Dehydration-responsive element-binding proteins (DREBs) belong to members of the AP2/ERF transcription factor superfamily, which has been reported to involve various abiotic-stress responses and tolerance in plants. However, research on the DREB -family is still limited in alfalfa ( Medicago sativa L.), a forage legume cultivated worldwide. The recent genome-sequence release of the alfalfa cultivar “XinJiangDaYe” allowed us to identify 172 DREBs by a multi-step homolog search. The phylogenetic analysis indicated that such MsDREB s could be classified into 5 groups, namely A-1 (56 members), A-2 (39), A-3 (3), A-4 (61) and 13 (A-5 (13), thus adding substantial new members to the DREB -family in alfalfa. Furthermore, a comprehensive survey in silico of conserved motif, gene structure, molecular weight, and isoelectric point (pI) as well as gene expression was conducted. The resulting data showed that, for cold-stress response, 33 differentially expressed MsDREB s were identified with a threshold of Log2-fold > 1, and most of which were transcriptionally upregulated within 48 h during a cold treatment(s). Moreover, the expression profiling of MsDREB s from two ecotypes of alfalfa subspecies i.e. M. sativa ssp. falcata (F56, from a colder region of Central Asia) and M. sativa ssp. sativa (B47, from Near East) revealed that most of the cold-stress responsive MsDREBs exhibited a significantly lower expression in F56, leading to a proposal of the existence of a distinct mechanism(s) for cold tolerance regulated by DREB-related action, which would have been evolved in alfalfa with a genotypic specificity. Additionally, by examining the transcriptome of a freezing-tolerance species ( M. sativa cv. Zhaodong), eight DREBs were found to be implicated in a long-term freezing-stress adaptation with a great potential. Taken together, the current genome-wide identification in alfalfa points to the importance of some MsDREB s in the cold-stress response, providing some promising molecular targets to be functionally characterized for the improvement of cold tolerance in crops including alfalfa.

Polyamine(s) (PA, PAs), a sort of N-containing and polycationic compound synthesized in almost all organisms, has been recently paid considerable attention due to its multifarious actions in the potent modulation of plant growth, development, and response to abiotic/biotic stresses. PAs in cells/tissues occur mainly in free or (non- or) conjugated forms by binding to various molecules including DNA/RNA, proteins, and (membrane-)phospholipids, thus regulating diverse molecular and cellular processes as shown mostly in animals. Although many studies have reported that an increase in internal PA may be beneficial to plant growth under abiotic conditions, leading to a suggestion of improving plant stress adaption by the elevation of endogenous PA via supply or molecular engineering of its biosynthesis, such achievements focus mainly on PA homeostasis/metabolism rather than PA-mediated molecular/cellular signaling cascades. In this study, to advance our understanding of PA biological actions important for plant stress acclimation, we gathered some significant research data to succinctly describe and discuss, in general, PA synthesis/catabolism, as well as PA as an internal ameliorator to regulate stress adaptions. Particularly, for the recently uncovered phenomenon of urea-antagonized NH4+-stress, from a molecular and physiological perspective, we rationally proposed the possibility of the existence of PA-facilitated signal transduction pathways in plant tolerance to NH4+-stress. This may be a more interesting issue for in-depth understanding of PA-involved growth acclimation to miscellaneous stresses in future studies.

Although recent physiological studies demonstrate that flue-cured tobacco preferentially utilizes nitrate (NO3−) or ammonium nitrate (NH4NO3), and possesses both high- and low-affinity uptake systems for NO3−, little is known about the molecular component(s) responsible for acquisition and translocation in this crop. Here we provide experimental data showing that NtNRT1.1B with a 1,785-bp coding sequence exhibited a function in mediating NO3− transport associated with tobacco growth on NO3− nutrition. Heterologous expression of NtNRT1.1B in the NO3− uptake-defective yeast Hp△ynt1 enabled a growth recovery of the mutant on 0.5 mM NO3−, suggesting a possible molecular function of NtNRT1.1B in the import of NO3− into cells. Transient expression of NtNRT1.1B::green fluorescent protein (GFP) in tobacco leaf cells revealed that NtNRT1.1B targeted mainly the plasma membrane, indicating the possibility of NO3− permeation across cell membranes via NtNRT1.1B. Furthermore, promoter activity assays using a GFP marker clearly indicated that NtNRT1.1B transcription in roots may be down-regulated by N starvation and induced by N resupply, including NO3−, after 3 days’ N depletion. Significantly, constitutive overexpression of NtNRT1.1B could remarkably enhance tobacco growth by showing a higher accumulation of biomass and total N, NO3−, and even NH4+ in plants supplied with NO3−; this NtNRT1.1B-facilitated N acquisition/accumulation could be strengthened by short-term 15N-NO3− root influx assays, which showed 15%–20% higher NO3− deposition in NtNRT1.1B-overexpressors as well as a high affinity of NtNRT1.1B for NO3− at a Km of around 30–45 µM. Together with the detection of NtNRT1.1B promoter activity in the root stele and shoot–stem vascular tissues, and higher NO3− in both xylem exudate and the apoplastic washing fluid of NtNRT1.1B-transgenic lines, NtNRT1.1B could be considered as a valuable molecular breeding target aiming at improving crop N-use efficiency by manipulating the absorption and long-distance distribution/transport of nitrate, thus adding a new functional homolog as a nitrate permease to the plant NRT1 family.

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