SYSTEMIC ALLOCATION OF TRACE ELEMENTS IN RICE

Several studies have shown that Cd, a non-essential and toxic metal, is taken up from soil and translocated in a root-to-shoot direction through transporters of essential elements such as Zn, suggesting that the two metal ions may compete for the same transporter protein on a membrane. However, the movement of Zn and Cd ions across several biological membranes involves a wide range of transport systems, each characterized by a specific selectivity. Although divergent results have been obtained, they suggest that Zn-independent pathways for Cd translocation in plants could be possible. The proteins belonging to the HMA (Heavy-Metal ATPases) family have been partially characterized as the main actors of the process of translocation of trace elements (essential or non-essential) to all organs of the plant. In particular, OsHMA2 is the main transport system so far described in rice as involved in the xylem loading of Zn and Cd, even though both its activity and function has not been unambiguously characterized. The research carried out in this PhD project took place in this context. Indeed, the general purpose was studying the main mechanisms involved in the systemic distribution of some trace elements in rice plants. In particular the activity was aimed at better understanding the Zn and Cd translocation pathways, and was focused on studying the possible competition between the two metal ions mainly for the root-to-shoot translocation, since these processes have been seen to be crucial in determining Cd accumulation in the shoots. Specifically, the aims of this study were: (i) to investigate the effects of the possible competition between Zn and Cd on their chelation and subcellular compartmentalization at the root level, thus in reducing the amount of the two metals potentially mobile through the plant. This was done using physiological techniques aimed at isolating and quantifying thiol based Zn- and/or Cd-binding complexes; (ii) to investigate the potential inhibitory effect exerted by Zn on Cd translocation in unstressed rice plants, performing a short-term positron-emitting tracer imaging system (PETIS) experiment using 107Cd as tracer; (iii) to identify genes encoding transporters involved in a putative Zn-insensitive Cd xylem loading, thus responsible for a possible Zn-independent Cd translocation pathway, by performing bioinformatic analysis. Our attention focused on the P1B-type ATPase (HMA) family in order to search for orthologs of the genes codifying the transporters that in the model plant Arabidopsis were found to mediate the xylem loading of Cd; (iv) to functional characterize the transporters encoded by the abovementioned genes by heterologous expression in Saccharomyces cerevisiae. A complete set of competition experiments were performed: in the first, rice plants (O. sativa L. ssp. japonica cv. Roma) were hydroponically grown and differentially exposed for a 10-day period to increasing Zn external concentrations, in the absence or presence of a steady amount of Cd, whilst, in the second, plants were exposed for 10 days to different Cd concentrations in the presence of a steady amount of Zn. The concentrations of Zn and Cd in xylem sap, roots and shoots were evaluated by inductively coupled plasma-mass spectrometry (ICP-MS), to determine their partitioning between plant organs. The results were related to the total Zn and Cd content in root fractions obtained by a sequential extraction procedure with buffer and acid. The procedure allowed to discriminate Zn and Cd ions potentially mobile (cationic) from those retained in complexes with thiol-peptides or other soluble molecules negatively charged in the extraction buffer (anionic), or tightly adsorbed to cellular matrices or apoplast components (acid soluble and ash); so, the last three fractions should be considered not available for root-to-shoot translocation. Moreover, the systemic movement of Cd in the whole rice plants was monitored by applying to the roots fresh marked (107Cd) culture solutions containing a steady amount of Cd and different concentrations of Zn in PETIS experiments. The main results clearly indicate the lack of a fully reciprocity considering the effect of Cd on Zn accumulation, and vice versa, since the accumulation of Zn in the shoot was significantly inhibited by Cd increases in all the analyzed conditions, whereas those of Cd was only partially impaired by Zn increases. Such a finding suggests that Cd ions may use at least two distinct pathways to be translocated from the root to the shoot. The first one \u2013 shared with Zn \u2013 is probably used for Zn translocation in physiological conditions, whilst the second one appears as a Zn-independent route that Cd may preferentially use when the first pathway is saturated with Zn. Moreover, the Zn-independent pathway seems constitutively expressed in rice plants since the partial inhibitory effect exerted by Zn on Cd translocation was also observed in short-term PETIS experiments performed with unstressed plants. Since OsHMA2 appears to play an important role in Zn/Cd root-to-shoot translocation, in this work we also contributed to elucidate some aspects related to the OsHMA2 transport activity and selectivity by comparing the inhibitory effects exerted by Zn or Cd on the growth of yeast cells expressing, or not, OsHMA2. The results indicate that OsHMA2 enhances Zn and Cd tolerance in yeast, so we can reasonably conclude that OsHMA2 may pump excess of cytosolic Zn or Cd into the apoplast and thus has all the requisites to be considered the xylem loading system potentially involved in mediating the translocation of Cd through the Zn-dependent pathway. In addition, this study represents one of the first examples of growth inhibition analysis applied to plant gene functional characterization. In conclusion, our data provide several evidence to support the hypothesis that at least two competing pathways may be interested in mediating root-to-shoot Cd translocation in rice. The first one, prevailing at relatively low Zn concentrations, could involve OsHMA2 as Zn2+/Cd2+ xylem loading system, while the second one appears to involve a Zn-independent system that still needs to be identified among the plethora of transporters involved in the metal homeostasis. The possible future identification of the transporter(s) responsible for the Zn-independent Cd translocation pathway(s) could allow the development of markers to select rice genotypes able to exclude Cd from the shoots. Furthermore, these activities could have important technological implications in the fields of food safety, especially in cases where the strategies used for containing Cd accumulation in the crops be founded on Zn fertilization

Gene Expression Biomarkers for Evaluating Nitrogen Nutritional Status in Rice

Over the last five decades the increase in rice yield has been associated with a dramatic increment in the use of nitrogen (N) fertilizers. Understanding of plant molecular responses to N is critical for our ability to improve the agricultural sustainability of rice cropping systems by developing a comprehensive approach that allows the selection of varieties with enhanced efficiency in their ability to use N and the development of new strategies to better manage N fertilization practices. In order to develop novel tools for real-time assessing of rice N nutritional status, we analyzed the expression profiles of seven putative gene expression markers in the shoots of rice plants grown under different N availability and environmental conditions. Our results suggest that five out of the seven genes analyzed have the potential to be used as agronomic tools to monitor and optimize the N nutritional status of rice

In vitro propagation and shoot encapsulation as tools for ex situ conservation of the aquatic plant Ludwigia palustris (L.) Ell.

Ludwigia palustris (L.) Ell. is an aquatic perennial herb present in several regions of Italy, which is one of its native countries. In this research, micropropagation and encapsulation protocols were established from axillary buds of L. palustris. Shoots proliferated on half-strength Murashige and Skoog medium without growth regulators. Different culture vessels were tested. Shoots in GROWTEK bioreactor showed the highest fresh and dry weight and total length while the plantlets grown in the RITA bioreactor showed the highest shoot number per explant. Encapsulation of L. palustris microcuttings with sodium alginate formed small and whitish beads which were stored for 14 or 28 days at 7° or 25°C. Storage for 14 days at both temperatures gave the best results but prolonged storage at 25°C decreased the shoot viability to 73%. After 4 weeks of recovery, all the plantlets showed the typical features of the species. Even though the latest Italian IUCN Red List does not mention L. palustris, conservation measures are proposed at local level because this species locally remains vulnerable mainly due to the loss of adequate habitats.Our protocol could be one of the methods for ex situ conservation of L. palustris particularly because its seed storage behavior is uncertai

Analysis and characterization of diffusive flows and movement of the human acinar region: An experimentally validated CFD model

This paper presents a study of the gas exchanges at the alveolar level to define the respiratory capacity of a subject. A numerical multidimensional approach is proposed for the prediction of the diffusive flows of gases (oxygen and carbon dioxide) in the alveoli accounting for the gas exchange phenomena including the surface variation due to the alveoli motion. The overset mesh technique is used to reproduce a realistic surface variation during inspiration and expiration and simulate the expansion and retraction of the alveolar sac. A gas-exchange model is implemented to predict the gas distribution in the alveolar sac wall by assigning a gas flow function through the membrane. The numerical analysis enables to evaluate the flow field within the single alveoli in terms of total and partial pressure of the considered alveolar gases, as well as the species distribution due to the diffusive flows. The predictive capabilities of the numerical model are addressed by comparing the calculations with the values obtained by means the mobile metabolic system K5 from COSMED. An experimental campaign on a set of healthy subjects is used to evaluate the diffusive flows through the membrane and a good agreement between the numerical results and the experimental measurements is found. These evaluations are suitable for the characterization of the breathing capacity in physiological or pathological conditions; therefore, the proposed model can be used as a valid support for bioengineering studies in terms of respiratory gas exchange prediction

Biomechanical analyses of professional ultramarathon athletes: The effect of repeated long distances on the gait kinematic and kinetics

Constant monitoring of an athlete allows to foresee any injuries by acting on the dynamics of the movements. For runners, the conduct of a correct athletic gesture according to the athlete's specific body biomechanics guarantees the minimization of non-accidental injury factors. For athletes who are engaged in endurance sports such as marathon runners, the long distances to which they are subjected increases the importance of this type of monitoring. This work reports the results of a study carried out on three IUTA (Italian Ultramarathon and Trail Association) athletes during a routine check carried out at a reference healthcare facility that takes care of their care. These athletes are all specialized in the 24-hour race in which they try to reach the most distance in this time. This type of effort can be made if the athlete undergoes an adequate training regime over long distances. The execution of the running pattern in such a repeated way can lead to the accentuation of postural and joint problems. It is therefore necessary to monitor the biomechanics parameters. In this work, therefore, various gestures are analyzed to show potential movement deficits in order to act in advance on the running technique

Early assessment of posture disorders through pre-adolescent monitoring: The case of volley academy modena

The pre-adolescent age is basic in the beginning of the development of body kinematics. During this age the body structure needs to continuously search for stabilization due to the modification in body segment lengths. This behavior can evolve into incorrect postural attitudes that, if not properly treated, can lead to behaviors that are difficult to recover with advancing age. These incorrect attitudes require an accurate and early diagnosis that can be put in place by expert doctors and clinicians. As well known, sporting activity in this age can help the development of proprioceptive apparatus and the musculoskeletal one. Volleyball is considered a complete sport thanks to the wide range of actions necessary for a match. Following this evaluation, it was decided to carry out a monitoring study of young volleyball players in what is one of the capitals of Italian volleyball, i.e. Modena. To achieve this objective, in this work, 97 female volleyball players have been analyzed during their adolescent and pre-adolescent age. The study has the objective to collect data and design an exercise routine for the training to prevent postural problems. Finally, the data have been analyzed and the most representative ones have been reported in this paper

Integration protocol of different measurement methods for the analysis of the physiological and biomechanical efficiency of a professional athlete

The paper focuses on the methodology for the analysis of the physiological and biomechanical efficiency of a professional athlete for integrating the standard preparation routine. The proposed methodology combines an in-house developed prototype of multiple uniaxial force plates for the measurement of the vertical component of ground reaction forces during movement and an infrared motion capture technique is adopted for measuring accurately the body motion. The procedure is applied on a top level professional volley player and integrates the working routines used for the training over an entire season. The dynamic performance of the athlete is measured in terms of fatigue threshold and the aerobic workload. The proposed methodology demonstrates to be an accurate and reliable instrument for quantifying, for both slow and fast movements, the efficiency with which the athlete reaches the defined training targets and the precision achieved in developing an exercises’ routine. Furthermore, the dynamic response of the athlete is also measured by evaluating the position of the body during the workload as well as the speed of the movements and the corresponding interaction with the ground. This analysis verifies if an asymmetrical loading of the lower limbs and the power exerted during the impulsive contact phase with the ground. The measurements carried out during the analysis provide a map of the athlete performances during an entire season training and the mono- and bi-podalic movements could be associated with the time evolution of the athletic results, such as jumping length and height, speed, precision. Therefore, inefficiencies in the postural and technical aspects during the training can be measured and thus corrected leading to an improvement of the performance and to a reduction of the possibility for injuries onset