SENTINEL PLANTS TO IMPROVE NUTRIENT USE EFFICIENCY: LIVING TOOLS FOR NONDESTRUCTIVE ANALYSIS UNDER FIELD CONDITIONS

Sulfur is an essential element for plant growth which availability affects both quantitative and qualitative traits of crop yield. Our aim was to generate specific bioassays based on the use of “sentinel plants” to quickly determine sulfate bioavailability and/or crop nutritional status which may represent a reliable and efficient strategy to obtain valuable, timely and low-cost information about changes in sulfate availabilities and nutritional requirements in a crop system. The characterization of two Arabidopsis gene-trap lines (FLAG and 718 lines) allowed the identification of two portions (440 and 1331-bp respectively) of the intergenic region (between At1g12030 and At1g12040), controlling as a bidirectional promoter the expression of both At1g12030 and GUS under sulfur limitation. Thus, these lines are able to provide information about the sulfur nutritional status of the plant and/or the sulfate concentration in the growing medium. For this purpose, the two lines were grown in agar plates under a continuous sulfate gradient ranging from 0 to 150 μM in order to describe the growth of both roots and shoots as a function of sulfate external concentration and to determine the critical concentration of sulfate (i.e. the minimum concentration of sulfate necessary to achieve maximum biomass) in the growing medium. The main results indicate that the pilot lines are able to correctly indicate the critical concentration of sulfate in the external medium also in the presence of interfering metal ions (such as cadmium) able to increase the plant metabolic demand for sulfur. Others experiments were done to better characterize the pilot bioindicators. Firstly, experiments were conducted in order to evaluate if the lines sense the lack of sulfate in the growth medium or indicates an alteration of the S nutritional status in the plant. For this purpose the response of the lines was analyzed growing the plants in the presence of cysteine or glutathione as sole sulfur source. The presence of cysteine as sole sulfur source in the agar medium did not produce any GUS activity in both shoots and roots, differently from glutathione which instead induced GUS activity only in the shoots. The results indicate that the promoters of the Arabidopsis pilot bioindicators sense the metabolic effect produced by sulfur starvation and not the presence/absence of sulfate in the growing medium. Moreover, GUS expression was studied in lines grown in hydroponic media containing different sulfate concentrations. Interestingly, both lines showed GUS expression when grown in agar media in sulfur starved conditions, whereas in hydroponics, a significant GUS expression was detectable only in the FLAG line, for sulfate concentrations ≤ 5 μM. To better analyze this effect we tested the effect of sucrose, only present in agar media, on GUS expression and results show that expression level of GUS is influenced not only by sulfur but also by the presence of sucrose in the external medium. Finally, we tested the bidirectional promoter- 2 3 function of the 440-bp intergenic region shared by the divergent genes At1g12030 and GUS using GUS reporter gene in both orientations in stably transformed transgenic Arabidopsis. The sulfur responsive functional nature of the bidirectional promoter was evaluated in independent transgenic Arabidopsis lines. The 440-bp bidirectional promoter in both orientations shows GUS expression under sulfur limitation which was detected in the leaves and the root tissues indicating that 440-bp fragment is able to modulate the expression of GUS in two orientations under sulfur starvation. Then, to confirm the response of 440-bp promoters to sense the metabolic effect produced by sulfur starvation, transgenic Arabidopsis plants were grown in complete agar medium in the presence of cadmium. Results of this experiment showed that also Cd was able to induce a strong GUS activity in both leaves (vascular tissues) and root tips. In conclusion, we identified putative bidirectional promoters involved in modulating the expression of GUS reporter gene in response to sulfur deficient conditions and suitable for developing specific bioindicators to monitor plant S nutritional status.