Switchgrass (Panicum virgatum L.) has been increasingly recognized as one of the most valuable perennial bioenergy crop. To improve its biomass production, especially under salt stress, we isolated a putative vacuolar Na+ (K+)/H+ antiporter gene from switchgrass and designated as PvNHX1. Subcellular localization revealed that this protein was localized mainly on the vacuole membrane. The PvNHX1 was found to be expressed throughout the entire growth period of switchgrass, exhibited preferentially expressed in the leaf tissue, and highly induced by salt stress. Transgenic switchgrass overexpressing PvNHX1 showed obvious advantages with respect to plant height and leaf development compared to the wild-type (WT) and transgenic control (EV, expressing the empty vector only) plants, suggesting PvNHX1 may serve as a promoter in switchgrass growth and development. Moreover, transgenic switchgrass were more tolerant than control plants with better growth-related phenotypes (higher shoot height, larger stem diameter, longer leaf length, and width) and physiological capacities (increased proline accumulation, reduced malondialdehyde production, preserved cell membrane integrity, etc.) under high salinity stress. Furthermore, the genes related to cell growth, flowering, and potassium transporters in transgenic switchgrass exhibited a different expression profiles when compared to the control plants, indicating a pivotal function of PvNHX1 in cell expansion and K+ homeostasis. Taken together, PvNHX1 is essential for normal plant growth and development, and play an important role in the response to salt stress by improving K+ accumulation. Our data provide a valuable foundation for further researches on the molecular mechanism and physiological roles of NHXs in plants.

Understanding the regulation of proline metabolism necessitates the suppression of two D 1-pyrroline-5-carboxylate synthetase enzyme (P5CS) genes performed in switchgrass (Panicum virgatum L.). The results reveal that overexpressing PvP5CS1 and PvP5CS2 increased salt tolerance. Additionally, transcript levels of spermidine (Spd) and spermine (Spm) synthesis and metabolism related genes were upregulated in PvP5CS OEtransgenic plants and downregulated in the PvP5CS RNAi transformants. According to salt stress assay and the measurement of transcript levels of Polyamines (PAs) metabolism-related genes, P5CS enzyme may not only be the key regulator of proline biosynthesis in switchgrass, but it may also indirectly affect the entire subset of pathway for ornithine to proline or to putrescine (Put). Furthermore, application of proline prompted expression levels of Spd and Spm synthesis and metabolism-related genes in both PvP5CS-RNAi and WT plants, but transcript levels were even lower in PvP5CS-RNAi compared to WT plants under salt stress condition. These results suggested that exogenous proline could accelerate polyamines metabolisms under salt stress. Nevertheless, the enzymes involved in this process and the potential functions remain poorly understood. Thus, the aim of this study is to reveal how proline functions with PAs metabolism under salt stress in switchgrass.

Genetic improvement through overexpressing PuP5CS in switchgrass is feasible for enhancing plant salt stress tolerance. Switchgrass (Panicum virgatum L.) has developed into a dedicated bioenergy crop. To improve the biomass production of switchgrass grown on different types of soil, abiotic stress tolerance traits are considered for its genetic improvement. Proline accumulation is a widespread response when plants are subjected to abiotic stresses such as drought, cold and salinity. In plants, P5CS gene encodes the key regulatory enzyme that plays a crucial role in proline biosynthesis. Here, we introduced the PuP5CS gene (from Puccinellia chinampoensis) into switchgrass by Agrobacterium-mediated transformation. Transgenic lines overexpressing the PuP5CS gene showed phenotypic advantages, in leaf width, internode diameter, internode length, tiller numbers and precocious flowering under normal conditions, and the transgenic lines displayed better regenerative capacity in forming more tillers after harvest. Moreover, the PuP5CS gene enhanced the salt tolerance of transgenic switchgrass by altering a wide range of physiological responses. In accordance with the physiological results, histological analysis of cross sections through the leaf blade showed that the areas of bulliform cells and bundle sheath cells were significantly increased in PuP5CS-overexpressing leaves. The expression levels of ROS scavenging-associated genes in transgenic plants were higher than in control plants under salt stress. The results show that genetic improvement through overexpressing PuP5CS in switchgrass is feasible for enhancing plant stress tolerance.

Melatonin is a well-known bioactive molecule with an array of health-promoting properties. Here, we detected the physiological function of melatonin in transgenic switchgrass overexpressing the homologous sheep arylalkylamine N-acetyltransferase and hydroxyindole O-methyltransferase genes, which catalyze the last two steps of melatonin synthesis. Compared to the wild-type (WT) and transgenic control (EV, expressing the empty vector only) plants, the transgenic switchgrass showed higher melatonin levels. Melatonin was detected in almost all switchgrass tissues, and relatively higher levels were detected in the roots and stems. Besides, melatonin showed diurnal or circadian rhythms in switchgrass similar to that in other species. Furthermore, we also found that melatonin positively affected switchgrass growth, flowering and salt tolerance. The genes related to flowering (APL3, SL1, FT1, FLP3, MADS6 and MADS15) and salt stress resistance (PvNHX1) in transgenic switchgrass exhibited a different expression profiles when compared to the control plants. Our study provided valuable findings that melatonin functions as a promoter in the regulation of switchgrass growth, flowering and salt tolerance.

Salt is among the most harmful agents that negatively influences crop yield. Alfalfa is an important perennial forage crop that exhibits wide cultivar variations in salt tolerance. Developing salt‐tolerant alfalfa plants is a promising way to utilize salinized land. A comprehensive method was developed to achieve reliable and effective evaluation of alfalfa salt resistance. This included principal components, membership functions and cluster and stepwise regression analyses. These were used to analyse the salt tolerance coefficients of 14 traits and to evaluate 20 diverse alfalfa cultivars at the seedling stage. The various morphological root parameters of six alfalfa cultivars with contrasting salt tolerance were also tested by a scanning apparatus. According to the comprehensive evaluation value (D value), one highly salt‐tolerant, two salt‐tolerant, four moderately salt‐tolerant and 13 salt‐sensitive alfalfa cultivars were screened. A mathematical equation for the evaluation of alfalfa salt tolerance was established: D′ = −0.126 + 0.667SFW + 0.377SDW + 1.089K+/Na+ + 0.172SFW/RFW (R2 = 0.988; average forecast accuracy of 96.95%), where four indices were closely related to the salt tolerance: shoot fresh weight, ratio of shoot fresh weight to root fresh weight, shoot dry weight and ratio of K+ to Na+ in the shoot. We also found that SSA correlated strongly with SFW, SDW, K+/Na+, D values, while SRV correlated obviously with SFW, SFW/RFW and D values after 150 mm NaCl treatment. In conclusion, the SFW, K+/Na+, SDW, SFW/RFW, SSA and SRV could be used as indicators of salt tolerance in alfalfa seedlings grown under 150 mm NaCl treatment.