Reducing cadmium accumulation in field grown tobacco plants

Patent metadata

Field	Value
Publication number	US-11213004-B2
Application number	US-201716072391-A
Country	US
Kind code	B2
Filing date	Jan 27, 2017
Priority date	Jan 29, 2016
Publication date	Jan 4, 2022
Grant date	Jan 4, 2022

Abstract

There is described herein a mutant plant or part thereof having at least partially reduced expression or activity of at least two heavy metal ATPases (HMAs), said two HMAs comprising, consisting or consisting essentially of: (i) polypeptides having at least 65% sequence identity to SEQ ID NO:1 and SEQ ID NO:2; (ii) polynucleotides encoding the polypeptides set forth in (i); or (iii) polynucleotides having at least 65% sequence identity to SEQ ID NO:3 and SEQ ID NO:4 encoding HMAs; wherein the expression or activity of one of the HMAs set forth in (i) or (ii) or (iii) is partially reduced or lost and the expression or activity of one of the HMAs set forth in (i) or (ii) or (iii) is lost as compared to a control plant; and wherein the mutant plant or part thereof exhibits at least a 27% reduction, as compared to the control plant, in the accumulation of cadmium in leaf when the mutant plant is grown in the field in the presence of naturally or non-naturally occurring cadmium.

First claim

The invention claimed is:

1. A non-naturally occurring and genetically modified plant or an artificially induced non-naturally occurring mutant plant or part thereof, having at least partially reduced expression or activity of at least two heavy metal ATPase (HMA) polypeptides encoded by at least two polynucleotide sequences, wherein said at least two polynucleotide sequences are selected from the group consisting of: (i) a polynucleotide sequences encoding a HMA polypeptides comprising an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1, and a polynucleotide sequence encoding a HMA polypeptide comprising an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2 and (ii) a polynucleotide sequences having at least 90% nucleotide sequence identity to the nucleotide sequence as set forth in SEQ ID NO: 3 and encoding a HMA polypeptide having at least 95% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1, and a polynucleotide sequence having at least 90% nucleotide sequence identity to the nucleotide sequence as set forth in SEQ ID NO: 4 and encoding a HMA polypeptide having at least 95% amino acid sequence identity to the amino acid sequence as set forth in SEQ ID NO: 2, and wherein said at least partially reduced expression or activity is by artificial mutagenesis or inhibitory RNAi construct mediated transformation of corresponding wild type plant or part thereof of the same species, wherein said non-naturally occurring and genetically modified plant or said artificially induced non-naturally occurring mutant plant or part thereof exhibits at least a 27% reduction in the accumulation of cadmium levels in leaf tissues of said non-naturally occurring and genetically modified plant or said artificially induced non-naturally occurring mutant plant or part thereof as compared to said corresponding wild type plant or part thereof of the same species and grown under identical growth conditions, wherein said non-naturally occurring and genetically modified plant or said artificially induced non-naturally occurring mutant plant or part thereof is grown under field conditions in the presence of cadmium, and wherein said non-naturally occurring and genetically modified plant or said artificially induced non-naturally occurring mutant plant or part thereof when grown under said field conditions in the presence of cadmium exhibits normal growth and development, and seed set which is identical and comparable to a wild type plant or part thereof of the same species grown under cadmium free conditions, and as compared to a hydroponically grown plant having said reduced expression or activity of said at least two heavy metal ATPase (HMA) polypeptides grown under said cadmium, and which shows stunted growth and does not produce seeds.

2. The non-naturally occurring genetically modified plant or said artificially induced non-naturally occurring mutant plant or part thereof according to claim 1 , wherein the expression or activity of one of the ATPase (HMA) polypeptides set forth in (i) or (ii) is lost as compared to the corresponding wild type plant or part thereof.

3. The non-naturally occurring genetically modified plant or said artificially induced non-naturally occurring mutant plant or part thereof according to claim 1 , wherein the phenotype of said non-naturally occurring genetically modified plant or said artificially induced non-naturally occurring mutant plant or part thereof at harvest time is the same as the corresponding wild type plant or part thereof at the same harvest time, wherein said non-naturally occurring genetically modified plant or said artificially induced non-naturally occurring mutant plant or part thereof does not show a biomass reduction at harvest time as compared to the corresponding wild type plant or part thereof at the same harvest time.

4. The non-naturally occurring genetically modified plant or said artificially induced non-naturally occurring mutant plant or part thereof according to claim 1 , comprising at least one mutation in coding sequences of polynucleotide sequences as set forth in (i) or (ii).

5. The non-naturally occurring genetically modified plant or said artificially induced non-naturally occurring mutant plant or part thereof according to claim 1 , comprising one or more of the following groups of mutations:
at least one mutation at a position corresponding to an amino acid position in the A-domain of the second cytoplasmic loop of the HMA polypeptide encoded by the non-mutated sequence set forth in SEQ ID NO: 3 and at least one mutation at a position corresponding to an amino acid position in the P/N-domain of the third cytoplasmic loop of the HMA polypeptide encoded by the non-mutated sequence set forth in SEQ ID NO: 4;
at least one mutation at a position corresponding to an amino acid position in the A-domain of the second cytoplasmic loop of the HMA polypeptide encoded by the non-mutated sequence set forth in SEQ ID NO: 3 and at least one mutation at a position corresponding to an amino acid position in the A-domain of the second cytoplasmic loop of the HMA polypeptide encoded by the non-mutated sequence set forth in SEQ ID NO: 4;
at least one mutation at a position corresponding to an amino acid position in the P/N-domain of the third cytoplasmic loop of the HMA polypeptide encoded by the non-mutated sequence set forth in SEQ ID NO: 3 and at least one mutation at a position corresponding to an amino acid position in the P/N-domain of the third cytoplasmic loop of the HMA polypeptide encoded by the non-mutated sequence set forth in SEQ ID NO: 4; and
at least one mutation at a position corresponding to an amino acid position in the P/N-domain of the third cytoplasmic loop of the HMA polypeptide encoded by the non-mutated sequence set forth in SEQ ID NO: 3 and at least one mutation at a position corresponding to an amino acid position in the A-domain of the second cytoplasmic loop of the HMA polypeptide encoded by the non-mutated sequence set forth in SEQ ID NO: 4.

6. The non-naturally occurring genetically modified plant or said artificially induced non-naturally occurring mutant plant or part thereof according to claim 1 comprising one or more of the following groups of mutations:
a nonsense mutation at a position corresponding to amino acid position 293 of SEQ ID NO: 1 and a nonsense mutation at a position corresponding to amino acid position 561 of the non-mutated sequence set forth in SEQ ID NO: 2;
a nonsense mutation at a position corresponding to amino acid position 293 of SEQ ID NO: 1 and a nonsense mutation at a position corresponding to amino acid position 265 of the non-mutated sequence set forth in SEQ ID NO: 2;
a missense mutation at a position corresponding to amino acid position 296 of SEQ ID NO: 1 and a nonsense mutation at a position corresponding to amino acid position 561 of the non-mutated sequence set forth in SEQ ID NO: 2;
a missense mutation at a position corresponding to amino acid position 402 of SEQ ID NO: 1 and a nonsense mutation at a position corresponding to amino acid position 561 of the non-mutated sequence set forth in SEQ ID NO: 2;
a missense mutation at a position corresponding to amino acid position 251 of SEQ ID NO: 1 and a nonsense mutation at a position corresponding to amino acid position 561 of the non-mutated sequence set forth in SEQ ID NO: 2;
a nonsense mutation at a position corresponding to amino acid position 464 of SEQ ID NO: 1 and a nonsense mutation at a position corresponding to amino acid position 561 of the non-mutated sequence set forth in SEQ ID NO: 2;
a nonsense mutation at a position corresponding to amino acid position 293 of SEQ ID NO: 1 and

Assignees

• Philip Morris Products Sa

Inventors

• Bovet Lucien
• Liedschulte Verena

Classifications

• C12N15/8218 Primary

  Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS] · CPC title

• A24B15/248

  Heavy metals · CPC title

• C07K14/415

  from plants · CPC title

• C12N15/8242 Primary

  with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits · CPC title

• A01G22/45

  Tobacco · CPC title