Genome-wide in-silico analysis of OsSWEET transporters indicate their involvement in abiotic and heavy metal(loid) stress responses in rice Anuska Sen⁎, Binayak Das⁎, Sanket Roy⁎, Surajit Bhattacharya (⁎contributed equally) Amity Institute of Biotechnology, Amity University Kolkata
ABSTRACT Background: The sugars will eventually be exported transporters (SWEETs) are a unique class of membrane bound sugar transporters that not only take part in hexose and sucrose transport across the biological membranes, but also are involved in diverse physiological function in plants. Rationale: Although the role of SWEETs in biotic stress response is well documented, their involvement in plants response to abiotic and heavy metal(loid) stress is poorly understood. Thus, to bridge this gap a comprehensive in silico study of the OsSWEET family was conducted to check their involvement in abiotic stresses and heavy metal(loid) stresses. Strategy and results: A total of 21 OsSWEET members showing elaborate gene duplication events were identified, and was classified in four evolutionary clades by phylogenetic analysis. Fascinatingly, an apparent preservation of gene and conserved motif structures in the members of the same phylogenetic clade was observed. Most interestingly, the promoter cis-regulatory element and miRNA target analysis indicated possible regulation of OsSWEET expression during abiotic as well as heavy metal(loid) stress. In-silico gene expression analysis further validated the involvement of OsSWEET transporters in abiotic and heavy metal(loid) stress responses in rice. Moreover, protein interaction analysis also indicated their possible interaction with some of the well-known heavy metal transporters proteins in rice. Taken together, this is the first comprehensive in-silico analysis of the involvement of OsSWEETs in abiotic, especially heavy metal(loid) stress responses in rice; however, further studies will be useful to understand their mode of action and regulatory mechanisms in heavy metal(loid) stress.
INTRODUCTION WHY RICE ?
WHY SWEET ?
• Second most important cereal crop in the world • Exposed to various abiotic stresses due to changing climatic conditions result in low productivity of rice grains • As and Cd cause bioaccumulation and hence toxicity in the consumers of rice
• Carry out bi-directional, pH-independent sugar transport across the cell or vacuolar membrane • Water-soluble carbohydrates can act as antioxidants by undertaking ROSscavenging during abiotic stress in plants cells •SWEET transporters are responsive to both biotic and abiotic stresses
RESULTS 1. Chromosomal distribution of SWEET genes in rice chromosomes shows block and tandem duplications
2. Gene structure and conserved motif analysis of OsSWEET genes shows diversification in no. of exons and motifs.
3. Presence of abiotic and heavy metal stress responsive cis-promoter elements indicate SWEETs role in stress tolerance
❑ Total 21 OsSWEET genes mapped to 8 out of 12 chromosomes in rice genome. ❑ Tandem duplications: black boxes Block duplications: connected through a black line.
Clade I: 7 OsSWEETs; 6 exons; 7 identical motifs (except LOC_Os01g40960 ). Clade II: 8 OsSWEETs; 1-5 exons; 5-10 motifs. Clade III: OsSWEET16; 6 exons; 6 motifs. Clade IV: 5 OsSWEETs; 4-6 exons; 6 motifs.
OsSWEET 7c contained the most number of abiotic and metal stress elements while OsSWEET6b has the least number of elements combined.
Cd-upregulated OsSWEET15 displayed interactions with metal(loid)responsive efflux proteins and heavy metal associated domain containing proteins .
OsSWEET11 OsSWEET11 OsSWEET7e OsSWEET7c OsSWEET7c
OsSWEET15 OsSWEET11 OsSWEET1b OsSWEET1b OsSWEET7c
OsSWEET3a OsSWEET 5 OsSWEET1b OsSWEET15
OsSWEET16
OsSWEET3a
OsSWEET16 OsSWEET15
OsSWEET1b OsSWEET1b OsSWEET14 OsSWEET12 OsSWEET16 OsSWEET11 OsSWEET11 OsSWEET7e OsSWEET7c OsSWEET7cOsSWEET12 OsSWEET3a
OsSWEET7c
OsSWEET7b
OsSWEET7c
OsSWEET7b OsSWEET15 OsSWEET16 OsSWEET16 OsSWEET7c OsSWEET3a OsSWEET3a OsSWEET15 OsSWEET14 OsSWEET7b OsSWEET5 OsSWEET15 OsSWEET15 OsSWEET 4 OsSWEET15 OsSWEET14 OsSWEET12
OsSWEET13 OsSWEET12 OsSWEET12
OsSWEET3b OsSWEET13
OsSWEET15 OsSWEET15 OsSWEET13 OsSWEET14 OsSWEET4 OsSWEET14 OsSWEET14
OsSWEET3b OsSWEET3b
OsSWEET7c OsSWEET7c OsSWEET6b OsSWEET14 OsSWEET7b OsSWEET7b OsSWEET14
OsSWEET13 OsSWEET13 OsSWEET2a
OsSWEET1a
LOC01g40960 LOC01g40960 OsSWEET2b OsSWEET2a OsSWEET1a OsSWEET2a OsSWEET3b OsSWEET3b LOC01g40960 OsSWEET14 OsSWEET14 OsSWEET6b OsSWEET6b OsSWEET6b OsSWEET6b
OsSWEET2a OsSWEET2a
Metal Stress Abiotic Stress
OsSWEET1b OsSWEET1b OsSWEET11 OsSWEET11 OsSWEET7e OsSWEET7c OsSWEET7c
OsSWEET5 OsSWEET15 OsSWEET15
OsSWEET16 OsSWEET16 OsSWEET3a OsSWEET3a
OsSWEET12 OsSWEET12
OsSWEET15 OsSWEET15 OsSWEET4 OsSWEET14 OsSWEET14
OsSWEET7c OsSWEET7c OsSWEET7bOsSWEET7b
OsSWEET14OsSWEET14 OsSWEET13OsSWEET13
OsSWEET3bOsSWEET3b
OsSWEET6bOsSWEET6b
OsSWEET2a OsSWEET2a
Metal stresses showed upregulation of OsSWEET3a, OsSWEET12, OsSWEET13 and OsSWEET14 under arsenic and cadmium stress and inhibition of OsSWEET2a
OsSWEET2bOsSWEET2b OsSWEET1a OsSWEET1a
6. Protein interaction network highlight the involvement of As-( and Cd- responsive OsSWEET transporters in heavy metal(loid) response
OsSWEET2bOsSWEET2b OsSWEET1aOsSWEET1a LOC01g40960LOC01g40960
•10 OsSWEET members predicted as potential targets of miRNAs involved in various abiotic, hormonal stress responses •OsSWEET7b : predicted as most suitable target of miRNA having 16 independent miRNA target sites
OsSWEET2b OsSWEET2b LOC01g40960LOC01g40960OsSWEET1a
5. Expression profiles of SWEET genes from rice under various metal and abiotic stress conditions
Metal Stress
4. Prediction reveals various heavy metal responsive miRNA target sites in OsSWEETs
Abiotic stresses showed significant upregulation of OsSWEET12, OsSWEET13 and OsSWEET15 genes and inhibition of OsSWEET3b
CONCLUSION & FUTURE PROSPECT
❖ Promoter cis-regulatory element and miRNA target analysis indicated possible regulation of OsSWEET expression during abiotic and heavy metal(loid) stress. ❖ In silico expression and protein-protein interaction analysis validated the involvement of OsSWEETs in abiotic and heavy metal(loid) stress responses. ❖ However, further studies are required to validate and analyze the mode of action and regulatory mechanisms of stress-responsive members (OsSWEET12, OsSWEET13 and OsSWEET 14 ) so that they can be used as potential candidates future genetic engineering approach for developing stress-resistant transgenic rice plants.
ACKNOWLEDGEMENT ❑ AIB, AUK.
❑ DST-SERB for research grant (SRG/2019/001247).