Industrial and Products 98 (2017) 130–138

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Industrial Crops and Products

jo urnal homepage: www.elsevier.com/locate/indcrop

Characterization of cuticular waxes and cutin monomers of

Camelina sativa and closely-related species

a b a a a c a,∗

P. Tomasi , H. Wang , G.T. Lohrey , S. Park , J.M. Dyer , M.A. Jenks , H. Abdel-Haleem

a

USDA-ARS, US Arid-Land Agricultural Research Center, 21881 North Cardon Lane, Maricopa, AZ 85138, USA

b

USDA-ARS, Genetics and Breeding Research Unit, 115 Coastal Way, Tifton, GA 31793, USA

c

Division of and Soil Sciences, West Virginia University, Morgantown, WV, 26505, USA

a r t i c l e i n f o a b s t r a c t

Article history: is an old world crop newly introduced to the semi-arid regions of the Southwestern US.

Received 26 September 2016

Recently, Camelina gained attention as a feedstock crop due to its relatively high oil content,

Received in revised form 13 January 2017

polyunsaturated fatty acids, very short growing season with fairly good adaption to marginal lands, and

Accepted 19 January 2017

low input agricultural systems. To expand Camelina growing zones into more arid regions, it is important

Available online 26 January 2017

to develop new drought resistant cultivars that can grow under water-limited conditions. hav-

ing cuticles with low permeability to water can possess elevated dehydration avoidance and improved

Keywords:

drought tolerance. To extend our understating of cuticle chemical composition among Camelina species,

Camelina sativa

Cuticle leaf wax and cutin monomers in seventeen accessions representing four Camelina species were ana-

Cutin lyzed. Camelina exhibited a wide range of wax and cutin contents. The primary alcohols and alkanes

Drought were the predominant classes of leaf wax, followed in abundance by wax esters, fatty acids, aldehy-

␣

Cuticular wax des, alkylguaiacols, methylalkylresorcinols, -amyrin and ß-sitosterol. Among primary alcohols, the

Stress tolerance dominant constituents were the C24, C26 and C28 homologues, while the C31 homologue was the most

abundant alkane among all Camelina accessions. Cutin monomers included monohydroxy monobasic

acids, phenolics, monobasic acids, monohydroxy epoxymonobasic acids, and dibasic acids. Among the

cutin monomers examined, the C16:0 diOH acid showed extensive variation among Camelina species.

Published by Elsevier B.V.

1. Introduction Vaughn, 2010), and very short growing season with good adaption

to marginal lands and low agricultural inputs (Putnam et al., 1993;

The genus Camelina belongs to the , a family com- Moser and Vaughn, 2010; Obour et al., 2015). To expand Camelina

posed of 11 known Camelina species (Warwick and Al-Shehbaz, production zones to more marginal regions, it will be important

2006). Currently five species, including C. alyssum (Mill.) Thell., C. to develop new cultivars that can grow under water-limited condi-

hispida Boiss, C. microcarpa Andrz. ex DC, C. rumelica Velen. and C. tions and still maintain comparably high yield and stability (French

sativa (L.) Crantz are available in public plant germplasm reposito- et al., 2009).

ries, such as The Leibniz Institute of Plant Genetics and Crop Plant The primary mechanisms for plant adaptation to drought can

Research, Gatersleben, Germany (http://www.ipk-gatersleben.de/ be grouped into four categories that include drought escape, avoid-

en/), U.S. National Plant Germplasm System (https://npgsweb. ance, tolerance and recovery (Fang and Xiong, 2015). The drought

ars-grin.gov/gringlobal/search.aspx) and The Centre for Genetic tolerance response in plants is a complex process likely triggered

Resources, the Netherlands (http://cgngenis.wur.nl/). Only C. sativa first by the plant’s perception of dehydration in soil (or air) followed

and C. microcarpa are currently cultivated for their oil. Commonly by targeted changes in metabolic responses and gene expres-

known as gold-of-pleasure or false flax, Camelina originated from sion, and ultimately to cellular and whole plant developmental

Northern Europe and Southeastern Asia, recently gaining attention changes (Bartels and Sunkar, 2005; Golldack et al., 2014). The avoid-

as a biofuel feedstock crop due to its high oil content (28–40%), ance mechanism is generally characterized as the plant’s ability

polyunsaturated fatty acids (54.3%) (Budin et al., 1995; Moser and to delay the onset of dehydration in its tissues as soil moisture

depletes. Plants better able to avoid tissue dehydration often pos-

sess more efficient root systems that increase soil water extraction,

and/or possess a higher capacity to reduce stomatal conductance, ∗

Corresponding author.

absorption of solar radiation, cuticle water permeability, and/or

E-mail address: [email protected] (H. Abdel-Haleem).

http://dx.doi.org/10.1016/j.indcrop.2017.01.030

0926-6690/Published by Elsevier B.V.

P. Tomasi et al. / Industrial Crops and Products 98 (2017) 130–138 131

evaporative surface area (Jones et al., 1981). Non-stomatal water 2.2. Leaf wax extractions and analyses

loss is controlled primarily by the cuticle, an extracellular, lipophilic

polymer that protects the aerial organs of plants from the surround- Three leaf subsamples (approximately seventh to twelfth leaf of

ing environment. It also provides protection from abiotic stress; for basal rosette) from each plant (four replications) were collected at

example, pathogens and insects (Jenks et al., 1994; Yeats and Rose, 35 days after planting (dap). Each leaf was individually submerged

2013) and stresses such as drought, heat, and supra-optimal solar in 10 ml hexane (Sigma-Aldrich, USA), capped and agitated for 45 s

radiation (Shepherd and Wynne Griffiths, 2006; Yeats and Rose, in a 20 ml glass scintillation vial. The leaf was removed from the

2013; Kosma et al., 2009). The cuticle itself is composed primarily of solvent with forceps and leaf area determined using a flatbed scan-

cuticular waxes and cutin monomers, the latter composed primar- ner, after which it was moved into a new vial containing 20 ␮l

ily of aliphatic components cross-linked into a polyester matrix. In isopropanol for use in subsequent cutin analysis. The wax extracts

◦

the model plant Arabidopsis, and its brassicaceous relatives, such were heated (70 C) and reduced under N2 until the volume could

as Eutrema (syn. Thellungiella) and Brassica napus L. (), the be transferred into a 2 ml glass vial. The scintillation vials were

cutin layer consists mainly of C16 and C18 ␻-hydroxy and ␣, ␻- rinsed once with a few milliliters of hexane, the volume transferred

dicarboxylic fatty acids monomers and is associated with both epi- again and then evaporated to dryness. For each wax sample, 90 ␮l of

and intra-cuticular waxes, generally dominated by derivatives of N,O-bis-(trimethylsilyl)-trifluoroacetamide (BSTFA, Sigma-Aldrich,

the saturated very long-chain fatty acids (VLCFA) and isoprenoids USA), 10 ␮l ISTD (2 ␮g hexadecane, Sigma-Aldrich, USA) and 110 ␮l

(von Wettstein-Knowles, 2001). Wax and cutin monomer compo- hexane was added for a total volume of 200 ␮l. The sample vials

sition often varies significantly between species, between organs were capped and loaded onto the GC–MS.

of the same plant (Lee and Suh, 2015; Bernard and Joubès, 2013), An Agilent 7890A gas chromatograph equipped with a 5975C

by developmental stage (Samuels et al., 2008; Lee and Suh, 2015), mass spectrometer was used for chemical identifications and quan-

and as a response to environmental conditions (Baker, 1974). The tifications. Thirty two min sample overlap was enabled to increase

◦

surface waxes of , seeds, and roots of Camelina sativa cul- throughput and barcode heat each vial at 80 C for 35 min, then mix

tivar ‘Celine’ are dominated by alkanes, primary alcohols, and 6 s at 2000 rpm for five cycles prior to a 1 ␮l splitless injection. An

free fatty acids (Razeq et al., 2014). To date, the cutin composi- HP-Ultra 1 capillary column (12 m length, 200 ␮m inner diameter,

tion of Camelina has not been reported. With the advancement in 0.33 ␮m film thickness, Agilent, USA) was used, with helium as the

◦

genomics and gene discovery tools, and increased knowledge about carrier gas at 1 ml per min and temperature settings of inlet 300 C,

◦ ◦ ◦

the physiological, metabolic, and genetic determinants of impor- detector 300 C, initial oven temperature 50 C, then increased 20 C

◦ ◦

tant traits like the composition of the cuticle, the application of per minute to 260 C, where it was held for 8 min, then 25 C per

◦

more powerful genomics-based crop improvement strategies has min to 325 C and held again for 13.9 min for a total run time of

great potential to improve stress tolerance in crops. The objective of 35 min.

the current study was to extend our understanding of cuticle lipid For cutin analysis, the isopropanol was decanted from each vial

−1

composition and variation in Camelina sativa cultivars and related, after two days and 20 ␮l of CHCl3: CH3OH (1:1 v/v) with 50 mg l

non-domesticated Camelina species. butylated hydroxytoluene was added to each leaf sample. Vials

were flushed with nitrogen for 1 min, capped, and stored again at

− ◦

20 C until delipidation. Before loading the soxhlets, the storage

solvent was decanted and the leaf sample was transferred to liquid

N2 in a cold mortar. A pestle was used to grind the leaves, which

2. Materials and methods

were then rinsed into a thimble with CHCl3: CH3OH (1:1 v/v) and

loaded into the soxhlets. After three days of heating, the CHCl3:

2.1. Plant material and growth conditions

CH3OH (1:1 v/v) solvent was mostly decanted, the sample poured

into a culture tube and any remaining solvent removed by glass

Seventeen accessions of four Camelina species, including C.

pipette. Samples were dried for three days under vacuum.

sativa, C. hispida, C. microcarpa and C. rumelica, initially collected

from different geographical regions, were acquired from IPK Gater-

sleben GenBank, Germany and/or USDA Germplasm Resources

2.3. Leaf cutin depolymerization and analysis

Information Network (GRIN) (Table 1). Accession 18097E was ini-

tially collected from Turkey and provided by Dr. Mark Beilstein

Fifty microliters of ISTD (10 ␮g methyl heptadecanoate, Sigma-

from the University of Arizona. Two transgenic camelina lines with

Aldrich, USA) was added to each delipidated sample as an internal

independent MYB96 TF insertions, C2x2.9.1 and R2x6.1.3, were

standard. Cutin monomers were solubilized by transesterification

also examined. C2x2.9.1 and R2x6.1.3 are improved Celine and

by first adding 6 ml of 3 N methanolic HCl (Sigma-Aldrich, USA),

◦

Robinson respectively. Camelina cultivars, Celine and Robinson,

sealing under N2,then heating for 20 h at 60 C. After incubation,

were transformed with A. tumefaciens GV3101 strain harboring the

samples were allowed to cool, caps were slowly released and 6 ml

binary construct pCAMBIA3301M1 containing AtMyb96 gene and

of saturated NaCl and 10 ml dichloromethane were added to each.

CaMV 35 prompter, using a transformation protocol described by

The sample tubes were then inverted ten times and spun down

Lu and Kang (2008). T1 plants were selected on one-half strength

at 2500 rpm for 3 min. The lower solvent phase was pipetted into a

Murashige and Skoog minimal organic medium supplemented

new culture tube, 5 ml of 0.9% NaCl was added, and then the samples

−1

with 1% agar and mg l glufosinolate. Multiple transgenic lines

were slightly mixed and centrifuged again. The top wash layer was

were obtained for both cultivars, and C2x2.9.1 and R2x6.1.3 were

pipetted off and the salt washing was done twice more for a total

reported in this study.

of three washes. The remaining lower phase was then evaporated

Seeds were planted in 29.29 cu. in. containers of Sunshine Mix

under N2 until ca. 500 ␮l remained, at which point it was trans-

#1/LC1 (Sun Gro Horticulture, Canada), covered and vernalized at

ferred to a smaller 2 ml vial. One milliliter of dimethoxypropane

◦

4 C in the dark. After five days, the containers were uncovered and

was added to each sample, vials were capped, vortexed and incu-

◦

moved to a growth chamber. The conditions were 12-h-light/12-h-

bated at 50 C for 5 min, after which the samples were evaporated to

◦ −2 −1

dark at 22/20 C and an intensity of ca. 175 ␮E m s and ambient

dryness. For each cutin sample, 100 ␮l of N,O-bis-(trimethylsilyl)-

humidity. Plants were regularly watered and fertilized with 20-20-

trifluoroacetamide (BSTFA, Sigma-aldrich, USA), 100 ␮l pyridine

20 fertilizer (Scotts Miracle-Grow, USA).

and 200 ␮l heptane: toluene (1:1 v/v) was added for a total vol-

132 P. Tomasi et al. / Industrial Crops and Products 98 (2017) 130–138

Table 1

Seventeen Camelina accessions representing four species from public gene banks including The Leibniz Institute of Plant Genetics and Crop Plant Research, Gatersleben,

Germany (http://www.ipk-gatersleben.de/en/) and U.S. National Plant Germplasm System (https://npgsweb.ars-grin.gov/gringlobal/search.aspx).

Line Accession name Genus species ‘subsp.’ accesion name (other) source orgin

1 Robinson C. sativa (L.) Crantz subsp. sativa – USA USA

2 Celine C. sativa (L.) Crantz subsp. sativa – France France

3 CAM212 C. sativa (L.) Crantz subsp. sativa STAMM 02X11A IPK Germany

4 CAM159 C. sativa (L.) Crantz subsp. sativa STAMM 13X15 IPK Germany

5 CAM23 C. sativa (L.) Crantz subsp. pilosa – IPK –

14 PI650152 C. sativa (L.) Crantz subsp. sativa CPS-CAM23 USDA Germany

9 C2x2.9.1 C. sativa (L.) Crantz subsp. sativa USA USA

10 R2x6.1.3 C. sativa (L.) Crantz subsp. sativa USA USA

8 CAM42 Camelina sativa (L.) Crantz subsp. pilosa IPK USA

13 PI650143 C. sativa (L.) Crantz subsp. sativa CS-CROO USDA Germany

15 PI650167 C. sativa (L.) Crantz subsp. sativa Index Seminum 144 USDA Poland

17 PI633186 C. microcarpa Andrz. ex DC. No. 61 USDA Hungery

6 CAM244 C.rumelica Velen. Leindotter IPK Russia

a

7 18097E C.rumelica Velen. USA Turkey

11 PI650138 C. rumelica Velen. 161-3724-75 USDA Iran

12 PI650139 C. rumelica subsp. transcaspica 162-3726-75 USDA Iran

16 PI650133 C. hispida var. grandiflora 158-6281-83 USDA Turkey, Nevsehir

a

18097E was collectedand donated Dr. Mark Beilstein, University of Arizona.

ume of 400 ␮l. The sample vials were capped and loaded onto the report, Razeq et al. (2014) characterized leaf wax constituents of the

GC–MS. Camelina sativa cultivar ‘Celine’. To greatly extend these studies,

The same GC–MS instrument was used for cutin monomer anal- we examined both leaf surface wax and cutin monomer compo-

ysis, but a different method was utilized (Jenkin and Molina, 2015; sition and variation in 17 different accessions of Camelina sativa

Parsons et al., 2013). Eighteen minute sample overlap was enabled and three related Camelina species collected from different geo-

◦

to increase throughput and barcode heat each vial at 80 C for graphical regions worldwide. As the MYB96 transcription factor

20 min, then mix 6 s at 2000 rpm for five cycles prior to a 1 ␮l split- was previously used to increase leaf wax content in Camelina (Lee

less injection. An HP-Ultra 1 capillary column (12 m length, 200 ␮m et al., 2014), two transgenic lines having independent insertions of

inner diameter, 0.33 ␮m film thickness, Agilent, USA) was used, MYB96 were also generated and examined in this study (Table 1).

with helium as the carrier gas at 1 ml per minute and temperature

◦ ◦

settings of inlet 300 C, detector 300 C, initial oven temperature

◦ ◦ ◦ 3.1. Cuticular wax characterization

50 C, then increased 15 C per minute to 320 C, where it was held

for 2 min for a total run time of 20 min.

The Camelina accessions examined displayed a wide range of

wax content, wherein the MYB96 transgenic line had the highest

−

2.4. Interpretation of wax and cutin constituents and statistical 2

total wax (288.10 ␮g dm ) and the Robinson variety exhibited the

analysis −2

lowest (72.19 ␮g dm ) (Table 2). Our results for the wax compo-

sitions of cultivar ‘Celine’ (Table 2) are similar to that reported by

Molecular identities of compounds were determined by char-

Razeq et al. (2014), however our total wax amounts were slightly

acteristic quadrupole electron impact mass spectra. Many of the

lower, potentially because leaves were younger at sampling. Our

waxes and cutin monomers were identified by NIST, however those

results showed that Camelina sativa leaves produced a wax load

−

missing from the library were compared to previously published 2

of 137.08 ␮g dm when averaged across all C. sativa accessions

spectra or elucidated from their ion chromatograms. Uncorrected

(excluding the MYB96 transgenic lines). C. rumelica had higher total

−

wax quantifications were based on total ion content and for cutin 2

wax amount averaging 201.65 ␮g dm across all C. rumelica acces-

−

monomers, AMDIS deconvoluted target ions were utilized, each 2

sions, while C. hispida and C. microcarpa averaged 85.66 ␮g dm

−

relative to the correspondingly added internal standard. Leaf sur- 2

and 84.57 ␮g dm , respectively. MYB96 is a transcription fac-

face areas were determined from scanned leaves using ImageJ

tor known to activate genes encoding very long chain

(Schneider et al., 2012). Each leaf value was multiplied by two to

(VLCFA)-condensing enzymes involved in cuticular wax biosynthe-

account for both surfaces and quantified wax values are expressed

sis (Seo et al., 2011; Lee et al., 2014). Interestingly, the two MYB96

−2

as ␮g dm . The three leaf subsamples were averaged to represent

overexpressing lines produced using the USDA GRIN seed accession

−

one replicate. Four replications were used for each accession. 2

PI650140 exhibited leaf wax loads of 288.10 and 141.21 ␮g dm ,

Each constituent was analyzed using analysis of variance and

with the C2x2.9.1 transgenic line producing the highest wax load

the GLM procedure of SAS software, version 9.4 (Statistical Analysis

of all C. sativa, amounts significantly higher than reported for the

System, SAS institute, 2001), to compare among accessions. Least

non-transgenic parent as previously reported in Lee et al. (2014).

significant differences (LSD) were calculated at significance level

By comparison, the non-transgenic CAM23C. sativa accession pro-

−

(p = 0.05). 2

duced 222.15 ␮g dm wax load, which was actually higher than

the lower MYB96 overexpressor (Table 2). It should be noted that

3. Results and discussion some of the C. sativa accessions produced wax loads that were

among the lowest of all Camelina examined, indicating a very broad

With the emergence of Camelina as a new biofuel crop for range of wax loads present in natural accessions of C. sativa. The

marginal, semi-arid and arid regions, a better understanding of the 18097E accession collected from Turkey exhibited the highest total

−2

adaptations it might possess to help support growth in such stress- wax amount (226.41 ␮g dm ) among the C. rumelica, followed by

−2

ful environments is needed. Increasing or otherwise modifying the the PI650138 accession from Iran, with 215.76 ␮g dm of total

Camelina cuticle to limit plant water loss could improve its capacity wax (Table 2). Besides MYB96, there are more than 20 Arabidopsis

for drought avoidance by delaying the onset of tissue dehydration genes encoding regulatory proteins involved in wax biosynthe-

(Fang and Xiong, 2015; Riederer and Schreiber, 2001). In a recent sis (Lee and Suh, 2015; Bernard and Joubès, 2013). In the current

P. Tomasi et al. / Industrial Crops and Products 98 (2017) 130–138 133 , 2 − dm

g ␮ LSD(0.05) 13.80 1.32 5.59 0.15 7.46 0.13 3.82 0.07 1.32 0.04 0.19 0.13 13.36 0.11 0.34 2.30 0.18 7.12 0.23 4.26 0.25 7.15 0.19 0.90 2.09 2.90 2.04 0.38 5.03 0.64 0.74 0.19 1.11 0.11 1.45 0.09 1.46 0.06 0.64 1.06 0.06 0.18 0.53 0.32 0.06 0.69 0.40 0.31 0.04 0.47 0.21 0.24 0.07 1.05 0.49 27.17

in

.hispida

Values,

C PI650133 37.27 0.55 14.27 0.26 14.62 0.07 5.57 0.11 1.82 0.00 0.00 0.00 37.63 0.00 0.45 4.64 0.21 19.92 0.28 11.51 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 3.15 1.33 1.67 0.14 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.47 0.14 0.13 0.20 0.00 0.00 4.77 2.84 1.93 0.00 0.41 0.00 0.41 0.00 2.17 0.20 86.07

replicate.

PI633186 38.40 4.62 13.51 0.41 8.72 0.04 6.26 0.00 3.70 0.00 0.69 0.47 9.07 0.00 0.23 1.06 0.39 4.44 0.72 2.39 0.47 23.54 1.06 3.45 8.96 8.01 2.06 0.00 6.19 0.56 0.95 0.20 0.56 0.00 1.66 0.00 1.68 0.00 0.57 0.35 0.12 0.05 0.10 0.05 0.02 3.34 1.10 2.24 0.00 3.52 0.96 1.81 0.75 0.00 0.01 84.42

one

PI650167 20.76 2.91 7.44 0.18 5.31 0.05 2.81 0.04 0.97 0.04 0.43 0.58 12.20 0.00 0.21 1.33 0.21 6.19 0.41 3.33 0.51 40.46 1.63 5.78 16.59 13.29 3.16 0.00 5.68 0.77 1.20 0.21 0.56 0.05 0.78 0.02 1.30 0.01 0.77 0.21 0.07 0.04 0.07 0.03 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.09 79.40 represent

to

PI650143 17.70 0.93 5.32 0.09 4.65 0.00 3.96 0.00 1.79 0.00 0.50 0.47 13.42 0.00 0.20 1.28 0.21 6.90 0.45 3.75 0.64 38.98 1.59 6.38 14.68 13.39 2.94 0.00 6.79 0.67 1.06 0.15 0.56 0.00 1.12 0.00 2.01 0.00 1.23 0.00 0.00 0.00 0.00 0.00 0.00 2.17 0.73 1.18 0.26 0.23 0.00 0.23 0.00 0.00 0.00 79.06

averaged

microcarpa

C. CAM42 16.25 0.89 3.43 0.08 3.55 0.00 4.24 0.00 2.68 0.00 0.67 0.71 35.15 0.00 0.45 4.88 0.55 17.96 0.94 9.14 1.21 34.48 1.17 4.71 14.64 11.38 2.58 0.00 6.90 0.91 1.55 0.10 0.44 0.00 1.22 0.00 1.82 0.00 0.86 0.00 0.00 0.00 0.00 0.00 0.00 2.62 0.89 1.47 0.26 0.01 0.00 0.01 0.00 0.00 0.00 95.39 were

PI650139 63.92 0.39 5.31 0.57 46.85 0.51 9.07 0.06 0.63 0.12 0.19 0.23 55.80 0.23 0.80 4.64 0.48 22.82 0.92 24.24 1.68 45.60 0.37 3.20 17.81 11.18 12.70 0.33 8.33 2.21 3.81 0.91 0.52 0.00 0.15 0.00 0.45 0.00 0.28 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.79 1.42 176.85

subsamples

leaves PI650138 79.37 0.66 14.32 0.62 29.43 0.34 23.10 0.31 7.59 0.21 1.61 1.17 81.21 0.56 1.77 10.69 0.49 38.01 1.00 27.45 1.25 32.70 0.68 4.68 7.50 12.09 6.61 1.15 13.04 2.51 4.07 0.83 1.72 0.00 1.34 0.22 1.50 0.02 0.83 1.94 0.24 0.48 0.91 0.23 0.08 1.41 0.29 0.94 0.18 0.00 0.00 0.00 0.00 6.00 0.10 215.76

then

18097E 100.85 0.97 19.53 0.86 39.69 0.49 28.99 0.33 6.80 0.19 1.42 1.59 71.57 0.82 2.03 9.24 0.51 33.99 1.01 22.68 1.29 34.67 0.82 4.75 8.90 12.42 6.73 1.05 14.74 3.05 5.38 1.19 1.82 0.14 1.09 0.08 1.22 0.00 0.77 2.68 0.31 0.81 1.25 0.22 0.09 1.72 0.39 1.12 0.21 0.00 0.00 0.00 0.00 0.00 0.18 226.41

analyzed,

rumelica

C. CAM244 75.60 0.65 10.33 0.44 24.24 0.21 24.72 0.39 11.97 0.24 1.60 0.83 71.18 0.35 0.98 11.11 0.63 39.55 0.94 16.78 0.84 25.23 0.67 3.77 8.40 9.33 2.87 0.20 12.56 2.67 4.54 0.70 1.23 0.04 1.73 0.04 1.23 0.00 0.38 1.23 0.20 0.24 0.49 0.31 0.00 1.42 0.38 0.85 0.19 0.22 0.05 0.08 0.08 0.00 0.12 187.56 a

separately

x6.1.3

R2 41.72 0.48 11.10 0.51 15.26 0.33 10.39 0.22 2.35 0.11 0.56 0.42 38.79 0.15 0.84 5.24 0.42 21.22 0.61 9.78 0.52 29.38 0.28 2.93 6.27 12.51 6.26 1.13 27.92 0.69 0.95 1.10 6.74 0.78 6.95 0.71 6.88 0.34 2.79 1.93 0.15 0.32 1.00 0.46 0.00 0.47 0.16 0.31 0.00 0.44 0.17 0.20 0.07 0.00 0.56 141.21 were

a

2.9.1

leaves

C2x 120.81 1.32 31.05 1.90 55.05 1.36 22.37 0.61 4.62 0.36 1.37 0.80 101.52 0.46 3.17 18.13 1.42 57.35 1.39 18.76 0.84 9.89 0.23 1.95 3.12 3.24 1.35 0.00 46.57 0.40 0.83 1.42 10.58 1.44 13.15 1.38 12.62 0.66 4.10 6.60 0.28 1.00 3.42 1.90 0.00 0.61 0.33 0.28 0.00 1.54 0.71 0.68 0.15 0.00 0.56 288.10

Three

PI650152 41.62 0.45 11.68 0.31 13.49 0.10 10.58 0.13 4.06 0.12 0.46 0.24 46.47 0.00 0.76 9.77 0.42 25.05 0.52 9.22 0.72 22.76 0.37 2.71 5.15 11.03 3.50 0.00 12.81 4.02 5.04 0.60 1.10 0.00 1.12 0.00 0.70 0.00 0.24 1.01 0.18 0.27 0.47 0.09 0.00 0.15 0.04 0.11 0.00 0.00 0.00 0.00 0.00 0.00 0.00 124.80

accessions.

CAM23 90.02 0.54 19.67 0.65 26.85 0.27 27.33 0.41 12.84 0.19 0.92 0.36 71.60 0.69 1.59 16.82 1.09 38.89 0.91 10.97 0.63 39.42 0.61 6.35 9.72 17.30 5.04 0.40 16.24 3.21 4.22 0.86 2.43 0.16 3.44 0.09 1.47 0.00 0.35 2.90 0.31 0.49 1.20 0.73 0.16 0.63 0.18 0.37 0.08 0.39 0.11 0.16 0.13 0.00 0.95 222.15

Camelina

CAM159 38.00 2.78 15.12 0.46 14.09 0.10 3.98 0.05 1.08 0.03 0.19 0.14 17.14 0.06 0.17 1.45 0.18 7.37 0.38 6.88 0.66 11.39 0.35 1.62 3.61 4.64 1.15 0.02 4.68 0.49 0.61 0.26 0.70 0.05 0.72 0.07 1.06 0.05 0.66 0.13 0.04 0.04 0.04 0.01 0.00 0.51 0.15 0.33 0.03 0.73 0.18 0.44 0.11 0.00 0.21 72.80 0.05).

=

analyzed ␣

CAM212 51.36 1.73 16.62 0.65 21.74 0.22 7.87 0.12 1.81 0.06 0.31 0.23 23.32 0.06 0.27 2.58 0.28 11.93 0.37 7.46 0.38 8.55 0.16 1.15 2.46 3.75 1.03 0.00 7.35 0.42 0.52 0.31 1.26 0.10 1.62 0.14 1.99 0.09 0.90 0.18 0.04 0.06 0.07 0.01 0.00 0.40 0.13 0.25 0.03 0.47 0.13 0.23 0.11 0.00 0.42 92.06 insertions.

means,

TF

seventeen

Celine 41.26 2.11 19.92 0.59 11.84 0.14 4.85 0.09 1.37 0.03 0.21 0.11 14.03 0.05 0.18 1.70 0.16 7.36 0.27 3.97 0.34 20.68 0.39 2.80 5.21 10.04 2.17 0.07 6.00 0.52 0.81 0.28 1.07 0.10 1.28 0.09 1.32 0.05 0.48 0.17 0.06 0.03 0.06 0.02 0.00 0.37 0.11 0.24 0.02 0.68 0.18 0.36 0.14 0.00 0.12 83.31 for

accessions

squares

MYB96

sativa classes

(least

Camelina C. Robinson 35.72 0.95 17.31 0.45 11.30 0.10 4.11 0.06 1.04 0.04 0.21 0.16 15.04 0.23 0.42 1.65 0.37 6.78 0.36 4.76 0.48 12.55 0.25 2.08 2.72 5.91 1.55 0.05 6.27 0.84 1.03 0.29 0.98 0.07 1.14 0.08 1.25 0.04 0.56 0.14 0.06 0.03 0.04 0.01 0.00 0.88 0.20 0.61 0.08 1.21 0.23 0.74 0.24 0.00 0.38 72.19

with

major

Abr.

by independent

and

with

length

Alc Alc Alc Alc Alc Alc Alc Alc Alc Alc Alk Alk Alk Alk Alk Alk Alk WE WE WE WE WE FA FA FA FA FA FA FA FA FA Ald Ald Ald Ald AG AG AG MAR MAR MAR Alc Alk WE FA Ald grouped replications,

lines Am

22 24 25 26 27 28 29 30 31 32 34 25 27 29 30 31 32 33 35 38 40 42 44 46 48 22 24 26 28 29 30 31 32 33 34 24 26 28 30 32 19 21 23 19 21 23 Chain C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C ␣ BSS four

) of

) ) ) 34

) ) ) ) (C

32 31 33

)

camelina )

)

) ) (C (C (C 29

24

26 constituents 30

28

) ) 34 mean

35 ) ) )

(C

(C acid ) 31

(C (C

22

)

(C

) )

)

) ) ) ) (C ) )

32 (C 32 ) )

) )

32 31 33 (C (C acid acid

acid

as

27

25

) (C 29 27 (C 24 26 24 30

26 30 25 wax

(C (C (C 29 28 28 30 acid

acid

acid

acid (C

(C

acid

(C (C (C (C

(C (C

(C (C 22 (C

(C (C

(C

(C

acid

Acids (C

leaf

Alcohols transgenic

2 Esters constituent Fatty ) ) ) ) ) ) ) ) ) ) ) )

reported

Two 38 40 42 44 46 48 19 21 23 19 21 23 -Amyrin a Docosanol Tetracosanol Pentacosanol Hexacosanol Heptacosanol Octacosanol Nonacosanol Triacontanol Hentriacontanol Dotriacontanol Tetratriacontanol Alkanes Pentacosane Heptacosane Nonacosane Triacontane Hentriacotane Dotriacontane Tritriacontane Pentatriacontane Wax (C (C (C (C (C (C Free Docosanoic Tetracosanoic Hexacosanoic Octacosanoic Nonacosanoic Triacontanoic Hentriacotanoic Dotriacontanoic Tritriacontanoic Tetratriacontanoic Aldehydes Tetracosanal Hexacosanal Octacosanal Triacontanal Dotriacontanal Alkylguaiacols (C (C (C Methylalkylresorcinols (C (C (C ␣ ß-Sitosterol Totals Wax Primary Cuticular Table were

134 P. Tomasi et al. / Industrial Crops and Products 98 (2017) 130–138

−2

study, we reveal an extremely wide range of total wax amounts and 40.46 ␮g dm , respectively (Table 2). The transgenic acces-

across these 17 accessions of more than 3-fold variation, indicating sions did not show a major impact of the MYB96 transgene on ester

a likelihood that different Camelina species and genotypes vary sig- profiles, except that the R2x6.1.3 line had the highest, and slightly

nificantly in the genetic regulation of their wax pathways. Recent elevated amounts of the longest C46 and C48 homologues relative

studies have revealed the feasibility for intercrossing C. sativa with to the line with the next most abundant C46 and C48 homologues of

its related Camelina species (Séguin-Swartz et al., 2013), indicating CAM23 (Table 2). Interestingly, the C. rumelica line PI650139 and all

that it may be possible to introgress more active cuticle genes from four of the C. microcarpa accessions had the C42 carbon homologue

one species to another, and/or create new combinations of cuticle- as their most abundant ester, rather than the C44 esters that domi-

associated genes that can increase or modify wax composition in nated the other 13 Camelina accessions. Whether an inter-specific

ways to improve plant stress tolerance. breeding approach might be used to modify ester chain lengths, and

Across all Camelina accessions, the primary alcohols and alkanes perhaps ester-associated cuticle permeability properties, would be

−2

␮

were the predominant wax classes having 53.57 and 42.07 g dm an intriguing subject for future research.

of average amount, respectively, trailed by wax esters, fatty The amounts of VLCFAs varied by more than 5-fold across

␣

acids, aldehydes, alkylguaiacols, methylalkylresorcinols, -amyrin all non-transgenic Camelina (Table 2), with the species C. hisp-

␤

and -sitostrol (Table 2, Fig. 1). Camelina accessions showed a ida exhibiting very low relative amounts. Especially notable was

wide range of primary fatty alcohols ranging from the lowest that VLCFAs on the two transgenic lines were increased nearly 15-

−2

␮

value of 16.25 g dm for C. microcarpa CAM42 to the high- fold above the lowest acid line (Table 2), which was accounted for

−2

␮

est 100.85 g dm for the non-transgenic C. rumelica 18097E, to mainly by the longer, even chained C28, C30 and C32 homologues.

−2

␮

120.81 g dm for the transgenic C2x2.9.1. Among the primary These results are comparable with the previous report by Lee et al.

alcohols, the dominant constituents were the C24, C26 and C28 (2014), except under our environmental conditions the transgenic

homologues that together accounted for an average 86% of total lines showed a much higher load of VLCFAs. These acid levels are in

primary alcohols across all lines. The CER4 gene is known to encode part an indicator of thioesterase activity associated with removal of

an alcohol-forming fatty acyl-CoA reductase (FAR) responsible for the Co-enzyme-A from the acyl chain during elongation reactions,

the synthesis of primary alcohols in the epidermis of Arabidop- as well as the relative competition for acyl-CoA chains from other

sis aerial tissues and roots (Rowland et al., 2006) and Wang et al. branches of the wax pathway. The very high variation among these

(2015) reported that overexpression of the TaFAR5 gene in accessions, as well as the high responsiveness to ectopic expression

tomato leaves resulted in greater accumulation of C26, C28 and C30 of MYB96, may indicate a strong potential for modifying VLCFAs

alcohols. Whether variation in the expression of CER4 homologues using a genetic approach.

in Camelina explains the large observed variation in primary alco- The majority of Camelina accessions in our panel had low or

hols across these Camelina species is uncertain. Interestingly, the undetectable amounts of aldehydes that accounted for 1% of the

−2

C2x2.9.1C. sativa MYB96 overexpressor produced very high levels total waxes ranging from 0.0–6.60 ␮g dm (Table 2). Several other

of primary alcohols (similar as in Lee et al., 2014), a result indi- compounds were marginally detected in the leaf surface extracts,

cating that MYB96 may activate genes involved in the synthesis of many which have not been reported previously for Camelina. While

␣

primary alcohols. -amyrin has been detected in other Brassicaceae (Xu et al., 2014),

Alkanes were the second most abundant wax class produced in it was only detected in three Camelina lines, specifically PI650138,

all Camelina accessions (30.5% of total waxes, excluding the trans- PI650133, and PI650139, but not in others. The phytosterol ß-

genic lines) with C29, C31 and C33 accounting for 93% of total alkanes sitosterol was reported in Camelina seeds (Mansour et al., 2014),

(Table 2). As with alcohols, the amount of alkanes on these acces- but not previously reported in leaf waxes. The accession C. rumel-

−2

␮

sions varied greatly, from as low as 9.07 g dm for C. microcarpa ica PI650139 exhibited the highest amounts of ß-sitosterol at

−2 −2 −2

␮

PI633186 to as high as 81.21 g dm for C. rumelica PI650138. The 1.42 ␮g dm , followed by CAM23 that produced 0.95 ␮g dm . A

−2

␮

transgenic line C2x2.9.1 produced 101.52 g dm of alkanes. The homologous series of alkylguaiacols (AGs) and methylalkylresor-

relative proportions of constituents within the alkane class were cinols (MARs) were detected at low levels in the leaf surface wax

very similar across all accessions, except for the C. rumelica line of saltcedar (Tamarix canariensis [Willd.]) (Basas-Jaumandreu et al.,

PI650139, which showed a shift in the major homologue. In all 2014), Secale cereale and Brachypodium distachyon (Luna, 2014), but

other Camelina accessions, the most abundant alkane was the C31 not previously for Camelina or any other member of the Brassi-

alkane. However, in the accession PI650139, the C33 homologue caceae. In Camelina, heptadecylguaiacol (C17) was detected on some

dominated (Table 2). These results indicate a significant shift in the Camelina accessions, but only in trace amounts (not shown). Many

elongation of wax precursors, such that PI650139 is able to gener- Camelina lines produced significant amounts of C19, C 21and C23 AGs,

−2

ate longer acyl chains feeding the alkane pathway of wax synthesis. with the accession PI650133 producing the most at 4.77 ␮g dm

No such genotypic variation occurs for the primary alcohol pathway (Supplemental Fig. 1), while PI633186 showed the highest amounts

−2

among these Camelina accessions since no such shift in chain length of MARs at 3.52 ␮g dm , with other accessions exhibiting less than

distribution for primary alcohols is observed. Alkanes are consid- half that amount (Table 2, Supplemental Fig. 2).

ered highly hydrophobic and effective in creating cuticles with very

low permeability to water, especially those having longer chain

lengths (Kosma and Jenks, 2007). Increasing alkanes was observed 3.2. Cutin monomers

in many species including alfalfa (Ni et al., 2012), Populus euphrat-

ica (Xu et al., 2016), sesame (Kim et al., 2007a) and (Kim Cuticular waxes and cutin monomers are the building blocks

et al., 2007b) when plants were grown under drought conditions. of the plant cuticle, with less being known about the synthe-

It would be interesting to explore the C. rumelica line PI650139 as sis of cutin, nor the role of cutin in plant stress tolerance. Cutin

a parent in a breeding effort to introgress longer chain alkanes into monomers include aliphatic C16-18, ␻-hydroxy and polyhydroxy

cultivated Camelina as a means to reduce non-stomatal water loss. fatty acids, and glycerol (Rautengarten et al., 2012). To date, no

Wax esters were the third most abundant class of waxes report of leaf cutin monomer constituents of Camelina has been

accounting for an average 20% of total waxes (excluding transgenic published. Cutin constituents were extracted from Camelina using a

lines). C. hispida accession PI650133 did not produce detectable chloroform/methanol-based method (Parsons et al., 2013), depoly-

amounts of esters using our protocol, while C. rumelica PI650139 merized, and then separated and analyzed using GC–MS and

and C. microcarpa PI650167 had the highest ester deposits of 45.60 quantified by characteristic quadrupole electron impact mass spec-

P. Tomasi et al. / Industrial Crops and Products 98 (2017) 130–138 135 , 2 − dm

g LSD0.05 4.71 4.67 0.24 17.49 5.85 1.29 1.48 12.36 0.40 0.41 0.34 5.00 2.06 0.32 0.57 1.50 2.27 10.19 3.81 3.24 0.42 0.44 1.24 5.53 0.62 3.80 3.80 39.29 36.85 2.65 1.45 0.66 3.35 3.66 1.45 8.22 0.48 3.71 1.01 5.04 5.48 2.33 1.03 3.06 21.95 16.82 2.18 10.06 2.80 78.42 ␮

in

hispida

C. PI650133 22.89 22.52 0.37 44.54 25.45 0.00 0.76 15.43 0.66 1.41 1.19 33.80 13.74 1.10 1.79 3.68 13.49 23.24 8.61 6.19 0.00 0.84 0.89 6.44 0.28 21.76 21.76 72.13 64.06 1.80 5.60 2.33 0.39 2.99 0.39 17.61 0.27 4.79 0.21 12.34 17.00 1.05 1.60 14.35 79.26 72.45 2.95 0.14 3.73 332.61 Values,

PI633186 20.81 20.50 0.30 47.13 19.77 2.37 1.66 20.03 1.17 1.06 0.72 37.76 14.95 1.07 0.89 12.22 8.63 70.54 24.08 31.72 0.68 1.83 8.29 3.52 0.44 21.85 21.85 211.47 187.95 10.09 9.12 3.05 0.54 2.90 0.54 11.47 2.64 0.82 0.84 7.18 9.31 4.98 0.25 4.08 116.55 71.96 15.46 14.72 14.42 547.43 replicate.

one

PI650167 19.19 18.63 0.56 39.88 14.78 0.00 1.09 20.61 1.23 1.69 0.47 26.02 10.70 2.76 1.23 7.55 3.78 73.24 22.54 23.79 2.96 2.11 3.36 16.62 1.87 15.49 15.49 203.08 173.83 15.66 10.28 2.02 4.48 2.06 4.48 9.19 1.85 3.19 0.22 3.92 13.23 5.82 1.23 6.19 46.75 23.83 6.51 7.91 8.50 450.54

represent

to

PI650143 17.96 16.86 1.10 57.73 23.97 0.00 1.87 29.48 0.86 0.58 0.77 23.82 12.97 1.57 0.00 5.91 3.37 81.58 26.36 22.09 1.40 1.70 3.28 25.40 1.36 21.31 21.31 224.29 203.98 10.54 6.55 1.00 5.73 3.44 5.73 14.98 2.22 5.11 0.85 6.80 19.39 9.26 1.22 8.93 43.00 25.26 4.68 6.31 6.75 509.80

microcarpa

C. CAM42 13.61 12.82 0.80 54.51 24.42 0.00 1.43 26.48 1.25 0.79 0.27 21.66 12.29 1.21 0.28 5.96 1.93 80.14 30.24 22.43 0.98 1.95 4.12 19.39 1.03 18.34 18.34 240.05 220.28 9.17 9.07 1.53 6.73 2.95 3.78 10.94 2.00 5.63 0.45 2.86 12.98 6.86 1.26 4.87 51.82 30.23 7.39 6.67 7.52 510.77 averaged

were

PI650139 11.40 10.65 0.74 110.80 46.82 0.00 7.38 52.96 0.46 0.82 2.35 13.99 6.91 0.58 0.65 3.70 2.15 48.79 16.33 9.39 0.11 0.44 3.31 18.36 0.85 16.28 16.28 111.88 99.14 6.60 5.41 0.72 1.34 0.00 1.34 14.27 1.67 3.13 0.71 8.76 4.66 1.75 0.00 2.91 31.02 7.35 1.59 17.66 4.43 364.41

subsamples

PI650138 15.44 14.89 0.56 59.42 28.01 3.15 3.49 21.14 0.80 0.98 1.86 24.95 10.77 1.42 0.00 5.49 7.27 67.79 14.82 12.29 0.22 0.70 4.02 33.27 2.47 23.51 23.51 139.76 128.83 6.60 3.85 0.49 6.18 2.23 3.95 20.11 1.34 3.38 1.81 13.59 23.83 8.98 1.25 13.60 25.10 17.55 0.81 5.79 0.96 406.09

leaves

18097E 18.94 18.52 0.42 46.33 18.91 2.54 1.78 20.37 0.82 1.39 0.98 30.63 12.61 1.70 0.76 6.00 9.56 64.91 18.84 13.52 0.26 1.04 5.18 23.49 2.58 24.41 24.41 170.89 151.43 12.02 6.24 1.21 2.77 1.44 1.33 30.45 2.16 10.89 2.88 14.52 14.03 3.54 1.02 9.47 66.71 40.78 1.93 20.26 3.75 470.06

then

rumelica

C. CAM244 12.58 12.09 0.49 40.43 19.10 3.17 3.46 12.07 0.61 1.73 1.04 15.81 5.51 0.64 0.57 3.87 5.22 49.80 7.12 9.90 0.18 0.63 5.78 24.23 1.97 10.70 10.70 49.08 37.09 6.33 4.91 0.76 5.08 4.10 0.97 12.92 0.88 3.07 1.29 7.68 17.13 6.90 1.28 8.96 49.77 12.40 1.20 34.67 1.50 263.29

analyzed,

R2x6.1.3 9.60 9.26 0.34 56.36 26.22 2.95 1.89 22.38 0.99 0.99 0.53 13.33 5.64 0.76 0.26 3.40 3.28 32.01 10.34 9.02 0.36 0.71 1.29 9.82 0.46 8.82 8.82 54.45 50.39 2.05 1.56 0.46 3.45 1.66 1.79 5.15 0.09 2.93 0.25 1.89 7.89 2.16 0.42 5.31 14.72 1.09 2.35 9.50 1.78 205.78

separately

C2x2.9.1 18.26 17.78 0.49 70.35 24.87 4.36 2.52 35.51 0.74 0.96 0.62 20.53 6.28 0.95 0.68 6.74 5.89 44.02 8.80 15.45 0.51 1.63 2.52 14.20 0.92 7.79 7.79 87.64 78.55 5.71 2.59 0.80 10.28 5.36 4.92 18.61 0.20 9.29 1.00 8.12 28.14 9.02 2.18 16.94 49.33 5.55 3.54 37.28 2.96 354.93

were

leaves PI650152 12.44 11.95 0.49 57.78 31.34 3.13 4.65 16.36 0.75 0.67 0.97 13.41 7.03 1.67 0.12 2.30 2.29 48.39 15.11 10.08 0.72 0.89 2.69 17.09 1.81 15.90 15.90 150.54 129.43 13.05 6.11 0.99 1.64 7.10 1.64 17.44 1.34 6.19 1.84 8.08 21.09 7.89 1.71 11.48 54.65 29.15 2.99 20.79 1.72 393.26

Three

CAM23 10.26 9.95 0.31 48.84 19.23 2.97 4.11 19.75 0.93 0.93 0.91 20.43 9.10 0.71 0.82 4.62 5.18 44.99 11.04 10.76 0.50 0.47 4.88 15.67 1.67 14.78 14.78 88.61 71.58 9.67 5.58 1.78 7.09 7.09 0.00 13.21 1.13 1.92 1.19 8.98 22.20 9.90 1.85 10.45 42.26 20.64 4.36 13.11 4.16 312.65

CAM159 15.72 15.47 0.24 21.77 7.74 1.28 1.51 8.94 0.52 1.26 0.54 29.38 10.75 1.04 0.68 7.89 9.02 59.74 17.75 16.66 0.26 1.07 3.03 19.30 1.68 12.92 12.92 74.20 66.21 4.97 2.66 0.37 8.43 3.15 5.28 6.11 1.43 1.42 0.16 3.11 23.11 9.04 1.80 12.27 38.40 7.39 3.25 26.34 1.42 289.76 accessions.

CAM212 13.16 12.78 0.39 24.63 10.24 1.46 0.64 10.01 0.35 1.43 0.50 40.40 17.13 1.60 0.98 11.11 9.58 57.47 16.90 19.15 0.38 0.84 2.94 16.57 0.70 11.40 11.40 106.97 95.72 7.70 3.05 0.50 3.77 0.00 3.77 3.32 0.46 1.17 0.00 1.70 20.29 9.27 1.35 9.67 34.64 6.94 1.85 22.93 2.94 316.04 Camelina

Celine 10.90 10.56 0.35 23.46 8.78 1.90 0.96 9.99 0.63 0.84 0.37 17.10 6.29 0.52 0.56 4.31 5.42 37.10 10.84 12.89 0.28 0.76 2.40 9.57 0.37 8.95 8.95 59.87 52.45 3.67 3.26 0.49 4.77 1.12 3.66 5.23 0.48 2.02 0.16 2.57 18.65 6.61 1.22 10.82 25.72 4.04 3.01 17.66 1.02 211.75 0.05).

=

analyzed

␣

sativa

C. Robinson 17.61 17.42 0.20 24.16 9.28 1.31 0.39 11.29 0.50 1.02 0.36 26.94 12.11 1.17 0.65 7.03 5.99 65.71 23.85 20.60 0.61 1.22 2.22 16.09 1.13 10.73 10.73 81.69 72.71 6.28 2.20 0.50 7.63 3.10 4.53 4.55 1.11 1.34 0.08 2.03 21.68 8.77 1.50 11.41 38.41 5.63 2.36 28.20 2.22 299.12

means, e t

seventeen

ME diC

diC diC

a

a a

OH OH OH a

for diC

a

␻ ␻ ␻

Abr. OH OH OH OH OH OH OH

ME ME diC ␻ ␻ ␻ ␻ diOH triOH triOH Ep Ep Diol Diol ME ME ME diC diC diC ␻ OH diOH diOH diOH diOH diOH triOH triOH Ep ME & squares

18:0 22:0 16:0 18:0 18:1 18:2&18:3 20:0 22:0 24:1 16:0 16:0 18:0 18:1 18:2&18:3 16:0 18:1 18:1 18:2 18:2 18:0 18:0 16:0 16:0 16:0 18:0 18:1 18:0 18:0 16:0 18:0 18:1 18:2 18:0 18:1 18:2 classes

Chain C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C pCA FA CA SA (least

(3))

major

with

ester 18

by

(2))

(3)) (C erythro threo

)) ester ) )

ester erythro

18 &

(1))

) TMS 1

) 18 (2)) ester

) ) (C

18 18 ester )

18 18 (C

18

18 TMS (C (C (2)) ) ) (1)) 16 16 TMS

(C (C 16 &

(C18( (C (C

TMS (C (C acid

TMS 16 18 18

(C 18

) )

grouped

(2)) (2)) acid acid

(C (C (C

(2)) (C ester acid acid

18 18 acid acid acid replications,

acid acid ) (1)) (1))

18 18

acid

(C (C 18 ester.

Me (C (C

acid acid acid

16

acid acids

18 18 ) ) (1))

(C

(C

)

(C (C four

acid acid 16 16

18 acid acid

18

(C (C ) (1))

(C of )

acids

acid

enoic

(C

acid acid

)

) − 16 diTMSi )

18 18

acids ) (1)) acid acid

acids constituents 18 (C acids

acid

22

(C (C

tri

20

acid or

22 (C

(C

24 &

(C

mean en-1,18-dioic

acids (C

(C − di

as

monobasic dibasic epoxymonobasic

tri

TMSi

&

a

monobasic acids

monobasic dibasic

di class monomer

acid

acid

18-Dihydroxy-octadecanoic 18-Dihydroxy-octadecenoic

acids hexadecanoate octadecanoate octadecenoate octadece- eicosanoate docosanoate nervonate 16-Dihydroxy-hexadecanoic 16-Dihydroxy-hexadecanoic

acid acid

16-Trihydroxyoctadecenoic 18-Trihydroxyoctadecenoic 18-Trihydroxyoctadecenoic 18-Trihydroxyoctadecadienoic Cutin 3

reported

cutin

Denote 10-Dihydroxyoctadecane-1,18-dioic 10-Dihydroxyoctadecane-1,18-dioic 10, 10, 10, 10, 10-Epoxy-18-hydroxyoctadecanoic 10-Epoxy-18-hydroxyoctadecenoic 10-Epoxy-18-hydroxyoctadecadienoic

a 1,18-Octadecanediol 1,22-Docosanediol Monobasic Methyl Methyl Methyl Methyl Methyl Methyl Methyl Dibasic Hexadecane-1,16-dioic Hexadecane-1,16-dioic Octadecan-1,18-dioic Octadecene-1,18-dioic Octadeca- Monohydroxy 16-Hydroxy-hexadecanoic 18-Hydroxy-octadecenoic 18-Hydroxy-octadecenoic 18-Hydroxy-octadecadienoic 18-Hydroxy-octadecadienoic 8/9/10-Hydroxy-octadecanoic 8/9/10-Hydroxy-octadecanoic Monohydroxy 7/8-Hydroxy-hexadecan-1,16-dioic Dihydroxy 8/9/10, 8/9/10, 9/10/11, 9/10/11, Dihydroxy 9, 9, Trihydroxy 9, 9, 9, 9, Monohydroxy 9, 9, 9, Phenolics Coumaric Ferulic Caffeic Sinapinic Total Monomer Alkanediols Leaf Table were

136 P. Tomasi et al. / Industrial Crops and Products 98 (2017) 130–138

Fig. 1. Total ion chromatogram of retention times 10.0–35.0 min for Camelina rumelica cuticular leaf wax. Cn denotes chain length with abbreviations as listed:

AG = alkylguaiacol, Alc = fatty alcohol, Ald = aldehyde, Alk = alkane, BSS = ß-Sitosterol, FA = free fatty acid and WE = wax ester. Of the seventeen analyzed accessions, ␣-amyrin

was detected in three and methylalkylresorcinols in thirteen (these compounds are not depicted as they were not present in this accession).

Fig. 2. Total ion chromatogram for Camelina rumelica leaf cutin. (A) Retention times 8.4–12.6 min, constituents numbered within the peaks at baseline are minor coelution

compounds extracted with AMDIS. 1 = SA. *Asterisks denote a TMSi or diTMSi ester. (B) Retention times 12.6–16.8 min, constituents numbered within the peaks at baseline

are minor coelution compounds extracted with AMDIS. 2 = C18:2 ␻OH, 3 = C18:0 diC, 4 = C22:0 Diol, 5 = C20:0 2OH, 6 = C18:1 ␻OH* and 7 = C18:0 triOH. *Asterisks denote a TMSi or

diTMSi ester.

−2

tra (Jenkin and Molina 2015). Since overexpression of MYB96 did amount of cutin monomers at 547.43 ␮g dm , and this was mainly

not have a notable effect on accumulation of cutin monomers in attributable to elevated phenolics and dihydroxy monobasic acids.

these accessions (Table 3), it appears that MYB96 has its primary The lowest of the seventeen accessions was C. sativa ‘Celine’

−2

effect on wax and not cutin pathways. Fig. 2, is a representative that produced 211.75 ␮g dm of total cutin monomers. Within

(C. rumelica) GC–MS total ion chromatogram of depolymerized each species, cutin constituents varied greatly as well. For exam-

Camelina leaf cutin. Camelina species showed significant variation ple, total cutin monomer amount on C. sativa accessions ranged

−2 −2

in cutin monomer content, wherein total cutin monomers in C. from 211.75 ␮g dm to 393.29 ␮g dm . The main Camelina cutin

−2

␮

microcarpa were the highest, averaging 504.64 g dm , followed monomers identified included the monohydroxy monobasic acids,

−2 −2

␮ ␮

by C. rumelica at 375.96 g dm , C. hispida at 332.61 g dm , phenolics, monobasic acids, monohydroxy epoxymonobasic acids

−2

␮

and then C. sativa at 297.91 g dm (Table 3). For individual and dibasic acids, and low abundance constituents included alka-

accessions, C. microcarpa PI633186 displayed the highest total nediols, monohydroxy dibasic acids, dihydroxy dibasic acids, and

P. Tomasi et al. / Industrial Crops and Products 98 (2017) 130–138 137

trihydroxy monobasic acids (Supplemental Figs. 3–6). The dihy- could involve a combination of different traits including those that

droxy monobasic acids group contains C16:0 diOH, C18:0 diOH and are morphological, physiological, and/or biochemical. Cuticle com-

C18:1 diOH acids, which accounted for 34% of total cutin monomers position is considered part of a drought avoidance mechanism that

(Table 3). Unlike Arabidopsis leaves, where the major monomer is delays the onset of dehydration as water becomes increasingly

octadeca-cis-6, cis-9-diene-1, C18:2 diOH acid (Bonaventure et al., limiting. The current study characterized leaf cuticular wax and

2004), the C16 carbon monomer, 16-dihydroxy-hexadecanoic acid cutin monomers of 17 Camelina accessions representing four dif-

(C16:0 diOH acid) was most abundant for Camelina (Table 3). The ferent species. Wide variations in total wax and cutin amount and

C16:0 diOH acid accounted for 30% of the total Camelina cutin chemical constituent profile within and among Camelina species

monomers. Camelina accessions had large variation in the amount were observed. Major wax and cutin constituents, such as primary

−2

of C16:0 diOH acids, ranging from 37.09 ␮g dm in CAM244 to alcohols, alkanes, and dihydroxy acids identify potential targets

−2

220.28 ␮g dm in CAM42 (40% of total cutin in CAM42). The C16:0 for breeding efforts to improve drought tolerance in Camelina,

diOH acid was the major cutin monomer in canola (Bonaventure potentially by the creation of interspecific hybrids or transgenic

et al., 2004) and cucumber (Gérard et al., 1994) leaves. Xu et al. approaches. Future studies to elucidate Camelina cuticle metabolic

(2016) demonstrated that the major cutin monomers in leaves of pathways, the ecological function of specific cuticle lipid profiles,

Populus euphratica were 10,16-diOH C16:0 acids, that were 1.5 fold and the genetic networks regulating their expression, are needed to

more abundant on leaves growing in a more arid than less arid loca- lay the groundwork for future application of new genomics-based

tion. These findings suggest that Camelina’s most abundant C16:0 strategies to improve environmental stress tolerance in Camelina.

diOH monomer could contribute to Camelina’s reported stress tol-

erance, and its levels among diverse genotypes could serve as a Acknowledgements

biomarker for drought stress tolerance in Camelina. Future cutin

characterization studies are warranted.

This work was supported by the Department of

−2

Phenolic acids ranged from 14.72 to 116.55 ␮g dm and

Agriculture National Institute of Food and Agriculture

accounted for 13% of total cutin monomers (Table 3). Coumaric

Research and Development Initiative Award (grant no. 11011027).

and caffeic acids contributed 47% and 36% of total phenolics.

Mention of trade names or commercial products in this article is

There tended to be an inverse relationship between coumaric and

solely for the purpose of providing specific information and does

caffeic acids between C. sativa and C. microphylla (r = −0.47107;

not imply recommendation or endorsement by the U. S. Depart-

Prob > 0.0563), wherein an accession that exhibited high caf-

ment of Agriculture. USDA is an equal opportunity provider and

feic amount would exhibit low coumaric acid amount, and vice employer.

versa (Table 3). Whether this is influenced by shunting within

related metabolic pathways is unknown. Ferulic and sinapinic acids

Appendix A. Supplementary data

accounted together for 16.6% of the total phenolics, wherein C.

microcarpa PI633186 exhibited the highest for both (at 15.46 and

−2 Supplementary data associated with this article can be found, in

14.42 ␮g dm , respectively), and also had highest total phenolic

−2 the online version, at http://dx.doi.org/10.1016/j.indcrop.2017.01.

acid class production (116.55 ␮g dm ) (Table 3).

030.

The monobasic acids were represented by a series including

C16:0 to C24:1 acids (as methylated depolymerization products)

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