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First published online June 26, 2003; 10.1104/pp.103.025361 Plant Physiology 132:1448-1463 (2003) © 2003 American Society of Plant Biologists Expression of Anthocyanins and Proanthocyanidins after Transformation of Alfalfa with Maize Lc1,2Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, Saskatchewan, Canada S7N 0X2 (H.R., M.Y., P.A., L.B.-B., N.W. B.C., M.Y.G.); Plant Biotechnology Institute, 110 Gymnasium Place, Saskatoon, Saskatchewan, Canada S7N 0W9 (H.R.); Department of Plant Agriculture, University of Guelph, Guelph, Ontario, Canada N1G 241 (S.B., B.M.); and Department of Botany, Institute for Cellular and Molecular Biology, University of Texas, Austin, Texas 78713 (A.L.)
Three anthocyanin regulatory genes of maize (Zea mays; Lc, B-Peru, and C1) were introduced into alfalfa (Medicago sativa) in a strategy designed to stimulate the flavonoid pathway and alter the composition of flavonoids produced in forage. Lc constructs included a full-length gene and a gene with a shortened 5'-untranslated region. Lc RNA was strongly expressed in Lc transgenic alfalfa foliage, but accumulation of red-purple anthocyanin was observed only under conditions of high light intensity or low temperature. These stress conditions induced chalcone synthase and flavanone 3-hydroxylase expression in Lc transgenic alfalfa foliage compared with non-transformed plants. Genotypes containing the Lc transgene construct with a full-length 5'-untranslated region responded more quickly to stress conditions and with a more extreme phenotype. High-performance liquid chromatography analysis of field-grown tissue indicated that flavone content was reduced in forage of the Lc transgenic plants. Leucocyanidin reductase, the enzyme that controls entry of metabolites into the proanthocyanidin pathway, was activated both in foliage and in developing seeds of the Lc transgenic alfalfa genotypes. Proanthocyanidin polymer was accumulated in the forage, but (+)-catechin monomers were not detected. B-Peru transgenic and C1 transgenic populations displayed no visible phenotypic changes, although these transgenes were expressed at detectable levels. These results support the emerging picture of Lc transgene-specific patterns of expression in different recipient species. These results demonstrate that proanthocyanidin biosynthesis can be stimulated in alfalfa forage using an myc-like transgene, and they pave the way for the development of high quality, bloat-safe cultivars with ruminal protein bypass.
The ability to manipulate flavonoid biosynthesis in crop plants is gaining rapidly in importance as new economically important uses are found in the areas of nutraceuticals, food quality, and feed quality. The introduction of proanthocyanidin (PA, a flavonoid polymer) into alfalfa (Medicago sativa) forage is particularly important to ruminant livestock producers. Proanthocyanidins eliminate pasture bloat, improve the efficiency of conversion of plant protein into animal protein (ruminal protein bypass), reduce greenhouse gases, reduce gastrointestinal parasites, and inhibit insect feeding (Waghorn, 1990  ; Neizen
et al., 1995, 1998;
Aerts et al., 1999;
Muir et al., 1999;
McMahon et al., 2000). Alfalfa
forage (leaf and stem tissues) accumulate anthocyanins only at senescence or
locally under some stress conditions such as insect feeding
(Goplen et al., 1980). No
known conditions induce proanthocyanidins in alfalfa forage, although they are
structurally related to anthocyanins. However, these compounds do accumulate
in seed coats (Koupai-Abyazani et al.,
1993).
Anthocyanins and proanthocyanidins share early and middle steps of the
flavonoid biosynthetic pathway, including chalcone synthase (CHS), chalcone
isomerase, flavanone 3
Myc-like genes that regulate anthocyanin biosynthesis are well
known and include the B series, the R series, and
Lc (leaf color) of maize (Zea mays), all of which have very
similar sequences (Ludwig et al.,
1989
Several of the anthocyanin myc and myb regulatory genes
have been tested for their ability to influence anthocyanin or
proanthocyanidin accumulation when expressed in heterologous plants, but the
success of this strategy has varied. For example, Sn was shown to
stimulate or inhibit proanthocyanidin accumulation in Lotus
corniculatus, depending on the tissue type, transformed line, and gene
copy number (Damiani et al.,
1998
We tested the ability of Lc to stimulate the flavonoid pathway and
to alter the composition of flavonoid end products produced in alfalfa forage
and seed coat. Two Lc constructs (each under the control of the same
single cauliflower mosaic virus [CaMV] 35S promoter) were tested, including
one with a nearly full-length leader region and a second one with the leader
region truncated by about 200 bp. Observations in Arabidopsis and tobacco
(Nicotiana tabacum) suggested that the longer leader region may limit
expression (Lloyd et al.,
1992
Development of Transgenic Alfalfa Genotypes After transformation and regeneration from tissue culture, five small populations of new alfalfa genotypes exhibiting resistance to kanamycin and a positive response in an nptII PCR assay were recovered. These populations contained either a 2.2-kb Lc gene, a 2.4-kb Lc gene, a B-Peru gene under one of two promoters, or a C1 gene from maize (Table I). Several of the Lc genotypes developed deep red-green leaves when the newly transformed plantlets were grown in a greenhouse under bright lights (Condition A, Table II), in contrast to the non-transformed parent (control) plantlets that remained bright green under these conditions. New leaflets of Lc plants were bright green and did not display the red phenotype when the young plantlets were transferred to lower light intensity (Condition B, Table II). Also, red color in older leaves of the Lc genotypes disappeared when the plants were removed from the brighter light conditions. Novel color phenotypes were not observed under either of these conditions for the B-Peru and C1 genotypes.
The presence of the introduced genes, and in some cases their promoters, was confirmed by Southern blotting (Fig. 1, top, illustrating the Lc genotypes; promoter and transgene data for B-Peru and C1 genotypes not shown). The blots indicated that a large number of these primary transgenic events (kanamycin resistance and nptII+ plants) had no flavonoid transgene or were missing a promoter (Table I). For example, in the C1 population, 50% of the nptII+ plants had a promoter, whereas only 30% had a flavonoid transgene and a promoter (Table I). In addition, only one-half of the nptII+ genotypes recovered in population 88 actually contained the Lc transgene, whereas a 2-fold larger number of genotypes were recovered with the smaller construct (population 90). Although intact independent transgenic events were easily recovered from Brassica napus using the same maize regulatory gene constructs used in this investigation (S. Wang and M.Y. Gruber, unpublished data), our experience suggests that alfalfa may be particularly sensitive to construct design, transgene type, or transgene size and may sometimes require the development of very large sets of primary transgenic events to select a range of suitable transgenic genotypes.
Northern blots of RNA extracted from immature (folded) and mature (open and expanded) leaf tissue were used to select those primary transgenic events confirmed to have both a flavonoid transgene and a promoter and to place them into transgene expression categories (Fig. 1, bottom; Table I). A similar number of genotypes expressing a maize flavonoid transgene were recovered for the B-Peru and Lc populations, whereas a smaller number were recovered for C1. Selected genotypes were multiplied by rooting shoot cuttings, and these established clones were used to assess response to a variety of conditions.
Several clones (i.e. rooted cuttings) of each of the selected alfalfa genotypes that expressed a maize transgene were tested for phenotypic color changes in response to several different environmental conditions. A minimum of two mature clones of each selected genotype were placed under Conditions C to H (Table II), and 24 young clones (4 months old) for each genotype were placed in the Saskatoon field trial (Condition I). Twenty-four clones of representative genotypes of B-Peru and C1, but not Lc, were also placed in a field trial in Elora (ON, Canada; Condition J). Lc genotypes were not developed early enough to be tested in the latter field trial. Throughout growth under all of the test conditions, B-Peru transgenic and C1 transgenic genotypes (populations 45, 47, and 100) remained green and did not show phenotypic color change (data not shown), although transcripts for these two transgenes were clearly detected on the northern blots (data not shown). Even after 1 month under cold conditions, C1+ genotypes had green leaves and stems, whereas B-Peru+ genotypes had green leaves and only a light-red stem coloration. The untransformed parent genotype A01 (Fig. 2A), clones of untransformed varieties Peace and Beaver, and several transgenic genotypes that poorly expressed the Lc transgene also remained green under all conditions tested (data not shown).
In contrast, genotypes that strongly expressed the 2.4-kb Lc transgene (from population 88, Table I) developed a light-red coloration on newly developed leaf tips under the increased light intensity of Condition C (data not shown), but plants expressing the 2.2-kb Lc transgene (from population 90, Table I) could not be detected under these latter conditions. Immature and mature leaves of the 2.4-kb Lc plants consistently developed a deep red-purple color when the plants were placed under cold temperature with moderate light intensity (Condition E) or under moderate temperature with either high-intensity artificial light (Conditions F and G) or natural sunlight conditions (Condition H, Fig. 2, A and C; Tables II and II). In one clone, 88-19, the red phenotype became visible within 6 h, whereas other selected plants of population 88 required about 24 h. The forage color was less intense when the 2.2-kb Lc plants (from population 90) were exposed to cold or high-intensity light. For example, genotypes 90 developed a faint red leaf phenotype and a red or deep red stem phenotype by 72 h under cold conditions, whereas leaves and stems from genotypes from population 88 turned deep red within 24 h under these conditions. Return to a green phenotype occurred quickly when plants were returned to Condition B. Usually, the "greening" occurred within 2 to 3 d of growth if the plants had been subjected to a photoperiod but took longer (approximately 7 d) if plants had been subjected to continuous light (condition F). In addition to phenotypic color changes under cold or high light conditions, all plants from both Lc+ populations developed a red phenotype while developing roots from cuttings under Condition B. The red coloration was only visible on surfaces of the Lc transgenic plants exposed to light and was particularly noticeable on the abaxial leaf surface of folded leaves, over veins, and on stems (Fig. 2C). In emerging leaflet clusters, the outer abaxial surface was deep red, whereas areas of inner leaflets protected from the light remained green with a sharp boundary between the red and green regions. When plants were placed in the cold room under low-fluence fluorescent light (Condition E), the red color was most intense close to the light source. Under strong natural sunlight either in pots (Condition H in May or September) or planted in a field trial (Condition I) or under high-fluence artificial light (Conditions F and G), the red hue of population 88 genotypes strengthened to a deep red-purple and was present on all exposed shoots (Fig. 2C). In contrast, genotypes of population 90 always displayed a lighter red color even under field conditions. Color development in leaves resulted primarily from pigment accumulated in mesophyll cells (Fig. 2, D and E). Under growth cabinet conditions where flowering was permitted (Conditions F and G), Lc transgenic flower buds had a distinct red color compared with the green buds of the non-transformed parent plant (data not shown). However, the natural deep purple coloration of the mature flower was not visibly altered under these latter conditions. Growth habit and morphology of the Lc transgenic clones at the Saskatoon field location (Condition I) were very similar to the A01 non-transformed parent plant, except for rooted clones of genotype 88-4. Clones of this genotype consistently grew more slowly than all the other plants in the field trial and appeared to have shortened internodes. One of the C1 genotypes (47-6) also had a short phenotype. Lc-expressing clones from genotypes that displayed a moderate or deep red-green phenotype indoors (Conditions C and E–H) consistently maintained a deeper coloration when grown outdoors throughout two field seasons in Saskatoon (Condition I) and through some light frost in the fall. Lc transgenic plants that did not express Lc remained green under field conditions and appeared phenotypically similar to the non-transformed parent clones and to the B-Peru-transgenic and C1-transgenic clones.
Because genotypes expressing B-Peru or C1 did not display any red color under laboratory or field conditions (a phenotype that signaled stimulation of the lower portion of the flavonoid pathway), expression of endogenous flavonoid structural genes was not examined in either of these populations. Instead, selected genotypes containing the Lc transgene were tested by northern blotting for accumulation of Lc, CHS, F3H, or DFR transcripts under conditions (outlined above) that generated the red phenotype. Lc transgenic genotypes that constitutively expressed either the 2.2- or 2.4-kb Lc transgene under Condition B did not display significant changes in expression of CHS, F3H, or DFR under these conditions (results not shown). After acclimation of plants expressing the 2.2-kb Lc gene for 7 d to cold temperature (Condition E), an altered pattern of flavonoid gene expression was observed in several of these genotypes Under the latter conditions, the red stems of these genotypes clearly accumulated more CHS and F3H transcripts compared with stems of plants under noninducing conditions (Table III). Genotypes in which stem F3H was not induced did not display a red stem phenotype, i.e. 90-3. Coordinate expression of F3H and CHS was difficult to assess in leaves after cold acclimation due to weak gene expression (data not shown). RNA accumulation and phenotypic changes were also very similar in plants of population 88 (data not shown), although CHS gene expression was stronger in population 88 genotypes than in plants from population 90, and the phenotype developed faster. DFR transcript levels were not detectably affected in any of these plants after cold acclimation (data not shown). Gene expression and color were also unchanged under cold conditions in the non-transformed parent (control) plants (data not shown).
Expression of flavonoid genes was next examined over 48 h in two of the Lc transgenic genotypes (88-19 and 90-5a) after a shift from growth under moderate light intensity (Condition B) into high light intensity (either Condition F or G). Northern blots indicated that CHS transcripts were moderately abundant by the earliest time period (5 h) in clones of the non-transformed A01 parent plant and maintained at that level until 24 h, after which transcript levels declined (Fig. 3, top). Initially, CHS was accumulated to a similar extent in clones of Lc transgenic genotypes 88-19 and 90-5a and the non-transgenic A01, but by 24 h CHS accumulation was stronger in the transgenic genotypes. A low abundance of F3H transcripts was detected by 24 h both in 90-5a and 88-19 clones, after which transcript levels declined. F3H did not accumulate at all in A01 plants. DFR transcripts were barely detectable in transgenic and non-transgenic clones.
Expression of flavonoid biosynthetic genes was also evaluated in acclimated tissues of Lc transgenic alfalfa forage collected from the field plot at the end of September 2001, 3 months after planting (Condition I). Little change was observed in RNA from the non-transformed parent plant after acclimation. However, plants that had expressed Lc under moderate or cold temperatures and moderate light intensities and had displayed a red or deep red-purple phenotype in the field plot accumulated CHS RNA when acclimated to these more extreme conditions (Fig. 3, bottom). CHS was more intensely expressed in population 88 containing the strongly expressed 2.4-kb Lc gene, compared with population 90 containing the more weakly expressed 2.2-kb construct without the 5'-untranslated region (Fig. 3, bottom). CHS was also weakly expressed under field conditions in transgenic genotypes that poorly expressed Lc, i.e. 88-7, 90-1, and 90-13 (Fig. 3, bottom). Neither F3H nor DFR accumulated significantly after acclimation under Condition I in any of the transgenic or non-transgenic populations (data not shown).
Quantification of total anthocyanin content of leaf was carried out on forage harvested from potted plants grown under Condition H and from field material harvested at the end of September (Condition I). Spectrophotometric scans had a very broad peak typical of anthocyanins and centered at about 523 nm. Total anthocyanins correlated strongly with field observations of the color phenotype (Table IV). Green plants, i.e. A01 and Lc transgenics with limited transgene expression such as 88-7, had little or no detectable anthocyanins. Deep red field plants expressing the Lc transgene had 3- to 5-fold higher anthocyanin compared with plants that poorly expressed Lc (e.g. 88-7 and 90-13). The weight of forage harvested at the end of September had no correlation to the amount of anthocyanin present in leaf (data not shown).
Anthocyanin composition was examined in aqueous extracts of one transgenic genotype, 88-19, using mild hydrolysis conditions. The extraction resulted in two small red spots that cochromatographed on thin-layer chromatography (TLC) plates with cyanidin chloride and pelargonidin chloride in 15% (w/v) acetic acid and Forestal's reagent (spot A, reagent flows [Rfs], 0.06 and 0.39; spot B, Rfs, 0.29 and 0.65, respectively). These spots also displayed identical colors to these standards when TLC plates were viewed under visible and UV light (data not shown). Under mild to moderate hydrolysis conditions, a large red spot that chromatographed with pelargonin chloride in 15% (w/v) acetic acid and Forestal's reagent (Rfs, 0.65 and 0.75, respectively), was visible in the 88-19 water extract. This spot decreased in size under harsh hydrolysis conditions and even under mild conditions did not exhibit the same color characteristics as pelargonin on the TLC plate.
Forage of three Lc transgenic genotypes (88-4, 88-19, and 90-5a) and the non-transformed parent genotype A01 was harvested from the Saskatoon field trial (Condition I) and then analyzed by C18-HPLC after acid hydrolysis and separation into four subfractions of increasing polarity on an Oasis C18 cartridge. Total flavonoid content was reduced in the three transformed plants compared with the non-transformed A01, as visualized in the 100% (w/v) MeOH subfraction by the overall reduction in UV absorbance on the HPLC traces (Fig. 4). In addition, luteolin was disproportionately reduced in leaves and stems of all three transgenic genotypes tested, whereas apigenin was only affected in leaves of two of the transgenic plants and not noticeably affected in stems (Table V). Other changes to the UV-absorbing HPLC peaks were also observed, including a reduction of a peak that had a luteolin-like spectrum but a retention time different from our available standards, suggesting that other flavonoids were also reduced. These other HPLC peaks will be the subject of a more comprehensive analysis at a later date using selective extraction and ion cyclotron mass spectroscopy to document metabolic changes in the Lc transgenic alfalfa in greater detail.
LCR activity was examined in leaves from two selected Lc transgenic genotypes, 88-19 and 90-5a, that were grown under Condition B, then shifted for up to 48 h into Condition F or G. Leaf LCR specific activity was detectable after 12 h of continuous light exposure but rose to a maximum in 88-19 leaves after 24 h (Fig. 5A; Table VI) and then declined but was still detectable after 48 h. LCR enzyme activity had the same profile but was lower in genotype 90-5a compared with 88-19 and could not be detected in A01 non-transgenic leaves under stringent assay conditions (see "Materials and Methods" and footnotes from Table VI).
LCR activity was also examined in developing seed of Lc transgenic alfalfa. Two batches of seeds (11 DAP) were dissected under sterile conditions from pods of 88-19 and 90-5a plants that had been acclimated to Condition F for 1 week before cross-pollination and then maintained under the same conditions during seed development. A third batch of 14-d-old seeds was dissected from plants that had been maintained under Condition B and then shifted into Condition G shortly before seed harvesting. After dissection, all harvested seeds were immediately incubated under sterile conditions on Murashige and Skoog medium for 48 h under supplemented light (Condition K), then frozen in liquid N2 and assayed for LCR activity. LCR activity was preferentially stimulated in 11-d-old developing seeds of the transgenic plants exposed to continuous high-intensity light. Seeds from genotype 88-19 displayed 4-fold higher activity than the A01 control plant, whereas seed activity from genotype 90-5a was 10-fold higher than the control (Fig. 5B; Table VI). LCR activity remained higher in the 14-d-old transgenic seed than in the control but displayed no difference between the two genotypes.
Forage tissue was also analyzed to determine proanthocyanidin polymers,
dimers, and flavan-3-ol monomer content. Initially, individual leaves from
field-grown material or from plants grown under continuous light (condition F)
were stained with dimethylaminocinnamylaldehyde (DMACA;
McMurrough and McDowell, 1978
Subsequently, plants grown for 32 or 48 h under continuous light were
tested for proanthocyanidin using more sensitive heat-dependent PVPP:butanol:
HCl assays after tissue fractionation into 80% (w/v) MeOH-extractable and
unextractable fractions (Watterson and
Butler, 1983
A low concentration of 80% (w/v) MeOH-extractable proanthocyanidin
(approximately 0.06%–0.1% fresh weight) was selectively bound to PVPP
and was detected in both transgenic genotypes after heating the PVPP-bound
material in butanol:HCl (Table
VI; Watterson and Butler,
1983
Stimulation of Anthocyanin Biosynthesis by Lc Is a Function of Environmental Conditions and Transgene Specificity Alfalfa was transformed with three maize anthocyanin regulatory genes, Lc, B-Peru, and C1, as part of a strategy to stimulate flux through the forage flavonoid pathway into late products such as anthocyanins and proanthocyanidins. Plants grown under moderate greenhouse conditions expressed abundant transcripts of these transgenes in leaves when tested by northern blotting (results not shown). However, no anthocyanin or proanthocyanidin was accumulated under these conditions, and the flavonoid structural genes CHS, F3H, and DFR were not induced. These data suggested that the maize regulatory transgenes were insufficient on their own to stimulate the lower branches of the flavonoid pathway. Induction of the flavonoid pathway was highly dependent on interactions between the environment and a specific transgene sequence. When the four transgenic populations were stressed by cold or strong light, only plants from the two Lc populations developed a red or deep red-purple color in leaves and stems, and the intensity of the color depended on the Lc construct within the plants. This phenotypic change was accompanied by a rapid accumulation of CHS and F3H transcripts, although the F3H was transiently induced. Our results imply that the flavonoid pathway in alfalfa is induced only in the presence of an unknown stress-responsive alfalfa factor and that the induction is strongly enhanced by Lc. The strong induction of CHS transcripts, coupled with transient induction of F3H, suggests that this stress-responsive factor in combination with Lc acts predominantly but not exclusively at the entrance into the flavonoid pathway (although we have not tested expression of late anthocyanin genes). The weak expression of CHS and F3H and the green phenotype in non-transgenic alfalfa under strong light conditions suggests that a critical level of gene expression is required to stimulate anthocyanin, and this may be particularly true for F3H. For example, genotypes from population 90 that induce both CHS and F3H under cold conditions display a red stem phenotype. In contrast, a green stem phenotype occurs in genotype 90-3 under cold conditions because stem CHS but not F3H is induced.
Lc may interact with a stress-induced myc-like factor to override
repression and to stimulate anthocyanin production in alfalfa forage, although
we have not excluded other factors such as inhibitory myb-like factors.
Inhibitory myb and myc genes, or those that may not be able
to form a functional complex, have been identified in the past
(Hope et al., 1988
Red color was visibly induced within hours by either cold temperatures or
bright light in most Lc transgenic alfalfa genotypes, and the plants
changed to the new phenotype completely within about 3 d. The red color faded
quite quickly when these stresses were removed. However, plants placed
outdoors in pots or in the field remained red to deep red on every occasion
they were observed over a 3- to 4-month period, including light frost. This
suggests that the stress of natural outdoor light is more than sufficient to
maintain a stress-responsive factor. The rapid appearance and subsequent
disappearance of red color is suggestive of a high turnover rate for
anthocyanin in transgenic alfalfa, possibly incorporating an active mechanism
of destruction. The anthocyanins (cyanidin and pelargonidin) that we detected
in the transgenic forage differ in composition from the 3,5-diglucoside
derivatives of delphinidin, petunidin, and malvidin previously detected in
petals of different alfalfa varieties and Medicago spp.
(Lesins, 1955
Lc has been somewhat unpredictable as a transgene among different
recipient species, with effects ranging from no phenotype to strong induction
of anthocyanin and ectopic effects. The 2.4-kb Lc construct in
transgenic alfalfa population 88 contained an additional 200 bp of upstream
transcribed, non-translated sequence that was absent in the 2.2-kb construct
in population 90. This additional sequence includes a short open reading frame
containing three in-frame start codons
(Damiani and Wessler, 1993
Populations expressing B-Peru or C1 did not display the red
phenotype under any indoor or outdoor conditions tested, even though
B-Peru is highly homologous to Lc
(Colliver et al., 1997
Because the maize Lc transgene was successful at stimulating anthocyanins in alfalfa forage, one genotype from each of the two Lc alfalfa populations was selected to test for LCR activity and proanthocyanidin content in forage after plant growth under high light intensity. LCR activity was selectively detected in leaves of genotypes 88-19 and 90-5a, but not in leaves of the non-transgenic parent A01. Activity profiles between the two transgenic genotypes tested had a similar pattern, but the activity of 88-19 was always higher than 90-5a. In addition, proanthocyanidin was accumulated in the transgenic leaves, and could be fractionated into extractable and unextractable fractions. When measured after 32 h of high-intensity light, all of the detectable proanthocyanidin of the transgenic plants was found in the extractable fraction, and a 3-fold higher concentration was recovered from genotype 88-19 leaves compared with 90-5a. After 48 h of exposure, a larger portion of detectable proanthocyanidin was unextractable except under extremely harsh conditions (butanol:HCL hydrolysis of the extracted and washed cell residue) or was partitioned into ethyl acetate.
Accurate determination of the concentration of different fractions of PA in
Lc alfalfa forage may not be possible if their behavior differs from
the alfalfa seed polymer standard (for review, see
Marles et al., 2003
LCR activity was also stimulated in developing alfalfa seed from
Lc transgenic genotypes 88-19 and 90-5a relative to A01
non-transgenic control seed when these three genotypes were cross-pollinated
with cv Peace. Seed coat is the only alfalfa tissue that normally accumulates
proanthocyanidin. The LCR activity we observed with our A01 non-transgenic
seed falls within the range detected for seed from cv Beaver
(Skadhauge et al., 1997
The stimulation of LCR activity and proanthocyanidin accumulation in
alfalfa forage is likely to raise strong interest in the worldwide forage seed
sector. LCR activity and proanthocyanidin has not been detected previously in
alfalfa leaf tissue (Skadhauge et al.,
1997
The effect of the Lc gene on alfalfa flavonoid biosynthesis
extended beyond anthocyanins and proanthocyanidins. Alfalfa forage normally
accumulates a range of small flavonoids, predominantly flavones and
isoflavones (Ingham, 1979 The total content of flavones was reduced in leaves and stems in field-grown clones of our Lc genotypes relative to the parent non-transformed genotype. Luteolin, a flavone with a di-hydroxylated B ring, was strongly reduced in all Lc genotypes tested. In some cases, apigenin (a monohydroxylated relative of luteolin), was also decreased, but distribution between apigenin and luteolin depended on individual transgenic genotypes and tissue type. The reduction in flavone content may be due to a draw by anthocyanin and proanthocyanidin biosynthesis on the metabolic capacity of the pathway. Alternatively, the Lc gene may displace or interfere with a gene that normally stimulates the flavone pathway in alfalfa. Because the flavonoid pathway is involved in plant defense, we are now conducting tests to determine whether the reduction in flavones has resulted in any changes to the stress-coping mechanisms of the Lc transgenic alfalfa plants.
The transgenic alfalfa populations expressing maize anthocyanin regulatory genes offer a unique opportunity to study the interactions between closely related introduced genes and the environment and to test strategies aimed at developing anthocyanins and proanthocyanins in forage crop species. We have shown that the Lc gene, but not B-Peru or C1, can stimulate both anthocyanin and proanthocyanidin biosynthesis in alfalfa forage but that stimulation only takes place in the presence of an unknown stress-responsive alfalfa factor. In addition, we have shown that the 200-bp Lc 5'-untranslated region sequence enhances expression of CHS and LCR and anthocyanin and proanthocyanin accumulation in alfalfa forage. These findings demonstrate that it will be possible to introduce proanthocyanidins into alfalfa and develop forage with improved ruminant digestibility, a technology that is highly sought by the forage industry worldwide. In the future, we will examine the interaction of Lc transgenic genotypes in combination with other transgenes to see if anthocyanins can be diverted into additional proanthocyanidin production. It may be possible to stimulate proanthocyanidin biosynthesis further by directly introducing a proanthocyanidin-specific regulatory transgene, such as TAN1 (Ray and Gruber, 2001 ) or the LCR and ANR biochemical transgenes
that have been cloned recently (Tanner et
al., 2002; Xie et al.,
2003).
Alfalfa (Medicago sativa) Transformation
Two Lc sense constructs, controlled by a single CaMV 35S promoter
in Agrobacterium tumefaciens vectors pAL69 and pAL144
(Lloyd et al., 1992
Initially, selected ntpII+ shoots were transferred to a medium of fine brick chips and grown hydroponically in Guelph (ON, Canada) in a greenhouse supplemented with high pressure halogen lights (Condition A, Table I). Later, plants were transferred to a soil-less potting mixture and maintained as stock plants in a Saskatoon greenhouse supplemented with high-pressure halogen lights (Condition B, Table I). Plants were propagated through rooted shoot cuttings to test growth and phenotype and gene expression under a range of environmental conditions. One or two internodes of stem were trimmed free of mature leaves, dipped in rooting powder (Root Stim No. 1, Plant Products Co. Ltd., Brampton, ON, Canada), and placed in florist's foam wetted with water. Rooted cuttings were grown in 3.5-x 3.5-inch pots under Condition B until they were well-established plants and then transferred to a range of additional growth conditions (C–J) outlined in Table I to test their response.
Established rooted cuttings (4–6-month-old clones) from selected primary transformed plants from all four populations were inoculated with a commercial preparation of Rhizobium meliloti and transplanted into dry land, dark-brown soil in a 16-x 24-m field plot in early July 2001 at the Agriculture and Agri-Food Canada farm (Saskatoon, SK, Canada; Condition I; Table I). Six clones per primary transgenic plant were evenly spaced within a 2-m distance in rows 1 m apart. Each planting was replicated in four locations within the plot (total of 24 clones per genotype). Seven centimeters of rainfall over the first field season was supplemented by hand watering three times while plantlets were establishing. Young flower buds and 4 cm of vegetative tissue were removed from each floral shoot before floral opening. Forage was clipped and discarded in late August. At the end of the growing season (late September 2001 and early October 2002), material longer than about 7 cm was harvested, weighed, and quick frozen in liquid nitrogen at the field site. The plants were then allowed to recover without additional harvesting before the onset of winter in November. A fence was erected after the growing season to trap a light layer of blowing snow on the plots over the winter, but plots were often bare. Plant phenotype was observed over two growth seasons. Winter temperatures averaged –20°C at the field site. Rainfall (less than 3 cm) in the second field season was not supplemented by hand watering. The C1 population and one of the B-Peru populations (45) were also grown for 3 consecutive years in a replicated trial under milder south-central Canadian conditions at the Elora farm (University of Guelph) beginning in the 1998 field season (Condition J, Table I). Six plants grown from rooted cuttings of each primary transgenic plant were transplanted evenly into short (2-foot) rows spaced 6 feet apart. Each of these plantings was replicated four times. Rainfall was plentiful, and plots were mowed when necessary to prevent flowering. Winter temperatures averaged –10°C at the Elora trial, and a heavy blanket of wet snow covered the trial in the winter.
Nucleic acids were extracted from quick-frozen young or mature leaf
material harvested from field- or greenhouse-grown plants using Qiagen plant
DNA or RNA kits (Qiagen USA, Valencia, CA). Except where indicated, all
molecular biology methods were conducted using standard techniques
(Sambrook et al., 1989
Deep frozen leaf tissue (approximately 1 g comprised mainly of leaf,
petiole, and a small amount of fine stem material) was ground in liquid
N2, heated to 55°C for 10 min in 10 mL of 2 N HCl,
then cooled and incubated overnight in the dark at 24°C to extract
anthocyanidins. Extraction under extremely harsh hydrolysis conditions
(90°C, 15 min) was avoided because it caused the red extract color to turn
brown. The extract was clarified by centrifugation and then an aliquot was
scanned from 240 to 600 nm in a Varian Cary 3 spectrophotometer using 2
N HCl as a blank. Anthocyanidin hydrolysates were also partitioned
with water-saturated ethyl acetate, after which the red aqueous phase was
extracted into isoamyl alcohol, dried, and reconstituted in methanol-1% (w/v)
HCl (MeOH-HCl; Harborne,
1998
For analysis of anthocyanin composition using TLC, moderately harsh
hydrolysis conditions were used to extract anthocyanidins from genotypes 88-19
and A01 according to Harborne
(1998
Deep-frozen forage collected from the field trial in October 2002 was
freeze dried and separated manually into stem and leaf samples for analysis of
flavonoids other than anthocyanins following an established protocol with
variations (Stochmal et al.,
2001
Proanthocyanidin and flavan-3-ol monomers were initially analyzed using the
DMACA method outlined by McMurrough and McDowell
(1978
Bulked leaf samples (1 to 2 g from genotypes A01 and 88-19 and 1 g from
genotype 90-5a) were subsequently extracted and analyzed by the more sensitive
PVPP:butanol:HCl method (Watterson and
Butler, 1983
Each of the MeOH-extracted or dried, aqueous-partitioned MeOH phases from
alfalfa leaves was reconstituted in 2 mL of MeOH and divided into two equal
1-mL samples. Each 1-mL sample was vortexed intermittently for 15 min with 50
mg of PVPP (Sigma, St. Louis) in a screw cap test tube in a protocol to
selectively bind longer chain proanthocyanins and distinguish them from
anthocyanins (Watterson and Butler,
1983 Dried ethyl acetate-partitioned fractions from MeOH-extracted leaves were reconstituted in 80% (w/v) MeOH, and a small portion was analyzed in an HP1100 HPLC on a Waters Novapack C18 column (3.9 x 150 mm, 5-µm particle size) at 1 mL min–1 using a 3% (w/v) acetic acid isocratic solvent system for 30 min. Chromatography was monitored with a PDA set at 230, 250, and 280 nm to monitor UV-absorbing compounds, such as (+)-catechin and small, extractable PA polymers (e.g. dimers), and at 550 nm to detect any residual contaminating anthocyanin. The remainder of the ethyl acetate-partitioned fraction was divided into two equal portions and hydrolyzed in 100% (w/v) butanol:100% (w/v) HCL (70:30 [v/v]) either at room temperature or at 70°C for 1 h to detect anthocyanidins arising from any small, extractable PA polymers. Cell residue, i.e. PM retained after the initial MeOH extraction to remove extractable PA, was washed at least five times with 100% (w/v) MeOH (2:1 [w/v] PM:MeOH) throughout a 24-h period until the supernatant was colorless. The PM was tested in a butanol:HCl assay as outlined above (but without the PVPP step) to detect unextractable PAs.
LCR activity assays were conducted on young and mature fresh leaf material
that was harvested from duplicate primary Lc transgenic plants that
had been grown in a growth cabinet under Condition B and then transferred to
Conditions F or G for 12, 24, or 48 h. Assays were also conducted on young
developing alfalfa seed harvested from genotypes 88-19, 09-5a, and A01 that
had been grown under Condition G and cross-pollinated on three separate
occasions with a clone of the western Canada-adapted alfalfa cv Peace. At 11
DAP, pods were dissected open, and seeds were removed under sterile
conditions. Young seeds were incubated for 2 d on sterile filter paper placed
on solid Murashige and Skoog media under Condition K. LCR enzyme activity was
assayed in the alfalfa tissues by measuring (+)[3H]catechin
extracted into ethyl acetate after incubating
[3H]cis-leucocyanidin, NADPH, and aqueous tissue extracts for 30
min at 30°C under reducing conditions
(Tanner and Kristiansen, 1993 Several control assays were included routinely in each experiment. These included negative controls (assay buffer, NADPH, and substrate without plant extract or with boiled plant extract), a strong positive control (assay buffer, substrate, NADPH, and Lotus uliginosis leaf extract), tissue-specific controls (including leaf and cross-pollinated seed extracts of A01), and extracts spiked with nonradioactive (+)catechin. Some of the control assays were necessary because the continuous use of acetic acid in the elution buffer slowly stripped away the C18 column coating and increased retention times. The control assays were also necessary to monitor products unrelated to LCR activity that accumulated in assays containing a high proportion of concentrated plant extract with weak LCR activity. Assays that contained (+)[3H]catechin HPLC peak heights <1,000 dpm above a smooth constructed baseline were regarded as "not detected." Plant extracts with high protein concentrations were developed to recover consistent, easily measured (+)[3H]catechin peaks in samples with low activity.
Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes, subject to the requisite permission from any third party owners of all or parts of the material. Obtaining any permissions will be the responsibility of the requestor.
We are grateful to Sandra Northrup, Terry Huebert, M.A. Susan Marles, Cecilio Gregorio, Ning Chen, Donna Hancock, Tim Nelson, Naomi Carriere, and Jim Elder for proficient technical assistance and advice. The authors acknowledge the gift of a pea ssRBC promoter and plasmid pBI525 containing the enhanced CaMV 35S promoter from the Plant Biotechnology Institute (National Research Council, Saskatoon, SK, Canada). Alfalfa seed polymer was purified earlier in the laboratory of Dr. Alister Muir at the Saskatoon Research Centre (Agriculture and Agri-Food Canada). Received December 27, 2002; returned for revision February 20, 2003; accepted April 15, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.025361.
1 This work was supported in part by grants from the Alberta Agriculture
Research Institute, the Alberta Cattle Commission, and the Saskatchewan
Agricultural Development Fund. This is Institute paper no. 1547.
2 This paper is dedicated to the memory of Dr. Bernie Goplen and Dr. Ron
Howarth. Together, they had a vision for improving alfalfa forage quality by
introducing proanthocyanidins and faith that it could be accomplished.
3 These authors contributed equally to the paper and to the assessment of the
transgenic plants.
4 P.A. has retired from Agriculture and Agri-Food Canada.
5 Present address: BASF Plant Science, 26 Davis Drive, Research Triangle
Park, Raleigh-Durham, North Carolina 27709-3528. * Corresponding author; e-mail gruberm{at}agr.gc.ca; fax 306–956–7247.
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ABSTRACT
RESULTS
-hydroxylase (F3H), and dihydroflavonol reductase
(DFR; Winkel-Shirley, 2001
