Legume Research, 40 (3) 2017 : 462-469
AGRICULTURAL RESEARCH COMMUNICATION CENTRE
Print ISSN:0250-5371 / Online ISSN:0976-0571
www.arccjournals.com/www.legumeresearch.in
Galactomannan content and key enzymes of its metabolism in seeds of cluster
bean [Cyamopsis tetragonoloba (L.) Taub.]
Neha Wadhwa* and Udai Narayan Joshi
Department of Chemistry and Biochemistry,
Chaudhary Charan Singh Haryana Agricultural University, Hisar-125 004, Haryana, India.
Received: 01-09-2015
Accepted: 10-12-2015
DOI:10.18805/lr.v0iOF.10758
ABSTRACT
The present investigation was carried out to estimate galactomannan content in mature seeds of 17 guar genotypes and activity of
enzymes involved in galactomannan metabolism. Galactomannan content was found in the range of 16.82 (in IC 310630) to
36.68 per cent (in HG 3-2). The developing pods were sampled at 25, 32, 39 and 46 days after flowering (DAF) for galactosyltransferase, ß-D-mannosidase & ß-1, 4-mannanase assay. The mean -galactosyltransferase specific activity increased
from 25 to 39 DAF (1557 to 3093 units) followed by decrease at 46 DAF (1484 units). The mean specific activity increased from
392 to 3166 units with the increase in galactomannan content from 16.82 to 36.68 per cent. Thus, this enzyme showed highly
positive correlation with the galactomannan content. The mean specific activity of ß-D-mannosidase increased gradually from
25 to 39 DAF (67 to 138 units) followed by sharp decrease at 46 DAF (32 units). The mean specific activity of ß-1, 4-mannanase
was found maximum at 25 DAF (102 units) and afterwards, it decreased continuously with advancement of days after flowering
up to 46 days (9 units). On the whole, it can be said that the ß-D-mannosidase requires prior activity of ß-1, 4-mannanase for
galactomannan catabolism while -galactosyltransferase activity is positively correlated with galactomannan content and play a
major role in guar gum synthesis and can be further used for gum improvement via genetic manipulation.
Key words: Clusterbean, Galactomannan, Galactosyltransferase, Guar, Mannanases.
INTRODUCTION
Guar [Cyamopsis tetragonoloba (L.) Taub.], also
known as cluster bean, is an important annual droughttolerant forage crop grown in semi-arid regions of India,
Pakistan and USA. It is a diploid plant (2n = 14) that belongs
to the family Leguminosae. Guar has been traditionally used
as forage, green manure and vegetable (Dwivedi et al., 1995).
Recently, it has attained the status of an important commercial
crop because of several industrial applications of guar gum.
Guar gum is obtained from the endosperm which constitute
up to 42 per cent of the whole seed. It is a viscosity builder
and natural polysaccharide; and has been variably used in
textile, paper, petroleum, mining, cosmetic, oil,
pharmaceuticals, explosion, tobacco and food industries
(Punia et al., 2009). It has been used in the production of
water proof biocide films (Das et al., 2011) and as a
controlling agent in oil wells to facilitate easy drilling.
Biosynthesis of galactomannan is a major metabolic
pathway which occurs during seed development in the
endospermic seeds of cluster bean. Sucrose (a diasaccharide)
acts as the building block for its biosynthesis. The enzyme
invertase (a hydrolytic enzyme) hydrolyzes sucrose into
glucose and fructose units, whereas sucrose synthase (a
glycosyl transferase), in the presence of UDP, converts
*Corresponding author’s e-mail: [email protected].
sucrose into UDP-glucose and fructose (Sturum and Tang,
1999). The glucose and fructose units, produced through the
activity of hexokinase, get converted into glucose-6phosphate and fructose-6-phosphate. Further, the enzyme
phosphomannoisomerase convert fructose-6-phosphate into
mannose-6-phosphate, which in turn is converted into
mannose-1-phosphate by phosphomannomutase (Lee and
Matheson, 1984). The precursors of galactomannan
biosynthesis GDP-D-mannose and UDP-D-galactose are
formed by the action of enzymes GDP-mannose
phosphorylase and UDP-galactose-4-epimerase. The GDPD-mannose is consumed by enzyme mannan synthase to form
a long mannan chain while enzyme galactosyltransferase
transfers UDP-D-galactose to the growing mannan chain.
The specificity of galactosyltransferase plays a key role in
the regulation of galactose substitution in galactomannan
biosynthesis and also the statistical distribution of galactosyl
substituents along the mannan backbone (Edwards et al.,
2002), which, in turn, affects the solubility of the
galactomannan (Edwards et al., 1992).
Galactomannans are water soluble, and thus more
accessible to enzymatic degradation, compared with cellulose
microfibrils. The released hexose sugars (mannose and
galactose) are easily fermentable compared with pentoses (Pauly
and Keegstra, 2008). Therefore, it is an attractive option to
Volume 40 Issue 3 (June 2017)
increase the galactomannan content in vegetative tissues of
bioenergy crop plants to enhance biofuel production. Recently,
mannan and its degradation products, i.e., mannooligosaccharides, attracted attentions of researchers in the field
of food and pharmaceutical industries because these poly- and
oligosaccharides are found to exhibit various beneficial effects
on human health (Dhawan and Kaur, 2007; Moreira and Filho,
2008). Consequently, a mannan-degrading enzyme, -1,4mannanase is also recognized as an important enzyme to
produce the above bioactive manno-oligosaccharides. In
addition, this enzyme is demonstrated to be useful in various
industrial processes, e.g., clarification of fruit juice, viscosity
reduction of coffee extract, improvement in the digestibility of
poultry feeds, and bleaching of pulp, where, degradation of
mannan improves the quality of products (Dhawan and Kaur,
2007). With the emerging need for increased plant biomass,
work on the identification of enzymes metabolizing and
modifying these polysaccharides has enormous potential for
future industrial applications. Therefore, understanding the role
of key enzymes for galactomannan metabolism should be the
priority for biochemists. Considering these points, the present
study was planned to screen the guar genotypes for
galactomannan content and to study the key enzymes of
galactomannan metabolism.
MATERIALS AND METHODS
463
Seeds of 17 guar genotypes, including the wild
species, i.e. C. serrata, were raised in pots filled in with
sandy loam soil mixed with compost (3 soil: 1 compost)
during Kharif, 2013 (Fig. 1). The characteristics of soil after
mixing with compost were : pH (1:2) 7.8, organic carbon
0.42%, N 8.0 mg kg-1 soil, P 26.0 mg kg-1 soil, K 632 mg kg-1
soil, Zn2+ 1.64 mg kg-1 soil, Fe2+ 2.74 mg kg-1 soil, Cu2+ 0.38
mg kg-1 soil and Mn2+ 3.65 mg kg-1 soil. The homogenized
soil was filled in earthen pots at the rate of 10 kg soil per
pot. Before sowing, the seeds were treated with Rhizobium
culture (strain-Rhizo-1305). Ten seeds per pot (in duplicate)
were sown at approximately uniform depth and distance, on
July 15, 2013. After the emergence of seedling and
subsequent week of growth, two uniform plants were
retained, per pot, for further studies.
Sampling: Flowers were tagged at anthesis and developing
pods were harvested at 25, 32, 39 and 46 days after flowering
(DAF) from the plants and brought to laboratory in ice buried
condition.
Galactomannan content estimation : The method developed
by Das et al. (1977) and improved by Joshi (2004) was used
for this estimation, which involves extraction and purification
of the galactomannan, which is then precipitated by alcohol,
further dissolved and measured spectrophotometrically.
Fig 1: Genotypes of cluster bean grown in naturally lit net house
464
LEGUME RESEARCH - An International Journal
Mature and ground seed sample weighing 100 mg
was mixed with 40 ml of 0.01M HgCl2. The samples were,
then, autoclaved at 15 psi for one hour. After cooling the
samples, volume was made up to 100 ml by using distilled
water. The samples were mixed thoroughly and 10 ml of
each was subjected to centrifugation at 3,500 x g for 15
minutes. From the supernatant obtained, 0.5 ml was taken
and absolute alcohol was added to it to make it 90 per cent
alcohol. The solution was kept overnight. Next day, the
samples were again centrifuged at 3,500 x g for 15 minutes
and the residue was dissolved in 5 ml of 0.01M HgCl2 in
boiling water bath for one hour. Afterward, the volume of
the extracts was made up to 5 ml and the samples were
subjected to sugar estimation using method of Dubois et al.
(1956). Standard and blank was run simultaneously &
galactomannan content was estimated by using a standard
curve prepared by using galactose: mannose (1:2) ratio.
Enzyme assays: Seeds weighing 500 mg were homogenized
in 5 ml phosphate buffer in a previously chilled mortar using
glass beads as abrasive. For extraction of -D-mannosidase
and -1, 4-mannanase, 0.1 M phosphate buffer (pH 7.0) while
for -galactosyltransferase, 2 mM phosphate buffer (pH 8.0)
was used. The homogenate, thus obtained, was then centrifuged
at 5,000 x g for 20 min in a refrigerated centrifuge. The
supernatant thus obtained was referred as crude extract and was
used on the same day for enzyme assay.
- Galactosyltransferase (EC 2.4.1.87): - Galactosyltrans
-ferase activity was assayed by a pH-sensitive method of
Deng and Chen (2004). For this assay, one ml of reaction
mixture was prepared containing 0.01 mM phenol red
(indicator), 0.1 mM MnCl 2 (activator), 10 mM Nacetylglucosamine (acceptor), and 100 µl of crude extract
mixed with phosphate buffer (2mM, pH 8). The reaction
was started by adding UDP-galactose (substrate) to a final
concentration of 2 mM and the absorbance was read at 557
nm for each sample at 30-sec interval for a total of 3 min.
The activity of enzyme was calculated from the calibration
curve prepared by using different amount of 10 mM HCl in
place of UDP-galactose. The enzyme activity has been
expressed in units, as 1 unit= 1.0 nmol protons liberated per
min/mg protein.
-D-Mannosidase (EC 3.2.1.25): This enzyme was assayed
by the method of Kestwal and Bhinde (2005) with some
modification. For determination of -D-mannosidase
activity, a system of p-nitrophenyl--D-mannopyranoside (2
mM) in citrate buffer (0.1 M, pH 4.6) and citrate buffer (0.1
M, pH 4.6), incubated at 30ºC was used. Reaction was started
by adding appropriate amount of the enzyme extract,
resulting in the liberation of p-nitrophenol. The reaction was
terminated after 30 minutes by addition of 2 ml of borate
buffer (0.1 M, pH 9.5). Liberated p-nitrophenol was
measured spectrophotometrically at 405 nm. A blank was
run simultaneously. The enzyme activity was expressed in
units, where 1 unit = 1.0 nmol p-nitrophenyl--Dmannopyranoside hydrolyzed per minute/mg protein.
-1, 4-Mannanase (EC 3.2.1.78): This mannan-chain
degrading enzyme was assayed by the method of Shimahara
et al. (1975). Assay for -1, 4-mannanase was carried out in
a medium containing 0.5 per cent (w/v) locust bean gum
and 10 mM phosphate buffer (pH 7.0). The reaction was
started by adding standardized amount of the enzyme extract,
resulting in the hydrolysis of the locust bean gum and
liberation of reducing sugar, which was measured by the
Nelson-Somogyi’s method after 30 minutes. A blank was
run simultaneously and the enzyme’s activity was calculated
in units. One unit of -1, 4-mannanase was defined as the
amount of enzyme that liberated 1.0 nmol of D-mannose
per minute per mg protein.
Soluble protein: The soluble protein in the enzyme extract
was precipitated by 20 per cent trichloro acetic acid (TCA),
centrifuged and residue dissolved in 0.1N sodium hydroxide
(NaOH) solution and was determined by the method of
Lowry et al. (1951).
Statistical analysis: The experimental data was analyzed
by the application of CRD design using OPSTAT software
available on CCSHAU home page (Sheoran).
RESULTS AND DISCUSSION
Screening of the guar genotypes for galactomannan
content: Seeds of 17 guar genotypes were used for
galactomannan content estimation. A wide range of
galactomannan content (16.82 to 36.68 per cent) was observed
Table 1: Galactomannan content in seeds of different guar
genotypes
S. No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
Group 1: less than 20%
Genotype
IC 310630
RGC 1055
IC 402296
C. serrata
Group 2: between 20-25%
RGC 1033
HVG 2-40
IC 421831
Group 3: between 25-30%
CAZG-97-1
Group 4: between 30-35%
HG 2-20
HG 563
RGC 936
HG 884
GAUG 0013
Group 5: more than 35%
GAUG 825
IC 402293
GAUG 815
HG 3-2
Content (%)
16.82
17.58
17.87
18.32
21.91
22.09
23.85
27.32
30.40
30.55
31.54
31.90
33.26
35.22
35.51
36.19
36.68
Volume 40 Issue 3 (June 2017)
in the seeds of 17 guar genotypes and genotypes were grouped
into five categories (Table 1).
Four genotypes were found to have very low (16.82
to 18.32 %), three genotypes have low (21.91 to 23.85 %), one
genotype with moderate (27.32 %), five genotypes with high
(30.04 to 33.26 %) and four genotypes with very high (35.22
to 36.68 %) galactomannan content.
Key enzymes of galactomannan metabolism in the
promising genotypes: Accumulation of galactomannan is
related to the mechanism of galactomannan metabolism; and
for its improvement, it is necessary to understand its
metabolic pathway, which involves anabolic (galactosyltransferase) as well as catabolic enzymes (ß-Dmannosidase & ß-1, 4-mannanase). Therefore, the
developing pods of 17 diverse genotypes were used for
enzymatic studies.
-Galactosyltransferase activity: The enzyme galactosyltransferase is responsible for the transfer of
galactose residues to mannose on the growing
mannan backbone. In present investigation, the galactosyltransferase specific activity was found to increase
from 25 to 39 DAF on an average as well as in each genotype
and thereafter, it decreased at 46 DAF (Table 2). Similar
results were observed by RT-PCR analysis in root, leaves,
465
stem and cotyledons of guar (Naoumkina et al., 2007) in
which active galactosyltransferase transcripts were observed
only in seeds, with maximal accumulation at 35 DAF. Peak
activity of this enzyme was also exclusively observed in the
guar endosperm in the later stage of seed maturation around
25-35 DAF (Edwards et al., 1999; Dhugga et al., 2004). At
25 DAF, maximum activity was found in HG 3-2 (2458 units)
and minimum activity was found in IC 310630 (123 units).
Similar results were found at 32 and 39 DAF, where
maximum specific activity was found in HG 3-2 (3786 and
4832 units) and minimum was found in IC 310630 (131 and
792 units), respectively. At 46 DAF, minimum specific
activity was again found in IC 310630 (522 units) but
maximum activity was found in GAUG 815 (2274 units).
The mean specific activity of -galactosyltransferase was
found to be significantly and positively correlated with the
galactomannan content (r = 0.944, Table 5), i.e. specific
activity in diverse genotypes increased with the increase in
galactomannan content. The specific activity (mean value)
increased from 392 to 3166 units, with the increase in
galactomannan content from 16.82 to 36.68 per cent.
Moreover, the transfer specificity of the
galactosyltransferase to the elongating mannan chain is
critical in regulating the degree of Gal substitution of the
mannan backbone of the primary biosynthetic product and
Table 2: -Galactosyltransferase activity at different days after flowering (DAF) in diverse guar genotypes varying in galactomannan
content
Genotype
IC 310630
RGC1055
IC 402296
C. serrata
RGC 1033
HVG 2-40
IC 421831
CAZG-97-1
HG 2-20
HG 563
RGC 936
HG 884
GAUG-0013
GAUG-825
IC 402293
GAUG-815
HG 3-2
Mean
C. D. @ 5%
SE (m)
SE (d)
Galactomannan* (%)
16.82
17.58
17.87
18.32
21.91
22.09
23.85
27.32
30.40
30.55
31.54
31.90
33.26
35.22
35.57
36.19
36.68
27.35
-Galactosyltransferase activity** (DAF)
25
32
39
46
Mean
123
685
1016
962
1230
1618
1576
2084
1610
1224
1686
1937
1816
2220
2280
1945
2458
1557
131
965
1219
1820
1850
1945
1916
2165
1980
2468
2457
2353
2934
2842
3060
2935
3786
2166
792
1464
1612
2622
2644
2966
2522
2971
3688
3780
3602
3629
3831
3932
3654
4038
4832
3093
522
1089
740
768
1040
1062
1686
1464
2056
2120
1912
1829
1575
1630
1876
2274
1588
1484
392
1051
1147
1543
1691
1898
1925
2171
2334
2398
2414
2437
2539
2656
2717
2798
3166
A
27.06
9.58
13.55
* Galactomannan content estimated in seeds at maturity
** 1 unit = 1.0 nmol protons liberated per min/mg protein
A= Stages, B= Genotypes, AXB = Interaction
B
57.41
20.32
28.74
AXB
114.82
40.64
57.48
466
LEGUME RESEARCH - An International Journal
ultimately the M/G ratio (Edwards et al., 2002; Reid et al.,
2003). The close relation of galactosyltransferase gene
(GMGT) expression with seed development is also depicted
by the observation made in the seeds of coffee (Pre et al.,
2008). In coffee grain, the GMGT expression was first
detected in large green (LG) grain tissue (RQ¼ 0.06), which,
then rose to a maximum transcript level at yellow stage (YG;
RQ¼ 0.11) and after that, it reduced sharply at maturity (RG;
RQ¼ 0.02).
The change in Man/Gal ratio was also observed in
the endosperm of developing coffee beans at 11, 15, 21, 26,
31 and 37 weeks after flowering. At 11 weeks after flowering,
galactomannan was highly substituted with the M/G ratio
value from 2: 1 to 7: 1. But near maturity at 31 weeks after
flowering, it became less substituted with M/G ratio of 7: 1
to 40: 1 during coffee grain development (Redgwell et al.,
2003). This indicated that the activity of galactosyltransferase
was developmentally regulated and led to low level of
galactosylation (Pre et al., 2008) at maturity due to its low
activity.
ß-D-mannosidase activity: ß-Mannosidase (EC 3.2.1.25)
catalyzes the successive removal of D-mannose residues
from the nonreducing end of ß-1,4-linked mannooligosaccharides, produced by ß-mannanase (EC 3.2.1.78)
(Reese and Shibata, 1965). This enzyme is generally regarded
as being dependent upon prior activity of -mannanase to
provide its oligomeric mannan substrate.
The mean specific activity of ß-D-mannosidase
increased gradually from 25 to 39 DAF followed by sharp
decrease at 46 DAF (Table 3). This reduction in activity could
be the result of metabolic inhibitors that also inhibited
galactomannan breakdown (Reid and Meier, 1973). Similar
results were earlier reported by RT-PCR studies in guar via
elevated transcript levels for this gene at 30-35 DAF
(Naoumkina et al., 2007). The change in ß-D-mannosidase
activity was also observed in transformed Aspergillus oryzae
containg manB gene, when cultured on a medium containing
4-methylumbelliferyl -D-mannopyranoside as substrate
(Kanamasa et al., 2001). A gradual increase in the ß-Dmannosidase activity was observed with time that reached a
maximum of 662 units/l culture broth on day 9, and then
began to decrease slowly. McCleary and Matheson (1975)
also studied the changes in galactomannan structure in
lucrene, carob, honey locust, guar and soyabean seeds during
hydrolysis of mannan chain by ß-mannosidase and observed
increase in the activity of ß-mannosidase followed by its
decrease.
At different DAF, maximum and minimum specific
activities of ß-D-mannosidase were observed in different
genotypes. On the whole, no relation was observed in
Table 3: -D-mannosidase activity at different days after flowering (DAF) in diverse guar genotypes varying in galactomannan content
Genotype
Galactomannan* (%)
-D-mannosidase activity**(DAF)
25
IC 310630
RGC1055
IC 402296
C. serrata
RGC 1033
HVG 2-40
IC 421831
CAZG-97-1
HG 2-20
HG 563
RGC 936
HG 884
GAUG-0013
GAUG-825
IC 402293
GAUG-815
HG 3-2
Mean
C. D. @ 5%
SE (m)
SE (d)
16.82
17.58
17.87
18.32
21.91
22.09
23.85
27.32
30.40
30.55
31.54
31.90
33.26
35.22
35.57
36.19
36.68
27.35
113.63
81.31
90.06
59.29
54.58
43.55
64.11
41.76
45.56
44.70
78.23
78.58
52.97
50.94
60.79
92.28
88.43
67.10
32
125.47
108.35
110.47
67.36
91.41
56.33
123.97
114.68
53.32
89.90
100.97
117.86
131.21
75.03
175.10
102.18
94.27
102.23
A
1.43
0.51
0.72
* Galactomannan content estimated in seeds at maturity
**1 unit=1.0 nmol p-nitrophenyl--D-mannopyranoside hydrolyzed per min/mg protein
A= Stages, B= Genotypes, AXB = Interaction
39
142.66
182.82
116.78
125.38
118.54
195.42
150.71
130.96
70.52
158.55
118.73
141.24
167.82
203.37
33.09
160.17
133.53
138.25
B
3.04
1.08
1.52
46
55.42
46.81
66.40
33.03
20.15
23.60
44.93
21.64
27.94
20.47
22.51
22.63
13.81
25.73
23.80
40.42
41.46
32.40
Mean
109.29
104.83
95.93
71.26
71.17
79.72
95.93
77.26
49.34
78.41
80.11
90.08
91.45
88.77
73.20
98.76
89.42
AXB
6.07
2.15
3.04
Volume 40 Issue 3 (June 2017)
galactomannan content and specific activity of
ß-D-mannosidase. But, mean value of specific activity of
ß-D-mannosidase and galactomannan content were
negatively correlated (r = -0.166, Table 5) with each
other.
 - 1, 4-Mannanase activity: - 1, 4-Mannanase randomly
hydrolyzes mannosidic linkages in mannans. The maximum
specific activity of ß-1,4-mannanase was observed at 25 DAF
and decreased continuously with advancement of days after
flowering up to 46 days (Table 4). Similar to ß-Dmannosidase activity, different genotypes differ for ß-1,4mannanase at various DAF. The active transcription of mannanases during early seed development was also
observed earlier in guar (Naoumkina et al., 2007), with high
steady-state levels. On the whole, no co-relation was
observed in galactomannan content and specific activity of
ß-1,4-mannanase and even no relevant literature is available
regarding the same. However, mean value of ß-1,4mannanase activity was positively correlated (r = 0.332,
Table 5) with galactomannan content.
467
Conclusively, it can be said that the enzymes ß1,4-mannanase and ß-D-mannosidase are potential
candidates for galactomannan depletion and can be used
for bio-fuel production with pr ior act ion of ß-1,
4-mannanase while -galactosyltransferase activity is
directly related to galactomannan content and plays a major
role in guar gum synthesis and this gene can be further used
for guar gum improvement programme via genetic
engineering.
ACKNOWLEDGEMENTS
Financial assistance from DST INSPIRE, New
Delhi as fellowship is thankfully acknowledged. The authors
are highly thankful to Dr. S. K. Pahuja from Forage Section
and Dr. G. S. Dahiya from Department of Genetics and
Plant Breeding, CCS Haryana Agricultural University,
Hisar for their co-operation and for providing all
the necessary facilities during investigation. The help of
NBPGR, Jodhpur is also acknowledged for supplying the
germplasm lines.
Table 4: -1,4-mannanase activity at different days after flowering (DAF) in diverse guar genotypes varying in galactomannan content
Genotype
IC 310630
RGC1055
IC 402296
C. serrata
RGC 1033
HVG 2-40
IC 421831
CAZG-97-1
HG 2-20
HG 563
RGC 936
HG 884
GAUG-0013
GAUG-825
IC 402293
GAUG-815
HG 3-2
Mean
Galactomannan* (%)
16.82
17.58
17.87
18.32
21.91
22.09
23.85
27.32
30.40
30.55
31.54
31.90
33.26
35.22
35.57
36.19
36.68
27.35
-1, 4-Mannanase activity** (DAF)
25
32
105.02
41.86
46.84
37.75
70.51
162.36
58.11
91.86
115.61
185.69
99.06
169.02
126.12
247.02
49.65
75.33
47.48
101.72
40.76
26.32
42.14
29.87
26.23
21.82
37.14
80.28
67.58
92.72
59.26
49.66
43.68
35.09
23.85
18.50
42.91
43.40
A
2.58
0.92
1.29
C. D. @ 5%
SE (m)
SE (d)
39
40.75
7.40
11.81
18.00
12.65
19.03
29.95
16.70
11.30
16.20
18.39
20.81
20.66
28.33
7.04
27.43
16.04
18.97
46
Mean
4.64
6.69
10.91
14.40
8.15
7.34
7.60
12.37
10.63
8.59
6.40
3.83
7.05
16.03
15.90
9.07
5.19
9.11
47.79
20.57
27.93
25.00
29.38
52.64
33.20
50.30
51.28
75.80
45.78
60.83
49.38
81.62
24.11
32.58
27.90
B
5.48
1.94
2.74
AXB
10.96
3.88
5.49
* Galactomannan content estimated in seeds at maturity
** 1 unit = 1.0 nmol D-mannose liberated per min/mg protein
A= Stages, B= Genotypes, AXB = Interaction
Table 5: Correlation between galactomannan content and corresponding metabolic enzymes’ specific activity
Galactomannan
Galactomannan
Galactosyltransferase
Mannosidase
Mannanase
1.000
0.944**
-0.166 NS
0.332NS
Galactosyltransferase
**
0.944
1.000
-0.333 NS
0.259NS
Mannosidase
NS
-0.166
-0.333 NS
1.000
-0.114NS
Mannanase
0.332NS
0.259NS
-0.114NS
1.000
468
LEGUME RESEARCH - An International Journal
REFERENCES
Das, B., Arora, S. K. and Luthra, Y. P. (1977). A rapid method for determination of gum in guar (Cyamopsis tetragonoloba
(L.) Taub.). In: Proc. First ICAR Guar Res. Workshop. CAZRI, Jodhpur. pp: 117-123.
Das, D., Ara, T., Dutta, S. and Mukherjee, A. (2011). New water resistant biomaterial biocide film based on guar gum.
Bioresour. Tech. 102: 5878-5883.
Deng, C. and Chen, R. R. (2004). A pH-sensitive assay for galactosyltransferase. Anal. Biochem. 330: 219-226.
Dhawan, S. and Kaur, J. (2007). Microbial mannanases: an overview of production and applications. Crit. Rev. Biotechnol.
27: 197-216.
Dhugga, K. S., Barreiro, R., Whitten, B., Stecca, K., Hazebroek, J., Randhawa, G. S., Dolan, M., Kinney, A. J., Tomes, D.,
Nichols, S. and Anderson, P. (2004). Guar seed ß-mannan synthase is a member of the cellulose synthase super
gene family. Sci. 303:363-366.
Dubois, M., Gilles, K. A., Hamilton, J. J., Rubress, P. A. and Smith, F. (1956). Colorimetric method for determination of
sugar and related substances. Anal. Chem. 28:350-356.
Dwivedi, N. K., Bhandari, D. C., Dubas, B. S., Agrawal, R. C., Mandal, S. and Rana, R. S. (1995). Catalogue on clusterbean
(Cyamopsis tetragonoloba (L.) Taub.) germplasm, part III NBPGR, New Delhi.
Edwards, M. E., Dickson, C. A., Chengappa, S., Sidebottom, C., Gidley, M. J. and Reid, J. S. G. (1999). Molecular
characterisation of a membrane bound galactosyltransferase of plant cell wall matrix polysaccharide biosynthesis.
Plant. J. 19:691-697.
Edwards, M. E., Marshall, E., Gidley, M. J. and Reid, J. S. G. (2002). Transfer Specificity of Detergent-Solubilized Fenugreek
Galactomannan Galactosyltransferase. Plant Physiol. 129: 1391-1397.
Edwards, M. E., Scott, C., Gidley, M. J. and Reid, J. S. G. (1992). Control of mannose/galactose ratio during galactomannan
formation in developing legume seeds. Planta. 187: 67-74.
Joshi, U. N. (2004). Advances in Chemistry, Biochemistry and Industrial Utilization of Guar Seed. In: Guar. (J.V. Singh
and B.S. Dahiya eds.), Indian Society of Forage Research, Hisar and Agricultural and Processed Food Products
Export Development Authority (APEDA), New Delhi, India. pp: 197-229.
Kanamasa, S., Takada, G., Kawaguchi, T., Sumitani, J. and Arai, M. (2001). Overexpression and purification of Aspergillus
aculeatus ß-mannosidase and analysis of the integrated gene in Aspergillus oryzae. J. Biosci. Bioengg.
92: 131-137.
Kestwal, R. M. and Bhide, S. V. (2005). Purification and partial characterization of -D-mannosidase from Erythrina
indica seeds. Ind. J. Biochem. Biophy. 42: 156-160.
Lee, B. T. and Matheson, N. K. (1984). Phosphomannoisomerase and phosphoglucoisomerase in seeds of Cassia coluteoides
and some other legumes that synthesize galactomannan. Phytochem. 23:983-987.
Lowry, D. H., Rosebrough, N. J., Farr, A. L. and Randall, J. L. (1951). Protein measurement with Folin-phenol reagent.
Biol. Chem. 193:265-275.
McCleary, B.V. and Matheson, N. K. (1975). Galactomannan structure and ß-mannanase and ß-mannosidase activity in
germinating legume seeds. Phytochem. 14: 1187-1194.
Moreira, L. R. and Filho, E. X. (2008). An overview of mannan structure and mannan-degrading enzyme systems. Appl.
Microbiol. Biotechnol. 79: 165-178.
Naoumkina, M., Torres-Jerez, I., Allen, S., He, J., Zhao, P. X., Dixon, R. A. and May, G. D. (2007). Analysis of cDNA
libraries from developing seeds of guar [Cyamopsis tetragonoloba (L.) Taub.]. BMC Plant Biol. 7: 62-73.
Nelson, N. (1944). A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol.
Chem.153:375-381.
Pauly, M. and Keegstra, K. (2008) Cell-wall carbohydrates and their modification as a resource for biofuels. Plant J.
54: 559-568.
Pre, M., Caillet, V., Sobilo, J. and McCarthy, J. (2008). Characterization and expression analysis of genes directing
galactomannan synthesis in coffee. Ann. Bot. 102: 207-220.
Punia, A., Arora, P., Yadav, R. and Chaudhary, A. (2009). Optimization and inference of PCR conditions for genetic
variability studies of commercially important clusterbean varieties by RAPD analysis. Asia Pac. J. Mol. Biol.
Biotechnol. 17: 33-38.
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Redgwell, R. J., Curti, D., Rogers, J., Nicolas, P. and Fischer, M. (2003). Changes to the galactose / mannose ratio in
galactomannans during coffee bean (Coffea arabica L.) development: implications for in vivo modification of
galactomannan synthesis. Planta. 217: 316-326.
Reese, E. T. and Shibata, Y. (1965). ß-mannanases of fungi. Can. J. Microbiol. 11: 167-183.
Reid, J. S. G. and Meier, H. (1973). Enzymic activities and galactomannan mobilisation in germinating seeds of fenugreek
(Trigonella foenum-graecum L. Leguminosae): Secretion of -galactosidase and -mannosidase by the aleurone
layer. Planta. 112: 301-308.
Reid, J. S. G., Edwards, M. E., Dickson, C. A., Scott, C. and Gidley, M. J. (2003). Tobacco transgenic lines that express
fenugreek galactomannan galactosyltransferase constitutively have structurally altered galactomannans in their
seed endosperm cell walls. Plant Physiol. 131:1487-1495.
Sheoran, O.P. Statistical Analysis Software at the site of CCSHAU. www.hau.ernet.in.
Shimahara, H., Suzuki, H., Sugiyama, N. and Nisizawa, K. (1975). Partial purification of beta-mannanase from the konjac
tubers and their substrate specificity in relation to the structure of konjac glucomannan. Agric. Biol. Chem.
39:301-312.
Somogyi, N. (1945). A new reagent for determination of sugars. J.Biol.Chem. 160:61-69.
Sturm, A. and Tang, G. Q. (1999). The sucrose-cleaving enzymes of plants are crucial for development, growth and carbon
partitioning. Trends Plant Sci. 4: 401-407.