Considering all these, we aim to characterize the role and regulation of galactinol and GolS enzymes in chickpea. Chickpea, being a rich source of proteins is considered as one of the major sources of human food for developing countries.
Unfortunately, the productivity of this grain legume is usually low and further reduced by environmental stresses Furthermore, chickpea seeds are sensitive to aging and a major concern for seed storage particularly in humid tropical climate.
Even though there is an enormous agronomic and nutritional importance, research in chickpea is rather restricted due to the lack of mutant resources and efficient and dependable transformation protocol.
In present study in chickpea, we report that galactinol synthase activity is differentially regulated in different organs. Further, galactinol synthase activity along with galactinol and raffinose content increase during seed maturation and seed aging.
Further analysis also revealed that both these isoforms exhibit similar yet distinct biochemical properties. Subcellular localization of these GolS isoforms has also been determined. Finally implication of these isoforms in seed vigor and longevity has been investigated through seed specific overexpression in Arabidopsis thaliana. Results GolS activity and consequent galactinol and raffinose content are markedly enhanced during seed development in chickpea In order to explore the role and regulation of galactinol synthase in chickpea, initially different tissues were analyzed for the galactinol synthase activity.
For this, total protein was extracted from different organs and was assayed as described in Methods section. As shown in Fig. The maximum level of GolS activity was detected in pod followed by stem, root, flower and seed Fig.
Further to obtain more detailed picture of galactinol synthase activity profile during the course of seed development in chickpea, flowers were tagged according to the day after pollination DAP Supplementary Fig.
S1 and subsequently total protein was extracted and activity was measured. However the activity sharply decreased at 40 DAP and in dry seeds. The activity was also monitored during the course of germination and data revealed that GolS activity gradually decreased during the course of germination and further reduced in germinated seed Fig. Simultaneously, galactinol along with myo-inositol and raffinose content was also quantified in different organs, during seed development and germination.
Galactinol accumulation was found to be maximum in pod followed by seed but undetectable in other organs. Raffinose was also undetectable in other organs except pod and seed. The tree is drawn to scale and bootstrap test was performed with replicates, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site.
All ambiguous positions were removed for each sequence pair. RFOs undoubtedly accumulate in many plant groups in response to diverse abiotic stresses Nishizawa et al.
Under extreme conditions, RFOs are thought to act as osmolytes to maintain cellular integrity and function Nishizawa et al. In ABA-deficient and ABA-insensitive double-mutant seeds of Arabidopsis aba-1, abi , which are viable but desiccation-intolerant, ABA treatment in vitro increased seed Raf content and promoted desiccation tolerance Ooms et al.
RFOs have significant report of accumulation in low temperature stress. Ajuga reptans Peters and Keller, , alfalfa Cunningham et al. Reports of accumulation of Raf in the unicellular organisms are not obtained. So, the role of Raf in a unicellular organism is unknown. A recent study demonstrated that starch hydrolysis results in hexose and Raf accumulation during the first 24 h after a cold shock treatment in Arabidopsis.
The importance of such accumulation remains unknown but it has been suggested that Raf may take part in chilling-induced ROS homeostasis Sicher, Nishizawa et al. Presence of a Raf transporter raf in chloroplast membrane has been established, although whether they help in maintaining chloroplast membrane integrity under oxidative stress, is not known Schneider and Keller, ; Valluru and Van den Ende, Haritatos et al.
The molecular handles of such differential accumulation are not clearly known; however, RFOs accumulate late in seed development, starting at about the beginning of seed fill and continuing up to maturation drying. Reduction of the content of myo-inositol in tubers of transgenic potato Solanum tuberosum L.
GolS is also purified from the cotyledons of kidney bean Phaseolus vulgaris, Liu et al. RFOs protect the embryo during the desiccation that occurs during seed maturation and thus play important role in prolonged seed survival Peterbauer et al. Raf and Sta also serve as the main transportable solute in the orders Lamiales, Cucurbitales, Cornales, and in one family of the Celastrales and are mechanistically linked with phloem loading Zimmermann and Ziegler, ; Haritatos et al.
The structural and anatomical specificities of plants that drive accumulation of RFOs as major transport sugar are reviewed elsewhere Lemoine et al. The phloem loading function of RFOs is best studied in Ajuga reptans common bugle. In this frost tolerant evergreen Labiatae, i Sta is the main carbon translocate; ii higher RFO oligomers are the main carbon store Bachmann et al.
There are two RFO pools in its leaves: a storage pool associated with leaf mesophyll and a transport pool associated with the phloem-loading sites Bachmann et al. These two pools rely on different GolS isoforms Sprenger and Keller, The expression pattern is consistent with the loading function, i. From the classic study of Rennie and Turgeon a species-specific pattern of transport sugars can be drawn.
The anatomy of phloem remains highly important in this pattern, especially the occurrence of intermediate companion cells Gunning and Pate, ; Turgeon et al.
To improve the stress resistances and reduce the possibly damages from abiotic stresses, plants have evolved complex strategies, such as the accumulation of certain small soluble molecules such as trehalose, proline, tryptophan, polyamines and glycyl betaine , to increase the osmotic pressure in cells [ 4 , 5 ]. Acting as osmoprotectants, raffinose family oligosaccharides RFOs , including raffinose, stachyose and verbascose, play key roles in plant growth and development.
Additionally, the accumulation of RFOs is associated with stressful environmental conditions, especially desiccation tolerance [ 6 , 7 , 8 , 9 , 10 ]. In addition, RFOs also act as signaling molecules during pathogen attack and wounding and are used in carbohydrate catabolism to generate energy during germination [ 11 , 12 , 13 , 14 , 15 ].
Galactinol synthase GolS; EC: 2. As a key regulator of carbon-partitioning, it also catalyzes the first step in the RFO biosynthesis, i. Previous researches revealed the positive relationship between GolS activity and plant abiotic stress tolerance.
Expression of OsGolS increase in response to cold treatments and osmotic stress in rice Oryza sativa seedlings [ 17 ]. In maize Zea mays , ZmGolS1 has low expression in most tissues, while ZmGolS2 accumulates in germinating seeds subjected to dehydration stress and ZmGolS3 predominated in seeds prior to maturation desiccation [ 18 ]. In seeds, RFOs can stabilize membranes during dehydration and extend the longevity of seeds. Raffinose protects sucrose by preventing crystallization during the withdrawal of water, and the vitrified cytoplasm is stabilized when a sufficient amount of raffinose is present Sun and Leopold The protective role of RFOs in vegetative tissues is less thoroughly explained.
The accumulation of galactinol and raffinose in vegetative tissues of different plant species occurs under cold, heat, drought and osmotic stresses ElSayed et al. Interestingly, the antioxidant activity of galactinol and raffinose has been discovered in some research Nishizawa et al. The participation of RFOs and fructans fructosyl sucrose oligosaccharides in response to oxidative stress was postulated by Van den Ende and Valluru Fructans and RFOs may contribute to an overall cellular homeostasis of reactive oxygen species ROS by specific ROS scavenging processes in the vicinity of organelle membranes e.
We have found that osmotic stress induces the activity of galactinol synthase GolS, EC 2. Similarly, dehydration induces expression and activity of GolS and RS in both epicotyl and root tissues of 7-day-old seedlings of pea Lahuta et al. Although seedlings accumulated galactinol and raffinose, the concentrations of both galactosides was several-fold lower than that of sucrose.Their high oligomeric length may serve to protect liposomes Cacela and Hincha, and probably also act as a free radical scavenger Nishizawa et al. OE of RFO synthesizing genes and increase in RFO levels improve stress tolerance in the plant, but loss of the gene does not compromise the normal physiology. Raffinose protects sucrose by preventing crystallization during the withdrawal of water, and the vitrified cytoplasm is stabilized when a sufficient amount of raffinose is present Sun and Leopold Apart from these functions, RFOs were recently shown to act as signaling molecules upon pathogen attack and wounding 5 , 6 , 7 , 8.
GolS has primarily been characterized biochemically from legume seeds and cucurbit leaves Keller and Pharr, The monocots are grouped into a small clade from the base of which dicots diverge and many distinct families form well defined clusters. Enzyme assays and kinetics Enzyme assays were based on the method of Ribeiro et al.
Galactinol accumulation was found to be maximum in pod followed by seed but undetectable in other organs. These RFOs participate in many physiological processes like translocation of photoassimilates, abiotic stress tolerance, seed desiccation tolerance etc. GolS is a member of glycosyltransferase 8 GT8 family and is usually encoded by a small gene family in higher plants. Simultaneously, galactinol along with myo-inositol and raffinose content was also quantified in different organs, during seed development and germination. As shown in Fig. The optimal trees with the sum of branch length equal to 0.
Results are summarized in Table 1. The experiment was repeated three times. RFOs undoubtedly accumulate in many plant groups in response to diverse abiotic stresses Nishizawa et al. Nishizawa et al. Haritatos et al. Sequence analysis revealed that CaGolS1 contains an open reading frame of bp encoding aa protein while CaGolS2 contains an open reading frame of bp encoding aa protein.
There are two RFO pools in its leaves: a storage pool associated with leaf mesophyll and a transport pool associated with the phloem-loading sites Bachmann et al. Phytozome accession numbers are provided for the Populus trichocarpa GolS proteins and GenBank accession number are provided for the remaining proteins. Nishizawa et al. B GolS proteins from different plant species. Tobacco Nicotiana tabacum is an important allopolyploid plant for evolution and gene function characterization [ 27 , 28 ]. Reverse genetic tools can be of better assistance, however sufficient knowledge of the functional areas of RFOs are necessary.
Ordinary companion cells OC , transfer cells TC , and intermediate cell IC are the three types of companion cells found in phloem. Our study provides a comprehensive evolutionary analysis of GolS genes among the Brassicaceae and Solanaceae as well as an insight into the biological function of GolS genes in hormone response in plants.
Error bars indicate the standard deviation. Biologically, it was shown that the transcript abundance of AtGolS1 and -2 were induced by drought and increasing salinity but not by cold. RFOs are loaded into phloem suggestively in symplastic type II plants using polymer trapping model. As shown in Fig.
StaS has been purified from seeds of kidney bean, adzuki bean and lentil Lens culinaris, Tanner and Kandler, ; Peterbauer and Richter, ; Hoch et al. As shown in Fig. During the early phase of seed development both galactinol and raffinose were undetectable, however became detectable during the mid phase of seed development and then gradually increased till 35 DAP.
Therefore to investigate this and towards exploring the potential role of galactinol and galactinol synthase in seed longevity, galactinol synthase activity along with galactinol, myo-inositol and raffinose were quantified after artificial aging. Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. RFOs are generally non-structural, non-reducing but soluble oligosaccharides present at high concentrations within the cell. GolS activity was determined: a in different organs; b during seed development; c during seed germination. There are two RFO pools in its leaves: a storage pool associated with leaf mesophyll and a transport pool associated with the phloem-loading sites Bachmann et al.
All ambiguous positions were removed for each sequence pair.
The trees are drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic trees. So, the role of Raf in a unicellular organism is unknown.
XvGolS1 Xerophyta viscosa had a low bootstrap value and was not consider part of this clade. The expression pattern is consistent with the loading function, i.
The expression of the key enzyme GolS is known to be linked to both abiotic stress and developmental stages. However, no detailed study of galactinol synthase and RFOs has been carried out in chickpea so far. Figure 5 Biochemical characterization a—d and sub-cellular localization e of CaGolS isoforms. The tree is drawn to scale and bootstrap test was performed with replicates, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Plant J. Schematic representation of the biosynthetic pathway of RFOs.