Lettuce Chloroplast Research Articles

Oral booster vaccine antigen-Expression of full-length native SARS-CoV-2 spike protein in lettuce chloroplasts.

Oral booster vaccine antigen-Expression of full-length native SARS-CoV-2 spike protein in lettuce chloroplasts.

Production of tetravalent dengue virus envelope protein domain III based antigens in lettuce chloroplasts and immunologic analysis for future oral vaccine development.

SummaryDengue fever is a mosquito (Aedes aegypti) ‐transmitted viral disease that is endemic in more than 125 countries around the world. There are four serotypes of the dengue virus (DENV 1‐4) and a safe and effective dengue vaccine must provide protection against all four serotypes. To date, the first vaccine, Dengvaxia (CYD‐TDV), is available after many decades’ efforts, but only has moderate efficacy. More effective and affordable vaccines are hence required. Plants offer promising vaccine production platforms and food crops offer additional advantages for the production of edible human and animal vaccines, thus eliminating the need for expensive fermentation, purification, cold storage and sterile delivery. Oral vaccines can elicit humoural and cellular immunity via both the mucosal and humoral immune systems. Here, we report the production of tetravalent EDIII antigen (EDIII‐1‐4) in stably transformed lettuce chloroplasts. Transplastomic EDIII‐1‐4‐expressing lettuce lines were obtained and homoplasmy was verified by Southern blot analysis. Expression of EDIII‐1‐4 antigens was demonstrated by immunoblotting, with the EDIII‐1‐4 antigen accumulating to 3.45% of the total protein content. Immunological assays in rabbits showed immunogenicity of EDIII‐1‐4. Our in vitro gastrointestinal digestion analysis revealed that EDIII‐1‐4 antigens are well protected when passing through the oral and gastric digestion phases but underwent degradation during the intestinal phase. Our results demonstrate that lettuce chloroplast engineering is a promising approach for future production of an affordable oral dengue vaccine.

Human granulocyte colony-stimulating factor (hG-CSF) expression in plastids of Lactuca sativa.

Human granulocyte colony-stimulating factor (hG-CSF) can serve as valuable biopharmaceutical for research and treatment of the human blood cancer. Transplastomic plants have been emerged as a new and high potential candidate for production of recombinant biopharmaceutical proteins in comparison with transgenic plants due to extremely high level expression, biosafety and many other advantages. hG-CSF gene was cloned into pCL vector between prrn16S promoter and TpsbA terminator. The recombinant vector was coated on nanogold particles and transformed to lettuce chloroplasts through biolistic method. Callogenesis and regeneration of cotyledonary explants were obtained by Murashige and Skoog media containing 6-benzylaminopurine and 1-naphthaleneacetic acid hormones. The presence of hG-CSF gene in plastome was studied with four specific PCR primers and expression by Western immunoblotting. hG-CSF gene cloning was confirmed by digestion and sequencing. Transplastomic lettuce lines were regenerated and subjected to molecular analysis. The presence of hG-CSF in plastome was confirmed by PCR using specific primers designed from the plastid genome. Western immunoblotting of extracted protein from transplastomic plants showed a 20-kDa band, which verified the expression of recombinant protein in lettuce chloroplasts. This study is the first report that successfully express hG-CSF gene in lettuce chloroplast. The lettuce plastome can provide a cheap and safe expression platform for producing valuable biopharmaceuticals for research and treatment.

Expression of dengue-3 premembrane and envelope polyprotein in lettuce chloroplasts.

Dengue is an acute febrile viral disease with >100 million infections occurring each year and more than half of the world population is at risk. Global resurgence of dengue in many urban centers of the tropics is a major concern. Therefore, development of a successful vaccine is urgently needed that is economical and provide long-lasting protection from dengue virus infections. In this manuscript, we report expression of dengue-3 serotype polyprotein (prM/E) consisting of part of capsid, complete premembrane (prM) and truncated envelope (E) protein in an edible crop lettuce. The dengue sequence was controlled by endogenous Lactuca sativa psbA regulatory elements. PCR and Southern blot analysis confirmed transgene integration into the lettuce chloroplast genome via homologous recombination at the trnI/trnA intergenic spacer region. Western blot analysis showed expression of polyprotein prM/E in different forms as monomers (~65 kDa) or possibly heterodimers (~130 kDa) or multimers. Multimers were solubilized into monomers using guanidine hydrochloride. Transplastomic lettuce plants expressing dengue prM/E vaccine antigens grew normally and transgenes were inherited in the T1 progeny without any segregation. Transmission electron microscopy showed the presence of virus-like particles of ~20 nm diameter in chloroplast extracts of transplastomic lettuce expressing prM/E proteins, but not in untransformed plants. The prM/E antigens expressed in lettuce chloroplasts should offer a potential source for investigating an oral Dengue vaccine.

Expression of cholera toxin B-proinsulin fusion protein in lettuce and tobacco chloroplasts--oral administration protects against development of insulitis in non-obese diabetic mice.

Lettuce and tobacco chloroplast transgenic lines expressing the cholera toxin B subunit-human proinsulin (CTB-Pins) fusion protein were generated. CTB-Pins accumulated up to ~16% of total soluble protein (TSP) in tobacco and up to ~2.5% of TSP in lettuce. Eight milligrams of powdered tobacco leaf material expressing CTB-Pins or, as negative controls, CTB-green fluorescent protein (CTB-GFP) or interferon-GFP (IFN-GFP), or untransformed leaf, were administered orally, each week for 7 weeks, to 5-week-old female non-obese diabetic (NOD) mice. The pancreas of CTB-Pins-treated mice showed decreased infiltration of cells characteristic of lymphocytes (insulitis); insulin-producing beta-cells in the pancreatic islets of CTB-Pins-treated mice were significantly preserved, with lower blood or urine glucose levels, by contrast with the few beta-cells remaining in the pancreatic islets of the negative controls. Increased expression of immunosuppressive cytokines, such as interleukin-4 and interleukin-10 (IL-4 and IL-10), was observed in the pancreas of CTB-Pins-treated NOD mice. Serum levels of immunoglobulin G1 (IgG1), but not IgG2a, were elevated in CTB-Pins-treated mice. Taken together, T-helper 2 (Th2) lymphocyte-mediated oral tolerance is a likely mechanism for the prevention of pancreatic insulitis and the preservation of insulin-producing beta-cells. This is the first report of expression of a therapeutic protein in transgenic chloroplasts of an edible crop. Transplastomic lettuce plants expressing CTB-Pins grew normally and transgenes were maternally inherited in T(1) progeny. This opens up the possibility for the low-cost production and delivery of human therapeutic proteins, and a strategy for the treatment of various other autoimmune diseases.

Efficient and stable transformation of Lactuca sativa L. cv. Cisco (lettuce) plastids.

Transgenic plastids offer unique advantages in plant biotechnology, including high-level foreign protein expression. However, broad application of plastid genome engineering in biotechnology has been largely hampered by the lack of plastid transformation systems for major crops. Here we describe the development of a plastid transformation system for lettuce, Lactuca sativa L. cv. Cisco. The transforming DNA carries a spectinomycin-resistance gene (aadA) under the control of lettuce chloroplast regulatory expression elements, flanked by two adjacent lettuce plastid genome sequences allowing its targeted insertion between the rbcL and accD genes. On average, we obtained 1 transplastomic lettuce plant per bombardment. We show that lettuce leaf chloroplasts can express transgene-encoded GFP to approximately 36% of the total soluble protein. All transplastomic T0 plants were fertile and the T1 progeny uniformly showed stability of the transgene in the chloroplast genome. This system will open up new possibilities for the efficient production of edible vaccines, pharmaceuticals, and antibodies in plants.

Preparation of Chloroplast DNA from Barley and Lettuce and Comparison of Restriction Fragments

Chloroplast DNA from three barley cultivars and from one lettuce cultivar was prepared from chloroplasts isolated by Conventional differential centrifugation. Barley chloroplast DNA size was sensibly lower (130 kpb) than lettuce chloroplast DNA (150 kpb). Chloroplast DNAs from the three barley cultivars showed similar restriction fragment patterns after digestion with: BamHI, EcoRI or HindIII. The lettuce chloroplast DNA restriction pattern was very different from the barley chloroplast DNA restriction pattern.

Chloroplast DNA from lettuce and Barnadesia (Asteraceae): structure, gene localization, and characterization of a large inversion

We have cloned into plasmids 17 of 18 lettuce chloroplast DNA SacI fragments covering 96% of the genome. The cloned fragments were used to construct cleavage maps for 10 restriction enzymes for the chloroplast genomes of lettuce (Lactuca sativa) and Barnadesia caryophylla, two distantly related species in the sunflower family (Asteraceae). Both genomes are approximately 151 kb in size and contain a 25 kb inverted repeat. We also mapped the position and orientation of 37 chloroplast DNA genes. The mapping studies reveal that chloroplast DNAs of lettuce and Barnadesia differ by a 22 kb inversion in the large single copy region. Barnadesia has retained the primitive land plant genome arrangement, while the inversion has occurred in a lettuce lineage. The endpoints of the derived lettuce inversion were located by comparison to the well-characterized spinach and tobacco genomes. Both endpoints are located in intergenic spacers within tRNA gene clusters; one cluster being located downstream from the atpA gene and the other upstream from the psbD gene. The endpoint near the atpA gene is very close to one endpoint of a 20 kb inversion in wheat (Howe et al. 1983; Quigley and Weil 1985). Comparison of the restriction site maps gives an estimated sequence divergence of 3.7% for the lettuce and Barnadesia genomes. This value is relatively low compared to previous estimates for other angiosperm groups, suggesting a high degree of sequence conservation in the Asteraceae.

Reconstitution of photophosphorylation with coupling factor 1 ATPases from the thermophilic bacterium PS3 and lettuce chloroplasts

(1) EDTA-resolved particles (partially depleted of CF 1) from lettuce chloroplast thylakoids were reconstituted with CF 1 from lettuce and with TF 1 from the thermophilic bacterium PS3. The binding affinity of both F 1 proteins for the membranal CF 0 is similar. (2) The phosphorylation activities of the EDTA-resolved particles after reconstitution with either the tentoxin-sensitive CF 1 or the tentoxin-resistant TF 1 are equally inhibited by tentoxin. The same degree of reconstitution of photophosphorylation is observed either with the native TF 1, the DCCD-modified TF 1 or the DCCD-modified CF 1. Particles reconstituted with native CF 1 always catalyzed a higher rate of ATP formation. (3) NaBr-resolved particles (CF 1-depleted) bind TF 1 with the reconstitution of the light-dependent H + uptake. However, no significant enhancement of ATP formation accompanies the binding of TF 1 to the membranal CF 0. (4) These findings suggest a close relationship between the F 0-F 1 complex of the prokaryotic thermophilic bacterium PS3 and that from lettuce, a eukaryotic organism. It is also apparent that the rebinding of added TF 1 or CF 1 to CF 1-depleted particles reforms ATP synthetase complexes that are not fully catalytically functional. In particular, the TF 1 incorporated into the membrane appears to have only a structural role of sealing the membrane and preventing protons from leaking out but does not participate in the catalytic process itself. The endogenous CF 1 that is present in the native undissociated ATP synthetase complex and still remains in these CF 1-partially depleted membranes is the catalytically active enzyme species.

Inverted repeats in chloroplast DNA from higher plants

The circular chloroplast DNAs from spinach, lettuce, and corn plants have been examined by electron microscopy and shown to contain a large sequence repeated one time in reverse polarity. The inverted sequence in spinach and lettuce chloroplast DNA has been found to be 24,400 base pairs long. The inverted sequence in the corn chloroplast DNA is 22,500 base pairs long. Denaturation mapping studies have shown that the structure of the inverted sequence is highly conserved in these three plants. Pea chloroplast DNA does not contain an inverted repeat. All of the circular dimers of pea chloroplast DNA are found to be in a head-to-tail confirmation. Circular dimers of spinach and lettuce were also found to have head-to-tail conformation. However, approximately 70-80% of the circular dimers in preparations of lettuce and spinach chloroplast DNA were found to be in a head-to-head conformation. We propose that the head-to-head circular dimers are formed by a recombination event between two circular monomers in the inverted sequence.

Tentoxin An uncompetitive inhibitor of lettuce chloroplast coupling factor 1

The interaction of tentoxin [cyclo (- l-leucyl -N- methyl-(Z)-dehydrophenyl-analyl-glycyl-N- methyl- l-alanyl-) ] with solubilized lettuce chloroplast coupling factor 1 was characterized by direct binding studies, measurement of the time course of ATPase inhibition, and steady-state enzyme kinetics. Neither substrates, products or Ca 2+ competed with the tentoxin binding site, nor did they induce any large change in tentoxin affinity. The inhibition of lettuce chloroplast coupling factor 1 ATPase was found to be the time dependent, and at equilibrium the affinities estimated by equilibrium ultrafiltration and enzyme inhibition were similar (1.8 · 10 8M −1). The steady-state kinetics best fit an uncompetitive pattern suggesting that the inhibited steps follow an irreversible step occurring after ATP binding.

Isolation of a chloroplast N,N'-dicyclohexylcarbodiimide-binding proteolipid, active in proton translocation.

The N,N'-dicyclohexylcarbodiimide-binding proteolipid from lettuce chloroplast membranes has been purified by a novel, rapid technique involving I-butanol extraction and ether precipitation. Reconstitution of this proteolipid into liposomes composed of chloroplast lipids and subsequent incorporation of bacteriorhodopsin resulted in the formation of liposomes exhibiting a light-dependent accumulation of protons. This accumulation was significantly enhanced upon addition of N,N'-dicyclohexylcarbodiimide at concentrations similar to those that inhibit chloroplast adenosinetriphosphatase activity. Radioactively labeled N,N'-dicyclohexylcarbodiimide was found to be incorporated essentially into the proteolipid of the reconstituted liposomes. These results suggest that the functional unit responsible for proton channeling in the chloroplast membrane has been isolated and reconstituted in the native state.

Inorganic sulfate and selenate as energy transfer inhibitors of photophosphorylation

1. Inorganic sulfate or selenate inhibits photophosphorylation in lettuce chloroplast fragments. The inhibition was found to be competitive with inorganic phosphate in all systems tested (photophosphorylation, post-illumination ATP formation, acid-base phosphorylation) with a K i for sulfate of around 2 mM. 2. Sulfate or selenate inhibited only the fraction of electron transport stimulated by coupled phosphorylation. The basal electron transport rate was nearly unaffected. Sulfate inhibited also the arsenate-stimulated electron transport rate, but not the stimulation caused by other uncouplers. 3. Sulfate and selenate inhibit light-triggered ATP-P i exchange, but not the light-triggered Mg-ATPase. The concentrations needed for 50% inhibition are higher than those needed to inhibit phosphorylation. 4. The results indicate that sulfate or selenate act as energy transfer inhibitors of photophosphorylation, competing with P i at its binding site.

OSCILLATIONS OF THE TRIGGERED LUMINESCENCES OF ISOLATED CHLOROPLASTS PREILLUMINATED BY SHORT FLASHES*

Abstract— Four types of triggered luminescence of isolated lettuce chloroplast (HCl‐induced, methanol‐induced, sodium benzoate‐induced and T‐jump‐induced) were examined after preillumination by a series (from 1 to 10) of short flashes. Oscillations were observed in the luminescence peaks, with a period of four flashes. These oscillations had maxima after the second and the sixth flash, similar to those of delayed light emission. The maxima were shifted forward two flashes by 50 μM hydroxylamine, as in oxygen evolution, and were abolished by 5 μM DCMU, as for delayed light. These results may show that the mechanism of triggered luminescence is influenced directly by the oxidation states (S1) on the donor side of photo‐system II.

Ribosomal ribonucleic acids of chloroplastic and mitochondrial preparations.

RNA prepared from fractions of chloroplasts and mitochondria sedimented at rates characteristic of ribosomal RNA. A predominance of the 18S species was frequently observed in preparations from chloroplasts from romaine lettuce and endive. The usual distribution, a preponderance of the 28S species, was observed in studies on tomato and spinach chloroplasts and mitochondria from mushroom and cauliflower. Comparisons of the base composition of RNA from organelles with their cytoplasmic ribosomal counterparts revealed that the 18S component from romaine lettuce chloroplast was different. A marginally significant difference was observed in the 28S particle from mushroom mitochondria preparations whereas distinct differences, reflected in all the bases, were seen when the 18S component of cauliflower mitochondria preparations was compared with cytoplasmic RNA.

The biosynthesis of long-chain fatty acids by lettuce chloroplast preparations

1. 1. It is demonstrated that the major site of biosynthesis of saturated fatty acids from C 10 to C 18 and of oleic acid in leaves is the chloroplast. 2. 2. The co-factors required in the presence of light are ATP, CoA, Mg 2+, CO 2 and inorganic phosphate. 3. 3. In the dark greatly diminished synthesis occurs despite the presence of a full complement of cofactors. 4. 4. As in photosynthetic phosphorylation the synthesis of fatty acids is inhibited by NH 3 and 3( p-chlorophenyl) 1,1-dimethyl urea but not by dinitrophenol. 5. 5. Under anaerobic conditions the biosynthesis of oleic acid drops markedly but that of the saturated acids is relatively unaffected. 6. 6. Isolated chloroplasts are less effective in utilising the C 8, C 10, C 12 and C 14 saturated fatty acids for the biosynthesis of oleic acid than is the intact leaf. Both malonic and acetic acids are, however, readily utilised.

The biosynthesis of long-chain fatty acids by lettuce chloroplast preparations

1. 1. It is demonstrated that the major site of biosynthesis of saturated fatty acids from C 10 to C 18 and of oleic acid in leaves is the chloroplast. 2. 2. The co-factors required in the presence of light are ATP, CoA, Mg 2+, CO 2 and inorganic phosphate. 3. 3. In the dark greatly diminished synthesis occurs despite the presence of a full complement of cofactors. 4. 4. As in photosynthetic phosphorylation the synthesis of fatty acids is inhibited by NH 3 and 3( p-chlorophenyl) 1,1-dimethyl urea but not by dinitrophenol. 5. 5. Under anaerobic conditions the biosynthesis of oleic acid drops markedly but that of the saturated acids is relatively unaffected. 6. 6. Isolated chloroplasts are less effective in utilising the C 8, C 10, C 12 and C 14 saturated fatty acids for the biosynthesis of oleic acid than is the intact leaf. Both malonic and acetic acids are, however, readily utilised.