NITRATE TRANSPORT IN ROOTS OF GRAPEVINE AND MAIZE: A BIOCHEMICAL AND MOLECULAR CHARACTERIZATION

Nitrogen (N) is the most important inorganic nutrient for plants, and its availability is often a limit for plant growth and production in agricultural systems. Thus the use of fertilizers has become indispensable. However, N use efficiency of crops (NUE) is often low. Therefore, there is an urgent need to increase NUE in order to decrease the costs of crop production and environment pollution. Nitrate (NO3-) is the main source of N in agricultural aerobic soils and plants have evolved two distinct systems for its uptake: the HATS (High-affinity transport system) and the LATS (Low-affinity transport system). The former operates when the external concentration of NO3- is below 1 mM, the latter when the anion is above that concentration. Both systems are characterized by the presence of two components, one constitutive and one inducible by NO3-. Two gene families, NRT1 and NRT2, encoding NO3- transporters involved in LATS and HATS transport respectively, are involved in root NO3- uptake. In order to sustain the inducible high-affinity NO3- transport the presence of NAR2 proteins would seem to be necessary. The anion is taken up by the roots through an active transport across the plasma membrane, coupled to a favorable H+ electrochemical gradient created by the plasma membrane PM H+-ATPase. Most of the knowledge on molecular physiology on NO3- uptake comes from works carried out on Arabidopsis thaliana and some cereals, while the studies regarding NO3- transport in perennial fruit tree roots are very few. The main goal of this work is the study of the role of NRT2 transporters, NAR2 proteins and PM H+-ATPase in the high-affinity NO3- transport in grapevine roots (V. vinifera cv. Corvina-clone 48 grafted onto SO4 rootstock-clone 102), an important crop for its economic impact on Italian agriculture. Due to the practical difficulties linked to the use of grapevine cuttings as an experimental system, physiological and molecular characterization of NO3- uptake has also been performed on maize (Pioneer, hybrid PR33T56). In this species NO3- uptake mechanism is at least known in part even if more knowledge is necessary in order to describe the role of each molecular entity involved in the process. The HATS transport rate of 4-week-old grapevine root cuttings was studied. After one week of N-starvation, grapevine cuttings were treated with NO3- (5 mM) for different lengths of time and high affinity NO3- uptake was characterized using 15N as tracer and IRMS (Isotope Ratio Mass Spectrometry) analysis. The highest uptake rate occurred after 18 h NO3- treatment, whereas the PM H+-ATPase activity (measured in isolated membrane vesicles) increased linearly within the first 24 h of contact with NO3-. Using the Grape Genome Browser, the putative genes encoding NRT2, NAR2 and PM H+-ATPase genes were identified and their transcriptional levels were analyzed by Real Time RT-PCR during NO3- treatment. The expressions of one NRT2 gene and two H+-ATPase ones were up-regulated by NO3- resupply. The expression of NAR2 genes increased during root exposure to 5 mM NO3-. At protein level, the PM H+-ATPase quantity linearly increased for up to 24 hours after the contact with the anion and this data matched with the enzyme activity trend. With regard to maize, the HATS and LATS responses were investigated in four-day-old etiolated seedlings grown hydroponically and subsequently put in contact with a NO3- containing solution. The HATS uptake rate increased for up to 8 hours after NO3- treatment. Thereafter the uptake rate decreased reaching values similar to those of untreated roots after 24 h; whereas LATS transport activity behaved differently being down-regulated later than HATS. The putative NRT2, NAR2 and PM H+-ATPase genes were identified using maize genome. In order to study the gene expression of these genes during HATS response, transcriptional analyses were performed by Real Time RT-PCR approach. ZmNRT2.1 and ZmNRT2.2 genes showed the highest induction following the HATS activity trend. Regarding NAR2 genes, only the NAR2.1 gene expression was up-regulated by NO3- treatment and it matched with the ZmNRT2.1 and ZmNRT2.2 transcript profiles. This result suggests a possible NAR2.1 involvement in the NO3- uptake. Three of the five PM H+-ATPase genes expressed in the roots were positively modulated by the treatment. This data suggests the involvement of a new isogene, in addition to ZmHA2 and ZmHA4, is involved in the NO3- uptake phenomena. NRT2, NAR2 and PM H+-ATPase protein levels were investigated by Western blot analysis. NRT2 and NAR2 protein levels displayed similar profiles and they were only partially related to the time course of NO3- uptake. PM H+-ATPase protein levels were in line with the trend of enzymatic activity reaching their peak 15 h after the beginning of the treatment. A 2D-gel electrophoresis analysis was performed in order to study a possible regulation of the high-affinity transporters and PM H+-ATPase via protein-protein interactions. Maize microsome vesicles extracted at the highest transport activity time point were solubilized in 1% α-D-maltoside. Then they were subjected to a DERIPHAT-PAGE with a 4% - 12% gel gradient in first dimension and to a Tris-Tricine SDS-PAGE in second dimension. Preliminary results showed the presence of a high-weight molecular mass protein complex (approximately 900 kDa) detected by the anti-ZmNRT2 antibody possibly due to the formation of not specific protein complex during the solubilisation phases. The possibility that ZmNRT2s could belong to a large protein complex is much less sound. The results of the immunoblotting carried out with anti-PM H+-ATPase antibody suggest the presence of a monomeric and hexameric form of the enzyme in native conditions, both were more abundant in microsomes extracted from NO3- -induced roots.