Physiol Mol Biol Plants

https://doi.org/10.1007/s12298-019-00720-1

RESEARCH ARTICLE

Comparative fatty acid profiling of Indian seabuckthorn showed

altitudinal gradient dependent species-specific variations
Bhavana Sharma1 • Shaweta Arora2 • Dinabandhu Sahoo3 • Renu Deswal1

Received: 2 May 2019 / Revised: 14 September 2019 / Accepted: 10 October 2019

Prof. H.S. Srivastava Foundation for Science and Society 2019

Abstract The present study provides the first comparative diversity analysis also formed two broad clusters of Trans-
fatty acid profiling of the three Indian seabuckthorn spe- Himalayan and Sikkim populations which correlated with
cies, collected from varying altitudes (2900–4300 masl) of earlier taxonomic studies.
Trans-Himalayas (Hippophae rhamnoides, H. tibetana)
and Sikkim Himalayas (H. salicifolia) regions. Gas chro- Keywords Altitudinal gradient Hippophae sp. FAMEs
matography–mass spectrometry analysis showed variabil- Different geographical locations Stress tolerance
ity in fatty acid composition of different seabuckthorn
populations. Sikkim populations showed higher (1.28–1.6
folds) palmitic acid than Trans-Himalayan populations Introduction
which possess higher linoleic (1.3–1.5 folds) and linolenic
(1.6–1.8 folds) acids. Interestingly, a strong altitudinal Hippophae (seabuckthorn) a cold hardy shrub is native to
gradient associated positive correlation was observed with Europe and Asia. The plant is well adapted to grow in
the degree of unsaturation and PUFA content while nega- harsh environments such as drought, salinity and extreme
tive correlation was observed with saturated fatty acids temperatures. In India, genus Hippophae comprises of
content of different seabuckthorn populations. H. salicifo- Hippophae rhamnoides, H. salicifolia and H. tibetana,
lia collected from Sikkim showed healthy x-6:x-3 ratio which grows naturally in high-altitude (2391–4300 masl)
(closer to 1:1) of functional lipids exhibiting its better Himalayas (Raina et al. 2011). H. rhamnoides has gained
nutraceutical potential than other commonly used seed oils. wide attention as a functional food with high pharmaco-
Interestingly, H. tibetana from Losar showed higher (5.81) logical value, owing to the presence of [ 200 bioactive
degree of unsaturation than Sikkim populations (3.5) sug- compounds (Suryakumar and Gupta 2011; Zielinska and
gesting its better stress tolerance trait. Chemo-taxonomic Nowak 2017). Several studies highlighting its health ben-
efits are available but the remaining two species H. sali-
cifolia and H. tibetana remain relatively unexplored and
Electronic supplementary material The online version of this underutilized.
article (https://doi.org/10.1007/s12298-019-00720-1) contains sup-
plementary material, which is available to authorized users. Seabuckthorn fruit, pulp, and seed oils are extensively
used in food, health care, medicinal and cosmetic industries
& Renu Deswal (Zielinska and Nowak 2017), yet studies pertaining to
[email protected] lipids and fatty acids are limited for Indian seabuckthorn
1
Molecular Physiology and Proteomics Laboratory,
(Fatima et al. 2013; Ding et al. 2018). In general, H.
Department of Botany, University of Delhi, New Delhi, rhamnoides pulp is a source of higher palmitoleic
Delhi 110007, India (32–53%) and palmitic (25–35%) acid while seeds possess
2
Department of Botany, University of Delhi, New Delhi, higher linoleic (30–40%) and a-linolenic (20–35%) acids
Delhi, India (Zheng et al. 2017). Interestingly, H. rhamnoides is a rich
3
Department of Biotechnology, Institute of Bioresources and source of essential fatty acids and antioxidants (toco-
Sustainable Development, Imphal, Manipur, India pherols, tocotrienols, phytosterols, polyphenols, flavonoids,

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Physiol Mol Biol Plants

carotenoids and organic acids). Further, it is also known to Therefore, the objective of the present study was to
prevent oxidative stress associated diseases like diabetes, provide a comparative analysis of fatty acid composition in
rheumatoid arthritis, ulcerative colitis, cancer, cardiovas- Indian seabuckthorn particularly Trans Himalayan (H.
cular, Crohn’s and autoimmune diseases (Zheng et al. rhamnoides and H. tibetana) and Sikkim Himalayan (H.
2017; Shukla et al. 2017) suggesting that seabuckthorn salicifolia) populations growing at different altitudes.
could be beneficial in promoting human health. Analysis of essential fatty acids would help in identifica-
Seabuckthorn being a non-cultivated plant, exhibit tion of elite populations with higher nutraceutical relevance
higher variability even amongst naturally growing popu- or better stress tolerance for large scale cultivation and
lation (Dolkar et al. 2017a). However, due to its high breeding in future.
nutritional value and role in soil and water conservation,
efforts have been made for large scale cultivation of
seabuckthorn (Stobdan et al. 2017). Although, several Materials and methods
studies are available on the nutraceutical relevance and
stress tolerance (Gupta and Deswal 2012) of H. rhamnoides Plant material
but studies on H. tibetana and H. salicifolia remains rela-
tively less explored. Moreover, most of these studies are Berries of all the three Indian seabuckthorn species from
restricted to region-specific analysis of different popula- major seabuckthorn growing areas in Trans-Himalayan (H.
tions. To the best of our knowledge, broader comparison of rhamnoides and H. tibetana) and Sikkim (H. salicifolia)
Trans-Himalayan (H. rhamnoides and H. tibetana), and Himalayan regions of India were collected. H. rhamnoides
Sikkim (H. salicifolia) populations is not available till date. orange (HrS1) and yellow (HrS2) colored ecotypes were
Lack of species specific comparative information on elite collected from Shey (34040 N 77370 E; 3200 m asl) and
germplasm with better nutraceutical and stress tolerance Thiksey (34310 N 78160 E; 3530 m asl) respectively from
poses a major hindrance in seabuckthorn large-scale cul- Leh, Jammu & Kashmir, India. H. rhamnoides (HrS3,
tivation and breeding programmes (Dolkar et al. 2017a; 31220 N 77070 E; 3300 m asl) and H. tibetana (HtS,
Saikia and Handique 2013). 32220 N 78080 E; 4300 m asl) were collected from Kaza
Plant membranes is the first organelle to sense envi- and Losar respectively, Lahaul and Spiti valley, Himachal
ronmental variations (temperature, salinity, drought and Pradesh, India. Different ecotypes of H. salicifolia (HsS1
pathogen attack) which affect the fluidity, permeability and and HsS2) were collected from Lachen (HsS1, 27430 N
stretching of membrane due to lipid remodelling (Zheng 88300 E; 2391 m asl) and Lachung (HsS2, 27410 N
et al. 2014). In plants growing in alpine and desert 88440 E; 3111 m asl) valley, North Sikkim. Geographical
ecosystems, maintenance of membrane fluidity is of par- locations and altitudes of all the natural populations were
ticular biological significance as these are exposed to fre- determined using a GPS device (eTrex Vista HCx, Tai-
quent environmental fluctuations throughout the day wan) (Fig. 1).
(Zheng et al. 2014; Barrero-Sicilia et al. 2017). Alterations As per the phenological data reported previously, the
in membrane lipid composition along with increased fatty tissue was collected at the same developmental stage i.e.
acid unsaturation contributes to acclimation ability. Fur- mature berries in the month of September (Trans-Hi-
ther, effort is also made to know if fatty acid methyl esters malayan populations) and November (Sikkim populations)
(FAMEs) analysis could help in analyzing the biochemical from morphologically comparable natural populations.
and phenotypic diversity of seabuckthorn and could serve Seabuckthorn not being cultivated shows a lot of varia-
as a chemotaxonomic marker (Choudhary Kumar et al. tions. Therefore, to minimize the variations even in a
2017). phenotypically similar population growing at same geo-
Being a stress tolerant plant, seabuckthorn may serve as graphical location, blocking of the area was done in three
a system to study resilience of Himalayan flora to extreme different parts using randomized block design. Samples
environmental conditions. Moreover, bioactive constituents (berries) were collected randomly from individuals
vary depending on the species, cultivar, harvesting time of (n = 50) distributed evenly at each site and pooled. These
fruits, agro-climatic conditions and geographical locations pooled samples collected from three different parts of the
(Yang and Kallio 2001). Interestingly, a recent ecophysi- area were considered as biological replicates. Further,
olomic study using naturally growing H. rhamnoides has berries were washed, frozen in liquid nitrogen and stored at
shown that variations in climatic factors may alter proteins, - 80 C. For FAMEs analysis intact seeds were separated
metabolites and fatty acids. The results also indicated the manually from these berries.
multifactorial stress acclimation strategies of seabuckthorn
in response to diverse environmental conditions (Sharma
and Deswal 2019).

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Physiol Mol Biol Plants

Fig. 1 Figure showing the geographical distribution of naturally growing Trans-Himalayan (H. rhamnoides, H. tibetana) and Sikkim Himalayan
(H. salicifolia) Indian seabuckthorn populations

GC–MS for fatty acid methyl ester analysis different (SPSS, STATS 21). Principle component analysis
(FAMEs) (PCA) was performed to study the relationship amongst
different Hippophae species on the basis of their seed fatty
Fatty acid methyl esters (FAMEs) were obtained from acid composition. Heat maps were used to represent the
lipids using acid-catalyzed transesterification procedure as fold changes (log10 transform) in FAs and agglomerative
described previously with minor modifications (Choudhary hierarchical clustering (AHC) using euclidean distance
Kumar et al. 2017). Seeds (10–15) were grounded to measure was performed to evaluate overall multivariate
powder, transferred to Teflon lined screw-capped glass phylogenetic relationship amongst different seabuckthorn
tube and incubated in 2% (w/v) HCl in methanol (1 mL) species. PCA, AHC and heatmaps were generated using
for 1 h at 80 C. After cooling, FAMEs were extracted by XLSTAT 2018 (New York) software.
addition of 0.9% NaCl and hexane (2 mL), followed by
centrifugation to allow phase separation. Hexane layer was
pooled, dried under nitrogen gas and resuspended in Results and discussion
(100 ll) hexane. FAMEs were determined by gas chro-
matography-mass spectrometry (GC–MS), using an Agi- Seabuckthorn is a stress tolerant shrub with immense
lent Technologies GC–MS (5977A MSD coupled with medicinal and nutraceutical properties. Seabuckthorn seed
7890B GC series) equipped with a DB-wax capillary col- oil is rich in essential fatty acids (linoleic, x-6 and a-
umn (30 m 9 0.25 mm 9 0.25 mm; Agilent Technolo- linolenic, x-3) (Fatima et al. 2013). Despite a plentiful
gies). Initial oven temperature was 50 C, increased to source of nutraceuticals, currently the area under
230 C at 3 C min-1. The carrier gas used was nitrogen seabuckthorn cultivation is limited which needs to be
with a flow rate of 1.8 mL min-1 and injection temperature expanded. Naturally growing seabuckthorn populations
was 230 C. Injection volume was kept at 1 ll with a split face extreme environmental conditions and are exposed to
ratio of 18:1. FAMEs were identified through comparison multiple abiotic stress. To survive these extreme condi-
of mass spectral data with the NIST 11 library. FA com- tions, it exhibits multifactorial stress adaptation including
position was expressed as the percentage (%) of total fatty the alterations in proteins, metabolites and fatty acids
acids. (Sharma and Deswal 2019).
Remodeling of membrane lipids, the essential compo-
Statistical analysis nents of cellular membranes via regulation of unsaturation
is an interesting acclimation strategy adapted by plants.
FA composition data were analysed and data from tripli- Alpine and desert plants show significant variations in lipid
cates is expressed as mean ± standard deviation. One-way species during stress conditions thus, indicating their
ANOVA using the Tukey post hoc range test was per- specific roles in signaling cascades and defense (Zheng
formed to find significant statistical differences amongst et al. 2014; Barrero-Sicilia et al. 2017).
the samples where p \ 0.05 were considered significantly

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Physiol Mol Biol Plants

Most of the studies on Indian seabuckthorn are focused composition with the global H. rhamnoides populations
on H. rhamnoides (Dolkar et al. 2017a) till date, whereas containing major fatty acids palmitic (7–13%), palmitoleic
H. tibetana and H. salicifolia populations remains rela- (\ 4%), oleic (\ 4%), linoleic (33–43%) and a-linolenic
tively less explored (Ranjith et al. 2006). Thus, compara- acid (28–39%) in the defined range (Fig. 3).
tive study of different seabuckthorn species may provide Interestingly, comparative analysis showed higher pal-
insights into their differential stress tolerance or nutraceu- mitic (1.15–2 folds) and linoleic (1.06–1.8 folds) acid in
tical relevance. Indian (Trans-Himalayan) H. rhamnoides than global
populations [Romanian (Dulf 2012), German (Burcová
Alterations in fatty acid profiles of seabuckthorn et al. 2017) and Finnish (Yang and Kallio 2001) and
populations collected from H.P, Leh and Sikkim Canadian (Fatima et al. 2013)] (Fig. 3). Moreover, the
Trans-Himalayan populations growing at higher altitudes
It is well established that variable environmental variations also contained higher linoleic (1.3–1.5 folds) and a-li-
(nutrient availability, salinity, moisture, rainfall, humidity, nolenic (1.5–1.8 folds) than Sikkim populations (Table 2).
and daylight) and geographical locations (latitude, longi- On the contrary, higher linoleic (1.76 folds), a-linolenic
tude, altitude) may contribute to alterations in the FA (1.5 folds) acids, PUFA (61–66%) and lower palmitoleic
content for maintenance of appropriate membrane fluidity (fourfolds) and oleic (1.8–2.6 folds) content was observed
(Johansson et al. 2000). In the present investigation, H. for Trans-Himalayan H. rhamnoides (3200 and 3530 masl)
tibetana (4300 masl), H. rhamnoides (3200–3530 masl), than H. rhamnoides seeds from Tabo, Lahaul-Spiti, HP
and H. salicifolia (2391, 3111 masl) berries were collected (3165 masl) thus supporting the altitude and geographical
from three diverse geographical locations and agro-cli- locations dependent variations in FA profile (Fig. 3).
matic zones across Indian Himalayas. Therefore, the prime Interestingly, H. salicifolia (HS1 and HS2) populations
interest of present study was to provide a broader com- collected from Sikkim Himalayas showed higher content of
parison of fatty acid composition in three seabuckthorn saturated fatty acids including palmitic (1.28–6 folds) and
species. Further, we also wanted to investigate the altitu- palmitoleic (2.75–7.6 folds) acid than Trans-Himalayan
dinal gradient associated variations in fatty acid composi- populations (Table 2). Further, H. salicifolia from Sikkim
tion of these populations. also showed higher essential fatty acid content [palmitic
Distribution, altitude and geographical location of (1.18 folds), palmitoleic (2.2 folds), stearic (2.36 folds),
seabuckthorn species is detailed in Fig. 1. Mature seeds linoleic (1.25 folds) and linolenic (1.5 folds)] than reported
from berries of H. rhamnoides (HrS1 and HrS2, Leh); H. previously for H. salicifolia collected from H.P (Kaushal
rhamnoides (HrS3, HP), and H. tibetana (HtS, H.P), and H. and Sharma 2011) suggesting altitudinal gradient associ-
salicifolia (HsS1 and HsS2, Sikkim) were used for Gas ated variations in FA composition (Fig. 3).
chromatography-mass spectrometry (GC–MS) analysis. Similar quantitative variations were observed in Oliva
GC chromatogram of seabuckthorn seeds showed total cuspidate seeds [oleic (69.3–74.5%), linoleic
eight major FAs including three saturated [Palmitic acid (11.2–15.2%), palmitic (11.2–14.0%), linolenic
(16:0), Stearic acid (18:0), Eicosanoic acid (20:0)], three (1.3–3.20%), palmitoleic (1.31–2.10%) stearic (0.1–0.2%),
monounsaturated [Palmitoleic acid (16:1), Oleic acid and linolenic acid (11.2–15.2%)] collected from different
(18:1D9), Cis-vaccenic acid (18:1D11)] and two polyunsat- geographical locations in Pakistan (Gulfraz et al. 2009).
urated [Linoleic acid (18:2), a-Linolenic acid (18:3)] Likewise, variations (14–17%) in total FAs including lau-
(Fig. S1). ric, myristic, oleic and linoleic acids are reported in 44
Biochemical profiling of FA composition revealed sig- different species of Cuphea due to adaptive radiation,
nificant variability within the seabuckthorn species col- speciation, and habitats (Graham et al. 2016). These
lected from diverse geographical locations or agro-climatic observations indicate that both Trans and Sikkim Hima-
conditions (Fig. 2a, b and Table 1). In the present inves- layan populations showed marked variations in their
tigation, linoleic acid (30–46%), a-linolenic acid respective FA metabolic signatures which suggested alti-
(18–32%), palmitic (11–19%), and oleic (10–19%) acids tudinal gradient associated differential acclimation
were observed as major FA while minor FA included responses.
palmitoleic (1.4–6%), stearic (2–3.6%), cis-vaccenic
(2.6–6%) and eicosanoic acid (0.5–0.85%) (Fig. 2). To the Degree of unsaturation in seabuckthorn populations
best of our knowledge, the present investigation is the first may contribute to differential stress adaptation
comparative study of Trans-Himalayan and Sikkim
Himalayan seabuckthorn (seeds) FA and also the first Increase in unsaturated fatty acids is an acclimation strat-
report on H. tibetana seed FA profile. Trans-Himalayan H. egy known as homeoviscous acclimation, in response to
rhamnoides populations showed comparable FA deviations from optimal temperature to maintain

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Physiol Mol Biol Plants

Fig. 2 Comparative FAMEs

analysis showing the a fatty acid
(FA) composition and b the
ratio of saturated (SFA),
monounsaturated (MUFA) and
polyunsaturated (PUFA) fatty
acids in seeds of different
seabuckthorn species collected
from Trans-Himalayan and
Sikkim populations. Data
correspond to averages ± SD of
3 replicates per sample. Mean
values marked with different
letters in each column are
significantly different
(p \ 0.05)

Table 1 Fatty acid composition (weight% of total fatty acids) of individual lipid classes from seed oils of different seabuckthorn species
Fatty acid content (%)
FAMEs HtS HP HrS1 Leh HrS2 Leh HrS3 HP HsS Lachen HsS Lachung
c c b c a
C16:0 11.23 ± 0.09 12.49 ± 0.25 14.85 ± 0.15 12.34 ± 1.36 18.03 ± 0.42 19.12 ± 0.09a
C18:0 2.88 ± 0.06b 3.58 ± 0.27a 3.23 ± 0.21a 3.43 ± 0.15a 2.63 ± 0.09c 2.15 ± 0.06c
b b b b a
C20:0 0.58 ± 0.03 0.58 ± 0.10 0.59 ± 0.05 0.54 ± 0.03 0.75 ± 0.05 0.84 ± 0.03a
P
SFA 14.69 ± 0.18c 16.66 ± 0.62b,c 18.68 ± 0.36b 16.31 ± 1.54b,c 22 – 0.56a 21.41 – 0.18a
C16:1 0.95 ± 1.24c 1.39 ± 0.20c 1.55 ± 0.19c 1.37 ± 1.01c 3.82 ± 1.79b 11.4 ± 1.24a
d a d a,b b,c
C18:149 11.43 ± 0.39 17.19 ± 0.35 10.38 ± 1.38 15.84 ± 1.54 14.45 ± 0.02 12.66 ± 0.39c,d
C18:1411 4.07 ± 0.28b 3.087 ± 0.41c 3.81 ± 0.33b 2.72 ± 0.45c 4.32 ± 0.28b 5.79 ± 0.28a
P d b,c d c b
MUFA 16.45 ± 2.21 21.68 ± 0.92 15.74 ± 2.49 19.92 ± 2.94 22.08 – 1.98 29.86 – 2.24a
c b a a c
C18:2 37.12 ± 1.20 40.8 ± 1.20 44.38 ± 0.24 45.87 ± 2.06 35.31 ± 1.83 30.16 ± 1.20d
a b b b b
C18:3 31.74 ± 0.17 20.86 ± 1.7 21.19 ± 0.62 17.88 ± 2.74 20.69 ± 0.21 17.87 ± 0.17b
P c
PUFA 68.86 – 1.37 a
61.66 – 2.90 b
65.58 – 0.86a
63.76 – 4.80 a
55.91 ± 2.04 48.03 ± 1.37d
P
UFA 85.31 – 3.58a 83.35 – 3.82a 81.31 – 3.35a 83.68 – 6.6a 78.5 ± 4.02b 77.89 ± 3.61b
c
UFA:SFA 5.81 a
5.0 a
4.35 b
5.13a
3.67 3.52c
b a a a c
x-6:x-3 1.68 2.09 1.95 2.56 1.19 1.69b
P
TFA 100 100 100 99.99 99.99 99.3
Results are expressed as mean ± standard deviation (n = 3). Mean values marked with different letters in each column are significantly different
(p \ 0.05)

appropriate membrane fluidity (Sakamoto and Murata Minimum temperature during the seed fill and matura-
2002). Literature studies have shown higher unsaturation tion periods during soybean seed development is consid-
index (ratio of unsaturated and saturated fatty acid) to be ered as major factor affecting FA composition, particularly
positively correlated with the better stress tolerance. relative amounts of saturated/unsaturated FA, due to
Therefore, comparative analysis of saturated (SFA), stimulatory effect of lower temperatures on desaturases
monounsaturated (MUFA) and polyunsaturated FA gene expression (Hou et al. 2006). The study also showed
(PUFA) contents was also performed to find whether that different geographical locations with varying altitudes
similar correlation exists between the fatty acid composi- accounted for higher variability in PUFA (linoleic and
tion and differential stress tolerance in different seabuck- linolenic) content than SFAs like palmitic, stearic, oleic
thorn populations (Table 1). acid (Hou et al. 2006).

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Physiol Mol Biol Plants

Fig. 3 Comparative analysis Fatty acid composition in different seabuckthorn seed germplasm
showing the variations in fatty Global Germplasm Indian Germplasm
60
acid composition of Global and
Literature reports Present investigation
Indian seabuckthorn seed 50
populations. Samples shown in
dotted box (red) shows the 40

FAMEs %
Indian Trans-Himalayan (H.
30
rhamnoides, H. tibetana) and
Sikkim Himalayan (H. 20
salicifolia) seed populations
analyzed in the present study. 10

FAMEs data for global and

0
other Indian germplasm was

d
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ia
a

ia
ia

ia

ia
da

ia
obtained from literature studies

an

di
an

di

di

nd
an

nd

nd

nd
nd
na

In

In
In
m

nl
m

,I

,I

,I

,I

,I
Ca

h,

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as mentioned in the text (color

er

P,
Fi

bo

HP

im

im
Ro

HP
Le

Le
G

H
S
S

kk

kk
Ta

S
S

S3
Hr

S
S
Hr

figure online)

S1

S2

Si

Si
Hr

Hs

Ht
Hr

Hr
Hr

Hr
Hr

S1

S2
Hs

Hs
Palmitic Palmitoleic Oleic Linoleic Linolenic

Table 2 Correlations between

Variables C16:0 C18:0 C20:0 C16:1 C18:149 C18:1411 C18:2 C18:3
seed fatty acid parameters in
Trans-Himalayan and Sikkim C16:0 1 - 0.779 0.928 0.822 - 0.174 0.779 - 0.666 - 0.530
seabuckthorn germplasm
C18:0 - 0.779 1 - 0.911 - 0.840 0.428 - 0.956 0.901 0.016
C20:0 0.928 - 0.911 1 0.919 - 0.157 0.908 - 0.889 - 0.327
C16:1 0.822 - 0.840 0.919 1 - 0.142 0.874 - 0.796 - 0.436
C18:149 - 0.174 0.428 - 0.157 - 0.142 1 - 0.499 0.177 - 0.429
C18:1411 0.779 - 0.956 0.908 0.874 - 0.499 1 - 0.898 - 0.024
C18:2 - 0.666 0.901 - 0.889 - 0.796 0.177 - 0.898 1 - 0.078
C18:3 - 0.530 0.016 - 0.327 - 0.436 - 0.429 - 0.024 - 0.078 1

Higher PUFA (1.2–1.4 folds) content was observed in Sikkim [HsS1 (3.6) = HsS2 (3.5)] populations suggesting
Trans-Himalayan populations (HtS) growing at higher stress adaptive changes in membranes (Fig. 2b, Table 1).
altitudes (4300 masl) than Sikkim (2391–3111 masl) pop- Further, linear regression (statistical) analysis was per-
ulations. Higher PUFA may help in mitigating environ- formed to strengthen the association between altitudinal
mental stress-induced damage due to harsher climatic gradients and the fatty acid composition of seabuckthorn
conditions prevalent at higher altitudes. On the contrary, populations. Interestingly, a strong altitudinal gradient
higher (1.15–1.47 folds) saturated FA was observed in H. associated correlation was observed with the unsaturation
salicifolia from Sikkim growing at lower altitude than index (strong, R2 = 0.8707, p \ 0.05), PUFA content
Trans-Himalayan populations (Fig. 2b, Table 1). (moderate, R2 = 0.77, p \ 0.05) and unsaturated (strong,
Changes in degree of unsaturation (ratio of UFA vs. R2 = 0.96, p \ 0.01) fatty acids content of different
SFA), of membrane lipids affect membrane fluidity with seabuckthorn populations. Similar, low temperature
temperature fluctuations. Cold acclimation increases the induced increase (15%) was also observed in degree of
degree of unsaturation. Multiple studies in Arabidopsis unsaturation due to PUFA accumulation in Cicer arietinum
have shown that defects in desaturases display inhibited (Bakht et al. 2006). Likewise, cold acclimated potato
growth or even death at non-freezing low temperatures of (Solanum commersonii) showed higher (twofolds) linoleic
6–12 C (Falcone et al. 2004). Similarly, over-expression acid in membrane glycerolipids of leaves, than non-accli-
of chloroplastic Cucurbita maxima and A. thaliana glyc- mated potato (Solanum tuberosum) during cold stress
erol-3-phosphate acyltransferase (GPAT) gene involved in (Upchurch 2008).
desaturation led to increase in UFAs with corresponding Interestingly, the above observations are in accordance
decrease in chilling sensitivity of genetically engineered with previous studies where altitudinal gradient associated
tobacco (Upchurch 2008). variations in phenotypic (decrease in leaf size and area,
In the present study, unsaturation index correlated with increase in leaf thickness) and metabolic signatures (in-
altitudinal gradient where higher unsaturation was crease in chlorophyll and proline content) were observed in
observed in Trans-Himalayan populations [HtS Trans-Himalayan H. rhamnoides populations (Dolkar et al.
(5.81) [ HrS3 (5.1) = HrS1 (5) [ HrS2 (4.35)] than 2017b, 2019). These variations are considered as adaptive

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Physiol Mol Biol Plants

Biplot (axes F1 and F2: 89.04 %)

A -4 -2 0
B Dendrogram
-3 -1 1 2 3 4 5
8 8

C18:3
6 6 HrS1 Leh

4 4 HrS3 HP H. rhamnoides
HtS HP
F2 (21.09 %)

2 C18:1△11 2 HrS2 Leh Trans-Himalayan germplasm

0
HrSY Leh
0 H. tibetana
C18:2 HtS HP
HsS Lachen HsS Lachung
HrSO Leh C20:0
-2 C18:0 HrS HP C16:1 -2 H. salicifolia
C16:0 HsS1 Sikkim
Sikkim-Himalayan germplasm
-4 -4
HsS2 Sikkim
C18:1△9
-6 -6
-4 -3 -2 -1 0 1 2 3 4 5
F1 (67.94 %) 0 100 200 300 400 500 600 700

Dissimilarity
Active variables Active observations

Fig. 4 Chemo-diversity represented by FA patterns using multivari- b Dendrogram showing agglomerative hierarchical clustering depict-
ate analysis. a Scatter biplots showing principal component analysis ing the relatedness of different seabuckthorn populations based on
of seed fatty acids from different seabuckthorn populations. their fatty acid profiles

responses contributing to better stress tolerance to high Seabuckthorn seed oils are considered excellent sources
altitudes plants growing in harsher environmental of PUFAs (48–69%), and EFAs due to unique (x-6:x-3
conditions. ratio close to 1:1) composition of linoleic (30–46%) and a-
linolenic acids (18–32%) (Fatima et al. 2013). Proportions
Exploring the nutraceutical potential (functional of PUFA from seeds obtained in this study were similar to
lipids) of different seabuckthorn populations those reported peviously for Finnish Hippophae ssp. si-
nensis and rhamnoides (Yang and Kallio 2001). Interest-
Present-day hectic lifestyle and high levels of stress affects ingly, both the seabuckthorn populations possess a healthy
health negatively. Deficiency of essential fatty acids x-6:x-3 ratio of fatty acids in the range 1.19–2.56. These
(EFAs) crucial for maintenance of good human health may ratios lie within the approved USFDA range (closer to 1:1)
occur during stress, due to inhibition of enzymes involved suggesting better nutraceutical potential than commonly
in formation of long chain PUFAs. EFAs (linoleic and a- used seed oils including flaxseed oil (4:1), hemp oil (1:3),
linolenic acid), can only be supplied by diet as humans sunflower (0:65), safflower (0:75), corn (0:59), olive oil
(due to lack of desaturase enzymes) are unable to synthe- (0:8), Canola (1:4) and soybean (1:7) (Erasmus 1993).
size these. Linoleic acid is precursor for functionally Based on the unique composition of x-6:x-3 values
essential longer-chain x-3, (eicosapentaenoic acid EPA closest to 1:1, Sikkim populations [HsS1 (1.19) and HsS2
and docosahexaenoic acid), x-6 (Gamma linoleic acid) (1.69)] exhibited better nutraceutical potential than other
fatty acids and eicosanoids (prostaglandins, thromboxanes, commonly used seed oils. Interestingly, Trans-Himalayan
and leukotrienes) biosynthesis in the human body (Alab- population particularly, HtS seeds also exhibited lower x-
dulkarim et al. 2012). Today’s diet is low in EFA while 6:x-3 (1.68) ratio with higher degree of unsaturation
rich in unwanted saturated and trans fatty acids showing x- (UFA:SFA ratio, 5.81) thus, representing its better
6:x-3 FAs ratio (1:10 to 1:50), higher than the recom- nutraceutical value and better stress tolerance (Table 1).
mended (1:1 to 4:1) ranges (Alabdulkarim et al. 2012).
Fish oils are considered abundant (60–70%) source of Multivariate analysis for chemotaxonomic studies
EFAs but growing concerns regarding accumulation of
environmental pollutants in fish oil and sustainability of In order to further understand the correlation between FA
marine fish stock demands other alternative sources. Plant variables and different seabuckthorn populations, principal
seeds (linseed, flaxseed) rich in x-3 (40–50%) FAs may component analysis (PCA) was performed using eight FAs
replace and offer a sustainable source of these lipids (Fa- as variables. First two principal components (PC1, PC2) in
tima et al. 2013). With the exception of flaxseed oil the PCA model explaining 89.04% of the total data vari-
([ 50% a-linolenic acid), higher levels of x-3 FAs are ance (Fig. 4a), were chosen on the basis of their eigen-
uncommon. Moreover, most of the seed oils have an values ([ 1, 5.436 and 1.687 respectively) (Fig. S2)
undesirable x-6:x-3 ratio. indicating that these reflect majority of the information.

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Contribution of PC1 was higher (67.94%) than PC2 fatty acids suggested the higher nutritional value of
(21.09%) (Fig. 4a). Pearson correlation data matrix of FA seabuckthorn than other seed oils.
variables for bi-plot demonstrated the correlation • Linear regression analysis confirmed the association
(p \ 0.05) between each FA variable and seabuckthorn between altitudinal gradient and fatty acid composition.
species (Fig. 4a, Table 2). PC1 elucidated high positive Interestingly, increase in altitude showed a strong
loading factor for C16:0 (0.891), C16:1 (0.934), C18:1411 positive correlation with the unsaturation index and
(0.966) and C20:0 (0.982) (Fig. 4a, Table S1). Hierarchical the PUFA content.
clustering analysis (HCA) revealed that HsS Lachen and • Higher PUFA (13–21%) content and highest unsatura-
HsS Lachung grouped together on the basis of higher tion index (5.81) of H. tibetana (Trans-Himalayan) than
abundances of C16:0, C16:1, C18:1411 and C20:0 FAs. H. rhamnoides [H.P (4.3), Leh (5)] and H. salicifolia
However, HrS HP and HrSO Leh exhibited closer corre- populations (3.85) suggested its better stress tolerance
lations owing to presence of higher C18:0, C18:149 and potential at higher altitude (4300 masl).
C18:2 (Figs. 4a, S3A). Fatty acid clustering performed • Further, multivariate PCA analysis showed a positive
using heat-maps supported PCA analysis and demarcated correlation between chemo-taxonomical analysis and
the variations in FA composition of seabuckthorn species earlier taxonomic studies suggesting that FA profiling
(Fig. S3B). may be used as a chemotaxonomic marker not only for
Algorithmic hierarchical clustering (AHC) dendrogram analysis of geographic origin associated diversity but
generated using Euclidean distance and Ward’s method also for nutritional value assessment for crop selection
showed marked intra and interspecies biochemical diver- in breeding programs.
sity dividing populations into two major clusters (Fig. 4b).
Based on the degree of relatedness, cluster I represent Acknowledgements This work was supported by financial assistance
Trans-Himalayan populations including both H. rham- from Department of Biotechnology (IBSD/A1/P(PH-2)/4), Govern-
noides (HP and Leh) along with H. tibetana. On the con- ment of India to RD. BS is thankful to UGC and DBT for providing
trary, cluster II represents Sikkim populations containing fellowship. Authors are thankful to Dr. Girish Mishra for kindly
extending GC–MS facility for FAMEs analysis and critical
both the ecotypes of H. salicifolia from Lachen and suggestions.
Lachung. However, cluster I was further divided into two
subclusters, wherein subcluster I include H. rhamnoides
from both HP and Leh, while subcluster II contained H. References
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