Final Report Summary - SENSOMETAL (Nanosensors to monitor metal dynamics in living plant cells)
Transition metals play central roles in all living cells as cofactors for enzymes, as redox acceptors and as structural elements necessary for adequate folding of proteins. Metals are highly compartmentalised in eukaryotic cells and investigating the dynamics of metal trafficking in cells is required to understand the mechanisms of plant cells metal homeostasis. Genetically encoded fluorescence resonance energy transfer (FRET) sensors are the most adequate tools to monitor in vivo the dynamics of cellular metal ion levels over a wide concentration of metals. The goal of the project was to design or use a set of genetically encoded sensors to monitor in vivo dynamics of Zn2+, Mn2+ or Fe2+ and to introduce these sensors in plants to assess metal homeostasis mechanisms.
A set of six high zinc sensitivity sensors, named eCALWY1 to 6, based on the metal binding domain of the Atox1 chaperone linked to the ATP7B domain of Cu-P type ATPase were developed in 2009 and made available (Vinkenborg, 2009). Their affinities for Zn2+ range from 2 pM to 3 nM and display maximum amplitude in FRET index of 2.4 indicative of high sensitivity and high dynamic range. These characteristics make them suitable for monitoring zinc in vivo.
These sensors were successfully used in plant cells either by being transiently expressed in mesophyll protoplasts with imaging coupled to a microfluidic perfusion platform (CellASIC-ONIX) and by stable expression in different genotypes: wild-type plants, Rdr6 mutants which are impaired in transgene silencing and enable a high expression of the transgenes, bzip19bzip23 mutants which are impaired in two transcription factors controlling the zinc deficiency response; hma2hma4 mutants which are impaired in zinc translocation from roots to shoots. Transgenic lines were isolated for Calwy sensor variants in Wild-type, Rdr6 and bzip19bzip23 but sufficient levels of fluorescence were only found for the Rdr6 lines. These Rdr6 lines expressing the Calwy variants were further investigated and used to estimate Zn cytosolic concentration under different Zn nutrition conditions. We estimated the Zn cytolic concentration at 420 ± 200 pM when plants are grown under Zn sufficient condition (5 micromole), 2 ± 0.6 nM under high Zn concentration (30 micromole) but could not estimate this value under starvation conditions (0 micromole). In addition, using a microfluidic chip platform developed in the outgoing lab (Grossmann, 2011), time-resolved zinc cytosolic fluctuations have been monitored in plant roots. Under sufficient Zn nutrition condition, and when providing 1 mM external Zn, the velocity of cytosolic Zn increase was estimated at 100 pmol.min-1 +/- 2 (n = 2) in the root tip area and 160 +/- 40 pmol.min-1 in the elongation zone (n = 2). Different components such as glucose, nitric oxide or hydrogen peroxide were tested for their ability to affect free cytosolic Zn concentration but the results were inconclusive. In a first attempt undertaken at generating Mn2+ or Fe2+ sensors, metal binding scaffold were based on periplasmic solute-binding proteins from Synechocystis: MntC for Mn2+ and FutA1 for Fe2+. A dozen of MntC variants and FutA1 were cloned, expressed, purified and ligand specificity was tested. None displayed any significant FRET index change after metal addition. In addition, the use of a polyhistidine tag for the protein purification led to non-specific metal binding effects. A new strategy to generate and purify the sensors was implemented and an Intein mediated purification system was used to produce tag free sensors. New binding scaffolds were cloned: MntR, a manganese transcription factor, SODA, a manganese dependant superoxide dismutase for manganese and PCBP1, a Fe2+ chaperone for iron. Unfortunately, the duration of the fellowship was not sufficient to test this second generation of putative metal sensors.
A set of six high zinc sensitivity sensors, named eCALWY1 to 6, based on the metal binding domain of the Atox1 chaperone linked to the ATP7B domain of Cu-P type ATPase were developed in 2009 and made available (Vinkenborg, 2009). Their affinities for Zn2+ range from 2 pM to 3 nM and display maximum amplitude in FRET index of 2.4 indicative of high sensitivity and high dynamic range. These characteristics make them suitable for monitoring zinc in vivo.
These sensors were successfully used in plant cells either by being transiently expressed in mesophyll protoplasts with imaging coupled to a microfluidic perfusion platform (CellASIC-ONIX) and by stable expression in different genotypes: wild-type plants, Rdr6 mutants which are impaired in transgene silencing and enable a high expression of the transgenes, bzip19bzip23 mutants which are impaired in two transcription factors controlling the zinc deficiency response; hma2hma4 mutants which are impaired in zinc translocation from roots to shoots. Transgenic lines were isolated for Calwy sensor variants in Wild-type, Rdr6 and bzip19bzip23 but sufficient levels of fluorescence were only found for the Rdr6 lines. These Rdr6 lines expressing the Calwy variants were further investigated and used to estimate Zn cytosolic concentration under different Zn nutrition conditions. We estimated the Zn cytolic concentration at 420 ± 200 pM when plants are grown under Zn sufficient condition (5 micromole), 2 ± 0.6 nM under high Zn concentration (30 micromole) but could not estimate this value under starvation conditions (0 micromole). In addition, using a microfluidic chip platform developed in the outgoing lab (Grossmann, 2011), time-resolved zinc cytosolic fluctuations have been monitored in plant roots. Under sufficient Zn nutrition condition, and when providing 1 mM external Zn, the velocity of cytosolic Zn increase was estimated at 100 pmol.min-1 +/- 2 (n = 2) in the root tip area and 160 +/- 40 pmol.min-1 in the elongation zone (n = 2). Different components such as glucose, nitric oxide or hydrogen peroxide were tested for their ability to affect free cytosolic Zn concentration but the results were inconclusive. In a first attempt undertaken at generating Mn2+ or Fe2+ sensors, metal binding scaffold were based on periplasmic solute-binding proteins from Synechocystis: MntC for Mn2+ and FutA1 for Fe2+. A dozen of MntC variants and FutA1 were cloned, expressed, purified and ligand specificity was tested. None displayed any significant FRET index change after metal addition. In addition, the use of a polyhistidine tag for the protein purification led to non-specific metal binding effects. A new strategy to generate and purify the sensors was implemented and an Intein mediated purification system was used to produce tag free sensors. New binding scaffolds were cloned: MntR, a manganese transcription factor, SODA, a manganese dependant superoxide dismutase for manganese and PCBP1, a Fe2+ chaperone for iron. Unfortunately, the duration of the fellowship was not sufficient to test this second generation of putative metal sensors.
