Somatic Embryogenesis Receptor Kinase (SERK) family of receptor kinases is functionally diverse, involved in cell-to-embryo transition and controlling a number of other fundamental aspects of plant development. The morphological transformation of somatic to embryonic cells has drawn scientific attention utmost due to remarkable genetic-switch system evolved across species. Receptor kinases having direct role in somatic embryogenesis (SE) and involved in other functions are designated as “SERK” and “SERK-like” genes, respectively. We aim for phylogenetic reconstruction to reveal major SERK groups across plant species (angiosperm to gymnosperm) for their functional diversification. Data indicate that the development of SERK proteins occurred prior to the divergence of monocots and eudicots. Also, the SERK orthology is not directly proportional to their functions. Structure prediction results identified novel transmembrane topologies, short linear motifs and O-glycosylation sites exclusively in SERK proteins than SERK-like proteins. Comparative temporal expression analyses of SERK and SERK-like genes provided significant accordance with their physiological function. The identification of intrinsic disordered regions (IDRs) exclusively in SERK proteins was assumed to perceive external stress-induced signals that may lead to rapid protein folding. In a result it switches-on the precise cellular signals essential for the acquisition of SE. Moreover, the regulatory sequences of SERK genes are evolved with unique cellular fate deciding AP2-like ethylene responsive transcription factor AINTEGUMENTA binding sites for their spatial expression in SE. Based on these analyses we suggest future avenues of research that may be imperative for elucidating the importance of SERK gene evolution in SE.
Among plant species, somatic embryogenesis (SE) is the transition of a somatic cell into embryonic cell that subsequently develops in to a somatic pro-embryo. Plant SE is one of the finest paradigms of developmental plasticity in which single cell destined for division comparable to its primogenitor, suddenly alter the develop- mental path and ensue towards embryogenesis. In general, SE involves with three distinct stages during embryo development including globular, heart-shaped and torpedo-shaped stage in dicot; globular, scutellar, and coleoptilar stages in monocots [
Somatic embryogenesis is extraordinarily complex and multistep process [
The endogenous and exogenous auxin levels have direct correlation with various stress-induced factors and genes which has been observed up-regulated during the initiation and progression of SE [
The SERK gene is one of the potential candidate genes those have been revealed for their direct role in SE in- duction [
Phobius online platform (http://phobius.sbc.su.se/) was used for transmembrane topology and signal peptide analysis in the AtSERK1 (AEE35238.1), AtSERK4 (AEC06259) and MtSERK1 (AAN64294.1). The protein sequences in FASTA format for each were obtained from NCBI protein database as already mentioned above and directly submitted to the Phobius online platform (http://phobius.sbc.su.se/). The prediction is based on a Hidden Markov Model (HMM) and algorithm called N-best performing the different sequence regions of a sig- nal peptide and the different regions of a transmembrane protein in a series of interconnected states [
AtSERK1, AtSERK4 and MtSERK1 protein sequences were obtained from NCBI protein database. AtSERK1 protein sequence obtained from NCBI which has reported as marker for SE were analysed with comparative analysis of AtSERK4 (non significant for SE) using “Eukaryotic Linear Motif” (ELM) resource at (http://elm.eu.org) which is a comprehensive database of known experimentally validated motifs, and an exploratory tool to dis- cover putative linear motifs published very recently it uses different logical filters (or rules) based on context information to discriminate between likely true and false positives to predict accurately [
NetOGlyc3.1 online server (http://www.cbs.dtu.dk/services/NetOGlyc/) was used for the prediction of glycosy- lation pattern in the AtSERK1, AtSERK4 and MtSERK1 protein sequences. For pattern recognition NetOG- lyc3.1 online server is based on artificial neural networks and weight matrix algorithms to determine the exact position of in vivo O-linked GalNAc-glycosylated serine and threonine residues from the primary sequence.
Sequences of different SERK proteins were obtained from NCBI protein database and used for phylogenetic analysis on Phylogeny.fr online platform [
Cotton (Gossypium hirsutum L. cv. Coker 310FR) plants [
Intrinsically unstructured/disordered pattern in the protein sequence of AtSERK1 (highly significant for SE) in comparison to AtSERK4 (non-significant for SE) along with MtSERK1 (highly significant for SE) for validation purpose were predicted using IUPred online platform (www.iupred.enzim.hu). Protein sequences were obtained from NCBI protein database and directly submitted in IUPred in FASTA format to get the result. Unstructured/ disordered regions of protein predicted were based on estimated energy content [
The detailed sequence analysis of SERK and SERK-like proteins was performed using different in silico tools. Studied SERK and SERK-like proteins comprised of signal peptide, leucine zipper, leucine rich repeats, Ser-Pro rich region, transmembrane domain, kinase domain and C-terminal region, however selective specific alterations were observed in the SERK proteins than SERK-like proteins. This might be important for confirmation of the precise role of SERK proteins in the acquisition of embryogenic potential in a somatic cell.
1. Transmembrane topology
The signalling molecules such as SERK proteins may reflect the signature of evolutionary patterns of trans- membrane proteins which evolved exclusively for their role in SE. To validate this assumption, transmembrane topology was performed using Phobius online server for SERK proteins including AtSERK1, MtSERK1, StSERK1, GhSERK1 and CuSERK1. This platform was also used to understand transmembrane topology on the SERK-like proteins (earlier reported to be non-significant for SE including AtSERK4, AtSERK5, GhSERK2 and GhSERK3). Prediction results showed conserved length of cytoplasmic domain of 362 amino acid across spe- cies for SERK whereas it varied for SERK-like and of greater length (
Medium | 1/2 MS | MST1 | MSOT2 | MSOT3 |
---|---|---|---|---|
Salt (MS) | 0.5× | 1× | 1× | 1× |
B5 Vitamins | 0.5× | 1× | 1× | 1× |
Carbohydrate Source | Sucrose (2%) | Glucose (3%) | Glucose (3%) | Glucose (3%) |
Phytohormones | - | 2,4-D (100 µg/l) Kinetin (500 µg/l) | - | - |
Gelling Agents | Agar (0.7%) | Phytagel (0.2%) | Phytagel (0.2%) | Phytagel (0.2%) |
pH | 5.80 | 5.80 | 5.80 | 5.80 |
Proteins | Plant Species | Signal Peptide | N-Region | H-Region | C-Region | Non Cytoplasmic Region | Transmembrane Region | Cytoplasmic Region |
---|---|---|---|---|---|---|---|---|
SERK Proteins | AtSERK1 | 1 - 26 | 1 - 5 | 6 - 16 | 17 - 26 | 27 - 233 | 234 - 262 | 263 - 625 = 362aa |
MtSERK1 | 1 - 28 | 1 - 6 | 7 - 20 | 21 - 28 | 29 - 240 | 241 - 264 | 265 - 627 = 362aa | |
StSERK1 | 1 - 30 | 1 - 8 | 9 - 22 | 23 - 30 | 31 - 242 | 243 - 266 | 267 - 629 = 362aa | |
GhSERK1 | 1 - 32 | 1 - 9 | 10 - 20 | 21 - 32 | 33 - 240 | 241 - 264 | 265 - 627 = 362aa | |
CuSERK1 | 1 - 22 | 1 - 4 | 5 - 14 | 15 - 22 | 23 - 234 | 235 - 258 | 259 - 621 = 362aa | |
SERK-Like Proteins | AtSERLK4 | 1 - 33 | 1 - 9 | 10 - 21 | 22 - 33 | 34 - 229 | 230 - 255 | 256 - 620 = 364aa |
AtSERLK5 | 1 - 24 | 1 - 7 | 8 - 16 | 17 - 24 | 25 - 215 | 216 - 236 | 237 - 601 = 364aa | |
GhSERLK2 | 1 - 32 | 1 - 8 | 9 - 20 | 21 - 32 | 33 - 227 | 228 - 251 | 252 - 620 = 368aa | |
GhSERLK3 | 1 - 32 | 1 - 8 | 9 - 20 | 21 - 32 | 33 - 227 | 228 - 251 | 252 - 620 = 368aa |
Interestingly, investigated SERK proteins contained smaller cytoplasmic domain of 362 amino acids (aa) than to SERK-like proteins comprising upto 368 aa. Consistency in the length of cytoplaasmic domain of SERK pro- teins and SERK-like protein may be accounted for their diverse functional evolution across species.
2. Short linear motifs
Short linear motifs (SLiMs) are the short amino acid chains earlier reported to be important in mediating the regulatory functionality in the cell, mainly in cell signalling [
Since the acquisition of SE is directly dependent upon the induction of cellular signalling mainly through SERK proteins, it becomes essential to explore the possibilities for the precise role of SLiMs in this mechanism. Therefore, SERK gene sequences of Arabidopsis (AtSERK1) and Medicago (MtSERK1) reported beforehand as robust biomarkers of SE [
3. O-GalNAc (mucin type) glycosylation sites
The process of an addition of a sugar chain to a protein is called glycosylation. Emerging genetic studies have revealed the function of a subset of glycosyltransferases gene family responsible for the formation of mucin- type-O-glycans essential for normal plant development. Mucin-type O-glycosylation, consisting of glycans at- tached via O-linked N-acetylgalactosamine (GalNAc) to serine and theronine residues, is one of the most abun- dant forms of protein glycosylation [
of SE, experimental validation of these important differences between SERK and SERK-like proteins would enhance our understanding to the key genes regulating different pathways in SE.
Members of receptor kinase protein family play diverse roles in different biological systems. Besides SE, they contribute in BR signalling pathway, cell death control pathway, disease resistance pathway, microsporogenesis and apomixes (
Name of Gene | Species | Gene Type | Characterized Function | References | Remarks |
---|---|---|---|---|---|
AtSERK1 | Arabidopsis | SERK | Somatic embryogenesis | [ | Total 5 members in SERK gene family of Arabidopsis |
AtSERK2 | Arabidopsis | SERK-like | Microsporgenesis, BR signalling pathway, cell death control pathway | [ | |
AtSERK3 (BAK1) | Arabidopsis | SERK-like | BR signalling pathway, cell death control pathway, disease resistance pathway | [ | |
AtSERK4 (BKK1) | Arabidopsis | SERK-like | BR signalling pathway, cell death control pathway | [ | |
AtSERK5 | Arabidopsis | SERK-like | [ | SERK5 might not be functional due to a natural point mutation at a highly conserved “RD” motif | |
OsSERK1 | Oryza sativa | SERK-like | Defence signal transduction, somatic embryogenesis | [ | |
MtSERK1 | Medicago truncatula | SERK | Somatic embryogenesis | [ | A total of nine SERK or SERK-like genes have been identified in M. truncatula |
SERK | Dactylis glomerata, | SERK | Somatic embryogenesis | [ | |
SERK | Helianthus annuus | SERK | Somatic embryogenesis | [ | |
SERK | Ocotea catharinensi | SERK | Somatic embryogenesis | [ | |
SERK | Theobroma cacao | SERK | Somatic embryogenesis | [ | |
PpSERK | Poa pratensis | SERK | Apomixis | [ | |
McSERK | Momordica charantia | SERK | Somatic embryogenesis | [ | |
DcSERK | Daucus carota | SERK | Somatic embryogenesis | [ | |
CitSERK1 | Citrus sinensis | SERK | Somatic embryogenesis | [ | |
SERK | Citrus unshiu | SERK | Somatic embryogenesis | [ | |
TaSERK1 | Triticum aestivum | SERK | Somatic embryogenesis | [ | |
GmSERK1 | Glycine max | SERK | Somatic embryogenesis | [ | A total of 17 SERK in soybean |
AaSERK1 | Araucaria angustifolia | SERK | Somatic embryogenesis | [ | Gymnosperm |
ClSERK | Cyrtochilum loxense | SERK | Somatic embryogenesis | [ | Gymnosperm |
CnSERK | Cocos nucifera | SERK | Somatic embryogenesis | [ |
In Group I of monocots, SERK and SERK-like genes of maize and wheat showed close relationship than co- conut and Ananas, and clustered into a single group with an average of 69% identity. The cluster includes both SERK and SERK-like protein sequences and has been reported to be involved in SE and other roles of the SERK proteins, alone or in combination with other members. Group II is constituted with two conifers Pinus and Araucaria aligned together with high amino acid similarity. However, all studied eudicots constructed group III harboring different species of Solanum, Cyclamen and Citrus spp. along with other taxa (
SERK protein variants from different species of genus Solanum clustered together highlighting high amino acid sequence similarity with most recent genic duplication of SERK and SERK-like genes. The Arabidopsis SERK protein has five variants of SERK and SERK-like genes involved in SE, brassinosteroid signalling pathways and male microsporogenesis, respectively [
Similarly, isolation and characterization of three cotton (Gossypium) homoelogous SERK and SERK-like genes with their full-length transcript sequence have been reported previously, exhibiting homology with other species SERK sequences, for example, five SERK genes from Arabidopsis [
Expression comparison of SERK with SERK-like genes firmly distinguished both class of receptor kinases. The SERK-like proteins appeared to revert the growth reduction by the loss of SERK-mediated cell-signaling required during SE. This indicates that differences in SERK and SERK-like proteins’ expression specificity may be true reflection of significant accordance with their physiological functions. Though these results are prelimi- nary and further work is required to determine if SERKs fully complements under growth and stress conditions. Given the high sequence similarity among different SERK members across taxa, it could also be assumed that this class of proteins are functionally redundant, thereby masking a synergistic effect with SERK-like variants.
Intrinsically disordered proteins (IDPs) have no sole explicit tertiary structure in their native functional state due to their intrinsic flexibility. Till recently, any protein is considered for a functional property based on its tertiary structure, and based on this conjecture more than 80 K protein sequences have been deposited in the protein data bank so far. Interestingly, out of these nearly 1000 structures are now among the registry of unstructured or dis- ordered chains which only convert itself into prerequisite structured forms in response to an appropriate signal/ stimuli received in the form of a protein/ligand [
dered regions are very important for the function of a protein and helps in protein folding within very short time- frame only in response to an appropriate signal [
Previously in our laboratory, it has been shown that application of diverse stress conditions in vitro induce SE among plant species. For example, micronutrient boron-mediated stress increased endogenous auxin level that in turn up-regulated the selected variant of signalling molecule SERK in SE-competent somatic cells [
To validate this conjecture, SERK protein sequences of Arabidopsis (AtSERK1) and Medicago (MtSERK1) reported beforehand as robust biomarkers of SE [
The disordered regions identified in AtSERK1 and MtSERK1 proteins may thus be assumed to perceive ex- ternal stress-induced signals those in result mediate quick protein folding bringing the property for switching-on the cellular signals essential for the acquisition of SE. However, it would be fascinating to search the fine bal- ance between type and intensity of stress that is required for the induction of proper folding of disordered re- gions of SERK protein and its contribution towards SE acquisition. Also, refolding of these disordered regions in SERK protein in response of stress may have affected more indigenous pathways which compelled somatic-to- embryo transition. Therefore, probably such flexible regions are one of the key regions of SERK proteins in- tended to trigger SE in plants.
The endogenous auxin-mediated induction of cell-signalling through up-regulated SERK genes has earlier been revealed at the onset of in vitro SE [
AINTEGUMENTA is an AP2-like ethylene responsive transcription factor, a family of proteins involved in ethylene signal transduction recognizing and binding to the DNA consensus sequence 5’-CAC[AG]N[AT] TNCCNANG-3’ [
However, this is still a concern whether such evolutionary changes are species-specific or global? To confirm this hypothesis, regulatory sequence of SERK variant of Medicago truncatula (MtSERK1) reported earlier as significant biomarker of SE [
The current study implicates the structural evolution of receptor kinases involved in the intricate process of SE, enhancing our understanding for the SERK-mediated cell-signalling during SE. We provide evidences for the evolution of precise structural components in the receptor kinases and their regulatory sequences through a comparison of SERK and SERK-like proteins, reported earlier for their direct role in embryogenic transitions and other metabolic pathways, respectively. In general, presence of SERKs across angiosperm to gymnosperm highlights the evolution of such proteins much before in the plant kingdom for the enhancement of occurrence and magnitude of embryogenesis through fine-tuning of cell-siganling genes. Remarkably, intrinsic disordered regions known for their role in protein folding in response to a stress signal, various SE-related components on the N-terminal part of the proteins along with SE-specific trans-factor binding sites are diagnosed as having be- come enhanced system-wide exclusively in SERK proteins. An exciting prospect for future work will be to dis- sect these structural modifications and their responsible constituents for their precise role in the complex me- chanism of SE.
The authors are thankful to the Council of Scientific and Industrial Research (CSIR), and the Department of Biotechnology (DBT), Government of India, for providing the financial support to carry out cotton research work in the laboratory.