FATTY ACID AND TRANSCRIPT PROFILING IN DEVELOPING SEEDS OF THREE BRASSICA NAPUS CULTIVARS

Communicated by Grzegorz Żurek

Mariana Petkova1, Wun S. Chao2, Leonard Cook2, Mark West3, Mukhlesur Rahman4_{, Michael E. Foley}2

1_{Agriculture University-Plovdiv, 400012 Mendeleev Blvd., Bulgaria;} 2_{USDA-Agricultural}

Research Service, 1605 Albrecht Blvd., Fargo, ND 58102 USA; 3USDA-Agricultural Research Service, 2150 Centre Ave., Bldg. D, Suite 300, Fort Collins, CO 80526 USA;

4

North Dakota State University, Department of Plant Sciences, P.O. Box 6050, Fargo, ND 58108 USA; michael.foley@ars.usda.gov

FATTY ACID AND TRANSCRIPT PROFILING IN DEVELOPING SEEDS OF THREE BRASSICA NAPUS CULTIVARS

ABSTRACT

Fatty acid levels and gene expression profiles for selected genes associated with the synthesis of fatty acids (FA), triacylglycerol, and oil body proteins were examined in three oilseed rape (Brassica napus) culti-vars that have utility for cultivar development in our spring canola breeding program. The seed oil content of Bronowski, Q2, and Westar was 39.0, 40.1, and 40.6%, respectively at 40 days after flowering (DAF). During the 20 to 40 day period of seed development, cultivars had varying levels of palmitic, stearic, oleic, linoleic, α -linolenic, eicosenoic, and erucic acid. In general, the percentage of each FA was similar among the cultivars during seed development. However, the level of oleic acid was lower and the levels of eicosenoic acid and erucic acid were higher in Bronowski than in Q2 and Westar seeds; linoleic acid also tended to be lower in Bronowski. Gene expression among the cultivars was similar from 10 to 40 DAF. The few exceptions were that expression of KAS1 and SAD were higher in Westar and Q2 than in Bronowski at 25 DAF, SAD was highest in Q2, intermediate in Westar, and lowest in Bronowski at 35 DAF, FAD2 was higher in Q2 than in Bronowski at 35 DAF, FAD3 was higher in Q2 than in Bronowski at 15 DAF and Q2 and Westar at 25 and 30 DAF, and FAE1 was higher in Westar and Q2 than in Bronowski at 30 DAF. Correlation analysis for gene expression against DAF for each genotype supported a common trend in gene expression among the three cultivars with gene expression tending to decrease over time; except for LPAAT, which tended to increase. The correlation between the level of FAs and expression of genes by genotype indicated no general trend; rather correlations seem to depend on the genotype.

Key words: Brassica napus, canola, fatty acid, gene expression, oilseed, rapeseed, seed.

DOI: 10.1515/plass-2015-0029

INTRODUCTION

Brassica napus (L.) is commonly referred to as canola, rapeseed, or

oil-seed rape. Canola itself was bred from rapeoil-seed in Canada to develop

a nutritious oil low in glucosinolates and erucic acid (Stefansson et al. 1961;

Stefansson and Kondra 1970), which are anti-nutritional components for

humans and livestock. Canola is the second largest vegetable oilseed crop

worldwide behind soybean (http://www.ers.usda.gov/data-products/oil-crops

-yearbook.aspx). The U.S. ranks eighth in worldwide oilseed rape

produc-tion (http://apps.fas.usda.gov/psdonline/psdQuery.aspx), valued at

approxi-mately $483 million in 2011/2013, yet the U.S. remains a primary importer

of canola oil and meal (http://usda.mannlib.cornell.edu/usda/current/

CropValuSu/CropValuSu-02-14-2014.pdf). Because 80% of the U.S. canola

production is in the state of North Dakota, a public spring canola

improve-ment project was initiated to develop germplasm adaptable to the Northern

Plains of the U.S.

High oil yield and quality are fundamental to developing adapted

germ-plasm. The biosynthesis and regulation of oil production in oilseeds is

com-plex

,

encompassing several steps and organelles within the cell (Baud and

Lepiniec 2010; Bates et al. 2013; Li-Beisson et al. 2013). Rapeseed or

ca-nola oil is a mixture of triacylglycerols (TAG) that account for about

40-45% of the seed dry weight (Troncoso-Ponce et al. 2011). Initially,

com-pounds like sucrose are imported into the plastid, and through a number of

enzymatically mediated steps beginning with a multisubunit heteromeric

acetyl-CoA carboxylase (HtACCase), free fatty acids (FA) of 16 to 18

car-bons are synthesized. Long-chain FA are then exported to the endoplasmic

reticulum (ER) for modification in the form of desaturation and elongation

and assembly of TAG, which are esters of glycerol and FA. The formation

of very long-chain FA (VLCFA) such as erucic acid (22:1), a major

compo-nent of non-canola quality rapeseed oil, is enzymatically mediated by the

fatty acid elongase complex (FAE), with fatty acid elongation1 (FAE1)

be-ing the first of four enzymes that comprise FAE. Synthesis of

polyunsatu-rated FA such as linoleic (18:2) and α-linolenic acid (18:3) is mediated by

fatty acid desaturase (FAD) enzymes. De novo assembly of TAG occurs by

various routes in the ER, with the relatively straight forward one being the

Kennedy pathway (Baud and Lepiniec 2010). This pathway encompasses

a series of sequential acylation of a glycerol-3-phosphate backbone

culmi-nating with the third acylation catalyzed by diacylglycerol acyltransferase

(DGAT). Finally, TAGs are stored in oil bodies composed of a matrix of

TAGs and various proteins such as oleosins and steroleosin (Baud and

Le-piniec 2009). The TAGs are important as they act as a reserve for

post-germination growth prior to achieving sufficient photosynthetic capacity and

comprise the tremendous economic value for oilseed crops such as canola.

Thus, it is important to understand the link between various genes involved

in the oil biosynthesis during development and composition of seeds as

a prelude to germplasm development, as well as to understand factors

re-lated to oilseed quality improvement.

While the FA composition in rapeseed oil has been documented (Canvin

1965), employing genomic techniques to evaluate expression of gene

tran-scripts in relation to FA composition during rapeseed development is more

recent. Hu et al. (2009) used quantitative reverse transcription (qRT-PCR) to

examine transcript levels of 32 genes involved in the biosynthesis of FA,

TAG, storage proteins, and in other physiological processes during seed

de-velopment of an older Chinese high erucic acid cultivar and a descendent,

low erucic acid cultivar. They determined that the transcription profiles were

similar for both cultivars, while selection pressure for no erucic acid, low

glucosinolates, high oleic acid, oil content, and yield affected the expression

levels of several genes. In turn, they determined FA levels during seed

de-velopment and correlated those with the gene transcripts. In another

investi-gation, comparative transcriptome analysis in developing oilseeds of

multi-ple species, including B. napus, relied on expressed sequence tag (EST)

da-tabase development through pyrosequencing (Troncoso-Ponce et al. 2011).

A notable outcome of this study was that regardless of the species ESTs

rep-resenting almost all reactions of FA biosynthesis had comparable

stoichiometry and consistent temporal profiles. This outcome and related

results from EST sequencing and gene and protein expression studies

sug-gest it is valid to make some cross species comparisons such as between

Arabidopsis thaliana and B. napus (Niu et al. 2009; Venglat et al. 2013).

The first canola-type cultivar of summer rape released in Canada (Stefansson and

Kondra 1975) was derived from a complex series of crosses that include selections

from Liho and Bronowski to impart low erucic acid and glucosinolates,

respec-tively. Likewise, subsequent canola quality summer rape cultivars, such as Westar

and Q2, with superior agronomic and disease resistance traits (Klassen et al. 1987;

Stringam et al. 1999) relied on series of crosses using germplasm with low erucic

acid and glucosinolates. Westar, released in 1982 by Agriculture and Agri-Food

Canada, has been widely cultivated, modified, and used as a baseline for subsequent

germplasm development (Juska et al. 1997). However, it is susceptible to a serious

disease of canola called blackleg caused by the fungus Leptosphaeria maculans. Q2

released in 1998 by University of Alberta is resistant to blackleg disease and

rela-tively resistant to lodging. We cultivated Bronowski, Westar, and Q2 in the

green-house to examine for potential traits that could be used in our spring canola

breed-ing program. Thus, the objectives of this research were to examine fatty acid levels

and expression profiles for selected genes associated with the synthesis of FA,

TAG, and oil body proteins during seed development in the three cultivars.

MATERIALS AND METHODS

Plant Materials

Oilseed rape (Brassica napus) cultivars, Bronowski, Westar, and Q2 were grown

in a greenhouse at North Dakota State University, Fargo, ND, USA, at 22±4°C (day

and night). The seeds were sown in 15 cm (diameter) by 15 cm (depth) pots filled

with Sunshine-Mix-1 (Sun Gro Horticulture). The plants were watered daily and

fertilized with water soluble 20 N–20 P–20 K fertilizer. Light in the greenhouse was

provided with a 16-h photoperiod by natural sunlight supplemented with 400 W

HPS PL 2000 lights (P.L. Light Systems Inc. ON, Canada). During the flowering

stage, plants were bagged (microperforated polybag, Crawford Provincial, ON,

Canada) and allowed to self-pollinate. Developing pods were harvested at 5-day

intervals, 10 to 40 days after flowering (DAF). Seeds were harvested into liquid

nitrogen and stored at -80°C for gas chromatographic analysis and RNA extraction.

There were three biological replications per treatment.

Determination of Oil and Fatty Acid Profile

At 40 d after flowering (DAF), oil was extracted from seeds with n-hexane using

accelerated solvent extraction (Dionex ASE 200, Thermo Scientific, Sunnyvale,

CA) according to the methods of Haagenson et al. (2010) for oil content

determina-tion. One gram canola seed was oven dried for 4 h at 70°C. Seed was milled in

a coffee grinder with 3.5 g diatomaceous earth, and samples were loaded into 11 ml

stainless steel cells. Any remaining extraction cell void volume was filled with

dia-tomaceous earth prior to extraction. Extractions were performed at 100°C, 6.7 MPa

with a 5 min equilibration time and three 10 min static cycles having a 100% flush

volume and 60 s purge time. The solvent containing extracted oil was collected in

pre-weighed vials, and solvent was evaporated to dryness with a stream of dry air

(-70°C dew point). Extracted samples were air dried, and reground for a second

ex-traction and the total oil recovery from the two exex-tractions was recorded. Oil is

re-ported as a percent of seed dry weight.

At 5 d intervals from 20 to 40 DAF, fatty acid profiles were determined on seeds

air dried overnight at room temperature in a fume hood. Samples of 0.1 to 0.3 g of

dried seed were ground in a mortar and pestle and vortexed in 0.5 to 2 ml of hexane

-chloroform-sodium methoxide (HCSM) derivatization reagent to produce fatty

acid methyl esters (FAMEs). The HCSM reagent was freshly prepared by mixing

75 ml hexane, 20 ml chloroform (pentene stabilized), and 5 ml 0.5 M sodium

meth-oxide in methanol (Sigma #403067). Analysis of the FAMEs was carried out on

a Hewlett-Packard 5890 Series II gas chromatograph with a flame-ionization

detec-tor. Split injections of 1 µl of the FAMEs were separated on a J & W Scientific

DB-23, 30 m by 0.25 mm, 0.25 µm film column with helium carrier gas at 29 psi (1.9

ml/minute) and split flow at 50-100 ml/minute. The column was temperature

pro-grammed at 190

C for 4 min then to 220°C at 15°C/min and held 1 min, then to

240°C at 25

C/min and held 1 min. Inlet temperature was 230°C and detector

tem-perature was 250°C with air at 345 ml/minute, hydrogen at 36 ml/minute, and

he-lium makeup gas at 35 ml/minute. Nu-Check 21A and 411 standards were used to

identify the FAMEs.

Template cDNA preparation and quantitative real-time PCR (qRT-PCR)

Total RNA was extracted at 5 d intervals from 10 to 40 DAF from canola

seeds using the pine tree extraction protocol (Chang et al. 1993), and these

sam-ples were used to prepare template cDNA through reverse transcription

follow-ing manufacturer’s instructions (Invitrogen). Briefly, 5 µg of total RNA was

DNase treated and then reverse transcription was performed in 20 µl total

vol-ume using a SuperScript First-Strand Synthesis Kit to produce total cDNA from

each sample. After cDNA synthesis, each 20 µl reaction was diluted to 800 µl

and stored at -80°C.

Gene expression by qRT-PCR was examined using template cDNA on

a Roche LightCycler® 480 real-time PCR system. Primer pairs were

synthe-sized based on sequences from Hu et al. (2009) (Electronic Supplementary

Ta-ble S1). qRT-PCR parameters were described previously by Chao (2008) with

some modification. The formula used to calculate the fold differences is similar

to the comparative C

method (ΔΔC

) except that no control sample is

incorpo-rated in the calculation. Thus, levels of different target gene expressions can be

compared based on the expression of a reference SAND family gene, which

served as a base line. A canola SAND family gene was used as a reference

be-cause it was verified to be stably expressed during seed development (see

Elec-tronic Supplementary Fig. S1 and Table S1 & S2). The modified formula for

fold difference in gene expression of target vs. reference gene is:

where, ΔC

is the C

value of the target gene, and ΔC

is the C

value of the reference gene. SYBR green chemistry was used to produce

fluo-rescent signal, and three technical replicates were used per sample for the

RT-qPCR experiments. The C

value of each gene is the average of three technical

replicates. The difference in gene expression is designated as log2 value.

Heat-map of the qRT-PCR results in Fig. S1 was created based on log2 values using

Eisen Lab software, Cluster and TreeView as described by Eisen et al. (1998).

Statistical Analysis

The standard error (SE) of the mean difference of Ct values between the

tar-get and reference (SAND family) genes were calculated based on

where SA and SB are the standard deviations and nA and nB are the sample

sizes for samples A and B.

is the estimated correlation of these pairs. The

95% confidence intervals

were obtained based on the mean difference ±t

-value × SE

; the t-value with 2 degrees of freedom and 95% confidence is

4.303. The variance sum law was applied in the calculation of each reference

gene normalized target gene SE and explains why the 95% confidence intervals

for most of the target genes appeared very large.

MANOVA was used to compare FA profiles among cultivars using the manova

function of the stats package in R (2015). The Wilks Lambda statistic was used to

determine significant difference for the FA profiles (Johnson and Wichern 2007).

Pearson correlation coefficients between the FA and the gene expression by

cul-tivar were computed using the cor function of the stats package in R (2015).

Be-cause only three biological replications were used in this study for each cultivar and

DAF, only large effects and/or strong associations could be expected to be detected

statistically.

RESULTS

Oil and Fatty Acids

The seed oil content of Bronowski, Westar, and Q2 grown in the

green-house was 39.0, 40.6, and 40.1%, respectively. During the 20 to 40 d period

of seed development, cultivars had varying levels of palmitic, stearic, oleic,

linoleic, α-linolenic, eicosenioc acid, and erucic acid (Fig. 1). Levels of the

two saturated FA, palmitic acid and stearic acid, were similar among the

cultivars averaging 7.5% and 4.4% and 2.5% and 1.9%, respective at 20 and

40 DAF. The level of oleic acid was the same in Westar and Q2 over the 20

to 40 d period, although there was a trend for slightly high levels in Westar.

In contrast, the mean level of oleic acid in Bronowski seeds was about 25%

lower over the 20 d period relative to Westar and Q2. In general, the levels

for two polyunsaturated FA, linoleic acid and α-linolenic acid, were similar

among the cultivars averaging 22.2% and 15.7% and 10.8% and 6.1%,

re-spective at 20 and 40 DAF. Nevertheless, the trend was for higher levels of

linoleic acid in Q2 > Westar > Bronowski seeds from 20 to 40 DAF. The

monounsaturated VLCFAs, eicosenioc acid and erucic acid, varied

tremen-dously among the cultivars, particularly for Bronowski. Eicosenioc acid

levels over the 20 to 40 d period averaged 16.3%, 1.5% and 1.4%,

respec-tive for Bronowski, Westar, and Q2 seeds. Erucic acid was not detected in

Westar seeds and the levels in Q2 were 3% and 1.1% at 35 and 40 DAF,

respectively. In contrast, erucic acid levels in Bronowski seeds increased

from 10.9% to 22.5% of total FAs over the 20 to 40 d period.

Fig. 1. Fatty acid accumulation during seed development.

The levels of 7 fatty acids (palmitic, stearic, oleic, linoleic, α-linolenic, eicosenioc, and erucic acid) were examined at 5-day intervals, 20 to 40 DAF in cultivars Bronowski, Westar, and Q2

Gene Expression

Gene designation, role, and location are provided in Table 1. Overall and as

determined by the 95% confidence intervals, gene expression among the three

cultivars was similar from 10 to 35 DAF (Fig. 2). We had only one data point

for Bronowski genes at 40 DAF so confidence intervals could not be calculated.

The few exceptions were that expression of KAS1 and SAD were higher in

Westar and Q2 than in Bronowski at 25 DAF, SAD was highest in Q2,

interme-diate in Westar, and lowest in Bronowski at 35 DAF, FAD2 expression was

higher in Q2 than in Bronowski at 35 DAF, FAD3 expression was higher in Q2

than in Bronowski at 15 DAF and Q2 and Westar at 25 and 30 DAF, and FAE1

expression was higher in Westar and Q2 than in Bronowski at 30 DAF. At its

peak, expression of the gene for the seed storage protein napin was nearly

33,000 fold higher (log

of 15) than the SAND gene. Conversely, lowest level

of expression was the caleosin gene at 1/64 (log

of -6) that of the SAND gene

at 10 DAF.

Table 1 Gene designation, role, and location

Gene name Gene annotation Role Location

ACCase Homeomeric acetyl CoA carboxylase Fatty acid biosynthesis Cytosol

α-C7 Alpha carboxyltransferase Fatty acid biosynthesis Plastid

β-C7 Beta carboxyltransferase Fatty acid biosynthesis Plastid

BC Biotin carboxylase Fatty acid biosynthesis Plastid

MCMT Malonyl-CoA:ACP malonyltransferase Fatty acid biosynthesis Plastid

KAS1 Beta-ketoacyl-ACP synthase 1 Fatty acid biosynthesis Plastid

KAS2 Beta-ketoacyl-ACP synthase 2 Fatty acid biosynthesis Plastid

KAS3 Beta-ketoacyl-ACP synthase 3 Fatty acid biosynthesis Plastid

HD/KACD 3-hydroxyacyl-ACP dehydratase Fatty acid biosynthesis Plastid

SAD Stearoyl-ACP desaturase Fatty acid biosynthesis Plastid

FatA Acyl-ACP thioesterasae Fatty acid biosynthesis Plastid

FatB Palmiottoyl-ACP thioesterase Fatty acid biosynthesis Plastid

FAD6 Oleate desaturase Acid editing Plastid

FAD2 Oleate desaturase Acid editing Endoplasmatic reticulum

FAD3 Linoleate desaturase Acid editing Endoplasmatic reticulum

LPATT Lysophosphatidic acid acyltransferase Triacylglycerol biosynthesis Endoplasmatic reticulum DGAT2 Acyl-CoA:diacylglycerol acyltransferase Triacylglycerol biosynthesis Endoplasmatic reticulum AAPT1 Aminoalcoholphosphotransferase Triacylglycerol biosynthesis Endoplasmatic reticulum

FAE1 Fatty acid elongase 1/3-ketoacyl-CoA synthase Very long chain fatty acid _biosynthesis Endoplasmatic reticulum

KCR2 3-ketoacyl-CoA reductase Very long chain fatty acid _biosynthesis Endoplasmatic reticulum

Oleosin Oil nody associated protein Storage protein Oil body Cruciferin 12S neutral oil body protein Storage protein Oil body Napir 1.7S oil body protein Storage protein Oil body Caleosin Ca2+ _{binding oil body surface protein Storage protein Oil body}

Fig. 2. Gene expression profiles during seed development.

The expression profiles of 24 genes were examined at 5-day intervals, 10 to 40 DAF in cultivars Bronowski, Westar, and Q2. Levels of different target gene expressions were compared based on the expression

of a reference SAND family gene, which also served as a base line here. The fold difference is designated as log2 value. Gene designation is in Table 1. The 95% confidence intervals

T ab le 2 T h e co rr el a ti o n c o ef fi ci en ts b et w ee n f a tt y a ci d ( F A ) a cc u m u la ti o n a n d l ev el s o f g en e ex p re ss io n in c u lt iv a rs B ro n o w sk i, W es ta r, a n d Q 2 a cr o ss d a y s a ft er f lo w er in g ( D A F ) G en o ty p e F at ty a ci d A C C as e α C T β C T B C M C A T K A S 1 K A S 2 K A S 3 H D /K A C D S A D F at A F at B B ro w n o w sk i P al m it ic 0 .6 9 9 0 .6 4 2 0 .7 2 4 0 .6 6 8 0 .6 7 7 0 .4 5 2 0 .7 1 -0 .0 8 4 0 .6 0 5 0 .4 8 9 0 .6 1 -0 .0 3 3 B ro w n o w sk i S te ar ic 0 .6 6 2 0 .5 8 9 0 .7 3 4 0 .9 3 8 0 .6 0 5 0 .5 4 3 0 .6 9 4 0 .1 1 7 0 .6 4 0 .4 9 9 0 .6 0 7 -0 .0 5 6 B ro w n o w sk i O le ic 0 .6 0 3 0 .6 0 3 0 .4 8 4 0 .6 1 0 .6 6 6 0 .4 5 7 0 .5 9 7 -0 .5 3 1 0 .5 0 1 0 .5 4 9 0 .5 3 0 .7 2 5 B ro w n o w sk i L in o le ic 0 .3 3 7 0 .3 3 2 0 .5 9 9 0 .3 7 1 0 .3 4 9 0 .2 2 0 .3 7 8 0 .2 1 0 .3 4 5 0 .4 3 3 0 .3 7 7 -0 .1 1 9 B ro w n o w sk i L in o le n ic 0 .9 7 8 0 .7 3 5 0 .8 8 5 0 .7 2 8 0 .7 6 5 0 .5 2 7 0 .7 5 1 -0 .1 9 3 0 .7 4 2 0 .8 0 3 0 .7 3 3 -0 .1 4 8 B ro w n o w sk i E ic o se n o ic -0 .5 6 5 -0 .6 2 7 -0 .7 5 8 -0 .6 2 3 -0 .6 4 7 -0 .2 8 6 -0 .6 1 8 0 .0 6 8 -0 .5 3 9 -0 .5 7 -0 .5 8 9 -0 .0 9 6 B ro w n o w sk i E ru ci c -0 .8 1 3 -0 .8 3 4 -0 .9 0 4 -0 .8 3 6 0 .8 6 5 -0 .6 4 6 -0 .8 2 8 0 .2 7 2 -0 .7 7 3 -0 .8 3 5 -0 .7 9 6 -0 .4 5 6 Q 2 P al m it ic 0 .7 0 8 0 .7 4 9 0 .7 8 9 0 .6 7 8 0 .7 2 2 0 .8 0 8 0 .7 9 2 0 .1 8 6 0 .7 8 3 0 .6 8 1 0 .7 2 9 -0 .1 3 2 Q 2 S te ar ic 0 .2 9 9 0 .3 1 4 0 .4 3 0 .3 6 9 0 .3 8 7 0 .4 8 5 0 .4 3 1 -0 .0 0 2 0 .3 4 2 0 .4 2 0 .3 6 4 -0 .3 2 7 Q 2 O le ic -0 .6 2 2 -0 .6 0 5 -0 .7 5 2 -0 .5 5 8 -0 .6 2 9 -0 .7 2 6 -0 .6 8 6 -0 .3 2 -0 .6 7 -0 .6 0 9 -0 .6 3 7 0 .0 6 2 Q 2 L in o le ic 0 .7 4 3 -0 .7 3 8 0 .6 7 3 0 .6 6 7 0 .7 2 0 .7 5 3 0 .7 6 8 0 .2 0 6 0 .7 8 7 0 .6 5 6 0 .7 4 6 -0 .0 0 4 Q 2 L in o le n ic 0 .4 9 9 0 .4 8 1 0 .8 3 9 0 .3 6 9 0 .4 5 1 0 .5 9 5 0 .5 3 5 0 .5 3 2 0 .5 5 7 0 .4 7 3 0 .4 8 1 -0 .1 6 9 Q 2 E ic o se n o ic -0 .6 8 2 -0 .7 3 8 -0 .7 2 6 -0 .5 7 1 -0 .6 0 1 -0 .6 4 3 -0 .6 8 -0 .2 4 1 -0 .7 4 7 -0 .5 5 8 -0 .6 5 5 0 .1 2 7 Q 2 E ru ci c -0 .5 3 7 -0 .6 2 3 -0 .4 5 7 -0 .4 4 2 0 .4 2 8 -0 .4 4 1 -0 .5 2 1 -0 .1 5 2 -0 .6 1 4 -0 .4 2 4 -0 .5 4 3 0 .2 8 1 W es ta r P al m it ic 0 .5 2 6 0 .5 6 2 0 .5 9 6 0 .7 1 0 .5 3 5 0 .5 8 5 0 .6 1 9 -0 .0 6 6 0 .5 6 1 0 .5 5 4 0 .5 2 2 -0 .3 2 6 W es ta r S te ar ic 0 .2 2 0 .3 1 7 0 .4 5 0 .4 4 3 0 .3 5 3 0 .4 2 9 0 .3 5 0 .0 6 5 0 .3 0 8 0 .4 2 5 0 .2 6 8 -0 .4 5 4 W es ta r O le ic -0 .0 7 6 -0 .1 7 -0 .4 1 2 -0 .1 0 6 -0 .1 4 4 -0 .1 1 9 -0 .2 0 3 0 .2 3 -0 .0 9 5 -0 .1 0 4 -0 .0 2 1 0 .4 0 5 W es ta r L in o le ic 0 .2 7 3 0 .3 2 3 0 .3 4 2 0 .3 6 3 0 .2 5 3 0 .2 6 6 0 .3 5 1 -0 .0 8 2 0 .2 8 4 0 .3 0 9 0 .2 7 3 -0 .3 8 8 W es ta r L in o le n ic 0 .4 5 0 .5 0 2 0 .5 1 0 .4 8 7 0 .4 1 2 0 .4 0 9 0 .5 2 2 -0 .0 9 9 0 .4 4 0 .4 7 5 0 .4 4 5 -0 .4 2 6 W es ta r E ic o se n o ic -0 .6 1 2 -0 .5 9 8 -0 .2 3 -0 .7 5 -0 .5 0 7 -0 .5 8 3 0 .5 7 9 -0 .1 9 5 -0 .6 2 4 -0 .6 7 1 -0 .7 1 6 0 .0 5 6 W es ta r E ru ci c -0 .5 6 6 -0 .5 0 7 -0 .1 4 4 -0 .6 9 1 -0 .4 4 9 -0 .5 2 4 0 .5 1 2 -0 .2 1 8 -0 .5 7 4 -0 .6 1 2 -0 .6 7 2 0

T ab le 2 C o n ti n u ed R ed a n d g re en c o lo re d c o ef fi ci en ts d is p la y s ta ti st ic al ly s ig n if ic an t (α = 0 .0 5 B o n fe rr o n i ad ju st ed f o r m u lt ip le t es ts w it h in e ac h g en e an d c u lt iv ar ) p o si ti v e an d n eg at iv e co rr e-la ti o n s, r es p ec ti v el y G en o ty p e F at ty a ci d F A D 6 F A D 2 F A D 3 L P A A T D G A T 2 A A P T 1 F A E 1 K C R 2 O le o si n C ru ci fe ri n N ap in C al eo si n B ro w n o w sk i P al m it ic 0 .8 0 5 0 .6 9 3 0 .5 8 6 -0 .0 7 0 .5 5 4 0 .4 6 9 0 .6 4 0 .6 9 7 0 .0 2 9 0 .5 7 2 0 .5 8 4 0 .1 0 7 B ro w n o w sk i S te ar ic 0 .7 4 5 0 .6 6 7 0 .5 7 -0 .1 5 7 0 .6 3 6 0 .4 2 2 0 .6 1 1 0 .6 4 -0 .0 1 8 0 .7 3 3 0 .6 0 4 -0 .0 1 3 B ro w n o w sk i O le ic 0 .6 8 5 0 .5 2 2 0 .5 7 7 -0 .0 3 9 0 .3 0 3 0 .7 5 4 0 .5 1 3 0 .5 8 4 0 .5 0 5 -0 .2 8 8 0 .4 9 6 0 .2 2 B ro w n o w sk i L in o le ic 0 .4 8 9 0 .3 7 0 .2 9 9 -0 .1 4 9 0 .3 5 5 0 .2 1 1 0 .4 0 .3 2 2 0 .0 0 7 0 .7 7 4 0 .4 1 9 0 .1 0 8 B ro w n o w sk i L in o le n ic 0 .7 7 7 0 .7 4 6 0 .6 8 8 -0 .3 7 3 0 .5 0 9 0 .6 0 1 0 .7 1 0 .7 0 8 0 .0 6 9 0 .5 5 2 0 .6 5 8 -0 .0 9 4 B ro w n o w sk i E ic o se n o ic -0 .7 4 5 -0 .5 8 6 -0 .5 2 9 0 .0 3 7 -0 .4 0 9 -0 .4 8 3 -0 .6 0 4 -0 .5 9 2 -0 .1 6 -0 .5 5 7 -0 .5 6 3 -0 .2 4 6 B ro w n o w sk i E ru ci c -0 .9 4 9 -0 .7 8 8 -0 .7 7 0 .2 1 7 -0 .5 9 4 -0 .8 -0 .7 8 1 -0 .7 9 1 -0 .3 4 8 -0 .3 9 9 -0 .7 6 7 -0 .1 1 8 Q 2 P al m it ic 0 .6 7 2 0 .7 0 5 0 .6 8 1 -0 .6 1 0 .7 0 4 -0 .1 2 5 0 .6 2 0 .7 4 6 0 .2 7 8 0 .3 3 8 0 .5 2 8 0 .2 9 8 Q 2 S te ar ic 0 .5 2 7 0 .3 9 5 0 .3 2 3 -0 .2 0 7 0 .1 5 1 -0 .1 8 7 0 .4 2 5 0 .3 8 1 0 .4 1 9 0 .2 3 7 0 .6 4 7 0 .3 3 3 Q 2 O le ic -0 .5 0 3 -0 .6 2 4 -0 .6 1 4 0 .5 6 2 -0 .6 5 1 0 .1 6 8 -0 .5 3 8 -0 .6 4 9 -0 .1 8 7 -0 .2 8 9 -0 .3 8 6 -0 .2 3 5 Q 2 L in o le ic 0 .5 1 3 0 .6 8 1 0 .7 1 9 -0 .7 5 0 .8 3 7 -0 .0 5 8 0 .5 8 0 .7 4 5 0 .1 5 0 .3 3 9 0 .3 0 7 0 .2 3 4 Q 2 L in o le n ic 0 .2 9 2 0 .4 8 6 0 .4 7 1 -0 .3 4 1 0 .5 4 4 -0 .3 3 2 0 .4 3 4 0 .4 8 3 0 .0 9 2 0 .2 9 7 0 .1 9 7 0 .1 8 4 Q 2 E ic o se n o ic -0 .4 3 1 -0 .5 8 -0 .6 0 7 0 .6 -0 .7 8 3 0 .1 2 3 -0 .5 2 1 -0 .6 4 9 -0 .1 0 5 -0 .3 8 8 -0 .1 6 6 -0 .2 1 6 Q 2 E ru ci c -0 .2 8 9 -0 .4 0 1 -0 .4 5 1 0 .5 0 5 -0 .6 5 4 0 .1 5 9 -0 .4 3 8 -0 .4 8 6 -0 .1 5 5 -0 .4 0 5 -0 .1 6 1 -0 .2 5 9 W es ta r P al m it ic 0 .6 7 8 0 .5 7 5 0 .5 1 2 -0 .3 0 6 0 .1 2 1 -0 .2 4 9 0 .3 2 2 0 .5 8 1 -0 .2 6 5 -0 .0 7 8 0 .2 4 3 -0 .2 1 W es ta r S te ar ic 0 .5 7 8 0 .3 9 0 .2 6 1 -0 .0 6 4 0 .0 0 4 -0 .0 4 2 0 .1 7 4 0 .3 6 6 -0 .2 4 6 0 .1 1 2 0 .1 7 7 -0 .1 1 6 W es ta r O le ic -0 .3 5 7 0 .1 8 1 -0 .1 4 2 0 .1 3 0 .1 4 8 0 .1 5 5 0 .0 3 2 -0 .2 2 3 0 .4 5 5 0 .2 3 7 0 .0 1 6 0 .2 9 4 W es ta r L in o le ic 0 .3 5 1 0 .2 6 6 0 .2 9 5 -0 .3 3 2 -0 .1 2 2 -0 .3 3 7 0 .2 4 2 0 .3 3 5 -0 .1 2 3 0 .0 0 1 0 .2 4 1 -0 .0 4 4 W es ta r L in o le n ic 0 .5 2 1 0 .4 5 2 0 .4 7 3 -0 .4 6 4 0 .0 5 -0 .3 2 3 0 .3 8 8 0 .5 3 4 -0 .0 3 6 0 .0 7 7 0 .3 5 9 -0 .0 0 4 W es ta r E ic o se n o ic -0 .3 8 9 -0 .4 7 6 -0 .5 4 4 0 .4 6 5 -0 .2 0 8 0 .3 5 8 -0 .6 4 -0 .5 2 -0 .4 7 8 -0 .4 0 7 -0 .5 7 2 -0 .3 3 9 W es ta r E ru ci c -0 .2 9 5 -0 .4 1 8 -0 .4 9 3 0 .4 2 9 -0 .2 1 2 0 .3 1 7 -0 .6 1 4 -0 .4 5 5 -0 .5 3 7 -0 .4 2 9 -0 .5 5 -0 .3 7 6

Correlations

We examined the correlation between FA levels and gene expression across

DAF for each cultivar. The data support a common trend in gene expression

among the three cultivars with gene expression tending to decrease over time;

except for LPAAT, which tended to increase (Electronic Supplementary Table

S3). In our subsequent determination of the correlation between the level of FAs

and expression of genes by individual cultivar, we observed no consistent

rela-tionship between FA and gene expression, rather these correlations seem to

de-pend on the individual cultivar (Table 2). Forty-eight (red) and 27 (green)

coef-ficients displayed significant (P<0.05) positive and negative correlations,

re-spectively. Of these, 43, 29, and 3 were associated with Bronowski, Q2, and

Westar, respectively. The correlation coefficients ranged from a positive

corre-lation of 0.88 for β-CT and α-linolenic acid in Bronowski, to no correcorre-lation

be-tween FatB and erucic acid in Westar, to a highly negative correlation of -0.95

between FAD6 and erucic acid in Bronowski. Interestingly, for Bronowski and

Q2, 52% (r

=0.72) or more of the variation in the expression of β-CT was

re-lated to variation in the level of palmitic acid, α-linolenic acid, and eicosenioc

acid. In this case for Bronowski and Q2, expression of β-CT was positively

cor-related with levels of palmitic acid and α-linolenic acid, whereas levels of

eicos-enioc acid were negatively correlated with β-CT. For oleic acid, the correlations

coefficients for most genes were positive for Bronowski but negative for Q2

and Westar; although most of the correlation coefficients were not significantly

different. There were no significant correlations between the level of any fatty

acid for the three cultivars and expression of KAS3, Oleosin, and Caleosin

genes when correlations were computed across DAF.

DISCUSSION

We designed this investigation of B. napus seed development after a similar

study by Hu et al. (2009). They examined an older high erucic acid cultivar

Zhongyou 821 (ZY821) and a low erucic acid descendant of ZY821,

Zhong-shuang 9 (ZS9). In turn, we employed several cultivars important for breeding

improved germplasm adapted to a region of the U.S. where 80% of the spring

canola is produced. The level of seed oil at maturity in our three greenhouse

grown cultivars was 2.7 to 7.7% lower than the content reported for seeds of

these cultivars from field grown plants (Bronowski 41.7%, Westar 43.3%, and

Q2 47.8%), but were similar to the level in rapeseed cultivars (ZY821 39.8% ,

ZS9 42%, respectively).

The abundance of FA in our three cultivars was typical relative to another

report with oleic acid > linoleic > α-linolenic (Vuorinen et al. 2014). The

VLCFAs, eicosenioc acid and erucic acid, as reported previously Finlayson et

al

., 1973, were higher in Bronowski at the expense of oleic acid, which is the

economically important FA component. In contrast to the level of eicosenioc

acid in Bronowski (17%), the level in ZY821 was reduced by about one-half

(9.75%); whereas, the level of erucic acid in Bronowski (21%) was reduced by

about one-half the level in ZY821 (42%) (Hu et al. 2009). These difference

likely reflect dissimilarity in genetic background of the cultivars.

We examined nearly the same set of genes as reported by Hu et al. (2009),

but direct comparison is problematic because different reference genes (β-actin

vs. SAND) were utilized for qRT-PCR normalization, different statistical

proce-dures were employed, and we expressed our data on a log2 scale. To compare

fold differences between relative copy number data (Hu et al. 2009) and our

log2 scale data would require numerical data for the qRT-PCR done by Hu et

al.

(2009). This is because fold estimates are not possible when the relative

copy number value is close to zero as is the case for many of the genes they

ex-amined. Nevertheless, similarities and differences in general trends can be

dis-cerned (see Fig. 2 (Hu et al. 2009)). For example, the biosynthesis of FAs

be-gins with ACCase catalyzing the carboxylation of acetyl-CoA to malonyl-CoA.

Expression of β-CT, a gene encoding for β-carboxyltransferase, one of four

components of the heteromeric ACCase, is nearly the same in the two studies,

increasing by about 2 fold from 10 to 15 DAF and thereafter decreasing about 2

fold by 35 DAF. Expression of FAE1, a component of a multienzyme complex

involved in VLCFA biosynthesis, increases by 5 fold from 25 to 40 DAF in

ZY821 and thereafter decreased to the 25 DAF level; whereas, the expression

peaks (log2 = 7) around 15 DAF in Bronowski and thereafter decreases 128 fold

by 40 DAF (log2 = 0).

The expression profile of the seed storage proteins was similar among

Westar, Q2, and Bronowski, which was similar to that observed based on

a comparison between Westar and Reston (Katavic et al. 2002), another high

erucic acid (26%) low oleic acid (30%) cultivar similar to Bronowski. However,

the expression profiles of the seed storage proteins between our cultivars and

ZY821 and ZS9 differed (see Fig. 2 (Hu et al. 2009)). For example, oleosin,

which is the major protein component of oil bodies, narrowly peaked at 40 DAF

in ZY821 with a relative copy number of 25,000 (log2 =14.6) and 12,500 for

ZS9 (log2 = 13.6); whereas in the cultivars we examined the broad peak

oc-curred around 25 DAF with a log2 = 7. However, the napin gene, which

ac-counted for over 75% of total transcription from all 32 genes assessed by Hu et

al.

(2009), and displayed the highest level of expression among the genes we

assessed, had nearly the same level of expression at its peak; ZY821 and ZS9

peaked at 40 and 35 DAF, respectively with a similar relative copy number of

175,000 (log2 =17.4); whereas in the cultivars we examined the broad peak

oc-curred around 25 DAF with a log2 = 13. In any event, the seed storage protein

genes in both studies generally displayed the highest level of expression of the

genes assessed.

Some of the genes we appraised were significantly correlated with fatty acid

accumulation, especially for the Bronowski and Q2 cultivars. In particular, the

level of several FAs was correlated with β-CT expression. β-CT encodes for one

of the subunits (α-CT, β-CT and BC) for the plastid localized heteromeric

AC-Case, which catalyzes the first committed step of fatty acid biosynthesis. This

gene is thought to be unique in that it is the only known lipid metabolism gene

that is encoded by the plastid genome (Elborough et al. 1996; Li-Beisson et al.

2013). In the high erucic acid cultivars Bronowski and ZY821, β-CT expression

was negatively correlated with erucic acid, whereas β-CT expression was

posi-tively correlated with palmitic acid, steric acid, α-linoleic acid in Bronowski,

but not in ZY821 (Hu et al. 2009). Perhaps there is a negative correlation

be-tween erucic acid levels and β-CT expression because expression of this gene is

declining, while erucic acid levels increase after 25 DAF.

Different patterns of gene expression exists for FAE1 between the high erucic

acid cultivars Bronowski and ZY821 (Hu et al. 2009). FAE1 is a component of

the multi-enzyme complex involved in VLCFA biosynthesis; mutations in the

FAE1

gene are responsible for the low erucic acid trait (Puyaubert et al. 2005).

Erucic acid levels peak by 30 DAF in Bronowski, whereas the levels

substan-tially increases in ZY821 until 40 DAF. Thus, the negative correlation

coeffi-cient (-0.78) that we observed for FAE1 and erucic acid in Bronowski is

consis-tent with a large fold decrease in gene expression and slightly increased level of

erucic acid as Bronowski seeds mature. However, the positive correlation (0.78)

between erucic acid and FAE1 for ZY821 is likely explained by the much

dif-ferent temporal pattern of FAE1 expression and erucic acid accumulation (see

Fig. 2 (Hu et al. 2009)). The high level of FAE1 expression in low erucic acid

cultivars such as Q2, ZS9 and other cultivars (Hu et al. 2009; Vuorinen et al.

2014) might seem inconsistent with the absence of VLCFAs. However, as

men-tioned, FAE1 gene contains a mutation that result in the absence of

3-ketoaacyl-CoA synthase protein, thus preventing the synthesis of VLCFAs (Puyaubert et

al.

2005; Wu et al. 2008). Interestingly, Westar contains a point mutation while

ZS9 contains a point mutation and four base pair deletion (Katavic et al. 2006;

Wu et al. 2008). Overall, appraisal of the correlation coefficients, which are

sometimes different between our cultivars and the Chinese cultivars

investi-gated by Hu et al. (2009), is instructive of the different patterns of gene

expres-sion in relation to a particular FA or storage protein.

The results of this investigation, which employed three publically available

cultivars from Canadian breeding programs, provide background data into the

transcriptional network for FA, TAG, and seed storage proteins. By comparing

the outcome of our investigation to that of Hu et al. (2009), we further

demon-strated that genetic background of the cultivars from different breeding

pro-grams affects important metabolic and molecular responses during oilseed

de-velopment. In any event, these insights and benchmark data will be important

for the success of the recent public spring canola improvement project we

initi-ated to develop germplasm adaptable to the Northern Plains of the U.S.

ACKNOWLEDGEMENTS

Research funded by the America for Bulgaria Foundation through the

USDA-Foreign Agricultural Service, which provided financial and administrative

sup-port for Dr. Mariana Petkova and the USDA-Agricultural Research Service

pro-ject #3060-21220-026. Dr. Darrin Haagenson conducted the oil analysis, and

Cheryl Huckle, Wayne Sargent, Angela Adsero, and Andrew Ross provided

technical support.

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SUPLEMENTARY FIGURE AND TABLES

S u p p le m en ta ry T a b le S 1 : P ri m er s u se d f o r q R T -P C R a n a ly si s P ri m er se t C an ol a P ri m er p ai rs P ri m er n am e P ri m er s eq ue nc e A m pl ic on si ze (b p) P C R ef fi ce nc y C at eg ry A cc es si on nu m be r G en e na m e G en e an no ta tio n 1 2 3 4 5 6 7 8 9 66 7a A C C as e X 77 38 2 A C C a se H om om er ic a ce ty l C oA c ar bo xy la se B n _ A C C a se _ F F :5 'A G G A C T T G C C A A T C T T C T A A A C 3' 15 7 0, 97 3 66 7 b B n _ A C C a se _ R R :5 'A G C T T C T T T C A C C G T A G G A C A C 3' 66 8a A Y 53 86 75 α -C T al ph a-ca rb ox yl tr an sf er as e B n _α -C T _F F :5 'C T T G T C C A C C C T A T T C T G A T T G 3' 10 6 0, 95 8 66 8 b B n _α -C T _R R :5 'A T G T C C A G C T T A G A T T T G A G G C 3' 66 9a Z 50 86 8 β -C T B et a-ca rb ox yl tr an sf er as e B n _ β -C T _F F :5 'C A G C A A G T T T G G G T A T G T T G G G 3 ' 11 6 1, 03 66 9 b B n _ β -C T _R R :5 'G T G A A C C T T C A G G C A C G G C T T T 3' 67 0a A Y 03 44 10 B C B io tin c ar bo xy la se B n _B C _F F :5 'A G G A C C C A T T C A A A G G A T T C A G 3' 11 8 1 67 0 b B n _B C _R R :5 'G C T T G G A G G A A C A A C A T A G T C G 3' 67 1a D es at ur as e A Y 64 25 37 S A D S te ar oy l-A C P d es at ur as e B n _ S A D _F F :5 'G T T T A C A C T G C C A A A G A C T A T G C G 3' 13 5 0, 93 7 67 1 b B n _ S A D _R R :5 'C C T G A T T C T C G G A G T C A A C C C A C 3' 67 2a A Y 59 29 75 F A D 2 O le at e de sa tu ra se B n _F A D 2_ F F :5 'A G G C G A T A A A G C C G A T A C T T G G 3' 10 7 1, 09 5 67 2 b B n _F A D 2_ R R :5 'C C T A T C C G G T T C A A C A T A G A T A C A C T 3' 67 3a A Y 59 98 84 F A D 3 L in ol ea te d es at ur as e B n _F A D 3_ F F :5 'T T C C C A C A A A T C C C T C A C T A T C A 3' 13 2 0, 93 6 67 3 b B n _F A D 3_ R R :5 'A C T T G C C A C C A A A C T T T C C A C C 3' 67 4a A Y 64 25 35 F A D 6 O le at e de sa tu ra se B n _F A D 6_ F F :5 'A T C A C A T A A G C C C A A G G A T A C C G 3' 11 6 0, 95 3 67 4 b B n _F A D 6_ R R :5 'T C G T C T T C A T C A A C C G C C A A T T 3'

S u p p le m en ta ry T a b le S 1 — co n ti n u ed 1 2 3 4 5 6 7 8 9 67 5a E lo ng as e A J0 07 04 6 M C M T M al on yl C oA -A C P m al on yl tr an sf er as e B n _M C A T _F F :5 'A T C A T A G G G T T G G A C T C A G A A A 3 ' 11 6 0, 95 5 67 5 b B n _M C A T _R R :5 'A C T G C G T A G T T A C C C G G A C A T A 3 ' 67 6a A F 24 45 19 K A S 1 B et a-ke to ac yl -A C P s yn th as e 1 B n _K A S 1_ F F :5 'A C A C G G T C G C A A A C G A G A A G A A 3' 20 4 0, 97 6 67 6 b B n _K A S 1_ R R :5 'G A A G A T A A T G G T G A T G G A G C A G 3' 67 7a A F 24 45 20 K A S 2 B et a-ke to ac yl -A C P s yn th as e 2 B n _K A S 2_ F F :5 'G G A G T A C C A A G C C C T T G C T C A C 3' 13 3 0, 81 2 67 7 b B n _K A S 2_ R R :5 'T C C T T A T G G C C T G C A C A G T T G C 3' 67 8a A F 17 98 54 K A S 3 B et a-ke to ac yl -A C P s yn th as e 3 B n _K A S 3_ F F :5 'G G A T G A T G G G T T A T T T A G T T T C 3' 10 8 0, 91 8 67 8 b B n _K A S 3_ R R :5 'C C A A A G G G T A A A G C A G G A G A A G 3' 67 9a A F 00 95 63 F A E 1 F at ty a ci d el on ga se 1 /3 -k et oa cy l-C oA s yn -th as e B n _F A E 1_ F F :5 'G T C A G G C T T T A A G T G T A A C A G T G C A 3' 15 9 0, 95 7 67 9 b B n _F A E 1_ R R :5 'T T A T T A G G A C C G A C C G T T T T G G 3' 68 0a A F 38 21 46 H D /K A C D 3-ke to -a cy l-A C P d eh yd ra ta se B n _K A C D _F F :5 'G A T A G C G A A A A T G G A A G G G A A A G 3' 11 5 0, 95 8 68 0 b B n _K A C D _R R :5 'A A A G C A A A A G G C A C G A G A A C A T A 3' 68 1a A Y 19 61 97 K C R 2 3-ke to ac yl -C oA r ed uc ta se B n _K C R 2_ F F :5 'T G A G T A C A A G A A A A G T G G G A T T G 3' 10 1 0, 98 3 68 1 b B n _K C R 2_ R R :5 'G A G A T G C C A C T A A G A A A G A T G C T 3' 68 2a T hi oe st er as e B R U 17 09 8 F a tA A cy l-A C P th io es te ra se B n _F at A _F F :5 'G G G A C C A A T G G C T C T G C A T C A T 3' 12 1 0, 96 5 68 2 b B n _F at A _R R :5 'G G C T T C T T T C T C C A C A G G G T T G 3' 68 3a D Q 84 72 75 F a tB P al m ito yl -A C P th io es te ra se B n _F at B _F F :5 'A G T T T G T G G G T G A T G A T G A A T A 3' 10 7 0, 94 4 68 3 b B n _F at B _R R :5 'G C A A G G A T A G G G T C A G A G T T C A 3'

S u p p le m en ta ry T a b le S 1 — co n ti n u ed 1 2 3 4 5 6 7 8 9 68 4a T A G s yn -th es is A F 15 52 24 D G A T 2 A cy l-C oA : d ia cy lg ly ce ro l a cy ltr an sf er as e B n _ D G A T 2_ F F :5 'C A T G A C C T G A T G A A C C G C A A A G 3' 11 1 0, 98 5 68 4 b B n _ D G A T 2_ R R :5 'A C G G C T A C C A A A A G G A T A C A A A A 3' 68 5a A F 11 11 61 L P A A T L ys op ho sp ha tid ic a ci d ac yl tr an sf er as e B n _L P A A T _F F :5 ' C G A A G A G G C G A G A A A C A A G A T A G 3' 10 0 0, 97 68 5 b B n _L P A A T _R R :5 'T G G T T T A G C C T T C T C A T T G T T C A 3' 68 6a A Y 17 95 60 A A P T 1 A m in oa lc oh ol ph os ph ot ra ns fe ra se B n _A A P T 1_ F F :5 'T G G T G C T T C T T G G T T A T T G T A T 3' 15 6 0, 82 1 68 6 b B n _A A P T 1_ R R :5 'G G A T T T G C A T T A T C C T C C C T T G 3' 68 7a O il bo dy pr ot ei n A Y 57 02 50 N a p in 1. 7S o il bo dy p ro te in B n _ N ap in _F F :5 'G A C C C T C G A T G G T G A G T T T G A 3' 14 8 0, 95 8 68 7 b B n _ N ap in _R R :5 'C T T T G G A T G C T C C T T T C A A G G T 3' 68 8a A Y 96 64 47 C a le o si n C a2 + -b in di ng o il bo dy s ur fa ce p ro te in B n _C al eo si n _F F :5 'G T A A T C A A T T T G G C C C T T A G C T 3' 11 6 0, 95 2 68 8 b B n _C al eo si n _R R :5 'C T C A A G A T T C A C A G G C A T A A A C 3' 68 9a X 58 00 0 O le o si n oi l b od y as so ci at ed p ro te in B n _ O le o si n _F F :5 'C T G G G A G G C A A A G T T C A G G A T A 3' 12 2 0, 96 9 68 9 b B n _ O le o si n _R R :5 'C A T G G C G T A A T T T A G G T A G T G T 3' 69 0a M 16 86 0 C ru ci fe ri n 12 S n eu tr al o il bo dy pr ot ei n B n _C ru ci fe ri n _F F :5 'G A G G A G T C A G A G A C C G C A G G A 3' 16 5 0, 96 9 69 0 b B n _C ru ci fe ri n _R R :5 'A A G G A A G C G A A G G A T G G G G A G A 3' 69 1a H ou se ke ep ge ne s A F 11 18 12 β -a ct in H ou se ke ep in g ge ne B n _ β -a ct in _F F :5 'C T G G A A T T G C T G A C C G T A T G A G 3 ' 14 5 1, 00 1 69 1 b B n _ β -a ct in _R R :5 'A T C T G T T G G A A A G T G C T G A G G G 3 ' 69 2a D Q 09 73 38 G A P D H G ly ce ra ld eh yd e- 3-ph os ph at e de hy dr og en as e B n _ G A P D H _F F :5 'G C T A T C A A G G A G G A A T C T G A G G A C 3' 14 6 0, 93 6 69 2 b B n _ G A P D H _R R :5 'C T T C A C G A A A T T G T C A C T C A A C G 3' 69 3a D Q 16 71 82 P 4 5 0 C yt oc hr om e P 45 0 B n _P 45 0_ F F :5 'A T G G A T C T C G G G A T C G G A C A G T 3' 15 6 0, 95 4 69 3 b B n _P 45 0_ R R :5 'G T C A A G C G A T G A C G G A G C A A A A 3' 69 4a X 93 01 5 G K T P G ly ox ys om al b et a-ke to ac yl -t hi ol as e pr ec ur so r B n _ G K T P _F F :5 'G T T G G T C C A G C A G T T G C C A T T C 3' 15 9 0, 93 4 69 4 b B n _ G K T P _R R :5 'C G C C T C C G T T G A C A T T G A T T T T 3' O th er s

S u p p le m en ta ry T a b le S 1 — co n ti n u ed 1 2 3 4 5 6 7 8 9 O th er s 69 5a A J2 23 49 7 P E P C P ho sp ho en ol py ru va te c ar bo xy la se B n _P E P C _F F :5 'G G T T G G G T T T A T T G G T T T G T T T A T G 3' 13 4 0, 93 4 69 5 b B n _P E P C _R R :5 'A T T C C C T T G C T C G G T T T T G T T A 3' 69 6a A J2 71 16 2 A G P a se A D P -g lu co se p yr op ho sp ho ry la se s m al l s ub -B n _A G P a se _F F :5 'A G A C A C C A C C A C C C C G T T T G A C 3' 12 9 0, 97 4 69 6 b B n _A G P a se _R R :5 ' T T T A G G G A T A A G G C A G G A G G A T 3' 69 7a A B 04 16 22 B cR K 6 R ec ep to r k in as e 6 B n _B cR K 6_ F F :5 'A G G T T A A G T G A C G G G C A A G A A A 3' 14 3 1, 01 9 69 7 b B n _B cR K 6_ R R :5 'T T G A A C G C A A C A G C C A A G A A G T 3' 69 8a A Y 06 58 39 S U C 1 S uc ro se tr an sp or te r B n _ S U C 1_ F F :5 'G C C A A G G A C T G T C G T T A G G A G T T T 3' 13 3 0, 97 69 8 b B n _ S U C 1_ R R :5 'T G C G A T T G C T C C G A C T A T A A A T G 3' 69 9a A J7 16 22 7 A R F 2 A ux in R es po ns e F ac to r2 B n _ A R F 2_ F F :5 'A C C A C T A G T A T T C C T C G C C C T G A T 3' 17 1 69 9 b B n _ A R F 2_ R R :5 'T G C C T T A G A T G A G C C T T C C C T T A T 3' 73 3a E V 11 60 54 A C T 7 A ct in A C T 7F 5’ -T G G G T T T G C T G G T G A C G A T 63 73 3 b A C T 7R 5’ - T G C C T A G G A C G A C C A A C A A T A C T 73 4a E V 08 69 36 U B C 2 1 U bi qu iti n co nj ug at in g en zy m e 21 U B C 21 F 5’ - C C T C T G C A G C C T C C T C A A G T 77 73 4 b U B C 21 R 5’ - C A T A T C T C C C C T G T C T T G A A A T G C 73 5a E V 05 10 05 P P 2 A R eg ul at or y su bu ni t o f pr ot ei n ph os ph at as e 2A P P 2A F 5’ - T G G C T T C A G T T A T A A T G G G A A T G G 75 73 5 b P P 2A R 5’ - G A A A G A T T G G A A G G A G A T G C T C A A T 73 6a E V 22 27 61 T IP 4 1 T IP 41 -l ik e fa m ily p ro te in T IP 41 F 5’ - A G A G T C A T G C C A A G T T C A T G G T T 69 73 6 b T IP 41 R 5’ - C C T C A T A A G C A C A C C A T C A A C T C T A A 73 7a E V 00 21 23 U B C 9 U bi qu iti n co nj ug at in g en zy m e 9 U B C 9F 5’ - G C A T C T G C C T C G A C A T C T T G A 68 73 7 b U B C 9R 5’ - G A C A G C A G C A C C T T G G A A A T G 73 8a E V 08 42 76 S A N D S A N D -f am ily p ro te in S A N D F 5’ - G C T G G G T C A C T C C A G A T T T T G 63 73 8 b S A N D R 5’ - C C A T C G C C T T G T C T G C A A G 73 9a E E 45 03 88 U P 1 U nk no w n pr ot ei n U P 1F 5’ - A G C C T G A G G A G A T A T T A G C A G G A A 87 73 9 b U P 1R 5’ - A T C T C A C T G C A G C T C C A C C A T 74 0a E V 11 67 50 U P 2 U nk no w n pr ot ei n U P 2F 5’ - A A A T T C C T G G G A G G G A A G C T A T 70 74 0 b U P 2R 5’ - T T C T G T C T C A G G A G C G A A G T C A T

S u p p le m en ta ry T a b le S 2 : C y cl e tr es h o ld ( C T ) v a lu es f o r re fe re n ce g en es 1 0 d v a lu e a s a b a se li n e G en e na m e P ri m er # B io R ep G en e n am e P ri m er # B io R ep V ar ie ty 1 0 d ay 1 5 d ay 2 0 d ay 2 5 d ay 3 0 d ay 3 5 d ay 4 0 d ay β -a ct in ( 69 1 B 5) β -a c ti n (6 9 1 B 5 ) B ro no w sk i 1 80 5 2 1 .8 3 3 2 0 .9 50 2 0 .7 70 2 1 .7 3 7 2 2 .2 5 0 2 2 .9 2 7 2 3 .2 8 0 β -a ct in ( 69 1 B 6) β -a c ti n (6 9 1 B 6 ) B ro no w sk i 1 80 6 2 0 .8 0 3 2 0 .5 73 2 2 .0 37 2 2 .6 9 0 2 4 .2 6 0 2 4 .5 8 0 n o cD N A β -a ct in ( 69 1 B 7) β -a c ti n (6 9 1 B 7 ) B ro no w sk i 1 80 7 2 1 .8 8 3 2 0 .4 90 2 0 .5 70 2 2 .1 1 0 2 1 .9 5 3 2 3 .8 7 0 n o cD N A β -a ct in ( 69 1 Q 6) β -a c ti n (6 9 1 Q 6 ) Q 2 19 66 2 0 .9 7 0 2 1 .2 90 2 1 .4 73 2 3 .0 7 0 2 4 .8 2 0 2 1 .9 2 0 2 2 .1 7 7 β -a ct in ( 69 1 Q 7) β -a c ti n (6 9 1 Q 7 ) Q 2 19 67 2 1 .0 6 0 n o cD N A n o cD N A 2 1 .3 2 7 2 1 .9 8 0 2 1 .4 6 3 2 2 .3 1 3 β -a ct in ( 69 1 Q 8) β -a c ti n (6 9 1 Q 8 ) Q 2 19 68 2 0 .8 7 0 2 1 .3 07 2 2 .4 43 2 1 .6 2 3 2 2 .0 1 0 2 2 .5 9 7 2 3 .3 1 3 β -a ct in ( 69 1 W 0) β -a c ti n (6 9 1 W 0 ) W es ta r 2 03 0 2 2 .1 7 3 2 1 .8 43 2 1 .6 63 2 1 .7 1 0 2 1 .9 8 7 2 2 .1 4 7 2 3 .3 5 0 β -a ct in ( 69 1 W 1) β -a c ti n (6 9 1 W 1 ) W es ta r 2 03 1 2 2 .2 2 0 2 1 .3 80 2 1 .2 07 2 1 .8 7 0 2 2 .0 2 0 2 2 .5 7 7 2 2 .7 8 3 β -a ct in ( 69 1 W 2) β -a c ti n (6 9 1 W 2 ) W es ta r 2 03 2 2 1 .1 8 7 2 1 .6 80 n o cD N A 2 1 .2 7 7 2 1 .8 1 7 2 1 .8 3 7 2 2 .9 7 0 G A P D H ( 69 2 B 5) G A P D H (6 9 2 B 5 ) B ro no w sk i 1 80 5 2 3 .1 1 0 2 1 .7 17 2 2 .0 17 2 2 .9 4 7 2 2 .7 6 7 2 4 .3 2 7 2 5 .2 5 7 G A P D H ( 69 2 B 6) G A P D H (6 9 2 B 6 ) B ro no w sk i 1 80 6 2 1 .9 8 0 2 1 .3 30 2 2 .9 70 2 4 .3 1 0 2 5 .3 4 3 2 5 .7 5 7 n o cD N A G A P D H ( 69 2 B 7) G A P D H (6 9 2 B 7 ) B ro no w sk i 1 80 7 2 2 .9 0 0 2 1 .7 20 2 1 .3 00 2 3 .5 0 0 2 3 .3 5 7 2 5 .7 7 0 n o cD N A 0 ! G A P D H ( 69 2 Q 6) G A P D H (6 9 2 Q 6 ) Q 2 19 66 2 2 .1 8 3 2 1 .8 13 2 2 .4 80 2 4 .0 3 7 2 6 .0 0 0 2 3 .8 0 3 2 4 .7 5 7 G A P D H ( 69 2 Q 7) G A P D H (6 9 2 Q 7 ) Q 2 19 67 2 2 .3 5 7 # D Z IE L /0 ! n o cD N A 2 2 .6 0 7 2 3 .5 6 7 2 3 .2 3 3 2 3 .7 8 0 G A P D H ( 69 2 Q 8) G A P D H (6 9 2 Q 8 ) Q 2 19 68 2 2 .4 4 3 2 1 .9 77 2 3 .4 30 2 2 .7 0 7 2 3 .4 1 0 2 4 .2 9 7 2 5 .4 9 3 G A P D H ( 69 2 W 0) G A P D H (6 9 2 W 0 ) W es ta r 2 03 0 2 3 .6 1 7 2 2 .5 03 2 2 .2 00 2 2 .4 4 3 2 2 .9 0 3 2 4 .1 9 3 2 5 .2 0 0 G A P D H ( 69 2 W 1) G A P D H (6 9 2 W 1 ) W es ta r 2 03 1 2 3 .4 5 0 2 2 .0 10 2 1 .9 60 2 2 .8 3 3 2 3 .6 2 0 2 4 .6 1 3 2 4 .7 5 7 G A P D H ( 69 2 W 2) G A P D H (6 9 2 W 2 ) W es ta r 2 03 2 2 2 .6 0 7 2 2 .1 77 n o cD N A 2 2 .3 7 0 2 3 .2 7 3 2 3 .6 7 3 2 5 .3 6 7 P 45 0 (6 93 B 5) P 4 5 0 (6 9 3 B 5 ) B ro no w sk i 1 80 5 2 7 .4 5 0 2 8 .6 80 2 7 .6 57 2 7 .5 1 7 2 9 .2 3 0 3 1 .2 1 0 3 2 .8 8 0

S u p p le m en ta ry T a b le S 2 — co n ti n u ed G en e na m e P ri m er # B io R ep G en e n am e P ri m er # B io R ep V ar ie ty 1 0 d ay 1 5 d ay 2 0 d ay 2 5 d ay 3 0 d ay 3 5 d ay 4 0 d ay P 45 0 (6 93 B 6) P 4 5 0 (6 9 3 B 6 ) B ro no w sk i 1 80 6 2 6 .0 0 7 2 7 .9 3 7 2 9 .4 4 0 2 9 .2 3 7 3 5 .0 0 0 3 5 .0 0 0 n o cD N A P 45 0 (6 93 B 7) P 4 5 0 (6 9 3 B 7 ) B ro no w sk i 1 80 7 2 7 .9 9 3 2 7 .4 3 7 2 8 .0 8 7 2 8 .4 1 0 2 8 .5 0 3 3 2 .3 8 3 n o cD N A P 45 0 (6 93 Q 6) P 4 5 0 (6 9 3 Q 6 ) Q 2 19 66 2 8 .0 8 0 2 8 .7 6 7 2 9 .8 0 0 3 0 .5 4 7 3 5 .0 0 0 3 0 .2 1 3 3 0 .7 5 0 P 45 0 (6 93 Q 7) P 4 5 0 (6 9 3 Q 7 ) Q 2 19 67 2 7 .8 6 3 n o cD N A n o cD N A 2 8 .2 6 0 2 8 .8 7 3 2 9 .2 9 0 2 9 .5 8 7 P 45 0 (6 93 Q 8) P 4 5 0 (6 9 3 Q 8 ) Q 2 19 68 2 7 .8 8 3 2 9 .0 4 0 3 0 .0 3 3 2 8 .5 1 5 2 9 .7 2 3 3 2 .2 6 7 3 2 .3 0 7 P 45 0 (6 93 W 0) P 4 5 0 (6 9 3 W 0 ) W es ta r 2 03 0 2 9 .0 9 0 2 9 .7 9 0 2 9 .2 7 7 2 8 .9 7 0 2 9 .2 5 7 3 1 .5 5 7 3 2 .0 5 0 P 45 0 (6 93 W 1) P 4 5 0 (6 9 3 W 1 ) W es ta r 2 03 1 2 9 .0 2 7 2 9 .8 1 0 2 9 .7 1 0 2 9 .7 0 3 2 9 .4 9 0 3 0 .5 6 0 2 9 .9 0 3 P 45 0 (6 93 W 2) P 4 5 0 (6 9 3 W 2 ) W es ta r 2 03 2 2 8 .1 7 3 2 9 .9 1 3 n o cD N A 2 8 .8 8 0 2 9 .6 6 0 3 0 .3 6 0 3 3 .1 9 3 G K T P ( 69 4 B 5) G K T P (6 9 4 B 5 ) B ro no w sk i 1 80 5 2 4 .6 2 0 2 4 .1 7 0 2 3 .3 9 3 2 3 .3 0 0 2 2 .3 7 0 2 1 .4 8 7 2 2 .2 4 0 G K T P ( 69 4 B 6) G K T P (6 9 4 B 6 ) B ro no w sk i 1 80 6 2 3 .5 3 7 2 4 .4 0 0 2 5 .5 2 3 2 0 .4 8 3 2 1 .6 4 7 2 2 .5 7 7 n o cD N A G K T P ( 69 4 B 7) G K T P (6 9 4 B 7 ) B ro no w sk i 1 80 7 2 4 .4 6 3 2 3 .4 7 0 2 3 .8 3 7 2 1 .1 2 7 2 2 .2 3 3 2 3 .4 7 0 n o cD N A G K T P ( 69 4 Q 6) G K T P (6 9 4 Q 6 ) Q 2 19 66 2 4 .3 1 7 2 5 .6 2 0 2 5 .2 0 7 2 6 .7 7 7 2 2 .6 5 0 2 2 .2 0 7 2 3 .5 1 3 G K T P ( 69 4 Q 7) G K T P (6 9 4 Q 7 ) Q 2 19 67 2 4 .0 5 0 n o cD N A n o cD N A 2 4 .2 8 3 2 3 .6 2 7 2 2 .3 4 7 2 3 .1 6 7 G K T P ( 69 4 Q 8) G K T P (6 9 4 Q 8 ) Q 2 19 68 2 4 .5 6 7 2 5 .1 4 0 2 6 .5 3 3 2 4 .6 6 3 2 4 .5 9 0 2 2 .8 4 3 2 4 .7 9 3 G K T P ( 69 4 W 0) G K T P (6 9 4 W 0 ) W es ta r 2 03 0 2 5 .6 5 7 2 5 .7 7 0 2 5 .5 5 3 2 5 .3 3 7 2 4 .6 7 3 2 2 .6 0 7 2 4 .1 8 7 G K T P ( 69 4 W 1) G K T P (6 9 4 W 1 ) W es ta r 2 03 1 2 5 .5 8 7 2 5 .6 5 3 2 5 .5 0 3 2 5 .3 3 7 2 4 .6 0 7 2 3 .1 8 0 2 4 .2 5 3 G K T P ( 69 4 W 2) G K T P (6 9 4 W 2 ) W es ta r 2 03 2 2 5 .0 2 3 2 5 .8 0 7 n o cD N A 2 4 .8 7 0 2 4 .0 2 3 2 3 .4 7 3 2 3 .9 7 7 P E P C ( 69 5 B 5) P E P C (6 9 5 B 5 ) B ro no w sk i 1 80 5 2 9 .1 0 7 2 8 .3 6 3 2 8 .0 6 3 2 8 .0 9 7 2 8 .9 0 7 2 8 .6 6 0 2 9 .9 5 3 P E P C ( 69 5 B 6) P E P C (6 9 5 B 6 ) B ro no w sk i 1 80 6 2 7 .6 2 7 2 8 .5 5 7 2 8 .9 4 7 2 8 .9 8 7 2 9 .6 4 7 3 0 .0 1 0 n o cD N A