Abstract:
Methods are provided for modifying and screening for carotenoid biosynthesis in a plant. The methods are useful for enhancing plant adaptation to climate change and food security, providing increased carotenoid content to a plant, improving stress resistance to climate changes in a plant, and for selecting plants having improved stress resistance to climate changes

Description:
[0001]    This application claims priority to U.S. Provisional Patent Application No. 61/683,494 filed Aug. 15, 2012. 
     
    
     BACKGROUND OF THE INVENTION 
       [0002]    Carotenoids are a large class of yellow, orange and red lipophilic structures synthesized by all photosynthetic organisms. In plants, carotenoids play multiple roles such as photosynthetic light harvesting, protection against light and heat stress, and as precursors to hormones that mediate stress and developmental signalling. Carotenoid antioxidants increase heat and light stress tolerance by protecting membranes from reactive oxygen species (ROS) and lipid peroxidation. Concentrated in fibrillar plastoglobuli of fruit chromoplasts, carotenoids are attractants to animals that serve as plant seed distributors. Certain carotenoids in the endosperm tissue provide nutritional value and have been targets for improvement, especially in cereal crops of the grass family. 
         [0003]    The biosynthetic pathway of carotenoids occurs in plastids where the lipophilic carotenoids accumulate in envelope/thylakoid membranes and plastoglobuli. Carotenoids are synthesized in both light and dark-grown tissues, such as leaves, endosperm, and roots. In the dark, leaf tissues develop etioplasts with rudimentary prolamellar bodies, which are the precursors for thylakoids and support low levels of carotenoid biosynthesis. In the light, leaves turn green due to development of highly specialized chloroplasts filled with complex photosynthetic systems. Carotenoids are distributed differently in etioplasts and chloroplasts that might require differential localization of their biosynthetic enzymes as well. 
         [0004]    Phytoene synthase (PSY) catalyzes the committed step to carotenoid biosynthesis and is a key target for pathway engineering. There are up to three PSY isozymes in evolutionarily distant plants, including all the major food staples in the grasses and other crops of agronomic importance. Different PSY isozymes mediate carotenogenesis in particular tissues, in response to developmental and physiological signals. Allele-specific variation accounts for yellow endosperm maize and yellow rooted cassava. 
         [0005]    The core carotenoid biosynthetic pathway consists of about 10 enzymes. However, the location of the biosynthetic pathway as a complete entity for controlling the unique spatial distribution of carotenoids is unknown. Moreover, this pathway must respond to environmental and developmental signals to link photomorphogenesis, photoprotection, and stress responses with location-specific carotenoid synthesis and degradation. It has long been desired to understand the nature of this dynamic pathway landscape and how isozymes and allelic variants fit into the picture. 
         [0006]    According to recent proteomic studies on  Arabidopsis  chloroplasts, many of the carotenoid biosynthetic pathway enzymes are exclusively localized to envelope membranes. Only a few carotenoid enzymes are found in thylakoids: xanthophyll cycle enzymes and phytoene desaturase (PDS). For example, in pepper fruit chromoplasts, most carotenoid enzymes are localized to plastoglobuli. In maize proteomic studies, the only carotenoid enzymes detected were PDS and □-carotene desaturase (ZDS) that were respectively found in membrane fractions of bundle sheath and mesophyll cells. Carotenoids are found in both cell types, yet other carotenoid biosynthetic enzymes were undetectable. Chloroplast suborganellar localization of the key pathway enzyme, PSY, has yet to be detected by proteomic analysis. 
         [0007]    As a result of this invention, it has been discovered that PSY isozymes differ in chloroplast suborganellar localization and that overexpression of naturally occurring allelic variants produces striking differences in localization and profound effects on chloroplast architecture. 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0008]      FIG. 1 . Transient expression of various PSY-GFP fusion constructs in leaf mesophyll protoplasts. 
           [0009]    A. Expression in etiolated maize protoplasts. All PSYs from maize and rice, except  zm PSY1, are localized to specific speckles.  zm PSY1 is localized to stroma and associated to prolamellar bodies. Chl: chlorophyll autofluorescence, concentrated in a partial area of an etioplast. 
           [0010]    B. Expression of  zm PSYs and  os PSYs in green maize protoplasts and of  at PSY-RFP in green bean protoplasts. All PSY from maize, rice and  Arabidopsis , except  zm PSY1, are localized to specific speckles.  zm PSY1 is localized to stroma. Chl: chlorophyll autofluorescence, occupying the entire area of a chloroplast. Bar=10 μm. 
           [0011]      FIG. 2 . Plastoglobuli localization of various proteins in mesophyll protoplasts. 
           [0012]    A. Transient expression of  zm PG2-RFP, suggesting localization to plastoglobuli. 
           [0013]    1. Expression in bean cotyledon protoplasts. 
           [0014]    2. Expression in green maize protoplasts. 
           [0015]    3. Expression in etiolated maize protoplasts. 
           [0016]    **1 and 2 show plastoglobular localization, 3 shows stromal localization. 
           [0017]    B. Transient co-expression of  zm PSY2-GFP (1) and  zm PSY3-GFP (2) with  zm PG2-RFP. 
           [0018]      zm PSYs and  zm PG2 are co-localized, as seen on merged image, indicating plastoglobular localization of  zm PSY2 and  zm PSY3. Chl: chlorophyll autofluorescence. Bar=10 μm. 
           [0019]      FIG. 3 . Import of proteins into chloroplasts. Protein precursors were made by in vitro transcription/translation. Protein precursors were incubated with isolated chloroplasts for import and processing to the mature forms. Mature proteins were resistant to thermolysin treatment of chloroplasts post-import and of smaller mass compared to the unimported precursors. 
           [0020]    A. import of proteins with known localization used as a control for fraction purity: 16/EGFP (a transit peptide of spinach OE16 of oxygen evolution system from thylakoid lumen, fused with EGFP), LHCP (light-harvesting chlorophyll a/b binding pea protein localized in thylakoid membranes), and Toc34 (a component of the protein transport complex from the outer envelope membrane). 
           [0021]    B. import of maize PSYs. Arrow—mature processed protein. Star—20 kDa band. 
           [0000]    kDa−molecular weight marker
 
P−precursor (1 μl of the translation mix)
 
I−import of radiolabelled precursor protein into intact chloroplasts
 
T−thermolysin treated chloroplasts
 
M−membrane fraction
 
MA−purified membrane fraction (after alkaline treatment)
 
S−soluble fraction
 
           [0022]      FIG. 4 . Alignment of PSY aminoacids for all enzymes used in experiments adjusted to secondary SQS structure. #168: aminoacid represented by asparagine (N) in  zm PSY1 and serine (S) in all other PSYs. #257: aminoacid represented by threonine (T) in  zm PSY1 and proline (P) in all other PSYs. #174, 190: respective aminoacids were shown to affect the activity of PSY in cassava and tomato. #285: mutation at this site inactivates SQS. S—putative cleavage site for chloroplast transit sequence, predicted by ChloroP.  XXXXX —psy and sqs signature motif 1 and 2 (conserved pattern). XXXXX—a-helix, predicted by Metaserver based on SQS structure. Box—DXXXD putative active site, mutagenesis of D 285  inactivates the enzyme. (*)—62 aa of C-terminus of SQS sequence were truncated. 
           [0023]      FIG. 5 . Alignment of phytoene synthase amino acid sequences. BLAST alignment of  zm PSY1 to all PSY sequences available from NCBI. Box—DXXXD putative active site, based on SQS alignment. Highlight—amino acids 168 and 257. Amino acid numbers refer to  zm PSY1 sequence. 
           [0024]      FIG. 6 . Transient expression of  zm PSY1-NP-GFP and its mutagenized variants in etiolated maize protoplasts.  zm PSY1-NP is forming characteristic spikes in 30% of cases (see central protoplast), the rest 70% it is soluble (top left corner).  zm PSY1-SP,  zm PSY1-NS and  zm PSY1-ST are localized evenly inside plastids, suggesting soluble stromal localization. Chl: chlorophyll autofluorescence. Bar=10 μm. 
           [0025]      FIG. 7 . Sequence of  zm PSY1s with transit peptide removed threaded onto crystal structure of squalene synthase. 
           [0026]    A. Graphical representation of RMSD data on  zm PSY1 modeling. Blue:  zm PSY1, green:  zm PSY1-NP. Star: amino acid #257. Red arrow: perturbed region. Black double arrow: regions  159 DELVD 163  and  285 DVGED 289  as putative active site. 
           [0027]    B. RMSD data showing mean distance (y-axis, in angstroms) between atoms of the three superimposed  zm PSY structures (numbered  zm PSY amino acids shown on x-axis). Red:  zm PSY1-NP, green:  zm PSY1-SP, blue: marker to show location in sequence of amino acids 168, 174, 190 and 257. 
           [0028]      FIG. 8 . Overexpression of proteins with known location in etiolated maize protoplasts. 
           [0029]    A. soluble GFP (protoplasts transformed with pUC35S-sGFP-Nos), GFP is localized to cell cytoplasm. 
           [0030]    B. LHCP-GFP (protoplasts transformed with pUC35S-LHCP-sGFP-Nos), LHCP is an integral protein from thylakoid membrane and colocalized with chlorophyll fluorescence. 
           [0031]    C. AtAPG1 (protoplasts transformed with pFB70), 
           [0032]    D. AtTic40 (protoplasts transformed with pFB71). AtAPG1 and AtTic40 are proteins from chloroplast inner envelope membrane and demonstrate half-moon or circle pattern correspondingly. 
           [0000]    Chl—chlorophyll autofluorescence. Bar=10 μm. 
           [0033]      FIG. 9 . Hidden 3D (“third eye”) stereogram of transiently expressed  zm PSY1-NP-GFP in etiolated maize protoplasts.  zm PSY1-NP-GFP is forming characteristic spikes. Stereogram prepared from original microscopic image by Hidden 3D studio, USA. It can be viewed by unfocusing the eyes and looking “through” the image. Additional instructions for viewing 3-D sterograms can be found at: http://www.hidden-3d.com/how_to_view_stereogram.php. 
           [0034]      FIG. 10 . Transient expression of  zm PSY-GFP variants in etiolated maize protoplasts from PSY1 knockout line. 
           [0035]      FIG. 11 . Spectral dye separation for transiently expressed GFP-fusion proteins in maize PSY1 knockout leaf protoplasts. Different fusion protein constructs were transiently expressed in protoplasts, isolated from etiolated leaves of maize PSY1 knockout plants. GFP and carotenoid fluorescent spectra were separated using Spectral Dye Separation tool in LAS AF software (Leica Microsystems). Carotenoids have a low fluorescence emission between 500 and 650 nm which can be excited by a 488 nm laser. The software separates the carotenoid emission from the GFP emission. To eliminate any background carotenoid fluorescence in the absence of a PSY transgene, we used protoplasts prepared form  zm PSY1-knockout maize y1-8549 which are unable to produce carotenoids in the absence of the transgenically supplied PSY. 
           [0036]    A. Soluble GFP without any protein or transit peptide attached, localized to protoplast cytoplasm Soluble cytoplasmic GFP, used as a negative control and GFP spectrum reference, showed no carotenoid emission 
           [0037]    B.  zm PSY1-NP-GFP—Fibrils formed by  zm PSY1-NP have bright fluorescence in the carotenoid channel suggesting presence of concentrated carotenoids. 
           [0038]    C.  zm PSY1-NP-E-GFP—Such fluorescence disappeared, showing no fibrils when non-active  zm PSY1-NP-E was overexpressed in etioplasts. 
           [0039]    D.  zm PSY1-NT-GFP—A low level of carotenoid fluorescence was observed in etioplasts carrying overexpressed  zm PSY1-NT which might be due to a basal level of carotenoid biosynthesis in leaves due to inactivity of  zm PSY when not attached to membranes. 
           [0040]    E.  zm PSY2-GFP—punctuated dots, formed in etioplasts by  zm PSY2-GFP show bright fluorescence in the carotenoid channel suggesting presence of concentrated carotenoids 
           [0041]    F.  zm PSY3-GFP—Punctuated dots, formed in etioplasts by  zm PSY3-GFP show bright fluorescence in the carotenoid channel suggesting presence of concentrated carotenoids. 
           [0042]    G. LHCP-GFP (negative control of plastid protein fused with GFP; LHCP is a not involved in carotenoid biosynthesis). In contrast, only background level of fluorescence is observed when a control chloroplast-localized noncarotenoid protein is introduced (integral thylakoid membrane protein LHCP. 
           [0000]    GFP, Carotenoids—maximum projection of corresponding isolated fluorescent spectra;
 
Bright field—bright field image of the protoplast. Bar=10 μm.
 
           [0043]      FIG. 12 .  E. coli  complementation test.  E. coli  Top 10 with pACCAR25□crtB carrying carotenoid biosynthetic enzymes for zeaxanthin production, but lacking the PSY enzyme, was transformed with different mutagenized variants of  zm PSY1. Active PSY enzymes were able to complement the pathway which resulted in zeaxanthin production. Zeaxanthin was extracted from pelleted cells by methanol and then analyzed by HPLC to detect the relative amount produced, which was calculated as peak area divided by OD of the  E. coli  culture.  zm PSY1,  zm PSY1-NP were active in this test,  zm PSY1-D 285 E was inactive. An empty expression vector was used as a negative control. 
       
    
    
     SUMMARY OF THE INVENTION 
       [0044]    The present invention related to a method for providing increased carotenoid content to a plant and for improving stress resistance to climate changes in a plant comprising. The method involves overexpressing naturally occurring allelic variants of phytoene synthase (PSY) in the plant. 
         [0045]    In one embodiment, the allelic variant is the NP variant of phytoene synthase 1 (PSY1). 
         [0046]    In another aspect, the invention provides a method for selecting plants with increased carotenoid content and/or having improved stress resistance to climate changes. The method involves selecting plants in which naturally occurring allelic variants of phytoene synthase (PSY) are overexpressed in said plant. 
         [0047]    In another embodiment, the allelic variant is the NP variant of phytoene synthase 1 (PSY1). 
       DETAILED DESCRIPTION OF THE INVENTION 
       [0048]    As a rate-controlling enzyme of the pathway, PSY has been used extensively for metabolic engineering of the carotenoid biosynthetic pathway in plants despite limited understanding of its plastid suborganellar location. Confocal microscopy of fluorescently tagged PSYs provided a glimpse into the carotenoid biosynthetic microenvironment in leaf mesophyll cells. 
         [0049]    PSYs are highly conserved in their amino acid sequence (See  FIG. 4 ). Yet, small variations in a region that must be important for activity have been discovered. This region lies adjacent to a common signature motif found in isoprenoid synthases. These natural variants include variations in amino acid residues at positions 168 and 257 of PSY. See Example 3 and Table 1 below. 
         [0050]    As will be discussed below,  zm PSY1 of B73 has the allele in yellow endosperm N 168 T 257  or “NT.” If T 257  is changed to proline, an “NP” variation is produced. If T 257  is changed to a serine an “NS” variation is produced. 
         [0051]    It has been discovered that the effect of the NP variation on fibril formation occurred in etioplasts from a subpopulation of protoplasts found among the complex population of cell-types shown to exist in leaves. Whole-tissue carotenoid or proteomic extraction would otherwise have masked these perturbations on enzyme location and carotenoid accumulation taking place in subpopulations of cells. 
         [0052]    The present invention elucidates the physical location of a key pathway enzyme that must interface with a complex and dynamic metabolon in the context of the suborganellar architecture that is unique to plastid type in specific tissues. More importantly, it has been discovered that not all PSYs localize identically. This discovery serves as a source of caution and also opportunity for improving carotenoid targets needed, for example, to improve seed nutritional quality or plant stress resistance to address challenges of food security and biofuels in the face of global climate change. 
         [0053]    It has been discovered that PSY isozymes target to unique chloroplast suborganellar sites and small sequence variation and enzyme activity of PSY1 alters enzyme localization. This is the first time that PSYs have been localized to plastoglobules. Plastoglobuli are found in all plastids although their specific function is not well understood. Plastoglobuli range in size from 60-4000 nm and their composition varies depending on plastid type and plant source. In chloroplasts, plastoglobuli associate with thylakoids, while details of plastoglobular association in non-photosynthetic plastids are sparse. 
         [0054]    In general, plastoglobuli contain carotenoids, plastoquinones, tocopherols, and various proteins surrounded by a distinctively-composed lipid monolayer that is contiguous with the outer layer of the thylakoid lipid bilayer. Fibrilins, the major proteins of plastoglobuli, maintain the structure of the globules and assist in the globular-fibril transition to store high amounts of carotenoids synthesized during chromoplast development. Plastoglobuli also enlarge and proliferate in response to abiotic stress (e.g. high light, drought, salt, heat and nitrogen starvation). These stress conditions induce accumulation of carotenoids and/or their apocarotenoid products, and the expression of core plastoglobuli protein genes correlates with the expression of several enzymes from the carotenoid pathway. The importance of plastoglobuli in modulating plant metabolism is beginning to gain attention. Overexpression of the tocopherol cyclase VTE1, a plastoglobular enzyme, resulted in proliferation of plastoglobuli and an increased level of tocopherols. 
         [0055]    As a result of the herein invention, it has been discovered that activity of the overexpressed PSY1, with the naturally-occurring NP sequence variation, may exert an effect on fibrillar plastoglobule architecture, since the active enzyme caused plastoglobular fibril formation, which disappeared when the PSY active site was mutated. In both VTE1 and PSY1 cases, overexpression of plastoglobular-associated enzymes caused physical changes in the site of carotenoid sequestration. Taken together, increased levels of rate-controlling plastoglobule-located vitamin E and carotenoid biosynthesis enzymes might drive plastid structural changes needed to provide a sink for the hydrophobic biosynthetic pathway products. 
         [0056]    Establishment of PSY localization leads to the question of how and where the entire pathway is reconstituted. Carotenoids are found on envelope and thylakoid membranes, implying that either the pathway forms on two membrane sites or that carotenoids are transported by an unknown mechanism. Carotenoid metabolons (enzyme complexes) are predicted to exist on the basis of high molecular weight complexes containing PSY or other carotenoid enzymes. A recent study showed that the capacity for enzymes to interact was associated with enhanced carotenoid pathway activity. Metabolon-associated enzymes could facilitate substrate channeling, as has been suggested by the absence of carotenoid pathway intermediates, except in cases where the pathway is artificially blocked. 
         [0057]    It is significant that PSY1 with NT or SP combinations behaved similarly in localization, in contrast to NP which was shown to have a dramatic effect on plastid architecture. The present invention indicates that use of the NP variation may be more effective in enhancing production of carotenoids in certain tissues. For example, PSY1 is naturally expressed in etiolated tissue and known to provide thermal tolerance which is lost in plants that are unable to make PSY1. Indeed, if the NP variant is more effective in promoting carotenoids in etioplasts, this allelic variant could be valuable in selecting plants that are more resilient to climate change. 
         [0058]    As a result of the herein invention, it has been discovered that  Arabidopsis  PSY is localized to plastoglobules. Based on proteomics studies of  Arabidopsis  chloroplasts, PDS is on the envelope and thylakoid and ZDS is in the stroma. Together with PSY, it is possible to form a complex to produce prolycopene, a pathway intermediate. The absence of detectable prolycopene suggests that additional enzymes are recruited, but these are not detected by proteomics which may be due to limitations of the proteomics methodologies. In contrast to the enzyme localization seen in  Arabidopsis  chloroplasts, in chromoplasts, which exhibit an exaggerated developmental induction of carotenoid accumulation, the proteomics analysis revealed that most of the enzymes were found in plastoglobules. Therefore, the possibility exists that the complexes are forming in a dynamic fashion and “recruited as needed.” 
         [0059]    The present invention provides methods for increasing nutritional value in a plant or enhancing stress resistance to climate change in a plant by metabolic engineering of carotenoids in plants. There are known connections between induction of carotenoid enzymes and physical changes at the subcellular level. For example, morphological changes associated with carotenogenesis in development of chromoplasts include increases in fibrillins, plastoglobules, and biosynthetic enzymes. The different suborganellar localizations exhibited by allelic variants suggest that PSYs might be involved in mobilization of carotenoid pathway enzymes to mediate carotenogenesis at distinct locations, may control carotenogenesis by altered localization of PSY, and that localization of an active PSY may influence plastid ultrastructure. Clearly, not all PSYs behave identically, representing an opportunity for metabolic engineering or breeding with specific allelic variants. 
       Example 1 
     PSY Isozymes Exhibited Differential Locations in Maize Chloroplasts 
       [0060]    To investigate localization of phytoene synthase isozymes, PSYs from two cereal crops, maize and rice, and a classical model plant  Arabidopsis  were chosen. Both maize and rice have three PSY isozymes and  zm PSY1,  zm PSY2,  zm PSY3 of  Z. mays  var. B73, and  os PSY1,  os PSY2 of  O. sativa  var. IR36 (Indica), and  os PSY3 of  O. sativa  var TP309 were tested. Maize variety B73 has yellow colored kernels due to carotenoid accumulation in the endosperm mediated by  zm PSY1 activity encoded by the maize yellow1 (y1) locus. Rice does not accumulate endosperm carotenoids.  Arabidopsis  has only one  at PSY. 
         [0061]    The localization of PSYs was studied by transient expression of fluorescent protein fusions in plant leaf protoplasts. Protoplasts retain their tissue specificity after isolation, thereby reflecting in vivo conditions to observe localization of transiently expressed PSY proteins. 
         [0062]    The above approach provides a great advantage for studying PSY, a low abundance protein that otherwise escapes detection in proteomic studies. Protoplast sources were chosen in consideration of different stages of plastid development. Protoplasts both from dark-grown tissues (etiolated protoplasts), or light-grown tissues (green protoplasts) were isolated. Also, monocot maize leaves as a protoplast source for expression of PSYs from monocotyledonous species maize and rice, and dicot beans for experiments on PSY from dicotyledonous  Arabidopsis  were chosen. Each PSY protein together with its chloroplast target peptide was C-terminally fused to synthetic green fluorescent (sGFP) or red fluorescent (RFP) protein, and transient expression was monitored by confocal microscopy. 
         [0063]    To confirm reliability of the approach, proteins of known localization using protoplasts prepared from etiolated maize leaves were tested ( FIG. 8 ). It was discovered that most, but not all, PSYs of all species studied were distributed in plastids the same way in both etiolated protoplasts ( FIG. 1A ), and green protoplasts ( FIG. 1B ), whether from monocots or dicots. These PSYs localized to chloroplasts in specific fixed speckles, distributed inside the plastid and attached to areas that displayed red chlorophyll fluorescence indicative of prolamellar bodies or thylakoids. The size and distribution of the speckles were suggestive of plastoglobuli: spherical lipid structures attached to thylakoid membranes of chloroplasts or distributed in chromoplast stroma. To define the nature of the speckles, transient expression with a protein from the fibrillin family was performed, since fibrillins are structural proteins of plastoglobuli. Maize plastoglobulin-2 ( zm PG2) was identified by BLAST search as a homolog to several fibrillins from  Arabidopsis  (AT4G04020, AT4G22240, AT2G35490, 80%-90% sequence similarity).  zm PG2 has a PAP-fibrillin domain, and is also homologous to the other  Arabidopsis  fibrillins of the superfamily (50-60% similarity). The isoelectric point (5.4) and hydrophobicity (GRAVY index, −0.142) of  zm PG2 were similar to  Arabidopsis  fibrillin FBN4, which is a core protein of plastoglobuli, although minor amounts of FBN4 are also identified by proteomic studies in chloroplast stroma.  zm PG2 was fused to RFP and expressed in bean and maize protoplasts ( FIG. 2A ). Indeed, in bean and maize green tissue protoplasts, the speckled pattern of  zm PG2-RFP was identical to the speckled pattern of the majority of PSYs. However, in etioplasts  zm PG2-RFP was distributed evenly throughout, suggesting a stromal localization for this fibrillin in dark-grown tissue.  zm PSY2-GFP and  zm PSY3-GFP along with  zm PG2-RFP in green protoplasts was also expressed ( FIG. 2B ). The GFP signal of the PSYs was distributed in speckles together with the RFP signal of  zm PG2. Merging of both signals confirmed co-localization of PSYs with  zm PG2; thus, the speckles are considered to be plastoglobuli. 
         [0064]      zm PSY1-GFP stood alone from the group of other PSYs. In etioplasts,  zm PSY1-GFP was distributed throughout, together with small bright (punctate) spots attached to membranes of red-fluorescent prolamellar bodies, very different in appearance from plastoglobuli in the case of all other PSYs. Homogeneous filling of plastids indicated a soluble, stromal location of  zm PSY1. In light-grown tissue,  zm PSY1-GFP was evenly distributed throughout the chloroplast. In chloroplasts, the association with membranes could not be seen, but should not be excluded due to limitations of image resolution. 
       Example 2 
     Import Experiments Confirmed Peripheral Membrane Binding of Chloroplast-Localized PSYs 
       [0065]    It has been discovered that by using transient expression,  zm PSY2 and  zm PSY3, as well as rice and  Arabidopsis  PSYs, localized to plastoglobuli structures, mostly attached to the surface of thylakoid membranes. Therefore, phytoene synthases were expected to associate with lipids/membranes. To confirm this, the three maize PSY isozymes were tested by chloroplast import assay. In vitro translated  35 S labeled  zm PSY precursor proteins were imported into isolated pea chloroplasts, followed by chloroplast fractionation into three parts: soluble, membrane, and alkaline treated membrane (to purify from peripherally bound proteins) ( FIG. 3 ). 
         [0066]    After import, chloroplasts were treated with thermolysin to remove unimported proteins. The unimported protein, seen in the import samples of  zm PSY2 and  zm PSY3 as an upper band, completely disappeared after thermolysin treatment, and only the imported mature protein remained, being protected by the envelope membrane ( FIG. 3B , arrow). Fractionation of these chloroplasts revealed that  zm PSY2 and  zm PSY3 were peripherally bound to chloroplast membranes. These results are consistent with association of these proteins with plastoglobuli, as was suggested by transient expression in protoplasts. The results of chloroplast import of  zm PSY2 and  zm PSY3 were similar to  os PSYs. In import experiments with pea chloroplasts,  os PSYs are known to be associated with the membrane fraction (although alkaline treatment of the membrane fraction was not performed, the lack of integral membrane helices in the reported structural predictions of  os PSYs suggested that they were likely to be peripherally bound). 
         [0067]    Compared to other PSYs,  zm PSY1 from yellow endosperm maize behaved uniquely in the import experiments, just as we found for  zm PSY1 localization in protoplasts. After thermolysin treatment, the envelope-associated precursor band disappeared as expected, leaving an undigested band of a mature protein ˜42 kDa ( FIG. 3B , arrow). However, a smaller band ˜20 kDa appeared ( FIG. 3B , star). This smaller peptide might be a part of  zm PSY1 that is located within the membrane and therefore is protected from protease treatment. The pattern after the thermolysin treatment looked similar to one of the integral proteins from the outer chloroplast membrane, Toc34. The inter-membrane and periplasm facing domains of Toc34 remained untouched by thermolysin. Fractionation of chloroplasts showed that  zm PSY1 is peripherally associated with membranes as found for the other PSYs. 
         [0068]    Altogether, the results indicate that  zm PSY1 was somehow localized to chloroplasts in two forms. One form of  zm PSY1 is bound to the envelope membrane. A second form of  zm PSY1 is peripherally bound to thylakoids. The peripheral membrane association of  zm PSY1 agrees with the results of transient expression in etiolated protoplasts, where punctate spots of  zm PSY1-GFP were observed around prolamellar bodies. 
       Example 3 
     Single Amino Acid Variants Displayed Altered PSY1 Localization and Transformed Plastid Architecture 
       [0069]    Transient expression and import experiments suggested that almost all investigated PSYs were localized to plastoglobuli, regardless of whether the plants were grown in light or dark.  zm PSY1 was unique and exhibited dual localization to stroma and attached to membranes, as clearly seen in etioplasts ( FIG. 1 ). Next, protein features responsible for differences in localization were identified. PSYs ( FIG. 4 ), and searched for amino acids that are shared by all PSYs except for  zm PSY1 from yellow endosperm maize were aligned. A striking difference was found in the highly conserved coding region, at amino acid residue 257 which was a threonine (T 257 ) in  zm PSY1, as compared to proline (P) in other PSYs. Another position, 168, was occupied by asparagine in  zm PSY1 (N 168 ), in contrast with serine (S) in all other PSYs. BLAST alignment of  zm PSY1 from yellow endosperm maize line B73 used in the experiments, against other PSY sequences available from the NCBI database, showed that T 257  was characteristic for  zm PSY1 from 99% of the 79 maize varieties with yellow endosperm. In addition, T 257  was found in 30% of the 50 maize lines with white endosperm and two species of teosinte, the wild ancestor of maize which has the ancestral characteristic of white endosperm. 70% of white maize varieties had either P 257  or S 257 ; PSYs from all other plants carried proline at the corresponding position. N 168  was found in  zm PSY1 from all maize varieties, as well as in PSY of teosinte and some grass species; PSYs from other plants carried serine at the corresponding position ( FIG. 5 ). 
         [0070]    More detailed analysis of PSY1 sequences from maize and other grasses revealed that indeed, the only difference between  zm PSY1 amino acid sequences from yellow and white endosperm varieties and teosinte, was T/P/S 257 . We also found some sequence differences within the chloroplast transit peptide around positions 52-55 (not shown). Since the transit peptide is processed after chloroplast import and does not affect enzyme activity, it was not included in this study. 
         [0071]    To test if amino acids in positions 168 and 257 are important for localization, a set of variants was generated from  zm PSY1 of B73 (for which the allele in yellow endosperm is N 168 T 257  or “NT”): with one amino acid change of N 168  to serine ( zm PSY1-ST) and an independent or additional change of T 257  to proline or serine ( zm PSY1-NP,  zm PSY1-NS, and  zm PSY1-SP). In addition, sites corresponding to 168 and 257, in  zm PSY2 and  os PSY1 (see Table 1 for explanation of all PSY variants) were mutated. PSY variant cDNAs were fused with GFP and expressed in maize protoplasts from both etiolated ( FIG. 6 ) and green tissues (not shown). With the exception of  zm PSY1-NP, the stromal location of  zm PSY1 GFP-fusions was unchanged. Also, all  zm PSY2 and  os PSY1 variants retained localization phenotype to plastoglobuli (not shown) as seen for the progenitor maize PSY2 or rice PSY1 proteins. 
         [0072]    The striking exception was seen in etiolated protoplasts, where  zm PSY1-NP, naturally found in some white varieties and teosinte, showed a surprising localization phenotype. In plastids of 30% of transformed protoplasts,  zm PSY1-NP-GFP formed unusual spikes, which stretched chloroplasts from inside causing a remarkable morphological change of plastid shape, from round elliptical to diamond with sharp corners where spikes touched the envelope membrane ( FIG. 6  and  FIG. 9 ). In the remaining 70% of protoplasts,  zm PSY1-NP-GFP was localized to stroma, similar to the phenotype exhibited by the progenitor yellow endosperm  zm PSY1. That is, a single residue change in the PSY1 protein altered PSY localization and plastid morphology. Remarkably, the double mutation of  zm PSY1-SP-GFP (where both 168 and 257 sites were mutated) restored stromal localization as exhibited by the progenitor  zm PSY1. The secondary mutation N 168  to S 168  appeared to counteract the effect of the single mutation T 257  to P 257 . Interestingly, when  zm PSY1-NP-GFP was expressed in protoplasts from green seedlings, no fluorescent spikes or drastic morphological change in plastid shape was observed; the phenotype was the same as found with  zm PSY1 ( FIG. 1B ). The dramatic effect of the single residue change was only apparent in non-photosynthetic plastids. 
         [0073]    To exclude the possible effect of the endogenous parent  zm PSY1 on localization pattern of overexpressed  zm PSY1, different PSY-GFP constructs in protoplasts of the y1-8549 maize line which lacks PSY1 were also expressed, and found no difference in localization of proteins to compare to ones in the B73 maize line ( FIG. 10 ). 
         [0074]    The fluorescent spikes observed in  zm PSY1-NP-GFP expression experiments were similar to fibrils seen in carotenoid-rich chromoplasts of  Solanum capsicastrum . In  Solanum , such fibrillar plastoglobuli initiate from globular plastoglobuli. This morphogenic change is observed together with an increase in carotenoid concentration, although it is unknown what triggers fibril formation. Capacity to accumulate large quantities of carotenoids is characteristic of non-photosynthetic plastids. For example, constitutive overexpression of  at PSY in  Arabidopsis  resulted in carotenoid bar-shaped crystals (spikes) formed in non-photosynthetic plastids of roots, while no changes were observed in photosynthetic tissues. Similarly, fibrils in green protoplasts were not observed, which might be explained by alternative mechanisms of carotenoid sequestration in chloroplasts as compared to non-photosynthetic plastids. Thus, the results indicate that  zm PSY1-NP was located in fibrillar plastoglobuli, which initiate from globular plastoglobuli in the presence of high concentrations of carotenoids. The presence of carotenoids in fibrils was supported by the use of Spectral Dye Separation tool in LAS AF software (Leica), applied to the fluorescence intensity spectra of  zm PSYs-GFP constructs expressed in protoplasts prepared from etiolated leaves of  zm PSY1-knockout maize ( FIG. 11 ). The Spectral Dye Separation tool extracted fluorescence of carotenoids from total fluorescence in fibrils (or plastoglobuli, as positive control) of transformed protoplasts, suggesting the presence of carotenoids in those locations. 
         [0075]    If fibrils formed as a consequence of high carotenoid production from overexpressed PSY, then inactivation of  zm PSY1-NP-GFP would be predicted to eliminate fibril formation. To test this, the enzyme was inactivated by mutagenesis of the active site. The choice of the active site was based on structural homology of  zm PSY1 to squalene synthase (SQS), as predicted online by Structure Prediction Meta Server. SQS has a similar catalytic mechanism to PSY and a known crystal structure. The PSY active site and other regions critical for enzyme activity are highly conserved among PSY/SQS family members. Meta Server (http://meta.bioinfo.pl/submit_wizard.pl) gave a significant 3D-Jury score of 211 (&gt;50 is considered significant) regarding structural similarity between PSY and SQS. Predicted structural similarities between PSYs and SQS are presented in  FIG. 4 . Mutagenesis of either of two highly conserved aspartate residues 219 and 223 to glutamate inactivates SQS. Thus, the corresponding aspartate residue 285 to glutamate was mutagenized and  zm PSY1 (Table 1) was inactivated, which was confirmed by testing for functional complementation in  E. coli . When the inactive enzyme  zm PSY1-NP-E-GFP was inactivated in etiolated protoplasts, the plastid morphology was now normal, fibrils no longer formed, and the inactive enzyme showed a stromal localization as found for the active, progenitor enzyme,  zm PSY1 ( FIG. 6 ). 
         [0076]    The Spectral Dye separation tool showed no carotenoid fluorescence signal when protoplasts expressed the inactive enzyme  zm PSY-NP-E as compared to the positive signal obtained from protoplasts expressing the active enzyme  zm PSY-NP ( FIG. 11 ). Thus, it is concluded that increased local carotenoid concentration, causing fibril spikes and plastid morphological change, was due to PSY1 enzyme activity. 
       Example 4 
     Computer Modeling of PSY Structures Provided Insight into Localization Phenotypes of Mutant Enzymes 
       [0077]    It was expected that changes in the localization phenotype of  zm PSY1-NP (compared to  zm PSY1 and  zm PSY1-NP-E) were related to structural changes in the protein. To study the effect of various residues at positions 168 and 257 on structure of  zm PSY1, the computational methods of structural homology modeling and molecular modeling were used. Homology modeling provided initial structural predictions for  zm PSY1 (“NT”),  zm PSY1-NP and  zm PSY1-SP. Selected structural predictions resulting from homology modeling calculations were then subjected to minimization and molecular dynamics techniques to derive final predicted structures. The latter two structural predictions against that of our predicted structure of  zm PSY1 were aligned. The aligned structures of the T 257  and P 257  variants of  zm PSY1 ( FIG. 7A ) clearly showed that the overall structure of the enzyme is preserved, in particular the length and relative positions of alpha helices, with some perturbations in a few of the loop domains. The overall root mean square deviation (RMSD) between the two structures was 3.8 angstroms ( FIG. 7B , red line). Most notable was the large variation in the loop region around residue 184. The loop is located in close proximity to the  159 DELVD 163  region of the enzyme ( FIG. 7A ), which together with  285 DVGED 289  is a conserved sequence among isoprenoid synthases, and forms an active site to bind phosphate groups of a substrate. The deviation between RMSD values of  zm PSY1 and  zm PSY1-NP at this region was noted to be significantly larger than 3.8 angstroms. The difference between  zm PSY1 and  zm PSY1-SP ( FIG. 7B , green line) in the same region, however, was not significant when taking into account the overall average RMSD values difference across the entire protein. This observation suggested that the structure of  zm PSY1-SP was similar to  zm PSY1. The similar structure was consistent with the common stromal localization of these two proteins. 
         [0078]    This modeling predicted that a change of threonine to proline at position 257 will cause remote structural alterations in PSY1 in the loop region around residue 184, where several mutations were shown to affect PSY activity. For example, a change of the amino acid corresponding to A174 to D increased PSY activity in cassava, while mutagenesis of the amino acid corresponding to P190 to L decreased the activity of PSY1 in tomato (all residue numbers are relative to  zm PSY1, and shown in  FIG. 4  and  FIG. 7B  in blue). 
         [0079]    Thus, as a result of the present invention, it has been shown that a single specific amino acid alteration could have functional and/or localization consequences. The change in residue at critical locations such as at position 257 could change protein folding at a location remote and thus either affect activity of the enzyme by altering substrate affinity, or affect interaction with an upstream enzyme that provides the PSY substrate. Indeed, the T 257  to P 257  mutation in the PSY1-GFP fusion caused formation of spikes and altered plastid morphology. A second amino acid change at S 168  (S 168 P 257 ), however, was able to counteract the structural perturbations caused by P 257 , restoring the structure and specific features of the progenitor  zm PSY1. Therefore, PSYs with NT or SP are predicted to be structurally similar whereas NP is predicted to cause a structural perturbation. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 PSY variants used in transient expression experiments. Numbers of amino acids are 
               
               
                 actual numbers in PSY amino acid sequences. Plastid localization is based on the PSY 
               
               
                 GFP-fusion experiments. Abbreviations: zm, maize; os, rice; at, Arabidopsis . 
               
             
          
           
               
                   
                 PSY 
                 Amino 
                   
                 Test of 
                   
               
               
                   
                 variant 
                 acid 
                 Is this a naturally found 
                 activity in 
                 Plastid 
               
               
                   
                 name 
                 variation 
                 variant? 
                 
                   E. coli 
                 
                 localization 
               
               
                   
                   
               
             
          
           
               
                 Maize 
                   zm PSY1 
                 N 168 ; T 257   
                 Yes, yellow endosperm 
                 active 
                 Stroma/spots 
               
               
                   
                   
                   
                 lines including B73, and 
               
               
                   
                   
                   
                 some white endosperm lines 
               
               
                   
                   zm PSY1-NP 
                 N 168 ; P 257   
                 Yes, white endosperm lines 
                 active 
                 fibrils 
               
               
                   
                   zm PSY1-NS 
                 N 168 ; S 257   
                 Yes, white endosperm lines 
                 — 
                 stroma 
               
               
                   
                   zm PSY1-NP-E 
                 N 168 ; P 257 ; 
                 no 
                 inactive 
                 stroma 
               
               
                   
                   
                 E 285   
               
               
                   
                   zm PSY1-SP 
                 S 168 ; P 257   
                 no 
                 — 
                 stroma 
               
               
                   
                   zm PSY1-ST 
                 S 168 ; T 257   
                 no 
                 — 
                 stroma 
               
               
                   
                   zm PSY2 
                 S 168 ; P 257   
                 yes 
                 active 1   
                 plastoglobuli 
               
               
                   
                   zm PSY2-ST 
                 S 168 ; T 257   
                 no 
                 — 
                 plastoglobuli 
               
               
                   
                   zm PSY2-NP 
                 N 168 ; P 257   
                 no 
                 — 
                 plastoglobuli 
               
               
                   
                   zm PSY2-NT 
                 N 168 ; T 257   
                 no 
                 — 
                 plastoglobuli 
               
               
                   
                   zm PSY3 
                 S 176 ; P 265   
                 yes 
                 active 3   
                 plastoglobuli 
               
               
                 Rice 
                   os PSY1 
                 S 179 ; P 268   
                 yes 
                 active 1   
                 plastoglobuli 
               
               
                   
                   os PSY1-ST 
                 S 179 ; T 268   
                 no 
                 — 
                 plastoglobuli 
               
               
                   
                   os PSY2 
                 S 163 ; P 253   
                 yes 
                 active 1   
                 plastoglobuli 
               
               
                 
                   Arabidopsis 
                 
                   at PSY 
                 S 181 ; P 270   
                 yes 
                 active 2   
                 plastoglobuli 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Plasmids and primers used for cloning 
               
             
          
           
               
                   
                 SEQ ID 
                   
                 Restriction sites  
               
               
                 Plasmid 
                 NO: 
                 Primers used for cloning 
                 used for cloning 
               
               
                   
               
               
                 pTnT- zm PSY1 
                  51 
                 F 5′ TCTCGAGATGGCCATCATACTCGTACGAG 
                 XhoI/XbaI 
               
               
                   
                  52 
                 R 5′ ATCTAGACTAGGTCTGGCCATTTCTCAATG 
                   
               
               
                   
               
               
                 pTnT- zm PSY2 
                  53 
                 F 5′ ACTCGAGAATGGCTGCGGGCTCGTCCG 
                 XhoI/NotI 
               
               
                   
                  54 
                 R 5′ GAT GTG ATC TAC GGA TGG TTC AT 
                   
               
               
                   
               
               
                 pTnT- zm PSY3 
                  55 
                 F 5′ AAGAATTCGCCACCATGATGTCTACGAGC 
                 EcoRI + Kozak 
               
               
                   
                  56 
                 R 5′ AAGCGGCCGCCTATGTTAGGGTGGAATAGC 
                 sequence, 
               
               
                   
                   
                   
                 NotI 
               
               
                   
               
               
                 pUC35S- zm PSY1-sGFP-Nos 
                  57 
                 F 5′ TTCTAGAATGGCCATCATACTCGTACGAG 
                 XbaI/BamHI 
               
               
                   
                  58 
                 R 5′ AGGATCCGGTCTGGCCATTTCTCAATGAA 
                   
               
               
                   
               
               
                 pUC35S- zm PSY2-sGFP-Nos 
                  59 
                 F 5′ ATCTAGAATGGCTGCGGGCTCGTCC 
                 XbaI/BamHI 
               
               
                   
                  60 
                 R 5′ AGGATCCTGGTGCAACCGCAGCCCTTGCA 
                   
               
               
                   
               
               
                 pUC35S- zm PSY3-sGFP-Nos 
                  61 
                 F 5′ ATCTCTAGAATGATGTCTACGAGCCGCGCGGTGAAGTCG 
                 XbaI/BamHI 
               
               
                   
                  62 
                 R 5′ ATCGGATCCTGTTAGGGTGGAATAGCGTCTCCGGCTC 
                   
               
               
                   
               
               
                 pUC35S- os PSY1-sGFP-Nos 
                  63 
                 F 5′ ATCTAGAATGGCCCATCACGCTCCTAC 
                 XbaI/BgII 
               
               
                   
                  64 
                 R 5′ A AGATCT CTT CTG GCT ATT TCT CAG TGA G 
                   
               
               
                   
               
               
                 pUC35S- os PSY2-sGFP-Nos 
                  65 
                 F S′ AAC TAG TTC CAC ACG AAC ACA CAA CCC CAA 
                 SpeI/BgII 
               
               
                   
                  66 
                 R 5′ AAGATCTTGATGCAACTGCCGCTCTTGCATA 
                   
               
               
                   
               
               
                 pUC35S-LHCP-sGFP-Nos 
                  67 
                 F 5′ ATCTCTAGAATGGCCGCTTCATCC 
                 XbaI/BamHI 
               
               
                   
                  68 
                 R 5′ ATCGGATCCCTTTCCGGGAACAAAGTTGGTAGC 
                   
               
               
                   
               
               
                 pSAT- at PSY-RFP 
                  69 
                 F 5′ ATCGAATTCATGTCTTCTTCTGTAGCAGTG 
                 EcoRI/BamHI 
               
               
                   
                  70 
                 R 5′ ATTGGATCCGTATCGATAGTCTTGAACTTG 
                   
               
               
                   
               
               
                 pSAT- zm PG2-RFP 
                  71 
                 F 5′ ATCGAATTCATGGCMCCTCCGCGTTCCTCAACG 
                 EcoRI/BcII 
               
               
                   
                  72 
                 R 5′ ATCTGATCAGGTATAGAAGAGTACTTCCC 
                   
               
               
                   
               
               
                 pUC35S- zm PSY1-NP-sGFP-Nos 
                  73 
                 F 5′ CCTGTGATGGGCATCGCACCCGAGTCTAAAG 
                 — 
               
               
                   
                  74 
                 R 5′ CTTTAGACTCGGGTGCGATGCCCATCACAGG 
                   
               
               
                   
               
               
                 pUC35S- zm PSY1-SP-sGFP-Nos 
                  75 
                 F 5′ CCTGTGATGGGCATCGCACCCGAGTCTAAAG 
                 — 
               
               
                   
                  76 
                 R 5′ CTTTAGACTCGGGTGCGATGCCCATCACAGG 
                   
               
               
                   
                  77 
                 F 5′ GATGGGCCAAACGCCAGCTACATTACACCAACAG 
                   
               
               
                   
                  78 
                 R 5′ CTGTTGGTGTAATGTAGCTSGCGTTTGGCCCATC 
                   
               
               
                   
               
               
                 pUC35S- zm PSY1-NS-sGFP-Nos 
                  79 
                 F 5′ CCTGTGATGGGCATCGCATCCGAGTCTAAAG 
                 — 
               
               
                   
                  80 
                 R 5′ CTTTAGACTCGGATGCGATGCCCATCACAGG 
                   
               
               
                   
               
               
                 pUC35S- zm PSY1-ST-sGFP-Nos 
                  81 
                 F 5′ GATGGGCCAAACGCCAGCTACATTACACCAACAG 
                 — 
               
               
                   
                  82 
                 R 5′ CTGTTGGTGTAATMAGOGGCGTTTGGCCCATC 
                   
               
               
                   
               
               
                 pUC35S- zm PSY2-ST-sGFP-Nos 
                  83 
                 F 5′ CCTGTCATGGGCATCGCTACCGACTCCAA 
                 — 
               
               
                   
                  84 
                 R 5′ TTGGAGTCGGTAGCGATGCCCATGACAGG 
                   
               
               
                   
               
               
                 pUC35S- zm PSY2-NT-sGFP-Nos 
                  85 
                 F 5′ CCTGTCATGGGCATCGCTACCGACTCCAA 
                 — 
               
               
                   
                  86 
                 R 5′ TTGGAGTCGGTAGCGATGCCCATGACAGG 
                   
               
               
                   
                  87 
                 F 5′ GACGGTCCCAACGCGAACTACATCACGCCGAC 
                   
               
               
                   
                  88 
                 R 5′ GTCGGCGTGATGTAGTTCGCGTTGGGACCGTC 
                   
               
               
                   
               
               
                 pSAT- zm PSY2-NP-RFP 
                  89 
                 F 5′ GACGGTCCCAACGCGAACTACATCACGCCGAC 
                 — 
               
               
                   
                  90 
                 R 5′ GTCGGCGTGATGTAGTTCGCGTTGGGACCGTC 
                   
               
               
                   
               
               
                 pUC35S- os PSY1-NT-sGFP-Nos 
                  91 
                 F 5′ GTTCCTGTGATGGGTATTGCAACCGAGTCGAAG 
                 — 
               
               
                   
                  92 
                 R 5′ 5′ CTTCGACTCGGTTGCAATACCCATCACAGGAAC 
                   
               
               
                   
               
               
                 pBS- zm PSY1-NP 
                  93 
                 F 5′ CCTGTGATGGGCATCGCACCCGAGTCTAAAG 
                 — 
               
               
                   
                  94 
                 R 5′ CTTTAGACTCGGGTGCGATGCCCATCACAGG 
                   
               
               
                   
               
               
                 pBS- zm PSY1-D 285 E 
                  95 
                 F 5′ CGAACATACTCCGGGAGGTTGGAGAGGATGCTA 
                 — 
               
               
                   
                  96 
                 R 5′ TAGCATCCTCTCCAACCTCCCGGAGTATGTTCG 
                   
               
               
                   
               
               
                 pUC35S- zm PSY1-NP-E-sGFP-Nos 
                  97 
                 F 5′ CGAACATACTCCGGGAGGTTGGAGAGGATGCTA 
                 — 
               
               
                   
                  98 
                 R 5′ TAGCATCCTCTCCAACCTCCCGGAGTATGTTCG 
                   
               
               
                   
                  99 
                 F 5′ CCTGTGATGGGCATCGCACCCGAGTCTAAAG 
                   
               
               
                   
                 100 
                 R 5′ CTTTAGACTCGGGTGCGATGCCCATCACAGG