Abstract
Fifteen transcription factors in the CAMTA (calmodulin binding transcription activator) family of soybean were reported to differentially regulate in multiple stresses; however, their functional analyses had not yet been attempted. To characterize their role in stresses, we first comprehensively analyzed the GmCAMTA family in silico and thereafter determined their expression pattern under drought. The bioinformatics analysis revealed multiple stress-related cis-regulatory elements including ABRE, SARE, G-box and W-box, 10 unique miRNA (microRNA) targets in GmCAMTA transcripts and 48 proteins in GmCAMTAs’ interaction network. We then cloned the 2769 bp CDS (coding sequence) of GmCAMTA12 in an expression vector and overexpressed in soybean and Arabidopsis through Agrobacterium-mediated transformation. The T3 (Transgenic generation 3) stably transformed homozygous lines of Arabidopsis exhibited enhanced tolerance to drought in soil as well as on MS (Murashige and Skoog) media containing mannitol. In their drought assay, the average survival rate of transgenic Arabidopsis lines OE5 and OE12 (Overexpression Line 5 and Line 12) was 83.66% and 87.87%, respectively, which was ~30% higher than that of wild type. In addition, the germination and root length assays as well as physiological indexes such as proline and malondialdehyde contents, catalase activity and leakage of electrolytes affirmed the better performance of OE lines. Similarly, GmCAMTA12 overexpression in soybean promoted drought-efficient hairy roots in OE chimeric plants as compare to that of VC (Vector control). In parallel, the improved growth performance of OE in Hoagland-PEG (polyethylene glycol) and on MS-mannitol was revealed by their phenotypic, physiological and molecular measures. Furthermore, with the overexpression of GmCAMTA12, the downstream genes including AtAnnexin5, AtCaMHSP, At2G433110 and AtWRKY14 were upregulated in Arabidopsis. Likewise, in soybean hairy roots, GmELO, GmNAB and GmPLA1-IId were significantly upregulated as a result of GmCAMTA12 overexpression and majority of these upregulated genes in both plants possess CAMTA binding CGCG/CGTG motif in their promoters. Taken together, we report that GmCAMTA12 plays substantial role in tolerance of soybean against drought stress and could prove to be a novel candidate for engineering soybean and other plants against drought stress. Some research gaps were also identified for future studies to extend our comprehension of Ca-CaM-CAMTA-mediated stress regulatory mechanisms.
Highlights
A successful sustainable agriculture should ensure food security, must be eco-friendly and safe to humans [1]
We demonstrated that the transgenic Arabidopsis and chimeric soybean (OE-Overexpressing GmCAMTA12) plants exhibited enhanced tolerance and performed better under drought stress than their non-transgenic counterpart at phenotypic, physiological and molecular level. qPCR of the downstream genes in Arabidopsis and soybean displayed altered expression as a result of GmCAMTA12 overexpression
We report that GmCAMTA12 as a transcription factor plays role in drought stress by regulating the downstream genes involved in drought tolerance and could be exploited in developing drought-tolerant crops
Summary
A successful sustainable agriculture should ensure food security, must be eco-friendly and safe to humans [1]. The abiotic stresses due to drought, salinity, water and temperature fluctuations are causing 50–80% losses in crop yield, and should be as effectively managed as possible [4]. The warmer earth will cause a more humid atmosphere but less humid soil, leading to more frequent drought that would negatively affect the rate of photosynthesis, uptake of CO2, accumulation of biomass and yield [5,6]. Developing stress-resistant crops with stable yields under adverse conditions is an important strategy to ensure future food security [2,7]. Deep insights into the mechanisms underlying signaling crosstalk mediating stress tolerance would aid plant researchers to upgrade the plant’s indigenous natural machinery through biotechnology and genetic engineering
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