Identification of Differential Drought Response Mechanisms in Medicago sativa subsp. sativa and falcata through Comparative Assessments at the Physiological, Biochemical, and Transcriptional Levels

Abstract

Alfalfa (Medicago sativa L.) is an extensively grown perennial forage legume, and although it is relatively drought tolerant, it consumes high amounts of water and depends upon irrigation in many regions. Given the progressive decline in water available for irrigation, as well as an escalation in climate change-related droughts, there is a critical need to develop alfalfa cultivars with improved drought resilience. M. sativa subsp. falcata is a close relative of the predominantly cultivated M. sativa subsp. sativa, and certain accessions have been demonstrated to exhibit superior performance under drought. As such, we endeavoured to carry out comparative physiological, biochemical, and transcriptomic evaluations of an as of yet unstudied drought-tolerant M. sativa subsp. falcata accession (PI 641381) and a relatively drought-susceptible M. sativa subsp. sativa cultivar (Beaver) to increase our understanding of the molecular mechanisms behind the enhanced ability of falcata to withstand water deficiency. Our findings indicate that unlike the small number of falcata genotypes assessed previously, falcata PI 641381 may exploit smaller, thicker leaves, as well as an increase in the baseline transcriptional levels of genes encoding particular transcription factors, protective proteins, and enzymes involved in the biosynthesis of stress-related compounds. These findings imply that different falcata accessions/genotypes may employ distinct drought response mechanisms, and the study provides a suite of candidate genes to facilitate the breeding of alfalfa with enhanced drought resilience in the future.

The CRISPR/Cas9-Mediated Modulation of SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 8 in Alfalfa Leads to Distinct Phenotypic Outcomes

“…This improvement in drought tolerance across SPL8-gRNA1 genotypes does not appear to be a direct result of decreased water loss from their leaves ( Figure 7 ). However, it is possible that reduced leaf size, which is known to be associated with superior drought tolerance in plants ( Quan et al, 2016 ; Singer et al, 2021b ), could be contributing to an overall reduction in water loss at the plant level. Intriguingly, enhancements in drought tolerance in RNAi alfalfa genotypes in which other SPL genes were down-regulated have been suggested to occur, at least in part, through the up-regulation of anthocyanin biosynthetic genes, thus resulting in an increased accumulation of this secondary metabolite and a consequent enhancement in ROS scavenging capacity ( Gou et al, 2018 ; Feyissa et al, 2019 ; Hanly et al, 2020 ).…”

“…Detached leaf water loss assays were carried out essentially as described previously ( Singer et al, 2021b ) using fully expanded trifoliate leaves (third from the shoot tip) from eight biological replicate plants of each SPL8-gRNA1 genotype (two leaves per plant; sixteen replicates total for each) and seventeen biological replicate control plants (derived from three independent transformants with two leaves sampled per plant; thirty-four replicates total) under well-watered conditions. Assays were carried out by weighing leaves immediately upon harvest (W initial ), placing them on a benchtop, and then weighing every 30 min for 240 min.…”

Identification of QTL and candidate genes associated with biomass yield and Feed Quality in response to water deficit in alfalfa (Medicago sativa L.) using linkage mapping and RNA-Seq

“…Ascertaining the DEGs that change upon water stress in alfalfa is of crucial in understanding the genetic base of drought tolerance. In alfalfa, although drought-responsive DEGs have been reported in previous studies ( Ma et al., 2021 ; Singer et al., 2021 ), there were some limitations due to the lack of a reference genome. The release of a high-quality alfalfa reference genome has given new power to transcriptional analysis ( Chen et al., 2020b ).…”

Combining QTL mapping and RNA-Seq Unravels candidate genes for Alfalfa (Medicago sativa L.) leaf development

“…‘Beaver’ and ‘PI 641,381’, appeared morphologically similar overall, and no significant differences were noted between genotypes in terms of plant height, aboveground biomass, or root length. ‘PI 641,381’ had a significant reduction in leaf area compared to 'Beaver' [ 37 ].…”

“…sativa subsp. falcata accession PI 641,381) was performed [ 37 ]. Significant difference was noted between genotypes in terms of leaf size, while they had no difference in plant height and biomass.…”