Patent Description:
During photosynthesis, at low sunlight intensities, all absorbed photons are utilized efficiently to drive electrons in the electron-transport chain. As the level of irradiance increases further, photosynthesis becomes saturated and reaches a plateau due to the fact that the carbon reactions cannot keep up with the linear increase in light absorption. Plant lines with a wild type light-harvesting antenna system reach this light intensity for saturation at lower levels of irradiance than their mutant counterparts. The sunlight harvested by the chlorophyll antenna exceeds the maximal operational capacity of the electron-transport chain and of the carbon reactions of photosynthesis, rendering the excess absorbed photons useless. Under bright sunlight conditions (<NUM>µmol photons m-<NUM>-<NUM>), wild type lines with their fully developed light-harvesting antenna utilize photons inefficiently; only about <NUM>% of the incoming sunlight energy is converted into useful photosynthesis, while excess absorbed energy is dissipated by the non-photochemical quenching (NPQ) process. <NPL>) relates to a recessive mutation in Arabidopsis, named chaos (for chlorophyll a/b binding protein harvesting-organelle specific; designated gene symbol CAO), which was isolated by using transposon tagging. Characterization of the phenotype of the chaos mutant reportedly revealed a specific reduction of pigment binding antenna proteins in the thylakoid membrane. <NPL>) relates to a truncated light-harvesting antenna size3 (tla3) DNA insertional transformant of Chlamydomonas reinhardtii which was reported to be a chlorophyll-deficient mutant with a lighter green phenotype, a lower chlorophyll (Chl) per cell content, and higher Chla/b ratio than corresponding wild-type strains. The work reportedly demonstrated that cpsrp43 deletion in green microalgae could be employed to generate tla mutants with a substantially diminished Chl antenna size which would exhibit improved solar energy conversion efficiency and photosynthetic productivity under mass culture and bright sunlight conditions. <NPL>) discusses the concept of the Truncated Light-harvesting chlorophyll Antenna (TLA) size as a tool by which to maximize sunlight utilization and photosynthetic productivity in microalgal mass cultures or high-density plant canopies. <NPL> reports a sequence described as "PREDICTED: signal recognition particle <NUM> kDa protein, chloroplastic [Nicotiana sylvestris]". <NPL>) discusses the relationships between observed photosynthetic efficiencies and theoretical efficiencies in relation to light saturation of photosynthesis.

Tobacco plants that exhibit altered photosynthesis are described herein, as well as methods of making and using such plants.

The invention is as defined in the claims appended hereto.

The invention provides a method of making a Nicotiana tabacum plant having increased biomass, which method comprises targeted mutation of an endogenous Truncated Light-harvesting Antenna (TLA) nucleic acid comprising a sequence having at least <NUM>% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: <NUM> and <NUM>, wherein the mutation in the TLA sequence results in reduced expression or activity of the encoded TLA polypeptide, wherein the plant comprising the mutation comprises an increased leaf biomass relative to the leaf biomass from a plant lacking said mutation. Described herein is a method of making a Nicotiana tabacum plant. Such a method typically includes inducing mutagenesis in N. tabacum cells to produce mutagenized N. tabacum cells; obtaining one or more N. tabacum plants from the mutagenized N. tabacum cells; and identifying at least one of the N. tabacum plants that comprises a mutated TLA sequence. Representative TLA sequences have at least <NUM>% sequence identity to a sequence shown in SEQ ID NOs: <NUM> or <NUM>.

Such a method further can include identifying at least one of the N. tabacum plants that exhibits reduced amount of TLA mRNA; reduced amount of TLA polypeptide; reduced activity of a TLA polypeptide; reduced concentration of thylakoid membranes in the photosystems; reduced amount of total chlorophyll; increased ratio of chlorophyll a to chlorophyll b; but at least increased biomass leaf relative to a N. tabacum plant lacking a mutated TLA sequence.

Leaf from the mutant N. tabacum plant may exhibit comparable quality than leaf from the plant lacking a mutated TLA sequence. In some embodiments, the N. tabacum plant is a Burley type, a dark type, a flue-cured type, or an Oriental type.

Described herein is a variety of Nicotiana tabacum. Such a variety typically includes plants having a mutation in an endogenous nucleic acid, where the wild type endogenous nucleic acid encodes the TLA sequence shown in SEQ ID NO: <NUM> or <NUM>. Typically, leaf from the mutant plants exhibits reduced amount of TLA mRNA; reduced amount of TLA polypeptide; reduced activity of a TLA polypeptide; reduced concentration of thylakoid membranes in the photosystems; reduced amount of total chlorophyll; increased ratio of chlorophyll a to chlorophyll b; but at least increased biomass relative to leaf from a plant lacking the mutation. In some embodiments, leaf from the mutant N. tabacum plant exhibits comparable quality than leaf from the plant lacking a mutated TLA sequence.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods and compositions of matter belong. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the methods and compositions of matter, suitable methods and materials are described below. In addition, the materials and methods are illustrative only and not intended to be limiting.

Photosynthetic organisms (e.g., green plants, algae, and many bacteria) contain reaction centers, which is a complex of proteins, pigments and co-factors that perform the photosynthetic conversion of light to energy via a multitude of electron transfer steps. Despite the evolutionary distances between such photosynthetic organisms, the reaction centers possess remarkable homology. In contrast, the light-harvesting complexes in the various photosynthetic organisms differ. The current methods used to measure light absorption and utilization in plants and microalgae are described in <NPL>) and <NPL>).

During the process of photosynthesis, the light-harvesting complex, which typically surrounds the reaction center, absorbs the light (e.g., sunlight). In plants, the light energy is absorbed by the light-harvesting antenna complex and is transferred to two chlorophyll a molecules, which are embedded in the reaction center. As described herein, light-harvesting antenna complex size can be inhibited or reduced in tobacco using, for example, mutagenesis or RNAi, to diminish over-absorption of sunlight at the higher canopy. Embodiments, including transgenic plants, relating to RNAi are described for demonstration only and do not per se form part of the claimed invention. Diminishing over-absorption of sunlight in the higher canopy of the plant can minimize wasteful dissipation of energy, while, at the same time, allowing for a far greater transmittance of sunlight deeper into the lower canopy by eliminating unwanted shading, particularly under high density growth conditions.

Tobacco genes and the encoded proteins were screened to identify those involved in harvesting light; those sequences identified in the screen were evaluated further to identify their mode of action. The sequences identified herein can be inhibited (e.g., by RNA interference and/or mutation) to result in smaller light-harvesting chlorophyll antenna size, which ultimately results in a plant that exhibits substantially improved photosynthetic efficiency. A number of tobacco sequences (e.g., truncated light-harvesting antenna (TLA) <NUM>, TLA3, TLA4 and CAO (Chlorophyllide a oxygenase)) as well as corresponding homologues from Chlamydomonas and/or Arabidopsis were obtained.

Four TLA-related genes, TLA2 and TLA2 Homo (encoding the CpFTSY protein), TLA3 and TLA3 Homo (encoding theCpSRP43 protein), and TLA4 (encoding the CpSRP54 protein) were obtained from Nicotiana tabacum, as well as three CAO genes (CAO-<NUM>, COA-<NUM> and CAO-<NUM>). Based on sequence alignment, CAO-<NUM> appears to have originated from Nicotiana tometosiformis, while both CAO-<NUM> and COA-<NUM> originated from Nicotiana sylvestris.

As described in more detail below, the expression of one or more of the sequences described herein can be inhibited or reduced using, for example, mutagenesis or inhibitory RNA (RNAi). The resulting plants can be evaluated for total chlorophyll, as well as the ratio of chlorophyll a : chlorophyll b and/or the photosynthetic apparatus size in Photosystem I (PSI) and/or Photosystem II (PSII). Sequences that, when their expression is knocked down or completely eliminated, result in a higher ratio of chlorophyll a : b and reduced antenna size in PSI and/or PSII systems were desired, as it is these sequences that will substantially improve photosynthetic efficiency.

As described herein, modification of TLA sequences and CAO sequences in tobacco results in smaller light-harvesting chlorophyll antenna complex size by reducing antenna number and a substantially improved photosynthetic efficiency. The modified tobacco lines further exhibit enhanced productivity (e.g., increased biomass).

Specifically, for example, knocking down TLA2 resulted in plants that grow slower than wild type plants, plants that have a ratio of Chlorophyll a : Chlorophyll b similar to wild type, plants that have an antenna size in PSI that is similar to wild type, plants that have an antenna size in PSII that is reduced compared to wild type plants, and plants that have a lighter leaf color than wild type plants due to the reduction of total chlorophyll content.

In addition, knocking down TLA3 resulted in plants that grow at a similar rate to wild type plants, plants that have a ratio of Chlorophyll a : Chlorophyll b that is increased relative to wild type plants, plants that have an antenna size in both PSI and PSII that is reduced compared to wild type plants, plants in which the amount of total chlorophyll increased from low to normal levels during maturation, relative to wild type plants.

Further, knocking down TLA4 resulted in plants in which the amount of total chlorophyll was reduced, but both the ratio of chlorophyll a : chlorophyll b and the antenna size in both PSI and PSII were unchanged.

Nucleic acids encoding TLA2, TLA2-homo, TLA3, TLA3-homo and TLA4 from N. tabacum are shown in SEQ ID NOs: <NUM>, <NUM>, <NUM>, <NUM>, and <NUM>, respectively, and nucleic acids encoding CAO2, CAO3 and CAO4 from N. tabacum are shown in SEQ ID NOs: <NUM>, <NUM>, and <NUM>, respectively. Unless otherwise specified, nucleic acids referred to herein can refer to DNA and RNA, and also can refer to nucleic acids that contain one or more nucleotide analogs or backbone modifications. Nucleic acids can be single stranded or double stranded, and linear or circular, both of which usually depend upon the intended use.

As used herein, an "isolated" nucleic acid molecule is a nucleic acid molecule that is free of sequences that naturally flank one or both ends of the nucleic acid in the genome of the organism from which the isolated nucleic acid molecule is derived (e.g., a cDNA or genomic DNA fragment produced by PCR or restriction endonuclease digestion). Such an isolated nucleic acid molecule is generally introduced into a vector (e.g., a cloning vector, or an expression vector) for convenience of manipulation or to generate a fusion nucleic acid molecule, discussed in more detail below. In addition, an isolated nucleic acid molecule can include an engineered nucleic acid molecule such as a recombinant or a synthetic nucleic acid molecule.

The sequence of the TLA2, TLA2-homo, TLA3, TLA3-homo and TLA4 polypeptides from N. tabacum are shown in SEQ ID NOs: <NUM>, <NUM>, <NUM>, <NUM>, and <NUM>, respectively, and the sequences of the CAO2, CAO3 and CAO4 polypeptides from N. tabacum are shown in SEQ ID NOs: <NUM>, <NUM>, and <NUM>, respectively. As used herein, a "purified" polypeptide is a polypeptide that has been separated or purified from cellular components that naturally accompany it. Typically, the polypeptide is considered "purified" when it is at least <NUM>% (e.g., at least <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>%) by dry weight, free from the polypeptides and naturally occurring molecules with which it is naturally associated. Since a polypeptide that is chemically synthesized is, by nature, separated from the components that naturally accompany it, a synthetic polypeptide is "purified.

Nucleic acids can be isolated using techniques well known in the art. For example, nucleic acids can be isolated using any method including, without limitation, recombinant nucleic acid technology, and/or the polymerase chain reaction (PCR). General PCR techniques are described, for example in<NPL>. Recombinant nucleic acid techniques include, for example, restriction enzyme digestion and ligation, which can be used to isolate a nucleic acid. Isolated nucleic acids also can be chemically synthesized, either as a single nucleic acid molecule or as a series of oligonucleotides.

Polypeptides can be purified from natural sources (e.g., a biological sample) by known methods such as DEAE ion exchange, gel filtration, and hydroxyapatite chromatography. A polypeptide also can be purified, for example, by expressing a nucleic acid in an expression vector. In addition, a purified polypeptide can be obtained by chemical synthesis. The extent of purity of a polypeptide can be measured using any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

Nucleic acids can be detected using any number of amplification techniques (see, e.g.,<NPL>; and <CIT>; <CIT>; <CIT>; and <CIT>) with an appropriate pair of oligonucleotides (e.g., primers). A number of modifications to the original PCR have been developed and can be used to detect a nucleic acid. Nucleic acids also can be detected using hybridization.

Polypeptides can be detected using antibodies. Techniques for detecting polypeptides using antibodies include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence. An antibody can be polyclonal or monoclonal. An antibody having specific binding affinity for a polypeptide can be generated using methods well known in the art. The antibody can be attached to a solid support such as a microtiter plate using methods known in the art. In the presence of a polypeptide, an antibody-polypeptide complex is formed.

Detection (e.g., of an amplification product, a hybridization complex, or a polypeptide) is oftentimes accomplished using detectable labels. The term "label" is intended to encompass the use of direct labels as well as indirect labels. Detectable labels include enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.

Described herein is a construct, sometimes referred to as a vector, containing a nucleic acid (e.g., a coding sequence or a RNAi nucleic acid molecule). Constructs, including expression constructs (or expression vectors), are commercially available or can be produced by recombinant DNA techniques routine in the art. A construct containing a nucleic acid can have expression elements operably linked to such a nucleic acid, and further can include sequences such as those encoding a selectable marker (e.g., an antibiotic resistance gene). A construct can encode a chimeric or fusion polypeptide (i.e., a first polypeptide operatively linked to a second polypeptide). Representative first (or second) polypeptides are those that can be used in purification of the other (i.e., second (or first), respectively) polypeptide including, without limitation, 6xHis (SEQ ID NO:<NUM>) tag or glutathione S-transferase (GST).

Expression elements include nucleic acid sequences that direct and regulate expression of nucleic acid coding sequences. One example of an expression element is a promoter sequence. Expression elements also can include introns, enhancer sequences, response elements, or inducible elements that modulate expression of a nucleic acid. Expression elements can be of bacterial, yeast, insect, mammalian, or viral origin, and vectors can contain a combination of elements from different origins. As used herein, operably linked means that a promoter or other expression element(s) are positioned in a vector relative to a nucleic acid in such a way as to direct or regulate expression of the nucleic acid (e.g., in-frame).

Constructs as described herein can be introduced into a host cell. Many methods for introducing nucleic acids into host cells, both in vivo and in vitro, are well known to those skilled in the art and include, without limitation, electroporation, calcium phosphate precipitation, polyethylene glycol (PEG) transformation, heat shock, lipofection, microinjection, and viral-mediated nucleic acid transfer. As used herein, "host cell" refers to the particular cell into which the nucleic acid is introduced and also includes the progeny or potential progeny of such a cell. A host cell can be any prokaryotic or eukaryotic cell. For example, nucleic acids can be introduced into bacterial cells such as E. coli, or into insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.

RNA interference (RNAi), also called post-transcriptional gene silencing (PTGS), is a biological process in which RNA molecules inhibit gene expression, typically by causing the destruction of specific mRNA molecules. Without being bound by theory, it appears that, in the presence of an antisense RNA molecule that is complementary to an expressed message (i.e., a mRNA), the two strands anneal to generate long doublestranded RNA (dsRNA), which is digested into short (< <NUM> nucleotide) RNA duplexes, known as small interfering RNAs (siRNAs), by an enzyme known as Dicer. A complex of proteins known as the RNA Induced Silencing Complex (RISC) then unwinds siRNAs, and uses one strand to identify and thereby anneal to other copies of the original mRNA. RISC cleaves the mRNA within the complementary sequence, leaving the mRNA susceptible to further degradation by exonucleases, which effectively silences expression of the encoding gene.

Several methods have been developed that take advantage of the endogenous machinery to suppress the expression of a specific target gene and a number of companies offer RNAi design and synthesis services (e.g., Life Technologies, Applied Biosystems). In transgenic plants, the use of RNAi can involve the introduction of long dsRNA (e.g., greater than <NUM> bps) or siRNAs (e.g., <NUM> to <NUM> bps) that have complementarity to the target gene, both of which are processed by the endogenous machinery. Alternatively, the use of RNAi can involve the introduction of a small hairpin RNA (shRNA); shRNA is a nucleic acid that includes the sequence of the two desired siRNA strands, sense and antisense, on a single strand, connected by a "loop" or "spacer" nucleic acid. When the shRNA is transcribed, the two complementary portions anneal intra-molecularly to form a "hairpin," which is recognized and processed by the endogenous machinery.

A RNAi nucleic acid molecule as described herein is complementary to at least a portion of a target mRNA (e.g., a TLA mRNA, a CAO mRNA), and typically is referred to as an "antisense strand". Typically, the antisense strand includes at least <NUM> contiguous nucleotides of the DNA sequence (e.g., the nucleic acid sequence shown in SEQ ID NO:<NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>); it would be appreciated that the antisense strand has the "RNA equivalent" sequence of the DNA (e.g., uracils instead of thymines; ribose sugars instead of deoxyribose sugars).

A RNAi nucleic acid molecule can be, for example, <NUM> to <NUM> nucleotides in length (e.g., <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, or <NUM> to <NUM> nucleotides in length).

The antisense strand (e.g., a first nucleic acid) can be accompanied by a "sense strand" (e.g., a second nucleic acid), which is complementary to the antisense strand. In the latter case, each nucleic acid (e.g., each of the sense and antisense strands) can be between <NUM> and <NUM> nucleotides in length (e.g., between <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, or <NUM> to <NUM> nucleotides in length).

A spacer nucleic acid, sometimes referred to as a loop nucleic acid, can be positioned between the sense strand and the antisense strand. The spacer nucleic acid can be an intron (see, for example, <NPL>). Although not required, the intron can be functional (i.e., in sense orientation; i.e., spliceable) (see, for example,<NPL>). A spacer nucleic acid can be between <NUM> nucleotides and <NUM> nucleotides in length (e.g., <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, or <NUM>-<NUM> nucleotides in length).

A construct can be produced by operably linking a promoter that is operable in plant cells; a DNA region, that, when transcribed, produces an RNA molecule capable of forming a hairpin structure; and a DNA region involved in transcription termination and polyadenylation. It would be appreciated that the hairpin structure has two annealing RNA sequences, where one of the annealing RNA sequences of the hairpin RNA structure includes a sense sequence identical to at least <NUM> consecutive nucleotides of a TLA or CAO nucleotide sequence, and where the second of the annealing RNA sequences includes an antisense sequence that is identical to at least <NUM> consecutive nucleotides of the complement of the TLA or CAO nucleotide sequence. In addition, as indicated herein, the DNA region can include an intron (e.g., a functional intron). When present, the intron generally is located between the two annealing RNA sequences in sense orientation such that it is spliced out by the cellular machinery (e.g., the splicesome). Such a construct can be introduced into one or more plant cells to reduce the phenotypic expression of a nucleic acid (e.g., a nucleic acid sequence that is normally expressed in a plant cell).

A construct (e.g., an expression construct) can include an inverted-duplication of a segment of a TLA or CAO gene, where the inverted-duplication of the TLA or CAO gene segment includes a nucleotide sequence substantially identical to at least a portion of the TLA or CAO gene and the complement of the portion of the TLA or CAO gene, respectively. It would be appreciated that a single promoter can be used to drive expression of the inverted-duplication of the TLA or CAO gene segment, and that the inverted-duplication typically contains at least one copy of the portion of the TLA or CAO gene in the sense orientation. Such a construct can be introduced into one or more plant cells to delay, inhibit or otherwise reduce the expression of a TLA or CAO gene in the plant cells.

Representative RNAi nucleic acid molecules directed toward TLA2, TLA3 and TLA4 are shown in SEQ ID NOs: <NUM>, <NUM> and <NUM>, respectively, and a representative RNAi nucleic acid molecule directed toward CAO2, CAO3 and CAO4 is shown in SEQ ID NO:<NUM>. The sense strand and antisense strand are identified with dashed underlining, and a spacer or loop sequence lies between. It would be appreciated by the skilled artisan that the region of complementarity, between the antisense strand of the RNAi and the mRNA or between the antisense strand of the RNAi and the sense strand of the RNAi, can be over the entire length of the RNAi nucleic acid molecule, or the region of complementarity can be less than the entire length of the RNAi nucleic acid molecule. For example, a region of complementarity can refer to, for example, at least <NUM> nucleotides in length up to, for example, <NUM> nucleotides in length (e.g., at least <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> nucleotides in length up to, e.g., <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> nucleotides in length). A region of complementarity can refer to, for example, at least <NUM> contiguous nucleotides in length up to, for example, <NUM> contiguous nucleotides in length (e.g., at least <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> nucleotides in length up to, e.g., <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> contiguous nucleotides in length).

It would be appreciated by the skilled artisan that complementary can refer to, for example, <NUM>% sequence identity between the two nucleic acids. In addition, however, it also would be appreciated by the skilled artisan that complementary can refer to, for example, slightly less than <NUM>% sequence identity (e.g., at least <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>% sequence identity). In calculating percent sequence identity, two nucleic acids are aligned and the number of identical matches of nucleotides (or amino acid residues) between the two nucleic acids (or polypeptides) is determined. The number of identical matches is divided by the length of the aligned region (i.e., the number of aligned nucleotides (or amino acid residues)) and multiplied by <NUM> to arrive at a percent sequence identity value. It will be appreciated that the length of the aligned region can be a portion of one or both nucleic acids up to the full-length size of the shortest nucleic acid. It also will be appreciated that a single nucleic acid can align with more than one other nucleic acid and hence, can have different percent sequence identity values over each aligned region.

The alignment of two or more nucleic acids to determine percent sequence identity can be performed using the computer program ClustalW and default parameters, which allows alignments of nucleic acid or polypeptide sequences to be carried out across their entire length (global alignment). ClustalW calculates the best match between a query and one or more subject sequences (nucleic acid or polypeptide), and aligns them so that identities, similarities and differences can be determined. Gaps of one or more residues can be inserted into a query sequence, a subject sequence, or both, to maximize sequence alignments. For fast pairwise alignment of nucleic acid sequences, the default parameters can be used (i.e., word size: <NUM>; window size: <NUM>; scoring method: percentage; number of top diagonals: <NUM>; and gap penalty: <NUM>); for an alignment of multiple nucleic acid sequences, the following parameters can be used: gap opening penalty: <NUM>; gap extension penalty: <NUM>; and weight transitions: yes. For fast pairwise alignment of polypeptide sequences, the following parameters can be used: word size: <NUM>; window size: <NUM>; scoring method: percentage; number of top diagonals: <NUM>; and gap penalty: <NUM>. For multiple alignment of polypeptide sequences, the following parameters can be used: weight matrix: blosum; gap opening penalty: <NUM>; gap extension penalty: <NUM>; hydrophilic gaps: on; hydrophilic residues: Gly, Pro, Ser, Asn, Asp, Gln, Glu, Arg, and Lys; and residue-specific gap penalties: on. ClustalW can be run, for example, at the Baylor College of Medicine Search Launcher website or at the European Bioinformatics Institute website on the World Wide Web.

The skilled artisan also would appreciate that complementary can be dependent upon, for example, the conditions under which two nucleic acids hybridize. Hybridization between nucleic acids is discussed in detail in <NPL>). Sambrook et al. disclose suitable Southern blot conditions for oligonucleotide probes less than about <NUM> nucleotides (Sections <NUM>-<NUM>). The Tm between a nucleic acid that is less than <NUM> nucleotides in length and a second nucleic acid can be calculated using the formula provided in Section <NUM>. Sambrook et al. additionally disclose Southern blot conditions for oligonucleotide probes greater than about <NUM> nucleotides (see Sections <NUM>-<NUM>). The Tm between a nucleic acid greater than <NUM> nucleotides in length and a second nucleic acid can be calculated using the formula provided in Sections <NUM>-<NUM> of Sambrook et al.

The conditions under which membranes containing nucleic acids are prehybridized and hybridized, as well as the conditions under which membranes containing nucleic acids are washed to remove excess and non-specifically bound probe, can play a significant role in the stringency of the hybridization. Such hybridizations and washes can be performed, where appropriate, under moderate or high stringency conditions. For example, washing conditions can be made more stringent by decreasing the salt concentration in the wash solutions and/or by increasing the temperature at which the washes are performed. Simply by way of example, high stringency conditions typically include a wash of the membranes in <NUM>. 2X SSC at <NUM>.

In addition, interpreting the amount of hybridization can be affected, for example, by the specific activity of the labeled oligonucleotide probe, by the number of probe-binding sites on the template nucleic acid to which the probe has hybridized, and by the amount of exposure of an autoradiograph or other detection medium. It will be readily appreciated by those of ordinary skill in the art that although any number of hybridization and washing conditions can be used to examine hybridization of a probe nucleic acid molecule to immobilized target nucleic acids, it is more important to examine hybridization of a probe to target nucleic acids under identical hybridization, washing, and exposure conditions. Preferably, the target nucleic acids are on the same membrane. A nucleic acid molecule is deemed to hybridize to a nucleic acid, but not to another nucleic acid, if hybridization to a nucleic acid is at least <NUM>-fold (e.g., at least <NUM>-fold, <NUM>-fold, <NUM>-fold, <NUM>-fold, <NUM>-fold, <NUM>-fold, <NUM>-fold, or <NUM>-fold) greater than hybridization to another nucleic acid. The amount of hybridization can be quantified directly on a membrane or from an autoradiograph using, for example, a PhosphorImager or a Densitometer (Molecular Dynamics, Sunnyvale, CA).

Constructs, including expression constructs, are described herein and are known to those of skill in the art. Expression elements (e.g., promoters) that can be used to drive expression of a RNAi nucleic acid molecule are known in the art and include, without limitation, constitutive promoters such as, without limitation, the cassava mosaic virus (CsMVM) promoter, the cauliflower mosaic virus (CaMV) <NUM> promoter, the actin promoter, or the glyceraldehyde-<NUM>-phosphate dehydrogenase promoter, or tissue-specific promoters such as, without limitation, root-specific promoters such as the putrescine N-methyl transferase (PMT) promoter or the quinolinate phosphosibosyltransferase (QPT) promoter. It would be understood by a skilled artisan that a sense strand and an antisense strand can be delivered to and expressed in a target cell on separate constructs, or the sense and antisense strands can be delivered to and expressed in a target cell on a single construct (e.g., in one transcript). As discussed herein, a RNAi nucleic acid molecule delivered and expressed on a single strand also can include a spacer nucleic acid (e.g., a loop nucleic acid) such that the RNAi forms a small hairpin (shRNA).

Described herein are transgenic N. tabacum plants that contain a transgene encoding at least one RNAi molecule, which, when transcribed, silences expression of any of the TLA or CAO sequences described herein. As used herein, silencing can refer to complete elimination or essentially complete elimination of the TLA or CAO mRNA, resulting in <NUM>% or essentially <NUM>% reduction (e.g., greater than <NUM>% reduction; e.g., greater than <NUM>%, <NUM>%, <NUM>% or <NUM>% reduction) in the amount of encoded TLA or CAO polypeptide; silencing also can refer to partial elimination of the TLA or CAO mRNA (e.g., eliminating about <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or more of the TLA or CAO mRNA), resulting in a reduction (e.g., about <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or more, but not complete elimination) in the amount of the encoded TLA or CAO polypeptide.

A RNAi nucleic acid molecule can be transcribed using a plant expression vector. Methods of introducing a nucleic acid (e.g., a heterologous nucleic acid) into plant cells (e.g., N. tabacum cells) are known in the art and include, for example, particle bombardment, Agrobacterium-mediated transformation, microinjection, polyethylene glycol-mediated transformation (e.g., of protoplasts, see, for example, <NPL>)), liposome-mediated DNA uptake, or electroporation.

Following transformation, the transgenic plant cells can be regenerated into transgenic tobacco plants. The regenerated transgenic plants can be screened for the presence of the transgene (e.g., a RNAi nucleic acid molecule) and/or one or more of the resulting phenotypes (e.g., reduced amount of TLA or CAO mRNA; reduced amount of TLA or CAO polypeptide; reduced activity of a TLA or CAO polypeptide; reduced concentration of thylakoid membranes in the photosystems; reduced amount of total chlorophyll; increased ratio of chlorophyll a to chlorophyll b; and/or increased biomass).

Methods of detecting alkaloids (e.g., nicotine) or TSNAs, and methods of determining the amount of one or more alkaloids or TSNAs are known in the art. For example, high performance liquid chromatography (HPLC) - mass spectroscopy (MS) (HPLC-MS) or high performance thin layer chromatography (HPTLC) can be used to detect the presence of one or more alkaloids and/or determine the amount of one or more alkaloids. In addition, any number of chromatography methods (e.g., gas chromatography/thermal energy analysis (GC/TEA), liquid chromatography / mass spectrometry (LC/MS), and ion chromatography (IC)) can be used to detect the presence of one or more TSNAs and/or determine the amount of one or more TSNAs.

As used herein, "reduced" or "reduction" refers to a decrease (e.g., a statistically significant decrease), in green leaf or cured leaf, of / in one or more of the following: a) the amount of TLA or CAO mRNA; b) the amount of TLA or CAO polypeptide; c) the activity of a TLA or CAO polypeptide; d) the concentration of thylakoid membranes in the photosystems measured spectrophotometrically from the amplitude of the light-minus-dark absorbance difference signal at <NUM> (P800) for PSI and <NUM> (QA) for PSII (see, for example, <NPL>; and <NPL>); and/or e) the amount of total chlorophyll. As used herein, "reduced" or "reduction" refers to a decrease in any of the above by at least about <NUM>% up to about <NUM>% (e.g., about <NUM>% to about <NUM>%, about <NUM>% to about <NUM>%, about <NUM>% to about <NUM>%, about <NUM>% to about <NUM>%, about <NUM>% to about <NUM>%, about <NUM>% to about <NUM>%, about <NUM>% to about <NUM>%, about <NUM>% to about <NUM>%, about <NUM>% to about <NUM>%, about <NUM>% to about <NUM>%, about <NUM>% to about <NUM>%, about <NUM>% to about <NUM>%, about <NUM>% to about <NUM>%, about <NUM>% to about <NUM>%, and about <NUM>% to about <NUM>%) relative to similarly-treated leaf (e.g., green or cured) from a tobacco plant lacking the transgene. As used herein, statistical significance refers to a p-value of less than <NUM>, e.g., a p-value of less than <NUM> or a p-value of less than <NUM>, using an appropriate measure of statistical significance, e.g., a one-tailed two sample t-test.

As used herein, "increased" refers to an increase (e.g., a statistically significant increase), in green leaf or cured leaf, of the ratio of chlorophyll a / chlorophyll b or in plant biomass. As used herein, "increased" refers to an increase in any of the above by at least about <NUM>% up to about <NUM>% (e.g., about <NUM>% to about <NUM>%, about <NUM>% to about <NUM>%, about <NUM>% to about <NUM>%, about <NUM>% to about <NUM>%, about <NUM>% to about <NUM>%, about <NUM>% to about <NUM>%, about <NUM>% to about <NUM>%, about <NUM>% to about <NUM>%, about <NUM>% to about <NUM>%, about <NUM>% to about <NUM>%, about <NUM>% to about <NUM>%, about <NUM>% to about <NUM>%, about <NUM>% to about <NUM>%, about <NUM>% to about <NUM>%, and about <NUM>% to about <NUM>%) relative to similarly-treated leaf (e.g., green or cured) from a tobacco plant lacking the transgene. As used herein, statistical significance refers to a p-value of less than <NUM>, e.g., a p-value of less than <NUM> or a p-value of less than <NUM>, using an appropriate measure of statistical significance, e.g., a one-tailed two sample t-test.

Leaf from progeny plants also can be screened for the presence of the transgene and/or the resulting phenotype, and plants exhibiting the desired phenotype can be selected. As described herein, leaf from such transgenic plants can exhibit reduced amount of TLA or CAO mRNA; reduced amount of TLA or CAO polypeptide; reduced activity of a TLA or CAO polypeptide; reduced concentration of thylakoid membranes in the photosystems; reduced amount of total chlorophyll; increased ratio of chlorophyll a to chlorophyll b; and/or increased biomass in the plant (e.g., compared to leaf from a plant lacking or not transcribing the RNAi). Leaf from regenerated transgenic plants can be screened (e.g., reduced amount of TLA or CAO mRNA; reduced amount of TLA or CAO polypeptide; reduced activity of a TLA or CAO polypeptide; reduced concentration of thylakoid membranes in the photosystems; reduced amount of total chlorophyll; increased ratio of chlorophyll a to chlorophyll b; and/or increased biomass), and the desired plants (e.g., having leaf that exhibit reduced amount of TLA or CAO mRNA; reduced amount of TLA or CAO polypeptide; reduced activity of a TLA or CAO polypeptide; reduced concentration of thylakoid membranes in the photosystems; reduced amount of total chlorophyll; increased ratio of chlorophyll a to chlorophyll b; and/or increased biomass), compared to the amount in a leaf from a corresponding non-transgenic plant, can be selected and, for example, used in a breeding program.

Transgenic plants exhibiting the desired phenotype can be used, for example, in a breeding program. Breeding is carried out using known procedures. Successful crosses yield Fi plants that are fertile and that can be backcrossed with one of the parents if desired. A plant population in the F<NUM> generation may be screened for the presence of a transgene and/or the resulting phenotype using standard methods (e.g., amplification, hybridization and/or chemical analysis of the leaf). Selected plants are then crossed with one of the parents and the first backcross (BC<NUM>) generation plants are self-pollinated to produce a BC<NUM>F<NUM> population that is again screened. The process of backcrossing, self-pollination, and screening is repeated, for example, at least four times until the final screening produces a plant that is fertile and reasonably similar to the recurrent parent. This plant, if desired, is self-pollinated and the progeny are subsequently screened again to confirm that the plant contains the transgene and exhibits variant gene expression. Breeder's seed of the selected plant can be produced using standard methods including, for example, field testing and/or chemical analyses of leaf (e.g., cured leaf).

The result of a plant breeding program using the transgenic tobacco plants described herein are novel and useful varieties, lines, and hybrids. As used herein, the term "variety" refers to a population of plants that share constant characteristics which separate them from other plants of the same species. A variety is often, although not always, sold commercially. While possessing one or more distinctive traits, a variety is further characterized by a very small overall variation between individual with that variety. A "pure line" variety may be created by several generations of self-pollination and selection, or vegetative propagation from a single parent using tissue or cell culture techniques. A "line," as distinguished from a variety, most often denotes a group of plants used non-commercially, for example, in plant research. A line typically displays little overall variation between individuals for one or more traits of interest, although there may be some variation between individuals for other traits.

A variety can be essentially derived from another line or variety. As defined by the<NPL>), a variety is "essentially derived" from an initial variety if: a) it is predominantly derived from the initial variety, or from a variety that is predominantly derived from the initial variety, while retaining the expression of the essential characteristics that result from the genotype or combination of genotypes of the initial variety; b) it is clearly distinguishable from the initial variety; and c) except for the differences which result from the act of derivation, it conforms to the initial variety in the expression of the essential characteristics that result from the genotype or combination of genotypes of the initial variety. Essentially derived varieties can be obtained, for example, by the selection of a natural or induced mutant, a somaclonal variant, a variant individual plant from the initial variety, backcrossing, or transformation.

Hybrid tobacco varieties can be produced by preventing self-pollination of female parent plants (i.e., seed parents) of a first variety, permitting pollen from male parent plants of a second variety to fertilize the female parent plants, and allowing Fi hybrid seeds to form on the female plants. Self-pollination of female plants can be prevented by emasculating the flowers at an early stage of flower development. Alternatively, pollen formation can be prevented on the female parent plants using a form of male sterility. For example, male sterility can be produced by cytoplasmic male sterility (CMS), nuclear male sterility, genetic male sterility, molecular male sterility where a transgene inhibits microsporogenesis and/or pollen formation, or self-incompatibility. Female parent plants having CMS are particularly useful. Where the female parent plants are CMS, the male parent plants typically contain a fertility restorer gene to ensure that the Fi hybrids are fertile. Where the female parents are CMS, male parents can be used that do not contain a fertility restorer. Fi hybrids produced from such parents are male sterile. Male sterile hybrid seed can be interplanted with male fertile seed to provide pollen for seed-set on the resulting male sterile plants.

Varieties and lines described herein can be used to form single-cross tobacco Fi hybrids. The plants of the parent varieties can be grown as substantially homogeneous adjoining populations to facilitate natural cross-pollination from the male parent plants to the female parent plants. The F<NUM> seed formed on the female parent plants is selectively harvested by conventional means. One also can grow the two parent plant varieties in bulk and harvest a blend of F<NUM> hybrid seed formed on the female parent and seed formed upon the male parent as the result of self-pollination. Alternatively, three-way crosses can be carried out wherein a single-cross Fi hybrid is used as a female parent and is crossed with a different male parent. As another alternative, double-cross hybrids can be created wherein the Fi progeny of two different single-crosses are themselves crossed. Self-incompatibility can be used to particular advantage to prevent self-pollination of female parents when forming a double-cross hybrid.

The tobacco plants used in the methods described herein can include, but are not limited to, a Burley type, a dark type, a flue-cured type, or an Oriental type. The tobacco plants used in the methods described herein typically are from N. tabacum, and can be from any number of N. tabacum varieties. A variety can be BU <NUM>, CC <NUM>, CC <NUM>, CC <NUM>, CC <NUM>, CC <NUM>, CC <NUM>,CC <NUM>, CC <NUM>, CC <NUM>, CC <NUM>, CC <NUM>, CC <NUM>, CC <NUM>, CC <NUM>, CC <NUM>, CC <NUM>, CC <NUM>, Coker <NUM>, Coker <NUM>, Coker <NUM> Gold, Coker <NUM>, CU <NUM>, DF911, Galpao tobacco, GL <NUM>, GL <NUM>, GL <NUM>, GL <NUM>, GL <NUM>, GL <NUM>, GL <NUM>, GL <NUM>, GF <NUM>, GF <NUM>, RJR <NUM>, HB 04P, K <NUM>, K <NUM>, K <NUM>, K <NUM>, K394, K <NUM>, K <NUM>, NC <NUM>, NC 37NF, NC <NUM>, NC <NUM>, NC <NUM>, NC2326, NC <NUM>, NC <NUM>, PVH <NUM>, PVH <NUM>, PVH <NUM>, PVH <NUM>, PVH <NUM>, VA <NUM>, VA <NUM>,, KDH <NUM>, KT <NUM>, KT204LC, KY <NUM>, KY <NUM>, KY <NUM>, KY <NUM>, KY <NUM>, KY <NUM>, KY907LC, KTY14 x L8 LC, Little Crittenden, McNair <NUM>, McNair <NUM>, msKY 14xL8, Narrow Leaf Madole, NC <NUM>, NC <NUM>, NC <NUM>, NC <NUM>, NC <NUM>, NC <NUM>, NC <NUM>, NC <NUM>, NC <NUM>, NC <NUM>, NC7, NC <NUM>, NC <NUM>, NC <NUM>, NC <NUM>, NC BH <NUM>, NC <NUM>, Neal Smith Madole, OXFORD <NUM>, Perique tobacco, PVH03, PVH09, PVH19, PVH50, PVH51, R <NUM>, R <NUM>, R <NUM>-<NUM>, R <NUM>-<NUM>, RG <NUM>, RG <NUM>, RG H51, RGH <NUM>, RGH <NUM>, RS <NUM>, Speight <NUM>, Speight <NUM>, Speight <NUM>, Speight <NUM>, Speight <NUM>, Speight <NUM>, Speight <NUM>, Speight <NUM>, Speight G-<NUM>, Speight G-<NUM>, Speight H-<NUM>, Speight H20, Speight NF3, TI <NUM>, TI <NUM>, TN <NUM>, TN86LC, TN <NUM>, TN90LC, TN <NUM>, TN97LC, TN D94, TN D950, TR (Tom Rosson) Madole, VA <NUM>, or VA359.

Methods of making a N. tabacum plant having a mutation are known in the art. Mutations can be random mutations or targeted mutations. The mutations introduced according to the presently claimed methods are targeted mutations.

For random mutagenesis, cells (e.g., N. tabacum cells) typically are mutagenized using, for example, a chemical mutagen or ionizing radiation. Representative chemical mutagens include, without limitation, nitrous acid, sodium azide, acridine orange, ethidium bromide, and ethyl methane sulfonate (EMS), while representative ionizing radiation includes, without limitation, x-rays, gamma rays, fast neutron irradiation, and UV irradiation. The dosage of the mutagenic chemical or radiation is determined experimentally for each type of plant tissue such that a mutation frequency is obtained that is below a threshold level characterized by lethality or reproductive sterility. The number of M<NUM> generation seed or the size of M<NUM> plant populations resulting from the mutagenic treatments are estimated based on the expected frequency of mutations.

For targeted mutagenesis, representative technologies include TALEN (see, for example,<NPL>) or zinc-finger (see, for example, <NPL>). Whether random or targeted, a mutation can be a point mutation, an insertion, a deletion, a substitution, or combinations thereof, which are discussed in more detail below.

The resultant Nicotiana tabacum of the method of the invention includes plants having a mutation in an endogenous TLA nucleic acid (e.g., SEQ ID NOs: <NUM> or <NUM>) encoding a TLA polypeptide sequence (e.g., SEQ ID NOs: <NUM> or <NUM>).

A mutation in a TLA sequence as described herein typically results in reduced expression or activity of TLA, which, in turn, results in one or more of the phenotypes described herein (e.g., reduced concentration of thylakoid membranes in the photosystems; reduced amount of total chlorophyll; increased ratio of chlorophyll a to chlorophyll b; but at least increased leaf biomass).

As discussed herein, one or more nucleotides can be mutated to alter the expression and/or function of the encoded polypeptide, relative to the expression and/or function of the corresponding wild type polypeptide. It will be appreciated, for example, that a mutation in one or more of the highly conserved regions would likely alter polypeptide function, while a mutation outside of those highly conserved regions would likely have little to no effect on polypeptide function. In addition, a mutation in a single nucleotide can create a stop codon, which would result in a truncated polypeptide and, depending on the extent of truncation, loss of function.

Suitable types of mutations in a TLA coding sequence include, without limitation, insertions of nucleotides, deletions of nucleotides, or transitions or transversions relative to the wild-type TLA coding sequence, respectively. Mutations in the coding sequence can result in insertions of one or more amino acids, deletions of one or more amino acids, conservative or non-conservative amino acid substitutions in the encoded polypeptide, or truncation of the protein (e.g., by introduction of a stop codon). In some cases, the coding sequence of a TLA comprises more than one mutation and/or more than one type of mutation.

Insertion or deletion of amino acids in a coding sequence, for example, can disrupt the conformation of the encoded polypeptide. Amino acid insertions or deletions also can disrupt sites important for recognition of binding ligand(s) or substrate(s) or for activity of the polypeptide. It is known in the art that the insertion or deletion of a larger number of contiguous amino acids is more likely to render the gene product non-functional, compared to a smaller number of inserted or deleted amino acids. In addition, one or more mutations (e.g., a point mutation) can change the localization of the TLA polypeptide, introduce a stop codon to produce a truncated polypeptide, or disrupt an active site or domain (e.g., a catalytic site or domain, a binding site or domain) within the polypeptide.

A "conservative amino acid substitution" is one in which one amino acid residue is replaced with a different amino acid residue having a similar side chain (see, for example, <NPL>), which provides frequency tables for amino acid substitutions), and a non-conservative substitution is one in which an amino acid residue is replaced with an amino acid residue that does not have a similar side chain. Non-conservative amino acid substitutions can replace an amino acid of one class with an amino acid of a different class. Non-conservative substitutions can make a substantial change in the charge or hydrophobicity of the gene product. Non-conservative amino acid substitutions can also make a substantial change in the bulk of the residue side chain, e.g., substituting an alanine residue for an isoleucine residue. Examples of non-conservative substitutions include a basic amino acid for a non-polar amino acid, or a polar amino acid for an acidic amino acid.

Following mutagenesis, M<NUM> plants can be regenerated from the mutagenized cells and those plants, or a subsequent generation of that population (e.g., M<NUM>, M<NUM>, M<NUM>, etc.), can be screened for those carrying a mutation in a TLA sequence. Screening for plants carrying a mutation in a TLA nucleic acid or polypeptide can be performed directly using methods routine in the art (e.g., hybridization, amplification, nucleic acid sequencing, peptide sequencing, combinations thereof) or by evaluating the phenotype (e.g., reduced amount of TLA mRNA; reduced amount of TLA polypeptide; reduced activity of a TLA polypeptide; reduced concentration of thylakoid membranes in the photosystems; reduced amount of total chlorophyll; increased ratio of chlorophyll a to chlorophyll b; but at least increased biomass). It would be understood that the phenotype of a mutant plant (e.g., reduced amount of TLA mRNA; reduced amount of TLA polypeptide; reduced activity of a TLA polypeptide; reduced concentration of thylakoid membranes in the photosystems; reduced amount of total chlorophyll; increased ratio of chlorophyll a to chlorophyll b; but at least increased biomass) would be compared to a corresponding plant (e.g., having the same varietal background) that lacks the mutation.

An M<NUM> tobacco plant may be heterozygous for a mutant allele and exhibit a wild type phenotype. In such cases, at least a portion of the first generation of self-pollinated progeny of such a plant exhibits a wild type phenotype. Alternatively, an M<NUM> tobacco plant may have a mutant allele and exhibit a mutant phenotype (e.g., reduced amount of TLA mRNA; reduced amount of TLA polypeptide; reduced activity of a TLA polypeptide; reduced concentration of thylakoid membranes in the photosystems; reduced amount of total chlorophyll; increased ratio of chlorophyll a to chlorophyll b; and/or increased biomass). Such plants may be heterozygous and exhibit a mutant phenotype due to a phenomenon such as dominant negative suppression, despite the presence of the wild type allele, or such plants may be homozygous due to independently induced mutations in both alleles.

As used herein, "reduced" or "reduction" refers to a decrease (e.g., a statistically significant decrease), in green leaf or cured leaf, of / in one or more of the following: a) the amount of TLA mRNA; b) the amount of TLA polypeptide; c) the activity of a TLA polypeptide; d) the concentration of thylakoid membranes in the photosystems measured spectrophotometrically from the amplitude of the light-minus-dark absorbance difference signal at <NUM> (P800) for PSI and <NUM> (QA) for PSII (see, for example, <NPL>; and <NPL>); and/or e) the amount of total chlorophyll. As used herein, "reduced" or "reduction" refers to a decrease in any of the above by at least about <NUM>% up to about <NUM>% (e.g., about <NUM>% to about <NUM>%, about <NUM>% to about <NUM>%, about <NUM>% to about <NUM>%, about <NUM>% to about <NUM>%, about <NUM>% to about <NUM>%, about <NUM>% to about <NUM>%, about <NUM>% to about <NUM>%, about <NUM>% to about <NUM>%, about <NUM>% to about <NUM>%, about <NUM>% to about <NUM>%, about <NUM>% to about <NUM>%, about <NUM>% to about <NUM>%, about <NUM>% to about <NUM>%, about <NUM>% to about <NUM>%, and about <NUM>% to about <NUM>%) relative to similarly-treated leaf (e.g., green or cured) from a tobacco plant lacking the transgene. As used herein, statistical significance refers to a p-value of less than <NUM>, e.g., a p-value of less than <NUM> or a p-value of less than <NUM>, using an appropriate measure of statistical significance, e.g., a one-tailed two sample t-test.

A tobacco plant carrying a mutant allele can be used in a plant breeding program to create novel and useful lines, varieties and hybrids. Such plant breeding programs do not per se form part of the claimed invention. Desired plants that possess the mutation can be backcrossed or self-pollinated to create a second population to be screened. Backcrossing or other breeding procedures can be repeated until the desired phenotype of the recurrent parent is recovered. DNA fingerprinting, SNP or similar technologies may be used in a marker-assisted selection (MAS) breeding program to transfer or breed mutant alleles into other tobaccos, as described herein.

An M<NUM>, M<NUM>, M<NUM> or later generation tobacco plant containing at least one mutation may be crossed with a second Nicotiana tabacum plant, and progeny of the cross are identified in which the mutation(s) is present. It will be appreciated that the second Nicotiana tabacum plant can be one of the species and varieties described herein. It will also be appreciated that the second Nicotiana tabacum plant can contain the same mutation as the plant to which it is crossed, a different mutation, or be wild type at the locus. Additionally or alternatively, a second tobacco line can exhibit a phenotypic trait such as, for example, disease resistance, high yield, high grade index, curability, curing quality, mechanical harvesting, holding ability, leaf quality, height, plant maturation (e.g., early maturing, early to medium maturing, medium maturing, medium to late maturing, or late maturing), stalk size (e.g., small, medium, or large), and/or leaf number per plant (e.g., a small (e.g., <NUM>-<NUM> leaves), medium (e.g., <NUM>-<NUM> leaves), or large (e.g., <NUM>-<NUM>) number of leaves).

The methods described herein allow for increasing tobacco biomass while still maintaining high leaf quality. The methods of the invention include the production of targeted-mutant plants.

Leaf quality can be determined, for example, using an Official Standard Grade published by the Agricultural Marketing Service of the US Department of Agriculture (<NUM> U. §<NUM>); Legacy Tobacco Document Library (Bates Document # <NUM>/<NUM>, July <NUM>, <NUM>, Memorandum on the Proposed Burley Tobacco Grade Index); and <NPL>. For dark-fired tobacco, leaves typically are obtained from stalk position C, and the average grade index determined based on Federal Grade and <NUM> Price Support for Type <NUM> Western dark-fired tobacco.

Leaf from the tobacco described herein can be cured, aged, conditioned, and/or fermented. Methods of curing tobacco are well known and include, for example, air curing, fire curing, flue curing and sun curing. Aging also is known and is typically carried out in a wooden drum (e.g., a hogshead) or cardboard cartons in compressed conditions for several years (e.g., <NUM> to <NUM> years), at a moisture content of from about <NUM>% to about <NUM>% (see, for example, <CIT> and <CIT>). Conditioning includes, for example, a heating, sweating or pasteurization step as described in <CIT> or <CIT>, while fermenting typically is characterized by high initial moisture content, heat generation, and a <NUM> to <NUM>% loss of dry weight. See, e.g., <CIT>; <CIT>; <CIT>; and <CIT>. The tobacco also can be further processed (e.g., cut, expanded, blended, milled or comminuted), if desired, and used in a tobacco product.

Tobacco products are known in the art and include any product made or derived from tobacco that is intended for human consumption, including any component, part, or accessory of a tobacco product. Representative tobacco products include, without limitation, cigarettes, smokeless tobacco products, tobacco-derived nicotine products, cigarillos, non-ventilated recess filter cigarettes, vented recess filter cigarettes, cigars, snuff, electronic cigarettes, e-vapor products, pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco, and cut tobacco. Representative smokeless tobacco products include, for example, chewing tobacco, snus, pouches, films, tablets, sticks, rods, and the like. Representative cigarettes and other smoking articles include, for example, smoking articles that include filter elements or rod elements, where the rod element of a smokeable material can include cured tobacco within a tobacco blend. In addition to the reduced-nicotine or reduced-TSNA tobacco described herein, tobacco products also can include other ingredients such as, without limitation, binders, plasticizers, stabilizers, and/or flavorings. See, for example, <CIT>, <CIT>, <CIT>, <CIT>, and <CIT> for examples of tobacco products.

In accordance with the present invention, there may be employed conventional molecular biology, microbiology, biochemical, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. The invention will be further described in the following examples.

Tobacco leaf sample from a burley variety, TN90, was collected. RNA from the sample was isolated using RNeasy Plant Mini Kit (Qiagen; MA) and its quality tested using Agilent Plant RNA Nano Kit and a <NUM> Bioanalyzer (Agilent Technologies, CA, USA). A cDNA library was constructed and indexed using a TrueSeq RNA Library Prep Kit v. <NUM> (Illumina). cDNAs were run on an Illumina HiSeq <NUM> under conditions for <NUM> bp single reads and a minimum of <NUM> million reads per sample. Leaf gene expression in TN90 tobacco was determined by RNA deep sequencing performed by ArrayXpress (Raleigh, NC).

TLA2, TLA3 and TLA4 gene sequences from Chlamydomonas and Arabidopsis were used to Blast the TN90 burley genomic sequence data base and leaf RNA sequence data. Five candidate genes were identified: TLA2, TLA2 Homo, TLA3, TLA3 Homo, and TLA4, and primers were designed to clone the five candidate genes. Leaf tissue was collected and a cDNA library was created using the In-Fusion® SMARTer® Directional cDNA Library Construction Kit from Clontech. Full length candidate genes were amplified using the gene specific primers designed from predicted full length cDNA sequences. The full length coding sequences were identified, cloned and confirmed by sequencing.

The sequence of the candidate genes are provided as indicated in Table <NUM>.

In order to investigate the function of the candidate genes, RNAi constructs were produced against TLA2, TLA3 and TLA4, and transgenic plant lines were generated. An Agrobacterium expression vector (SEQ ID NO:<NUM>) was used, which has a CsVMV promoter and a NOS terminator, as well as a cassette having a kanamycin selection marker (NPT II) under direction of the actin2 promoter and a NOS terminator. The nucleic acid constructs carrying each RNAi construct were introduced into tobacco leaf disc using an Agrobacterium transformation approach. See, for example, <NPL>; and <NPL>.

Briefly, ascetical tobacco plants (Narrow Leaf Madole (NLM)) were grown in magenta boxes, and leaf discs were cut onto <NUM> x <NUM> plates. Agrobacterium tumefaciens containing each nucleic acid construct were collected by centrifugation of <NUM> cell suspension in <NUM> centrifuge tube at <NUM> rpm for <NUM> minutes. Supernatant was removed and the Agrobacterium cell pellet was re-suspended in <NUM> liquid re-suspension medium. About <NUM> of the solution was transferred to each <NUM> x <NUM> petri plates. In those <NUM> x <NUM> plates, tobacco leaves were cut into <NUM> discs (avoiding the midrib).

Leaves were placed upside down, a thin layer of MS/B5 liquid re-suspension medium was added, and leaf discs were produced using a #<NUM> razor blade. The leaf discs were poked uniformly with a fine point needle. Eight discs were placed in each regeneration plate (<NUM> x <NUM>). Agrobacterium tumefaciens suspension was added and incubated with the leaf discs for <NUM> minutes. Leaf discs were transferred to co-cultivation plates (<NUM>/<NUM> medium) and discs were placed upside down in contact with filter paper overlaid on the co-cultivation TOM medium (MS medium with <NUM> sucrose/L; <NUM>/L IAA and <NUM>/L BAP). The plate was sealed with parafilm and labeled appropriately.

Plates were incubated in dim light (<NUM>-<NUM> mE/ms) under <NUM>/<NUM> photoperiods at <NUM> for three days. Leaf discs were transferred to regeneration/selection medium plates with TOM K media (TOM medium with <NUM>/L kanamycin) and sub-cultured biweekly in the same fresh medium in dim light at <NUM> until shoots become excisable. Shoots from leaves were removed with forceps and inserted in MS basal medium with <NUM>/L kanamycin for rooting at <NUM> under <NUM>/<NUM> photoperiods in dim light (<NUM>-<NUM> mE/ms). When plantlets having both shoots and roots grew large enough (e.g., reached over half of the height of the GA7 box), they were transferred to soil for acclimatization. During the transfer, any gel remaining on the root tissue was washed off with tap water. Established seedlings were transferred to the greenhouse to set seed and for further analysis.

An RNAi sequence against TLA2 is provided in SEQ ID NO:<NUM>. The sense, spacer and antisense portions of the RNAi molecule are provided in SEQ ID NOs: <NUM>-<NUM>, respectively. An RNAi sequence against TLA3 is provided in SEQ ID NO:<NUM>. The sense, spacer and antisense portions of the RNAi molecule are provided in SEQ ID NOs: <NUM>-<NUM>, respectively. An RNAi sequence against TLA4 is provided in SEQ ID NO:<NUM>. The sense, spacer and antisense portions of the RNAi molecule are provided in SEQ ID NOs: <NUM>-<NUM>, respectively. An RNAi sequence against CAO-<NUM>, CAO-<NUM> and CAO-<NUM> is provided in SEQ ID NO:<NUM>. The sense, spacer and antisense portions of the RNAi molecule are provided in SEQ ID NOs: <NUM>-<NUM>, respectively.

<FIG>, <FIG> and <FIG> are photographs of the transgenic plants described herein. <FIG> shows a T0 transgenic tobacco plant containing a TLA2 RNAi construct (right) next to a wild type tobacco plant (left), while <FIG> shows a T1 transgenic tobacco plant containing a TLA2 RNAi construct. <FIG> shows T0 transgenic tobacco plants containing different integration events for a TLA3 RNAi construct (the three plants on the left) next to a wild type tobacco plant (the plant on the right). <FIG> and <FIG> show two T1 transgenic tobacco plants containing a TLA3 RNAi construct. <FIG> shows transgenic tobacco plants containing a TLA4 RNAi construct (right) next to wild type tobacco plants (left).

<FIG>, <FIG> and <FIG> shows that transgenic tobacco plants expressing a RNAi construct against a TLA gene exhibit lower chlorophyll levels depends on the target gene and integration event. As described herein, chlorophyll levels can be qualitatively determined by observing the color of the leaf and/or quantitatively determined by measuring the amount of total chlorophyll and/or the ratio of chlorophyll a to chlorophyll b.

RealTime PCR analysis: To confirm the expression pattern of selected candidate genes, relative changes in transcripts from <NUM> different samples were measured. In brief, total RNA was isolated using TRI Reagent (Sigma-Aldritch). To remove DNA impurities, purified RNA was treated with RNase free DNase (Turbo DNA-free; Ambion). To synthesize the first cDNA strand, approximately <NUM>µg of total RNA was transcribed utilizing the High Capacity cDNA Kit (Applied Biosystems). To measure the level of selected gene transcripts in the samples, RT PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems). Gene specific primers are shown below.

Antigenic domains were identified from the sequences of the TLA2, TLA3 and TLA4 proteins. The oligopeptides shown in the Table below were synthesized and injected into rabbit to generate polyclonal antibodies. Western blots then were used to confirm the protein expression level for the target knock down genes.

Oligopeptides (solid underlining or dashed underlining represents different charges for the amino acids), and the ratio is the ratio of polar amino acids to total amino acids:.

RRSESRKQFADSGSTRPGPR [SEQ ID NO:<NUM>; corresponds to positions <NUM>-<NUM> of SEQ ID NO:<NUM>] (<NUM>:<NUM>)
LKEVKRVLNPTEVLLVVD [SEQ ID NO:<NUM>; corresponds to positions <NUM>-<NUM> of SEQ ID NO:<NUM>] (<NUM>:<NUM>).

Total tobacco leaf protein extracts from TLA3-RNAi transgenic plants (e.g., TLA3-<NUM>, <NUM> WT, and TLA3-<NUM>) were loaded (on the basis of equal Chl) and run on an SDS-PAGE gel. Western blotting was performed on the SDS-PAGE gel and the membrane was probed with specific polyclonal antibodies raised against two TLA3 oligopeptides (primary anti-TLA3 antibody diluted at <NUM>:<NUM>). Substantially lower amounts of the TLA3 protein was observed in the extracts from the TLA3-<NUM> and TLA3-<NUM> plants compared to that of the wild type plants.

Interestingly, the TLA3 protein from N. tabacum has a predicted molecular weight of <NUM> kD, but, under electrophoretic conditions, the protein migrates to a position of about <NUM> kD. This faster electrophoretic mobility is attributed to the fact that TLA3 from N. tabacum has about <NUM> negatively charged amino acids and, thus, migrates faster under the influence of the electrophoresis field.

<FIG> is a graph showing the real time PCR results from T0 generation plants transgenic for TLA2 RNAi. The T2-<NUM>, T2-<NUM>, T2-<NUM>, T2-<NUM> and T2-<NUM> designations represent individual TLA2 RNAi transgenic plants while CK refers to a transgenic control tobacco plant (transformed with an empty vector). The Y axis (<NUM>^-ΔCt) shows the relative expression level, based on mRNA levels, of the TLA2 gene. There were different levels of knock-down of the TLA2 gene depending upon the particular integration event. Among them, the T2-<NUM> line exhibited plants with stunted growth; most of the plants died before they matured. Overall, regenerating transgenic tobacco plants deficient in TAL2 was difficult, and the growth was inhibited by the level of TLA2 knock-down.

<FIG> is a graph showing the real time PCR results in T0 generation plants transgenic for TLA3 RNAi. T3-<NUM> to T3-<NUM> designations represent individual TLA3 RNAi transgenic plants, whereas CK refers to transgenic control tobacco (e.g., transformed with an empty vector). As with TLA2 transformants, there were different levels of knock down of TLA3 gene in TLA3 RNAi transformants.

According to the TLA concept proposed by the University of California - Berkeley group (<NPL>;<NPL>; and <NPL>), the mutants described herein should result in smaller light-harvesting chlorophyll antenna size and a substantially improved photosynthetic efficiency, as well as a higher [chlorophyll a / chlorophyll b] ratio. The chlorophyll from tobacco leaf samples was extracted in <NUM>% acetone, and cell debris was removed by centrifugation at <NUM>,<NUM> xg for <NUM>. The absorbance of the supernatant was measured with a Shimadzu UV-<NUM> spectrophotometer, and the Chl concentration of the samples was determined according to <NPL>), with equations corrected as described by <NPL>). Total carotenoid content was determined according to the method of <NPL>).

The antenna size of tobacco leaf samples was calculated by measuring photosynthetic activity. The oxygen evolution activity of the tobacco leaf (punched from fresh leaf tissue sample) was measured at <NUM> with a Clark-type oxygen electrode illuminated with light from a halogen lamp projector. A Corning <NUM>-<NUM> filter (<NUM>-nm cutoff filter) defined the yellow actinic excitation via which photosynthesis measurements were made. Samples of <NUM>-mL cell suspension containing <NUM> Chl were loaded into the oxygen electrode chamber. Sodium bicarbonate (<NUM> of <NUM> solution, pH <NUM>) was added to the cell suspension prior to the oxygen evolution measurements to ensure that oxygen evolution was not limited by the carbon supply available to the cells. After registration of the rate of dark respiration by the cells, samples were illuminated with gradually increasing light intensities. The rate of oxygen exchange (uptake or evolution) under each of these irradiance conditions was recorded continuously for a period of about <NUM>.

The following Table shows total chlorophyll measurement and Chlorophyll a / Chlorophyll b ratios of T0 lines transgenic for TLA2, TLA3 or TLA4 RNAi.

Tobacco lines exhibit a particular ratio of chlorophyll a / b, which can be used as an index for total antenna size. Therefore, changes in the chlorophyll a / b ration can be used to measure changes in antenna size. As shown in the Table above, the UC Berkeley mutant line increased the chlorophyll a/b ratio to about <NUM>, from a wild type ratio of about <NUM>. In the NLM tobacco lines described herein, the wild type ratio of chlorophyll a / b is about <NUM>, but in T0 generations of NLM plants transgenic for a TLA3 RNAi, most of the mutant lines reached a chlorophyll a / b ratio of about <NUM> to about <NUM>. There was no obvious increase in chlorophyll a / b ratio in the T0 generation of plants transgenic for TLA2 RNAi and TLA4 RNAi. These results indicate that TLA3 likely is the initial candidate gene to knock down and decrease antenna size in photosynthetic light harvesting centers in tobacco.

T1 generation of TLA3 RNAi line <NUM> (<NUM>-<NUM> to <NUM>-<NUM>) and <NUM> (data not shown) were harvested and chlorophyll a / b ratios were determined. The following Table shows chlorophyll data for the T1 generation of the TLA3 RNAi plants. The chlorophyll a / b ratios in the T1 generation of the TLA3 RNAi plants were higher compared to wild type ratios, and the change in the ratio was stable in the T1 generation.

Antenna size of the photosynthetic light harvesting centers (both PSI and PSII) were measured in the T1 generation for TLA3 RNAi plants, TLA2 RNAi plants, and TLA4 RNAi plants. The following Table shows the antenna size measurement of PSI and PSII in the T1 generation for four TLA2 RNAi line <NUM> plants (e.g., <NUM>-<NUM>-<NUM>, <NUM>-<NUM>-<NUM>, <NUM>-<NUM>-<NUM>, and <NUM>-<NUM>-<NUM>). The data showed that total antenna size in PSI for mutant lines was similar to wild type, but that PSII antenna size decreased in TLA2 knock out lines.

The antenna size for PSI and PSII in the T1 generation of TLA3 RNAi line <NUM> plants (e.g., <NUM>-<NUM>-<NUM>, <NUM>-<NUM>-<NUM>, <NUM>-<NUM>-<NUM>, <NUM>-<NUM>-<NUM>, <NUM>-<NUM>-<NUM>, <NUM>-<NUM>-<NUM>, and <NUM>-<NUM>-<NUM>) is shown in the following Table. Notably, the total number of photoreceptor antenna was knocked down in the transgenic plants from <NUM> to <NUM> for PSII and from <NUM> to <NUM> for PSI. The decrease in antenna size was correlated with the increase in chlorophyll a / b ratio in the T1 generation.

The following Table shows antenna size in PSI and PSII from T1 generation plants transgenic for TLA4 RNAi line <NUM> and line <NUM>. This data demonstrated that total antenna size of the mutant lines was similar to that of the wild type in both PSI and PSII.

Tobacco plants transgenic for the CAO RNAi nucleic acid shown in SEQ ID NO:<NUM> were produced as described herein, and chlorophyll a and chlorophyll b was measured as described herein. The data is shown in the following Table.

Decreasing, or truncating, the chlorophyll antenna size of the photosystems should improve photosynthetic solar energy conversion efficiency and productivity in mass cultures of algae or plants by up to <NUM>-fold. A Truncated Light-harvesting chlorophyll Antenna size (TLA) in photosynthetic organisms should help alleviate excess absorption of sunlight and the ensuing wasteful non-photochemical dissipation of excitation energy and, thus, would increase solar-to-biomass energy conversion efficiency and photosynthetic productivity in high density cultures.

Tobacco was grown under conditions that result in high-density canopies to evaluate the TLA plants described herein. The T0 stage of multiple NL Madole TLA3-RNAi transformants were screened and selected to identify lines for generating T1 seeds. The latter were germinated and T1 leaves were subjected to phenotypic and functional analysis. Plants were grown in high density canopies, with the canopy layout of <NUM> plants in a <NUM> x <NUM> configuration, and the distance between individual plants set at <NUM> inches. Biochemical analysis and biomass accumulation was performed.

This work showed a <NUM>% improvement in stem and leaf biomass accumulation for the TLA tobacco canopies over that of their wild-type counterparts grown under the same ambient conditions. Distinct differences were observed in the appearance of the canopy between plants containing a TLA RNAi and wild type tobacco plants. For example, the TLA3-RNAi canopy was a light-green color, while the wild type canopy was a much darker green. The results described herein can lead to significant improvements in agronomy, agricultural productivity, and the optimization of photosynthesis in commodity crops (e.g., tobacco) or parts thereof (e.g., leaves).

The average biomass values were determined for four different canopies. The results demonstrated that canopy interior plants performed better than plants in the periphery, as would be expected from the greater transmittance of sunlight. Significantly, an increase in leaf biomass of <NUM>% was observed for the canopy interior TLA3-RNAi plants as compared to that of the corresponding wild type plants.

Claim 1:
A method of making a Nicotiana tabacum plant having increased biomass,
which method comprises targeted mutation of an endogenous Truncated Light-harvesting Antenna (TLA) nucleic acid comprising a sequence having at least <NUM>% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: <NUM> and <NUM>,
wherein the mutation in the TLA sequence results in reduced expression or activity of the encoded TLA polypeptide,
wherein the plant comprising the mutation comprises an increased leaf biomass relative to the leaf biomass from a plant lacking said mutation.