2.2. Characterization of Flavan-3-ols and Extension Units of Proanthocyanidins
HPLC-PAD analysis coupled with the use of authentic standards has been shown an effective approach to characterize flavan-3-ols [
19,
26]. In this study, leaf extracts in methanol were analyzed by HPLC-PAD. Chromatography of separated metabolites was recorded at 280 nm to characterize flavan-3-ols. Four peaks were identified with the same retention times as authentic standards, (+)-catechin, (−)-epicatechin, (−)-gallocatechin and (−)-epigallocatechin, respectively (
Figure 3a). These four peaks were also showed the same UV spectra as authentic standards, respectively. These data show that 2, 3-
trans and
cis-flavan-3-ols are synthesized in leaves.
HPLC-PAD and TLC analyses have been used to characterize products derived from the butanol-HCl boiling of PAs [
19,
26]. Red pigments produced in the butanol-HCl boiling of PAs were separated on cellulose-based TLC plates. Components that were at the positions with the same R
f values as authentic standards of cyanidin, delphinidin and pelargonidin were determined in leaf extracts from different developmental stages (
Figure 3b). This result demonstrated that these three red pigment metabolites were produced from the butanol-HCl boiling of PAs. In addition, other red pigment components with higher R
f values than pelargonidin R
f value were obviously detected in leaf extracts (
Figure 3b). To further demonstrate the properties of red pigments, metabolites separated by HPLC was recorded at 550 nm to compare their retention time and UV-spectra with authentic standards. The resulting data showed that three peaks were eluted at the same retention times (
Figure 3c) and exhibited the same spectra (data not shown) as cyanidin, pelargonidin and delphinidin, respectively. Therefore, cyanidin, delphinidin and pelargonidin were released from the butanol-HCl boiling cleavage of PAs, demonstrating that the extension units of PAs are composed of 2-(3',4'-dihydroxyphenyl)-3,4- dihydro-2H-chromene-3,5,7-triol, e.g. (+)-catechin or (-)-epicatechin; 2-(4-hydroxyphenyl)-3,4- dihydro- 2H-chromene -3,5,7-triol, e.g. (+)-afzelechin or (-)-epiafzelechin; and 2-(3',4',5'-hydroxyphenyl) -3,4-dihydro-2H-chromene-3,5,7-triol, e.g. (+)-gallocatechin or (-)-epigallocatechin.
Figure 3.
HPLC profiling of flavan-3-ols and TLC analysis of proanthocyanidins in leaves of Vitis bellula. (a). A HPLC profile shows (±)-catechin (CA), (-)-epicatechin (EP), (−)-gallocatechin (GC) and (−)-epigallocatechin (EGC) in crude extracts of leaves (stage 3); (b). A TLC image shows anthocyanidin profiles released from the butanol: HCl boiling of crude extracts of proanthocyanidins from leaves (stage 3), authentic standards including: cyanidin (Cy), pelargonidin (Pel) and delphinidin (Del); (c). A HPLC profile shows anthocyanidins released from the butanol: HCl boiling of crude extracts of proanthocyanidins from leaves (stage 3), authentic standards including: cyanidin (Cy), pelargonidin (Pel) and delphinidin (Del).
It was interesting that, in addition to cyanidin, delphinidin and pelargonidin, there were additional red pigment components detected from the butanol-HCl boiling of PAs by both TLC and HPLC (
Figure 3b,c). Images of TLC showed that main red pigments were characterized by higher R
f values in comparison with cyanidin, delphinidin and pelargonidin. Meanwhile, HPLC showed two main additional peaks between cyanidin and pelargonidin. Although the properties of these additional pigments remains further identified, these results reveal that additional structures likely exists in PAs of leaves.
2.3. Expression Patterns of ANR, LAR and DFR Homologs During Leaf Development
ANR from
V. vinifera has been functionally and in crystal characterized to convert anthocyanidins to three different isomers of flavan-3-ols featured by configurations of 2R, 3R-2,3-
cis, 2S, 3S-
cis and 2S, 3R-
trans-flavan-3-ols, e.g. (−)-epicatechin, (+)-epicatechin and (−)-catechin (
Figure 1) [
22]. Based on the sequence of
V. vinifera ANR (
Vv ANR), one pair of degenerated primers (
Table 1) was created to amplify
ANR homologs from leaves of
V. bellula. The open reading frame (ORF) were cloned and termed as
VbANR in this study. Its sequence (gi|381392348|gb|JQ308620.1) was shown to possess very high similarity to
VvANR [
19]. Semi-quantitative PCR analysis showed that its expression level was higher at stage #3 than at any of the other four stages in the leaf development (
Figure 4), which supported the observation of the peak value of E-PAs at the stage 3 and consistent synthesis of flavan-3-ol isomers, e.g. (−)-epicatechin and (−)-epigallocatechin extracted in methanol and (
Figure 2b and
Figure 3a).
Figure 4.
Images of semi quantitative RT-PCR. RT-PCR results show expression patterns of three late pathway genes and three MYB genes at five points in the leaf development of V. bellula. ANR: anthocyanidin reductase, LAR: leucoanthocyanidin reductase, DFR: dihydroflavonol reductase, MYBA1, A2 and 5a: MYB transcription factors, EF1-γ: elongation factor gamma as reference gene.
Two
LAR members,
VvLAR1 and
2, have been identified in the genome of
V. vinifera [
22]. These two isomers have been shown to convert 2R, 3S-2, 3-
trans-3, 4-
cis-leucocyanidin to (+)-catechin. Based on sequences of
VvLAR 1 and
2, degenerated premier pairs (
Table 1) were designed to amplify homologs from leaves of
V. bellula. Consequently, two ORF cDNA fragments were amplified and sequence analysis showed that these two homologs were highly similar to
VvLAR1 and
2, respectively. As result, we termed these two
LAR homologs as
VbLAR1 and
2 and deposited these two sequences to the gene bank curated at NCBI (
VbLAR1: gi|381392360|gb|JQ308626.1|;
VbLAR2: gi|381392362|gb|JQ308627.1|). A phylogeny analysis with 11
LAR homolog sequences revealed that
VbLAR1 and
VvLAR1 were in the same clade, while
VbLAR2 and
VvLAR2 were in the same clade (
Figure 5a). Semi-quantitative RT-PCR analysis revealed differentiated expression patterns of these two genes. The expression of
VbLAR1 occurred in the early times but was hardly detected in the late times of leaf development. The expression level of
VbLAR2 was similar at stages #1 to #2 and then increased at stage #3 followed by a decrease at stages #4 and 5 of leaf development (
Figure 4).
An ORF cDNA fragment of
DFR homolog was amplified by PCR and its sequence (>gi|381392350|gb|JQ308621.1|) was deposited to the gene bank curated at NCBI. We termed this cDNA as
VbDFR. Semi-quantitative RT-PCR analysis showed that its expression levels were similar at stages #1 and 2, and then increased at stage #3 followed by a decrease at stage #4, but hardly detected at stage #5 during leaf development (
Figure 4). A phylogeny analysis with 22
DFR homologs revealed that
VbDFR and other homologs from
Vitis species were in the same clade (
Figure 5b).
2.4. Expression Patterns of VbMYBPA1, VbMYB5a, VbMYBA1 and VbMYBA2 During Leaf Development
VvMYBPA1 cloned from berries of
V. vinifera encodes a R2R3-MYB member, a homolog of TT2 regulating PA biosynthesis in seeds of
A. thaliana [
25]. A cDNA homolog of
VvMYBPA1 was amplified from leaves of
V. bellula, which is termed
VbMYBPA1. Its ORF sequence (gi|381392354|gb|JQ308623.1) has been deposited in the gene bank curated at NCBI. Semi-quantitative RT-PCR analyses showed that the expression level of
VbMYBPA1 was higher at stage 3 than those at any of the other four stages tested during leaf development (
Figure 4), which was consistent to the trend of the PA level (
Figure 2c). Given that VvMYBPA1 has been demonstrated to specifically regulate PA biosynthesis by controlling promoter activities of
VvLAR and
VvANR [
21,
25,
27], we hypothesize that
VbMYBPA1 most likely is involved in the regulation of PA biosynthesis in leaves of
V. bellula.
A cDNA homolog of
VvMYB5a was amplified from leaves of
V. bellula, which is termed as
VbMYB5a. We have deposited its OFR sequence (gi|381392352|gb|JQ308622.1|) to the gene bank curated at NCBI. Semi-quantitative RT-PCR analyses showed that the expression level of
VbMYB5a was higher at stage 3 than those at any of the other four stages tested (
Figure 4), the trend of which was corresponding to that of PA levels (
Figure 2c).
VvMYB5a has been cloned and demonstrated to encode another R2R3-MYB member regulating the general phenylpropanoid pathway. Its overexpression leads to production of anthocyanins and flavonols with a cost of lignin alternation in transgenic tobacco plants [
28]. The expression patterns of
VvMYB5a have been demonstrated to be consistent to the expression of
VvLAR and
VvANR in the beginning of the development of berries [
21]. As a result, we hypothesize that
VbMYB5a is likely involved in PA and other flavonoid biosynthesis in leaves of
V. bellula.
Two cDNA homologs of the grape
VvMYBA family were amplified from leaves of
V. bellula using one pair of primers (
Table 1 and
Figure 4). The full sequence of ORF for one cDNA was obtained and deposited to the public gene bank (gi|381392354|gb|JQ308623.1|) curated at NCBI. Sequence blast analysis showed that this ORF sequence was highly similar to the ORF of
VvMYBA1, which is one main member of the small
VvMYBA family regulating anthocyanin biosynthesis associating with red and white berry features of different
V. vinifera varieties [
29]. Here, this cDNA homolog was termed
VbMYBA1. In addition, the second PCR fragment amplified was smaller than
VbMYBA1 (
Figure 4)
. Sequence analysis showed that it had approximately 90% of the identity compared to
VvMYBA2, which is another member of the grape
VvMYBA family, and thus was termed
VbMYBA2 in this study. Semi-quantitative RT-PCR analysis showed that these two DNAs shared a very similar expression pattern, which was featured by from undetectable levels at stage #1 to a relatively high expression level at stage #5 tested in leaf development (
Figure 4). Multiple studies have shown that VvMYBA1 likely is a master regulator associating with red pigmentation in berries of grapes and VvMYBA2 is also involved in certain specific accumulation of anthocyanin pigmentation [
29,
30,
31,
32,
33]. We hypothesize that
VbMYBA1 and
2 are also involved in anthocyanin biosynthesis and other flavonoids biosynthesis in leaves of
V. bellula.
2.5. Discussion
Vitis has approximately 60 species [
34]. In addition to
V. vinifera, 15 other species are cultivated for berry products and wine production, such as
V. rotundifolia that is another economically important crop. To date, only berries of
V. vinifera have gained most intensive investigations to understand biosynthesis of PAs. This is because the biosynthetic properties and structures of PAs in berries of this species are fundamentally associated with nutritional values of grape berries and quality of wine products [
15,
35,
36,
37]. Numerous investigations have showed that the structures of PAs in wine products made from berries of
V. vinifera are mainly characterized by procyanidins consisting of either (+)-catechin or (−)-epicatechin or both [
6,
12,
38,
39,
40,
41]. In contrast, little has been reported about the presence of (+)-afzelechin (derived from leucopelargonidin) or (−)-epiafzelechin (derived from pelargonidin) units in grape seed PAs. Several experiments have demonstrated that grape skins synthesize pelargonidin-glycosides [
42,
43,
44,
45,
46]. These discoveries imply that (−)-epiafzelechin units likely exist in grape PAs. In addition, compared to berries, leaves have gained very limited studies to understand PA biosynthesis. Meanwhile, whether leaves can produce propelargonidin or miscellaneous oligomeric or polymeric PAs consisting of (−)-epiafzelechin and (−)-epicatechin remains unclear. We believe that studies on berries and leaves of different species of
Vitis can enhance a better understanding of properties of PA biosynthesis and structures in this genus. In present study, the butanol-HCl boiling of crude PAs in leaf extract of
V. bellula released pelargonidin (
Figure 3b,c). These results imply the presence of (−)-epiafzelechin or (+)-afzelechin units in foliage PAs of
V. bellula because these units after butanol-HCl boiling are converted to pelargonidin [
1,
19]. As more experiments for elucidation of PA structures using LC-MS and NMR analyses will be carried out, (−)-epiafzelechin or (+)-afzelechin units in PAs can be characterized in the future. In addition to pelargonidin, cyanidin and delphinidin were produced from the butanol-HCl boiling of crude PAs (
Figure 3b,c), showing that epicatechin or catechin and epigallocatechin or gallocatechin are two other types of extension units. These results indicate that the foliage PAs contain extension units featured with one, two and three groups of –OH in the B-ring of the flavan-3-ols, potential examples of which are epiafzelechin, (−)-epicatechin and (−)-epigallocatechin. Furthermore, both HPLC and TLC analyses show additional pigment molecules (
Figure 3b,c). Although the structures of these pigment molecules remain to be elucidated, our data indicate the structural diversity of PAs in leaves of
V. bellula.
Figure 5.
Cladogram style phylogeny trees of DFR and LAR from certain species. (a). a phylogeny tree developed from 11 cDNA homolog sequences of LAR. (b). a phylogeny tree developed from selected 22 cDNA homolog sequences of DFR.
In this study, we focus on gene expression analysis of late pathway genes (
VbDFR,
VbANR,
VbLAR1 and VbLAR2) of PA biosynthesis. ORFs of cDNAs for these four genes were sequenced for phylogeny analysis. Our previous phylogenetic analysis showed that all available nucleotide sequences of
ANR cDNAs in the
Vitis genus were clustered in the same clade [
19]. Here, a phylogenetic analysis also showed that
VvLAR1 and
VbLAR1 were in the same clade, while
VbLAR2 and
VvLAR2 were in the same clade (
Figure 5a). In addition,
VbDFR was in the same clade as
VvDFR and
VaDFR cloned from
V. vinifera and
V. amurensis, respectively (
Figure 5b). These analyses show that the nucleotide sequences of
DFR,
ANR and
LAR ORF cDNAs are conserved in the
Vitis genus. Semi-quantitative analyses have been performed to understand their expression patterns during leaf development examined from young to fully expanded stages (
Figure 4). Based on intensity of amplified cDNA fragment indicated by signal of EB-DNA binding (
Figure 4), the expression patterns of
VbANR,
VbDFR and
VbLAR2 were corresponding to the trend of levels of PAs extracted in methanol (
Figure 2c). Our observation on
VbANR and
VbLAR2 expression patterns is different from the report about the
VvANR and
VvLAR2 expression patterns in leaf development of
V. vinifera [
22]. In our experiments, the expression pattern of
VbANR and
VbLAR2 was characterized by a similar level at the stages 1 and 2, followed by a level increase at the stage 3 and then a level decrease at stages 4 and 5 (
Figure 4). The expression pattern of
VvANR in leaves of
V. vinifera was featured by a trend consisting of a level decrease from stage 1 to stage 2 and then a continuous level increase from stage 2 to stage 5 [
22]. The expression pattern of
VvLAR2 was characterized by a trend of a continuous decrease from stage 1 to stage 4 and then an increase at stage 5 [
22]. Unlike
VbANR and
VbLAR2, the expression of
VbLAR1 followed a unique pattern in leaf development, which was featured by relatively high levels in the early two stages followed by a decrease to an undetectable level at stage 5 (
Figure 4). This expression pattern is also different from that of
VvLAR1 that was showed a very low expression level in leaves of
V. vinifera [
22]. We propose two possibilities associating with these different expression patterns between two species. On the one hand, these distinct expression patterns in two species are likely associated with different patterns of biosynthesis of PAs and sampling times. On the other hand, these differences most likely result from two genotypes and other environmental conditions in their growth areas. More importantly, our data show that the ANR and LAR pathways co-exist in leaves of
V. bellula. In summary, these observations support the general hypothesis that the co-existence of the ANR and LAR pathways of PA biosynthesis is common biochemical phenomenon in the vascular plants [
19]. To date, in addition to
V. vinifera and
V. bellula, many vascular plants, such as tea (
Camellia sinensis) [
47], apple [
24],
Gingko biloba [
48] and other species [
19], have been demonstrated to include these two branches toward the formation of PAs.
The expression patterns of four MYB transcription factor homologs (
VbMYBPA1,
VbMYB5a,
VbMYBA1 and
VbMYBA2) involved in biosynthesis of grape PAs and anthocyanins were analyzed by semi-quantitative RT-PCR (
Figure 4). VbMYBPA1 is a homolog of VvMYBPA1, which has been demonstrated to regulate the expression of both
VvANR and
VvLAR1, but not
VvLAR2 during berry development of
V. vinifera [
25,
49]. In our study, we also observed that the expression pattern of
VbMYBPA1 was similar to those of
VvANR and
VvLAR2, but not
VvLAR1 (
Figure 4).
VvMYB5a encodes a R2R3-MYB member that has been demonstrated to regulate several branches of the phenylpropanoid pathway leading to PAs, anthocyanins and flavonols during berry development of
V. vinifera [
21,
28,
50]. The expression pattern of
VvMYB5a has been shown to associate that of
VvLAR1 [
21]. In our study, a
VbMYB5a with a high sequence similarly to
VvMYB5a was cloned from leaves of
V. bellula. Its expression profile was very similar to
VbANR,
VbLAR2 and
VbMYBPA1 (
Figure 4), indicating that
VbMYB5a most likely is associated with the regulation of
VbANR and
VbLAR2. Two R2R3-MYB homologs associated with anthocyanin biosynthesis, VbMYBA1 and VbMYBA2, were identified from berry of
V. vinifera [
29,
30,
31,
32,
33,
51,
52]. These two transcription factors regulate the biosynthesis of anthocyanins in the skin of berries. RT-PCR analysis revealed the expression of two homologs,
VbMYBA1 and
VbMYBA2, in late two stages but not in the early three stages of leaf development examined (
Figure 4). Compared to the expression patterns of
VbLAR and
VbANR, this result does not indicate an involvement of these two transcription factors in the PA biosynthesis. In addition, their expression profiles were not consistent with that of
VvDFR. Based on these observations, we suggest that
VbMYBA1 and
2 in leaf of
V. bellula are involved in regulation of other branches of the phenylpropanoid pathway.
