Natural variation of wheat cuticular waxes in relation to drought tolerance
Aims. The aim of our research is to investigate the role of wheat leaf cuticle in drought and heat protection by characterizing natural variability of cuticle components and revealing genetic and biochemical background of this variability in Australian wheat varieties. Methods. Cultivars with diff...
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Bi, H. Kovalchuk, N.V. Dias, D. Roessner, U. Langridge, P. Lopato, S. Borisjuk, N.V. 2021-02-15T13:50:23Z 2021-02-15T13:50:23Z 2015 Natural variation of wheat cuticular waxes in relation to drought tolerance / H. Bi, N.V. Kovalchuk, D. Dias, U. Roessner, P. Langridge, S. Lopato, N.V. Borisjuk // Фактори експериментальної еволюції організмів: Зб. наук. пр. — 2015. — Т. 16. — С. 174-178. — Бібліогр.: 9 назв. — англ. 2219-3782 https://nasplib.isofts.kiev.ua/handle/123456789/177382 Aims. The aim of our research is to investigate the role of wheat leaf cuticle in drought and heat protection by characterizing natural variability of cuticle components and revealing genetic and biochemical background of this variability in Australian wheat varieties. Methods. Cultivars with different levels of drought tolerance were analyzed for cuticle wax composition by gas chromatography (GC–MS) and for cuticle structure properties by scanning electron microscopy (SEM). Results. Metabolomics analyses demonstrated signifi cant quantitative differences between drought-sensitive and drought-tolerant cultivars in several types of wax components. These data are complemented by differences in cuticle structure revealed by SEM. Ten wheat genes potentially involved in regulation of cuticle biosynthesis have been cloned and analysed for expression profi les in wheat by RT-qPCT. Conclusions. Acquired data are currently being used for identifi cation of enzymes and genes responsible for drought-related cuticular components. Keywords: wheat, drought tolerance, cuticle waxes, gene expression. The reported study has been partially supported by an ACPFG — DuPont/Pioneer collaborative grant. The China Scholarship Council is acknowledged for providing a fellowship to PhD student Huihui Bi. The authors thank Yuan Li for conducting RT-qPCR analysis and Gwen Mayo for advises and help with SEM analyses. en Інститут молекулярної біології і генетики НАН України Фактори експериментальної еволюції організмів Прикладна генетика і селекція Natural variation of wheat cuticular waxes in relation to drought tolerance Article published earlier |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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DSpace DC |
| title |
Natural variation of wheat cuticular waxes in relation to drought tolerance |
| spellingShingle |
Natural variation of wheat cuticular waxes in relation to drought tolerance Bi, H. Kovalchuk, N.V. Dias, D. Roessner, U. Langridge, P. Lopato, S. Borisjuk, N.V. Прикладна генетика і селекція |
| title_short |
Natural variation of wheat cuticular waxes in relation to drought tolerance |
| title_full |
Natural variation of wheat cuticular waxes in relation to drought tolerance |
| title_fullStr |
Natural variation of wheat cuticular waxes in relation to drought tolerance |
| title_full_unstemmed |
Natural variation of wheat cuticular waxes in relation to drought tolerance |
| title_sort |
natural variation of wheat cuticular waxes in relation to drought tolerance |
| author |
Bi, H. Kovalchuk, N.V. Dias, D. Roessner, U. Langridge, P. Lopato, S. Borisjuk, N.V. |
| author_facet |
Bi, H. Kovalchuk, N.V. Dias, D. Roessner, U. Langridge, P. Lopato, S. Borisjuk, N.V. |
| topic |
Прикладна генетика і селекція |
| topic_facet |
Прикладна генетика і селекція |
| publishDate |
2015 |
| language |
English |
| container_title |
Фактори експериментальної еволюції організмів |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| format |
Article |
| description |
Aims. The aim of our research is to investigate the role of wheat leaf cuticle in drought and heat protection by characterizing
natural variability of cuticle components and revealing genetic and biochemical background of this variability in Australian
wheat varieties. Methods. Cultivars with different levels of drought tolerance were analyzed for cuticle wax composition
by gas chromatography (GC–MS) and for cuticle structure properties by scanning electron microscopy (SEM). Results.
Metabolomics analyses demonstrated signifi cant quantitative differences between drought-sensitive and drought-tolerant
cultivars in several types of wax components. These data are complemented by differences in cuticle structure revealed
by SEM. Ten wheat genes potentially involved in regulation of cuticle biosynthesis have been cloned and analysed for
expression profi les in wheat by RT-qPCT. Conclusions. Acquired data are currently being used for identifi cation of
enzymes and genes responsible for drought-related cuticular components.
Keywords: wheat, drought tolerance, cuticle waxes, gene expression.
|
| issn |
2219-3782 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/177382 |
| citation_txt |
Natural variation of wheat cuticular waxes in relation to drought tolerance / H. Bi, N.V. Kovalchuk, D. Dias, U. Roessner, P. Langridge, S. Lopato, N.V. Borisjuk // Фактори експериментальної еволюції організмів: Зб. наук. пр. — 2015. — Т. 16. — С. 174-178. — Бібліогр.: 9 назв. — англ. |
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2025-11-24T02:53:40Z |
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2025-11-24T02:53:40Z |
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| fulltext |
174 ISSN 2219-3782. Ôàêòîðè åêñïåðèìåíòàëüíî¿ åâîëþö³¿ îðãàí³çì³â. 2015. Òîì 16
Bread wheat, Triticum aestivum, represents
about 30 % of the world’s cereal cultivation area
and provides 20 % of the calories for the human
population. Wheat is cultivated over 220 million
ha of soil worldwide, which is often under the
infl uence of abiotic stresses such as limited water
supply, high salinity and heat that signifi cantly
impair crop’s yield. It is the growing consensus
among the scientifi c community, that the need to
compensate the potential yield losses associated
with these challenges could be achieved through
selection and adaptation of cultivars with improved
genetic potential [1]. Understanding how plant
copes with the environmental challenges has a vital
signifi cance for improvement of crop tolerance
and yield. Plant cuticle, a continuous protective
sheet that covers aerial surfaces of plant organs
has evolved as an exterior extension of epidermal
cell walls. The biochemical composition of cuticle
is not only species/cultivar specifi c, but differs
also between organs of the same species and is
modulated by environmental conditions, defi ning
the plant tolerance level to drought and excessive
UV radiation. While there is a range of scientifi c
indications about a connection of plant cuticle with
plant stress tolerance, these data, obtained mostly on
model plant Arabidopsis [2]. As a consequence, little
is known about the biochemical details of cuticle
composition and regulation in wheat and its relation
to drought. The new knowledge on the biodiversity
in cuticle composition of wheat varieties, the cuticle
composition changes during the drought stress and
particularly, the differences between tolerant and
intolerant varieties, would be a great support for the
wheat breeding programmes.
Materials and methods
Plant material. Wheat plants for all
experiments were grown from seeds available at
the ACPFG collection in greenhouse under well-
watered and drought conditions in large containers.
BI H.1, KOVALCHUK N. V.1, DIAS D.2, ROESSNER U.2, LANGRIDGE P.1, LOPATO S.1,
BORISJUK N. V.1
1 Australian Centre for Plant Functional Genomics, University of Adelaide,
Australia, Adelaide, Hartley Grove, Urrbrae SA 5064, e-mail: mykola.borysyuk@acpfg.com.au
2 School of Botany, University of Melbourne,
Australia, Melbourne, e-mail: u.roessner@unimelb.edu.au
NATURAL VARIATION OF WHEAT CUTICULAR WAXES IN RELATION
TO DROUGHT TOLERANCE
© BI H., KOVALCHUK N. V., DIAS D., ROESSNER U., LANGRIDGE P., LOPATO S., BORISJUK N. V.
Containers were equipped with an automatic
watering system and four soil water tensiometers
were installed at 0.1 and 0.3 m soil depths, and
connected to a data logger for continuous monitoring
of soil water tension. Cuticular wax metabolomics.
For wax component analysis, 6.5 cm long fl ag leave
blades from the base at 24 days after anthesis were
used. Leaf samples were weighted before placing
into liquid nitrogen, after which samples were stored
at –80 freezer until wax extraction with chloroform
or hexane. Following extraction, waxes were dried
using stream of nitrogen and redissolved in a small
amount of n-hexane prior to analysis using gas
chromatography-mass spectrometry. Mass spectra
of eluted waxes were identifi ed using commercial
mass spectra library NIST08 (http:/www.nist.
gov) and the in-house Metabolomics Australia
(Schholl of Botany, Melbourne) mass spectral
library. Scanning electron microscopy (SEM).
Flag leaf blades collected at 10 days after anthesis
were examined using Philips XL30 Field Emission
Scanning Electron Microscope, equipped with a
Gatan CT1500 HF Cryotransfer Stage (Adelaide
Microscopy). Samples attached to the holder were
frozen in nitrogen slush, and transferred under
vacuum to the preparation chamber where it was
coated with platinum under low temperature of
–110 °C. It was then loaded onto the microscope
chamber (held at a temperature lower than –150 °C)
and examined. Gene expression analysis. Wheat
genes coding for 10 cuticle biosynthesis related
transcription factors were amplifi ed by PCR from
cDNA of wheat cv RAC875 using homology
cloning based on previously characterized genes
in Arabidopsis, Medicago and maize. Following
cloning and sequencing the confi rmed wheat gene
sequences were used to design specifi c primers for
gene expression analysis by quantitative RT-PCR
using a series of cDNA from different tissues of T.
aestivum cv. Chinese Spring available at the ACPFG
qPCR facility.
ISSN 2219-3782. Ôàêòîðè åêñïåðèìåíòàëüíî¿ åâîëþö³¿ îðãàí³çì³â. 2015. Òîì 16 175
Natural variation of wheat cuticular waxes in relation to drought tolerance
Results and discussion
Due to recent technological advances in plant
genomics and metabolomics, cuticle emerged as
a crucial barrier that control water lose and help
plant to survive under drought and high UV radia-
tion conditions [3]. Accumulation of epicuticular
wax on plant surfaces often results in a bluish-
white coloration termed glaucousness (fi g. 1 A),
which is the visible form of densely distributed
epicuticular wax crystals. Glaucousness increases
light refl ectance and reduces leaf temperatures and
transpiration, thereby enhancing leaf survival under
water stress and improving water use effi ciency
[4]. As a classical genetic marker and agricultural
trait, glaucousness has been intensively studied in
association with drought/heat tolerance and yield
in Australian wheat varieties [5, 6]. However, the
precise value of this trait in biomass production and
grain yield remains somewhat uncertain because
of the complex biochemical/genetic nature of the
phenotype. Based on solubility in organic solvents,
the cuticle components can be divided into insoluble
cutin and soluble cuticle waxes. Intracuticular wax
is embedded in the underlying cutin framework and
epicuticular wax is overlaid on the cutin matrix and
intracuticular wax.
The waxes are typically a complex mixture
of derivatives of very-long-chain (24–34 carbons)
saturated fatty acids, such as alkanes, aldehydes,
ketones, primary alcohols, and secondary alcohols
[2]. Fig. 1 B schematically summarizes the gene
Fig. 1. Glaucous (waxy) and non-glaucous (waxless) wheat varieties (A), and schematic overview of pathways involved
in cuticle wax biosynthesis (B). CER1–4: cuticular wax mutations; FAS: Fatty Acid Synthase complex; FAE — Fatty Acid
Elongation complex; WRI, Shine, MYB, HD-Zip — Transcription Factors involved in biosynthesis pathways (reviewed
in Borisjuk et al., 2014) [7]
networks and biochemical pathways responsible
for biosynthesis of major cuticle wax species which
have been deciphered through characterization of
plant mutants, mostly in Arabidopsis, barley and
maize.
In order to get insights into the nature of cuticle
components variations in spring wheats in relation to
drought tolerance, we initiated a project on targeted/
quantitative analysis of cuticle lipophilic waxes and
the genes related to wax biosynthesis. Primarily, fi ve
elite Australian wheat varieties: Kukri, Excalibur,
RAC875, Gladius and Drysdale, which are under
intensive genetic [5, 6] investigation related to
drought tolerance at the ACPFG, have been included
in the project. Earlier comparative metabolite of
Kurki (nonglaucous, drought intolerant, high quality
grain) vs Gladius, Excalibur and RAC875 (all
are glaucose, drought tolerant cultivars) revealed
signifi cant differences in their stress reactions.
Other wheat cultivars which have been included in
the analysis are commercial varieties cultivated in
different regions of Australia: Alsen, Baxter, Berkut,
Chara, Pastor, Volkani, Westonia and Wialkat [8].
Altogether, twelve Australian wheat cultivars with
different levels of tolerance to drought and heat were
compared for cuticle structure properties (Light and
Electron Microscopy) and wax composition (GC–
MS). Metabolomics analyses of chloroform and
hexane extracted waxes using GC–MS demonstrated
signifi cant quantitative differences between
drought-sensitive and drought-tolerant cultivars in
176 ISSN 2219-3782. Ôàêòîðè åêñïåðèìåíòàëüíî¿ åâîëþö³¿ îðãàí³çì³â. 2015. Òîì 16
Bi H., Kovalchuk N. V., Dias D., Roessner U., Langridge P., Lopato S., Borisjuk N. V.
several types of wax components. Table 1 represents
the cumulative relative amounts (chloroform
plus hexane extraction) of wax component in the
analysed cultivars compared to cv. Kukri. While the
majority of wax components, many of them still not
properly identifi ed, are relatively even represented
in all analysed cultivars within two-folds range of
variation, the unidentifi ed components 7–9 show
much higher representation in most cultivars (up to
186.9 fold difference for unknown 8 in cv Gladius)
compared to Kukri. The chromatogram in fi g. 2,
where the peaks between 36 and 43 min represent
unknown components 7–9 in the table 1, further
details the cuticular wax components difference
between Kukri and Gladius. While we were not able
to precisely identify the 36–43 min components, there
are good reasons to assume that those components
belong to beta-diketones, which has been recently
identifi ed as key cuticle compounds contributing to
wheat drought tolerance [9]. The revealed variation
in wax component biochemistry is also translated
in the drastic difference of the morphology of leaf
surface wax crystal as revealed by SEM (fi g. 2, right
block).
The SEM has been useed to assess cuticular
waxes in cv Gladius grown in greenhouse under
well watered and drought conditions. As evident
from fi g. 3, the load of wax crystals on wheat leaves
Fig. 2. GC–MS chromatograms of wax components and SEM image of fl ag leaf surface crystals in wheat cultivars Kukri
(waxless, not tolerant; top) and Gladius (glaucous, drought tolerant; bottom)
grown under drought is higher compared to well
watered plants. The wax crystals under drought also
are bigger and are differently shaped. The detailed
comparative metabolomics analysis of waxes
extracted from well-watered and drought treated
plants is in progress.
Twelve Australian wheat cultivars with
different levels of tolerance to drought and heat were
compared for cuticle structure properties (SEM)
and wax composition (GC–MS). Metabolomics
analyses of chloroform and hexane extracted
waxes using GC–MS demonstrated signifi cant
quantitative differences between drought-sensitive
and drought-tolerant cultivars in several types of
wax components. Acquired data are currently being
used for identifi cation of enzymes and upstream
regulatory genes responsible for the biosynthesis of
cuticular components.
Conclusions
Abiotic stresses such as drought, heat and high
UV-irradiation adversely infl uence crop growth and
productivity. While the nonglaucous phenotypes
dominate among wild wheat ancestors, glaucousness
positively correlates with yield in cultivated wheat
varieties, especially under drought conditions. The
trait depends on cuticle wax composition. Using
GC–MS analysis we have identifi ed wheat cuticle
ISSN 2219-3782. Ôàêòîðè åêñïåðèìåíòàëüíî¿ åâîëþö³¿ îðãàí³çì³â. 2015. Òîì 16 177
Natural variation of wheat cuticular waxes in relation to drought tolerance
Table
Comparison of wax components in 12 selected Australian cultivars
Fig. 3. Cryo-scanning electron micrographs of the abaxial side of the wheat fl ag leaf (cv. Gladius) grown under well
watered (top) and drought conditions (bottom)
178 ISSN 2219-3782. Ôàêòîðè åêñïåðèìåíòàëüíî¿ åâîëþö³¿ îðãàí³çì³â. 2015. Òîì 16
Bi H., Kovalchuk N. V., Dias D., Roessner U., Langridge P., Lopato S., Borisjuk N. V.
wax components that are present in drought resistant
wheat cultivars and absent in the sensitive cultivar.
Most likely these components represent beta-
diketones which have been previously suggested to
affect drought tolerance in wheat. The variation in
wax components composition defi ne shape of wax
crystals on leaf surface as revealed by scanning
electron microscopy. A detailed biochemical and
molecular biology study of wheat cuticular wax
regulation in relation to drought tolerance is in
progress.
The reported study has been partially supported by
an ACPFG — DuPont/Pioneer collaborative grant. The
China Scholarship Council is acknowledged for providing
a fellowship to PhD student Huihui Bi. The authors thank
Yuan Li for conducting RT-qPCR analysis and Gwen Mayo
for advises and help with SEM analyses.
LITERATURE
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2. Yeats T. H., Rose J. K.C. The formation and function of plant cuticles // Plant Physiol. — 2013. — 163. — P. 5–20.
3. Javelle M., Vernoud V., Rogowsky P. M., Ingram G. C. Epidermis: the formation and functions of a fundamental plant
tissue // New Phytol. — 2011. — 189. — P. 17–39.
4. Richards R. A., Rawson H. M., Johnson D. A. Glaucousness in wheat: its development and effect on water-use effi ciency
gas exchange and photosynthetic tissue temperatures // Aust. J. Plant Physiol. — 1986. — 13. — P. 465–473.
5. Bennett D., Izanloo A., Reynolds M., Kuchel H., Langridge P., Schnurbusch T. Genetic dissection of grain yield and
physical grain quality in bread wheat under water-limited environments // Theor. Appl. Genet. — 2012. — 25. —
P. 255–271.
6. Izanloo A., Condon A. G., Langridge P., Tester M., Schnurbusch T. Different mechanisms of adaptation to cyclic water
stress in two South Australian bread wheat cultivars // J. Exp. Bot. — 2008. — 59. — P. 3327–3346.
7. Borisjuk N., Hrmova M., Lopato S. Transcriptional regulation of cuticle biosynthesis // Biotechnol. Advances. —
2014. — 32. — P. 526–540.
8. Wheeler R. Wheat Variety Sowing Guide. — 2013. http://www.nvtonline.com.au/wp-content/ uploads/2013/03/ Crop-
Guide-SA-Wheat-Variety-Sowing-Guide-2012.pdf.
9. Adamski N. M., Bush M. S., Simmonds J., Turner A. S., Mugford S. G., Jones A., Findlay K., Pedentchouk N., von
Wettstein-Knowles P., Uauy C. The inhibitor of wax 1 locus (Iw1) prevents formation of β- and OH-β-diketones
in wheat cuticular waxes and maps to a sub-cM interval on chromosome arm 2BS // Plant J. — 2013. — 74, N 6. —
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BI H.1, KOVALCHUK N.V.1, DIAS D.2, ROESSNER U.2, LANGRIDGE P.1, LOPATO S.1, BORISJUK N.V.1
1 Australian Centre for Plant Functional Genomics, University of Adelaide,
Australia, Adelaide, Hartley Grove, Urrbrae SA 5064, e-mail: mykola.borysyuk@acpfg.com.au
2 School of Botany, University of Melbourne,
Australia, Melbourne, e-mail: u.roessner@unimelb.edu.au
NATURAL VARIATION OF WHEAT CUTICLE WAXES IN RELATION TO STRESS TOLERANSE
Aims. The aim of our research is to investigate the role of wheat leaf cuticle in drought and heat protection by characterizing
natural variability of cuticle components and revealing genetic and biochemical background of this variability in Australian
wheat varieties. Methods. Cultivars with different levels of drought tolerance were analyzed for cuticle wax composition
by gas chromatography (GC–MS) and for cuticle structure properties by scanning electron microscopy (SEM). Results.
Metabolomics analyses demonstrated signifi cant quantitative differences between drought-sensitive and drought-tolerant
cultivars in several types of wax components. These data are complemented by differences in cuticle structure revealed
by SEM. Ten wheat genes potentially involved in regulation of cuticle biosynthesis have been cloned and analysed for
expression profi les in wheat by RT-qPCT. Conclusions. Acquired data are currently being used for identifi cation of
enzymes and genes responsible for drought-related cuticular components.
Keywords: wheat, drought tolerance, cuticle waxes, gene expression.
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