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Plant Physiology and Biochemistry 48 (2010) 764e771

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Plant Physiology and Biochemistry

journal homepage: www.elsevier .com/locate/plaphy

Research article

Metabolic response to cold and freezing of Osteospermum ecklonisoverexpressing Osmyb4

Marina Laura a,1, Roberto Consonni b,1, Franca Locatelli c, Elisabetta Fumagalli b, Andrea Allavena a,Immacolata Coraggio c, Monica Mattana c,*

aUnità di Ricerca per la Floricoltura e le Specie Ornamentali, Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Corso Degli Inglesi 508, 18038 Sanremo (IM), Italyb Istituto per lo Studio delle Macromolecole, Laboratorio NMR, Consiglio Nazionale delle Ricerche, Via Bassini 15, 20133 Milano, Italyc Istituto di Biologia e Biotecnologia Agraria, CNR, Via Bassini 15, 20133 Milano, Italy

a r t i c l e i n f o

Article history:Received 6 October 2009Accepted 10 June 2010Available online 18 June 2010

Keywords:Cold toleranceFreezing toleranceNMR spectroscopyOsmyb4OsteospermumTranscription factor

Abbreviations: BAP, 6-benzylaminopurine; IAA,sodium trimethylsilyl [2,2,3,3e2H4] propionate.* Corresponding author. Tel.: þ39 0223699677; fax

E-mail address: mattana@ibba.cnr.it (M. Mattana)1 The two authors contributed equally to this work

0981-9428/$ e see front matter � 2010 Elsevier Masdoi:10.1016/j.plaphy.2010.06.003

a b s t r a c t

The constitutive expression of the rice Osmyb4 gene in Arabidopsis plants gives rise to enhanced abioticand biotic stress tolerance, probably by activating several stress-inducible pathways. However, the effectof Osmyb4 on stress tolerance likely depends on the genetic background of the transformed species.

In this study, we explored the potential of Osmyb4 to enhance the cold and freezing tolerance ofOsteospermum ecklonis, an ornamental and perennial plant native to South Africa, because of anincreasing interest in growing this species in Europe where winter temperatures are low.

Transgenic O. ecklonis plants were obtained through transformation with the Osmyb4 rice gene underthe control of the CaMV35S promoter.

We examined the phenotypic adaptation of transgenic plants to cold and freezing stress. We alsoanalysed the ability of wild-type and transgenic Osteospermum to accumulate several solutes, such asproline, amino acids and sugars. Using nuclear magnetic resonance, we outlined the metabolic profile ofthis species under normal growth conditions and under stress for the first time. Indeed, we found thatoverexpression of Osmyb4 improved the cold and freezing tolerance and produced changes in metaboliteaccumulation, especially of sugars and proline. Based on our data, it could be of agronomic and economicinterest to use this gene to produce Osteospermum plants capable of growing in open field, even duringthe winter season in climatic zone Z9.

� 2010 Elsevier Masson SAS. All rights reserved.

1. Introduction

Osteospermum (Compositae) is an ornamental genus native toSouth Africa. It includes species that are either evergreen subshrubsor herbaceous plants, which are characterised by vigorous growth,abundant flowers, and various habits, such as erect, prostate, ordecumbent. The flower is a capitulum with one or more rings ofligulated ray florets. While Osteospermum is an attractive spring-summer ground cover plant, it has recently been developed intoa successful flowering pot plant, with some cultivars even beingsuitable for the production of cut flowers. The economic potentialfor this species in Europe is highlighted by the number of

3-indolyl-acetic acid; TSP,

: þ39 0223699411...

son SAS. All rights reserved.

applications for community protection (an average of 49 annuallyfor the last five years, including 70 in 2005).

Genetic engineering can add novel traits to a species of interest,but it requires efficient methods for plant regeneration fromsomatic tissues and for genetic transformation. Tissue culturetechniques and transformation protocols suitable for geneticallyengineering Osteospermum species are available [10]. The riceOsmyb4 gene (accession number Y11414) has been isolated in ourlaboratory [26], and its role in cold acclimation and drought toler-ance has been shown through its overexpression in Arabidopsisthaliana plants. Transgenic Arabidopsis plants show an increasedtolerance to cold and drought stresses as well as multiplebiochemical changes commonly observed during adaptation tothese adverse environmental conditions [19,36]. Moreover, tran-scriptome comparison between wild-type (WT) and Osmyb4-expressing plants revealed that Myb4 affects the expression ofseveral genes known to be regulated by other abiotic stresses (e.g.,salt and photo-oxidation), as well as genes involved in pathogenresistance [37]. The importance of this gene in abiotic stress

M. Laura et al. / Plant Physiology and Biochemistry 48 (2010) 764e771 765

tolerance was confirmed by its overexpression in other species,such as tomato and apple. In these two species, various levels oftolerance to different stresses were induced according to thegenetic background of the transformed plant, possibly dependingon the -specific genes acting downstream of theMyb4 transcriptionfactor in the transformed species [27,38]. For example, Osmyb4-expressing tomato plants acquired a higher tolerance to droughtstress and to viral disease, but not to cold [38].

In this work, we transformed an Osteospermum heterozygousgenotype with the Osmyb4 gene to enhance cold and freezingtolerance. These traits would add value to cultivars grown in openfield during the winter season in climatic zone Z9 (minimumwinter temperature around �7 �C), such as in the northern coastalregions of the Mediterranean basin, and would possibly extendOsteospermum cultivation into climatic zone Z8 (minimumwintertemperature between �7 and �12 �C). In addition to the inducedresistance, we measured some biochemical and physiological traitsknown to be involved in the response to cold and freezing, such asthe proline and total amino acid content [31,33]. Since it has beenrecently reported that changes at transcriptomic and proteomiclevels should be reflected in the metabolome [8,32], we analysedwith 1H NMR techniques the metabolic content in untreated andcold-stressed WT and transgenic Osteospermum leaves. Through1H NMR and principal component multivariate statistical analysis(PCA), we investigated the differences in the metabolome of WTand transgenic lines, the modulation induced by different treat-ments and the contribution of individual compound to thesemodifications. This study on the metabolic profile of Osteo-spermum is here described for the first time.

Our results indicate that Osmyb4 increased both the level ofstress tolerance as well as the osmolyte accumulation in transgenicOsteospermum in comparison to the WT.

2. Materials and methods

2.1. Reagents and kits for bacterial engineering

All restriction and DNA-modifying enzymes were from NewEngland Biolabs� (Ipswich, MA, USA). The oligonucleotides andprimers were purchased from TIB MOLBIOL (Berlin, Germany).Recombinant Taq polymerase was obtained from Invitrogen (Invi-trogen, Carlsbad, CA, USA). The following kits provided by QIAGEN(Milan, Italy) were used: “QIAprep Spin Miniprep Kit” for plasmidextraction; “QIAquick Gel Extraction Kit” for DNA elution fromagarose gels; “QIAquick PCR Purification Kit” for enzyme inactiva-tion and DNA purification; and “DNeasy Plant Mini Kit” for plantDNA extraction.

2.2. Plasmid construct and transgenic plant production

The cassette containing the Myb4 coding sequence (1200 bp)under the control of the CaMV35S promoter (450 bp) and the Nosterminator (250 bp) was isolated from the plasmid pCaMVMyb4[26] by digestionwithHindIII. The obtained fragment (1900 bp) wascloned into the HindIII site of pGreenI 0029 (http://www.pgreen.ac.uk), which contains an nptII gene under the Nos promoter/termi-nator that confers kanamycin resistance. The pGreenIMyb4recombinant plasmid was introduced into Agrobacterium tumefa-ciens strain AGL1 and used to transform leaves from Osteospermumecklonis plants following the method of Giovannini et al. [10].

One hundred and fifty leaf pieces were wounded and immersedin the bacterial culture for 30 min. Explants were then brieflyblotted dry on sterile filter paper and placed on the co-cultivationmedium composed of MS medium [23], 30 g L�1 sucrose, 2 mg L�1

3-indolyl-acetic acid (IAA), 1 mg L�1 6-benzylaminopurine (BAP),

and 8 g L�1 agar. After 2e3 days, the leaf pieces were blotted andtransferred to selective regeneration medium (co-cultivationmedium with 100 mg mL�1 cefotaxime and 100 mg mL�1 kana-mycin). Non-inoculated explants were prepared as a control andgrown directly on co-cultivation medium.

Regenerated shoots (about 1.5-cm long) with a clear independentorigin on the leaf explants were excised and cultivated on propaga-tionmedium (MSmedium, 30g L�1 sucrose, 8 g L�1 agar,100 mgmL�1

cefotaxime and 100 mg mL�1 kanamycin). Each shoot representinga separate regeneration event was subsequently propagated throughaxillary shoot proliferation to provide plant clones. All in vitro plantculturesweremaintained in a growth roomat 23e25 �Cwith a 16/8 hlight/dark photoperiod. The transformed plants were transferred tosoil and grown in a containment greenhouse.

2.3. Analysis of regenerated plants

The presence of the Osmyb4 transgene in kanamycin-resistantplants was confirmed by PCR analysis. Genomic DNAwas extractedfrom leaves of in vitro-grown transgenic and control plants. Foramplification of the Osmyb4 fragment, the forward 50-AGGGAAGGAGCAAGCACAATG-30 and reverse 50-TCGGCTTCTTGTGCTTCTTGC-30 primers were used, generating a 420-bp fragment. The PCRreaction was performed in a PTC 100 thermal cycler (MJR ResearchInc., USA) with the following conditions: 5 min at 94 �C, 40 cycles at94 �C for 30 s, 60 �C for 45 s, 72 �C for 30 s; and a final extension at72 �C for 5 min.

The absence of A. tumefaciens contamination in the putativepositive plants was checked by PCR amplification of a 326-bpfragment of the virC1 gene following the method of Vaira et al. [34].To assess the presence of at least one full-length insert copy in thePCR-positive clones 466, 467, 468, 469, 470, and 471, a 1200-bpfragment spanning the entire ORF of Osmyb4 was amplified witha forward 50-CCTCCCTTCCAAGAACACAC-30 and a reverse 50-TCAAATGAGAAACGCAGAGC-30 primer.

Total RNA was extracted from WT and transgenic Osteo-spermum plants grown for 4 weeks in the greenhouse using TRIzolreagent (Invitrogen, Carlsbad, CA, USA). Two micrograms of DNase-treated RNA were retrotranscribed to first-strand cDNA with theSuperScript First-Strand Synthesis System for RT-PCR kit (Invi-trogen, Carlsbad, CA, USA). The PCR reaction was carried out withthe Osmyb4-specific primers specified above.

The samples were subjected to 30 amplification cycles with anannealing temperature of 60 �C. The amplification of 18S rDNAwasused as the control. The amplification products were separated byelectrophoresis on a 1% agarose gel.

2.4. Chilling and freezing treatments

Chilling and freezing treatments were performed on WT andtransgenic Osteospermum plants grown normally until 20 cm tall.The plants were transferred into a controlled chamber at 4 �C, witha 16/8 h light/dark regime for ten days, at which point some plantswere placed at �5 �C overnight. The leaf samples were collectedimmediately before the stress treatment (control), after two andten days at 4 �C and after the overnight freezing treatment. All leafsamples were harvested in triplicate and immediately frozen inliquid nitrogen for subsequent use for 1H NMR analysis or prolineand amino acid measurements.

2.5. High-resolution magic angle spinning 1H NMR

Leaf tissues (typically 15 mg of dry weight) were packed intoa magic angle spinning 4-mm zirconium rotor with a 50-mLspherical insert; then 200 mL of D2O were carefully added without

Fig. 1. Semi-quantitative RT-PCR of Osmyb4 gene expression in wild-type and sixindependent transgenic lines. Lane 1: wild-type plants (WT); lanes 2, 4: high-expressing plants (HE); lanes 3, 5: not-expressing plants (NE); lanes 6, 7: low-expressing plants (LE). For all RT-PCR analyses, 18S rRNA was used as a control.

M. Laura et al. / Plant Physiology and Biochemistry 48 (2010) 764e771766

introducing air bubbles. Samples were spun at 6 kHz during theexperiment. Spectra were recorded on a Bruker Avance600 spec-trometer operating at 600.13 MHz at 300 K and with a 90� protonflip angle pulse of 4 ms. Solvent suppression was achieved byapplying a pre-saturation scheme with low-power radiofrequencyirradiation [3,20].

2.6. Extraction of plant material and NMR spectra measurements

Osteospermum leaves were harvested and pooled fromdifferent plants in order to account for single plant differences. Leaftissues were ground in an ice-cold mortar with quartz sand with1 mL of H2O added for each 0.5 g of fresh weight. Samples werecentrifuged at 13 000�g for 10 min and the water-soluble fractionswere buffered at pH 6.5 with 0.5 M NaH2PO4 to avoid NMR signalshifts among samples [4,21,32]. The extracts were lyophilised anddissolved in 0.5 mL of D2O and transferred to a 5-mm NMR tube.

Multidimensional homonuclear and heteronuclear NMR exper-iments were acquired on a Bruker Advance DMX500 spectrometeroperating at 11.7 T with a proton frequency of 500.13 MHz andequipped with z-gradient coils. All spectra were recorded at 300 K,with a spectral width of 6000 Hz over 32k data points, and refer-enced to sodium trimethylsilyl [2,2,3,3e2H4] propionate (TSP).Solvent suppression was achieved by applying a pre-saturationscheme with low-power radiofrequency irradiation. Total Correla-tion Spectroscopy (TOCSY), Heteronuclear Single Quantum Coher-ence (HSQC) and Heteronuclear Multiple Bond Coherence (HMBC)experiments from Bruker standard pulse sequences were used forspin system assignment [6,9,13,30]. One-dimensional 1H NMRspectra were Fourier transformed and corrected for phase andbaseline before integration of resonance signals.

For statistical analysis 1H NMR one-dimensional spectra werereduced to integrated regions (buckets) of equal width (0.04 ppm)over a spectral region spanning from 10.5 to 0.5 ppm by an intel-ligent bucketing macro, with the exclusion of the residual watersignal. An additional manual correction of bucket size was appliedfor shifted signals with ACD/SpecManager 8.12. The NMR datawerenormalised to fresh weight and imported into SIMCA-Pþ12(Umetrics, Umea, Sweden) for multivariate statistical analysis.Normalised integrals were pre-treatedwith “mean centring” beforethe application of the PCA statistical algorithm [16].

2.7. Proline and total amino acid content

Proline and free amino acid concentrations were determined foruntreated and treated Osteospermum plant leaves at 4 �C and�5 �C.Leaves were detached from plants about 20 cm tall after two and tendays of cold treatment and after one night at�5 �C, and immediatelyfrozen in liquid nitrogen. The experiment was repeated three times.

Proline and total amino acids were extracted from frozen leaveswith 0.6 N perchloric acid and then centrifuged at 16 000�g for15 min. The supernatant was used for the analysis.

The proline concentration was determined as previouslydescribed [36], according to the procedure of Bates et al. [2]. Prolinecontent was calculated on the basis of an L-Pro standard curve.The free amino acid concentrations were determined following themethod described by Moore [22] using the acid ninhydrin reagentand dimethyl sulfoxide.

3. Results

3.1. Analysis of regenerated plants

Osteospermum plants were transformed with the pGreenIMyb4recombinant plasmid carrying the Osmyb4 coding sequence under

the control of the CaMV35S promoter. The presence of the trans-gene in explants resistant to kanamycin was confirmed by PCR.

Ten randomly selected plants were positive to the Osmyb4-specific region (420-bp fragment) amplification. The presence ofone full-length copy of Osmyb4 (1200-bp fragment) was verified insix of the ten transgenic clones (466, 467, 468, 469, 470, and 471),The expression analysis of these clones was evaluated by RT-PCR,and the positive transgenic clones were classified on the basis ofthe level of transgene expression as: high-expressing (HE, 466 and468), low-expressing (LE, 470 and 471) and not-expressing (NE, 467and 469) clones (Fig. 1). The phenotype of the two transgenic lineswas very similar to that of the wild-type (WT): no plants showeda dwarf phenotype as in tomato (38), contrarily to what happens inArabidopsis and apple (27, 36).

3.2. Tolerance to cold and freezing stress

To test cold tolerance, four-week-old WT, LE and HE plants wereplaced at 4 �C for ten days. As shown in Fig. 2, during the first twodays of cold treatment, no differences were displayed among theWT, LE and HE plants. However, after ten days of cold stress, theWTand LE plants were wilted, whereas the HE plants were healthy.

It has been reported that some species improve freezing toler-ance after an acclimation period of several days at 6e8 �C [17,39].The increase in freezing tolerance in Osteospermum transgenicplants after cold acclimation is shown in Fig. 3. Non-acclimatedplants did not survive overnight at �5 �C (Fig. 3A), but enhancedfreezing tolerance was evident in the HE cold-acclimated plants(Fig. 3B). Both theWTand LE acclimated plants were damaged afterthe freezing treatment.

3.3. NMR analysis of Osteospermum leaves extracts

The metabolic content of WT, LE and HE Osteospermum leaveswere investigated with liquid-state high-resolution NMR spec-troscopy. The 1H and 13C chemical shifts of TOCSY, HSQC and HMBCexperiments enabled us to assignmost of the resonances present inthe recorded spectra, and we identified different water-solublecompounds, such as amino acids, sugars and polysaccharides.Metabolites were assigned based on reported chemical shifts and insome cases by addition of standard compounds. The resonances ofOsteospermum leaves are listed in Table 1. In particular, thealiphatic region (between 0.8 and 3.00 ppm), of the 1H NMR spectraof Osteospermum leaves showed the presence of several aminoacids, including valine, threonine, alanine, leucine, proline, gluta-mate, glutamine, and asparagine, and other compounds, such ascitrate and choline. Moreover, the anomeric sugar region (between3.5 and 5.5 ppm) revealed the presence of sucrose, fructose andboth isomeric forms of glucose. In addition, a small broad signal dueto the fructan inulin was detected at lower field. This naturalcarbohydrate is mainly found in the Compositae family and can be

Fig. 2. Phenotype of cold-treated (4 �C), wild-type and transgenic Osteospermumplants. The wild-type (WT) and transgenic (LE, HE) plants were normally grown forthree weeks (T0) and then treated for two (2d) and ten days (10d).

Fig. 3. Phenotype of wild-type and transgenic Osteospermum after an overnightfreezing treatment at �5 �C. The wild-type (WT) and transgenic (LE, HE) plants werestress-treated without acclimation (A) and after the ten days acclimation period (B).

M. Laura et al. / Plant Physiology and Biochemistry 48 (2010) 764e771 767

present in different forms, as a mixture of oligo- and poly-saccharides composed by fructose units of various lengths, andgenerally terminating with a glucose unit [28]. In the aromaticregion (between 7.14 and 7.35 ppm), very low resonance intensitiesmost likely due to phenylpropanoid derivatives were detected, andshikimate was identified among them.

During the stress treatments, the aliphatic compounds werepresent at higher levels in theWT plants than in the HE plants (datanot shown). However, along the cold and overnight �5 �C treat-ments, a difference in sugar accumulation was detected betweenHE and WT plants; the HE plants showed a higher accumulation ofsucrose, whereas the WT plants accumulated more glucose andinulin (Fig. 4).

Wild-type leaves under normal growth conditions were alsoinvestigated with HR MAS solid-state NMR experiments to analysethe polar and non-polar compounds simultaneously. The HR MASNMR spectra revealed few lipid and polymer resonances in theintact tissue. Comparison with liquid-state NMR spectra showedthe presence of sharp peaks belonging to soluble metabolites in thesolid-state, which were also observed in liquid-state NMR, togetherwith few broad resonances due to lipids (1.5 and 2.7 ppm) andpolymers (from 7 to 7.5 ppm). These broad peaks overshadowedthe resonances of small metabolites and thus the liquid-state NMRspectra were chosen to analyse the metabolic changes.

3.4. Statistical analysis of stress-treated Osteospermum

Liquid-state NMR spectra were processed and coupled withmultivariate statistical analysis to study the metabolic changes ofwild-type and transgenic Osteospermum plants during cold andfreezing treatments. Principal Component Analysis (PCA) wasinitially applied to bucketed spectra to identify a possible sampleclustering due to biochemical similarity. The PCA resulted in a linearcombination of the original variables to yield principal componentswith higher variance, thus reducing the number of variables.

Table 11H chemical shifts values (d ppm) of metabolites identified in Osteospermum.

Compound Assignment 1H ppm 13C ppm

Leucine d,d0 CH3 0.94 22.0g CH 1.70 24.9b CH2 1.70 40.5a CH 3.73 54.2

Valine g,g0 CH3 1.02/0.97 17.6/18.4b CH 2.26 29.8a CH 3.60 61.0

Threonine g CH3 1.31 19.9b CH 4.25 66.9a CH 3.58 61.0

Alanine b CH3 1,46 16.8a CH 3.77 51.1

Gaba b CH2 1.88 24.3a CH2 2.29 34.7

Glutamate g CH2 2.35 33.9b CH2 2.06 27.4a CH 3.75 55.1

Glutamine g CH2 2.44 31.4b CH2 2.13 26.8a CH 3.76 54.9

Citrate CH2 2.75/2.58 42.5

Asparagine b b0 CH 2.94/2.85 35.0a CH 4.00 51.9

Choline NeCH3 3.18 54.6

b Glucose H1 4.63 96.5H2 3.22 74.9H3,5 3.41/3.46 76.3H4 3.45 70.0H6,60 3.87/3.70 61.6

a Glucose H1 5.21 92.7H2 3.58 72.0H3 3.67 73.4H4 3.39 70.3H5 3.82 72.6H6 3.80 60.9

b Fructofuranose H1 3.55 63.1H3,4 4.09 75.5H5 3.81 81.5H6 3.80 63.1

b Fructopyranose H1 3.69/3.55 64.3H3 3.77 68.1H4 3.88 70.1H5 3.98 69.9H6 4.01 64.0

Sucrose H1 G 5.39 93.1H2 3.54 71.9H3,5 3.74 73.3H4 3.45 69.9H6,60 3.82 72.8H1,10 F n.d. n.d.H3 4.19 77.3H4 4.03 74.7H5 3.86 81.8H6,60 3.78 63.1

Inulin H1 G 5.43 93.1H2 3.52 71.9H3 3.75 73.3H4 3.45 69.9H5,6 3.83 72.8H1,10 F n.d. n.d.H3,5 4.26 77.5H4 4.03 74.7H5 3.86 81.8H6,60 3.80 63.1

Table 1 (continued)

Compound Assignment 1H ppm 13C ppm

Shikimate H3 6.45 131.3H4 4.47 n.d.H5 4.35 n.d.H6 3.98 n.d.H7,70 3.00/2.19 n.d.

Fig. 4. NMR content of some sugars in Osteospermum samples. (A) glucose, (B)sucrose, and (C) inulin in WT and transgenic Osteospermum (LE, HE) during cold andfreezing treatments. The glucose and sucrose contents are reported as mg.g FW �1,inulin content is reported as the integrated value (peak area) due to its unknownmolecular weight. Plants were grown under control conditions (t ¼ 0), cold-treated at4 �C for two (2d) and ten days (10d), and overnight treated at �5 �C (O.N. �5 �C). Dataare the mean of three independent experiments, bars represent standard deviation.

M. Laura et al. / Plant Physiology and Biochemistry 48 (2010) 764e771768

M. Laura et al. / Plant Physiology and Biochemistry 48 (2010) 764e771 769

In the PCA model, 99% of the overall variance was explainedwith three PCs and Q2 ¼ 96,7% (Fig. 5). The score plot revealed thatOsteospermum samples were distributed according to genotype(WT, LE and HE) and stress treatment. The untreated (t ¼ 0) andshort-term treated samples (2 days at 4 �C) grouped in the sameregion independently of the genotype, whereas the long-termtreated (ten days at 4 �C) and the overnight frozen samples (O.N.�5 �C) separated into different regions based on genotype. Thesample distribution during the stress treatments accounted fordifferent amounts of sugars and amino acids accumulated by WT,LE and HE. The ten-day treated WT samples displayed a positivecontribution to both first and second components of the PCA andwere characterised by larger amounts of amino acids and sugars.The HE plants affected negatively the second component, resultingin a slightly larger sucrose content (peak at 5.39 ppm). The LE plantsshowed an intermediate behaviour between the WT and HE plantsunder all treatments. The analysis of the score contribution plot,which represents the weighted differences of metabolic contentamong observations, revealed that WT plants contained a largeramount of amino acids (NMR region between 0.8 and 3.00 ppm),sugars (between 3.5 and 5.5 ppm) and aromatics (between 7.14 and7.35 ppm) under normal growth conditions (t ¼ 0) (Fig. 6A). Thetwo-day cold treatment gave rise to a slight increase in the meta-bolic content in all genotypes (data not shown). The score contri-bution plot of the ten-day treated plants highlighted the highermetabolite content of WT with respect to HE (Fig. 6B). The scorecontribution plot of the WT plants treated overnight at �5 �Cshowed the accumulation of the same metabolites observed for theten-day cold-treated WT plants, but at lower concentrations(Fig. 6C).

Interestingly, in the HE samples treated overnight at �5 �C thesugar content increased in comparison to the ten-day cold-treatedplants (Fig. 6B,C). The corresponding score contribution plot of theWT plants showed a larger amount of amino acids, glucose andfructose, whereas the HE samples were characterised by a highersucrose concentration (Fig. 6C). The PCA model showed that the�5�C-treated WT and HE clustered in the same region as theirrespective ten-day treated genotypes (Fig. 5).

The PCA model considering only the aliphatic and anomericsugar regions of the bucketed spectra were equivalent to the full-spectrum model, indicating that the aromatic region of the spectradid not affect sample clustering (data not shown).

Fig. 5. PCA score plot of Osteospermum samples. Wild-type (WT), low-expressing (LE),and high-expressing (HE) Osteospermum leaves grown under control conditions(circle), and cold-treated for two days (diamond), ten days (triangle) at 4 �C andovernight at �5 �C (square).

Fig. 6. Score contribution plot of wild-type and transgenic Osteospermum withdifferent stress treatments. (A) Score contribution plot of Osteospermum grown undercontrol conditions; comparison of spectra from WT and HE transgenic plants. Spectralregion spans from 9.5 to 0.8 ppm without the water region. (B) Score contribution plotof Osteospermum after a ten-day cold treatment, comparison of spectra from WT andHE transgenic plants. Spectral region spans from 9.5 to 0.8 ppm without the waterregion. (C) Score contribution plot of Osteospermum after overnight treatment at�5 �C, comparison of spectra from WT and HE transgenic plants. Spectral region spansfrom 9.5 to 0.8 ppm without the water region.

M. Laura et al. / Plant Physiology and Biochemistry 48 (2010) 764e771770

3.5. Proline and amino acid accumulation

The accumulation of proline has been associated with increasedcold and freezing tolerance. In this study, we monitored theconcentration of proline after two and ten days of cold stress andafter the overnight freezing treatment. The proline concentrationwas measured in Osteospermum leaf extracts and expressed bothas mg. mg�1 FWand the ratio of proline to amino acids. As shown inFig. 7A, the concentration of proline increased in all three geno-types during the stress treatment. The proline concentration washigher in HE plants even before the stress and increased withtreatment. Higher proline levels were also observed in WT and LEplants, but to a lower extent. The LE plants exhibited an interme-diate behaviour between WT and HE plants.

The proline/amino acids ratio followed the same trend, reachingthe maximumvalue in HE genotypes after ten days of cold (Fig. 7B).

4. Discussion

Osteospermum is an ornamental plant native to South Africathat grows as a perennial species in that environment. However, inthe last few years, it has been cultivated in Europe, where it behavesas an annual plant due to the cold winter. Due to the economicpotential of this species in Europe, altering the cold/freezingtolerance to this plant genus may be advantageous.

In previous studies, we reported the ability of the Osmyb4 gene toconfer abiotic (chilling, freezing drought) and biotic (bacteria, fungi,viruses) stress tolerance to several plant species [19,36e38].However, the overexpression of Osmyb4 in different plant speciesgave rise to different levels of tolerance according to the geneticbackground. Indeed, we reported that Arabidopsis plants

Fig. 7. Proline levels and proline/amino acids ratio in Osteospermum leaves duringcold and freezing treatment. (A) proline levels and (B) proline/amino acids ratios ofWT, LE, and HE plants grown under control conditions (t ¼ 0), cold-treated at 4 �C fortwo (2d) and ten days (10d), and overnight treatment at �5 �C (O.N. �5 �C). Data arethe mean of three independent experiments, bars represent standard deviation.

overexpressingOsmyb4weremore tolerant to someabiotic andbioticstress whereas transgenic tomato plants acquired a higher toleranceto drought stress and to virus disease but not to cold (36, 38).

Based on the results previously observed in transgenic plants, westudied the effect of ectopic Osmyb4 expression on the cold andfreezing tolerance of Osteospermum species with the aim ofobtaining plants able to grow in open field during the winter inclimatic zones Z9 and Z8. To evaluate the tolerance of the transgenicplants to cold and freezing stress, we have grown WT and twotransgenic clones (LE, HE) at 4 �C and �5 �C during a time courseexperiment. A short-term cold stress (two days) did not damage theWT Osteospermum plants (Fig. 2B). We previously described thatOsmyb4 ectopic expression in Arabidopsis and apple led to a bettertolerance to cold [19,27,36]. Accordingly, we found that after ten daysof cold treatment, theWTand LE plantswerewilted in comparison tothe HE plants (Fig. 2C), suggesting that Osmyb4 overexpression canmediate low temperatures in this species. Overnight exposure to�5 �C resulted in general plant death in all of the analysed genotypes(Fig. 3A),whereas the same treatment performedafteranacclimationperiod at 4 �C resulted in a marked tolerance increase in the HEgenotype only (Fig. 3B). These results are in agreementwith previousreports that the freezing tolerance of plants is not constitutive, but isinduced in response to a period of low, non-freezing temperature,a phenomenon known as cold acclimation [33,39]. The cold accli-mation period is important for inducing several physiologicalchanges, including altered lipid composition in plasma membranes,accumulation of protectant compounds, such as carbohydrates, freeamino acids or other osmolytes, and induction of new gene activity,altogether leading to a better freezing tolerance [1,12,33]. In Arabi-dopsis, a plant able to acclimate, the ectopic expression ofOsmyb4 ledto several physiological changes mimicking plant cold acclimation[36]. In the case of Osteospermum, which does not acclimate underour experimental conditions, the overexpression of OsMyb4 did notprotect theplant fromthe freezing treatment (Fig. 3A).However, afteran acclimation period, the HE transgenic plants showed a betterfreezing tolerance than acclimated WT plants, suggesting a synergicactivity of Myb4 transcription factor and cold treatment for freezingtolerance.

Because the levels of severalmetabolites are higher inArabidopsistransgenic plants than inWT plants under normal growth conditionsand further increaseduringcold stress,weanalysed theaccumulationofmetabolites byWTand transgenicOsteospermumwith control andstress treatments. The leaf sampleswere analysed by 1HNMRand themetabolites were assigned based on reported chemical shifts and byaddition of standard compounds. The obtained metabolic profile,which is the first reported one for Osteospermum, is presented inTable 1. The metabolic profiling can be useful for studying theconsequences of changes in gene expression, to follow the metabolicresponse under different environmental conditions and to improveknowledge of the connection between genes, proteins and metabo-lites. In the last years, metabolomics studies have received increasingattention as a mean of acquiring a better insight into the completebiological processes, combining this information with that obtainedthrough genomics, transcriptomics and proteomics [25].

The multivariate data analysis (PCA) performed using 1H NMRdata clustered the samples based on treatment and genotypes. Thecontrol and two-day cold samples grouped independently of thegenotype, whereas the most severely treated samples separatedbased on genotype (Fig. 5). Our results indicate that for short-termtreatments differences among genotypes did not involve differ-ences in soluble compounds, whereas for long-term treatments themetabolites content played the major role in separating thegenotypes.

The differences in metabolic content of WT and HE-treatedsamples suggest a correlation between the level of metabolites and

M. Laura et al. / Plant Physiology and Biochemistry 48 (2010) 764e771 771

the different behaviour of the two genotypes under stress. Indeed,the concentration of several compounds that increased duringstress in WT remained almost constant in the HE genotype, and theLE plants showed an intermediate behaviour between WT and HE.In particular, the level of inulin strongly increased in WT, reachinga concentration 5 and 9 times higher than in HE after ten days ofcold treatment and overnight freezing, respectively. The inulinincrease in WT leaves supports its role as a membrane protectantduring drought and frost stress, as previously proposed by severalauthors [7,14], even if some contradictions have been reportedabout the relationship between fructan accumulation and abioticstress [18]. It has been suggested that fructans have less effect oncellular water potential than simple sugars, such as sucrose [35]; assuch, we found that HE-treated Osteospermum plants accumulatedmore sucrose during cold and freezing stress (Fig. 4).

As far as proline is concerned, its level was higher in HE plantseven before each treatment and reached the highest value after tendays of cold treatment and after freezing. The proline content of HEwas almost twice that of WT, and the HE proline/amino acids ratiowas 7 and 4 times higher after ten days of cold and after freezing,respectively (Fig. 7). The total amino acid content was higher in WTthan in HE plants, thus lowering the proline/amino acids ratio of theWT. Higher total amino acid content has been reported to increasesalt sensitivity in barley [5]. The LE genotype showed an intermediatebehaviour between WT and HE. Proline accumulation during coldand freezing has been extensively studied and is assumed to enhanceprotein and membrane stability in addition to adjusting the intra-cellular osmotic potential [11,15,29]. Moreover, it has been demon-strated that antisense transgenic Arabidopsis with AtProDH cDNA(encoding proline dehydrogenase) accumulated more proline thanWT and was constitutively tolerant to freezing, suggesting a key roleof this amino acid in mediating stress [24]. The Arabidopsis eskimomutant, which accumulates more proline than WT and becomesconstitutively freezing-tolerant [40], further highlights the strongrole of this amino acid during cold and freezing stress. The clearcorrelation between the cold and freezing tolerance and the accu-mulation of proline suggest the involvement of Osmyb4 in prolinemetabolism/catabolism. On the basis of our experimental data, itseems that the enhanced cold and freezing tolerance of HE could bemainly attributed to the higher content of proline and sucrose of thisgenotype with respect to the WT. However, we cannot exclude thatother physiological or biochemical changes could be involved in theincreased stress tolerance of transgenic Osteospermum.

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