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Chloroplast Genetic Engineering

Authors:
Henry Daniell at University of Pennsylvania
Henry Daniell
Paul Cohill at University of Central Florida
Paul Cohill
Shashi Kumar at International Centre for Genetic Engineering and Biotechnology
Shashi Kumar
Nathalie Dufourmantel
Nathalie Dufourmantel
Nathalie Dufourmantel
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Abstract

Chloroplasts are ideal hosts for the expression of transgenes. Once integrated via homologous recombination, these transgenes express large amounts of protein (up to 46% of total leaf protein) due to the high copy number of the chloroplast genome in each plant cell. Foreign proteins that are toxic when present in the cytosol, such as vaccine antigens, trehalose and xylanase, are non-toxic when sequestered within transgenic plastids. Because transgenes are maternally inherited in most crops, there is little danger of cross-polination with wild-type plants. By using chloroplast DNA sequences that flank transgenes, higher plants have efficiently and stably integrated transgenes imbuing important agronomic traits, including herbicide, insect and disease resistance, drought and salt tolerance, and phytoremediation. More recently, highly efficient, soybean, carrot and cotton plastid transformation have been accomplished via somatic embryogenesis using species-specific vectors. Chloroplast transgenic carrot plants withstand salt concentrations that only halophytes could tolerate. Previously an exclusively mitochondrial-encoded trait, cytoplasmic male sterility is now possible through β-ketothiolase expression via the chloroplast genome. This is a valuable tool towards transgene containment, in addition to the maternal inheritance of transgenes integrated into the chloroplast genome, in most crops. Crops such as tobacco have expressed transgenes for a variety of biopharmaceuticals, vaccines and biomaterials. Due to the high biomass of tobacco plants (~40 mtons/acre), large amounts of vaccines preventing anthrax, plague, tetanus and cholera, and pharmaceuticals like human somatotropin, serum albumin, interferons and insulin-like growth factor have been produced in transgenic chloroplasts. The chloroplast also contains machinery that allows for correct folding and disulfide bond formation, resulting in fully functional human blood proteins or vaccine antigens. Additionally, expression of the Rubisco small subunit gene (RbcS) via the chloroplast genome restored normal photosynthetic activity in a nuclear RbcS antisense line, a goal that has been elusive for decades. Multigene operons engineered into the chloroplast genome do not require processing of polycistrons to monocistrons for efficient translation. Secondary structures formed by intergenic spacer regions in bacterial operons are efficiently recognized by the chloroplast processing machinery; when such processing occurs, 3’ UTRs are not required for transcript stability. Extension of chloroplast genetic engineering technology to other useful crops will depend on the availability of the plastid genome sequences and the ability to regenerate transgenic events and advance them towards homoplasmy. In addition to biotechnology applications, plastid transformation system has been extensively used to study chloroplast biochemistry and molecular biology.
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© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 31
Biotechnol. J. 2006, 1, 26–33 www.biotechnology-journal.com
The Daniell laboratory has pioneered and advanced the
concept of chloroplast genetic engineering. Plant genetic
engineering research was revolutionized with the accu-
mulation of Bacillus thuringiensis (Bt) cry2Aa2 protein at
46.1% in transgenic chloroplasts [1]. It was not only the
highest accumulation of protein in transgenic plants but
a complete bacterial operon was successfully expressed,
for the first time, resulting in the formation of stable
cry2Aa2 crystals. Transgenes express large amounts of
foreign protein due to the high copy number of the chloro-
plast genome in each plant cell—up to 10,000 copies of
transgene per cell [2–8]. Transgenes are integrated into
the spacer regions of the chloroplast genome by homolo-
gous recombination of flanking sequences. This allows
site-specific integration and eliminates concerns of posi-
tion effect, frequently observed in nuclear transgenic
plants. As a result, it is not necessary to screen large num-
bers of putative transgenic lines to choose for high-level
expression of transgenes. In contrast, all chloroplast trans-
genic lines express similar levels of foreign proteins, with-
in the range of physiological variations [9]. In addition,
site-specific integration by homologous recombination
eliminates introduction of vector sequences, which is
often a concern in nuclear transformation achieved by
non-homologous recombination. Yet another advantage
is the lack of transgene silencing in chloroplast transgenic
plants, which is a serious concern in nuclear transforma-
tion. There is no gene silencing in chloroplast transgenic
lines at the transcriptional levels despite accumulation of
transcripts 169-fold higher than nuclear transgenic plants
[10, 11]. Similarly, there is no post-transcriptional gene
silencing despite accumulation of foreign proteins up to
47% of the total plant protein in cp transgenic lines [1].
In most angiosperms, plastids are maternally inherit-
ed, which minimizes the concern of outcrossing of trans-
genes [12, 13] and reduces the potential toxicity of trans-
genic pollen to non-target insects [1]. Maternal inheri-
tance of transgenes offers containment because of lack of
gene flow through pollen [13] and this has been demon-
strated in different plant species [7, 12, 14]. The Daniell
laboratory developed a chloroplast genome derived cyto-
plasmic male sterility system in which the tapetal layers
are destroyed in developing pollen, making transgenic
plants male sterile without affecting other metabolic func-
tions [15]. This provides yet another strategy for trans-
gene containment. Chloroplast transgenic carrot plants
have been engineered to withstand salt concentrations
only halophytes could tolerate [16]. The Daniell laboratory
has conferred several other plant traits, including herbi-
cide [12], insect [17], and disease [18] resistance, drought
[10], phytoremediation [19] and production of biopolymers
[20].
The chloroplast is an ideal cellular location to express
and accumulate certain proteins or their biosynthetic
products that would otherwise be harmful to the plant if
they were expressed in the cytoplasm. This has been
demonstrated by the non-toxic effect of cholera toxin B
subunit (CTB), a candidate oral subunit vaccine for
cholera, when it was accumulated in large quantities
within transgenic plastids; in contrast, even very small
quantities of CTB were toxic when expressed in the cyto-
plasm [9, 21]. Also, trehalose, which is a pharmaceutical
preservative, was very toxic when it was accumulated in
the cytosol but was non-toxic when it was compartmen-
talized within plastids [10].
Oral delivery of vaccine antigens has been shown to
yield high systemic IgG titers and high mucosal IgA
titers, enabling the immune system to fight germs at their
portals of entry. Therefore, the Daniell laboratory has
demonstrated expression and assembly of several vac-
cine antigens, including the cholera toxin B subunit (CTB)
[9], the F1~V fusion antigen for plague [6]; the 2L21 pep-
tide from the canine parvovirus (CPV) [21], the anthrax
protective antigen (PA) [22], and the NS3 protein as a vac-
cine antigen for hepatitis C [6]. Cytotoxicity measure-
Henry Daniell: Chloroplast genetic engineering
Henry Daniell is Pegasus Professor and
Trustee Chair at the University of Cen-
tral Florida (UCF). He is an elected
member of the Italian Academy of Sci-
ences. Daniell is the technical founder
of Chlorogen, Inc. (which received mul-
ti-million dollar investment) and an
inventor on more than 95 patents
(awarded or in prosecution) in the US
and abroad. Daniell has served as a
consultant to the United Nations on biotechnology for many years and
to major biotechnology companies and appears often on television
shows or newspaper articles around the world. Prof. Daniell also
served on numerous panels on Biodefense and Bioterrorism Risk
Assessment. Daniell is recognized for his pioneering inventions on
chloroplast genetic engineering. He developed and advanced this con-
cept, which overcomes concerns of transgene containment. He used
this concept to confer plant traits, including herbicide, insect, disease
resistance, drought/salt tolerance or phytoremediation. Furthermore,
this concept has been used in his laboratory to express several thera-
peutic proteins, including vaccine antigens and biopharmaceuticals.
Daniell has published over 150 articles in premier scientific journals
(cited over 1500 times).
026_BIOT_SeniorEditors.qxd 22.12.2005 7:34 Uhr Seite 31
Biotechnology
Journal
Biotechnol. J. 2006, 1, 26–33
32 © 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ments in macrophage lysis assays showed that chloro-
plast-derived anthrax protective antigen (PA) was equal
in potency to PA produced in B. anthracis. Subcutaneous
immunization of mice with partially purified chloroplast-
derived or B. anthracis-derived PA with adjuvant both
yielded IgG titers up to 1:320 000, and both groups of mice
survived (100%) challenge with lethal doses of toxin. It
was reported that an average yield of about 150 mg of PA
per plant should produce 360 million doses of a purified
vaccine free of the bacterial toxins EF and LF, from one
acre of land [23]. However, these examples only demon-
strate the use of single vaccine antigens derived from
chloroplasts. Recent transcript analyses conducted on
chloroplast transgenic lines showed that the engineered
multigenic operons were transcribed mostly as poly-
cistrons and were efficiently translated, demonstrating
that transcripts need not be monocistronic to be translat-
ed [24], thus facilitating the development of multivalent
vaccines.
In addition to its use for the hyper-expression of vac-
cine antigens, the tobacco chloroplast has been used in
the Daniell laboratory for production of valuable therapeu-
tic proteins, such as human elastin-derived polymers for
various biomedical applications [25], human serum albu-
min [26], magainin, a broad spectrum topical agent, sys-
temic antibiotic, wound healing stimulant and a potential
anticancer agent [18], interferon and insulin-like growth
factor [3, 6]. These examples show that chloroplast also
contains the machinery that allows for correct folding and
disulfide bond formation, resulting in fully functional pro-
teins. Finally, the transformation of non-green tissue plas-
tids like cotton [14] and carrot [16] were recently achieved
[6], further facilitating oral delivery of therapeutic pro-
teins. To advance the concept of oral delivery of thera-
peutic proteins, an antibiotic free cp transformation sys-
tem has been developed [27]. In order to advance chloro-
plast genetic engineering to other crops, ten new crop
chloroplast genomes have been sequenced recently,
including soybean [28].
References
[1] DeCosa, B., Moar, W., Lee, S.B., Miller, M., Daniell, H., Overexpres-
sion of the Bt Cry2Aa2 operon in chloroplasts leads to formation of
insecticidal crystals. Nat. Biotechnol. 2001, 19, 71–74
[2] Daniell, H, Molecular strategies for gene containment in transgenic
crops. Nat. Biotechnol. 2002, 20, 581–587.
[3] Daniell, H., Carmona-Sanchez, O., Burns, B., Chloroplast derived
antibodies, biopharmaceuticals and edible vaccines. In: Schillberg,
S. (Ed.), Mol. Farming, Wiley–VCH Verlag, Germany 2004, Chapter
8, pp 113–133.
[4] Daniell, H., Cohill, P. R., Kumar, S., Dufourmantel, N., Chloroplast
genetic engineering. In: Daniell, H. and Chase, C.D. (Eds.), Molecu-
lar Biology and Biotechnology of Plant Organelles, Springer, the
Netherlands 2004, pp 437–484.
[5] Daniell, H., Dhingra, A., Ruiz, O.N. Chloroplast genetic engineering
to confer desired plant traits. Methods Mol. Biol. 2004, 286, 111–137
[6] Daniell, H., Chebolu, S., Kumar, S., Singleton, M., Falconer, R.,
Chloroplast- derived Vaccine antigens and other Therapeutic pro-
teins. Vaccine 2005, 23, 1779–1783.
[7] Daniell, H., Kumar, S., Duformantel, N., Breakthrough in chloroplast
genetic engineering of agronomically important crops. Trends
Biotechnol. 2005, 23, 238–245.
[8] Grevich, J., Daniell, H., Chloroplast genetic engineering: Recent
advances and perspectives. Crit. Rev. Plant Sci. 2005, 24, 1–25.
[9] Daniell, H., Lee, S.B., Panchal, T., Wiebe, P.O., Expression of the
native cholera toxin B subunit gene and assembly as functional
oligomers in transgenic tobacco chloroplasts. J. Mol. Biol. 2001, 311,
1001–1009.
[10] Lee, S.B., Byun, M.O., Daniell, H., Accumulation of trehalose within
transgenic chloroplasts confers drought tolerance. Mol. Breed. 2003,
11, 1–13.
[11] Dhingra, A., Portis, A.R., Daniell, H., Enhanced translation of a
chloroplast expressed RbcS gene restores SSU levels and photosyn-
thesis in nuclear antisense RbcS plants. Proc. Natl. Acad. Sci. U.S.A.
2004, 101, 6315–6320.
[12] Daniell, H., Datta, R., Varma, S., Gray, S., Lee, S.B., Containment of
herbicide resistance through genetic engineering of the chloroplast
genome. Nat. Biotechnol. 1998, 16, 345¯348.
[13] Daniell, H., Khan, M.S. Allison, L., Milestones in chloroplast genetic
engineering: an environmentally friendly era in biotechnology.
Trends Plant Sci. 2002, 7, 84–91.
[14] Kumar, S., Dhingra, A., Daniell, H., Manipulation of gene expression
facilitates cotton plastid transformation of cotton by somatic
embryogenesis & maternal inheritance of transgenes. Plant Mol.
Biol. 2004, 56, 203–216.
[15] Ruiz, O. N., Daniell, H., Engineering cytoplasmic male sterility via
the chloroplast genome. Plant Physiol. 2005, 138, 1232–1246, fea-
tured on the cover and in Nature as a News & Views article.
[16] Kumar, S., Dhingra, A., Daniell, H., Plastid expressed betaine alde-
hyde dehydrogenase gene in carrot cultured cells, roots and leaves
confers enhanced salt tolerance. Plant Physiol. 2004, 136, 2343–2354.
[17] Kota, M., Daniell, H., Varma, S., Garczynski, S.F., Gould F., William,
M.J., Overexpression of the Bacillus thuringiensis (Bt) Cry2Aa2 pro-
tein in chloroplasts confers resistance to plants against susceptible
and Bt-resistant insects. Proc. Natl. Acad. Sci. U.S.A. 1999, 96,
1840¯1845.
[18] DeGray, G., Kanniah, R., Franzine, S., John, S., and Daniell H.,
Expression of an antimicrobial peptide via the chloroplast genome
to control phytopathogenic bacteria and fungi. Plant Physiol. 2001,
127, 852¯862.
[19] Ruiz, O.N., Hussein, H., Terry N., Daniell, H., Phytoremediation of
organomercurial compounds via chloroplast genetic engineering.
Plant Physiol. 2003, 132, 1344–1352.
[20] Vitanen, P.V., Devine, A. L., Kahn, S., Deuel, D. L., Van Dyk, D. E.,
Daniell, H., Metabolic engineering of the chloroplast genome using
the E. coli ubiC gene reveals that corismate is a readily abundant
precursor for 4-hydroxybenzoic acid synthesis in plants. Plant Phys-
iol. 2004, 136, 4048–4060.
[21] Molina, A., Daniell, H., Mingo-Castel, A., Veramendi, J., High-yield
expression of a viral peptide animal vaccine in transgenic tobacco
chloroplasts. Plant Biotechnol. J. 2004, 2, 141–153.
[22] Watson, J., Koya, V., Leppla, S., Daniell, H., Expression of Bacillus
anthracis protective antigen in transgenic chloroplasts of tobacco, a
non-food/feed crop. Vaccine 2004, 22, 4374–4384.
[23] Koya, V., Moayeri, M., Leppla, S.H., Daniell, H., Plant based vaccine:
mice immunized with chloroplast-derived anthrax protective anti-
gen survive anthrax lethal toxin challenge. Infect. Immun. 2005, 73,
1–9.
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 33
Biotechnol. J. 2006, 1, 26–33 www.biotechnology-journal.com
[24] Quesada-Vargas, T., Ruiz, O.N., Daniell, H., Characterization of het-
erologous multigene operons in transgenic chloroplasts: transcrip-
tion, processing and translation. Plant Physiol. 2005, 138, 1746–1762.
[25] Guda, C., Lee, S.B., Daniell, H., Stable expression of biodegradable
protein based polymer in tobacco chloroplasts. Plant Cell Rep. 2000,
19, 257¯262.
[26] Fernandez-San, M., Mingeo-Castel, A., Miller M., and Daniell H., A
chloroplast transgenic approach to hyper-express and purify Human
Serum Albumin, a protein highly susceptible to proteolytic degra-
dation. Plant Biotechnol. J. 2003, 1, 71–79.
[27] Daniell, H., Muthukumar, B., Lee, S.B., Marker tree transgenic
plants: engineering the chloroplast genome without the use of
antibiotic selection. Curr. Genet. 2001, 39, 109–116.
[28] Saski, C., Lee, S.B., Daniell, H. Tomkins, J., Kim, H.G. Jansen, R.K.,
Complete chloroplast genome sequence of Glycine max and com-
parative analysis with other legumes. Plant Mol. Biol. 2005, in press.
Note:
Our Senior Editors Pushpa M. Bhargava, Theo Dinger-
mann, Marc Blondel and Zhuan Cao are introduced with
their original articles hereafter. The remaining Senior Edi-
tors will be introduced in a coming issue of Biotechnolo-
gy Journal.
... Plastid transformation has also aided the study of plastid biogenesis and function. It has been used to investigate such areas as: plastid DNA replication origins, RNA editing elements, promoter elements, RNA stability determinants, intron maturases, translation elements, protein import machinery, proteolysis, transgene movement and evolution, and transcription and translation of polycistrons (Daniell, Cohill et al., 2004) IV. PLASTID GENOMICS The original concept of a "universal vector" containing plastid DNA flanking sequences from one plant species to transform another species (of unknown genome sequence) was proposed several years ago (Daniell et al., 1998). ...
... Tobacco plastid transformation has also aided in the study of plastid biogenesis and function. Tobacco has been used to investigate plastid DNA replication origins, RNA editing elements, promoter elements, RNA stability determinants, intron maturases, translation elements, protein import machinery, proteolysis, transgene movement and evolution, and transcription and translation of polycistrons (Daniell, Cohill, et al., 2004). Even though plastid transformation is quite efficient in tobacco and used routinely in several laboratories, this is not true in other solanaceous crops (such as potato and tomato) for reasons discussed above. ...
... Such folding and correct assembly minimizes the need for in vitro processing, which can account for up to 30 percent of the production cost and 70 percent of the setup cost (Petridis, Sapidou, and Calandranis, 1995). Functionality of chloroplast-derived vaccine antigens and therapeutic proteins has been demonstrated by several assays including the macrophage lysis assay (Watson et al., 2004), GM1-ganglioside binding assay Molina et al., 2004), protection of HeLA cells or human lung carcinoma cells against encephalomyocarditis virus, systemic immune response, and growth or inhibition of cell cultures (Chebolu and Daniell, 2005;Daniell, Cohill et al., 2004). Purification of human proinsulin has been achieved using novel purification strategies (inverse temperature transition property) that do not require expensive column chromatography techniques (Daniell, Carmona-Sanchez, and Burns, 2004). ...
Chloroplast Genetic Engineering: Recent Advances and Future Perspectives
Article
Full-text available
  • Oct 2005
  • CRIT REV PLANT SCI
Chloroplast genetic engineering offers a number of unique advantages, including a high-level of transgene expression, multi-gene engineering in a single transformation event, transgene containment via maternal inheritance, lack of gene silencing, position and pleiotropic effects, and undesirable foreign DNA. Thus far, over forty transgenes have been stably integrated and expressed via the tobacco chloroplast genome to confer important agronomic traits, as well as express industrially valuable biomaterials and therapeutic proteins. The hyperexpression of recombinant proteins within plastid engineered systems offers a cost effective solution for using plants as bioreactors. Additionally, the presence of chaperones and enzymes within the chloroplast help to assemble complex multi-subunit proteins and correctly fold proteins containing disulfide bonds, thereby drastically reducing the costs of in vitro processing. Oral delivery of vaccine antigens against cholera, tetanus, anthrax, plague, and canine parvovirus are made possible because of the high expression levels and antibiotic-free selection systems available in plastid transformation systems. Plastid genetic engineering also has become a powerful tool for basic research in plastid biogenesis and function. This approach has helped to unveil a wealth of information about plastid DNA replication origins, intron maturases, translation elements and proteolysis, import of proteins and several other processes. Although many successful examples of plastid engineering have set a foundation for various future applications, this technology has not been extended to many of the major crops. Highly efficient plastid transformation has been recently accomplished via somatic embryogenesis using species-specific chloroplast vectors in soybean, carrot, and cotton. Transgenic carrots were able to withstand salt concentrations that only halophytes could tolerate; more than twice the effectiveness of other engineering attempts. Recent advances in plastid engineering provide an efficient platform for the production of therapeutic proteins, vaccines, and biomaterials using an environmentally friendly approach. This review takes an in-depth look into the state of the art in plastid engineering and offers directions for further research and development.
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... Chloroplast targets genetic construct having Guy's 13 gene under the transcriptional control of psbA promoter. This strategy was successful in expressing IgA-G, a humanized chimeric form of Guy's 13 antibody, with uniformly folded disulfide bonds [115]. ...
9. Chloroplast genetic engineering: Concept and industrial applications
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Full-text available
  • Oct 2019
Chloroplast engineering has emerged as an environment-friendly tool and is rather favored over nuclear engineering in some crops. Genome of plastid is highly polyploidy, and therefore it offers introduction of multiple copies of foreign gene in plant cells. Transformation of chloroplast has many inherent advantages. These advantages include higher level of foreign gene expression, multi-gene engineering in a single transformation process, absence of gene silencing and position effect variation, minimal outcrossing of transgene and precise regulation and sequestration of foreign protein. In recent years, successful chloroplast genome engineering has resulted in improved resistance to insect disease, herbicides and drought and production of biopharmaceuticals, including vaccine antigen. Furthermore, development and advancements in chloroplast transformation will help in genetic modification, genetic improvements in plants and cost-effective production of pharmaceutical components with ecofriendly approach. This chapter focuses on basic concepts of genetic engineering and its commercial prospects.
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... Since then, tobacco also became the most preferred model for understanding the evolutionary relationship among photosynthetic organisms and cp (plastid)-engineering or chloroplast transformation technology (CTT) for agricultural biotechnology applications (Daniell et al. 2002;. CTT brings advantages such as control of the site of gene insertion, high rates of transgene expression and protein accumulation, lack of transmission of the transgene via pollen due to the fact that plastid genes are maternally inherited and an absence of epigenetic effects (Daniell et al. 2004). Improving agricultural traits such as herbicide and pathogen resistance, resistance to drought and salt tolerance, phytoremediation and molecular-pharming are all promising and potential applications of CTT (Daniell et al. 2002;Řepková 2010;Adem et al. 2017). ...
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This study examines the early and late age at initiation of tobacco consumption in India. It uses Global Adult Tobacco Survey (2009-10) data to this end. Bivariate and multivariate statistical techniques are used to analyze the data set. The findings reveal that the respondents living in rural areas were highly initiated in different forms of tobacco as compared with urban space. Education was one of the primary factors for initiation of tobacco use. Respondents having secondary and above education were highly initiated in late age. However, the age of tobacco initiation either early or late was significantly higher (7 per cent) among males as compared with females. Respondents belonging to the central (4.9 per cent) and northern regions (4.6 per cent) were highly initiated to tobacco as compared with other regions. Considering a decline in the initiation of tobacco consumption, local level awareness and catchy programmes play an effective role. In this, the involvement of children, women and leaders can be help to reach the goal.
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... Third, during genetic transformation experiments, plasma membranes and the walls of some cell types are the first biological barriers to be pierced or to pass, depending on the biological system being targeted and the cell compartment one wants to attain (nuclear, mitochondria or chloroplast organelles in eukaryotic cells) [95,98,[136][137][138][139][140][141][142]. Plant cell differentiation leads to the increased mechanical strength of the plant wall [130]. ...
Delporte F. Regenerative property and transgenesis competency of plant cell : physiological, histological and molecular approaches in wheat (Triticum aestivum L.)
Thesis
  • Dec 2018
Regenerative property and transgenesis competency of plant cell : physiological, histological and molecular approaches in wheat (Triticum aestivum L.) Thesis Gembloux, Belgium University of Liege – Gembloux Agro-Bio Tech - 143 p., 5 tabl., 27 fig. Summary: Widely used in plant biotechnology for over fifty years, the property of cell totipotency is central to various issues of current interest in biology, and in particular, it has significant potential for medical application. Transgenesis is a biological research tool and a valuable investigative tool in plant physiology; it is also an appropriate strategy for varietal improvement. While only partially understood, some fundamental mechanisms involved in the regenerative property and in cellular receptiveness to gene transfer are common to animal and plant cells, highlighting the principle of the unity of life. The historical difficulties encountered in designing genetically modified cereals, particularly wheat, brought to light the importance of the notion of competence for regeneration and transformation. With regard to the difficulty of combining these two capabilities in the same cell line, this species, considered to be recalcitrant, is a system of choice for exploring the physiological and genetic complexity of both characteristics. This thesis introduces: (1) the development of a novel tissue culture methodology that is simple, functional, applicable to varieties of agricultural interest and is based on the cultivation of fragmented mature zygotic embryos (in place of the immature zygotic embryos traditionally used); (2) the development of a methodology for genetic transformation by means of direct transfer based on the particle bombardment technology, combined with the method of tissue culture described above; (3) the preparation of a documented framework for understanding the fundamental biological mechanisms that regulate regeneration and transformation phenomena in wheat, by means of further analyses at differing levels of observation (macroscopic, microscopic and molecular). For this purpose, we characterised the impact of genetic variability between cultivars on the regenerative process and the temporal sequencing of morphogenetic processes; determined the chronology and ontogenesis by means of a detailed analysis of histological sections; and conducted spatial-temporal analysis of relative levels of target gene expression. Lastly, the originality of this doctoral approach lies in the fact that it at once addresses the processes of regeneration and genetic transformation and, based on an extensive review of the literature, suggests an original, integrated vision of the fundamental bases for these competences, which we propose to consider as being central to dynamic adaptation, defence and survival mechanisms that occur during ‘critical’ transitions between undifferentiated cellular proliferation and cellular organisation Propriété régénérative et compétence à la transgénèse de la cellule végétale : approches physiologique, histologique et moléculaire chez le blé (Triticum aestivum L.) Résumé: Largement exploitée depuis plus de cinquante ans en biotechnologie végétale la fascinante propriété de totipotence cellulaire sous-tend diverses questions d'actualité en biologie, avec notamment d'importantes potentialités médicales. Outil de recherche capital en biologie, la transgénèse constitue un instrument précieux d'investigation en physiologie végétale ; elle représente également une stratégie pertinente en amélioration variétale. Partiellement connus, certains mécanismes fondamentaux impliqués dans la propriété régénérative et la réceptivité cellulaire à la transgénèse sont communs aux cellules animales et végétales; ils soulignent le principe de l'unité du vivant. Les difficultés rencontrées historiquement dans la création de céréales génétiquement modifiées, le blé en particulier, mirent en lumière l’importance de ces notions essentielles de compétences à la régénération et à la transformation. Dans la perspective problématique de combiner ces deux aptitudes dans la même lignée cellulaire, cette espèce réputée récalcitrante constitue un système privilégié pour explorer la complexité physiologique et génétique de ces deux caractères. Notre dissertation présente : (1) la mise au point d’une méthodologie originale de culture de tissus, simple, fonctionnelle, applicable à des variétés d’intérêt agronomique, fondée sur la mise en culture d’embryons matures fragmentés (au lieu d’embryons zygotiques immatures alors classiquement utilisés) ; (2) la mise au point d’une méthodologie de transformation génétique par transfert direct fondée sur la technique de bombardement de particules, combinée à la méthode de culture tissulaire décrite précédemment ; (3) l’élaboration d’un cadre documenté de compréhension de mécanismes biologiques fondamentaux régulant les phénomènes de régénération et de transformation chez le blé, par le biais d’analyses complémentaires menées à différents niveaux d’observation (macroscopique, microscopique et moléculaire). A cette fin, nous avons caractérisé l’incidence de la variabilité génétique inter-cultivars sur le processus régénératif et la séquence temporelle des processus morphogénétiques, identifié la chronologie et l’ontogénèse moyennant une analyse fine de coupes histologiques, et réalisé une analyse spatio-temporelle des niveaux relatifs d’expression de gènes cibles. Enfin, l’originalité de notre démarche doctorale réside dans le fait d’aborder simultanément les processus de régénération et de transformation génétique, et de proposer, sur base d’une large revue de la littérature, une vision intégrative originale des bases fondamentales de ces compétences, que nous proposons de considérer au cœur de mécanismes dynamiques d'adaptation, de défense et de survie intervenant lors de transitions « critiques », entre prolifération cellulaire indifférenciée et organisation.
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... The chloroplast genome typically has a quadripartite organization, with a LSC region, a SSC region and two identical copies of IR regions [1]. In angiosperms, the most complete cp genome sizes range from 120 to 160 kb [2]. Apart from its quadripartite structure, about 100-130 genes were included in chloroplast genome, and therefore the performance in their composition and arrangement are very conservative [3]. ...
Complete Chloroplast Genome Sequence of Malus hupehensis: Genome Structure, Comparative Analysis, and Phylogenetic Relationships
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  • Nov 2018
  • MOLECULES
Malus hupehensis belongs to the Malus genus (Rosaceae) and is an indigenous wild crabapple of China. This species has received more and more attention, due to its important medicinal, and excellent ornamental and economical, values. In this study, the whole chloroplast (cp) genome of Malus hupehensis, using a Hiseq X Ten sequencing platform, is reported. The M. hupehensis cp genome is 160,065 bp in size, containing a large single copy region (LSC) of 88,166 bp and a small single copy region (SSC) of 19,193 bp, separated by a pair of inverted repeats (IRs) of 26,353 bp. It contains 112 genes, including 78 protein-coding genes (PCGs), 30 transfer RNA genes (tRNAs), and four ribosomal RNA genes (rRNAs). The overall nucleotide composition is 36.6% CG. A total of 96 simple sequence repeats (SSRs) were identified, most of them were found to be mononucleotide repeats composed of A/T. In addition, a total of 49 long repeats were identified, including 24 forward repeats, 21 palindromic repeats, and four reverse repeats. Comparisons of the IR boundaries of nine Malus complete chloroplast genomes presented slight variations at IR/SC boundaries regions. A phylogenetic analysis, based on 26 chloroplast genomes using the maximum likelihood (ML) method, indicates that M. hupehensis clustered closer ties with M. baccata, M. micromalus, and M. prunifolia than with M. tschonoskii. The availability of the complete chloroplast genome using genomics methods is reported here and provides reliable genetic information for future exploration on the taxonomy and phylogenetic evolution of the Malus and related species.
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Methods and Tools for Plant Organelle Genome Sequencing, Assembly, and Downstream Analysis
Chapter
Full-text available
  • Jan 2020
Organelles play an important role in a eukaryotic cell. Among them, the two organelles, chloroplast and mitochondria, are responsible for the critical function of photosynthesis and aerobic respiration. Organellar genomes are also very important for plant systematic studies. Here we have described the methods for isolation of the mitochondrial and plastid DNA and its subsequent sequencing with the help of NGS technology. We have also discussed in detail the various tools available for assembly, annotation, and visualization of the organelle genome sequence.
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OBPC SYMPOSIUM: MAIZE 2004 & BEYOND – RECENT ADVANCES IN CHLOROPLAST GENETIC ENGINEERING
Article
Full-text available
  • Jun 2005
The chloroplast genetic engineering approach offers a number of unique advantages, including high-level transgene expression, multi-gene engineering in a single transformation event, transgene containment via maternal inheritance, lack of gene silencing, position and pleiotropic effects and undesirable foreign DNA. Thus far, more than 40 transgenes have been stably integrated and expressed via the tobacco chloroplast genome to confer several agronomic traits and produce vaccine antigens, industrially valuable enzymes, biomaterials, and amino acids. Functionality of chloroplast-derived vaccine antigens and therapeutic proteins have been demonstrated by in vitro assays and animal studies. Oral delivery of vaccine antigens has been facilitated by hyperexpression in transgenic chloroplasts (leaves) or non-green plastids (carrots) and the availability of antibiotic-free selectable markers or the ability to excise selectable marker genes. Additionally, the presence of chaperones and enzymes within the chloroplast help to assemble complex multi-subunit proteins and correctly fold proteins containing disulfide bonds, thereby drastically reducing the costs of in vitro processing. Despite such significant progress in chloroplast transformation, this technology has not been extended to major crops. This obstacle emphasizes the need for plastid genome sequencing to increase the efficiency of transformation and conduct basic research in plastid biogenesis and function. However, highly efficient soybean, carrot, and cotton plastid transformation has been recently accomplished via somatic embryogenesis using species-specific chloroplast vectors. Recent advancements facilitate our understanding of plastid biochemistry and molecular biology. This review focuses on exciting recent developments in this field and offers directions for further research and development.
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Chloroplast Transformation 245 245 Chloroplast Genetic Engineering Via Organogenesis or Somatic Embryogenesis
Article
Full-text available
  • May 2004
  • Meth Mol Biol
Chloroplast genetic engineering offers a number of unique advantages, including high-level trans-gene expression, multigene engineering in a single transformation event, transgene containment via maternal inheritance, lack of gene silencing, position and pleiotropic effects and undesirable foreign DNA. More than 40 transgenes have been stably integrated and expressed via the tobacco chloro-plast genome to confer desired agronomic traits or express high levels of vaccine antigens and biopharmaceuticals. Despite such significant progress, this technology has not been extended to other important plant species. For example, Arabidopsis may be an ideal model system for chloro-plast functional genomics. The employment of chloroplast transformation technology in Arabidopsis has been hampered by the lack of an efficient and reproducible protocol that provides fertile chloro-plast transgenic plants. Transformation of the Arabidopsis chloroplast genome was achieved via organogenesis but the efficiency was at least a 100-fold lower than in tobacco and had the drawback of polyploidy in the leaf tissue that resulted in sterile transgenic plants. This problem can be overcome by adapting procedures that are now available to regenerate plants from both diploid and tetraploid explants via callus. In addition, it is feasible to regenerate Arabidopsis via somatic embryogenesis. Recent breakthroughs in highly efficient plastid transformation of recalcitrant crops such as cotton and soybean have opened the possibility of engineering Arabidopsis plastid genome via somatic embryogenesis. Therefore, protocols of recent improvements in tissue culture, DNA delivery, and the novel vector designs are provided here in order to achieve highly efficient plastid transformation in Arabidopsis.
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Plastome Engineering: Basics Principles and Applications
Chapter
  • Mar 2017
Genetic material in plants is distributed into the nucleus, plastids, and mitochondria. Plastid has a central role of carrying out photosynthesis in plant cells. Plastid transformation is an advantage to nuclear gene transformation due to higher expression of transgenes, absence of gene silencing and position effect, and transgene containment by maternal inheritance, i.e., plastid gene inheritance via seed not by pollen prevents transmission of foreign DNA to wild relatives. Thus, plastid transformation is a viable alternative to conventional nuclear transformation. Many genes encoding for industrially important proteins and vaccines, as well as genes conferring important agronomic traits, have been stably integrated and expressed in the plastid genome. Despite these advances, it remains a challenge to achieve plastid transformation in non-green tissues and recalcitrant crops regenerating via somatic embryos. In this chapter, we have summarized the basic requirements of plastid genetic engineering and discuss the current status and futuristic potential of plastid transformation.
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Chloroplast Genetic Engineering: Recent Advances and Future Perspectives
Article
Full-text available
  • Oct 2005
  • CRIT REV PLANT SCI
Chloroplast genetic engineering offers a number of unique advantages, including a high-level of transgene expression, multi-gene engineering in a single transformation event, transgene containment via maternal inheritance, lack of gene silencing, position and pleiotropic effects, and undesirable foreign DNA. Thus far, over forty transgenes have been stably integrated and expressed via the tobacco chloroplast genome to confer important agronomic traits, as well as express industrially valuable biomaterials and therapeutic proteins. The hyperexpression of recombinant proteins within plastid engineered systems offers a cost effective solution for using plants as bioreactors. Additionally, the presence of chaperones and enzymes within the chloroplast help to assemble complex multi-subunit proteins and correctly fold proteins containing disulfide bonds, thereby drastically reducing the costs of in vitro processing. Oral delivery of vaccine antigens against cholera, tetanus, anthrax, plague, and canine parvovirus are made possible because of the high expression levels and antibiotic-free selection systems available in plastid transformation systems. Plastid genetic engineering also has become a powerful tool for basic research in plastid biogenesis and function. This approach has helped to unveil a wealth of information about plastid DNA replication origins, intron maturases, translation elements and proteolysis, import of proteins and several other processes. Although many successful examples of plastid engineering have set a foundation for various future applications, this technology has not been extended to many of the major crops. Highly efficient plastid transformation has been recently accomplished via somatic embryogenesis using species-specific chloroplast vectors in soybean, carrot, and cotton. Transgenic carrots were able to withstand salt concentrations that only halophytes could tolerate; more than twice the effectiveness of other engineering attempts. Recent advances in plastid engineering provide an efficient platform for the production of therapeutic proteins, vaccines, and biomaterials using an environmentally friendly approach. This review takes an in-depth look into the state of the art in plastid engineering and offers directions for further research and development.
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Molecular Strategies for Gene Containment in Transgenic Crops
Article
Full-text available
  • Aug 2002
The potential of genetically modified (GM) crops to transfer foreign genes through pollen to related plant species has been cited as an environmental concern. Until more is known concerning the environmental impact of novel genes on indigenous crops and weeds, practical and regulatory considerations will likely require the adoption of gene-containment approaches for future generations of GM crops. Most molecular approaches with potential for controlling gene flow among crops and weeds have thus far focused on maternal inheritance, male sterility, and seed sterility. Several other containment strategies may also prove useful in restricting gene flow, including apomixis (vegetative propagation and asexual seed formation), cleistogamy (self-fertilization without opening of the flower), genome incompatibility, chemical induction/deletion of transgenes, fruit-specific excision of transgenes, and transgenic mitigation (transgenes that compromise fitness in the hybrid). As yet, however, no strategy has proved broadly applicable to all crop species, and a combination of approaches may prove most effective for engineering the next generation of GM crops.
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Accumulation of trehalose within transgenic chloroplasts confers drought tolerance
Article
Full-text available
  • Jan 2003
Yeast trehalose phosphate synthase(TPS1) gene was introduced into the tobacco chloroplast or nuclear genomes to study resultant phenotypes. PCR and Southern blots confirmed stable integration of TPS1 into the chloroplast genomes of T1, T2 and T3 transgenic plants. Northern blot analysis of transgenic plants showed that the chloroplast transformant expressed 169-fold more TPS1 transcript than the best surviving nuclear transgenic plant. Although both the chloroplast and nuclear transgenic plants showed significant TPS1 enzyme activity, no significant trehalose accumulation was observed in T0/T1 nuclear transgenic plants whereas chloroplast transgenic plants showed 15–25 fold higher accumulation of trehalose than the best surviving nuclear transgenic plants. Nuclear transgenic plants (T0) that showed even small amounts of trehalose accumulation showed stunted phenotype, sterility and other pleiotropic effects whereas chloroplast transgenic plants (T1, T2,T3) showed normal growth and no pleiotropic effects. Transgenic chloroplast thylakoid membranes showed high integrity under osmotic stress as evidenced by retention of chlorophyll even when grown in 6% PEG whereas chloroplasts in untransformed plants were bleached. After 7 hr drying, chloroplast transgenic seedlings (T1, T3) successfully rehydrated while control plants died. There was no difference between control and transgenic plants in water loss during dehydration but dehydrated leaves from transgenic plants (not watered for 24 days) recovered upon rehydration turning green while control leaves dried out. These observations suggest that trehalose functions by protecting biological membranes rather than regulating water potential. In order to prevent escape of drought tolerance trait to weeds and associated pleiotropic traits to related crops, it may be desirable to engineer crop plants for drought tolerance via the chloroplast genome instead of the nuclear genome.
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Stable expression of biodegradable protein based polymer in tobacco chloroplasts
Article
Full-text available
  • Jan 2000
 Bioelastic protein-based polymers (PBP) have several medical (prevention of post-surgical adhesions) and non-medical (biodegradable plastic) applications. This study compares expression levels of PBP genes (synthetic) integrated into the nuclear genome or the large single-copy (LSC) or inverted repeat (IR) region of the chloroplast genome in transgenic tobacco plants. Polymer transcripts accumulated up to 100-fold higher in the IR plants than in those of nuclear transgenic plants. Integration of foreign genes into all of the chloroplast genomes (homoplasmy) and higher levels of polymer transcripts were observed only in the IR and not in LSC transgenic plants. Expression of the polymer protein was further confirmed by Western blot analysis.
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Overexpression of the Bacillus Thuringiensis (Bt) Cry2Aa2 Protein in Chloroplasts Confers Resistance to Plants Against Susceptible and Bt-resistant Insects
Article
Full-text available
  • Apr 1999
Evolving levels of resistance in insects to the bioinsecticide Bacillus thuringiensis (Bt) can be dramatically reduced through the genetic engineering of chloroplasts in plants. When transgenic tobacco leaves expressing Cry2Aa2 protoxin in chloroplasts were fed to susceptible, Cry1A-resistant (20,000- to 40,000-fold) and Cry2Aa2-resistant (330- to 393-fold) tobacco budworm Heliothis virescens, cotton bollworm Helicoverpa zea, and the beet armyworm Spodoptera exigua, 100% mortality was observed against all insect species and strains. Cry2Aa2 was chosen for this study because of its toxicity to many economically important insect pests, relatively low levels of cross-resistance against Cry1A-resistant insects, and its expression as a protoxin instead of a toxin because of its relatively small size (65 kDa). Southern blot analysis confirmed stable integration of cry2Aa2 into all of the chloroplast genomes (5,000–10,000 copies per cell) of transgenic plants. Transformed tobacco leaves expressed Cry2Aa2 protoxin at levels between 2% and 3% of total soluble protein, 20- to 30-fold higher levels than current commercial nuclear transgenic plants. These results suggest that plants expressing high levels of a nonhomologous Bt protein should be able to overcome or at the very least, significantly delay, broad spectrum Bt-resistance development in the field.
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Containment of herbicide resistance through genetic engineering of the chloroplast genome
Article
Full-text available
  • May 1998
Glyphosate is a potent herbicide. It works by competitive inhibition of the enzyme 5-enol-pyruvyl shikimate-3-phosphate synthase (EPSPS), which catalyzes an essential step in the aromatic amino acid biosynthetic pathway. We report the genetic engineering of herbicide resistance by stable integration of the petunia EPSPS gene into the tobacco chloroplast genome using the tobacco or universal vector. Southern blot analysis confirms stable integration of the EPSPS gene into all of the chloroplast genomes (5000-10,000 copies per cell) of transgenic plants. Seeds obtained after the first self-cross of transgenic plants germinated and grew normally in the presence of the selectable marker, whereas the control seedlings were bleached. While control plants were extremely sensitive to glyphosate, transgenic plants survived sprays of high concentrations of glyphosate. Chloroplast transformation provides containment of foreign genes because plastid transgenes are not transmitted by pollen. The escape of foreign genes via pollen is a serious environmental concern in nuclear transgenic plants because of the high rates of gene flow from crops to wild weedy relatives.
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Regeneration and genetic transformation of grain legumes: An overview
Article
  • Feb 2003
  • CURR SCI INDIA
Grain legume crops like pigeonpea, chickpea, mungbean, groundnut and soybean are extensively grown in the rainfed and dryland areas of India. These legume crops are a source of dietary protein, especially for the largely vegetarian population of sub-tropics. Despite large acreag e under these crops, total productivity remains low and has been stagnating for the last few decades. A number of biotic and abiotic stresses are severely affecting full reali - zation of the yield potential of these crops. There is need to increase productivity and enhance the nut ritional value of these pulse crops. Cultivars resistant to biotic and abiotic stresses and which have better protein quality and quantity are needed. Grain legumes have a narrow gene- tic base since they are essentially self -pollinated (although cross-pollination does take place, it is at very low fre - quency). Thus, there is need to widen the genetic base and incorporate desirable characters. There is an urgent need to use transgenic technologies for improve ment of legu - minous crops. Worldwide, soybean is the only transgenic grain legume being cultivated in nearly 63% of the total area under transgenics 1 . Routine transformation protocols are limited in most grain legumes. The low success has been attributed to poor regeneration ability (especially via callus) and lack of compatible gene delivery methods, although some success has been achieved in soybean. This review is an attempt to summarize the studies on rege - neration and genetic transformation in soybean, pigeon pea, chickpea, pea, groundnut, and Vigna spp. and to identify the hurdles being faced in the effi cient recovery of transgenic plants. The review presents a comparative account of explants used, mode of regenera tion (organo- genesis v/s embryogenesis), gene delivery techniques and recovery of transgenics in crops considered here. Plant tissues regenerate in vitro through two pathways, namely 'organogenesis' wherein shoot buds are orga- nized by concerted meristematic activity of a number of cells and 'embryogenesis', where usually single cell o r a small cluster of cells undergo differentiation to produce somatic embryos similar to zygotic embryos. The regene- ration of complete plants via tissue culture has made it possible to introduce foreign genes into plant cells and recover transgenic plants. Morphogenesis could occur directly from the explant or indirectly via the formation of a dedifferentiated callus (Figure 1). However the dif - ferent pathways of regeneration, viz. organogenesis from callus (pathway I), embryogenesis from callus (pathway IV), organogenesis directly from explants (pathway II) and embryogenesis from explants in a direct mode (path- way III) vary in their amenability to different gene deli - very techniques. Although many different techniques (electroporation of intact tissues, silicone carbide whiskers, etc.) have been tested for gene delivery to plant cells, two major methods , namely Agrobacterium-mediated and particle bombard- ment, have been extensively employed for genetic trans - formation of crop plants. Regeneration via the callus lends itself easily (compared to explants regenerating directly) to Agrobacterium-mediated transformations, while direct regeneration is more amenable for particle bombardment. Organogenesis from an unorganized callus (pathway I) has only been reported in soybean 2 and pea 3 , where shoots were recovered from callus tissues at a low frequency. Thus, this pathway, although amenable to Agro-
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Molecular Farming: Plant-Made Pharmaceuticals and Technical Proteins
Chapter
  • Jun 2005
IntroductionExpression of Therapeutic and Human Proteins in PlantsThe Transgenic Chloroplast System Chloroplast-derived Human AntibodiesChloroplast-derived Biopharmaceuticals Human Serum AlbuminHuman Insulin-like Growth Factor-1Human Interferon (IFNα2b)Anti-Microbial Peptides (AMPs): MSI-99Chloroplast-derived Vaccine Antigens Cholera Toxin B Subunit (CTB)2Bacillus anthracis Protective Antigen3Yersinia pestis F1∼V Fusion Antigen Canine Parvovirus (CPV) VP2 ProteinAdvances in Purification Strategies for BiopharmaceuticalsConclusion AcknowledgementsReferences Chloroplast-derived Human AntibodiesChloroplast-derived Biopharmaceuticals Human Serum AlbuminHuman Insulin-like Growth Factor-1Human Interferon (IFNα2b)Anti-Microbial Peptides (AMPs): MSI-99Chloroplast-derived Vaccine Antigens Cholera Toxin B Subunit (CTB)2Bacillus anthracis Protective Antigen3Yersinia pestis F1∼V Fusion Antigen Canine Parvovirus (CPV) VP2 Protein Human Serum AlbuminHuman Insulin-like Growth Factor-1Human Interferon (IFNα2b)Anti-Microbial Peptides (AMPs): MSI-99 Cholera Toxin B Subunit (CTB) Canine Parvovirus (CPV) VP2 Protein
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Processing and degradation of chloroplast mRNA
Article
  • Jun 2000
  • BIOCHIMIE
The conversion of genetic information stored in DNA into a protein product proceeds through the obligatory intermediate of messenger RNA. The steady-state level of an mRNA is determined by its relative synthesis and degradation rates, i.e., an interplay between transcriptional regulation and control of RNA stability. When the biological status of an organism requires that a gene product’s abundance varies as a function of developmental stage, environmental factors or intracellular signals, increased or decreased RNA stability can be the determining factor. RNA stability and processing have long been known as important regulatory points in chloroplast gene expression. Here we summarize current knowledge and prospects relevant to these processes, emphasizing biochemical data. The extensive literature on nuclear mutations affecting chloroplast RNA metabolism is reviewed in another article in this volume (Barkan and Goldschmidt-Clermont, this issue).
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