Drought Effects on Proanthocyanidins in Sainfoin (Onobrychis viciifolia

Article pubs.acs.org/JAFC

Drought Effects on Proanthocyanidins in Sainfoin (Onobrychis viciifolia Scop.) Are Dependent on the Plant’s Ontogenetic Stage Carsten S. Malisch,†,# Juha-Pekka Salminen,*,§ Roland Kölliker,‡ Marica T. Engström,§ Daniel Suter,† Bruno Studer,# and Andreas Lüscher† †

Forage Production and Grassland Systems and ‡Molecular Ecology, Agroscope, 8046 Zurich, Switzerland § Laboratory of Organic Chemistry and Chemical Biology, Department of Chemistry, University of Turku, 20500 Turku, Finland # Molecular Plant Breeding, Institute of Agricultural Sciences, ETH Zurich, 8092 Zurich, Switzerland S Supporting Information *

ABSTRACT: Sainfoin (Onobrychis viciifolia Scop.) is a forage legume, which improves animal health and the environmental impact of livestock farming due to its proanthocyanidin content. To identify the impact of drought on acetone/water-extractable proanthocyanidin (PA) concentration and composition in the generative and vegetative stages, a rain exclosure experiment was established. Leaves of 120 plants from 5 different sainfoin accessions were sampled repeatedly and analyzed by UPLC-ESI-MS/ MS. The results showed distinct differences in response to drought between vegetative and generative plants. Whereas vegetative plants showed a strong response to drought in growth (−56%) and leaf PA concentration (+46%), generative plants showed no response in growth (−2%) or PA concentration (−9%). The PA composition was stable across environments. The five accessions varied in PA concentrations and composition but showed the same pattern of response to the experimental treatments. These results show that the ontogenetic stage at which drought occurs significantly affects the plant’s response. KEYWORDS: Onobrychis viciifolia, sainfoin, drought stress, sainfoin rust, MS/MS quantification, polyphenols, condensed tannins, proanthocyanidins

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INTRODUCTION Proanthocyanidins (syn. condensed tannins) are plant secondary metabolites (PSMs) and, as such, synthesized in plants without being directly essential for basic functions of survival of the plant cell, such as photosynthesis, respiration, and growth.1 PSMs are, however, supposedly produced as protection against abiotic stresses, such as drought, and biotic stresses, such as insect herbivores and pathogens, and are therefore crucial for the survival of the whole plant.2 Recently, there has been a growing interest in PSMs in general and, particularly, in proanthocyanidins, which originates from the fact that they have been discovered to be highly useful in ruminant-based livestock agriculture. They have been found to act as anthelmintic compounds, reduce methane emissions, and increase nitrogen use efficiency in ruminants.3−5 Additionally, proanthocyanidins shift nitrogen excretion partially to the feces, which reduces volatilization as nitrous oxide, as well as urea excretion. This in turn reduces the environmental impact from subsequent ammonia loads.6 Proanthocyanidins are typically present in plants as mixtures of tens and hundreds of individual tannins that are difficult or even impossible to quantify individually. Instead, they are most often quantified as total proanthocyanidin concentration and characterized for summary characteristics, for example, a ratio of procyanidin (PC) units to prodelphinidin (PD) units present in all proanthocyanidins or average tannin size for all proanthocyanidins, expressed as the mean degree of polymerization (mDP). Both total proanthocyanidin concentration and the structural characteristics of the proanthocyanidins are important for bioactivity. For instance, it has been shown that a © XXXX American Chemical Society

high mDP and a large share of PDs in the proanthocyanidins were characteristic of high anthelmintic activity and high protein precipitation capacity.7−9 On the other hand, PDs may bind proteins in the rumen so strongly that the desired dissociation of the tannin−protein complex in the small intestine is inhibited and, therefore, high relative proportions of PD may result in an antinutritional effect.10 Consequently, the ideal PC/PD ratio may be a delicate balance between desired bioactive effects and nutritional quality. Additionally, the anthelmintic effects of proanthocyanidins have previously been shown to be enhanced by flavonoids, such as rutin.3,11 Arbutin, on the other hand, had previously been linked to the drought tolerance of the resurrection plant (Myrothamnus flabellifolia).12 Whereas a large variability in proanthocyanidin concentration and composition across a large array of sainfoin genotypes has been previously reported,13 nothing is known about their stability across different environments and across different ontogenetic stages of the plant. This is important because one of the most pressing issues for agriculture is the impact of climate change, which is projected to increase the occurrence of drought periods.14 Moreover, even the ambitious target to limit the increase in global mean temperatures to 2 °C translates into a 3 °C increase for certain regions, such as the Mediterranean and central Europe.15 Therefore, understanding the mechaReceived: Revised: Accepted: Published: A

May 24, 2016 November 7, 2016 November 21, 2016 November 21, 2016 DOI: 10.1021/acs.jafc.6b02342 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Table 1. Developmental stage of Onobrychis viciifolia Plants over the Entire Drought Period and the Recovery Phase

nisms that drought stimulates in plants, and implementing adaptation in agriculture through measures such as selection of drought tolerant crops, will be essential to navigate the challenge of climate change. To study the effect of drought on acetone/water-extractable proanthocyanidin (PA) concentration and composition, we selected sainfoin (Onobrychis viciifolia Scop.) because it has good forage quality and higher proanthocyanidin concentrations than the other common proanthocyanidin-containing forage legumes birdsfoot trefoil (Lotus corniculatus) and white clover (Trifolium repens L.).16,17 Also, there is some indication that sainfoin is drought-tolerant.18 Furthermore, several cultivars have been shown to contain an appropriate concentration and composition of PAs to reduce methane and nitrous oxide emissions from ruminant farming while maintaining high weight gains in ruminants.6,19 Sainfoin was found to have high potential to be implemented in livestock systems to help improve their sustainability.20,21 However, very little is known about how the proanthocyanidin concentration and structural characteristics of sainfoin are specifically affected by drought stress. Generally, previous field experiments on the effect of drought in many species often lacked comparability, due to the use of different parameters to determine drought intensity, as well as differences in timing of drought application.22 These factors made generalization of the results difficult. Therefore, this study examined how the plants’ ontogenetic stage (generative or vegetative) specifically affects their response to drought stress under otherwise comparable conditions. This work aimed at answering the following questions: (i) What is the impact of drought stress on plant yield, concentration, and structural characteristics of extractable proanthocyanidins (PAs) as well as on selected flavonoids in sainfoin: (ii) Is the impact of drought affected by the ontogenetic stage at which it occurs or the presence of selected flavonoids, such as arbutin: (iii) Are there differences in the drought response among different sainfoin accessions? And, (iv) do the PAs provide protection against biotic stresses, such as pathogens, in the field?

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ontogenetic stage in treatments without additional cut

with additional cut

3

vegetative

vegetative

6 10

flowering immature seeds ripe seeds vegetative

flowering vegetative

week after beginning of drought

14 23

vegetative vegetative

cutting type and time point 12 days prior to droughta week 7b

week 17a

a

Indicates the cutting of all plants. bIndicates the timing of the additional cut during the drought period (half of the plants to reset the ontogenetic stage back to vegetative).

MATERIALS AND METHODS

Experimental Setup. The experimental site was described in detail previously.13 Briefly, the field experiment was located near Zurich, Switzerland (47°44′ N 8°53′ E, 482 m asl), and the soil was a calcic cambisol with a pH of approximately 7.1 and a depth of at least 0.75 m. In the experiment, five sainfoin accessions were subjected to four drought-related treatments: drought and control conditions, each with or without an additional cut during the drought stress period. During most of the first 7 weeks of the drought period, plants grew in their vegetative stage and then started to flower (generative stage, Table 1). At week 7, half of the plants were subjected to an additional cut (removal of entire aboveground biomass; Figure 1), changing their ontogenetic stage back to vegetative, whereas the uncut plants continued their generative growth (Table 1). In this context, the term “uncut” refers only to the drought period (with these plants still having been cut prior to and post drought period). This cut treatment during drought allowed comparison of the effects of drought on sainfoin in either the vegetative or the generative stage under identical environmental conditions for the remaining 11 weeks of the drought period. The accessions consisted of the registered cultivars ‘Perly’, ‘Taja’, ‘Esparsette’, and ‘Visnovsky’, as well as an accession from Turkey, ‘CPI 63750’ (for details see Malisch et al.13). In an open field, nine experimental plants were sown in rows per accession and replicate,

Figure 1. Environmental data during experimental period, with soil water potential (SWP) at 20 cm depth, soil temperature at 5 cm depth, and air temperature at 1 m height for drought and control conditions. with 0.25 m between individual plants of the same accession and 0.5 m between different accessions. The experiment was sown in late May 2012. Drought was simulated by the installation of stationary rainout shelters once plants were fully established and lasted 127 days, from June 12 to October 17, 2013, and was followed by a 9-week recovery period. The shelters were 4 m wide, 23 m long, and 2.4 m high at the center. The cover of the shelters was made of low-density polyethylene (LDPE) with 90% light transmission, a UV-A transmission from 340 nm, and a transmission throughout the entire spectrum of UV-B of at least 70% (UV5, Folitec Agrarfolien-Vertriebs GmbH, Westerburg, Germany). Light inhibition of the covers was not a limiting factor for photosynthesis in the drought treatment, as was confirmed with photosynthetic active radiation (PAR) measurements. At the top B

DOI: 10.1021/acs.jafc.6b02342 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry of the tents, there was a 0.3 m wide gap for ventilation, which was covered with a secondary, 0.9 m wide, roof, mounted 0.2 m above the gap, to prevent rain intrusion. On the sides, the covers ended at a height of 0.5 m, to act as the inlet for cooler, ambient air; the heated air was vented at the opening in the roof. This additionally allowed exposure of plants to wind and to maintain plant movements similar to field conditions. There were no significant differences in either soil temperature (at both 5 and 40 cm depths) or air temperature (at 1 m above the surface) between the drought and control treatments, thus allowing for quantification of a drought effect without additional temperature stress (Figure 1). To prevent influence of possible lateral water movement in the soil on the experimental plants, there was a 1 m buffer zone, split into 0.75 m on the weather-exposed side and 0.25 m on the more sheltered side and planted with perennial ryegrass (Lolium perenne L.). Additionally, there was 0.5 m of Onobrychis viciifolia buffer plants on both sides to prevent effects from reduced intraspecific competition in the experimental plants at the margins. This resulted in a total buffer of 1.25 m on the weather exposed side and 0.75 m on the more sheltered side. All plants (including buffer plants) were cut with electrical scissors to 7 cm height. Individual plants were utilized instead of swards due to the limited seed availability of several accessions and to ensure the largest possible control over the growing conditions. Distance between individual plots was 5 m to prevent shading or other interactions between plants with or without additional cut. The experimental design was a split plot with the eight mainplots derived from the four treatment combinations of the factors cut and drought, which were replicated twice, whereas the five accessions were arranged as subplots. This design, with big mainplots for cut and drought, allowed the use of large borders to prevent effects of lateral water movements and effects of uncut plants on cut plants via shading. The disadvantage of this design, however, is a weak statistical power to test drought and cut effects of an individual harvest. However, the statistical test of the temporal development of the treatment combinations (cut × drought × time interaction), which was the main focus of the present study, is very powerful with this design. Sampling. Of each of the five accessions, three plants were selected in the two replicates of the four treatment combinations (drought/no drought × additional cut/no additional cut), resulting in a total of 120 plants. A small leaf fraction of each plant was sampled at five different sampling events (Figure 1), by removing five leaves of comparable age, and from a comparable position at the plant (around the center of the entire stem length) including leaflets, petiole, and rachis. Leaf age was determined by a visual comparison based on leaf size and color (for details, see Malisch et al.13), whereby young (freshly unfolded, small and light green) and old (very dark green, very large) leaves, as well as damaged or sick leaves, were omitted to prevent a distortion of PA concentrations due to leaf size or plant responses to pathogens. Whereas all leaves of the individual plants were pooled, no leaves from different plants were pooled. Samples were stored in refrigerated coolers immediately after sampling, before transferring them to a −70 °C freezer within the first hour after sampling. Infections with sainfoin rust were scored 2 days prior to the fourth sampling event. Scoring was based on visual estimation of the percentage of the overall leaf area covered with fungal spores. Additionally, a mixed sample of single leaves from various plants was taken to identify the fungus species. Sample Preparation and Chemicals. Sample preparation and extraction of soluble polyphenols were conducted as described in Malisch et al.13 Briefly, freeze-drying of plant samples was conducted in a Freeze-Drying Plant Sublimator 3x4x5 (ZIRBUS technology GmbH, Bad Grund, Germany). Plant material was milled for 1 min at 25 Hz in a MM 400 ball mill (Retsch Technology GmbH, Haan, Germany) in 25 mL tungsten carbide containers with four tungsten carbide balls (7 mm diameter). Extractions were made with 1.4 mL of 80:20 (v/v) acetone/H 2O solvent and were repeated twice. Accordingly, this study does refer to acetone/water extractable proanthocyanidins (PAs) and does not account for the level of bound proanthocyanidins that may exist in the plants. The extracts were freeze-dried with a Christ Alpha 2-4 (B. Braun Biotech

International, Melsungen, Germany) Lyophilisator and were stored at −20 °C. Prior to analysis, freeze-dried extracts were dissolved in 1 mL of H2O, filtered with 0.2 μm PTFE syringe filters (VWR International, Radnor, PA, USA) and diluted to four mL of extract with H2O. Chemicals used for extraction and compound calibration were identical to those, utilized in Malisch et al.13 Additionally, pentagalloyl glucose was purified by J.-P. Salminen of Turku University, with a confirmed purity of >98%. UPLC-MS/MS Analysis. The quantitative and qualitative analysis of PAs and the main compounds of sainfoin was conducted according to the protocol of Engström et al.23 for UPLC-MS/MS analysis under the identical setup as the Acquity UPLC system (Waters Corp., Milford, MA, USA), connected to a Xevo TQ triple-quadrupole mass spectrometer (Waters Corp., Milford, MA, USA) with electrospray ionization. The UPLC system utilized a 100 mm × 2.1 mm i.d., 1.7 μm, Acquity UPLC BEH Phenyl column (Waters Corp., Wexford, Ireland). Quantification occurred via both a diode array detector and the Xevo TQ mass spectrometer, using multiple reaction monitoring methods of Engström et al.23 as described by Malisch et al.13 Operating conditions of the system were identical to those reported by Malisch et al.13 Before each run, a flavonoid mix stock solution was injected twice to determine the system’s performance at the start of analysis. Additionally, prior to and after each 10 samples, 5 samples of a catechin stock solution (1.0 μg mL−1) were run to determine the stability of the system’s ionization efficiency for polyphenols over the course of the analysis. Each sample was analyzed once and MRM responses of polyphenols were corrected against MRM responses of corresponding five plus five catechin stock solutions and quantified by using the calibration curves obtained from the calibration standards described below. Calibration Curves. The calibration curve for chlorogenic acid was produced from a stock solution of 20 μg mL−1, with the range of the dilution series from 1.25 to 20 μg mL−1. Arbutin and rutin were prepared in stock solutions of 40 μg mL−1, and the dilution series ranged from 0.625 to 40 μg mL−1. Dilution of all compounds occurred in H2O. As in Malisch et al.,13 solutions for PA calibration curves were produced from stock solutions (1.0 mg mL−1) of a PC-rich sample (95% purity, determined by thiolysis) and a PD-rich sample (98% purity, determined by thiolysis) and by dilution with acetonitrile/water (20:80, v/v). Dilutions ranged from 1.0 to 0.01 mg mL−1 of the stock solution. Calibration curves were utilized to determine the linear range for quantitation. The mDP was obtained by calculating the ratio of terminal units and extension units of PCs and PDs, as obtained by four different cone voltages to optimize for different molecule weights, according to the method of Engström et al.23 (see the Supporting Information). Statistical Analysis. Linear mixed regression24 was used to determine the impact of treatments on the response variables y (plant weight, PA concentration, PA structural characteristics, and flavonoids), of which plant weight was log-transformed prior to analysis to meet the assumptions of the applied models. At any individual sampling event, the model was yikl = α × cut + β × drought + γ × accession + λ1 × plotk + λ 2 × subplotl + ei

(1)

where y is the estimated response at any given cut × drought × accession combination and yikl is the response of the ith plant in plot k and subplot l. The fixed parameters α, β, and γ estimate the effects of the variables cut, drought, and accession, respectively, including their interactions. The random coefficients λ model the multilevel structure of the design. λ1 estimates the variance across the plot level, whereas λ2 estimates the variance across the subplot level, that is, across the five accession subplots nested within the plots. The error ei is assumed to be normally distributed with zero mean and variance σ2. Inference on differences among treatment levels and accessions were derived from the model contrasts. For the effect of treatments across the entire experimental period, eq 1 was extended to C


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Table 2. Onobrychis viciifolia Biomass Yield (Dry Matter, DM) per Plant, Leaf Share of Harvested Biomass, and Amount of Extractable Proanthocyanidins (PAs) per Harvested Biomass of Whole Plant at 7 and 17 Weeks after Beginning of the Drought Period, As Affected by Drought and Additional Cut (Ontogenetic Stage) Averaged over the Five Accessions (acc)a 7 weeks DM yield (g plant−1)

treatment mean

Cut+/Ctr (n = 30) Cut+/Drt (n = 30) Cut−/Ctr (n = 30) Cut−/Drt (n = 29) mean SE

LMM cut drought acc cut × drought cut × acc drought × acc cut × drought × acc

Dfnum/Dfden NA/NA 1/2 4/8 NA/NA NA/NA 4/8 NA/NA

leaf share (%)

17 weeks PA amount (mg plant−1)

39.9 25.2 NA NA 3.4

29.3 34.5 NA NA 2.8

147.9 68.4 NA NA 11.3

NA 4.63 ns 1.81 ns NA NA 1.29 ns NA

NA 0.22 ns 1.06 ns NA NA NA NA

NA 4.13 ns 1.58 ns NA NA 1.30 ns NA

DM yield (g plant−1) 14.7 6.4 36.4 35.4 2.9 Dfnum/Dfden 1/4 1/4 4/16 1/4 4/16 4/16 4/16

31.66 ** 12.28 * 7.62 *** 4.90 † 2.09 ns 4.96 ** 2.67 †

leaf share (%) 89.4 98.3 32.1 38.2 2.0 490.50 9.20 3.21 0.64 1.28 1.17 NA

PA amount (mg plant−1) 228.2 111.0 210.7 196.3 29.4

*** * * ns ns ns

1.35 3.81 3.32 1.26 1.41 4.40 3.21

ns † * ns ns * *

a The treatments consist of the factor cut, with (Cut+) or without (Cut−) an additional cut, and the factor drought, with the levels control (Ctr, rainfed) and drought (Drt, rain exclusion). Mean SE is the mean standard error over all treatments. NA values result for the Cut− treatments in week 7 result from the lack of a biomass harvest for these treatments at this time. The LMM table identifies the F value, as well as the significance level of the factors and their interactions on the different variables, as calculated by the linear mixed model described in eq 1. Degrees of freedom (Df) are given for both numerator (Dfnum) and denominator (Dfden). ns, P ≥ 0.1; †, P < 0.1; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

drought from vegetative plants (cut × drought interaction, f1,4 = 4.90, P = 0.09): at the end of the drought period (week 17), stressed generative plants achieved the same dry matter (DM) yield (36.4 g DM plant−1) as those of the rainfed control (35.4 g DM plant−1, Table 2; t4 = 0.81, P = 0.46). Also, the leaf share of the harvested biomass was not significantly affected by drought (t4 = 1.37, P = 0.24) in generative plants. The biomass reduction of 37% at the additional cut demonstrates that drought stress was already effective after 7 weeks of rain exclusion. This is in line with other studies, in which biomass reductions of approximately 25−85% were observed after 6−10 weeks of imposed drought.27−29 Although the SWP at a depth of 20 cm remained high during the first 4 weeks, superficial soil drying started earlier, and at 5 cm deep the SWP was beginning to drop rapidly after 2.5 weeks, reaching −0.72 MPa at week 7 (results not shown), compared to −0.19 MPa at 20 cm deep (Figure 1). To our knowledge, no study had ever subjected plants from temperate grasslands to rain exclusion for 18 weeks. Even though the biomass reduction was −56% in plants with the additional cut, sainfoin still regrew under very low water availability, highlighting its potential as agricultural forage in water-limiting conditions. Direct comparisons to other forages are difficult due to differences in response based on the timing of drought and the duration of drought, as well as soil conditions. Nevertheless, the results from an experiment that was performed directly adjacent to our experimental area (site Reckenholz, Switzerland) and which exposed four forages to a 9 week drought period, including a cut during the drought period, does provide a reference. In this study, yield losses after the additional cut under drought were approximately −75% for Lolium perenne, −60% for Cichorium intybus, −30% for Trifolium repens, and −20% for Trifolium pratense.29 Our results demonstrate that the plant’s drought response clearly depended on its ontogenetic stage. This helps to explain why the observed response of grassland to drought has been so diverse among experiments.22 The smaller growth reduction of

yjklm = α × cut + β × drought + γ × accession + δ × sampling + λ1 × plotk + λ 2 × subplotl + λ3 × plant m + ej

(2)

where the fixed parameter δ denotes the effect of sampling time on y and yjklm being the response of a particular plant m in plot k and subplot l at a specific sampling time j. The random coefficient λ3 estimates the variance of individual plants across the repeated samplings; thus, λ3 represents a further sublevel of the hierarchical design and rules out pseudoreplication of plants over time. All remaining parameters have meanings as explained for eq 1. Unless otherwise stated, we restricted eq 2 to sampling events 2, 3, and 4 to prevent a dilution of effects from sampling event 1, at which the drought effect was generally very weak, as well as from sampling event 5, which was the recovery period. Linear mixed models were calculated with the R package “nlme”,25 whereas statements about correlations were determined using the Pearson correlation coefficient. All analyses were computed using the software R.26

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RESULTS AND DISCUSSION From the time of installation of the rainout shelters through week 7, the drought stress increased from no stress to a moderate restriction of plant growth with a soil water potential (SWP) of −0.8 MPa at 20 cm depth (Figure 1). In subsequent weeks, until the end of testing at week 18, water limitation continued to increase and SWP reached values below −1.5 MPa, the threshold of plant accessible soil water. Effects of Drought on Aboveground Biomass. In the first 7 weeks of the drought period, when all plants grew vegetatively and drought stress was moderate, the drought treatment resulted in a yield reduction of 37% (t2 = 2.14, P = 0.17) compared to the rainfed control (Table 2). In the subsequent weeks (8−17) of the drought period, vegetative plants had a yield reduction of 56% (t4 = 3.55, P < 0.05, plants with additional cut) compared to the rainfed control. Simultaneously, the leaf share of the harvested biomass, assessed at week 17, was significantly greater (t4 = 3.06, P < 0.05) under drought (98%) as compared to the control (89%). Generative plants (uncut) responded differently to D


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Journal of Agricultural and Food Chemistry generative plants might help to explain why less frequently defoliated systems were less affected by drought than frequently defoliated systems.27,30,31 in which plants are primarily in their vegetative stage. An increased leafiness under drought has been reported previously for other legumes, with a mean leaf/stem ratio of 5.1 for drought-stressed plants and 1.7 for well-watered plants.32 This shift to a higher leafiness is most likely the result of a reduction in stem length, combined with delayed plant maturation, and is thus a size effect, in which leafiness is obtained not by an increase in leaf production but is a relative increase due to shorter and lighter stems. This is in accordance with previous research, which, irrespective of drought, has identified that smaller plants have increased leafiness. As for larger plants, the stratification of leaves over height is important and, therefore, longer stems are required. Consequently, with increased biomass, the leaf/stem ratio decreases to maintain the balance between optimized light utilization and mechanical requirements.33 This increase in leafiness of vegetative plants is highly beneficial in terms of the bioactivity of the forage, as leaves are almost 3.5 times richer in PAs than stems, with a mDP more than twice as high and an almost 30% higher PD share.13 According to our results, the drought-induced increase in leafiness was significant only in the vegetative stage, which is most likely a combination of the facts that (a) plant size was not affected in the generative stage and (b) stem formation is increased with increasing plant maturity and plant size. Effect of Drought on Concentration and Structure of Extractable Proanthocyanidins. During the first 7 weeks, drought-stressed plants showed no significant difference in concentrations of PAs compared to the rainfed controls (Figure 2). However, after the additional cut, the development of PA concentration over time due to drought was significantly different between cut and uncut plants (cut × drought × time, f 2,191 = 28.54, P < 0.001, Figure 2). The PA concentrations of the vegetatively regrowing plants increased dramatically under drought stress and, thus, were 24% (t4 = 1.81, P = 0.14) and 46% (t4 = 2.97, P < 0.05) higher in stressed plants than in control plants at weeks 10 and 14, respectively. In contrast, plants in the generative stage showed no difference in PA concentrations in drought conditions compared to the rainfed control (week 10, t4 = 0.27, P = 0.80; week 14, t4 = 0.47, P = 0.67). As a consequence of this different response of vegetative and generative plants to drought (cut × drought, f 2,4 = 6.70, P < 0.05), the PA concentration at week 14 was equal (t2 = 0.17, P = 0.88) for vegetative and generative plants, when grown under rainfed control, whereas under drought, vegetative plants had a 66% higher (t2 = 4.81, P < 0.05) PA concentration than generative plants (24.8 and 15.0 mg g−1, respectively). Under growth conditions without water limitations, the main reactive oxygen species (ROS), superoxide radicals, and hydrogen peroxide are formed in the chloroplasts as part of the electron chain in the Mehler reaction. As ROS are highly reactive and can oxidize lipids, proteins, or even DNA and RNA, excess ROS can result in cell death. This mechanism can be utilized by the plant as a tool against invading pathogens, but it can also severely reduce plant health. Therefore, in the absence of biotic stresses, the plant controls the amount of ROS using a group of antioxidant enzymes, namely, ascorbate peroxidase, superoxide dismutase, and catalases.34 This allows the plant to maintain a critical balance between the required amounts of ROS for messenger and electron transport

Figure 2. Concentration of extractable proanthocyanidins (PAs), their prodelphinidin (PD) share, and mean degree of polymerization (mDP) in sainfoin leaves as affected by drought and additional cut (ontogenetic stage) averaged over the five accessions. The treatments consist of the factor cut, with (Cut+) or without (Cut−) an additional cut at week 7, and the factor drought, with the levels control (Ctr, rainfed) and drought (Drt, rain exclusion). The time point of a cut is indicated by an arrow, with filled arrows indicating a general cut (all plants) and empty arrows an additional cut during the drought period (half of the plants) to reset the ontogenetic stage to vegetative. Error bars are standard errors of the mean. Significance of levels for the effects of experimental factors (LMM) are are: P ≥ 0.1 ns; 651 P < 0.1 †; P < 0.05 *; P < 0.01 **; P < 0.001 ***. NA refers to not applicable, as no cutting treatment was applied prior to week 7.

purposes, without resulting in harm to the plant’s cells.35 Under drought stress, plants respond via abscisic acid (ABA)mediated signaling to induce stomatal closure in the leaves.36 The stomatal closure results in reduction of carbon fixation, which, in turn, decreases the amount of energy that is required to reduce CO2.37 The excess energy results in both photorespiration and excess accumulation of ROS.38 Because the vast increments in ROS are toxic to the plant itself, proanthocyanidins and other antioxidants can act as radical scavengers, reducing ROS levels and preventing cell damage in the plant.35 The difference in the PA response to drought stress between vegetative and generative plants matches the optimal defense hypothesis (ODH). According to the ODH, plants invest most resources in protection of those parts, for which their loss would incur the greatest cost to plant vitality.39 Because the most vulnerable plant parts differ depending on the stage of ontogenetic development, the relative importance of leaves to E


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Journal of Agricultural and Food Chemistry the plant changes.40 During the vegetative stage, and especially in the first weeks after a cut when the plants have only small amounts of leaves, leaves are the most important part for the plant, as they are required to photosynthesize and produce the energy required for growth. In the generative stage, less energy is required and a lot of leaves are present; thus, the protection of flowers becomes relatively more important, to ensure reproduction. This may explain why vegetative plants after the additional cut protected their leaves through increased PA concentrations, whereas the generative plants did not. Although drought stress has often been linked to increased PA concentrations41 due to the lack of phenotypic observations, increases in PA concentrations have frequently been misinterpreted as increases in the rate of synthesis. However, it is important to incorporate biomass when stating changes in the plant’s production of secondary metabolites due to drought (or any other factor). This is because the biomass is likely to be most severely affected by drought. Thus, increases in PA concentration are often the result of the same amount of metabolites in plants with reduced biomass, but without the metabolic rates changing at all.37 Therefore, in addition to the PA concentration, we calculated the PA amount per harvested biomass of whole plant (calculation in the Supporting Information). For the vegetative stage at week 17 of the drought period, plants grown under drought had 51% lower (t4 = 2.37, P = 0.08) PA amounts, with 228.2 and 111.0 mg plant−1 for control and drought, respectively (Table 2)). Consequently, the suppression of the amount of PAs due to drought was in the same range as the suppression of biomass. Over the entire observation period, structural characteristics of PAs were generally not affected by the experimental factors. There was only one exception: at week 14, plants in the generative stage (uncut) had a higher (t4 = 5.25, P < 0.01) mean degree of polymerization under drought (mDP = 11.2) than in the rainfed control (mDP = 9.2), whereas the vegetative plants showed no effect (t4 = 0.00, P = 1.0; Figure 2). A limited response in the structural characteristics was also observed in the distribution of the tannin size, as also the largest average mDP (maxmDP)13 was not affected by the treatments in either week 10 (drought × cut, f1,4 = 0.50, P = 0.52) or week 14 (f1,4 = 2.07, P = 0.22). This indicates a stable distribution of the degree of polymerization, which is supported by the correlation of mDP with the maxmDP, which was very high across all treatments (r = 0.86, P < 0.001; result not shown). This hypothesis about the stability is supported when calculating the ratio between the maxmDP and mDP: this ratio was not affected by the treatments in either week 10 (drought × cut, f1,4 = 1.82, P = 0.25) or 14 (f1,4 = 2.31, P = 0.20). Nevertheless, very little is known, and the link between effect of different polymer sizes and the various bioactive effects has still not been well-established. Therefore, further research with more sophisticated in vivo experiments will be required to satisfactorily evaluate the importance of these seemingly small changes for achieving the desired bioactivity. Effect of Drought on Flavonoids. Before the additional cut, no drought effect on arbutin concentration was observed (Figure 3). After the additional cut, the plants reacted differently to drought in the vegetative and generative stages, as evident from the highly significant cut × time × drought interaction ( f 2,191 = 11.72, P < 0.001). Whereas drought increased the arbutin concentration in vegetative plants in week 14 by +23% (t4 = 2.20, P = 0.09), it decreased the arbutin leaf concentration in generative plants by −22% (t4 = 1.93,

Figure 3. Rutin and arbutin concentration in sainfoin leaves as affected by drought and additional cut (ontogenetic stage) averaged over the five accessions. For abbreviations (treatments) and significance levels, see Figure 2.

P = 0.13) and −36% (t4 = 3.41, P < 0.05) for weeks 10 and 14, respectively. Rutin concentrations were strongly and consistently reduced by drought ( f1,4 = 19.34, P < 0.01) over the entire drought period (Figure 3). With −41%, the extent of this reduction was largest during the first weeks (average of weeks 3 and 6) when all of the plants grew vegetatively. During the second half of the drought period this extent was reduced to −35% (t4 = 2.78, P < 0.05) for the plants regrowing vegetatively after the additional cut and to −16% (t4 = 0.11, P = 0.92) for plants growing generatively. Arbutin has previously been identified to increase drought tolerance in the resurrection plant (Myrothamnus flabellifolia) via membrane stabilization.12 However, further studies found that arbutin can have this effect only in the presence of nonbilayer-forming lipids, such as monogalactosyldiacylglycerol or phosphatidylethanolamine. 42 In our experiment, such a drought-induced increase was only observed after the additional cut in the vegetative stage. The drought effect on rutin was surprising for two reasons: (i) because the observed effect was strongest when drought was still weak and (ii) because rutin concentrations were lower under drought. The strong response of rutin to drought after only 7 weeks of drought can be explained by the fast-drying topsoil, as mentioned under Effect on Aboveground Biomass. Therefore, this is in accordance with previously observed rapid increases in rutin concentration after drought for wild privet (Ligustrum vulgare).43 Although rutin concentrations were higher in drought-stressed wild privet, rutin concentrations in water-stressed cherry tomatoes were found to be up to 40% lower when compared to well-watered plants.44 Therefore, changes in rutin under drought stress may be variable and more research is required. Effect of Drought on Accession. Two aspects of the accessions are important: First, the overall PA properties of an accession (averaged over all environmental conditions; i.e., genotype effect), and, second, the pattern of change of the PA F


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between weeks 7 and 14 of 35% (drought, t4 = 1.86, P = 0.14; Figure 4) and, importantly, this trend did not differ among accessions (accession × drought, f4,8 = 0.64, P = 0.65). In their generative stage, accessions did not increase PA concentration (drought, t4 = 0.86, P = 0.44), and this response once again did not differ significantly among accessions (accession × drought, f4,8 = 1.66, P = 0.25), despite ‘Taja’ being the only accession to increase its PA concentration under drought. For vegetative plants and their structural characteristics of the PAs, the response to drought did not differ among accessions, including mDP (accession × drought, f4,8 = 0.08, P = 0.99) and PD share (accession × drought, f4,8 = 0.38, P = 0.82). With regard to the main flavonoids, the mean changes due to drought were also consistent during the same period in all five accessions in the vegetative stage and entailed a 34% decrease (t4 = 2.44, P = 0.07; Figure 5) in rutin (accession × drought, f4,8 = 0.75, P = 0.58) and a 10% increase (t4 = 0.63, P = 0.56) in arbutin (accession × drought, f4,8 = 0.29, P = 0.88).

properties of an accession as a response to the environment (i.e., genotype × environment effect). With respect to the genotype effect, averaged over all of the treatments, CPI 63750 had a PA concentration of 22.0 mg g−1, which was 23 and 45% higher (accession, f4,16 = 10.1, P < 0.001) than that of Visnovsky and Perly, the accessions with the second highest and the lowest PA concentrations, respectively (Figure 4). When PA structures were compared,

Figure 4. Concentration of extractable proanthocyanidins (PAs), their prodelphinidin (PD) share, and mean degree of polymerization (mDP) in sainfoin leaves as affected by drought and additional cut (ontogenetic stage) for each of the five accessions (acc). For abbreviations (treatments) and significance levels, see Figure 2. Values are the means of the two measurements at weeks 10 and 14 after the beginning of the drought period, and accordingly, linear mixed model (LMM) estimates are based on eq 2, yet with only including measurements from sampling events 3 and 4. Error bars are standard errors of the mean.

Figure 5. Rutin and arbutin concentration in sainfoin leaves as affected by drought and additional cut (ontogenetic stage) for each of the five accessions (acc). For abbreviations (treatments) and significance levels, see Figure 2. Values are the means of the two measurements at weeks 10 and 14 after the beginning of the drought period, and, accordingly, linear mixed model (LMM) estimates are based on eq 2, yet with only including measurements from sampling events 3 and 4. Error bars are standard errors of the mean.

The absence of genotype × environment effects has two implications: first, it tests whether the drought-induced PA increase of vegetative sainfoin plants, as described above, is comparable for all of the accessions and can thus be generalized; second, it tests whether the same accession was the best in terms of PA properties in all environments. On the basis of our results, both of these aspects can be confirmed. Therefore, sainfoin can generally be expected to increase its PA concentration in the vegetative stage under drought, and the order of PA concentrations and structural characteristics over several cultivars is unlikely to change across different environments. This is in accordance with recent findings in poplar (Populus spp.), in which genetic composition was identified as the major determinant for structural characteristics of PAs.45 Both of these findings, the high genetic variability (significant accession effect) and the comparable responses of the

‘Taja’ had the highest mDP with 11.1, which was 20% higher than Visnovsky and CPI 63750 (accession, f4,16 = 22.9, P < 0.001). With regard to the PD share, Perly had a mean PD share of 55%, which was significantly lower (accession, f4,16 = 27.3, P < 0.001) than the mean PD shares of any other accession, which were in the range of 76−79% (Figure 4). With these results, this set of five commercial accessions confirms the findings of high genetic variability in PA concentration and structural characteristics within sainfoin, which was observed on a much broader set including cultivars and wild accessions.13 All five accessions demonstrated the same response to the environment (genotype × environment effect) for PA concentrations and their structural characteristics, as well as rutin. Concentrations of PA increased under drought stress in their vegetative stage with the mean increase over all accessions G


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Table 3. Pearson Correlation Coefficients of Rust Infection in Onobrychis viciifolia at Week 14 of the Drought Period, with the Leaf Concentration of the Main Polyphenols and the Main Phenotypic Characteristicsa Ctr/Cut− (n = 30) Ctr/Cut+ (n = 30) Drt/Cut− (n = 30) Drt/Cut+ (n = 29)

PA (mg g−1 DM)

chlorogenic acid (mg g−1 DM)

arbutin (mg g−1 DM)

rutin (mg g−1 DM)

DM yield (g plant−1)

plant height (cm)

0.17 −0.59 *** −0.04 −0.42 *

0.21 −0.42 * −0.22 0.12

0.24 −0.21 0.08 −0.02

−0.01 −0.16 −0.26 0.10

−0.21 −0.19 −0.13 0.11

−0.01 −0.02 −0.24 0.06

a Treatments consist of factors cut, with the levels with (Cut+) or without (Cut−) additional cut and the factor drought, with the levels control (Ctr, rainfed) and drought (Drt, rain exclusion). *, P < 0.05; ***, P < 0.001.

genotypes to the environment (not significant accession × environment interaction), are important for the practical application to ameliorate PA properties of sainfoin by breeding. The former implies that selection of genotypes (cultivars) with desired PA properties is feasible, and the latter implies that such ameliorated cultivars will perform their advantages over a wide range of environments. Practical Implications. As identified under Effect of Drought on Concentration and Structure of Extractable Proanthocyanidins, the PA concentration was higher under drought in the second half of the drought period, yet the PA amount was still significantly lower due to the dramatic reduction in biomass. This is important from a utilitarian point of view, as drought resulted in higher PA concentrations with identical structural characteristics, and therefore potentially higher bioactivity per feed intake, while not changing too dramatically to, for example, cause antinutritional effects.10 On the other hand, control conditions resulted in higher PA amount per harvested biomass of whole plant and, therefore, potentially higher PA amounts per land area. However, it is not safe to assume that higher PA concentrations in individual plants directly translate to higher PA concentrations in swards, because in swards there is a high level of intraspecific competition. Under controlled conditions, plants may not grow as large as other individual plants, and under drought conditions, several small plants may have a higher density and therefore provide the same PA yield per area as fewer large plants. Despite limitations in scaling to the sward level, our results are promising for the cultivation of sainfoin as a source of PA for its desired bioactive effects. This is because, on the basis of current knowledge, the nature of bioactivity of the PAs is predominantly influenced by structural characteristics. Consequently, the lack of effect from environmental stress on the structural characteristics of sainfoin PAs is helpful, as the mode of action of the bioactivity also appears to be unaffected,10 thereby maintaining predictability of the bioactive effects. Secondary Metabolites Are Related to Reduced Sainfoin Rust. Because the experimental plants were growing in the field, there was a natural presence of biotic stresses, such as spores of sainfoin rust (Uromyces onobrychidis (Desmazières) Léveillé). In the vegetative treatment during week 14 of the drought period, PA concentrations in sainfoin leaves were negatively correlated with the coverage of leaves with sainfoin rust, under both control (r = −0.59, P < 0.001, Table 3) and drought (r = −0.42, P < 0.05) conditions. Furthermore, the concentration of chlorogenic acid was correlated to the rust coverage in control conditions (r = −0.42, P < 0.05), but not under drought (r = 0.12, P = 0.50). However, the concentrations of PAs and chlorogenic acid were autocorrelated (r = 0.58, P < 0.001, result not shown). The rust infection was not significantly correlated with any morphologic parameter

(i.e., yield, plant height, etc.) or concentrations of any other compound (Table 3). With leaves for PA analysis having been selected to express low levels of rust infection, the correlation indicates a systemic response, rather than a local response in which the infected leaf forms increased PA concentrations. These results are in accordance with previous studies, which have identified an effect of PAs against at least some pathogenic fungi.46 Similarly, chlorogenic acid did increase plant resistance against Verticillium albo atrum, Phytophthora infestans, and Phlyctaena vagabunda.2 With regard to their mode of defense, PAs and chlorogenic acid belong to the constitutive defense, meaning that they are not synthesized after a pathogen attack, but rather their naturally high concentration increases resistance to fungal infections.2,47 However, although it seems that PAs, chlorogenic acid, or both had an effect on the rust infection, the observed correlation between the two makes it impossible to differentiate between the extents with which each of them has been involved in the increased resistance of sainfoin to sainfoin rust. In conclusion, this work identified that the response of extractable proanthocyanidin (PA) concentration and yield to drought stress depends on the plant’s ontogenetic stage. Contrary to that, the structural characteristics of PAs were not predominantly affected by either drought or ontogenetic stage. The general pattern in PAs and their structural characteristics were consistent over several accessions. The combination of the similar response over several accessions and the robustness of the structural characteristics of PAs across several environments suggest high predictability of the PA-based bioactivity of sainfoin. Still, despite the similarity of the treatment response across accessions, differences existed among the PA concentration and polymer sizes in the five accessions, indicating a genetic influence that could be very beneficial to breeding. With regard to biotic stresses, the infection of the plant with sainfoin rust was significantly negatively correlated to its PA and chlorogenic acid concentrations, but it remains unclear which of the two was responsible for an increased resistance to sainfoin rust.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b02342. Calculations for the amount of PAs and mDP and pictures of the drought shelters (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(J.P.S.) Mail: Department of Chemistry, University of Turku, Vatselankatu 2, 20500 Turku, Finland. Phone: +358 2 333 6753. Fax: +358 2 333 6700. E-mail: j-p.salminen@utu.fi. H


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Journal of Agricultural and Food Chemistry ORCID

(8) Williams, A. R.; Fryganas, C.; Ramsay, A.; Mueller-Harvey, I.; Thamsborg, S. M. Direct anthelmintic effects of condensed tannins from diverse plant sources against Ascaris suum. PLoS One 2014, 9, e97053. (9) Brunet, S.; Jackson, F.; Hoste, H. Effects of sainfoin (Onobrychis viciifolia) extract and monomers of condensed tannins on the association of abomasal nematode larvae with fundic explants. Int. J. Parasitol. 2008, 38, 783−790. (10) Mueller-Harvey, I. Unravelling the conundrum of tannins in animal nutrition and health. J. Sci. Food Agric. 2006, 86, 2010−2037. (11) Barrau, E.; Fabre, N.; Fouraste, I.; Hoste, H. Effect of bioactive compounds from sainfoin (Onobrychis viciifolia Scop.) on the in vitro larval migration of Haemonchus contortus: role of tannins and flavonol glycosides. Parasitology 2005, 131, 531−538. (12) Suau, R.; Cuevas, A.; Valpuesta, V.; Reid, M. S. Arbutin and sucrose in the leaves of the resurrection plant Myrothamnus f labellifolia. Phytochemistry 1991, 30, 2555−2556. (13) Malisch, C. S.; Lüscher, A.; Baert, N.; Engström, M. T.; Studer, B.; Fryganas, C.; Suter, D.; Mueller-Harvey, I.; Salminen, J.-P. Large variability of proanthocyanidin content and composition in sainfoin (Onobrychis viciifolia). J. Agric. Food Chem. 2015, 63, 10234−10242. (14) Trnka, M.; Olesen, J. E.; Kersebaum, K. C.; Skjelvag, A. O.; Eitzinger, J.; Seguin, B.; Peltonen-Sainio, P.; Rotter, R.; Iglesias, A.; Orlandini, S.; Dubrovsky, M.; Hlavinka, P.; Balek, J.; Eckersten, H.; Cloppet, E.; Calanca, P.; Gobin, A.; Vucetic, V.; Nejedlik, P.; Kumar, S.; Lalic, B.; Mestre, A.; Rossi, F.; Kozyra, J.; Alexandrov, V.; Semeradova, D.; Zalud, Z. Agroclimatic conditions in Europe under climate change. Glob. Change Biol. 2011, 17, 2298−2318. (15) Seneviratne, S. I.; Donat, M. G.; Pitman, A. J.; Knutti, R.; Wilby, R. L. Allowable CO2 emissions based on regional and impact-related climate targets. Nature 2016, 529, 477−483. (16) Wang, Y.; McAllister, T. A.; Acharya, S. Condensed tannins in sainfoin: composition, concentration, and effects on nutritive and feeding value of sainfoin forage. Crop Sci. 2015, 55, 13−22. (17) Scharenberg, A.; Arrigo, Y.; Gutzwiller, A.; Soliva, C. R.; Wyss, U.; Kreuzer, M.; Dohme, F. Palatability in sheep and in vitro nutritional value of dried and ensiled sainfoin (Onobrychis viciifolia) birdsfoot trefoil (Lotus corniculatus), and chicory (Cichorium intybus). Arch. Anim. Nutr. 2007, 61, 481−496. (18) Koch, D. W.; Hinze, G. O.; Dotzenko, A. D. Influence of three cutting systems on yield, water use efficiency, and forage quality of sainfoin. Agron. J. 1972, 64, 463−467. (19) Hatew, B.; Stringano, E.; Mueller-Harvey, I.; Hendriks, W. H.; Carbonero, C. H.; Smith, L. M. J.; Pellikaan, W. F. Impact of variation in structure of condensed tannins from sainfoin (Onobrychis viciifolia) on in vitro ruminal methane production and fermentation characteristics. J. Anim. Physiol. Anim. Nutr. 2016, 100, 348−360. (20) Häring, D. A.; Scharenberg, A.; Heckendorn, F.; Dohme, F.; Lüscher, A.; Maurer, V.; Suter, D.; Hertzberg, H. Tanniferous forage plants: agronomic performance, palatability and efficacy against parasitic nematodes in sheep. Renew. Agric. Food Syst. 2008, 23, 19−29. (21) Häring, D. A.; Suter, D.; Amrhein, N.; Lüscher, A. Biomass allocation is an important determinant of the tannin concentration in growing plants. Ann. Bot. 2007, 99, 111−120. (22) Vicca, S.; Gilgen, A. K.; Serrano, M. C.; Dreesen, F. E.; Dukes, J. S.; Estiarte, M.; Gray, S. B.; Guidolotti, G.; Hoeppner, S. S.; Leakey, A. D. B.; Ogaya, R.; Ort, D. R.; Ostrogovic, M. Z.; Rambal, S.; Sardans, J.; Schmitt, M.; Siebers, M.; van der Linden, L.; van Straaten, O.; Granier, A. Urgent need for a common metric to make precipitation manipulation experiments comparable. New Phytol. 2012, 195, 518− 522. (23) Engström, M. T.; Pälijärvi, M.; Fryganas, C.; Grabber, J. H.; Mueller-Harvey, I.; Salminen, J.-P. Rapid qualitative and quantitative analyses of proanthocyanidin oligomers and polymers by UPLC-MS/ MS. J. Agric. Food Chem. 2014, 62, 3390−3399. (24) Pinheiro, J.; Bates, D. Mixed-Effects Models in S and S-Plus, 2nd ed.; Springer: New York, 2009; p 528.

Carsten S. Malisch: 0000-0002-2905-8885 Funding

These investigations were supported by the European Commission (PITN-GA-2011-289377, “LegumePlus” project). Sample analyses by UPLC-MS/MS were made possible by the strategic grant of the University of Turku (Ecological Interactions). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are very grateful to the USDA ARS-GRIN germplasm database for the provision of the seeds of the sainfoin accessions. We thank Matthias Suter for his consultancy regarding the statistical analysis. We also thank H.-U. Hirschi, E. Rosenberg, and F. Johnigk for their support with the field work and sample preparation, as well as J. Suvanto, A. Koivuniemi, and J. Kim for their guidance and assistance in the laboratory.

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ABBREVIATIONS USED ABA, abscisic acid DM, dry matter maxmDP, largest average mean degree of polymerization LMM, linear mixed model LDPE, low-density polyethylene mDP, mean degree of polymerization MRM, multiple reaction monitoring ns, not significant ODH, optimal defense hypothesis PSMs, plant secondary metabolites PAR, photosynthetic active radiation PAs, acetone/water extractable proanthocyanidins PCs, procyanidins PDs, prodelphinidins ROS, reactive oxygen species SWP, soil water potential

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Drought Effects on Proanthocyanidins in Sainfoin (Onobrychis viciifolia

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