Relative Expression of a Salinity Stress-Responsive Na+/H+ Exchanger (NHX) in Root and Leaf Tissues of the African Leafy Vegetable, Amaranthus dubius

Abstract

Amaranthus dubius, an African leafy vegetable (ALV), is an easy-to-grow, annual shrub and a highly nutritious food source, containing elevated levels of essential nutrients in the leaves. Many ALVs, including A. dubius, can tolerate salinity stress, enabling their cultivation on marginal land. However, the widespread propagation of A. dubius as a stable food source has thus far not been realised due partially to the high frequency at which hybridisation occurs, resulting in high genotypic and phenotypic variability. Therefore, to increase the agricultural output capacity of this species on salt-affected marginal lands, it is important to screen, select and then clonally propagate the identified salinity-tolerant genotypes to ensure true-to-type fidelity in the regenerated population. It is also important, thereafter, to elucidate their underlying gene expression of the stress response. The present study exposed 4-week-old A. dubius seedlings to 100, 200 and 400 mM NaCl to determine their degree of salt tolerance. Genotypes were then screened, selected and clonally propagated through cuttings, based on high growth rates and biomass, and salt tolerance. Generally, growth and physiological parameters decreased as substrate salinity increased. However, individual salt-stressed genotypes demonstrated similar vigour to nonstressed plants and were able to maintain total protein and chlorophyll concentrations despite increasing salinity. The relative expression of an NHX1-like transcript was quantified in 15 genotypes using degenerately primed real-time qPCR. The relative expression of the putative NHX1 gene was 6.7 times greater in root tissues of seedlings treated with 400 mM NaCl (10.7 ± 1.8) compared to the roots of untreated seedlings (1.6 ± 1.3), and 2.8-fold more than leaf tissues harvested from seedlings treated with 400 mM NaCl. Furthermore, the relative electrical conductivity (EC) of root tissues was 10 times greater than the EC of leaf tissues from the same 400 mM NaCl treatment. Numerous genotypes yielded similar chlorophyll content between 200 and 400 mM NaCl treatments, with genotypes salinity-1 (S1) (3.5 ± 0.2 μg/cm²) and S34 (4.0 ± 0.4 μg/cm²) having the highest concentrations of chlorophyll in the 400 mM group, which was positively correlated with total protein content. Following micropropagation through direct organogenesis, selected clones maintained true-to-type traits such as similar chlorophyll, protein and NHX1-like expression as their parent plants when exposed to 400 mM NaCl. This study revealed that some genotypes demonstrated salt stress tolerance capabilities rivalling established halophytes by regulating the constitutive or inducible expression of an NHX1-like protein in roots and leaves. The correlation between protein content and NHX1-like expression was nonlinear and nonproportional, demonstrating the complexity of this response and necessitating further exploration of specific protein families or functional groups conferring salinity tolerance in this species.