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 – shared with Zn – 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.

SYSTEMIC ALLOCATION OF TRACE ELEMENTS IN RICE / L. Fontanili ; supervisor: G.A. Sacchi ; cosupervisor: F.F. Nocito. - : . DIPARTIMENTO DI SCIENZE AGRARIE E AMBIENTALI - PRODUZIONE, TERRITORIO, AGROENERGIA, 2015 Jan 23. ((27. ciclo, Anno Accademico 2014. [10.13130/fontanili-laura_phd2015-01-23].

SYSTEMIC ALLOCATION OF TRACE ELEMENTS IN RICE

L. Fontanili
2015

Abstract

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 – shared with Zn – 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.
SACCHI, GIAN ATTILIO
NOCITO, FABIO FRANCESCO
SACCHI, GIAN ATTILIO
Oryza sativa ; heavy metals ; HMA ; Cd ; Zn
Settore AGR/13 - Chimica Agraria
SYSTEMIC ALLOCATION OF TRACE ELEMENTS IN RICE / L. Fontanili ; supervisor: G.A. Sacchi ; cosupervisor: F.F. Nocito. - : . DIPARTIMENTO DI SCIENZE AGRARIE E AMBIENTALI - PRODUZIONE, TERRITORIO, AGROENERGIA, 2015 Jan 23. ((27. ciclo, Anno Accademico 2014. [10.13130/fontanili-laura_phd2015-01-23].
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