Published February 26, 2016
o r i g i n a l r es e a r c h
Evolutionary Profiling of Group II PyridoxalPhosphate-Dependent Decarboxylases Suggests
Expansion and Functional Diversification of
Histidine Decarboxylases in Tomato
Rahul Kumar,* Gitanjali Jiwani, Amit Pareek, Thula SravanKumar,
Ashima Khurana, and Arun K. Sharma
Abstract
P
(a derivative of vitamin B6) is
a pivotal organic cofactor and used by diverse range
of enzymes in all living organisms (Facchini et al., 2000).
Pyridoxal-phosphate-dependent enzymes catalyze the
transfer of amino groups, decarboxylation, and formation of L- and D-amino acids and removal of chemical
groups attached to b- and g-carbon. Their enzymatic
versatility arises as a result of their ability of binding of
PLP group covalently to the substrate. Their electrophilic
catalytic property further assists them stabilize a chemical reaction by stabilizing different carbon ionic intermediates (John, 1995; Schneider et al., 2000).
Depending on distinct structural groups, PLP
enzymes are grouped in at least five evolutionary independent families (Milano et al., 2013; Percudani and
Peracchi, 2003). One of such important classes is group
II PLP-dependent decarboxylase (PLP_deC) enzymes.
This class includes aromatic L-amino-acid decarboxylase
(AAD), catalyzes the conversion of L-phenylalanine to
phenylamine), glutamate decarboxylase (GAD, catalyzes
the conversion of L-glutamate to g-aminobutyric acid
yridox al phosphate
Pyridoxal phosphate (PLP)-dependent enzymes are one of the
most important enzymes involved in plant N metabolism. Here,
we explored the evolution of group II PLP-dependent decarboxylases (PLP_deC), including aromatic L-amino acid decarboxylase,
glutamate decarboxylase, and histidine decarboxylase in the
plant lineage. Gene identification analysis revealed a higher
number of genes encoding PLP_deC in higher plants than in
lower plants. Expression profiling of PLP_deC orthologs and
syntelogs in Arabidopsis thaliana (L.) Heynh., pepper (Capsicum
annuum L.), and tomato (Solanum lycopersicum L.) pointed toward
conserved as well as distinct roles in developmental processes
such as fruit maturation and ripening and abiotic stress responses.
We further characterized a putative promoter of tomato ripeningassociated gene (SlHDC10) operating in a complex regulatory
circuit. Our analysis provides a firm basis for further in-depth exploration of the PLP_deC gene family, particularly in the economically important Solanaceae family.
R. Kumar and T. SravanKumar, Repository of Tomato Genomics Resources, Dep. of Plant Sciences, Univ. of Hyderabad, Hyderabad,
500046, India; G. Jiwani, A. Pareek, and A.K. Sharma, Dep. of
Plant Molecular Biology, Univ. of Delhi South Campus, New Delhi,
110021, India; A. Khurana, Botany Dep., Zakir Husain Delhi College, Univ. of Delhi, 110002, India; R. Kumar and G. Jiwani contributed equally to this work. Received 28 Mar. 2015. Accepted 31
Aug. 2015. *Corresponding author ([email protected]).
Abbreviations: AAD, aromatic-L-amino acid decarboxylase; ABA,
abscisic acid; BAP, 6-benzylamonopurine; GA, gibberellic acid;
GABA, g -aminobutyric acid; GAD, glutamate decarboxylase;
GADC, glutamate decarboxylase; HDC, histidine decarboxylase;
HMM, hidden Markov models; IAA, indole-3-acetic acid; MS, Murashige and Skoog; PCR, polymerase chain reaction; PLP, pyridoxal
phosphate; PLP_deC, pyridoxal-phosphate-dependent decarboxylase; qPCR, quantitative polymerase chain reaction; SDC, serine-like
decarboxylase.
Published in The Plant Genome 9
doi: 10.3835/plantgenome2015.07.0057
© Crop Science Society of America
5585 Guilford Rd., Madison, WI 53711 USA
An open-access publication
All rights reserved.
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[GABA]), and histidine decarboxylase (HDC, catalyzes
the conversion of histidine to histamine) enzymes. Studies on characterization of PLP_deCs, mainly GADs and
AADs, have implicated them in the production of secondary metabolites and flavor volatiles in plants (Akihiro
et al., 2008; Clayton, 2006; Facchini et al., 2000; Gallego
et al., 1995; Sakai et al., 2007; Sorrequieta et al., 2010;
Tieman et al., 2006). Two genes, namely SlGAD2 and
SlGAD3, are positively associated with GABA accumulation in maturing fruits of tomato (Akihiro et al., 2008;
Gallego et al., 1995). Recently, two serine-like decarboxylases (SDCs) have been implicated in generation of
acetaldehydes through decarboxylation and oxidative
deamination of phenylalanine, methionine, leucine, and
tryptophan (Torrens-Spence et al., 2014). In spite of their
importance in plant metabolism, surprisingly, we found
limited information available on PLP_deCs in tomato
and other plant species. Moreover, published evidence
suggests that there is a scope for further improvement
on the existing annotations of previously identified PLP_
deC orthologs, as it was based on a limited sequence data
available on plant genomes (Picton et al., 1993; Rontein et
al., 2001; Tieman et al., 2006; Torrens-Spence et al., 2014).
In this study, we identified PLP_deC members from
18 species from plant kingdom, ranging from algae to
lower and higher plants. The analysis clearly demonstrated the expansion of class II PLP_deC family genes
during evolution of higher plants. The subgroup HDC
showed a clear expansion in tomato. Transcript profiling
of these genes identified five ripening-associated HDC
genes in tomato. Nonetheless, transcript levels of PLP_
deC genes are also affected by nitrate, phytohormones,
and abiotic stresses. Altogether, our results not only
improve the current understanding of the mechanisms
associated with the evolutionary expansion, sequence
conservation, and functional divergence of PLP_deC
genes in tomato but also provide a foundation for further
research on them in plants, in general, and Solanaceae
family, in particular.
Experimental Procedure
Identification of Pyridoxal-PhosphateDependent Decarboxylase
To identify PLP_deC genes, protein sequences of full
complement of human, Arabidopsis, rice (Oryza sativa
L.), Escherichia coli, and yeast pyridoxal-phosphatedependent decarboxylases (Interpro entry, IPR002129;
pfam entry, PF00282) were retrieved from EMBLEBI webpage (http://www.ebi.ac.uk/interpro/entry/
IPR002129/taxonomy). Additionally, name searches for
histidine decarboxylase, aromatic-L-amino decarboxylase, glutamate decarboxylase, and pyridoxal phosphatedependent decarboxylase were conducted on the Sol
Genomics website (http://solgenomics.net/search/loci)
for tomato and pepper genes and protein sequences
for all the output entries were downloaded. All the
unique protein sequences were clubbed with the earlier
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retrieved proteins and combined sequences were used to
generate hidden Markov models (HMM) profile using
HMMERv3.1b1 (http://hmmer.janelia.org/) (Supplemental Table S1 in Supplemental File S1). Additionally,
protein.fasta files containing all the proteins encoded
by the respective genomes, included in this study, were
downloaded from either ENSEBML Plants (http://plants.
ensembl.org/index.html) or Phytozome v9.1 (http://www.
phytozome.net/) repository databases. Already generated HMM profiles were used to search the protein.fasta
files and the entries listed in output files (score ³100
and e-value £10−10) were saved. The protein sequences
of the listed entries were retrieved by using a custom
sequence.extraction perl script. The retrieved protein
sequences were then checked for the typical PLP_deC
domain architecture by using pfam (http://pfam.xfam.
org/search#tabview = tab1) and SMART (http://smart.
embl-heidelberg.de/) tools. The protein sequences lacking
the characteristics PLP_deC domain were discarded and
not included in further analyses. Promoter architecture
of tomato PLP_deCs was studied by extracting 1-kb promoter region from their start codon. The cis-elements
were analyzed by using online web tools such as PLACE
(http://www.dna.affrc.go.jp/PLACE/) and PlantCARE
(bioinformatics.psb.ugent.be/webtools/plantcare/html/).
Multiple Sequence Alignment, Phylogenetic
Analysis, and Gene Nomenclature
ClustalX v2.0 was employed to generate a multiple
sequence alignment file of the retrieved PLP_deC proteins (Thompson et al., 2002). Phylogenetic analysis was
performed by using Molecular Evolutionary Genetic
Analysis (MEGA 6) tool. Neighbor-joining algorithm
with p-distance method and pair wise deletion of gaps,
using default parameters was adopted to generate an
unrooted phylogenetic tree. A total number of 1000
iterations were tested per analysis and bootstrap statistical analysis for these replicates was employed to test
the phylogeny (Tamura et al., 2013). Nomenclature of
the identified genes, belonging to the three subcategories, including HDC, AAD, and GAD, was performed
on the basis of either their respective positions on the
chromosomes from 1 to 12 (first priority) or increasing
ENSEMBL PAC IDs used for their annotation. Tomato
PLP_deCs were named based on their position on 12
pseudo molecules, whereas pepper homologs were designated as per their relative homology with tomato genes
(Supplemental Table S1 in Supplemental File S1).
Plant Growth Conditions and Treatments
with Phytohormones and Growth Regulators
and Abiotic Stresses
Tomato plants (S. lycopersicon ‘Pusa Ruby’) were grown
and maintained under a daily photoperiodic regime of
16 h light and 8 h dark in a greenhouse or culture room,
unless mentioned specifically. The temperature of green
house was maintained at 28 ± 2°C. Different stages
of fruits were scored by tagging them at very young
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developmental stage (7 d after pollination with fruit
size approximately 1 cm). Fruits were harvested from
mature green (MG), breaker (B), 3 d postbreaker (B+3),
7 d postbreaker (B+7), and 20 d postbreaker (B+20)
stages, as described previously (Kumar et al., 2011). The
harvested tissues were flash frozen in liquid nitrogen to
avoid any injury. Three-week-old tomato seedlings grown
in culture room were used for harvesting root, shoot,
and leaf tissues. Various chemical treatment experiments were performed exactly in the same manner as
reported earlier (Kumar et al., 2012a, 2015). Ten-day-old
seedlings grown in culture room were immersed in the
50-µM solutions of abscisic acid (ABA; Sigma-Aldrich),
6-benzylamonopurine (BAP; Sigma-Aldrich), gibberellic acid (GA; Sigma-Aldrich), and ethylene (ethrel, Sisco
Research Laboratories Pvt. Ltd) for two time points, that
is, 1 and 3 h. Same stage seedlings treated with water for
the same time periods served as their controls. Similarly,
10-d-old seedlings grown on 0.5´ Murashige and Skoog
(MS) agar were exposed to various stress treatments
including cold, desiccation, heat, and salt. For cold stress,
culture tubes with tomato seedlings were transferred to
a cold chamber maintained at 4 ± 1°C for 2- and 24-h
time periods. Similarly, heat stress was given by incubating tomato seedlings in culture tubes in an incubator
maintained at 42 ± 1°C, for 30-min and 8-h time points.
Seedlings were removed from culture tubes and left on 3
mm blotting paper for 8 h for desiccation stress. For salt
stress, seedlings were removed from solid medium without disturbing roots and kept in liquid 0.5´ MS medium
with 200 mM NaCl for 8 h. Untreated seedlings were
used as control for stress treatments. Tissue samples were
harvested after the stress treatments and flash frozen
in liquid nitrogen. Auxin (50 µM indole-3-acetic acid;
IAA) treatment to the KPSC buffer (10 mM potassium
phosphate, pH 6.0; 2% sucrose; 50 µM chloramphenicol)
treated (12 h) apical portion (10 mm) of 3-d-old etiolated
tomato seedlings was performed as described earlier
(Kumar et al., 2012a). The KPSC buffer was changed
every 2 h of the treatment. Seedlings treated with only
KPSC buffer (without IAA) for the same time period
were used as a control for this experiment.
Gene Expression Analysis
In-house Affymetrix GeneChip (Affymetrix, Inc.)
tomato microarray data of five fruit ripening stages,
MG, B, B+3, B+5, and B+20 in Indian tomato Pusa
Ruby was used to identify the differentially expressed
genes (P-value £ 0.05, FC ³ 2) during the four transition phases (MG to B, B to B+3, B+3 to B+5, and B+5 to
B+20), as described earlier (Kumar et al., 2012b). Additionally, the available RNA sequencing expression data
of tomato (Sato et al., 2012) and pepper (Kim et al., 2014)
and publicly available Affymetrix GeneChip (Affymetrix,
Inc.) Arabidopsis microarray data (Schmid et al., 2005)
were also analyzed. The expression profiles of different
developmental stages were imported and final heat maps
were plotted using gplots package in R. Quantitative
polymerase chain reaction (qPCR) analysis was employed
to validate the expression of selected tomato PLP_deC
genes during different stages of development and also
to study their expression under various treatments. In
the case of cold and heat stresses, 1 µg RNA each from
the two stages of treatments were mixed in equimolar
concentration before cDNA preparation. The expression
of gene was quantified by SYBR-green-based standard
method following the methodology reported earlier
(Kumar et al., 2015).
Isolation and Cloning of SlHDC10 Promoter
Promoter architecture of SlHDC10 was studied by
performing in silico prediction of various cis-regulatory elements present in 1599-bp upstream region,
upstream to the start codon. This region was amplified
from BAC C08HBa0018C13 using the primers named
HDC10-fw: 5¢-TTCAGGATCCGAAAGACAAAATTTTATCTTGACC-3¢ and HDC10-rv: 5¢-TTCACCCGGGGAAGTTCTGGCATGGACTAGC-3¢ using DNA
polymerase enzyme (iMax II; Intron). The amplified PCR
fragment was digested with BamHI and SmaI restriction enzymes and then purified product was cloned
upstream to gus (b-glucuronidase) gene in PBI101 vector. Same strategy was used for making two SlHDC10deletion::GUS constructs. The sequences of primers used
for making these constructs are listed in Supplemental
Table S2 in Supplemental File S1. The final constructs
were used for subsequent transient as well as stable
tomato transformation using Agrobacterium tumefaciens
AGL1 strain, as described earlier (Sharma et al., 2009).
Briefly, cotyledons from 11-d-old 0.5´ MS mediumgrown Pusa Ruby seedlings were used for cocultivation
with Agrobacterium cultures containing binary vector
pBI101-SlHDC10-Fl::GUS (Murashige and Skoog 1962).
The cocultivated cotyledons were transferred to 1´ MS
supplemented with 1 mg L−1 transzeatin and 100 mg L−1
kanamycin for regeneration in Petri-plates (9 cm). Plates
were cultured under a 16 h light vs. 8 h dark cycle at 28 ±
2°C and explants with callus formation were subcultured
onto fresh medium every 15 d. The differentiated shoots
were excised from callus and transferred to rooting
medium (1´ MS supplemented with 100 mg L−1 kanamycin). Those plantlets with healthy shoots and dense roots
were transferred to pots containing 50% Soilrite (Kelperilite; 1:1:1 ratio of vermiculite, perlite, and Sphagnum
moss) mixed with soil for hardening. The young plantlets
were transferred to green house (as described earlier)
and characterized for the presence of transgene by using
nptII-specific primers. Kanamycin-resistant (1´ MS supplemented with 500 mg L−1 kanamycin) transgenic lines
were propagated for T2 generation and further analysis.
Transient Expression of the SlHDC10 Promoter
Expression Constructs in Nicotiana benthemiana
For transient assays, Nicotiana benthemiana Domin
plants were grown in growth chamber at 26 and 22°C
day and night temperature cycles in the condition of
kum ar et al .: histi dine decarbox yl ases are expanded in tom ato 3
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cycle of 16 h light and 8 h dark. For each set of experiment, 6- to 8-wk-grown six-leaf staged plants were used.
Mostly, the fourth and older true leaves of tobacco were
used for the experiment. Independent A. tumefaciens
AGL1 cultures containing both full length and deletion promoter constructs were separately grown in YEB
medium containing rifampicin and kanamycin at 28°C.
The grown culture was then transferred to induction
medium (0.5% beef extract, 0.1% yeast extract, 0.5% peptone, 0.1% sucrose, 2 mM MgSO4, 20 mM acetosyringone,
10 mM MES pH 5.6.) with appropriate antibiotics and
grown overnight. Next day, culture were centrifuged and
resuspended in infiltration medium (10 mM MgCl2, 10
mM MES, 200 mM acetosyringone, pH 5.6, O.D. = 1).
This culture was incubated at room temperature with
gentle agitation of 20 rpm for 2 h and infused against the
lower side of leaf lamina using a syringe with the needle
removed. The infiltration was done at 3 to 4 places within
a leaf with some distance between each of them. In case
of nitrate treatment, the tobacco leaves were detached
from the plant after 4 to 5 h to prevent nitrate induction from the soil. Following this, the leaves were given
nitrate treatment and the control leaf was incubated in
Milli-Q water or KCl solution free from any nitrate contaminations. Ethylene treatment was given to detached
tobacco leaves, after agroinfiltration, at different concentrations in air-tight containers for 5 h. Similarly, the
control was incubated in Milli-Q water.
Histochemical and Fluoremetric Analysis
of b-glucuronidase Activity
Histochemical assay for gus gene expression in different
organs of transgenic plant was performed as per protocol
given by Jefferson and colleagues (Jefferson et al., 1987).
The plant organs were dissected and incubated in GUS
histochemical buffer [50mM sodium phosphate pH 7.0,
50 mM EDTA pH 8.0, 0.5 mM K3Fe(CN)6, and 0.5 mM
K4Fe(CN)6, 0.1% Triton X-100, 1 mM X-Gluc (5-bromo4-chloro-3-indolyl-b-D-glucuronic acid), 20% methanol]
at 37°C for 16 to 24 h. The chlorophyll was removed from
the tissue using a destaining solution of acetone–ethanol
(1:3). The activity of GUS enzyme was measured using
the same protocol with a few modifications. Frozen tissue (100 mg) was homogenized using a glass rod in a tube
with addition of a pinch of glass powder for proper grinding. Next, 150 mL of the extraction buffer (50 mM sodium
phosphate buffer pH 7.0, 10 mM disodium EDTA, 0.1%
Triton X-100, 0.1% N-lauryl sarcosine, and 10 mM b-mercaptoethanol) was added to the minced tissue, mixed gently and centrifuged at 18,213 g for 20 min at 4°C to pellet
the debris. The supernatant was then filtered through 40
filter cell strainer to remove residual debris. The supernatant obtained was quantified for protein by using Bradford’s method (Bradford, 1976). The specific activity of
GUS was expressed as nmol of 4-MU (mg protein)−1 h−1
and GUS activity of the control plants was deducted from
the experimental samples to obtain the final values.
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Results
We analyzed the in-house fruit transcriptome microarray data (GSE20720) of five stages during ripening in
tomato and identified 145 differentially expressed genes
belonging to gene ontology functional category ‘Cellular
amino acid metabolic processes’ (Supplemental Fig. S1 in
Supplemental File S2). The previously identified histidine
decarboxylase (annotated as HDC) gene was present in
this category. Chromosome localization study showed
that HDC is present on chromosome 8 and flanked by
three other highly similar paralogous genes on either side
(Table 1). Based on this observation, we further explored
the tomato genome and identified the full complement of
this gene family in tomato (Fig. 1).
Identification of Pyridoxal-Phosphate-Dependent
Decarboxylase Complement in Plants
Complementation analysis using reference genome of
Chlamydomonas reinhardtii, two nonflowering lower
plants, Physcomitrella patens (Hedw.) Bruch & Schimp.
and Selaginella moellendorffii, 15 higher plant species,
bacteria (E. coli), yeast (Saccharomyces cerevisae) and
human identified full PLP_deC complements in these
species (Fig. 1; Supplemental Table S3 in Supplemental
File S1). While both bacterial and yeast genomes encoded
only GAD genes, three genes, including one gene each
for HDC, GAD, and AAD subclasses were encoded by
C. reinhardtii genome. The most number of PLP_deC
members was identified in tomato (30), whereas the
least number (10) was shared by Arabidopsis and papaya
(Carica papaya L.). Surprisingly, 20 homologs were found
to be present in S. moellendorffii (Supplemental Table
S3 in Supplemental File S1). Members of the AAD and
GAD subclasses were the major contributors of PLP_deC
genes in higher plants. In contrast, HDC subclass was
the major contributor of the PLP_deC complement in
Leguminaceae and Solanaceae (Fig. 1). A total of 20
HDC genes were found to be encoded by tomato genome,
whereas only eight homologs were identified in pepper.
Only two HDC genes each could be identified in most
of the remaining fruit bearing species. Gene structure
analysis of tomato PLP_deCs showed that SlAAD2 and
SlAAD3 were devoid of introns, whereas SlAAD5 had
the most number of introns (12) (Table 1). Protein sizes
of tomato AADs and GADs mostly ranged in between
53 and 57 kDa with an isoelectric point <6.0. In contrast,
HDC proteins exhibited great variation in their sizes,
ranging from 12 to 53 kDa, and had higher isoelectric
points (mostly >6.0) (Table 1).
The syntenic analysis using SynFind (http://genomevolution.org/CoGe/SynFind) identified seven syntelogs,
including two each for AADs (SlAAD3-4:CaAAD3-4)
and GADs (SlGAD3:CaGAD3 and SlGAD5:CaGAD4,
CaGAD5) and three HDCs (SlHDC1:CaHDC1,
SlHDC9:CaHDC9, and SlHDC15:CaHDC15) in pepper
and tomato (Supplemental Fig. S2 in Supplemental File
S2; Supplemental Table S3 in Supplemental File S1).
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Table 1. Characteristics of tomato group II pyridoxal-phosphate-dependent decarboxylases (PLP_deC) and their
orthologs and syntelogs in pepper.
Sol Genomics ID
(version Sl2.40)
Cellular
Protein size and Chromosome localization
isoelectric point (Sl2.40)
(TargetP)
Gene name†
Exon/
intron
Protein
(AA)
Solyc03g044120.1.1
Solyc03g045020.2.1
Solyc07g054280.1.1
Solyc07g054860.1.1
Solyc09g064430.2.1
Solyc01g005000.2.1
Solyc03g098240.2.1
Solyc04g025530.2.1
Solyc05g054050.2.1
Solyc11g011920.1.1
SlAAD1
SlAAD2
SlAAD3
SlAAD4
SlAAD5
SlGAD1
SlGAD2
SlGAD3
SlGAD4
SlGAD5
2/1
5/4
1/0
1/0
13/12
7/6
7/6
7/6
7/6
8/7
477
375
505
505
469
485
503
494
498
504
kDa
53.2 (7.1)
41.7 (6.0)
56.8 (5.9)
56.5 (6.5)
52.3 (5.9)
55.2 (5.9)
56.8 (6.1)
56.2 (5.2)
56.2 (5.7)
57.0 (5.4)
3
3
7
7
9
1
3
4
5
11
Solyc04g071140.2.1
Solyc05g007330.1.1
Solyc07g041260.1.1
Solyc07g041960.1.1
Solyc07g043220.1.1
Solyc08g006740.2.1
Solyc08g006750.2.1
Solyc08g016770.2.1
Solyc08g066220.2.1
Solyc08g066240.2.1
SlHDC1
SlHDC2
SlHDC3
SlHDC4
SlHDC5
SlHDC6 (AADC2)
SlHDC7
SlHDC8
SlHDC9
SlHDC10 (HDC)
6/5
2/1
3/2
3/2
2/1
6/5
6/5
5/4
6/5
7/6
483
424
236
145
190
466
460
456
445
445
53.8 (6.1)
12.0 (5.2)
27.2 (10.1)
16.1 (9.0)
21.8 (10.2)
52.6 (6.7)
52.4 (6.7)
51.4 (7.4)
50.2 (6.7)
50.9 (6.8)
Solyc08g066250.2.1
Solyc08g066260.2.1
Solyc08g068600.2.1
Solyc08g068610.2.1
Solyc08g068620.1.1
Solyc08g068630.2.1
Solyc08g068640.2.1
Solyc08g068670.2.1
Solyc08g068680.2.1
Solyc12g062790.1.1
SlHDC11
SlHDC12
SlHDC13
SlHDC14
SlHDC15
SlHDC16
SlHDC17
SlHDC18
SlHDC19 (AADC1A)
SlHDC20
6/5
7/6
5/4
5/4
5/4
5/4
5/4
5/4
5/4
2/1
403
350
472
472
350
477
410
456
472
218
46.1 (6.7)
39.4 (6.8)
53.1 (7.1)
53.1 (6.9)
39.3 (7.3)
54.0 (6.9)
46.2 (6.6)
51.1 (6.5)
53.2 (6.9)
25.4 (10.3)
†
Cis-elements in
SlPLP promoter‡
Tomato PLP_deC syntelogs
in pepper
Other
Other
Other
Other
Other
Other
Other
Other
Other
Other
ABRE, GA, LECPLEACS2
ERE, MBS, LTR,
ERE, HSE, LECPLEACS, LTR, MBS
ARFAT, ERE, GARE, HSE, MBS, TGA
ARFAT, ERE, LECPLEACS2
na
ABRE, HSE
ABRE, ERE, GARE HSE, MBS
ARFAT, ABRE, LECPLEACS2, MBS
ERE, GA, HSE, TGA
4
5
7
7
7
8
8
8
8§
8§
Other
Other
Other
Other
Other
Other
Other
Other
Other
Other
8§
8§
8¶
8¶
8¶
8¶
8¶
8¶
8¶
12
Other
Other
Other
Other
Other
Other
Other
Other
Other
Other
ARFAT, LTR, MBS
GARE, HSE, MBS
ARFAT, HSE, LECPLEACS2, MBS
na
na
ABRE, ERE, HSE
na
ABRE, ERE, GA, HSE, TGA
ARFAT, ERE, HSE
ARFAT, GA, GARE, ERE, HSE,
LECPLEACS2
ERE, HSE
ARFAT, GARE, HSE, LECPLEACS2
GARE, GA, ERE, LECPLEACS2
ERE, HSE, LECPLEACS2
TGA
ARFAT, ERE
ARFAT, GA, GARE, HSE, LTR
GA, HSE, LECPLEACS2, MBS
GARE, LECPLEACS2, MBS
na
–
–
CA07g12870 (CaAAD3)
CA07g12150 (CaAAD4)
–
CA08g00850 (CaGAD3)
–
–
–
CA05g16090 (CaGAD4),
CA12g01020 (CaGAD5)
CA04g11330 (CaHDC1)
–
–
–
–
–
–
–
CA08g04940 (CaHDC9)
–
–
–
–
–
CA08g05780 (CaHDC15)
–
–
–
–
–
Names represented in parentheses depict names used for noted genes at chromosome 8, as noted in column 6, in published literature (Picton et al., 1993; Kumar et al., 2012b; Tieman et al., 2006).
ARFAT and TGA, auxin-responsive element; ABRE, abscisic-acid-responsive element; GA and GARE, gibberellin-responsive element; ERE, ethylene-responsive element; LECPLEACS2, ethylene-biosynthesis element;
HSF, heat-responsive element; LTR, cold-stress-related element; MBS, drought-responsive element; na, no putative promoter sequence could be retrieved for these genes.
‡
§
and ¶ symbols indicate tandem duplication of two sets of genes at chromosome 8.
Phylogenetic analysis further grouped all PLP_deCs
into three subclasses (Fig. 1). The AAD and HDC members of dicot origin largely showed higher sequence
similarity with each other than that of monocot origin.
In contrast, GAD sequences among higher plants were
found to be relatively more conserved (Fig. 1). Rice
PLP_deC members grouped with their sorghum [Sorghum bicolor (L.) Moench] homologs. Members of lower
plants from all three subclasses were well interspersed
with their counterparts originated from higher plants
(Fig. 1). Furthermore, phylogenetic study among fruit
bearing species showed that PLP_deC members belonging to different species of the same taxon such as tomato
and pepper from Solanaceae or apple (Malus domestica
Borkh.), peach [Prunus persica (L.) Batsch], and strawberry (Fragaria ×ananassa Duchesne ex Rozier) from
Rosaceae are comparatively more similar in their protein
sequences than their homologs originating from a species
from another taxon (Fig. 2). In many cases, more than
single homologs for several PLP_deC genes such as peach
PpAAD4 with four putative apple homologs (MdAAD1,
5, 6 and 8), pepper CaHDC15 genes with seven putative
tomato homologs (SlHDC13–19), or papaya CpGAD3
genes with four putative citrus GAD genes (CsGAD4-7)
were also identified (Fig. 2).
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Figure 1. Evolutionary relationship of tomato PLP_deCs with other plants. The evolution was studied using PLP_deC proteins from an
algae (Chlamydomonas reinhardtii; CrPLP), nonflowering plants, such as Physcomitrella patens (PpPLP) and Selaginella moellendorffii
(SmPLP), and flowering plants, including monocots such as Poaceae members rice (OsPLP) and sorghum (SbPLP) and various dicots
such as Brassicaceae members Arabidopsis (AtPLP), brassica (BrPLP), Solanaceae member tomato (SlPLP), Saliaceae member Populus
trichocarpa (PtPLP), Leguminaceae members Medicago (MtPLP), and soybean (GmPLP). Multiple alignment of the sequences was done
by ClustalX v2.0 and the phylogeny was studied by generating an unrooted tree using MEGA6 program by neighbor-joining method.
Bootstrap supports are indicated at each branch.
Gene Expression Studies on PLP_deC Genes
during Plant Development
Previously, a ripening-associated histidine decarboxylase has been described in tomato (Kumar et al., 2012b;
Picton et al., 1993). To predict the plausible role of the
remaining genes in fruit development and ripening,
first we studied transcript profiling of PLP_deC genes
using RNA sequencing data of tomato available in public
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domain. Four HDC genes, including SlHDC9, SlHDC10,
SlHDC11, and SlHDC12 showed sharp induction in their
transcript accumulation at the early stages of ripening (mostly at breaker stage), whereas SlGAD2 showed
such induction even earlier, at mature green stage (Fig.
3). SlAAD5, SlHDC1, SlHDC13, SlHDC14, SlGAD1, and
SlGAD5 maintained high expression levels in most of the
organs, tissues, and stages studied. SlGAD4 was found
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Figure 2. Evolution of PLP_deCs in fruit bearing plants. The evolutionary trends associated PLP_deCs in fruit bearing plants were analyzed using proteins from members of different group of plants such as Solanaceae (tomato and capsicum), Rosaceae (peach, apple,
and strawberry), Vitaceae (grapes), Rutaceae (citrus) and Cariaceae (papaya). These proteins were retrieved from their protein.fa files
present at SGN, ENSENBL, and Phytozome v9.1 databases. Multiple-sequence alignment was performed by ClustalX v2.0 and then the
output .aln file was used to study their phylogeny by generating an unrooted tree. The tree was generated by neighbor-joining method
in MEGA6. Bootstrap supports are indicated at each branch.
to be highly expressed in floral tissues (Fig. 3). Transcripts of SlAAD1-3, SlHDC2-5, SlHDC8, and SlHDC20
remained at low level ubiquitously (Fig. 3). Further,
expression profiles of seven SlHDC genes (SlHDC1, 6,
9–12, and 16) and two genes each of SlAAD (SlAAD4
and 5) and SlGAD (SlGAD2 and 5) were validated by
qPCR (Fig. 4). Despite their ripening-associated nature,
SlHDC6 and SlHDC12 exhibited high, whereas SlHDC9
showed moderate transcript levels in at least one of the
vegetative tissue (Fig. 4). The qPCR analysis majorly supported the expression profiles observed in RNA sequencing data and established SlHDC10 and SlHDC11 as
fruit-ripening-associated genes (Fig. 3, 4).
Three tomato syntelog pairs (SlAAD3:CaAAD3,
SlGAD5:CaGAD5, and SlHDC1:CaHDC1a) exhibited almost similar expression patterns, whereas
paired members of the remaining four syntelogs
(SlGAD1:CaGAD3, SlAAD4:CaAAD4, SlHDC9:CaHDC9,
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Figure 3. Expression profiles of (A) tomato, (B) pepper and, (C) Arabidopsis PLP_deC genes during plant development. Heatmaps show
expression profiles of members of the three subclasses in various vegetative and reproductive tissues and stages. Color scale represents
log2 (RPKM) values in case of tomato and pepper and log2 (signal intensities on Affymetrix microarray GeneChip) values in case of
Arabidopsis. L, leaf; R, root; S; shoot; FL, flower; 1, 2, 3CM, fruit diameter in centimeter; MG, mature green; B, breaker; B.5, and B.10,
5, and 10-d postbreaker; DPA, days postanthesis; COT, cotyledon; HYP, hypocotyl; INF, inflorescence; PL, pollen; SIL, silique.
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Figure 4. Quantitative polymerase chain reaction analysis of tomato PLP_deC genes in a total of seven vegetative and reproductive
tissues and stages. The x-axis represents the different tissues and stages of development. The y-axis represents average values of relative messenger-RNA levels of genes. Expression of GAPDH was used as internal control to normalize the expression of PLP_deC genes.
L; leaf, S; shoot, R; root, MG, mature green; B, breaker; B+7 and B+20, 7- and 20-d postbreaker. Error bars show the standard error
between values from three each biological and technical replicates.
and SlHDC15:CaHDC15) exhibited mutually distinct
expression profiles during fruit development in tomato
and pepper (Fig. 4). Whereas transcription of SlGAD1
was found to be inhibited at the ripening initiation
in tomato; the expression of CaGAD3 appeared to be
induced at the similar stages in pepper. On the contrary,
expression of SlHDC9 is ripening linked in tomato while
its pepper syntelog CaHDC9 expression remained null
at these stages. The other two close homologs, HDC6
and HDC7, showed the highest transcript accumulation in root, whereas their expression was undetectable
at fruit ripening stages in both tomato and pepper (Fig.
4). GAD2 homologs showed very high expression levels from mature-green to red-ripe stages in pepper and
tomato fruits. Expression profiling of PLP_deC genes in
Arabidopsis showed that transcripts of all but AtGAD6
remained at high levels throughout development (Fig.
4). Surprisingly, transcript level of AtGAD6 was highest
in pollens, whereas its expression remained low in other
tissues and stages (Fig. 4).
Expression Studies on PLP_deC Genes under
Hormonal Treatments and Abiotic Stresses
We further preformed qPCR and investigated the effect
of different plant hormones and abiotic stresses on the
expression of selected tomato PLP_deC genes. While
ABA and BAP treatments mostly repressed these genes,
GA and IAA treatments, on the other hand, induced
their transcript levels (Table 2). In silico analysis of the
promoter architecture of tomato PLP_deCs identified
many important cis-elements (Table 1). We observed very
good correlation between auxin responsive element in
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Table 2. Expression profiling of selected group II pyridoxal-phosphate-decarboxylase genes under different phytohormone and stress treatments. Tomato seedlings were subjected to all such treatments following the protocol
described earlier (Kumar et al. 2012a). Water-treated seedlings for the same time periods were used as controls.
The DDCT method was used to calculate the relative mRNA levels. ABA, abscisic acid; BAP, 6-benzylaminopurine;
C2H4, ethylene; GA, gibberellins; IAA, auxin; A activation, R repression on hormone treatment; −, <2.5-fold; +,
2.5- to fivefold; ++, 5- to 10-fold; +++, 10- to 20-fold; ++++, >20-fold.
ABA
Gene
SlAAD4
SlAAD5
SlGAD2
SlGAD5
SlHDC-1
SlHDC-6
SlHDC-9
SlHDC10
SlHDC11
SlHDC12
SlHDC16
BAP
C 2H4
GA
of
Cold
Desiccation
Salt
Heat
1h
3h
1h
3h
1h
3h
1h
3h
1h
3h
2h
2h
2h
2h
R+++
R+
−
R+
R+++
R++
R++
R+
R+
−
A+
R+++
R+
A+
−
R+++
R++
R+
R++
R++
A++
−
R++++
R+
A+
R+
R+++
R++
R++
A++++
R+
A+
A+
R+
R+
−
−
R+++
R+
−
A++++
R++
A++
A+
R+
−
−
−
R+
−
−
A+++
A+
−
−
R+
−
−
−
−
−
−
A++
−
−
−
A++++
A+++
A+++
R+
A+++
A+
A+
−
A+
A++
−
A++
A++
A++
−
A++
−
A+
A+
−
A+
R+
A++
A++++
−
A+
−
−
A+
A++
A+
−
A++
A+
−
−
−
−
−
−
A++
A+++
A+
A+++
R++
R++
A+
−
R++
R+
R+
A+
A++
−
A+
−
R++
R+++
R+++
R+++
R+
R+++
R+
R+
−
R++
−
−
A+
−
R+
A+
A+
R++
R++
R+
R+
−
R+
−
−
R+
−
−
−
−
R+
R+
SlAAD4, SlAAD5, SlGAD5, SlHDC9, SlHDC10, SlHDC12,
and SlHDC16 promoters with their induction by this
hormone (Table 1, 2). Despite the presence of ethyleneresponsive elements in several tomato PLP_deC promoters, ethylene exerted a limited effect on the modulation
in their transcript accumulation, except SlHDC10 and
SlHDC11, which were elevated by its treatment (Table 1,
2). Transcript level of SlHDC10 was found to be elevated
under most treatments except under ABA, desiccation,
and salt stresses. Desiccation predominantly had a negative effect, similar to ABA treatment, on the expression
of most of genes studied (Table 2). Overall, stress treatments downregulated the expression of PLP_deC genes
in tomato. However, a few exceptions, such as induced
expression of SlGAD2, SlHDC10, SlHDC11, and SlHDC16
under cold and SlGAD2, SlHDC6, and SlHDC9 under salt
stress, were also observed (Table 2). Despite the presence
of heat shock element in the promoters of most of tomato
PLP_deCs, SlAAD5, SlHDC1, SlHDC12, and SlHDC16
were the only genes that showed a change (mild repression) in their transcripts after heat stress (Table 1, 2).
On the basis of previous studies (Turano and Fang,
1998; Wang et al., 2000), we selected three different concentrations (250 µM, 5 mM, and 10 mM) of potassium
nitrate (KNO3) and analyzed their effect on the transcript accumulation of these genes and found that such
nitrate treatment induces several tomato PLP_deC genes,
except SlGAD2 and SlGAD5 (Fig. 5). Transcript levels of
SlAAD1, SlAAD6, SlHDC1, SlHDC9–12, and SlHDC16
were found to be induced in all nitrate treatments. We
noticed an increase in transcript level of SlHDC6 only in
250 µM nitrate treatment, whereas higher nitrate level
(10 mM) was found to inhibit its expression (Fig. 5).
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15
Functional Characterization
of SlHDC10 Promoter
We next characterized putative promoter of SlHDC10
by retrieving 1.6-kb upstream sequence. Only 1.6-kb
region was selected, as information of further upstream
sequence was not available at the time of cloning.
Besides many other cis-elements, in silico examination
of this sequence showed presence of one each of auxinresponsive element (AuxRE, at −1282 bp), putative fruitspecific element (TCCAAAA at −444 bp), and putative
nitrate response element (TGACCTTT, at −909 bp),
two ethylene biosynthesis elements LECPLEACS2 (at
−1480 and −656 bp), three each MADS-box transcription factors binding CArG elements (at −1587, −994,
and −718 bp) and ethylene-responsive element (at −402,
−390, and −211 bp). In contrast to the very high levels of
SlHDC10 transcripts, surprisingly, we could not detect
an equivalent GUS activity in fruits of SlHDC10-Fl::GUS
transcriptional fusion transgenic tomato lines. GUS
accumulation was found to be higher in mature green
fruits than red fruits. Additionally, roots of these transgenic plants were found to have maximum GUS expression (Supplemental Fig. S3A–C in Supplemental File
S2). To find out the reason for these unexpected results,
we further made two deletion constructs to identify the
minimum promoter sequence (Fig. 6A; Supplemental
Fig. S3A in Supplemental File S2). SlHDC10-DI::GUS
and SlHDC10-DII::GUS deletion constructs in tobacco
leaves resulted in an enhanced activity of shorter promoter region under both nitrate and ethylene treatments. At least three times more GUS accumulation
was observed in SlHDC10-DI::GUS (−968 to −1599 bp
deletion) or SlHDC10-DII::GUS (−668 to −1599 bp deletion) infiltrated tobacco leaves in comparison to those
infiltrated with SlHDC10-Fl::GUS construct. Similarly,
the enhanced promoter activity, at least five times and
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Figure 5. Expression pattern of tomato PLP_deC gene family members in response to nitrate treatment (KNO3) at different concentrations. Transcript levels of two genes each belonging to aromatic L-amino acid decarboxylases and glutamate decarboxylase subclasses
and seven genes of histidine decarboxylase subclass were studied in response to nitrate treatment. Milli-Q water–KCl treated seedlings
were the control for this treatment. At least two biological and three technical replicates were used in the analysis.
10-fold higher, was recorded in SlHDC10-DI::GUS and
SlHDC10-DII::GUS infiltrated leaves, respectively, under
ethylene treatment (Fig. 6A, 6B).
Discussion
Type II PLP_deC enzymes, especially belonging to AAD
and GAD subclasses, play important roles in amino
acid metabolism during plant growth and development
(Akihiro et al., 2008; Facchini et al., 2000; Gutensohn et
al., 2011; Tieman et al., 2006). These enzymes are known
to have stringent substrate specificities, which suggests
toward their functional evolution from a common evolutionary origin. The availability of limited information on
their protein sequences and biochemical properties has
prevented an understanding their actual roles of many
PLP_deCs in plant development, to date. In the present
study, we identified full complement of PLP_deC genes
in many plants (both lower and higher plants) belonging
to different taxa and classified them in GAD, AAD, and
HDC subclasses (Fig. 1, 2; Table 1). The nomenclature of
these genes is strictly based on their high sequence homology, however, we cannot rule out the possibility that some
of these genes might have evolved for diverse functions.
For example, the already characterized LeAADC1A and
LeAADC2, annotated as SlHDC19 and SlHDC6, respectively, in the present study, do not act on histidine but
prefer tyrosine as their substrate (Tieman et al., 2006).
Further, these tomato enzymes are reported to share
higher homology (57%) to the characterized plant AtSDCs
than AADs (10–15% identity). Similarly, many SDCs, initially annotated as HDCs, upon biochemical analysis, were
found to prefer serine rather than histidine (Rontein et al.,
2001). Recent biochemical characterization of SDC-like
enzymes from Medicago truncatula Gaertn. and Cicer
arietinum L. further indicates toward their novel aldehyde
synthase activity (Torrens-Spence et al., 2014). Therefore,
despite of their high sequence similarity, PLP_deCs have
evolved for diverse substrate specificities and their precise
function will be known only after in-depth biochemical
characterization (Torrens-Spence et al., 2014).
Gene identification analysis showed presence of
maximum 30 PLP_deC members in tomato, whereas
papaya and Arabidopsis genomes were found to encode
the minimum set of such genes among 15 plant species
used in this study (Fig. 1). Presence of more PLP_deC
genes in all plant genomes than in bacteria, yeast, and
Chlamydomonas is in accordance with the earlier results
and supports that number of these genes increases with
the increased complexity of genomes (Facchini et al.,
2000). However, a few exceptions for the ratio of genome
size and number of encoded group PLP_deC genes exist
in organisms such as Chlamydomonas, pepper, and
Selaginella (Supplemental Table S3 in Supplemental File
S1). This presumably supports the earlier observation that
the number of such genes in a genome also depends on
the nutrient requirements of organisms and the external
environment might have contributed to their evolution
and diverse substrate preferences (Facchini et al., 2000).
The comprehensive phylogenetic analyses of PLP_deC
genes are further in accordance with the divergence time
of different groups during evolution of plants, as land
plants are expected to have diverged from green algae 750
million yr ago, whereas lower land plants are suggested to
have diverged from their flowering counterparts by more
than 400 million yr on evolutionary time scale (Fig. 1, 2)
(Guo et al., 2013; Nickrent et al., 2000). Comparatively
lower degree of sequence similarity of PLP_deC members
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Figure 6. Histochemical analysis of GUS activity in Nicotiana benthemiana leaves infiltrated with different SlHDC10::GUS promoter
constructs under nitrate and ethylene treatments. (A) In case of nitrate treatment at least four independent plants for each construct were
taken. After agroinfilteration, the tobacco leaves were detached from plants after 5 h and incubated in Milli-Q water and 5 mM KNO3.
The leaves were immersed in nitrate medium for 5 h. For each construct, four leaves were incubated in GUS buffer while the other four
leaves were used for fluorimetric analysis. Similar procedure was adopted for ethrel (ethylene source at different concentration) treatment;
however, following agroinfilteration, plants were left for 12 h. Control was incubated in KCl and Milli-Q water in air tight containers. (B)
The fluorimetric values of control samples were deducted from the experimental samples and final graphs were plotted in Microsoft Excel.
between monocots and eudicots, than that between members of either eudicots or monocots, further supports
the earlier observations on their divergence as these two
groups are proposed to have diverged at least 150 million
yr ago (Fig. 1) (Chaw et al., 2004; Guo et al., 2013; Salinas
et al., 2012). Further, predicted protein size of PLP_deCs
is in accordance with the previously published reports
(Molina-Rueda et al., 2010; Tieman et al., 2006).
Presence of seven syntelog pairs (SlAAD34:CaAAD3-4; SlGAD3:CaGAD3, SlGAD5:CaGAD4,
CaGAD5, SlHDC1:CaHDC1, SlHDC9:CaHDC9, and
SlHDC15:CaHDC15) in tomato and pepper (Fig. 2) suggests that these genes might have originated from a
common ancestor before divergence of Solanum and
Capsicum lineages (Wu and Tanksley, 2010). Two pepper
syntelogs (CaGAD4 and CaGAD5) for SlGAD5 and presence of seven putative tomato homologs (SlHDC13–19)
for CaHDC15 suggests that the additional homologs in
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either species would have evolved through gene duplication event after the speciation (Kim et al., 2014). Altogether, gene identification and phylogenetic analyses
revealed that HDC subclass has moderately expanded in
most of Leguminaceae and Solanaceae members; however, in tomato, mainly tandem duplication events have
contributed in their abrupt expansion (Fig. 1, 2).
The expression data of tomato syntelogs in pepper
is very interesting as the conservation and divergence
of their function may contribute to quantitative and
qualitative differences observed under climacteric and
nonclimacteric ripening programs. The similar expression pattern for SlGAD1:CaGAD3, SlHDC9:CaHDC9,
and SlHDC15:CaHDC15 syntelogs during fruit developmental stages in both tomato and pepper suggests
conservation in their functions in the two species
(Fig. 4). In contrast, nonsimilar expression profiles of
SlGAD1:CaGAD3, SlAAD4:CaAAD4, SlHDC9:CaHDC9,
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and SlHDC15:CaHDC15 syntelogs indicate that these
genes have evolved to perform different functions in the
two species (Kim et al., 2014). Lack of any syntelog for
fruit-specific ripening-associated SlHDC10 and SlHDC11
and almost undetected levels of CaHDC9 transcripts in
pepper is interesting and indicates that function of these
genes is not required for maintaining fruit quality in the
latter species (Fig. 4).
Presence of at least four ripening-induced genes
(SlHDC9–12) (Fig. 3, 4) further indicates that these genes
might have overlapping functions during fruit ripening
in tomato (Tieman et al., 2006). Histamine levels are
known to increase during fruit ripening (Martin et al.,
2005; Wojtaszek, 2003). Further, presence of more HDC
genes in Solanaceae could be a reason for the relatively
higher amount of histamine (mostly >1.5 mg 100 mg−1
fresh weight) reported in these plants (Feldman, 1983;
Kumar et al., 2009). Presence of fruit-specific elements
viz. TCTTCACA and TCCAAAA in the putative promoters of SlHDC10 and SlHDC11, respectively, supported
the expression data and provided an additional evidence
for their fruit-specific nature (Table 1) (Yin et al., 2009;
Zhao et al., 2009). The higher GUS expression in mature
green, whereas very low in red fruits in SlHDC10Fl::GUS tomato transgenic lines, is intriguing and could
be due to either higher nitrate levels in mature green
tomatoes or an insufficient length of the putative promoter used, respectively, and most likely be corrected by
using a >1.6-kb promoter sequence (Schaart et al., 2002).
Moreover, ectopic expression of GUS in the roots could
be due to exposure of roots to nitrate source present in
soil (Wang et al., 2000). Previous evidence in tomato,
tobacco, and Arabidopsis further support the present
findings (Eyal et al., 1993; Twell et al., 1990). The analysis
of the GUS activity of two deletion constructs established
that nitrate responsiveness of SlHDC10-DII::GUS deletion construct could be due to some yet to be characterized nitrate-response element present in the first 668-bp
region of this promoter (Das et al., 2007).
The results presented in this study further established that members of PLP_deC family are under the
control of multiple signals as transcript level of several
members of this gene family in tomato were found to
be affected by plant hormones such as auxin, ABA, ethylene, cytokinin, and gibberellins and abiotic stresses
including cold, heat, salt, and desiccation and supports
earlier findings (Gutensohn et al., 2011; Hyun et al., 2014;
Kanwal et al., 2014; Kumar et al., 2012a, 2014; Picton
et al., 1993). High correlation between auxin-induced
increase in transcript levels of these genes and AuxRE
(ARFAT) in their promoters is in accordance with previously published studies (Table 1, 2; Guilfoyle and Hagen,
2007; Ulmasov et al., 1997). However, lack of same level
of tight correlation between presences of other phytohormone-responsive elements, vis-à-vis expression of genes
under specific phytohormone treatment, suggest toward
involvement of complex molecular mechanisms underlying their transcriptional regulation.
Overall, the present study provides an initial glimpse
into the evolution of PLP_deC gene family members in
plants and improves the current understanding on their
plausible role during plant growth and development.
Moreover, the expression data under various phytohormones, nitrate treatments, and abiotic stresses in tomato
suggests their involvement in contributing toward modified metabolic activities under such conditions in plants.
In silico promoter data and functional characterization
of SlHDC10 promoter further supports the expression
data and suggests that these genes operate under complex
molecular circuits. Finally, the comprehensive identification, expression, and functional characterization of
group II PLP_deC genes in tomato will further help in
assigning them functions in Solanaceae, in general, and
tomato, in particular.
Acknowledgments
This work was financially supported by grants received from the Department of Science and Technology (DST), and Department of Biotechnology (DBT), Government of India. GJ, AP, TSK acknowledge CSIR, UGC,
and DST, respectively, for the fellowship granted during their tenure as
research fellows. RK is grateful to DST for INSPIRE-Faculty Award (grant
number IFA-LSPA-15). RK also thanks Prof. R. P. Sharma, Department of
Plant Sciences, University of Hyderabad, for his support in carrying out
the research work. Authors are grateful to Dr. Hriyadesh Prakash, University of Hyderabad, and Dr. Pavel Kerchev, Department of Plant System
Biology/VIB, University of Gent for critical reading of the manuscript.
Authors acknowledge the Solanaceae Genomics Network for the use of
Tomato BAC Sequence Database to retrieve putative promoter sequences.
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