Overexpression of a nitrate transporter NtNPF2.11 increases nitrogen accumulation and yield in tobacco.

Nitrogen (N) is the key essential macronutrient for crop growth and yield. Over-application of inorganic N fertilizer in fields generated serious environmental pollution and had a negative impact to human health. Therefore, improving crop N use efficiency (NUE) is helpful for sustainable agriculture. The biological functions of nitrogen transporters and regulators have been intensively studied in many crop species. However, only a few nitrogen transporters have been identified in tobacco to date. We reported the identification and functional characterization of a nitrate transporter NtNPF2.11 from tobacco (Nicotiana tabacum). qRT-PCR assay revealed that NtNPF2.11 was mainly expressed in leaf and vein. Under middle N (MN, 1.57 kg N/100 m2) and high N (HN, 2.02 kg N/100 m2) conditions, overexpression of NtNPF2.11 in tobacco greatly improved N utilization and biomass. Moreover, under middle N and high N conditions, the expression of genes for nitrate assimilation, such as NtNR1, NtNiR, NtGS and NtGOGAT, were upregulated in NtNPF2.11 overexpression plants. Compared with WT, overexpression of NtNPF2.11 increased potassium (K) accumulation under high N conditions. These results indicated that overexpression of NtNPF2.11 could increase tobacco yield, N and K accumulation under higher N conditions. Overall, these findings improve our understanding the function of NtNPF2.11 and provide useful gene for sustainable agriculture.

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.

Transcription factor NtWRKY33a modulates the biosynthesis of polyphenols by targeting NtMYB4 and NtHCT genes in tobacco.

There are abundant polyphenols in tobacco leaves mainly including chlorogenic acid (CGA), rutin, and scopoletin, which not only influence plant growth, development, and environmental adaptation, but also have a great impact on the industrial utilization of tobacco leaves. Few transcription factors regulating the biosynthesis of polyphenols have been identified in tobacco so far. In this study, two NtWRKY33 genes were identified from N. tabacum genome. NtWRKY33a showed higher transcriptional activity than NtWRKY33b, and encoded a nuclear localized protein. Overexpression and knock-out of NtWRKY33a gene revealed that NtWRKY33a inhibited the accumulation of rutin, scopoletin, and total polyphenols, but meanwhile promoted the biosynthesis of CGA. Chromatin immunoprecipitation and Dual-Luc assays indicated that NtWRKY33a could directly bind to the promoters of NtMYB4 and NtHCT, and thus induced the transcription of these two genes. The contents of polyphenols in ntwrky33a, ntmy4, and ntwrky33a/ntmyb4 mutants further confirmed that the repression of NtWRKY33a on the biosynthesis of rutin, scopoletin, and total polyphenols depends on the activity of NtMYB4. Moreover, the promotion of NtHCT by NtWRKY33a modulates the distribution of metabolism flux into the synthesis of CGA. Ectopic expression of NtWRKY33a inhibit the expression of NtSAUR14, NtSAUR59, NtSAUR66, NtIAA4, NtIAA17, and NtIAA19 genes, indicating that NtWRKY33a might be involved in the regulation of plant auxin response. Our study revealed new functions of NtWRKY33a in regulating the synthesis of polyphenols, and provided a promising target for manipulating polyphenols contents in tobacco.

Polyamine: A Potent Ameliorator for Plant Growth Response and Adaption to Abiotic Stresses Particularly the Ammonium Stress Antagonized by Urea.

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.

Evidence that exogenous urea acts as a potent cue to alleviate ammonium-inhibition of root system growth of cotton plant (Gossypium hirsutum).

Many plants grown with low-millimolar concentration of NH4 + as a sole nitrogen source develop NH4 + -toxicity symptoms. To date, crucial molecular identities and a practical approach involved in the improvement of plant NH4 + -tolerance remain largely unknown. By phenotyping of upland cotton grown on varied nitrogen forms, we came across a phenomenon that caused sub-millimolar concentrations of urea (e.g., up 50 µM) to repress the growth inhibition of roots and whole plant cultivated in a NH4 + -containing nutrient solution. A growth-recovery assay revealed that the relief in NH4 + -inhibited growth required only a short-term exposure (≧12 h) of the roots to urea, implying that urea could elicit an internal signaling and be involved in antagonizing NH4 + -sensitivity. Intriguingly, split-root experiments demonstrated that low urea occurrence in one root-half could efficaciously stimulate not only supplied root but also the root-half grown in NH4 + -solution without urea, indicating the existence of urea-triggered local and systemic long-distance signaling. In the split-root experiment we also observed high arginase activity, strong arginine reduction and remarkable upregulation of polyamine biosynthesis-related genes (ADC1/2, SPDS and SPMS). Therefore, we suggest that external urea might serve as an effective cue (signal molecule) in an arginine-/polyamine-related process for ameliorating NH4 + -suppressed root growth, providing a novel aspect for deeper exploring and understanding plant NH4 + -tolerance.