Cloning and functional characterization of a p-coumaroyl quinate/shikimate 3′-hydroxylase from potato (Solanum tuberosum)

Article abstract of DOI:10.1016/j.bbrc.2018.01.075

Chlorogenic acid (CGA) plays an important role in protecting plants against pathogens and promoting human health. Although CGA accumulates to high levels in potato tubers, the key enzyme p-coumaroyl quinate/shikimate 3′-hydroxylase (C3′H) for CGA biosynthesis has not been isolated and functionally characterized in potato. In this work, we cloned StC3′H from potato and showed that it catalyzed the formation of caffeoylshikimate and CGA (caffeoylquinate) from p-coumaroyl shikimate and p-coumaroyl quinate, respectively, but was inactive towards p-coumaric acid in in vitro enzyme assays. When the expression of StC3′H proteins was blocked through antisense (AS) inhibition under the control of a tuber-specific patatin promoter, moderate changes in tuber yield as well as phenolic metabolites in the core tuber tissue were observed for several AS lines. On the other hand, the AS and control potato lines exhibited similar responses to a bacterial pathogen Pectobacterium carotovorum. These results suggest that StC3′H is implicated in phenolic metabolism in potato. They also suggest that CGA accumulation in the core tissue of potato tubers is an intricately controlled process and that additional C3′H activity may also be involved in CGA biosynthesis in potato.

Full text of DOI:10.1016/j.bbrc.2018.01.075

Biochemical and Biophysical Research Communications xxx (2017) 1e6
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
Cloning and functional characterization of a p-coumaroyl quinate/
shikimate 30-hydroxylase from potato (Solanum tuberosum)
,
,
, e, f, *
Benjamin J. Knollenberg a b, Jingjing Liu a c, Shu Yu a, Hong Lin d, Li Tian a
a Department of Plant Sciences, University of California, Davis, CA, 95616, USA
b The Huck Institutes of the Life Sciences, Pennsylvania State University, State College, PA, 16801, USA
c State Key Laboratory of Subtropical Silviculture, Zhejiang A & F University, Hangzhou, Zhejiang, 311300, China
d San Joaquin Valley Agricultural Sciences Center, USDA-ARS, Parlier, CA, 93658, USA
e Shanghai Key Laboratory of Plant Functional Genomics and Resources, Shanghai Chenshan Botanical Garden, Shanghai, 201602, China
f Shanghai Chenshan Plant Science Research Center, Chinese Academy of Sciences, Shanghai, 201602, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Chlorogenic acid (CGA) plays an important role in protecting plants against pathogens and promoting
human health. Although CGA accumulates to high levels in potato tubers, the key enzyme p-coumaroyl
quinate/shikimate 30-hydroxylase (C30H) for CGA biosynthesis has not been isolated and functionally
characterized in potato. In this work, we cloned StC30H from potato and showed that it catalyzed the
formation of caffeoylshikimate and CGA (caffeoylquinate) from p-coumaroyl shikimate and p-coumaroyl
quinate, respectively, but was inactive towards p-coumaric acid in in vitro enzyme assays. When the
expression of StC30H proteins was blocked through antisense (AS) inhibition under the control of a tuber-
specific patatin promoter, moderate changes in tuber yield as well as phenolic metabolites in the core
tuber tissue were observed for several AS lines. On the other hand, the AS and control potato lines
exhibited similar responses to a bacterial pathogen Pectobacterium carotovorum. These results suggest
that StC30H is implicated in phenolic metabolism in potato. They also suggest that CGA accumulation in
the core tissue of potato tubers is an intricately controlled process and that additional C30H activity may
also be involved in CGA biosynthesis in potato.
Received 1 January 2018
Accepted 10 January 2018
Available online xxx
Keywords:
C30H
Chlorogenic acid
Potato
Antisense
Ferulic acid conjugate
© 2018 Elsevier Inc. All rights reserved.
1. Introduction
health-promoting benefits of CGA in potato, a better understanding
of its biosynthetic mechanism is needed in this plant.
Potato (Solanum tuberosum) is widely consumed in the world.
Chlorogenic acid (CGA), a conjugate of caffeic acid and quinic acid,
constitutes the most abundant phenolic compound found in potato
tubers [1]. A high-level accumulation of CGA in plants has been
linked to their increased resistance to a variety of microbial path-
ogens [2]. Additionally, CGA has also been associated with multiple
bioactivities in humans, such as lowering the risks of cancer and
type 2 diabetes [3]. To exploit the pathogen defense and human
Three possible routes have been proposed for CGA biosynthesis
in plants, all of which involve the action of p-coumaroyl quinate/
shikimate 30-hydroxylase (C30H) while using p-coumaric acid
(route 1), p-coumaroyl shikimate (route 2) or p-coumaroyl quinate
(route 3) as a substrate (Fig. 1A) [4]. To date, several C30H enzymes
from different plant species have been biochemically characterized
[5e8]. These C30Hs exhibited substantial activities towards p-cou-
maroyl shikimate and p-coumaroyl quinate, but very little or no
activity towards p-coumaric acid, suggesting that routes 2 and 3
likely lead to the formation of CGA (Fig.1A). When the expression of
hydroxycinnamoyl CoA quinate:hydroxycinnamoyl transferase (HQT)
was downregulated via RNAi, up to a 90% reduction in CGA was
observed in tubers of the transgenic potato plants [9]. This result
suggests that route 2 involving p-coumaroyl shikimate may play a
major role in CGA biosynthesis in potato tubers (Fig. 1A) [9].
However, C30H genes have not been cloned and biochemically
characterized in potato.
Abbreviations: AS, antisense; CGA, chlorogenic acid; C30H, p-coumaroyl quinate/
shikimate 30-hydroxylase; EV, empty vector; HPLC, high performance liquid chro-
matography; HQT, hydroxycinnamoyl CoA:quinate hydroxycinnamoyl transferase;
MS, Mass Spectrometry; MUSCLE, Multiple Sequence Comparison by Log-Expec-
tation; RACE, rapid amplification of cDNA ends; RT, reverse transcription; WT, wild
type.
* Corresponding author. Department of Plant Sciences, Mail Stop 3, University of
California, Davis, Davis, CA, 95616, USA.
E-mail address: ltian@ucdavis.edu (L. Tian).
https://doi.org/10.1016/j.bbrc.2018.01.075
0006-291X/© 2018 Elsevier Inc. All rights reserved.
Please cite this article in press as: B.J. Knollenberg, et al., Cloning and functional characterization of a p-coumaroyl quinate/shikimate 30-
hydroxylase from potato (Solanum tuberosum), Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/
j.bbrc.2018.01.075

2
B.J. Knollenberg et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e6
Fig. 1. A. Three proposed routes for chlorogenic acid (CGA) formation in potato. PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; C30H, p-coumaroyl quinate/
shikimate 30-hydroxylase; 4CL, p-coumaroyl:CoA ligase; HCT, hydroxycinnamoyl CoA shikimate/quinate:hydroxycinnamoyl transferase; HQT, hydroxycinnamoyl CoA quinate:hy-
droxycinnamoyl transferase. Dotted arrows denote multiple enzymatic reactions. B. A neighbor-joining phylogenetic tree of selected plant C30H proteins. StC30H is highlighted in
bold. AtCYP97A3 is used as an outgroup for the analysis.
To investigate CGA biosynthesis and assess the role of C30H on
phenolic metabolism in potato, we cloned a StC30H gene and
determined its activity towards p-coumaric acid, p-coumaroyl
shikimate, and p-coumaroyl quinate in in vitro enzyme assays. In
addition, an antisense (AS) StC30H was expressed in potato tubers
under the control of a tuber specific patatin promoter. Tuber yield,
soluble phenolic accumulation, and response to a bacterial path-
ogen were determined in the AS StC30H transgenic as well as wild
type and vector-transformed control potato tubers.
(AAU44038), Panicum virgatum (AB723823), Pinus taeda
(AAL47685), Selaginella moellendorffii isoform 1 (XM_002982586),
Sesamum indicum (AAL47545), S. lycopersicum (XP_004230046),
S. tuberosum (XM_006347618), Sorghum bicolor (AAC39316), Trifo-
lium pratense (GQ497816), and Triticum aestivum (CAE47489). The
accession number for StC30H is a predicted mRNA sequence in
GenBank, which is identical to the cDNA sequence obtained in this
work. AtCYP97A3 (NP_564384) was used as an outgroup for the
phylogenetic analysis.
2. Materials and methods
2.3. Recombinant protein expression in yeast and in vitro enzyme
assays
2.1. Cloning of StC30H
The open reading frame of StC30H was cloned into the BamHI
and EcoRI restriction sites of the yeast expression vector pYeDP60
[12]. pYeDP60-StC30H and the empty pYeDP60 vector were trans-
formed into yeast (Saccharomyces cerevisiae) WAT11 cells [12]. Yeast
microsomes were prepared as described [13]. Protein concentra-
tions of the microsomes were determined using the Bradford assay
[14].
A partial StC30H cDNA sequence was retrieved from the SOL
Genomics Network (https://solgenomics.net/). Total RNA was
extracted from leaves of potato cv. Atlantic using TRIzol reagent
(Invitrogen, Carlsbad, CA) and used for the synthesis of first strand
cDNA. The full-length StC30H was obtained by 50 rapid amplification
of cDNA ends (RACE) PCR using the SMARTer RACE kit (Clontech,
Mountain View, CA). Primer sequences used for RACE PCR, yeast
expression, cloning of the patatin promoter, and generation of AS
StC30H for expression in potato, as well as amplification of AS StC30H
transgene and StC30H/AS StC30H transcripts are listed in Table S1.
Reactions using p-coumaric acid (Sigma Aldrich, St. Louis, MO)
and p-coumaroyl shikimate (provided by Prof. John Ralph, Univer-
sity of Wisconsin, Madison) were performed as follows. The 100
reaction included 400 M p-coumaric acid or p-coumaroyl shiki-
mate, 600 M NADPH, and approximately 100 g of microsomal
proteins in 100 mM sodium phosphate buffer, pH 7.5. The reaction
was incubated at 30 ꢀC for 3 h and stopped by addition of 10
L of
mL
m
m
m
2.2. Phylogenetic analysis
m
Selected plant C30H protein sequences were aligned using
Multiple Sequence Comparison by Log-Expectation (MUSCLE) [10].
A neighbor-joining tree was constructed using the sequence
alignment with MEGA7 [11]. The GenBank accession numbers are:
Ammi majus (AAT06912), Arabidopsis thaliana (NP850337), Coffea
canephora isoform 1 (DQ269126), Cynara cardunculus (KU711508),
Daucus carota isoform 1 (KU757478), Glycine max (AAB94587),
Lithospermum erythrorhizon (BAC44836), Lonicera japonica
(KC765076), Ocimum basilicum (AAL99200), Oryza sativa
17.5 M acetic acid. The reaction mixture was centrifuged at
13,000 ꢁ g for 10 min. The supernatant was collected and mixed
with an equal volume of methanol for high performance liquid
chromatography (HPLC) analysis.
Since p-coumaroyl quinate was not commercially available,
coupled enzyme assays were carried out using the A. thaliana p-
coumaroyl:CoA ligase (At4CL1; for formation of p-coumaroyl CoA
from p-coumaric acid and coenzyme A) [15], the P. virgatum
hydroxycinnamoyl transferase (PvHCTa1; for formation of p-
Please cite this article in press as: B.J. Knollenberg, et al., Cloning and functional characterization of a p-coumaroyl quinate/shikimate 30-
hydroxylase from potato (Solanum tuberosum), Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/
j.bbrc.2018.01.075

B.J. Knollenberg et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e6
3
coumaroyl quinate from p-coumaroyl CoA and quinic acid) [5], and
pYeDP60-StC30H- or pYeDP60-expressing yeast microsomes. The
initial reaction (1 mL) included 0.35 mg p-coumaric acid, 0.2 mg
cycle of 5 min at 94 ꢀC, 28 cycles of 30 s at 94 ꢀC, 30 s at 63 ꢀC, and
90 s at 72 ꢀC, followed by 1 cycle of 10 min at 72 ꢀC. The PCR prod-
ucts were separated on a 1% agarose gel.
coenzyme A, 11.5 mL of 100 mM ATP, and 5.5 mg of purified, His-
tagged At4CL1 proteins in 100 mM sodium phosphate buffer, pH
2.6. HPLC and MS/MS analyses
7.5. After incubating at room temperature for 1 h, an additional
0.2 mg coenzyme A and 11.5
m
L of 100 mM ATP were added to the
StC30H enzyme assay products and metabolite extracts from
potato tubers were separated on a reverse-phase HPLC using a
gradient between A (0.1% formic acid in water) and B (acetonitrile)
at a flow rate of 1 mL minꢂ1. For analysis of assay products, the
gradient was 0e15 min, 88-65% A and 15e15.5 min, 65e88% A. The
retention time and absorption spectra of reaction products were
compared to the authentic p-caffeoyl shikimate (provided by Prof.
John Ralph) and CGA (Sigma Aldrich) standards. For analysis of
potato metabolite extracts, the HPLC gradient was 0e5 min, 95% A;
5e15 min, 95-80% A; 15e34.5 min, 80-45% A; 34.5e35 min,
45e95% A. Individual peaks were collected, concentrated in a vac-
uum centrifuge, and subjected to tandem Mass Spectrometry (MS)
analysis as described in Ref. [19].
reaction mixture, followed by continued incubation at room tem-
perature overnight. On the next day, the above reaction was divided
into 100
PvHCTa1 proteins (at 2 mg mLꢂ1), 500
DTT were added. This reaction was incubated at room temperature
for 2 h. To the above reaction approximately 100 g of pYeDP60-
StC30H- or pYeDP60-containing microsomal proteins were added
along with 600
M NADPH. The reaction was incubated at 30 ꢀC for
3 h before adding 10 L of 17.5 M acetic acid. After centrifugation at
L of methanol was added to the
m
L aliquots, to which 0 (used as control) or 5
mL of purified
mM quinic acid, and 500
mM
m
m
m
13,000 ꢁ g for 10 min, 100
m
collected supernatant prior to HPLC analysis.
2.4. Potato transformation
2.7. Disease assay
A 2.5-kb B33 patatin promoter [16] was amplified from potato
cv. Atlantic and cloned into the pCAMBIA2300 vector. The StC30H
coding sequence was then cloned downstream from the patatin
promoter in the AS orientation in pCAMBIA2300. The recombinant
pCAMBIA2300-patatin promoter-AS StC30H plasmid and the empty
pCAMBIA2300 vector were transformed into the electrocompetent
Agrobacterium tumefaciens LBA4404 cells (Invitrogen).
Agrobacterium-mediated potato transformation was carried out
using microtuber discs and internode explants following an
established method [17]. Nine transgenic potato lines were pro-
duced, including 2 empty vector controls (EV) and 7 AS StC30H lines.
Incorporation of the AS StC30H transgene was confirmed by PCR
amplification using primers that are specific for the pCAMBIA2300
vector and StC30H in the AS orientation; the PCR products were
verified by DNA sequencing.
Disease assays with Pectobacterium carotovorum subsp. car-
otovorum were performed following a previously established
method [20]. Briefly, each tuber was inoculated with the bacterial
suspension at three points from the basal to apical end to a depth of
1 cm. The tubers were placed on paper towels wetted with sterile
water and incubated in dark at room temperature for 4 d. At the end
of the incubation period, tubers were cut longitudinally, photo-
graphed, and lesions were measured with a ruler at their widest
diameter to the nearest 0.5 mm. All lesions for each tuber were
averaged. There were four biological replicates for the transgenic
and control potato lines.
2.8. Statistical analysis
The tuber yield, metabolite, and disease assay data were
analyzed by one-way ANOVA followed by Tukey's HSD test using
JMP vers. 11 (SAS Institute, 2014).
2.5. Molecular characterization of transgenic potato plants
Potato seedlings were grown in
a temperature-controlled
greenhouse in a randomized complete block design. Potato tubers
were harvested at 110 d after planting in soil, which is approxi-
mately when CGA concentrations stabilize in the tuber tissue [18].
Total tuber yield at harvest was measured for four biological rep-
licates of the transgenic and control plants. After the tubers were
cleaned with water and 75% ethanol, the peel/cortex tissue
(approximately 4 mm from the surface) was removed using a po-
tato peeler. A core borer was then used to collect a plug of the
medullary/core tissue (diameter ¼ 1 cm, length ¼ 2 cm). Tissues
from three tubers were pooled for each plant, which constitute one
biological replicate; three biological replicates were collected for
metabolite and gene expression analyses. The tuber tissues were
ground in liquid nitrogen and stored at ꢂ80 ꢀC until further
analysis.
Total RNA was extracted from the ground tuber tissue using
Trizol reagent (Invitrogen) and treated with DNase I (Fermentas,
Glen Burnie, MD). Reverse transcription (RT) reactions were carried
out using 650 ng of total RNA with the Superscript IV cDNA syn-
thesis kit (Invitrogen). The oligo(dT)12-18 primer was used in the RT
reaction to amplify both sense and AS StC30H transcripts. To reverse
transcribe specifically the AS StC30H transcript, a gene specific
primer (50-ATGGCTATTCCCATTTCTTTACCTG-30) was used in the RT
reaction. The sense and/or AS StC30H transcripts were amplified
3. Results
3.1. StC30H is closely related to dicot C30Hs
StC30H, a C30H homolog, was identified in potato through
homology-based searches and its full-length sequence was ob-
tained using RACE-PCR. To understand the evolutionary relation-
ship of StC30H with other C30Hs, phylogenic analysis was conducted
using StC30H and (putatively) identified plant C30Hs (Fig. 1B). The
lycophyte S. moellendorffii C30H (SmC30H) is ancestral to both
monocot and dicot C30Hs. StC30H falls in the dicot C30H clade, and
associates most closely with the tomato C30H (SlC30H) that is 99%
identical to StC30H (Fig. 1B).
3.2. StC30H demonstrated p-caffeoyl shikimate and CGA forming
activities in in vitro enzyme assays
To determine the catalytic activity of StC30H, the recombinant
StC30H protein was expressed in yeast strain WAT11, which contains
an A. thaliana cytochrome P450 reductase for testing the activities
of cytochrome P450 enzymes. The StC30H-expressing yeast micro-
somes were incubated with substrates of three possible routes,
including p-coumaric acid, p-coumaroyl shikimate, and p-cou-
maroyl quinate (derived from sequential activities of At4CL1 and
PvHCTa1 using p-coumaric acid as substrate). Although StC30H did
using 1.5
mL of cDNA template and AmpliTaq DNA polymerase
(Thermo Scientific, Waltham, MA). The cycling parameters were 1
Please cite this article in press as: B.J. Knollenberg, et al., Cloning and functional characterization of a p-coumaroyl quinate/shikimate 30-
hydroxylase from potato (Solanum tuberosum), Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/
j.bbrc.2018.01.075

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B.J. Knollenberg et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e6
not exhibit any activity towards p-coumaric acid (Fig. 2A), it con-
verted p-coumaroyl shikimate near completely to p-caffeoyl shi-
kimate (Fig. 2B). CGA was formed as the final product when p-
coumaric acid was incubated with At4CL1, PvHCTa1, and StC30H,
indicating that StC30H is active towards p-coumaroyl quinate and
can convert it to the hydroxylated derivative CGA (Fig. 2C). These
in vitro assay results collectively suggested that routes 2 and 3
involving p-coumaroyl shikimate and p-coumaroyl quinate can lead
to the formation of CGA in potato (Fig. 1A).
3.3. AS StC30H transcripts accumulated in transgenic potato tubers
To understand the in vivo function of StC30H, an AS StC30H
construct was transformed in potato tubers to block its translation
into proteins. To prevent pleiotropic effects from downregulating
StC30H expression in aboveground tissues, a tuber-specific patatin
promoter was used to direct AS StC30H expression. Incorporation of
AS StC30H in potato plants was confirmed by PCR amplification of
the AS StC30H transgene, using primers located in the patatin pro-
moter and the StC30H gene in AS orientation, in the seven AS StC30H
transgenic, but not wild type (WT) or EV control lines (Fig. 3A).
To determine whether AS StC30H transcripts were expressed in
transgenic potato lines, AS strand-specific RT-PCR was performed
(Fig. 3B). AS StC30H transcripts accumulated in all seven lines, with
strong AS StC30H expression in the core tissues of AS5, AS6, AS14,
and AS16 tubers (Fig. 3B). In a separate set of reactions intended to
detect both sense and AS StC30H transcripts, a similar trend of AS
StC30H transcript accumulation/enrichment was also observed in
the AS StC30H transgenic potato lines (Fig. 3C).
3.4. AS StC30H potato plants showed moderate changes in tuber
yield and soluble phenolics in the core tuber tissue, but similar
responses to a bacterial pathogen, compared to the controls
Although no obvious changes were observed in the growth of
aboveground tissues (data not shown), there was an around 26%
Fig. 3. Molecular characterization of the antisense (AS) StC30H transgenic and control
potato plants. A. Amplification of the AS StC30H transgene using primers located on the
patatin promoter and StC30H in the AS orientation. B. Amplification of AS StC30H
transcripts. A primer that specifically amplifies the AS StC30H transcript was used in the
reverse transcription (RT) reaction. C. Detection of sense and AS StC30H transcripts. The
oligo(dT)12-18 primer was used in the RT reaction. Each experiment was performed
with three biological replicates and a representative image of each experiment is
shown. The expected StC30H gene/transcript amplification product is indicated with an
arrow.
Fig. 2. Enzyme activity assays of StC30H using p-coumaric acid (A), p-coumaroyl shi-
kimate (B), and p-coumaroyl quinate (C) as substrates. HPLC elution profiles of the
authentic standards and reaction mixtures are shown. Reaction products are high-
lighted with arrows. The p-coumaroyl quinate substrate in (C) was derived from
coupled reactions catalyzed by the Arabidopsis thaliana p-coumaroyl:CoA ligase
(At4CL1) and the Panicum virgatum hydroxycinnamoyl transferase (PvHCTa1).
Please cite this article in press as: B.J. Knollenberg, et al., Cloning and functional characterization of a p-coumaroyl quinate/shikimate 30-
hydroxylase from potato (Solanum tuberosum), Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/
j.bbrc.2018.01.075

B.J. Knollenberg et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e6
5
reduction in total tuber yield for AS5 (238.7 g/plant) and a trend of
decreased tuber yield for AS4, AS6, AS16, and AS17 lines (Fig. 4A).
Overall, the AS StC30H lines on average (269.3 g/plant) had a 17%
decrease in total tuber yield compared to the EV control plants
(324.4 g/plant) (Fig. 4A).
significantly decreased in AS5 and to a less extent in four other AS
StC30H lines (Fig. 4A). The reduction in tuber yield could be a result
of metabolic perturbations in this tissue. It is also possible that, in
maintaining normal aboveground tissue growth, carbons were re-
allocated between the source (aboveground) and sink (tuber) tis-
sues. As such, less photosynthate was available to drive tuber
growth, which led to decreased tuber yield in AS StC30H lines.
Interestingly, while most soluble phenolics accumulated at
comparable levels between the AS StC30H and control plants, CGA
and feruloyl conjugates were moderately increased in the AS StC30H
plants (Fig. S1). A search of potato transcriptome sequences (Potato
Genomics Resources; http://solanaceae.plantbiology.msu.edu/)
identified four open reading frames that are 62e74% identical to
StC30H at the protein sequence level (Fig. S2). In the future, these
StC30H-like proteins can be biochemically and genetically (e.g. via
overexpression and downregulation of gene expression) charac-
terized to determine whether they can functionally compensate for
the missing C30H activity in AS StC30H plants. Another possible
explanation for the increased CGA and feruloyl conjugates could be
that the initial reduced CGA production from AS StC30H expression
limits carbon flux towards lignin (wall-bound phenolics; uses caf-
feoyl CoA as a precursor) biosynthesis (Fig. 1A). As a result, the
backed up carbons positively feedback regulate CGA biosynthesis.
The intricate interplay of soluble and wall-bound potato phenolics
should be further investigated in future research.
To determine the impact of AS StC30H expression on CGA accu-
mulation and phenolic metabolism in potato tubers, metabolite
analysis was performed on soluble phenolics extracted from the
core tissue of potato tubers. When the peak areas were compared
among the control (WT and EV) and AS StC30H lines, only peaks 1
and 2 showed significant differences (in particular, increases) from
the controls in 2 and 4 of the 7 AS lines, respectively (Fig. 4B and
Fig. S1). Peak 1 was determined to be CGA based on the UV ab-
sorption spectra and MS fragmentation patterns, which are similar
to the authentic standard as well as those shown in previous re-
ports (Fig. S1) [21]. Peak 2 was determined to be a mix of trans-5-O-
feruloyl quinic acid and an unknown feruloyl conjugate, with a
larger contribution from trans-5-O-feruloyl quinic acid, based on
comparison to previous work in potato (Fig. S1) [22].
To evaluate the disease resistance property of the transgenic
plants, tubers from the control and AS StC30H lines were subjected
to disease assays with P. carotovorum subsp. carotovorum, one of
the main disease-causing agents of soft rot in potato tubers
(Fig. 4C). Despite changes in potato tuber yield and soluble phe-
nolics, AS StC30H lines showed similar lesion sizes to the control
lines when inoculated with P. carotovorum (Fig. 4C).
CGA and ferulic acid conjugates have shown to be associated
with potato resistance to P. carotovorum [2]. However, the slight
increases in these metabolites in AS StC30H tubers may not be
sufficient for suppression of P. carotovorum growth as observed in
the disease assays (Fig. 4C).
4. Discussion
Stunted growth was reported for Arabidopsis and poplar plants
with reduced C30H gene expression in all tissues [23,24]. To avoid
such pleiotropic effect on plant development, AS StC30H was
expressed specifically in potato tubers and we did not observe any
obvious reduction in the growth of aboveground tissues in the
transgenic potato plants. However, total tuber yield was
In summary, this work elucidated the biochemical activity of
StC30H towards p-coumaroyl shikimate/quinate. AS down-
regulation of StC30H protein expression in potato tubers provided
interesting new insights into the complex control of CGA and
phenolic metabolism in potato and posed new questions for future
Fig. 4. A. Total tuber yield of the transgenic and control potato lines. Data shown are the average of four biological replicates and the standard deviation. B. A representative HPLC
elution profile of phenolic metabolites extracted from the core tissue of potato tubers. C. Disease assays with Pectobacterium carotovorum subsp. carotovorum. The average lesion
sizes of four biological replicates and standard deviations are shown.
Please cite this article in press as: B.J. Knollenberg, et al., Cloning and functional characterization of a p-coumaroyl quinate/shikimate 30-
hydroxylase from potato (Solanum tuberosum), Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/
j.bbrc.2018.01.075

6
B.J. Knollenberg et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e6
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Conflicts of interest
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The authors have no conflict of interest.
Author contribution
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D.A. Navarre, Synthesis and regulation of chlorogenic acid in potato: rerouting
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formed the experiment; BJK, JJL, SY, HL and LT analyzed the data;
BJK and LT wrote the draft; All authors read and approved the
manuscript.
Acknowledgments
This work was supported by the USDA ARS cooperative agree-
ment 58-5302-0-223. We thank Prof. John Ralph for providing us p-
coumaroyl shikimate and p-caffeoyl shikimate, Prof. Norman Lewis
for the At4CL1 plasmid, Prof. Richard Dixon for the PvHCTa1
plasmid, and Prof. Philip Urban for the yeast WAT11 strain and the
pYeDP60 vector. We would also like to thank Alex Wilson for his
assistance with MS analysis. BJK was supported by a fellowship of
the International Agricultural Development Graduate Group at
University of California, Davis. JJL was supported by Top Priority and
First Class Discipline in Forestry of Zhejiang province. SY was sup-
ported by a Chinese Scholarship Council Fellowship, a Graduate
Research Fellowship of the Department of Plant Sciences, Univer-
sity of California, Davis, and the Henry A. Jastro Research
Fellowship.
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Appendix A. Supplementary data
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Please cite this article in press as: B.J. Knollenberg, et al., Cloning and functional characterization of a p-coumaroyl quinate/shikimate 30-
hydroxylase from potato (Solanum tuberosum), Biochemical and Biophysical Research Communications (2017), https://doi.org/10.1016/
j.bbrc.2018.01.075