Morphological, physiological and molecular characterization of drought tolerance in cassava (Manihot esculenta Crantz)

Abstract

Cassava (Manihot esculenta Crantz) is inherently drought tolerant. Nevertheless, substantial genotypic variation exists for this trait. Characterization of these morphological, physiological and molecular differences establish an essential foundation for future development of drought associated molecular markers for cassava. In the present study, multi-seasonal and locational field-based drought trials were carried out in Kenya with the purpose of identifying drought tolerant and drought susceptible cassava genotypes. These were then subjected to further physiological and molecular characterization under controlled water deficit assays at ETH-Zurich, Switzerland. Field drought stress generally reduced cassava vegetative growth and productivity. In addition to other phenotypic parameters, storage root fresh weight was used as a primary criterion to discriminate between drought tolerant and drought susceptible genotypes. Selected drought tolerant cassava genotypes were M98/0068, 94/0039, 95/0306, 98/0002 and I92/0067, while drought susceptible genotypes comprised PYT, 92/0427, TME-419, I96/1439 and 96/0409. Under water deficit treatment across seasons and regardless of the locations, tolerant candidates bulked above average fresh root weight, whereas susceptible candidates performed below average. Field data also showed significant and positive correlations between root yield, harvest index, number of storage roots and above ground biomass but not leaf retention. The genotypes were physiologically assessed under controlled water deficit assays through stomatal conductance (Gs). More pronounced Gs decrease was observed from one tolerant (94/0039) and two susceptible (TME-419 and 92/0427) genotypes, lower Gs decrease recorded from susceptible I96/1439, while tolerant 98/0002, I92/0067, M98/0068 and 95/0306 generally showed moderate Gs decrease. Cessation of leaf Gs after 10 days of water deficit (WD) and increased Gs rates after 1 day of re-watering (WDR) treatments respectively mimicked drought-induced stomatal closure and stomatal re-opening. Thus we hypothesized drought avoidance mechanism in cassava. Genes with roles or functions in molecular pathways such as phosphatidic acid biosynthesis (PLDα1, ABI1, GPA1), positive and negative regulators of ABA-induced stomatal closure in 9 guard cells (KAT1, ERA1, PYR1, Aquaporins, PLDα1, ABI1), polyamine biosynthesis (ADC2), transcription factors (TFs) involved in ABA signalling and stomatal movement (MYB44, MYB60, NFYA5), Caleosin biosynthesis (RD20/AtCLO3) and other transcription factors (NAC002 and ABF2) were selected from literature reviews. Using RT-qPCR, mathematical model developed by Pfaffl (2001) and Student Paired Sample t-test, the relative expression profiles of these genes were evaluated from three biological replicates of two drought tolerant (94/0039 & 95/0306) and two drought susceptible (I96/1439 & 92/0427) cassava genotypes subjected to well-watered (WW), WD and WDR treatments. The set of genes that showed consistent differential regulation between tolerant and susceptible cassava genotypes under WD and WDR could provide further molecular insights into cassava’s drought response and subsequent recovery from the effects of drought, were ABI1, GPA1, Aquaporin (RWC3), MYB44 and MYB60. Other genes such as PLDα1, PYR1, ADC2, ATAF1, RD20, NFYA5 and ABF2 exhibited discrepant or inconsistent expression between treatments and cassava genotypes. Genes that provide insights into pathways regulating stomatal function may be used to produce plants with enhanced water-usage efficiency, maintain high yield and drought tolerance (Wang et al., 2007; Cominelli et al., 2010). Most of the genes we characterized in this project were involved in stomatal aperture movement. In general, the genes could primarily be used to interpret tolerance to drought or water stress in tolerant cassava genotypes. Further gene validation through genetic engineering or mutants would be required to conclude on their contribution to drought tolerance trait in cassava.