Organoselenides from Nicotiana tabacum genetically modified to accumulate selenium

Article abstract of DOI:10.1016/j.phytochem.2009.06.001

Nicotiana tabacum L. (tobacco) plants were transformed to overexpress a selenocysteine methyltransferase gene from the selenium hyperaccumulator Astragalus bisulcatus (Hook.) A. Gray (two-grooved milkvetch), and an ATP-sulfurylase gene from Brassica oleracea L. var. italica (broccoli). Solvent extraction of leaves harvested from plants treated with selenate revealed five selenium-containing compounds, of which four were identified by chemical synthesis as 2-(methylseleno)acetaldehyde, 2,2-bis(methylseleno)acetaldehyde, 4-(methylseleno)-(2E)-nonenal, and 4-(methylseleno)-(2E,6Z)-nonadienal. These four compounds have not previously been reported in nature.

Full text of DOI:10.1016/j.phytochem.2009.06.001

Phytochemistry 70 (2009) 1098–1106
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Organoselenides from Nicotiana tabacum genetically modified
to accumulate selenium
*
Adam J. Matich , Marian J. McKenzie, David A. Brummell, Daryl D. Rowan
The New Zealand Institute for Plant and Food Research Limited (Plant and Food Research), Private Bag 11600, Palmerston North 4442, New Zealand
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 April 2009
Received in revised form 28 May 2009
Available online 29 June 2009
Nicotiana tabacum L. (tobacco) plants were transformed to overexpress a selenocysteine methyltransfer-
ase gene from the selenium hyperaccumulator Astragalus bisulcatus (Hook.) A. Gray (two-grooved milk-
vetch), and an ATP-sulfurylase gene from Brassica oleracea L. var. italica (broccoli). Solvent extraction of
leaves harvested from plants treated with selenate revealed five selenium-containing compounds, of
which four were identified by chemical synthesis as 2-(methylseleno)acetaldehyde, 2,2-bis(methylse-
leno)acetaldehyde, 4-(methylseleno)-(2E)-nonenal, and 4-(methylseleno)-(2E,6Z)-nonadienal. These four
compounds have not previously been reported in nature.
Keywords:
Nicotiana tabacum
Tobacco
Ó 2009 Elsevier Ltd. All rights reserved.
Selenium
Volatiles
Methylselenocysteine
Selenocysteine methyltransferase
ATP-sulfurylase
1. Introduction
quire the SMT enzyme. An exchange reaction between MeS2Me
and MeSe2Me produces dimethylselenosulfide (MeSeSMe)
Selenium (Se) and sulfur (S) are chemically similar and hence
flowering plants do not differentiate in their uptake of these two
elements from the soil (White et al., 2007). Thus selenium (as sel-
enate) is taken up by the plant’s roots and processed via the sulfur
assimilation pathway to ultimately form the selenium analogues of
cysteine and methionine; selenocysteine (SeCys) and selenomethi-
onine (SeMet) (Brown and Shrift, 1981). In plants not adapted to
higher levels of selenium in the soil (non-accumulating plants),
these seleno-amino acids are mis-incorporated into proteins
resulting in Se toxicity with symptoms such as stunting, necrotic
lesions on the leaves, and reduced root growth.
Selenium accumulators or hyperaccumulators are plants that
tolerate high levels of selenium uptake and assimilation. They
are able to take up and metabolise inorganic Se to organoselenides
that are not harmful to the plant and which may in part be
removed from the plant by volatilisation (Sors et al., 2005; Terry
et al., 2000). This Se tolerance is created by the selenocysteine
methyltransferase (SMT) enzyme which methylates selenocysteine
(SeCys) to the well-tolerated methylselenocysteine (MeSeCys)
(Neuhierl et al., 1999; Sors et al., 2005; Terry et al., 2000), which
in turn is catabolised to produce the volatile, dimethyl diselenide
(MeSe2Me). Dimethyl selenide (MeSeMe) is also produced in large
quantities, but from methylselenomethionine, which does not re-
(Kubachka et al., 2007; McKenzie et al., 2009; Meija et al., 2002).
Se-accumulating (de Souza et al., 1998, 2002) and non-accumu-
lating plants, which have been genetically engineered for overex-
pression of
a
SMT transgene, have potential for the
phytoremediation of Se-contaminated soils (Ellis et al., 2004;
LeDuc et al., 2004, 2006). For phytoremediation, the discharge of
volatile organoselenides from the plant is more important than
their accumulation, since volatilisation removes Se from the local
food chain and the plants also achieve a longer useful life of remov-
ing Se from the soil rather than needing to be harvested, removed,
and then replanted (Tagmount et al., 2002). Interest in enhancing
organoselenide concentrations in plants has also arisen from the
importance of an appropriate selenium intake for human health
(Rayman, 2008) and the apparent linkage of selenium intake to a
reduced risk of cancer (Ellis et al., 2004; McKenzie et al., 2009).
In a previous study (McKenzie et al., 2009), we demonstrated
the accumulation of MeSeCys and c-glutamyl-MeSeCys (GluMeSe-
Cys) in tobacco plants overexpressing a constitutively controlled
SMT transgene from Astragalus bisulcatus (Hook.) A. Gray. Accumu-
lation of MeSeCys and GluMeSeCys was greater again in tobacco
plants overexpressing the SMT gene as well as an ATP-sulfurylase
transgene (ATPS) from broccoli (Brassica oleracea L. var italica). In
addition to seleno-amino acid production, the transformed tobacco
plants produced the volatile organoselenides, MeSeMe, MeSe2Me,
and MeSSeMe, which were measured in the headspace above the
plants.
* Corresponding author. Tel.: +64 6 9537679; fax: +64 6 3517701.
E-mail address: amatich@hortresearch.co.nz (A.J. Matich).
0031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2009.06.001

A.J. Matich et al. / Phytochemistry 70 (2009) 1098–1106
1099
In the above study, solvent extracts of the tobacco leaves also
contained organoselenides, which were not found in the head-
space. We have not found reports of such ‘non-volatile’, non-amino
acid organoselenides in Se-accumulating plants. This study em-
ployed chemical synthesis to identify four of the ‘non-volatile’
organoselenides detected in the two tobacco populations. The
identification of 2-(methylseleno)acetaldehyde 1, in particular,
suggests the operation of a new biosynthetic pathway for selenium
mobilisation occurring in these Se-accumulating plants.
Zhang and Chasteen, 1994). MeSeSMe may arise from dispropor-
tionation of MeSe2Me and MeS2Me (Chasteen, 1993). However,
while MeSe2Me was present in all transgenic plant lines, only
traces of MeS2Me were detected in extracts of two of the four plant
lines (SMT2 and ATPS/SMT24, data not shown). These two organo-
selenides are highly volatile, as is evidenced by their detection in
the headspace above the live plants (McKenzie et al., 2009). There-
fore the concentrations given in Table 1 are likely to be lower limit
estimates as there will have been loss of these compounds during
solvent extraction and concentration of their solvent extracts.
MeSeMe was not found in the solvent extracts, presumably be-
cause of its volatility.
2. Results and discussion
In addition to MeSe2Me, and MeSeSMe (McKenzie et al., 2009),
five organoselenium compounds ((1)–(4) and Unknown 1), that
were not found in the headspace above the plants, were identified
in the solvent extracts (Table 1) based on their distinctive Se iso-
tope patterns. Only line ATPS/SMT24 produced all compounds,
including the single occurrence of Unknown 1. As too little sample
was available to allow isolation and identification by NMR, possi-
ble structures for these volatiles were deduced from the mass spec-
tral data and candidate compounds were prepared by organic
synthesis for direct comparison of GC retention times and MS
fragmentation patterns with the compounds extracted from the
tobacco plants.
2.1. Chemical analysis of SMT and ATPS/SMT transformed tobacco
plants
The first two organoselenides to elute from the GC–MS were
MeSeSMe (RT 9.12 min) and MeSe2Me (RT 12.62 min), which were
previously identified in the headspace above these plants
(McKenzie et al., 2009). MeSe2Me, derived from MeSeCys, was
the major selenium compound found in the tobacco leaf extracts
(Table 1), and in some extracts it was a significant contributor to
the total volatile profile (Fig. 1). MeSe2Me is the most commonly
reported organoselenide in selenium accumulating organisms
(Kubachka et al., 2007; Meija et al., 2002, 2003; Shah et al., 2007;
Table 1
Semi-quantitative analysis of organoselenium compounds in solvent extracts of the leaves from tobacco plants overexpressing an AbSMT (SMT) transgene or BoATPS and AbSMT
(ATPS/SMT) transgenes measured by GC–MS as M sodium selenate for 10 (SMT) and
l
g equivalents of MeSe2Me kgꢀ1 (FW). Leaves were harvested following treatment with 300
l
15 (ATPS/SMT) days. Each value is the average of two samplings from the same plant. Selenium compounds were not found in selenate-treated wild type plants or in transgenic
plants that were not treated with selenate (McKenzie et al., 2009).
Compound
Ret. time (min)
Plant line
SMT37
SMT2
ATPS/SMT24
ATPS/SMT47
MeSeSMe
MeSe2Me
MeSeCH2CHO (1)
Unknown 1
(MeSe)2CHCHO (2)
4-(Methylseleno)-(2E)-nonenal (3)
4-(Methylseleno)-(2E,6Z)-nonadienal (4)
9.12
12.62
17.36
19.34
27.5
5
121
31
0
0
0
9
58
39
0
2
0
11
860
49
9
10
18
40
41
750
97
0
12
29.7
30.4
47
119
5
0
Fig. 1. TIC GC–MS traces showing the selenium metabolites found in solvent extracts of tobacco plants overexpressing both BoATPS and AbSMT, and treated with 300 lM
sodium selenate. Not all metabolites were found in each sample. Sections of the GC–MS traces of extracts of different plants are shown to the same vertical and horizontal
scale.

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A.J. Matich et al. / Phytochemistry 70 (2009) 1098–1106
The third eluting compound, 2-(methylseleno)acetaldehyde (1
C-atoms as oxalic acid (HOOCCOOH), to give 1 (2-(methylse-
leno)acetaldehyde). Aldehyde 1 might also be formed from MeSe-
Cys by a mechanism analogous to that for the catabolism of
branched-chain amino acids, such as isoleucine, by yeast (Dickin-
son et al., 2000) as proposed in Fig. 3. Thus, transamination pro-
duces a 2-oxo carboxylic acid which can be decarboxylated
directly to 1. Alternatively, the 2-oxo acid may undergo decarbony-
lation, oxidation and CoA formation, and finally reduction to 1
(Fig. 3). A similar pathway has been proposed for the catabolism
of methionine to 3-(methylthio)propanal in Saccharomyces cerevi-
siae (Perpète et al., 2006).
The formation of aldehyde 1 in the transformed tobacco plants
is probably a consequence of introducing a new substrate into a
pre-existing metabolic pathway. This new substrate might be
either MeSeCys or SeMet, however the nature of the pathway is
presently unknown. Certainly, a methionine catabolism pathway
to dimethyl sulfide appears to exist in tobacco, as wild-type tobac-
co plants produce dimethylselenide when treated with selenate
(McKenzie et al., 2009). There is no evidence for catabolism of
in Fig. 2, RT 17.36 min), was the second most prevalent organosel-
enide in the solvent extracts (Table 1 and Fig. 1). The molecular for-
mula was determined as C3H6OSe by high resolution EI-GC–MS,
with the fragment ion m/z 108.9554 (C2H5Se+) indicating loss of
an aldehydic group (Fig. 3). A dominant feature of the fragmenta-
tion of alkylselenides is the loss of the alkyl group as an alkene,
such as the loss of C2H4 from an ethyl selenide or C3H6 from a pro-
pyl selenide (Kubachka et al., 2007; Meija et al., 2002; Shah et al.,
2007). No such fragments were observed (Fig. 3) and so the com-
pound was unlikely to have an ethyl or propyl moiety. The mass
spectrum did not match that reported for acetylselenomethane
(Tsai et al., 1998), which has a very simple mass spectrum with
prominent ions centred around m/z 138 (M+) and m/z 93 (SeCH+),
and a base peak at m/z 43, but lacks the prominent ion cluster
found around m/z 109 ðCH3SeCHþÞ (Fig. 3). The reported high mass
2
ions for aldehyde 1 (Adlington and Barrett, 1981), m/z 138, 136,
109, 107, 95, and 93, were in good agreement with the clusters
of ions observed for the tobacco compound, although the lower
mass ions (m/z 83, 57, and 43) did not match convincingly. Never-
theless, we synthesised aldehyde 1 and its GC retention time and
mass spectrum (Fig. 3) agreed with those of the tobacco leaf com-
pound. While 1 is a known synthetic compound (Adlington and
Barrett, 1981), we have not found this compound reported as a nat-
ural product, and so 1 represents a new compound in the metabo-
lism of selenium-accumulating plants.
MeSeCys by
a naturally-occurring MeCys-specific catabolic
pathway and the existence of such a pathway might be disputed
since MeCys was only detected in the SMT-transformed plants
(McKenzie et al., 2009).
The fourth organoselenide (Unknown 1, RT 19.34 min) to elute
from the GC–MS system was present at less than 1% of the organo-
selenides in a single solvent extract (Table 1). The molecular for-
mula was C4H6O2Se (m/z 165.9533), with the base peak at m/z
43.0188 (C2H3O+) (Fig. 4). Significantly, Unknown 1 showed m/z
137.9589 (C3H6OSe+), which resulted from loss of CO rather than
C2H4 (Kubachka et al., 2007; Meija et al., 2002). Therefore, this
compound does not have an alkyl moiety larger than CH3. The mass
Biosynthetically, aldehyde 1 may arise from the catabolism of
SeMet in a manner analogous to that of methionine by Lactococcus
lactis (Bonnarme et al., 2004). The first step would be transamina-
tion of SeMet to the 2-oxo acid, 4-(methylseleno)-2-oxobutanoic
acid, which is then oxidatively cleaved, with the removal of two
Fig. 2. Structures of selenium-containing volatiles identified in solvent extracts of leaves from transgenic tobacco plants, and of synthetic candidate compounds.

A.J. Matich et al. / Phytochemistry 70 (2009) 1098–1106
1101
Fig. 3. MS fragmentation pattern of 2-(methylseleno)acetaldehyde (1) found in solvent extracts of the tobacco plants overexpressing both BoATPS and AbSMT, and treated
with 300 M sodium selenate and possible pathways in transgenic tobacco leaves for the metabolism of MeSeCys to 1. E1, branched-chain amino acid amino transferase; E2,
l
2-oxoacid decarboxylase (cf. pyruvate decarboxylase); E3, 2-oxoacid dehydrogenase; E4, acyl-CoA hydrolase (Dickinson et al., 2000); E5, carboxylic acid and acyl-CoA
reductases.
spectrum of Unknown 1 (Fig. 4) also had features in common with
acetylselenomethane (Tsai et al., 1998). Therefore, the candidate
compounds, 2-(seleno)bis(acetaldehyde) (5) (Blount and Robinson,
1932; Ficken et al., 1968), 2-(acetylseleno)acetaldehyde (6), and
bis(acetyl)selenide (7) (Kageyama et al., 1989) were synthesised.
The mass spectra of 5 and 7 are not available, and (6) has not been
reported in the literature. Neither the GC retention times nor the
mass spectra of these compounds matched that of Unknown 1
(Fig. 4). Other candidate compounds included methylseleno pyru-
vate (10), and 2-(methylseleno)malonaldehyde. If Unknown 1 is
methylseleno pyruvate, rearrangement is required to produce m/
Se–Se bond, and therefore the above-mentioned m/z 175 ðMeSeþ2 Þ
fragment probably arose from rearrangement of (MeSe)2CH+ (m/z
203) to MeSeSe+CHMe prior to loss of 28 (C2H4). Similarly, our
hydrolysis of (2,2-dimethoxyethyl)(methyl)selane produced
2
identical with the compound found in tobacco leaves. Aldehyde 2
would seem to arise from an acid-catalysed reaction between the
keto and enol forms of 2-(methylseleno)acetaldehyde (Fig. 5). As
with 1, 2 has been prepared synthetically (Adlington and Barrett,
1981), but has not been reported from natural sources.
The sixth (3, C10H18OSe, RT 29.7 min) and seventh (4,
C10H16OSe, RT 30.4 min) eluting organoselenides were found par-
ticularly in leaves overexpressing both the ATPS and SMT genes
(Table 1). Compounds 3 and 4 both showed losses of MeSeÅ and
MeSeH to give fragment ions C9H15O+ and C9H14O+ (3 DBE) at m/
z 139 and 138, and C9H13O+ and C9H12O+ (4 DBE) at m/z 137 and
136, respectively. This, together with the 2 amu difference in the
masses at m/z <110, suggested that these compounds differed by
one double bond. The shorter retention time of the more saturated
3 on the polar GC column indicated it was not the alcohol deriva-
tive of 4. Structures related to the reaction of methylselenol with
unsaturated nonenals were indicated and this was supported by
the identification of a number of aldehydes, including (2E,6Z)-non-
adienal (Fig. 1) and (2E)-nonenal in the tobacco leaf extracts.
The fragmentation pattern of aldehyde 4 (C10H16OSe) included
two groups of Se-containing fragment ions centred at m/z 163
(C5H7SeO), corresponding to loss of C5H9, and at m/z 135
(C4H7Se), corresponding to an additional loss of CO. These MS frag-
mentations suggest that this compound has a 4-(methylseleno)-
2,6-nonadienal type structure. To confirm this, (2E,6Z)-nonadienal
was added to (2,2-dimethoxyethyl)(methyl)selane prior to its acid
hydrolysis, with the expectation that during the hydrolysis, a
mechanism akin to that shown in Fig. 5 for the production of (Me-
Se)2CH2CHO (2), would produce a MeSe adduct of (2E,6Z)-nonadi-
enal. Examination of the 1H NMR spectrum of the reaction product
z 138 (M+-CO). Rearrangement of
a-keto esters via decarbonylation
is known (March, 1985), but we are not aware of a precedent in se-
leno-carbonyl compounds.
The fifth eluting Se-compound (2, RT 27.5 min) was the disele-
nide C4H8OSe2 (m/z 231.8935, Fig. 5). The fragments m/z 202.8878
ðC3H7SeþÞ and m/z 175 ðCH3SeþÞ indicated the loss of CHO and of
2
2
C2H4. C2H5Se+ (m/z 109) corresponded to M-SeC2H3O (M+-123).
Thus there appeared to be a terminal carbonyl and an ethyl group.
2-(Ethyldiseleno)acetaldehyde (8) and 3-(methyldiseleno)prop-
anal (9) were synthesised as candidate compounds, but their mass
spectra and RT did not match those of the tobacco compound (2).
Most notably, their m/z 43 (CH2CHO+) and 160 ðSe2þÞ fragments
were not present for 2, which itself had a cluster of ions at around
m/z 203 (C3H7Seþ, Fig. 5) that was not present in the mass spec-
2
trum of 8 and 9. The absence of m/z 160 from the mass spectrum
of 2 suggests that this diselenide does not in fact contain a Se–Se
unit.
A by-product (13%) from the synthesis of aldehyde 1, by acid
hydrolysis of 2,2-dimethoxyethylselenomethane, had a similar
fragmentation to that of 2,2-bis(methylseleno)acetaldehyde (2).
This by-product of the acid hydrolysis of (2,2-diethoxyeth-
yl)(methyl)selane was characterised by NMR and GC–MS
(Adlington and Barrett, 1981). This compound does not have a

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A.J. Matich et al. / Phytochemistry 70 (2009) 1098–1106
for H2 appeared as a doublet of doublet of doublets (ddd, J = 0.7,
7.7, and 15.4 Hz), rather than as a doublet of doublet of triplets
(ddt, J = 1.4, 7.7, and 15.6 Hz) as seen in 2E-nonenal, thus indicating
the presence of a substituent at C4. The multiplicity of H3 was sim-
ilarly reduced to a doublet of doublets (J = 9.7 and 15.3 Hz). H4 ap-
peared as a complex quartet at d 3.05. Compounds 3 and 4 have not
previously been reported.
Given the ready conversion of aldehyde 1 to aldehyde 2 and the
reactivity of 1 with (2E)-nonenal and (2E,6Z)-nonadienal, under
acid conditions in vitro, we cannot be certain whether 2–4 are of
biosynthetic origin or arise as a result of spontaneous chemical
reaction in the plants or in the solvent extracts. Aldehydes 1–4
were not found using headspace SPME, but given their lower vola-
tility with respect to MeSe2Me, this was not unexpected.
Generally, the tobacco plant lines expressing both the AbSMT
and BoATPS transgenes had higher levels of organoselenium com-
pounds than those expressing the AbSMT gene alone (Table 1). In
particular, the ATPS/SMT plants had around 10-fold more Me-
Se2Me than the SMT-expressing plants (Table 1). Also, the doubly
transformed plants had more of the aldehydic organoselenides.
However, as leaves were harvested from the doubly transformed
plants after 15 days of watering with selenate fertiliser, whereas
the harvest from the singly transformed plants was after 10 days,
it is possible that these differences relate more to the amount of
selenium accumulated in the plants.
Transformation with the AbSMT transgene not only converts to-
bacco from a Se non-accumulating species into an accumulator of
MeSeCys (McKenzie et al., 2009, and data presented here), it has
also resulted in the formation of new Se-containing metabolites.
These metabolites may arise both through the catabolism of Se-
amino acids, and by chemical reactions occurring in planta or in
the plant extracts. The identification of aldehyde 1 as 2-(methylse-
leno)acetaldehyde, in particular, suggests the operation of a new
pathway for Se mobilisation in plants with enhanced Se metabo-
lism. The observation of such novel metabolites leads to a caution
regarding the consequences of increasing the accumulation of
organoselenium compounds in plants.
Fig. 4. Mass spectral fragmentation patterns of Unknown 1 from a tobacco plant
overexpressing both BoATPS and AbSMT, and treated with 300 lM sodium selenate,
and the three candidate synthetic compounds.
3. Experimental
showed that this had occurred, with resonances for H2 and H3 of 4
fully resolved from those of the excess of (2E,6Z)-nonadienal also
present. Resonances for 4 were fully assigned using COSY and
selective TOCSY experiments. The signal for H3 appeared as a dou-
blet of doublets (J = 9.6 and 15.4 Hz), rather than as a doublet of
triplets (J = 6.4 and 15.7 Hz) as seen in (2E,6Z)-nonadienal, thus
indicating the presence of a substituent at C4. This was confirmed
by the reduced multiplicity observed for H2 of 4 (ddd, J = 0.7, 7.7,
and 15.4 Hz) compared with H-2 of (2E,6Z)-nonadienal (ddt,
J = 1.3, 6.4, and 15.7 Hz). The resonance assigned to H4 now ap-
peared as a complex quartet at d 3.08 for 4 rather than at d 1.90
for (2E,6Z)-nonadienal. The mass spectrum (Fig. 6) and GC–MS
retention time of the synthetic compound matched those of the
compound in tobacco leaves.
The mass spectrum of 3 was less definitive, including only one
set of weak Se-containing fragmentation ions centred at m/z 133
(C4H7Se78) and m/z 135 (C4H7Se80). However, given the presence
of (2E)-nonenal in the tobacco leaf extracts, it was proposed that
3 might result from reaction of (2E)-nonenal with aldehyde 1. Thus
(2E)-nonenal was added to (2,2-dimethoxyethyl)(methyl)selane
prior to its acid hydrolysis to give a product with the same GC
retention time and MS (Fig. 6) as aldehyde 3 found in the plant ex-
tracts. Examination of the 1H NMR of the synthetic sample showed
resonances for H2, but not H3, of 3 were fully resolved from those
of the excess of (2E)-nonenal present in the sample. Further reso-
nances for 3 were identified using COSY and selective TOCSY
experiments and used to confirm the structure of 3. The signal
3.1. Plant material
The tobacco plants (Nicotiana tabacum L. ‘Samsun’), over-
expressing the SMT-A gene from A. bisulcatus (AbSMTA, GenBank
accession number AJ13143) under the control of the 35S promoter,
either alone or in combination with the constitutively expressed
broccoli BoATPS1 (GenBank accession number U05218) gene were
prepared as described previously (McKenzie et al., 2009). Three
clonal plants of each line were ex-flasked into pots of medium
grade vermiculite (Nuplex, Auckland, New Zealand) and acclima-
tized in a mist tent for 1–2 weeks before moving to a glasshouse,
where they were placed in trays and watered from the bottom
with half strength liquid Hoaglands solution (Hoagland and Arnon,
1950). After a further 2 weeks, when the plants were at the 5–6 leaf
stage, sodium selenate (0 or 300
lM) was added to the watering li-
quid. Plants were watered from below with Se for 10 days (SMT
plants) or 15 days (ATPS/SMT plants) before young (the two top
fully expanded leaves below the meristem leaf cluster) and old
leaves (the second and third fully expanded leaves at the bottom
of the plant) were harvested.
3.2. Solvent extraction of tobacco leaves
Leaf material was removed from the plant, frozen immediately
in liquid nitrogen and stored at ꢀ80 °C until extraction. Frozen

A.J. Matich et al. / Phytochemistry 70 (2009) 1098–1106
1103
Fig. 5. MS fragmentation patterns of 2,2-bis(methylseleno)acetaldehyde (2) extracted from tobacco plants overexpressing BoATPS and AbSMT treated with 300
l
M sodium
selenate, and produced synthetically. Synthesis of 2-(methylseleno)acetaldehyde (1) and a possible mechanism for the acid-catalysed conversion of 1–2 during acetal
hydrolysis.
Fig. 6. MS fragmentation patterns of 3, 4-(methylseleno)-(2E)-nonenal and 4, 4-(methylseleno)-(2E,6Z)-nonadienal, which were identified in solvent extracts of tobacco
plants overexpressing BoATPS and AbSMT, and treated with 300 lM sodium selenate, and of synthetic compounds.
tissue (10 g) was ground in a liquid nitrogen-cooled mortar and
pestle and the cold powder mixed to a slurry in pentane:Et2O
(1:1, v/v, 20 ml). The slurry was stored at 4 °C for 6 days in a Schott
bottle and frozen at ꢀ20 °C before the organic phase was decanted
off. Prior to GC–MS analysis the solvent extracts were dried by pas-
sage through anhydrous MgSO4, and their volumes reduced to
2.0 ml under a gentle stream of nitrogen.
3.3. GC–MS analysis of solvent extracts
GC–MS separations were carried out on an Agilent 6890 N GC
using a 20 m ꢁ 0.18 mm i.d. ꢁ 0.18
lm film thickness DB-Wax
(Agilent) capillary column with a He flow of 1 ml minꢀ1 coupled
to a Waters GCT time of flight (TOF) mass spectrometer with EI
of 70 eV, a scan time of 0.4 s, and for HRMS lock masses of

1104
A.J. Matich et al. / Phytochemistry 70 (2009) 1098–1106
121.0014 and 265.9964 (2,4,6-tris(trifluoromethyl)1,3,5-triazine).
The oven temperature program was 2 min at 30 °C, 2 °C minꢀ1 to
50 °C, 5 °C minꢀ1 to 90 °C, 10 °C minꢀ1 to 180 °C, hold 5 min. Injec-
tions were made into a PTV (Gerstel) injection port at ꢀ40 °C,
which was held splitless for 30 s. Three seconds after the injection,
the injection port temperature was ramped up to 260 °C at
700 °C sꢀ1. This injection port temperature and splitless regime im-
proved the peak shape of the earlier eluting peaks. For the compar-
isons between the tobacco and synthetic compounds, 1-min
splitless injections were made at 220 °C onto a 30 m ꢁ 0.25 mm
tion of the above mixture (81 mg) was taken up in THF (3 ml),
H2O (200 l) and 25 mg of HCl (conc.) and left to stand overnight.
l
The acid was neutralized with satd. aq. NaHCO3 (3 ml) and the
product extracted with pentane (4 ꢁ 25 ml) and dried (MgSO4) to
give 1 (87%) EI-MS, m/z (rel. int.): 138 (80) M+, 136 (35), 109
(100), 107 (51), 96 (26), 95 (55), 94 (50), 93 (74), 92 (31), 91
(34), 83 (23), and 80 (19). 1H NMR (400 MHz, CDCl3): d 2.03 (3H,
s, MeSe), 3.16 (2H, d, J = 4.2 Hz, CH2Se), 9.37 (1H, t, J = 4.2 Hz,
CHO) (Adlington and Barrett, 1981), and a by-product (13%) 2,2-
bis(methylseleno)acetaldehyde (2) EI-MS, m/z (rel. int.): 232 (27)
M+, 230 (23), 203 (21), 137 (19), 109 (51), 107 (38), 96 (28), 95
(56), 94 (51), 93 (100), 92 (30), 91(32), and 80 (23). 1H NMR
(400 MHz, CDCl3): d 1.93 (6H, s, MeSe), 4.44 (1H, d, J = 4.0 Hz,
CHSe), and 9.12 (1H, d, J = 3.9 Hz, CHO) (Adlington and Barrett,
1981).
i.d. ꢁ 0.25
lm film thickness HP-5MS (Agilent) capillary column.
The oven temperature programme was 1 min at 35 °C, 5 °C minꢀ1
to 240 °C, hold 10 min.
3.3.1. Mass spectral data of selenium compounds in tobacco leaves
3.3.1.1. 2-(Methylseleno)acetaldehyde (1). Accurate Mass EI-MS, m/z
(D
) [fragment]: 137.9586 (0.2 mDa) [C3H6O80Se, M+], 108.9554
3.4.2. 4-(Methylseleno)-(2E)-nonenal (3) and 4-(methylseleno)-
(2E,6Z)-nonadienal (4)
(ꢀ0.2 mDa) [C2H80Se, M+-CHO], and 94.9395 (ꢀ0.2 mDa) ½CH830Seꢂ.
5
Crude 2,2-(dimethoxyethyl)(methyl)selane was prepared as for
the synthesis of aldehyde 1, above. For the acid hydrolysis of this
compound, 10 mg of either (2E,6Z)-nonadienal (Aldrich) or (2E)-
nonenal (Aldrich) was added before adding 25 mg of HCl (conc.).
The reaction mixtures were analysed by GC–MS. For the reaction
containing (2E,6Z)-nonadienal, the products were MeSe2Me (82%),
aldehydes 1 (11.5%) and 2 (1.5%), and 4-(methylseleno)-(2E,6Z)-
nonadienal (4) (4.8%). EI-MS, m/z (rel. int.): 232 (14, M+), 217
(3), 163 (15), 137 (29), 136 (27), 135 (23), 121 (14), 107 (41),
96 (52), 95 (63), 94 (56), 93 (64), 91 (53), 80 (45), 79 (76), 68
(75), 67 (100), 55 (31), and 41 (28). 1H NMR (400 MHz, d6-ben-
zene) d 9.42 (1H, d, J = 7.7 Hz, H1), 6.28 (1H, dd, J = 9.6 and
15.4 Hz, H3), 5.75 (1H, ddd, J = 0.7, 7.7, and 15.4 Hz, H2), 5.50
(1H, m, H7), 5.29 (1H, m, H6), 3.08 (1H, m, H4), 2.31 (m, H5),
1.96 (m, H8), and 0.96 (t, J = 7.6 Hz, H9). For the reaction obtained
after addition of (2E)-nonenal, the products were MeSe2Me (31%),
aldehydes 1 (29%) and 2 (10%), 4-(methylseleno)-(2Z)-nonenal
(3%), and 4-(methylseleno)-(2E)-nonenal (3) (27%). EI-GC–MS, m/
z (rel. int.): 234 (4, M+), 232 (2), 219 (1), 139 (6), 135 (5), 108
(10), 97 (11), 96 (30), 95 (29), 94 (23), 93 (26), 92 (9), 84 (22),
83 (41), 81 (62), 69 (65), 55 (100), and 53 (30). 1H NMR
(400 mHz, d6-benzene) d 9.45 (1H, d, J = 7.7 Hz, H1), 6.25 (1H,
dd, J = 9.7 and 15.3 Hz, H3), 5.75 (1H, ddd, J = 0.7, 7.7, and
15.4 Hz, H2), 3.05 (1H, m, H4), 1.45 (m, H5), 1.1–1.55 (m, H6–
H8), and 0.98 (t, J = 7.1 Hz, H9).
3.3.1.2. 2,2-bis(Methylseleno)acetaldehyde (2). Accurate Mass EI-MS,
m/z
(D)
[fragment]: 231.8935 (2.9 mDa) [C4H8O80Se2, M+],
202.8878 (0.0 mDa) [C3H80Se2, M+-CHO], and 108.9548
7
(ꢀ0.8 mDa) ½C2H80Seꢂ.
5
3.3.1.3. 4-(Methylseleno)-(2E)-nonenal (3). Accurate Mass EI-MS, m/
z (D
) [fragment]: 234.0564 (4.1 mDa) [C10H18O80Se, M+], 219.0365
(7.7 mDa) [C9H15O80Se, M+-CH3], and 139.1135 (1.2 mDa)
[C9H15O].
3.3.1.4. 4-(Methylseleno)-(2E,6Z)-nonadienal (4). Accurate Mass EI-
MS, m/z (D
) [fragment]: 232.0366 (1.0 mDa) [C10H16O80Se, M+],
217.0112 (ꢀ2.0 mDa) [C9H13O80Se, M+-CH3], 162.9693 (+3.1 mDa)
[C5H7Se80O], 137.0966 (ꢀ1.1 mDa) [C9H13O], 136.0878 (ꢀ1.0
mDa) [C9H12O], 134.9896 (+10.6 mDa) [C4H7Se80], 108.9580
(2.4 mDa) ½C2H80Seꢂ, and 67.0541 (ꢀ0.7 mDa) [C5H7].
5
3.3.1.5. Unknown 1. EI-MS, m/z (rel. int.): 166 (1.3), 138 (4), 136
(1.4), 96 (2), 95 (6.5), 94 (2.5), 93 (6), 91 (3), 80 (2.4, Se), and 43
(100). Accurate Mass EI-MS, m/z
(D) [fragment]: 165.9533
(0.0 mDa) [C4H6O80Se, M+], 137.9589 (0.5 mDa) [C3H6O80Se, M+-
2
CO], 94.9408 (0.8 mDa) CH830Se, and 43.0188 (0.4 mDa) C2H3O.
3.4. Synthesis of candidate compounds
All Se-containing compounds are presumed to be highly toxic
and should be handled with appropriate precautions. To minimise
risk while handling these volatile and potentially toxic materials,
chemical reactions were conducted on a small scale and designed
to minimise handling, purification steps and the generation of
chemical wastes.
3.4.3. 2-(Seleno)bis(acetaldehyde) (5)
Sodium chips (100 mg, 4.3 mmol) were added to Se powder
(171 mg, 2.1 mmol) and naphthalene (55.3 mg, 0.43 mmol) in dry
distilled THF (7 ml) under N2. The mixture was stirred for 24 h to
yield a pale pink soln. of NaSeNa (Thompson and Boudjouk,
1988). Bromoacetaldehyde dimethyl acetal (500 ll, 4.2 mmol)
was added and the mixture refluxed for 3 h to produce a cream
ppt. in a yellow soln. The reaction was filtered to give a soln. of
unreacted bromoacetaldehyde dimethyl acetal (29%), bis(2,2-
dimethoxyethylselenide) (26%), and bis(2,2-dimethoxyethyl)sele-
nide (45%). EI-MS, m/z (rel. int.): 258 (3) M+, 195 (8), 137 (12),
109 (7), 76 (13), 75 (100), 58 (16), 47 (11), and 43 (14). Solvent
was removed and the residue taken up in THF (3 ml) and water
3.4.1. 2-(Methylseleno)acetaldehyde (1)
NaBH4 (60 mg) was added to a solution of MeSe2Me (100 mg,
530 lmol, Acros) in absolute EtOH (5 ml) under N2. After the colour
of the reaction mixture changed from yellow to white, bromoacet-
aldehyde dimethyl acetal (2,2-dimethoxyethylbromide, 127 mg,
750 lmol, Aldrich) was added. After 90 min of stirring under N2,
the colour reverted to yellow. Further NaBH4 (20 mg) was added
and the reaction stirred overnight. Water (5 ml) was added and
the solution extracted with distilled pentane (4 ꢁ 25 ml) and dried
with MgSO4. The solvent was removed in vacuo and to give a yel-
low oil (108 mg), which by GC–MS consisted of MeSe2Me (65%)
and (2,2-dimethoxyethyl)(methyl)selane (35%). EI-MS, m/z (rel.
int.): 184 (25) M+, 153 (35), 137 (3), 109 (15), 93 (13), 75 (100),
58 (17), 47 (15), and 43 (12). 1H NMR (400 MHz, CDCl3): d 2.07
(s, MeSe), 2.71 (d, J = 5.6 Hz, CH2Se), 4.57 (t, J = 5.6 Hz, CH). A por-
(200 ll). HCl (25 mg, conc.) was added and the sample stirred un-
der argon for 24 h at room temperature to produce 5. NaHCO3
(5 ml, satd. aq.) was added, the mixture extracted with 3 ꢁ 50 ml
of pentane:Et2O (50:50), and dried (MgSO4). The mixture contained
an unknown organoselenide (26%), the non-hydrolysed bis(2,2-
dimethoxyethylselenide) (48%), and 5 (26%), reported (Blount
and Robinson, 1932; Ficken et al., 1968) but MS and NMR data
are not available. The MS was consistent with the target compound
5. EI-GC–MS, m/z (rel. int.): 166 (16) M+, 109 (74), 124 (43), 123

A.J. Matich et al. / Phytochemistry 70 (2009) 1098–1106
1105
(21),107 (52), 95 (64), 94 (100), 93 (88), 92 (43), 91 (40), 85 (31), 43
(50), and 42 (27).
gon for 18 h at room temperature, during which time the dark red
NaSe2Na soln. changed to a pale orange. The reaction mixture was
extracted with Et2O (3 ꢁ 25 ml) and dried (MgSO4). MeSe2Me
3.4.4. 2-(Acetylseleno)acetaldehyde (6)
(30 ll) was added and the solution stirred under argon for 2 h.
To Se powder (30 mg, 375
der argon was added a solution of NaBH4 (30 mg, 790
gassed water (2.5 ml). After stirring for 20 min a colourless soln. of
NaSeH formed. Se powder (30 mg) was added and the flask placed
in hot water (65 °C) to speed the formation of the red/brown Na-
Se2Na (Klayman and Griffin, 1973). The soln. was allowed to cool
l
mol) in degassed water (2.5 ml) un-
GC–MS showed the major constituents to be the unreacted
bis(2,2-dimethoxypropylselenide), bis(2,2-dimethoxypropyl)sele-
nide, and a poor yield (1%) of 1-(3,3-dimethoxypropyl)-2-methyl-
diselane. EI-MS, m/z (rel. int.): 278 (11), 276 (12), 151 (16), 103
(12), 75 (100), 73 (10), 71 (80), 57 (11), 55 (8), 47 (38), 45 (63),
41 (42), and 39 (10). The above sample was filtered and stirred
for 1 h, under argon, with p-toluenesulfonic acid (10 mg) to give
a sample of aldehyde 9 (26% of a mixture of organoselenides). EI-
MS, m/z (rel. int.): 232 (100), 230 (85), 229 (32), 228 (54), 176
(54), 175 (33), 174 (56), 173 (40), 172 (43), 160 (48), 158 (47),
109 (25), 107 (22), 95 (31), 93 (60), and 57 (86).
lmol) in de-
and bromoacetaldehyde dimethyl acetal (88 ll, 750 lmol) was
added and the mixture stirred for 22 h under argon, after which
the product was taken up in 100 ml of pentane, filtered and dried
(MgSO4). The mixture contained unreacted bromoacetaldehyde di-
methyl acetal (81%), the by-product bis(2,2-dimethoxyethyl)sele-
nide (6%), and bis(2,2-dimethoxyethylselenide) (13%). EI-MS, m/z
(rel. int.): 338 (1) M+, 275 (0.5), 217 (2), 191 (1), 160 (2), 138 (6),
107 (6), 93 (5), 75 (100), 58 (34), and 43 (27).
Acknowledgements
Solvent was removed and the sample was taken up in dry THF
(4 ml) and stirred with of 1 M Li(Et)3BH (1.5 ml, 1.5 mmol, Acros)
for 30 min under argon. Acetyl chloride (200 ll) was added and
We thank Ian King for care of the plants, Martin Hunt for GC–
MS, and Barry Bunn for NMR. This work was funded by the New
Zealand Foundation for Research, Science and Technology (FRST)
contract CO6X0207 and by Vital VegetablesÒ, a research project
jointly funded by Horticulture Australia Ltd., The New Zealand
Institute for Plant and Food Research Ltd., FRST, the Victorian
Department of Primary Industries, the New Zealand Vegetable
and Potato Growers Federation Inc., and the Australian Vegetable
and Potato Growers Federation Inc.
stirred for 30 min, when satd. aq. NaHCO3 (10 ml) was added and
the mixture extracted with Et2O (4 ꢁ 25 ml), and dried (MgSO4)
to produce 1-acetylseleno-2,2-dimethoxyethane. EI-MS, m/z (rel.
int.): 181 (2) M+-OCH3, 138 (18), 107 (10), 93 (5), 75 (85), 58
(29), 47 (21), and 43 (100). The above ethereal solution was stirred
with p-toluenesulfonic acid (40 mg) under argon for 19 h to give 6
(96% hydrolysis). EI-GC–MS, m/z (rel. int.): 166 (1) M+, 124 (0.5), 93
(8), 80 (4), 57 (4), and 43 (100).
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