Molecular identification and functional characterization of GhAMT1.3 in ammonium transport with a high affinity from cotton (Gossypium hirsutum L.)

Abstract

Ammonium (NH4+) represents a primary nitrogen source for many plants, its effective transport into and between tissues and further assimilation in cells determine greatly plant nitrogen use efficiency. However, biological components involved in NH4+ movement in woody plants are unclear. Here, we report kinetic evidence for cotton NH4+ uptake and molecular identification of certain NH4+ transporters (AMTs) from cotton (Gossypium hirustum). A substrate‐influx assay using 15N‐isotope revealed that cotton possessed a high‐affinity transport system with a Km of 58 μM for NH4+. Sequence analysis showed that GhAMT1.1–1.3 encoded respectively a membrane protein containing 485, 509 or 499 amino acids. Heterologous functionality test demonstrated that GhAMT1.1–1.3 expression mediated NH4+ permeation across the plasma membrane (PM) of yeast and/or Arabidopsis qko‐mutant cells, allowing a growth restoration of both mutants on NH4+. Quantitative PCR measurement showed that GhAMT1.3 was expressed in roots and leaves and markedly up‐regulated by N‐starvation, repressed by NH4+ resupply and regulated diurnally and age‐dependently, suggesting that GhAMT1.3 should be a N‐responsive gene. Importantly, GhAMT1.3 expression in Arabidopsis improved plant growth on NH4+ and enhanced total nitrogen accumulation (∼50% more), conforming with the observation of 2‐fold more NH4+ absorption by GhAMT1.3‐transformed qko plant roots during a 1‐h root influx period. Together with its targeting to the PM and saturated transport kinetics with a Km of 72 μM for NH4+, GhAMT1.3 is suggested to be a high‐affinity NH4+ permease that may play a significant role in cotton NH4+ acquisition and utilization, adding a new member in the plant AMT family.

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

“…Quantitative real-time PCR (qPCR) was performed with total RNA from split roots (3 biological replicates) derived from the experiment as described in Figure 4a; relative mRNA abundance of target genes relative to that of internal reference gene UBQ7 (set to 1. Sun et al 2018) in each sample was calculated (Schmittgen and Livak, 2008). The relative expression level (log2 fold change) was presented in a false color scale, where blue or red color indicates respectively a lowest or highest expression with an absolute mean value from −6 to 6.…”

“…Considering ammonium (NH 4 + ) nutrition, although it represents a major inorganic‐N source available to plants in most soils and its assimilation in cells consumes less metabolic energy than assimilation of nitrate (NO 3 − ), NH 4 + does exert paradoxical effects on plant growth and development. In low concentrations (e.g., <1 mM), roots of many species prefer absorption of NH 4 + rather than NO 3 − (Gazzarrini et al ., 1999; Sun et al ., 2018), whereas higher NH 4 + occurrence often induces stress or toxicity in plants (Britto and Kronzucker, 2002; Fan et al ., 2018). The growth inhibition caused by higher NH 4 + concentrations is often associated with symptoms like leaf chlorosis, tawny and short/less roots (Britto and Kronzucker, 2002; Fan et al ., 2018); and this toxicity is proposed to be physiologically linked to, for example, deficiency of mineral cations, perturbation of pH‐gradient across cellular membranes, oxidative stress with increased ROS generation, repression of protein N‐glycosylation, or futile NH 4 + cycling at the plasma membrane (PM) of root and shoot cells (Chaillou et al ., 1991; Szczerba et al ., 2008; Chen et al ., 2013; Esteban et al ., 2016; Liu and Wirén, 2017).…”

“…qPCR was carried out in 20 μl volume (consisting of 2 μl of 1:10 diluted original cDNAs, 200 nM of given gene-specific primers, and iQ™ SYBR Green Supermix from Bio-Rad using a Bio-Rad iCycler).The PCR program was as follows: 95 C for 5 min, 40 cycles of 30 s at 94 C, 30 s at 57 C, signal acquisition, and then a final melting curve of 65-95 C. The expression of genes involved in certain nitrogen transporters (e.g., AMT, DUR3, and NRT), arginine breakdown and polyamine biosynthesis was analyzed. The relative geneexpression level was normalized to that of two stable internal reference genes (i.e., UBQ7 [Ubiquitin7] and SAD1 [Stearoyl-Acyl-Carrier Protein Desaturase1]) were used as described inSun et al, 2018). qPCR reactions were conducted with 3 biological replicates, together with three "no template control" to check out cross contamination of reagents.…”

Function and Regulation of Ammonium Transporters in Plants

“…Additionally, many root-expressed functional AMTs have been identified, although only through one method among the three methods mentioned above. For example, overexpression of OsAMT1;1 in rice [34]; or NtAMT1.3 in tobacco [35]; or ZmAMT1.1a and ZmAMT1.3 [36], GhAMT1;3 [37], and PutAMT1;1 [38] in Arabidopsis can enhance the absorption of ammonium by plants under the condition of limiting ammonium supply, consequently increasing the biomass, which indicates that these proteins participate in obtaining ammonium from soil solution. The finding of either restoration of the growth of yeast mutants under restricted ammonium supply [1,26,[39][40][41][42][43][44][45] or mediation of ammonium influx in oocytes in a heterologous system [46][47][48][49][50][51][52][53] suggests that these root-expressed AMTs potentially obtain ammonium nitrogen from soil solution.…”

“…The leaf-expressed LjAMT1;1 and BnAMT1;2 are conceived to play a similar role as that of LeAMT1;2 [39,62]. OsAMT1;1 [33], NtAMT1.3 [35], ZmAMT1.1a [36], ZmAMT1.3 [36], and GhAMT1.3 [37] are all detected in the leaf. Amongst the three leaf types of young, mature, and senescent, transcript abundances of PtrAMT1;6 and PtrAMT3;1 are strongly induced in the senescent leaf.…”