Structure And Expression Analysis of Sucrose Synthase Gene

Across Plant Taxa: An In Silico Approach

Project report submitted in the partial fulfilment of the requirement for the
degree of M.Sc. in Botany at University of Calcutta

Project report submitted by

SOUMITA MITRA

ROLL NO: 221/BOT/191053

REGISTRATION NO: 136-1221-0245-16

POST GRADUATE DEPARTMENT OF BOTANY

BETHUNE COLLEGE

UNIVERSITY OF CALCUTTA
2021
Structure And Expression Analysis of Sucrose Synthase Gene
Across Plant Taxa: An In Silico Approach

Project report submitted in the partial fulfilment of the requirement for the
degree of M.Sc in Botany at University of Calcutta

Project report submitted by

SOUMITA MITRA

ROLL NO: 221/BOT/191053

REGISTRATION NO: 136-1221-0245-16

POST GRADUATE DEPARTMENT OF BOTANY

BETHUNE COLLEGE

UNIVERSITY OF CALCUTTA
2021

SUPERVISED BY –

DR. SRITAMA MUKHERJEE

ASSISTANT PROFESSOR

BETHUNE COLLEGE

KOLKATA

WEST BENGAL
 ACKNOWLEDGEMENT
The project work entitled “Structure And Expression Analysis of Sucrose Synthase Gene Across Plant
Taxa: An In Silico Approach” was suggested by Dr. Sritama Mukherjee, Assistant Professor, Department
of Botany, Bethune College, Kolkata. I express my heartfelt gratitude for their endless inspiration and
incredible co-operation during the dissertation work.
I would like to express my deepest gratitude to the Dr. Ashok Kumar Das, Associate Professor,
Head of the department of Botany and all my teachers for extending all the necessary help for the
completion of my project work.
I would have not finished this project without constant co-operation of my dear classmates and my family,
especially my parents who were always there for me whenever I needed them, the encouragement they gave me
to keep me going, and their love empowering me, which never fails at any point of time. I am also grateful to our
respected Principal, Prof. Krishna Roy for her endless support to carry out the project
work

.............................................................. .....
(Soumita Mitra)
 1

TABLE OF CONTENTS

1. Abstract: ............................................................................................……….…………………………. 2
2. Introduction: ...........................................................................................…………………………..……3
2.1 Regulation of Sucrose Synthase.: .......................................… ….………………………………….4
2.2. Significance of Sucrose Synthase: ..................................................………………………………..5

3. Review of literature: ..................................................................................………………..……………6

3.1. Role of SuSy in water stress: ..........................………………………………………………………………………………6

3.2. Role of Susy in low oxygen: .............................................…………………………………………………………….7.
3.3. Role of SuSy in absence of Oxygen: …………………………………………………………………………………………… 8.
3.4 Role of SuSy in cold: …………………………………………………………………………………………………………………….8
3.5. Role of SuSy in high temperature: ……………………………………………………………………………………………….9
3.6. Role of SuSy in Salinity: ……………………………………………………………………………………………………………….9
. 3.7 Role of SuSy in different Sugar level: …………………………………………………………………………………………..9.

4. Aim and objectives: ………………………………………………………………………………………………………………..………11

5. Materials and method: …………………………………………………………………………………………………………..……… 12
5.1. Derivation of sequence data: ……………………………………………………………………………………………....……...12
5.2. Building of phylogenetic tree: ………………………………………………………………………………………………...…...13
5.3. Searching of evolutionary conserved stretches: …………………………………………………………………...……..13
5.4. Prediction of gene structure and protein domain structure: ………………………………………………...………14
5.5. Microarray based expression analyses: ………………………………………………………………………………....……..15

6. Results: ……………………………………………………………………………………………………………………………………...……16
6.1. Sequence data mining of SuSy sequences: ……………………………………………………………………………....… 16
6.2. Phylogenetic analyses of SuSy sequences: ……………………………………………………………………………....…..26
6.2.1. Preference for gene/protein for phylogenetic tree construction: …………………………………...........…28
6.2.2. Comparative study of nucleotide phylogenetic tree with APG phylogeny: …………………...........……28
6.3. Position analysis of intron/exon structure of SuSy gene: …………………………………………………....………..30
6.4 Protein domain analysis of SuSy: ………………………………………………………………………………………....………..30
6.5 Identification of conserved amino acid stretch: ..................................................................................34
6.6. Microarray based expression profiling of SuSy in model plants: ........................................................36

7. Discussion: ………………………………………………………………………………………………………………..........................39

8. Conclusion and Future prospective: ......................................................................................................40

9. References: ............................................................................................................................................41
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1. ABSTRACT
Sucrose synthase (SuSy) performs a vital role in sucrose metabolism and plant development and response to
abiotic stress. The SuSy gene family has been recognized in many plants, but the SuSy gene has not been clearly
studied in the whole plant kingdom. Here,114 SuSy genes were identified and extensively analysed using
bioinformatics tools. The analysed parameters included arrangements of species in APGIV classification, gene
structures, protein domain and phylogenetic as well as genes expression profiles. Phylogenetic analysis revealed
that the 114 members could be allocated into two clades and eight subclades. The gene phylogenetic tree shows
huge diversification of angiosperm SuSy gene and curious relationship with lower group of species where as in
protein phylogenetic tree the species are highly organized and lower group are branches off before divergence of
monocots and dicots. The exon/intron structure of the SuSy gene indicates that these genes contain conserved
intron variants, which is the result of intron loss under different selection pressures during evolution. The
expression patterns of the SuSy isoforms differed from each other in various tissues and developmental stage.
Our results provide an evolutionary and an empirical molecular genetic foundation of the SuSy gene family in
entire plant kingdom including its role in response to abiotic stress condition in different experimental species
will be beneficial for further investigations of SuSy gene functions in the processes of crop productivity and its
improvement.

Keywords: Sucrose synthase; Phylogeny; abiotic stress; conserved; gene expression.

Abbreviations

SuSy Sucrose synthase

Inv Invertase
NDP Nucleoside diphosphate
UDP Uridine diphosphate
ADP Adenosine diphosphate
CDS Coding sequence
G Glucose
 3

2. INTRODUCTION
In plants, Sucrose synthase is a key sucrose metabolizing enzyme. It is mainly produced by photosynthesis
through kelvin cycle in the source tissues such as mature leaves and transported to sink tissues where it serves as
a carbon and energy source for the various metabolic pathways. In non- photosynthetic tissue, the transported
sucrose serves as a raw material for various metabolic pathways, providing energy as well as carbon skeletons for
the production of organic matter such as amino acid, nucleotides and structural carbohydrates.

Fig.1 SuSy converts sucrose

into NDP-Glucose and fructose.

Inside sink cell sucrose is

metabolized by cleaving into
NDP-G (UDP -G commonly; and
ADP-G rarely) and fructose by
Sucrose Synthase (SuSy) enzyme.
The sucrose synthase is the only
sucrose metabolic enzyme that can catalyze both the synthesis of sucrose from fructose and UDP G and cleavage
of sucrose in presence of UDP to fructose and UDP-G (Fig.1).

Sucrose Synthase

Sucrose synthase (Susy) is a glycosyltransferase enzyme (EC NO 2.4.1.13), belongs to CAZy database and
Glycosyl transferase family 4. It may exist as a homotetramer as well as a heterotetramer. Single SuSy has two
domains: N terminal and C terminal domain. The N terminal domain participate in cellular targeting and the C
terminal domain of GT-B consist of 500 amino acids and is responsible for enzymes’ glycosyltransferase activity
(Zheng et al., 2011). Each Susy enzyme has two phosphorylation sites. The 1st one is serine phosphorylation site
at the position 11 to 15 have a pivotal role in membrane association and 2nd one is also serine at position around
170 have a role in protein degradation (Hardin et al., 2003). The activity of sucrose synthase is best at alkaline
pH and its degradation is mediated by acidic pH.

IUBMB Enzyme Nomenclature

SUCROSE SYNTHASE: EC.2.4.1.13

EC: Enzyme commission
EC2: Transferase (major class)
EC2.4: Glycosyl transferase(subclass)
EC2.4.1: Hexosyltransferase (sub- sub class)
EC2.4.1.13: Sucrose Synthase (serial no)
Accepted name: sucrose synthase

Reaction:  NDP-glucose + D-fructose = NDP + sucrose

Other name(s):  UDP-glucose-fructose glucosyltransferase; sucrose synthetase; sucrose-UDP

glucosyltransferase; sucrose-uridine diphosphate glucosyltransferase; uridine
diphosphoglucose-fructose glucosyltransferase

Systematic name: NDP-glucose: D-fructose 2-α-D-glucosyltransferase

Comments: Although UDP is generally considered to be the preferred nucleoside diphosphate for sucrose
synthase., ADP serves as an effective acceptor molecule to produce ADP-glucose. Sucrose
 4

synthase has a dual role in producing both UDP-glucose (necessary for cell wall and glycoprotein
biosynthesis) and ADP-glucose (necessary for starch biosynthesis)

CAZy Database

CAZy is a database of Carbohydrate-Active enZymes (CAZymes) (http://www.cazy.org/). The database contains

classification and related information about enzymes involved in the synthesis, metabolism, and recognition of
complex carbohydrates i.e. disaccharides, oligosaccharides, polysaccharides, and glycoconjugates. Enzyme
classification is a sequence-based classification that synthesizes or degrades saccharides, originated with the
seminal grouping of glycoside hydrolases (Bernard et al., 2010). CAZy contains about 300 protein families with
the following enzyme activity categories: 1. Glycosyltransferase (GT) families , 2. Glycoside Hydrolase (GH)
families, 3. Polysaccharide Lyase (PL) families, 4. Carbohydrate Esterase (CE) families, 5. Auxiliary Activity
(AA) families. 

Significance
 It reflects the structural characteristics of these enzymes better than their specificity for a single substrate.
 It helps to determine the evolutionary relationships between these enzymes.
 Provides a practical framework for understanding mechanical properties.

2.1 Regulation of Sucrose Synthase (SuSy)

At the biochemical level, nodule Sucrose synthase (SuSy) is known to be a homotetrameric enzyme composed
of four subunits with a molecular weight of approximately 92 kDa (Gonzalez et al.,1999)

 By the control of the steady state level of enzyme and the concentration of hexose sugars, which inhibit the
cleavage reaction in vitro.
 Disulfides may reversibly inhibit SuSy in the cleavage direction.
 The activity of Sucrose synthase (SuSy) can be regulated by the free heme in the nodules.
 Sucrose Synthase activity could be regulated in vivo by phosphorylation, which led to a to a reversible
modification of the affinity for sucrose.

At the molecular level, the expression of Sucrose synthase (SuSy) gene is regulated by carbohydrate levels.
At the physiological level, the regulation is controlled by

 Sucrose concentration i.e., there is a linear, inverse correlation between SuSy activity and sucrose
concentration.
 SuSy activity is known to be triggered by a hypoxic environment.
 The activity of SuSy can be adjusted by water potential.
 The Plant hormone, abscisic acid (ABA), indirectly control sucrose synthase activity in root nodule.
 5

2.2 Significance of Sucrose Synthase (SuSy)

Recent studies have found that sucrose is a signal that regulate various processes in plants by regulating the
expression level of genes encoding enzymes. Sucrose synthase is present in all plant organs, but it is more
common in receiving organs, where it functions primarily as a sucrose degrading enzyme (Avigad et al., 1982). It
is assumed that SuSy plays an important role in loading and unloading phloem (Nolte & Koch., 1993) and in
determining sink strength (Zrenner et al., 1995). The positive correlation between SuSy activity and sink size has
been confirmed in many plant storage organs, including potato tuber (Zrenner et al., 1995; Baroja-Fernández et 
al., 2009), pea (Craig et al., 1999), maize seed (Chourey et al., 1998; Li et al., 2013) and cotton fibres (Ruan et
al., 2003).
In addition, Sucrose synthase has several other biological functions. It is assumed that cytosolic form of SuSy is
believed to supply the products of sucrose decomposition for energy metabolism and starch synthesis (Baroja-
Fernández et al., 2009), whereas membrane-bound form may directly provide the precursor UDPG for cellulose
or callose synthesis (Amor et al., 1995; Haigler et al., 2001; Ruan et al., 2003). It plays an important role in cell
division (Gaudin et al., 2000), seed germination (Ohto et al., 2001), vascular tissue differentiation (Stein and
Granot, 2019), response to abiotic stresses (Harada et al., 2004, 2005) and the development of shoot apical
meristem (Hanggi and Fleming, 2001). The study of sucrose metabolism is the basis for understanding many
aspects of plant physiology.
Earlier studies have shown that Sucrose synthase is encoded by many small gene families with different
overlapping expression patterns. Identifying the gene encoding Sucrose synthase is the first step in understanding
its physiological role and participating in various metabolic processes. To date, the research on the structure and
function of SuSy gene mainly focuses on model plants and important crops such as Arabidopsis (Baud et al.,
2004), Oryza (Hirose et al., 2008), Solanum lycopersicum (Goren et al., 2017), Zea mays (Barratt et al., 2001),
Pisum sativum (Duncan et al., 2006) etc.

In this work we tried to clarify the specificity and conservation of Sucrose synthase enzymes across different
plant families. In order to further expand the knowledge of the Sucrose synthase gene family in different plants,
we analysed the gene structure, phylogenetic relationship between different plants, their tissue- and
developmental-dependent expression patterns, and their potential roles in response to environmental stresses. Our
results provide precise information on Sucrose synthase gene among diverse taxa that will be supportive for
ongoing research on this important gene family predominantly in the field of evolutionary origin, biochemistry,
molecular biology and biotechnology.

3. REVIEW OF LITERATURE
 6

Water availability, nitrogen availability and temperature are the major factors controlling the distribution of
vegetation over the earth's surface. To cope with these environmental stresses, plants execute several
physiological and metabolic responses, including the expression of numerous stress-specific genes. Sucrose
Synthase enzyme participates in many stress responses of plants, such as low temperature, drought, anoxia, high
salinity and wounding all had been shown to induce the transcription of SuSy gene (Baud et al., 2004; Klotz &
Haagenson., 2008; Barrero-Sicilia et al., 2011). In the present review, we have searched for the effect of different
abiotic stresses on sucrose synthase gene expression in experimental organisms and its regulation in response to
stress conditions in a nutshell.

3.1 Role of SuSy in water stress

Water stress affect plant growth. Continuous monitoring of molecular changes and tissue response to water
stress exhibited the accumulation of sucrose synthase in response.
Biological nitrogen fixation (BNF) in angiosperms occurs in legumes in symbiosis with Rhizobia, in root
nodules are primarily dependent on the import and metabolism of Sucrose to provide the energy and C skeletons
for biological N fixation, the assimilation of ammonia, and the export of nitrogenous fixation products.
Moreover, nitrogen fixation in legume root nodules is dependent on their water status and it decreases steadily
throughout the water deficiency period (Fig.2) (Gonzalez et al.,1999)
The initial hydrolysis of sucrose is a key step of nitrogen fixation. The genes encoding sucrose synthase is
termed “Nodulin” which is unique and strongly expressed in root nodule (Gonzalez et al.,1999). Water
deficiency causes down regulation of Sucrose synthase gene and reduce metabolic capacity.

Fig.2 Scheme representing the

possible sequence of events leading
creased nitrogen fixation in nodules
subjected to water stress (Gonzalez et
al.,1999)

Under stressed conditions, Sucrose

synthase (SuSy) was the enzyme whose activity declined, coincident with BNF (Fig.3) The decline in activity
was directly related to SuSy protein levels, as determined by Western blot analysis (Gonzalez et al.,1999).

Fig.3 A) Relationship between nitrogen

fixation and Sucrose synthase (SuSy)
activity, both expressed as percentage of
control nodules, indicating that
increasing sucrose synthase activity is
linearly proportional to the biological
nitrogen fixation (BNF) in legume
nodules. B) Sucrose accumulation in
inversely proportional to Sucrose
synthase activity (Gonzalez et al.,1999).

The decrease in Sucrose synthase (SuSy) activity leads to an accumulation of sucrose causing depletion of
substrate for bacteroid respiration as a result, there is a transient accumulation of oxygen in the infected region,
 7

leading to a closure of the oxygen diffusion barrier which would cause the observed decline in biological
nitrogen fixation. It can be hypothesized that stress-mediated signal a decline of SuSy activity could render
nodules less capable of N fixation and loss of SuSy activity would ultimately cause the complete cessation of N
fixation.

3.2 Role of SuSy in low oxygen (hypoxia)

Hypoxia in soil produces major abiotic stress in plant, slowing down the efficiency of photosynthesis, the
production of singlet oxygen and reduce plant growth. The remarkable increase in Sucrose cleaving by SuSy
after the onset of hypoxia, has been well documented for a variety of plant species and organs including cereal
seeds. All available data indicate that SuSy is essential for survival at low O2 levels. (Wang et al., 2013).
Oxygen deprivation led to the expression of SuSy gene in the root of Cucumis sativus figures out its role in the
hypoxic period through following mechanisms: (Wang et al., 2013):
 The expression of Sucrose synthase can reduce the consumption of energy in roots under hypoxia stress by
inhibiting invertase activity which require two ATP molecules for each Sucrose molecule converted into
hexose monophosphates while SuSy only requires only one inorganic pyrophosphate (PPi) per molecule
(Guglielminetti et al., 1995; Rolletschek et al., 2002).
 The increased SuSy activity in the cytosol can supply sugars to glycolysis to supply energy for maintaining
the metabolism in plants under hypoxia conditions (Guglielminetti et al., 1995).
 In addition, the activity of membrane-bound SuSy can save energy by reducing polysaccharide biosynthesis
(Bologa et al., 2003).
 The increased SuSy activity in the cytosol could supply the necessary amounts of substrates to glycolysis
(UDPG is a substrate of glycolysis) under hypoxia conditions.

Another study has shown that the increase in SuSy activity has been correlated with increased cellulose
content (UDP-G is the precursor of cellulose biosynthesis) (Fig.4) and with changes in the cell wall structure of
wheat (Triticum aestivum) roots in response to hypoxic stress (Albrecht and Mustroph, 2003).

Fig.4 The precursor for cellulose biosynthesis

is UDP-Glucose which is produced from
sucrose clevage by Sucrose synthase (SuSy)
(Glucose Phosphate: Bernardheath.us).

This article investigated that the high amount of

carbon channelled to secondary cell wall
biosynthesis in relation to the increasing SuSy
activity in hypoxia. SuSy ranks high among
hypoxia-induced proteins because it catalyzes
the absorption of input sucrose from cells and
maintains the absorption intensity during
periods of low oxygen levels. Association of SuSy with the plasma membrane can promote Sucrose partitioning
into cellulose synthesis during low-O2 pressure. The potato tubers of the transgenic antisense line SuSy are more
sensitive to hypoxia (Biemelt et al., 1999).

3.3 Role of SuSy in the absence of oxygen (anoxia)

 8

Plant anaerobiosis has been widely studied, mainly in major crops with an emphasis on various aspects
including energy metabolism, maintenance of cell homeostasis, and the regulation of gene and protein
expression. SuSy activation is thought to be a metabolic adaptation to anaerobic conditions (Guglielminetti et al.,
1995; Bologa et al., 2003). SuSy is an anaerobic protein (ANPs) that are expressed in maize roots under
anaerobic conditions (Sachs et al., 1980; Sachs et al., 1996).

After continuously submerging the maize seedling root extracts for 2hr, the activity of Sucrose synthase
increased significantly in a Ca2 + dependent manner, indicating that it plays a role in anoxia tolerance. Sucrose is
the principal Carbon source and it is the main enzyme active in Sucrose breakdown in roots of maize seedlings
deprived of O2. SuSy play role in anoxic tolerance by providing an adequate sugar supply during the stress period.
A study of rice seedlings showed anoxic germination (Ricard et al., 1991) and SuSy activity has also been found
to be correlated with rice coleoptile length under submerged conditions (Fukuda et al., 2008). In anoxic period,
Invertase (INV) activities decreases and SuSy activities increases indicating that SuSy is the main enzyme that
breaks down sucrose during anoxia (Guglielminetti et al., 1995). A probable mechanism of SuSy action of maize
root tip under anoxic stress is shown in (Fig.5) (Subbaiah and Sachs, 2001).

Fig.5 Probable steps of Sucrose

metabolism and SuSy activity in
root tips of maize under anoxic
stress (Subbaiah and Sachs., 2001).

A study in aquatic angiosperm

Potamogeton, had shown elongation of stem of under anoxic stress was supported by active glycolytic pathway
(Sato et al., 2002; Harada et al., 2005). Remarkably, the rising in the SuSy activity in the cytosol is thought to
supply UDP-glucose and fructose (produced by sucrose cleavage) for glycolysis, and possibly starch synthesis
(Giegenberger and Stitt, 1993). In addition to this, increasing association of SuSy with plasma membrane is
considered to contribute to growth by supplying sugars for cellulose synthesis under anoxic stress (Amor et al.,
1995; Carlson and Chourey., 1996; Winter et al., 1997; Haigler et al., 2001). Oxygen deficiency has also been
shown to increase SuSy protein levels in Arabidopsis roots (Bieniawska et al., 2007) and leaves (Dejardin et al.,
1999)

3.4 Role of SuSy in cold stress

A sudden drop in plant growth temperature (called cold stress) will cause changes in plant metabolism, which
may vary from plant to plant. When wheat seedlings are exposed to a cold temperature 2-40°C, accumulation of
sucrose increases, causing an increase in SuSy activity as a response to cold acclimatization (Pontis et al., 1991).
The increase in SuSy transcription level indicates a positive effect in response to cold stress but the actual
mechanism is still unclear.
Another potential cold tolerant chickpea (Cicer arietinum) breeding line had shown high SuSy activity in
comparison to cold intolerant line. SuSy plays important role in increasing the vegetative growth as well as
increasing crop yield by modulating source and sink and by providing sugar nucleotides for their growth and
continuous starch synthesis during unfavourable low temperature activities (Chopra et al.,2000, Kaur et al.,
2005). Thus the increased sucrolytic activity in seeds of cold stress tolerant/resistant strain might result in
establishment

of greater sink strength in them and they also grow more under low temperature stress seed filling and crop
yield in chickpea. (Kaur et al.,2005)
 9

3.5 Role of SuSy in high temperature stress

Sucrose synthase may play a role under heat stress metabolism. Recent studies have shown that a SuSy- 3 allele
which is highly expressed during seed maturation can confer resistance to the chalky grain phenotype of brown
rice induced by heat stress (Takehara et al., 2018). Recently it is shown that Starch accumulation is reduced, in
Hordeum vulgare when endosperms develop at elevated temperatures (above 37℃) due to an increase of
endosperm sucrose level. Elevated temperature causes excessive down regulation of sucrose synthase found in
endosprem. When developing barley ears are exposed to thermal stress, there is an irreversible reduction in the
capacity of the endosperm to convert sucrose to starch due to decreasing activity of SuSy enzymes (MacLeod
and Duffus. ,1988).

Another potential heat-resistant SuSy was purified from a heat-resistant strain of Cicer arietinum (chickpea)
(ICC15614, ICCV. 92944) have the potential to maintain their reproductive function under stressed conditions
with continuous synthesis, degradation and transport of sucrose into sink tissue whereas two heat sensitive strain
(ICC10685, ICC5912) exhibit poor fertilization and poor pod set due to down regulation of SuSy (Kaushal et al.,
2013). Heat stress also downregulates the sucrose transporters in leaves, as observed in wheat (Qin et al., 2008),
which may disrupt the sucrose availability to anthers and pollen. SuSy exhibit optimum activity at 37°C and is
stable at temperatures up to 50°C (Verma et al., 2018).

3.6 Role of SuSy in salinity stress

Sucrose synthase may also play a role in the metabolism under salt stress. Salt tolerant callus and cell
suspension cultures of Brassica oleracea L. var. Botrytis was shown to accumulate Sucrose synthase after
exposure in medium supplemented with 85, 117, 270 mM of NaCl (Martin and Moreto., 2003) (Fig.6). The sugar
accumulation in cultures with increasing salinity is a common adaptive mechanism in response to salt stress.
Sugar accumulation could be related with high sucrose synthase activity which fulfills the requirement of hexose
uptake by the cultured cell under stress condition. SuSy by fulfilling the metabolic requirement set a criterion to
tolerate salt stress.

Fig.6 In vitro SuSy activity in

control and salt tolerant callus
and cell suspension cultures of
Brassica oleracea L. var. Botrytis
in different NaCl concentration
after 1 and 3 weeks of subculture
shown as vertical bar (Adapted
from Martin and Moreto., 2003).

3.7 Role of SuSy in different sugar levels

Under normal physiological conditions in mature leaves, the activity of SuSy and the expression of the gene
encoding the enzyme are both low or below the detection limit. During stress conditions, however, SuSy activity
and expression of the corresponding gene(s) were frequently stimulated in leaves and other organs.
 10

(Ciereszko et al., 2000; Dejardin et al., 1999). Specific sugar molecules can be used as signals to initiate
transduction pathways that control the expression of SuSy genes, and can play a role in the adaptation of the
whole plant to changes in sugar supply.
The examination of maize root tip had shown the expression of two SuSy genes (Sh1 and Sus1.) have reciprocal
responses in tissue carbohydrate status. The Sh1 mRNA was maximally expressed under conditions of limited
carbohydrate supply (~0.2% glucose). In contrast, Sus1 mRNA were low or non-detectable under sugar-depleted
conditions and peaked when sugar concentration was increased (2.0%) (Fig.7); (Karen Koch et al., 1992).

Fig.7 The column graph showing

reciprocal expression of Sh1 and Sus1
mRNA in Zea mays different glucose
concentration. (Koch et al., 1992)

SuSy performs various metabolic actions

in carbohydrate limiting condition. The greater activities of this enzyme meet the demand of nucleotide sugars
for cellulose biosynthesis (Hendrix et al., 1990; Maas et al., 1990). Another potentially important aspect of sugar
regulated gene expression may be that of adjustment in the respiratory rate in response to sugar availability. SuSy
activity could undoubtedly be key to the rate of carbon entry into the respiratory path (Huber and Akazawa.,
1986; Black et al., 1987). The differentially expressed sugar-responsive SuSy genes is the possible role of Sh1 in
low sugar tolerance recovery. Its involvement in surviva1 may lie in its capacity to preserve (Baysdorfer et al.,
1988) or possibly enhance (Huber and Akazawa., 1986) the capability for import or utilization of low levels of
sugars. SuSy may have an undefined role in phloem transport and control of phloem sucrose level (Claussen et
al., 1985).
 11

4. AIM AND OBJECTIVES

The aim of the present work is to establish a phylogenetic relationship of key Sucrose metabolizing enzyme i.e
Sucrose synthase (SuSy) among diverse group of plant taxa.
To fulfil the aim following objectives and plan of work has been followed:

1. Searching sequences of SuSy gene and protein among different organisms.

2. Characterization of SuSy gene across diverse taxa to establish evolutionary lineage.
3. Searching for conserved stretches and motifs.
4. Searching for gene architecture and conserved domains.
5. Expression profiling of SuSy gene in model plants.

PLAN OF WORK

To achieve the mentioned objectives the following work plan has been followed:

1. Alignment of the sequences (both gene and protein) in FASTA format as Text file on the basis of SuSy in
different organisms.
2. Analysis of the SuSy sequences (both gene and protein) and aligned them by using MEGAX software.
3. Generation of phylogenetic tree and evolutionary analysis of SuSy genes and proteins.
4. Determination of evolutionary conserved stretches of SuSy and analysis of motifs and stretches using
CONSURF software.
6. Construction and analysis of gene structure of selective species using GSDS server.
7. Construction and analysis of protein domain structure of selective species using INTERPRO software.
8. Analysis of expression profile (microarray based) of SuSy gene by GENEVESTIGATOR software.
 12

5. MATERIALS AND METHODS

5.1 Derivation of sequence data

The nucleotide and protein sequences were collected from NCBI (https://www.ncbi.nlm.nih.gov/)
Phytozome v12.1(Goodstein et. al., 2012). The gene for SuSy in Arabidopsis thaliana was searched in NCBI and
CDs was downloaded in fasta format. BLAST search was conducted with Arabidopsis thaliana SuSy sequence in
NCBI (X70990) and the sequences for SuSy across divergent plant taxa were documented. For a detailed study
of the gene across the plant kingdom, nucleotide and peptide sequences were derived from Phytozome v12.1.

 NCBI (NATIONAL CENTRE FOR BIOTECHNOLOGY INFORMATION)

NCBI serves as an international resource for the scientific research community - providing access to public
databases and software tools for analyzing biological data, as well as performing research in computational
biology. It is the largest sequence repository bank for gene and other biological data.

 BLAST (BASIC LOCAL ALIGNMENT SEARCH TOOL)

BLAST is a program of NCBI that finds regions of local similarity between sequences. BLAST can be used to
infer functional and evolutionary relationships between sequences. In our present work we used nucleotide
BLAST and protein BLAST program to find similarity between nucleotide sequence of DNA and amino acid
sequence of protein respectively.
The flow chart is shown in. (Fig.8)

Fig.8 Flow chart of Blast

programme.

 PHYTOZOME

Phytozome is a plant genome based database and provides a view of the evolutionary history of every plant
gene including algae, bryophyte and pteridophyte, at the level of sequence, gene structure, gene family and
genome organization providing
access to the sequences and
functional annotation. It is available
at phytozome portal.
(http:/www.phytozome.net/). The
flow chart is shown in (Fig.9)

Fig.9 Flow chart of

PHYTOZOME.
 13

5.2 Building of phylogenetic tree

All the derived sucrose synthase gene and protein sequences were imported into MEGA X (Kumar et al., 2018)
and multiple sequence alignments were performed using Clustal W tool.The alignment file was subjected to
create an unrooted phylogenetic tree based on the neighbor-joining method; after the bootstrap analysis for 1,00
replicates, the final tree(s) was generated. Trees were edited using iTOL (Interactive Tree of Life) software
(https://itol.embl.de/).

 MEGA X (MOLECULAR EVOLUTIONARY GENETIC ANALYSES)

MEGA is an integrated computer software for conducting automatic or manual multiple sequence alignment,
inferring phylogenetic trees to conduct statistical analysis of molecular evolution between different group of
organism. The detailed protocol is mentioned in Fig.10

Fig.10 Flow chart of

MEGA X.

5.3 Searching for evolutionary conserved stretch

Nine sequences (1 alga- Botryococcus braunii; 1 bryophyte- Physcomitrella patens; 1 pteridophyte-
Selaginella moellendorffii; 3 monocots- Zostera marina, Oryza sativa, Zea mays and 3 dicots- Vigna
unguiculata, Arabidopsis thaliana, Kalanchoe laxiflora) among the 114 obtained sequences were shortlisted as
representative organism from different domain of plant kingdom for further experiments.

5.3.1 Multiple sequence alignment of selected protein sequences

The .txt file of protein sequences of the shortlisted organisms were uploaded in Tcoffee (Notredame et al.,
2000) (http://tcoffee.crg.cat/) to obtain an easy alignment of sequences

 T-COFFEE

T-coffee is a consistency-based widely used bioinformatic tool for multiple sequence alignment that provides
a dramatic improvement in accuracy with a modest sacrifice in speed as compared to the most commonly used
alternatives. It provides us with a library of alignment information that can be used to guide the progressive
alignment. T coffee has come up with several variation like M-coffee, PSI-coffee, PRO-coffee, R-coffee etc. The
protocol is mentioned in Fig.11
 14

Fig.11 Flowchart showing

steps for obtaining MSA
file using T-COFFEE.

 CONSURF

Consurf is a web based bioinformating tool used for high throughput characterization of functional important
regions in proteins (Glaser et al., 2013). It automatically calculates evolutionary conservation scores and maps
them on protein structures via a user-friendly interface (Landu et al., 2005).
The conservation scores are made based on the evolutionary relations among the Protein and the scores are
subsequently translated into a discrete colouring scale that is used to project them on a known3D structure of one
of the homologous protein A short description of the methodology is provided in Fig.12 and a more detailed
description is available at (http://consurf.tau.ac.il/)

Fig.12 Flow chart of

CONSURF.

5.4 Prediction of gene structure and protein structure

The gene structure was predicted using the online Gene Structure Display Server 1 (Guo et al., 2015) and
protein structure was displayed by Interpro. The search engines are (http://gsds.gao-lab.org) and
(https://www.ebi.ac.uk/interpro).

 GSDS (GENE STRUCTURE DISPLAY SERVER)

GSDS is a web based bioinformatic tool for drawing gene structure schematic diagrams. GSDS provides
visualization of gene features such as composition and position of exons and introns for genes offers visual
 15

presentation for biologists to integrate annotation (Guo et al., 2015). To facilitate evolutionary analysis, a
user-specified phylogenetic tree can be added to the figure. Finally, the generated figure can be exported as either
vector graphic (in SVG and PDF format), or raster graphic (in PNG format). Methodology is provided in Fig.13

Fig.13 Flowchart of GSDS.

 INTERPRO

InterPro is an integrated software for protein families, domains and functional sites. Interpro is combined with
the effort of other database project such as PROSITE, PRINTS, Pfam and ProDom. InterPro provides internal
consistency checks and deeper coverage.The database is accessible for text- and sequence-based searches at (
http://www.ebi.ac.uk/interpro/ ) (Apweiler et al., 2001)

5.5 Microarray based expression analyses

Expression profile of Sucrose Synthase gene in Oryza sativa and Arabidopsis thaliana was obtained from
publicly available microarray data using Genevestigator.

 GENEVESTIGATOR

GENEVESTIGATOR is a database and user-friendly online tool for large-scale expression data analysis. The
database works as a “data warehouse” containing experimental and annotation data, pre-processed data, as well
as diverse tables for control of workflow and analysis (Zimmermann et al., 2004).. GENEVESTIGATOR has
come up with covering model organisms such as Arabidopsis thaliana, Oryza sativa, Physcomitrella patens,
Saccharomyces cerevisiae, Escherichia coli as the No. 1 gene expression tool in the field.
 16

6. RESULTS
6.1 Sequence data mining of Sucrose synthase gene across evolutionary diverse taxa of plant
kingdom
Sequence search with Arabidopsis SuSy gene from NCBI and PHYTOZOME reported 120 putative SuSy
genes. Short and/or low identity sequences were excluded and finally reported 114 species of SuSy gene/
proteins were reported across the plant kingdom including Algae: 3, Bryophyte: 3, Pteridophyte: 1,
Gymnosperm: 0 and Angiosperm: 107 (Basal angiosperm: 1, Monocot: 30, Dicot: 76). Table 1 shows the
distribution of SuSy gene and proteins across evolutionary diverse taxa, the angiosperms being classified
according to the APG IV system of classification (2016) (Chase et al., 2016). The cDNA size of these genes
varied from 2.0 to 3 kb in length and the putative polypeptides varied between 750 and 850 amino acids.
 17

Table 1: Distribution of Sucrose synthase gene in plant taxa. The angiosperms are distributed according
to APG IV system of classification (Angiosperm Phylogeny Group, 2016)

S ORGANISM FAMILY GENE NUCLEOTIDE PROTEIN FIGURE

L POSITION
.
N ACCESSION SIZE ACCESSION SIZE
O
ALGAE
Botryococcus Chlorophyceae scaffold_174_2: Bobra.174_2s 2592 - 863
1 braunii 99248.217314 0018.1

Chromochloris Chromochloridace Chr01: Cz01g28110.t 2253 - 884

2 zofingiensis ae 2842767.28480 1
28
Coccomyxa Coccomyxaceae scaffold_21: - 2655 - 750
3 subellipsoidea 674944.681514

BRYOPHYTE
Marchantia Marchantiateae scaffold_122: Mapoly0122s 2499 - 832
4 polymorpha L. 515825.523248 0057.1

Physcomitrella Funariaceae Chr10: Pp3c10_1133 2574 - 857

5 patens (Hedw.) 7610372.76155 0V3.1
Bruch & 98
Schimp.
Sphagnum Sphagnaceae Super_102: Sphfalx0102s 2538 - 845
6 fallax H. 154456.161243 0015.1
Klinggr.

PTERIDOPHYTE
Selaginella Selaginellaceae scaffold_40: - 2520 - 839
7 moellendorffii 543660.547339
Hieron.
BASAL ANGIOSPERM
AMBORELLALES
Amborella Amborellaceae Scaffold 00106: evm_27.mode 2433 - 810
8 trichopoda 82144.86042 l.AmTr_v1.0_
Baill. scaffold00106
.5

MESANGIOSPERMAE
MONOCOT
ALISMATALES
9 Spirodela Araceae pseudo2: Spipo2G0031 2409 - 802
polyrrhiza (L.) 2867518.28726 600
Scheild. 98

1 Zostera marina Zosteraceae scaffold_38: LFYR010010 2529 KMZ65710 842

0 L. 344477.347314 14

1 Potamogeton Potamogetonacea 23….2551 AB193516 2529 BAE06059 842

1 distinctus  e
A.Benn.
ARECALES
1 Cocos Arecaceae - - - KAG1366377 815
2 nucifera L.

1 Elaeis Arecaceae Chr13: XM_0109389 2451 XP_0109372 816

3 guineensis  24346695.2435 75 77
A.Chev. 5705
1 Phoenix Arecaceae Chr6: XM_0087848 2451 XP_0087830 816
4 dactylifera L. 4433343.44408 76 98
84

ASPARAGALES
1 Apostasia Orchidaceae - KZ451906 2451 PKA64018 816
5 shenzhenica 
Z.J.Liu &
L.J.Chen.
 18

1 Bletilla striata  Orchidaceae 258-2708 KU566805 2451 ANW09660 806

6 Rchb.f.

1 Dendrobium Orchidaceae 81…2408 KU169126 2328 ALS55906 775

7 officinale 
Kimura &
Migo.
1 Asparagus Asparagaceae Chr6: XM_0204145 2448 XP_0202701 815
8 officinalis L. 5825070.58320 98 87
81

1 Albuca Hyacinthaceae 44……2496 KT833617 3130 AMN16535 815

9 bracteata 
(Thunb.)
J.C.Manning &
Goldblatt.
LILIALES
2 Lilium davidii  Liliaceae 41….2581 KC261286 2541 AGW23638 846
0 Duch.

Tulipa Liliaceae 53….2470 X96938 2418 CAA65639 805

2 gesneriana L..
1

POALES
2 Ananas Bromeliaceae Chr1: XM_0202389 2460 XP_0200945 819
2 comosus (L.) 1677866.16844 42 31
Merr. 22

2 Bambusa Poaceae 44……2496 KJ525746 2536 AJA91786 816

2 emeiensis 
L.C.Chia &
H.L.Fung,
2 Brachypodium Poaceae Bd 1: Bradi1g46670 2427 - 808
3 distachyon (L.) 45376532.4538 .5
P.Beauv. 2751

2 Hordeum Poaceae - X65871 2667 CAZ64535 808

4 vulgare L..

2 Lolium Poaceae - BAE79815.1 2658 AB232656.1 885

5 perenne L..

2 Miscanthus Poaceae Chr 18: Misin18G061 2409 - 802

6 sinensis  13283238.1328 100.1
Andersson. 9731

2 Oryza sativa  Poaceae Chr3: Z15028 2627 CAA78747 806

7 L.. 16301277.1630
6143

2 Oryza sativa  Poaceae Chr6_4796545. HQ895720 2427 XP_0156430 808 -

8 subsp.  4804283 25.1
japonica 
Shig.Kato.
2 Oryza Poaceae Chr6_3424266. XM_0066558 2427 XP_0066558 808 -
9 brachyantha  3430446 14 77.1
A.Chev. &
Roehrich.
3 Panicum Poaceae Chr4K_731597 KF729953 2409 AIO11845 802
0 virgatum L.. 9.7323622

3 Panicum hallii  Poaceae Chr04 : Pahal.4G2945 2409 - 802

1 Vasey. 48403377..4841 00.1
0605
 19

3 Saccharum Poaceae - AY118266 7771 AAM68126 802

2 officinarum L..

3 Setaria italica  Poaceae Chr4: XM_0049645 2409 XP_0049646 802

3 Roem. & 2942954.29488 54 11
Schult. 06

3 Sorghum Poaceae Chr10: JQ062975 2409 AEX98033 802

4 bicolor (L.) 5859225.58672
Moench. 38
3 Triticum Poaceae ta_iwgsc_7ds_v Traes_7DS_5 2427 - 808
5 aestivum L.. 1_3893149: 29BAB150.2
245.5766
3 Zea mays L. Poaceae Chr9: X02382 2409 CAA26229 802
6 12838008.1284
3823
ZINGIBERALES
3 Musa Musaceae Chr10: JF682523 2451 AEO09338 816
7 acuminata  25290100.2529
Colla.. 5740
EUDICOTS
PROTEALES
3 Nelumbo Nelumbonaceae Chr unknown: XM_0102491 2421 XP_0102474 806
8 nucifera  72902.77856 56 58
Gaertn.
RANUNCULALES
3 Aquilegia Ranunculaceae Chr_07: Aqcoe7G438 241 - 803
9 coerulea  43163104.4316 900.1 2
E.James. 7409

SUPERROSIDS
SAXIFRAGALES
4 Kalanchoe Crassulaceae scaffold_236: Kalax.0236s0 2439 - 812
0 laxiflora Baker 69573.75273 011.1

ROSIDS
VITALES
4 Vitis riparia  Vitaceae Chr11: XM_0348434 2673 XP_0346993 806
1 Michx. 19375802.1938 33 24
0287

4 Vitis vinifera  Vitaceae Chr 11: GSVIVT0101 2421 - 806

2 L.. 490468.494415 5018001

FABIDS
CUCURBITALES
4 Cucumis Cucurbitaceae Chr1: NM_0013057 2421 NP_0012926 806
3 sativus L.. 26362508.2636 09 38
7667

4 Cucurbita Cucurbitaceae Chr unknown: XM_0231109 2421 XP_0229667 806

4 maxima Lam. 3559718.35643 86 54
94

4 Momordica Cucurbitaceae Chr unknown: XM_0222918 2430 XP_0221475 809

5 charantia L.. 472711.477931 91 83

FABALES
4 Abrus Fabaceae Chrun: XM_0274778 2418 XP_0273336 805
6 precatorius L.. 31682465.3168 00 01
7962

4 Arachis Fabaceae Arahy.01: JF346233 2421 AEF56625 806

7 hypogaea L.. 93589438.9359
3687
4 Cajanus cajan  Fabaceae Chr11_4370699 XM_0203732 2813 XP_0202288 805
8 (L.) Huth. 2.43712558 46 35
 20

4 Cicer Fabaceae Ca 1: XM_0045079 2421 XP_0045080 806

9 arietinum L. 24762031.2476 78 35
7519

5 Glycine max  Fabaceae Chr 13: NM_0012505 2418 NP_0012375 805

0 (L.) Merr. 21795249.2180 96 25
0589

5 Medicago Fabaceae Chr 4: AF049487 2760 O65026 805

1 sativa L.. 51648884.5165
3878

5 Mucuna Fabaceae - QJKJ0100846 2415 RDX79692 805

2 pruriens (L.) 2
DC.

5 Phaseolus Fabaceae 52…2459 AF315375 2738 AAN76498 805

3 vulgaris L..

5 Pisum sativum  Fabaceae - AJ012080 2652 AYD78246 806

4 L..

5 Spatholobus Fabaceae - QUWT01000 2418 TKY54375 806

5 suberectus  006
Dunn.

5 Vicia faba L.. Fabaceae 24……… X69773 2647 CAA49428 806

6 2454

5 Vigna Fabaceae Chr 03: XM_0280642 2800 XP_0279200 805

7 unguiculata  60835963.6084 05 06
(L.) Walp. 1355

FAGALES
5 Quercus suber  Fagaceae Chr unknown: XM_0240524 2421 XP_0239082 806
8 L.. 312932.320396 80 48

5 Alnus Betulaceae - X92378 2783 CAA63122 806

9 glutinosa (L.)
Gaertn.

6 Betula Betulaceae - KC204974 2424 AGV22112 807

0 luminifera 
H.J.P.Winkl.

ROSALES
6 Malus Rosaceae Chr9: XM_0083831 2513 XP_0083813 837
1 domestica  35765410.3577 71 93
subsp.  0146
cerasifera 
(Spach)
Likhonos
6 Prunus Rosaceae ChrG3: KJ493334 2502 AHZ90141 833
2 persica (L.) 1011213.10156
Batsch. 68
6 Rosa chinensis  Rosaceae Chr2: XM_0243247 2436 XP_0241805 806
3 Jacq. 8882049.88863 53 21
79
6 Ziziphus Rhamnaceae Chrunknown: XM_0160121 2427 XP_0158676 806
4 jujuba Mill. 61964.68135 99 85

6 Cannabis Cannabaceae Chr1: XM_0306328 2918 XP_0304886 806

5 sativa L. 68154480.6816 23 83
0992
6 Parasponia Cannabaceae - JXTB010000 2424 PON66060 807
6 andersonii  83
Planch.

MALPIGHIALES
 21

6 Populus Salicaceae Chr06: KF471021 2418 AHA41509 805

7 deltoides  25291305.2529
W.Bartram ex 5732
Marshall,

6 Populus Salicaceae Chr06: Potri.006G13 2412 - 803

8 trichocarpa  11283727.1129 6700.1
Torr. & A.Gray 0774
ex Hook.

6 Salix purpurea  Salicaceae Chr06: SapurV1A.02 2412 - 806

9 L. 12679932.1268 49s0050.1
7684

7 Jatropha Euphorbiaceae - NM_0013087 2541 NP_0012956 805

0 curcas L. 61 90

7 Hevea Euphorbiaceae Chr unknown: KJ000530 2715 XP_0216651 806

1 brasiliensis  93860.100285 96
(Willd. ex
A.Juss.)
Müll.Arg.
7 Manihot Euphorbiaceae Chr 03: XM_0217505 2421 XP_0216062 806
2 esculenta  3583270.35885 66 58
Crantz 87

7 Ricinus Euphorbiaceae Chr unknown: XM_0157174 2418 XP_0155729 805

3 communis L. 804916.811348 57 43

7 Linum Linaceae Scaffold 149: Lus10012454 2370 - 789

4 usitatissimum  259027.262359
L.

MALVIDS
BRASSICALES
7 Carica papaya  Caricaceae supercontig_82: evm.model.su 2418 - 805
5 L. 1095128.10986 percontig_82.
75 65
7 Arabidopsis Brassicaceae Chr5: X70990 2424 CAA50317 807
6 thaliana (L.) 7050217.70553
Heynh. 98

7 Arabidopsis Brassicaceae Scaffold 12632: Araha.12632s 2427 - 807

7 halleri (L.) 3569.7358 0001.1
O'Kane & Al-
Shehbaz.
7 Boechera Brassicaceae Scaffold 26527: Bostr.26527s 2427 - 808
8 stricta  838451.842632 0127.1
(Graham) Al-
Shehbaz.
7 Brassica rapa  Brassicaceae chrA10: XM_0091225 2418 XP_0091207 805
9 L. 14524194.1452 18 66
9197
8 Brassica Brassicaceae Chr 03: Bol025773 2421 - 806
0 oleracea var. 4429435.44333
capitata L. 66
8 Camelina Brassicaceae Chr20: XM_0104948 2427 XP_0104931 808
1 sativa Boiss. 11147593.1115 93 95
2741

8 Capsella Brassicaceae Chr unknown: XM_0062869 2427 XP_0062870 808

2 rubella Reut. 7062166.70672 97 59
53

8 Eutrema Brassicaceae scaffold_2: Thhalv10012 2421 - 806

3 salsugineum  7199478.72047 719m
(Pall.) Al- 09
Shehbaz &
Warwick.
 22

8 Raphanus Brassicaceae Chrunknown:22 XM_0185902 2418 XP_0184457 806

4 sativus L. 23863.2228758 28 30

MALVALES
8 Gossypium Malvaceae Chr9: NM_0013300 2418 NP_0013169 805
5 arboreum L. 57299396.5730 54 83
3842
8 Gossypium Malvaceae Chr13: Gorai.013G2 2448 - 812
6 raimondii  54230252.5423 22400.1
Ulbr. 4271

8 Herrania Malvaceae Chr un: XM_0214204 2809 XP_0212761 852

7 umbratica  1072858210733 69 44
R.E.Schult. 346

8 Durio Malvaceae Chr un: XM_0228942 2751 XP_0227500 806

8 zibethinus L. 7530270.75348 79 14
45

8 Theobroma Malvaceae Chr9: XM_0070124 2559 XM_0070124 852

9 cacao L. 3626470.36312 84 84
23

MYRTALES
9 Eucalyptus Myrtaceae Chr3: NM_0013027 2418 NP_0012896 805
0 grandis W.Hill. 8519212.85249 26 55
64

9 Syzygium Myrtaceae Chr un: XM_0305923 2418 XP_0304482 805

1 oleosum  1421975.14277 58 18
(F.Muell.) 46
B.Hyland.
SAPINDALES
9 Anacardium Anacardiaceae scaffold_13: Anaoc.0013s 2049 - 682
2 occidentale L. 6093024.60979 0864.1
88
9 Pistacia vera  Anacardiaceae Chr un: XM_0314275 2418 XP_0312833 806
3 L. 672273.677156 19 79

9 Dimocarpus Sapindaceae 97…2514 KP769776 2418 AJW82916 805

4 longan Lour.

9 Litchi Sapindaceae 242….2701 JQ773415 2460 AFP23359 819

5 chinensis Sonn.

9 Citrus sinensis  Rutaceae Chr4: XM_0064745 2418 XP_0064745 805

6 Pers. 4312952.43184 17 80
32

SUPERASTERIDS
CARYOPHYLLALES
9 Dianthus Caryophyllaceae 92….2497 AB543810 2406 BAJ10424 801
7 caryophyllus L.

9 Amaranthus Amaranthaceae Scaffold: AH014306- 2412 - 803

8 hypochondriacu 10729922.1073 RA
s L. 3538

9 Beta vulgaris  Chenopodiaceae 143…2560 AY457173 2418 AAR19769 805

9 L.

1 Chenopodium Chenopodiaceae Chr un: XM_0219010 2436 XP_0217567 811

0 quinoa Willd. 2251471.22596 33 25
0 26

1 Oxybasis Chenopodiaceae 76….2487 X82504 2412 CAA57881 803

0 rubra (L.)
1 S.Fuentes,
Uotila &
Borsch. 
 23

ASTERIDS
LAMIIDS
GENTIANALES
1 Coffea Rubiaceae Chr1e: AM087675 2436 CAJ32597 811
0 arabica L. 47993041.4799
2 9005
SOLANALES

1 Ipomoea Convolvulaceae Chr11: XM_0312358 2816 XP_0310916 805

0 triloba L. 5423480.54283 22 82
3 22

1 Capsicum Solanaceae Chr9: XM_0166848 2418 XP_0165403 805

0 annuum L. 5411184.54176 24 10
4 51
1 Solanum Solanaceae _ STU24087 2418 AAA97571 805
0 tuberosum L.
5
1 Nicotiana Solanaceae - KF977579 2954 AHL84158 805
0 tabacum L.
6

LAMIALES
1 Olea europaea  Oleaceae Chr9: XM_0230273 2793 XP_0228830 805
0 L. 15799572.1580 04 72
7 3995

1 Craterostigma Linderniaceae 106..2535 AJ131999 2430 CAB38021 809

0 plantagineum 
8 Hochst.

1 Sesamum Pedaliaceae ChrLG6_70283 XM_0110816 2448 XP_0110799 815

0 indicum L. 5.708443 60 62
9

1 Mimulus Phrymaceae Scaffold_10: Migut.J00882 2532 - 843

1 guttatus DC. 5309732.53151 .1
0 09
CAMPANULIDS
APIALES
1 Daucus carota  Apiaceae 139….2525 X75332 2427 CAA53081 808
1 L.
1
ASTERALES
1 Cichorium Asteraceae 89……2509 DQ400357 2707 ABD61653 806
1 intybus L.
2

1 Lactuca sativa  Asteraceae Chr un: XM_0238949 2418 XP_0237507 805

1 L. 620563.624724 95 63
3

1 Cynara Asteraceae ChrLG8: XM_0251234 2816 XP_0249792 808

1 carduncellus  531366.535410 71 39
4 Willd. ex Steud.

1 Helianthus Asteraceae Chr10: XM_0221349 2424 XP_0219906 807

1 annuus L. 181092907.181 10 02
5 096287

6.2 Phylogenetic analyses across green lineages

 SuSy gene
A phylogeny of SuSy genomic sequences from the 114 sequences of the diverse plant groups under study
suggests the existence of two clades comprising of 18 and 95 taxa respectively. Almost 50% of the unrooted
circular phylogenetic tree (Fig.14A.) shows the distribution of dicotyledon species and the rest 50% consist of
monocotyledons, algae, bryophytes, and pteridophytes respectively (Clade II). Out group is represented by
 24

Trifolium pratense, a typical dicot of family Fabaceae closely resembling other dicotyledon members of the main
clade. Clade I contain 50% Poaceae, and the rest are dicotyledonous plants with moss (Marchantia polymorpha)
in the middle. There are eight subclades under clade II consist mainly of dicots, few monocots (Musa acuminata,
Ananas comosus, Asparagus officinalis) bryophytes (Sphagnum fallax, Physcomitrella patens), algae
(Chromochloris zofingiensis) and one pteridophyte (Selaginella moellendorffii). Subclade I contain both dicots
and monocots belonging to family Brassicaceae, Linderniaceae, Cucurbitaceae, Rutaceae, Musaceae,
Orchidaceae and Aracaceae respectively with ambiguous phylogenetic position. Here surprisingly, Spirodela
polyrhiza, common fresh water free- floating duckweed from Araceae having thalloid fronds similar to algal
character shares a node with Momordica sp, a member of Cucurbitaceae showing close phylogenetic relationship
possibly due to their same genome size with dicots. Subclade II represent a diverse array of taxa with distant
evolutionary relationship, Botrycoccus braunii, an algal species and Mimulus guttatus a dicot share homology in
SuSy reinforcing their close phylogenetic relationship.
Another node demonstrating that two individuals, Selaginella moellendorffii and Kalanchoe laxiflora showed
common ancestry though they belong to two different classes, one from pteridophyte and the other from
succulent respectively. It is very striking that SuSy is highly conserved among two distantly related species and
the gene has not passed through much diversification to date. Physcomitrella patens and Brassica rapa have
occupied a distinct node and they have not clustered under any clade. This might be due to their sequence
variability from other members leads to recognizing them as an outgroup taxon. It is interesting to note that
Marchantia polymorpha being a thallophyte has separated from its close relatives, Sphagnum fallax and
Physcomitrella patens due to their sequence dissimilarity and evolutionary pattern. Sphagnum shares a node with
typical monocot, Lilium. It can be stated that being a true moss, Sphagnum has distinct physiology, anatomy,
morphology, and reproduction that distinguishes it from other bryophytes. It might lead to a conclusion that the
green plants have been the recipients of this enzyme from moss due to evolution.
The rest of the subclades from IV to VIII are entirely occupied by dicot from several families. There are 7 major
families dominating in this domain viz. Fabaceae, Euphorbiaceae, Myrtaceae and Solanaceae respectively from
left to right in the tree. The SuSy gene is highly conserved among the members of these families so they have
been clustered together supported by modest bootstrap values. All monocots (except Tulipa sp, Lilium sp.) are
clustered in a narrow clade whereas the dicots members of families Asteraceae (Helianthus annuus), Rosaceae
(Rosa odorata), Malvaceae (Theobroma cacao), Brassicaceae (Brassica rapa) etc. are positioned separately and
distributed throughout the tree suggesting that the gene have been greatly diversified may be due to their
sequence variability, mutation rate or changing environment. Monocots and dicots are separated having greater
bootstrap values.

 SuSy protein
An unrooted circular cladogram constructed from the SuSy sequences of 114 green plants suggested the
existence of two small and one large clade comprising of 2,3 and 112 species respectively (Fig.14B). The
cladogram showed distinct separation of monocots, dicots and other green lineages. Here the members of
Rosaceae family separately lay hold of clade I, possibly due to the presence of single nucleotide polymorphism
which leads to amino acid substitutions in their protein sequence and made their separation from other dicots
(Boris et al., 2014). Following them, the algal proponents of SuSy have occupied the clade II supporting the algal
origin of sucrose synthase enzyme. The rest of the clade is covered by more than 60% of the plant species from
dicot families Fabaceae, Brassicaceae, Malvaceae, Euphorbiaceae, Solanaceae,Asteraceae and Myrtaceae and
showing sequence homology suggesting the fact that the amino acid sequence didn’t change over a long period.
All monocots are clustered in a narrow monophyletic clade (subclade III). The subclade II has the bryophyte
sequence Physcomitrella patens at the base of monocot (subclade III) suggesting similar origin and evolution
with angiosperm. Nishiyama et al.,2003 proposed that P. patens are unique among bryophyte as it has 20%
similar genome frequency with Arabidopsis thalliana and Oryza sativa. Selaginella being a lycophyte share
common ancestry for SUSY protein with a thallophyte Marchantia suggesting a high degree of conservancy due
to slow mutation rate/ synonymous mutations and/ or invariant codons sites. Subclade IV to VI is entirely
occupied by dicots Malvaceae, Asteraceae, Oleaceae, Fabaceae, Euphorbiaceae, Solanaceae and Brassicaceae
from left to right over circular cladogram.
Hence, it can be concluded that the SUSY is ubiquitously distributed in the plant kingdom and the gene/protein is
highly conserved among different groups of green plants from algae to dicots.
25
 26

Fig.14 Circular cladograms showing evolutionary relationships among 114 taxa for SuSy gene/protein. Coloured
ranges is the colour code for different group of plants. Evolutionary analyses are conducted in MEGA10. The
evolutionary history was inferred using the Neighbour-joining method. The phylogenetic tree is an ITOL circular
visualization with bootstrap values inferred from 100 replicates which is equal to 1. (A) The tree was constructed
from nucleotide sequences. The optimal tree with sum of branch length=50.38655483 is shown. The evolutionary
distances were computed using the Maximum likelihood composite method. (B) The tree was constructed from
protein sequences. The optimal tree with sum of branch length=68.120034 is shown. The evolutionary distances
were computed using the Poisson model method.

6.2.1 Preference for a gene/protein for phylogenetic tree construction??

There are some differences in clading pattern of species between the amino acid sequence-based trees and the
nucleotide-based trees. Most of the differences are in the branching order of the clades and the position of
organism on circular cladogram. The phylogenetic tree based on nucleotide sequences are more informative as
the nucleotide sequences of a pair of homologous genes having a higher information content than the amino
acid sequences of the corresponding proteins, because mutations cause non-synonymous changes in the DNA
sequence but do not change the amino acid. The degeneracy nature of amino acids also supports the fact. Amino
acid sequences are always used to decipher the homology pattern between two closely related species. Since
DNA is consist of 4 characters: A, G, C, T and two unrelated DNA sequences are expected to have 25%
similarity. In contrast, the protein sequence is composed of 20 characters (AA), which increases the sensitivity of
comparison. It is also accepted that convergence of proteins is rare, meaning that high similarity between two
proteins always means homology (Hall et al., 2004). Proteins rarely mutate during evolution and due to their
conservation, searching for them reveals remote evolutionary relationships. It has also been observed that the
protein phylogenetic tree satisfies the APG phylogeny to a greater extent than the nucleotide one.
In the present work, phylogenetic trees were built from both the nucleotide and amino acid sequences to detect
homology and divergence (Fig.14A). In nucleotide tree, Trifolium pratens being a member of Fabaceae lies
separately as outgroup taxon from other member of Fabaceae, whereas in protein tree Trifolium pratens remain
in grouped with other Fabaceae members. The members of Rosaceae recognized as outgroup species and in
nucleotide tree the members of Rosaceae are scattered throughout the tree. In nucleotide tree algal ancestors of
SuSy gene are scattered and it has shared a node with angiosperms whereas in protein tree algal ancestors are in
clustered and occupied a separate node. In nucleotide tree Marchantia polymorpha has been positioned distantly
from other bryophyte members at the base of monocot whereas in protein tree bryophytes are remain clustered
just before the primitive angiosperm Amborella trichopoda. In nucleotide tree, Potamogeton distinctus, Albuca
bracteata, Asparagus officinale, Lilium davidii have been located so far from monocot clade and has been fallen
among dicot group. But in protein tree the mentioned species have been arranged in monocot clade side by side.
In nucleotide tree surprisingly Arabidopsis thaliana and Arabidopsis halleri two closely related species have
been positioned in distance whereas in protein phylogenetic tree they remain as sister species, sharing a common
node for SuSy protein. In nucleotide tree, Oryza sativa is sister to O. brachyantha and with both sister to O.
japonica, in contrast to protein tree where Oryza sativa is sister to O. japonica and with both sister to O.
brachyantha.

6.2.2 A comparative study of nucleotide phylogenetic tree with APG system of classification
(version IV)
The study of Phylogenetic trees fulfills many purposes and are an indispensable tool for explaining the
evolutionary trends and trait changes. Phylogenetic reconstruction is usually based on the comparison of
informative homologous characters, which can be morphological data, and/or molecular (sequence) data. Plant
molecular phylogenetic studies, based on representative plastid, mitochondrial, or nuclear genes, have greatly
improved our knowledge and understanding of the evolution of angiosperms. For example, with the development
of high-throughput sequencing technology, the Angiosperm Phylogenetic Group (APG) provides a widely
accepted classification of flowering plants and the development of molecular phylogeny has entered a new era
marked by the large-scale use of genomic data. In such a typical ‘phylogenomics’ approach we generated and
analysed a phylogentic tree from 114 species from 45 families across 24 orders of flowering plants and inferred
with phylogenies obtained with more traditional, sequence-based methods. We compared our nucleotide
phylogenetic tree to the current APG phylogeny (version IV) and summarized.
 27

Our phylogenetic tree strongly favours that the vast majority (~99.95%) of angiosperms that make up the clade
called Mesangiospermae. Our tree favours Trifolium pratense as a basal angiosperm instead of Amborella
trichopoda. APG classification shows that the monocots form a monophyletic group (clade) but our phylogenetic
tree have shown vast deviation from that and it has been grouped with eudicots such as Alismatales
(Potamogeton distinctus) and Liliales (Tulipa gensneriana) are not sister to the rest of other orders as they have
shared clade with eudicots. Arecales (Phonix dactylifera), Asparagales (Dendrobium officinale) and Zingiberales
are proved to be sister between themselves have gene conservancy partially similar to APG phylogeny. Our trees
favor Viatales (Vitis vinifera) to be sister of Proteales (Nelumbo nucifera) instead of Ranuncuales (Aquilegia
corulea) which should be actually and they are not early diverging eudicot too. Saxifragales (Kalanchoe
laxiflora) being a eudicot are sister of lycophyte (Selaginella moellendorfii). For orders within Fabids trees
support a clustered relationship of [{Malpigiales (Manihot esculenta), fabales (Arachis hypogaea)},
(Cucurbitales (Cucurbita maxima), Rosales (Cannabis sativa)}] except some members. For orders within
Malvids, Sapindales (Anacardium occidentale) and Myrtales (Syzygium oleosum) are clusterd but Malvales
(Theobroma cacao) and Brassicales (Brassica rapa) are highly diverged. Caryophyllales (Amaranthus) are
considered as sister of Poales (Triticum aestivum). There are subtle ambiguity including the position
of Musa (Zingiberales), Arabidopsis (Brassicales), Boechera (Brassicales), Coffea (Gentianales), Tulipa
(Liliaceae), Daucas (Apiales). Our phylogenetic tree is partially showing conservancy with APG phylogeny.

6.3 Intron-Exon position analysis of SuSy gene

SuSy gene size varied between 3kb-18kb (Fig.15A). All introns were started with 5′ G–T and ended with 3′ A–G
following the GT–AG rule for a splice site. The exon/intron structure of the SuSy gene varied among different
groups. The intron loss events may have resulted in the formation of larger exons in some species. The
comparison of length, position and number of the introns/exons of SuSy in a diverse group of plants represents
the evolutionary relationship among them and indicates the diversity of SuSy structure among them.
In all dicot species, the length and position of introns and exons are almost similar and the exon at 11th position
is generally larger than others (Table 2; Fig.15A). Physcomitrella patens followed almost the same trend as in
dicot but the 3’ exon at 12th position is quite large than other angiosperms. In Botryococcus braunii, the length,
position and number of both intron and exon are different from all other members. Botryococcus has a high no of
introns and the length of these introns varying from 50 to 600 bp with the exception of two at 11 th and 17th (~
larger than 1 kb). Oryza sativa and Zea mays, contain a large 5’ intron at 1st position comprising of 410 and 502
bp and characteristic tiny 14th 3’exon of size 47 and 29 bp respectively. In Zostera marina, the number of intron
is 1, both the UTR are very small and 3’exon is characteristically very large. UTRs are known to play crucial
roles in the post-transcriptional regulation of gene expression, including modulation of the transport of mRNAs
out of the nucleus and translation efficiency, subcellular localization and stability. The importance of UTRs in
regulating gene expression is underlined by the finding that mutations that alter the UTR can lead to serious
pathology (Mignone et al., 2003). As Zostera is a marine angiosperm, loss and gain of multiple genes in
comparison to other group plants, facilitate adaptation to marine life. These adaptations include morphological,
physiological and breeding pattern modifications along with the ability to tolerate high salt levels of marine
environments (Shivaraj et al., 2017). Arabidopsis contains a small 5’ UTR of 88 bp whereas the usual length in
other species is 1kb. Lycophytes bridge the gap between the green algal and bryophyte lineages, which branched
off earlier in plant evolution, and the fern, gymnosperm, and angiosperm lineages (together referred to as
euphyllophytes), which evolved after the lycophytes had branched off. Selaginella moellendorffii, a lycophyte
represents the earliest evolutionary branch of vascular plants for which whole genome information is available
(Gramzow et al., 2014). In Selaginella, significantly there were no noncoding nucleotides before the 5′-terminal
start codon hence it may be cited as a leaderless gene, however precise information about the UTR loss is still
missing. Selaginella possesses 14 introns similar to monocots.

6.4 Protein domain analyses of SuSy

Protein domains are the structural and functional units of proteins. It is now well established that proteins carry
out their functions primarily through their constituent domains. They can be gained by proteins to acquire new
 28

functions or lost via mutations resulting in loss of function. Domains, therefore the evolutionary units of protein
architecture are well suited to study divergent evolutions.
In addition to the nucleotide sequences, amino acid sequences were analysed from the same group of organisms.
SuSy protein size varied between 802-863 amino acids. There are two characteristic conserved domains of SuSy
proteins domains, N-terminal Sucrose synthase and C- terminal Glycosyl transferease-1. Sucrose synthase, which
catalyzes the reversible conversion of sucrose and uridine diphosphate (UDP) into fructose and UDP-glucose, is
a key enzyme in sucrose metabolism in higher plants. As the sucrose synthase enzyme from all diverse group of
organisms are functionally similar, the protein domains are also found to be conserved among them, though the
stretches of domain may vary between species (Fig.15B). It is revealed from the results (Fig 15.B) that although
the gene structure may vary among a similar or diverse groups of organisms, the proteins are highly conserved
and rarely undergo mutation.
 29

Table 2: Details of intron- exon and UTR structure of nine representative taxa obtained from GSDS tool and INTER-PRO
 30

(A) (B)

Fig.15 Gene structure analysis of SUSY. (A) Intron-exon structures of SuSy genes of nine representative taxa. (B) Protein domain structure of SuSy gene.
 31

6.5 Identification of conserved amino acid stretches

SuSy amino acid sequences from nine representative taxa, green alga (Botryococcus braunii), bryophyte
(Physcomitrella patens), pteridophyte (Selaginella moellendorffii), dicots (Arabidopsis thaliana, Kalanchoe
laxiflora, Vigna unguiculata), and monocots (Zostera marina, Zea mays and Oryza sativa) were subjected to
multiple alignment with Consurf tool. The amino acid length ranges from 1- 810 and were found to be highly
conserved among nine representative taxa (Table 3), highlighted in Fig.16. A detailed study of multiple sequence
alignment has shown ten common binding motif stretches: FLN, VVI, PDTGGQ, VYILDQVRAL, EML, TTC,
SRF, PDLII, IAHALEKTKY, AMN, QEIA, FT which are rectangularized in different colours in Fig.16 that are
involved in the enzyme activity and stability (Dhar & Chakrabarti., 2019).

Table 3: Conserved amino acids and their position in all representative taxa aligned through Consurf tool.

CONSERVED CONSERVED AMINO ACIDS WITH POSITION

STRETCH
1-150 M1, N24, G37, Q42, F64, Q72, E73, V76, R86, P87, G90, Y94, L102, L107, L113, F115, K116, E117,
L119, L130, E131, D133, F137, I150
151-300 V154, F156, L157, N158, S162, L165, F166, L175, F178, L179, M190, L205, L212, G232, G236, W237,
G238, L251, A258, P259, D260, F266, L267, V276, I278, H282, F285, Q287, L291, G292, P294, D295,
T296, G297, G298, Q299
301-450 V301, Y302, I303, L304, D305, Q306, V307, R308, A309, L310, E311, E313, M314, L315, Q321, L323,
I325, I329, R334, P337, A339, G341, T342, T343, C344, E349, I359, R361, P363, F364, S376, R377,
F378, V380, P381, L383, E384, E394, P403, D404, L405, I406, N409, Y410, D412, N414, V416, L417,
P427, Q428, C429, I431, A432, H433, A434, L435, E436, K437, T438, K439, Y440, D444,
451-600 Y454, H455, F 456, S457, C458, Q459, F460, T461, A462, D463, A466, M467, N468, D471, F472, I473,
T475, S476, T477, Q479, E480, I481, A482, V488, Q490, Y491, E492, H494, F497, T498, L502, Y503,
R504, V505, V511, F512, D513, K515, F516, N517, I518, V519, S520, A523, D524, Y528, R537, L538,
T539, H542, E546, L548, D564, K567, F571, M573, A574, R575, L576, D577, K580, N581, L585, E587,
L595, R596
601-750 L601, V602, V604, S612, D614, E616, E617, E620, K623, M624, Y631, L633, R638, I640, Q643, R646,
V647, N649, E651, Y653, R654, I656, D658, F663, V664, Q665, A667, Y669, E670, F672, L674, T675,
V676, E678, M680, T681, L684, A688, T689, A695, E696, I697, I698, S703, H706, I707, D708, F722,
F723, S737, L741, R743, I744, T749,
751-810 Y753, R756, L757, T759, L760, V763, Y764, F766, K768, R775, E777, R780, Y781, E783, M784, F785,
Y786, L788, K789, R791, L793, V797
 32

Fig.16 A colour-coded conserved Multiple sequence alignment for the predicted amino acid sequences of the
eight SuSy genes of the 9 representative taxa (Botryococcus braunii, Physcomitrella patens, Selaginella
moellendorffii, Arabidopsis thaliana, Kalanchoe laxiflora, Vigna uniguiculata, Zostera marina, Zea mays and
Oryza sativa). Sequence alignment was performed using The Consurf Server. Identical amino acids are shaded
and gaps are indicated by dashes. The ten conserved Motif stretches are wrapped with rectangular boxes: red
(141-190)(SIG-LM); yellow (257-306)(QAPD-LDQ); orange (307-356)(RAL-TE); deep green (374-414)(SRF-
DCN); purple (454-503)(YHF-LY); grey (504-553)(RVV-EL); blue (558-607)(HVK-VGG); light green (638-
687)(RWI-LPT); black (689-738)(AT-SG) and pink (742-791)(RI-LK).
 33

6.6 Microarray based expression profiling of SuSy in plants

Most terrestrial plants possess more than one isoform of Sucrose synthase, encoded by different genes located
on the same chromosome or on different chromosomes generated by alternative splicing (Mukherjee et al., 2012).
In order to understand the expression pattern, we used publicly available microarray data to analyze the
expression profile of SuSy members in Arabidopsis and rice under different developmental stages, tissues and
specific stress conditions. The microarray-based expression data were analyzed during different developmental
stages, different tissue samples as well as during different abiotic stresses and clustered heat maps were
generated (FiG.17 and Fig.18.). In the present analysis, six probe sets each for Oryza and Arabidopsis isoforms
are mentioned in Table 4. In Arabidopsis, three alternative splice variants are found for respective SuSy gene on
same chromosome, they are denoted by separate probe-id also. They are following 834978, 823393 and 833692
respectively. Oryza Sucrose synthase contain only one splice variant i.e 4332788 on chromosome 3.
Fig.17 and Fig.18 summarizes that in Arabidopsis and rice some of their isoforms get upregulated whereas
other remain as down regulated upon exposure to stress. In Arabidopsis, AtSUS1(245998_at) is the most sensitive
toward stress, it is upregulated in all stresses such as cold, drought, hypoxia, anoxia, salt, wounding etc. AtSUS2
(248647_at), AtSUS5 (249633_at) and AtSUS6 (245725_at) are not upregulated in any of the stress treatments but
AtSUS4 (252746_at) is upregulated in anoxia and hypoxia stress; AtSUS3 (25521_at) is upregulated in osmotic
and salinity stress. Remarkably all SuSy gene isoforms, in the root endodermis under salt stress are all
downregulated. In Oryza, OsSUS1 (LOC_Os03g28330), OsSUS2 (LOC_Os06g09450) and OsSUS3
(LOC_Os7g42490) is upregulated but OsSUS4 (LOC_Os03g22120) is downregulated in anoxic stress; in cold
stress all of the SuSy gene (in Oryza) isoforms are roughly downregulated; OsSUS4 (LOC_Os03g22120) is only
upregulated in drought, dehydration and submerged stress period.
Most of the SuSy gene isoforms in both Arabidopsis and rice show low level expressions in their entire
developmental stages. In Arabidopsis, AtSUS2 (248647_at) and AtSUS3 (25521_at) is only expressed in mature
siliques and AtSUS3 (25521_at) is the only isoform whose expression is detected in senescence stage. Oryza
being a typical monocot its developmental stages are different where OsSUS1 (LOC_Os03g28330) is expressed
at the time of germination providing a continuous supply of hexoses as energy source; OsSUS4
(LOC_Os03g22120) is upregulated at both milk and dough stage. Sucrose synthase expression remain mostly
unaltered in different anatomical tissues, a basal level of expression is observed in the tissues starting from callus
to root except in some cases. For example, In Arabidopsis, AtSUS2 (248647_at) show high expression in mature
siliques and raceme inflorescence with no expression in roots. In Oryza, OsSUS4 (LOC_Os03g22120) shows
high expression only in endosperm.

Table 4: The respective Probe-ID for each gene in Arabidopsis and Oryza generated through
GENEVESTIGATOR
 34

Fig.17 Clustered heat maps of expression profile of SuSy isoforms in Arabidopsis thaliana.

(A) Showing expression profile under abiotic stresses.

(B) Showing expression profile at different stages of development.
(C) Showing expression profile at different anatomical tissues.
 35

Fig.18 Clustered heat maps of expression profile of existent SuSy isoforms in Oryza sativa.
(A) Showing expression profile under abiotic stresses.
(B) Showing expression profile at different stages of development.
(C) Showing expression profile at different anatomical tissues
 36

7.DISCUSSION

In plants, Sucrose synthase has been shown to play important roles in plant sugar metabolism, primarily in sink
tissues. They are shown to be involved in a plethora of metabolic pathways such as starch synthesis (Craig et al.,
1999), callose and cellulose synthesis (Fujii et al., 2010) and to play developmental and possibly signalling roles
in sink carbohydrate flux, vascular tissues(Yang and Russell., 1990) and meristem functioning(Goren et al.,
2017). In addition, SuSy may be a key component in the perception and response to stress, including drought,
hypoxia, anoxia, salinity and wounding (Subbaiah and Sachs, 2001; Martin and Moreto, 2003). It has been
shown that, the down-regulation of the SS gene is the cause or the consequence of the decline in nitrogen fixation
under water stress conditions (Gonzalz et al.,1999). It has been reported from Arabidopsis mutant analysis that
Sucrose synthase have a role in callose deposition in response to leaf wounding. The role of SUS under low
oxygen has previously been studied from providing substrate for glycolysis (Guglielminetti, Pereta et al., 1995);
or increasing the cellulose content to change the cell wall structure (Albrecht & Mustroph., 2003).
In the present study, the search of SuSy sequences from different domains of plant kingdom, expanded our
knowledge on their molecular structures, evolutionary relationships, and expression patterns. In our gene
phylogenetic tree, monocots and eudicots have not separated even they have shared same node with some lower
group of organism exhibiting that the SuSy sequence has not undergo much changes during the course of
evolution and the sequences are conserved at their gene level (Fig.14A). In the phylogenetic tree with amino acid
sequences a higher degree of conservation is observed. Monocots and dicots have possessed distinct clade and
the lower group of organism have clustered separately prior to the base of angiosperm (Fig.14B). In gene
phylogenetic tree, there is a clear indication of genetic divergence in Arabidopsis/Asteraceae/Poaceae due to
recombination or genetic drift in course of evolution. Comprehensive analysis of exon / intron gene structure
between eight representative species of variety domain of plant kingdom have documented that the length and
positional characteristics of introns and exons of the SuSy genes are variable (Fig.15A). Moreover, the genes of
monocot group contain more introns than the genes of the eudicot group. This led to the supposition that intron
loss events took place at least twice in the evolution of the eudicots. SuSy genes under selection pressure (Chen
et al., 2012). Before the split of monocots and eudicots, duplication of the ancestral gene gave rise to the two
progenitors (Selaginella and Botryococcus) of the two SuSy groups with 14 and 18 conserved introns
respectively. The multiple alignment of the amino acid sequences also reflected the same pattern, the sequences
being highly conserved and the distribution of all conserved motifs along the sequences. The conserved domains
are almost similar in all groups of plants resembling the conservation of core catalytic structure of the
protein/enzyme (Fig.15B). Through a ‘phylogenomics’ approach the phylogentic tree from 114 species from 45
families across 24 orders of flowering plants (Fig.14A) was compared and inferred with the phylogenies of more
traditional, sequence-based methods of APG phylogeny (version IV). Our sequence based phylogenetic tree
have yielded conflicting topologies over APG phylogeny due to complications associated with frequent gene
duplication and loss, ancestral hybridization and lateral gene transfer between species. Besides, misspecifications
in nucleotide substitution can be the cause of differentiation. These forces can cause genetic variation and
contribute to develop divergent evolution between species.
Analysis of gene expression patterns can be used to some extent to predict the molecular functions of genes
involved in different physiological processes. In our present study, more than one isoform is expressed in all
organs examined. The apparent redundancy displayed by the isoforms of SuSy under normal growth conditions
might occur because a significant proportion of the sucrose in Arabidopsis is metabolized via invertase rather
than SuSy (Barratt et al.,2007). Previous studies have shown that Sus activity is highly correlated with sink
strength (Zrenner et al., 1995). In this study, we also demonstrated that the overall expression of AtSus and
OsSus genes is much higher in sink tissues, such as root, and flower (inflorescence, stamen, fruit etc), than in the
source tissue (mature leaf) (Fig.17 and Fig.18). The expression data of SuSy isoforms of Arabidopsis and rice
from the publicly available microarray has been found to be upregulated under several abiotic stresses like
hypoxia, drought and temperature. These results suggested divergent functions and requirement of SuSy genes
throughout the life cycles of plant.
 37

8. CONCLUSION AND FUTURE PERSPECTIVES

Plants are being sessile greatly influenced by abiotic stresses, due to gradual changes in the environment. Water
deficit stress, salt stress, imbalances in nutrients (including mineral toxicity and deficiencies) and temperature
extremes are significant environmental limitations on the productivity of crops all over the world. The crops
undergo various kinds of modifications due to abiotic stresses, which can cause antagonistic effects on the
growth and development of a plant. A better understanding of the molecular basis of major genes involved in
stress responses and tolerance and their exploration may pave the way for biotechnological interventions. The
current analysis is done on one of such pathways, sucrose metabolism to understand the evolution, diversification
and expression of the key gene SuSy. The present study reveals that the gene is ubiquitously distributed in plant
kingdom from algae to angiosperms. Sucrose is an essential primary metabolite in the plant kingdom. The
homology between the gene/protein structures across the diversified taxa denotes its conserved nature.
Microarray data reveals that basal expression level is always maintained in the cells to fulfil the requirement.
Altered expressions during the abiotic stress specific conditions may shed some light on sugar signalling and the
role of sugars in stress tolerance.
The present work is an initial, in silico approach to study the SuSy gene throughout the plant kingdom. More
genomic data from different plant groups would be required to search for the ancestry of the gene, relevance of
its preferential evolution and diversification in higher plants. Investigating the in planta expression level of SuSy
in model plants under different growth and stress conditions and protein expression studies can provide possible
avenues for future biotechnological inquiries.
 38

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