Abstract
Sugarcane (Saccharum spp.) is a prominent source of sugar and serves as bioenergy/biomass feedstock globally. Multiple biotic and abiotic stresses, including drought, salinity, and cold, adversely affect sugarcane yield. G-protein-coupled receptors (GPCRs) are components of G-protein-mediated signaling affecting plant growth, development, and stress responses. Here, we identified a GPCR-like protein (ShGPCR1) from sugarcane and energy cane (Saccharum spp. hybrids) and characterized its function in conferring tolerance to multiple abiotic stresses. ShGPCR1 protein sequence contained nine predicted transmembrane (TM) domains connected by four extracellular and four intracellular loops, which could interact with various ligands and heterotrimeric G proteins in the cells. ShGPCR1 sequence displayed other signature features of a GPCR, such as a putative guanidine triphosphate (GTP)-binding domain, as well as multiple myristoylation and protein phosphorylation sites, presumably important for its biochemical function. Expression of ShGPCR1 was upregulated by drought, salinity, and cold stresses. Subcellular imaging and calcium (Ca2+) measurements revealed that ShGPCR1 predominantly localized to the plasma membrane and enhanced intracellular Ca2+ levels in response to GTP, respectively. Furthermore, constitutive overexpression of ShGPCR1 in sugarcane conferred tolerance to the three stressors. The stress-tolerance phenotype of the transgenic lines corresponded with activation of multiple drought-, salinity-, and cold-stress marker genes, such as Saccharum spp. LATE EMBRYOGENESIS ABUNDANT, DEHYDRIN, DROUGHT RESPONSIVE 4, GALACTINOL SYNTHASE, ETHYLENE RESPONSIVE FACTOR 3, SALT OVERLY SENSITIVE 1, VACUOLAR Na+/H+ ANTIPORTER 1, NAM/ATAF1/2/CUC2, COLD RESPONSIVE FACTOR 2, and ALCOHOL DEHYDROGENASE 3. We suggest that ShGPCR1 plays a key role in conferring tolerance to multiple abiotic stresses, and the engineered lines may be useful to enhance sugarcane production in marginal environments with fewer resources.
Highlights
Membrane-localized receptors play key roles in signal perception and transduction in downstream intra- and intercellular signaling networks (Khatri et al, 2012)
We report the identification, isolation, and functional characterization of a G-protein-coupled receptors (GPCRs) gene from Saccharum spp. hybrids (ShGPCR1) and show that genetically modified sugarcane plants overexpressing ShGPCR1:OE and NT leaf cells (ShGPCR1) are more resistant to drought, salinity, and cold stresses
To examine the accumulation of ShGPCR1 transcripts in the sugarcane ShGPCR1-OE lines, we performed RT-PCR on a ProFlex PCR System in a total reaction volume of 25 μl using 1 μl of cDNA, 0.1 μM of each target-specific primer, and 1.0 U of Taq DNA polymerase and ThermoPolTM buffer (New England BioLabs)
Summary
Membrane-localized receptors play key roles in signal perception and transduction in downstream intra- and intercellular signaling networks (Khatri et al, 2012). Members of the GPCR protein family share a common central core domain composed of 7–9 transmembrane (TM) helices connected by three N-terminal extracellular loops and three C-terminal intracellular loops, a distinct characteristic not seen in other classes of cell membrane receptors (Ding et al, 2013; Ofoe, 2021). Studies of the few plant GPCRs characterized to date (Pandey and Assmann, 2004; Liu et al, 2007; Pandey et al, 2009; Ma et al, 2015) have provided evidence that plants use similar mechanisms to other eukaryotes to regulate G-protein-mediated signaling, the signal inputs are different. GPCRs are involved in diverse abiotic stress responses. The role of GPCRs in crop plants, such as rice (Oryza sativa) and maize (Zea mays), has been investigated (Ma et al, 2015; Ferrero-Serrano and Assmann, 2016)
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