The Open Plant Science Journal, 2009, 3, 1-6
Open Access
Triacylglycerol Metabolization during Germination of Sea Buckthorn
Seeds
Vasily Pchelkin, Vladimir Tsydendambaev and Andrei Vereshchagin*
Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya ul. 35, 127276 Moscow,
Russia
Abstract: Dark germination of sea buckthorn seeds was characterized by an initial 3-day-long lag-period, when
the contents of triacylglycerols (TAGs) and total acyl-containing lipids (ACLs) remained nearly the same
dueretardation in the lipid metabolization. Subsequently, TAG content decreased rapidly, and by the 10th day of
germination, it did not exceed 5% of total lipids. In this process, total saturated (S) and total unsaturated fatty acids
(U), as well as various TAG types such as S2U, SU2, and U3, were consumed at nearly similar relative rates. At
the same time, separate TAG groups, which included one of the individual fatty acids, such as palmitic (P), stearic
(St), oleic (O), linoleic (L), or linolenic (Le), differed from each other in the intensity of degradation. For L- and
Le-TAGs, initial and final concentrations were similar, while initial concentrations of St- and O-TAGs by the 10th
day of germination increased 2.3- and 1.5-fold, respectively, and as regards P-TAGs, this value decreased 3.5-fold.
Thus, P-TAGs considerably exceeded other TAG groups in their consumption rate in seedlings, while St- and OTAGs ranked below them in this respect
INTRODUCTION
Sea buckthorn belongs to a group of higher plants (e.g.oil
palm, avocado, olive, etc.), which contain a considerable
portion of their fruit oil not only in the seeds of a fruit, butalso
in its mesocarp and therefore are designated as the plants with
an oil-bearing mesocarp (POM) [1].
rapid decrease in the dry matter [9] and TAG [10] contents,
while other ones claimed that such decrease was slow [11,12].
Furthermore, according to some evidence, these changes were
preceded by a several-day-long lag-period [10, 11], whereas
no such evidence was found elsewhere [12, 13].
In our earlier studies devoted to the mature fruit oils of
several sea buckthorn genotypes, we have established the
contents and quantitative composition of fatty acids (FAs) and
triacylglycerols (TAGs) in the oils of both mesocarp [2-5] and
seeds [6, 7], as well as the changes in these parameters during
fruit maturation [1, 3, 4, 7]. It was found that the seed oils
dramatically differed from the mesocarp ones as regards the
concentration, accumulation pattern, FA and TAG
compositions, as well as the effects of genotype factors on
these compositions [5, 8].
As regards the selectivity of catabolism of definite FA and
TAG types and molecular species during germination, one
must stress, first of all, that, at present, there is no respective
information concerning TAG types and very few data on FA
types [14, 15]. In many cases, the FA species composition of
TAGs and the molecular species composition of the TAGs
themselves remained unchanged [10, 11, 13,16], while in
other ones, the concentration of a given individual FA in total
TAGs either increased, or decreased depending on a plant
species under investigation [15, 17-19]. Moreover, the
patterns of germination-induced quantitative dynamics of
TAG molecular species were shown to be in conflict to each
other [20, 21].
It is well known, that, in POM fruits, mesocarp oil cannot be
used by seedlings for “making” a new organism, and so their
seed oil serves here as the only source of carbon and energy
during germination before the onset of photosynthesis in this
organism. However, the pattern of oil TAG consumption in
the course of germination has not been studied in any POM
species up to now, and therefore it was of interest to perform
such study using sea buckthorn seeds, which contain about
10% oil in the cotyledons
[1].
At the same time, there are many oleiferous and nonoleiferous
plant species other than POM, in which the dynamics of dry
weight, oil content, and TAG degradation in seedlings has
been investigated, but the evidence obtained remains in many
cases a rather contradictory one. Thus, some authors asserted
that seed germination was accompanied by a
*Address correspondence to this author at the Timiryazev Institute of
Plant Physiology, Russian Academy of Sciences, Botanicheskaya ul.
35, 127276 Moscow, Russia; Tel: 7 (499) 231 8351; Fax: 7 (495) 977
8018; E-mail: [email protected]
1874-2947/09 2009
Taking into account such situation, the objectives of our work
were to obtain some initial evidence on oil metabolism in the
seedlings of a POM species as well as to contribute to
resolving the contradictions regarding the dynamics of dry
mater and oil mobilization during seed germination in
general. The results of our investigation are outlined in the
present communication.
MATERIALS AND METHODS
Sea buckthorn (Hippophae rhamnoides L., cv. Katunskaya24) mature fruits were collected in a local botanical garden;
the seeds were separated and stored at a chilling temperature.
The seeds were germinated in darkness at 25oC on moist filter
paper, and their germinability was close to 100%. Intact seeds
(0th day) and their 1-, 2-, 3-, 4-, 5-, 6-, 8-, and 10-day-old
seedlings were used in the experiments. Each time point
comprised 60 seeds or their seedlings; 20 of them were used
for determining the changes in the cotyledon fresh and dry
Bentham Open
2 The Open Plant Science Journal, 2009, Volume 3
Pchelkin et al
weights, and the cotyledon material from 40 seedswas fixed
with boiling isopropanol and homogenized. At the 10th day,
the seedling development was at the stage of the first true leaf
emergence; their oil reserves were nearly exhausted, and so
the experiment was terminated.
gasflow linear velocity in the column, 15 cm/s; sample size,
10 μg; flow split ratio, 1:100; starting column temperature,
80oC; temperature program, from 80oC at a rate of 30o/min
to 140oC, and from 140oC at a rate of 10oC/min to 170oC;
finally, the temperature was raised at 4oC/min to 220oC.
Total lipids from the homogenate were extracted as described
earlier [6]. Absolute contents and composition of FAs in the
total acyl-containing lipids (ACLs) of the extract and in the
TAGs isolated from the extract by preparative adsorption
chromatography [6] were established by GLC of FA methyl
esters in the presence of an internal standard [8] using the
Tracor 540 Gas Chromatograph apparatus (Tracor,
USA) equipped with a 50-m-long capillary column having an
internal diameter of 0.25 mm and containing a grafted
Positional-species composition (PSC) and positional-type
composition (PTC) of TAGs were calculated from their FA
composition [6] using the Ekokhrom computer program
complex software [5].
The means of five replications each containing 20 and 40
seeds (see above) and their standard errors were determined at
P = 0.05
RESULTS AND DISCUSSION
In the course of two-day-long imbibition, the seeds acquired softer coats, and the radicle emerged from the coat. This was
followed by an intense growth of seedling axial organs. The cotyledon fresh weight content steadily increased, and by the 10th
day exceeded 5-fold the initial one (Fig. 1).
Earlier, similar results were obtained in the experiments with
cotton, hazel, and yellow foxtail seeds, in which this value
increased 3-8-fold during germination [16, 22, 23]. Atn the
same time, a dry weight decrease, caused by reserve
consumption during the sea buckthorn seedling growth,
proceeded steadily, but at a slow rate. A slow mobilization of
cotyledon reserves was also observed in the experiments with
linseed [12], hazel [16], tobacco [24], yellow foxtail [23], and
two cruciferous species [11]; however, in corn [25], cotton
[22], nut pine [9], and water melon [26] seeds, their dry
weight contents decreased two- or even threefold by the end
of germination. The causes for differences between
various plant species in this respect are still unknown at
present.
As shown in Fig. (2), the absolute TAG and ACL contents in the
seedling cotyledons remained nearly constant during the first
three days-long imbibition. Growth induction following three
days after the onset of germination was accompanied by a rapid
decrease in the cotyledon total TAG level, but at the 8th day, i.e.
after the dark seedling growth slowed down (Fig. 1), TAG
content did not change any longer. Thus, intense shifts in the
TAG level took place only during the period of active growth.
Cotyledon ACLs included, along with TAGs, also polar lipids,
and their predomination at the later stages of germination
reflected an intense formation of cotyledon membrane lipids
accompanying the TAG breakdown.
Fig. (1). Developmental changes in the fresh and dry weights
(FWand DW) of sea buckthorn seedling cotyledons. The
means of five replications each containing 20 seeds and their
37 °C for 60 minutes in a 10 μl volume. In vivo ubiquitination assays
were performed as described [23].
CK2 Regulates PDEF
RESULTS
Production and Characterization of an Anti-PDEF
Polyclonal Antiserum
To reduce the likelihood of cross-reactivity with other ETS
factors, the N-terminal 141 amino acids of human PDEF were
chosen as the antigen (Fig. 1A). PDEF1-141 does not bear
significant homology to other ETS factors or other human
proteins, however, there is 89% homology in this region
between mouse and human PDEF. This region was cloned
into the bacterial expression vector pEQ80L, and expressed in
BL21 cells. The protein was enriched from bacterial lysates
by nickel affinity and anion exchange chromatography to
~90% purity. New Zealand White rabbits were immunized
following a standard boost/bleed regimen, and postimmunization sera were screened by Western blot analysis of
LNCaP cells transfected with exogenous PDEF (data not
shown) and LNCaP cells treated with siRNA specific to
PDEF (Fig. 1B). A prominent 46 kDa species was observed,
and was specifically diminished in the PDEF siRNA-treated
cells. This apparent molecular weight is consistent with
previous analyses of PDEF by SDS-PAGE [9, 10, 24]. Other
molecular weight species were variably observed in Western
blot analyses (Fig. 1B) however these were not diminished by
treatment with PDEF siRNA and represent cross-reactivity of
The Open Cancer Journal, 2010, Volume 3 111
the polyclonal sera. To further verify the specificity of the
anti-PDEF1-141, parallel Western blots with commercially
available (Zymed, Santa Cruz Biotechnology), and existing
anti-PDEF antibodies [8, 24] were performed (data not
shown). This comparison indicated that our anti-PDEF
antibody recognized a 46 kDa isoform PDEF
indistinguishable from that recognized by extant antibodies.
Protein Kinase CK2 Phosphorylates PDEF
To determine if PDEF can be phosphorylated by CK2, in vitro
kinase reactions were carried out with full-length recombinant
PDEF or the N-terminal domain (amino acids 1-141) with
CK2 holoenzyme or CK2α alone in the presence of γ-32PATP. Robust phosphorylation was observed after gel
electrophoresis followed by autoradiography (data not
shown). To determine specific sites of CK2 phosphorylation
in PDEF, in vitro kinase reactions were analyzed by LCMS/
MS. Products of kinase reactions were resolved by
twodimensional SDS-PAGE, silver stained, excised, and
digested with trypsin. Mass spectra obtained from nonphosphorylated PDEF were compared with spectra generated
from PDEF phosphorylated by CK2. These data revealed
evidence for three CK2 phosphoacceptor sites in PDEF:
Serine 187, in a fragment spanning amino acids 182-192,
ELCAMS*EEQFR, with a mass of 1342.5766, and Threonine
144 and Serine 151, in a fragment spanning amino
Fig. (1). Production of an anti-PDEF polyclonal antibody. A. The N-terminal region of PDEF (amino acids 1-141) was used to
immunize rabbits to generate polyclonal anti-PDEF antibodies. B. Western blot detection of endogenous PDEF protein in LNCaP
cells 24 hours after transfection with a PDEF siRNA or a negative control siRNA. The 46 kDa species is significantly diminished
specifically in the PDEF siRNA-treated lanes. The blots were stripped and probed with an antibody against α-tubulin as a loading
control.
112 The Open Cancer Journal, 2010, Volume 3
Chisholm et al.
Table 1. Peptides Identified by Mass Spectrometric Analysis of Recombinant PDEF Phosphorylated in vitro
by CK2
1SEQUEST data identifying the three phosphoacceptor sites in the pointed domain of PDEF: Thr144, Ser151, and Ser187. highlighted in red are
peptides containing the Ser187 phosphoacceptor site and in green are the Thr144, and Ser151 containing peptides. A +80 amu and/or -18 amu
Shift in MH+ in the phospho-peptide over the non-phosphorylated peptide confirms phosphorylation at these sites. ‘*’Denotes phosphorylated
site identified through gain of 80 amu and ‘#’denotes phosphorylated site identified through loss of 18 amu.
acids 140-157, LLNIT*ADPMWS*PSNVQK, with a mass
of 2029.0059 (Table 1).
Inhibition of CK2 Reduces the Steady State Level
of PDEF in Prostate Cancer Cells
Apigenin (4',5,7,-trihydroxyflavone) is a common
flavonoid distributed widely in fruits and vegetables [25],
and is a potent inhibitor of CK2 and several other kinases
[26]. To determine if apigenin exposure affects PDEF
stability in prostate cancer cells, LNCaP cells were cultured
in the presence or absence of apigenin and the steady-state
level of PDEF was monitored by Western blot analysis
after 4 hours of exposure. Significant down-regulation of
PDEF was observed in LNCaP cells after 4 hours of
apigenin exposure, raising the possibility that CK2
blockade altered PDEF accumulation (Fig. 2A). However,
while apigenin is a potent inhibitor of CK2, it has also been
shown to block the activity of other kinases in vitro [26].
To further investigate the possibility that CK2 inhibition
may affect PDEF level, LNCaP cells were treated with the
highly CK2-selective inhibitor TBB [27]. After 4 hours of
TBB treatment, the steady-state level of PDEF in LNCaP
cells was reduced to an extent similar to that observed after
apigenin treatment (Fig. 2C). Parallel Northern blots
conducted on apigenin (Fig. 2A, bottom panel) and TBBtreated LNCaP cells (data not shown) revealed that the
effect occurs post-transcriptionally, since PDEF mRNA
accumulation was not affected by CK2 pharmacologic
inhibition. In contrast, NKX3.1 has been shown to be
affected at both the protein and mRNA level in similar CK2
pharmacologic inhibition studies [16]. Morphologic
analysis by light microscopy revealed that approximately
10-30% of the cells lost contact with the cell culture vessel
and were rounded and floating during the course of
apigenin and TBB treatment. This effect was seen as early
as 60 minutes after exposure to CK2 inhibitory agents.
To determine the role of both CK2 catalytic subunits in
maintaining the steady-state level of PDEF, specific
siRNAs were used to knock down either CK2α or CK2α′.
In LNCaP cells transfected with a CK2α′-specific siRNA,
PDEF accumulation was diminished after 24 hours (Fig.
2D). In contrast, knockdown of CK2α did not affect the
protein level of PDEF (Fig. 2D).
Blocking the 26S Proteasome Reverses the Effect of CK2 Inhibition on PDEF Accumulation
Based on the results of pharmacologic and siRNAmediated CK2 inhibition, we hypothesized that phosphorylation by CK2
stabilized PDEF, and that the abrogation of CK2 activity resulted in PDEF degradation, possibly by the 26S proteasome pathway.
To determine if this was the case, LNCaP cells were treated with apigenin in the presence or absence of the proteasome inhibitor
MG132. The effect of apigenin on PDEF accumulation was reversed in the presence of MG132 (Fig. 2B), suggesting that PDEF
is degraded by the 26S proteasome in prostate cells and that phosphorylation by CK2 prevents this degradation.