Abscisic acid influences ammonium transport via regulation of kinase CIPK23 and ammonium transporters.

Ammonium uptake at plant roots is regulated at the transcriptional, posttranscriptional, and posttranslational levels. Phosphorylation by the protein kinase calcineurin B-like protein (CBL)-interacting protein kinase 23 (CIPK23) transiently inactivates ammonium transporters (AMT1s), but the phosphatases activating AMT1s remain unknown. Here, we identified the PP2C phosphatase abscisic acid (ABA) insensitive 1 (ABI1) as an activator of AMT1s in Arabidopsis (Arabidopsis thaliana). We showed that high external ammonium concentrations elevate the level of the stress phytohormone ABA, possibly by de-glycosylation. Active ABA was sensed by ABI1-PYR1-like () complexes followed by the inactivation of ABI1, in turn activating CIPK23. Under favorable growth conditions, ABI1 reduced AMT1;1 and AMT1;2 phosphorylation, both by binding and inactivating CIPK23. ABI1 further directly interacted with AMT1;1 and AMT1;2, which would be a prerequisite for dephosphorylation of the transporter by ABI1. Thus, ABI1 is a positive regulator of ammonium uptake, coupling nutrient acquisition to abiotic stress signaling. Elevated ABA reduces ammonium uptake during stress situations, such as ammonium toxicity, whereas ABI1 reactivates AMT1s under favorable growth conditions.

Concentration-dependent physiological and transcriptional adaptations of wheat seedlings to ammonium.

Conventional wheat production utilizes fertilizers of various nitrogen forms. Sole ammonium nutrition has been shown to improve grain quality, despite the potential toxic effects of ammonium at elevated concentrations. We therefore investigated the responses of young seedlings of winter wheat to different nitrogen sources (NH4 NO3 = NN, NH4 Cl = NNH4 + and KNO3 = NNO3 - ). Growth with ammonium-nitrate was superior. However, an elevated concentration of sole ammonium caused severe toxicity symptoms and significant decreases in biomass accumulation. We addressed the molecular background of the ammonium uptake by gathering an overview of the ammonium transporter (AMT) of wheat (Triticum aestivum) and characterized the putative high-affinity TaAMT1 transporters. TaAMT1;1 and TaAMT1;2 were both active in yeast and Xenopus laevis oocytes and showed saturating high-affinity ammonium transport characteristics. Interestingly, nitrogen starvation, as well as ammonium resupply to starved seedlings triggered an increase in the expression of the TaAMT1s. The presence of nitrate seamlessly repressed their expression. We conclude that wheat showed the ability to respond robustly to sole ammonium supply by adopting distinct physiological and transcriptional responses.

A pore-occluding phenylalanine gate prevents ion slippage through plant ammonium transporters.

Throughout all kingdoms of life, highly conserved transport proteins mediate the passage of ammonium across membranes. These transporters share a high homology and a common pore structure. Whether NH3, NH4+ or NH3 + H+ is the molecularly transported substrate, still remains unclear for distinct proteins. High-resolution protein structures of several ammonium transporters suggested two conserved pore domains, an external NH4+ recruitment site and a pore-occluding twin phenylalanine gate, to take over a crucial role in substrate determination and selectivity. Here, we show that while the external recruitment site seems essential for AtAMT1;2 function, single mutants of the double phenylalanine gate were not reduced in their ammonium transport capacity. Despite an unchanged ammonium transport rate, a single mutant of the inner phenylalanine showed reduced N-isotope selection that was proposed to be associated with ammonium deprotonation during transport. Even though ammonium might pass the mutant AMT pore in the ionic form, the transporter still excluded potassium ions from being transported. Our results, highlight the importance of the twin phenylalanine gate in blocking uncontrolled ammonium ion flux.

Agrobacterium tumefaciens-mediated transformation of common bean (Phaseolus vulgaris) var. Brunca

Common bean is a crop recalcitrant to in vitro regeneration and therefore it lacks an efficient transformation protocol that can be reproduced using A. tumefaciens. The main goal of this study was to establish a protocol for A. tumefaciens mediated transformation of Phaseolus vulgaris var. Brunca by marker genes (gusA and nptII) together with the gene for trehalose-6-phosphate synthase from Saccharomyces cerevisiae (TPS1) used in other species to increase tolerance to abiotic stress. The β-glucuronidase activity was detected in 45 % of the LBA4404 ElectroMAX® pCAMBIA1301 infected explants. Transformed explants regenerated new shoots after four to five months period in a kanamycin rich media. Surviving plants were evaluated by PCR and presented an 0.5 % efficiency of transformation. The established protocol for genetic transformation of common bean has two additional advantages with respect to previous reports: (1) it allows for obtaining transformed regenerants and (2) the genetic transformation was stable for the selective gene.

El frijol común en un cultivo recalcitrante a la regeneración in vitro y se carece de un protocolo eficiente y reproducible de transformación genética usando A. tumefaciens. Desarrollamos un protocolo de transformación genética mediada por A. tumefaciens de frijol común variedad Brunca utilizando genes marcadores (gusA y nptII) junto con el gen de la trehalosa-6-fosfato sintasa de levadura (TPS1) utilizado para incrementar tolerancia a estrés abiótico. La actividad de la β-glucoronidasa fue detectada en 45 % de los explantes infectados con la cepa LBA4404 de A. tumefaciens transformada con pCAMBIA1301. Después de 4 o 5 meses se regeneraron tallos en un medio adicionado con kanamicina. Los explantes supervivientes se evaluaron mediante PCR y presentaron una eficiencia de transformación de 0.5 %. El protocolo de transformación genética de frijol común establecido tiene dos ventajas adicionales con respecto a los reportes previos: (1) permite la obtención de regenerares transformados y (2) la transformación genética fue estable para el gen selectivo.

Expression analysis of two SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) genes during in vitro morphogenesis in Spanish cedar (Cedrela odorata L.).

Somatic embryogenesis (SE) is one of the most important steps during regeneration, but the molecular mechanism of SE remains unclear for Cedrela odorata. SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) is one of the genes associated with induction of SE and is considered a marker of cells competent to form somatic embryos. Our objective was to clone and characterize the SERK1 and SERK2 gene homologues and analyze their expression patterns during in vitro morphogenesis in Spanish cedar. CoSERK1 and CoSERK2 were isolated from cedar, both share domains characteristic of the SERK family, including leucine-rich repeats, a proline-rich motif, a transmembrane domain, and kinase domains. Embryogenic cultures were established from callus cultures induced on medium supplemented with 1 mg/L dicamba. Histological sections were studied to determine the embryogenic nature of the samples. The CoSERK1 gene was highly expressed during the acquisition of embryogenic competence. The expression level of SERK1 was lower in non-embryogenic tissues and organs than in embryogenic calli, and it was higher in 3-week old embryogenic calli. CoSERK2 gene was highly expressed in leaves and shoots but no difference in expression was obtained between somatic and embryogenic tissues. These results suggest that the expression of CoSERK1 is associated with somatic embryogenesis induction and could be used as a potential marker to monitor the transition from competent to embryogenic cells and tissues in Spanish cedar.