Effect of growth temperature on glucosinolate profiles in Arabidopsis thaliana accessions

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Phytochemistry xxx (2016) 1e13

Contents lists available at ScienceDirect

Phytochemistry journal homepage: www.elsevier.com/locate/phytochem

Effect of growth temperature on glucosinolate proﬁles in Arabidopsis thaliana accessions Ralph Kissen a, Franziska Eberl a, 1, Per Winge a, Eivind Uleberg b, Inger Martinussen b, Atle M. Bones a, * a b

Department of Biology, Norwegian University of Science and Technology, Høgskoleringen 5, NO-7491, Trondheim, Norway NIBIO, Norwegian Institute of Bioeconomy Research, Box 115, NO-1431, Ås, Norway

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 February 2016 Received in revised form 23 May 2016 Accepted 5 June 2016 Available online xxx

Glucosinolates are plant secondary metabolites with important roles in plant defence against pathogens and pests and are also known for their health beneﬁts. Understanding how environmental factors affect the level and composition of glucosinolates is therefore of importance in the perspective of climate change. In this study we analysed glucosinolates in Arabidopsis thaliana accessions when grown at constant standard (21 C), moderate (15 C) and low (9 C) temperatures during three generations. In most of the tested accessions moderate and pronounced chilling temperatures led to higher levels of glucosinolates, especially aliphatic glucosinolates. Which temperature yielded the highest glucosinolate levels was accession-dependent. Transcriptional proﬁling revealed also accession-speciﬁc gene responses, but only a limited correlation between changes in glucosinolate-related gene expression and glucosinolate levels. Different growth temperatures in one generation did not consistently affect glucosinolate composition in subsequent generations, hence a clear transgenerational effect of temperature on glucosinolates was not observed. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Arabidopsis thaliana Brassicaceae Secondary metabolites Glucosinolates Temperature Chilling Abiotic stress Transgenerational effect Parental effect Epigenetics

1. Introduction The Earth’s climate change is a regular issue in politics and society. But also natural scientists, among them plant biologists, are dealing with the potential consequences of climate change. With a predicted overall temperature increase, but with high interregional variability, and an increased frequency of weather extremes, plants all over the world risk to experience a change of their abiotic environment. While crop production in current key producing regions will be negatively impacted, some high-latitude regions currently too cold to grow crops might become more suitable in the future (Porter et al., 2014). Those changes will affect plant growth and phenology as well as the chemical composition of plant tissues, and thereby impact biological processes such as

* Corresponding author. Department of Biology, Norwegian University of Science and Technology, Realfagbygget, Høgskoleringen 5, NO-7491, Trondheim, Norway. E-mail address: [email protected] (A.M. Bones). 1 Present address: Department of Biochemistry, Max Planck Institute for Chem€ ll-Strasse 8, D-07745 Jena, Germany. ical Ecology, Hans-Kno

plant-insect and plant-pathogen interactions (Ahuja et al., 2010a). Understanding how environmental factors affect the level and composition of secondary metabolites in plants is therefore of importance and has been actively studied (Hannah et al., 2010; Nakabayashi and Saito, 2015; Ramakrishna and Ravishankar, 2011). One important group of plant secondary metabolites with a well-documented role in plant defence against pathogens and pests but which are also known for their health beneﬁts are glucosinolates, produced by plants of the Brassicales (Ahuja et al., €rkman et al., 2011). 2010b; Bjo Glucosinolate biosynthesis is known to be inﬂuenced by developmental parameters (Brown et al., 2003), genetic variation (Bellostas et al., 2007; Kliebenstein et al., 2001a), biotic stressors (Hopkins et al., 2009; Mewis et al., 2006) as well as numerous exogenous abiotic factors. Those factors include nutrition (Falk et al., 2007; Gerendas et al., 2009), photoperiod and light quality (Engelen-Eigles et al., 2006), air composition (Himanen et al., 2008; Schonhof et al., 2007b), water (Mewis et al., 2012) and temperature. The effect of temperature on glucosinolates has been studied under ﬁeld or controlled growth conditions in some Brassicaceae species

http://dx.doi.org/10.1016/j.phytochem.2016.06.003 0031-9422/© 2016 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Kissen, R., et al., Effect of growth temperature on glucosinolate proﬁles in Arabidopsis thaliana accessions, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.06.003

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R. Kissen et al. / Phytochemistry xxx (2016) 1e13

such as cabbage (Rosa, 1997; Rosa et al., 1994; Rosa and Rodrigues, 1998), kale (Steindal et al., 2015; Velasco et al., 2007), broccoli (Guo et al., 2014; Mølmann et al., 2015; Steindal et al., 2013), turnip (Francisco et al., 2012) and watercress (Engelen-Eigles et al., 2006). Accessions of the model plant Arabidopsis thaliana (L.) Heynh. present a natural variation in their response to growth temperature (Hasdai et al., 2006; Lefebvre et al., 2009; Zhen and Ungerer, 2008) and in their glucosinolate composition (Kliebenstein et al., 2001a). Therefore different A. thaliana accessions were used in our study to investigate the effect of the growth temperature on glucosinolate levels. Another important aspect in the context of plant adaptation to changing conditions that has gained attention lately is the question whether plants possess a transgenerational stress memory, what the underlying mechanisms might be and how such a memory might affect plant traits (Hauser et al., 2011; Heard and Martienssen, 2014; Holeski et al., 2012). Little is known about the transgenerational effect of abiotic (stress) conditions on the levels of secondary metabolites in plants. Recently, the effect of drought stress in the parent generation on the levels of glucosinolates in the € ve & D. offspring was reported for Boechera stricta (A. Gray) A. Lo €ve (Alsdurf et al., 2013). Similarly, in our experiments we did not Lo

only assess the effect of different growth temperatures on glucosinolates in A. thaliana for one generation, but also for subsequent generations (Fig. 1) experiencing a different temperature than the parents.

2. Results 2.1. Effects of growth temperature on seed germination of Arabidopsis thaliana accessions The germination of A. thaliana F1 plants was assessed after seven days (Fig. 1 and Supplementary Fig. 1). The accessions C24, Col-0, Cvi and Ler had similar germination rates, ranging from 75 to 91%, at 21 C, while Ru-0 showed much lower germination at this temperature than the other accessions. At 15 C, the germination rate of all ﬁve accessions was reduced compared to 21 C, but to different extents. While the germination rate of Col-0 was only slightly affected, Cvi showed a germination that was reduced to 22%. Ru-0 was most affected in germination by the temperature, with less than 4% of Ru-0 seeds having germinated after 4 day at 15 C. None of the seeds had germinated at 9 C after seven days. Eventually, germination rates at 9 C and 15 C reached levels

Fig. 1. Schematic overview of the setup for growth experiments of the ﬁve A. thaliana accessions at three growth temperatures described in this study, indicating at what generation plants were monitored for seed germination and ﬂowering or tissue was harvested for glucosinolate analysis or transcriptional proﬁling. Numbers in brackets refer to sections of the Results part.

Please cite this article in press as: Kissen, R., et al., Effect of growth temperature on glucosinolate proﬁles in Arabidopsis thaliana accessions, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.06.003

R. Kissen et al. / Phytochemistry xxx (2016) 1e13

ranging from 66 to 98% (data not shown). Seed germination was also assessed in F2, twenty-two days after sowing (Supplementary Fig. 2). Analysis of the germination rates in F2 by ANOVA revealed an effect of the accession, the growth temperature in F1, the growth temperature in F2 and the interaction between accession and growth temperature in F1. All accessions had reduced germination when F1 seeds from 9 C were sown out again at 9 C in F2 (Supplementary Fig. 2A). C24 and Cvi F1 seeds from plants grown at 9 C had also low germination rates at 15 C and 21 C in F2 (Supplementary Fig. 2A). Cvi showed the same behaviour for F1 seeds obtained at 15 C (Supplementary Fig. 2B). The accession Ru-0 showed a somewhat peculiar behaviour as F1 seeds from plants grown at 9 C had a high germination rate in F2 at 15 C, but low rates at 9 C and 21 C (Supplementary Fig. 2A), which was different from the results in the F1 for this accession (Supplementary Fig. 1). When F2 seeds of C24 and Col-0 plants grown either for two generations at 9 C or for two generations at 21 C in the phytotron were sown on in vitro culture medium and grown at 9 C or 21 C, seeds from 9 C had lower germination rates than seeds from 21 C for both accessions (Supplementary Fig. 3). The temperature at which the seeds were germinating did not have a clear effect. A reduced germination of C24 seeds when previously grown at 9 C as seen in the F2 on soil (Supplementary Fig. 2) was not observed in the F3 on in vitro medium (Supplementary Fig. 3). 2.2. Effects of growth temperature on ﬂowering time of Arabidopsis thaliana accessions During growth of the F1 plants, ﬂowering was monitored by registering the percentage of plants that had bolted at each of ﬁve dates (Supplementary Table 1). As expected the ﬂowering time depended on the temperature, with bolting occurring earlier at higher temperatures. All accessions reached 100% ﬂowering 36 days after sowing when they were growing at 21 C. Bolting was retarded when plants were grown at 15 C, with three out of ﬁve accessions reaching 100% bolting at day 42. Only Ru-0 and Cvi were ﬂowering just partly, both showing almost no bolting at day 36. While Ru-0 reached over 90% ﬂowering at day 42, the percentage of ﬂowering for Cvi did not exceed 28%. Plants grown at 9 C bolted even later, with four accessions only bolting after day 52. Only in C24 did bolting at 9 C occur distinctly earlier and reach 100% at the last time point that was monitored. Analysis of the ﬂowering by ANOVA did not reveal any statistical signiﬁcance among the accessions and growth temperatures at F1 at the last ﬂower registration date. 2.3. Effects of growth temperature on glucosinolate proﬁles of Arabidopsis thaliana accessions over several generations Different experiments were performed in order to monitor the effect of the temperature during the growth period of A. thaliana plants on their glucosinolate proﬁle (Fig. 1). In this section results from offspring grown at the same temperature as the parent generation(s) are reported. 2.3.1. “2nd generation”-experiment First, glucosinolate proﬁles were determined in shoot tissue of offspring plants (hereafter called F2) grown in a phytotron at one of three different temperatures (9 C, 15 C or 21 C). These F2 plants were offspring from a parental generation (called F1) grown previously in a phytotron at the same temperature, i.e. the F1-F2 temperature regimes were either 9 C-9 C, 15 C-15 C or 21 C-21 C (Fig. 2). MANOVA and GLM analysis showed signiﬁcant effects of accession, temperature and interaction between accession and

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temperature. The four A. thaliana accessions C24, Col-0, Ler and Ru0 showed different behaviours towards growth temperatures. For three accessions shoot tissue of plants growing at 15 C had generally higher amounts of glucosinolates than those growing at 21 C (Supplementary Fig. 4). In Ru-0 all glucosinolates, except 3methylsulﬁnylpropylglucosinolate (3MSP), showed higher levels at 15 C than at 21 C (Fig. 2D). Similarly, ﬁve out of eight aliphatic glucosinolates of C24 were present in higher amounts at 15 C than at 21 C (Fig. 2A). Other glucosinolate levels in C24 were not signiﬁcantly different between 15 C and 21 C but showed a similar trend, except for 4-methoxyindol-3-ylmethylglucosinolate (4MeO-I3M) which was the only glucosinolate with higher amounts at 21 C. Glucosinolate levels in C24 seemed to be the lowest at 9 C, but this should be interpreted with caution as only one 9 C-9 C biological replicate could be analysed for this accession (Fig. 2A). In Ler the growth temperature did not signiﬁcantly affect the levels of most glucosinolates, although there was a trend that the highest levels were observed at 15 C, followed by 21 C and 9 C (Fig. 2C). Only Col-0 seemed to diverge from this general trend, as six of the eight detected glucosinolates signiﬁcantly showed the highest level in plants grown at 9 C, while they did not differ in plants grown at 15 C or 21 C (Fig. 2B). Also, under these conditions aliphatic glucosinolates were more affected by growth temperatures than indolic glucosinolates in C24 and Col-0, while the fold changes between these glucosinolate groups were similar in Ler and Ru-0 (data not shown). 2.3.2. “3rd generation”-experiment In a second experimental setup, the offspring (F3) of Col-0 and C24 plants grown over two generations (F1 and F2) at either 9 C or 21 C in a phytotron were grown in vitro under the same temperatures and glucosinolate analysis was performed (compare 9 C9 C-9 C with 21 C-21 C-21 C Fig. 3). MANOVA showed signiﬁcant effects of accession and temperature, as well as interaction between accession and temperature. For both accessions total glucosinolate levels were higher in shoot tissue of plants growing at 9 C than in those growing at 21 C. These differences were signiﬁcant for Col-0 but not for C24 (Supplementary Fig. 5). At the level of individual glucosinolates in C24 the two aliphatic glucosinolates 4-methylsulﬁnylbutylglucosinolate (4MSB) and 7methylthioheptylglucosinolate (7MTH) and the indolic glucosinolate 4MeO-I3M were higher at 21 C (Fig. 3A). None of the other glucosinolates were signiﬁcantly affected in C24 by the difference in temperatures. In Col-0 three of the ﬁve detected aliphatic glucosinolates [i.e. 4MSB, 7-methylsulﬁnylheptylglucosinolate (7MSH) and 8-methylsulﬁnyloctylglucosinolate (8MSO)] and I3M were present in higher amounts at 9 C than at 21 C (Fig. 3B). The indolic glucosinolate 4MeO-I3M and the aliphatic glucosinolate 4methylthiobutylglucosinolate (4MTB) had signiﬁcantly lower levels in Col-0 shoot tissue grown at 9 C than at 21 C. This experiment where F3 plants were grown in vitro at either 9 C or 21 C was repeated once and gave similar results as to the temperature effect on glucosinolate proﬁles, but with more changes revealed as signiﬁcant for C24 (Supplementary Fig. 6 and Supplementary Fig. 7). 2.4. Effects of growth temperature on glucosinolate-related gene expression proﬁles Plant material harvested from Col-0 and C24 shoot tissue during the “3rd generation”-experiment described above were submitted to transcriptional proﬁling by microarray to assess the effect of growth temperature on (1) glucosinolate-related gene expression in particular and (2) gene expression proﬁles in general in these two accessions.

Please cite this article in press as: Kissen, R., et al., Effect of growth temperature on glucosinolate proﬁles in Arabidopsis thaliana accessions, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.06.003

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R. Kissen et al. / Phytochemistry xxx (2016) 1e13

Fig. 2. Glucosinolates (expressed as m mol g1 of fresh weight) in F2 shoot tissue of the A. thaliana accessions C24 (A), Col-0 (B), Ler (C) and Ru-0 (D) when grown in a phytotron for two subsequent generations at the same temperature (i.e. F1-F2 either 9 C-9 C, 15 C-15 C or 21 C-21 C). When differences in glucosinolate levels among the different temperatures within each accession were revealed as signiﬁcant, these are indicated by different letters (n ¼ 4 except n ¼ 1 for C24 9 C-9 C and n ¼ 3 for Ru-0 15 C-15 C).

As temperatures of around 21 C are commonly used experimental growth temperatures for A. thaliana, this could be considered as a standard (even though not necessarily natural) growth temperature. Hence, the effect of temperature on gene expression in our experiment would be a long term cold response inﬂicted by growing the plants at 9 C. Growing plants at 9 C seemed to affect a larger number of genes involved in glucosinolate biosynthesis in the C24 accession than in Col-0 (Table 1). The difference between accessions was particularly striking for the biosynthesis of aliphatic glucosinolates. Most of the genes in this pathway showed a moderate increase in expression levels in C24 plants at 9 C, but four genes were upregulated more than two-fold: FMOGS-OX1 (At1g65860), FMOGS-OX4 (At1g62570), MYB28 (At5g61420) and MYB29 (At5g07690). In Col-0 fewer genes were affected and only FMOGS-OX4 (At1g62570) was highly upregulated in plants grown at 9 C. In C24 two genes were downregulated at 9 C: AOP2 (At4g03060) and GGP1 (At4g30530). In Col0 four genes were downregulated, including GGP1 (At4g30530) and MAM3 (At5g23010). In contrast to the aliphatic glucosinolate pathway, most of the genes of the indolic glucosinolate pathway showed lower expression levels in C24 when grown at 9 C compared to 21 C. These included the transcription factors MYB51/HIG1 (At1g18570) and MYB122 (At1g74080), as well as key genes of the ﬁrst committed steps of this pathway such as CYP79B2 (At4g39950), CYP79B3 (At2g22330), CYP83B1 (At4g31500) and those involved in the

conversion of I3M into 4MeO-I3M [CYP81F2 (At5g57220), IGMT1 (At1g21100), IGMT2 (At1g21120)]. All of the above mentioned genes, except IGMT1, were also downregulated at the lower temperature for Col-0. Notable exception in indolic glucosinolate pathway was CYP81F3 (At4g37400) which was highly upregulated in both C24 and Col-0 at 9 C. The expression of genes encoding enzymes involved in glucosinolate transport and degradation was also affected by a difference in growth temperature, again with a stronger response in C24 than in Col-0. These genes all showed lower expression levels at 9 C compared to 21 C and included the glucosinolate transporter GRT1 (At3g47960), the typical TGG1/2 (At5g26000/At5g25980) and atypical PEN2 (At2g44490) myrosinases, several nitrile-speciﬁer proteins (NSPs) and nitrilases (NITs).

2.5. Transgenerational effect of growth temperature on glucosinolate proﬁles We have shown in Section 2.3. that glucosinolate levels changed in plant tissue exposed to different growth temperatures. In addition, we wanted to assess whether the growth temperature in one generation had an effect on glucosinolate proﬁles of offspring in the subsequent generation (i.e. from F1 to F2 and from F2 to F3). To detect a potential transgenerational effect of growth temperature on glucosinolates, different experimental setups were used.

Please cite this article in press as: Kissen, R., et al., Effect of growth temperature on glucosinolate proﬁles in Arabidopsis thaliana accessions, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.06.003

R. Kissen et al. / Phytochemistry xxx (2016) 1e13

Fig. 3. Glucosinolates (expressed as m mol g1 of fresh weight) in F3 shoot tissue of the A. thaliana accessions C24 (A) and Col-0 (B) grown in vitro at either 9 C or 21 C from seeds of plants grown in a phytotron for two subsequent generations at the same temperature, generating the four cases F1-F2-F3: 9 C-9 C-9 C, 9 C-9 C-21 C, 21 C21 C-9 C and 21 C-21 C-21 C. When differences in glucosinolate levels among the different temperatures within each accession were revealed as signiﬁcant, these are indicated by different letters (n ¼ 3).

2.5.1. “2nd generation”-experiment First we investigated a potential transgenerational effect of a temperature shift from F1 to F2 by growing C24 and Col-0 F2 plants in vitro at 21 C, the seeds having been obtained from F1 plants grown at either 9 C or 21 C in the phytotron. MANOVA analysis showed effects of accession, temperature in F1 and an interaction between accession and temperature in F1 on glucosinolate proﬁles. The level of 8MTO in C24 shoots was signiﬁcantly higher when previously grown at 21 C compared to 9 C (Fig. 4A; Supplementary Fig. 8). Interestingly, six of eight detected glucosinolates were present in signiﬁcantly higher levels in Col-0 F2 plants where the F1 had been grown at 21 C instead of 9 C (Fig. 4B). 2.5.2. “3rd generation”-experiments Next, the presence of a transgenerational effect of growth temperature on glucosinolates was tested in the F3. For this the offspring of A. thaliana accessions grown in a phytotron for two generations either at 9 C, 15 C or 21 C was grown on soil in a growth room at 21 C. The analysis of glucosinolate levels in shoot tissue of these F3 plants did not reveal a signiﬁcant inﬂuence of growth temperatures in previous generations on glucosinolate levels in the subsequent generation (MANOVA), but there was an interaction between accession and temperature in the previous generations (Fig. 5; Supplementary Fig. 9). C24 plants showed

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higher levels of 3MTP and 8MTO, or I3M, when F1 and F2 were grown at 15 C compared to 9 C or 21 C, respectively (Fig. 5A). A signiﬁcant change of the level of 8MTO was also observed in Ru-0, with more 8MTO in the shoot tissue of plants whose F1 and F2 had been grown at 21 C than at15 C (Fig. 5D). Overall, aliphatic, indolic and total glucosinolate levels were slightly (but not signiﬁcantly) higher in plants previously grown on 15 C than in plants grown on 9 C or 21 C for the C24, Col-0 and Ler accessions (Supplementary Fig. 9). To verify these results the experiment was repeated once under similar conditions with C24 and Col-0 seeds from plants previously grown for two generations at either 9 C or 21 C. The effect of previous temperatures on glucosinolates in soil grown C24 was conﬁrmed to be small and not signiﬁcant by MANOVA, although somewhat higher levels of the short chain aliphatic glucosinolate 2P and the long chain aliphatic glucosinolates 6methylsulﬁnylhexylglucosinolate (6MSH), 7MSH and 8MSH in plants previously grown at 21 C were revealed as signiﬁcant by GLM analysis (Fig. 6A; Supplementary Fig. 10A). As observed during the ﬁrst experiment different temperatures during the two previous generations did not have any effect on any of the detected glucosinolates of Col-0 F3 plants growing on soil at 21 C (Fig. 6B; Supplementary Fig. 10B). As the two experiments using soil-grown F3 plants showed slightly different results for glucosinolate levels, the absence/ presence of a transgenerational effect of a temperature shift from F2 to F3 was also tested under more controlled in vitro conditions. In addition to more homogenous growth conditions, this allowed us to test temperatures of 9 C and 21 C. Hence C24 and Col-0 F3 plants were grown at a different temperature than that during the two ﬁrst generations (i.e. F1-F2-F3 of 9 C-9 C-21 C and 21 C-21 C9 C). HPLC analysis of glucosinolates under these conditions showed no (or little) signiﬁcant temperature effect in two independently run experiments (Fig. 3, Supplementary Fig. 5). Indeed, growth in the phytotron at 9 C or 21 C during previous generations did not inﬂuence glucosinolate proﬁles in in vitro plant shoots of the third generation, in opposition to the effect of the growth temperature within a generation seen before (Fig. 3) and to small changes observed in soil-grown plants (Figs. 5 and 6). 3. Discussion Arabidopsis thaliana is a chilling-tolerant plant, able to grow at temperatures as low as 5 C (Tokuhisa et al., 1997), although the chilling growth temperatures used in our study had strong effects on the phenological and biochemical parameters that we assessed. In addition, the effects we saw were accession-dependent. It is well known that a natural variation in the response to growth temperature exists in A. thaliana (e.g. Balasubramanian et al., 2006; Box et al., 2015; Edwards et al., 2005; Hannah et al., 2006; Hasdai et al., 2006; Lefebvre et al., 2009; Zhen and Ungerer, 2008). The accessions in our study were chosen as they present genetic and phenotypic variation, as well as geographical variation in their original habitat and differ in their glucosinolate composition. 3.1. Effect of growth temperature on seed germination For four of the accessions 75e91% of the seeds had germinated after 4 day at 21 C. Only Ru-0 presented a lower germination rate at this temperature, although the reason for this is not known. All accessions showed a delay in germination at 15 C compared to 21 C, with Ru-0 again showing lowest germination after 4 days. But also the germination of Cvi at this time point seemed to be more affected by lower temperature than the three other accessions. The delay in germination was even stronger at 9 C as no

Please cite this article in press as: Kissen, R., et al., Effect of growth temperature on glucosinolate proﬁles in Arabidopsis thaliana accessions, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.06.003

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Table 1 Genes involved in glucosinolate biosynthesis, transport and degradation whose expression was signiﬁcantly (adjusted P-value < 0.05) changed in C24 and/or Col-0 in response to the growth temperature. Ratios 9 C/21 C for each accession are those of plants grown for three generations at 9 C compared to those grown at 21 C. Gene ID

Gene description

Aliphatic glucosinolate pathway At3g19710 BCAT4 At4g12030 BAT5 At5g23010 MAM1 At5g23020 MAM3 At4g13430 IIL1 At3g58990 IPMI1 At1g31180 IPMDH3 At3g49680 BCAT3 At1g16410 CYP79F1 At4g13770 CYP83A1 At3g03190 GSTF11 At1g78370 GSTU20 At4g30530 GGP1 At2g20610 SUR1 At1g24100 UGT74B1 At2g31790 UGT74C1 At1g74090 SOT18 At1g65860 FMO GS-OX1 At1g62540 FMO GS-OX2 At1g62560 FMO GS-OX3 At1g62570 FMO GS-OX4 At4g03060 AOP2 At2g25450 GS-OH At5g61420 HAG1/MYB28 At5g07700 HAG2/MYB76 At5g07690 HAG3/MYB29 Indolic glucosinolate pathway At4g39950 CYP79B2 At2g22330 CYP79B3 At4g31500 CYP83B1 At2g30870 GSTF10 At4g30530 GGP1 At2g20610 SUR1 At1g24100 UGT74B1 At1g74100 SOT16 At4g37430 CYP81F1 At5g57220 CYP81F2 At4g37400 CYP81F3 At1g21100 IGMT1 At1g21120 IGMT2 At1g21110 O-methyltransferase At1g21130 O-methyltransferase At5g60890 MYB34 At1g18570 HIG1/MYB51 At1g74080 MYB122 Glucosinolate transport At3g47960

GRT1

Glucosinolate degradation At5g26000 TGG1 At5g25980 TGG2 At2g44490 PEN2 At1g54040 ESP At3g16400 NSP1 At2g33070 NSP2 At5g48180 NSP5 At3g44300 NIT2 At3g44320 NIT3

C24 9 C/21 C

Col-0 9 C/21 C

log2 fold-change

adj.P-value

0.502 0.720 0.216 0.557 0.699 0.661 0.466 0.565 0.935 0.611 0.563 0.715 0.882

0.0348 0.0040 0.0407 0.0013 0.0125 0.0004 0.0009 0.0313 0.0002 0.0256 0.0287 0.0002 0.0069

0.460 0.913 1.029 0.609 0.982 1.289 0.750 0.918 1.218 0.812 1.731

0.0017 0.0007 0.0003 0.0146 0.0026 1.48E-07 0.0115 0.0038 1.48E-07 0.0021 0.0002

1.561 1.895 1.517 1.086 0.715 0.882

6.80E-06 5.02E-07 8.86E-07 7.71E-06 0.0002 0.0069

0.314 0.407 2.911 2.319 1.652 2.283 1.343 2.413

0.0134 0.0067 3.30E-06 1.57E-08 2.10E-05 6.42E-07 0.0010 5.50E-07

3.393 2.147

7.07E-08 1.50E-06

1.003

3.82E-05

0.395

0.0139

1.195

2.66E-06

0.985 0.823 0.995 2.893

0.0115 0.0161 0.0090 0.0009

germinated seeds were detected for any of the accessions after 4 days, but eventually normal germination rates were also attained for this temperature. The effect of parental temperature preconditioning on germination of the next generation (F2) was also assessed. Preconditioning of F1 at 21 C led to equally high germination rates for

log2 fold-change

adj.P-value

0.485

0.0421

1.009

0.0212

1.361

2.62E-05

0.968

0.0002

2.111

0.0003

0.473

0.0244

0.780

0.0098

0.630 1.228 0.851 0.799 1.361

0.0111 0.0004 0.0010 0.0040 2.62E-05

0.968 0.505

0.0002 0.0334

1.874 1.665

0.0020 0.0001

0.787 0.985 0.926 0.600 1.934

0.0093 0.0018 0.0094 0.0195 0.0003

0.688

0.0027

0.787 0.949 0.652

0.0045 0.0007 0.0411

1.036

0.0123

0.591

0.0141

all accessions and all temperatures in F2 while the ﬁve accessions showed a different response in germination to the preconditioning at lower temperatures. This is in accordance with a previous survey of 73 accessions, where for most accessions preconditioning at 14 C led to a lower and more diverse germination rate at 10, 18 and 26 C than a preconditioning at 22 C (Schmuths et al., 2006).

Please cite this article in press as: Kissen, R., et al., Effect of growth temperature on glucosinolate proﬁles in Arabidopsis thaliana accessions, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.06.003

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9 C than the other accessions. 3.3. Effect of long-term chilling on glucosinolate levels

Fig. 4. Glucosinolates (expressed as m mol g1 of fresh weight) in F2 shoot tissue of the A. thaliana accessions C24 (A) and Col-0 (B) grown in vitro at 21 C from seeds of F1 plants grown in a phytotron at either 9 C or 21 C, generating the two cases F1-F2: 9 C-21 C and 21 C-21 C. When differences in glucosinolate levels between the two cases within each accession were revealed as signiﬁcant, these are indicated by different letters (n ¼ 4).

Preconditioning at 15 C and 9 C led to the strongest reduction in germination in F2 for the accessions Cvi and C24. However, when C24 seeds preconditioned at 9 C for two generations were sown on in vitro medium and grown at 9 C in the F3 a greatly reduced germination was no longer observed.

3.2. Effect of growth temperature on ﬂowering The three different growth temperatures also affected the time plants needed to reach the bolting stage. F1 plants of all ﬁve accessions bolted earlier at higher temperatures (Supplementary Table 1), with most of them attaining full ﬂowering at 36, 42 and later than 70 days after sowing at 21 C, 15 C and 9 C, respectively. That ﬂowering time is delayed by low growth temperatures is known (Balasubramanian et al., 2006; Hasdai et al., 2006). Our results differ however from a study of 74 A. thaliana accessions where those from colder locations [e.g. Ler and Ru-0 (Ruppachtal, Germany) in our case] developed much faster at 22 C than at 14 C, but not those from warmer locations [e.g. Cvi (Cape Verde Islands) in our case](Hoffmann et al., 2005). Interestingly, we also observed that the ﬂowering percentage for Cvi at 15 C was only 28% at day 70 and that C24 (Coimbra, Portugal) showed an earlier ﬂowering at

In a ﬁrst stage, to assess the long term effect of growth temperatures on glucosinolates, different A. thaliana accessions were grown at constant standard (21 C), moderate chilling (15 C) and pronounced chilling temperatures (9 C) using different experimental set-ups. Natural variation in the metabolic response of A. thaliana accessions to temperature has previously been shown (Cook et al., 2004; Hannah et al., 2010), but we are not aware of any report that showed a natural variation in the response to temperature affecting glucosinolate levels and proﬁles. The data presented here show that temperature during the growth period affected the glucosinolate composition of A. thaliana rosettes in an accessiondependent manner. Total glucosinolate levels were higher in plants grown at 15 C than at 21 C increasing more than two-fold for C24, Ler and Ru0 and 1.6-fold (not signiﬁcant) for Col-0 (Supplementary Fig. 4). Total glucosinolate levels were also higher at 9 C than at 21 C when Col-0 and C24 were grown in vitro, with a slightly bigger foldchange for Col-0 than for C24 (Supplementary Figs. 5 and 7). Previous studies to evaluate short-term and/or long-term effects of temperature on glucosinolate content and composition performed on other Brassicaceae species report varying results. Similar to our observations for the total glucosinolate content, EngelenEigles et al. (2006) reported higher levels of gluconasturtiin (2phenethylglucosinolate) in water cress (Nasturtium ofﬁcinale) when grown at 10 C or 15 C than at 20 C or 25 C. A signiﬁcant effect of temperature was also reported for Brassica oleracea L. where total glucosinolate contents in leaves increased when plants were moved to 32 C or 12 C compared to when they were kept at 22 C (Charron and Sams, 2004). Similarly, in broccoli sprouts (B. oleracea L. var. italica L. cv. DeCicco) higher glucosinolate levels were obtained at constant temperatures below (11 and 16 C) and above (29 and 33 C) normal growth conditions (i.e. 21.5 C) (Pereira et al., 2002). Guo et al. (2014) reported however higher levels of the major glucosinolate, glucoraphanin (4MSB), in broccoli sprouts grown at 25 C than at 20 C or 30 C. Factors such as age of plant material or growth conditions, but also cultivar-dependent responses, might explain these differences within the same species. In B. oleracea L. var. capitata L. (pointed cabbage) Rosa and Rodrigues (1998) did not observe signiﬁcant differences in total levels of leaf glucosinolates when comparing two week old seedlings grown at 20 C or 30 C for two days, while they observed higher glucosinolate levels at the higher temperature in roots. Charron and Sams (2004) on their side reported higher total glucosinolate levels in B. oleracea roots at 22 C than at 32 C. Also several ﬁeld trials have been run (e.g. Cartea et al., 2008; Rosa et al., 1996; Velasco et al., 2007), but to assess the effect of temperature on glucosinolates under these setups is difﬁcult due of the interference of e.g. other climatic factors, soil type, plant developmental stage, insect and pathogen attack. In addition, it has been shown that a large portion of the metabolomes of leaves is differently affected in situations where plants are shifted from normal to cold temperature or where plants develop at low temperature (Gray and Heath, 2005). Hence, there may be quantitative and qualitative differences in the temperature effect on glucosinolates between studies where plants are moved from a common temperature to different temperatures at one time point in their growth (e.g. Rosa and Rodrigues, 1998; Schonhof et al., 2007a) and those where plants are grown at constant temperatures throughout their growth period (Guo et al., 2014; Pereira et al., 2002), the latter setup being used also in our experiments. Similarly to previous reports on glucosinolate proﬁles of

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Fig. 5. Glucosinolates (expressed as m mol g1 of fresh weight) in F3 shoot tissue of the A. thaliana accessions C24 (A), Col-0 (B), Ler (C) and Ru-0 (D) grown on soil at 21 C from seeds of plants grown in a phytotron for two subsequent generations at 9 C, 15 C or 21 C generating the three cases F1-F2-F3: 9 C-9 C-21 C, 15 C-15 C-21 C and 21 C-21 C21 C. When differences in glucosinolate levels among the different temperatures within each accession were revealed as signiﬁcant, these are indicated by different letters (n ¼ 4).

A. thaliana (Brown et al., 2003; Kliebenstein et al., 2001a; Petersen et al., 2002) aliphatic glucosinolates were much more abundant than indolic glucosinolates in the shoot tissue we analysed, and changes in aliphatic glucosinolates were therefore responsible for the differences in total glucosinolate levels. In general, aliphatic glucosinolate levels were more affected by the growth temperature, although changes in indolic glucosinolate levels showed a similar trend to those of aliphatic glucosinolates in most cases (Supplementary Figs. 4, 5 and 7). This is in opposition to the situation in broccoli sprouts observed by Pereira et al. (2002), where total aliphatic glucosinolates and indolic glucosinolates were differently affected by temperature. Charron and Sams (2004) reported that aliphatic but not indolic glucosinolate levels varied with temperature in B. oleracea leaves. One of the reasons why the accessions were chosen for our experiments were their different glucosinolate proﬁle. Some glucosinolates, such as 3-hydroxypropylglucosinolate (3OHP), 7MTH and 3B were just detected in one accession or under certain growth conditions. Others, such as the three aliphatic glucosinolates, 3MSP, 8MTO and 8MSO were common to all or most of the accessions we used. Changes in 3MSP and 8MTO levels showed the same trend in response to temperature in all three accessions where these glucosinolates were detected, although the amplitude differed (Fig. 2). I3M and 8MSO levels were (signiﬁcantly) higher at 15 C than at 21 C in all four accessions, but the effect of 9 C on these glucosinolates was different between Col-0 and Ler. Also 4MeO-I3M was

differently affected at 9 C in Col-0 and Ler (Fig. 2). The glucosinolate 2P was present in both C24 and Ru-0 leaves, as a minor and the major glucosinolate respectively, and its levels are higher at 15 C than at 21 C in both (Fig. 2). 4MSB was the major glucosinolate in Col-0 rosettes but only lowly abundant in C24 when grown in vitro and was in addition differently affected in these accessions when plants were grown at either 9 C or 21 C (Fig. 3; Supplementary Fig. 6). Hence, our data indicate that the effect of growth temperature on individual glucosinolates is highly dependent on the accession. It is thus not surprising that differences are also observed when looking at the temperature effect on a given glucosinolate in different species. As mentioned above, 4MSB was more abundant in broccoli sprouts when grown at 25 C than at 20 C or 30 C (Guo et al., 2014). In B. oleracea leaves the level of 4MSB was lower at 22 C than at 12 C and 32 C (Charron and Sams, 2004) opposite to what we observed for C24 at 9 C and 21 C (Fig. 3; Supplementary Fig. 6). Glucosinolates are not the only secondary metabolites whose levels change with the growth temperature in an accessiondependent manner in A. thaliana. Anthocyanin levels increased in Col-0 and Ler with lower temperatures (14 C and 6 C compared to 22 C) while C24 exhibited the opposite behaviour (Hasdai et al., 2006). On a more general metabolite level, Hannah et al. (2006) revealed differences in metabolite changes after 14 days acclimation at 4 C among A. thaliana accessions. We also observed some notable differences within accessions in

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Fig. 6. Glucosinolates (expressed as m mol g1 of fresh weight) in F3 shoot tissue of the A. thaliana accessions C24 (A) and Col-0 (B) grown on soil at 21 C from seeds of plants grown in a phytotron for two subsequent generations either at 9 C or at 21 C generating the two cases F1-F2-F3: 9 C-9 C-21 C and 21 C-21 C-21 C. When differences in glucosinolate levels among the different temperatures within each accession were revealed as signiﬁcant, these are indicated by different letters (n ¼ 3).

different experimental setups, such as the differential effect of temperature on 4MTB and 4MeO-I3M in Col-0 when grown on soil or in vitro (compare Figs. 2 and 3). This points towards the inﬂuence of parameters other than temperature, such as growth stage, light, photoperiod and nutrition. These have indeed been shown to affect glucosinolates in several species (e.g. Alnsour et al., 2013; Brown et al., 2003; Charron and Sams, 2004; Engelen-Eigles et al., 2006). 3.4. Expression levels of glucosinolate-related genes after long-term chilling In order to see if and in how far temperature effects on glucosinolate levels could be related to differences in gene expression levels, transcriptional proﬁling was performed on C24 and Col0 grown in vitro at 9 C and 21 C. The difference in growth temperature affected a larger number of glucosinolate biosynthesis genes in C24 than in Col-0 (Sønderby et al., 2010). The difference in numbers was especially striking for the biosynthesis of aliphatic glucosinolate (Table 1). Here, genes involved in all major steps of the core pathway were affected in C24 while only a few changed expression in Col-0. In addition, for C24 all except two showed increased expression levels at 9 C. The genes encoding three MYB transcription factors called HAG1 to HAG3 (for high aliphatic

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glucosinolate) identiﬁed as positive regulators of aliphatic glucosinolates (Gigolashvili et al., 2007b, 2008) were thus strongly induced at 9 C. Also four of ﬁve members of a subclade of ﬂavincontaining monooxygenases (FMOGS-OX) that catalyse the conversion of methylthio- to methylsulﬁnyl-alkyl glucosinolates (Hansen et al., 2007; Li et al., 2008), were among the most highly induced genes in C24. One of the two genes in the aliphatic glucosinolate pathway with lower expression levels in C24 at 9 C encodes the 2oxoglutarate dependent dioxygenase AOP2 (At4g03060), which is responsible for the further conversion of short-chain methylsulﬁnyl-alkyl glucosinolates to alkenyl glucosinolates (Kliebenstein et al., 2001b). These changes in gene expression levels in C24 at 9 C could therefore be expected to lead to increased levels of total aliphatic glucosinolates, especially that of methylsulﬁnyl- at the expense of methylthio-alkyl and alkenyl glucosinolates at 9 C. This was however only observed to a certain extent at the glucosinolate level with e.g. higher total aliphatic and higher 7MSH/7MTH and 8MSO/8MTO ratios (Fig. 3). Regarding genes involved in the indolic glucosinolate pathway, most of these showed lower expression levels at the lower temperature in both C24 and Col-0. These included the MYB transcription factors MYB51/HIG1 (for high indolic glucosinolate 1), a positive regulator of indolic glucosinolate biosynthesis (Gigolashvili et al., 2007a) and the cytochrome P450 cytochromes CYP79B3 (At2g22330) and CYP83B1 (At4g31500) catalysing the ﬁrst two committed step of this pathway (Hansen et al., 2001; Mikkelsen et al., 2003). Also the cytochrome P450 CYP81F2 (At5g57220) and the O-methyltransferase IGMT2 (At1g21120) catalysing the two steps that convert I3M to 4MeO-I3M (Pfalz et al., 2011) were downregulated at 9 C. Interestingly, the gene encoding CYP81F3 (At4g37400), which catalyses the same reaction as CYP81F2 (At5g57220), was highly upregulated in both C24 and Col0, indicating that these two genes might be differently regulated by abiotic stress conditions. Notable differences in gene expressions between C24 and Col-0 were the fact that the positive regulator of indolic glucosinolate biosynthesis MYB122 (At1g74080) (Gigolashvili et al., 2007a) and the O-methyltransferase IGMT1 (At1g21100) (Pfalz et al., 2011) were strongly downregulated in C24 but not affected in Col-0. From the overall reduced expression levels of indolic glucosinolate-related genes at 9 C in both accessions, one could expect lower total indolic glucosinolate levels and a lower proportion of 4MeO-I3M at 9 C. However, total indolic glucosinolate levels at 9 C were either not signiﬁcantly or slightly higher compared to 21 C (Supplementary Fig. 5), which was due to the fact that I3M levels were higher while the levels of 1MeO-I3M and 4MeO-I3M were lower. Genes encoding enzymes that catalyse reactions common to both indolic and aliphatic glucosinolate pathways, such as GGP1 (At4g30530), SUR1 (At2g20610), UGT74B1 (At1g24100) were either not, up- or downregulated, making it difﬁcult to interpret their effect on the metabolic outcome. In summary, there was only a limited correlation between changes in biosynthesis gene expression levels and glucosinolate levels under pronounced chilling growth conditions. This behaviour has been observed for other metabolic processes under cold acclimation (Kaplan et al., 2007). Also, an uncoupling between glucosinolate transcript and metabolite levels has been reported previously in A. thaliana under other conditions (Moussaieff et al., 2013; Sønderby et al., 2010). In addition to de novo synthesis, changes in glucosinolate concentrations and proﬁles might be due to transport and degradation of glucosinolates. Also for these two processes, the responses were affecting more genes and reached higher fold changes in C24 than in Col-0. Of the two transporters likely involved in the transport of glucosinolates between rosette leaves and roots at certain

Please cite this article in press as: Kissen, R., et al., Effect of growth temperature on glucosinolate proﬁles in Arabidopsis thaliana accessions, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.06.003

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developmental stages (Andersen et al., 2013), GRT1 (At3g47960) was downregulated at 9 C compared to 21 C in both Col-0 and C24. The expression of genes encoding enzymes involved in glucosinolate degradation was also lower at 9 C. Interestingly, different members of the small gene families encoding myrosinases (TGG (Barth and Jander, 2006)), nitrile-speciﬁer proteins (NSP (Kong et al., 2012)) and nitrilases (NIT (Vorwerk et al., 2001)) were affected in Col-0 and C24. The atypical myrosinase PEN2 (At2g44490) (Bednarek et al., 2009) on the other hand, was downregulated in both accessions. As the turn-over of glucosinolates in intact plant tissue is still under debate, it is difﬁcult to ascertain whether these changes in gene expression contributed to overall higher glucosinolate levels at 9 C. 3.5. Transgenerational effect of growth temperature on glucosinolates Growing A. thaliana accessions at different constant temperatures had a marked effect on glucosinolate levels, as discussed above. In addition, our experiments were designed so that we would be able to test a potential transgenerational effect of temperature on glucosinolates under different set-ups (Fig. 1). It has been reported that stressful environments can induce changes (maternal effect and/or epigenetic changes) in the treated plant that can be transmitted to subsequent generations (Hauser et al., 2011; Holeski et al., 2012; Molinier et al., 2006). This may affect a range of phenotypic characteristics of the progeny, although a transgenerational effect may not happen for all stress conditions (Pecinka et al., 2009; Rahavi and Kovalchuk, 2013). Temperature was used as an abiotic stress in several of such studies and shown to €dner et al., 2007; Boyko affect various traits of A. thaliana (e.g. Blo et al., 2010; Lang-Mladek et al., 2010; Migicovsky et al., 2014; Suter and Widmer, 2013; Whittle et al., 2009). Although the progeny of plants grown at different temperatures in our experiments showed some small changes in long chain aliphatic glucosinolates under some experimental conditions, no consistent and unequivocal effect of the parental growth temperature on glucosinolate contents in subsequent generations was revealed in our study. An exception to this were lower levels of six glucosinolates in F2 plants of the Col-0 accession grown in vitro at 21 C when the parent generation had been grown at 9 C instead of 21 C (Fig. 4). As a similar behaviour was not observed in the F3 generation of Col0 when grown in vitro at 21 C (Fig. 3) it remains to be investigated whether the differences in F2 were generation-speciﬁc, related to the particular nature of Col-0 as a lab strain, inﬂuenced by other parameters than temperature speciﬁcally present under in vitro conditions or an interaction of these factors. 4. Conclusions In this study we monitored the effect of three different growth temperatures on glucosinolate levels in A. thaliana accessions through three generations. Growth at moderate (15 C) and pronounced (9 C) chilling temperatures led to important quantitative and qualitative glucosinolate changes at the within-generation level. Although these glucosinolate changes where highly accession-speciﬁc, a general trend of higher glucosinolate levels at the lower growth temperatures was observed. Changes in glucosinolate-related gene expressions also showed accessionspeciﬁc patterns but correlated only poorly with the observed glucosinolate changes. Except for Col-0 after one generation at 9 C, no clear memory effect of chilling growth temperatures on glucosinolate levels of subsequent generations, i.e. at the transgenerational level, was detected in our study.

5. Experimental 5.1. Plant material and chemicals Seeds of the Arabidopsis thaliana accessions C24, Col-0, Cvi, Ler and Ru-0 were obtained from the European Arabidopsis Stock Centre (NASC) and propagated for several generations in our lab. All chemicals unless otherwise stated were purchased from Sigma-Aldrich (Saint Louis, USA). 5.2. Phytotron growth conditions The ﬁve A. thaliana accessions were grown on soil (1/2 peat and 1/2 perlite) for two consecutive years (i.e. 2009 and 2010) in a phytotron under natural light at the Bioforsk Nord-Holt facilities in Tromsø (Norway). Seeds were sown on March 16th, 2009, incubated for 3 day at 4 C and moved to three identical growth rooms where the growth temperature was either kept at 9 C, 15 C or 21 C throughout the growth period. Seeds from this ﬁrst generation (called F1 hereafter) were harvested in bulk at maturity at the end of the 2009 growth period and kept at 4 C until further use. In March 2010 seeds from F1 were sown in the same three growth rooms and grown under the same temperatures as in 2009. For this, seeds from F1 grown at any of the three temperatures were grown at all three temperatures in this second generation (called F2 hereafter; Fig. 1). Shoot tissue was harvested at maturity for glucosinolate analysis. At 9 C the plantlets were so small that rosettes from as many as ﬁve individual plants were pooled. Seeds of F2 plants were harvested in bulk at maturity at the end of the 2010 growth period and kept at 4 C until further use. Germination rates and ﬂowering time in F1 and F2 were monitored as described below. Due to varying germination efﬁciency, different numbers of plants were grown. 5.3. Growthroom conditions Seeds were stratiﬁed for 3 days at 4 C in 0.1% agarose before being sown on soil [5 parts S-JORD (Hasselfors Garden AB, Hasselfors, Sweden): 1 part perlite (LOG, Oslo, Norway)] in controlled growth rooms under an 8 h light/16 h dark photoperiod (100 mmol m2 sec1) at 21 C/18 C (day/night). Two rounds of growth experiments were performed under these conditions. In a ﬁrst round, F2 seeds of C24, Col-0, Ler and Ru0 plants grown for two generations either at 9 C, 15 C or 21 C in the phytotron were sown and grown in the growthroom at 21 C. Cvi was not used due to the limited amount of material. Shoot tissue of this third generation (called F3 hereafter) was harvested after 33 days (4 biological replicates consisting of a pool of 10 plants each) and submitted to glucosinolate analysis. In the second round, F2 seeds of C24 and Col-0 plants grown either for two generations at 9 C or for two generations at 21 C in the phytotron were sown and grown at 21 C. Shoot tissue (F3) was harvested after 29 days (4 biological replicates consisting of a pool of 10 plants each) and submitted to glucosinolate analysis. 5.4. In vitro growth conditions Seeds were surface sterilized by chlorine gas treatment for 3 h and sown on Petri dishes (14 cm diameter; 38 seeds per dish) containing ½ x Murashige and Skoog Basal Salt Mixture (SigmaAldrich) (pH 5.7) supplemented with sucrose (20 g L1) and agar (6 g L1; 1:2 Bactoagar (BD Biosciences): Phytoagar (Duchefa)). Seeds were stratiﬁed by putting the Petri dishes for 4 days at 4 C and then transferred to controlled in vitro growth chambers (Percival E-36LX) and grown under a 16 h photoperiod

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(100 mmol m2 sec1) at either 9 C or 21 C as detailed below. These differences in growth temperatures led to differences in plant development. As the developmental stage of A. thaliana plants impacts on glucosinolates (Brown et al., 2003; Petersen et al., 2002), we opted for a comparison of glucosinolates when plants grown at different temperatures reached the same developmental stage. Therefore plants were harvested at the stage 1.06e1.07 (Boyes et al., 2001), which they reached after 16 days when grown at 21 C and 51 days when grown at 9 C under the described conditions. Shoot tissue was harvested by pooling material from all the plants of a dish and by harvesting separate replicates from 4 dishes. Plant tissue was immediately ﬂash frozen in liquid nitrogen and stored at 80 C until further processing. Two types of growth experiments were performed in vitro. In one, seeds of C24 and Col-0 from F1 plants grown at 9 C and 21 C in the phytotron were sown on in vitro medium and grown at 21 C. The harvested tissue (called F3 hereafter) was submitted to glucosinolate analysis. In the second type of experiment, F2 seeds of C24 and Col-0 plants grown either for two generations at 9 C or for two generations at 21 C in the phytotron were divided into two groups and grown in two identical growth chambers, one being kept at 21 C and one kept at 9 C. The harvested tissue (F3) was used for transcriptional proﬁling by microarray and submitted to glucosinolate analysis. This setup was repeated once and the material was submitted to glucosinolate analysis. 5.5. Phenological observations For F1, 104 seeds were sown out for each of the 15 accession temperature combinations and germination rates were monitored after 7 days. The ﬂowering of F1 plants was monitored by registering the percentage of plants that had bolted at ﬁve dates (31, 36, 42, 52 and 70 days after sowing). For F2, 240 seeds were sown out for each of the accession temperature combinations, except for one combination (Cvi 9 C e> 15 C) due to the reduced amount of seeds harvested from F1. Germination rates were assessed after 22 days. The germination rates of C24 and Col-0 F2 seeds from plants grown either for two generations at 9 C or for two generations at 21 C in the phytotron and sown in vitro were assessed based on a total of 152 seeds per accession temperature combination at the end of the experiment. 5.6. Glucosinolate extraction Plant material (about 100 mg) collected as described in Sections 5.2, 5.3 and 5.4 was freeze-dried in 2 ml Eppendorf tubes before glucosinolate extraction. Sinigrin (2-propenylglucosinolate) was used as glucosinolate standard for Col-0 and Ler samples while benzylglucosinolate was used as glucosinolate standard for C24 and Ru-0 samples. To the freeze-dried plant material 750 ml boiling methanol (80% v/v) was added and incubated for 3 min at 80 C. Tissue disruption was performed on a TissueLyser II (Qiagen, Hilden, Germany) for 1 min at 25 Hz. The extract was incubated again for 3 min at 80 C and then centrifuged at maximum speed in a benchtop centrifuge for 1 min. The supernatant was transferred into a new tube and kept on ice. The pellet was reextracted by adding 750 ml boiling methanol (80% v/v), vortexing and centrifuging at maximum speed in a benchtop centrifuge for 1 min. The supernatant was pooled with the ﬁrst supernatant, 200 ml barium acetate (0.4 M) was added and mixed, and the extract was centrifuged at 4000 g for 10 min. Sephadex DEAE A25 (GE Life Sciences, Uppsala, Sweden) equilibrated in 0.02 M acetate buffer (pH 5) was used to prepare 1 ml-columns and rinsed with H2O. The glucosinolate supernatant

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was loaded on the column and the column was rinsed ﬁrst with H2O, then with 0.02 M acetate buffer (pH 5). Sulfatase (75 ml; Helix pomatia Type H-1 sulfatase) prepared as previously described (Graser et al., 2000) was added to the column, and the column was capped and incubated at room temperature overnight in the dark. Desulfoglucosinolates were eluted in 3 0.5 ml H2O and frozen at 20 C until the next day before being freeze dried for 24 h. The dry extract was resuspended in H2O at 400 ml per mg of dry extract.

5.7. Glucosinolate analysis by HPLC-UV Desulfoglucosinolates (referred to as their corresponding glucosinolates throughout the text) were separated by HPLC (Agilent HPLC 1200 series) on a C-18 reversed phase column (Supelcosil LC18, L i.d. 250 2.1 mm; particle size 3 mm) at 25 C using a H2Oacetonitrile (solvent A-solvent B) gradient (0e2 min 3% B; 2e17 min 3e40% B, 17e22 min 40% B, 22e22.1 min 40e100% B, 22.1e32 min 100% B, 32e32.1 100-3% B, 32.1e60 min 3% B) at a ﬂow rate of 0.3 ml min1. Desulfoglucosinolates were monitored by UV diode array detection, identiﬁed based on retention times and LCMS conﬁrmation of peaks, and quantiﬁed by A229nm relative to the standard and taking into account published response factors (Brown et al., 2003). Depending on the accession, the following aliphatic (desulfo)glucosinolates: 3-methylthiopropylglucosinolate (3MTP), 3-methylsulﬁnylpropylglucosinolate (3MSP), 4-methyl thiobutylglucosinolate (4MTB), 4-methylsulﬁnylbutylglucosino late (4MSB), 6-methylsulﬁnylhexylglucosinolate (6MSH), 7methylthioheptylglucosinolate (7MTH), 7-methylsulﬁnylhepty lglucosinolate (7MSH), 8-methylthiooctylglucosinolate (8MTO), 8methylsulﬁnyloctylglucosinolate (8MSO), 3-hydroxypropylgl ucosinolate (3OHP), 2-propenylglucosinolate (2P), 3butenylglucosinolate (3B), and the three indolic (desulfo)glucosinolates: indol-3-ylmethylglucosinolate (I3M), 1-methoxyindol-3ylmethylglucosinolate (1MeO-I3M), 4-methoxyindol-3ylmethylglucosinolate (4MeO-I3M) were detected. Statistical analysis of glucosinolate data for each individual experiment (tested for normality and equal variance) was done by multivariate analysis of variance (MANOVA), where glucosinolates present in all four accessions were included. Glucosinolates detected in two or three accessions were analysed by General Linear Models (GLM). Models used for MANOVA or GLM analysis included main effects of accession and temperature in one or several generations, as well as interactions between the main effects. Next, the data was analysed by Student’s t-test in the case of pairwise comparisons or by One Way ANOVA for multiple comparisons followed by pairwise comparisons (Holm-Sidak). Depending on the experimental setup either three or four biological replicates were analysed.

5.8. RNA extraction Total RNA was extracted with the Spectrum Plant Total RNA kit (Sigma-Aldrich, Saint Louis, USA) basically as described by the supplier (protocol A) from 100 mg of frozen plant tissue. Lysis solution was added to the plant tissue between two tissue disruption cycles on a TissueLyser II (Qiagen, Hilden, Germany) for 2 min at 25 Hz. On-column DNase digestion was performed using the RNase-Free DNase Set (Qiagen, Hilden, Germany) to eliminate genomic DNA. Recombinant RNasin Ribonuclease Inhibitor (Promega, Madison, USA) was added to the ﬁnal concentration of 1 U/ml. Total RNA was quantiﬁed with a NanoDrop ND-1000 (Nanodrop, Wilmington, USA) and RNA quality was veriﬁed by formaldehyde gel electrophoresis.

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5.9. Microarray analysis For microarray experiments, total RNA (200 ng) isolated as described above was reverse transcribed, ampliﬁed and labelled using the Low Input Quick Amp Labeling Kit, One-Color (Agilent Technologies, Santa Clara, USA). 1650 ng cRNA from each sample was fragmented and hybridized on 4 44 K Arabidopsis (V4) Gene Expression Microarray (Agilent Technologies, Santa Clara, USA) in an Agilent G2545A Hybridization rotary oven (10 rpm, 65 C, 17.5 h). Hybridization was performed with the Gene Expression Hybridization Kit (Agilent Technologies, Santa Clara, USA). The slides were washed with buffer 1 & 2 from Gene Expression Wash Buffer kit (Agilent Technologies, Santa Clara, USA) and scanned twice at 5 mm resolution on a laser scanner (Agilent Technologies, Santa Clara, USA), using the “dynamic range expander” option in the scanner software. The resulting images were processed using Agilent Feature Extraction software v9.5. 5.10. Statistical analysis of microarray data The microarray study is MIAME-compliant. Data were preprocessed using the Limma package (version 3.2.3) as implemented in R (Smyth, 2005). Spots identiﬁed as feature outliers were excluded from analysis, and weak or non-detected spots were given reduced weight (0.5). The data were normalized using quantile normalization and no background subtraction was performed. The Benjamini and Hochberg’s method was used to estimate the false discovery rate (Benjamini and Hochberg, 1995). Values are an average of all probes mapping to the gene in question. Genes with an adjusted p-value below 0.05 were considered to be statistically signiﬁcant differentially expressed. Acknowledgements This work was supported by the Research Council of Norway through the grant “Plant metabolites for healthy plants and healthy people (SIP 186903)”. We thank Torﬁnn Sparstad for performing the microarray experiment. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.phytochem.2016.06.003. References Ahuja, I., de Vos, R.C., Bones, A.M., Hall, R.D., 2010a. Plant molecular stress responses face climate change. Trends Plant Sci. 15, 664e674. Ahuja, I., Rohloff, J., Bones, A.M., 2010b. Defence mechanisms of Brassicaceae: implications for plant-insect interactions and potential for integrated pest management. A review. Agron. Sustain. Dev. 30, 311e348. €chter, M., Bo €hme, J., Selmar, D., 2013. Sulfate determines the Alnsour, M., Kleinwa glucosinolate concentration of horseradish in vitro plants (Armoracia rusticana Gaertn., Mey. & Scherb.). J. Sci. Food Agric. 93, 918e923. Alsdurf, J.D., Ripley, T.J., Matzner, S.L., Siemens, D.H., 2013. Drought-induced transgenerational tradeoff between stress tolerance and defence: consequences for range limits? Aob Plants 5, plt038. Andersen, T.G., Nour-Eldin, H.H., Fuller, V.L., Olsen, C.E., Burow, M., Halkier, B.A., 2013. Integration of biosynthesis and long-distance transport establish organspeciﬁc glucosinolate proﬁles in vegetative Arabidopsis. Plant Cell 25, 3133e3145. Balasubramanian, S., Sureshkumar, S., Lempe, J., Weigel, D., 2006. Potent induction of Arabidopsis thaliana ﬂowering by elevated growth temperature. PLoS Genet. 2, e106. Barth, C., Jander, G., 2006. Arabidopsis myrosinases TGG1 and TGG2 have redundant function in glucosinolate breakdown and insect defense. Plant J. 46, 549e562. Bednarek, P., Pislewska-Bednarek, M., Svatos, A., Schneider, B., Doubsky, J., Mansurova, M., Humphry, M., Consonni, C., Panstruga, R., Sanchez-Vallet, A., Molina, A., Schulze-Lefert, P., 2009. A glucosinolate metabolism pathway in living plant cells mediates broad-spectrum antifungal defense. Science 323,

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Please cite this article in press as: Kissen, R., et al., Effect of growth temperature on glucosinolate proﬁles in Arabidopsis thaliana accessions, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.06.003

Effect of growth temperature on glucosinolate profiles in Arabidopsis thaliana accessions

Effect of growth temperature on glucosinolate profiles in Arabidopsis thaliana accessions

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