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We describe simple and efficient plastid transformation methods for suspension-cultured cells and sporelings of the liverwort, Marchantia polymorpha L. Use of rapidly proliferating cells such as suspension-cultured cells and sporelings, which are immature thalli developing from spores, as targets made plastid transformation by particle bombardment efficient. Selection on a sucrose-free medium and linearization of the transformation vector significantly improved the recovery rate of plastid transformants. With the methods described here, a few plastid transformants are obtained from a single bombardment of sporelings, while more efficient plastid transformation is expected in suspension-cultured cells, ~60 transformants from a single bombardment. Homoplasmic transformants of thalli are obtained immediately after primary selection, whereas homoplasmic transformants from suspension-cultured cells are obtained after 12–16 weeks of repeated subculture. [ABSTRACT FROM AUTHOR]

The availability of routine techniques for the genetic manipulation of the chloroplast genome of Chlamydomonas reinhardtii has allowed a plethora of reverse-genetic studies of chloroplast biology using this alga as a model organism. These studies range from fundamental investigations of chloroplast gene function and regulation to sophisticated metabolic engineering programs and to the development of the algal chloroplast as a platform for producing high-value recombinant proteins. The established method for delivering transforming DNA into the Chlamydomonas chloroplast involves microparticle bombardment, with the selection of transformant lines most commonly involving the use of antibiotic resistance markers. In this chapter we describe a simpler and cheaper delivery method in which cell/DNA suspensions are agitated with glass beads: a method that is more commonly used for nuclear transformation of Chlamydomonas. Furthermore, we highlight the use of an expression vector (pASapI) that employs an endogenous gene as a selectable marker, thereby avoiding the contentious issue of antibiotic resistance determinants in transgenic lines. [ABSTRACT FROM AUTHOR]

Although plastid transformation has attractive advantages and potential applications in plant biotechnology, for long time it has been highly efficient only in tobacco. The lack of efficient selection and regeneration protocols and, for some species, the inefficient recombination using heterologous flanking regions in transformation vectors prevented the extension of the technology to major crops. However, the availability of this technology for species other than tobacco could offer new possibilities in plant breeding, such as resistance management or improvement of nutritional value, with no or limited environmental concerns. Herein we describe an efficient plastid transformation protocol for potato (Solanum tuberosum subsp. tuberosum). By optimizing the tissue culture system and using transformation vectors carrying homologous potato flanking sequences, we obtained up to one transplastomic shoot per bombardment. Such efficiency is comparable to that usually achieved in tobacco. The method described in this chapter can be used to regenerate potato transplastomic plants expressing recombinant proteins in chloroplasts as well as in amyloplasts. [ABSTRACT FROM AUTHOR]

The amounts of specific transcripts that accumulate in chloroplasts are determined by the rates of synthesis and degradation of the transcripts. The 3′ untranslated region of transcripts is a major determinant of the stability of transcripts in chloroplasts. The half-lives of specific transcripts can be determined by northern blot analysis of a time course of transcripts in detached tobacco leaves incubated with actinomycin D, a potent transcription inhibitor. This analysis may be applied to transcripts of endogenous genes or of transgenes introduced into the chloroplast genome in transplastomic plants. Sequence determinants of transcript stability can be identified by analysis of transplastomic plants containing constructs of the green fluorescent protein (gfp) reporter gene fused to the sequences of interest. [ABSTRACT FROM AUTHOR]

Tomato (Solanum lycopersicum) is one of the most important vegetable crops and has long been an important model species in plant biology. Plastid biology in tomato is especially interesting due to the chloroplast-to-chromoplast conversion occurring during fruit ripening. Moreover, as tomato represents a major food crop with an edible fruit that can be eaten raw, the development of a plastid transformation protocol for tomato was of particular interest to plant biotechnology. Recent methodological improvements have made tomato plastid transformation more efficient and facilitated applications in metabolic engineering and molecular farming. This article describes the basic methods involved in the generation and analysis of tomato plants with transgenic chloroplast genomes and summarizes current applications of tomato plastid transformation. [ABSTRACT FROM AUTHOR]

While chlorophyll has served as an excellent label for plastids in green tissue, the development of fluorescent proteins has allowed their ready visualization in all tissues of the plants, revealing new features of their morphology and motility. Gene regulatory sequences in plastid transgenes can be optimized through the use of fluorescent protein reporters. Fluorescent labeling of plastids simultaneously with other subcellular locations reveals dynamic interactions and mutant phenotypes. Transient expression of fluorescent protein fusions is particularly valuable to determine whether or not a protein of unknown function is targeted to the plastid. Particle bombardment and agroinfiltration methods described here are convenient for imaging fluorescent proteins in plant organelles. With proper selection of fluorophores for labeling the components of the plant cell, confocal microscopy can produce extremely informative images at high resolution at depths not feasible by standard epifluorescence microscopy. [ABSTRACT FROM AUTHOR]

Expression of transgenes from the plastid genome offers a number of attractions to biotechnologists, with the potential to attain very high protein accumulation levels arguably being the most attractive one. High-level transgene expression is of particular importance in resistance engineering (e.g., via expression of insecticidal proteins) and molecular farming. Over the past years, the production of many commercially valuable proteins in chloroplast-transgenic (transplastomic) plants has been attempted, including pharmaceutical proteins (such as subunit vaccines and protein antibiotics) and industrial enzymes. Although, in some cases, spectacularly high foreign protein accumulation levels have been obtained, expression levels were disappointingly poor in other cases. In this review, I summarize our current knowledge about the factors influencing the efficiency of plastid transgene expression and highlight possible optimization strategies to alleviate problems with poor expression levels. [ABSTRACT FROM AUTHOR]

Overall translational machinery in plastids is similar to that of E. coli. Initiation is the crucial step for translation and this step in plastids is somewhat different from that of E. coli. Unlike the Shine-Dalgarno sequence in E. coli, cis-elements for translation initiation are not well conserved in plastid mRNAs. Specific trans-acting factors are generally required for translation initiation and its regulation in plastids. During translation elongation, ribosomes pause sometimes on photosynthesis-related mRNAs due probably to proper insertion of nascent polypeptides into membrane complexes. Codon usage of plastid mRNAs is different from that of E. coli and mammalian cells. Plastid mRNAs do not have the so-called rare codons. Translation efficiencies of several synonymous codons are not always correlated with codon usage in plastid mRNAs. [ABSTRACT FROM AUTHOR]

Chloroplasts, the sites of photosynthesis and sources of reducing power, are at the core of the success story that sets apart autotrophic plants from most other living organisms. Along with their fellow organelles (e.g., amylo-, chromo-, etio-, and leucoplasts), they form a group of intracellular biosynthetic machines collectively known as plastids. These plant cell constituents have their own genome (plastome), their own (70S) ribosomes, and complete enzymatic equipment covering the full range from DNA replication via transcription and RNA processive modification to translation. Plastid RNA synthesis (gene transcription) involves the collaborative activity of two distinct types of RNA polymerases that differ in their phylogenetic origin as well as their architecture and mode of function. The existence of multiple plastid RNA polymerases is reflected by distinctive sets of regulatory DNA elements and protein factors. This complexity of the plastid transcription apparatus thus provides ample room for regulatory effects at many levels within and beyond transcription. Research in this field offers insight into the various ways in which plastid genes, both singly and groupwise, can be regulated according to the needs of the entire cell. Furthermore, it opens up strategies that allow to alter these processes in order to optimize the expression of desired gene products. [ABSTRACT FROM AUTHOR]

Abstract Chloroplasts and mitochondria both evolved from endosymbionts and therefore share many physiologic, biochemical, and genetic features. This chapter reviews a selection of chloroplast RNA-binding proteins, specifically those from land plants that meet the following criteria: (1) the encoding gene is known; (2) an association with RNA has been demonstrated; and (3) the protein does not have any (known) catalytic activity. This set of RNA-binding proteins predominantly consists of members from five protein families: the pentatricopeptide repeat (PPR) protein family; the chloroplast ribonucleoprotein (cpRNP) family; the chloroplast ribosome maturation (CRM) and RNA splicing protein family; the Whirly (Why) protein family; and the plant organelle RNA recognition (PORR) protein family. The functions of representative members of these families are summarized, and their protein–RNA interactions are discussed. [ABSTRACT FROM AUTHOR]

Recently, confocal microscope has become a routine technique and indispensable tool for cell biological studies and molecular investigations. The light emitted from the point out-of-focus is blocked by the pinhole and can not reach the detector, which is one of the critical features of the confocal microscope. In present studies, the probes acridine orange (AO) and rhodamine-123 were used to research stoma and mitochondria of mung bean leaves, respectively. The results indicated that the stomatal guard cells and mitochondria were clearly seen in epidermic tissue of mung bean leaves. Taken together, it is a good method to research plant cells with confocal microscope and fluorescence probes. [ABSTRACT FROM AUTHOR]