Journal of Plant Physiology ](]]]])]]]—
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Differential expression of four Arabidopsis PAL genes;PAL1and PAL2have functional
specialization in abiotic environmental-triggered ?avonoid synthesis
Kristine M.Olsen a ,Unni S.Lea a ,Rune Slimestad b ,Michel Verheul c ,Cathrine Lillo a,?
a
Faculty of Science and Technology,University of Stavanger ,N-4036Stavanger ,Norway b
PlantChem,Saerheim Research Center ,N-4353Klepp stasjon,Norway c
Bioforsk,Saerheim Research Center ,N-4353Klepp stasjon,Norway
Received 7September 2007;received in revised form 30October 2007;accepted 5November 2007
KEYWORDS Arabidopsis;Flavonoids;
Nitrogen depletion;PAL;
Temperature
Summary
Phenylalanine ammonia-lyase (PAL,EC 4.3.1.5)catalyzes the ?rst step in the phenylpropanoid pathway,and is considered an important regulation point between primary and secondary metabolism.In the present work we analyzed expression of the PAL genes in leaves of Arabidopsis thaliana rosette-stage plants in response to nitrogen depletion at temperatures ranging from 5to 301C.Only PAL1and PAL2responded strongly to both environmental factors,nitrogen and temperature.Regardless of nitrogen treatments,PAL1and 2transcript levels increased at 5and 101C.Averaged across all temperatures,nitrogen depletion led to a two-fold increase in PAL1and PAL2transcripts.PAL activity was correlated with PAL transcript levels (R ?0.94).Accumulation of major soluble phenylpropanoids,sinapic acid esters and ?avonoids,increased in response to lowering temperature.The ?avonoids,kaempferols,quercetins and anthocyanins,showed signi?cantly in-creased levels as a result of nitrogen depletion (two-,?ve-and six-fold increases,respectively)when averaged across all temperatures.PAL1,PAL2and PAL4have previously been shown to be related with tissue-speci?c lignin synthesis,and the present work shows that PAL1and PAL2also have functional specialization in abiotic environmental-triggered ?avonoid synthesis.&2007Elsevier GmbH.All rights reserved.
www.elsevier .de/jplph
0176-1617/$-see front matter &2007Elsevier GmbH.All rights reserved.doi:10.1016/j.jplph.2007.11.005
Abbreviations:CAD,cinnamyl alcohol dehydrogenase;CCR,cinnamoyl-CoA reductase;PAL,phenylalanine ammonia-lyase.?Corresponding author .Tel.:+14751831875;fax:+14751831750.E-mail address:Cathrine.Lillo@uis.no (C.Lillo).
Introduction
Phenylpropanoids have many functions in plants, for example as protectants against stressful factors such as UV-light or pathogen attack.Flavonoids constitute a sub-group of the phenylpropanoids and are known to accumulate in response to nutrient depletion,although the physiological function of this accumulation is unclear(Stewart et al.,2001; Misson et al.,2005;Lea et al.,2007,Lillo et al., 2008).Phenylpropanoids are also the precursors in lignin synthesis.The phenylpropanoids are of great importance for the quality of commercial plants, including fruits and vegetables,and?avonoids are considered of interest for human health.Phenyla-lanine ammonia-lyase(PAL)commits the?ux of primary metabolism into secondary metabolism in the phenylpropanoid pathway.Thus,formation of lignin,sinapic acid esters and?avonoids all depend on PAL activity(Figure1).PAL has long been known to be highly regulated at the transcriptional level in response to pathogen attack,abiotic factors,and a demand for lignin in special tissue(Anterola and Lewis,2002;Khan et al.,2004).It has also been shown that silencing a key enzyme of lignin synthesis increases accumulation of?avonoids, indicating competition between?avonoid and lignin synthesis for precursors(Besseau et al., 2007).Generally,PAL genes constitute a small gene family in plants,as found in bean,parsley,rice, potato,tomato and Arabidopsis.This can enable differential gene expression,a widely accepted explanation for maintenance of duplicated genes (Gachon et al.,2005b;Shuf?ebottom et al.,1993). In the present work,effects of nitrogen depletion on the four Arabidopsis PAL genes were studied in order to examine their possible differential roles in response to nitrogen depletion over a wide range of temperatures.
Raes et al.(2003)showed that all four Arabi-
dopsis PAL genes were expressed in in?orescent stems,a tissue rich in lignifying cells.PAL1and4 were most strongly expressed,PAL2at a somewhat lower level,and PAL3showed very low transcript levels.PAL1and PAL2were also identi?ed as related to the ligni?cation process by Oh et al. (2003).The characterization of pal1,pal2and pal1 pal2double mutants further con?rmed that both PAL1and PAL2are important for lignin synthesis (Rohde et al.,2004).The high level of PAL4in in?orescence stems pointed to PAL4as also being an important gene for lignin synthesis(Raes et al., 2003;Rohde et al.,2004).PAL1expression has been shown to be induced by environmental factors such as light,wounding and pathogen attack(Cochrane et al.,2004;Mauch-Mani and Slusarenko,1996;Ohl et al.,1990;Rookes and Cahill,2003).Generally, environmental induction has been reported for the PAL1gene,and often the other three PAL genes have not been examined.However,in work performed by Wanner et al.(1995)and Mizutani et al.(1997),PAL2and3were also included in their investigations and showed that PAL1and2were co-expressed in different plant organs,and also in response to inductive treatments with phytopatho-genic pseudomonas.PAL1and4were the two genes showing a positive(about two-fold)response to nitrogen depletion in microarray experiments (Scheible et al.,2004),whereas PAL2and3showed a small negative response.Altogether,these in-vestigations have shown that PAL2is responsive to some environmental factors,but possibly not
L-phenylalanine
4-coumarate
PAL
sinapic acid esters
kaempferols
quercetins
FLS
CHS
dihydroquercetin
anthocyanidins
ANS
naringenin chalcone
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Figure1.Simpli?ed scheme of phenylpropanoid synth-esis in Arabidopsis with pathways leading to lignin and major soluble phenolic compounds,sinapic acid esters, kaempferols,quercetins and anthocyanins.Key enzymes marked are PAL(phenylalanine ammonia-lyase),CHS (chalcone synthase),FLS(?avonol synthase),ANS(antho-cyanidin synthase).4-coumaroyl-CoCA is established as a precursor for sinapic acid esters and lignin,but additional routes may exist(Raes et al.2003,Gachon et al.2005a, Besseau et al.2007).
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to nitrogen status(Wanner et al.,1995;Mizutani et al.,1997;Scheible et al.,2004).
To investigate the function of different PAL genes,transcript levels of all four PAL genes were tested by RT-PCR,in response to environmental inductive as well as non-inductive conditions with respect to phenylpropanoid synthesis.Transcript levels are often found to be poorly correlated with enzyme activities of the corresponding proteins (Osuna et al.,2007).PAL activity was,therefore, tested and con?rmed that this activity correlated with transcript levels.The experiments revealed that when?avonoid synthesis was induced by nitrogen depletion or lowering the growth tem-perature,expression of PAL1and PAL2were highly induced.
Materials and methods
Plant material
Seeds of Arabidopsis thaliana ecotype Columbia(Col1) were sown on soil with complete Hoagland solution (Hoagland and Arnon,1950).After approximately3 weeks,seedlings were transferred into rockwool cubes and grown in a growth chamber at a temperature of201C in a12h light/dark regimen.Light was provided by ?uorescent lamps(Osram L58W/21)with a photon?ux density(PPFD)of100m mol mà2sà1PAR.Plants were watered when needed using a complete Hoagland solution.After1week,plants were transferred randomly to growth chambers at temperatures of5,10,15,20,25 or301C.Plants were given either complete Hoagland or Hoagland without nitrogen.A water vapor pressure de?cit of3.5–1.0g mà3and a CO2level of350m mol were maintained at all temperatures.Temperature?uctua-tions in the growth chambers were kept within70.51C. Samples were harvested in the morning,2h after onset of light.The?rst samples were harvested before exposure of plants to different treatments(day0),and then4and 7d after the change.
Measurements of phenolic compounds
About0.100g of plant tissues were exactly weighed and transferred to Eppendorf tubes.One milliliter methanol(1%tri?uoroacetic acid(TFA),v/v)was added to each tube,and phenolics were extracted for18h at ambient temperature and in darkness.The extracts were ?ltered through45m m nylon?lters prior to HPLC analyses.A liquid chromatograph(Agilent1100-system, Agilent Technologies)supplied with an autosampler and a photodiodearray detector was used for the analysis of individual phenolics.The phenolics were separated on an Eclipse XDB-C8(4.6?150mm,5m m)column(Agilent Technologies)by use of a binary solvent system consisting of(A)0.05%TFA in water and(B)0.05%TFA in acetonitrile.The gradient(%B in A)was linear from5to 10in5min,from10to25for the next5min,from25to 85in6min,from85to5in2min,and?nally recondition of the column by5%in2min.The?ow rate was0.8 mL/min,10m L samples were injected on the column,and separation took place at301C.Detection was made over the interval230–600nm in steps of2nm in order to obtain full absorbance spectrum of the compounds of interest,whereas quantitative data were obtained at 320nm(sinapic acid derivatives),370nm(?avonols)and 520nm(anthocyanins).The individual peaks were char-acterized as derivatives of kaempferol,quercetin, sinapic acid ester and anthocyanins according to their UV absorbance spectra(Lehfeld,2001;Mabry et al., 1970;Veit and Pauli,1999;Wintersohl et al.,1979).In addition,acid hydrolysis of the compounds was per-formed according to Markham(1982).The hydrolyzed products were determined by co-chromatography with authentic kaempferol,quercetin and sinapic acid (Carl Roth GmbH,Germany).Peak areas were adjusted to exact sample weights,and pooled together as kaempferols,quercetins,sinapic acid esters or antho-cyanins for the individual samples.
PAL assay
Approximately50mg leaf tissue was thoroughly ground with cold mortar and pestle in2ml100mM Tris–HCl (pH8.8)with12mM b-Mercaptoethanol and transferred to a centrifugation tube.The samples were centrifuged at41C for5min at16,000g.The supernatant was passed through a Sephadex G25column.The eluate was used for PAL assay similar to Saunders and McClure(1974).The PAL assay was performed at371C for1h in an assay mixture containing500m L enzyme extract,450m L100mM Tris–HCl(pH8.8)and50m L100mM L-phenylalanine. Assays were run in triplicate.The reaction was termi-nated by adding50m L5M HCl,centrifuged at16,000g for 15min and absorbance recorded at290nm against blanks made in the same way as the assays,only50m l5M HCl was added before L-phenylalanine.The activity was expressed as nmol trans-cinnamic acid formed per gram plant tissue,per hour.
RT-PCR
Total RNA was isolated using RNeasy s Plant Mini Kit (Qiagen,Chatswort,CA).RNA was quanti?ed by a spectrophotometer and cDNA synthesised using the High Capacity cDNA Archive Kit(Applied Biosystems)following the manufacturer’s instructions(concentration of RNA in the reaction tube was 4.6m g mL–1).Real-time PCR reactions were assayed using an ABI7300Fast Real-Time PCR System.The reaction volume was25m L containing 12.5m L TaqMan buffer(Applied Biosystems,includes ROX as a passive reference dye),8.75m L H2O, 2.5m L cDNA and 1.25m L primers.Primers were pre-designed TaqMan s Gene Expressions assays obtained for the following genes(TaqMan identi?cation number is given in parentheses): A.thaliana PAL1AT2G37040 (At02323251_g1),PAL2AT3G53260(At02188099_g1),
PAL1and PAL2in abiotic-triggered?avonoid synthesis3
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PAL3AT5G04230(At02180826_m1),PAL4AT3G10340(At02291526_m1),ACT 8AT1G49240(At02270958_gH),CCR1AT1G15950(At02156281_m1),CCR2AT1G80820(At02290107_g1),CAD4AT3G19450(At02254828_ml)(Applied Biosystems).Standard cycling conditions (2min at 501C,10min at 951C and 40cycles altering between 15s at 951C and 1min at 601C)were used for product formation.Real-time PCR products were analyzed by Sequence Detection Software version https://www.doczj.com/doc/1116476300.html,parative CT method for relative quanti?cation has been used with ubiquitine as endogenous control and the sample taken before change of nutrient solution (day 0)as calibrator.Relative quantity (RQ ?2àDD CT )of any gene is given as fold change related to day 0.This method,which is based on a light signal from each transcript copy being formed,also allows comparison of expression levels between the PAL genes (Figure 2).
Results
Expression of two of the PAL genes,PAL1and PAL2,was clearly enhanced after 4–7d at 5and 101C compared with temperatures 151C or higher .A positive effect of nitrogen depletion was seen from 5to 201C (Figure 2).PAL3and 4were expressed at a much lower level,and no positive effect of nitrogen depletion was seen.At 51C and nitrogen depletion the transcript levels,when PAL1was set to 100,were 96%for PAL2,1.5%for PAL3and 0.1%for PAL4based on CT values and ubiquitine as endogenous control.
PAL activity was strongly increased at 5and 101C compared with higher temperatures (Figure 3).PAL activity also increased in response to nitrogen depletion (Figure 3),i.e.by 83%when calculating a mean across all temperatures.At the two higher temperatures,25and 301C,PAL activity and PAL transcript levels were low.PAL activity was clearly correlated with the total amount of PAL transcripts as shown in Figure 2F .
CCR1,CCR2and CAD4were tested to examine whether genes of the lignin pathway were induced in response to different treatments.According to CT values,CCR1was expressed at much higher levels than CCR2;mRNA levels were 40times higher when comparing samples from 201C and complete nutrient solution.The results showed that none of these genes were induced by nitrogen depletion in
the present experiment,and for two of the genes CCR2and CAD4,transcript levels were higher in leaves from plants given full nutrient solution compared with starved plants (Figure 4).
Measurements of sinapic acid esters,?avonols and anthocyanins showed that all these compounds increased at lower temperatures (Figure 5).Whereas the ?avonols and anthocyanins were present at very low or undetectable levels at 20–301C,sinapic acid esters stayed at a high level at all temperatures tested.In contrast to kaemp-ferols,quercetins and anthocyanins,there was no consistent increase in sinapic acid esters due to nitrogen depletion,and this was con?rmed when calculating the mean nitrogen effect across all temperatures,which showed that nitrogen depletion led to two-,?ve-and six-fold increases in kaempferols,quercetins and anthocyanins,
temperature.Plants were grown in rockwool with Hoagland solution containing 15mM KNO 3at 201C.Rosette-stage Arabidopsis plants were then (day 0)placed at various growth temperatures between 5and 301C,and exposed to Hoagland solution with (dots)or without (squares)nitrogen.Leaves were harvested on days 4and 7after the variable treatments had started.The data are presented as fold change relative to day 0.There were no clear difference between results obtained at days 4and 7,therefore the data were pooled.n ?4,SE is given.(A)PAL1,(B)PAL2,(C)PAL3,(D)PAL4and (E)ACT8.(F)Correlations of PAL activity and total PAL transcript
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5Figure 3.PAL activity as in?uenced by nitrogen deple-tion and growth temperature.Plants were grown in rockwool with Hoagland solution containing 15mM KNO 3at 201C.Rosette-stage Arabidopsis plants were then (day 0)placed at various growth temperatures between 5and 301C,and exposed to Hoagland solution with (dots)or without (squares)nitrogen.Leaves were harvested on days 4and 7after the variable treatments had started.There were no clear difference between results obtained at days 4and 7;therefore,the data were pooled,n ?4,SE is given.
PAL1and PAL2in abiotic-triggered ?avonoid synthesis
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respectively.In contrast,the content of sinapic acid derivatives did not change signi?cantly.
Discussion
It was previously shown for tobacco leaf tissue that accumulation of soluble phenylpropanoids was correlated with PAL activity in transgenic plants exhibiting a range of different PAL levels.The major soluble tobacco phenylpropanoids,chloro-genic acid and rutin (a quercetin derivative),were both reduced in parallel with decreased PAL activity (Bate et al.,1994).In the present experi-ments with Arabidopsis rosette leaves and various environmental inductive treatments,all major groups of phenylpropanoid metabolism,i.e.sinapic acid esters,kaempferols,quercetins and anthocya-nins accumulated at high PAL activity.As PAL activity decreased to very low levels at 20–301C (Figure 3),this was paralleled with low ?avonoid levels (Figure 5).Sinapic acid derivatives were,however ,still present at relatively high levels.This is likely a result of control of ?avonoid synthesis further downstream of PAL prohibiting ?ux into ?avonoids,and thereby ?ux into sinapic acid derivatives is preserved even at very low PAL activity.
In the work by Leyva et al.(1995),the PAL1promoter linked to a reporter gene was activated at 41C.However ,in a study of the low-temperature transcriptome in Arabidopsis ,PAL2,but no other PAL genes,was identi?ed as a gene being up-regulated in leaves in response to 1or 7d at 41C (Vogel et al.,2005).The experiments presented here showed that both PAL1and PAL2genes were clearly responding positively to low temperatures (Figure 2).An essential difference in the experi-mental set-ups was that,in the present work,normal growth light was used,whereas Vogel et https://www.doczj.com/doc/1116476300.html,ed dim light at 41C.The results presented in Figure 2also showed that PAL1and 2not only responded positively at 51C,but a gradual response was observed at less extreme temperatures.
PAL1and PAL2genes,but none of the other PAL genes,responded positively to nitrogen deple-tion in our experiments with rosette leaves
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(Figure 2).Transcriptome analysis using microar-rays (Affymetrix ATH1)has provided indications that nitrogen depletion leads to increased PAL1and PAL4expression (Scheible et al.,2004).The apparently divergent results are most likely caused by different tissue and growth conditions applied.In the experiments by Scheible et al.(2004),an in vitro growth system containing sugar was used,and whole seedlings were harvested.The ATHI micro-array data showed that CCR1and CCR2were induced 2-fold,and CAD41.4-fold,indicating that the lignin pathway was also activated in whole seedlings when nitrogen was depleted in the presence of sucrose (Scheible et al.,2004,Supple-mentary data).In our experiments with rosette leaves,PAL4was not induced by nitrogen deple-tion,but on the contrary,showed higher levels in leaves from plants given full nutrient solution.The correlation between expression of PAL4and the genes of the lignin pathway is in agreement with both the data presented here (Figures 2,4)and the data provided by Scheible and co-workers.
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5Figure 5.Relative concentrations of the four major subclasses of soluble phenolic compounds as in?uenced by nitrogen depletion and growth temperature.Plants were grown in rockwool with Hoagland solution containing 15mM KNO 3at 201C.Rosette-stage Arabidopsis plants were then (day 0)placed at various growth temperatures between 5and 301C,and exposed to Hoagland solution with (dots)or without (squares)nitrogen.The ?gure shows data for samples harvested on days 4and 7after the variable treatments had started.For sinapic acids,the values were lower for day 4;thus,the data could not be pooled as for the other compounds.For sinapic acids the spread of measurements is given by vertical bars when exceeding the size of the symbol.The data point for 51C minus N represents only one sample.For other compounds there were no clear difference between results obtained at days 4and 7;therefore,the data were pooled,n ?4,SE is given.
PAL1and PAL2in abiotic-triggered ?avonoid synthesis
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Accumulation of quercetins and anthocyanins was strongly increased by nitrogen depletion, especially in combination with low temperatures. The shapes of the curves(Figure5)were similar for accumulation of anthocyanins and quercetins, suggesting a possible common way of regulation, different from the branch to kaempferols.
In accordance with the expression pattern of PAL1and2,their promoters contain well-conserved AC elements that specify vascular expression of phenylpropanoid genes,hence expression in tissue with lignifying cells.Following genome-wide transcriptome analysis Raes et al.(2003)con-cluded that PAL1and PAL2were extra-quali?ed within their gene family for playing a role in developmental ligni?cation of vascular tissue. All four PAL promoters contain the so-called PAL boxes within their500bp upstream region of the transcription start site.PAL1,2,3and4have four,three,one and four such boxes,respectively (Higo et al.,1999;Mizutani et al.,1997).Note, however,that a slight change of consensus motif may also allow a different number of boxes to be detected.PAL boxes have been found to be necessary,though not suf?cient,for elici-tor and light responsiveness in activation of parsley PAL genes(Logemann et al.,1995).PAL1 and PAL2promoters,but none of the other Arabidopsis PAL promoters,have SORLIP motifs within the330bp upstream region of the transcrip-tion start site(Zimmermann et al.,2004).Such motifs have been identi?ed as being involved in light responsiveness(Hudson and Quail,2003). Although the signi?cance of various motifs in differentiation of Arabidopsis PAL genes is not clear,microarray analysis no doubt shows close-coordinated expression of PAL1and2related to development and tissue,whereas PAL4expression was largely different(Zimmermann et al.,2004). The nitrogen and temperature effects presented here(Figure2)add these abiotic treatments as inducers of coordinated expression of PAL1and2, as opposed to PAL3or4,which were not greatly in?uenced.
With nitrogen depletion and low temperatures, both PAL1and PAL2transcripts were enhanced, PAL activity increased and?avonoids accumu-lated(Figures2,3,5).This implies that coordi-nated PAL1and PAL2expression has a speci?c role not only in lignin synthesis(Raes et al.,2003),but the genes are also co-expressed for?avonoid synthesis in response to environmental factors. The underlying signaling pathway responsible for PAL1and2induction in response to nitrogen depletion and temperature still remains to be elucidated.Acknowledgements
This work was supported by a grant from the Nansen foundation.
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