Patent Application
20230136941
A Sequence Listing is provided herewith as a text file, “2131055.txt” created on Apr. 6, 2021 and having a size of 172,032 bytes. The contents of the text file are incorporated by reference herein in their entirety.
Traditionally, agricultural scientists concentrated on breeding plants with high nutritional yield. Typically, these new varieties were richer in carbohydrates but usually poorer in essential proteins and amino acids than the wild type varieties from which they were derived. Previous attempts on fortifying crops with high yield of essential amino acids have not been successful, largely due to accompanying penalties in plant growth. Hence, it has been difficult to generate plants with a complete range and adequate amounts of essential amino acids.
Described herein are plants, plant cells, and seeds with increased essential amino acid content relative to wild type or in some cases relative to plants with a knockout IPMS gene. In particular, described herein are plants, plant cells, and seeds with a modified IPMS gene, having one or more mutations. The mutations can be in the IPMS catalytic domain or in the IPMS1 allosteric domain. For example, plants with a modified IPMS catalytic domain (e.g., those with eva1 mutations) can have a conserved aspartic acid replaced by another amino acid, and plants with a modified IPMS in the allosteric domain include plants with ipms1-1D mutations. Plants with such modifications or mutations can have significantly higher levels of any and all amino acids. In some cases, the plants with such modifications or mutations can have significantly higher levels of Gln, His, Ile, Leu. Lys, Met, Phe, Thr, Trp, Val, or any combination thereof. The increased amino acid levels can occur in vegetative tissues (e.g., leaves) and seeds. In some cases, various plant tissues can have an increased content of just one, or just two, or just three, or just four, or just five, just six, just seven, just eight, just nine, or just ten amino acids. For example, in seeds with ipms1-1D mutations in their allosteric domain Ile, Leu, His, Lys, Met, Phe, and Thr levels are significantly increased. In the vegetative tissues of plants with ipms1-1D mutations in their allosteric domain leucine and other amino acids are present in increased amounts. Similarly, plants with a modified IPMS1 in the catalytic domain include plants with mutations such as the eva1 mutations. These plants with catalytic domain mutations can, for example, have significantly higher levels of valine in their seeds than wild type seeds. In their vegetative tissues, plants with modified catalytic domains such as the eva1 mutations can higher levels of valine and other amino acids. Plants with knockout ipms1 mutations (e.g., ipms1-4 or ipms1-5) can have modified or even increased content of some amino acids compared to wild type, but modification of the catalytic or allosteric domains typically provide higher levels of key amino acids such as the branched amino acids.
In addition, plants with such modifications or mutations can have significant increases in their biomass compared to wild type or compared to plants with a knockout IPMS gene. For example, plants with modifications to their IPMS1 allosteric domain can have significantly increased biomass. In some cases, plants with modifications to their catalytic domains tend to be smaller and generally have lower biomass.
Hence, described herein are plant cells, plant seeds, and/or plants that include a modified isopropylmalate synthase (IPMS) protein. The isopropylmalate synthase (IPMS) protein can be encoded by a modified or mutant endogenous isopropylmalate synthase (IPMS) gene within the plant cells, plant seeds, and/or plants. In some cases, an expression cassette having a promoter operably linked to a nucleic acid segment encoding a modified isopropylmalate synthase (IPMS) protein can be used to introduce expression of the modified isopropylmalate synthase (IPMS) protein into the plant cell, plant seed, or plant.
The modified isopropylmalate synthase protein can have isopropylmalate synthase activity. In some cases, the modified isopropylmalate synthase protein has a modification within its catalytic domain, a modification within its allosteric domain, or a combination thereof. For example, the modified isopropylmalate synthase protein can have an aspartic acid within its catalytic domain that is replaced by another amino acid. Such an aspartic acid can be at a position within the isopropylmalate synthase protein that corresponds to position 228 of SEQ ID NO:2. In some cases, the modified isopropylmalate synthase protein has a glycine within its allosteric domain that is replaced by another amino acid. Such a glycine can be at a position corresponding to position 606 of SEQ ID NO:2 in the modified isopropylmalate synthase protein. The modified isopropylmalate synthase protein can have a sequence with at least one amino acid modification to any of SEQ ID NO: 2, 3, 5, 7, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 28, 30, 32, 33, 35, 36, or 38. The plant or a plant generated from the plant cell or the plant seed with the modified isopropylmalate synthase protein can have increased amino acid content.
The plant cell, plant seed, or plant with the modified isopropylmalate synthase protein can be a forage species, starch species, oil species, grain species, grass species, sugar producing species, vegetable species of plant, plant cell or plant seed. The modified isopropylmalate synthase (IPMS) protein can be generated from a forage species, starch species, oil species, grain species, grass species, sugar producing species, or vegetable species of isopropylmalate synthase (IPMS). In some cases, the plant cell, plant seed, plant, and/or the isopropylmalate synthase (IPMS) protein can be a canola, corn, soybean, sunflower, walnut, or olive species.
Methods are also described herein that involve cultivating one or more seeds or seedlings, where the seeds or seedlings have a modified isopropylmalate synthase (IPMS) nucleic acid that can express a modified isopropylmalate synthase (IPMS) protein, to generate one or more mature plants, and harvesting vegetative tissues and/or seeds from the one or more mature plants.
Methods are also described herein that involve modifying a plant cell by introducing a mutation or modification in an endogenous isopropylmalate synthase (IPMS) gene, generating a plant from the plant cell, cultivating the plant, analyzing the amino acid content and/or biomass of the plant, and selecting one or more plants that have increased biomass or increased amino acid content. The increased biomass or the increased amino acid content can be measured relative to an average amino acid content, or an average biomass, of wild type plants, or parental plants, or plants with a knockout IPMS gene that do not express the modified isopropylmalate synthase protein.
Described herein are modified plants, plant cells, and plant seeds that provide improved amino acid content, for example, higher levels of branched-chain amino acids (BCAAs) and other amino acids. Examples of amino acids that can be at higher levels in the modified plants, plant cells, and plant seeds include Gln, His, Ile. Leu, Lys, Met, Phe. Thr. Trp, Val, or a combination thereof. In some cases, the BCAAs that are increased in the modified plant tissues are leucine, isoleucine, and valine. However, in some cases, one or two of leucine, isoleucine, or valine may not be increased in the modified plant tissues.
Methods for making and using such modified plants, plant cells, and plant seeds are also described herein. The modified nucleic acids, expression cassettes, plants, seeds and methods described herein can also be used to improve the growth and quantity of plant biomass even while having improved amino acid content. Methods of making and producing such plant seeds and plants can include, for example, cultivating seeds or seedlings, harvesting the plants, seeds, or the tissues of the plants. Such methods can also include isolating proteins and/or amino acids from the plants, seeds, or the tissues of the plants.
The plants, seeds, and plants cells described herein can have a modified or mutant isopropylmalate synthase (IPMS) gene. Surprisingly, the IPMS gene or IPMS nucleic acids that provide increased biomass and increased amino acid content can have a modification in its catalytic domain, in its allosteric domain, or in both domains. The modification in the catalytic domain can be in the acetyl-CoA binding surface near the pocket for the substrate. The modification in the allosteric domain can be located within about 20 amino acids of the IPMS protein C-terminus.
The IPMS1 and IPMS2 genes encode isopropylmalate synthase (IPMS, classified as EC 2.3.3.13) that catalyzes the first dedicated step in Leu biosynthesis. An alternate name for the IPMS1 enzyme is methylthioalkylmalate synthase-like 4 (MAML-4). The IPMS1 (MAML-4) protein is naturally expressed constitutively throughout the plant.
Examples of IPMS nucleic acids include the Arabidopsis thaliana IPMS1 (At1g18500) and IPMS2 (At1g74040) cDNAs. In Arabidopsis thaliana the IPMS1 gene is located on chromosome 1. A cDNA sequence that encodes an Arabidopsis thaliana isopropylmalate synthase IPMS1 protein is shown below as SEQ ID NO: 1.
The amino acid sequence for the Arabidopsis thaliana isopropylmalate synthase protein encoded by the SEQ ID NO:1 cDNA is shown below as SEQ ID NO:2.
One example of a modified IPMS1 protein that provides plants with significantly higher levels of Gln, His, Ile, Leu, Lys. Met, Phe, Thr, Trp, Val, or a combination thereof in their leaves and seeds, and significant increases in their biomass or amino acid content compared to wild type, parental, or IPMS knockout leaves and/or seeds, can have a mutation located in the acetyl-CoA binding surface near the pocket for 2-oxoisovalerate substrate. One example of a modified IPMS1 protein that has an altered amino acid content is the eva1 protein, which has a point mutation at position 228 of SEQ ID NO:2, where the aspartic acid (D) can, for example, be an asparagine (N). This modification is identified above in SEQ ID NO:2 in bold and with underlining. The eva1 protein with the asparagine (N) substitution for aspartic acid (D) at position 228 (D228N) has the sequence shown below as SEQ ID NO:3.
Compared to the SEQ ID NO:1 IPMS11 cDNA, the cDNA encoding the SEQ ID NO:3 eva1 protein can have the following sequence (SEQ ID NO:4), where the guanine at position 682 is an adenine (highlighted in bold with underlining).
Another example of a modified IPMS1 protein that provides plants with significantly higher levels of Gln, His, Ile, Leu, Lys, Met, Phe, Thr, Trp. Val, or a combination thereof in their leaves and seeds, and significant increases in their amino acid content or biomass compared to wild type, parental, or IPMS knockout leaves and/or seeds, is a modified IPMS1 protein with a modification in the IPMS1 allosteric domain, which can be located within about 20 amino acids of the C-terminus. One example, of an IPMS1 protein with a modification in the allosteric domain is dominant ipms1-1D feedback-insensitive mutant, which can provide small Val decreases but increases in Leu. In Arabidopsis, the ipms1-1D protein can, for example, have a point mutation at position 606 where the glycine (G) can be substituted with another amino acid. For example, the glycine at position 606 can be a glutamic acid (E). The position of this modification is identified in SEQ ID NO:2 in bold and with underlining. The SEQ ID NO:2 with the glutamic acid (E) substitution for glycine (G) at position 606 (G606E) has the sequence shown below as SEQ ID NO:5.
Compared to the SEQ ID NO:1 IPMS1 cDNA, the coding region DNA for IPMS1-1D can have the following sequence (SEQ ID NO:6), where the guanine at position 1817 is an adenine (highlighted in bold with underlining).
Another Arabidopsis thaliana isopropylmalate synthase IPMS1 protein sequence is shown below as SEQ ID NO:7, where two positions (226 and 604) are highlighted that can be modified to increase the production of various amino acids.
A cDNA encoding the Arabidopsis thaliana IPMS1 protein sequence with SEQ ID NO:7 is shown below as SEQ ID NO:8, where modification of the guanine at position 825 (highlighted in bold with underlining) to an adenine can provide a protein like the eva1 protein, and/or modification of the guanine at position 1960 (highlighted in bold with underlining) to an adenine can provide a protein like the IPMS1-1D protein.
Hence, a modified IPMS1 protein that provides plant with significantly higher levels of Gln, His, Ile, Leu, Lys, Met, Phe, Thr, Tim, Val, or a combination thereof in their leaves and seeds, and significant increases in their amino acid content or biomass compared to wild type, parental, or IPMS knockout leaves and/or seeds, the SEQ ID NO:7 protein can have a substitution at position 226. For example, the sequence of the SEQ ID NO:7 IPMS1 protein can be modified to have an asparagine at position 226 instead of an aspartic acid (D226N), which has the following sequence (SEQ ID NO:9).
Also, a modified IPMS1 protein that provides plants with significantly higher levels of Gln, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Val, or a combination thereof in their leaves and seeds, and significant increases in their biomass or amino acid content compared to wild type, parental, or IPMS knockout leaves and/or seeds, the SEQ ID NO:7 protein can have a substitution at position 604. For example, the sequence of the SEQ ID NO:7 IPMS1 protein can be modified to have a glutamic acid at position 604 instead of a glycine (G604E), which has the following sequence (SEQ ID NO:10).
Various plant species have IPMS1 genes that can be modified to provide those plant species with significantly higher levels of Gln, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Val, or a combination thereof, in their leaves and seeds, and significant increases in their biomass or amino acid content compared to wild type, parental, or IPMS knockout leaves and/or seeds. Examples of sequences from various plant species are described below.
For example, a sequence for a Brachypodium distachyon (Purple false brome) IPMS protein is shown below as SEQ ID NO:11, where two positions (229 and 604) are highlighted that can be modified to increase the production of various amino acids.
The Brachypodium distachyon (Purple false brome) IPMS protein with SEQ ID NO:11 is encoded by the 2-isopropylmalate synthase A gene on chromosome 4 (LOC100832390; locus tag BRADI_4g43130; see NCBI webpage at ncbi.nlm.nih.gov/gene/100832390).
To generate a modified Brachypodium distachyon IPMS protein that provides plants with significantly higher levels of Gln, His, Ile, Leu, Lys, Met. Phe, Thr, Trp, Val, or a combination thereof in their leaves and seeds, and significant increases in their biomass or amino acid content compared to wild type, parental, or IPMS knockout leaves and/or seeds, the SEQ ID NO: 11 protein can be modified to have a substitution at position 229. For example, the sequence of the SEQ ID NO:11 IPMS protein can be modified to have an asparagine at position 229 instead of an aspartic acid (D229N), which has the following sequence (SEQ ID NO:12).
To generate a modified Brachypodium distachyon IPMS protein that provides plants with significantly higher levels of Gln, His. Ile, Leu, Lys. Met, Phe, Thr. Trp. Val, or a combination thereof in their leaves and seeds, and significant increases in their biomass or amino acid content compared to wild type, parental, or IPMS knock-out leaves and/or seeds, the SEQ ID NO:11 protein can have a substitution at position 604. For example, the sequence of the SEQ ID NO:11 IPMS1 protein can be modified to have a glutamic acid at position 604 instead of a serine (S604E), which has the following sequence (SEQ ID NO: 13).
A sequence for a Glycine max (soybean) IPMS1 protein is shown below as SEQ ID NO: 14, where two positions (167 and 545) are highlighted that can be modified to increase the production of various amino acids.
The Glycine max (soybean) IPMS1 protein IPMS protein with SEQ ID NO: 14 is encoded by the 2-isopropylmalate synthase gene on chromosome 3 (LOC100816439; locus tag GLYMA_03(3005700: see NCBI website).
The SEQ ID NO: 14 IPMS1 protein has about 67% sequence identity with the SEQ ID NO:2 IPMS1 protein as illustrated below.
To generate a modified IPMS1 protein that provides Glycine max (soybean) plants with significantly higher levels of Gln, His Ile Leu Lys, Met, Phe, Thr, Trp Val, or a combination thereof in their leaves and seeds, and significant increases in their biomass or amino acid content compared to wild type, parental, or IPMS knockout leaves and/or seeds, the SEQ ID NO: 14 protein can have a substitution at position 167. For example, the sequence of the SEQ ID NO:14 IPMS1 protein can be modified to have an asparagine at position 167 instead of an aspartic acid (D167N′), which has the following sequence (SEQ ID NO: 15).
To generate a modified IPMS1 protein that provides Glycine max (soybean) plant with significantly higher levels of Gln, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Vat, or a combination thereof in their leaves and seeds, and significant increases in their biomass or amino acid content compared to wild type, parental, or IPMS knockout leaves and/or seeds, the SEQ ID NO: 14 protein can have a substitution at position 545. For example, the sequence of the SEQ ID NO: 14 IPMS1 protein can be modified to have a glutamic acid at position 545 instead of a threonine (T545E), which has the following sequence (SEQ ID NO: 16).
Another example of a Glycine max (soybean) IPMS1 protein is shown below as SEQ ID NO: 17, where 2-3 positions (225, 226, and 595) are highlighted as amino acids that can be modified to increase the production of various amino acids.
The Glycine max (soybean) IPMS1 protein IPMS protein with SEQ ID NO: 17 is encoded by the 2-isopropylmalate synthase gene on chromosome 10 (LOC100788955; locus tag GLYMA_10G295400; see NCBI website).
The SEQ ID NO: 17 IPMS1 protein has about 75% sequence identity with the SEQ ID NO:2 IPMS1 protein as illustrated below.
To generate a modified IPMS protein that provides Glycine max (soybean) plants with significantly higher levels of Gln, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Val, or a combination thereof in their leaves and seeds, and significant increases in their biomass or amino acid content compared to wild type, parental, or IPMS knockout leaves and/or seeds, the SEQ ID NO:17 protein can have a substitution at position 225 and/or 226. For example, the sequence of the SEQ ID NO:17 IPMS protein can be modified to have an asparagine at 216 instead of an aspartic acid (D216N), which has the following sequence (SEQ ID NO:18).
To generate a modified IPMS protein that provides Glycine max (soybean) plants with significantly higher levels of Gln, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Vat, or a combination thereof in their leaves and seeds, and significant increases in their biomass or amino acid content compared to wild type, parental, or IPMS knock-out leaves and/or seeds, the SEQ ID NO: 17 protein can have a substitution at position 595. For example, the sequence of the SEQ ID NO:17 IPMS1 protein can be modified to have a glutamic acid at position 595 instead of an alanine (A595E), which has the following sequence (SEQ ID NO: 19).
An example of Zea mays (corn) IPMS1 protein is shown below as SEQ ID NO:20, where two positions (235 and 612) are highlighted that can be modified to increase the production of various amino acids.
The Zea mays (corn) IPMS1 protein IPMS protein with SEQ ID NO:20 is encoded by the 2-isopropylmalate synthase gene on chromosome 4 (LOC100280189; locus tag ZEAMMB73_Zm00001d052472; see NCBI website).
The SEQ ID NO:20 corm IPMS1 protein has about 75% sequence identity with the SEQ ID NO:2 Arabidopsis IPMS1 protein as illustrated below.
To generate a modified IPMS1 protein that provides corn plants with significantly higher levels of Gln, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Val, or a combination thereof in their leaves and seeds, and significant increases in their biomass compared to wild type, parental, or IPMS knockout leaves and/or seeds, the SEQ ID NO:20 protein can have a substitution at position 235. For example, the sequence of the SEQ ID NO:20 IPMS11 protein can be modified to have an asparagine at 235 instead of an aspartic acid (D235N), which has the following sequence (SEQ ID NO:21).
To generate a modified IPMS1 protein that provides corn plants with significantly higher levels of Gln, His, Ile, Leu, Lys, Met, Phe, Thr, Trp. Val. or a combination thereof in (heir leaves and seeds, and significant increases in their biomass compared to wild type, parental, or IPMS knock-out leaves and/or seeds, the SEQ ID NO:20 protein can have a substitution at position 612. For example, the sequence of the SEQ ID NO:20 IPMS1 protein can be modified to have a glutamic acid at position 612 instead of a serine (S612E), which has the following sequence (SEQ ID NO:22).
Another Zea mays (corn) IPMS1 protein is shown below as SEQ ID NO:23, where two positions (235 and 612) are highlighted that can be modified to increase the production of various amino acids.
The Zea mays (corn) IPMS1 protein IPMS protein with SEQ ID NO:23 is encoded by the 2-isopropylmalate synthase B gene on chromosome 2 (LOC100281571; locus tag ZEAMMB73_Zm00001d004960; see NCBI website).
The SEQ ID NO:23 corn IPMS1 protein has about 70% sequence identity with the SEQ ID NO:2 Arabidopsis IPMS1 protein as illustrated below.
To generate a modified IPMS1 protein that provides corn plants with significantly higher levels of Gln, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Val, or a combination thereof in their leaves and seeds, and significant increases in their biomass compared to wild type, parental, or IPMS knockout leaves and/or seeds, the SEQ ID NO:23 protein can have a substitution at position 235. For example, the sequence of the SEQ ID NO:23 IPMS1 protein can be modified to have an asparagine at 235 instead of an aspartic acid (D235N), which has the following sequence (SEQ ID NO:24).
To generate a modified IPMS1 protein that provides corn plants with significantly higher levels of Gln, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Val, or a combination thereof in their leaves and seeds, and significant increases in their biomass compared to wild type, parental, or IPMS knockout leaves and/or seeds, the SEQ ID NO:23 protein can have a substitution at position 612. For example, the sequence of the SEQ ID NO:23 IPMS1 protein can be modified to have a glutamic acid at position 612 instead of a serine (S612E), which has the following sequence (SEQ ID NO:25).
A Solanum lycopersicum (tomato) IPMS1 protein is shown below as SEQ ID NO:26, where two positions (210 and 588) are highlighted that can be modified to increase the production of various amino acids.
The Solanum lycopersicum (tomato) IPMS protein with SEQ ID NO:26 is encoded by the 2-isopropylmalate synthase B gene on chromosome 6 (LOC101245066, see NCBI website).
As illustrated below the SEQ ID NO:26 Solanum lycopersicum IPMS1 sequence has about 75% sequence identity with the Arabidopsis IPMS1 SEQ ID NO: 2 sequence.
A cDNA encoding the Solanum lycopersicum (tomato) IPMS1 protein with SEQ ID NO:26 is shown below as SEQ ID NO:27.
Another example of a Solanum lycopersicum (tomato) IPMS1 protein sequence is shown below as SEQ ID NO:28, where two positions (212 and 590) are highlighted that can be modified to increase the production of various amino acids.
The Solanum lycopersicum (tomato) IPMS protein with SEQ ID NO:28 is encoded by the 2-isopropylmalate synthase B gene on chromosome 8 (Gene ID: 101251907; see NCBI website).
As illustrated below the SEQ ID NO:28 Solanum lycopersicum IPMS1 sequence has about 76% sequence identity with the Arabidopsis IPMS1 SEQ ID NO: 2 sequence.
A cDNA encoding the SEQ ID NO:28 tomato IPMS1 protein is shown below as SEQ ID NO:29.
An Oryza sativa (rice) IPMS1 protein is shown below as SEQ ID NO:30, where two positions (231 and 607) are highlighted that can be modified to increase the production of various amino acids.
The Oryza sativa (rice) IPMS protein with SEQ ID NO:30 is encoded by the 2-isopropylmalate synthase A gene on chromosome 11 (LOC4349745; locus tag OSNPB_110142500; see NCBI website).
As illustrated below the SEQ ID NO:30 Oryza sativa IPMS1 sequence has about 72% sequence identity with the Arabidopsis IPMS1 SEQ ID NO:2 sequence.
A cDNA encoding the SEQ ID NO:30 rice IPMS1 protein is shown below as SEQ ID NO:31.
Another Oryza sativa IPMS1 protein is shown below as SEQ ID NO: 32, where two positions (231 and 607) are highlighted that can be modified to increase the production of various amino acids.
The Oryza sativa (rice) IPMS protein with SEQ ID NO:32 is encoded by the 2-isopropylmalate synthase A gene on chromosome 12 (LOC4351460; locus tag OSNPB_120138900; see NCBI website).
As illustrated below the SEQ ID NO:32 Oryza sativa IPMS1 sequence has about 72% sequence identity with the Arabidopsis IPMS1 SEQ ID NO:2 sequence.
A Sorghum bicolor IPMS1 protein is shown below as SEQ ID NO:33, where two positions (215 and 592) are highlighted that can be modified to increase the production of various amino acids.
The Sorghum bicolor IPMS protein with SEQ ID NO:33 is encoded by the 2-isopropylmalate synthase A gene on chromosome 5 (LOC8085635; locus tag SORBI_-3005G030100; see NCBI website).
As illustrated below the Sorghum bicolor IPMS11 protein with SEQ ID NO:33 has about 71%-7-1% sequence identity with the Arabidopsis IPMS1 SEQ ID NO:2 sequence.
A cDNA encoding the Sorghum bicolor IPMS1 protein with SEQ ID NO:33 is shown below as SEQ ID NO:34.
A Brassica napus IPMS protein is shown below as SEQ ID NO: 35, where two positions (219 and 598) are highlighted that can be modified to increase the production of various amino acids.
As illustrated below the Brassica napus IPMS protein with SEQ ID NO:35 has about 90%-91% sequence identity with the Arabidopsis IPMS1 SEQ ID NO:2 sequence.
Another Brassica napus IPMS protein sequence is shown below as SEQ ID NO:36, where two positions (223 and 601) are highlighted that can be modified to increase the production of various amino acids.
G
ALNKMLDFK ENAPTKVPSQ NNNVPA
The Brassica napus IPMS protein with SEQ ID NO:36 is encoded by the 2-isopropylmalate synthase 1 gene on chromosome A6 (LOC106346910; locus tag SORBI_30050030100; see NCBI website). A cDNA that encodes the Brassica napus IPMS protein with SEQ ID NO:36 is shown below as SEQ ID NO:37.
As illustrated below the Brassica napus IPMS protein with SEQ ID NO:36 has about 89-90% sequence identity with the Arabidopsis IPMS1 SEQ ID NO:2 sequence.
Another Brassica napus IPMS protein sequence is shown below as SEQ ID NO:38, where two positions (223 and 601) are highlighted that can be modified to increase the production of various amino acids.
Table 1 lists some of accession numbers for IPMS homologs.
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Brachypodium distachyon
Chlamydomonas reinhardtii
The sequences used in the plans, plant cells, seeds and methods described herein can have less than 100% sequence identity to any of SEQ ID NO:1-38. For example, the sequences can have about at least 40% sequence identity, or at least 50% sequence identity, or at least 60% sequence identity, or at least 70% sequence identity, or at least 80% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity, or at least 99.5% sequence identity, or 60-99%, sequence identity, or 70-99% sequence identity, or 80-99% sequence identity, or 90-95% sequence identity, or 90-99% sequence identity, or 95-97% sequence identity, or 97-99% sequence identity, or 100% sequence identity with any of SEQ ID N A1-38.
The modified IPMS proteins described herein can have a variety of amino acids, and a variety of mutations. For example, the modified IPMS1 proteins described herein can have any of the amino acids listed in Table 2.
The modified plant cells, plants, and seeds described herein can have genomic mutations that alter one or more amino acids in the encoded IPMS protein. For example, one or more amino acids in the IPMS polypeptide can be replaced or deleted. In some cases, one or more amino acids in the IPMS can have be replaced by a conservative amino acid. In other cases, one or more amino acids having physical and/or chemical properties that are different from the amino acid(s) that are present in the parental or wild type plant cells, plants, or seeds. For example, to change the physical and/or chemical properties of amino acids, the amino acids can be deleted or replaced by amino acids of another class, where the classes are identified in the following Table 3.
Different types of amino acids can therefore be employed in the modified IPMS proteins.
For example, in some cases an acidic amino acid in a catalytic domain of an IPMS protein can be replaced with a polar amino acid. Such acidic amino acids can, for example, be aspartic acid (D) or glutamic acid (E). In some cases, the acidic amino acid in a catalytic domain of an IPMS protein that is modified, mutated, or replaced can be aspartic acid, for example, in the catalytic domain sequence shown in
In another example, an apolar amino acid in the allosteric domain can be modified. Such an apolar amino acid can be a glycine, methionine or proline amino acid. In some cases, the apolar amino acid in an allosteric domain of an IPMS protein that is modified, mutated, or replaced can be glycine, for example, in the allosteric domain shown in
Modified IPMS1 nucleic acids and/or modified IPMS1 proteins are introduced into plant cells, plants, and seeds to provide higher levels of Gln, His, Ile, Leu, Lys, Met. Phe, Thr, Trp, Val. or a combination thereof in their vegetative tissues (e.g., leaves, roots, stems, branches) and seeds. In some cases, at least one native IPMS1 gene is modified or mutated to induce expression of one or more modified IPMS1 proteins. In other cases, and one or more expression cassettes is introduced that includes an expression cassette for expressing a modified IPMS1 protein, where the expression cassette encodes a modified IPMS1 coding region under the control of a promoter. One of skill in the art can generate genetically modified plant cells, plants, and/or seeds that contain nucleic acids encoding a modified IPMS1 within their somatic and/or germ cells. Such genetic modification can be accomplished by various procedures.
Non-limiting examples of methods of introducing a modification into the genome of a cell can include use of microinjection, viral delivery, recombinase technologies, homologous recombination, TALENS, CRISPR, and/or ZFN, see, e.g. Clark and Whitelaw Nature Reviews Genetics 4:825-833 (2003); which is incorporated by reference herein in its entirety.
For example, nucleases such as zinc finger nucleases (ZFNs), transcription activator like effector nucleases (TALENs), and/or meganucleases can be employed with a guide nucleic acid that allows the nuclease to target the genomic IPMS1 site(s). In some cases, a targeting vector can be used to introduce a deletion or modification of one or more genomic IPMS1 site(s).
A “targeting vector” is a vector generally has a 5′ flanking region and a 3′ flanking region homologous to segments of the gene of interest. The 5′ flanking region and a 3′ flanking region can surround a DNA sequence comprising a modification and/or a foreign DNA sequence to be inserted into the gene. For example, the foreign DNA sequence may encode a selectable marker. In some cases, the targeting vector does not comprise a selectable marker, but such a selectable marker can facilitate identification and selection of cells with desirable mutations. Examples of suitable selectable markers include antibiotics resistance genes such as chloramphenicol resistance, gentamycin resistance, kanamycin resistance, spectinomycin resistance (SpecR), neomycin resistance gene (NEO), and/or the hygromycin p-phosphotransferase genes. The 5′ flanking region and the 3′ flanking region can be homologous to regions within the gene, or to regions flanking the gene to be deleted, modified, or replaced with the unrelated DNA sequence.
The targeting vector is contacted with the native gene of interest in vivo (e.g., within the cell) under conditions that favor homologous recombination. For example, the cell can be contacted with the targeting vector under conditions that result in transformation of the cyanobacterial cell(s) with the targeting vector.
A typical targeting vector contains nucleic acid fragments of not less than about 0.1 kb nor more than about 10.0 kb from both the 5′ and the 3′ ends of the genomic locus which encodes the gene to be modified (e.g. the genomic IPMS site(s)). These two fragments are separated by an intervening fragment of nucleic acid which encodes the modification to be introduced. When the resulting construct recombines homologously with the chromosome at this locus, it results in the introduction of the modification, e.g. a deletion of a portion of the genomic IPMS site(s), replacement of one or more amino acids in the genomic IPMS coding region site(s), or the insertion of non-conserved codon into the IPMS coding region.
In some cases, a Cas9/CRISPR system can be used to create a modification in genomic IPMS1 site(s). Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems are useful for, e.g. RNA-programmable genome editing (see e.g., Marraffini and Sontheimer. Nature Reviews Genetics 11: 181-190 (2010); Sorek et al. Nature Reviews Microbiology 2008 6: 181-6; Karginov and Hannon. Mol Cell 2010 1:7-19; Hale et al. Mol Cell 2010:45:292-302; Jinek et al. Science 2012 337:815-820; Bikard and Marraffini Curr Opin Immunol 2012 24:15-20; Bikard et al. Cell Host & Microbe 2012 12: 177-186; all of which are incorporated by reference herein in their entireties). A CRISPR guide RNA can be used that can target a Cas enzyme to the desired location in the genome, where it generates a double strand break. This technique is described, for example, by Mali et al. Science 2013 339:823-6; which is incorporated by reference herein in its entirety. Kits for the design and use of CRISPR-mediated genome editing are commercially available, e.g. the PRECISION X CAS9 SMART NUCLEASE™ System (Cat No. CAS900A-1) from System Biosciences, Mountain View, Calif.
In other cases, a cre-lox recombination system of bacteriophage P1, described by Abremski et al. 1983. Cell 32:1301 (1983). Sternberg et al., Cold Spring Harbor Symposia on Quantitative Biology, Vol. XLV 297 (1981) and others, can be used to promote recombination and alteration of the genomic MinC and/or MinD site(s). The cre-lox system utilizes the cre recombinase isolated from bacteriophage P1 in conjunction with the DNA sequences that the recombinase recognizes (termed lox sites). This recombination system has been effective for achieving recombination in plant cells (see, e.g., U.S. Pat. No. 5,658,772), animal cells (U.S. Pat. Nos. 4,959,317 and 5,801,030), and in viral vectors (Hardy et al., J. Virology 71:1842 (1997).
Another method for generating modified plant cells, plants and/or seeds include introducing an expression cassette or expression vector that can express modified IPMS1 polypeptides. Plant cells can be transformed by the expression cassette or expression vector, and whole plants (and their seeds) can be generated from the plant cells that were successfully transformed with the IPMS nucleic acids. Some procedures for making such genetically modified plants and their seeds are described below.
Promoters: The modified IPMS nucleic acids described herein can include a modified IPMS1 coding region operably linked to a promoter, which provides for expression of mRNA from the modified IPMS coding region. The promoter is typically a promoter functional in plants and/or seeds and can be a promoter functional during plant growth and development. A modified IPMS nucleic acid is operably linked to the promoter when it is located downstream from the promoter, to thereby form an expression cassette.
Most endogenous genes have regions of DNA that are known as promoters, which regulate gene expression. Promoter regions are typically found in the flanking DNA upstream from the coding sequence in both prokaryotic and eukaryotic cells. A promoter sequence provides for regulation of transcription of the downstream gene sequence and typically includes from about 50 to about 2,000 nucleotide base pairs. Promoter sequences also contain regulatory sequences such as enhancer sequences that can influence the level of gene expression. Some isolated promoter sequences can provide for gene expression of heterologous DNAs, that is a DNA different from the native or homologous DNA.
Promoter sequences are also known to be strong or weak, or inducible. A strong promoter provides for a high level of gene expression, whereas a weak promoter provides for a very low level of gene expression. An inducible promoter is a promoter that allows gene expression to be turned on and off in response to an exogenously added agent, or to an environmental or developmental stimulus. For example, a bacterial promoter such as the Ptac promoter can be induced to vary levels of gene expression depending on the level of isothiopropylgalactoside added to the transformed cells. Promoters can also provide for tissue specific or developmental regulation. An isolated promoter sequence that is a strong promoter for heterologous DNAs is advantageous because it provides for a sufficient level of gene expression for easy detection and selection of transformed cells and provides for a high level of gene expression when desired.
Expression cassettes generally include, but are not limited to, a plant promoter such as the CaMV 35S promoter (Odell et al., Nature, 313:810-812 (1985)), or others such as CaMV 19S (Lawton et al., Plant Molecular Biology, 9:315-324 (1987)), nos (Ebert et al., Proc. Natl. Acad. Sci. USA. 84:5745-5749 (1987)), Adh1 (Walker et al., Proc. Natl. Acad. Sci. USA. 84:6624-6628 (1987)), sucrose synthase (Yang et al., Proc. Natl. Acad. Sci. USA. 87:4144-4148 (1990)), α-tubulin, ubiquitin, actin (Wang et al., Mol. Cell. Biol. 12:3399 (1992)), cab (Sullivan et al., Mol. Gen. Genet. 215:431 (1989)), PEPCase (Hudspeth et al., Plant Molecular Biology. 12:579-589 (1989)) or those associated with the R gene complex (Chandler et al., The Plant Cell. 1:1175-1183 (1989)). Further suitable promoters include the poplar xylem-specific secondary cell wall specific cellulose synthase 8 promoter, cauliflower mosaic virus promoter, the Z10 promoter from a gene encoding a 10 kDa zein protein, a Z27 promoter from a gene encoding a 27 kDa zein protein, inducible promoters, such as the light inducible promoter derived from the pea rbcS gene (Coruzzi et al., EMBO J. 3:1671 (1971)) and the actin promoter from rice (McElroy et al., The Plant Cell. 2:163-171 (1990)). Seed specific promoters, such as the phaseolin promoter from beans, may also be used (Sengupta-Gopalan, Proc. Natl. Acad. Sci. USA. 83:3320-3324 (1985). Other examples of seed specific promoters that can be used include the P1, P3, P4, P6, P7, P9, P13, P14, P15, P16, P17, and P19 promoters described in U.S. Pat. No. 7,081,565 (which information about the P1, P3, P4, P6, P7, P9, P13. P14, P15, P16, P17, and P19 promoters is incorporated by reference herein in its entirety).
A modified IPMS1 nucleic acid can be combined with the promoter by standard methods to yield an expression cassette, for example, as described in Sambrook et al. (M
In some embodiments, a cDNA clone encoding a modified IPMS1 protein is isolated from plant tissue, for example, a root, stem, leaf, seed, or flower tissue. For example, cDNA clones from selected species (that encode an IPMS1 protein with homology to any of those described herein) are made from isolated mRNA from selected plant tissues. In another example, a nucleic acid encoding a mutant or modified IPMS1 protein can be prepared by available methods or as described herein. For example, the nucleic acid encoding a mutant or modified IPMS1 protein can be any nucleic acid with a coding region that hybridizes to a segment of a SEQ ID NO:1, 4, 6, 8, 27, 29, 31, 34, or 37 nucleic acid. For example, any of the modified IPMS1 nucleic acids can have one or more nucleotide differences to any of the SEQ ID NO:1, 4, 6, 8, 27, 29, 31, 34, or 37 nucleic acid sequences. Such a nucleic acid can encode an enzyme with isopropylmalate synthase activity and/or protein folding activity. Using restriction endonucleases, the entire coding sequence for the modified IPMS1 nucleic acid segment is subcloned downstream of the promoter in a 5′ to 3′ sense orientation.
Targeting Sequences: Additionally, expression cassettes can be constructed and employed to target the modified IPMS1 proteins to an intracellular compartment within plant cells, into a membrane, or to direct an encoded protein to the extracellular environment. This can generally be achieved by joining a DNA sequence encoding a transit or signal peptide sequence to the coding sequence of the modified IPMS1 nucleic acid. The resultant transit, or signal, peptide will transport the protein to a particular intracellular, or extracellular destination, respectively, and can then be posttranslational removed. Transit peptides act by facilitating the transport of proteins through intracellular membranes, e.g., vacuole, vesicle, plastid and mitochondrial membranes, whereas signal peptides direct proteins through the extracellular membrane. By facilitating transport of the protein into compartments inside or outside the cell, these sequences can increase the accumulation of a particular gene product in a particular location. For example, see U.S. Pat. No. 5,258,300.
3′Sequences: When the expression cassette is to be introduced into a plant cell, the expression cassette can also optionally include 3′ nontranslated plant regulatory DNA sequences that act as a signal to terminate transcription and allow for the polyadenylation of the resultant mRNA. The 3′ nontranslated regulatory DNA sequence preferably includes from about 300 to 1.000 nucleotide base pairs and contains plant transcriptional and translational termination sequences. For example, 3′ elements that can be used include those derived from the nopaline synthase gene of Agrobacterium tumefaciens (Bevan et al., Nucleic Acid Research. 11:369-385 (1983)), or the terminator sequences for the T7 transcript from the octopine synthase gene of Agrobacterium tumefaciens, and/or the 3′ end of the protease inhibitor I or II genes from potato or tomato. Other 3′ elements known to those of skill in the art can also be employed. These 3′ nontranslated regulatory sequences can be obtained as described in An (Methods in Enzymology. 153:292 (1987)). Many such 3′ nontranslated regulatory sequences are already present in plasmids available from commercial sources such as Clontech, Palo Alto, Calif. The 3′ nontranslated regulatory sequences can be operably linked to the 3′ terminus of the IPMS1 nucleic acids by standard methods.
Selectable and Screenable Marker Sequences: To improve identification of transformants, a selectable or screenable marker gene can be employed with the expressible IPMS1 nucleic acids. “Marker genes” are genes that impart a distinct phenotype to cells expressing the marker gene and thus allow such transformed cells to be distinguished from cells that do not have the marker. Such genes may encode either a selectable or screenable marker, depending on whether the marker confers a trait which one can ‘select’ for by chemical means, e.g., by use of a selective agent (e.g., an herbicide, antibiotic, or the like), or whether it is simply a trait that one can identify through observation or testing. i.e., by ‘screening’ (e.g., the R-locus trait). Of course, many examples of suitable marker genes are known to the art and can be employed in the practice of the invention.
Included within the terms selectable or screenable marker genes are also genes which encode a “secretable marker” whose secretion can be detected as a means of identifying or selecting for transformed cells. Examples include markers which encode a secretable antigen that can be identified by antibody interaction, or secretable enzymes that can be detected by their catalytic activity. Secretable proteins fall into a number of classes, including small, diffusible proteins detectable. e.g., by ELISA; and proteins that are inserted or trapped in the cell wall (e.g., proteins that include a leader sequence such as that found in the expression unit of extensin or tobacco PR-S).
With regard to selectable secretable markers, the use of a gene that encodes a polypeptide that becomes sequestered in the cell wall, where the polypeptide includes a unique epitope may be advantageous. Such a secreted antigen marker can employ an epitope sequence that would provide low background in plant tissue, a promoter-leader sequence that imparts efficient expression and targeting across the plasma membrane and can produce protein that is bound in the cell wall and yet is accessible to antibodies. A normally secreted wall protein modified to include a unique epitope would satisfy such requirements.
Examples of proteins suitable for modification in this manner include extensin or hydroxyproline rich glycoprotein (HPRG). For example, the maize HPRG (Stiefel et al., The Plant Cell. 2:785-793 (1990)) is well characterized in terms of molecular biology, expression, and protein structure and therefore can readily be employed. However, any one of a variety of extensins and/or glycine-rich wall proteins (Keller et al., EMBO J. 8:1309-1314 (1989)) could be modified by the addition of an antigenic site to create a screenable marker.
Numerous other possible selectable and/or screenable marker genes will be apparent to those of skill in the art in addition to those forth herein below. Therefore, it will be understood that the discussion herein is exemplary rather than exhaustive. In light of the techniques disclosed herein and the general recombinant techniques that are known in the art, the present invention readily allows the introduction of any gene, including marker genes, into a recipient cell to generate a transformed plant cell, e.g., a monocot cell or dicot cell.
Possible selectable markers for use in connection with the present invention include, but are not limited to, a neo gene (Potrykus et al., Mol. Gen. Genet. 199:183-188 (1985)) which codes for kanamycin resistance and can be selected for using kanamycin, G418, and the like; a bar gene which codes for bialaphos resistance; a gene which encodes an altered EPSP synthase protein (Hinchee et al., Bio/Technology. 6:915-922 (1988)) thus conferring glyphosate resistance; a nitrilase gene such as brn from Klebsiella ozaenae which confers resistance to bromoxynil (Stalker et al., Science. 242:419-423 (1988)); a mutant acetolactate synthase gene (ALS) which confers resistance to imidazolinone, sulfonylurea or other ALS-inhibiting chemicals (European Patent Application 154.204 (1985)); a methotrexate-resistant DHFR gene (Thillet et al., J. Biol. Chem. 263:12500-12508 (1988)); a dalapon dehalogenase gene that confers resistance to the herbicide dalapon; or a mutated anthranilate synthase gene that confers resistance to 5-methyl tryptophan. Where a mutant EPSP synthase gene is employed, additional benefit may be realized through the incorporation of a suitable chloroplast transit peptide, CTP (European Patent Application 0 218 571 (1987)).
An illustrative embodiment of a selectable marker gene capable of being used in systems to select transformants is the gene that encode the enzyme phosphinothricin acetyltransferase, such as the bar gene from Streptomyces hygroscopicus or the pat gene from Streptomyces viridochromogenes (U.S. Pat. No. 5,550,318). The enzyme phosphinothricin acetyl transferase (PAT) inactivates the active ingredient in the herbicide bialaphos, phosphinothricin (PPT). PPT inhibits glutamine synthetase, (Murakami et al., Mol. Gen. Genet. 205:42-50 (1986); Twell et al., Plant Physiol. 91:1270-1274 (1989)) causing rapid accumulation of ammonia and cell death. The success in using this selective system in conjunction with monocots was surprising because of the major difficulties that have been reported in transformation of cereals (Potrykus, Trends Biotech. 7:269-273 (1989)).
Screenable markers that may be employed include, but are not limited to, a p-glucuronidase or uidA gene (GUS) that encodes an enzyme for which various chromogenic substrates are known; an R-locus gene, which encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues (Dellaporta et al., In: Chromosome Structure and Function: Impact of New Concepts, 18th Stadler Genetics Symposium. J. P. Gustafson and R. Appels, eds. (New York: Plenum Press) pp. 263-282 (1988)); a β-lactamase gene (Sutcliffe, Proc. Natl. Acad. Sci. USA. 75:3737-3741 (1978)), which encodes an enzyme for which various chromogenic substrates are known (e.g., PADAC, a chromogenic cephalosporin); a xvlE gene (Zukowsky et al., Proc. Natl. Acad. Sri. USA. 80:1101 (1983)) which encodes a catechol dioxygenase that can convert chromogenic catechols; an α-amylase gene (Ikuta et al., Bio/technology 8:241-242 (1990)); a tyrosinase gene (Katz et al., J. Gen. Microbiol. 129:2703-2714 (1983)) which encodes an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone which in turn condenses to form the easily detectable compound melanin; a β-galactosidase gene, which encodes an enzyme for which there are chromogenic substrates; a luciferase (lux) gene (Ow et al., Science. 234:856-859.1986), which allows for bioluminescence detection; or an aequorin gene (Prasher et al., Biochem. Biophys. Res. Comm. 126:1259-1268 (1985)), which may be employed in calcium-sensitive bioluminescence detection, or a green or yellow fluorescent protein gene (Niedz et al., Plant Cell Repons. 14:403 (1995)).
For example, genes from the maize R gene complex can be used as screenable markers. The R gene complex in maize encodes a protein that acts to regulate the production of anthocyanin pigments in most seed and plant tissue. Maize strains can have one, or as many as four. R alleles that combine to regulate pigmentation in a developmental and tissue specific manner. A gene from the R gene complex does not harm the transformed cells. Thus, an R gene introduced into such cells will cause the expression of a red pigment and, if stably incorporated, can be visually scored as a red sector. If a maize line carries dominant alleles for genes encoding the enzymatic intermediates in the anthocyanin biosynthetic pathway (C2, A1, A2, Bz1 and Bz2), but carries a recessive allele at the R locus, transformation of any cell from that line with R will result in red pigment formation. Exemplary lines include Wisconsin 22 that contains the rg-Stadler allele and TR112, a K55 derivative that is r-g, b, Pl. Alternatively any genotype of maize can be utilized if the C1 and R alleles are introduced together.
The R gene regulatory regions may be employed in chimeric constructs to provide mechanisms for controlling the expression of chimeric genes. More diversity of phenotypic expression is known at the R locus than at any other locus (Coe et al., in Corn and Corn Improvement, eds. Sprague, G. F. & Dudley, J. W. (Am. Soc. Agron., Madison, Wis.), pp. 81-258 (1988)). It is contemplated that regulatory regions obtained from regions 5′ to the structural R gene can be useful in directing the expression of genes, e.g., insect resistance, drought resistance, herbicide tolerance or other protein coding regions. For the purposes of the present invention, it is believed that any of the various R gene family members may be successfully employed (e.g., P, S, Lc, etc.). However, one that can be used is Sn (particularly Sn:bol3). Sn is a dominant member of the R gene complex and is functionally similar to the R and B loci in that Sn controls the tissue specific deposition of anthocyanin pigments in certain seedling and plant cells, therefore, its phenotype is similar to R.
A further screenable marker contemplated for use in the present invention is firefly luciferase, encoded by the lux gene. The presence of the lux gene in transformed cells may be detected using, for example, X-ray film, scintillation counting, fluorescent spectrophotometry, low-light video cameras, photon counting cameras or multiwell luminometry. It is also envisioned that this system may be developed for population screening for bioluminescence, such as on tissue culture plates, or even for whole plant screening.
Other Optional Sequences: An expression cassette of the invention can also further comprise plasmid DNA. Plasmid vectors include additional DNA sequences that provide for easy selection, amplification, and transformation of the expression cassette in prokaryotic and eukaryotic cells, e.g., pUC-derived vectors such as pUC8, pUC9, pUC18, pUC19, pUC23, pUC19, and pUC120, pSK-derived vectors, pGEM-derived vectors, pSP-derived vectors, or pBS-derived vectors. The additional DNA sequences include origins of replication to provide for autonomous replication of the vector, additional selectable marker genes, preferably encoding antibiotic or herbicide resistance, unique multiple cloning sites providing for multiple sites to insert DNA sequences or genes encoded in the expression cassette and sequences that enhance transformation of prokaryotic and eukaryotic cells.
Another vector that is useful for expression in both plant and prokaryotic cells is the binary Ti plasmid (as disclosed in Schilperoort et al., U.S. Pat. No. 4,940,838) as exemplified by vector pGA582. This binary Ti plasmid vector has been previously characterized by An (Methods in Enzymology. 153:292 (1987)) and is available from Dr. An. This binary Ti vector can be replicated in prokaryotic bacteria such as E. coli and Agrobacterium. The Agrobacterium plasmid vectors can be used to transfer the expression cassette to dicot plant cells, and under certain conditions to monocot cells, such as barley, corn, rice, or wheat cells. The binary Ti vectors preferably include the nopaline T DNA right and left borders to provide for efficient plant cell transformation, a selectable marker gene, unique multiple cloning sites in the T border regions, the colE1 replication of origin and a wide host range replicon. The binary Ti vectors carrying an expression cassette of the invention can be used to transform both prokaryotic and eukaryotic cells but is preferably used to transform dicot plant cells.
In Vitro Screening of Expression Cassettes: Once the expression cassette is constructed and subcloned into a suitable plasmid, it can be screened for the ability to substantially inhibit the translation of an mRNA coding for a seed storage protein by standard methods such as hybrid arrested translation. For example, for hybrid selection or arrested translation, a preselected antisense DNA sequence is subcloned into an SP6MT containing plasmids (as supplied by ProMega Corp.). For transformation of plants cells, suitable vectors include plasmids such as described herein. Typically, hybrid arrest translation is an in vitro assay that measures the inhibition of translation of an mRNA encoding a particular seed storage protein. This screening method can also be used to select and identify preselected antisense DNA sequences that inhibit translation of a family or subfamily of zein protein genes. As a control, the corresponding sense expression cassette is introduced into plants and the phenotype assayed.
DNA Delivery of the DNA Molecules into Host Cells: The present invention generally includes steps directed to introducing IPMS1 nucleic acids, such as a preselected cDNA encoding the modified IPMS1 enzyme, into a recipient cell to create a transformed cell. In some instances, the frequency of occurrence of cells taking up exogenous (foreign) DNA may be low. Moreover, it is most likely that not all recipient cells receiving DNA segments or sequences will result in a transformed cell wherein the DNA is stably integrated into the plant genome and/or expressed. Some may show only initial and transient gene expression. However, certain cells from virtually any dicot or monocot species may be stably transformed, and these cells regenerated into transgenic plants, through the application of the techniques disclosed herein.
Another aspect of the invention is a plant with isopropylmalate synthase activity, increased amino acid content, normal to increased biomass, wherein the plant has a modified IPMS nucleic acid. The modified IPMS nucleic acid can be from any species. This application provides examples of modified IPMS nucleic acids and proteins that can be used.
The plants and seeds can be monocotyledon or dicotyledon plants and seeds. Another aspect of the invention includes plant cells (e.g., embryonic cells or other cell lines) that can regenerate fertile transgenic plants and/or seeds. The cells can be derived from either monocotyledons or dicotyledons.
Suitable examples of plant and IPMS species include grasses, softwoods, hardwoods, or agricultural crop species. For example, the species of the IPMS nucleic acids and proteins employed as well as the species of modified plant cells, plants, and seeds can be a species of alfalfa, canola, corn, wheat, rice, maize, barley, rye, Brachypodium, Arabidopsis, oats, sorghum, millet, miscanthus, switchgrass, poplar, eucalyptus, sugarcane, bamboo, bean, tobacco, cucumber, tomato, lettuce, pea, soybean, and the like. In some embodiments, the IPMS, plant or cell is a monocotyledon IPMS, plant, seed, or cell. For example, the IPMS, plant or cell can be a grass IPMS, plant, seed, or cell. In some embodiments, the IPMS, plant, seed, or cell is a dicotyledon IPMS, plant, seed, or cell. For example, IPMS, plant, seed, or cell can be a hardwood IPMS, plant, seed, or cell. The cell(s) may be in a suspension cell culture or may be in an intact plant part, such as an immature embryo, or in a specialized plant tissue, such as callus, such as Type I or Type II callus.
Transformation of the cells of the plant tissue source can be conducted by any one of a number of methods can be used. Examples are: Transformation by direct DNA transfer into plant cells by electroporation (U.S. Pat. Nos. 5,384,253 and 5,472,869, Dekeyser et al., The Plant Cell. 2:591-602 (1990)); direct DNA transfer to plant cells by PEG precipitation (Hayashimoto et al., Plant Physiol. 93:857-863 (1990)); direct DNA transfer to plant cells by microprojectile bombardment (McCabe et al., Bio/Technology. 6:923-926 (1988); Gordon-Kamm et al., The Plant Cell. 2:603-618 (1990); U.S. Pat. Nos. 5,489,520; 5,538,877; and 5,538,880) and DNA transfer to plant cells via infection with Agrobacterium. Methods such as microprojectile bombardment or electroporation can be carried out with “naked” DNA where the expression cassette may be simply carried on any E. coli-derived plasmid cloning vector. In the case of viral vectors, it is desirable that the system retain replication functions, but lack functions for disease induction.
One method for dicot transformation, for example, involves infection of plant cells with Agrobacterium tumefaciens using the leaf-disk protocol (Horsch et al., Science 227:1229-1231 (1985). Monocots such as Zea mays can be transformed via microprojectile bombardment of embryogenic callus tissue or immature embryos, or by electroporation following partial enzymatic degradation of the cell wall with a pectinase-containing enzyme (U.S. Pat. Nos. 5,384,253; and 5,472,869). For example, embryogenic cell lines derived from immature Zea mays embryos can be transformed by accelerated particle treatment as described by Gordon-Kamm et al. (The Plant Cell. 2:603-618 (1990)) or U.S. Pat. Nos. 5,489,520; 5,538,877 and 5,538,880, cited above. Excised immature embryos can also be used as the target for transformation prior to tissue culture induction, selection and regeneration as described in U.S. application Ser. No. 08/112,245 and PCT publication WO 95/06128. Furthermore, methods for transformation of monocotyledonous plants utilizing Agrobacterium tumefaciens have been described by Hiei et al. (European Patent 0 604 662, 1994) and Saito et al. (European Patent 0 672 752, 1995).
Methods such as microprojectile bombardment or electroporation are carried out with “naked” DNA where the expression cassette may be simply carried on any E. coli-derived plasmid cloning vector. In the case of viral vectors, it is desirable that the system retain replication functions, but lack functions for disease induction.
The choice of plant tissue source for transformation will depend on the nature of the host plant and the transformation protocol. Useful tissue sources include callus, suspension culture cells, protoplasts, leaf segments, stem segments, tassels, pollen, embryos, hypocotyls, tuber segments, meristematic regions, and the like. The tissue source is selected and transformed so that it retains the ability to regenerate whole, fertile plants following transformation, i.e., contains totipotent cells. Type I or Type II embryonic maize callus and immature embryos are preferred Zea mays tissue sources. Similar tissues can be transformed for softwood or hardwood species. Selection of tissue sources for transformation of monocos is described in detail in U.S. application Ser. No. 08/112,245 and PCT publication WO 95/06128.
The transformation is carried out under conditions directed to the plant tissue of choice. The plant cells or tissue are exposed to the DNA or RNA carrying the IPMS1 nucleic acids for an effective period of time. This may range from a less than one second pulse of electricity for electroporation to a 2-3 days co-cultivation in the presence of plasmid-bearing Agrobacterium cells. Buffers and media used will also vary with the plant tissue source and transformation protocol. Many transformation protocols employ a feeder layer of suspended culture cells (tobacco or Black Mexican Sweet corn, for example) on the surface of solid media plates, separated by a sterile filter paper disk from the plant cells or tissues being transformed.
An Example of Production and Characterization of Stable Transgenic Maize: After effecting delivery of a modified IPMS1 nucleic acid to recipient cells by any of the methods discussed above, the transformed cells can be identified for further culturing and plant regeneration. As mentioned above, to improve the ability to identify transformants, one may desire to employ a selectable or screenable marker gene as, or in addition to, the expressible IPMS1 nucleic acids. In this case, one would then generally assay the potentially transformed cell population by exposing the cells to a selective agent or agents, or one would screen the cells for the desired marker gene trait.
Selection: An exemplary embodiment of methods for identifying transformed cells involves exposing the bombarded cultures to a selective agent, such as a metabolic inhibitor, an antibiotic, herbicide or the like. Cells which have been transformed and have stably integrated a marker gene conferring resistance to the selective agent used, will grow and divide in culture. Sensitive cells will not be amenable to further culturing.
To use the bar-bialaphos or the EPSPS-glyphosate selective system, bombarded tissue is cultured for about 0-28 days on nonselective medium and subsequently transferred to medium containing from about 1-3 mg/l bialaphos or about 1-3 mM glyphosate, as appropriate. While ranges of about 1-3 mg/l bialaphos or about 1-3 mM glyphosate can be employed, it is proposed that ranges of at least about 0.1-50 mg/l bialaphos or at least about 0.1-50 mM glyphosate will find utility in the practice of the invention. Tissue can be placed on any porous, inert, solid or semi-solid support for bombardment, including but not limited to filters and solid culture medium. Bialaphos and glyphosate are provided as examples of agents suitable for selection of transformants, but the technique of this invention is not limited to them.
An example of a screenable marker trait is the red pigment produced under the control of the R-locus in maize. This pigment may be detected by culturing cells on a solid support containing nutrient media capable of supporting growth at this stage and selecting cells from colonies (visible aggregates of cells) that are pigmented. These cells may be cultured further, either in suspension or on solid media. The R-locus is useful for selection of transformants from bombarded immature embryos. In a similar fashion, the introduction of the C1 and B genes will result in pigmented cells and/or tissues.
The enzyme luciferase is also useful as a screenable marker in the context of the present invention. In the presence of the substrate luciferin, cells expressing luciferase emit light which can be detected on photographic or X-ray film, in a luminometer (or liquid scintillation counter), by devices that enhance night vision, or by a highly light sensitive video camera, such as a photon counting camera. All of these assays are nondestructive and transformed cells may be cultured further following identification. The photon counting camera is especially valuable as it allows one to identify specific cells or groups of cells which are expressing luciferase and manipulate those in real time.
It is further contemplated that combinations of screenable and selectable markers may be useful for identification of transformed cells. For example, selection with a growth inhibiting compound, such as bialaphos or glyphosate at concentrations below those providing 100% inhibition followed by screening of growing tissue for expression of a screenable marker gene such as luciferase would allow one to recover transformants from cell or tissue types that are not amenable to selection alone. In an illustrative embodiment embryogenic Type II callus of Zea mays L, can be selected with sub-lethal levels of bialaphos. Slowly growing tissue was subsequently screened for expression of the luciferase gene and transformants can be identified.
Regeneration and Seed Production: Cells that survive the exposure to the selective agent, or cells that have been scored positive in a screening assay, are cultured in media that supports regeneration of plants. One example of a growth regulator that can be used for such purposes is dicamba or 2,4-D. However, other growth regulators may be employed, including NAA, NAA+2,4-D or perhaps even picloram. Media improvement in these and like ways can facilitate the growth of cells at specific developmental stages. Tissue can be maintained on a basic media with growth regulators until sufficient tissue is available to begin plant regeneration efforts, or following repeated rounds of manual selection, until the morphology of the tissue is suitable for regeneration, at least two weeks, then transferred to media conducive to maturation of embryoids. Cultures are typically transferred every two weeks on this medium. Shoot development signals the time to transfer to medium lacking growth regulators.
The transformed cells, identified by selection or screening and cultured in an appropriate medium that supports regeneration, can then be allowed to mature into plants. Developing plantlets are transferred to soilless plant growth mix, and hardened, e.g., in an environmentally controlled chamber at about 85% relative humidity, about 600 ppm CO2, and at about 25-250 microeinsteins/sec·m2 of light. Plants can be matured either in a growth chamber or greenhouse. Plants are regenerated from about 6 weeks to 10 months after a transformant is identified, depending on the initial tissue. During regeneration, cells are grown on solid media in tissue culture vessels. Illustrative embodiments of such vessels are petri dishes and Plant Con™. Regenerating plants can be grown at about 19° C. to 28° C. After the regenerating plants have reached the stage of shoot and root development, they may be transferred to a greenhouse for further growth and testing.
Mature plants are then obtained from cell lines that are known to express the trait. In some embodiments, the regenerated plants are self-pollinated. In addition, pollen obtained from the regenerated plants can be crossed to seed grown plants of agronomically important inbred lines. In some cases, pollen from plants of these inbred lines is used to pollinate regenerated plants. The trait is genetically characterized by evaluating the segregation of the trait in first and later generation progeny. The heritability and expression in plants of traits selected in tissue culture are of interest if the traits are to be commercially useful.
Regenerated plants can be repeatedly crossed to inbred plants to introgress the IPMS1 nucleic acids into the genome of the inbred plants. This process is referred to as backcross conversion. When a sufficient number of crosses to the recurrent inbred parent have been completed to produce a product of the backcross conversion process that is substantially isogenic with the recurrent inbred parent except for the presence of the introduced IPMS1 nucleic acids, the plant is self-pollinated at least once to produce a homozygous backcross converted inbred containing the modified IPMS1 nucleic acids. Progeny of these plants are true breeding.
Alternatively, seed from transformed monocot plants regenerated from transformed tissue cultures is grown in the field and self-pollinated to generate true breeding plants.
Seed from the fertile transgenic plants can then be evaluated for the presence and/or expression of the IPMS1 nucleic acids (or IPMS1 proteins). Transgenic plant and/or seed tissue can be analyzed for modified IPMS1 expression using standard methods such as SDS polyacrylamide gel electrophoresis, liquid chromatography (e.g., HPLC) or other means of detecting a product of IPMS1 activity (e.g., increased amino acid content and/or biomass).
Once a transgenic seed expressing the modified IPMS1 sequence and having an increase in amino acid content in the plant is identified, the seed can be used to develop true breeding plants. The true breeding plants are used to develop a line of plants with an increase in the percent of amino acid content (e.g., of various amino acids) and growth or biomass of the plant while still maintaining other desirable functional agronomic traits. Adding the trait of increased amino acid content (with or without normal to improved biomass) of the plant can be accomplished by back-crossing with this trait and with plants that do not exhibit this trait and studying the pattern of inheritance in segregating generations. Those plants expressing the target trait in a dominant fashion are preferably selected. Back-crossing is carried out by crossing the original fertile transgenic plants with a plant from an inbred line exhibiting desirable functional agronomic characteristics while not necessarily expressing the trait of an increased percent of isopropylmalate synthase activity, normal to improved growth, and/or protein folding in the plant. The resulting progeny are then crossed back to the parent that expresses the increased IPMS1 trait (increased amino acids, with or without normal to improved biomass). The progeny from this cross will also segregate so that some of the progeny carry the trait and some do not. This back-crossing is repeated until an inbred line with the desirable functional agronomic traits, and with expression of the trait involving an increase in amino acid content with or without normal to improved biomass of the plant. Such expression of the increased amino acid content with or without normal to improved biomass of a plant can be expressed in a dominant fashion.
Subsequent to back-crossing, the new transgenic plants can be evaluated for an increase in the weight percent of various amino acids, increased modified isopropylmalate synthase activity, and/or normal to improved biomass of the plant. This can be done, for example, by immunofluorescence analysis of whole plant cell walls (e.g., by microscopy), isopropylmalate synthase activity assays, amino acid content analyses, biomass measurements, and any of the assays described herein or available to those of skill in the art.
The new transgenic plants can also be evaluated for a battery of functional agronomic characteristics such as lodging, kernel hardness, yield, resistance to disease, resistance to insect pests, drought resistance, and/or herbicide resistance.
As described herein, expression of IPMS1 can not only increase the amino acid content of plant tissues but such expression can also increase the biomass, growth or height of plants. Hence it is useful to modify a variety of plant types to express IPMS1.
Plants that can be improved include but are not limited to forage plants (e.g., alfalfa, clover, soybeans, turnips, bromegrass, bluestem, and fescue), starch or oil plants (e.g., canola, potatoes, lupins, sunflower, soybean, and cottonseed), grains (maize, wheat, barley, oats, rice, sorghum, millet and rye), grasses (switchgrass, prairie grass, wheat grass, sudangrass, sorghum, straw-producing plants, miscanthus, switchgrass), sugar producing plants (sugarcane, beets), vegetable plants (e.g., cucumber, lettuce, tomato), Brachypodium, Arabidopsis, bamboo, softwood, hardwood and other woody plants (e.g., those used for paper production such as poplar species, pine species, and eucalyptus). In some embodiments the plant is an agricultural crop species, or a species useful for foraging by agricultural animals. Plants useful for generating dairy forage include legumes such as alfalfa, as well as clover, soybeans, turnips, Brachypodium, Arabidopsis, and forage grasses such as bromegrass, and bluestem. In some cases, the plant is an oil producing plant such as canola, corn, soybean, sunflower, walnut, olive, or the like.
The IPMS nucleic acids or IPMS proteins that are modified can be from the same species as the plant, plant cell, or plant seed that is modified to include the modified IPMS1 nucleic acids or modified IPMS1 proteins. In other cases, the IPMS nucleic acids or IPMS proteins that are modified can be from a different species from the plant, plant cell, or plant seed that is modified to include the modified IPMS1 nucleic acids or the modified IPMS1 proteins.
Determination of Stably Transformed Plant Tissues: To confirm the presence of the IPMS1 nucleic acids in the regenerating plants, or seeds or progeny derived from the regenerated plant, a variety of assays may be performed. Such assays include, for example, molecular biological assays available to those of skill in the art, such as Southern and Northern blotting and PCR; biochemical assays, such as detecting the presence of a protein product. e.g., by immunological means (ELISAs and Western blots) or by enzymatic function; plant part assays, such as leaf, seed or root assays; and also, by analyzing the phenotype of the whole regenerated plant.
Whereas DNA analysis techniques may be conducted using DNA isolated from any part of a plant. RNA may only be expressed in particular cells or tissue types and so RNA for analysis can be obtained from those tissues. PCR techniques may also be used for detection and quantification of RNA produced from introduced IPMS1 nucleic acids. PCR also be used to reverse transcribe RNA into DNA, using enzymes such as reverse transcriptase, and then this DNA can be amplified by use of conventional PCR techniques. Further information about the nature of the RNA product may be obtained by Northern blotting. This technique will demonstrate the presence of an RNA species and give information about the integrity of that RNA. The presence or absence of an RNA species can also be determined using dot or slot blot Northern hybridizations. These techniques are modifications of Northern blotting and also demonstrate the presence or absence of an RNA species.
While Southern blotting and PCR may be used to detect the IPMS1 nucleic acid in question, they do not provide information as to whether the preselected DNA segment is being expressed. Expression may be evaluated by specifically identifying the protein products of the introduced modified IPMS1 nucleic acids or evaluating the phenotypic changes brought about by their expression.
Assays for the production and identification of specific proteins may make use of physical-chemical, structural, functional, or other properties of the proteins. Unique physical-chemical or structural properties allow the proteins to be separated and identified by electrophoretic procedures, such as native or denaturing gel electrophoresis or isoelectric focusing, or by chromatographic techniques such as ion exchange, liquid chromatography or gel exclusion chromatography. The unique structures of individual proteins offer opportunities for use of specific antibodies to detect their presence in formats such as an ELISA assay. Combinations of approaches may be employed with even greater specificity such as Western blotting in which antibodies are used to locate individual gene products that have been separated by electrophoretic techniques. Additional techniques may be employed to absolutely confirm the identity of the modified IPMS1 sequences such as by amino acid sequencing following purification of IPMS1 nucleic acid or IPMS1 protein. The Examples of this application also provide assay procedures for detecting and quantifying IPMS1 activity. Other procedures may be additionally used.
The expression of a gene product can also be determined by evaluating the phenotypic results of its expression. These assays also may take many forms including but not limited to analyzing changes in the chemical composition, morphology, or physiological properties of the plant. Chemical composition may be altered by expression of preselected DNA segments encoding storage proteins which change amino acid composition and may be detected by amino acid analysis.
As used herein, the term “plant” is used in its broadest sense. It includes, but is not limited to, any species of grass (e.g. forage, grain-producing, turf grass species), ornamental or decorative, crop or cereal, fodder or forage, fruit or vegetable, fruit plant or vegetable plant, herb plant, woody plant, flower plant or tree. It is not meant to limit a plant to any particular structure. It also refers to a unicellular plant (e.g. microalga) and a plurality of plant cells that are largely differentiated into a colony (e.g. volvox) or a structure that is present at any stage of a plant's development. Such structures include, but are not limited to, a seed, a tiller, a sprig, a stolen, a plug, a rhizome, a shoot, a stem, a leaf, a flower petal, a fruit, et cetera.
As used herein, “isolated” means a nucleic acid or polypeptide has been removed from its natural or native cell. Thus, the nucleic acid or polypeptide can be physically isolated from the cell or the nucleic acid or polypeptide can be present or maintained in another cell where it is not naturally present or synthesized.
The term “transgenic” when used in reference to a plant or leaf or fruit or seed or plant biomass, for example a “transgenic plant.” transgenic leaf,” “transgenic fruit.” “transgenic fruit,” “transgenic seed,” “transgenic biomass,” or a “transgenic host cell” refers to a plant or leaf or fruit or seed or biomass that contains at least one heterologous or foreign gene in one or more of its cells. The term “transgenic plant material” refers broadly to a plant, a plant structure, a plant tissue, a plant seed or a plant cell that contains at least one heterologous gene in one or more of its cells.
The term “transgene” refers to a foreign gene that is placed into an organism (e.g. a plant) or host cell by the process of transfection. The term “foreign gene” or heterologous gene refers to any nucleic acid (e.g., gene sequence) that is introduced into the genome of an organism or tissue of an organism or a host cell by experimental manipulations, such as those described herein, and may include gene sequences found in that organism so long as the introduced gene does not reside in the same location, as does the naturally occurring gene.
As used herein, a “native” nucleic acid or polypeptide means a DNA. RNA or amino acid sequence or segment that has not been manipulated in vitro, i.e., has not been isolated, purified, and/or amplified.
As used herein, the term “wild-type” when made in reference to a gene refers to a functional gene common throughout an outbred population. As used herein, the term “wild-type” when made in reference to a gene product refers to a functional gene product common throughout an outbred population. A functional wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designated the “normal” or “wild-type” form of the gene. As used herein, the term “wild-type” when made in reference to a plant refers to the plant type common throughout an outbred population that has not been genetically manipulated to contain an expression cassette, e.g., any of the expression cassettes described herein.
The following Examples illustrate some of the experimental work performed and materials used in the development of the invention. Appendix A may provide further information.
This Example illustrates some of the materials and methods used in the development of the invention.
Table 4 below lists some of the resources used in the experiments described herein.
The T-DNA insertional mutants ipms1-4 (SALK_101771), ipms1-5 (WiscDsLoxHs221_05F), ipms2-1 (WiscDsLox426A07), ipms2-2 (SALK_046876), ahass2-7 (WiscDsLoxHs009_02G), ahass2-8 (WiscDsLoxHs110_12G), ahass1-1 (SALK_096207), and ahass1-2 (SALK_108628) can be obtained from ABRC (see website at abrc.osu.edu).
An EMS mutant line was first identified in a screen for vacuolar phenotypes (Avila et al., 2003) was crossed with wild type (Col-0) for three times to obtain a progeny with consistently inherited vacuolar and growth phenotypes, which was designated as eva1.
Except for chronical treatments with specified chemicals, Arabidopsis seeds were stratified and grown on medium containing half-strength Linsmaier and Skoog nutrients (½ LS; Caisson Labs, LSP03), 1% sucrose and 0.4% phytagel (Sigma-Aldrich, P8169) in chambers configured with 21° C. and 16-hour light: 8-hour dark cycle.
To examine the effect of latrunculin B (Lat B) on root elongation (
Photographs were also acquired 8 days after the transplant.
In another pharmaceutical examination using AZD-8055, wortmannin and Lat B (
Exogenous feeding of 1 mM BCAA was performed by stratification and germination of seeds on ½ LS, 1% sucrose and 1% Agar medium containing 1 mM equal concentrations of Ile, Val and Leu. L-Isoleucine (Sigma-Aldrich, I2752), L-Valine (Sigma-Aldrich, V0500) and L-Leucine (Sigma-Aldrich, L8000) were dissolved in water to prepare 1 M stock solutions, which were filtered by Millex-GS 0.22 μm filter units (Millipore, SLGS033SS).
A Zeiss LSM 510 META and a Nikon A1Rsi laser scanning confocal microscope were used for imaging. Acquired images were handled by NIS-Elements Advanced Research (Nikon), ZEN (Zeiss) and Fiji (ImageJ) (Schindelin et al., 2012). The fluorescent protein fusions used in this study are GFP-δTIP (Cutler et al., 2000), ERYK (Nelson et al., 2007), YFP-ABD2 (Sheahan et al., 2004), GFP-CASP (Renna et al., 2005), SEC-RFP (Faso et al., 2009) and -TIP-YFP (Nelson et al., 2007). Transformation of Arabidopsis plants were conducted using floral dip method (Clough and Bent, 1998).
Image acquisition and further evaluation of the ER cisternae was conducted using a previously described method (Cao et al., 2016) that measures the occupancy of ER area in a region of interest. Analyses of the actin cytoskeletal organization were performed following a previously described procedure (Lu and Day, 2017). Briefly, Z-stack images with 0.5 μm intervals were acquired to cover the whole epidermal cell. The Z-stack series were converted to maximal projection images using NIS-Elements Advanced Research (Nikon) and Fiji (ImageJ) (Schindelin et al., 2012). Utilizing two ImageJ macros that were previously generated (Lu and Day, 2017), skewness was measured to present the distribution of YFP-ABD2 fluorescence intensity and occupancy was measured for the density of skeletonized YFP-ABD2 fluorescence signal.
All temporal chemical treatments were performed using 10-day old seedlings. Each of the following chemicals was first dissolved in DMSO to prepare a stock solution, and then diluted in Arabidopsis growth medium (½ LS and 1% sucrose) to reach the specific working concentration. 33 μM Wortmannin (Sigma-Aldrich, W1628) and 100 μM LY294002 (MedChemExpress, HY-10108) were used to treat seedlings for 2 hr. Latrunculin B (Sigma-Aldrich, L5288) and Oryzalin (Chem Service Inc, N-12729) were diluted to 25 μM and 40 μM, respectively, for 2-hour treatments. For TOR inhibition, seedlings were incubated with 5 μM AZD-8055 (MedChemExpress, HY-10422) or 1 μM Torin2 (MedChemExpress, HY-13002) for 2 or 4 hours as the figure legends indicated. 10 μM solution of β-estradiol (Sigma-Aldrich, E8875) was used to induce gene silencing.
Plants used for amino acid extraction were grown under standard conditions for 10 or 20 days. The aerial tissue (fresh weight around 10 mg) was harvested into a 2 mL tube with two 3 mm steel beads and flash frozen in liquid N2. Tissue was either used immediately or stored at −80° C. until extraction. Tissue was pulverized using a mixer mill (Retsch Mill, MM400) for 1 min at 30 times per second. Amino acids were extracted as previously reported (Xing and Last, 2017; Angelovici et al., 2013). Briefly, an amino acid extraction buffer was prepared with ˜2 μM heavy labeled amino acids standards (13C, 15N, Sigma-Aldrich), 10 μM 1,4-dithiothreitol (DTT, Sigma-Aldrich), and 10 mM perfluoroheptanoic acid (PFHA, Sigma-Aldrich). To the ground tissue, 350 μL of extraction buffer was added, vortexed for 10 s and heated at 90° C. for 10 min. Tubes were cooled on ice for 5 min and centrifuged for 10 min at 4° C. at 13,000×g. The supernatant was applied to a low-binding hydrophilic 0.2 μm centrifugal polytetrafluoroethylene (PTFE) filter (Millipore, UFC30LG25) and centrifuged for 5 min at 3,500×g. 150 μL flow through was transferred to 2 mL glass vials with glass insert for LC-MS analysis.
Amino acid detection and quantification by LC-MS/MS was performed as previously reported (Xing and Last, 2017; Angelovici et al., 2013). Briefly, a dilution series (12.2 nM to 250 μM) of each individual amino acid standard was made containing the same concentration of the heavy standards as was in the amino acid extraction buffer. Samples were injected into a Quattro micro API LC/MS/MS (Waters) equipped with an Acquity UHPLC HSS T3 1.8 μm column (Waters) using a three-function method. A 13 min LC method was used with solvent A (10 mM PFHA) and solvent B (acetonitrile) at a flow rate of 0.3 mL/minute. Amino acids were quantified by comparison to their standard curves using QuanLynx.
EdU (5-ethynyl-2′-deoxyuridine) staining of root apical meristem was performed using Click-iT EdU Alexa Fluor 488 Imaging Kit (Invitrogen, C10337), following a protocol that was adapted for plant tissues (Kotogdny et al., 2010). Labeling was performed by incubating 10-day old Arabidopsis seedlings in 10 μM EdU in Arabidopsis growth medium (½ LS and 1% sucrose) for 30 min in a Percival chamber. All samples were then incubated with a fixation buffer (4% formaldehyde, 0.1% Triton X-100, 1×PBS) for 30 min. All samples were washed for three times, 10 min each, with 1×PBS after fixation. The EdU detection was conducted by 30 min incubation in dark with the Click-iT cocktail, which was prepared according to the manual of Click-iT EdU Alexa Fluor 488 Imaging Kit. Each sample was immediately washed for three times, 10 min each, with 1×PBS before imaging.
PI (propidium iodide) staining was conducted by 3 min incubation of Arabidopsis seedlings in propidium iodide (Invitrogen. P3566) diluted to 1 μg/mL using Arabidopsis growth medium (½ LS and 1% sucrose). After staining, all samples were immediately washed for 1 min and then subjected to imaging. Confocal images of propidium iodide stained root tips were analyzed using Cell-O-Tape (French et al., 2012), which is a plugin of ImageJ that automatically segments three zones in a root tip (the meristem, the transition zone and the mature zone) by comparing the lengths of adjacent cells in the same cortical layer. Adjacent cells with significant increase in cell length belong to the transition zone. Cells before and after the transition zone are categorized as cells in the meristem and the mature zone respectively. The program records the length of each cell and the cell number in each zone.
To detect the phosphorylation status of S6K, 50 mg plant aerial tissue was used for protein extraction using 1.5 mL extraction buffer of 1×PBS, pH 7.4, containing 250 mM sucrose. Protease Inhibitor Cocktail (Sigma-Aldrich, P9599) and PhosSTOP phosphatase inhibitor (Roche. 4906845001). Three times of centrifugation, 1 k×g for 5 min, 14 k×g for 5 min and 135 k×g for 30 min, were conducted to separate the soluble proteins. The supernatant from the last centrifugation was separated, concentrated to 200 μL using an Amicon Ultra centrifugal unit (Millipore, UFC501024), and then mixed with 40 μL 6× Laemmli buffer. Proteins were denatured by incubation at 95° C. for 10 min. Protein samples were separated on 15% SDS-PAGE with 8M urea and blotted to PVDF membranes (Bio-Rad, 1620177). Blots were blocked with 5% milk for 1 hour at room temperature. Blots were incubated with primary antibodies of either anti-S6K (Agrisera, AS12 1855) or anti-S6K-phosphorylated (Abcam, ab207399) overnight at 4° C. and subsequently with secondary HRP conjugated goat anti-rabbit antibody (Sigma-Aldrich, A0545) for 1 hour at room temperature.
The aerial parts of 10-day old Arabidopsis seedlings were collected, and then lyophilized and measured for dry weight. Total anthocyanins were extracted using 1 μL extraction buffer (50% methanol containing 3% formic acid) per 50 μg dry weight. After overnight incubation with extraction buffer at room temperature, the supernatant was collected and measured absorbance of 532 nm.
The electron microscopic imaging of the endomembrane structures and chloroplasts were performed following an established protocol (Kim et al., 2018). In brief, 1 mm×1 mm pieces of cotyledon samples were cut and fixed in TEM fixative buffer (2.5% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4) with vacuum infiltration. The fixed samples were stained with 1% osmium tetroxide overnight at 4° C. After series of dehydration with acetone, the samples were infiltrated and embedded in Spurr's Resin. Sections with 50 nm thickness were cut and mounted on the copper grid and 10 well slides. For TEM, the grids were post-stained in 2% uranyl-acetate for 30 min and then treated with 1% lead citrate for 15 min. JEOL 100CX TEM (JEOL USA) was used to observe the ultrastructure of cotyledon.
The thickness of cotyledons was measured as previously described (Weraduwage et al., 2016). Briefly, 2 mm xl mm samples cut from the center of the cotyledons were fixed in fixative buffer (4% paraformaldehyde and 0.5% glutaraldehyde in 1×PBS, pH 7.4) with vacuum infiltration. The fixed samples were stained with 1% osmium tetroxide overnight at 4° C. After series of dehydration with acetone, the samples were infiltrated and embedded in Spurr's Resin. Sections with 500 nm thickness were cut and mounted on the copper grid and 10 well slides. For leaf thickness analysis, the sections were stained with 1% toluidine blue for 1 min and washed with running water. Images were taken using Axio Imager M2 (Zeiss), and measurement of leaf thickness was performed using AxioVision SE64 Rel. 4.9.1 (Zeiss) software. Three biological samples with three technical replicates were used to measure leaf thickness.
A confocal microscopy-based screen was performed on an EMS-mutagenized population to identify mutants with defects in the subcellular distribution of a GFP-tagged tonoplast intrinsic protein (TIP), GFP-δTIP (Avila et al., 2003; Cutler et al., 2000). The inventors identified mutant line eva1, a mutant characterized by severe defects in vacuole morphology early in development. During the first 10 days after germination, in wild-type (WT) cotyledon epidermal cells, small vacuoles undergo membrane fusion to form a single large central vacuole (Zhang et al., 2014) (
The inventors next identified the causative mutation in eva1. Bulked segregant analysis and whole-genome resequencing narrowed down the eva1 mutation to a G-to-A transition in IPMS1 (ATIG18500) causing an aspartate (Asp)-to-asparagine (Asn) residue substitution (
The role of IPMS1 in BCAA biosynthesis has been characterized as directing flux towards Leu biosynthesis, and away from the competing product Val (Xing and Last, 2017; de Kraker et al., 2007; Field et al., 2004) (
This Example describes experiments designed to evaluate whether the transient changes in BCAA accumulation and vacuole morphology affected early plant growth.
At 10 days following germination, the IPMS1 loss-of-function mutants displayed retardation of growth and development (
The inventors then examined the development of cotyledons by confocal microscopy. The cotyledons constituted most of the aerial tissue for amino acid profiling and were used for analyses. Cotyledons of ipms1 mutants were thicker and larger than WT (
This Example describes analysis of other endomembrane compartments by experiments designed to provide more insights into the eva1 vacuolar phenotypes.
The endoplasmic reticulum (ER) is the most extensively distributed organelle of the plant secretory pathway, and it is closely associated with several other membrane-bound organelles, including the vacuole (Ueda et al., 2010).
In the eva1 mutant, the ER luminal marker ERYK (Nelson et al., 2007) revealed a pronounced appearance of the cortical ER network with strikingly thickened strands compared to WT (
These results and the absence of retention of the vacuolar marker in the ER document that the morphology of the vacuole, organization of the Golgi and the ER network are markedly affected by the eva1 mutation, while bulk-flow secretion is unaffected.
Collectively, the root-related defects of the ipms1 mutants, including delayed formation of root hairs and reduced number of lateral roots, are reminiscent of mutants with impaired actin depolymerization or promoted actin bundling (Ketelaar et al., 2004; Deeks et al., 2005), consistent with the possibility that reorganization of actin cytoskeleton may be causative of the observed developmental phenotypes. The establishment and maintenance of the trans-vacuolar strands, ER network and Golgi subcellular distribution are dependent on the actin cytoskeleton. The inventors hypothesized that the organization of actin cytoskeleton may be altered in eva1.
Confocal microscopy in cells expressing the actin filament (F-actin) marker YFP-ABD2 (Sheahan et al., 2004) revealed coalescence of actin cables compared to WT (
Experiments were designed to validate this hypothesis by testing the sensitivity of the ipms1 alleles to the F-actin depolymerizing reagent latrunculin B (Lat B) (Cao et al., 2016). The primary root length of 10-day old ipms1-4 and ipms1-5 was approximately 50% of WT (
This Example describes experiments involving chemicals that alter the vacuolar morphogenesis and cytoskeleton integrity to provide insights into the mechanisms by which eva1 defects in BCAA biosynthesis led to alteration of the organization of subcellular structures. The inventors hypothesized that the persistence of small vacuoles in eva1 could be due to delayed vacuole membrane fusion during vacuole morphogenesis. To test this, wortmannin (Wm), an inhibitor of phosphoinositide 3-kinases (PI3Ks), was first employed to disrupt the balance of phosphoinositides and promotes homotypic tonoplast fusion (Zheng et al., 2014; Wang et al., 2009; Mayer et al., 2000).
As shown in
The relationship between trans-vacuolar strands and integrity of the cytoskeleton in eva1 was then investigated. After a two-hour treatment with Lat B, the trans-vacuolar strands disappeared but the small vacuoles persisted in eva1 cotyledon epidermal cells (
As described above, homotypic membrane fusion and F-actin bundling are two processes directly involved in the eva1 Leu biosynthetic mutant phenotypes (
To test this hypothesis, two TOR inhibitors with high selectivity for TOR over PI3Ks were employed: AZD-8055 and Torin2 (Benjamin et al., 2011; Liu et al., 2011; Chresta et al., 2010), which also effectively inhibit plant TOR (Li et al., 2017; Wang et al., 2018; Dong et al., 2017; Pu et al., 2017).
Ten-day old wild type and eva1 seedlings were transferred to liquid growth medium containing 5 μM AZD-8055. Compared to untreated samples, wild type cells did not exhibit significant changes in the morphology of the central vacuole and the few thin trans-vacuolar strands after 2 or 4 hours of incubation, although numerous fluorescent punctae appeared (
In addition to the effects of temporal treatment on vacuolar phenotype (
The inventors next sought to confirm these results by testing the activation status of TOR in ipms1 mutants. Based on the evidence that TOR inhibition rescued the ipms1 subcellular phenotypes, we predicted to find an increased level of TOR activity in ipms1 mutants compared to wild type. S6K is a conserved substrate of TOR protein kinase and its phosphorylation status has been adopted as an indicator of TOR activity in plants (Pfeiffer et al., 2016; Wang et al., 2018; Dong et al., 2017; Xiong and Sheen, 2012). Indeed, immunoblot analyses with specific antisera for either phosphorylated or total S6K (Pfeiffer et al., 2016; Wang et al., 2018; Dong et al., 2017; Xiong and Sheen, 2012) revealed increased levels of TOR-phosphorylated S6K in eva1 and ipms1-4 compared to WT, despite similar levels of total S6K in three genotypes (
To validate this conclusion, DNA synthesis was monitored in root tips because stimulated TOR signaling promotes cell proliferation in the root apical meristem. Such cellular proliferation can be detected by EdU staining of newly synthesized DNA (Li et al., 2017; Dong et al., 2017; Xiong et al., 2013). Consistent with the inventors' hypothesis, EdU staining displayed enhanced labeling in the root apical meristem of ipms1-4 and ipms1-5 compared to wild type (
The foregoing results show suppression of vacuole phenotypes by TOR inhibition, increased levels of S6K phosphorylation, and root apical meristem activity (i.e., increased DNA synthesis and cell number) in the ipms1 mutants. These results support the hypothesis that TOR signaling is up-regulated in the IPMS1 loss-of-function mutants.
This Example describes testing of the role of TOR signaling and its specificity in the verified BCAA over-accumulation-induced phenotypes.
An estradiol-inducible TOR mutant (tor-es) (Xiong and Sheen, 2012) and a loss-of-function mutant of AtRAPTOR1B (raptor1b, SALK_022096) (Salem et al., 2017; a locus encoding the functional TORC1 component RAPTOR in Arabidopsis (Salem et al., 2018: Anderson et al., 2005)) were used in these experiments.
Before induction of TOR silencing, like wild type seedlings, for-es seedlings grown on BCAA-supplemented medium exhibited induced F-actin bundling compared to for-es grown on normal medium (compare
Next, experiments were performed to test the generality of the connection between over-accumulation of BCAAs, morphological alteration of cellular structures, and functional TOR signaling. To do so, a variety of BCAA mutants (Xing and Last. 2017) were used, combined with BCAA feeding. For example, an ipms1-1D mutant was chosen because it exhibits a modest decrease in Val with an increase in Leu. The ahass1-1 mutant exhibits a small increase in Val. The ahass2-7 mutant exhibits decreased Val and Leu. The omr1-11D mutant has a greater than 140-fold increase in Ile compared to WT.
Confocal microscopy analyses of cotyledon epidermal cells revealed that the organization of F-actin in ipms1-1 and ahass1-1 mutants resembled that of wild type (
The striking phenotype of enhanced ER strands in omr1-11D was recovered by a 2-hour Torin2 treatment. In addition to bundling of F-actin and enhancement of ER strands, supplementation of BCAAs also induced the formation of prominent trans-vacuole strands.
Together, these results support a general correlation between over-accumulation of BCAAs and distorted actin cytoskeleton and endomembranes.
All patents and publications referenced or mentioned herein are indicative of the levels of skill of those skilled in the art to which the invention pertains, and each such referenced patent or publication is hereby specifically incorporated by reference to the same extent as if it had been incorporated by reference in its entirety individually or set forth herein in its entirety. Applicants reserve the right to physically incorporate into this specification any and all materials and information from any such cited patents or publications.
The following statements describe some of the elements or features of the invention. The statements provide features that can be claimed in the application and the dependencies of the statements illustrate combinations of features that can be present when included in the claims.
The specific plants, plant cells, seeds, methods, and compositions described herein are representative of preferred embodiments and are exemplary and not intended as limitations on the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideration of this specification, and are encompassed within the spirit of the invention as defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.
The invention illustratively described herein suitably may be practiced in the absence of any element or elements, or limitation or limitations, which is not specifically disclosed herein as essential. The methods and processes illustratively described herein suitably may be practiced in differing orders of steps, and the methods and processes are not necessarily restricted to the orders of steps indicated herein or in the claims.
Under no circumstances may the patent be interpreted to be limited to the specific examples or embodiments or methods specifically disclosed herein. Under no circumstances may the patent be interpreted to be limited by any statement made by any Examiner or any other official or employee of the Patent and Trademark Office unless such statement is specifically and without qualification or reservation expressly adopted in a responsive writing by Applicants.
The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, it will be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims and statements of the invention.
The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.
This application claims the benefit of U.S. Provisional Application Ser. No. 63/006,543, filed Apr. 7, 2020, which application is incorporated by reference herein its entirety.
This invention was made with government support under 1714561 awarded by the National Science Foundation. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/026166 | 4/7/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63006543 | Apr 2020 | US |
