Partial Gene Sequence of Soluble Acid Invertase Gene fromSaccharumspontaneum: A First Report

RESEARCH ARTICLE

Partial Gene Sequence of Soluble Acid Invertase Genefrom Saccharum spontaneum: A First Report

Amaresh Chandra • Kriti Roopendra •

Amita Sharma • Radha Jain • Sushil Solomon

Received: 30 May 2013 / Revised: 20 August 2013 / Accepted: 30 October 2013 / Published online: 19 July 2014

� The National Academy of Sciences, India 2014

Abstract Sucrose is an important component of sugar-

cane yield and the enzymes, sucrose phosphate synthase,

sucrose synthase and invertases, work synergistically to

influence its metabolism, translocation and storage. Of the

invertases, soluble acid invertase (SAI) plays a pivotal role

in controlling the sucrose content in cane stalk vacuoles

and consequently, regulates the stored sucrose levels.

However, notably, no SAI gene sequence had yet been

reported for Saccharum spontaneum, one of the progenitors

of present day sugarcane, and thus, an endeavour was made

in this direction. Utilizing one (SAIF1/R1) of the six primer

pairs designed for SAI gene, in an earlier study, the first

ever nucleotide sequence was determined, specifically for

S. spontaneum SES34 (accession No.: KC570328). Addi-

tionally, the SAI gene sequence for Saccharum spp.

hybrids CoJ64 (an early maturing and high sucrose accu-

mulating variety of sugarcane) and for Saccharum offici-

narum 28NG210 (accession nos.: KC570326 and

KC570327, respectively) were also determined. Sequence

based phylogenetic tree showed close relatedness between

the SAI sequences for various Saccharum cultivars, thus

pointing to little genetic change while that for sorghum and

maize were relatively distantly related. Results help in

understanding source-sink process using species specific

genes.

Keywords Saccharum officinarum �Saccharum spontaneum � Sucrose � Invertase �Soluble acid invertase (SAI)

Introduction

Sugarcane (Saccharum spp. hybrids) is an inter-specific

hybrid derived from crosses of the domesticated species

Saccharum officinarum (a group that has sweet canes with

thick and juicy culms), natural hybrids (Saccharum sinense

and Saccharum barberi) and Saccharum spontaneum (a

wild species with no sugar and thin culms) [1]. It is an

important, sugar producing, C4 crop (family Poaceae) and

hence sucrose is an important component of its yield. It is

capable of accumulating up to 50 % of the total dry matter

of stalk as sucrose and storage parenchyma cells hoard

sucrose, which at maturity, usually attains levels of about

80 % of the dry weight and about 20 % of the fresh weight

[2]. High sucrose content in cane stalks is the utmost

important trait for farmers, and consequently for breeders

and agronomists as well. Thus, the crop is mainly valued in

terms of commercial cane sugar (CCS), in which sucrose

content is the dominant factor. At harvest, the quality of the

sugarcane juice is determined in terms of the concentration

of sucrose, such that high concentration of sucrose is a

prime. Sucrose begins to accumulate in the sugarcane in-

ternodes, when they start elongating and continues even

after elongation ceases [3]. During ripening, sucrose con-

centration increases along the entire stalk, while, in pro-

portion, the glucose and fructose concentrations decrease

significantly [4]. Thus, it is evident that sucrose metabo-

lism in the stalk also changes during development.

Sucrose is synthesized only in the cytosol by sucrose

phosphate synthase (SPS) or sucrose synthase (SS) enzymes,

but its distribution to various degrees between the apoplast,

the cytosol and the vacuole of the storage parenchyma is

monitored and controlled by invertases (b-fructofuranosid-

ase), thus, influencing the overall sucrose metabolism,

translocation and storage. Invertases are specifically known

A. Chandra (&) � K. Roopendra � A. Sharma � R. Jain �S. Solomon

Division of Plant Physiology and Biochemistry, Indian Institute

of Sugarcane Research, Lucknow 226002, India

e-mail: [email protected]

123

Natl. Acad. Sci. Lett. (July–August 2014) 37(4):317–323

DOI 10.1007/s40009-014-0242-7

to catalyze the irreversible cleavage of sucrose into the two

hexoses i.e. glucose and fructose, utilizing ATP in the pro-

cess. Thus, the primary function of invertases is considered

to be that of supplying carbohydrates to the sink tissues and

in particular, acting as a key regulator of sucrose accumu-

lation in sugarcane stem parenchyma (culm/stalk) [5–8],

thus in turn, helping plant development and crop produc-

tivity. Based on their sub-cellular locations, invertases are

categorized into cell wall (CWI), vacuolar soluble acid

(SAI) and cytoplasmic neutral (NI) subgroups. The two

kinds of acid invertase, the soluble acid invertase, SAI

(vacuole) and the cell wall invertase, CWI (apoplast), both

exhibit optimum activity between pH 5.0 and 5.5 and cleave

fructose residue from disaccharides. They are also known to

hydrolyse other b-fructose containing disaccharides like

raffinose and stachiose [9].

SAI apparently plays a role in the remobilization of stored

sucrose from the vacuole and can hence be important in the

regulation of hexose levels in certain tissues [5, 6]. It is

known to play a prominent role in both sucrose import and

sugar signaling, particularly during the initiation of sink

growth and cell wall expansion, when there is a high need for

sucrose hydrolysis [10, 11]. Thus, SAI gene(s) contribute to

controlling the sucrose content in cane stalk vacuoles as well

as developmental processes during growth and maturation

of the plants. The SAI activity has been observed to go up at

times when growth is rapid, particularly in storage tissues

that are rapidly growing during internode growth and

development, and low at other times. Perhaps, SAI must be

low for sucrose accumulation and thus mature sucrose-

storing internodes of sugarcane have been found to contain

negligible SAI levels [9, 12, 13].

The expression and regulation of SAI during various

stages of cane growth development, maturity and post-

harvest sucrose inversion is an important determinant of

the sucrose yield and hence SAI is probably the most

extensively studied of the invertases. A variety of gene

isoforms exist for it, which have been shown to have dif-

ferent developmental and tissue specific expression pat-

terns, in various species. However, the gene sequence of

SAI had not yet been reported for the S. spontaneum spe-

cies, although, it being a progenitor of current day sugar-

cane, any information in its regard would be of benefit to

sugarcane research as a whole.

Materials and Methods

Plant Materials and Isolation of Total RNA

Utilizing normal agronomical cultural practices S. sponta-

neum SES34, Saccharum spp. hybrids CoJ64 (an early

maturing and high sucrose accumulating variety of

sugarcane) and S. officinarum 28NG210 were grown at

Indian Institute of Sugarcane Research farm, India, for the

present study. For isolation of total RNA, leaves were

collected and frozen immediately in liquid nitrogen. Ten

months old field grown plants were used to isolate the total

RNA utilizing Qiagen RNeasy mini kit following the

manufacturer’s instructions. The DNA contamination was

removed by using RNase free DNase (QIAGEN) solution.

The quality and integrity of isolated RNA was checked on

1.0 % agarose gel. The purified RNA was stored at -20 �C

until further use.

cDNA Synthesis and qRT-PCR Analysis

cDNA synthesis and quantitative reverse transcriptase

reactions were performed with all three RNA samples

isolated from S. spontaneum SES34, Saccharum spp.

hybrids CoJ64 and S. officinarum 28NG210 lines using

one-step RT-PCR kit (QIAGEN) wherein cDNA is initially

synthesized based on the gene sequences of soluble acid

invertases (SAI) designed by Chandra et al. [14]. The

primers (SAIF1/R1) sequences were F:50ATGGCCCGG

TGTACTACAAG30 and R:50AGCGCGTAGTAGTCATG

TCG30. A fragment of 650 bps of the SAI gene was

amplified. For internal control, actin gene primer pairs

(F-50GGACATCCAGCCTCTTGTC30/R-50GCAAGATCC

AAACGAAGAATGG30) were used. The reaction condi-

tions for qRT-PCR were as follows: 50 �C for 30 min

(cDNA synthesis step), 95 �C for 15 min (denaturation of

reverse transcriptase), 32 cycles consisting of 95 �C for

1 min, 58 �C for 1 min and 72 �C for 1 min and finally an

extension step of 72 �C for 20 min. PCR was performed in

PTC 200 thermal cycler (MJ Research/BioRad, USA). The

amplified DNA products from varieties of sugarcane were

eluted and cloned into pGEM-T Easy vector according to

the manufacturer’s instructions. After purification, recom-

binant clones were directly sequenced using suitable pri-

mer with an automated sequencer ABI 3730XL (Applied

Biosystems, Foster City, CA, USA).

Data Analysis

Jalview (http://www.jalview.org/) [15] was used to visu-

alize the alignment of these three DNA sequences. Before

doing the sequence analysis, all DNA sequences were

screened for any plasmid sequence contamination using

VacScreen software available at NCBI web site. Also, the

online tool BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi)

was employed to run a nucleotide blast (blastn) for each of

the three sequences, in order to assess their degree of

similarity and homology using non redundant database.

Using the SAI sequence information available at NCBI, for

various Saccharum species as well as some closely related

318 A. Chandra et al.

123

http://www.jalview.org/

http://blast.ncbi.nlm.nih.gov/Blast.cgi

species like sorghum and maize, a phylogenetic tree was

generated using the online tool ClustalW2 (http://www.

ebi.ac.uk/Tools/clustalw2/index.html) to get an insight into

their evolutionary relationship.

Results and Discussion

In our earlier study, DNA sequences of soluble acid inver-

tases (SAI) of 13 crops species were analysed for sequence

homology and based on the most conserved gene region, six

primer-pairs (forward and reverse) were designed by

Chandra et al. [14]. Following trail from this study, we

employed the SAIF1/R1 primer F: ATGGCCCGGTGT

ACTACAAG R: AGCGCGTAGTAGTCATGTCG (from

Saccharum spp.; accession No.: AY302083) and isolated and

amplified a 650 bp fragment from two species namely S.

spontaneum SES34 and S. officinarum 28NG210 and a sug-

arcane variety namely Saccharum spp. hybrids CoJ64. The

DNA sequences obtained from these species and variety

KC570328.1_SacspontSES34SAI/1-344KC570326.1_SachybridCoJ64SAI/1-344

KC570327.1_Sacoffic28NG210SAI/1-344

Consensus

KC570328.1_SacspontSES34SAI/1-344

KC570326.1_SachybridCoJ64SAI/1-344


Consensus


KC570326.1_SachybridCoJ64SAI/1-344KC570327.1_Sacoffic28NG210SAI/1-344

Consensus



Consensus




Consensus



Consensus

11

1

2323

23

A C T A CG CG C T CG G G A G G T A TG A CA C T A CG CG C T CG G G A G G T A TG A C

A C T A CG CG C T CG G G A G G T A TG A C

A C T A CG CG C T CG G G A G G T A TG A C

24

24

24

46

46

46

G CG G C CG C C A A CG CG TG G A CG C C

G CG G C TG C C A A CG CG TG G A CG C C



47

4747

69

6969

G C TG G A CG C CG A G A A G G A CG T CG

G C T CG A CG C CG A G A A G G A CG T CGG C T CG A CG C CG A G A A G G A CG T CG

G C T CG A CG C CG A G A A G G A CG T CG

7070

70

9292

92

G C A C CG G C C TG CG G T A CG A C TG GG C A C CG G C C TG CG G T A CG A C TG G

G C A C CG G C C TG CG G T A CG A C TG G

G C A C CG G C C TG CG G T A CG A C TG G

93

93

93

115

115

115

G G C A A G T T C T A CG CG T C C A A G A C




116

116116

138

138138

G T T C T A CG A C C CG G C C A A G CG C C

G T T C T A CG A C C CG G C C A A G CG C CG T T C T A CG A C C CG G C C A A G CG C C

Fig. 1 Multiple sequence

alignment (MSA) result for

nucleotide sequences of SAI for

Saccharum spontaneum SES34,

Saccharum spp. hybrids CoJ64

and Saccharum officinarum

28NG210, using Jalview

Partial Gene Sequence of Soluble Acid Invertase Gene 319

123

http://www.ebi.ac.uk/Tools/clustalw2/index.html


were presumably belonging to SAI gene. Thus, we deter-

mined the first ever nucleotide sequence of SAI, specifically

for S. spontaneum SES34, now available at GenBank (NCBI)

(accession No.: KC570328). In addition, we have also

reported the first SAI gene sequence for Saccharum spp.

hybrid CoJ64 (an early maturing and high sucrose accumu-

lating variety of sugarcane) and for S. officinarum 28NG210

(accession nos.: KC570326 and KC570327, respectively).

Putative DNA sequences obtained from the amplified

products from two species namely S. spontaneum SES34

and S. officinarum 28NG210 and a sugarcane variety namely

Saccharum spp. hybrids CoJ64, were aligned using the

multiple sequence alignment viewer, Jalview (the alignment

result is shown in Fig. 1). The results showed high sequence

conservation; however, the sequence corresponding to S.

spontaneum mismatched with that of the other two, at



Consensus




Consensus



Consensus



Consensus




Consensus



Consensus

G T T C T A CG A C C CG G C C A A G CG C C

139139

139

161161

161

G C CG CG TG C T C TG G G G A TG G G T CG C CG CG TG C T C TG G G G A TG G G T C

G C CG CG TG C T C TG G G G A TG G G T C

G C CG CG TG C T C TG G G G A TG G G T C

162

162

162

184

184

184

G G CG A G A C CG A C T CG G A G CG CG C




185

185185

207

207207

TG A CG T C T C C A A G G G A TG G G C A T

TG A CG T C T C C A A G G G A TG G G C A TTG A CG T C T C C A A G G G A TG G G C A T

TG A CG T C T C C A A G G G A TG G G C A T

208208

208

230230

230

CG C TG C A G G G G A T C C C C CG G A CGCG C TG C A G G G T A T C C C C CG G A CG

CG C TG C A G G G T A T C C C C CG G A CG

CG C TG C A G G G T A T C C C C CG G A CG

231

231

231

253

253

253

G TG C TG C TG G A C A C C A A G A CG G G




254

254254

276

276276

C A G C A A C C TG C TG C A G TG G C C CG

C A G C A A C C TG C TG C A G TG G C C CGC A G C A A C C TG C TG C A G TG G C C CG

Fig. 1 continued


123

Table 1 Pair-wise alignment scores for nucleotide sequences of SAI for S. spontaneum SES34, Saccharum spp. hybrids CoJ64 and S. offici-

narum 28NG210, generated using ClustalW2

SeqA Name Length SeqB Name Length Score

1 SAI_S._spontaneum 344 2 SAI_S._officinarum 344 99.0

1 SAI_S._spontaneum 344 3 SAI_S._spp. hybrids 344 98.0

2 SAI_S._officinarum 344 3 SAI_S._spp. hybrids 344 99.0

Table 2 Prediction results for nucleotide sequences of SAI for Saccharum spontaneum SES34, Saccharum officinarum 28NG210 and Sac-

charum spp. hybrids CoJ64 using BLAST

Query No. of BLAST hits Top hits (2) Bit score E value Identities

SAI_S._spontaneum

SES34

71 Saccharum hybrid cultivar FN-28 soluble

acid invertase (SAI) mRNA, complete

cds

634 bits (343) 1e-178 343/343 (100 %)

Saccharum hybrid cultivar H65-7052

soluble acid invertase (scinvh3’2)

mRNA, partial cds

634 bits (343) 1e-178 343/343 (100 %)

SAI_S._officinarum

28NG210

66 Saccharum officinarum soluble acid

invertase mRNA, partial cds

625 bits (338) 8e-176 341/343 (99 %)

Saccharum robustum soluble acid


625 bits (338) 8e-176 341/343 (99 %)

SAI_S. spp. hybrids

CoJ64

66 Saccharum officinarum soluble acid


616 bits(333) 5e-173 338/341(99 %)

Saccharum robustum soluble acid


616 bits(333) 5e-173 338/341(99 %)



Consensus




Consensus



Consensus

C A G C A A C C TG C TG C A G TG G C C CG

277277

277

299299

299

TG G A G G A A G TG G A G A CG C TG CG CTG G A G G A A G TG G A G A CG C TG CG C

TG G A G G A A G TG G A G A CG C TG CG C

TG G A G G A A G TG G A G A CG C TG CG C

300

300

300

322

322

322

A C C A A C T C C A C CG A C C T C A G CG G

A C C A A C T C C A CG G A C C T C A G CG G



323

323323

344

344344

C A T C A C C A T CG A C T A CG G C T C A

C A T C A C C A T CG A C T A CG G C A C AC A T C A C C A T CG A C T A CG G C T C A

C A T C A C C A T CG A C T A CG G C T C AFig. 1 continued


123

residue No. 50, 218 and 311 while the sequence for Sac-

charum spp. hybrids CoJ64 mismatched with that of the

other two at residue No. 29 and 342. Using ClustalW2

(http://www.ebi.ac.uk/Tools/clustalw2/index.html) the

overall MSA (Multiple Sequence Alignment) score, was

found to be 8153 while the pair-wise alignment scores were,

as shown in Table 1. Also, the online tool BLAST

(http://blast.ncbi.nlm.nih.gov/Blast.cgi) was employed to

run a nucleotide blast (blastn) for each of the three sequen-

ces, in order to assess their degree of similarity and

homology using non redundant database. BLAST results are

summarized in Table 2. The phylogenetic tree generated,

using ClustalW2 (shown in Fig. 2), as expected, showed

close relatedness between the SAI sequence for various

Saccharum cultivars, thus pointing to little genetic change

while that for sorghum and maize were relatively distantly

related. This sequence information will be useful and may

henceforth be utilized in conducting expression analysis of

genes involved in metabolism of sucrose, which would

otherwise, in the absence of such DNA sequence informa-

tion, require construction and employment of primer-pairs

using gene sequences from related crops.

The major setback to yield from stale cane is due to

inversion of sucrose into glucose and fructose, leading to

significant loss of sucrose. Thus, future research attempts

should be oriented towards examining the role of sugar

metabolising systems, for employment in transgenic

manipulation of sucrose accumulation in sugarcane [16].

The reduction of invertase activity soon after harvest of

sugarcane crop could be useful in minimizing the post

harvest sucrose losses [17]. With more and more sequence

information being generated, the challenge of increasing

sucrose yield in sugarcane may be met by careful down/up

regulation of these enzymes involved in sucrose metabo-

lism. Thus, RNAi approach may be employed for exploring

possibilities of controlling the level of invertases at suitable

locations [18].

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Fig. 2 Phylogenetic tree

generated by ClustalW2 tool

(UPGMA clustering), using SAI

nucleotide sequence of various

Saccharum species, maize and

sorghum


123


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Documents

Partial Gene Sequence of Soluble Acid Invertase Gene fromSaccharumspontaneum: A First Report