 BABA was not able to alter the activities of sucrose

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metabolism-related enzymes; however, treatment with 100 or 500 mmol L-1 BABA

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enhanced the SS-cleavage activity but suppressed the SS-synthesis, SPS and SPP activities.

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In particular, 10 mmol L-1 BABA treatment did not detectably change SPS and SPP

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activities throughout the observation period compared with the controls; however, the fruits

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that were treated with 10 mmol L-1 BABA showed significantly higher SS-synthesis

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activity and significantly lower SS-cleavage activity than the controls during the storage.

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Effects of BABA treatment on soluble sugar contents and sweetness index in

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strawberries. Treatment with BABA at the lowest concentration of 1 mmol L-1 did not

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significantly affect soluble sugar accumulation compared with the controls. In contrast, the

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two highest BABA concentrations (100 and 500 mmol L-1) led to marked decreases in the

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contents of sucrose and glucose. Treatment with 10 mmol L-1 BABA yielded the highest

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concentrations of all three soluble sugars among all treatments. After the storage, the

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contents of sucrose, fructose and glucose in the 10 mmol L-1 BABA-treated fruits were 13.1,

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24.7, and 28.2 %, respectively, higher than those in the controls (Figure 6A).

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Correspondingly, treatment with 10 mmol L-1 BABA improved the sensory attributes of the

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strawberries, and the sweetness scores were the highest obtained among all the treatments

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after the storage (Figure 6B). ACS Paragon Plus Environment

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Effects of BABA treatments on strawberry sensory qualities. The sensory qualities of

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firmness, visual appearance, juiciness, flavor and taste in strawberries after storage are

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shown in Figure 7. After the storage, the control and the 1 mmol L-1 BABA-treated fruits

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presented lower visual appearance scores and were evaluated as being of the poorest quality

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(4.81 and 5.13, respectively); these berries were considered unacceptable for consumers

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probably owing to severe fungal decay. Meanwhile, the taste score (including the sweetness

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assay) of the fruits treated with 10 mmol L-1 BABA were 31.9 % and 43.3 % higher than

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those in the 100 or 500 mmol L-1 BABA-treated fruits, respectively, which paralleled the

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changes in sweetness index and soluble sugar contents.

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Effects of BABA on the growth of B. cinerea in vitro. The treatment with 1 mmol L-1

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BABA did not detain the growth of B. cinerea in PDB tubes during the incubation at 26 °C

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for 48 h. However, an effective inhibitory was observed when BABA was supplied to the

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medium at concentrations higher than 10 mmol L-1; the spore germination and germ tube

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elongation of the samples were lower than the controls throughout the whole incubation

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(Table 2).

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Similarly, treatment with BABA treatment at 1 mmol L-1 failed to cause obvious

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increase on B. cinerea cellular leakage over the 4-h incubation period. In contrast, treatment

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with BABA at concentrations higher than 10 mmol L-1 constantly stimulated increases in

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the amounts of leaked soluble proteins (Figure 8A) and carbohydrates (Figure 8B) during

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the whole incubation.

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DISCUSSION

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Recently, BABA has been investigated intensively as an inducer of host resistance in a

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wide variety of fruits.34 In the present study, the obtained data also revealed that a threshold

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level of 10 mmol L-1 BABA effectively induced defense responses in strawberries, as

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evidenced by a H2O2 burst, the stimulation of defensive enzyme activities, and the

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enhanced expression of several PR genes such as FaPR1, FaChi3, Faβglu and FaPAL,

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corresponding to the suppression of B. cinerea gray mold rot during the incubation at 20 °C

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(Figure 1). Furthermore, BABA exhibited direct antifungal activity against B. cinerea at

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the lowest studied concentration of 10 mmol L-1 (Table 2), possibly due to direct

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stimulation by BABA of plasma membrane disintegration and the cellular material leakage

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(Figure 8). Thus, we deduced that the induced disease resistance and direct antifungal

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activity were both involved in the mechanisms by which BABA inhibited the B. cinerea

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infection in postharvest strawberries.

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Ample evidence indicates that a strong burst of H2O2 performs several vital functions

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in the early cellular defense responses of plants against fungal infection, including the

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enhancement of hypersensitive (HR) response, cell wall reinforcement, defensive gene

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activation and the induction of antimicrobial compounds.35 It has been reported that

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transient H2O2 generation is closely connected with the BABA-induced disease resistance

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in plant-pathosystems.9, 10, 36 In the present study, the treatment with 1 mmol L-1 BABA did

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not stimulate H2O2 accumulation in either mock- or B. cinerea-inoculated strawberries at

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all time points during the incubation. In contrast, increasing the concentration of BABA to

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100 or 500 mmol L-1 directly induced a transient and strong H2O2 burst within the first day

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of the incubation regardless of whether the fruits were challenged with the pathogen B.

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cinerea. Treatment with 10 mmol L-1 BABA alone could not trigger H2O2 production in the

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absence of pathogen infection. However, in strawberries that were treated with 10 mmol L-1

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BABA and subsequently inoculated with B. cinerea, a stronger H2O2 burst was observed ACS Paragon Plus Environment

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within 1 day after inoculation, indicating that at relatively low concentration BABA could

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induce resistance at least in part by priming fruits for an increase in H2O2 accumulation

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following pathogen infection (Figure 2). This response was similar to a previous finding

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showing that the BABA-induced priming defense in Arabidopsis was required for the H2O2

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burst that occurs in the initial steps of plant-pathogen interactions.15 Moreover, our previous

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findings also indicated that H2O2 accumulation during early pathogen infection was a key

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step in the priming response underpinning a series of elicitor-induced resistances in grape

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berries19 and Chinese bayberries37 against the pathogens P. citrinum and B. cinerea,

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respectively. Thus, we suggested that the burst of H2O2 elicited by BABA was critical for

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the induced disease resistance observed in strawberries against B. cinerea infection.

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It is well documented that the plant restriction of pathogen penetration during infection

358

is generally based on an accurate perception of the pathogen infection time-course by the

359

host cells and the concomitant activation of a set of PR proteins that can bring out an

360

adequate defense against the infection.38 Chitinase and β-1,3-glucanase, which can

361

hydrolyze cell-wall, are the most frequently detected PR proteins in studies of induced

362

disease resistance.39 PAL is thought as the initial gateway enzyme in the plant’s

363

phenylpropanoid pathway for the secretion of phenolics, flavonoids, lignins and

364

phytoalexins, which restrict further pathogen growth around the infected area.40 We

365

analyzed changes in PR gene expression levels in strawberries following BABA treatment

366

and/or B. cinerea inoculation during incubation; in particular, we studied the PR1 gene,

367

FaPAL and two defense genes (FaChi3 and Faβglu) encoding chitinase and

368

β-1,3-glucanase.41 Application of chemical elicitors including acybenzolar-S-methyl,

369

chitosan and jasmonates has been reported to induce the activities of defensive enzymes

370

and/or up-regulate the transcript levels of PR genes, which are closely linked to induced

371

disease resistance and reduced disease severity in strawberries.42 Based on our data, the ACS Paragon Plus Environment


372

relationship between the suppression of B. cinerea infection and the activation of the

373

mentioned defense markers in strawberries after effective BABA elicitation is clear.

374

Increases in the activities of chitinase, β-1,3-glucanase and PAL and expression levels of

375

PR1, FaChi3, Faβglu and FaPAL were obtained with 10 to 500 mmol L-1 BABA. In

376

particular, we showed that treatment with BABA at concentrations of 100 or 500 mmol L-1

377

increased PR genes expressions and the corresponding defensive enzyme activities in both

378

mock- and B. cinerea-inoculated strawberries throughout the incubation, suggesting that

379

this BABA-induced resistance did not require a challenge inoculation and should be

380

considered a direct induction of defense responses. On the other hand, treatment with 10

381

mmol L-1 BABA stimulated little expression of phenotypic defense traits; however,

382

subsequent B. cinerea inoculation activated pronounced defensive responses and increased

383

the levels of PR genes transcriptions (Figures 3 and 4). According to the priming concept,

384

the pathogen-dependent resistance induced by 10 mmol L-1 BABA was a typical priming

385

defense because the BABA-treated fruits had the ability to express augmented molecular

386

defense responses upon pathogen attack. Collectively, these results indicate that BABA

387

caused a priming defense mechanism at 10 mmol L-1 but directly activated defense

388

responses at higher concentrations (100 and 500 µmol L-1) in harvested strawberries.

389

Similar concentration-dependent defense responses have been reported in BABA-, MeJA-

390

and riboflavin-induced disease resistance in Arabidopsis,15 Chinese bayberries37 and

391

grapevine,43,44 respectively. Previous studies have shown that the defense response of

392

priming by elicitors appears different from several known pathways of hormone signal

393

transduction but is dependent on NPR1 (Nonexpressor of pathogenesis-related genes 1),

394

which acts as a specific transcription factor that activates the promoter regions of several

395

PR genes.45 NRP1 can be in directly response to the SA-elicitation at low concentrations.46

396

However, other defense genes (those encoding hydroxyproline-rich glycoproteins) were ACS Paragon Plus Environment

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Page 17 of 37


397

only sensitive to the inducers at relative high concentrations.46,47 Thus, the dependence of

398

the mode of induced resistance on the BABA-applied concentration might be attributed to

399

be a dual mechanism of elicitations: low concentrations primed the augmented activation of

400

NPR1 gene, whereas higher concentrations directly induced the expression of another set of

401

defense-related genes. Moreover, we should note that in the cases of the strawberries

402

expressing BABA-induced resistance by either a direct or a priming defense mechanism as

403

described above, the transcript level of the PR1 gene in the two samples was consistently

404

enhanced. Because the PR1 gene is recognized as a representative molecular of the SAR

405

response that is associated with SA-dependent signaling pathways,48 it is possible that the

406

same SA-pathway exists in the different forms of defense responses against B. cinerea

407

following effective BABA elicitations under a wide range of concentrations. Thus, further

408

investigations are supposed to understand signal transduction pathways and molecular

409

mechanism(s) underlying BABA-induced direct or primed defense reactions associated

410

with SAR in postharvest fruits.

411

Although the defense response induced by various chemicals efficiently provides an

412

enough level of resistance to suppress pathogenic infection in plants, several studies have

413

shown that the expression of inducible defenses incurs metabolic losses that reduced plant

414

growth and seed reproduction.49 For example, applications of BTH at 200-500 mg L-1

415

directly enhanced the expression levels of a series of PR genes in Arabidopsis,15

416

grapevine50 and strawberries,51 but simultaneously reduced the growth parameters of plants

417

or leaves. We also found that this apparent cost occurred in grape cell suspensions, where

418

BTH-induced direct resistance greatly limited cellular fitness in terms of reduced cell

419

weight and soluble sugar contents.52 However, one investigation exhibited that the benefits

420

of priming was higher compared with that of the direct defensive elicitation because the

421

priming responses remained dormant under low disease pressure and resultantly conferred ACS Paragon Plus Environment


422

inconsiderable fitness costs15. Because soluble sugars including fructose, glucose and

423

sucrose are important factors that can affect fruit sensory quality in postharvest strawberries,

424

we analyzed the changes in sucrose metabolism in response to BABA treatment to

425

explicate the influence of the induced resistance on sugar accumulation. Among sucrose

426

metabolizing enzymes, SS-cleavage can cleave sucrose to uridine diphospho-glucose

427

(UDP-glucose) and fructose.28 In contrast, sucrose synthesis involves SPS, which can

428

catalyze a reaction by which fructose-6-phosphate and UDP-glucose are conversed to

429

sucrose-6-phosphate,29 and SPP, which subsequently hydrolyzes sucrose-6-phosphate to

430

sucrose.30 Likewise, SS-synthesis can catalyze a reaction by which fructose and

431

UDP-glucose are conversed to sucrose.28 In this study, the induction of direct resistance by

432

100 or 500 mmol L-1 BABA elicitation lowered the activities of SPS, SPP and SS-synthesis

433

while enhanced the SS-cleavage activity, thus decreasing contents of sucrose and glucose

434

as well as sweetness index and sensory scores compared with the control fruits. In contrast,

435

the priming defense-inducing treatment of BABA at 10 mmol L-1 significantly enhanced

436

the activities of SPS, SPP and SS-synthesis, which were responsible for the higher contents

437

of individual soluble sugar, sweetness evaluation and sensory scores in primed strawberries

438

(Figure 5-7). Thus, these results indicate that the induction of direct defense can cause

439

considerable quality losses in terms of impaired soluble sugar accumulation and sensory

440

qualities compared with the induction of priming. The observations made here are similar

441

to those of previous studies showing that the priming defense induced by low

442

concentrations of BABA or MeJA incurred few negative costs by minimizing plant growth

443

cost and fruit quality loss in Arabidopsis15 and grape berries,19 respectively, when

444

compared with the direct induction of defenses. However, because soluble sugars such as

445

sucrose and glucose also serve as primary substrates in glycolysis and the TCA

446

(tricarboxylic acid) cycle for generating ATP that supplies the energy for plant ACS Paragon Plus Environment

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Page 19 of 37


447

physiological metabolism,53 we postulated that the reduction of sugar costs in primed

448

strawberries might occur in part through suppressing glycolysis and the TCA cycle.

449

Briefly, in addition to its direct toxicity against B. cinerea, BABA at concentrations of

450

higher than 10 mmol L-1 induced disease resistance against gray mold rot in strawberries.

451

The defense responses in this study were either induced directly or primed for potentiated

452

expression when the strawberries suffered from pathogen attack. Moreover, we deduced

453

that the low concentration of BABA (10 mmol L-1) induced a defense through priming

454

mode, whereas higher concentrations of BABA (100 or 500 mmol L-1) activated a direct

455

defense in strawberries. Resistance through the form of direct activation was not an optimal

456

management for disease protection, due to this form of induced resistance negatively

457

affected soluble sugar accumulation in strawberries during the incubation period.

458

Conversely, priming defense offered an efficient form of protection with higher soluble

459

sugar contents, sweetness index and sensory qualities. The disease resistance from the

460

priming mechanism saved a significant amount of the energy and other resources required

461

for the production of defense-related compounds, presumably by avoiding the incurring of

462

unnecessary costs under pathogen-free conditions15. Thus, the priming defense appears an

463

optimal strategy that balances disease protection and maintaining sensory quality in

464

postharvest strawberry industry.

465 466

AUTHOR INFORMATION

467 468

Corresponding Authors

469

* E-mail: [email protected]. Tel: +86-25-84346256. Fax: +86-25-84346256

470

Funding

471

This study was supported by the National Natural Science Foundation of China (No. ACS Paragon Plus Environment


the

China

472

31201440),

473

2014M552300),

474

cstc2015jcyjA80028), the Scientific Research Innovation Team Project of Chongqing Three

475

Gorges University (No. 201302), and the Funding Project for 2nd Young Key Teachers in

476

the Universities of Chongqing City (2014046).

the

Postdoctoral

Natural

Science

Science

Foundation

Page 20 of 37

Foundation

Project

Funded of

CQ

Project

(No.

CSTC

(No.

477 478 479

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carbon dioxide concentration on PAL activity and phenolic contents in ripening

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cherimoya fruit. Postharvest Biol. Technol. 2001, 23, 33-39.

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quantities of protein utilizing the principle-dye binding. Anal. Biochem. 1976, 72,

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pear fruit. Food Chem. 2014, 159, 29-37.

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(25) Livak, K. J.; Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-∆∆C(T) method. Methods 2001, 25, 402-408.

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jasmonate on quality and antioxidant activity of postharvest loquat fruit. J. Sci. Food

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invertase as determinants of sucrose concentration in developing muskmelon (Cucumis

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activities in developing fruit of Lycopersicon esculentum Mill. and the sucrose

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accumulating Lycopersicon hirsutum Humb. and Bonpl. Plant Physiol. 1991, 95,

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Decreased sucrose-6-phosphate phosphatase level in transgenic tobacco inhibits

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combination with ethanol treatment on postharvest decay and antioxidant capacity in

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Chinese bayberries. J. Agric. Food Chem. 2010, 58, 9597-9604.

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on disease resistance against anthracnose rot in postharvest loquat fruit. Sci.

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Botrytis cinerea on table grapes and its possible mechanisms of action. Int. J. Food

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Poinssot, B. β-Aminobutyric acid primes an NADPH oxidase-dependent reactive

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oxygen species production during grapevine-triggered immunity. Mol. Plant-microbe

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Interactions 2010, 23, 1012-1021.

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(37) Wang, K. T.; Jin, P.; Han, L.; Shang, H. T.; Tang, S. S.; Rui, H. J.; Duan, Y. F.; Kong, F.

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Y.; Kai, X.; Zheng, Y. H. Methyl jasmonate induces resistance against Penicillium

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citrinum in Chinese bayberry by priming of defense responses. Postharvest Biol.

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Technol. 2014, 98, 90-97.

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(38) Van Loon, L. C.; Rep, M.; Pieterse, C. M. J. Significance of inducible defense-related proteins in infected plants. Annu. Rev. Phytopathol. 2006, 44, 135-162.

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(39) Wally, O.; Jayaraj, J.; Punja, Z. Comparative resistance to foliar fungal pathogens in

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transgenic carrot plants expressing genes encoding for chitinase, β-1,3-glucanase and

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peroxidise. Eur. J. Plant Pathol. 2009, 123, 331-342.

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(40) Ngadze, E.; Icishahayo, D.; Coutinho, T. A.; Van der Waals, J. E. Role of polyphenol

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oxidase, peroxidase, phenylalanine ammonia lyase, chlorogenic acid, and total soluble

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(41) Pombo, M. A.; Rosli, H. G.; Martínez, G. A.; Civello, P. M. UV-C treatment affects the

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expression and activity of defense genes in strawberry fruit (Fragaria × ananassa,

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Duch.). Postharvest Biol. Technol. 2011, 59(1), 94-102.

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(42) Landi, L.; Feliziani, E.; Romanazzi, G. Expression of defense genes in strawberry fruits treated with different resistance inducers. J. Agric. Food Chem. 2014, 62, 3047-3056.

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(43) Belhadj, A.; Telef, N.; Saigne, C.; Cluzet, S.; Barrieu, F.; Hamdi, S.; Mérillon, J. M.

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Effect of methyl jasmonate in combination with carbohydrates on gene expression of

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PR proteins, stilbene and anthocyanin accumulation in grapevine cell cultures. Plant

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Physiol. Biochem. 2008, 46, 493-499.

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(44) Boubakri, H.; Chong, J.; Poutaraud, A.; Schmitt, C.; Bertsch, C.; Mliki, A.; Masson, J.

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E.; Soustre-Gacougnolle, I. Riboflavin (vitamin B2) induces defence responses and

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resistance to Plasmopara viticola in grapevine. Eur. J. Plant Pathol. 2013, 136,

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837-855.

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(45) Dong, X. NPR1, all things considered. Curr. Opin. Plant Biol. 2004, 7, 547-552.

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(46) Thulke, O.; Conrath, U. Salicylic acid has a dual role in the activation of

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defence-related genes in parsley. Plant J. 1998, 14, 35-42.

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(47) Ton, J.; D'Alessandro, M.; Jourdie, V.; Jakab, G.; Karlen, D.; Held, M.; Mauch-Mani,

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B.; Turlings, T. C. Priming by airborne signals boosts direct and indirect resistance in

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maize. Plant J. 2007, 49, 16-26.

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(48) Van Loon, L. C.; Van Strien, E. A. The families of pathogenesis-related proteins, their

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activities, and comparative analysis of PR-1 type proteins. Physiol. Mol. Plant

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Pathol. 1999, 55, 85-97.

619 620 621

(49) Cipollini, D.; Purrington, C. B.; Bergelson, J. Costs of induced responses in plants. Basic Appl. Ecol. 2003, 4, 79-89. (50) Le Henanff, G.; Farine, S.; Kieffer-Mazet, F.; Miclot, A. S.; Heitz, T.; Mestre, P.; ACS Paragon Plus Environment


622

Bertsch, C.; Chong, J. Vitis vinifera VvNPR1.1 is the functional ortholog of AtNPR1

623

and its overexpression in grapevine triggers constitutive activation of PR genes and

624

enhanced resistance to powdery mildew Vitis vinifera VvNPR1.1 is the functional

625

ortholog of AtNPR1 and its overexpression in grapevine triggers constitutive activation

626

of PR genes and enhanced resistance to powdery mildew. Planta 2011, 234, 405-417.

627

(51) Hukkanen, A. T.; Kokko, H. I.; Buchala, A. J.; McDougall, G. J.; Stewart, D.;

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Kärenlampi, S. O.; Karjalainen, R. O. Benzothiadiazole induces the accumulation of

629

phenolics and improves resistance to powdery mildew in strawberries. J. Agric. Food

630

Chem. 2007, 55, 1862-1870.

631

(52) Wang, K. T.; Liao, Y. X.; Cao, S. F.; Di, H. T.; Zheng, Y. H. Effects of benzothiadiazole

632

on disease resistance and soluble sugar accumulation in grape berries and its possible

633

cellular mechanisms involved. Postharvest Biol. Technol. 2015, 102, 51-60.

634 635

(53) Fernie, A. R.; Carrari, F.; Sweetlove, L. J. Respiratory metabolism: glycolysis, the TCA cycle and mitochondrial electron transport. Curr. Opin. Plant Biol. 2004, 7, 254-261.

636 637 638

FIGURE CAPTIONS

639 640

Figure 1. Disease incidence (A) in strawberries treated with different concentrations of

641

BABA and inoculated with distilled water as well as disease incidence (B) and lesion

642

diameter (C) in grape berries treated with different concentrations of BABA and then

643

inoculated with B. cinerea during the incubation at 20 °C for 5 days. Each column

644

represents the mean of triplicate assays from one independent experiment. Vertical bars

645

represent the standard errors of the means. Different letters above the bars indicate

646

statistically significant differences (P < 0.05) within the same panel. ACS Paragon Plus Environment

Page 26 of 37

Page 27 of 37


647 648

Figure 2. H2O2 production in mock- (A) and B. cinerea-inoculated (B) strawberries during

649

5 days of incubation at 20 °C after BABA treatments (1-500 mmol L-1). Data are expressed

650

as the mean ± SE of triplicate assays from one independent experiment. Vertical bars

651

represent the standard errors of the means.

652 653

Figure 3. Activities of defense-related enzymes such as chitinase (A, B), β-1,3-glucanase

654

(C, D) and PAL (E, F) in mock- (A, C, E) and B. cinerea-inoculated (B, D, F) strawberries

655

during 5 days of incubation at 20 °C after BABA treatments (1-500 mmol L-1). Data are

656

expressed as the mean ± SE of triplicate assays from one independent experiment. Vertical

657

bars represent the standard errors of the means.

658 659

Figure 4. Expression of representative defense-related genes such as FaPR1 (A, B),

660

FaChi3 (C, D), Faβglu (E, F) and FaPAL (G, H) in mock- (A, C, E, G) and B.

661

cinerea-inoculated (B, D, F, H) strawberries during 5 days of incubation at 20 °C after

662

BABA treatments (1-500 mmol L-1). A quantitative real-time PCR (qRT-PCR) was

663

conducted by using 18S-rRNA as the internal control. Values were normalized to control at

664

each time point, arbitrarily set to 1. Data are expressed as the mean ± SE of triplicate assays

665

from one independent experiment. Different letters above the bars indicate statistically

666

significant differences between treatments (P < 0.05).

667 668

Figure 5. Activities of sucrose metabolism related enzymes such as SS-synthesis (A),

669

SS-cleavage (B), SPS (C) and SPP (D) in strawberries during 5 days of incubation at 20 °C

670

after BABA treatments (1-500 mmol L-1). Data are expressed as the mean ± SE of triplicate

671

assays from one independent experiment. Vertical bars represent the standard errors of the ACS Paragon Plus Environment


672

means.

673 674

Figure 6. Individual soluble sugar contents (A) and sweetness index (B) in strawberries at

675

the end of the 5 days of incubation at 20 °C after BABA treatments (1-500 mmol L-1). Data

676

are expressed as the mean ± SE of triplicate assays from one independent experiment.

677

Vertical bars represent the standard errors of the means. Different letters above the bars

678

indicate statistically significant differences (P < 0.05).

679 680

Figure 7. The effects of BABA treatments at different concentrations (1-500 mmol L-1) on

681

sensory scores of firmness, color, juiciness, flavor, taste, and visual appearance in

682

strawberries at the end of the 5 days of incubation at 20 °C after BABA treatments (1-500

683

mmol L-1). Data are expressed as the mean of triplicate assays from one independent

684

experiment.

685 686

Figure 8. Cytoplasmic leakage of soluble proteins (A) and carbohydrates (B) from

687

mycelium of B. cinerea during the incubation at 26 °C for 4 h after BABA treatments

688

(1-500 mmol L-1). Data are expressed as the mean ± SE of triplicate assays from one

689

independent experiment. Vertical bars represent the standard errors of the means.


Page 28 of 37

Page 29 of 37


Figure 1 50 Control 1 mmol/L BABA 10 mmol/L BABA 100 mmol/L BABA 500 mmol/L BABA

Disease incidence (%)

A 40

30 a a

20

b bc bc

10 d d d d d

b b

c c c

0 1

3

5

80

Disease incidence (%)

B 60 a a 40 b b c 20 d e e

c c

d d

e e e

0 1

3

5

6

Lesion diameter (mm)

C 4

a a

b b bc c

c c c c

c

c c

2

0 1

3

Time of incubation (days)


5

bc bc


Page 30 of 37

Figure 2 500

500

400

300

300

200

200

100

100

0

0 0

1

2

3

4

5

0

1



2

3

4

5

(μmol Kg-1FW)

(μmol Kg-1FW)

H2O2 content

400

H2O2 content

A

B

Control 1 mmol/L BABA 10 mmol/L BABA 100 mmol/L BABA 500 mmol/L BABA

Page 31 of 37


Figure 3 60

50

40

50 40

30

30 20

20 10

10 0

0 0

1

2

3

4

5

0

1

2

3

4

5

50

40

40 30

30 20

20 10

10

0

0 0

1

2

3

4

5

0

1

Time of incubation (days) ACS Paragon Plus Environment

2

3

4

5

β-1,3-glucanase activity (Units mg -1 protein)

D

C β-1,3-glucanase activity (Units mg -1 protein)

Chitinase activity (Units mg -1 protein)

A Chitinase activity (Units mg -1 protein)

B



Page 32 of 37


6

bcbc

ab

bb

bc bc

bbb

b

b

a

a

ab

ab ab bb

bc

a

c

a

bc bc c c

bb

bb

bb

8

a

6 cc

c c

c

c

4

d dd

dd d

2

0

0 1

Fold changes in FaChi3 mRNA values (relative values)

a

a

bb

4

2

B

A

2

3

4

1

5

2

3

4

5

30

15 a

C 12 b

cc

a ab

c cc

a

a

a a ab

b c

c

D

b

b

9

6

a

a ab

cc c

c c

c

c

c

b b

25

aa a

ab

a

a ab

a ab a

20

b b

b

bb

bb

15

b

10 3

5 0

0 1

2

3

4

5

1

2


3

4

Fold changes in FaPR1 mRNA values (relative values)

10

8

5

Fold changes in FaChi3 mRNA values (relative values)

Fold changes in FaPR1 mRNA values (relative values)

Figure 4

12

8

6 a

a

ab b

ab

ab

a

ab

ab b

b

b

ab b

ab b

a

a

c

b

b

c 2

c

c

c

cc

cc

d

c

cc

c c c

3

4

5

8 bc

c

4 c

ab

d

d d

d

d

d d

d

d

4

0

0 2

1

2

3

4

5

12

5

H

G 4 aa

aa

a

3

2

b

a

a

a

a

aa

a

ab

ab

ab

ab bb

bbb

b bb

b

bb

bb

8 ab b

b

b c

b

d

dd

dd

d

1

d

d

c

4

dd

0

0 1

2

3

4

5

1

2



3

4

5

Fold changes in FaPAL mRNA values (relative values)

a

1

Fold changes in FaPAL mRNA values (relative values)

F

E

Fold changes in Faβglu mRNA values (relative values)


Fold changes in Faβglu mRNA values (relative values)

Page 33 of 37


Page 34 of 37

A 8

40

6

30

4

20

2

10

0

0 0

Sucrose phosphate synthase activity (Units mg -1 protein)

B


1

2

3

4

5

0

1

2

3

4

4

10

D

C 8

3

6

2 4

1 2

0

0 0

1

2

3

4

5

0

1

Time of incubation (days) ACS Paragon Plus Environment

2

3

Sucrose synthase-cleavage activity (Units mg -1 protein)

50

10

4

5

Sucrose-6-phosphate phosphatase activity (Units mg -1 protein)

Sucrose synthase-synthesis activity (Units mg -1 protein)

Figure 5

Page 35 of 37


Figure 6 80

A a

a

B

Sucrose Fructose Glucose

Sweetness

a b

20 b

c cd cd

cd cd

10

c c

c d

cd

b

60

c 40

c cd d

20

0

0 Control

1 mmol/L BABA

10 mmol/L 100 mmol/L 500 mmol/L BABA BABA BABA

500 mmol/L 100 mmol/L 10 mmol/L BABA BABA BABA

Treatments


1 mmol/L BABA

Control

Sweetness index

Individual soluble sugar content (mg Kg-1 FW)

30


Table 1. Sequence of primers used for real-time quantitative PCR in strawberries Accession number Gene Forward Primer 5'→3' (NCBI)

Page 36 of 37

Reverse Primer 5'→3'

FaPR1

AB462752.1

CGGCGACTTATCAGGCACA

CACCCACAGGTTCACAGCAGA

Faβglu

AY989819.1

AAGCCCTTAGAGGTTCCA

CTTGACATCAGCCGAGTA

FaChi3

AF134347.1

GTGTCCCTTTCCTGATGCTA

CCTAAGAAGACCTGCTGTGC

FaPAL

AB360394.1

AGTGCTTGTTGAACACGCCTTGA

TGGAATCGCCGCATTACCG

Fa18s rRNA

AF163494.1

GCTTTGCCGTTGCTCTGATGAT

TTTGCCGATGGTGTAGGTTCCT


Page 37 of 37


Table 2 Effects of BABA on spore germination and germ tube length of B. cinerea in PDB tubesa

BABA treatment concentration ( mmol L-1)

Spore germination rate (%)

Germ tube length (μm)

After 24 h

After 48 h

After 24 h

After 48 h

Control

23.15±2.81a

67.12±4.58a

2.67±0.36a

6.71 ±0.20a

1

21.35±1.12a

65.19±3.71a

2.58±0.24a

5.98±0.21a

10

11.12±0.78b

41.24±4.14b

1.84±0.19b

3.65±0.24b

100

10.23±0.67b

37.51±3.83b

1.65±0.11b

3.21±0.15b

500

6.98±0.41c

31.65±2.85c

0.93±0.09c

2.87±0.21b

Germination rate and germ tube length were measured microscopically using approximate 100 spores of the pathogen during 36 h incubation at 26 C. Data are expressed as mean ± SD of triplicate assays. Means in a column followed by a different letter differ significantly at P = 0.05 by Duncan’s multiple range tests. a


Induction of Direct or Priming Resistance against Botrytis cinerea in

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