Metabolic fingerprinting of dormant and active flower primordia of

Article Cite This: J. Agric. Food Chem. 2017, 65, 10123-10130

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Metabolic Fingerprinting of Dormant and Active Flower Primordia of Ribes nigrum Using High-Resolution Magic Angle Spinning NMR Majken Pagter,*,† Christian Clement Yde,‡,§ and Katrine Heinsvig Kjær‡,∥ †

Department of Chemistry and Bioscience, Aalborg University, Fredrik Bajers vej 7H, DK-9220, Aalborg East, Denmark Department of Food Science, Aarhus University, Kirstinebjergvej 10, DK-5792 Aarslev, Denmark § DuPont Nutrition Biosciences ApS, Edwin Rahrs vej 38, DK-8220 Brabrand, Denmark ∥ Danish Technological Institute, Gregersensvej 1, DK-2630 Taastrup, Denmark ‡

ABSTRACT: Global warming may modify the timing of dormancy release and spring growth of buds of temperate fruit crops. Environmental regulation of the activity−dormancy cycle in perennial plants remains poorly understood at the metabolic level. Especially, the fine-scale metabolic dynamics in the meristematic zone within buds has received little attention. In this work we performed metabolic profiling of intact floral primordia of Ribes nigrum isolated from buds differing in dormancy status using high-resolution magic angle spinning (HR-MAS) NMR. The technique proved useful in monitoring different groups of metabolites, e.g., carbohydrates and amino acids, in floral primordia and allowed metabolic separation of primordia from endoand ecodormant buds. In addition, due to its nondestructive character, HR-MAS NMR may provide novel insights into cellular compartmentation of individual biomolecules that cannot be obtained using liquid-state NMR. Out results show that HR-MAS NMR may be an important method for metabolomics of intact plant structures. KEYWORDS: asymmetric warming, blackcurrant, climate change, dormancy, HR-MAS NMR, metabolomics

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INTRODUCTION The earth’s mean global surface temperature is increasing and is projected to increase further in the foreseeable future.1 Except for some tropical regions, meteorological records and climate model projections have shown that the rate of warming varies on a seasonal time scale, with temperatures increasing faster in winter and spring than in summer and autumn.2 In winter and spring, temperature has a dual effect on bud development of woody perennials from temperate climate regions. On the one hand, low temperatures are necessary to break endodormancy and, on the other hand, higher temperatures are necessary to promote bud growth afterward. One likely impact of warming during dormancy is a delay in the fulfilment of chill requirements and consequently a delay in the time at which perennials become receptive to warm temperatures for spring growth.3 Insufficient chilling may additionally cause uneven bud break and a reduction in flowering and/or reproductive output of perennial fruit crops.4,5 Inversely, the advances in spring phenology, which have dominated climate-warming responses thus far, have been explained by increasing temperatures during ecodormancy, leading to a more rapid fulfilment of the heat requirement.6 Environmental regulation of the activity− dormancy cycle in perennial plants remains poorly understood at the metabolic level. Especially, the transition from endodormancy to ecodormancy has not received much attention.7,8 Also, few studies have addressed the fine-scale metabolic dynamics during dormancy in the meristematic zone within buds, but rather they focused on entire buds, which may contain both vegetative and floral primordia and nearby structures (i.e., the cushion beneath the primordia, scales),9,10 making it difficult to determine which of the observed changes occur in which structures. © 2017 American Chemical Society

Metabolomics confers high-resolution snapshots of physiological and biological aspects of cellular responses to environmental stimuli. These snapshots may facilitate the identification of patterns or metabolite markers that are characteristic for a species, a genotype, a certain stage of development, or conditions such as disease state, stress, or daily or seasonal changes.11 At present, NMR and GC/LC−MS techniques dominate the data acquisition strategies in plant metabolomics studies.12 However, the intrinsic structure of plants poses the major challenge for many NMR- and GC-based approaches. NMR and GC methods requiring extraction of different plant components can cause biases resulting from the differential extraction efficiencies and from the loss of volatile metabolites, such as ethylene, ethanol, and methanol.13 The high-resolution magic angle spinning (HR-MAS) technique combines the advantages of analysis in the solid, gel-like, and liquid phases. In HR-MAS NMR a sample is spun very fast at the magic angle (54.74°), which reduces line-broadening effects and results in highly resolved NMR spectra.11 The HR-MAS NMR technique is nondestructive, requires a small amount of tissues, and does not require an extraction procedure, as compared with liquidstate NMR or MS. Hence, it allows measurements of intact plant tissue or organ samples in their natural swollen state. HRMAS has mainly been used for biomedical applications;11 however, the technique has proved valuable in profiling metabolites in leaves, stems, roots, or intact plants of various species.14−20 Received: Revised: Accepted: Published: 10123

August 14, 2017 October 19, 2017 October 27, 2017 October 30, 2017 DOI: 10.1021/acs.jafc.7b03788 J. Agric. Food Chem. 2017, 65, 10123−10130

Article

Journal of Agricultural and Food Chemistry

‘Ben Hope’ plants. During the treatment period, plants were exposed to natural precipitation, whereas after the removal of the polyethylene net tunnel and bud break, natural precipitation was supported by automatic irrigation when needed. Dormancy Status. To determine dormancy status, six plants per treatment were moved to forcing conditions in a greenhouse (20 °C, 16 h photoperiod) on November 18, December 16, January 20, and February 10. Budburst of the terminal and the four uppermost lateral buds were separately observed two to three times per week for 1 month on one shoot per plant. Budburst was recorded using a rating of 0 to 4, where 0 = no budburst, 1 = green tip visible, 2 = visible leaves, 3 = grape stage, and 4 = flowers open, as shown in ref 25. Bud Break, Flowering, and Cropping Performance. The timing of the bud break in the field was recorded on 12 plants per treatment by recording bud development from March 4 to April 28. Bud development was recorded using the same scale as for the evaluation of dormancy status. The total number of flowers per plant was recorded on April 28. Cropping performance of the same plants was evaluated on July 8 by recording the total berry yield per plant and the number of berries per plant. Sampling of Buds and Preparation of Floral Primordia for HR-MAS NMR Analysis. Flower buds were sampled monthly from October until mid-March, when buds started to break. At each time point, 50−100 mg of the uppermost axillary buds from six plants per treatment was sampled, immediately weighed, and then flash frozen in liquid nitrogen. To prepare floral primordia for HR-MAS NMR spectroscopy analysis, the buds were placed on dried ice and dissected free of the bud base, bud scales, and adjacent leaf primordia to uncover the flower primordia. Then the flower primordia were fitted into disposable preweighed 50 μL inserts (Bruker Biospin, Rheinstetten, Germany) followed by addition of 10 μL of D2O containing 0.05% (w/v) trimethylasilylpropionic acid sodium salt (TMSP-d4). The inserts containing TMSP-d4 and plant material were weighed again to obtain the exact weight of the primordia before being frozen at −80 °C until HR-MAS NMR analysis. In two bud samples of warmed plants, one harvested in December and one in March, only vegetative primordia were found. These samples were therefore discarded. HR-MAS NMR. All HR-MAS NMR measurements were performed at 280 K on a Bruker AVANCE 600 NMR spectrometer operating at 14.1 T, observing 1H at 600.13 MHz and equipped with a four channel (1H/2H/13C/31P) HR-MAS probe (Bruker BioSpin, Rheinstetten, Germany). Upon measurement, the inset (sample) was put into a 4 mm zirconium rotor (Bruker BioSpin, Rheinstetten, Germany) and 1H NMR spectra were acquired with a CPMG (Carr−Purcell− Meiboom−Gill) pulse sequence [Bruker cpmgpr1d; RD−90°x(−τ− 180°y−τ)n−FID] including presaturation to suppress signals from water molecules and attenuation of broad signals from macromolecules. The acquisition parameters for the spectra were as follows: 5 kHz spin rate, 64 scans, a spectral width of 12.15 ppm with 32K data points providing a FID resolution of 0.44 Hz, an acquisition time of 2.25 s, a total spin−spin relaxation delay of 100 ms (2nτ), a spin−echo delay of 1 ms (τ), and a recycle time of 3 s. The spectra were processed by the application of an exponential multiplication of the FIDs by a factor of 0.3 Hz prior to Fourier transformation. Each spectrum was automatically phased, baseline corrected, and referenced to the TMSP-d4 signal at 0.00 ppm as an internal reference. To aid spectral assignment, a 2D 1H−13C heteronuclear singlequantum coherence (HSQC) experiment on a selected sample was performed. The HSQC correlating experiment were acquired with a spectral width of 12.15 ppm in the 1H dimension and 180.00 ppm in the 13C dimension, a data matrix with a size of 2048 × 512 data points, 128 transients per increment, and a recycle delay of 3 s. Signal assignments was performed according to earlier literature and available databases; the Human Metabolome Database26 and the Biological Magnetic Resonance Data Bank, University of Wisconsin (http://www.bmrb.wisc.edu27). The Chenomx NMR Suite 8.1 software was applied to assign and quantify metabolites by determining the area of each metabolite and comparing the area with the integral of the TMSP-d4 signal. Afterward, the metabolites

Blackcurrant (Ribes nigrum L.) is a soft fruit species widely grown in cold and temperate regions. Floral primordia are initiated in late summer or early autumn in the year prior to that in which flowering occurs and is causally and temporally associated with growth cessation and dormancy induction.21 Blackcurrant has a relatively high chilling requirement and is one of the fruit crops that is potentially at risk in parts of Europe due to the lack of winter chilling forecast in projections of future climatic conditions.4,22 Accordingly, seasonal asymmetric warming can decrease fruit yield and modify the timing of dormancy release and spring growth of blackcurrant.23,24 Using GC−MS it was recently shown that primary metabolism of intact flower buds of blackcurrant is largely unaffected by seasonal asymmetric warming, but bud break is associated with a dramatic remodelling of the metabolome.24 Here we performed metabolic profiling of isolated floral primordia of R. nigrum in order (1) to examine the applicability of HR-MAS NMR spectroscopy in establishing a metabolic fingerprint of blackcurrant floral primordia directly in their swollen natural state that can be used to distinguish different stages in the dormancy−activity cycle and (2) to further investigate the effect of seasonal asymmetric warming on perennating organs. The samples analyzed were harvested in December 2014, when the dormancy status of age-matched plants exposed to ambient or slightly elevated temperatures differed significantly, and in March, when plants of both treatments were no longer dormant.

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MATERIALS AND METHODS

Experimental Site and Plant Material. The experimental area was established outdoors at the Department of Food Science, Aarhus University in Denmark (55°18′ N, 10°26′ E) and consisted of a control plot (ambient temperature) and a warming plot (elevated temperature), which were adjacent to each other and had identical sizes (5 × 5.5 m). Warming was conducted from late October 2013 to late March 2014 with 480 m of temperature-controlled heating cable (producing a maximum of 83 W m−2) distributed in rows directly on the soil surface between the plants. The air temperature in the plots was monitored with Pt100 temperature sensors covered by radiation shields 20, 50, and 80 cm above the soil surface. Temperature means over 5 min intervals were logged throughout the treatment period. Whenever the temperature at 20 cm in the warmed plot was more than 3 °C higher than the corresponding temperature in the control plot the heating cables were switched off until the temperature difference fell below 3 °C. The soil temperature was measured in each plot at a depth of ca. 10 cm using Tinytag Talk temperature loggers (Gemini Data Loggers, Chichester, England). The positioning of the heating cables on the ground did not only increase the air temperature, it also led to an undesirable increase in the soil temperature. Hence, early January 2013 the heating cables were raised approximately 5 cm from the soil surface by laying them on metal holders. From midNovember onward, after leaf abscission, both plots were covered with a transparent polyethylene net tunnel, providing some wind shelter. The net had a shading effect of 15% and was removed in early March, to avoid any shading effects during the growing season. The net tunnel allowed the plants to be exposed to the natural rainfall. The experiment was carried out using one-year old vegetatively propagated plants of Ribes nigrum L. ‘Ben Hope’. Plants were propagated as single cane plants in 3.5 L pots containing a sandy loam. Before the start of the experiment, plants were grown outside and exposed to natural precipitation supported with automatic irrigation with a standard liquid fertilizer solution (Ferti-Hvid). Before initiation of the warming treatment ca. 150 plants of ‘Ben Hope’ were placed in each of the experimental plots with pots buried in the soil to avoid root frost injuries. In each plot, plants were placed in 12 rows with plants of another R. nigrum cultivar, ‘Zusha’, interspersed between the 10124

DOI: 10.1021/acs.jafc.7b03788 J. Agric. Food Chem. 2017, 65, 10123−10130

Article

Journal of Agricultural and Food Chemistry were normalized to the weight of the primordia in each sample. A first step multivariate data analysis was performed on the whole NMR spectra, excluding the water signal. Misalignments of the spectra were corrected using the icoshift procedure.28 The regions 0.2−4.80 and 5.02−10.0 ppm of the 1H NMR spectra were segmented into 0.0132 ppm bins and integrated. The binned NMR data were normalized to the total area. Principal components analysis (PCA) was applied to the NMR data and indicated that one sample was an outlier. Hence, this replicate was excluded from further analysis. Data Analysis. Floral bud break, defined as the point at which green leaves were visible on the buds (stage 2), in response to forcing at permissive temperature (20 °C) or under natural conditions in the field was tested using logistic regression in R (R Core Team, 2015). To evaluate the dormancy status of the plants, the material was first analyzed by using the three experimental factors: treatment, harvest time point, and duration of forcing. Further analyses were carried out separately for each harvest time point as a two-way experiment with treatment and forcing duration as experimental factors. In addition, the number of days to 50% budburst was estimated in December, January, and February by interpolation for each treatment. November was omitted in this analysis, because only approximately half of the buds developed to stage 2. Differences in the timing of bud break in the field were analyzed with the treatment and observation duration as experimental factors. The effects of treatment and harvest time point on metabolite pool sizes were analyzed using a two-way ANOVA in R with correction for multiple testing using the Benjamini−Hochberg method.29 Statistical significance of differences in number of flowers or berries per plant, the berry yield, and average berry weight was tested by one-way ANOVA. PCA was performed on centered, Pareto-scaled metabolite spectra using the pcaMethods package in R.30

Figure 1. Daily mean air (A) and soil (B) temperatures at 20 cm height or ∼15 cm depth, respectively, in the control and warming plots during the experimental period.

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RESULTS AND DISCUSSION Asymmetric Warming Advances Spring Phenology of ‘Ben Hope’ but Does Not Affect Cropping Performance. The winter season 2013−2014 was mild, with the average daily mean temperature in the two plots at 20 cm above the soil surface being 6.0 °C in November, 5.3 °C in December, 2.1 °C in January, 5.0 °C in February, and 6.7 °C in March (Figure 1A). This is considerably warmer than the temperature recordings obtained in a similar setup the year before23 and also warmer than the winter climate (December, January, and February) the following year.24 The temperature at 20 cm height was on average 1.0 °C higher in the warming than in the control plot, with the daily mean temperature difference between the plots varying between 0.3 and 2.6 °C. The effect of the warming treatment was greatest closest to the heating cables. Hence, at approximately 50 and 80 cm above the soil surface the daily average temperature difference decreased to 0.6 and 0.3 °C, respectively. The soil temperature at 15 cm depth was on average 1.9 °C higher in the warming than in the control plot (Figure 1B). Asymmetric warming had no overall effect on dormancy status, evaluated as bud break following forcing in a permissive warm environment, indicating that chilling was sufficient to break bud dormancy of ‘Ben Hope’. However, the dormancy status of the plants differed significantly between seasonal harvest dates (P ≤ 0.001) and following different durations of forcing (P ≤ 0.001). When analyzing the dormancy status at individual harvest dates, warming significantly delayed floral budburst in December (P ≤ 0.05), indicating deeper dormancy of warmed than control plants, but it had no effect on the dormancy status of floral buds in November, January, and February. Accordingly, after 25 days at permissive temperature (20 °C) bud development of control plants was advanced compared to that of warmed plants in December, whereas in

November, January, and February the numbers of buds reaching each developmental stage were similar (Figure 2). That asymmetric warming caused a difference in dormancy status of floral buds in December but not in January and February was also evident from the number of days to 50% budburst (Figure 3). Deeper dormancy of warmed plants than control plants in December was probably offset by plenty of chilling hours in late January and early February. According to Rose and Cameron,31 the optimum chilling temperature for ‘‘Ben Hope’ is 2.2 °C, which is relatively high compared to other blackcurrant cultivars. Hence, mild winter temperatures may delay the fulfilment of chill requirements less in ‘Ben Hope’ than in most other genotypes. Also, ‘Ben Hope’ has proved adaptable in terms of chilling requirement in the United Kingdom, where recent warm winters have lowered yields of some cultivars, and is also of use for growing in the low-chill environment of New Zealand,4,22 indicating a low chilling requirement. Flower buds of plants at both ambient and elevated temperature started to break in early March (Figure 4), but asymmetric warming significantly advanced bud break (P < 0.001). This indicates a more rapid fulfilment of the heat requirement in warmed plants and that ‘Ben Hope’ is more responsive to warming during the heat accumulation phase in spring than during the dormancy period. This is in line with observations in the cultivars ‘Narve Viking’, ‘Titania’, and ‘Zusha’ exposed to asymmetric warming in other years.23,24 In the statistical analysis, timing of bud break was defined as the point at which green leaves were visible (stage 2). However, flower development (from stage 2 to 4) also seemed advanced in warmed plants as compared to the control plants (Figure 4). Compared to other years and cultivars, the dates of budburst observed in the field were approximately 1 month earlier than in the cold winter of 2012−201323 and about 1 week earlier 10125


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Figure 2. Number of lateral buds of R. nigrum ‘Ben Hope’ that had attained each of the four bud stages following 25 days of forcing in a permissive warming environment at different times during the winter season, where 0 = no budburst, 1 = green tip visible, 2 = visible leaves, 3 = grape stage and 4 = flowers open. Prior to forcing, plants were exposed to ambient or slightly elevated winter temperatures. The four uppermost lateral buds on one shoot of six plants per treatment were evaluated at each time point.

Table 1. Number of Flowers and Cropping Performance of R. nigrum ‘Ben Hope’ Exposed to Ambient or Slightly Elevated Temperatures during the Winter Seasona ambient temperature no. of flowers (plant−1) yield (g plant−1) no. of berries (plant−1) average berry weight (g berry−1) a

42.2 24.7 30.3 0.78

± ± ± ±

9.1 5.3 6.3 0.03

elevated temperature 40.1 24.4 29.4 0.85

± ± ± ±

7.5 4.6 6.1 0.02

Values given are means ± SE of n = 12 plants.

cultivars,23,24 where winter warming decreased fruit set and crop yield. However, the lack of a negative impact of asymmetric warming on cropping performance indicates sufficient chilling of both control and warmed plants4 and is thus in line with the results concerning floral bud break. Identification of Metabolites through HR-MAS NMR. According to the evaluations of dormancy status and bud break in spring, the dormancy status of the plants exposed to ambient and elevated temperatures differed significantly in December, although both groups of plants remained endodormant, and in early March, plants of both treatments were ecodormant and had just started to develop, approaching the green tip visible stage (stage 1). Hence, in order to try to identify metabolites that can be used to distinguish different stages in the dormancy−activity cycle and between dormancy statuses of otherwise age-matched plants, we performed a metabolite analysis of floral primordia from buds sampled in December and March. A representative one-dimensional 1H HR-MAS NMR spectrum obtained from intact floral primordia from a single blackcurrant bud is shown in Figure 5, and a list of identified metabolites is given in Table 2. In total, three sugars (sucrose, glucose, fructose) and one sugar alcohol (myo-inositol) were identified in the carbohydrate region, and seven amino acids (alanine, arginine, asparagine, citrulline, isoleucine, lysine, and valine) were identified in the aliphatic region. Intermediates from the tricarboxylic acid (TCA) cycle (malic acid, fumarate, citric acid, and succinic acid), along with choline and Ophosphocholine, were also identified from the HR-MAS NMR spectra. In addition, maltose, proline, and quinic acid were tentatively assigned. Most of these metabolites were also detected when analyzing primary metabolites of intact buds

Figure 3. Days to 50% floral budburst in plants of R. nigrum ‘Ben Hope’ as affected by harvest date. Depth of dormancy was assessed on December 15, January 20, and February 10. Prior to forcing, plants were exposed to ambient or slightly elevated winter temperatures. The four uppermost lateral buds on one shoot of six plants per treatment were evaluated at each time point.

Figure 4. Average attainment of each of the five bud stages of lateral buds of R. nigrum ‘Ben Hope’ during bud break in the field. During the winter season, plants were grown at ambient or slightly elevated temperatures. Values are means ± SE of n = 12 shoots.

than in 2014−2015,24 implying that the warming phase was advanced irrespective of the treatment. Warming had no effect on the number of flowers, fruit yield, number of berries per plant, or individual berry size (Table 1). This is in contrast to previous observations in other 10126


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Figure 5. A representative 1H HR-MAS NMR spectrum (0.0−6.6 ppm) of R. nigrum ‘Ben Hope’ flower primordia dissected free of bud base, bud scales, and adjacent leaves (small inset in left corner). Abbreviations: alanine (Ala), arginine (Arg), asparagine (Asp), glutamate (Glu), glutamine (Gln), isoleucine (Ile), lysine (Lys), proline (Pro), and valine (Val). The x-axis represents the chemical shift (ppm).

bud oil.32 The content of terpenes in bud oil shows large variations in relation to bud development. High terpenes content in bud oil is found in dormant buds, whereas the break of dormancy in early spring and bud development is associated with large transformations in the content of terpenes.33 Similar patterns were observed for the signals in the area between 0.2 and 1.2 ppm in the present study. Metabolic Changes Associated with Dormancy Release of Floral Primordia. PCA was used to identify the largest variance components in the metabolite 1H HR-MAS NMR spectra (Figure 6). Principal component 1 (PC1) explained 52.4% of the total variance and separated profiles of primordia collected in December from profiles of primordia collected in March, indicating that dormant floral primordia are clearly distinguishable from primordia that have started to become active. PC2 explained 20.8% of the total variance and tended to separate the metabolite profiles of flower primordia

Table 2. 1H HR-MAS NMR Data of the Metabolites Assigned by Chenomx in Floral Primordia of R. nigrum ‘Ben Hope’ compd alanine arginine asparagine choline citrate citrulline fructose fumarate glucose glutamate glutamine isoleucine lysine malic acid maltosea myo-inositol Ophosphocholine prolinea quinic acid succinic acid sucrose valine a

proton chemical shift in ppm and multiplicity 1.47 (d, 1, CH), 3.77 (q, 3, CH3) 1.71 (m, 2, CH2), 1.90 (m, 2, CH2), 3.23 (t, 2, CH2), 3.76 (t, 1, CH) 2.86 (m, 1, CH), 2.95 (m, 1, CH), 4.00 (dd, 1, CH) 3.18 (s, 9, N(CH3)), 3.51 (dd, 2, CH2), 4.10 (ddd, 2, CH2) 2.53 (d, 2, α-CH2), 2.68 (d, 2, α′-CH2) 1.87 (m, 2, CH2), 3.14 (q, 2, CH2), 3.74 (dd, 1, CH) 3.89 (dd, 1, CH), 4.02 (t, 2, CH2) 6.50 (s, 2, CHCH) 3.23 (dd, 1, CH), 3.48 (t, 1, CH), 3.52 (dd, 1, CH), 3.70 (t, 1, CH), 4.64 (d, 1, CH), 5.22 (d, 1, CH) 2.12 (m, 2, CH2), 2.32 (m, 2, CH2), 3.74 (dd, 1, CH) 2.11 (m, 2, CH2), 2.43 (m, 2, CH2) 0.93 (t, 3, CH3), 0.99 (d, 3, CH3) 1.71 (m, 2, CH2), 1.89 (m, 2, CH2), 3.74 (t, 1, CH) 2.37 (dd, 1, α-CH2), 2.67 (dd, 1, α′-CH2), 4.30 (dd, 1, CH) 4.64 (d, 1, CH), 5.22 (d, 1, CH), 5.40 (d, 1, CH) 3.52 (dd, 1, CH), 4.05 (t, 1, CH) 3.21 (s, 9, N(CH3)) 1.97 (m, 2, CH2), 2.06 (m, 1, CH2), 2.34 (m, 1, CH2), 3.33 (dt, 1, CH2) 1.84 (dd, 1, CH2), 1.96 (ddd, 1, CH2), 2.05 (m, 2, CH), 3.54 (dd, 1, CH) 2.40 (s, 4, CH2) 3.55 (dd, 1, CH), 4.22 (d, 1, CH), 5.40 (d, 1, CH) 0.98 (d, 3, CH3), 2.26 (m, 1, CH)

Tentative assignment.

using GC−MS;24 however, glutamate, choline, and Ophosphocholine have not previously been detected in perennating organs of blackcurrant. Furthermore, chemical shifts in the region from 0.2 to 1.2 ppm indicated the presence of terpenes in the flower primordia. Though this cannot be confirmed by the present literature, it is well-known that monoand sesquiterpenes are constituents of essential blackcurrant

Figure 6. Score plots from principal components analysis applied to the metabolite spectra from flower primordia of R. nigrum ‘Ben Hope’ exposed to ambient (black symbols) or slightly elevated (gray symbols) temperatures from late October to April 23 of the following year. Buds were sampled in December (closed symbols) or early March (open symbols). 10127


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Figure 7. Levels of metabolites in flower primordia of R. nigrum ‘Ben Hope’ exposed to ambient or elevated temperatures during the winter season. Flower primordia were analyzed in December, when floral buds were dormant, or in March, when buds had just started to develop. Values given are means ± SE of n = 5−6. The metabolic profiles collected in December and March were statistically analyzed using a two-way ANOVA at FDR P < 0.05 by comparing all conditions against each other. Different letters indicate significant differences between sampling time points.

obtained from buds of control and warmed plants. However, the separation was not clear-cut, suggesting that metabolically it is not possible to distinguish between age-matched flower primordia differing slightly in dormancy status. According to the ANOVA analysis, eight of the identified metabolites showed significantly changed contents between the two harvest time points, but none of the metabolites varied significantly between

control and warmed plants (Figure 7). The lack of significant differences in metabolite contents between flower primordia of control plants and plants exposed to asymmetric warming is in line with GC−MS-based analyses of intact flower buds.24 Hence, higher structural resolution does not alter the proposition that primary metabolism is largely unaffected by mild asymmetric warming. 10128


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choline also in flower organ development. Phosphocholine is additionally an essential metabolite for plant development because it is the precursor for the biosynthesis of phosphatidylcholine, which is a major lipid component in plant cell membranes. Interestingly, biochemical analysis showed that FLOWERING LOCUS T binds to diurnally changing molecular species of phosphatidylcholine to promote flowering,41 indicating a role of phophatidylcholine in the timing of flowering. In conclusion, HR-MAS NMR proved useful in monitoring different groups of metabolites, e.g., carbohydrates and amino acids, in intact floral primordia of blackcurrant, hence obtaining a higher structural resolution in metabolic profiling of flower buds than usually acquired. The technology allowed metabolic separation of floral primordia of dormant buds and buds that have started to become active, whereas it was not possible to distinguish between age-matched floral primordia differing slightly in dormancy status. The latter supports the suggestion that primary metabolism of flower buds is largely unaffected by mild asymmetric warming. In addition, due to its nondestructive character, HR-MAS NMR may provide novel insights into cellular compartmentation of individual biomolecules that cannot be obtained using solution NMR.

Significantly increased concentrations of succinic acid, fumarate, and malic acid in March indicate that dormancy release and development of floral primordia were associated with increased TCA cycle activity. Increased TCA cycle activity may be fueled by an increased production of glucose and fructose that can be metabolized during glycolysis. Hence, the concentration of glucose was significantly higher in March than in December, whereas the increase in fructose was nonsignificant. The concentration of sucrose did not change significantly from December to March, indicating that sucrose catabolism is not the source of energy and carbon skeletons in developing flower primordia. Rather, hexose sugars may be provided by photosynthetic activity of green buds. Alternatively, increased concentrations of fumarate, malic acid, and succinic acid may derive from β-oxidation and activity of the glyoxylate cycle, as observed in cambial meristem cells of Populus aspen,7 with succinic acid, fumarate, and malic acid replenishing the TCA cycle or functioning as precursors for amino acid or carbohydrate biosynthesis. Earlier studies of other species have shown that HR-MAS NMR resonance signals of malic acid in intact leaves are shifted in comparison to signals of malic acid in water.16,19 A similar shift in resonance frequency was not observed for malic acid in blackcurrant floral primordia, suggesting that whereas a large quantity of malic acid in leaves is stored in the acidic environment of the vacuole (stomatal regulation), the smaller fraction of malic acid in primordia possibly applies mainly to the mitochondrial fraction of citric cycle intermediates and that the vacuolar fraction is small or absent. Metabolites found in significantly higher concentrations in flower primordia in March than in December also included glutamate, glutamine, and valine. Glutamate is a central compound in amino acid metabolism in higher plants, and the glutamine synthetase−glutamate synthase (GS/GOGAT) cycle is subject to seasonal transcriptional regulation. Hence, in apical buds of Camellia sinensis, the gene encoding GS, which catalyze the transfer of the amide amino group of glutamine to 2-oxoglutarate to yield two molecules of glutamate, is downregulated during winter dormancy.34 In Lotus japonica, plants with metabolic lesions reducing the glutamate content in flowers were sterile, suggesting that high concentrations of glutamate and of amino acids derived from it, such as proline, may be required for adequate development of floral tissue.35 The important role of proline in plant growth and differentiation across the life cycle, including flower development, is well established.36 The concentration of proline did not change significantly in this study. However, as both the carbon skeleton and the α-amino group of glutamate form the basis for the synthesis of proline, the increase in glutamate may precede an increase in proline. In accordance with the present results, the levels of pyroglutamic acid+glutamic acid+glutamine and proline also increased in spring in intact buds of blackcurrant.24 Branched-chain amino acids have been implicated in shoot organogenesis in white spruce.37 Thus, accumulation of valine in March may also be associated with development of flowers. In Arabidopsis, endogenous phosphocholine content increases during plant development and affects root meristem size, cell division, and cell elongation.38 Recently, it has additionally been shown that the regulation of choline levels may be crucial for phloem and plasmodesmata development in plants.39,40 Accumulation of choline and a nonsignificant increase in the concentrations of phosphocholine in flower primordia of blackcurrant in March suggests a functional role of

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AUTHOR INFORMATION

Corresponding Author

*Phone: +45 30229330. E-mail: [email protected]. ORCID

Majken Pagter: 0000-0001-5441-1508 Funding

This work was supported by the Danish Council for Independent Research | Technology and Production Sciences (Grant No. DFF-1335-00182 to M.P.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Annette Steen Brandsholm, Connie Damgaard, and Nina Eggers for outstanding technical assistance and Elin Rosenstrøm for taking care of the plants.

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REFERENCES

(1) IPCC Working Group. Climate Change 2013: The Physical Science Basis; WHO and UNEP, 2013; 1535 pp. (2) Xia, J.; Chen, J.; Piao, S.; Ciais, P.; Luo, Y.; Wan, S. Terrestrial carbon cycle affected by non-uniform climate warming. Nat. Geosci. 2014, 7 (3), 173−180. (3) Luedeling, E.; Girvetz, E. H.; Semenov, M. A.; Brown, P. H. Climate change affects winter chill for temperate fruit and nut trees. PLoS One 2011, 6 (5), e20155. (4) Atkinson, C. J.; Brennan, R. M.; Jones, H. G. Declining chilling and its impact on temperate perennial crops. Environ. Exp. Bot. 2013, 91, 48−62. (5) Bonhomme, M.; Rageau, R.; Lacointe, A.; Gendraud, M. Influences of cold deprivation during dormancy on carbohydrate contents of vegetative and floral primordia and nearby structures of peach buds (Prunus persica L. Batch). Sci. Hortic. (Amsterdam, Neth.) 2005, 105 (2), 223−240. (6) Cleland, E. E.; Chuine, I.; Menzel, A.; Mooney, H. A.; Schwartz, M. D. Shifting plant phenology in response to global change. Trends in Ecology and Evolution 2007, 22, 357−365. (7) Druart, N.; Johansson, A.; Baba, K.; Schrader, J.; Sjödin, A.; Bhalerao, R. R.; Resman, L.; Trygg, J.; Moritz, T.; Bhalerao, R. P. Environmental and hormonal regulation of the activity-dormancy cycle

10129


Article


overwintering and spring phenology of blackcurrant. Environ. Exp. Bot. 2017, 140 (May), 96−109. (25) Winde, J.; Andersen, U. B.; Kjaer, K. H.; Pagter, M. Variation in freezing tolerance, water content and carbohydrate metabolism of floral buds during deacclimation of contrasting blackcurrant cultivars. Acta Physiol. Plant. 2017, 39, 201. (26) Wishart, D. S.; Tzur, D.; Knox, C.; Eisner, R.; Guo, A. C.; Young, N.; Cheng, D.; Jewell, K.; Arndt, D.; Sawhney, S. HMDB: The human metabolome database. Nucleic Acids Res. 2007, 35, D521. (27) Ulrich, E. L.; Akutsu, H.; Doreleijers, J. F.; Harano, Y.; Ioannidis, Y. E.; Lin, J.; Livny, M.; Mading, S.; Maziuk, D.; Miller, Z. BioMagResBank. Nucleic Acids Res. 2008, 36, D402. (28) Savorani, F.; Tomasi, G.; Engelsen, S. B. icoshift: A versatile tool for the rapid alignment of 1D NMR spectra. J. Magn. Reson. 2010, 202 (2), 190−202. (29) Benjamini, Y.; Hochberg, Y. Controlling the false discovery rate: A practical and powerful approach to multiple testing. J. R. Stat. Soc. Ser. B 1995, 57, 289−300. (30) Stacklies, W.; Redestig, H. pcaMethods; Bioconductor, 2013. (31) Rose, G. A.; Cameron, R. W. Chill unit models for blackcurrant (Ribes nigrum L.) cultivars “Ben Gairn”, “Ben Hope” and “Ben Tirran. Sci. Hortic. (Amsterdam, Neth.) 2009, 122 (4), 654−657. (32) Le Quere, J.-L.; Latrasse, A. Composition of the essential oils of blackcurrant buds (Ribes nigrum L.). J. Agric. Food Chem. 1990, 38 (1), 3−10. (33) Dvaranauskaite, A.; Venskutonis, P. R.; Raynaud, C.; Talou, T.; Viškelis, P.; Sasnauskas, A. Variations in the essential oil composition in buds of six blackcurrant (Ribes nigrum L.) cultivars at various development phases. Food Chem. 2009, 114 (2), 671−679. (34) Rana, N. K.; Mohanpuria, P.; Kumar, V.; Yadav, S. K. A CsGS is regulated at transcriptional level during developmental stages and nitrogen utilization in Camellia sinensis (L.) O. Kuntze. Mol. Biol. Rep. 2010, 37 (2), 703−710. (35) Suárez, R.; Márquez, J.; Shishkova, S.; Hernández, G. Overexpression of alfalfa cytosolic glutamine synthetase in nodules and flowers of transgenic Lotus japonicus plants. Physiol. Plant. 2003, 117 (3), 326−336. (36) Kavi Kishor, P. B.; Hima Kumari, P.; Sunita, M. S. L.; Sreenivasulu, N. Role of proline in cell wall synthesis and plant development and its implications in plant ontogeny. Front. Plant Sci. 2015, 6, 544. (37) Dowlatabadi, R.; Weljie, A. M.; Thorpe, T. a; Yeung, E. C.; Vogel, H. J. Metabolic footprinting study of white spruce somatic embryogenesis using NMR spectroscopy. Plant Physiol. Biochem. 2009, 47 (5), 343−350. (38) Alatorre-Cobos, F.; Cruz-Ramírez, A.; Hayden, C. A.; PérezTorres, C. A.; Chauvin, A. L.; Ibarra-Laclette, E.; Alva-Cortés, E.; Jorgensen, R. A.; Herrera-Estrella, L. Translational regulation of Arabidopsis XIPOTL1 is modulated by phosphocholine levels via the phylogenetically conserved upstream open reading frame 30. J. Exp. Bot. 2012, 63 (14), 5203−5221. (39) Dettmer, J.; Ursache, R.; Campilho, A.; Miyashima, S.; Belevich, I.; O’Regan, S.; Mullendore, D. L.; Yadav, S. R.; Lanz, C.; Beverina, L. CHOLINE TRANSPORTER-LIKE1 is required for sieve plate development to mediate long-distance cell-to-cell communication. Nat. Commun. 2014, 5, 4276. (40) Kraner, M. E.; Link, K.; Melzer, M.; Ekici, A. B.; Uebe, S.; Tarazona, P.; Feussner, I.; Hofmann, J.; Sonnewald, U. Choline transporter-like1 (CHER1) is crucial for plasmodesmata maturation in Arabidopsis thaliana. Plant J. 2017, 89 (2), 394−406. (41) Nakamura, Y.; Andrés, F.; Kanehara, K.; Liu, Y.; Dörmann, P.; Coupland, G. Arabidopsis florigen FT binds to diurnally oscillating phospholipids that accelerate flowering. Nat. Commun. 2014, 5, 3553.

in the cambial meristem involves stage-specific modulation of transcriptional and metabolic networks. Plant J. 2007, 50 (4), 557− 573. (8) Fennell, A. Y.; Schlauch, K. A.; Gouthu, S.; Deluc, L. G.; Khadka, V.; Sreekantan, L.; Grimplet, J.; Cramer, G. R.; Mathiason, K. L. Short day transcriptomic programming during induction of dormancy in grapevine. Front. Plant Sci. 2015, 6, 834. (9) Zhong, W.; Gao, Z.; Zhuang, W.; Shi, T.; Zhang, Z.; Ni, Z. Genome-wide expression profiles of seasonal bud dormancy at four critical stages in Japanese apricot. Plant Mol. Biol. 2013, 83 (3), 247− 264. (10) Mazzitelli, L.; Hancock, R. D.; Haupt, S.; Walker, P. G.; Pont, S. D. A.; McNicol, J.; Cardle, L.; Morris, J.; Viola, R.; Brennan, R.; et al. Co-ordinated gene expression during phases of dormancy release in raspberry (Rubus idaeus L.) buds. J. Exp. Bot. 2007, 58 (5), 1035− 1045. (11) Kim, H. K.; Choi, Y. H.; Verpoorte, R. NMR-based plant metabolomics: Where do we stand, where do we go? Trends Biotechnol. 2011, 29 (6), 267−275. (12) Tian, H.; Lam, S.; Shui, G. Metabolomics, a Powerful Tool for Agricultural Research. Int. J. Mol. Sci. 2016, 17 (11), 1871. (13) Wheeler, H. L.; Soong, R.; Courtier-Murias, D.; Botana, A.; Fortier-Mcgill, B.; Maas, W. E.; Fey, M.; Hutchins, H.; Krishnamurthy, D.; Kumar, R.; et al. Comprehensive multiphase NMR: A promising technology to study plants in their native state. Magn. Reson. Chem. 2015, 53 (9), 735−744. (14) Marino, G.; Righi, V.; Simoni, A.; Schenetti, L.; Mucci, A.; Tugnoli, V.; Muzzi, E.; Francioso, O. Effect of a peat humic acid on morphogenesis in leaf explants of Pyrus communis and Cydonia oblonga. Metabolomic analysis at an early stage of regeneration. J. Agric. Food Chem. 2013, 61 (21), 4979−4987. (15) De Oliveira, C. S.; Carlos, E. F.; Vieira, L. G. E.; Lião, L. M.; Alcantara, G. B. HR-MAS NMR metabolomics of “Swingle” citrumelo rootstock genetically modified to overproduce proline. Magn. Reson. Chem. 2014, 52 (8), 422−429. (16) Bharti, S. K.; Bhatia, A.; Tewari, S. K.; Sidhu, O. P.; Roy, R. Application of HR-MAS NMR spectroscopy for studying chemotype variations of Withania somnifera (L.) Dunal. Magn. Reson. Chem. 2011, 49 (10), 659−667. (17) Sidhu, O. P.; Annarao, S.; Pathre, U.; Snehi, S. K.; Raj, S. K.; Roy, R.; Tuli, R.; Khetrapal, C. L. Metabolic and histopathological alterations of Jatropha mosaic begomovirus-infected Jatropha curcas L. by HR-MAS NMR spectroscopy and magnetic resonance imaging. Planta 2010, 232 (1), 85−93. (18) Mori, T.; Tsuboi, Y.; Ishida, N.; Nishikubo, N.; Demura, T.; Kikuchi, J. Multidimensional High-Resolution Magic Angle Spinning and Solution-State NMR Characterization of 13C-labeled Plant Metabolites and Lignocellulose. Sci. Rep. 2015, 5, 11848. (19) Augustijn, D.; Roy, U.; Van Schadewijk, R.; De Groot, H. J. M.; Alia, A. Metabolic profiling of intact Arabidopsis thaliana leaves during circadian cycle using 1H high resolution magic angle spinning NMR. PLoS One 2016, 11 (9), e0163258. (20) Choze, R.; Alcantara, G. B.; Alves Filho, E. d. G.; e Silva, L. M. A. E.; Faria, J. C.; Liao, L. M. Distinction between a transgenic and a conventional common bean genotype by 1H HR-MAS NMR. Food Chem. 2013, 141 (3), 2841−2847. (21) Sønsteby, A.; Opstad, N.; Heide, O. M. Effects of summer temperature on growth and flowering in six black currant cultivars (Ribes nigrum L.). J. Hortic. Sci. Biotechnol. 2012, 87 (2), 157−164. (22) Jones, H. G.; Hillis, R. M.; Gordon, S. L.; Brennan, R. M. An approach to the determination of winter chill requirements for different Ribes cultivars. Plant Biol. 2013, 15, 18−27. (23) Pagter, M.; Andersen, U. B.; Andersen, L. Winter warming delays dormancy release, advances budburst, alters carbohydrate metabolism and reduces yield in a temperate shrub. AoB Plants 2015, 7 (0), plv024−plv024. (24) Andersen, U. B.; Kjaer, K. H.; Erban, A.; Alpers, J.; Hincha, D. K.; Kopka, J.; Zuther, E.; Pagter, M. Impact of seasonal warming on 10130


Metabolic fingerprinting of dormant and active flower primordia of

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