ORIGINAL PAPER

Vacuolar invertases in potato (Solanum tuberosum L.):molecular cloning, characterization, sequence comparison,and analysis of gene expression in the cultivars

Vijay Kumari • Niranjan Das

Received: 20 June 2012 / Revised: 5 February 2013 / Accepted: 6 February 2013 / Published online: 20 February 2013

� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2013

Abstract In plants, vacuolar invertase (b-fructofuranosidase,

EC 3.2.1.26) is known to play as a key modulator for

hexose accumulation and cell expansion. In this study, two

cDNA clones (2,013 and 1,945 bp, with 99 % sequence

identity) encoding vacuolar invertase isoforms were isolated

from a commercially important Indian potato cultivar, Kufri

Chipsona-1 by RT-PCR. The corresponding predicted pro-

teins consisted of 635 amino acids (designated as KC-VIN1,

lacking a few amino acids at N-terminus) and 639 amino

acids (designated as KC-VIN2), respectively. They showed

99 % identity, and found to vary at several locations with

mostly non-conservative substitutions. Multiple sequence

alignment of vacuolar invertase homologs covering four

Solanaceae family members revealed some notable distin-

guishing sequence features (signature-type sequences). A

consensus sequence was predicted using 45 vacuolar

invertase sequences from 27 taxonomically different plant

species, and a phylogenetic tree was generated to know the

evolutionary relation between them. Hydrophobic characters

were predicted, and compared in different plant species. All

these data are presented in a comprehensive manner which

were not documented in the earlier reports. As a preliminary

study, vacuolar invertase expression patterns in the tubers of

some Indian potato cultivars were analyzed by semi-quan-

titative RT-PCR and extractable enzyme assay. In all the

potato cultivars, the overall expression level of invertase was

found to be considerably higher after storage at low tem-

perature as compared to the freshly harvested tubers.

Keywords Indian potato cultivars � Vacuolar invertase

cDNA cloning � Sequence comparison � Phylogenetic tree �Invertase expression

Introduction

Sucrose is a soluble nonreducing disaccharide in which Glc

and Fru are linked (a1 ? b2). In most plants, sucrose is the

common form of sugar transported from photosynthetically

active tissues (i.e. source, mainly mature leaves) to non-

photosynthetic sinks such as flower, fruit, seed, tuber and

root. In the sink tissues sucrose must be degraded into

hexoses and other derivatives to act as a source of carbon

and energy for various metabolic and biosynthetic pro-

cesses. Moreover, sucrose and hexoses have important

signaling roles in regulating gene expression and plant

development (Koch 2004; Ruan et al. 2010). In higher

plants, cleavage of sucrose into hexoses is catalyzed by

either sucrose synthase (Sus, EC 2.4.1.13) or invertase

(INV, EC 3.2.1.26). Both these enzymes exist in several

isoforms (Tymowska-Lalanne and Kreis 1998; Sturm et al.

1999). Sucrose synthase converts sucrose in the presence of

UDP into UDP-glucose and fructose, whereas invertase

hydrolyses sucrose into glucose and fructose. According to

the classification of different enzyme families, plant

invertases (b-fructofuranosidase) belong to glycoside hydro-

lase family 32 (GH32, Clan GH-J) (http://www.cazy.org).

There are several isoenzymes of plant invertases

involved in a wide range of regulatory functions in growth

and development apart from their major roles in primary

carbon metabolism. The plant invertases (INVs) are clas-

sified as vacuolar, apoplasmic (cell wall) and cytoplasmic

isoforms named as VIN, CWIN, and CIN, respectively,

based on their optimum pH, solubility and subcellular

Communicated by S. Abe.

V. Kumari � N. Das (&)

Department of Biotechnology and Environmental Sciences,

Thapar University, Patiala 147004, Punjab, India

e-mail: ndas@thapar.edu

123

Acta Physiol Plant (2013) 35:2055–2068

DOI 10.1007/s11738-013-1240-y

locations. Both CWIN and VIN are N-glycosylated forms

with acid pH optima (between pH 4.5 and 5.0) and attack

the disaccharide from the Fru residue. These b-fructofur-

anosidases also hydrolyze other b-Fru-containing oligo-

saccharides such as raffinose and stachiose. On the

contrary, neutral and alkaline invertases (CIN), having pH

optima 7.0–7.8, appear to be sucrose specific. CWIN and

VIN have been purified from several plant species. These

acid invertases have low Km values for Suc, and activity is

inhibited by heavy metals such as Hg2? and Ag? sug-

gesting the presence of a sulfhydryl group at the catalytic

site. Glc acts as a non-competitive inhibitor and Fru as

competitive inhibitor for acid invertases. In most of the

cases molecular masses of the mature N-glycosylated

polypeptides are between 55 and 70 kDa. Based on the

growing body of evidences it is generally believed that

CWIN plays crucial role during flower, seed, and fruit

development; VIN as a key modulator for hexose accu-

mulation and cell expansion, but the role of CIN in plant

development still remains to be elucidated (Sturm 1999;

Fotopoulos 2005; Barratt et al. 2009; Ruan et al. 2010).

Sturm and Chrispeels (1990) first reported plant cell

wall invertase cDNA clone from carrot. Since its isolation

a number of cDNA sequences encoding vacuolar invertase

from taxonomically different plant species including eco-

nomically important crops have been deposited in the

databases and reported in the literature, such as tomato

(Ohyama et al. 1992; Elliott et al. 1993; Sato et al. 1993),

mung bean (Arai et al. 1992), potato (Zhou et al. 1994;

Zrenner et al. 1996; Draffehn et al. 2010), grape berries

(Davies and Robinson 1996), Arabidopsis (Haouazine-

Takvorian et al. 1997), maize (Kim et al. 2000), sweet

potato (Huang et al. 2003; Wang et al. 2005), rice (Ji et al.

2005), muskmelon (Tian et al. 2009), Pachysandra termi-

nalis (Buxaceae) (Van den Ende et al. 2011). By linkage

and association studies, quantitative trait loci (QTLs) and

quantitative trait alleles (QTAs) have been identified for

potato tuber yield and starch content (Schafer-Pregl et al.

1998) and chip quality (i.e., cold sweetening) (Menendez

et al. 2002). All these loci were found to colocalize with

three independent potato invertase loci encoding five

invertase genes: Pain-1(present in chromosome III encodes

vacuolar invertase), InvGE, InvGF, InvCD141 and

InvCD111. Draffehn et al. (2010) carried out functional

studies on natural variants of invertase genes in some tet-

raploid/diploid potato cultivars and clones, isolated and

sequenced a number of cDNA alleles corresponding to

each of these genes. Likewise, considerable progress has

been made on cell wall invertase both at biochemical and

molecular levels in different plant species, such as potato,

carrot, tomato, and Arabidopsis (Hedley et al. 1993; Unger

et al. 1994; Ohyama et al. 1998; Sherson et al. 2003). There

were some exciting advances in elucidating the three-

dimensional (3D) structures of the glycoside hydrolase

family 32 (GH32) and 68 (GH68) enzymes (Verhaest et al.

2005; Lammens et al. 2009).

Some of the focus areas of the aforesaid studies include

isolation of cDNA/genomic clones, sequence analysis and

comparison of the invertases for understanding the struc-

ture–function relationships; gaining knowledge on a vari-

ety of intracellular and extracellular factors that influence

invertase gene expression, and regulation of enzyme

activity at post-translational level through interactions with

the endogenous inhibitors (Greiner et al. 1999; Rausch

and Greiner 2004); understanding the emerging roles of

invertases in plant development; elucidating cross-talks

between invertase-mediated sugar signaling and hormonal

control of development (Rolland et al. 2006) along with

sugar and invertase-mediated responses to the abiotic

stresses. It is commonly believed that in potato, invertases,

together with other proteins, are involved in the undesirable

‘cold sweetening’ process in the potato tubers during

storage at low temperatures. An important applied aspect of

invertase research includes crop improvement through

various transgenic approaches (Zrenner et al. 1996; Greiner

et al. 1999; Bhaskar et al. 2010).

Potato is an important member of the Solanaceae family

that includes several other economically important species

such as tomato, eggplant, petunia, pepper and tobacco.

Potato cultivars are autotetraploid, and show the high

degree of heterozygosity. Therefore, in potato it is likely

that more allelic variants and the corresponding isoforms

are involved in sucrose metabolism depending on the cul-

tivar genotype, kind of tissue and the subcellular location.

There are number of high-yielding Indian potato cultivars

suitable to different agro climatic zones of the Indian

subcontinent. There is no report available on invertases

from these potato cultivars at molecular and biochemical

levels till date. Here, we report the isolation and charac-

terization of two cDNAs (one nearly full-length and other

one is full-lengh) encoding vacuolar invertase isoforms

through RT-PCR approach from the potato cultivar Kufri

Chipsona-1 (a processing variety). The deduced amino acid

sequences were analyzed, and compared with their homo-

logs from different plant species. Based on the vacuolar

invertase sequences, in silico approaches were adopted to

examine the presence of distinguishing sequence features

(signature-type sequences) between the Solanaceae family

members, and to predict segment-wise hydrophobic char-

acters between the plant species. Vacuolar invertase

sequences from a large number of taxonomically different

plant species were used in predicting the consensus

sequence, and also for generating a phylogenetic tree. All

these data are presented in a comprehensive manner which

were not documented in the earlier reports. The overall

expression pattern of vacuolar invertase was analyzed in

2056 Acta Physiol Plant (2013) 35:2055–2068

123

the freshly harvested and cold-stored tubers from some of

the Indian potato cultivars using semi-quantitative RT-PCR

and assaying total extractable activities of vacuolar

invertase enzyme and described in this report.

Materials and methods

Plant materials and growth conditions

In this study, six Indian potato cultivars namely Kufri

Chipsona-1, Kufri Chipsona-2, Kufri Chandramukhi, Kufri

Jyoti, Kufri Ashoka, and Kufri Pukhraj were used. The

germplasms were procured from Central Potato Research

Institute (CPRI), Shimla, India. These potato cultivars vary

with regard to genetic make up, maturation time, and

suitability to different agro-climatic zones of the Indian

subcontinent. The cultivars Kufri Chandramukhi and Kufri

Ashoka are early maturing, whereas the remaining cultivars

are medium maturing. All these cultivars along with

Desiree (a late maturing exotic cultivar used as a reference)

were routinely micropropagated in our laboratory under

controlled conditions (16 h light/8 h dark, 25–27 �C, 70 %

relative humidity) for 4–5 weeks on MS-Basal medium.

After proper hardening and acclimatization of the asepti-

cally grown micropropagated potato plantlets, all these

cultivars were grown in the field. Mature tubers were col-

lected from potato plants, stored at room temperature for

3 weeks, referred to as freshly harvested, and then trans-

ferred to 4 �C for 4 weeks. All the tuber samples were

immediately frozen in liquid nitrogen for further molecular

and biochemical studies.

RNA extraction, RT-PCR, and vacuolar invertase

cDNA cloning

Total RNA was isolated from 5–6 week-old aseptically

grown micropropagated potato plantlets, freshly harvested,

and cold-stored tubers from different potato cultivars by

SDS-Phenol method essentially as described by Gilman

(1987). Briefly, the plant materials (0.5–2.0 g) were frozen

in liquid nitrogen and pulverized to a fine powder, and

homogenized further in a buffer containing lithium chlo-

ride and SDS (the composition of the RNA extraction

buffer: 100 mM LiCl, 100 mM Tris–HCl pH 8.0, 10 mM

EDTA pH 8.0, 1.0 % SDS, 0.2 % b-mercaptoethanol)

followed by direct extraction with phenol:chloroform (1:1).

To remove DNA impurities, RNA was selectively precip-

itated from the aqueous phase by adding one-third volume

of 8.0 M LiCl under ice-cold condition followed by incu-

bation for at least two hours. The RNA pellet was further

purified by another round of solvent extraction followed by

ethanol precipitation. To remove the DNA impurities, the

aqueous RNA solution was treated with RNase-free DNase,

followed by solvent extraction. Finally, the RNA was

precipitated with ethanol, washed and dissolved in deion-

ized water, and kept in aliquots at -70 �C. The A260/A280

ratio of the RNA samples was measured spectrophoto-

metrically. For checking the intactness of RNA, apart from

normal and formaldehyde agarose gel electrophoresis,

RT-PCR was carried out using different potato gene-specific

primers.

Based on the available potato vacuolar acid invertase

cDNA sequence in the database (GenBank Accession No.

X70368) corresponding to the potato cultivar Desiree, the

following oligonucleotide primers were designed: the forward

primer K20-AI, 50-AGTACCATTCCAGTTATGAC-30 (cor-

responding to the bases 1–20); the reverse primer M20-AI,

50-CAATAGCATAGTGATCTTGC-30 (complementary to

the bases 995–1014), and the other reverse primer AI-2016,

50-TAAGTAGAGTATAACACTAC-30 (complementary to

the bases 1997–2016). Likewise, based on another vacuolar

acid invertase cDNA sequence from potato (GenBank

Accession No. L29099) the following oligonucleotide

primers were designed: the forward primer AI-F01, 50-GCACGAGTATGGCCACGCAG-30 (corresponding to the

bases 1–20); the reverse primer AI-R1950, 50-GAA

GAAGATATGGCTTGATG-30 (complementary to the

bases 1931–1950). For gene expression analysis, the fol-

lowing actin gene-specific primers were used: Actin-FW

(forward primer), 50-ATTCAGATGCCCAGAAGTCTTGT

TC-30; Actin-RV (Reverse primer), 50-GCAAGTGCTGTG

ATTTCTTTGCTCA-30. Reverse transcriptions (RT) were

performed using the RevertAidTM H Minus First Strand

cDNA Synthesis Kit from Fermentas Life Sciences con-

taining M-MuLV reverse transcriptase and the cDNA-spe-

cific reverse primers, AI-2016 and AI-R1950. For each RT

reaction, approx. 2.0 lg of total RNA from the microprop-

agated plantlets of two Indian potato cultivars, Kufri Chip-

sona-1 and Kufri Chandramukhi was used as template. All

the steps of reverse transcription were carried out according

to the manufacturer’s instructions. PCR was carried out

using the individual RT product as template, the following

primer pairs: K20-AI and AI-2016, AI-F01 and AI-R1950,

and 1.0 unit of Taq DNA polymerase (Bangalore Genei).

After initial denaturation at 94 �C for 1 min 30 s, the ther-

mal cycling parameters during PCR were: denaturation at

94 �C for 1 min, annealing at 55 �C for 2 min, polymeri-

zation at 72 �C for 3 min for 30 cycles followed by final

extension at 72 �C for 5 min. The RT-PCR products

(*2.0 kb), corresponding to the individual primer pairs and

specific to the cultivar Kufri Chipsona-1 were treated with

Klenow enzyme, purified, and cloned into the Sma1 site of

pUC19 vector according to the protocols as described by

Sambrook et al. (1989). E. coli DH5a was used as host for

routine molecular cloning experiments. Each cloned cDNA

Acta Physiol Plant (2013) 35:2055–2068 2057

123

was sequenced in both the directions by the commercial

company Bangalore Genei, Bangalore.

Sequence analyses, and construction

of phylogenetic tree

The nucleotide sequences of the cDNAs were analyzed by

NCBI Blast tools. The deduced amino acid sequences were

predicted by the open reading frame (ORF) finder available

at the National Center for Biotechnology Information

website (http://www.ncbi.nlm.nih.gov). To calculate the

theoretical molecular weight, isoelectric point (pI), and

amino acid composition of the predicted amino acid

sequences, the ProtParam tool of ExPASy (Expert Protein

Analysis System) proteomics server of the Swiss Institute

of Bioinformatics (SIB; http://expasy.org/tools/) was used.

Likewise, the different ProtScale tools of ExPASy were

used for prediction of the hydrophobic character (Kyte and

Doolittle 1982), and the various secondary structures such

as a-helix, b-sheet, b-turn, and random coil. G ? C content

analysis was carried out by DNADynamo software

(http://www.bluetractorsoftware.co.uk/). Isochore plots were

generated by another EMBL-EBI sequence analysis tool

(http://www.ebi.ac.uk/Tools/ emboss/cpgplot/). For multi-

ple sequence alignment the ClustalW2 tool, an EMBL-EBI

sequence analysis tool with its default parameters (http://

www.ebi.ac.uk/Tools/) was used. Both the ClustalW2 and

the MultAlin software (http://www.multalin.toulouse.inra.

fr/multalin/; Corpet 1988) tools were used in predicting the

consensus sequence. To generate phylogenetic tree, mul-

tiple sequence alignment was done first by the MultAlin

software, followed by the neighbor-joining method (with

bootstrap consensus) using MEGA 5.0 software (Saitou

and Nei 1987; Tamura et al. 2011). For this purpose, a total

of 45 predicted vacuolar invertase sequences covering 27

plant species from different taxonomic groups were used:

Solanum tuberosum cultivars (ACC93584, ACC93585,

ABF18956, AAQ17074, ADM47340, AAA50305, CAA4

9831); Solanum lycopersicum cultivars (NP_001234843,

NP_001234618, BAA01954, CAA78060, CAA78061);

Capsicum annum (AAB48484); Nicotiana tabacum (CAC

83577); Cucumis melo (ABX55832); Ipomoea batatas

(AAK71505, AAK71504, AAD01606); Oryza sativa Japon-

ica Gr. (AAK72492, AAD10239); Oryza sativa Indica Gr.

(CAH67112); Coffea canephora (ABI17894); Daucus carota

(CAA53097, CAA53098, CAA53099, CAA47636); Glycine

max (XP_003533514); Citrus sinensis (BAF34363, AAL2

7709); Gossypium hirsutum (ACQ82802); Ricinus communis

(XP_002510944); Pachysandra terminalis (CBM41476);

Sorghum bicolor (XP_002446857); Vitis vinifera (AAB4

7172); Cichorium intybus (CAD12104); Arabidopsis lyrata

(XP_002888009); Arabidopsis thaliana (NP_564798); Bras-

sica oleracea (AAG36943, AAG36942); Pyrus pyrifolia

(BAF35859); Populus trichocarpa (XP_002303519); Vigna

radiata (BAA01107); Phaseolus vulgaris (AAB68679); Vicia

faba (CAA89992) and Pisum sativum (AAM52062).

Semi-quantitative RT-PCR

Semi-quantitative RT-PCR was carried out to know the

vacuolar invertase expression pattern in the freshly har-

vested and cold-stored tubers from the seven field-grown

potato cultivars. 2.0 lg of total RNA (free from DNA

impurities) from each potato sample was used for reverse

transcription in a reaction volume of 20 lL using oligo

(dT)18 primer, and the cDNA Synthesis Kit from Fer-

mentas Life Sciences. 3.0 lL of each RT mixture was

used as template in PCR (50 lL reaction volume) using

the vacuolar invertase cDNA specific forward and reverse

primers, K20-AI and M20-AI, and 1.0 unit of Taq DNA

polymerase (Bangalore Genei). The thermal cycling

parameters for this PCR were kept same as mentioned

earlier except polymerization at 72 �C for 2 min. As a

control, the primers Actin-FW (forward primer) and

Actin-RV (reverse primer) specific to the housekeeping

actin gene were used to amplify *250 bp fragment using

the same 3.0 lL individual RT mixture as template. In

this case, polymerization step at 72 �C was for 1 min in

each thermal cycle. The invertase and actin specific

RT-PCR products were resolved in 0.8 and 1.2 % agarose

gel electrophoresis, respectively. The quantification tool

of the gel documentation system (Bio-Rad, USA) was

used to assess the relative expression levels between the

potato cultivars.

Vacuolar invertase assay

For determination of vacuolar invertase activity in the

freshly harvested and cold-stored potato tubers, a protocol

was adopted as described by Greiner et al. (1999). Nearly

500 mg of tuber sample, quickly frozen in liquid nitrogen,

was homogenized in 1.0 mL of extraction buffer [30 mM

MOPS (3-(N-morpholino)-propanesulphonic acid), 250 mM

sorbitol, 10 mM MgCl2, 10 mM KCl, 1 mM phenylmethyl

sulphonyl fluoride (PMSF)], followed by centrifugation for

10 min (6,000g, 4 �C). From each supernatant an aliquot of

20 lL was added to 200 lL reaction buffer containing

30 mM sodium acetate (pH 4.7) and 30 mM sucrose,

followed by incubation at 30 �C for 1 h. The reaction was

then stopped by addition of 1.0 mL alkaline copper tarta-

rate solution; liberated hexoses were assayed by Nelson–

Somogyi’s method as described by Sadasivam and

Manickam (1996) with proper controls and D-glucose

standard. Here one unit of vacuolar invertase activity refers

to the amount of enzyme which liberates 1.0 nmol of

hexose min-1 per mg of tuber FW.

2058 Acta Physiol Plant (2013) 35:2055–2068

123

Results

RT-PCR and molecular cloning of vacuolar

invertase cDNA

The A260/A280 ratio of the total RNA preparations from

different potato cultivars was found to be nearly 2.0. The

reverse transcription (RT) products corresponding to the

cultivars Kufri Chipsona-1 and Kufri Chandramukhi were

used to carry out PCR using two sets of the primer pairs as

mentioned earlier. The sizes of the amplified products were

around 2.0 kb for both the cultivars (Fig. 1a, b). Here, the

nucleotide sequences of only two cDNA clones cor-

responding to Kufri Chipsona-1, designated as AI-01

(2,013 bp; specific to the primer pair, K20-AI and

AI-2016), and AI-02 (1,945 bp; specific to the primer pair,

AI-F01 and AI-R1950), respectively, were analyzed by

NCBI BLAST tool. The sequence information of AI-01

and AI-02 that encoded two different forms of vacuolar

invertase were submitted to the GenBank data base under

the Accession Numbers EU622806 (protein id ACC93584)

and EU622807 (protein id ACC93585), respectively.

Sequence analyses

The 2,013 bp AI-01 cDNA truncated at the 50 terminus,

contained 1,910-bp ORF (bases 1–1910), the correspond-

ing predicted protein consisted of 635 amino acids, des-

ignated as KC-VIN1 which lacks four amino acid residues

at the N-terminus. The 1,945-bp AI-02 cDNA contained a

complete 1,920-bp ORF (bases 4–1,923) encoding

639-amino acid protein, designated as KC-VIN2. NCBI

BLAST search at nucleotide level revealed that AI-01 and

AI-02 share 99 % sequence identity (variations at 13 places

confined towards the 50 and 30 regions). AI-02 shares 99 %

sequence identity with vacuolar invertase cDNA sequences

from the other potato cultivars, namely May Queen

(DQ478950), Russet Burbank (L29099), an unknown cul-

tivar (AY341425), and 98 % identity for the potato cv.

Desiree (X70368). In the BLAST output data, few gaps

were noted only in the cases of Russet Burbank and

Desiree indicating more divergence. In case of tomato,

AI-02 showed 96 % sequence identity with the following

cultivars, such as Castlemart (M81081), Trujillo accession

LA 722 (Z12026), and UC82B (Z12025). For other mem-

bers of the Solanaceae family, AI-02 shares 89 and 87 %

sequence identity with capsicum (U87849) and tobacco cv.

SNN (AJ305044), respectively. The sequence divergence

was prominent for tomato, capsicum and tobacco as more

gaps were found in the BLAST search output data. AI-01

also showed similar sequence identity if compared with the

above sequences. The sequence divergence was found to be

more prominent for the plant species other than the Sola-

naceae family members. In these cases, the query coverage

length during NCBI BLAST search was considerably

reduced; only the coding region of AI-01 or AI-02 showed

sequence identity in the range of 70–80 %. Therefore, none

of the vacuolar invertase cDNA clones as isolated and

characterized in this study, was found to be identical with

the other cDNA sequences reported to date. AI-01 and

AI-02 represent two different cDNA alleles from the potato

cultivar Kufri Chipsona-1. The overall G ? C content of

the coding region and 30 UTR in either cDNA allele were

found to be *46 and *35 %, respectively. The vacuolar

invertase coding regions from other potato cultivars and

plant species showed considerable segment-wise variations

as examined by isochore plot using EBI tools for G ? C

content analyses (data not shown).

NCBI protein–protein BLAST search (blastp) revealed

that the 639-amino acid KC-VIN2 and the 635-amino acid

KC-VIN1 (truncated at N-terminus) share 99 % sequence

identity. Based on the ProtParam tool (http://web.expasy.

org/cgi-bin/protparam), the calculated molecular weight of

either of the predicted proteins, KC-VIN2 and KC-VIN1,

was found to be nearly 70.0 kDa with a predicted pI of 5.69.

However, once 100 amino acid residues are excluded from

the N-terminal region, the molecular weight becomes

*60.0 kDa with a predicted pI of 5.57 indicating approx-

imate values for the mature proteins. Out of a total 639

amino acids of KC-VIN2, 54 are strongly basic (?) (K, R),

66 are strongly acidic (-) (D, E), 221 are hydrophobic (A, I,

L, F, W, V), and 183 are polar (N, C, Q, S, T, Y). For the

entire predicted protein, the instability index (II) was

computed as 41.11, which classified the protein as unstable;

kb

2.0

A1 2 3 1 2 3

kb

2.0

B

Fig. 1 RT-PCR amplification products (*2.0 kb) using total RNA

from the micropropagated plantlets of different potato cultivars, and

the vacuolar invertase cDNA specific primers. a The primers used

K20-AI and AI-2016; lane 1, 500 bp DNA ladder; lanes 2 and 3

correspond to total RNA from the cultivars Kufri Chipsona-1 and

Kufri Chandramukhi, respectively, b The primers used AI-F01 and

AI-R1950; lanes 1 and 2 correspond to total RNA from the cultivars

Kufri Chipsona-1 and Kufri Chandramukhi, respectively, lane 3,

500 bp DNA ladder

Acta Physiol Plant (2013) 35:2055–2068 2059

123

but the value was computed as 35.81 for the predicted

mature protein and classified it as stable one. The amino

acid composition data also revealed that some of the amino

acids such as Asp (6.9 %), Pro (6.4 %), Ser (9.2 %), Trp

(3.0 %), Tyr (4.5 %), and Val (7.4 %) occurred more fre-

quently as compared to their average occurrence; whereas,

the amino acids, namely Arg (3.1 %), Cys (0.6 %), Glu

(3.4 %), Met (1.3 %) occurred less frequently (Doolittle

1989). Similar amino acid composition data were also

obtained in case of KC-VIN1. BLAST search (blastp) also

revealed that KC-VIN2 shared 97–99 % identity with the

corresponding sequences from other potato cultivars

(CAA49831, AAA50305, ABF18956, AAQ17074); and

*95, 94, 87 and 84 % for the tomato cultivars (NP_0012

34843, CAA78061, CAA78060), muskmelon (ABX55

832), Capsicum (AAB48484), and tobacco (CAC83577),

respectively. If compared with the invertase sequences from

other plant species, the sequence identity was found to be

considerably decreased.

Multiple sequence alignment, signature-type sequences,

and phylogenetic tree

To examine sequence similarities, nature and location of

the amino acid substitutions in the vacuolar invertases,

multiple sequence alignment was done using a total of

eleven homologs from four economically important

members of the Solanaceae family: six from potato cul-

tivars (KC-VIN2 and KC-VIN1 of this study, and the

remaining four from other potato cultivars), three from

tomato cultivars, one from capsicum, and one from

tobacco (Fig. 2). Nearly 100-amino acid N-terminal

regions of these sequences appeared to be more variable;

however, some small segments were found to be con-

served in this region. But most of the conserved segments

of varying lengths were found in the remaining major

parts of the vacuolar invertase sequences. KC-VIN2 and

KC-VIN1 were found to vary at eight locations: P6H,

Y29H, P95S, S108T, T536A, R568H, F607V, and R632Q.

A total of six positions represented nonconservative

substitutions. All these substitutions are confined to

N- and C-terminal regions only. Vacuolar invertase

sequences in potato differ significantly with that of

tomato. In case of KC-VIN2, apart from the insertion of a

3-amino acid segment, i.e. YPS near N-terminus, it

showed variations at 29 other positions if compared with

Sl-Tj or Sl-UC: H17R, F20L, Y29H, S43V, S64I, N65D,

V89A, N90G, P95S, S108T, T213A, Y226F, I271V,

E288G, E310K, T348G, K356N, K366R, Q384E, A439V,

I443T, N448D, H458R, V509I, F525Y, A552G, R554Q,

R568H, and R632Q. All these substitutions (mostly non-

conservative) occurred throughout the entire sequence.

More sequence divergence was noticed if compared with

the other members of the Solanaceae family, such as

capsicum, tobacco. Some important sequence features of

invertase such as three well-conserved motifs: the

WMNDPNG-motif (also known as b-fructosidase motif),

the RDP-motif (transition-state stabilizer), and the

WECVDF-motif, i.e. EC-motif or catalytic site were also

shown in Fig. 2.

Multiple sequence alignment between the Solanaceae

family members also revealed a few distinguishing

sequence features (signature-type sequences) in the vacu-

olar invertases. Here the well-conserved segments and

amino acid residues inserted in the conserved regions were

only considered, such as a 3-amino acid residue, Y(P/H)S

in potato near the amino terminus. Likewise, a 3-amino

acid residue, SAG could be found only in tobacco if we

proceed further towards the C-terminus. Moreover, some

segments including single amino acid insertions were

found to occur in more than one member of the Solanaceae

family, such as a 2-amino acid residue, LN in potato,

tomato, and capsicum; a 6-amino acid residue (S/H)

(A/S)SETL in capsicum and tobacco; a single Pro residue

in potato, tomato, and capsicum; a 3-amino acid residue,

FT(S/N) in potato, tomato and tobacco; a single His residue

in potato and tomato.

A total of 45 vacuolar invertase sequences from 27

taxonomically different plant species were used to predict

the consensus sequence as shown in Fig. 2. A number of

segments of varying lengths including single amino acid

residues were found to be conserved, and mostly they were

confined to the predicted mature proteins. A phylogenetic

tree was generated using the same sequences to know

evolutionary relatedness between the different plant

Fig. 2 Comparison of the predicted amino acid sequences of eleven

soluble vacuolar invertase homologs from the Solanaceae family:

KC-VIN2 (ACC93585) and KC-VIN1 (ACC93584) from the potato

cv. Kufri Chipsona-1, St-MQ (ABF18956), St-De (CAA49831),

StPain1 (ADM47340) and St-RB (AAA50305) are from other potato

cultivars; Sl-Tj (CAA78061), Sl-UC (CAA78060), and Sl-SF

(NP_001234843) are from the tomato cultivars Trujillo, UC82B

and SuperFirst, respectively; C. annum (AAB48484) from capsi-

cum; Nt-SNN (CAC83577) from the tobacco cv. SNN. This multiple

sequence alignment is based on ClustalW2 tool along with minor

manual adjustments. Dashes indicate gaps that arise during align-

ment. Asterisks indicate the conserved amino acids between the

sequences from the Solanaceae family members. The distinguishing

sequence features i.e. signature type sequences are highlighted. Six

potential N-linked glycosylation sites (Asn-X-Ser/Thr) are single

overlined in KC-VIN2 sequence; the WMNDPNG-motif, the RDP-

motif, and the WECVDF-motif are double overlined (the aspartate

in the WMNDPNG-motif, the aspartate in the RDP-motif, and the

glutamate in the EC-motif are shown by the downward arrows). For

consensus sequence (shown as CONS), single letter code in upper

case is used for the amino acids conserved in most of the plant

species (more than 90 %); the amino acids conserved in more than

50 % plant species are shown by the respective lower cases; square

is used for the variable amino acids

c

2060 Acta Physiol Plant (2013) 35:2055–2068

123

Acta Physiol Plant (2013) 35:2055–2068 2061

123

species (Fig. 3). The vacuolar invertase sequences from

different plant species were divided into many distinct

groups according to their sequence relatedness. KC-VIN1

and KC-VIN2 were found to occupy distinct branches in

the phylogenetic tree.

Hydropathy plot, prediction of secondary structures

The hydropathy profiles were generated for the vacuolar

invertases from six different plant species, namely potato,

sweet potato, oilseed crop Brassica, orange, poplar, and rice

with nine amino acids running window using the ProtScale

tool based on the Kyte-Doolittle scale (Fig. 4a–f). For

segment-wise comparison between the invertases, the

positions of the WMNDPNG-motif, the RDP-motif, and the

WECVDF-motif in each hydropathy plot were clearly

indicated. As revealed in each plot, some common features

were found such as the first two motifs belonged to the

hydrophilic regions, whereas, the catalytic EC-motif

appeared on the midpoint of the scale. The flanking regions

Fig. 2 continued

2062 Acta Physiol Plant (2013) 35:2055–2068

123

S. tuberosum cv. K Chipsona 1 (ACC93584)

S. tuberosum cv. K Chipsona 1 (ACC93585)

S. tuberosum cv. May Queen (ABF18956)

S. tuberosum cv. unknown (AAQ17074)

S. tuberosum cv. unknown (ADM47340)

S. tuberosum cv. Russet Burbank (AAA50305)

S. tuberosum cv. Desiree (CAA49831)

S. lycopersicum cv. Superfirst (NP_001234843)

S. lycopersicum cv. unknown (NP_001234618)

S. lycopersicum cv. H Odoriko (BAA01954)

S. lycopersicum cv. UC82B (CAA78060)

S. pimpinellifolium strain Trujillo (CAA78061)

Cucumis melo (ABX55832)

Capsicum annum (AAB48484)

Nicotiana tabacum (CAC83577)

Ipomoea batatas (AAK71505)

Oryza sativa Japonica Gr (AAK72492)

Oryza sativa Japonica Gr (AAD10239)

Ipomoea batatas (AAK71504)

Ipomoea batatas (AAD01606)

Coffea canephora (ABI17894)

Daucus carota (CAA53097)

Daucus carota (CAA53098)

Daucus carota (CAA53099)

Glycine max (XP_003533514)

Citrus sinensis (BAF34363)

Citrus sinensis (AAL27709)

Gossypium hirsutum (ACQ82802)

Ricinus communis (XP_002510944)

Pachysandra terminalis (CBM41476)

Sorghum bicolor (XP_002446857)

Oryza sativa Indica Gr (CAH67112)

Vitis vinifera (AAB47172)

Daucus carota (CAA47636)

Cichorium intybus (CAD12104)

Arabidopsis lyrata (XP_002888009)

Arabidopsis thaliana (NP_564798)

Brassica oleracea (AAG36943)

Brassica oleracea (AAG36942)

Pyrus pyrifolia (BAF35859)

Populus trichocarpa (XP_002303519)

Vigna radiata (BAA01107)

Phaseolus vulgaris (AAB68679)

Vicia faba (CAA89992)

Pisum sativum (AAM52062)

60

56

100

94

100

100

68

61

95

68

64

65

61

93

100

99

100

100

100

100

100

100

100

100

60

85

99

93

85

93

99

87

61

60

88

93

100

Fig. 3 The phylogenetic tree was generated by the MEGA 5.0

software using the Neighbor-Joining method. This represents a

bootstrap consensus tree. The analysis involved 45 vacuolar acid

invertase sequences from the different plant species as available in the

published reports and/or databases (the name of the plant species and

the GenBank accession numbers are indicated at each branch). The

number at each node represented the bootstrap value, with 1000

replicates. The predicted amino acid sequences of KC-VIN1

(ACC93584) and KC-VIN2 (ACC93585) of this study appeared to

occupy distinct positions in the phylogenetic tree (shown in bold cases)

Acta Physiol Plant (2013) 35:2055–2068 2063

123

of the individual motifs were found to vary in terms of their

hydropathic characters between the invertases. Some of

the segments in the *100-amino acid N-terminal regions

showed similar hydropathic characters. Segment-wise

analyses of the *300-amino acid C-terminal regions also

showed similar trends. With the help of ProtScale tool,

a-helix, b-sheet, b-turn, and random coil in KC-VIN2 were

also predicted (data not shown). Small segments containing

the individual motif of the catalytic triad appeared to show

fewer propensities towards the formation of regular sec-

ondary structures such as a-helix or b-sheet; however, after

the EC-motif, presence of some a-helices and b-sheet

structures can be predicted towards the C-terminal regions.

Analysis of vacuolar invertase expression

The overall vacuolar invertase gene expression pattern was

studied in the freshly harvested and cold-stored tubers from

some of the field-grown Indian potato cultivars along with

cv. Desiree. By semi-quantitative RT-PCR, *1.0 kb

cDNA could be amplified corresponding to the 50-end of

the transcript using total tuber RNA samples. The vacuolar

invertase expression was found to be low in the freshly

harvested tubers (Fig. 5a), but during storage at low tem-

perature (4 �C), the level of transcripts was considerably

increased in the tubers of all the cultivars (Fig. 5b). In the

cold-stored tubers of Kufri Jyoti, Kufri Chipsona-2 and

Kufri Chipsona-1, relatively more accumulation of tran-

scripts was noticed, but at moderate level in case of the

cultivar Kufri Chandramukhi. The remaining cultivars

namely Kufri Ashoka, Kufri Pukhraj, and the cv. Desiree

showed relatively less accumulation of the invertase tran-

scripts. The level of actin-specific transcripts (as internal

control) was found to be nearly uniform in all the potato

cultivars. In the freshly harvested tubers, total extractable

activities of vacuolar invertase in the different potato cul-

tivars were found to be negligible, ranging from 0.048 ±

0.003 nmol (mg min)-1 (Kufri Chipsona-1) to 0.089 ±

0.008 nmol (mg min)-1 (Desiree); whereas, in the cold-

stored tubers vacuolar invertase activity was increased sig-

nificantly, ranging from 0.935 ± 0.034 nmol (mg min)-1

(Desiree) to 3.411 ± 0.028 nmol (mg min)-1 (Kufri Jyoti)

as shown in Table 1. The results of vacuolar invertase

assay were more or less consistent with that of semi-

quantitative RT-PCR.

Discussion

Most of the potato cultivars are autotetraploid

(2n = 4x = 48), highly heterozygous, and suffer inbreed-

ing depression (Genova et al.2011). High level of DNA

polymorphism in the genome of Solanum tuberosum is well

known. For example, natural allelic variation is common in

potato invertase genes (Draffehn et al. 2010). It is also

known that multiple allelism occurs for potato genes that

affect morphological characteristics and various metabolic

pathways (van de Wal et al. 2001). Therefore, for the

improvement of this important food crop, advanced

molecular breeding needs to be facilitated instead of clas-

sical breeding approaches. In-depth understanding of dif-

ferent invertase genes and their allelic variants, various

factors that influence their expression in different tissues,

and establishing structure–function relationships in the

invertases would be quite useful in such efforts. The copy

number of acid invertases is low but their allelic compo-

sition is not known clearly in the individual potato culti-

vars. Different potato cultivars/clones with their rich

genetic resources have become quite attractive systems

both in terms of basic and applied aspects of invertase

research.

With regard to the Indian potato cultivars, no report was

available on invertases both at biochemical and molecular

level till to date. Keeping this in view, we report here

molecular cloning and characterization of two cDNAs

encoding vacuolar invertase isoforms from one commer-

cially important Indian potato cultivar, Kufri Chipsona-1

based on RT-PCR approach. These isoforms are likely to

be conserved in the cultivar Kufri Chandramukhi as evi-

dent from the RT-PCR amplified products (Fig. 1a, b). The

genetic resources of other potato cultivars could be further

explored to obtain more such allelic variants. It was known

that in the glycoside hydrolase family of enzymes, the

N-terminal fivefold b-propeller domain consists of three

common conserved motifs in the active sites that accom-

modate three crucial amino acid residues. More specifi-

cally, these are the nucleophile (the aspartate in the

WMNDPNG-motif), transition-state stabilizer (the aspar-

tate in the RDP-motif), and the acid/base catalyst (the

glutamate in the WECVDF-motif) (Verhaest et al. 2005;

Lammens et al. 2009). In the vacuolar invertases of the

Solanaceae family members, apart from these three motifs,

their flanking regions were also found to be well-conserved

as shown in Fig. 2. All these important motifs occurred in a

span of nearly 190 amino acid residues. The WMNDPNG-

motif and the RDP-motif were separated by *120 amino

Fig. 4 Hydropathy plots of the deduced amino acid sequences of

vacuolar invertases from plants belonging to different taxonomic

groups based on the Kyte and Doolittle scale. a Potato (cv. Kufri

Chipsona-1, KC-VIN2, ACC93585), b Sweet potato (cv. Tainong 57,

AAK71505), c Brassica oleracea (cv. Shogun, AAG36943), d Orange

(cv. Washington, BAF34363), e Populus trichocarpa (XP_002303

519), f Oryza sativa Indica Gr. (CAH67112). The beginning of the

WMNDPNG-motif, the RDP-motif, and the WECVDF-motif in each

hydropathy plot are indicated by the following upward arrows, arrow,

open arrow and filled arrow respectively

c

2064 Acta Physiol Plant (2013) 35:2055–2068

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Acta Physiol Plant (2013) 35:2055–2068 2065

123

acid residues, whereas the catalytic site occurred more

close to the RDP-motif, and separated by *50 amino acid

residues. Similar patterns were also found in the other plant

vacuolar invertases. It was known that aspartate in the

WMNDPNG-motif, aspartate in the RDP-motif, and glu-

tamate in the EC-motif were indispensable for the func-

tionality of acid invertases i.e. for substrate binding and

catalysis. These three amino acid residues were also

referred to as ‘the catalytic triad’. It is now believed that

the aspartate residue in RDP-motif is not directly involved

in the catalytic mechanism and probably acts as a transi-

tion-state stabilizer. Apart from glutamate, a conserved

cysteine residue is also present in the EC-motif. But the

precise role of this polar cysteine residue during catalysis is

yet to be understood. It is likely that apart from these

conserved motifs and their sequences, the distance between

these motifs and the amino acid sequences therein may also

be crucial for overall functionality of the acid invertases.

Six potential N-linked glycosylation sites (Asn-X-Ser/Thr)

were found in KC-VIN2, KC-VIN1 and the other potato

sequences. In tomato only five such sites were found. Only

three N-linked glycosylation sites were found to be com-

mon between the Solanaceae family members as examined

in this study.

One of the focus areas of this study remained on the

analyses and comparison of vacuolar invertase sequences

between the plant species of different taxonomic groups.

Sequence alignment between the four members of the

Solanaceae family (namely potato, tomato, capsicum and

tobacco) clearly revealed the sequence relatedness between

them along with nature and location of the variations in

their primary sequences. Most of the amino acid substitu-

tions were found to be nonconservative. Therefore, it is

likely that such changes may influence in the structure–

function relationships and the overall functionalities of the

individual vacuolar invertases within and between the plant

species. In some families of homologous proteins, cer-

tain segments of a protein sequence may be found in the

organisms of one taxonomic group but not in other groups;

these segments can be referred to as signature sequences for

the group in which they are found. Sequence alignment of

the vacuolar invertases from the Solanaceae family mem-

bers revealed the presence of some signature-type of

sequences not documented in the earlier reports. All these

short sequences appeared to confer some sort of identity

features of the vacuolar invertases in the different members

of this family. Moreover, they may provide some bio-

chemical clues in establishing the evolutionary relatedness

at different taxonomic levels. The biochemical roles of such

distinguishing sequence features remain to be elucidated.

Based on the large number of available plant invert-

ase sequences from different plant species, a consensus

sequence was predicted that presented variable, moderately

conserved and highly conserved amino acid residues and

segments. Hydrophobic characters were also predicted and

compared between some of the taxonomically unrelated

INV

1 2 3 4 5 6 7

B Tubers stored at 40C

Actin

1 2 3 4 5 6 7

INV

Actin

A Freshly harvested tubers

Fig. 5 Semi-quantitative RT-PCR approach for vacuolar invertase

expression analysis in the tubers from the mature field-grown potato

cultivars using the primers K20-AI and M20-AI. a Freshly harvested

tubers from the different potato cultivars; lanes 1–7 correspond to the

potato cultivars Kufri Chipsona-1, Kufri Chandramukhi, Kufri

Chipsona-2, Kufri Jyoti, Kufri Ashoka, Kufri Pukhraj, and the cv.

Desiree, respectively; b Potato tubers stored at 4 �C for 4 weeks;

lanes 1–7 correspond to the potato cultivars in the same order as

mentioned in a; the size of the vacuolar invertase-specific amplified

product was found to be *1.0 kb in each case. Actin-specific primers

were used as control (the size of the amplified product *0.25 kb)

Table 1 Vacuolar invertase activity in the freshly harvested, and

cold-stored tubers (after 4 weeks of storage at 4 �C) from different

potato cultivars

Potato cultivars Vacuolar invertase activity [nmol (mg�min)-1 ]

Freshly harvested 4 �C (4 weeks)

Kufri chipsona-1 0.048 ± 0.003 1.586 ± 0.096

Kufri chipsona-2 0.055 ± 0.005 3.317 ± 0.072

Kufri jyoti 0.051 ± 0.002 3.411 ± 0.028

Kufri

chandramukhi

0.064 ± 0.007 1.265 ± 0.016

Kufri pukhraj 0.076 ± 0.013 1.018 ± 0.012

Kufri ashoka 0.058 ± 0.006 0.951 ± 0.023

Desiree 0.089 ± 0.008 0.935 ± 0.034

Values are the mean ± SD of n = 3 independent tubers

2066 Acta Physiol Plant (2013) 35:2055–2068

123

plant species. Essentially, all these data could help in

identifying the crucial amino acids for proper understanding

the structure–function relationships, and improving the

invertase alleles through site-directed mutagenesis. A phy-

logenetic tree was generated to see the sequence relatedness

between a large number of plant species from different

taxonomic groups.

The overall expression patterns of vacuolar invertase

were analyzed in the freshly-harvested and cold-stored

tubers from some of the Indian potato cultivars along with

cv. Desiree using semi-quantitative RT-PCR and assaying

total extractable enzyme activities. The level of vacuolar

invertase expression was found to be considerably higher

after storage at low temperature as compared to the freshly

harvested tubers. But the levels of expression particularly in

the cold-stored tubers were found to vary between the

potato cultivars. Richardson et al. (1990) and Zrenner et al.

(1996) demonstrated earlier that the activity of acid

invertase increased in the potato tubers stored at low tem-

peratures, but the level of enzyme activities was found to

vary between the cultivars. Zrenner et al. (1996) also

showed that the level of soluble acid invertase determined

the hexose-to-sucrose ratio in the cold-stored potato tubers.

We also measured the level of accumulation of both

reducing and total soluble sugars in the freshly harvested

and cold-stored tubers from these cultivars. During storage

at *4 �C for 4–8 weeks, the tubers from all the cultivars

showed significantly increased but at varying levels of

reducing and total sugars (data not shown); the observations

were quite consistent with the published reports. However,

this was only a preliminary approach for gene expression

analysis. Further molecular and biochemical studies are

required to know the expression pattern of the individual

vacuolar invertase isoforms in the potato cultivars at various

stages of their growth and development. Matsuura-Endo

et al. (2004) worked on some Japanese potato cultivars.

They observed mainly three types of changes in the potato

tubers stored at 4 �C: (a) increased levels of reducing sugars

during storage, i.e. type-1; (b) almost similar pattern as in

type-1, but relatively lower levels of reducing sugars

throughout storage, i.e. type-2; and (c) increased levels of

sucrose, but not reducing sugars, i.e. type-3. They also

showed that during storage at 4 �C the vacuolar invertase

activity increased only in the type-1; but in the type-2 and 3

cultivars, invertase levels remained very low. Our data

clearly indicated that the potato cultivars namely Kufri

Jyoti, Kufri Chipsona-2, Kufri Chipsona-1, and Kufri

Chandramukhi belonged to type-1. Probably, the remaining

potato cultivars also belong to the same category.

In conclusion, apart from molecular cloning and analy-

sis of gene expression in the Indian potato cultivars, this

study also revealed some important sequence features of

vacuolar acid invertases particularly in the Solanaceae

family members. Acid invertases not only play important

biological roles, they are also commercially important

enzymes. Therefore, the data as presented in this report

would be useful in crop improvement, protein engineering,

and other biotechnological applications.

Author contribution (1) Vijay Kumari, designed and

carried out the experiments, analyzed the results, compiled

the data, and wrote the manuscript initially. (2) Niranjan

Das conceived the research area, provided scientific advice,

supervised the project, analyzed the results, corrected and

revised the manuscript.

Acknowledgments We gracefully thank the Council of Scientific

and Industrial Research (CSIR), Govt. of India for providing fel-

lowship to V. Kumari; Department of Biotechnology (DBT), Govt. of

India for providing research funding to N. Das.

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