Constructs and methods for enhancing protein levels in photosynthetic organisms

This invention provides novel gene constructs which enhance the efficiency of plant cells and cells of other photosynthetic organisms. Also provided are transgenic plants and seeds which overexpress proteins. Methods to elevate the amount of plastid proteins in plants and photosynthetic organisms are exemplified.

BACKGROUND OF THE INVENTION
 All photosynthetic organisms depend on the light-harvesting reactions of
 photosynthesis for energy to produce important compounds for growth and
 metabolism. Energy-rich carbohydrates, fatty acids, sugars, essential
 amino acids, and other compounds synthesized by photosynthetic organisms
 are the basis of the food chain on which all animal life depends for
 existence. Photosynthetic organisms are also the major source of oxygen
 evolution in the atmosphere, recycling carbon dioxide in the process. Thus
 life on earth is reliant on the productivity of photosynthetic organisms,
 especially plants.
 Plant productivity is limited by the amount of resources available and the
 ability of plants to harness these resources. The conversion of light to
 chemical energy requires a complex system which combines the light
 harvesting apparatus of pigments and proteins. The value of light energy
 to the plant can only be realized when it is efficiently converted into
 chemical energy by photosynthesis and fed into various biochemical
 processes.
 The thylakoid protein apparatus responsible for the photosynthetic
 conversion of light to chemical energy is one of the most complex
 mechanisms in the chloroplast and remains one of the most difficult
 biological systems to study. Oxygen-evolving photosynthetic organisms,
 such as cyanobacteria, algae and plants, possess two photosystems, PSI and
 PSII, which cooperate in series to acquire electrons from H.sub.2 O and
 deliver them energetically up a gradient to NADP.sup.+. The photosynthetic
 production of NADPH and ATP then, in turn, feeds into all biochemical
 pathways. The force driving the uphill flow of these electrons comes from
 the light energy absorbed by the 100-300 chlorophyll molecules associated
 with the two photosystems. An important pair of chlorophyll a molecules in
 the center of each photosystem modulates the movement of electrons. The
 remaining chlorophyll molecules are associated with proteins which in turn
 are organized into light gathering antennae that surround the reaction
 centers and transfer the light energy to them (Green et al. (1991) TIBS
 16:181).
 The capacity to absorb light, especially in shade, depends largely on the
 size and organization of the light harvesting complexes (Lhc) in the
 thylakoid membranes. The LhcII light harvesting complex is the major
 ensemble of chlorophyll a/b binding protein (Cab) acting as an antenna to
 photosystem II (PSII) and plays a key role in harvesting light for
 photosynthesis (Kuhlbrandt, W. (1984) Nature 307:478). Plants are capable
 of adjusting the size of the antennae in accordance with the light
 intensity available for growth. In shade, the allocation of nitrogen is
 shifted from polypeptides in the stroma, by decreasing ribulose
 1,5-bisphosphate carboxylase (Rbc or Rubisco) levels, to the thylakoidal
 proteins. Nitrogen redistribution is a compensating response to low
 irradiance, balancing light harvesting and CO.sub.2 fixation (Evans, J. R.
 (1989) Oecologia 78:9); Stitt, M. (1991) Plant, Cell and Environment
 14:741).
 In addition to the shift in the investment of nitrogen into different
 proteins, photosynthetic organisms can adapt to low light conditions by
 molecular reorganization of the light harvesting complexes (Chow et al.
 (1990) Proc. Natl. Acad. Sci. USA 87:7502; Horton et al. (1994) Plant
 Physiol. 106:415; Melis, (1991) Biochim. Biophys. Acta. 1058:87). A
 plant's reorganizational ability to compensate for changes in the
 characteristics of the light limits its productivity. Although a mechanism
 is in place to adapt to low light conditions, photosynthesis in plants
 grown in suboptimal illumination remains significantly lower due to a
 limited capacity to generate ATP and NADPH via electron transport (Dietz,
 K. J. and Heber, U. (1984) Biochim. Biophys. Acta. 767:432; ibid (1986)
 848:392). Under such conditions the capacity to generate ATP and NADPH,
 the assimilatory force, will dictate the capacity to reduce CO.sub.2. When
 light is limiting, plants reorganize to maximize their photosynthetic
 capacity; however, the ability to adapt is limited by molecular parameters
 ranging from gene expression to complex assembly to substrate and cofactor
 availability.
 If productivity of a plant or other photosynthetic organism is to be
 increased, methods to enhance the light-gathering capacity without
 restricting CO.sub.2 fixation must be developed.
 SUMMARY OF THE INVENTION
 The present invention provides a chimeric gene construct comprising a
 promoter region, a 5' untranslated region containing a translational
 enhancer, DNA encoding a plastid-specific transit peptide which enhances
 protein import, a gene encoding a plastid protein, and a 3' untranslated
 region containing a functional polyadenylation signal. This construct
 produces a high level of expression and importation of the functional
 protein to the site of its function.
 In one embodiment of the present invention the promoter is a 35S
 cauliflower mosaic virus (CaMV) promoter. In another embodiment, the
 translational enhancer is from the 5' untranslated region of the pea small
 subunit of ribulose-1,5-bisphosphate carboxylase. In another embodiment,
 the transit peptide is from the pea small subunit of
 ribulose-1,5-bisphosphate carboxylase. In a further embodiment, the gene
 encoding a protein is the pea cab gene, encoding a chlorophyll a/b binding
 protein. In yet another embodiment, the 3' untranslated region containing
 a functional polyadenylation signal is from the pea cab gene.
 This invention also provides a method for enhancing the light harvesting
 capability of a photosynthetic plant or organism comprising: preparing a
 gene construct comprising a promoter, a 5' untranslated region containing
 a translational enhancer, DNA encoding a plastid-specific transit peptide
 which enhances protein import, DNA encoding a protein, preferably a
 structural gene encoding a chlorophyll a/b binding protein, and a 3'
 untranslated region containing a functional polyadenylation signal;
 inserting the gene construct into a suitable cloning vector; and
 transforming a photosynthetic plant or other photosynthetic organism with
 the recombinant vector. Alternatively, the gene construct is coated
 directly on biolistic particles with which the cells are bombarded.
 This invention provides a DNA construct which can increase the amount of
 one or more proteins in a plastid, especially a chloroplast, or in the
 cells of photosynthetic prokaryotes. These constructs can alter the
 photosynthetic apparatus to increase the ability of the plant to harvest
 light, especially under conditions of low illumination.
 This invention also provides methods of increasing the light-harvesting
 efficiency of photosynthesis and the yield of photosynthetic products
 (such as carbohydrates) in plants and other photosynthetic organisms.
 These methods can be used to increase the commercial value of plants and
 seeds, and be used to increase the yields of products produced from
 fermentation and plant tissue culture operations.
 This invention also provides a transgenic (TR) plant or photosynthetic
 organism containing the construct described above. These transgenic plants
 and photosynthetic organisms have enhanced photosynthetic capacity and
 enhanced growth capabilities useful for increased yield, tissue culture,
 fermentation and regeneration purposes. Compared to wild-type (WT) plants,
 transgenic plants of this invention demonstrate increased yield, enhanced
 pigmentation, increased carbohydrate content, increased biomass, more
 uniform growth, larger seeds or fruits, increased stem girth, enhanced
 photosynthesis, faster germination, and increased ability to withstand
 transplant shock. Seeds produced from these plants are also provided by
 this invention, as well as plant parts useful for production of
 regenerated plants and other derived products.

DETAILED DESCRIPTION OF THE INVENTION
 This invention relates to a DNA construct which, when incorporated into a
 plant or cell of a photosynthetic organism, increases the efficiency of
 plastids or a photosynthetic cell, and to methods for increasing or
 improving the products of plastid metabolism via enhancement of protein
 expression and import. The present invention also relates to transgenic
 plants, seeds, plant cells and tissues, and other photosynthetic organisms
 incorporating these constructs.
 A DNA construct of this invention comprises a promoter, a 5' untranslated
 region containing a translational enhancer, DNA encoding a
 plastid-specific transit peptide which can enhance and direct import of a
 gene product to a plastid or photosynthetic apparatus, a gene encoding a
 plastid protein, and a 3' untranslated region containing a functional
 polyadenylation signal. Insertion of this construct results in increased
 expression and importation of proteins in plastids and the photosynthetic
 apparatus. These elements are usually provided as operably-joined
 components in the 5' to 3' direction of transcription. A preferred
 embodiment of the invention is a construct comprising a 5' constitutive
 promoter (such as the 35S cauliflower mosaic virus promoter), the 5'
 untranslated region of pea small subunit of ribulose-1,5-bisphosphate
 carboxylase containing a translational enhancer which has a nucleotide
 sequence consisting of residues 1 to 29 of SEQ ID NO:3, DNA encoding a
 transit peptide which is from pea small subunit of
 ribulose-1,5-bisphosphate carboxylase, a structural gene encoding a
 chlorophyll a/b binding protein and a 3' untranslated region containing a
 functional polyadenylation signal is from a pea cab gene.
 This novel gene construction scheme permits simultaneous high level
 transcription, high level translation, greater mRNA stability and a high
 level of protein importation into the plastid or photosynthetic apparatus
 of an organism, producing overproduction of the selected protein (in this
 case, a light harvesting Cab protein) in plants and other photosynthetic
 organisms. The multi-level gene construction, especially the enhancement
 of protein importation, can be widely used to enhance the import and
 expression of any protein. The gene construct exemplified achieves a high
 level of expression and importation of the functional protein (chlorophyll
 a/b binding protein) at the site of its function.
 The activity of many different proteins and polypeptides involved in the
 process of photosynthesis can be enhanced by the methods of this
 invention. In addition to increased levels of endogenous proteins, the DNA
 constructs of this invention can be used to import and express foreign
 proteins in the photosynthetic apparatus of plants and other
 photosynthetic organisms. Further, the DNA construct can contain a single
 protein encoding region, or can contain additional encoding regions so
 that several proteins can be imported and expressed. Thus, plastids of
 plants and cells of photosynthetic organisms can be altered to enhance the
 light-harvesting reactions of photosynthesis and/or to vary the level and
 kind of products of the photosynthetic dark reactions.
 To produce the chimeric constructs provided in this invention, an effective
 chimeric Rbcs-Cab coding region was created by combining coding sequences
 for appropriate portions of Rbcs and type I LhcIIb Cab. Transgenic tobacco
 plants containing the gene construct of this invention overproduce type I
 Cab of LhcIIb and possess enhanced low light photosynthetic activity and
 growth capabilities. The transgenic plants also demonstrate one or more
 morphological, developmental, biochemical and physiological modifications.
 These modifications have commercial value in crop plants where more rapid
 germination and growth, higher yields and improved appearance are highly
 preferred. The desired modifications are achieved through the elevated
 gene expression and protein import via this novel gene construction.
 Enhanced expression at the level of de novo transcription was facilitated
 by attaching the Rbcs-Cab gene construct to the strong CaMV 35S promoter.
 Further enhancements were obtained by increasing mRNA stability, thus
 increasing the magnitude of the steady state pool of transgene
 transcripts. This was accomplished by inclusion of the functional 3'
 untranslated nucleic acid sequence of the cab gene and the nucleic acid
 sequence encoding the Rbcs transit peptide. Both nucleic acid sequences
 play a role in increasing mRNA stability. Higher levels of translation or
 protein synthesis were achieved by the inclusion of the Rbcs 5'
 untranslated sequence containing a translational enhancer, thereby
 increasing the pool of protein precursors for importation into the
 plastidic compartment. The level of Cab assembled into thylakoid membranes
 and LhcIIb complexes was further elevated by using a more efficient
 transit peptide. Switching the type I LhcIIb Cab transit peptide with the
 one from the small subunit of ribulose-1,5-bisphosphate carboxylase
 enhanced the level of import into the chloroplast. The increase in type I
 LhcIIb Cab content inside the chloroplast allowed the LhcIIb antennae to
 incorporate the extra proteins and as a result increased the size of the
 antennae. Any transit peptide that will cause an increase in type I LhcIIb
 Cab in the chloroplast by replacing the Cab transit peptide can produce in
 a similar elevating effect. It is also possible to achieve lower levels of
 importation by using less efficient transit peptides and thus regulate the
 amount of protein expression. The experiments described herein show that
 the presence of the Rbcs transit peptide enhances importation of a wide
 range of plastid-destined protein precursors and probably represents the
 highest efficiency transit peptide characterized to date.
 In another embodiment, the transit peptide is constructed artificially by
 combining transit and signal peptides and/or functional domains from
 transit/signal peptides derived from different sources. The sources can be
 from other organisms either eukaryotic or prokaryotic in origin. Sources
 of transit peptides useful in these constructs can also encompass cryptic
 peptides from various parts of proteins as well as peptides made by
 chemical means (non-biological sources), e.g. via peptide synthesis
 reactions. Examples of peptides which are used in the construction of the
 transit peptides of this invention can be transit peptides taken from
 transit/signal peptides normally utilized to direct the transport of
 proteins across or into mitochondrial membranes, plastidic membranes,
 endoplasmic reticulum membranes, peroxisomal membranes, vacuolar
 membranes, plasma membranes, cell membranes, bacterial membranes, nuclear
 membranes, viral envelope membranes, artifical membranes, artificial
 liposomes, microbody membranes, and other such membranes.
 The term "promoter" or "promoter region" refers to a sequence of DNA,
 usually upstream (5') to the coding region of a structural gene, which
 controls the expression of the coding region by providing recognition and
 binding sites for RNA polymerase and any other factors required for
 transcription to start at the correct site.
 There are generally two types of promoters, inducible and constitutive
 promoters. The term "constitutive" as used herein does not necessarily
 indicate that a gene is expressed at the same level in all cell types, but
 that the gene is expressed in a wide range of cell types, although some
 variation in abundance is often detected.
 An inducible promoter is a promoter that is capable of directly or
 indirectly activating transcription of one or more DNA sequences or genes
 in response to an inducer. In the absence of an inducer the DNA sequences
 or genes will not be transcribed. Typically a protein factor (or factors),
 that binds specifically to an inducible promoter to activate
 transcription, is present in an inactive form which is then directly or
 indirectly converted to an active form by the inducer. The inducer can be
 a chemical agent such as a protein, metabolite, growth regulator,
 herbicide or phenolic compound, or a physiological stress imposed directly
 by heat, cold, salt, or toxic elements or indirectly through the action of
 a pathogen or disease agent such as a virus. The inducer can also be an
 illumination agent such as light, darkness and light's various aspects,
 which include wavelength, intensity, fluence, direction and duration. A
 plant cell containing an inducible promoter may be exposed to an inducer
 by externally applying the inducer to the cell or plant such as by
 spraying, watering, heating or similar methods. If it is desirable to
 activate the expression of a gene at a particular time during plant
 development, the inducer can be applied at that time.
 Examples of such inducible promoters include heat shock promoters, such as
 the inducible hsp70 heat shock promoter of Drosphilia melanogaster
 (Freeling, M. et al. (1985) Ann. Rev. of Genetics 19:297-323); a cold
 inducible promoter, such as the cold inducible promoter from B. napus
 (White, T. C. et al. (1994 Plant Physiol. 106:917); and the alcohol
 dehydrogenase promoter which is induced by ethanol (Nagao, R. T. et al.,
 Miflin, B. J., Ed. Oxford Surveys of Plant Molecular and Cell Biology,
 Vol. 3, p 384-438, Oxford University Press, Oxford 1986).
 Among sequences known to be useful in providing for constitutive gene
 expression are regulatory regions associated with Agrobacterium genes,
 such as nopaline synthase (Nos), mannopine synthase (Mas) or octopine
 synthase (Ocs), as well as regions regulating the expression of viral
 genes such as the 35S and 19S regions of cauliflower mosaic virus (CaMV)
 (Brisson et al. (1984) Nature 310:511-514), or the coat promoter of TMV
 (Takamatsu et al. (1987) EMBO J. 6:307-311).
 Other useful plant promoters include promoters which are highly expressed
 in phloem and vascular tissue of plants such as the glutamine synthase
 promoter (Edwards et al. (1990) Proc. Natl. Acad. Sci. USA 87:3459-3463),
 the maize sucrose synthetase 1 promoter (Yang et al. (1990) Proc. Natl.
 Acad. Sci. USA 87:4144-4148), the promoter from the Rol-C gene of the
 TLDNA of Ri plasmid (Sagaya et al., Plant Cell Physiol., 3:649-653), and
 the phloem-specific region of the pRVC-S-3A promoter (Aoyagi et al., Mol.
 Gen. Genet., 213:179-185 (1988)). Alternatively, plant promoters such as
 the small subunit of Rubisco (Rbcs) promoter (Coruzzi et al., EMBO J.,
 3:1671-1679 (1984); Broglie et al., Science, 224:838-843 (1984)), or heat
 shock promoters, e.g., soybean HPS17.5-E or HPS17.3-B (Gurley et al.
 (1989) Mol. Cell. Biol. 6:559-565 (1986)) may be used.
 Other useful promoters which can be used according to the present invention
 include: the low temperature and ABA-responsive promoter Kin1, cor6.6
 (Wang et al. (1995) Plant Mol. Biol. 28:605; Wang and Cutler (1995) Plant
 Mol. Biol. 28:619); the ABA inducible promoter from EM gene wheat
 (Marcotte Jr. et al. (1989) Plant Cell 1:969); the phloem-specific sucrose
 synthase promoter, ASUS1, from Arabidopsis (Martin et al. (1993) Plant J.
 4:367); the root and shoot promoter, ACS1 (Rodrigues-Pousada et al. (1993)
 Plant Cell 5:897); the seed-specific 22 kDa zein protein promoter from
 maize (Unger et al. (1993) Plant Cell 5:831); the ps1 lectin promoter in
 pea (de Pater et al. (1993) Plant Cell 5:877); the phas promoter from
 Phaseolus vulgaris (Frisch et al. (1995) Plant J. 7:503); the late
 embryo-abundant lea promoter (Thomas, T. L. (1993) Plant Cell 5:1401); the
 fruit-specific E8 gene promoter from tomato (Cordes et al. (1989) Plant
 Cell 1:1025); the meristematic tissue-specific PCNA promoter (Kosugi et
 al. (1995) Plant J. 7:877); the NTP303 pollen-specific promoter (Weterings
 et al. (1995) Plant J. 8:55); the late embryogenesis stage-specific OSEM
 promoter (Hattori et al. (1995) Plant J. 7:913); the ADP-glucose
 pyrophosphorylase tissue-specific promoter for guard cells and tuber
 parenchyma cells (Muller-Rober et al. (1994) Plant Cell 6:601); the Myb
 conductive tissue-specific promoter (Wissenbach et al.(1993) Plant J.
 4:411); and the plastocyanin promoter from Arabidopsis (Vorst et al.
 (1993) Plant J. 4:933).
 The construct of the present invention also includes a 5' untranslated
 leader sequence, which acts as a translational enhancer. Specific
 initiation signals may be required for efficient translation of the coding
 sequences. These signals include the ATG initiation codon and adjacent
 sequences. The initiation codon must be in phase with the reading frame of
 the coding sequence to ensure translation of the sequence. The translation
 control signals and initiation codon can be of a variety of origins, both
 natural and synthetic. Translational control signals and initiation codon
 can be provided from the source of the transcriptional initiation region,
 or from the structural gene. This sequence can also be derived from the
 promoter selected to express the gene, and can be specifically modified so
 as to increase translation of the mRNA.
 An example of a translational enhancer of the present invention is the 5'
 untranslated region of the pea small subunit of ribulose-1,5-bisphosphate
 carboxylase. Other nucleic acid sequences demonstrating translational
 enhancing activity have been reported for leader or 5' untranslated
 sequences such as from the ferrodoxin-binding protein gene psaDb (Yamamoto
 et al. (1995) J. Biol Chem. 270:12466), ferredoxin (Dickey et al. (1994)
 Plant Cell 6:1171), the 68 base leader from tobacco mosaic virus (TMV)
 (Gallie et al. (1987) Nucleic Acids Res. 15:3257) and the 36 base leader
 from alfalfa mosaic virus (Jobling et al. (1987) Nature 325:622). These
 translational enhancers can be used in place of the Rbcs translational
 enhancer signals in the present invention. Translational enhancing
 activity is most likely to be present in the 5' untranslated nucleic acid
 sequence of most other genes and their corresponding transcripts and can
 vary in strength and efficiency (see review by Gallie. 1993 Ann. Rev.
 Plant Physiol. Plant Mol. Biol. 44, 77). Such nucleic acid sequences, if
 demonstrated to contain translational enhancing effects, can also be used
 in the present invention. A translational enhancer demonstrating
 appropriate levels of enhancement can be selected to obtain a suitable
 level of translational enhancement in the constructs of the invention.
 The construct of the present invention also includes a transit peptide. A
 "transit peptide" refers to a peptide which is capable of directing
 intracellular transport of a protein joined thereto to a plastid in a
 plant host cell. The passenger protein may be homologous or heterologous
 with respect to the transit peptide. Chloroplasts are the primary plastids
 in photosynthetic tissues, although plant cells are likely to have other
 kinds of plastids, including amyloplasts, chromoplasts, and leucoplasts.
 The transit peptide of the present invention is a transit peptide which
 will provide intracellular transport to the chloroplasts as well as other
 types of plastids. In many cases, transit peptides can also contain
 further information for intraorganellar targeting within the plastid to
 sites of function such as outer and inner envelope membranes, stroma,
 thylakoid membrane or thylakoid lumen. Depending on the source of the
 transit peptide, the precursor proteins may display differences in import
 behavior and import activity such as efficiency. These differences in
 import behavior are not attributed solely to the function of the transit
 peptide but also to the passenger protein and are most likely due to
 interactions between the two portions (Ko and Ko, 1992 J. Biol. Chem. 267,
 13910). In photosynthetic prokaryotes, such as the cyanobacteria, proteins
 can be targeted to the photosynthetic and plasma membranes or to
 biochemical pathways involving the reduction of sugars and formation of
 photosynthetic products.
 The transit peptide for the constituent polypeptide of the light-harvesting
 chlorophyll a/b-protein complex is rich in serine, especially near the
 NH.sub.2 -terminus, where 7 of the first 13 residues are serine. An
 abundance of serine also occurs near the NH.sub.2 -terminus of the transit
 peptide for the small subunit of Rbc from pea (Cashmore, A. R., Genetic
 Engineering of Plants, Eds. Kosuge, T. et al. (Plenum Press, New York, pp.
 29-38 (1983)), soybean (Berry-Lowe, S. L. et al. (1982) J. Mol. Appl.
 Genet. 1:483-498), and Chlamydomonas (Schmidt, G. W. et al. (1989) J. Cell
 Biol. 83:615-623). Both the transit peptides for the light-harvesting
 chlorophyll a/b-protein complex and for the small subunit of Rbc function
 in the specific translocation of polypeptides across the chloroplast
 envelope. However, the final destination of these polypeptides is quite
 distinct, with the light-harvesting chlorophyll a/b-protein complex
 residing as integral membrane proteins in the chloroplast thylakoid and
 the small subunit of Rbc residing as a component of a soluble protein in
 the chloroplast stroma.
 In one embodiment, the transit peptide is from the small subunit of Rbc.
 The level of Cab assembled into the thylakoid membrane and the LhcIIb
 complex was further elevated by using a more efficient, heterologous
 transit peptide. The switching of the type I LhcIIb Cab transit peptide
 with the one from the small subunit of ribulose-1,5-bisphosphate
 carboxylase enhanced the level of Cab import into the chloroplast. The
 increase in type I LhcIIb Cab content inside the chloroplast allowed the
 LhcIIb antennae to incorporate the extra proteins and as a result
 increased the size of the antennae.
 The gene encoding the protein to be transcribed and incorporated into a
 plastid or cell of a photosynthetic organism is not particularly limited.
 Those of skill in the art will recognize that other genes encoding
 pigments (such as the phycobiliproteins) or pigment-binding proteins (such
 as carotenoid-binding proteins) could be utilized to enhance the
 efficiency of the light-harvesting reactions. Many processes of
 photosynthesis could be similarly enhanced. For example, genes encoding
 the subunits of ATP synthase and ferredoxin involved in electron transport
 could be incorporated into the constructs of this invention to enhance
 electron transport. Alternatively, the expression and import of pyruvate
 kinase, acetyl-CoA carboxylase and acyl carrier proteins could be
 increased, thus amplifying a biosynthetic pathway, such as carbon/lipid
 metabolism.
 Any gene encoding a chlorophyll a/b binding (Cab) protein can be selected
 as a structural gene. The chlorophyll a/b binding proteins include LhcI of
 four different types, LhcII of types I to III, CP29, CP26, CP24 and early
 light-induced proteins (Green B. R. (1991) Trends Biochem. Sci.
 16:181-186). These include genes or cDNAs encoding chlorophyll a/b binding
 proteins that can belong to the complexes LhcIIa, LhcIIb, LhcIIc, LhcIId,
 LhcIIe and any other uncharacterized subcomplexes of LhcII. The same gene
 construction scheme can be applied as well to genes or cDNAs encoding
 chlorophyll a/b binding proteins of LhcI which include chlorophyll a/b
 binding proteins of LhcIa, LhcIb, and LhcIc of photosystem 1.
 LhcII is the major complex comprising the most abundant members of the
 family of chlorophyll a/b binding proteins, accounting for approximately
 50% of total chlorophyll in the biosphere, and for the most chlorophyll b
 in green plants. Thus, a gene encoding a LhcII chlorophyll a/b binding
 protein would be a preferred gene for targeting to increase the amount of
 chlorophyll a/b binding proteins.
 In all plant species examined to date, chlorophyll a/b binding proteins of
 LhcII are encoded by a multi-gene family, comprising at least five genes
 in Arabidopsis, six genes in Nicotiana tabacum, eight genes in N.
 plumbaginifolia, and up to 15 genes in tomato (Jansson, S. et al. (1992)
 Plant Mol. Biol. Rep. 10:242-253). Thus, any of these genes would be a
 suitable target for increasing the amount of chlorophyll a/b binding
 protein. Table 1 provides a more complete list of genes encoding
 chlorophyll a/b binding proteins, including those presently in the nucleic
 acid sequence data banks such as that represented and listed in Table 2 of
 the publication by Jansson, et al. (1992) supra.
 In another embodiment, the gene encoding the chlorophyll a/b protein in the
 constructs of this invention can also encode a polyprotein consisting of
 two or more pigment-binding proteins. Such polyproteins, which are cleaved
 to produce mature proteins, are described in Enomoto, T., et al. (1997)
 Plant Cell Physiol. 38(6):743-746. The polyprotein can consist of all
 identical parts or of heterologous parts. For example, the DNA encoding
 the polyprotein can comprise multiple copies of genes encoding LhcIIb type
 I. Alternatively, the DNA can comprise a several genes encoding a mixture
 of proteins selected from, but not limited to, those included in Table I
 and transit peptides taken from transit/signal peptides normally utilized
 to direct the transport of proteins across or into mitochondrial
 membranes, plastidic membranes, endoplasmic reticulum membranes,
 peroxisomal membranes, vacuolar membranes, plasma membranes, cell
 membranes, bacterial membranes, nuclear membranes, viral envelope
 membranes, artifical membranes, artificial liposomes, microbody membranes,
 and other such membranes.
 TABLE 1
 Genes encoding chlorophyll a/b binding proteins, and their relation to
 designations for chlorophyll-protein
 complexes.
 Gene Product/Pigment-Protein Complex
 Green et al. 1991
 Trends Biochem Thornber et al. Bassi et References
 References
 Gene Sci. 16, 181 1991 al. 1990 (genes)
 (proteins)
 Lhca1 Type I LhcI Lhc Ib LhcI-730 Hoffman et al., 1987
 Ikeuchi et al., 1991
 Jansson & Gustafsson 1991
 Knoetzel et al., 1992
 Palomares et al., 1991
 Lhca2 Type II LhcI Lhc Ia LhcI-680 Stayton et al., 1987
 Ikeuchi et al., 1991
 Pichersky et al., 1988
 Knoetzel et al., 1992
 Jansson & Gustafsson 1991
 Lhca3 Type III LhcI Lhc Ia LhcI-680 Pichersky et al., 1989
 Ikeuchi et al., 1991
 Jansson & Gustafsson 1991
 Knoetzel et al. 1992
 Lhca4 Type IV LhcI Lhc Ib LhcI-730 Schwartz et al., 1991a
 Ikeuchi et al., 1991
 Zhang et al., 1991
 Schwartz et al., 1991a

photosynthesis
 B) Test tube studies
 Translational Transit Import
 Promoter Enhancer Signal Passenger Translation Level Comments
 -- Rbcs Rbcs Rbcs high high normal for Rbcs
 -- Rbcs Rbcs Cab high high 50% higher than
 Cab alone
 -- Cab Cab Cab good/high good normal for Cab
 -- Cab Cab Rbcs good/high good 50% lower than
 Rbcs
 alone
 -- Oeel.sup.3 Oeel Oeel moderate good normal for Oeel
 -- Rbcs Rbcs Oeel high high folds greater
 than Oeel
 alone
 -- Rbcs Rbcs/ Oeel high high folds greater
 than Oeel
 Oeel alone
 -- Com44.sup.4 Com44 Com44 low low normal for
 Com44
 -- Rbcs Com44 Com44 high low normal for
 Com44
 -- Rbcs Rbcs/ Com44 high high good import
 Com44
 -- Rbcs Rbcs Com44 high high good import
 -- Com70.sup.5 Com70 Com70 low normal normal for
 Com70
 -- Rbcs Com70 Com70 high normal normal for
 Com70
 -- PetA.sup.6 PetA PetA low low normal for PetA
 -- Rbcs Rbcs PetA high high high
 -- Rbcs -- PetA high low none due to
 loss of signal
 -- Rbcs Rbcs/ PetA high high high
 PetA
 -- Oeel Oeel Dhfr.sup.7 moderate good a foreign
 protein
 -- Dhfr -- Dhfr low no lacks transit
 signal
 -- Rbcs Rbcs Dhfr high high high
 -- Rbcs Rbcs Pka.sup.8 high high higher than
 Pka itself
 -- Rbcs Rbcs Pkg.sup.9 high high higher than
 Pkg itself
 -- Pka Pka Pka moderate good normal for Pka
 -- Pkg Pkg Pkg moderate good normal levels
 for Pka
 -- Pkg Pkg Rbcs moderate good resembles Pkg
 levels
 .sup.1 pea
 .sup.2 pea
 .sup.3 Arabidopsis thaliana
 .sup.4 Brassica napus
 .sup.5 Spinacea oleracea (spinach)
 .sup.6 Vicia faba
 .sup.7 mouse
 .sup.8 Ricinus cummunis (castor)
 .sup.9 Nicotiana tabacum (tobacco)
 Example 2
 Construction of Rbcs-Cab Gene Construct
 To produce transgenic tobacco plants with enhanced low light photosynthetic
 capacity through elevation of type I LhcIIb Cab protein levels, an
 enhancement of transcription, mRNA stability, translation and protein
 import was attained. The coding portion of the gene construct was a fusion
 of a DNA sequence (FIG. 1, SEQ ID NO:1) encoding the mature portion of the
 type I LhcIIb Cab protein (FIG. 1, SEQ ID NO:2) from pea. The coding
 sequence for native transit peptide was removed and replaced with a
 sequence for the transit peptide from the pea small subunit of Rbc (A. R.
 Cashmore, in Genetic Engineering of Plants, T. Kosugi, C. P. Meredith, A.
 Hollaender, Eds. (Plenum Press, New York, 1983) pp. 29-38). The 5' and 3'
 ends of the type I LhcIIb cab gene sequence used in the present construct
 are shown in FIG. 1. The Rbcs transit peptide (SEQ ID NO:4), and
 corresponding gene sequence (SEQ ID NO:3), are shown in Table 3, together
 with a short linker sequence linking the transit peptide to the Cab
 peptide. A 29 base pair 5' untranslated DNA sequence (5'UTR) originating
 immediately upstream of the pea Rbcs transit peptide coding region was
 used as a translation enhancer. This sequence is shown in Table 3 and is
 included within SEQ ID NO:3 (nucleotides 1 to 29). Expression of the gene
 construct was facilitated by the strong CaMV 35S promoter (Odell, J. T. et
 al. (1985) Nature 313:810) and transcriptional termination signals
 originated from the pea Cab gene (A. R. Cashmore (1984) Proc. Natl. Acad.
 Sci. USA 81:2960. A summary of the gene construct is shown in Table 3.
 TABLE 3
 Summary of the 35SCAMV-Rbcs-Cab Gene Construct
 Key structural parts:
 Rbcs (5'untranslated sequence)-pea Rbcs transit peptide-pea Cab protein
 body
 Published genetic names of key parts:
 SS3.6 (5'untranslated sequence)-SS3.6 Rbcs transit peptide-AB80 Cab protein
 body
 Sequence of key parts:
 ACGTTGCAATTCATACAGAAGTGAGAAAA ATG GCT TCT ATG ATA TCC
 M A S M I S
 TCT TCC GCT GTG ACA ACA GTC AGC CGT GCC TCT AGG GGG CAA TCC GCC
 S S A V T T V S R A S R G Q S A
 GCA GTG GCT CCA TTC GGC GGC CTC AAA TCC ATG ACT GGA TTC CCA GTG
 A V A P F G G L K S M T G F P V
 AAG AAG GTC AAC ACT GAC ATT ACT TCC ATT ACA AGC AAT GGT GGA
 K K V N T D I T S I T S N G G
 AGA GTA AAG TGC ATG GAT CCT GTA GAG AAG TCT . . .
 R V K C M D P V E K S
 Rbcs .rarw. .fwdarw. Cab
 Promoter:
 35S CaMV
 Terminator:
 Cab termination sequences (Cashmore (1984) Proc. Nat. Acad. Sci.
 USA 81:2960-2964)
 Binary vector:
 EcoRI-PvuII CAMV-Rbcs-Cab into BamHI/blunt end site of pEND4K (kanamycin
 resistance)
 Klee et al. (1985)
 Biotechnology 3:637-642).
 Agrobacterium strain:
 LBA4404
 Agrobacterium transformation:
 Freeze-thaw method (Holsters et al. (1978) Mol. Gen. Genet. 163:181-187)
 Transformation protocol:
 Leaf disc procedure (Horsch et al. (1985) Science 227:1229-1231)
 Cloning was initiated by the construction of the pSSTP vector containing a
 DNA sequence encoding the Rbcs 5'UTR and transit peptide (FIG. 2). The DNA
 fragment containing the required components was retrieved from plasmid
 pSSNPT (A. R. Cashmore, Univ. Pennsylvania, Philadelphia, Pa.) by
 digestion with HindIII. Phenol and chloroform:isoamyl alcohol extraction
 and ethanol precipitation in the presence of 0.1 M NaCl followed by a 70%
 ethanol wash were applied after each step of DNA manipulation as described
 in Example 1 to inactivate enzymes and to concentrate the DNA. The DNA
 precipitate was collected by centrifugation, dried and redissolved in 10
 .mu.l water. The HindIII end was rendered blunt utilizing the Klenow
 fragment of E. coli DNA polymerase I (Promega). The reaction consisted of
 1 unit of Klenow, 0.1 mM each of dATP, dCTP, dGTP and dTTP, 50 mM Tris-HCl
 pH7.5, 10 mM MgCl.sub.2, 5 mM DTT, and the DNA from the above step, and
 was incubated at 37.degree. C. for 1 h. After repurification by organic
 solvent extractions, the DNA was digested with BamHI, separating the
 required DNA fragment from the rest of the pSSNPT plasmid. The
 HindIII-BamHI DNA fragment was gel purified and ligated into the SmaI and
 BamHI sites of pGEM4 (Promega) that had been cleaved and subsequently
 dephosphorylated by calf intestinal alkaline phosphatase (Pharmacia). The
 purification of DNA was carried out using the standard low melting agarose
 gel and phenol extraction method (Sambrook et al. 1989, supra.). The low
 melting agarose was purchased from BRL (Gaithersburg, Md., USA). DNA was
 recovered from appropriate low melting agarose slices by heating at
 65.degree. C. followed by extraction with phenol, prewarmed initially at
 37.degree. C., and centrifugation. The phenol extraction was repeated and
 the aqueous DNA layer was then adjusted to 0.1 M NaCl and centrifuged for
 10 min in a microfuge. The supernatant was subjected to chloroform:isoamyl
 alcohol extraction followed by precipitation in ethanol as described
 above. The DNA pellet was collected by centrifugation, washed with 70%
 ethanol, dried and resuspended in water.
 The mature type I LhcIIb Cab coding DNA sequence (pea AB80), contained in a
 XbaI-PstI DNA fragment, was retrieved by digesting plasmid pDX80 (A. R.
 Cashmore, Univ. Pennsylvania, Philadelphia, Pa.) with XbaI and PstI (FIG.
 3). The DNA fragment was also gel purified and inserted into the plasmid
 vector pSSTP (FIG. 2) via the XbaI and PstI sites. Prior to ligation,
 these sites had been dephosphorylated by adjusting the restriction
 digestion reaction with 3.5 .mu.l 1M Tris-HCl, pH 8.0 and adding 0.5 units
 of calf intestinal alkaline phosphatase. Following a 30 minute incubation
 at 37.degree. C., the dephosphorylated vector was repurified by organic
 solvent extraction and precipitated with ethanol. The resulting plasmid
 was designated pRBCS-CAB (FIG. 3).
 The Rbcs-Cab chimeric gene was fused to the 35S CaMV constitutive promoter
 by inserting a gel-purified EcoRI-HindIII fragment carrying the 35S CaMV
 promoter from plasmid pCAMV (A. R. Cashmore, Univ. Pennsylvania,
 Philadelphia, Pa.) into the EcoRI-Asp718 sites of pRBCS-CAB (FIG. 4). The
 corresponding HindIII and Asp718 restriction sites were made blunt using
 the Klenow fragment of DNA polymerase I. The 35S CaMV-Rbcs-Cab construct
 was then transferred as an EcoRI-PvuII DNA fragment to the BamHI site of
 the binary vector pEND4K (FIGS. 5 and 6) (Klee, H. et al. (1985)
 Biotechnology 3:637). All of the restriction enzyme-generated ends were
 made blunt by Klenow fragment in this step.
 All ligation steps were carried out at 15.degree. C. overnight using T4 DNA
 ligase. The ligation reactions consisted of the two appropriate target DNA
 molecules, ligase buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl.sub.2, 1 mM
 DTT, 1 mM ATP) and 1-3 units of enzyme. All appropriate steps of the gene
 construction process were introduced into bacteria using standard
 CaCl.sub.2 bacterial transformation protocol (Sambrook et al. 1989, supra)
 and the E. coli host strain HB101. All recombinant plasmids were
 propagated in HB101 and isolated using standard techniques (Sambrook et
 al. 1989, supra). The resolution of DNA fragments was facilitated by using
 standard agarose and polyacrylamide gel electrophoresis techniques.
 Example 3
 Transformation and Selection of Plants
 The pEND4K-CAMV-Rbcs-Cab plasmid was introduced into Agrobacterium using
 the freeze-thaw method (Holsters et al. (1978) Mol. Gen. Genet.
 163:181-187). Competent LBA4404 cells were obtained by inoculating 50 ml
 of LB broth (50 .mu.g/ml rifampicin) with 500 .mu.L of an overnight
 culture, followed by incubation at 28.degree. C. with vigorous shaking
 until the cell density measured 0.7.sub.OD at 650 nm. Cells were harvested
 by centrifugation at 2000.times.g for 5 min at 4.degree. C., washed in ice
 cold 0.1M CaCl.sub.2 and resuspended in 1 ml of ice cold 20 mM CaCl.sub.2.
 A 150 .mu.l aliquot of competent LBA4404 cells was mixed with 1 .mu.g of
 plasmid DNA in a microfuge tube, and immediately frozen in liquid
 nitrogen. These cells were incubated at 37.degree. C. in a water bath or
 thermostat block for 5 min, 1 ml of LB broth added, and the mixture
 incubated at 28.degree. C. with shaking for 3 h. The cells were recovered
 by centrifugation at 2000.times.g for 5 min and resuspended in 100 .mu.l
 of LB broth. Cells were plated on LB plates containing 100 .mu.g/ml
 kanamycin and 50 .mu.g/ml rifampicin and incubated for 2 days at
 28.degree. C. The presence of the pEND4K-CAMV-Rbcs-Cab plasmid was
 confirmed by Southern blot analysis of plasmid preparations obtained from
 single kanamycin-resistant colonies. Three ml of YEB broth containing 50
 .mu.g/ml rifampicin and 100 .mu.g/ml kanamycin were inoculated with a
 kanamycin-resistant colony and incubated overnight at 28.degree. C. with
 shaking. The overnight culture (1.5 ml) was centrifuged for 30 sec in a
 microfuge. The cells were resuspended in 0.1 ml of GTE solution (50 mM
 glucose, 10 mM Na.sub.2 EDTA, 25 mM Tris-HCl pH 8.0) with 4 mg/ml of
 lysozyme, and incubated at room temperature for 10 min. Phenol (30 .mu.l)
 previously equilibrated with 2 vols of 1% (w/v) SDS, 0.2N NaOH was added.
 The mixture was vortexed gently until viscous and incubated at room
 temperature for 10 min. The lysed cells were neutralized with 3M sodium
 acetate, pH 4.8 (150 .mu.l) and incubated at -20.degree. C. for 15 min.
 before the mixture was centrifuged for 3 min in a microfuge. The
 supernatant was transferred to a fresh microfuge tube, two volumes of
 ethanol added, and the mixture was incubated at -80.degree. C. for 15 min
 to precipitate the DNA. Following centrifugation, the DNA pellet was
 resuspended in 90 .mu.l of water. Ten .mu.l of 3M sodium acetate pH 7.0
 were added, followed by an equal volume of phenol/chloroform, and the
 mixture was vortexed. After centrifuging for 5 min in a microfuge, the
 supernatant was transferred to a fresh tube and the DNA precipitated by
 adding 2 volumes of 100% ethanol. After centrifugation, the pellet was
 washed with 70% ethanol, dried and resuspended in 50 .mu.L of TE (10 mM
 Tris-HCl pH 8.0, 1 mM Na.sub.2 EDTA).
 The integrity of the pEND4K-CAMV-Rbcs-Cab plasmid in Agrobacterium was
 verified by restriction and Southern blot analysis of the plasmid isolated
 as described above and in Sambrook et al. 1989, supra. One of the
 Agrobacterium selected colonies containing an intact pEND4K-CAMV-Rbcs-Cab
 was used for plant transformation.
 Tobacco plants were transformed with plasmid pEND4K-CAMV-Rbcs-Cab following
 the leaf disc transformation protocol essentially as described by Horsch
 et al. (1985) Science 227:1229). Only young, not fully expanded leaves,
 3-7" length, from one month old plants were used. Excised leaves were
 surface-sterilized in 10% (v/v) sodium hypochlorite, 0.1% (v/v) Tween and
 rinsed 4 times with sterile deionized water. From this point on, standard
 aseptic techniques for the manipulation of the sterile material and media
 were used. Leaf discs, 6 mm in diameter, were made with the aid of a
 sterile paper punch and incubated for 10-20 min in a 1:5 dilution of an
 overnight culture of Agrobacterium harbouring the pEND4K-CaMV-Rbcs-Cab
 construct. After inoculation, excess bacteria were removed from the discs
 by briefly blotting on sterile filter paper and the discs transferred to
 petri dishes containing "shoot medium" (Horsch et al. (1988) in Plant
 Molecular Biology Manual, (Eds. S. B. Gelvin, R. A. Schilperoort) Kluwer
 Acad. Publishers, A5:1-9). Petri plates were sealed with parafilm and
 incubated in a growth chamber (24.degree. C. and equipped with "grow"
 mixed fluorescent tubes). After two days, Agrobacterium growing on the
 discs were killed by washing in 500 mg/ml Cefotaxime in liquid "shoot
 medium" and the discs were transferred to fresh "shoot medium" containing
 500 mg/ml Cefotaxime and 100 mg/ml kanamycin to select for growth of
 transformed tobacco cells.
 Leaf discs were incubated under the same growth conditions described above
 for 3-5 weeks, and transferred to fresh medium on a weekly basis. During
 this period of time, approximately 40 green shoots emerging from the 60
 discs were excised and transferred to "root medium" (Horsch et al. (1988)
 supra) containing 100 .mu.g/ml kanamycin. Shoots which rooted in the
 presence of kanamycin and were verified to possess high levels of NptII
 activity (McDonnell, R. E. et al. (1987) Plant Mol. Biol. Rep. 5:380) were
 transferred to soil. Selected transformants were selfed and seeds
 collected. T1 seeds from seven transgenic tobacco lines displaying high
 levels of NptII activity were propagated at low light parameters (50-100
 .mu.mol.m.sup.-2.s.sup.-1) to determine which lines contained high levels
 of steady state transgene mRNA.
 The same construct has been introduced into two cultivars of Arabidopsis,
 three cultivars of Brassica, tomato, lettuce and alfalfa. All of these
 species demonstrate increased growth in culture compared to their
 wild-type counterparts, especially under low light intensities. These
 plants have a better shade avoidance response. They grow faster, bigger
 and seek light more responsively than their wild-type counterparts. This
 is evident at 65 .mu.moles/meter.sup.2 /sec of illumination in tobacco and
 lettuce, and 5 .mu.mole/meter.sup.2 /sec of illumination for Arabidopsis.
 Example 4
 RNA Analysis
 Isolation of total RNA and subsequent blot hybridization analyses were
 carried out as described in A. R. Cashmore, 1982 in Methods in Chloroplast
 Molecular Biology, M. Edelman, R. B. Hallick, N. H. Chua, Eds. (Elsevier
 Biomedical Press, pp. 533-542) and Maniatis, T., Fritsch, E. F., and
 Sambrook, J., Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor
 Laboratory, Cold Spring Harbor, N.Y., 1982)). Total RNA was isolated from
 twenty individual T1 plants from each primary transformant. Leaf samples
 were collected between 11:00 a.m. and 1:00 p.m. Formaldehyde denaturing
 gels were used to resolve RNA and transferred onto nitrocellulose as
 described (Sambrook et al. 1989, supra). The RNA blot (FIG. 7A) was probed
 with the pea Cab DNA probe (marked PROBE 1), stripped and rehybridized
 with the pea Rbcs transit peptide-specific DNA probe (marked PROBE 2).
 Numbers 1-7 indicate the primary transformants carrying the Rbcs-Cab
 transgene and exhibiting high levels of NptII activity. Plants 7-12
 represent plants that have been transformed with a control pea Cab
 construct. Plant 13 represents a wild-type nontransformed tobacco plant
 (Nicotiana tabacum cv. Petit Havana SRI). Transcript levels detected by
 the pea Cab DNA probe were normalized and quantitated by laser
 densitometry. Plants arising from transformant lines 3 and 5 contained the
 lowest amount of pea cab mRNA, whereas lines 1, 2 and 7 contained the
 highest levels. The same results were obtained with the pea Rbcs transit
 peptide-specific DNA probe (FIG. 7A). Total RNA from wild-type plants (WT)
 did not hybridize to either of the DNA probes.
 Individual T1 plants from the lines with the highest observed transgene
 mRNA levels were self-crossed and subjected to segregation analysis. The
 transgene mRNA levels of subsequent homozygous lines were analyzed in the
 same manner as the primary transformant lines (FIG. 7B). Numbers indicate
 the derivation of the homozygous lines. Seeds were germinated at
 24.degree. C. on moist filter paper, transferred to pots containing a
 mixture of soil and vermiculite (1:1) and propagated in growth chambers
 set at 50-100 .mu.mol.m.sup.-2.s.sup.-1 lighting with a 14 h light/10 h
 dark photoperiod and 24/18.degree. C. day/night temperature. The pots were
 watered daily with a complete nutrient solution containing 10 mM nitrate
 and 2mM ammonium. Several subsequent homozygous lines exhibited high
 levels of transgene mRNA and displayed the same phenotype.
 Example 5
 Protein and Chlorophyll Analysis
 Thylakoid protein profiles of homozygous lines derived from transgenic line
 2 (TR) were compared to wild-type (WT) to determine if additional
 transgene transcripts translated into an overall increase in steady state
 Cab levels. Nine different leaf disc samples were collected from
 homozygous transgenic lines derived from primary transformant 2 (marked
 TR) and from wild-type plants (marked WT). Samples were collected between
 11:00 a.m. and 1:00 p.m. Thylakoids were isolated (K. E. Steinback, et
 al., in Methods in Chloroplast Molecular Biology, M. Edelman, R. B.
 Hallick, N. H. Chua, Eds. (Elsevier Biomedical Press, Amsterdam, 1982) pp.
 863-872; Vernon, L. P. (1960) Anal. Chem. 32:1144) and analyzed using
 standard immunoblotting techniques. Cab/Oee1 ratios were determined by
 laser densitometry of the corresponding immunostained bands. The bands
 corresponding to Oee1 and Cab are marked. Increases in overall Cab protein
 levels were detected by simultaneously probing the blots with antibodies
 against the 33 kDa protein of the PSII oxygen-evolving complex (Oee1) and
 Cab (FIG. 7C). The ratio of Cab to Oee1 was used to determine Cab levels
 relative to PSII units by using Oee1 as an internal marker of PSII levels.
 The densitometry results indicated that the level of Cab protein is
 enhanced 2-3.times. relative to Oee1, suggesting that there is more Cab
 protein per PSII. Parallel enhancement of chlorophyll content was evident
 when the LhcII complexes were isolated (K. E. Steinback, (1982) supra; L.
 P. Vernon (1960) supra). Approximately 1.5.times. more chlorophyll and
 about 2-3.times. more LhcII complexes were recovered per gram of fresh
 weight leaf, indicating that the additional Cab proteins are functionally
 binding chlorophyll.
 Example 6
 Morphological and Developmental Analysis
 The TR plants, both primary transformants and subsequent selfed homozygous
 lines, exhibit growth and morphological differences relative to WT under
 all conditions tested, e.g., greenhouse or growth chambers. All plants
 shown were at the same developmental age and were propagated as described
 above. The TR plants display a higher level of vigor under low light
 regimes (50-80 .mu.mol.m.sup.-2.s.sup.-1) (FIG. 8A).
 The high light responses of the TR plants are enhanced. They produce more
 biomass and more robust growth patterns, depending on the intensity of the
 lighting conditions during propagation. The TR plants are bigger than
 their WT counterparts under high light intensities, such as in
 greenhouses. Among other characteristics, TR plants, compared to WT plants
 show increased stern girth and less variability in growth pattern.
 Further, field trials show that TR plants grow as well as WT plants in
 full sunlight field conditions in terms of biomass and size. No
 detrimental effects were observed in TR plants under these conditions.
 The elevation of Cab appears to induce a series of changes, the most
 prominent ones being broader leaves with a smooth blade, a continuous edge
 around the leaves, higher vegetative biomass and delayed flowering time.
 In addition to the overall enlargement of leaf size, the base of the
 petiole is more expanded relative to the WT leaves (the 7th fully
 developed leaf from both WT and TR were compared) (FIG. 8B). The TR leaves
 are thicker with relatively larger intercellular spaces (FIG. 8C). The
 light micrographs (FIG. 8C) represent samples from the intermediary area
 of the leaf blade. Leaf pieces were fixed in FAA50 and examined using a
 light microscope (D. A. Johansen, Plant Microtechnique, (McGraw-Hill Book
 Co., New York, 1940)).
 Leaf samples were selected as above and processed for electron microscopy
 by fixing in 2.5% glutaraldehyde buffered with 0.1M phosphate buffer (pH
 7.5) and post-fixing with 1% osmium tetroxide for 2 h. Following
 dehydration in an ethanol series, the leaf samples were embedded in Spurr
 resin, sectioned and further stained in uranyl acetate and plumb citrate
 (Spurr, A. R. (1969) J. Ultrastr. Res. 26:31; Reynolds, E. S. (1963) J.
 Cell Biol. 17:208; Watson, M. L. (1958) J. Biophys. Biochem. Cytol.
 4:475). The same scale in the applies to both WT and TR mesophyll tissue
 photographs. The TR leaf cells contain higher numbers of chloroplasts on a
 per cell basis and the plastids are larger with a strikingly rounder shape
 (FIG. 11). Differences in the internal organization of the plastids, e.g.
 stacking of the thylakoids, were not detected at the level of resolution
 used. Differences were not detected at any level with respect to the
 mitochondria, vacuole or nucleus.
 The germination rate of TR seeds was strikingly different than that of WT
 seeds. TR seeds germinate on average 1-3 days earlier than WT seeds on
 solid MS media (FIG. 9), and the newly emerged TR seedlings are already
 green and grow faster upon emergence than WT. The WT seedlings emerge
 yellowish and begin greening within the day. Callus tissue comprising TR
 cells grows 2-3.times. faster than callus comprising WT cells (FIG. 10).
 Transplants of TR plants also withstand transplant shock better than
 transplants of WT plants. They recover and establish normal growing
 patterns more rapidly.
 Example 7
 Physiological and Biochemical Analysis
 Functionality and enhancement of photosynthetic activity as a result of the
 extra Cab protein was assessed using four different criteria:
 1) Gas exchange characteristics;
 2) Metabolite level changes;
 3) Carbohydrate content; and
 4) PSII electron transport efficiency.
 Photosynthetic rates of TR and WT plants propagated under limiting light
 conditions were compared. Plants were cultivated under two different light
 intensities, 50 .mu.mol.m.sup.-2.sub..cndot.S.sup.-1 (referred to as low,
 (FIG. 12A)) and 500 .mu.mol.m.sup.-2.s.sup.-1 (referred to as high, (FIG.
 12B)). Photosynthesis was measured using a leaf disc oxygen electrode
 (LD2/2 Hansatech, UK) under saturating 5% CO.sub.2 at 25 .degree. C. The
 5% CO.sub.2 was supplied from 200 .mu.l of a 2M KHCO.sub.3 /K.sub.2
 CO.sub.3 mixture (pH 9.3) on felt in the base of the leaf disc electrode
 (Walker, D. A. (1987) The use of the oxygen electrode and fluorescence
 probes in simple measurements of photosynthesis University of Sheffield,
 Sheffield, U.K.). Illumination was provided by a slide projector Novomat
 515 AF (Braun, Germany). The data are means of 5 plants of each phenotype.
 Standard deviations were less than 10% of the means.
 Photosynthetic response curves of TR plants display a behavior distinct
 from WT plants (FIG. 12A). In low light (between 20-100
 .mu.mol.m.sup.-2.s.sup.-1), the TR plants exhibit a higher rate of
 photosynthesis than in WT plants; whereas, the reverse situation occurs in
 higher light intensities (FIG. 12A). As the light intensity increases, the
 response curves become more similar, intersecting at approximately 300
 .mu.mol.m.sup.-2.s.sup.-1, where TR tissue reaches saturation at a lower
 rate. At the same light intensity, the increase in photosynthesis is
 higher and has not reached saturation in WT tissue. Saturation in WT
 tissue occurs at about 450 .mu.mol.m.sup.-2.s.sup.-1.
 The same response was displayed by plants grown in higher irradiance (500
 .mu.mol.m.sup.-2.s.sup.-1) (FIG. 12B). The rate in TR tissue is higher
 than WT tissue in the range of 20-500 .mu.mol.m.sup.-2.s.sup.-1, reaching
 saturation in higher light intensities, while WT remains unsaturated at
 1000 .mu.mol.m.sup.-2.s.sup.-1. The increased low light photosynthetic
 capacity of TR tissue was also evident in air CO.sub.2 levels and at a
 light intensity of 100 .mu.mol.m.sup.-2.s.sup.-1, where TR tissue
 exhibited an average photosynthetic rate 50% higher than WT tissue
 (3.3.+-.0.8 vs. 2.2.+-.0.8 .mu.mol O.sub.2.m.sup.-2.s.sup.-1,
 respectively).
 Alterations in metabolite and adenylate levels are also indicators of
 changes in photosynthetic capacity. In low light, photosynthesis is mainly
 limited by the capacity of electron transport to generate ATP and NADPH,
 the assimilatory force F.sub.A (Heber, U. et al. (1986) Biochim. Biophys.
 Acta 852:144; Heber et al., in Progress in Photosynthesis Research, J.
 Biggins, Ed., (Martinus Nijhoff, Dordrecht) Vol. 3 (1987) pp. 293-299).
 The strength of F.sub.A can be estimated by the ratio of PGA
 (3-phosphoglyceric acid) to TP (triose phosphate) (Dietz, K. J. and Heber,
 U. (1984) Biochim. Biophys. Acta. 767:432); ibid 848:392 (1986)).
 Measurements were obtained under 100 and 1000 .mu.mol.m.sup.-2.s.sup.-1
 lighting, and in 850 .mu.bar external CO.sub.2 concentration to minimize
 the effects of photorespiration (Table 4). When photosynthesis achieved
 steady state, the leaves were freeze-clamped and prepared for metabolite
 extraction. The CO.sub.2 assimilation rate of TR leaves in 100
 .mu.mol.m.sup.-2.s.sup.-1 lighting was 53% higher than WT leaves. The
 levels of PGA were similar between TR and WT plants, however, the TP level
 was 33% higher in TR. Thus, the PGA/TP ratio is higher in WT plants,
 indicating a limitation in the reduction of PGA to TP by the supply of ATP
 and NADPH in WT plants. The changes in adenylates indicate that the ATP
 content in TR leaves was twice the value observed in WT leaves, whereas
 the ADP content in both plants was similar. The ATP/ADP ratio in the
 chloroplast is lower than in the cytosol, typically calculated to be
 between 1.5 and 3.0 (Stitt, M. et al. (1982) Plant Physiol. 70:971;
 Giersch, C. et al. (1980) Biochim. Biophys. Acta 590:59; Neuhaus, N. E.
 and Stitt, M. (1989) Planta 179:51). As light intensity increases, the
 ratio decreases even further (Dietz and Heber, supra). The ATP/ADP ratio
 is higher in TR plants than WT plants (2.2 vs. 0.8). These results
 indicate that TR plants have an increased capacity to generate ATP in low
 light, leading to an enhancement of PGA reduction and a higher
 photosynthetic rate.
 Changes in the level of hexose phosphate were also observed, with more
 hexose phosphate in WT than TR plants. The G6P/F6P ratio is an indicator
 of hexose distribution in a cell, with values of 1-2 indicating
 chloroplastic compartmentalization and predominantly starch synthesis, and
 ratios of 3-5 indicating a cytoplasmic location with sucrose synthesis
 being dominant (Gerhardt, R. et al. (1987) Plant Physiol. 83:399). Thus,
 the low G6P/F6P values for both WT and TR plants grown in low light
 indicate that the carbon fixed is being partitioned mainly into starch.
 The enhanced capacity to absorb light has a negative effect on
 photosynthetic metabolism of TR plants in high irradiance regimes. The
 photosynthetic rate of WT plants was instead 37% higher than that of TR
 plants. The changes in metabolite levels and ratios indicate that major
 alterations in the regulatory mechanisms of photosynthesis have occurred
 in TR plants to compensate for the enhanced light-absorbing capacity in
 high light. The PGA/TP ratio was identical in both plants and the ATP/ADP
 ratio was lower in TR plants indicating that photophosphorylation was
 limiting photosynthesis. The change in partitioning indicated by the
 G6P/F6P ratio increase (2.9 vs. 3.9 in WT and TR plants, respectively)
 points to an increase in sucrose synthesis to compensate for the elevated
 demand for inorganic phosphate (Pi) in TR plants. The TR plants appear to
 be less efficient in the recycling of Pi via sucrose synthesis in high
 light.
 TABLE 4
 Photosynthesis and metabolite content in plants grown under 50 .mu.mol
 .multidot. m.sup.-2 .multidot. s.sup.-1 lighting.

SEQUENCE LISTING
 &lt;160&gt; NUMBER OF SEQ ID NOS: 4
 &lt;210&gt; SEQ ID NO: 1
 &lt;211&gt; LENGTH: 1166
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Pisum sativum
 &lt;220&gt; FEATURE:
 &lt;221&gt; NAME/KEY: CDS
 &lt;222&gt; LOCATION: (184)...(990)
 &lt;400&gt; SEQUENCE: 1
 tccatgaacg gattctagaa ttgcaaagaa aatctccaac tagccatagc tttagataac 60
 acacgataag agcatctgca ttataaatac agactcatat tcatcttaca aaatcaccat 120
 tgataaggat acaattatca aaagcataac aatcttttca atttcattgc aatataatac 180
 acg atg gcc gca tca tca tca tca tcc atg gct ctc tct tct cca acc 228
 Met Ala Ala Ser Ser Ser Ser Ser Met Ala Leu Ser Ser Pro Thr
 1 5 10 15
 ttg gct ggc aag caa ctc aag ctg aac cca tca agc caa gaa ttg gga 276
 Leu Ala Gly Lys Gln Leu Lys Leu Asn Pro Ser Ser Gln Glu Leu Gly
 20 25 30
 gct gca agg ttc acc atg agg aag tct gct acc acc aag aaa gta gct 324
 Ala Ala Arg Phe Thr Met Arg Lys Ser Ala Thr Thr Lys Lys Val Ala
 35 40 45
 tcc tct gga agc cca tgg tac gga cca gac cgt gtt aag tac tta ggc 372
 Ser Ser Gly Ser Pro Trp Tyr Gly Pro Asp Arg Val Lys Tyr Leu Gly
 50 55 60
 cca ttc tcc ggt gag tct cca tcc tac ttg act gga gag ttc ccc ggt 420
 Pro Phe Ser Gly Glu Ser Pro Ser Tyr Leu Thr Gly Glu Phe Pro Gly
 65 70 75
 gac tac ggt tgg gac act gcc gga ctc tct gct gac cca cag aca ttc 468
 Asp Tyr Gly Trp Asp Thr Ala Gly Leu Ser Ala Asp Pro Gln Thr Phe
 80 85 90 95
 tcc aag aac cgt gag ctt gaa gtc atc cac tcc aga tgg gct atg ttg 516
 Ser Lys Asn Arg Glu Leu Glu Val Ile His Ser Arg Trp Ala Met Leu
 100 105 110
 ggt gct ttg gga tgt gtc ttc cca gag ctt ttg tct cgc aac ggt gtt 564
 Gly Ala Leu Gly Cys Val Phe Pro Glu Leu Leu Ser Arg Asn Gly Val
 115 120 125
 aaa ttc ggc gaa gct gtg tgg ttc aag gca gga tct caa atc ttt agt 612
 Lys Phe Gly Glu Ala Val Trp Phe Lys Ala Gly Ser Gln Ile Phe Ser
 130 135 140
 gag ggt gga ctt gat tac ttg ggc aac cca agc ttg gtc cat gct caa 660
 Glu Gly Gly Leu Asp Tyr Leu Gly Asn Pro Ser Leu Val His Ala Gln
 145 150 155
 agc atc ctt gcc ata tgg gcc act cag gtt atc ttg atg gga gct gtc 708
 Ser Ile Leu Ala Ile Trp Ala Thr Gln Val Ile Leu Met Gly Ala Val
 160 165 170 175
 gaa ggt tac cgt att gcc ggt ggg cct ctc ggt gag gtg gtt gat cca 756
 Glu Gly Tyr Arg Ile Ala Gly Gly Pro Leu Gly Glu Val Val Asp Pro
 180 185 190
 ctt tac cca ggt gga agc ttt gat cca ttg ggc tta gct gat gat cca 804
 Leu Tyr Pro Gly Gly Ser Phe Asp Pro Leu Gly Leu Ala Asp Asp Pro
 195 200 205
 gaa gca ttc gca gaa ttg aag gtg aag gaa ctc aag aac ggt aga tta 852
 Glu Ala Phe Ala Glu Leu Lys Val Lys Glu Leu Lys Asn Gly Arg Leu
 210 215 220
 gcc atg ttc tca atg ttt gga ttc ttc gtt caa gct att gta act gga 900
 Ala Met Phe Ser Met Phe Gly Phe Phe Val Gln Ala Ile Val Thr Gly
 225 230 235
 aag ggt cct ttg gag aac ctt gct gat cat ctt gca gac cca gtc aac 948
 Lys Gly Pro Leu Glu Asn Leu Ala Asp His Leu Ala Asp Pro Val Asn
 240 245 250 255
 aac aat gca tgg tca tat gcc acc aac ttt gtt ccc gga aaa 990
 Asn Asn Ala Trp Ser Tyr Ala Thr Asn Phe Val Pro Gly Lys
 260 265
 taaacactct tatatttata tgtttttgtg atagtaatct tcttcccaat tcaatgtgaa 1050
 ttattatcat tatcattatc atgtgggtat gcataggttc actaatacaa gatgatggat 1110
 gctttttttt taccaaattt taaattttat gtttcatgct ttccattgct agacat 1166
 &lt;210&gt; SEQ ID NO: 2
 &lt;211&gt; LENGTH: 269
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Pisum sativum
 &lt;400&gt; SEQUENCE: 2
 Met Ala Ala Ser Ser Ser Ser Ser Met Ala Leu Ser Ser Pro Thr Leu
 1 5 10 15
 Ala Gly Lys Gln Leu Lys Leu Asn Pro Ser Ser Gln Glu Leu Gly Ala
 20 25 30
 Ala Arg Phe Thr Met Arg Lys Ser Ala Thr Thr Lys Lys Val Ala Ser
 35 40 45
 Ser Gly Ser Pro Trp Tyr Gly Pro Asp Arg Val Lys Tyr Leu Gly Pro
 50 55 60
 Phe Ser Gly Glu Ser Pro Ser Tyr Leu Thr Gly Glu Phe Pro Gly Asp
 65 70 75 80
 Tyr Gly Trp Asp Thr Ala Gly Leu Ser Ala Asp Pro Gln Thr Phe Ser
 85 90 95
 Lys Asn Arg Glu Leu Glu Val Ile His Ser Arg Trp Ala Met Leu Gly
 100 105 110
 Ala Leu Gly Cys Val Phe Pro Glu Leu Leu Ser Arg Asn Gly Val Lys
 115 120 125
 Phe Gly Glu Ala Val Trp Phe Lys Ala Gly Ser Gln Ile Phe Ser Glu
 130 135 140
 Gly Gly Leu Asp Tyr Leu Gly Asn Pro Ser Leu Val His Ala Gln Ser
 145 150 155 160
 Ile Leu Ala Ile Trp Ala Thr Gln Val Ile Leu Met Gly Ala Val Glu
 165 170 175
 Gly Tyr Arg Ile Ala Gly Gly Pro Leu Gly Glu Val Val Asp Pro Leu
 180 185 190
 Tyr Pro Gly Gly Ser Phe Asp Pro Leu Gly Leu Ala Asp Asp Pro Glu
 195 200 205
 Ala Phe Ala Glu Leu Lys Val Lys Glu Leu Lys Asn Gly Arg Leu Ala
 210 215 220
 Met Phe Ser Met Phe Gly Phe Phe Val Gln Ala Ile Val Thr Gly Lys
 225 230 235 240
 Gly Pro Leu Glu Asn Leu Ala Asp His Leu Ala Asp Pro Val Asn Asn
 245 250 255
 Asn Ala Trp Ser Tyr Ala Thr Asn Phe Val Pro Gly Lys
 260 265
 &lt;210&gt; SEQ ID NO: 3
 &lt;211&gt; LENGTH: 221
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Pisum sativum
 &lt;220&gt; FEATURE:
 &lt;221&gt; NAME/KEY: CDS
 &lt;222&gt; LOCATION: (30)...(221)
 &lt;400&gt; SEQUENCE: 3
 acgttgcaat tcatacagaa gtgagaaaa atg gct tct atg ata tcc tct tcc 53
 Met Ala Ser Met Ile Ser Ser Ser
 1 5
 gct gtg aca aca gtc agc cgt gcc tct agg ggg caa tcc gcc gca gtg 101
 Ala Val Thr Thr Val Ser Arg Ala Ser Arg Gly Gln Ser Ala Ala Val
 10 15 20
 gct cca ttc ggc ggc ctc aaa tcc atg act gga ttc cca gtg aag aag 149
 Ala Pro Phe Gly Gly Leu Lys Ser Met Thr Gly Phe Pro Val Lys Lys
 25 30 35 40
 gtc aac act gac att act tcc att aca agc aat ggt gga aga gta aag 197
 Val Asn Thr Asp Ile Thr Ser Ile Thr Ser Asn Gly Gly Arg Val Lys
 45 50 55
 tgc atg gat cct gta gag aag tct 221
 Cys Met Asp Pro Val Glu Lys Ser
 60
 &lt;210&gt; SEQ ID NO: 4
 &lt;211&gt; LENGTH: 64
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Pisum sativum
 &lt;400&gt; SEQUENCE: 4
 Met Ala Ser Met Ile Ser Ser Ser Ala Val Thr Thr Val Ser Arg Ala
 1 5 10 15
 Ser Arg Gly Gln Ser Ala Ala Val Ala Pro Phe Gly Gly Leu Lys Ser
 20 25 30
 Met Thr Gly Phe Pro Val Lys Lys Val Asn Thr Asp Ile Thr Ser Ile
 35 40 45
 Thr Ser Asn Gly Gly Arg Val Lys Cys Met Asp Pro Val Glu Lys Ser
 50 55 60