Molecular identification and physiological functional analysis of NtNRT1.1B that mediated nitrate long-distance transport and improved plant growth when overexpressed in tobacco.

Although recent physiological studies demonstrate that flue-cured tobacco preferentially utilizes nitrate ( NO 3 - ) or ammonium nitrate (NH4NO3), and possesses both high- and low-affinity uptake systems for NO 3 - , 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 NO 3 - transport associated with tobacco growth on NO 3 - nutrition. Heterologous expression of NtNRT1.1B in the NO 3 - uptake-defective yeast Hp△ynt1 enabled a growth recovery of the mutant on 0.5 mM NO 3 - , suggesting a possible molecular function of NtNRT1.1B in the import of NO 3 - 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 NO 3 - 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 NO 3 - , 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, NO 3 - , and even NH 4 + in plants supplied with NO 3 - ; this NtNRT1.1B-facilitated N acquisition/accumulation could be strengthened by short-term 15N- NO 3 - root influx assays, which showed 15%-20% higher NO 3 - deposition in NtNRT1.1B-overexpressors as well as a high affinity of NtNRT1.1B for NO 3 - at a K m 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 NO 3 - 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.

Genome-wide identification and expression analysis of DREB genes in alfalfa (Medicago sativa) in response to cold stress.

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 MsDREBs 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 MsDREBs 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 MsDREBs 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 MsDREBs in the cold-stress response, providing some promising molecular targets to be functionally characterized for the improvement of cold tolerance in crops including alfalfa.