Split-plot microarray design allows sensitive detection of expression differences after ultraviolet...

Plant Cell and Environment

(2004)

27

1374ndash1386

copy 2004 Blackwell Publishing Ltd

1374

Blackwell Science LtdOxford UKPCEPlant Cell and Environment0016-8025Blackwell Science Ltd 2004 2004

271113741386Original Article

UV alters expression profiles in maize mapping line inbredsJ E Blum

et al

Correspondence Ann E Stapleton Fax

+

1 910 962 4066 e-mailstapletonauncwedu

Split-plot microarray design allows sensitive detection of expression differences after ultraviolet radiation in the inbred parental lines of a key maize mapping population

J E BLUM

1

P CASATI

2

V WALBOT

2

amp A E STAPLETON

3

1

Department of Mathematics and Statistics and

3

Department of Biological Sciences University of North Carolina at Wilmington 601 S College Road Wilmington NC 28403 USA and

2

Department of Biological Sciences Stanford University Stanford CA 94304 USA

ABSTRACT

Gene expression levels were quantified after ultravioletradiation treatment in the parental inbred lines of the maizemapping (IBM) population This allows us to take advan-tage of natural variation between maize lines to analysevariation in gene expression Using a statistically soundsplit-plot experiment cDNAs were identified with differ-ently regulated expression in B73 and Mo17 after UV treat-ment Fewer genes were down-regulated in B73 this globalstrain difference in the number of genes up- and down-regulated does not appear to reflect general hybridizationdifferences Contrary to our expectation there was a higherproportion of highly expressed genes (based on EST recov-ery) that were differently expressed by UV between linesGenes affected by UV (but not significantly differentbetween B73 and Mo17) include gene types proposed tofunction in UV acclimation and adaptation based on exper-iments in other species or other experiments in maize Sev-eral new functional classes were identified as UV-regulatedincluding genes encoding proteins that modulate chromatinstructure

Key-words

B73 Mo17 gene expression inbred linesmaize microarray ultraviolet radiation

INTRODUCTION

Understanding of the mechanisms of UV responses inplants has lagged behind our understanding of plant per-ception of visible light wavelengths (Jordan 2002 Ballareacute2003) There are multiple damage and photoreceptor path-ways for perception of UV radiation signal transductionafter chromophore perception or damage leads to complexmulti-pathway responses including changes in gene expres-sion (Herrlich

et al

1997 Jordan 2002 Broscheacute amp Strid2003 Gyula Schafer amp Nagy 2003) Measurement of geneexpression of individual response genes in mutants defi-cient in hormones or in plants treated with inhibitors has

implicated at least five different pathways of signal trans-duction after UV irradiation (Jordan 2002 Broscheacute amp Strid2003) nitric oxide calciumcalmodulin superoxide jas-monic acid and ethylene

In surveys of plants of various accessions ecotypes orpopulations differences in responses to UV are oftenreported These differences could result in differential com-petitive ability in wild populations as terrestrial UVincreases and in crop plants resistant lines could providematerials for selective breeding for UV tolerance in cropspecies (Caldwell 1997) These differences also allowexploitation of natural variation for mechanistic under-standing (Alonso-Blanco amp Koornneef 2000 Jansen 2003)

Transcriptome profiles comparing treatments or compar-ing mutant to wild-type are entry points for making thelinkage between conditions and responding processesbecause a global assessment is conducted Of great interestis whether different strains or genotypes activate the samesignal transduction pathways to elicit identical responses orwhether some of the transduction pathways andor some ofthe responses are line-specific Comparison of differentstrains or lines has been carried out in yeast mouse Droso-phila fish and human cell culture (Sandberg

et al

2000 Jin

et al

2001 Oleksiak Churchill amp Crawford 2002 Schadt

et al

2003 Townsend Cavalieri amp Hartl 2003) but to ourknowledge this work is the first application of DNA micro-arrays to compare inbred lines in plants

We are interested in the mechanism of ultraviolet radia-tion stress responses especially in changes in the transcrip-tional program To address this question we have lookedfor genes with differences in expression that also show vari-ation in the mapping parent lines for future genetic analysisWe focused our comparison on lines that have appropriatemapping resources publicly available the IBM lines (Lee

et al

2002) A second rationale is that these two lines havedistinctive alleles because among US inbreds they are dis-tantly related (Liu

et al

2003) These IBM recombinantinbred lines are exceptionally valuable in mapping loci forquantitative traits (Lee

et al

2002) Expression profiling ofsegregating populations such as the B73 and Mo17 materi-als has been suggested as a way to increase the statisticalpower of arrays (Jansen amp Nap 2001 Cheung amp Spielman

UV alters expression profiles in maize mapping line inbreds

1375



27

1374ndash1386

2002) Regulation of protein expression was the first molec-ular trait mapped using a natural variationQTL approach(Damerval

et al

1994) Examples of the use of gene expres-sion levels for mapping of regulatory loci in several specieshave been recently described (Brem

et al

2002 Schadt

et al

2003) For example control of most expression differencesin normal unstressed yeast maps to

trans

-acting loci thatwould be classified as QTL (Yvert

et al

2003)Here parents of the key maize mapping lines are com-

pared to identify groups of co-expressed genes and to testwhether there was adequate variation in controlling locibetween these lines with regard to UV responses It is bestto start a mapping project with target genes that differ inthe parents as this ensures that variation will be present forthe loci that control those traits in future QTL mappingexperiments Thus we designed a statistically rigorousexperiment to find genes that differed in expression afterUV treatment in two inbred lines that are the foundationfor many mapping studies in maize The design of thisexperiment emphasized the detection of UV-specific effectsand interactions between UV exposure and line Ourdesign is akin to the classic split-plot with lines as the wholeplot factor and UV exposure as the split-plot factor Split-plots are commonly used in agriculture as line and treat-ment interactions are a key focus in breeding Overall 3of the cDNAs spotted on the array were shown to exhibitexpression differences resulting from interactions betweenthe lines and the UV treatment These interactions canpotentially be classified in three ways related to the changein expression from control to UV the expression is in oppo-site directions for the two lines it is in the same directionfor both lines but is significantly larger in one line than theother it is present in one line but not the other

Discovery of genes with expression alterations by UV isefficiently accomplished using our experimental design Wefound that 175 of the cDNAs on the array were signifi-cantly affected by UV treatment this group had no signifi-cant difference between the two lines Consequently UVtreatment elicits major changes in the transcriptome ofmaize leaves

MATERIALS AND METHODS

Maize lines

Inbred lines B73 and Mo17 from the same stocks used tocreate the IBM mapping lines were kindly supplied by MLee (Lee

et al

2002) These lines were increased in the fieldnursery at Clayton NC USA The seedling growth habit ofthese two lines is sufficiently distinct to allow error-check-ing in seed handling steps There is no obvious difference inseedling leaf insertion angle with the sheath in these twolines although B73 leaf blades are narrower (have a smallermargin-to-margin distance) than Mo17 blades

Growth conditions and ultraviolet treatment

Individual seeds were placed one per 6 cm pot filled withvermiculite at a density of 36 pots per flat and grown in the

greenhouse without supplemental lighting for 10 d to thethree-leaf stage Germination of seed from these two lineswas similar under these conditions so the two lines were atthe same growth stage upon collection The greenhousephotosynthetically active radiation levels are approxi-mately 20 less than outdoor radiation The two lines werethen exposed to 4 h of ultraviolet radiation from UV313bulbs (Q-Panel Lab Products Cleveland OH USA) sus-pended about 30 cm above the plants These bulbs arewidely used for UV supplementation the UV spectralpower output (available from the bulb manufacturer) issimilar to the Phillips FS40 bulbs that have been used forUV supplementation studies for many years (Sisson ampCaldwell 1975) The UV dose was measured at plant heightusing the UV-B sensor on an International Light radiome-ter Model IL1400A (International Light NewburyportMA USA) the treatment plants received a total UV doseof 86 J m

-

2

at a rate of 0024 W m

-

2

Outdoor solar UV-Blevels measured with this radiometer at peak annual UVover 3 years are 0038

plusmn

001 W m

-

2

(R Whitehead per-sonal comm) The output of the bulbs was similar whenmeasured at positions corresponding to all the positions ofthe plants in the experiment thus we have no evidence fora gradient of UV within the experiment Control plantswere placed under UV313 bulbs that were covered withpolyester (MylarD US Plastics Lima OH USA) whichtransmits visible light but excludes UV-B The UV-A sensorfor the IL1400A was used to measure UV-A levels in bothtreatment and control and there was no difference in UV-Alevels After UV irradiation the UV bulbs were turned offand the plants were allowed to recover for 4 h in the green-house A simplified experimental design is shown in Fig 1this figure is not drawn to scale and in order to make thedrawing less cluttered only one plant from each set isshown

The second and third seedling leaf blade from each offour plants was harvested and dropped immediately intoliquid nitrogen Pooling assured sufficient RNA and alsodecreases biological variability pooling of specimens hasbeen recommended for microarrays (Peng

et al

2003)

RNA preparation mRNA purification and probe synthesis

Total RNA was extracted from frozen tissue using Trizol(Invitrogen Co Carlsbad CA USA) as described inthe microarray manufacturerrsquos protocol (httpwwwzmdbiastateedu) Poly(A)

+

RNA was isolated usingOligotex (Qiagen Inc Valencia CA USA) and 4

m

g ofpoly (A)

+

RNA was used for each cDNA synthesis usingSuperscript II reverse transcriptase (Life TechnologiesCarlsbad CA USA) The cDNA was labelled using 100

m

M

Cy5-dUTP or Cy3-dUTP (Amersham Pharmacia BiotechPiscataway NJ USA) Excess nucleotides and primerswere removed using QIAquick PCR Purification Kit(Qiagen Inc)

1376

J E Blum

et al



27

1374ndash1386

Hybridization scanning and gridding

Eight maize cDNA Unigene 01-01-04 slides were usedMaize Unigene I arrays fabricated by the Maize Gene Dis-covery Project contain 5376 ESTs (for more informationsee httpgremlin3zooliastateeduzmdbmicroarray)About 95 of the spotted cDNAs showed significanthybridization when leaf mRNA was used for the experi-ments Within these arrays the cDNA samples are printedat least three times The experimental and reference sam-ples were labelled with either Cy5-dUTP or Cy3-dUTPfluorescent dye (Amersham Pharmacia Biotech) Two sam-ples one labelled with each dye were mixed and thenhybridized to a microarray for 15 h at 60

infin

C The slides werewashed in three wash steps 2

yen

SSC 05 SDS 05

yen

SSCand 005

yen

SSC The three washes were at room temperaturefor 5 min each with gentle shaking The slides were scannedwith a GenePix 4000B Scanner (Axon Instruments IncUnion City CA USA) The levels of the image intensitiesbetween the Cy3 and Cy5 fluorescent dye emission chan-nels was adjusted according the manufacturerrsquos instructionsso that both channels had maximum dynamic range Theexperiments were repeated four times with samples fromdifferent experiments as biological replicates in two differ-ent dye-swapping experiments In these dye swappingexperiments the RNA samples from different experimentswere labelled reciprocally

S

CANALYZE

(Eisen 2002) was used to quantify the imagefiles from the scanner We also repeated the gridding usingthe SPOT program (Yang

et al

2002) and the SPOT datawere statistically analysed using the same methods withlittle difference between the number and type of the signif-icance calls of the genes (data not shown) As the SPOTgrids cannot be individually adjusted and thus more spotswere flagged as poor quality we used the more completeS

CANALYZE

data set for our analyses

Statistical analysis

Determination of significant differential expression wasundertaken with analysis of variance (

ANOVA

) style mod-

els From the ideas provided by Kerr amp Churchill (2001)and Wolfinger

et al

(2001) and based on the specific designof this experiment the following

ANOVA

models were con-structed

(1)

(2)

In model (1)

y

is the logarithm (base 2) of the intensity fora particular spot

L

T

and

D

are global effects due todifferences in lines (Mo17 and B73) treatments (controland UV exposure) and dyes respectively while

LT

repre-sents interaction between lines and treatments accordingto convention

m

is overall mean and

Œ

represents residualerror The

AD

interaction term is present as suggested byWolfinger

et al

to account for intensity scaling done on thetwo channels The nested term

A

(

L

) accounts for variationacross replicate arrays in a slightly different fashion thanthat set forth in Kerr and Churchillrsquos

ANOVA

models Theirapproach considered connected designs however in thisexperiment lines are not connected because no array hassamples from two different lines hybridized to it Model (2)takes residuals from model (1) as normalized response val-ues and includes gene specific effects (

G

) for each term inthe previous model Additionally it models replicate spotvariation via the

GS

(

A

) termBoth models were fitted using the SASSTATreg soft-

warersquos MIXED procedure with model (2) being fitted geneby gene Observed significance levels for all effects testswere adjusted for multiplicity of testing by use of the Sidakmethod Significance was assessed based on an experiment-wise significance level of 005

The levels of expression of the SP10 negative controlswere used to determine the log-ratio level for expressionabove background SP10 is a human gene sequence the SPseries spots are recommended by the microarray slide pro-ducer as negative controls with little cross-hybridization tomaize We chose SP10 in particular as the levels of signalwere the most stable All cDNAs with all levels below the

y L A L T LT D ADgijklr i j i k ik l jl gijklr= + + + + + + +m e( ) ( )

r GL GA L GT GLT

GD GS Agijklr gi gj i gk gik

gl gr j gijklr

= + + + ++ +

( )

( )( )

( ) e

Figure 1

Simplified design of experi-ment Four replicates were done for each line with UV treatment and control RNA from one batch of plants from each line hybridized to one slide Dye-switch dupli-cates were done to remove the effect of differential incorporation of label For clarity only one plant is shown in each group


1377



27

1374ndash1386

SP10 median were set to background thus diminishingtheir influence on the final list of genes Ten genes that mayhave been otherwise called significant were removed fromthe 5366 total using this criterion There are still 201 geneswith at least one very low level however these wereretained for significance testing as some may have signifi-cant expression level alterations in one line or conditionThere were six cDNAs with significant differences betweenlines but not interaction or treatment these genes are indi-cated in Table S1 (GEO Accession GSE1353) and were notanalysed further In compliance with MIAME standards(Brazma

et al

2001) raw data standardized residuals frommodel (1) and a table listing all genes with expressionlevels

P

-values and significance calls are available fromNCBI GEO at httpwwwncbinlmnihgov as accessionsGPL1208 GSE1353 GSM21917 GSM21918 GSM21919GSM21921 GSM21922 GSM21923 GSM21924 andGSM21925

Cluster analysis on gene expression levels was performedusing the SAS version 8 E

NTERPRISE

M

INER

software Clus-ter node using the default settings except for specifyinginput cluster number as equal to the total sample numberClustering was performed separately on genes significantby interaction (Table S1) and genes significant by treatment(Table S2 GEO Accession GSE1353) the output clusternumber is listed for each gene name in these tables

Determination of significantly over-represented words inthe homolog lists was done using the automated lexicalanalysis program ALACK (Kim amp Falkow 2003) To avoidartificial over-representation for high-homology geneseach of the three homolog lists was compared separatelyThe list for all genes that passed quality control (5366 totalgenes) was compared to the interaction list and the treat-ment list Terms with

P

-values greater than 90 are listedas described in Kim amp Falkow (2003) (Table 3) The termprotein was abundant and a

P

-value could not be calculatedusing the program

RESULTS

Experimental design and statistical considerations

In prior work using B73 and Mo17 we found that a trendtoward increased whole-plant responses to UV began at adose of 82 J m

-

2

(Cartwright

et al

2001) Here we utilized asimilar dose of UV 86 J m

-

2

to capture changes in geneexpression most likely to be relevant to the morphologicalchanges we had previously reported Our goal was to iden-tify gene expression affected by a specific ultraviolet radia-tion treatment particularly those genes affected differentlyin the two mapping parent inbred lines A simplified dia-gram of the experimental design is given in Fig 1 Eacharray was hybridized to a treatment and a control samplefrom the same line with four replicates of each line Ourdesign provides high sensitivity for detection of effects ofUV exposure and the interactions between lines and UVexposure

A two-stage statistical analysis procedure was used firststandardization then significance testing Determination ofsignificant differential expression was undertaken withanalysis of variance (

ANOVA

) models We used a techniquesimilar to that suggested by Kerr amp Churchill (2001) in theirdiscussion of

ANOVA

models for microarray experimentsTheir approach considered connected designs however inthis experiment lines are not connected because no arrayhas samples from two different lines hybridized to it Froma classical experimental design perspective our design issimilar to a split-plot design with line being the whole plotfactor and treatment the split-plot factor The result of thisstatistical analysis is the placement of each gene that passedquality control into one of three significance groups (1) thegroup lsquononersquo which has no significant difference betweencontrol and ultraviolet radiation treatment and no signifi-cant difference between expression in two lines (2) thegroup lsquointeractionrsquo or (3) the group lsquotreatmentrsquo Thesegroups are described in more detail below

Expression differences resulting from interaction of lines and the UV treatment

Genes in the interaction class include those with change inexpression from control to UV in opposite directions forthe two lines those in the same direction for both lines butsignificantly larger in one line than the other and thosewith expression differences present in one line and not theother The list cDNAs of these types is given in Table 1 andS2 (GEO Accession GSE1353) 110 of the 165 cDNAs inthe list were called significant by treatment and interactionand 55 were significant by interaction Figure 2 shows theoverall pattern of expression in these genes comparing thegene expression after UV in each line for each cDNAFigure 2 also shows the pattern of alterations in geneexpression as each of the four quadrants of the graph indi-cates an expression grouping (increased in both lines todifferent extents decreased in both lines to differentextents differently expressed with B73 increased and Mo17decreased and differently expressed with B73 decreasedand Mo17 increased)

To view the gene list in simplified form we clustered thesedata by expression level reasoning that highly correlatedgenes could represent a specific process or related pro-cesses SASF

AST

C

LUS

reg software was used to generateoptimal clusters resulting in 16 clusters that contain 1ndash26members (Table 1 S2 and S3 GEO Accession GSE1353)There was no significant grouping or trend toward similar-ity of homolog terms in the cluster groups Cluster 14 con-tained most of the genes with very low expression levels

When we compared interaction gene expression in thetwo lines there were significantly more of the lsquointeractionrsquocDNAs down-regulated after UV irradiation in Mo17 thanin B73 (Table 2) Down-regulation could result from ahigher received UV dose if Mo17 leaf tissue is more UV-penetrant To date however no significant differences inUV-induced DNA damage levels have been detected usingsensitive antibody assays for pyrimidine dimers (Cartwright

1378

J E Blum

et al



27

1374ndash1386

Table 1

Selected genes with expression significantly affected by ultraviolet radiation

Accessionnumber orclone ID

Sidak adjusted

P

-valueSignificancecall

Ratio of UV minuscontrol B73 log2 median values (expression level)

Ratio of UV minuscontrol Mo17 log2median values (expression level)

Homolog description (of sequence most similar to this maize sequence) Cluster ID

Genes with expression affected by interactionInteraction

P

-value Interactionclusters

Translation

BE128853 187

yen

10

-

5

treatment 003

-

030 ribosomal protein L30 6interaction (low) (low)

AW787726 491

yen

10

-

3

treatment

-

095

-

033 putative chloroplast 50S 13interaction (lowmedium) (medium) ribosomal protein

AW066279 634

yen

10

-

3

treatment

-

123

-

084 chloroplast ribosomal 13interaction (medium) (medium) L1-like protein

AW147172 757

yen

10

-

4

treatment 152 112 protein disulphide 12interaction (high) (high) isomerase

AW352521 141

yen

10

-

6

treatment

-

118

-

027 chloroplast RelA 13interaction (medium) (medium) homologue 2

Transcription factors

treatment

-

092

-

029 squamosa promoter AW066393 155

yen

10

-

3

interaction (medium) (medium) binding protein

treatment

-

042 005 bZIP protein-likeAW400081 307

yen

10

-

4

interaction (medium) (medium) putative elongin protein 411

041

-

070AW787437 939

yen

10

-

10

interaction (medium) (low) 16

Signal transduction

029

-

022 protein phosphatase 2 A AW216078 156

yen

10-2 interaction (medium) (low) regulatory subunit 16

-026 040 Ras-related GTP-AW331062 778 yen 10-3 interaction (medium) (medium) binding protein 13

016 -032 putative proteinAW191188 489 yen 10-3 interaction (low) (low) kinase 6

-024 022 putative receptor-likeAW424524 909 yen 10-5 interaction (high) (high) protein kinase 11

025 -053 protein-serineAW256121 506 yen 10-6 interaction (medium) (low) threonine kinase 16

-035 025 serinethreonineAW313159 150 yen 10-3 interaction (high) (high) kinase 11

treatment -175 -079 calcium-dependent AW231338 113 yen 10-2 interaction (mediumhigh) (high) protein kinase 9

Proteinases

treatment 304 155 putative Bowman-BirkAW574456 0 interaction (mediumhigh) (medium) serine proteinase 5

treatment -106 -032 methionine aminopeptidase-AW927396 775 yen 10-5 interaction (medium) (medium) like p 13

Photosynthesis

treatment -163 -075 pyruvate orthophosphateAW256115 271 yen 10-3 interaction (mediumhigh) (high) dikinase 15

interaction 011 -060

AW062052 117 yen 10-2 (medium) (low) Psst70 16

treatment -163 -088 probable photosystem II BE025281 932 yen 10-3 interaction (highmedium) (high) oxygen-evolving complex 9

UV alters expression profiles in maize mapping line inbreds 1379

copy 2004 Blackwell Publishing Ltd Plant Cell and Environment 27 1374ndash1386

treatment -170 -082BE128865 154 yen 10-3 interaction (highmedium) (high) PEPCase 9

treatment -221 -090AW288857 193 yen 10-5 interaction (highmedium) (medium) ferredoxin 10

treatment -176 -088BE025333 187 yen 10-7 interaction (highmedium) (low) carbonic anhydrase 10

treatment -151 -078 chlorophyll ab-binding AW289151 563 yen 10-4 interaction (medium) (medium) protein 13

DNARNA binding proteins

treatment -067 -011AW330583 158 yen 10-2 interaction (medium) (medium) putative topoisomerase 13

034 -030PAUTRE1 335 yen 10-4 interaction (low)

014(low)-029

MudrA transposaseputative RNA-binding like

AW224979 114 yen 10-5 interaction (low) (low) protein 6

Stress-related

treatment 396 246BE056094 521 yen 10-10 interaction (lowhigh) (mediumhigh) PRm 3 5

-023 035 NADPH-dependent HC-AW438100 572 yen 10-4 interaction (medium) medium) toxin reductase 13

treatment -026 058 DNA-damage inducible AW927431 220 yen 10-6 interaction (medium) (mediumhigh) protein DDI1 11

treatment 083 009AW399958 468 yen 10-5 interaction (high) (low) heat shock protein 82 8

Genes with expression affected by treatment TreatmentP-value

UV effect(expression level)

Treatmentclusters

DNA binding

AW065905 138 yen 10-2 decrease(medium)

DNA cytosine methyltransferase 41

AW257943 185 yen 10-5 increase(low)

DNA methyltransferase ZMET4 69

AW216033 136 yen 10-4 increase(medium)

nucleosomechromatin assembly factor 53

AW231627 387 yen 10-3 increase(high)

putative helicase 32

AW438057 410 yen 10-15 decrease(high)

putative transposase 76

BE012167 195 yen 10-11 increase(medium)

SAR DNA binding protein 51

Ratios were truncated at two decimal places The complete summary tables are available from GEO (httpwwwncbinlmnih) as seriesaccession number GSE1353 Blue indicates increase in expression after UV exposure and peach indicates decrease in expression after UVexposure Expression levels were grouped into low (-3 to -099 log2 median) medium (-1 to +099 log2 median) and high (+1 to +3 log2

median) groups for clarity


Sidak adjusted P-value

Significancecall




Genes with expression affected by interactionInteractionP-value

Interactionclusters

Table 1 Continued

1380 J E Blum et al


et al 2001) by measuring UV-B-induced chlorophyll fluo-rescence (Mazza et al 2000) or by quantifying absorbanceof leaf methanol extracts (A Stapleton and C Ballareacute datanot shown) in the two lines Determination of ultravioletoptical density differences in these lines may require moresensitive measurements of UV penetrance B73 and Mo17synthesize very low levels of flavonoids in seedling leaftissue because the regulatory transcription factors aremutant low-expression alleles (E Coe personal communi-cation E Bruggeman personal communication)

Overall comparison of the genes that were significant byinteraction shows that the distribution of expression levelsdiffers in B73 and Mo17 (Fig 3) Centering of the expres-sion level measurements demonstrates that the Mo17downB73 up genes have a narrower distribution than genesincreased in Mo17 and decreased in B73 but that the distri-bution does not differ strikingly above and below the cen-

troid line To determine if some genes failed to hybridize tocDNA from one line we examined the control channelexpression levels for all 67 of the genes in the list of genesthat were increased in one line and decreased in the otherIn this comparison there was no significant difference inthe number of genes with levels higher in Mo17 comparedto the number with levels higher in B73 (P = 054) Thus

Figure 2 Genes with expression levels signif-icantly altered in UV that show interaction The log2-transformed normalized expression levels are plotted for genes with expression significantly altered by UV in one line and not the other or in both lines but in different direc-tions (ie increase in one decrease in the other) These genes are labelled as significant by lsquointeractionrsquo in the statistical analysis The median level from each line is shown for each gene

Table 2 Comparison of genes regulated in opposite directions

B73 Mo17 Number of genes

Expression + ndash 47Pattern ndash + 20

The number of genes with expression increased in one line anddecreased in the other line is shown A Fisher exact test gaveP lt 00001 for these data

Figure 3 Distribution of expression lev-els for genes significant by interaction This plot allows comparison of the general dis-tribution of genes increased by UV in one line compared to the other line These data are centred so that the plot does not show the expression level (high or low) of each gene instead this plot shows the way the expression levels are distributed between lines on a comparable scale



failure of messages from Mo17 to hybridize because ofmismatches between the target and probe does not seem toexplain why there are more down-regulated genes in Mo17after UV treatment

Expression differences after UV treatment

We found 937 cDNAs significantly altered in expressionafter ultraviolet radiation treatment as listed in Table S3This is 175 of the cDNAs on the array The treatment listdoes not include any genes that were significantly differentby interaction only genes with significant expressionchange in the comparison of control to UV treatment Theoverall pattern of expression differences between lines isshown in Fig 4 We found that 512 genes increased afterultraviolet treatment and 425 decreased in expression afterUV treatment Clustering of the expression level measure-ments gave 91 different clusters this grouped the treat-ment-affected genes into clusters containing an average of10 genes with a range of one to 70 cDNAs per cluster Therewas no obvious (or significant) grouping of homologdescription terms within the cluster groups

Functional analysis

We are especially interested in identifying genes withknown functions that are over-represented in our lists ofgenes affected by ultraviolet radiation As structured ontol-ogy terms (GO terms) or EBI protein accession numbersare available for only 10 of the maize genes included inour array (496 of 5366 total) we used a more general tool toexamine the descriptors for each gene The gene descrip-tions are much more complete 3210 of the 5376 genes onthe array have some annotation available Significantlyover-represented terms in the interaction and treatment

gene description lists were identified with the ALACK pro-gram (Kim amp Falkow 2003) This program automaticallycompares the selected gene list with the list from all thegenes on the array and provides P-values for any terms thatare present in excess in the selected gene list With the 165ndashmember interaction gene list we found only one signifi-cantly over-represented term the word lsquochloroplastrsquo withP = 099 This suggests that genes encoding proteins des-tined for the chloroplast are more likely to be transcribeddifferently in the two lines In the treatment gene list wefound 36 terms with P-values greater than 090 (Table 3)The genes with these descriptors are thus more likely to befound in the genes with expression altered by UV radiationthese groups include genes involved in translation signaltransduction secondary metabolism and chloroplast func-tion Examination of the expression level differences inthese lexical groups shows that the more precise terms withsmaller numbers of genes have the most consistent expres-sion levels (Fig 5) Some terms such as kinase includegenes with both up-regulated and down-regulated mem-bers (above and below the line in Fig 5) One conspicuousdifference is seen in expression levels of genes for the largesubunit as compared to the small subunit of the ribosomeThe 40S protein genes tend to be increased less in expres-sion after UV than the 60S genes

The proportions of rare and unknown genes affected byUV are of particular interest line-specific expression pat-terns could contain transcripts that are rare in the existingEST collections andor are more likely to be species-spe-cific Genes defined by multiple ESTs that cluster into asingle contig are by definition represented multiple times inthe EST sequence collection Thus assignment to a contig isa proxy for more widespread transcription or higher tran-script level We compared the number of genes representedby singleton EST clones to the number of cDNAs that

Figure 4 Genes with expression levels signifi-cantly altered after UV exposure Log-trans-formed normalized gene expression levels are plotted for genes with expression significantly altered by ultraviolet radiation These genes show no significant interaction with line this class of genes included those with expression sig-nificantly increased by UV in both lines or decreased by UV in both lines The median level for each line is shown for each gene

ndash

ndash

ndash

ndash

ndash ndash

1382 J E Blum et al


Table 3 All significantly over-represented terms in gene homolog descriptions from treatment-affected genes

Homolog 1 Homolog 2 Homolog 3

Term P-value Term P-value Term P-value

40S 098 40S 091 40S 09660S 099 60S 097 60S 096ammonia 091 EF 098 cytochrome 093beta 092 adenosyl 093 acidic 091binding 096 ammonia 091 aldehyde 091calmodulin 094 calmodulin 091 ammonia 091choloroplast 098 chloroplast 0973 binding 099cytochrome 095 family 091 chloroplast 090eukaryotic 094 lyase 091 ligase 092family 092 methionine 094 lyase 096guanine 099 phenylalanine 094 phenylalanine 094isomerase 098 phosphate 097 phosphate 099kinase 091 pyrophosphate 096 ribosomal 099nucleotide 096 ribosomal 099 subunit 091phenylalanine 094 ribulose 091 thaumatin 094phosphate 099 translation 097 unknown 095ribosomal 099 translational 098sugar 098translation 098transporter 091transporting 093

Figure 5 Expression levels of genes sig-nificantly altered by treatment and grouped by homolog term Homolog terms that were significantly over-represented in the list of genes affected by UV treatment (Table 4) were selected and the expression level differences (median UV-median con-trol for each gene) were plotted

Table 4 Comparison of contig assignment frequency in gene lists

Treatment genes Interaction genes No-significant-change genes

TUC ID present 644 134 2765No TUC assignment 293 (31) 31 (19) 1349 (33)

The number of genes in the treatment interaction and no-significant-change gene lists that are clustered into contig groups (tentative uniquecontig TUC) or not clustered (singletons) are shown A 2 yen 3 chi-squared table test gave P = 000067 Fisher exact tests comparing treatmentto no-change gave P = 0375 whereas comparing interaction to no-change gave P = 00001



belong to a cluster (a tentative unique contig or TUC) Inthe interaction gene list there were 81 with a TUCassignment as compared to 66 in the no-significant-change gene list (Table 4) Thus contrary to our expecta-tions there are significantly more clustered transcripts andhence more commonly expressed transcripts in the interac-tion list

In order to determine if genes with known homologs orfunctions are different in the treatment or interactiongroups we compared the frequency of genes with a blankannotation (assigned if there was no similar BLAST hitwith an E-value of less than 10-20) or with the annotationcontaining the word lsquounknownrsquo to those genes with ahomolog assigned The treatment list had 39 and the no-significant-change list had 53 such no-homolog genes(Table 5) this difference was significant (P = 00001) Thusthe treatment gene list has more genes with known closehomologs than the other lists

DISCUSSION

Overall 165 (3) of the cDNAs spotted on the array wereshown to exhibit significant expression differences whenconsidering interactions between line and UV treatmentOur experimental design also allowed efficient discovery ofgenes with significant expression alterations by UV Wefound that 937 (175) of the cDNAs on the array weresignificantly affected by UV treatment this group had nosignificant difference between the two lines Thus UV treat-ment elicits substantial alteration in the transcriptome pro-file of maize seedling leaf blades

Overview of functional analysis

In the analysis of UV-B impact on gene expression reportedhere all seven of the top-20 largest expression alterationgenes that have a known homolog are metabolic enzymesor chaperone proteins These results indicate that UV-Btreatment elicits changes in the expression of core cellularfunctions conducted by abundant proteins Transcriptionfactors and signal transduction pathway members tend tohave smaller differences in expression after treatmentHomolog names that are over-represented in the treatmentgene list include members with both increased anddecreased expression levels (Fig 5) As verification that thehomolog word list methods are useful in highlighting pro-

cesses specific names such as calmodulin identify arrayelements that are quite similar to each other in expressionlevels (Table S3)

To examine the functional assignments in more detail wemanually grouped the treatment and interaction gene listsinto functional groups (Table 1 S2 and S3) The no-signifi-cant-change gene list was not manually grouped When weexamined the genes that are manually classifiable we seeno striking differences in the proportion of genes in thetreatment list subcategories (such as transcription factors ormetabolic enzymes) compared to the interaction list subcat-egories Additional large-scale automatic annotation of themaize gene space would be very useful in further classifica-tion of differentially expressed genes

New gene classes differing after UV and across lines in maize

Manual classification of the list of interaction genes allowsus to compare gene classes with the previous maize micro-array experiment on near-inbred lines We focus on geneclasses those with several genes of the same functional typeincluded because annotations can have rather high errorrates for individual genes Gene classes newly identified asdiffering in their UV response between lines includekinases proteinases and transcription factors (Table 1)

Many of the genes that differ in expression betweenthese two maize inbred lines have no close similarity thatwould allow functional classification for example thecDNA (AW144870) with the largest difference in expres-sion is a gene with no known homolog

New classes of genes affected by UV

In this set of experiments we have identified a large numberof genes with transcript levels significantly increased afterultraviolet radiation but with no significant differencesbetween UV-affected expression in B73 and Mo17 Severalclasses of genes that are UV-regulated in our experimenthave not been previously identified as modulated by ultra-violet radiation treatment of plants In particular we foundcalcium-binding and guanine nucleotide exchange proteinsfrom the signal transduction class (Table 1 and S3) to haveexpression alterations these protein classes have not beenpreviously identified as UV-affected in maize

Table 5 Comparison of gene annotation frequency in gene lists


Homolog description present 571 85 1993No homolog description ordescription of lsquounknownrsquo

366 (39) 80 (48) 2265 (53)

Number of genes in the treatment and interaction gene lists that have homolog annotation compared those without annotation or with theannotation of lsquounknownrsquo A 2 yen 3 chi-squared table test gave P = 0 Fisher exact tests gave P = 00001 for the comparison of treatment tono-change and P = 026 for interaction compared to no-change

1384 J E Blum et al


Responding to information conveyed by signal transduc-tion transcription factors chromatin remodelling proteinsand DNA repair functions control the level of expression ofregulated genes We found several new types of transcrip-tion factors whose expression is altered by UV treatmentOne of the most striking new gene classes in our experi-ment was the chromatin-related group DNA cytosinemethyltransferase nucleosome assembly factor and SARbinding protein all increased in expression after ultravioletradiation treatment No genes of this class have been previ-ously reported as UV-affected in plants

New members of known gene classes

We found two bZIP protein-like cDNAs in the transcrip-tion factor class one was differently expressed in the twolines (Table 1 and S2) These genes are transcriptional fac-tors with a basic region for DNA binding followed by aleucine zipper region for dimerization Binding sites foranother class of bZIP factors the CPRFs are found in thepromotor region of the UV-B-regulated parsley chalconesynthase gene however the CPRFs in parsley are regu-lated by protein phosphorylation rather than by alterationsin transcription of their genes (Schafer Kunkel amp Frohnm-eyer 1997 Kircher et al 1998) Thus there are at least twodifferent modes of ultraviolet radiation regulation of eventhis one structural class of transcription factors

Ribosomal protein genes have recently been identified asUV-regulated in plants (Broscheacute et al 2002 Casati amp Wal-bot 2003 Izaguirre et al 2003) Additional ribosomal pro-tein genes from this work include S2 S3 S10 S24 L1 L2L11 L13 L15 L30 The ribosomal protein response is quiteUV-specific in plants and in cultured keratinocytes the onlyreport of differential up-regulation of expression of thisgroup is in development and differentiation in human andrat cells (Angelastro Torocsik amp Greene 2002 KirschnerPujol amp Radu 2002 Takao et al 2002) Ribosomal proteingenes tend to be co-ordinately expressed in all speciesexamined (Stuart et al 2003) Therefore the UV-regulatedstep is likely to be a regulatory function that controls anentire suite of ribosomal protein genes

Comparison with previous array experiments to examine UV-induced expression in maize

We compared our experiment with a recent microarrayexperiment designed to define genes differentially regu-lated in near isogenic W23 maize lines differing in flavonoidsunscreen pigments (Casati amp Walbot 2003) The flavonoidcomparison was done with array type 606 and this experi-ment was done with unigene1ndash1-4 Of the 82 genes presenton both arrays (54 control genes which are present on allthe public maize arrays plus 28 additional genes) 66 genesare regulated in the same manner in the flavonoid compar-ison experiment as in the B73 to Mo17 comparisonreported here Eight genes that were UV-regulated in theflavonoid experiment were not UV-regulated significantlyin this work and a further eight cDNAs significant by treat-

ment in this work were not called as regulated in the fla-vonoid comparison experiment Given that theexperiments used different ultraviolet fluences and treat-ment lengths as well as analysing different types of tissuethe 80 congruence is quite impressive

Comparison with array experiments performed in dicotyledonous plants

Approximately 150ndash200 mya separate maize a monocotfrom the dicotyedonous flowering plants To determine ifUV responses in these two major branches of theAngiosperms are similar we have compared the maizeresults to existing data from dicots A small-scale experi-ment using a single microarray and confirmatory RNA blothybridization to examine gene expression after UV in Ara-bidospis (Broscheacute et al 2002) also matches the resultsreported here in a general way Of the 41 genes identified asUV-regulated in Arabidopsis after 15 kJ m-2 over 3 h (a 17-fold difference in fluence rate compared to the maizeexperiment) there were nine genes with homologs on ourmaize unigene arrays Six of the nine were significantlyregulated in our experiment the remaining three were notcalled as significant in our experiment Specifically proteinphosphatase 2C chitinase caffeoyl CoA O-methyltrans-ferase phosphoribosylformylglycinamidine cyclo-ligaseprecursor 40S ribosomal S10 and A thaliana MGD826has maize homologs and were identified as UV-regulated inboth experiments One particular kinase homolog was notcalled as significant in our experiment (the P-value was justbelow the cutoff) and the maize homologs of A thalianagenes MAH2020 and T6H20230 were not identified assignificantly regulated by UV in our experiment

We compared our list of UV-affected genes with the listof genes affected by solar UV in an experiment with anarray of herbivory-affected genes from Nicotiana attenuataFew of the UV-affected N attenuata genes on the array hadhomologs on our Unigene slides Of the five that were incommon three (Rubisco small subunit photosystem II10 kDa and glyceraldehyde-3-phosphate dehydrogenase)were regulated in the same way in both experiments AnRNA-binding glycine-rich protein was not significantly reg-ulated in our maize experiment although the homolog wasdecreased in the tobacco experiment and a ribosomal 40SS4 protein homolog was increased in maize and decreasedin tobacco

The group of genes defined by ANOVA as exhibiting sig-nificant differences between Mo17 and B73 identifies geneswith expression that is altered differently after ultravioletradiation stress in the two mapping lines These genes aregood candidates for future mapping of the regulatory locithat control the expression of these genes It will be espe-cially interesting to see how the overall proportions of up-regulated and down-regulated genes are controlled that ishow many lsquomasterrsquo regulatory switches are distinctivebetween the two lines

We were able to detect many genes with altered expres-



sion after UV because of the high power of our experimen-tal design Some functional classes were identified for thefirst time in this work and our results also confirm priorwork for both maize and dicot genes Collectively the avail-able data on plant responses to UV indicate that althoughmany gene functional classes are responsive to diverse envi-ronmental influences there are genes that are specificallymodulated by UV-B At the moment grouping genes byfunction is still crude but as annotation and analysis meth-ods improve MIAME data sets can be analysed retrospec-tively to correlate gene expression patterns with specificregulatory states and pathways

ACKNOWLEDGMENTS

We appreciate the superb field nursery support provided toAS by Jim Holland and Major Goodman Department ofCrop Science North Carolina State University We aregrateful to Rob Whitehead Department of Chemistry andBiochemistry UNCW for contributing his data on UV-Blevels and for allowing us to use the radiometer This workwas supported by a new faculty grant from UNCW to ASthe Stanford component of the project was supported inpart by a grant from the USDA (NRI2003-00745) to VWand PC was a post-doctoral fellow of Fundacioacuten Antorchasand a member of the Research Career of the ConsejoNacional de Investigaciones Cientiacuteficas y Teacutecnicas(CONICET) JB performed the statistical analysis PCcarried out the microarray hybridizations and scans VWedited the manuscript AS designed this study grew andtreated the plants assisted in the statistical analysis andwrote the manuscript

REFERENCES

Alonso-Blanco C amp Koornneef M (2000) Naturally occurringvariation in Arabidopsis an underexploited resource for plantgenetics Trends in Plant Science 5 22ndash29

Angelastro J Torocsik B amp Greene L (2002) Nerve growth factorselectively regulates expression of transcripts encoding riboso-mal proteins BMC Neuroscience 3 3

Ballareacute CL (2003) Stress under the sun spotlight on ultraviolet-Bresponses Plant Physiology 132 1725ndash1727

Brazma A Hingamp P Quackenbush J et al (2001) Minimuminformation about a microarray experiment (MIAME) -towardstandards for microarray data Nature Genetics 29 365ndash371

Brem RB Yvert G Clinton R amp Kruglyak L (2002) Geneticdissection of transcriptional regulation in budding yeast Science296 752ndash755

Broscheacute M Schuler MA Kalbina I Connor L amp Strid Aring (2002)Gene regulation by low level UV-B radiation identification byDNA array analysis Photochemical and Photobiological Science1 656ndash664

Broscheacute M amp Strid Aring (2003) Molecular events following percep-tion of ultraviolet-B radiation by plants Physiologia Plantarum117 1ndash10

Caldwell MM (1997) Alterations in competitive balance InPlants and UV-B Responses to Environmental Change (ed PJLumsden) pp 305ndash315 Cambridge University Press Cam-bridge UK

Cartwright HN Baucom C Singh P Smith KL amp StapletonAE (2001) Intraspecific comparisons reveal differences in thepattern of ultraviolet radiation responses in four maize (Zeamays L) varieties Journal of Photochemistry and PhotobiologyB Biology 62 88ndash96

Casati P amp Walbot V (2003) Gene expression profiling inresponse to ultraviolet radiation in maize genotypes with vary-ing flavonoid content Plant Physiology 132 1739ndash1754

Cheung VG amp Spielman RS (2002) The genetics of variation ingene expression Nature Genetics Suppl 32 522ndash525

Damerval C Maurice A Josse JM amp de Vienne D (1994) Quan-titative trait loci underlying gene product variation a novel per-spective for analyzing regulation of genome expression Genetics137 289ndash301

Eisen M (2002) Scanalyze v 250 httpranalblgovEisenSoft-warehtm

Gyula P Schafer E amp Nagy F (2003) Light perception and signal-ling in higher plants Current Opinion in Plant Biology 6 446ndash452

Herrlich P Blattner C Knebel A Bender K amp Rahmsdorf HJ(1997) Nuclear and non-nuclear targets of genotoxic agents inthe induction of gene expression shared principles in yeastrodents man and plants Biological Chemistry 378 1217ndash1229

Izaguirre MM Scopel AL Baldwin IT amp Ballareacute CL (2003)Convergent responses to stress Solar ultraviolet-B radiation andManduca sexta herbivory elicit overlapping transcriptionalresponses in field-grwon plants of Nicotiana longiflora PlantPhysiology 132 1755ndash1767

Jansen RC (2003) Studying complex biological systems using mul-tifactorial perturbation Nature Reviews Genetics 4 145ndash151

Jansen RC amp Nap J-P (2001) Genetical genomics the addedvalue from segregation Trends in Genetics 17 388ndash391

Jin W Riley RM Wolfinger RD White KP Passador-GurgelG amp Gibson G (2001) The contributions of sex genotype andage to transcriptional variance in Drosophila melanogasterNature Genetics 29 389ndash395

Jordan BR (2002) Molecular response of plant cells to UV-Bstress Functional Plant Biology 29 909ndash916

Kerr MK amp Churchill GA (2001) Statistical design and the anal-ysis of gene expression microarray data Genetical Research 77123ndash128

Kim CC amp Falkow S (2003) Significance analysis of lexical bias inmicroarray data BMC Bioinformatics 4 12

Kircher S Ledger S Hayashi H Weisshaar B Schafer E ampFrohnmeyer H (1998) CPRF4a a novel plant bZIP protein ofthe CPRF family comparative analysis of light-dependentexpression post-transcriptional regulation nuclear import andheterodimerization Molecular and General Genetics 257 595ndash605

Kirschner M Pujol G amp Radu A (2002) Oligonucleotidemicroarray data mining search for age-dependent gene expres-sion Biochemical and Biophysical Research Communications298 772ndash778

Lee M-LT Sharopova N Beavis WD Grant D Katt M BlairD amp Hallauer A (2002) Expanding the genetic map of maizewith the intermated B73 x Mo17 (IBM) population PlantMolecular Biology 48 453ndash461

Liu K Goodman M Muse S Smith JS Buckler IVES ampDoebley J (2003) Genetic structure and diversity among maizeinbred lines as inferred from DNA microsatellites Genetics 1652117ndash2127

Mazza CA Boccalandro HE Giordano CV Battista DScopel AL amp Ballareacute CL (2000) Functional significance andinduction by solar radiation of ultraviolet-absorbing sunscreensin field-grown soybean crops Plant Physiology 122 117ndash126

Oleksiak MF Churchill GA amp Crawford DL (2002) Variation

1386 J E Blum et al


in gene expression within and among natural populationsNature Genetics 32 261ndash266

Peng X Wood C Blalock E Chen K Landfield P amp StrombergA (2003) Statistical implications of pooling RNA samples formicroarray experiments BMC Bioinformatics 4 26

Sandberg R Yasuda R Pankratz DG Carter TA Del Rio JAWodicka L Mayford M Lockhart DJ amp Barlow C (2000)From the cover regional and strain-specific gene expressionmapping in the adult mouse brain Proceedings of the NationalAcademy of Sciences of the USA 97 11038ndash11043

Schadt EE Monks SA Drake TA et al (2003) Genetics ofgene expression surveyed in maize mouse and man Nature 422297ndash302

Schafer E Kunkel T amp Frohnmeyer H (1997) Signal transductionin the photocontrol of chalcone synthase gene expression PlantCell and Environment 20 722ndash727

Sisson WB amp Caldwell MM (1975) Lampfilter systems for sim-ulation of solar UV irradiance under reduced atomosphericozone Photochemistry and Photobiology 21 453ndash456

Stuart JM Segal E Koller D amp Kim SK (2003) A gene-coex-pression network for global discovery of conserved genetic mod-ules Science 302 249ndash255

Takao J Ariizumi K Dougherty II amp Cruz PDJ (2002)Genomic scale analysis of the human keratinocyte response tobroad-band ultraviolet-B radiation Photodermatology Photo-immunology and Photomedicine 18 5ndash13

Townsend JP Cavalieri D amp Hartl DL (2003) Populationgenetic variation in genome-wide gene expression MolecularBiology and Evolution 20 955ndash963

Wolfinger RD Gibson G Wolfinger ED Bennett L HamadehH Bushel P Afshari C amp Paules RS (2001) Assessing genesignificance from cDNA microarray expression data via mixedmodels Journal of Computational Biology 8 625ndash638

Yang YH Buckley MJ Dudoit S amp Speed TP (2002)Comparison of methods for image analysis on cDNA microarraydata Journal of Computational and Graphical Statistics 11 108ndash136

Yvert G Brem RB Whittle J Akey JM Foss E Smith ENMackelprang R amp Kruglyak L (2003) Trans-acting regulatoryvariation in Saccharomyces cerevisiae and the role of transcrip-tion factors Nature Genetics 35 57ndash63

Received 16 December 2003 received in revised form 14 May 2004accepted for publication 1 June 2004


1375



27

1374ndash1386

2002) Regulation of protein expression was the first molec-ular trait mapped using a natural variationQTL approach(Damerval

et al

1994) Examples of the use of gene expres-sion levels for mapping of regulatory loci in several specieshave been recently described (Brem

et al

2002 Schadt

et al

2003) For example control of most expression differencesin normal unstressed yeast maps to

trans

-acting loci thatwould be classified as QTL (Yvert

et al

2003)Here parents of the key maize mapping lines are com-

pared to identify groups of co-expressed genes and to testwhether there was adequate variation in controlling locibetween these lines with regard to UV responses It is bestto start a mapping project with target genes that differ inthe parents as this ensures that variation will be present forthe loci that control those traits in future QTL mappingexperiments Thus we designed a statistically rigorousexperiment to find genes that differed in expression afterUV treatment in two inbred lines that are the foundationfor many mapping studies in maize The design of thisexperiment emphasized the detection of UV-specific effectsand interactions between UV exposure and line Ourdesign is akin to the classic split-plot with lines as the wholeplot factor and UV exposure as the split-plot factor Split-plots are commonly used in agriculture as line and treat-ment interactions are a key focus in breeding Overall 3of the cDNAs spotted on the array were shown to exhibitexpression differences resulting from interactions betweenthe lines and the UV treatment These interactions canpotentially be classified in three ways related to the changein expression from control to UV the expression is in oppo-site directions for the two lines it is in the same directionfor both lines but is significantly larger in one line than theother it is present in one line but not the other

Discovery of genes with expression alterations by UV isefficiently accomplished using our experimental design Wefound that 175 of the cDNAs on the array were signifi-cantly affected by UV treatment this group had no signifi-cant difference between the two lines Consequently UVtreatment elicits major changes in the transcriptome ofmaize leaves

MATERIALS AND METHODS

Maize lines

Inbred lines B73 and Mo17 from the same stocks used tocreate the IBM mapping lines were kindly supplied by MLee (Lee

et al

2002) These lines were increased in the fieldnursery at Clayton NC USA The seedling growth habit ofthese two lines is sufficiently distinct to allow error-check-ing in seed handling steps There is no obvious difference inseedling leaf insertion angle with the sheath in these twolines although B73 leaf blades are narrower (have a smallermargin-to-margin distance) than Mo17 blades

Growth conditions and ultraviolet treatment

Individual seeds were placed one per 6 cm pot filled withvermiculite at a density of 36 pots per flat and grown in the

greenhouse without supplemental lighting for 10 d to thethree-leaf stage Germination of seed from these two lineswas similar under these conditions so the two lines were atthe same growth stage upon collection The greenhousephotosynthetically active radiation levels are approxi-mately 20 less than outdoor radiation The two lines werethen exposed to 4 h of ultraviolet radiation from UV313bulbs (Q-Panel Lab Products Cleveland OH USA) sus-pended about 30 cm above the plants These bulbs arewidely used for UV supplementation the UV spectralpower output (available from the bulb manufacturer) issimilar to the Phillips FS40 bulbs that have been used forUV supplementation studies for many years (Sisson ampCaldwell 1975) The UV dose was measured at plant heightusing the UV-B sensor on an International Light radiome-ter Model IL1400A (International Light NewburyportMA USA) the treatment plants received a total UV doseof 86 J m

-

2

at a rate of 0024 W m

-

2

Outdoor solar UV-Blevels measured with this radiometer at peak annual UVover 3 years are 0038

plusmn

001 W m

-

2

(R Whitehead per-sonal comm) The output of the bulbs was similar whenmeasured at positions corresponding to all the positions ofthe plants in the experiment thus we have no evidence fora gradient of UV within the experiment Control plantswere placed under UV313 bulbs that were covered withpolyester (MylarD US Plastics Lima OH USA) whichtransmits visible light but excludes UV-B The UV-A sensorfor the IL1400A was used to measure UV-A levels in bothtreatment and control and there was no difference in UV-Alevels After UV irradiation the UV bulbs were turned offand the plants were allowed to recover for 4 h in the green-house A simplified experimental design is shown in Fig 1this figure is not drawn to scale and in order to make thedrawing less cluttered only one plant from each set isshown

The second and third seedling leaf blade from each offour plants was harvested and dropped immediately intoliquid nitrogen Pooling assured sufficient RNA and alsodecreases biological variability pooling of specimens hasbeen recommended for microarrays (Peng

et al

2003)

RNA preparation mRNA purification and probe synthesis

Total RNA was extracted from frozen tissue using Trizol(Invitrogen Co Carlsbad CA USA) as described inthe microarray manufacturerrsquos protocol (httpwwwzmdbiastateedu) Poly(A)

+

RNA was isolated usingOligotex (Qiagen Inc Valencia CA USA) and 4

m

g ofpoly (A)

+

RNA was used for each cDNA synthesis usingSuperscript II reverse transcriptase (Life TechnologiesCarlsbad CA USA) The cDNA was labelled using 100

m

M

Cy5-dUTP or Cy3-dUTP (Amersham Pharmacia BiotechPiscataway NJ USA) Excess nucleotides and primerswere removed using QIAquick PCR Purification Kit(Qiagen Inc)

1376

J E Blum

et al



27

1374ndash1386



infin


yen

SSC 05 SDS 05

yen

SSCand 005

yen


S

CANALYZE


et al


CANALYZE




ANOVA

) style mod-


et al


ANOVA


(1)

(2)

In model (1)

y


L

T

and

D


LT


m

is overall mean and

Œ


AD


et al


A

(

L


ANOVA


G


GS

(

A





r GL GA L GT GLT


gl gr j gijklr

= + + + ++ +

( )

( )( )

( ) e

Figure 1



1377



27

1374ndash1386


et al


P



NTERPRISE

M

INER



P


P


RESULTS



-

2

(Cartwright

et al


-

2



ANOVA


ANOVA





AST

C

LUS



1378

J E Blum

et al



27

1374ndash1386

Table 1



Sidak adjusted

P






P


Translation

BE128853 187

yen

10

-

5

treatment 003

-


AW787726 491

yen

10

-

3

treatment

-

095

-


AW066279 634

yen

10

-

3

treatment

-

123

-


AW147172 757

yen

10

-

4


AW352521 141

yen

10

-

6

treatment

-

118

-



treatment

-

092

-


yen

10

-

3


treatment

-


yen

10

-

4


041

-

070AW787437 939

yen

10

-

10


Signal transduction

029

-


yen








Proteinases



Photosynthesis














014(low)-029



Stress-related







Treatmentclusters

DNA binding

















Significancecall





Interactionclusters

Table 1 Continued

1380 J E Blum et al
















Functional analysis





ndash

ndash

ndash

ndash

ndash ndash

1382 J E Blum et al















DISCUSSION














366 (39) 80 (48) 2265 (53)


1384 J E Blum et al

















ACKNOWLEDGMENTS


REFERENCES





























1386 J E Blum et al















1376

J E Blum

et al



27

1374ndash1386



infin


yen

SSC 05 SDS 05

yen

SSCand 005

yen


S

CANALYZE


et al


CANALYZE




ANOVA

) style mod-


et al


ANOVA


(1)

(2)

In model (1)

y


L

T

and

D


LT


m

is overall mean and

Œ


AD


et al


A

(

L


ANOVA


G


GS

(

A





r GL GA L GT GLT


gl gr j gijklr

= + + + ++ +

( )

( )( )

( ) e

Figure 1



1377



27

1374ndash1386


et al


P



NTERPRISE

M

INER



P


P


RESULTS



-

2

(Cartwright

et al


-

2



ANOVA


ANOVA





AST

C

LUS



1378

J E Blum

et al



27

1374ndash1386

Table 1



Sidak adjusted

P






P


Translation

BE128853 187

yen

10

-

5

treatment 003

-


AW787726 491

yen

10

-

3

treatment

-

095

-


AW066279 634

yen

10

-

3

treatment

-

123

-


AW147172 757

yen

10

-

4


AW352521 141

yen

10

-

6

treatment

-

118

-



treatment

-

092

-


yen

10

-

3


treatment

-


yen

10

-

4


041

-

070AW787437 939

yen

10

-

10


Signal transduction

029

-


yen








Proteinases



Photosynthesis














014(low)-029



Stress-related







Treatmentclusters

DNA binding

















Significancecall





Interactionclusters

Table 1 Continued

1380 J E Blum et al
















Functional analysis





ndash

ndash

ndash

ndash

ndash ndash

1382 J E Blum et al















DISCUSSION














366 (39) 80 (48) 2265 (53)


1384 J E Blum et al

















ACKNOWLEDGMENTS


REFERENCES





























1386 J E Blum et al
















1377



27

1374ndash1386


et al


P



NTERPRISE

M

INER



P


P


RESULTS



-

2

(Cartwright

et al


-

2



ANOVA


ANOVA





AST

C

LUS



1378

J E Blum

et al



27

1374ndash1386

Table 1



Sidak adjusted

P






P


Translation

BE128853 187

yen

10

-

5

treatment 003

-


AW787726 491

yen

10

-

3

treatment

-

095

-


AW066279 634

yen

10

-

3

treatment

-

123

-


AW147172 757

yen

10

-

4


AW352521 141

yen

10

-

6

treatment

-

118

-



treatment

-

092

-


yen

10

-

3


treatment

-


yen

10

-

4


041

-

070AW787437 939

yen

10

-

10


Signal transduction

029

-


yen








Proteinases



Photosynthesis














014(low)-029



Stress-related







Treatmentclusters

DNA binding

















Significancecall





Interactionclusters

Table 1 Continued

1380 J E Blum et al
















Functional analysis





ndash

ndash

ndash

ndash

ndash ndash

1382 J E Blum et al















DISCUSSION














366 (39) 80 (48) 2265 (53)


1384 J E Blum et al

















ACKNOWLEDGMENTS


REFERENCES





























1386 J E Blum et al















1378

J E Blum

et al



27

1374ndash1386

Table 1



Sidak adjusted

P






P


Translation

BE128853 187

yen

10

-

5

treatment 003

-


AW787726 491

yen

10

-

3

treatment

-

095

-


AW066279 634

yen

10

-

3

treatment

-

123

-


AW147172 757

yen

10

-

4


AW352521 141

yen

10

-

6

treatment

-

118

-



treatment

-

092

-


yen

10

-

3


treatment

-


yen

10

-

4


041

-

070AW787437 939

yen

10

-

10


Signal transduction

029

-


yen








Proteinases



Photosynthesis














014(low)-029



Stress-related







Treatmentclusters

DNA binding

















Significancecall





Interactionclusters

Table 1 Continued

1380 J E Blum et al
















Functional analysis





ndash

ndash

ndash

ndash

ndash ndash

1382 J E Blum et al















DISCUSSION














366 (39) 80 (48) 2265 (53)


1384 J E Blum et al

















ACKNOWLEDGMENTS


REFERENCES





























1386 J E Blum et al
























014(low)-029



Stress-related







Treatmentclusters

DNA binding

















Significancecall





Interactionclusters

Table 1 Continued

1380 J E Blum et al
















Functional analysis





ndash

ndash

ndash

ndash

ndash ndash

1382 J E Blum et al















DISCUSSION














366 (39) 80 (48) 2265 (53)


1384 J E Blum et al

















ACKNOWLEDGMENTS


REFERENCES





























1386 J E Blum et al















1380 J E Blum et al
















Functional analysis





ndash

ndash

ndash

ndash

ndash ndash

1382 J E Blum et al















DISCUSSION














366 (39) 80 (48) 2265 (53)


1384 J E Blum et al

















ACKNOWLEDGMENTS


REFERENCES





























1386 J E Blum et al




















Functional analysis





ndash

ndash

ndash

ndash

ndash ndash

1382 J E Blum et al















DISCUSSION














366 (39) 80 (48) 2265 (53)


1384 J E Blum et al

















ACKNOWLEDGMENTS


REFERENCES





























1386 J E Blum et al















1382 J E Blum et al















DISCUSSION














366 (39) 80 (48) 2265 (53)


1384 J E Blum et al

















ACKNOWLEDGMENTS


REFERENCES





























1386 J E Blum et al



















DISCUSSION














366 (39) 80 (48) 2265 (53)


1384 J E Blum et al

















ACKNOWLEDGMENTS


REFERENCES





























1386 J E Blum et al















1384 J E Blum et al

















ACKNOWLEDGMENTS


REFERENCES





























1386 J E Blum et al


















ACKNOWLEDGMENTS


REFERENCES





























1386 J E Blum et al















1386 J E Blum et al















Split-plot microarray design allows sensitive detection of expression differences after ultraviolet...

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