Arabidopsis UDP-glycosyltransferase 78D1-overexpressing plants accumulate higher levels of kaempferol 3-O-β-d-glucopyranoside than wild-type plants

Flavonols are a class of flavonoids that are found in most plants. Certain flavonols exhibit anticancer, antioxidant, and antimicrobial functions. An array of genes plays a role in the regulation of flavonoid biosynthetic pathways, including the MYB–bHLH (basic helix-loop-helix-WD40 transcription factor complex. Flavonoids often display altered bioactivities after being glycosylated by the action of glycosyltransferases. These enzymes catalyze the transfer of sugars from a donor to various acceptors. In this study, we generated several transgenic lines of Arabidopsis that overexpress UDP-glycosyltransferase 78D1 (UGT78D1), which are hereafter referred to as UGT78D1-OX, to address three questions: (1) Can UGT78D1-OX seedlings accumulate more flavonols? (2) Can UGT78D1-OX seedlings accumulate more flavonols in the presence of sucrose? and (3) Will UGT78D1-OX be more sensitive to abiotic stresses? We observed that UGT78D1-OX seedlings accumulated specific types of kaempferol, while they had a decreased content of flavonols in the presence of sucrose. Contrary to our expectation, more anthocyanins accumulated in the UGT78D1-OX lines, although the expression of production of anthocyanin pigment 1 was slightly reduced in UGT78D1-OX seedlings compared with that in wild-type seedlings. It appeared that the overexpression of UGT78D1 did not interfere with abiotic stress tolerance in the mutant plants.


Introduction
Flavonoids, including anthocyanins and flavonols, are compounds that are well established to protect plants against pathogen attacks, ultraviolet irradiation, and free radicals [1][2][3][4]. The MYB-bHLH (basic helix-loop-helix)-WD40 complex plays crucial roles in the activation of flavonol biosynthesis by regulating several flavonoid biosynthesis genes [5]. Among those genes, the expression of Arabidopsis thaliana production of anthocyanin pigment 1 can result in a high level of anthocyanin production in various plants [6,7]. Thus, metabolic engineering strategies aimed at enhancing the contents of specific types of flavonoids in plants may involve the manipulation of certain genes in the flavonoid biosynthesis pathways.
Flavonoid biosynthesis routes diverge into different pathway branches that lead to the synthesis of specific classes of molecules such as proanthocyanins, flavonols, and lignols. One of these branches converts dihydrokaempferol either into flavonols via the action of flavonol synthase (FLS) or into anthocyanins via the action of dihydroflavonol 4-reductase (DFR). Many flavonoids are glycosylated by glycosyltransferases, which often leads to altered bioactivities following glycosylation [8]. Glycosyltransferases catalyze the transfer of sugars from a donor to various acceptors. Glycosylated flavonols can be sequestered in the vacuole [9], which results in a reduced flavonol content in the cytoplasm. If they are not sequestered in the vacuole, they can inhibit flavonol synthesis via a negative feedback mechanism. In our previous study, we observed that the overexpression of flavonol synthase, which synthesizes flavonols, did not lead to the overaccumulation of flavonols in Arabidopsis. Therefore, in the present study, we wanted to determine whether we could increase flavonol synthesis specifically in Arabidopsis by overexpressing a glycosyltransferase alone. If UGT78D1-OX would accumulate more glycosylated flavonols, then we were also curious about whether the increased flavonols may confer an enhanced tolerance toward abiotic stresses in Arabidopsis. To address this question, we generated several Arabidopsis lines of overexpressing UGT78D1, which are hereafter referred to as UGT78D1-OX. We observed that UGT78D1-OX accumulated specific types of kaempferol under control conditions and also accumulated kaempferol in sucrose-treated conditions, under which anthocyanins typically accumulate at high levels because of a shift in the utilization of metabolic pathways. We also examined the performance of UGT78D1-OX seedlings in response to osmotic stress.

Construction of UGT78D1-OX and transformation
The PCR TM /GW/TOPO Ò TA Cloning Ò Kit (Invitrogen, Carlsbad, CA, USA) was used to clone the PCR product from the AtUGT78D1 cDNA. AtUGT78D1 was then cloned into the pFLAG vector (E. coli, kanamycin resistance plant, Basta Ò resistance) by producing the construct 35Spro: AtUGT78D1, following a previously reported protocol [10]. Cells were centrifuged and suspended in liquid containing 1/2 MS media and 2% sucrose at an OD 650 = 0.5. Col-0 plants that were one month old and contained flower buds were soaked in Agrobacterium media for approximately 40-60 s and were then maintained in the dark for one night.
Anthocyanin measurement and ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry analysis Anthocyanin measurements were taken following a previously described detection method [11]. Ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry analysis was conducted following a previously described method for flavonol detection [10].

qRT-PCR and gene accession numbers
Nine-day-old seedlings were collected for RNA extraction [11]. cDNA synthesis and qRT-PCR were performed according to the manufacturer's protocols (Applied Biological Materials, Richmond, Canada). ACTIN2 was used as an internal control. The gene sequences were obtained from the Arabidopsis Information Resource (www.arabi dopsis.org) with the following accession numbers: DFR (AT5G42800), FLS1 (AT5G08640), PAP1 (AT1G56650). Two independent experimented data were performed and subjected to statistical analysis by Tukey's test (p \ 0.001). The asterisks above the columns denote significant differences. The error bars represent standard error.

Results and discussion
Flavonoids serve multifarious roles in the plant life cycle. Thus, their regulation is controlled via diverse transcription factors (TFs) that can be manipulated by scientists in the field of plant metabolic engineering. For instance, PAP1 overexpression increased the contents of various volatile compounds in rose [12]. Moreover, the interruption of the flavonol synthesis pathway directed the production of high levels of anthocyanins in Arabidopsis [13]. These reports prompted us to examine whether we could manipulate flavonol content by increasing flavonol glycosylation via the overexpression of Arabidopsis UGT78D1. Moreover, we investigated the type of flavonols that would be produced. To address these questions, several UGT78D1-OX lines were generated.
First, we attempted to determine the glycosylated flavonol contents of the UGT78D1-OX lines and the WT plants. As shown in Fig. 1(A), five different types of glycosylated flavonols were detected as follows: The seedlings of UGT78D1-OX line #7 produced 20% more K3 than those of WTs or other the UGT78D1-OX lines ( Fig. 1(A)). This increase became more prominent in the presence of sucrose or naringenin. Among the compounds that can elicit flavonoid biosynthesis, sugars are known to be one of the most potent inducers of flavonoid biosynthesis [14]. Therefore, we expected that supplying sucrose would reduce the flavonol content in the UGT78D1-OX lines. On the contrary, we observed that the content of K3 in UGT78D1-OX #7 remained the same regardless of the presence of the absorbance of sucrose (Fig. 1(A)). This indicates that an enhanced glycosylation activity directs the metabolic flux toward the accumulation of flavonols even in the presence of a strong inducer of anthocyanin synthesis such as sucrose. In addition, we examined whether the application of naringenin, which is the precursor of kaempferol and quercetin, could alter the flavonol contents of the UGT78D1-OX lines. The supply of a high concentration of exogenous naringenin caused an overall decrease in flavonol contents. However, K3 remained the most abundant flavonol in the UGT78D1-OX #7 line ( Fig. 1(A)). Next, we determined the anthocyanin contents of the WT and UGT78D1-OX seedlings in response to sucrose. As shown in Fig. 1(B), all three UGT78D1-OX lines accumulated more anthocyanins in the presence of a high concentration of sucrose. This observation suggests that as long as flavonols are glycosylated, sucrose can increase the contents of anthocyanins and flavonols. It would be plausible to expect that glycosylated flavonols are taken up by the vacuoles, thereby reducing the actual flavonol concentration available in the cytoplasm.
The expression of PAP1, FLS1, and DFR was lower in UGT78D1-OX seedlings than in WT seedlings Since we observed different responses in terms of flavonol accumulation among the three UGT78D1-OX lines, we were curious about whether the transcript level of UGT78D1 may vary among those transgenic lines. In accordance with our expectation, UGT78D1-OX #7 produced almost 10 times more UGT78D1 transcript (Fig. 2(A)) and its expression level further increased in response to 300 mM sucrose. As the overexpression of the cloned UGT78D1 cDNA was driven by the 35S CAMV promoter, the further increase in the expression of the UGT78D1 transcript upon the addition of sucrose indicates that posttranscriptional regulation plays a significant role in determining the transcript level (Fig. 2(A)). The posttranscriptional regulation of gene expression is often observed. For example, when a strong promoter was used to drive the overexpression of PAP1 in a previous study, the transcript level of PAP1 still altered when seedlings were exposed to sucrose [15]. Next, we examined whether the elevated levels of anthocyanins and certain flavonols in UGT78D1-OX seedlings were due to enhanced transcript levels of flavonoid biosynthesis genes. The transcripts of PAP1, FLS1, and DFR accumulated much less in UGT78D1-OX seedlings than in WT seedlings when the plants were exposed to 0, 200, and 300 mM sucrose for 6 h (Fig. 2(B)). Many flavonoid biosynthesis genes are known to respond to sucrose [16]. Because UGT78D1 is not a transcription factor, the reduced transcript levels of PAP1, FLS1, and DFR are most likely to result from the induction of feedback mechanisms by the altered flavonoid levels in UGT78D1-OX seedlings compared with those in WT plants.
UGT78D1 is highly induced by sucrose and high salt, but UGT78D1-OX seedlings did not exhibit a similar abiotic stress tolerance in comparison with WT seedlings The high responsiveness of UGT78D1 to sucrose led us to examine the transcript levels of UGT78D1 in response to The transcript levels of flavonoid synthesis genes were quantified, including DFR, FLS1, and the transcription factor PAP1. Three replicates were performed and subjected to statistical analysis by Tukey's test (p \ 0.01). The asterisks above the columns denote significant differences. The error bars represent standard error other plant growth stimulators and inhibitors. As shown in Fig. 3(A), the transcript level of UGT78D1 was highly increased in response to sucrose, high salt, and jasmonic acid. Plants that are metabolically engineered to obtain high levels of certain flavonoids sometimes exhibit an enhanced tolerance to abiotic stresses [17]. However, when the growth and performance of the WT and UGT78D1-OX seedlings were tested in various abiotic stress media, we did not observe any differences between them (Fig. 3B, C). These results imply that the enhanced levels of certain flavonols may not enough to increase the overall rate of plant growth or the degree of abiotic stress tolerance. Flavonoids are well known for having antioxidant activities, and there have been many attempts to synthesize flavonoids in microbial systems [4,17,18]. Recently, K3 was revealed to confer anti-hyperalgesic properties through the activation of cholinergic receptors [19]. Taken together, our results suggest that various pathways involved in the secondary metabolism of plants can be altered to manipulate specifically targeted metabolites. Thus, it should be possible to produce crops that accumulate a high content of flavonoids for human use. Wildtype and UGT78D1-OX #3, #7, and #12 seedlings were grown on 1/2 Murashige-Skoog medium for 9 days, at which point RNA was isolated after treatment with abiotic (sucrose, NaCl, mannitol, abscisic acid, indole-3yl-acetic acid) and biotic (jasmonic acid, salicylic acid) stress reagents for 6 h. The transcript levels of UGT78D1 were examined with quantitative real-time polymerase chain reaction using the UGT78D1 primer. ACTIN2 was included as an internal control. Three independent replicates were performed and subjected to statistical analysis by Tukey's test (p \ 0.001). The asterisks above the columns denote significant differences. The error bars represent standard error. (B) Four-day-old wild-type and UGT78D1-OX #3, #7, and #12 seedlings, which were initially grown on Hoagland's medium containing 20 mM KNO 3 , were transferred to solutions of NaCl (150 and 300 mM) and mannitol (300 and 400 mM). (C) Root length measurements 7 days after transfer. Five independent samples were included and subjected to statistical analysis by Tukey's test (p \ 0.05). The asterisks above the columns denote significant differences. The error bars represent standard error