Changes in ABA, IAA and JA levels during calyx, fruit and leaves development in cape gooseberry plants (Physalis peruviana L.)

Plant Physiology and Biochemistry 115 (2017) 174e182

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Research article

Changes in ABA, IAA and JA levels during calyx, fruit and leaves development in cape gooseberry plants (Physalis peruviana L.)

rez a, C. Lo
pez-Cristoffanini b, O. Ja
uregui c, L.M. Melgarejo a, F. Alvarez-Fl o
pez-Carbonell b, * M. Lo
, Colombia Department of Biology, Faculty of Science, Universidad Nacional de Colombia, Bogota Department of Evolutive Biology, Ecology and Environmental Sciences, Plant Physiology Section, University of Barcelona, Av. Diagonal 643, 08028, Barcelona, Spain c gics, Universitat de Barcelona, c/ Baldiri i Reixac 10-12, 08028, Barcelona, Spain Unitat de T ecniques Separatives, Centre Científics i Tecnolo a

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 14 February 2017 Received in revised form 24 March 2017 Accepted 28 March 2017 Available online 30 March 2017

Changes in abscisic acid (ABA), indole-3-acetic acid (IAA) and jasmonic acid (JA) content in developing calyx, fruits and leaves of Physalis peruviana L. plants were analysed. Plant hormones have been widely studied for their roles in the regulation of various aspects related to plant development and, in particular, into their action during development and ripening of fleshly fruits. The obtained evidences suggest that the functions of these hormones are no restricted to a particular development stage, and more than one hormone is involved in controlling various aspects of plant development. Our results will contribute to understand the role of these hormones during growth and development of calyx, fruits and leaves in cape gooseberry plants. This work offers a good, quickly and efficiently protocol to extract and quantify simultaneously ABA, IAA and JA in different tissues of cape gooseberry plants. © 2017 Elsevier Masson SAS. All rights reserved.

Keywords: Abscisic acid (ABA) Indole-3-acetic acid (IAA) Jasmonic acid (JA) Physalis peruviana Fruit development

1. Introduction Physalis peruviana L., also known as cape gooseberry, is an herbaceous plant native to the Andes that belongs to the Solanaceae family and the second export fruit of Colombia (Fischer et al., 2007). It is a shrub, upright, and perennial plant that grows continuously (from 1.0 to 1.5 m in height, up to 2 m in height when plants are staked). Both vegetative and reproductive meristems remain active during the plant life cycle (Ramirez et al., 2013). The purplish, spreading branches are ribbed and covered with fine hairs. Leaves are simple, petiolated, alternate, heart shaped and highly pubescent (Fischer, 2000). Flowers are bell-shaped, nodding and arise in leaf axils. The fruit is a yellow juice berry, to about 2 cm in diameter and 4e10 g in weight, containing inside small seeds (150e300) (Fischer, 1995). During ripening, the fruit colour turns from green to orange due to chlorophyll breakdown and carotenoid accumulation, and their size and weight increased linearly with fruit development (Fischer and Martínez, 1999). The calyx (or husk) completely encloses the ripening fruit, grows to a papery and

* Corresponding author.
pez-Carbonell). E-mail address: [email protected] (M. Lo http://dx.doi.org/10.1016/j.plaphy.2017.03.024 0981-9428/© 2017 Elsevier Masson SAS. All rights reserved.

blader-like organ during fruit development and it is a protection against insects, birds, diseases and solar radiation (Fischer and Lüdders, 1997). Moreover, this structure represents an essential source of carbohydrates during the first 20 days of growth and development (Puente et al., 2011). Cape gooseberry fruit contains high levels of vitamins A, B-complex, C, E and K1, as well as antioxidants, phytosterols, polyunsaturated fatty acids and several essential minerals and anti-inflammatory compounds (Puente et al., 2011). This study revealed that P. peruviana is a source of health-related compounds found in the fruit and other parts of the plant including leaves and steams. For this reason, in Colombia this fruit has become promissory and even with high demand in European markets. This is mainly due to its unique taste, attractive colour and shape, nutritional importance and potential medical value. Nevertheless, still exist considerable gaps in the control of hormonal networks among different hormones during fruit expansion, maturation and other aspects of ripening. Plant hormones have a key role in most physiological processes and play a central role in the integration of diverse environmental cues with the plant genetic program and in shaping the morphological structures. They are directly involved in fruit set, ripening and development (Klee and Giovannoni, 2011; Seymour et al., 2013)



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and sink-related processes (Roitsch and Ehness, 2000). Specially, fruit development and maturation are physiological process affected by external environmental conditions (including light, moisture, and temperature) as well as internal factors (hormones). However, interactions between developmental control genes and hormones involved in the origin of morphological novelties are not well understood (Khan et al., 2012). Because of the medicinal importance of cape gooseberry, exogenous plant growth regulators have been widely used at different developmental stages to enhance the growth, yield and quality of its fruits (Majumder and Mazumdar, 2001; De Jong et al., 2009; El-Tohamy et al., 2012; Rodrigues et al., 2013; Kaur et al., 2013; Ramar et al., 2014; Albacete et al., 2015). Abscisic acid (ABA) modulates numerous aspects of the plant life, including seed dormancy, embryo maturation, and plant responses to different kinds of abiotic stresses such as drought, high temperature, chilling, and salinity (Seki et al., 2007). The ripening process of fruits is controlled by several hormones such as ethylene, auxins, brassinosteroids and ABA (Karppinen et al., 2013). ABA is also an important ripening control factor since: (i) there is a sharp increase in ABA accumulation during the onset of fruit ripening in climacteric fruits (Buesa et al., 1994); (ii) ABA accumulates preceding ethylene release in climacteric fruits (Zhang et al., 2009); (iii) the application of exogenous ABA enhances the production of several metabolites involved in promoting fruit ripening (Ban et al., 2003; Jeong et al., 2004; Giribaldi et al., 2010); (iv) in ABA-deficient tomato mutants, the fruit did not show the normal growth pattern observed in wild type (Galpaz et al., 2008); and (v) the de-greening stage began later in ABA deficient orange mutants (Rodrigo et al., 2003). In addition, indole-3-acetic acid (IAA) and jasmonic acid (JA) play a crucial role in plant growth and development and IAA have a major role in the regulation of fruit set by increasing the levels at fruit set and growth (Dorcey et al., 2009). Earlier molecular studies had established a role for an Auxin Response Factor (ARF) by controlling gibberellic acid (GA) levels (Dorcey et al., 2009) and its interaction with Aux/IAA proteins also govern the fate of fruit initiation events (Kumar et al., 2014). JA interacts with the other hormones in eliciting biological activity, and play a prominent role in signaling plant defenses (Farmer and Ryan, 1990). Jasmonates and ABA have in many cases similar functions (Kondo and Fukuda, 2001). JA is involved in many plant responses to biotic and abiotic stresses (Wasternack, 2007), sex determination, leaf senescence and root growth and fruit ripening (Shan et al., 2001; Concha et al., 2013). Moreover, interactions between ABA and JA biosynthesis have been reported (Melan et al., 1993; Brossa et al., 2011). However, as far as we know, there is a lack of works on endogenous changes of other plant hormones such as ABA, IAA and JA obtained from the same extract of cape gooseberry plants. The aim of this work was to quantify the changes of IAA, JA and ABA concentrations in fruit, leaves and calyx of cape gooseberry during four stages of ripening development. 2. Results and discussion Analytical approaches to detect JA, IAA and ABA must be extremely selective and sensitive. Liquid chromatography coupled to mass spectrometry (LCeMS) is the most widely employed technique for the simultaneous qualitative and quantitative profiling of multiple classes of plant hormones because of its high sensitivity and selectivity (Du et al., 2012). LCeMS (low-resolution mass spectrometry) may be combined with single quadrupole or triple-quadrupole instruments (QqQ), quadrupole linear ion trap (QqLIT). In order to better understand the organ distribution of several hormones influencing plant growth and development, ABA, IAA and JA were identified by HPLC-MS/MS in MRM mode (Figs. 1

175

and 2). This technique has the main advantage of high sensitivity and has become a versatile tool to analyze trace plant hormones. The present work offers a good, quickly and efficiently protocol to extract and quantify simultaneously ABA, IAA and JA in different tissues of cape gooseberry plants. 2.1. Analysis of ABA in fruits, leaves and calyx of cape gooseberry The identification of ABA, IAA and JA both in standards and in different tissues of cape gooseberry plants by LC-ESI-MS/MS are presented in Figs. 1 and 2. Changes in total endogenous concentrations of ABA, IAA and JA in different organs of P. peruviana are shown in Figs. 3e5. In this study, three organs (calyx, fruit and leaves) and four stages of organ development (stage III, stage IV, stage V and stage VI) were chosen for analysis and statistical differences between organs and hormone concentrations were detected. Changes in total ABA concentrations are shown in Fig. 3. ABA concentrations show important changes especially in fruits, increasing five-fold from stage III to stage IV (from 405 ng g
1, f.w. to 2352 ng g
1, f.w.); then, once maximum values are reached, ABA progressively decreases (to 1100 ng g
1, f.w.) in the last development stage (stage VI). This behavior has been reported in other works where it was found that ABA continuously changed at the different development stages of seeds and fruits: once ABA reached its peak, seriously physiological fruit dropping begins (Fan et al., 2004; Liu et al., 2007). The role of ABA in influencing fruit development is well documented and ABA biosynthesis in plants has been well established (Nambara and Marion-Poll, 2005; North et al., 2007). Its biosynthesis is closely related to that of carotenoids; therefore, we suggest that the decreasing levels of ABA are related to the increasing orange color of ripening fruits because of a high carotenoid biosynthesis. Nevertheless, although considerable progresses in regards to the role of ABA in the regulation of fleshly fruit ripening have been made, the molecular mechanisms still remain to be elucidated. The ABA content of calyx is similar between stage III and IV (around 552 ng g
1, f.w.), and remained constant between these stages but it slightly decreases from stage IV to stage VI (357 ng g
1, f.w.), suggesting the end of growth period and the increase in carotenoid synthesis in this ripening tissue. It is worth to note the purple color (results not shown) of calyx extracts before being injected into the LC-MS/MS equipment. As already reported (Caldwell et al., 1998), there is an increased synthesis of phenylpropanoids (e.g. anthocyanins) in the epidermis that absorb this radiation and act as antioxidants. Since ABA also may have a major role in regulation of anthocyanin biosynthesis (Jeong et al., 2004; Loreti et al., 2008; Jia et al., 2011), the purple color observed de visu in calyx extracts suggest us the presence of phenolic compounds such as anthocyanins, which are known to have, among others, an important antioxidant role in tissues. The endogenous ABA content in leaves show not so remarkable changes as that observed in fruits. On the contrary, the ABA content of young leaves at stage III was higher (500 ng g
1, f.w.) than that observed at stage IV (200 ng g
1, f.w.). Then, there was a small increase in ABA levels at stage V (250 ng g
1, f.w.) and finally ABA levels drop at the end of the development period at the same values as those of stage IV (to 188 ng g
1, f.w.). ABA is needed to modulate responses to environmental stresses but also growth and development, particularly seed development and fruit ripening (Zhang et al., 2009; Sun et al., 2012). Thus, once leaves are totally expanded and the growth is accomplished, high ABA levels are not needed, unless a stress situation appears. The dynamic balance between biosynthesis and catabolism determines the endogenous levels of ABA; both processes are regulated by 9-cis-epoxycarotenoid dioxygenase (NCED) and ABA 8’-hydroxylase (CYP707A), respectively. Moreover, the

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Fig. 1. Trace chromatograms for a standard solution. A) IAA; B) IAA-d5; C) JA; D) JA-d5; E) ABA; F) ABA-d6.



rez et al. / Plant Physiology and Biochemistry 115 (2017) 174e182 F. Alvarez-Fl o

Fig. 2. Trace chromatograms for a fruit sample of cape gooseberry. A) IAA; B) IAA-d5; C) JA; D) JA-d5; E) ABA; F) ABA-d6.

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Fig. 3. Changes of ABA concentration (ng$g
1, f.w.) in calyx, fruits and leaves of cape gooseberry plants at four stages of development (III, IV, V and VI).

Fig. 4. Changes of IAA concentration (ng$g
1, f.w.) in calyx, fruits and leaves of cape gooseberry plants at four stages of development (III, IV, V and VI).

ABA conjugation by cytosolic UDP-glucosyltransferases or release by b-glucosidases is also important for maintaining ABA homeostasis (Leng et al., 2014) and distribution throughout the plant.

2.2. Analysis of IAA in fruits, leaves and calyx of cape gooseberry Changes in IAA levels in the three organs studied are shown in Fig. 4 and statistical differences between them are observed. The highest levels of IAA in leaves corresponded to young leaves at stage III, when they were growing (500 ng g
1, f.w.). Then IAA levels decreased at stage IV and afterwards they increased again until leaves were adult at stage V (around 300 ng g
1, f.w.); finally, IAA content decreased at the end of the growth period. IAA is essential in regulating plant growth, especially when tissues are young. Therefore, auxins (IAA) play an important role in ripening fruits

(Epstein et al., 2002). Several studies with other Solanaceae plants like tomato showed that IAA levels changed during the fruit maturation process (Buta and Spaulding, 1994) and so does the capacity of the tissue to metabolize IAA (Riov and Bangerth, 1992;
et al., 1994). On the contrary, fruits and calyx showed Catala lower IAA levels along the four periods of development, in comparison to those of leaves (Fig. 4). Fruits showed progressive increases of IAA from the beginning to the end of the growth period (from 100 to 150 ng g
1, f.w.). This is in accordance with other works suggesting that low auxin is required for the initiation of ripening (Devoghalaere et al., 2012; Kumar et al., 2012). It has been reported (Kumar et al., 2014) that GH3 class of proteins, which are required for auxin conjugation, maintain physiologically active concentrations of auxins. These proteins have been found at high €ttcher levels in mature fruits of several fruit-bearing species (Bo



rez et al. / Plant Physiology and Biochemistry 115 (2017) 174e182 F. Alvarez-Fl o

179

Fig. 5. Changes of JA concentration (ng$g
1, f.w.) in calyx, fruits and leaves of cape gooseberry plants at four stages of development (III, IV, V and VI).

et al., 2010). Looking at the example of their involvement in fruit development, over-expression of a capsicum GH3 gene was found to reduce auxin levels in tomato fruits and eventually this reduction was thought to be responsible for the early fruit-ripening phenotype of these transgenic tomatoes (Liu et al., 2005). In the calyx structure, IAA was only detected at the end of the growth period (100 ng g
1, f.w.). Fruits presented small changes in IAA content along the four stages of development. Nevertheless, leaves are the organs that showed the most important changes in IAA concentrations. It has been reported that in fleshy fruit, levels of IAA, decline towards the onset of ripening and the application of auxins € ttcher et al., to immature fruit can delay the ripening processes (Bo 2010). However, the mechanisms by which the decrease in endogenous IAA concentrations and the maintenance of low auxin €ttcher et al., levels in maturing fruit are achieved remain elusive (Bo 2010). The transcript of a GH3 gene (GH3-1), encoding for an IAAamido synthetase which conjugates IAA to amino acids, was detected in grape berries Thus, GH3-1 expression increased at the onset of ripening (termed veraison by viticulturists), suggesting that it might be involved in the establishment and maintenance of low IAA concentrations in ripening berries. Furthermore, this grapevine GH3 gene responded positively to the combined application of ABA and sucrose and to ethylene, linking it to the control of ripening processes (Kumar et al., 2014). Levels of IAA-aspartic acid (IAAAsp), an in vitro product of recombinant GH3-1, rose after veraison and remained high during the following weeks of the ripening phase when levels of free IAA were low. A similar pattern of changes in free IAA and IAA-Asp levels were detected in developing tomatoes (Solanum lycopersicum Mill.), where low concentrations of IAA and an increase in IAA-Asp concentrations coincided with the onset of ripening in this climacteric fruit. Since IAA-Asp might be involved in IAA degradation, the GH3 catalyzed formation of this conjugate at, and after, the onset of ripening could represent a common IAA inactivation mechanism in climacteric and nonclimacteric fruit which enables ripening. However, IAA concentrations have also been claimed to be low and relatively constant throughout berry development (Symons et al., 2006). Common to all these reports is that, like in tomato, IAA levels are low at the onset of ripening, and here defined as the last time point before the

start of sugar accumulation. 2.3. Analysis of JA in fruits, leaves and calyx of cape gooseberry The total content in JA show statistical differences between the organs studied (Fig. 5). Leaves showed a very high increase in JA levels, rising form the stage III (4163 ng g
1, f.w.) to the stage IV (14,702 ng g
1, f.w.) when they reach the mature stage. This fact can suggest us that during this period of time plant needs to have a good defensive capacity, since JA is known to have an important role in biotic stresses when needed (Farmer and Ryan, 1992). Then, foliar JA content progressively decreased when stages V and VI are reached (Fig. 5). By contrast, no changes in JA concentrations were detected in fruits along the four developmental stages and were the lowest concentrations (Fig. 5) since JA levels remained around 180 ng g
1, f.w. Endogenous JA content in calyx tissue showed some differences along the developmental stages. It was observed that JA levels were high at the stage III (4019 ng g
1, f.w.) and V (4500 ng g
1, f.w.), a little bit lower in stage IV (3000 ng g
1, f.w.) and they finally drop at the stage VI (to 1487 ng g
1, f.w.), coincident with the end of the growth period (Fig. 3). Several works €ttcher et al., 2015) (Gansser et al., 1997; Kondo and Fukuda, 2001; Bo have demonstrated that high levels of jasmonates are detected in early stages of development in grapes and berries, which decrease before the initiation of ripening and remained low during it. However, our results showed no remarkable changes of endogenous JA in cape gooseberry fruits along the different developmental stages. In the opposite way, leaves showed an important increase in JA between stage III and stage IV. As ABA did, JA showed an important peak in leaves, increasing more than five-fold the content showed in stage III. This behavior can be related to the jasmonate lipooxygenase activation, which is involved in ABA synthesis from carotenoids (Melan et al., 1993). Several works have reported the production of carotenoids, sesquiterpens and phenolic compounds in ripening fruits, such as anthocyanins and tannins (Kondo et al., 2000; Wang et al., 2008; D'Onofrio et al., 2009). This fact can explain the purple color observed in the calyx extracts obtained (results not shown), since most of these compounds have a defensive role in front of, among others, UV light and biotic stress,

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which are especially important in the first stages of development. Cape gooseberry is well adapted to high altitudes where there is an increase in UV-radiation by developing several adaptation mechanisms. Among them, developing a dense pubescence that covers all the green parts of the plant and an increase in leaf thickness and specific leaf weight (Fischer and Melgarejo, 2014). Nevertheless, natural UV-B radiation levels can have favorable effects on several species, favoring secondary metabolism, reducing abundant vegetative growth and pathogen incidence (Fischer et al., 2016). The relationship between ABA and JA biosynthesis, especially under stress conditions, has been already reported (Bandurska et al., 2003; Brossa et al., 2011). Moreover, there is a marked relationship between JA and antioxidant compounds (Shan and Liang, 2010). Therefore, our results lead us to think that the increase in JA could help tissues to synthesize antioxidant compounds that can be crucial to the maintenance of redox homeostasis inside the cells. Moreover, ABA, IAA, and JA play important roles in fruit, leaves and calyx development and provide a better understanding of the plant hormone crosstalk in physiological processes. 3. Experimental 3.1. Plant material Plants of cape gooseberry (P. peruviana L., ecotype “Colombia”) were grown in the experimental fields of the National University of Colombia (Bogot
a, Colombia) on a peat substrate, under a photosynthetic active radiation (PAR) of 474 mmol m
2$s
1 and a temperature of 20.7  C (±0.5  C). Cape gooseberry growth is continuous and both vegetative and reproductive meristems remain active during the plant life cycle. Fruit development periods and growth features of the selected stages in this study were the following: stage III: green color at 45 days after anthesis; stage IV: yellowinggreen color at 50 days after anthesis; stage V: yellow color at 55 days after anthesis; and stage VI: orange color at 60 days after anthesis. 3.2. Hormone analysis Leaves, calyx and fruits at four developmental stages were collected, lyophilized and stored at
80  C until hormone analysis. Each sample was tested in triplicate. Young and old leaves from the uppermost and lowest part of the plant respectively were taken. Concentrations of ABA, IAA and JA were analysed by liquid chromatography coupled to mass spectrometry in tandem mode (HPLC
pez-Carbonell et al. (2009) and Brossa et al. MS/MS) according to Lo (2011) with slight modifications. Lyophilized samples were ground in liquid nitrogen and 60 mg of each powdered sample was extracted with 1 mL of methanol:isopropanol (20:80, v/v, with 1% of glacial acetic acid) as extraction solvent. Deuterium labelled standards of ABA and d6ABA were purchased from Plant Biotechnology Institute (National Research Institute, Canada); JA, d5-JA, IAA and d5-IAA were obtained from OlChemIm Ltd. (Olomouc, Czech Republic). Before starting the extraction procedure, deuterium-labelled internal standards (20 ng mL
1 of d6-ABA, and 50 ng mL
1 of d5-JA and d5IAA) were added to each of the samples and replicates. After centrifugation (10,000 rpm for 15 min at 4  C), the supernatants were collected and the pellets were re-extracted with 0.5 mL of extraction solvent and centrifuged again. Then, supernatants were combined and dried completely under a nitrogen stream and redissolved in 200 mL of water/acetonitrile/acetic acid (90:10:0.05, v/v), vortexed, centrifuged (10,000 rpm for 10 min at 4  C) and filtered through a 0.22 mm PTFE filter (Waters, Milford, MA, USA). Finally, 5 mL of each sample were injected into the LC-ESI-MS/MS.

Hormones were determined in three independent samples for each treatment. Quantification was done by the creation of calibration curves including each of the unlabeled analyte compounds (ABA, JA, IAA,). Calibration curves for each analyte were generated using Analyst™ software (Applied Biosystems, Inc., California, USA). The limit of detection (LOD, S/N ¼ 3) and the limit of quantification (LOQ, S/N ¼ 10) were also calculated with the aid of this software. 3.3. Chromatography LCeESIeMSeMS procedure: The HPLC system consisted of an Agilent Infinity 1260 (Waldbronn, Germany) binary pump equipped with a thermostated (4  C) autosampler. For the analysis of the extracts, a Kinetex C18 column (50  2.1 mm, 2.6 mm) (Phenomenex, Torrance, CA, USA) equipped with a Securityguard C18 Phenomenex (4  3 mm, i.d.) was used. Gradient elution was done with water with 0.05% acetic acid (solvent A) and acetonitrile (solvent B) at a constant flow-rate of 400 mL min-1. A linear gradient profile with the following proportions of solvent B was applied (t, %B): (0, 2), (5, 2), (10, 100), (11, 100), (13, 2), (20, 2). LCeESIeMS/MS system: MS and MS/MS experiments were performed on an API 4000QTRAP mass spectrometer (ABSciex, Concord, Ontario, Canada). All the analyses were performed using the Turbo V source in negative ion mode with the following settings: capillary voltage
4500 V, nebulizer gas (N2) 50 (arbitrary units), curtain gas (N2) 30 (arbitrary units), collision gas High (arbitrary units), declustering potential (DP), focusing potential (FP), entrance potential 10 V, collision energy (CE); drying gas (N2) 50 (arbitrary units) was heated to 550  C. All the MS and MS/MS parameters were optimized in infusion experiments: individual standard solutions of ABA, ABA-d6, JA, d5-JA, IAA, d5-IAA, (0.1 ng mL1 ) were infused at a constant flow-rate of 5 mL min-1 into the mass spectrometer using a Model 11 syringe pump (Harvard Apparatus, Holliston, MA, USA). Full scan data acquisition was performed by scanning from m/z 100 to 800 in profile mode and using a cycle time of 2 s with a step size of 0.1 ms and a pause between each scan of 2 ms. In product ion scan experiments MS/MS product ions were produced by collision-activated dissociation (CAD) of selected precursor ions in the collision cell of the mass spectrometer and mass analyzed using the second analyzer of the instrument. Multiple reaction monitoring (MRM) acquisition was done monitoring each transition with a dwell time of 75 ms. Quantitation used MS/ MS (multiple reaction monitoring method, MRM). The MRM mode was required because many compounds could present the same nominal molecular mass, but the combination of the parent mass and unique fragment ions was used to selectively monitor ABA, JA and IAA in crude plant extracts. (see Table 1). 3.4. Statistical analysis Analyses were performed taking into account normality, homoscedasticity and randomness in the experimental design. Data for the analytical determinations were subjected to a factorial ANOVA (three-way ANOVA) where the variable to be evaluated was Table 1 MS/MS parameters optimized and transitions used to identify and quantify in MRMa. DP, declustering potential; CE, collision energy; CXP cell exit potentially.

ABA d6-ABA JA d5-JA IAA d5-IAA

MRM transitions

DP (V)

CXP (V)

CE (V)

263.0/152.9 269.0/159.0 209.0/58.9 214.2/62.2 174.1/129.8 178.9/134.8


70
75
70
60
60
50


9
9
9
9
9
7


16
16
25
16
16
17



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the concentration of the hormones under the maturity stage and the corresponding organ. The formula of the model was: Concentration ¼ Hormones * Maturation * Organ. Mean comparisons were performed using Fischer HSD post-hoc test to examine if differences were significant at P < 0.05. Analyses were performed with R software package (R Core Team, 2016). Contributions Each author has participated sufficiently, intellectually or practically, in the work to take public responsibility for the content of the article, including the conception, design, and conduction of the experiment and for data interpretation. FA, CLC and MLC carried out the experiments, including sample analysis, results discussion and helped to draft the manuscript. OJ carried out the LC-ESI-MS-MS procedure. FA, LMM and MLC participated in the design of the study, drafted the manuscript, and revised it critically. Acknowledgements The present study was financially supported by grant BFU201239527 from the Spanish Government. We thank N. Flechas (Universidad Nacional de Colombia) for support in statistical analysis. References Albacete, A., Cantero-Navarro, E., Balibrea, M.E., Grosskinsky, D.K., de la Cruz
lez, M., Martínez-Andújar, C., Smigocki, A.C., Roitsch, T., Pe
rez-Alfocea, F., Gonza 2015. Hormonal and metabolic regulation of tomato fruit sink activity and yield under salinity. J. Exp. Bot. 65, 6081e6095. Ban, T., Ishimaru, M., Kobayashi, S., Shiozaki, S., Goto-Yamamoto, N., Horiuchi, S., 2003. Abscisic acid and 2,4-dichlorophenoxyacetic acid affect the expression of anthocyanin biosynthetic pathway genes in “Kyoho” grape berries. J. Hortic. Sci. Biotechnol. 78, 586e589. Bandurska, H., Stroinski, A., Kubis, J., 2003. The effect of jasmonic acid on the accumulation of ABA, proline and spermidine and its influence on membrane injury under water deficit in two barley genotypes. Acta Physiol. Plant. 25, 279e285. €ttcher, C., Keyzers, R.A., Boss, P.K., Davies, C., 2010. Sequestration of auxin by the Bo indole-3-acetic-amido synthetase GH 3-1 in grape berry (Vitis vinifera L.) and the proposed role of auxin conjugation during ripening. J. Exp. Bot. 61, 3615e3625. €ttcher, C., Burbidge, C.A., di Rienzo, V., Boss, P.K., Davies, C., 2015. Jasmonic acidBo isoleucine formation in grapevine (Vitis vinifera L.) by two enzymes with distinct transcription profiles. J. Integr. Plant Biol. 57, 618e627.
pez-Carbonell, M., Jubany-Marí, T., Alegre, L., 2011. Interplay between Brossa, R., Lo abscisic acid and jasmonic acid and its role in water-oxidative stress in wildtype, ABA-deficient, JA-deficient, and ascorbate-deficient Arabidopsis plants. J. Plant Growth Regul. 30, 322e333. Buesa, C., Dominguez, M., Vendrell, M., 1994. Abscisic acid effects on ethylene production and respiration rate in detached apple fruits at different stages of development. Rev. Esp. Cienc. Tecnol. Alim 34, 495e506. Buta, J.G., Spaulding, D.W., 1994. Changes in indole-3-acetic acid and abscisic acid levels during tomato (Lycopersicon esculentum Mill.) fruit development and ripening. J. Plant Growth Regul. 13, 163e166. €rn, L.O., Bornman, J.F., Flint, S.D., Kulandaivelu, G., 1998. Effects of Caldwell, M.M., Bjo increased solar ultraviolet radiation on terrestrial ecosystems. J. Photochem. Photobiol. B. Biol. 46, 40e52.
, C., Crozier, A., Chamarro, J., 1994. Decarboxylative metabolism of (1’-14C) Catala indole-3-acetic acid by tomato pericarp discs during ripening: effects of wounding and ethylene. Planta 193, 508e513. ~ ate, F.A., Schwab, W., Figueroa, C.R., Concha, C.M., Figueroa, N.E., Poblete, L.A., On 2013. Methyl jasmonate treatment induces changes in fruit ripening by modifying the expression of several ripening genes in Fragaria chiloensis fruit. Plant Physiol. Biochem. 70, 433e444. De Jong, M., Mariani, C., Vriezen, W.H., 2009. The role of auxin and gibberellin in tomato fruit set. J. Exp. Bot. 60, 1523e1532. Devoghalaere, F., Doucen, T., Guitton, B., Keeling, J., Payne, W., Ling, T.J., Ross, J.J., Hallett, I.C., Gunaseelan, K., Dayatilake, G.A., Diak, R., Breen, K.C., Tustin, D.S., Costes, E., Chagne, D., Schaffer, R.J., David, K.M., 2012. A genomics approach to understanding the role of auxin in apple (Malus x domestica) fruit size control. BMC Plant Biol. 12 http://dx.doi.org/10.1186/1471-2229-12-7 article n 7. D'Onofrio, C., Cox, A., Davies, C., Boss, P.K., 2009. Induction of secondary metabolism in grape cell cultures by jasmonates. Funct. Plant Biol. 36, 323e338. Dorcey, E., Urbez, C., Blazquez, M.A., Carbonell, J., Perez-Amador, M.A., 2009.

181

Fertilization-dependent auxin response in ovules triggers fruit development through the modulation of gibberellin metabolism in Arabidopsis. Plant J. 58, 318e332. Du, F., Ruan, G., Liu, H., 2012. Analytical methods for tracing plant hormones. Anal. Bioanal. Chem. 403, 55e74. El-Tohamy, W.A., El-Abagy, H.M., Badr, M.A., Ghoname, A.A., Abou-Hussein, S.D., 2012. Improvement of productivity and quality of Cape gooseberry (Physalis peruviana L.) by foliar application of some chemical substances. J. Appl. Sci. Res. 8, 2366e2370. Epstein, E., Cohen, J.D., Slovin, J.P., 2002. The biosynthetic pathway for indole-3acetic changes during tomato fruit development. Plant Growth Regul. 38, 15e20. Fan, W.G., An, H.M., Liu, G.Q., He, S.T., Liu, J.P., 2004. Changes of endogenous hormone contents in fruit, seeds and their effects on the fruit development of Rosa rox-burghii. Sci. Agric. Sin. 37, 728e733. Farmer, E.E., Ryan, C.A., 1990. Interplant communication airborne methyl jasmonate induces synthesis of proteinase inhibitor in plant leaves. Proc. Natl. Acad. Sci. U. S. A. 87, 7713e7716. Farmer, E.E., Ryan, C.A., 1992. Octadecanoid precursors of jasmonic acid activate the synthesis of wound-inducible proteinase inhibitors. Plant Cell 4, 129e134. Fischer, G., 1995. Effect of Root Zone Temperature and Tropical Altitude on the Growth, Development and Fruit Quality of Cape Gooseberry (Physalis Peruviana L.) (Ph.D thesis). Humboldt University, Berlin. Fischer, G., Lüdders, P., 1997. Developmental changes in carbohydrates in cape gooseberry (Physalis peruviana L.) fruits in relation to the calyx and the leaves. Agron. Colomb. 14, 95e107. Fischer, G., Martínez, O., 1999. Quality and maturity of cape gooseberry (Physalis peruviana L.) in relation to fruit coloring. Agron. Colomb. 16, 35e39. Fischer, G., 2000. Crecimiento y desarrollo. In: Florez, V.J., Fischer, G., Sora, A.D.
n, Poscosecha y Exportacio
n de la Uchuva (Physalis peruviana (Eds.), Produccio
, pp. 9e26. L.). Unibiblos, Universidad Nacional de Colombia, Bogota Fischer, G., Ebert, G., Lüdders, P., 2007. Production, seeds and carbohydrate contents of Cape gooseberry (Physalis peruviana L.) fruits grown at two contrasting Colombian altitudes. J. Appl. Bot. 81, 29e35. Fischer, G., Melgarejo, L.M., 2014. Ecofisiología de la uchuva (Physalis peruviana L. In: Carvalho, C.P., Moreno, D.A. (Eds.), Physalis peruviana: fruta andina para el mundo. Programa Iberoamericano de Ciencia y Tecnología para el desarrolloCYTED, cap. II, pp. 29e47 (Limencop SL, Alicante, Spain). Fischer, G., Ramírez, F., Casierra-Posada, F., 2016. Ecophysiological aspects of fruit crops in the era of climate change. A review. Agron. Colomb. 34 (2), 190e199. Galpaz, N., Wang, Q., Menda, N., Zamir, D., Hirschberg, J., 2008. Abscisic acid deficiency in the tomato mutant high-pigment 3 leading to increase plastid number and higher fruit lycopene content. Plant J. 53, 717e730. Gansser, D., Latza, S., Berger, R.G., 1997. Methyl jasmonates in developing strawberry fruit (Fragaria ananassa Duch. cv. Kent). J. Agric. Food Chem. 45, 2477e2480. Giribaldi, M., Geny, L., Delrot, S., Schubert, A., 2010. Proteomic analysis of the effects of ABA treatments on ripening Vitis vinifera berries. J. Exp. Bot. 61, 2447e2458. Jeong, S.T., Goto-Yamamoto, N., Kobayashi, S., Esaka, A., 2004. Effects of plant hormones and shading on the accumulation of anthocyanins and the expression of anthocyanin biosynthetic genes in grape berry skins. Plant Sci. 167, 247e252. Jia, H.F., Chai, Y.M., Li, C.L., Lu, D., Luo, J.J., Qin, L., Shen, Y.Y., 2011. Abscisic acid plays an important role in the regulation of strawberry fruit ripening. Plant Physiol. 157, 188e199. Karppinen, K., Hirvela, E., Nevala, T., Sipari, N., Suokas, M., Jaakola, L., 2013. Changes in the abscisic acid levels and related gene expression during fruit development and ripening in bilberry (Vaccinium myrtillus L.). Phytochemistry 95, 127e134. Kaur, G., Kaur, A.P., Singh, B., Singh, S., 2013. Effect of plant growth regulators on fruit quality of cape gooseberry (Physalis peruviana L.) cv. Aligarh. Int. J. Agr. Sci. 9, 633e635. Khan, M.R., Hu, Y.J., He, C.Y., 2012. Plant hormones including ethylene are recruited in calyx inflation in Solanaceous plants. J. Plant Physiol. 169, 940e948. Klee, H.J., Giovannoni, J.J., 2011. Genetics and control of tomato fruit ripening and quality attributes. Annu. Rev. Genet. 45, 41e59. Kondo, S., Fukuda, K., 2001. Changes of jasmonates in grape berries and their possible roles in fruit development. Sci. Hortic. 91, 275e288. Kondo, S., Tomiyama, A., Seto, H., 2000. Changes of endogenous jasmonic acid and methyljasmonate in apples and sweet cherries during fruit development. J. Am. Soc. Hort. Sci. 125, 282e287. Kumar, R., Agarwal, P., Tyagi, A.K., Sharma, A.K., 2012. Genome-wide investigation and expression analysis suggest diverse roles of auxin-responsive GH3 genes during development and response to different stimuli in tomato (Solanum lycopersicum). MGG Mol. Genet. Genomics 287, 221e235. Kumar, R., Khurana, A., Sharma, A.K., 2014. Role of plant hormones and their interplay in development and ripening of fleshy fruits. J. Exp. Bot. 65, 4561e4575. Leng, P., Yuan, B., Guo, Y., 2014. The role of abscisic acid in fruit ripening and responses to abiotic stress. J. Exp. Bot. 65, 4577e4588. Liu, K., Kang, B.C., Jiang, H., Moore, S.L., Li, H., Watkins, C.B., Setter, T.L., Jahn, M.M., 2005. A GH3-like gene, CcGH3, isolated from Capsicum chinense L. fruit is regulated by auxin and ethylene. Plant Mol. Biol. 58, 447e464. Liu, B.H., Jiang, Y.M., Peng, F.T., Sui, J., Zhao, F.X., Wang, H.Y., 2007. The dynamic changes of endogenous hormones in sweet cherry (Prunus avium L.) pulp. Acta Hortic. Sin. 34, 1535e1538.
pez-Carbonell, M., Gabasa, M., Ja
uregui, O., 2009. Enhanced determination of Lo abscisic acid (ABA) and abscisic acid glucose ester (ABA-GE) in Cistus albidus

182



rez et al. / Plant Physiology and Biochemistry 115 (2017) 174e182 F. Alvarez-Fl o

plants by liquid chromatography-mass spectrometry in tandem mode. Plant Physiol. Biochem. 47, 256e261. Loreti, E., Povero, G., Novi, G., Solfanelli, C., Alpi, A., Perata, P., 2008. Gibberellins, jasmonate and abscisic acid modulate the sucrose-induced expression of anthocyanin biosynthetic genes in Arabidopsis. New Phytol. 179, 1004e1016. Majumder, K., Mazumdar, B.C., 2001. Effects of auxin and gibberellin on pectic substances and their degrading enzymes in developing fruits of capegooseberry (Physalis peruviana L.). J. Hortic. Sci. Biotechnol. 76, 276e279. Melan, M.A., Dong, X.N., Endara, M.E., Davis, K.R., Ausubel, F.M., Peterman, T.K., 1993. An Arabidopsis thaliana lipoxygenase gene can be induced by pathogens, abscisic acid, and methyl jasmonate. Plant Physiol. 101, 441e450. Nambara, E., Marion-Poll, A., 2005. Abscisic acid biosynthesis and catabolism. Annu. Rev. Plant Biol. 56, 165e185. North, H.M., De Almeida, A., Boutin, J.P., Frey, A., To, A., Botran, L., Sotta, B., MarionPoll, A., 2007. The Arabidopsis ABA-deficient mutant aba4 demonstrates that the major route for stress-induced ABA accumulation is via neoxanthin isomers. Plant J. 50, 810e824. ~ oz, C.A., Castro, E.S., Corte
s, M., 2011. Physalis peruviana Puente, L.A., Pinto-Mun Linnaeus, the multiple properties of a highly functional fruit: a review. Food Res. Int. 44, 1733e1740. R Core Team, 2016. A Language and Environment for Statistical Computing. R. Foundation for Statistical Computing, Vienna. https://www.R-project.org. Ramar, K., Ayyadurai, V., Arulprakash, T., 2014. In vitro shoot multiplication and plant regeneration of Physalis peruviana L., an important medicinal plant. Int. J. Curr. Microbiol. Appl. Sci. 3, 456e464. Ramirez, F., Fischer, G., Davenport, T.L., Pinzon, J.C.A., Ulrichs, C., 2013. Cape gooseberry (Physalis peruviana L.) phenology according to the BBCH phonological scale. Sci. Hortic. 162, 39e42. Riov, J., Bangerth, F., 1992. Metabolism of auxin in tomato fruit tissue: formation of high molecular weight conjugates of oxindole-3-acetic acid via the oxidation of indole-3-acetylaspartic acid. Plant Physiol. 100, 1396e1402.
rez, F., Mallent, M.D., Zacarías, L., 2003. CharacterRodrigo, M.J., Marcos, J.F., Alfe ization of Pinalate, a novel Citrus sinensis mutant with a fruit-specific alteration that results in yellow pigmentation and decreased ABA content. J. Exp. Bot. 54,

727e738. Rodrigues, F.A., Penoni, E.D., Soares, J.D.R., Silva, R.A.L., Pasqual, M., 2013. Phenological characterization and productivity of Physalis peruviana cultivated in greenhouse. Biosci. J. 29, 1771e1777. Roitsch, T., Ehness, R., 2000. Regulation of source/sink relations by cytokinins. Plant Growth Regul. 32, 359e367. Seki, M., Umezawa, T., Urano, K., Shinozaki, K., 2007. Regulatory metabolic networks in drought stress responses. Curr. Opin. Plant Biol. 10, 296e302. Seymour, G.B., Ostergaard, L., Chapman, N.H., Knapp, S., Martin, C., 2013. Fruit development and ripening. Annu. Rev. Plant Biol. 64, 219e241. Shan, X., Wang, J., Chua, L., Jiang, D., Peng, W., Xie, D., 2001. The role of Arabidopsis rubisco activase in jasmonate-induced leaf senescence. Plant Physiol. 155, 751e764. Shan, C., Liang, Z.S., 2010. Jasmonic acid regulates ascorbate and glutathione metabolism in Agropyron cristatum leaves under water stress. Plant Sci. 178, 130e139. Sun, L., Sun, Y.F., Zhang, M., Wang, L., Ren, J., Cui, M.M., Wang, Y.P., Ji, K., Li, P., Li, Q., Chen, P., Dai, S.J., Duan, C.R., Wu, Y., Leng, P., 2012. Suppression of 9-cis-epoxycarotenoid dioxygenase which encodes a key enzyme in abscisic acid biosynthesis, alters fruit texture in transgenic tomato. Plant Physiol. 158, 283e298. Symons, G.M., Davies, C., Shavrukov, Y., Dry, I.B., Reid, J.B., Thomas, M.R., 2006. Grapes on steroids. Brassinosteroids are involved in grape berry ripening. Plant Physiol. 140, 150e158. Wang, S.Y., Bowman, L., Ding, M., 2008. Methyl jasmonate enhances antioxidant activity and flavonoid content in blackberries (Rubus sp) and promotes antiproliferation of human cancer cells. Food Chem. 107, 1261e1269. Wasternack, C., 2007. Jasmonates: an update on biosynthesis, signal transduction and action in plant stress response, growth and development. Ann. Bot. 100, 681e697. Zhang, M., Leng, P., Zhang, G.L., Li, X.X., 2009. Cloning and functional analysis of 9cis-epoxycarotenoid dioxygenase (NCED) genes encoding a key enzyme during abscisic acid biosynthesis from peach and grape fruits. J. Plant Physiol. 166, 1241e1252.