Genotype influences sulfur metabolism in broccoli (Brassica oleracea L.) under elevated CO2 and NaCl stress.

Plant and Cell Physiology Advance Access published September 22, 2014

Genotype Influences Sulphur Metabolism In Broccoli (Brassica oleracea L.) Under Elevated CO2 And NaCl Stress

Running head (short title): Sulphur metabolism in different broccoli cultivars

*Corresponding author: Dr. M.C. Martínez-Ballesta. Department of Plant Nutrition. Centro de Edafologia y Biologia Aplicada del Segura (CEBAS-CSIC). Campus de Espinardo, Edificio 25, EMurcia, 30100, Spain. Telephone number: 968396308. Email address: [email protected]

Number of black and white figures: 2

Tables: 5

© The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For Permissions, please e-mail: [email protected]

1

Downloaded from http://pcp.oxfordjournals.org/ at Thammasat University on October 9, 2014

Subject areas: Environmental and stress responses.

Genotype Influences Sulphur Metabolism In Broccoli (Brassica oleracea L.) Under Elevated CO2 And NaCl Stress

Running head (short title): Sulphur metabolism in different broccoli cultivars

1

2

1

María del Carmen Rodríguez-Hernández , Diego A. Moreno , Micaela Carvajal , María del Carmen Martínez-Ballesta1*.

Edafologia y Biologia Aplicada del Segura (CEBAS-CSIC). Campus de Espinardo, Edificio 25, EMurcia, 30100, Spain.

Abbreviations: APS, adenosine-5-phosphosulfate; ATP-S, ATP sulphurylase; BSA, Bovine Serum

Albumin; DAD, Diode Array Detector; DTT, Dithiothreitol; GB, Glucobrassicin; GE, Glucoerucin; ɣECS, γ-glutamylcysteine synthetase; GI, Glucoiberin; GR, Glucoraphanin; GS, Glutathione synthetase; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HGB, 4-OH-glucobrassicin; LC-MS,

Liquid

chromatography–Mass

Spectrometry;

MGB,

4-MeO-glucobrassicin;

NGB,

Neoglucobrassicin; OAS-TL, O-acetyl-L-serine(thiol)lyase; PEP, Phosphoenolpyruvic acid; Pi, inorganic

phosphorus;

PVP,

polyvinylpyrrolidone;

acetyltransferase;

2

RH,

Relative

humidity;

SAT,

Serine


(1) Department of Plant Nutrition. (2) Department of Food Science and Technology. Centro de

Abstract Climatic change predicts elevated salinity in soils as well as increased carbon dioxide [CO2], in the atmosphere. The present study aims to determine the effect of combined salinity and elevated [carbon dioxide (CO2)] on sulphur (S) metabolism and S-derived phytochemicals in green and purple broccolis (cultivar (cv.) Naxos and cv. Viola, respectively). Elevated [CO2] entailed the amelioration of salt stress, especially in cv. Viola, where a lower biomass reduction by salinity was accompanied by higher sodium (Na+) and chloride (Cl-) compartmentation in the vacuole. Moreover, salinity and elevated [CO2] affected the mineral and glucosinolate contents and the activity of biosynthetic enzymes of S-

the related amino acids and the antioxidant glutathione (GSH). In cv. Naxos, elevated [CO2] may trigger the antioxidant response to saline stress by means of increased GSH concentration. Also, in cv. Naxos indolic glucosinolates were more influenced by the NaCl x CO2 interaction whereas in cv. Viola the aliphatic glucosinolates were significantly increased by these conditions. Salinity and elevated [CO2] enhanced the S cellular partitioning and metabolism affecting the myrosinaseglucosinolate system.

Key words: Brassica oleracea, elevated [CO2], glucosinolates, myrosinase, salinity.

3


derived compounds and the degradative enzyme of glucosinolate metabolism, myrosinase, as well as

Introduction Broccoli quality and composition can be affected by environmental and growth conditions (Rosa et al. 1996, Wszelaki and Kleinhenz 2003, Charron et al. 2005, Padilla et al. 2007, Pérez-Balibrea et al.

2008) such as temperature and photoperiod (Charron and Sams 2004), season (Rosa and Rodrigues 2001) and salt stress (Lopez-Berenguer et al. 2009). Salinity is one of the most important environmental limiting factors for crop production in many parts of the world (Bernstein 1975) and this abiotic stress is becoming even more prevalent as the intensity of agriculture increases (Zhu 2002). Salt stress induces ion toxicity, osmotic stress, mineral

plants (Neumann 1997, Yeo 1998, Hasegawa et al. 2000, Munns 2002) as a response to high sodium chloride (NaCl) concentrations. Also, salinity may directly inhibit photosynthesis (Stepien and Johnson 2009) as a consequence of stomata closing to prevent water loss (Martínez-Ballesta et al. 2006). Furthermore, atmospheric [CO2] has implications for agriculture and the environment as a whole and represents an environmental factor that is currently undergoing great changes, from 280 parts per million (ppm) in the preindustrial era to 365 ppm currently (Craigon et al. 2002, Krupa 2003), with further peaks in urban areas of up to 480 ppm (Pataki et al. 2006). In climate simulations a further increase or even a doubling of today’s concentration is envisaged (Gerber et al. 2004, Norby and Luo 2004, Carter et al. 2007). An enriched CO2 atmosphere results in greater photosynthetic rates, alleviating crop losses and improving the tolerance to salinity by enabling greater osmotic adjustment or improving the plant water balance (Pérez-López et al. 2012, 2013, Zaghdoud et al. 2013). However, relatively few studies have targeted the effect of elevated [CO2] alone or in combination with an additional stress (i.e NaCl) on crop quality and antioxidant capacity, and the results of these investigations are often a matter of debate (Levine and Paré 2009). Thus, carbon (C) and nitrogen (N) metabolism have been intensively investigated in the past, but many aspects of sulphur (S) metabolism remain unclear. So far, progress has been achieved in the areas of sulphate uptake, assimilation as well as storage and mobilisation, especially under restricted S conditions. In general, alterations in the S status provoked similar effects on S-uptake and -assimilation effectors, increasing transport sulphate to the roots through sulphate-transporters, which were regulated at transcriptional, post-transcriptional and post-translational levels. Also, the activity of adenosine 5´-phosphosulphate reductase, APR, a key enzyme of sulphate assimilation, was modulated in order to exert a tight control

4


deficiencies, physiological and biochemical perturbations, and combinations of these stresses on

on S-metabolism (Dadivian and Kopriva 2010). Moreover, a downstream regulatory mechanism has been reported for cysteine synthase complex (CSC), involved in the cysteine synthesis, depending on the S availability in the cell, where CSC may act as a sensor of the sulphide status and where fluctuations in the SAT and OAS-TL activities may support the cysteine synthesis regulation (Wirtz and Hell 2006). -

Plant S -remobilisation from vegetative to reproductive tissues has been also evaluated during sulphate deprivation. Thus, in oiseed rape (Brassica napus L.), a SO42- mobilisation to the youngest leaves was associated with an up-regulation of two transporters involved in the efflux of sulphate from

been reported that long-term sulphate deprivation affected the root-shoot ratio in favour of root production as a sulphur-deficiency strategy by the plant, rather than an increased expression of sulphate transporters, pointing out a lack of an efficient shoot to root signalling in this cultivar (Buchner et al. 2004). However, open questions still remain on the synthesis and function of secondary Smetabolites under abiotic stress conditions. Glucosinolates, nitrogen-sulphur bioactive compounds, may be affected by atmospheric CO2 enrichment (Klaiber et al. 2013). However, the production of N-containing secondary metabolites is species-specific (Karowe et al. 1997). Thus, it has been reported that glucosinolate levels were not affected by elevated [CO2] in radish Raphanus sativus, turnip Brassica rapa var. rapa (Karowe et al. 1997), oilseed rape Brassica rapa var. oleifera, cabbage Brassica oleracea var. capitata (Reddy et al. 2004), or Arabidopsis thaliana (Bidart-Bouzat et al. 2005), whereas in rapeseed Brassica napus (Himanen et al. 2008), and mustard Brassica juncea (Karowe et al. 1997) glucosinolates decreased. In broccoli Brassica oleracea var. italica (Schonhof et al. 2007) and Chinese kale Brassica alboglabra (La et al. 2009), glucosinolates increased. In addition, the synthesis of cysteine and methionine, precursors of glucosinolates, increased because of increasing photosynthesis at elevated [CO2] (Habash et al. 1995) affecting the glucosinolate content (Klaiber et al. 2013). Nevertheless, the combined effect of elevated [CO2] and salinity on glucosinolates has not been addressed, as well as the potential genotype-dependent response of these compounds under such conditions. Glutathione (GSH), the most abundant thiol compound, is synthesized from cysteine (Kimura and Kimura 2004) and plays an important role in the response of plants to environmental stresses (Bailly 2004, Rouhier et al. 2008) that stimulate synthesis of GSH and place an increased demand

5


vacuole (Dobousset et al. 2009, 2010, Kataoka et al. 2004c). However, in Brassica oleracea, it has

upon S-assimilation into cysteine. Fediuc et al. (2005) showed that the cytosolic isoform of O-acetyl-Lserine(thiol)lyase (OAS-TL), one of the enzymes that catalyses cysteine biosynthesis, was induced in leaves of Arabidopsis thaliana exposed to salt stress. Another study showed the level of cysteine in plants determined the cellular concentrations of GSH through kinetic restriction of the reaction catalysed by γ-glutamylcysteine synthetase (ɣ-ECS) (Noctor et al. 1997). Available studies in the literature are focused on the joint effects of salinity and [CO2] on plant productivity and there is limited information about the changes in S metabolism and its effects on plant physiology. In addition, changes in phytochemicals, including secondary sulphated compounds such

approached. Thus, the aim of this work was to determine the effects of the interaction of salinity and elevated atmospheric [CO2] on S-assimilation pathways in green and purple broccoli cv. in order to evaluate the production of sulphated metabolites under these environmental conditions. Total and individual glucosinolate contents and key enzymes involved in S-assimilation (O-acetyl-Lserine(thiol)lyase (OASTL), Serine acetyltransferase (SAT) activity, ATP sulphurylase (ATP-S), γglutamylcysteine synthetase (γ-EC Synthetase), and Glutathione synthetase (GS)) and glucosinolate hydrolysis (myrosinase) were determined, together their precursor amino acids and the natural antioxidant glutathione (GSH).

Results Biomass content, shoot/root ratio, dry matter and soluble protein content Similar salinity growth patterns were shown for both cultivars (Fig. 1). Thus, differences between plant biomass caused by salt stress were higher as increasing treatment duration. However, the effect of [CO2] differed in both cultivars; in cv. Naxos, a progressive biomass increase by elevated [CO2] was observed under non-saline (0 mM NaCl) and saline (80 mM NaCl) conditions. However, in cv. Viola, under non-saline conditions, the effect of elevated [CO2] on plant biomass was higher at short-term, inducing a biomass increase of 73.7%, whereas at middle (7d) and long term (14d) the biomass increase was around ~40%. Under saline conditions, the increment of biomass by elevated [CO2] was similar (~78%) at short and middle term and lower (51%) at long term. In cv. Naxos, salinity significantly decreased the biomass under both ambient (by 26.4 %) and elevated [CO2] (by 27.2%). In cv. Viola, a greater reduction (by 46.2%) due to salt stress was found

6


as glucosinolates, under conditions of salinity and elevated atmospheric [CO2] have not been

under ambient [CO2] compared to that under elevated [CO2] (by 29.1%). In both cultivars, under nonsaline and saline conditions, the dry weight (DW) was higher in plants grown at elevated [CO2] with respect to those grown under ambient [CO2] (Table 1). Although, the combined NaCl x CO2 interaction was not significant, an ameliorative effect of [CO2] on DW reduction under saline treatment was observed. In both cultivars, the shoot/root ratio did not show significance changes under all the treatments (Table 1). However, whereas in cv. Naxos dry matter was not significantly modified by the different treatments, in cv. Viola, an increase of the dry matter by elevated [CO2] was observed under saline conditions.

salinity increased the protein content under both [CO2] concentrations, whereas elevated [CO2] decreased the protein content under non-saline conditions and had no effect under salinity. By contrast, in cv. Viola, whereas salinity increased the protein content at ambient [CO2], it was reduced at elevated [CO2]. Also, the protein content was unmodified and decreased, by elevated [CO2], under non-saline and saline conditions, respectively. A higher influence of the NaCl x CO2 interaction on the soluble protein content resulted in cv. Viola with respect cv. Naxos.

Sulphate (SO42-), sulphur (S), sodium (Na+) and chloride (Cl-) content and C/N and N/S ratios, 2-

+

-

-

Sulphate (SO4 ), sulphur (S), sodium (Na ), chloride (Cl ), nitrate (NO3 ), carbon (C), and nitrogen (N) content as well as the C/N and N/S ratios were determined in the leaves of broccoli 2-

cultivars (Table 2). In cv. Naxos, salinity significantly increased the sulphate (SO4 ) content under elevated [CO2] but had no effect under ambient [CO 2] (Table 2). However, the SO42- content was 2-

higher at elevated [CO2] for both saline conditions. In cv. Viola, the SO4 content was increased by salt stress and elevated [CO2], but the NaCl x CO2 interaction was not significant. In cv. Naxos, salinity had no effect on sulphur (S) content at both ambient and elevated [CO2] (Table 2). By contrast, in cv. Viola, 80 mM NaCl increased the S content at ambient [CO2] but at elevated [CO2] had no effect. Furthermore, the S content was significantly influenced by the interaction NaCl x CO2 in cv. Viola. In both cultivars, under non-saline conditions, the S content increased at elevated [CO2], whereas under salt-stress [CO2] had no effect on the S level. In cv. Naxos, under salt stress, no influence of [CO2] on the Na+ content was observed (Table +

2). However, in cv. Viola, the Na concentration was significantly influenced by the interaction NaCl x

7


Soluble protein content, expressed as mg g-1 DW, was determined (Table 1). In cv. Naxos,

CO2, and plants grown under 80 mM NaCl showed much higher Na+ content at elevated [CO2] than those grown at ambient [CO2]. In cv. Naxos, the Cl- content showed no influence of [CO2], whereas the highest Cl- content values were observed in plants of cv. Viola grown at 80 mM NaCl and elevated [CO2] (Table 2). Thus, in cv. Viola, a significant effect of the NaCl x CO2 interaction on Cl- content was observed. -

In cv. Naxos, salinity decreased the NO3 content at ambient [CO2] whereas it remained unchanged at elevated [CO2] (Table 2). Similarly, elevated [CO2] decreased the NO3- content under -

non-saline conditions whereas no effect on the NO3 concentration was observed under salinity. In cv.

-

NO3 concentration only under saline conditions. No influence of the NaCl x CO2 interaction on the NO3- content was observed for both cultivars. In cv. Naxos, the C content was increased by salinity at ambient [CO2] but not at elevated [CO2] (Table 2). Moreover, under saline and non-saline conditions, elevated [CO2] increased the C content. In cv. Viola, salinity had no effect on the C content under both CO2 levels and similarly to cv. Naxos, elevated [CO2] increased the C concentration independently of the saline conditions. No influence of the NaCl x CO2 interaction on the C content was observed for both cultivars. In cv. Naxos, the N content was reduced by salinity at ambient [CO2] but not at elevated [CO2] (Table 2). Elevated [CO2] decreased the N concentration at non-saline conditions but not under salinity. By contrast, in cv. Viola, salinity decreased the N content at elevated [CO2] and elevated [CO2] reduced the N concentration only under salt stress. Thus, no influence of the NaCl x CO2 interaction on the N content was observed for both cultivars. Salinity had no effect on the C/N ratio in cv. Naxos, whereas in cv. Viola, 80 mM NaCl increased the C/N ratio at elevated [CO2] (Table 2). In general, the C/N ratio was higher at elevated [CO2] and saline conditions in both cultivars compared to ambient [CO2] due to a higher C assimilation, with the exception of non-saline plants of cv. Viola, where there was no difference between the C/N ratios at the two CO2 concentrations. Furthermore, in cv. Viola, an influence of the NaCl x CO2 interaction on the C/N ratio was observed. Finally, in cv. Naxos, salinity increased the N/S ratio at both CO2 levels (Table 2), whereas in cv. Viola the N/S ratio was significantly decreased by salinity at elevated [CO2]. In both cultivars, under saline and non-saline conditions, the N/S ratio decreased at elevated [CO2].

8


Viola, salt stress decreased the NO3- content only at elevated [CO2] and elevated [CO2] reduced the

Glucosinolate content Aliphatic glucosinolates (Glucoiberin (GI) and Glucoerucin (GE)) and indolic glucosinolates (Glucobrassicin

(GB),

4-MeO-glucobrassicin

(MGB),

4-OH-glucobrassicin

(HGB)

and

Neoglucobrassicin (NGB)) were detected and quantified in the leaves of cvs. Naxos and Viola (Table 3). Salinity increased the amount of total glucosinolates (Fig. 2) except in cv. Viola plants grown at ambient [CO2], where salinity had no effect on total glucosinolate levels. Furthermore, the total glucosinolate content was increased by elevated [CO2], with the exception of non-saline treated plants of cv. Viola where elevated [CO2] had no significant effect on total glucosinolate content.

[CO2] condition (Table 3). Similar results were found for the rest of the glucosinolates under ambient [CO2] but not at elevated [CO2] where salinity increased glucosinolate concentration. No influence of combined salinity and [CO2] on GE, GB and NGB contents was found in cv. Naxos. In cv. Viola, with the exception of GB that was increased by salt stress at elevated [CO2], no significant effect of the salinity was found for indolic glucosinolates at either [CO2], with similar results for aliphatic glucosinolates at ambient [CO2]. However, at elevated [CO2], salinity increased the aliphatic glucosinolate content. In this cultivar, no influence of the NaCl x CO2 interaction on indolic glucosinolates, excepting GB, was observed. In cv. Naxos, significant enhancements of individual glucosinolates were found at elevated [CO2], with the exception of GI, GE and GB that were not significantly enhanced by elevated [CO2] under non-saline conditions. In cv. Viola the increase at elevated [CO2] was only significant under saline conditions and for GB and aliphatic glucosinolates.

Foliar enzymes The enzymatic activities involved in the S assimilation pathway as well as the glucosinolate hydrolysis enzyme, myrosinase, were determined in the leaves of green and purple broccolis (Table 4). In both cultivars, all the enzymatic activities were increased by salinity under ambient and elevated [CO2], with the exception of myrosinase that decreased under salt stress. Also, elevated [CO2] enhanced the levels of the activities compared to ambient [CO2], again excluding myrosinase. On the other hand, the NaCl x CO2 interaction significantly affected the OAS-TL, SAT and ATPS activities in cv. Naxos,

9


In cv. Naxos, NaCl treatment had no significant effect on the content of NGB under either

whereas in cv. Viola the combined factors salinity and CO2 significantly affected the OAS-TL, SAT, γECS and myrosinase activities.

Amino acids and glutathione The S-amino acids cysteine (Cys) and methionine (Met) as well as glutamic acid (Glu), tryptophan (Trp), serine (Ser), and glutathione (GSH) were determined (Table 5) and increased in NaCl-treated plants also subjected to elevated [CO2], except for Ser, which remained practically unchanged. In cv. Naxos the NaCl x CO2 interaction did not affect Glu, Trp and Ser, whereas in cv. Viola no significant

GSH was higher under salt stress in both cultivars and [CO2] levels. In cv. Naxos, elevated [CO2] only had a significant effect on GSH at 80 mM NaCl, but in cv. Viola, the NaCl x CO2 interaction did not significantly influence the GSH concentration.

Discussion It has been observed that elevated [CO2] may alleviate the repressive effects of salinity on plant growth through enhanced photosynthesis that leads to carbohydrate accumulation and higher

productivity (Mavrogianopoulos et al. 1999, Poorter and Navas 2003, del Amor 2013). Therefore, studying the interactive effect of salinity and elevated [CO2] concerning S metabolism in broccoli and its related bioactive compounds in S assimilation is of essential importance. Similar to previous reports (Pérez-López et al. 2009, 2010), in our work elevated [CO2] increased the plant biomass under nonsalinized and salinized conditions and also entailed the amelioration of NaCl stress, especially in cv. Viola. A genotypic influence has been described in the growth response to [CO2] under saline conditions for tomato (del Amor 2013) and barley (Pérez-López et al. 2009) and, although an equal growth response of these broccoli cultivars to salt stress has been previously observed (RodríguezHernández et al. 2013), a genotype-dependence of NaCl x CO2 on plant growth can be deduced. Root exudation has been reported to increase in plants grown under enriched [CO2] (Norby et al. 1987), which might enhance nutrient acquisition, especially under stress conditions where nutrition is limited (Uren and Reisenauer 1988, Schulte et al. 2002). Robredo et al. (2011) observed that the values for N uptake by barley plants were higher under elevated [CO2], while CO2 enrichment inhibited nitrate assimilation in wheat, Arabidopsis and sunflower (Bloom et al. 2012, De la Mata et al. 2013,

10


effect of the NaCl x CO2 interaction was found on its Cys, Met and Trp contents.

Tausz et al. 2013). In this work, the combination of salinity and elevated [CO2] decreased N content -

compared to control in cv. Viola according to the greater reduction in NO3 content. A decreased shoot capacity to assimilate NO3- under elevated [CO2] has been previously suggested (Bloom et al. 2010), conditioning the total N and protein content, which were drastically reduced in cv. Viola by elevated [CO2] under salinity. Several studies have shown that elevated [CO2] may increase, decrease or have no effect on the protein concentration (Taub and Wang 2008). In this work, genotypic differences on protein content were observed, with reductions produced by elevated [CO2] under non-saline and saline conditions in cv. Naxos and cv. Viola, respectively. Previous reports showed that elevated [CO2]

biomass increments. However, the initial stimulation of photosynthesis by elevated [CO2] use to be gradually declined during [CO2] time-exposition, a phenomenon known as photosynthesis acclimation (Seneweera et al. 2011). This acclimation was attributed to a reduction in the Rubisco activity accompanied by a decrease in foliar soluble proteins (Rogers and Humphries 2000) that could be occurring in our broccoli plants. However, a dilution effect on protein content derived from a higher biomass growth and dry matter production (Taub and Wang 2008) must not be discarded in cv. Viola -

under elevated [CO2] and salinity. The reduced NO3 assimilation justifies the significant effect on the C/N ratio by the NaCl x CO2 interaction in cv. Viola, since under salt stress elevated [CO2] increased the total C content compared to ambient [CO2] to similar percentages (~33%) in both cultivars (Table 2). However, under salt stress, plant biomass was enhanced 52.3% by elevated [CO2] in cv. Viola, whereas the increase was 36.3% in cv. Naxos. This fact may be an indication of carbohydrate production in cv. Naxos in response to NaCl x CO2, but structural from non-structural dry matter and organic from inorganic forms of the nutrient must be distinguished. Also, an additive effect of the NaCl x CO2 interaction was exerted on Na+ and Cl- levels in cv. Viola, indicating that elevated [CO2] may +

contribute to compartmentation of Na in the vacuoles of this cultivar. An influence of the NaCl x CO2 interaction on S content and N/S relation was observed, and under non-saline conditions there was a 2-

correlation between SO4 and total S levels that was not observed under saline conditions The lack of correspondence between the increased SO42- concentration and the unchanged total S levels produced by elevated [CO2] regarding ambient [CO2], under salinity, could be explained by the low (in cv. Naxos) or nil effect (in cv Viola) of the NaCl x CO2 interaction on ATP-S activity. In fact, the 2-

incorporation of S into organic compounds, requiring the binding of SO4 to ATP to form adenosine-5-

11


increased leaf photosynthesis in broccoli plants (Zaghdoud et al. 2013) providing shoot and root

phosphosulfate (APS), is catalysed by ATP-S and is one of the rate limited steps in S-assimilation (Leustek et al. 1994, Ahmad et al. 2001). In our work, elevated [CO2] increased ATP-S 17.35 and 30.9 folds in cvs. Naxos and Viola, respectively, under non-saline conditions, whereas under salt stress the increments were 3.40 and 4.25 for cvs. Naxos and Viola, respectively. In addition, other structural Scompounds must be considered in the partitioning of S vs extractable SO42-. 2-

Moreover, the increase in the SO4 content produced by elevated [CO2] under salinity could affect the osmotic status of the plants through the changes in other ions concentration such as 2-

selenate and molybdate (Schiavon et al. 2012). Also, increased SO4 content may determine salinity

content, have been involved in redox processes through the biological enzymes regulation, and had an important role in the protection against salt stress (Nazar et al. 2011b). Among them, GSH, the major soluble antioxidant in the cell compartments was strongly affected by S nutrition and metabolism (Khan et al. 2009). Also, it has been reported that the S-assimilation rate, the biosynthesis of Cys and GSH as well as the S-assimilation enzymes were increased by salinity in Brassica napus and Brassica oleracea plants (Ruiz and Blumwald 2002, López-Berenguer et al. 2007) contributing to the acclimation of these cultivars to salt stress. In this sense, elevated [CO2] favoured the S-assimilation in both, cvs. Naxos and Viola, in their response to salinity. It has been reported that glucosinolates may be affected by atmospheric [CO2] enrichment (Klaiber et al. 2013) as this enhances the plants’ carbon supply, increasing the C/N ratio (Cotrufo et al. 1998). Schonhof et al. (2007) found that total glucosinolate content was enhanced in broccoli inflorescences at elevated [CO2] as the result of strong increases in both glucoraphanin (GR) and glucoiberin (GI). By contrast, indole glucosinolates simultaneously decreased, mainly due to reductions in glucobrassicin (GB) and 4-methoxy-glucobrassicin (MGB) levels. However, in our broccoli plants, under non-saline conditions, either an increase [MGB] or no change [GB] were observed upon elevating the [CO2]. Differences from the above study could be due to distinct genotypes or plant developmental stage and organ, as well as experimental conditions. To the best of our knowledge, no previous studies have been reported on the interactive effect of NaCl x CO2 on glucosinolate content. In general, both conditions dramatically increased the total and most individual glucosinolates, according to the increased SO42- and Met and Trp contents. Thus, the well-known enhancement of glucosinolates upon NaCl treatment in broccoli cultivars (Dominguez-Perles et al.

12


tolerance in broccoli plants. The S-containing thiol groups, which ultimately depend on the SO42-

2011) was also higher under elevated [CO2]. The NaCl x CO2 interaction was evident for indolic glucosinolates in cv. Naxos and for aliphatic glucosinolates in cv. Viola, making these cultivars of interest to grow under elevated [CO2]. It has been reported that a reduced N/S ratio provoked an increase in the synthesis of Cys (Hesse et al. 2004), the precursor of Met, needed for the synthesis of glucosinolates (Mikkelsen et al. 2

2002). In the present study, there was a negative and weak (R = 0.65) correlation between the N/S ratio and total glucosinolate content in cv. Viola, suggesting that the glucosinolate changes induced by the treatments were influenced by the modifications of N and S content in this cultivar. However, in cv.

with previous reports where the reduced N/S ratio under elevated [CO2] was not correlated to glucosinolates (Schonhof et al. 2007, La et al. 2009). Moreover, an adequate N/S ratio is positive for glucosinolate content, but when N/S > 10/1 a decreased glucosinolate concentration is produced (Schonhof et al. 2007). In oilseed rape, glucosinolate levels were higher when N/S < 10/1. However, in our green and purple broccolis (Table 2), although a N/S > 10/1 was found, glucosinolate synthesis was according to previous reports (López-Berenguer et al. 2009). A positive correlation between the 2

2

C/N ratio and glucosinolate for cv. Naxos (R = 0.724) and cv. Viola (R = 0.999), pointing out that the relation C/N relation could be a bioindicator of the changes in glucosinolates seen in the NaCl x CO2 interaction, especially in cv. Viola. Amino acid accumulation under conditions of salinity (Mansour et al. 2001) and elevated [CO2] has been reported in wheat and Arabidopsis plants (Habash et al. 1995, Romero et al. 2001). Similarly, in broccoli (Table 5), the amino acid pools increased with the NaCl x CO2 interaction with the exception of Ser, which was reduced. This decrease in Ser concentration may be a consequence of the induced activity of SAT and OAS-TL under these conditions to produce Cys, using Ser as the primary substrate (Dominguez et al. 2003). In cv. Naxos, the NaCl x CO2 interaction significantly altered GSH, which acted as an antioxidant under salt stress (Barroso et al. 1999, Ruiz and Blumwald 2002, Kocsy et al. 2004), demonstrating the ability of elevated [CO2] to promote the antioxidant capacity of this cultivar under +

salt stress. In addition, it has been reported that the accumulation of excess Na in vacuoles in salt stress-tolerant transgenic plants minimized the GSH response (Ruiz and Blumwald 2002). Similarly, +

under salt stress, the concentration of Na in the leaves of cv. Viola was higher than in cv. Naxos,

13


Naxos, there was a lack of correlation between the N/S ratio and total glucosinolates in agreement

reaching maximum values at elevated [CO2], which was in consonance with the 51.5% increase in GSH under conditions of salinity compared to cv. Naxos (where the increase was 71.3%). It has been reported that Cys and γ-ECS are the limiting precursors for GSH synthesis and, although it has been shown that an increase in Cys (Noctor et al. 1999, Bloem et al. 2004, 2007, Zechmann et al. 2007, 2008) as well as overexpression of genes and enzymes involved in Cys synthesis (Harms et al. 2000, Noji and Saito 2002, Wirtz and Hell 2007) will enhance GSH in plants, this response did not occur under salt stress in cv. Viola under elevated [CO2] in spite of an enhanced Cys level and increased γ-ECS activity. Other factors limiting the production of GSH such as the

levels do not always correlate well with the components of the GSH synthesis pathway, as seen in two transformed plants overexpressing γ-ECS activity in different cellular compartments that showed similar GSH levels but distinct γ-ECS activities (Noctor et al. 1998). In our broccoli plants, GSH production may depend on genotype. In cv. Viola the limiting step in GSH synthesis seemed to be the GSH biosynthetic pathway rather than Cys availability when plants were grown under NaCl x CO2. In any case, the crosstalk between S, N and C metabolism required for GSH synthesis is complexly regulated, and in broccoli plants the C/N and N/S ratios showed distinct genotype-dependent responses to the NaCl x CO2 interaction. It has been reported that cytosolic OAS-TL is involved in the defence responses of Arabidopsis against salinity (Barroso et al. 1999, Dominguez-Solis et al. 2001, Romero et al. 2001). The fact that elevated [CO2] increased this enzymatic activity under saline conditions may be an indication of the beneficial effects of CO2 under salt stress in inducing S-metabolism. It is well known that myrosinase is susceptible to abiotic conditions such as salt stress (Kim et al. 2006, Yan and Chen 2007, Yuan et al. 2009). However, no data concerning the effect of the NaCl x CO2 interaction have been reported, to the best of our knowledge. In this study, myrosinase activity was significantly reduced, maintaining the availability of glucosinolate, which compromised the plants’ defences without producing isothiocyanates. Further studies of the glucosinolate-myrosinase system and the NaCl x CO2 interaction merit attention. Summarizing, elevated [CO 2] entailed the amelioration of salt stress, especially in cv. Viola, by means of biomass reduction, and higher Na+ and Cl- sequestration in the vacuole. Although under salt stress the C content was raised similarly in both cultivars by elevated [CO2], different biomass

14


availability of ATP, glutamate and glycine could be involved (Keys et al. 1978). Furthermore, GSH

increments under these conditions were observed, indicating that carbohydrate production in cv. 2-

Naxos should be considered. A lack of correlation between SO4 and total S levels under the NaCl x CO2 interaction may be an indication of the deleterious effect of salt stress on ATP-S activity. The differences in the cultivars’ response to the NaCl x CO2 interaction with regard to S level, the N/S and C/N ratios as well as GSH and glucosinolates were significant. While the C/N ratio could be an indicator of modified glucosinolate levels under the NaCl x CO2 interaction in both cultivars, there was only a correlation between the N/S ratio and glucosinolate content in cv. Viola pointing out that glucosinolate levels showed a greater reliance on N and S content in this cultivar. Also, the amino acid

SAT and OAS-TL activities under these conditions to produce Cys. In cv. Naxos, elevated [CO2] may enhance the antioxidant capacity of the salt-treated plants by increasing the GSH concentration. The reduced response of GSH in cv. Viola compared to cv. Naxos at elevated [CO2] may be related to a higher Na+ accumulation in the vacuole. GSH production may depend on the cultivar, with the GSH biosynthetic pathway being a limiting step in cv. Viola rather than Cys availability under the NaCl x CO2 interaction. Finally, although elevated [CO2] may ameliorate salt stress favouring S-metabolism, the NaCl x CO2 interaction may compromise the myrosinase-glucosinolate system; this needs further characterization.

Materials and methods

Plant material and growth conditions The experiment was carried out with two commercial varieties of broccoli (Brassica oleracea L. [Italica Group]): cv. Naxos and Viola, the first variety being obtained from Spain (Sakata Seed Ibérica S.L, Valencia, Spain) while Viola was obtained from the UK (Thompson & Morgan (UK) Ltd., Cedar Lane, Ipswich, England). First, broccoli seeds (Brassica oleracea L. var. italica) were pre-hydrated with aerated, deionised water for 24 h and then germinated in vermiculite at 28°C in darkness in a germination chamber for 3 d. They were then transferred to a controlled-environment growth chamber with a 16 h light-8 h dark cycle and air temperatures of 25 and 20°C, respectively. The relative humidity (RH) was -2

-1

60% (day) and 80% (night), and the photosynthetically active radiation (PAR) was 400 µmol m s ,

15


pools increased with salinity and elevated [CO2] except for Ser, which was in consonance with induced

provided by a combination of 44 fluorescent tubes (Philips TLD 36 W/83 and Sylvania F36 W/GRO) for every 2 metal halide lamps (Osram HQI.T 400 W). After 4 d, the seedlings were transplanted to hydroponic cultivation (Hoagland nutrient solution) in 15 L containers. The solution was replaced completely every week. After 15 days (when plants were 21 days old), the plants were treated with 0 mM NaCl and 80 mM NaCl. Half of these plants were grown at elevated [CO2] (800 ppm) and the other half at ambient [CO2] (380 ppm). The CO2 concentration was supplied and regulated by injection of external compressed CO2 (bottled [CO2] ≥ 99.9%), controlled by an infrared gas analyser (Aerasgard RCO2, S+S Regeltechnik, Nuremberg,

desired level. Biomass, shoot/root ratio, dry matter, soluble protein content, mineral analysis, glucosinolates, different enzymatic activities involved in S-metabolism and amino acid analysis were measured after 15 days of treatment, when the plants were 36 days old. Measurements and harvesting were all performed in the middle of the light period. Six replications of each treatment and cultivar of two independent experiments were used for determinations.

Biomass content and dry matter production The plants were collected 15 days after treatment, the aerial parts (leaves and stems) separated, and the fresh weight (FW) directly recorded with a portable balance. The total dried biomass (DW) of the aerial part was obtained after drying in an oven at 70°C until a constant weight was attained. Biomass content, shoot/root ratio and dry matter production were determined. For the growth curve representation, the total biomass was measured at 0, 7 and 14, days after NaCl addition, when plants were 14, 21 and 36 days old, respectively.

Soluble protein content For soluble protein content determination, 0.5 g of leaf tissue were frozen with liquid N and ground in 50 mM sodium-phosphate buffer (pH= 7.4). The homogenate was centrifuged (10,000g, 20 min) and the buffer-soluble proteins were obtained from the supernatant by heat denaturation (60 min, 100ºC) and collected by centrifugation (10,000g, 20 min). Buffer-soluble protein content was determined by

16


Germany) equipped with an automatic switching solenoid to maintain the CO2 concentration at the

the Bio-Rad kit assay (Bio-Rad Laboratories, Hercules, CA, USA) which is based on the Lowry method (Lowry et al. 1951).

Mineral content analysis For mineral analysis the third leaf of broccoli plants collected 15 days after starting the salt treatments was dried at 65°C until unchanged dry weight. Minerals were analysed in samples (ca. 100 mg DW) of the oven-dried plant material, which were ground finely in a mill grinder (IKA model A10) resulting in test samples of 0.5 to 0.7 mm.

were diluted with H2O and centrifuged at 3000 g for 20 min (4°C) using a Hettich-Universal 132R centrifuge (Tuttlingen, Germany). The supernatants were collected and filtered through a 0.2-µm inorganic membrane filter (ANOTOP 10 plus, Whatman, Maidstone, UK). The anions were determined by injection into a Dionex-D-100 ion chromatograph (Sunnyvale, CA). An Ionpac AS12A (4 x 250 mm) (10–32) column and guard column were used. The flow rate was 1.5 ml min with an eluent of 2.7 mM Na2CO3/0.3 mM NaHCO3. The contents were expressed as µmol g-1 DW.

Cations and total C and N analysis. The concentrations of sulphur (S) and sodium (Na) were analysed. For that, the samples were digested in a microwave oven (CEM Mars Xpress, North Carolina, USA) by nitric acid (HNO3):perchloric acid (HClO4) (2:1) digestion. The cation analysis was carried out by a Perkin–Elmer (Waltham, MA) model ICP 5500 emission spectrophotometer at 589 nm. The ions were detected with a conductivity detector and quantified by comparing peak areas with -1

those of known standards. The cation content was expressed as g 100g

DW. The total C and N

concentrations of leaf samples were analysed using a Thermo-Finnigan 1,112 EA elemental analyzer (Thermo-Fisher Scientific, Milan, Italy).

Extraction and determination of intact glucosinolates Glucosinolates were analysed according to the procedure described by Martinez-Sanchez et al. (2006) with modifications (Dominguez-Perles et al. 2010). A freeze dried powder of the fully-expanded leaves (50 mg) was extracted in 1.5 ml of 70% methanol (MeOH) for 30 min at 70°C, vortexing every 5 min to improve extraction, followed by centrifugation (15 min, 13000 rpm, Sigma 1-13, B. Braun

17


Anions. The concentrations of sulphate (SO42-) and chloride (Cl-) were analysed. For that, the samples

Biotech International, Osterode, Germany) to pellet insoluble material. The supernatants were collected and methanol was removed using a rotary evaporator; the dried residue was reconstituted in ultrapure water to the initial volume of the supernatant and filtered through a 0.2 µm inorganic membrane filter (ANOTOP 10 plus, Whatman, Maidstone, UK). The samples were kept on ice during the whole procedure. Each sample (20 µL) was analysed in a Waters HPLC-Diode Array Detector (DAD) system (Waters Cromatografía S.A., Barcelona, Spain) consisting of a W600E multisolvent delivery system, in-line degasser, W717 plus autosampler, and W2996 photodiode array detector at 330 nm. The

Phenomenex, Macclesfield, UK) with a security guard C18-ODS (4 x 30 mm) cartridge system (Phenomenex). The mobile phase was a mixture of water/trifluoroacetic acid (99.9:0.1, v/v) (A) and a -1

mixture of acetonitrile/trifluoroacetic acid (99.9:0.1, v/v) (B). The flow rate was 1 mL min in a linear gradient, starting with 1% B for 5 min until reaching 17% B at 15 min, which was then maintained for 2 min, then 25% B at 22 min, 35% B at 30 min, 50% B at 35 min and 99% B at 40 min. Glucosinolates (227 nm) were eluted off the column at 35 min, identified using a previously

described Liquid chromatography–mass spectrometry (LC-MS) method (Martinez-Sanchez et al. 2006) and quantified using sinigrin as standard (sinigrin monohydrate from Sinapis nigra, Phytoplan Diehm & Neuberger, Gmbh, Heidelberg, Germany). The content of glucosinolates was expressed as milligrams per 100 g of fresh weight.

Determination of S- metabolism enzyme activities O-acetyl-L-serine(thiol)lyase (OAS-TL) activity. OAS-TL activity was measured according to the method described by Nakamura et al. (1987) modified by Stuiver and De Kok (2001). For this, 1 g of fresh leaf tissue (third leaf) was homogenized with 10 mL of 0.2 M potassium phosphate buffer (pH 8) containing 10 mM 2-mercaptoethanol and 0.5 mM EDTA. The homogenate was filtered through one layer of miracloth and centrifuged for 15 min (10000 g, 0°C). The filtered extract was used for OAS-TL determination. The reaction mixture contained 0.2 M Tris-Hydrochloric acid (Tris-HCl) (pH 7.5), 0.01 M DTE, 7.8 mM O-acetil-L-serina-HCl, 2.7 mM Na2S and 20 µl of extract in a final volume of 480 uL. Sodium sulphide (Na2S) was added to start the reaction that was continued for 30 min at 25°C. The reaction was stopped by putting the glass tubes on ice and adding 400 µL of 4 M HCl. Then the

18


compounds were separated in a Luna C18 column (25 cm x 0.46 cm, 5 µm particle size;

mixture was centrifuged for 3 min (15000 g) and 200 µL of acid ninhydrine reagent was added to 200 µL of the supernatant (Gaitonde 1967). The mixture was heated at 100°C for 30 min and then rapidly cooled on ice. A volume of 400 µL of ethanol (99.5%) was added and the absorbance determined after 15 min at 560 nm. A calibration curve was established by adding known concentrations of L-cysteine to the reaction mixture and measuring without incubation.

Serine acetyltransferase (SAT) activity. SAT activity was measured according to the method described by Nakamura et al. (1987). For this, 0.25 g of fresh leaf tissue (third leaf) was homogenized with 2.5

EDTA. The homogenate was filtered through one layer of miracloth and centrifuged for 10 min (10000 g, 0°C). The filtered extract was used for SAT determination. The incubation mixture contained 2 mM potassium phosphorus (KPO4), 2 mM serine, 30 µg Bovine Serum Albumin (BSA), 250 µM acetylCoA, 500 µΜ Na2S, and 50 µL of extract in a final volume of 300 µL. The reaction was started by adding the extract and was continued for 20 minutes at 25°C. After this time, the reaction was stopped by putting the glass tubes on ice and adding 400 µL of 4 M HCl. The tubes were centrifuged at 15000 g for 3 min and 200 µL of acid ninhydrine reagent was added to a 200 µL aliquot of the supernatant (Gaitonde 1967). The mixture was heated at 100°C for 10 min and then rapidly cooled on ice. A volume of 400 µL of ethanol (99.5%) was added and the absorbance was determined at 560 nm after 15 min. The calibration curve was established by adding known amounts of L-cysteine to the assay mixture and measuring without incubation.

ATP sulphurylase (ATPS) activity. ATPS activity was measured according to the method described by Lappartient and Touraine (1996). For this, 0.5 g of fresh leaf tissue (third leaf) was homogenized with 2.5 mL of 0.2 M maceration buffer consisting of 20 mM Tris-HCl (pH 8) containing 10 mM

Ethylenediaminetetraacetic Acid, disodium Salt (Na2EDTA), 2 mM Dithiothreitol (DTT) and 0.025 g mL-1 insoluble polyvinylpyrrolidone (PVP). The homogenate was filtered through one layer of miracloth and centrifuged for 10 min (20000 g, 4°C). The filtered extract was used for ATP-S determination. The incubation mixture contained 80 mM Tris-HCl (pH 8), 7 mM magnesium chloride (MgCl2), 5 mM Sodium molybdate (Na2MoO4), 2 mM ATP, 0.032 units mL

-1

of sulphate-free inorganic

pyrophosphatase dissolved in 80 mM Tris-HCl (pH 8) and 50 µl of extract in a final volume of 300 µL.

19


mL of 0.2 M potassium phosphate buffer (pH 8) containing 10 mM 2-mercaptoethanol and 0.5 mM

5 mM Na2MoO4 was added to start the reaction, and the reaction was continued for 5 min at 37°C. Then, the reaction was stopped by putting the glass tubes on ice and adding 500 µL 0.5 M sodium acetate (pH 4) and 100 µL of developer solution (Na2MoO4, sodium-L-ascorbate and Sulfuric acid (H2SO4)). After vortexing, the absorbance was determined at 660 nm after 10 min. A calibration curve was established by adding known amounts of inorganic phosphorus (Pi) to a maceration buffer. Furthermore, 50 µL of extract was added to 250 µL of incubation mixture except for the 5 mM Na2MoO4, which was substituted by 80 mM Tris-HCl buffer (pH 8). The ATP-S activity was the difference between the complete reaction mixture and the reaction mixture without 5 mM Na2MoO4.

according to the method described by Rüegsegger and Brunold (1992) and the quantification was performed according to the method described by Cobbett et al. (1998). For measurement of γ-EC synthetase activity, 0.5 g of fresh leaf tissue (third leaf) was homogenized with 2.5 mL of 0.2 M maceration buffer consisting of 0.1 M Tris-HCl (pH 8) containing 5 mM EDTA. The homogenate was filtered through one layer of miracloth and centrifuged for 10 min (30000 g, 4°C). The filtered extract was used for γ-EC synthetase determination. The incubation mixture contained 100 mM 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid-Sodium hydroxide (HEPES-NaOH) (pH 8), 40 mM Magnesium Chloride (MgCl2), 30 mM L-glutamine, 0.8 mM L-cysteine, 0.4 mM DTT, 5 mM ATP, 5 mM Phosphoenolpyruvic acid (PEP) and 100 µL of extract in a final volume of 450 µL. Then, the samples were incubated for 45 min at 37°C and the reaction was stopped by putting the glass tubes on ice and by adding 500 µL of 0.5 M sodium acetate (pH 4) and 100 µL of developer solution (Na2MoO4, sodium-L-ascorbate and H2SO4). After vortexing, the absorbance was determined at 660 nm after 10 min. A calibration curve was established by adding known amounts of Pi to a maceration buffer. Furthermore, a standard of each sample was performed, where the reaction was stopped immediately after the extract addition, without incubating at 37°C for 45 min. γ-EC synthetase activity was the difference between the values for the incubated reaction mixture and the reaction mixture without incubation.

Glutathione synthetase (GS) activity. The GS activity was measured according to the method described by Rüegsegger and Brunold (1992) and quantified according to the method described by

20


γ-glutamylcysteine synthetase (γ-EC Synthetase) activity. The γ-EC synthetase activity was measured

Cobbett et al. (1998). For this, 0.5 g of fresh leaf tissue (third leaf) was homogenized with 2.5 mL of 0.2 M maceration buffer consisting of 0.1 M Tris-HCl (pH 7.5) containing 10 mM MgCl2 and 1 mM EDTA. The homogenate was filtered through one layer of miracloth and centrifuged for 20 min (30000 g, 4°C). The filtered extract was used for GS determination. The reaction mixture contained 90 mM Tris-HCl (pH 8.4), 20 mM MgCl2, 45 mM KCl, 2 mM glycine, 0.5 mM γ-glutamylcysteine, 4 mM DTT, 7 mM ATP, 5 mM PEP and 100 µL of extract in a final volume of 500 µL. The samples were incubated for 45 min at 37°C and the reaction was stopped by putting the glass tubes on ice and adding 500 µL of 0.5 M sodium acetate (pH 4) and 100 µL of developer solution (Na2MoO4, sodium-L-ascorbate and

was established by adding known amounts of Pi to a maceration buffer. Furthermore, a standard for each sample was performed, where the reaction was stopped immediately after the extract addition, without incubating at 37°C for 45 min. GS activity was the difference in values between the incubated reaction mixture and the reaction mixture without incubation.

Myrosinase activity. Myrosinase was analysed by a previously reported spectrophotometric method (Charron and Sams 2004). Myrosinase activity was determined by the decrease in absorbance at 227 nm measured on a UV160U spectrophotometer (Shimadzu, Kyoto, Japan) resulting from the enzymatic hydrolysis of sinigrin (Palmieri et al. 1982). A 1.5 mL reaction mixture (25 ± 1°C) was prepared containing 50 µL of crude extract, 1.35 mL of 30 mM citrate/phosphate buffer (pH 7) with 1 mM EDTA, and 100 µL of 37.50 mM sinigrin. At the same time, 1.5 mL of a reference reaction mixture was prepared in the same way except that 100 µL of water was used instead of the sinigrin solution. In preliminary experiments it was determined that substrate saturation was achieved with 2.5 mM sinigrin. The reaction and reference mixtures were each vortexed for 5 s in a 13 × 100 mm polypropylene culture tube (Fisher Scientific, Pittsburgh, PA, USA) and transferred to separate 1.5 mL semimicro cuvettes (BrandTech Scientific, Essex, CT, USA). After 1 min, absorbance was measured over a period of 5 min. Activity was determined from the linear slope representing the disappearance of sinigrin from the reaction mixture. The following formula was used (Li and Kushad 2004):

Ucm− 3 =

∆A 1 VA × × × 103 ∆t εl VE 21


H2SO4). After vortexing, the absorbance was determined at 660 nm after 10 min. A calibration curve

A molar extinction coefficient (ε) of 6780 M−1cm−1 at 227 nm was used as reported for sinigrin (Palmieri et al. 1982). The cuvette path length (l) was 1 cm. The total volume of the assay mixture (VA) was 1.5 cm3. The reaction time is given in minutes (t) and was 5 min. The volume of enzyme solution (VE) is given in µL. A unit (U) of activity was defined as the disappearance of 1 µmol sinigrin per min under the conditions described above. The protein concentration of the crude extracts was determined by the Bio-Rad kit assay (Bio-Rad Laboratories, Hercules, CA, USA), which is based on the Lowry

protein concentrations expressed as mg of protein per g fresh weight and used in the determination of specific activity (U mg−1 protein). Activity on a fresh weight basis (Activity-FW) was expressed as U g−1 FW.

Amino acid analysis The amino acids were extracted from fresh leaf tissue using a methanol: chloroform: water (MCW) mixture (5: 12: 3 v/v/v). Pre-weighed plant samples (0.2 to 1.0 g FW) were ground to a fine powder with liquid nitrogen in a mortar and pestle and extracted with MCW. After 1 h at 4°C, the upper aqueous phase (containing amino acids and onium compounds) was concentrated to dryness under a stream of N2. The analyses were carried out with an HPLC/MS system consisting of an Agilent 1100 Series HPLC (Agilent Technologies, Santa Clara, CA, USA) equipped with a thermostatised µ-well plate auto sampler and a capillary pump, and connected to an Agilent Ion Trap XCT Plus Mass Spectrometer (Agilent Technologies) using an electrospray (ESI) interface. Standards with known concentrations of each amino acid (0.1, 0.5, 1, 10, 25, and 50 µM) and samples were prepared in mobile phase A, consisting of water/acetonitrile/formic acid (89.9:10:0.1), and passed through 0.22 µm filters. Then 5 µL of each standard or sample was injected onto a Zorbax SB-C18 HPLC column (5 -

µm, 150 x 0.5 mm, Agilent Technologies), thermostated at 40°C, and eluted at a flow rate of 5 µL min 1

. Mobile phase B, consisting of water/acetonitrile/formic acid (10:89.9:0.1), was used for the

chromatographic separation. The elution consisted of 5 min of 0% B, a linear gradient from 0 to 10% B in 10 min, and 10% B during 5 min. The column was equilibrated with the starting composition of the mobile phase for 20 min before each analytical run. The UV chromatogram was recorded at 210 nm

22


method (Lowry et al. 1951). Bovine serum albumin was used for the standard curve to determine

with the DAD module (Agilent Technologies, Santa Clara, CA, USA). The mass spectrometer was operated in the positive mode with a capillary spray voltage of 3500 V and a scan speed of 26000 (m z-1) s-1 from 50–250 m/z. The nebulizer gas (He) pressure was set to 15 psi, whereas the drying gas -1

was set to a flow of 5 L min at a temperature of 350°C. The chromatogram of each amino acidic ion from both standards and samples was extracted and the peak area was quantified using the Data Analysis program for LC/MSD Trap Version 3.2 (Bruker Daltonik, GmbH, Germany). The peak area data of the standards were used for the calculation of the calibration curve from which the concentration of each amino acid in each sample was obtained.

Total GSH (measured as total thiol) was determined according to Ruiz and Blumwald, 2002. For determination of total GSH, leaf tissue was extracted in 6% m-phosphoric acid (pH 2.8) containing 1 mM EDTA. Total GSH was measured in a reaction mixture consisting of 400 µl of reagent A [110 mM Sodium Monohydrogen Phosphate Heptahydrate (Na2HPO4.7H2O), 40 mM sodium dihydrogen phosphate monohydrate (NaH2PO4.H2O), 15 mM EDTA, 0.3 mM 5,5-dithiobis (2-nitrobenzoic acid)], 320 µl reagent B [1 mM EDTA, 50 mM imidazole, an equivalent of 1.5 units of glutathione reductase activity (baker’s yeast, Type III; Sigma)], and 400 µl of a 1:50 dilution of the leaf extract in 5% Na2HPO4 (pH 7.5) prepared immediately prior to starting the assay. The reaction was initiated by adding 80 µl of NADPH and the absorbance changes measured at 412 nm.

Statistical analysis The data were subjected to a two-way analysis of variance (ANOVA) (SPSS v. 20.0) within each broccoli cultivar. The variance was related to the main treatments (salinity and CO2 concentration) and to the interaction between them. Means ±SD were calculated and, when the F ratio was significant, least significant differences were evaluated by the Tukey-b test as available in the SPSS statistical package (version 20.0). The test was considered significant at P≤0.05.

Funding

23


Glutathione content

This work was supported by the Seneca Foundation – Regional Agency for Science and Technology of the Autonomous Community of the Murcia Region [11909/PI/09]; and the Excellence in Research Grant [04486/GERM/06].

Acknowledgements The authors would also thank Proof-Reading-Service for correcting the English language and style. Finally, the authors would like to thank Dr. Idoia Garmendia phD. from the University of Alicante for her insights and suggestions.

The authors have no conflicts of interest to declare.

References Ahmad, A., Abraham, G. and Abdin, M.Z. (2001) Biochemical evaluation of sulfur and nitrogen assimilation potential of mustard (Brassica juncea L. Czern. & Coss.) under application of slow-release sulfur fertilizer. Appl Biochem Biotech. 96: 167-172. Bailly, C. (2004) Active oxygen species and antioxidants in seed biology. Seed Sci Res. 14: 93-107. Barroso, C., Romero, L.C., Cejudo, F.J., Vega, J.M. and Gotor, C. (1999) Salt specific regulation of the cystosolic O-acetylserine(tilo)lyase gene from Arabidopsis thaliana is dependent on abscisic acid. Plant Mol Biol. 40: 729-736. Bernstein, L. (1975) Effects of salinity and sodicity on plant growth. Annu Rev Phytopathol. 13: 295312. Bidart-Bouzat, M.G., Mithen, R. and Berenbaum, M.R. (2005) Elevated CO 2 influences herbivoryinduced defense responses of Arabidopsis thaliana. Oecologia. 145: 415-424. Bloem, E., Haneklaus, S. and Schnug, E. (2007) Sulphur-induced resistance (SIR) - Sulphur fertilization as a sustainable strategy for keeping plants healthy. [Schwefel-induzierte Resistenz (SIR) - Schwefeldüngung als nachhaltige Strategie zur Gesunderhaltung von Pflanzen]. J Consum Prot Food Saf. 2: 7-12.

24


Disclosures

Bloem, E., Riemenschneider, A., Volker, J., Papenbrock, J., Schmidt, A., Salac, I., Haneklaus, S. and Schnug, E. (2004) Sulphur supply and infection with Pyrenopeziza brassicae influence Lcysteine desulphydrase activity in Brassica napus L. J Exp Bot. 55: 2305-2312. Bloom, A.J., Asensio, J.S.R., Randall, L., Rachmilevitch, S., Cousins, A.B. and Carlisle, E.A. (2012) CO2 enrichment inhibits shoot nitrate assimilation in C3 but not C4 plants and slows growth under nitrate in C3 plants. Ecol Soc Am. 93: 355-367. Bloom, A.J., Burger, M., Asensio, J.S.R. and Cousins, A.B. (2010) Carbon dioxide enrichment inhibits nitrate assimilation in wheat and Arabidopsis. Science. 328: 899-903.

Kok, L.J. (2004) Regulation of sulphate uptake and expression of sulphate transporter genes in Brassica oleracea as affected by atmospheric H2S and pedospheric sulphate nutrition. Plant Physiol. 136: 3396-3408. Carter, T.R., Jones, R.N., Lu, X., Bhadwal, S., Conde, C., Mearns, L.O., O’Neill, B.C., Rounsevell, M.D.A. and Zurek, M.B. (2007) New assessment methods and the characterization of future

conditions. In Climate change 2007: impacts, adaptation and vulnerability. Contribution of working group II to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change. Edited by Parry, M.L., Canziani, O.F., Palutikof, J.P., van der Linden, P.J. and Hanson, C.E. pp. 133-171. Cambridge University Press, Cambridge. Charron, C.S. and Sams, C.E. (2004) Glucosinolare content and myrosinase activity in rapid-cycling Brassica olearacea grown in a controlled environment. J Am Soc Hort Sci. 129: 321-330. Charron, C.S., Saxton, A.M. and Sams, C.E. (2005) Relationship of climate and genotype to seasonal variation in the glucosinolate-myrosinase system. I. Glucosinolate content in ten cultivars of Brassica oleracea grown in fall and spring seasons. J Sci Food Agric. 85: 671-681. Cobbett, C.S., May, M.J., Howden, R. and Rolls, B. (1998) The glutathione-deficient, cadmiumsensitive mutant, cad2-1, of Arabidopsis thaliana is deficient in g-glutamylcysteine synthetase. Plant J. 16: 73-78. Cotrufo, M.F., Ineson, P. and Scott, A. (1998) Elevated CO2 reduces the nitrogen concentration of plant tissues. Global Change Biol. 4: 43-54.

25


Buchner, P., Elisabeth, C., Stuiver, E., Westerman, S., Wirtz, M., Hell, R., Hawkesford, M.J. and De

Craigon, J., Fangmeier, A., Jones, M., Donnelly, A., Bindi, M., De Temmerman, L., Persson, K. and Ojanpera, K. (2002) Growth and marketable-yield responses of potato to increased CO2 and ozone. Eur J Agron. 17: 273-289. Dadivian, J.C. and Kopriva, S. (2010) Regulation of sulphate uptake and assimilation-the same or not the same?. Mol Plant 3: 314-325. De la Mata, L., De la Haba, P., Alamillo, J.M., Pineda, M. and Agüera, E. (2013) Elevated CO2 concentrations alter nitrogen metabolism and accelerate senescence in sunflower (Helianthus annuus L.) plants. Plant Soil Environ. 59: 303-308.

CO2 concentration. J Plant Physiol. 170: 283-290. Dobousset, L., Abbdallah, M., Desfeux, A.S., Etienne, P., Meuriot, F., Hawkesford, M.J., Gombert, J., Ségura, R., Bataillé, M.P., Rezé, S., Bonnefoy, J., Ameline, A.F., Ourry, A., Le Dily, E. and Avice, J.C. (2009) Remobilization of leaf S compounds and senescence in response to restricted sulphate supply during the vegetative stage of oilseed rape are affected by mineral N availability. J Exp Bot 60: 3293-3253. Dobousset, L., Etienne, P. and Avice, J.C. (2010) Is the remobilization of S and N reserves for seed filling of winter oilseed rape modulated by sulphate restrictions occurring at different growth stages? J Exp Bot. 61: 4313-4324. Dominguez, J., Gutierrez, F., Leon, R., Vilchez, C., Vega, J. and Vigara, J. (2003) Cadmium increase the activity levels of glutamate dehydrogenase and cysteine synthase in Chlamydomonas reinhardtii. Plant Physiol Bioch. 41: 828-832. Dominguez-Perles, R., Martinez-Ballesta, M.C., Carvajal, M., García-Viguera, C. and Moreno, D.A. (2010) Broccoli-derives by products-A promising source of bioactive ingredients. J Food Sci. 75: C383-C392. Dominguez-Perles, R., Moreno, D.A., Carvajal, M. and Garcia-Viguera, C. (2011) Composition and antioxidant capacity of a novel beverage produced with green tea and minimally-processed byproducts of broccoli. Innov Food Sci Emerg. 12: 361-368. Dominguez-Solis, J.R., Gutierrez-Alcala, G., Vega, J.M., Romero, L.C. and Gotor, C. (2001) The cytosolic O-acetylserine(thiol)lyase gene is regulated by heavy metals and can function in cadmium tolerance. J Biol Chem. 276: 9297-9302.

26


del Amor, F.M. (2013) Variation in the leaf δ13C is correlated with salinity tolerance under elevated

Fediuc, E., Lips, S.H. and Erdei, L. (2005) O-acetylserine (thiol) lyase activity in Phragmites and Typha plants under cadmium and NaCl stress conditions and the involvement of ABA in the stress response. J Plant Physiol. 162: 865-872. Gaitonde, M.K. (1967) A spectrophotometric method for the direct determination of cysteine in the presence of other naturally occurring amino acids. Biochem J. 104: 627. Gerber, S., Joos, F. and Prentice, I.C. (2004) Sensitive of a dynamic global vegetation model to climate and atmospheric CO2. Glob Chang Biol. 10: 1223-1239. Habash, D., Paul, M., Parry, M.A.J., Keys, A.J. and Lawlor, D.W. (1995) Increased capacity for

and carbon metabolism. Planta. 197: 482-489. Harms, K., von Ballmoos, P., Brunold, C., Hofgen, R. and Hesse, H. (2000) Expression of a bacterial serine acetyltransferase in transgenic potato plants leads to increased levels of cysteine and glutathione. Plant J. 22: 335-343. Hasegawa, P.M., Bressan, R.A., Zhu, J.K. and Bohnert, H.J. (2000) Plant cellular and molecular responses to high salinity. Annu Rev Plant Physiol Plant Mol Biol. 51: 463-499. Hesse, H., Nikiforova, V., Gakiere, B. and Hoefgen, R. (2004) Molecular analysis and control of cysteine biosynthesis: integration of nitrogen and sulphur metabolism. J Exp Bot. 55: 12831292. Himanen, S.J., Nissinen, A., Auriola, S., Poppy, G.M., Stewart, C.N., Holopainen, J.K. and Nerg, A.M. (2008) Constitutive and herbivore-inducible glucosinolate concentrations in oilseed rape (Brassica napus) leaves are not affected by Bt Cry1Ac insertion but change under elevated atmospheric CO2 and O3. Planta. 227: 427-437. Karowe, D.N., Seimens, D.H. and Mitchell-Olds, T. (1997) Species-specific response of glucosinolate content to elevated atmospheric CO 2. J Chem Ecol. 23: 2569-2582. Kataoka, T., Watanabe-Takahashi, A., Hayashi, N., Ohnishi, M., Mimura, T., Buchner, P., Hawkesford, M.J., Yamaya, T. and Takahashi, H. (2004c) Vacuolar sulphate transporters are essential determinatns controlling internal distribution of sulphate in Arabidopsis. Plant Cell. 16: 26932704. Keys, A.J., Bird, I.F., Cornelius, M.J., Lea, P.J., Miﬂin, B.J. and Wallsgrove, R.M. (1978) Photorespiratory nitrogen cycle. Nature. 275: 741-743.

27


photosynthesis in wheat grown at elevated CO2: the relationship between electron transport

Khan, N.A., Nazar, R. and Anjum, N.A. (2009) Growth, photosynthesis and antioxidant metabolism in mustard (Brassica juncea L.) cultivars differing in ATP-Sulfurylase activity under salinity stress. Sci Hort. 122: 455-460. Kim, H.J., Chen, F., Wang, X. and Choi, J.H. (2006) Effect of methyl jasmonate on phenolics, isothiocyanate, and metabolic enzymes in radish sprout (Raphanus sativus L.). J Agr Food Chem. 54: 7263-7269. Kimura, Y. and Kimura, H. (2004) Hydrogen sulﬁde protects neurons from oxidative stress. The FASEB J. 18: 1165-1167.

constitutive glucosinolate levels of Brassica plants and affects the performance of specialized herbivores from contrasting feeding guilds. J Chem Ecol. 39: 653-665. Kocsy, G., Szalai, G. and Galiba, G. (2004) Effect of osmotic stress on glutathione and hydroxymethylglutathione accumulation in wheat. J Plant Physiol. 161: 785-794. Krupa, S. (2003) Atmosphere and agriculture in the new millennium. Environ Pollut. 126: 293-300. La, G.X., Fang, P., Teng, Y.B., Li, Y.J. and Lin, X.Y. (2009) Effect of CO2 enrichment on the glucosinolate contents under different nitrogen levels in bolting stem of chinese kale (Brassica alboglabra L.). J Zhejiang Univ-Sc B. 10: 454-464. Lappartient, A.G. and Touraine, B. (1996) Demand-driven control of root ATP sulfurylase activity and SO4 22 uptake in intact canola: the role of phloem-translocated glutathione. Plant Physiol. 111: 147-157. Leustek, T., Murillo, M. and Cervantes, M. (1994) Cloning of cDNA encoding ATP sulfurylase from Arabidopsis thaliana by functional expression in Saccharomyces cerevisiae. Plant Physiol. 105: 897-902. Levine, L.H. and Paré, P.W. (2009) Antioxidant capacity reduced in scallions grown under elevated CO2 independent of assayed light intensity. Adv Space Res. 44: 887-894. Li, X. and Kushad, M.M. (2004) Correlation of glucosinolate content to myrosinase activity in horseradish (Armoracia rusticana). J Agr Food Chem. 52: 6950-6955. López-Berenguer, C., Carvajal, M., García-Viguera, C. and Alcaraz C. F. (2009) Nitrogen, phosphorus, and sulfur nutrition in Broccoli plants grown under salinity. J Plant Nutr. 30:18551870.

28


Klaiber, J., Dorn, S. and Najar-Rodriguez, A.J. (2013) Acclimation to elevated CO2 increases

López-Berenguer, C., Martínez-Ballesta, M.D. and Moreno, D.A. (2009) Growing hardier crops for better health: salinity tolerance and the nutritional value of broccoli. J Agr Food Chem. 57: 572-578. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Fohn phenol reagent. J Biol Chem. 193: 265-275. Mansour, M.M.F., Al-Mutawa, M.M., Salama, K.H.A. and Abou Hadid, A.M.F. (2001) Salt acclimation of wheat sensitive cultivar by polyamines. In Proceedings of International Seminar on Prospects for Saline Agriculture, Edited by Ahmad, R. and Malik, K.A. pp. 155-160. Islamabad.

Plants aquaporins: New perspectives on water and nutrient uptake in saline environment. Plant Biol. 8: 535-546. Martinez-Sanchez, A., Allende, A., Bennett, R.N., Ferreres, F. and Gil, M.I. (2006) Microbial, nutritional and sensory quality of rocket leaves as affected by different sanitizers. PostharVest Biol Technol. 42: 86-97. Mavrogianopoulos, G.N., Spanakis, J. and Tsikalas, P. (1999) Effect of carbon dioxide enrichment and salinity on photosynthesis and yield in melon. Sci Hortic-Amsterdam. 79: 51-63. Mikkelsen, M.D., Petersen, B., Olsen, C. and Halkier, B.A. (2002) Biosynthesis and metabolic engineering of glucosinolates. Amino Acids. 22: 279-295. Munns, R. (2002) Comparative physiology of salt and water stress. Plant Cell Environ. 25: 239-250. Nakamura, K., Hayama, A., Masada, M., Fukushima, K. and Tamura, G. (1987) Measurement of serine acetyltransferase activity in crude plant extracts by a coupled assay system using cysteine synthase. Plant Cell Physiol. 28: 885-891. Nazar, R., Iqbal, N., Masood, A., Syeed, S. and Khan, N.A. (2011b) Understanding the significance of sulfur in improving salinity tolerance in plants. Environ Exp Bot. 70: 80-87. Neumann, P.M. (1997) Salinity resistance and plant growth revisited. Plant Cell Environ. 20: 11931199. Noctor, G., Arisi, A.C.M., Jouanin, L. and Foyer, C.H. (1999) Photorespiratory glycine enhances glutathione accumulation in both the chloroplastic and cytosolic compartments. J Exp Bot. 50: 1157-1167.

29


Martinez-Ballesta, M.C., Silva, C., López-Berenguer, C., Cabañero, F.J. and Carvajal, M. (2006)

Noctor, G., Arisi, A.C.M., Jouanin, L., Kunert, K.J., Rennenberg, H. and Foyer, C.H. (1998) Glutathione: biosynthesis, metabolism and relationship to stress tolerance explored in transformed plants. J Exp Bot. 49: 623-647. Noctor, G., Arisi, A.C.M., Jouanin, L., Valadier, M.H., Roux, Y. and Foyer, C.H. (1997) The role of glycine in determining the rate of glutathione synthesis in poplar: possible implications for glutathione production during stress. Physiol Plant. 100: 255-263. Noji, M. and Saito, K. (2002) Molecular and biochemical analysis of serine acetyltransferase and cysteine synthase towards sulfur metabolic engineering in plants. Amino Acids. 22: 231-243.

warming in a multi-factor world. New Phytol. 162: 281-293. Norby, R.J., O'Neill, E.G., Hood, W.G. and Luxmoore, R.J. (1987) Carbon allocation, root exudation and mycorrhizal colonization of Pinus echinata seedlings grown under CO2 enrichment. Tree Physiol. 3: 203-210. Padilla, G., Cartea, M.E. and Velasco, P. (2007) Variation of glucosinolates in vegetable crops of Brassica rapa. Phytochemistry. 68: 536-545. Palmieri, S., Leoni, O. and Iori, R. (1982) A steady state kinetics study of myrosinase with direct ultra violet spectrophotometric assay. Anal Biochem. 123: 320-324. Pataki, D.E., Bowling, D.R., Ehleringer, J.R. and Zobitz, J.M. (2006) High resolution monitoring of urban carbon dioxide sources. Geophys Res Lett. 33: 1-5. Pérez-Balibrea, S., Moreno, D.A. and García-Viguera, C. (2008) Influence of light on health-promoting phytochemicals of broccoli sprouts. J Sci Food and Agr. 88: 904-910. Perez-Lopez, U., Miranda-Apocada, J., Mena-Petite, A., Muñoz-Rueda, A. (2013) Barley growth and its underlying components are affected by elevated CO2 and salt concentration. J Plant Growth Regul. 32: 732-744. Perez-Lopez, U., Robredo, A., Lacuesta, M., Mena-Petite, A., Muñoz-Rueda, A. (2012) Elevated CO2 reduces stomatal and metabolic limitations on photosynthesis caused by salinity in Hordeum vulgare. Photosynth Res. 111: 269-283. Pérez-López, U., Robredo, A., Lacuesta, M., Sgherri, C., Mena-Petite, A., Navari-Izzo, F. and MuñozRueda, A. (2010) Lipoic acid and redox status in barley plants subjected to salinity and elevated CO2. Physiol Plantarum. 139: 256-268.

30


Norby, R.J. and Luo, Y. (2004) Evaluating ecosystem responses to rising atmospheric CO2 and global

Pérez-López, U., Robredo, A., Lacuesta, M., Sgherri, C., Munoz-Rueda, A., Navari-Izzo, F. and MenaPetite, A. (2009) The oxidative stress caused by salinity in two barley cultivars is mitigated by elevated CO2. Physiol Plantarum. 135: 29-42. Poorter, H. and Navas, M.L. (2003) Plant growth and competition at elevated CO2: on winners, losers and functional groups. New Phytol. 157: 175-198. Reddy, G.V.P., Tossavainen, P., Nerg, A.M. and Holopainen, J.K. (2004) Elevated atmospheric CO2 affects the chemical quality of Brassica plants and the growth rate of the specialist Plutella xylostella, but not the generalist, Spodoptera littoralis. J Agr Food Chem. 52: 4185-4191.

Rueda, A. (2011) Elevated CO2 reduces the drought effect on nitrogen metabolism in barley plants during drought and subsequent recovery. Environ Exp Bot. 71: 399-408. Rodríguez-Hernández, M.C., Moreno, D.A., Carvajal, M. and Martínez-Ballesta, M.C. (2013) Interactive effects of boron and NaCl stress on water and nutrient transport in two broccoli cultivars. Funct Plant Biol. 40: 739-748. Rogers, A. and Humphries, S.W. (2000) A mechanistic evaluation of photosynthetic acclimatation at elevated CO2. Glob Change Biol. 6: 1005-1011. Romero, L.C., Domínguez-Solís, J.R., Gutiérrez-Alcalá, G. and Gotor, C. (2001) Salt regulation of Oacetylserine(thiol)lyase in Arabidopsis thaliana and increased tolerance in yeast. Plant Physiol Bioch. 39: 643-647. Rosa, E.A.S. and Rodrigues, A.S. (2001) Total and individual glucosinolate content in 11 broccoli cultivars grown in early and late seasons. HortScience. 36: 56-59. Rosa, E.A.S., Heaney, R.K., Portas, C.A.M. and Fenwick, G.R. (1996) Changes in glucosinolate concentrations in brassica crops (Brassica oleracea and Brassica napus) through growth seasons. J Sci Food Agr. 71: 237-244. Rouhier, N., Lemaire, S.D. and Jacquot, J.P. (2008) The Role of Glutathione in Photosynthetic Organisms: Emerging Functions for Glutaredoxins and Glutathionylation. Annu Rev Plant Biol. 59: 143-166. Rüegsegger, A. and Brunold, C. (1992) Effect of cadmium on ϒ-glutamylcysteine synthesis in maize seedlings. Plant Physiol. 99: 428-433.

31


Robredo, A., Pérez-López, U., Miranda-Apodaca, J., Lacuesta, M., Mena-Petite, A. and Muñoz-

Ruiz, J.M. and Blumwald, E. (2002) Salinity-induced glutathione synthesis in Brassica napus. Planta. 214: 965-969. Schiavon, M., Pittarello, M., Pilon-Smits, E.A.H., Wirtz, M., Hell, R. and Malagoli, M. (2012) Selenate and mlybdate alter sulphur transport and assimilation in Brassica juncea L. Czern.: Implications for phytoremediation. Environ Exp Bot. 75: 41-51. Schonhof, I., Kläring, H.P., Krumbein, A. and Schreiner, M. (2007) Interaction between atmospheric CO2 and glucosinolates in broccoli. J Chem Ecol. 33: 105-114. Schulte, M., Von Ballmoos, P., Rennenberg, H. and Herschbach, C. (2002) Life-Long growth of

metabolism of the progeny to elevated pCO2. Plant Cell Environ. 25: 1715-1727. Senerweera, S., Makino, A., Hirotsu, N., Norton, R. and Suzuki, Y. (2011) New insights into photosynthetic acclimation to elevated CO2: the role of leaf nitrogen and ribulose-1,5biphosphate carvboxylase/oxygenase content in rice leaves. Environ Exp Bot. 71: 128-136. Stepien, P. and Johnson, G.N. (2009) Contrasting responses of photosynthesis to salt stress in the glycophyte Arabidopsis and the halophyte Thellungiella: role of the plastid terminal oxidase as an alternative electron sink. Plant Physiol. 149: 1154-1165. Stuiver, C.E.E. and De Kok, L.J. (2001) Atmospheric H2S as sulfur source for Brassica oleracea: kinetics of H2S uptake and activity of Oacetylserine (thiol)lyase as affected by sulfur nutrition. Environ Exp Bot. 46: 29-36. Taub, D.R. and Wang, X.Z. (2008) Why are nitrogen concentrations in plant tissues lower under elevated CO2?. A critical examination of the hypothesis. J Integr Plant Biol. 50: 1365-1374. Tausz, M., Tausz-Posch, S., Norton, R.M., Fitzgeral, G.J., Nicolas, M.E. and Seneweer, S. (2013) Understanding crop physiology to select breeding targets and improve crop management under increasing atmospheric CO2 concentrations. Environ Exp Bot. 88: 71-80. Uren, N.C. and Reisenauer, H.M. (1988) The role of root exudates in nutrient acquisition. Adv Plant Nutr. 3: 79-114. Wirtz, M. and Hell, R. (2006) Functional analysis of the cysteine synthase protein complex from plants: Structural, biochemical and regulatory properties. J Plant Physiol. 163: 273-286. Wirtz, M. and Hell, R. (2007) Dominant-negative modification reveals the regulatory function of the multimeric cysteine synthase protein complex in transgenic tobacco. Plant Cell. 19: 625-639.

32


Quercus ilex L. at natural CO2 springs acclimates sulphur, nitrogen and carbohydrate

Wszelaki, A. and Kleinhenz, M.D. (2003) Yield and relationships among head traits in cabbage as influenced by planting date and cultivar. Hortscience. 38: 1355-1359. Yan, X.F. and Chen, S.X. (2007) Regulation of plant glucosinolate metabolism. Planta 226: 13431352. Yeo, A. (1998) Molecular biology of salt tolerance in the context of whole-plant physiology. J Exp Bot. 49: 915-929. Yuan, G.F., Sun, B., Yuan, J. and Wang, Q.M. (2009) Effects of different cooking methods on healthpromoting compounds of broccoli. J Zhejiang Univ-Sc B. 10: 580-588.

(2013) Elevated CO2 alleviates negative effects of salinity on broccoli (Brassica oleracea L. var Italica) plants by modulating water balance through aquaporins abundance. Environ Exp Bot. 95: 15-24. Zechmann, B., Müller, M., and Zellnig, G. (2008) Modified levels of cysteine affect glutathione metabolism in plant cells. In Sulfur Assimilation and Abiotic Stress in Plants. Edited by Khan, N.A., Singh, S. and Umar, S. pp. 193-206. Springer Verlag, Berlin. Zechmann, B., Zellnig, G., Urbanek-Krajnc, A. and Müller, M. (2007) Artificial elevation of glutathione affects symptom development in ZYMV-infected Cucurbita pepo L. plants. Arch Virol. 152: 747-762. Zhu, J.K. (2002) Salt and drought stress signal transduction in plants. Annu Rev Plant Physiol Plant Mol Biol. 53: 247-273.

33


Zaghdoud, C., Mota-Cadenas, C., Carvajal, M., Muries, B., Ferchichi, A. and Martínez-Ballesta, MC.

Table 1. Biomass (g DW plant-1), shoot/root biomass ratio, dry matter (g) and soluble protein (mg g-1 DW) content in the broccoli plants grown under non-saline (0mM NaCl) and saline (80 mM NaCl) conditions for 2 weeks at ambient [CO2] (380 ppm) or elevated [CO2] (800 ppm) differentiating between cultivars (cvs. Naxos and Viola).

CO2 (ppm)

Biomass

Shoot/root biomass ratio

Dry matter

Soluble protein content

0

380

2.80±0.20b

5.12±0.86aa

8.07±0.14a

11.91±1.1.8b

800

4.16±0.36a

5.04±0.41a

10.75±1.67a

8.92±0.7c

380

2.06±0.23c

4.92±0.17a

8.86±0.11a

15.77±1.3a

800

3.02±0.40b

5.02±0.89a

9.49±1.23a

16.94±1.4a

Naxos 80

** ** ns

ns ns ns

ns ns ns

*** * *

380

3.05±0.30b

3.48±0.46a

6.74±0.55bc

14.48±1.1b

800

4.73±0.25a

5.01±1.07a

8.05±0.39b

13.71±1.1b

380

1.564±0.10c

3.56±0.70a

5.47±0.17c

20.33±1.8a

800

3.35±0.22b

5.49±0.44a

9.62±0.47a

8.68±0.9c

NaCl CO2 NaCl x CO2 0 Viola 80

b

NaCl *** ns ns *** CO2 *** * *** * NaCl x CO2 ns ns * * a Different letters are statistically different (Tukey, P 0.05 (ns), significant at P< 0.05 (*), P< 0.01 (**) and P< 0.001 (***).

34


NaCl (mM)

Table 2 Sulphate (SO42-), sulphur (S), sodium (Na+), chloride (Cl-), nitrate (NO3-), carbon (C) nitrogen (N) (µmol g-1 DW) and the C/N and N/S ratios, in the broccoli leaves of plants grown under non-saline (0mM NaCl) and saline (80 mM NaCl) conditions for 2 weeks at ambient [CO2] (380 ppm) or elevated [CO2] (800 ppm) differentiating between cultivars (cvs. Naxos and Viola). NaCl

CO2

(mM)

(ppm)

0

Naxos 80

Viola 80

S total

Na

+

Cl

-

NO3

-

C

N

N/S

5.2±0.8b

21.4±3.1b

10.2±2.8ba

185.3±20.6b

30.3±4.6.b

12.6±1.8b

419.4±40.3a

20458 103±2250c

800

13.8±3.4b

284.5.5±40a

20.5±4.6b

14.6±1.7b

179.0±43.5b

38608 10 ±3227a

3242 10 ±207b

11.01±1.2a

10.2±1.1c

380

14.5±4.0b

136.2±22.3b

829.3±68.2a

52.7±4.5a

272.6±14.5b

29350 103±2305b

3314 103±172b

7.91±0.9b

26.2±2.1a

800

22.2±4.3a

210.6±35.4ab

963.2±40.5a

48.9±3.8a

211.56±38.7b

39433 103±2401a

3325 103±184b

12.02±0.4a

17.4±2.2b

ns *** ns

*** ns ns

*** ns ns

** *** ns

* ** ns

** ** ns

ns *** ns

*** * ns

3

5.3±0.07b

25.8±2.3a

3

b

*** *** ns

3

4471 103±214a

C/N

380

NaCl CO2 NaCl x CO2 0

2-

SO4

3

3

4622 10 ±43.8a

33758 10 ±2769a

3

4587 10 ±124a

6.2±0.9b

15.86±1.0b

201.6±25.8a

26175 103±1806b

4288 103±67.7a

6.0±0.03b

15.09±0.6b

79.7±12.9b

34466 10 ±2416a

3261 10 ± 205b

11.0 ±0.5a

9.13±1.8c

380

9.2±1.9c

170.6±15.6b

37.5±4.6c

13.2±2.9b

209.7±32.2a

24683 10 ±2315 b

800

14.3±2.8b

280.9±18.5a

25.3±3.9c

18.5±4.7b

272.6±45.2a

380

13.4±1.9b

272.6±14.4a

1010.2±93.6b

19.8±2.9b

800

19.9±4.3a

299.3±18.8a

1520.2±96.5a

66.3±5.8a

3

3

NaCl

***

***

***

***

**

ns

***

***

***

CO2

***

**

**

***

**

***

**

***

**

NaCl x CO2

ns

***

**

**

ns

ns

ns

**

***

a

Different letters are statistically different (Tukey, P 0.05 (ns), significant at P< 0.05 (*), P< 0.01 (**) and P< 0.001 (***).

35


-1

Table 3 Individual glucosinolates (mg 100g FW), in the broccoli leaves of plants grown under non-saline (0 mM NaCl) and saline (80 mM NaCl) conditions for 2 weeks at ambient [CO2] (380 ppm) or elevated [CO2] (800 ppm) differentiating between cultivars (cvs. Naxos and Viola). NaCl (mM)

CO2 (ppm)

Aliphatic glucosinolates GI

GB

MGB

HGB c

NGB

a

0.19±0.02b

3.02±0.70b

2.10±0.10c

n.d.

1.2±0.03b

0

380

0.51±0.02b

80

800 380

5.68±1.44b 0.62±0.07b

0.37±0.08b 0.35±0.04b

10.22±1.56b 5.12±0.09b

6.85±1.25b 3.70±0.30c

0.90±0.22c 4.78±0.53b

9.78±1.90a 1.42±0.30b

800

29.10±7.40a

0.82±0.12a

23.42±5.55a

14.98±0.84a

8.32±0.93a

11.23±2.05a

** *** **

*** *** ns

* *** ns

*** *** ***

*** *** *

ns *** ns

3.02±0.90b

0.53±0.05b

0.20±0.0b

6.84±2.40ab

0.40±0.05b

2.48±0.51a

Naxos

b

NaCl CO2 NaCl x CO2 0 Viola

GE

Indolic glucosinolates

380

6.05±0.37b 0.54±0.09b 1.22±0.62b 11.42±0.85a 3.15±0.97ab 3.92±1.52a 80 10.20±1.14b 0.620±0.120b 0.24±0.08b 3.03±0.82b 1.70±0.20ab 8.34±1.96a 49.62±2.93a 4.12±0.98a 13.94±1.50a 8.96±1.82ab 12.05±1.31a 13.05±2.25a NaCl *** ** *** ns * * CO2 *** ** *** * * ns *** ** *** ns ns ns NaCl x CO2 GI: Glucoiberin, GE: Glucoerucin, GB: Glucobrassicin, MGB: 4-MeO-Glucobrassicin, HGB: 4-OH-Glucobrassicin, NGB: Neoglucobrassicin.(F.W.=Fresh weight) a Different letter are statistically different (Tukey, P 0.05 (ns), significant at P< 0.05 (*), P< 0.01 (**) and P< 0.001 (***). c (n.d. = not detected). 800 380 800

36


Table 4 Heat map of a selected set of enzymes related to the different synthesis and degradation pathways. The different enzymatic activities OAS-TL and SAT (mmol Cys mg protein-1 min-1), ATPS, GS and γ-ECS (mmol Pi mg protein-1 min-1) and Myrosinase (U g−1 FW)were measured in the leaves of broccoli plants grown under non-saline (0mM NaCl) and saline (80 mM NaCl) conditions for 2 weeks ,at ambient [CO2] (380 ppm) or elevated [CO2] (800 ppm) differentiating between cultivars (cvs. Naxos and Viola). NaCl (mM)

CO2 (ppm)

S-derived metabolites synthesis OAS-TL 0.7d

ATPS 0.17d

γ-ECS 0.09d

GS 4.32c

Glucosinolate hydrolysis Myrosinase 0.32a

1.45c 2.46b

2.95b 1.36c

0.27b 0.18c

5.76b 6.50b

0.18b 0.13c

4.26a

4.62a

0.36a

7.01a

0.09d

*** ***

***

***

***

***

***

***

***

***

***

***

380

*** 1.65c

*** 0.62d

* 0.17d

ns 0.01c

ns 3.21c

ns 0.32a

800

2.03b

2.01b

5.26b

0.08b

5.86b

0.15c

380

2.25b

1.02c

1.35c

0.09b

5.20b

0.17b

800

3.51a

3.74a

5.74a

0.28a

7.39a

0.08d

NaCl

***

***

***

***

***

***

CO2

***

***

***

***

***

***

SAT 0 Naxos 80

1.42c 2.01b 2.16b

800

3.56a b

NaCl

CO2 NaCl x CO2 0

Viola 80

a

380 800 380

*** *** ns *** ns *** NaCl x CO2 SAT: serine acetyltransferase, OAS-TL: O-acetyl-L-serine(thiol)lyase, ATPS: ATP sulphurylase, γ-ECS: γ-Glutamylcysteine synthetase, GS: Glutathione synthetase. a Different letter are statistically different (Tukey, P 0.05 (ns), significant at P< 0.05 (*), P< 0.01 (**) and P< 0.001 (***).

37


Table 5 Amino acids concentration and glutathione (µmol g-1 FW) in the broccoli leaves of plants grown under non-salinized (0 mM NaCl) and salinized (80 mM NaCl) conditions for 2 weeks at ambient [CO2] (380 ppm) or elevated [CO2] (800 ppm) differentiating between cultivars (cvs. Naxos and Viola).

NaCl (mM)

CO2 (ppm)

CYS

0

380 800

80

380 800

Naxos

NaCl CO2 NaCl x CO2 0 Viola

380 800

GLU

MET

TRP

SER

GSH

0.92±0.03c

1.44±0.05c

0.06±0.01c

0.01±0.01c

0.09±0.02ab

0.53±0.09c

1.70±0.12b 1.57±0.15b 2.96±0.04a b *** *** * 1.10±0.10c

2.63±0.16bc 3.36±0.43b 5.59±1.12a *** ** ns 1.82±0.05c

0.12±0.01b 0.11±0.01b 0.23±0.04a *** *** * 0.06±0.03c

0.09±0.01b 0.09±0.01b 0.20±0.02a *** *** ns 0.01±0.003c

0.09±0.01ab 0.13±0.02a 0.06±0.01b ns * ns 0.02±0.01b 0.02±0.01b

0.68±0.06c 1.35±0.13b 1.94±0.11a *** * * 0.56±0.09b 0.63±0.04b

a

1.44±0.03bc 4.98±0.30b 0.39±0.04b 0.32±0.02b 0.08±0.02a 1.01±0.18a 1.69±0.18b 2.22±0.41c 0.15±0.02c 0.09±0.005c 2.58±0.10a 7.05±0.20a 0.72±0.06a 0.55±0.04a 0.04±0.02b 1.30±0.18a NaCl *** ** *** *** ** ** CO2 * ns *** *** *** *** NaCl x CO2 ns * ns ns * ns CYS: Cysteine, GLU: Glutamic acid, MET: Methionine, TRP: Tryptophan, SER: Serine, GSH: Glutathione. a Different letter are statistically different (Tukey, P 0.05 (ns), significant at P< 0.05 (*), P< 0.01 (**) and P< 0.001 (***). 380 800

38


80

LEGENDS TO FIGURES -1

Fig. 1 Biomass production (g DW plant ) of broccoli plants grown under non-salinized (0 mM NaCl) and salinized (80 mM NaCl) conditions for 2 weeks at ambient [CO2] (380 ppm) or elevated [CO2] (800 ppm) differentiating between cultivars (cvs. Naxos and Viola). Bars with different letter are statistically different (Tukey, P

Selenium-Induced Toxicity Is Counteracted by Sulfur in Broccoli (Brassica oleracea L. var. italica).

Chromosome Doubling of Microspore-Derived Plants from Cabbage (Brassica oleracea var. capitata L.) and Broccoli (Brassica oleracea var. italica L.).

Ultrasound assisted immersion freezing of broccoli (Brassica oleracea L. var. botrytis L.).

Efficient plant regeneration from hypocotyl protoplasts of broccoli (Brassica oleracea L. ssp. italica Plenck).

Agrobacterium tumefaciens-mediated transformation of broccoli (Brassica oleracea var. italica) and cabbage (B. oleracea var. capitata).

Changes in C-N metabolism under elevated CO2 and temperature in Indian mustard (Brassica juncea L.): an adaptation strategy under climate change scenario.

Sulfur Protects Pakchoi (Brassica chinensis L.) Seedlings against Cadmium Stress by Regulating Ascorbate-Glutathione Metabolism.

Variation in bioactive content in broccoli (Brassica oleracea var. italica) grown under conventional and organic production systems.

RNA-seq analysis of transcriptome and glucosinolate metabolism in seeds and sprouts of broccoli (Brassica oleracea var. italic).

The effects of UV radiation during the vegetative period on antioxidant compounds and postharvest quality of broccoli (Brassica oleracea L.).

Genome-Wide Identification and Characterization of bZIP Transcription Factors in Brassica oleracea under Cold Stress.

Effect of cooking on the concentration of bioactive compounds in broccoli (Brassica oleracea var. Avenger) and cauliflower (Brassica oleracea var. Alphina F1) grown in an organic system.

Distribution of S-alleles and breeding structure of Cape broccoli (Brassica oleracea var.'italica').

Compositional and proteomic analyses of genetically modified broccoli (Brassica oleracea var. italica) harboring an agrobacterial gene.

Plant regeneration from leaf protoplasts of Brassica oleracea var. italica CV Green Comet broccoli.

Segregation of genetic markers among plants regenerated from cultured anthers of broccoli (Brassica oleracea var. 'italica').

Effect of water content and temperature on inactivation kinetics of myrosinase in broccoli (Brassica oleracea var. italica).

Ultrasonic-assisted enzymatic extraction of phenolics from broccoli (Brassica oleracea L. var. italica) inflorescences and evaluation of antioxidant activity in vitro.

Metabolic differentiation of diamondback moth ( Plutella xylostella (L.)) resistance in cabbage ( Brassica oleracea L. ssp. capitata).

Evaluation of different cooking conditions on broccoli (Brassica oleracea var. italica) to improve the nutritional value and consumer acceptance.

Extracts from Brassica oleracea L. convar. acephala var. sabellica inhibit TNF-α stimulated neutrophil adhesion in vitro under flow conditions.

Detection of the Diversity of Cytoplasmic Male Sterility Sources in Broccoli (Brassica Oleracea var. Italica) Using Mitochondrial Markers.

Comparative Transcriptome Analysis between Broccoli (Brassica oleracea var. italica) and Wild Cabbage (Brassica macrocarpa Guss.) in Response to Plasmodiophora brassicae during Different Infection Stages.

Health-promoting compounds of broccoli (Brassica oleracea L. var. italica) plants as affected by nitrogen fertilisation in projected future climatic change environments.