Engineering plastid genomes: methods, tools, and applications in basic research and biotechnology.

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ANNUAL REVIEWS

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Further

Annu. Rev. Plant Biol. 2015.66:211-241. Downloaded from www.annualreviews.org Access provided by Columbia University on 07/17/17. For personal use only.

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Engineering Plastid Genomes: Methods, Tools, and Applications in Basic Research and Biotechnology Ralph Bock Max-Planck-Institut fur ¨ Molekulare Pflanzenphysiologie, D-14476 Potsdam-Golm, Germany; email: [email protected]

Annu. Rev. Plant Biol. 2015. 66:211–41

Keywords

First published online as a Review in Advance on December 1, 2014

plastid transformation, chloroplast transformation, reverse genetics, metabolic engineering, molecular farming, experimental evolution, horizontal gene transfer

The Annual Review of Plant Biology is online at plant.annualreviews.org This article’s doi: 10.1146/annurev-arplant-050213-040212 c 2015 by Annual Reviews. Copyright All rights reserved

Abstract The small bacterial-type genome of the plastid (chloroplast) can be engineered by genetic transformation, generating cells and plants with transgenic plastid genomes, also referred to as transplastomic plants. The transformation process relies on homologous recombination, thereby facilitating the site-specific alteration of endogenous plastid genes as well as the precisely targeted insertion of foreign genes into the plastid DNA. The technology has been used extensively to analyze chloroplast gene functions and study plastid gene expression at all levels in vivo. Over the years, a large toolbox has been assembled that is now nearly comparable to the techniques available for plant nuclear transformation and that has enabled new applications of transplastomic technology in basic and applied research. This review describes the state of the art in engineering the plastid genomes of algae and land plants (Embryophyta). It provides an overview of the existing tools for plastid genome engineering, discusses current technological limitations, and highlights selected applications that demonstrate the immense potential of chloroplast transformation in several key areas of plant biotechnology.

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Contents


INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PLASTID TRANSFORMATION METHODS FOR ALGAE AND PLANTS . . . . . Transformation Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Selectable Markers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Integration of Foreign DNA into the Plastid Genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cotransformation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transformable Species and Bottlenecks in Extending the Species Range . . . . . . . . . . . Transfer of Transgenic Plastids Between Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . THE TOOLBOX FOR PLASTID GENOME ENGINEERING . . . . . . . . . . . . . . . . . . . Vector Backbones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Promoters and Untranslated Regions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reporter Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Operon Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inducible and Repressible Gene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Expression in Nongreen Tissues. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Marker Excision . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PLASTID TRANSFORMATION IN BASIC RESEARCH . . . . . . . . . . . . . . . . . . . . . . . . . Reverse Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In Vivo Analysis of Gene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Experimental Evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PLASTID TRANSFORMATION IN PLANT BIOTECHNOLOGY . . . . . . . . . . . . . . Engineering Resistances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Metabolic Engineering. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular Farming . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SUMMARY AND OUTLOOK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

212 215 215 218 218 220 221 222 223 223 223 224 224 225 225 226 226 226 227 228 229 229 229 230 231

INTRODUCTION

Embryophyta: a subkingdom of green plants (Plantae) that comprises bryophytes, ferns and their allies, and seed plants

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The chloroplasts of all eukaryotic algae and all embyrophytes (bryophytes, ferns, and seed plants) are the product of a singular endosymbiotic event that happened approximately 1.5 billion years ago and occurred through the uptake of a cyanobacterium by a heterotrophic protist. The subsequent gradual evolutionary optimization of the relationship between the host cell and the endosymbiont involved several key innovations, including (a) the establishment of metabolite exchange systems that facilitate the transport of reduced carbon compounds across the double membrane of the endosymbiont, (b) genome streamlining by elimination of dispensable and redundant genetic information, (c) massive gene transfer from the endosymbiont genome to the host nuclear genome, and (d ) the evolution of a protein import machinery that reroutes the gene products of transferred genes into the chloroplast. The combined action of genome streamlining and gene transfer resulted in a dramatic shrinkage of the genome of the cyanobacterial endosymbiont, in that thousands of genes disappeared and were either deleted or moved to the nucleus (23, 139). Consequently, present-day plastid (chloroplast) genomes of photosynthetic eukaryotes are much reduced and typically harbor only 100–250 genes (approximately 130 genes in seed plants) (Figures 1 and 2). From its structure and sequence, the genome is still clearly recognizable as a remnant of the genome of the ancestral cyanobacterial endosymbiont (16, 183). The plastid DNA (or plastome) Bock

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Chloroplast genome 203,828 base pairs

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psbA 2 trnSf12 yc tpE a s7 4 rpps1 r sbM p sbZ p

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Photosystem I Photosystem II Cytochrome b/f complex ATP synthase RuBisCO large subunit RNA polymerase Ribosomal proteins (small subunit) Ribosomal proteins (large subunit) clpP, matK Other gene Hypothetical chloroplast reading frame (ycf) Open reading frame Transfer RNA Ribosomal RNA Intron

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Figure 1 Physical map of the Chlamydomonas reinhardtii chloroplast genome, drawn using the complete genome sequence as input (GenBank accession number NC_005353.1) in version 1.1 of the OrganellarGenomeDRAW software tool (106, 107). The gray arrows denote the direction of transcription for the two DNA strands of the (circularly mapping) genome, and the interior circle shows its tetrapartite structure. Abbreviations: IRA , inverted repeat A; IRB , inverted repeat B; LSC, large single-copy region; SSC, small single-copy region.

www.annualreviews.org • Plastid Genetic Engineering

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p p sb p sb J psbsbF L O E trnRF10 rpl2 rps1 0 trnPW 3 2 clpP

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rpoA rps11

rpl36 ΨinfA rps8 rpl14 rpl16 rps3 rpl2129 rps 3 2 s rp rpl2 trnI

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Chloroplast genome 155,943 base pairs

trnK matK

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A

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rrn 4 rr .5 trnn5 ORF R 35 oriB0

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Photosystem I Photosystem II Cytochrome b/f complex ATP synthase NADH dehydrogenase RuBisCO large subunit RNA polymerase Ribosomal proteins (small subunit) Ribosomal proteins (large subunit) clpP, matK Other gene Hypothetical chloroplast reading frame (ycf) Open reading frame Transfer RNA Ribosomal RNA Origin of replication Intron

214

ycf1

0B F7 rnV OR t

ndhF

trn ORFN 75

OR

SSC

rrn 4.5 rrnrrn5 R trn

OR

F7

16 0B rrn rnI t nA F7 rnV tr OR t

L trn

hB nd 7 s 2 rp ps1 r

rpl2 trnI 3

ycf2

IR

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trnR trnG psbI psbK

trnS trnQ

petD

rps15 ndhH ndhA ndhI


ycf3 ORF74 rps4 trnT

trnV E atppB at

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of most embryophyte plants and green algae shows a tetrapartite genome organization, with a large single-copy region (LSC) and a small single-copy region (SSC) separating two inverted repeat regions. The two inverted repeats are identical in their nucleotide sequence and differ only in their relative orientation (Figures 1 and 2). The availability of technology for chloroplast genome engineering led to a major upsurge in research on chloroplast genomes, gene functions, and gene expression. This article describes the methodology of plastid transformation and the toolbox that has been assembled by the community over the years, discusses current limitations and future challenges in plastid genome engineering, and provides an overview of applications of the technology in basic research and biotechnology.

Mesophyll: parenchyma cells (L2 + L3 layers) that lie between the upper and lower epidermis (L1 layer) of the leaf


PLASTID TRANSFORMATION METHODS FOR ALGAE AND PLANTS The key innovation that made organelle transformation possible was the invention of the gene gun, a device that allows researchers to bombard living cells and tissues with accelerated DNA-covered microparticles. The technology became known as biolistic (biological + ballistic) transformation, and because it relies entirely on physical principles, it provides a universal method for introducing naked (purified or synthetic) nucleic acids into essentially any organism or cell type. Amazingly, the technology works for cell organelles as well, even though they are in the same size range as (chloroplasts) or even smaller than (mitochondria) the standard particle size used for shooting (0.4–1.7 μm). Transformation of the chloroplast genome was first accomplished in Chlamydomonas reinhardtii, a unicellular green alga harboring a single chloroplast that occupies approximately half the cell volume and contains approximately 80 identical copies of the plastid genome (27) (Figure 1). Chloroplast transformation was thought to be much more challenging in seed plants (and was even believed to be impossible by quite a few researchers in the field) because a typical leaf mesophyll cell contains 1,000–2,000 copies of the plastid genome (Figure 2) and approximately 100 chloroplasts (68). However, soon after the initial success with Chlamydomonas, chloroplast transformation was achieved in tobacco (Nicotiana tabacum) (169). For many years, Chlamydomonas and tobacco remained the only two species that were routinely transformable (Tables 1 and 2). In these two model systems, the basic principles of plastid genome engineering were worked out, essentially all currently available tools were developed, and most proof-of-concept applications were conducted. The genome size, coding capacity, and genome organization of the two model systems for chloroplast transformation are similar but not identical. For example, as a result of recent (lineagespecific) endosymbiotic gene transfer events, a number of genes encoded in the plastid genome of tobacco are encoded in the nuclear genome of Chlamydomonas and vice versa (Figures 1 and 2).

Transformation Methods Although biolistic transformation has remained the method of choice for plastid transformation in both algae and embryophyte plants (Table 2), a few alternative transformation protocols have ←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Figure 2 Physical map of the tobacco (Nicotiana tabacum) plastid genome, drawn using the complete genome sequence as input (GenBank accession number NC_001879.2) in version 1.1 of the OrganellarGenomeDRAW software tool (106, 107). The gray arrows denote the direction of transcription for the two DNA strands of the (circularly mapping) genome, and the interior circle shows its tetrapartite structure. Abbreviations: IRA , inverted repeat A; IRB , inverted repeat B; LSC, large single-copy region; SSC, small single-copy region. www.annualreviews.org • Plastid Genetic Engineering

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Escherichia coli Acinetobacter baumannii Escherichia coli Plastid genome

Plastid genome

Plastid genome

Plastid genome

Plastid genome

Plastid genome

Neomycin phosphotransferase

Aminoglycoside phosphotransferases

Chloramphenicol acetyltransferase

β subunit of ATP synthase

H subunit of photosystem II

Plastid 16S rRNA (spectinomycin-resistant mutant alleles)

Plastid 16S rRNA (streptomycin-resistant mutant allele)

Plastid 23S rRNA (erythromycin-resistant mutant allele)

D1 protein of photosystem II (herbicide-resistant alleles)

nptII

aphA6

cat

atpB

psbH

rrn16

rrn16

rrn23

psbA

216

Bock Metribuzin resistance, DCMU resistance

Erythromycin resistance

Streptomycin resistance

Spectinomycin resistance

Photoautotrophy

Photoautotrophy

Chloramphenicol resistance

Kanamycin resistance (or amikacin resistance)

Kanamycin resistance

Spectinomycin resistance (and/or streptomycin resistance)

Selection



Chlamydomonas reinhardtii, Nicotiana plumbaginifolia (double selection with spectinomycin)

Chlamydomonas reinhardtii, Nicotiana tabacum, Solanum lycopersicum



Nicotiana tabacum

Chlamydomonas reinhardtii, Nicotiana tabacum

Nicotiana tabacum

Chlamydomonas reinhardtii, Marchantia polymorpha, Physcomitrella patens, Nicotiana tabacum, several other seed plant species

Examples of transformed species Remarks

Not applicable in multicellular plants

Recessive and therefore less efficient than dominant antibiotic resistance genes



Requires an appropriate mutant as recipient strain; not applicable in multicellular plants

Requires an appropriate mutant as recipient strain; not applicable in multicellular plants

Requires higher expression levels than aadA for efficient selection



The most efficient selectable marker known to date; not applicable in cereals

128, 138

127

127, 134

127, 131, 169

54

27, 91

104

11, 83

30

70, 170

Reference(s)

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Note that this is not a complete list of published marker genes; for example, markers that are based only on a single report and/or could not be confirmed by the author’s laboratory (or other laboratories; cf., e.g., 115) were not included. Abbreviation: DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea.

Escherichia coli

Aminoglycoside 3 -adenylyltransferase

aadA

Gene source

Gene product

ARI

Gene

Table 1 Selectable marker genes for plastid transformation


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Table 2 Selected species for which plastid transformation protocols have been developed and confirmed by at least two published reports


Species

Taxonomic classification

Selectable markers

Reference(s)


Chlorophyta, Chlamydomonaceae

Biolistic bombardment of cells, glass-bead-assisted DNA uptake

Transformation methods

Photosynthesis genes; aadA, aphA-6, rRNA alleles

See Table 1

Marchantia polymorpha

Marchantiophyta, Marchantiaceae

Biolistic bombardment of suspension culture cells (thalli)

aadA

37, 176

Physcomitrella patens

Bryophyta, Funariaceae

PEG treatment of protoplasts

aadA

166

Brassica oleracea (cauliflower, cabbage)

Spermatophyta, Brassicaceae

Biolistic bombardment of leaves, PEG treatment of protoplasts

aadA

105, 130

Glycine max (soybean)

Spermatophyta, Fabaceae

Biolistic bombardment of embryogenic callus tissue

aadA

51, 52

Nicotiana tabacum (tobacco)

Spermatophyta, Solanaceae

Biolistic bombardment of leaves (also suspension cells), PEG treatment of protoplasts

aadA, nptII, aphA-6, cat, rRNA alleles

Solanum tuberosum (potato)


Biolistic bombardment of leaves

aadA

155, 177

Solanum lycopersicum (tomato; formerly Lycopersicon esculentum)



aadA, rRNA alleles

131, 146

Lactuca sativa (lettuce)

Spermatophyta, Asteraceae


aadA

87, 101

See Table 1; 69, 100, 134

Species in which no integration of the transforming DNA into the plastid genome could be demonstrated (e.g., the alga Euglena gracilis), stable homoplasmy could not be achieved (e.g., rice), or no fertile plants could be recovered (e.g., Arabidopsis) have been omitted. Abbreviation: PEG, polyethylene glycol.

been developed. Typically, they require cell cultures and removal of the cell wall prior to transformation (or, alternatively, use of cell wall–deficient mutant strains), which makes the procedures technically more demanding, labor intensive, and time consuming. In Chlamydomonas, agitating cell wall–deficient cells in the presence of glass beads and transforming plasmid DNA can result in chloroplast transformation (54). Similarly, in multicellular plants, incubation of isolated protoplasts with the polyether polyethylene glycol (PEG) and plasmid DNA allows selection of stable chloroplast transformants (Table 2). In both cases, DNA uptake is likely to be facilitated by the close proximity of chloroplasts to the plasma membrane, which may allow the passage of DNA through three tightly appressed layers of membranes if their structure is sufficiently loosened by physical or chemical means. In multicellular plants, PEG-mediated plastid transformation requires subsequent regeneration of plants from (wall-less) protoplasts, which is a sensitive and lengthy process that is not well established for many plant species. In summary, biolistics is at present unrivaled in both speed and transformation efficiency. Although alternatives to the biolistic protocol are available, unless the costs of the instrumentation and/or intellectual property issues are a serious consideration, there is currently no compelling reason to move away from particle gun–mediated chloroplast transformation. www.annualreviews.org • Plastid Genetic Engineering

Protoplast: a wall-less plant cell, typically produced by enzymatic removal of the cell wall

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Selectable Markers


Aminoglycoside: a class of antibiotics that contain an aminomodified glycoside (sugar) and act as inhibitors of bacterial protein biosynthesis Polyploidy: the state of possessing more than two complete copies of the genetic information in a cell or a genetic compartment Homologous recombination: reciprocal exchange of nucleotide sequences between two similar or identical DNA molecules

Table 1 provides an overview of established selectable marker genes for primary selection of transplastomic cells. Historically, chloroplast transformation was developed by using endogenous chloroplast sequences as selectable markers: a photosynthesis-related gene that restores photoautotrophic growth in Chlamydomonas, and point mutations in the chloroplast 16S rRNA that confer antibiotic insensitivity to plastid translation in tobacco (Table 1). However, especially in seed plants, these markers quickly fell out of fashion when much more efficient transgene-based selectable markers were developed. A single antibiotic resistance marker, initially developed for Chlamydomonas and subsequently adapted for tobacco (70, 170), turned out to be a lucky strike: the aadA gene from the gut bacterium Escherichia coli. This gene encodes an aminoglycoside 3 -adenylyltransferase, an enzyme that inactivates several antibiotics of the aminoglycoside type through covalent modification (i.e., attachment of an AMP residue). Importantly, spectinomycin and streptomycin, two aminoglycoside antibiotics that act as potent inhibitors of plastid translation, are efficient substrates of the AadA enzyme. Spectinomycin selection turned out to be particularly effective because of the high specificity of the drug to the chloroplast 70S ribosome and its low mutagenic and other side effects (Figure 3). Although several alternative selectable marker genes have been developed over the years (Table 1), the aadA gene is still superior to all of them. Besides the high specificity of spectinomycin as an inhibitor of plastid translation, the high enzymatic activity of the AadA protein is likely to also contribute to the unparalleled efficiency of the aadA marker gene in combination with spectinomycin selection. In addition to positive selectable markers that facilitate the selection of transplastomic cells, a few negative selectable marker genes have been established in chloroplasts (66, 151). These confer conditional lethality and are likely to become useful in genetic screens for regulators of plastid gene expression.

Integration of Foreign DNA into the Plastid Genome Stable transformation of chloroplasts requires (a) integration of the transforming DNA into the resident plastid DNA and (b) elimination of all untransformed copies of the highly polyploid plastid genome. Integration of foreign DNA into the plastid genome appears to occur exclusively by homologous recombination. This is reflected in the design of vectors for plastid transformation: The vectors must contain flanking regions of homology to the targeted integration site in the plastid genome (Figure 4). The efficiency of targeting is positively correlated to the length of these flanking regions (41), but it is generally believed that flank sizes of 0.5–1 kb are sufficient and that the transformation frequency does not increase much if larger flanks are used. Homologous recombination in chloroplasts is remarkably efficient, and usually only the final recombination products resulting from double-crossover events are detectable (Figure 4). A specific challenge associated with chloroplast transformation lies in the high degree of polyploidy of the plastid genome, with up to 1,000 copies or more of the plastid DNA being present in a single cell. It is generally assumed that the primary transformation event changes only one (or at most a few) genome copies. Consequently, transplastomic cells are initially heteroplasmic and contain a mixed population of wild-type genome copies and transformed genome copies. The genomes segregate freely upon subsequent rounds of cell division and organelle division. In the absence of selection, heteroplasmy of randomly segregating DNA molecules is genetically unstable, and sooner or later homoplasmic cells (harboring only one of the two genome types) will arise. To prevent the loss of the transgenic plastid genome (or transplastome), transplastomic cell lines are continuously propagated under selection pressure until all residual wild-type genomes are 218

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a

b

c

d

e

f

Figure 3 Generation of transplastomic potato (Solanum tuberosum) plants. (a) Preparation of leaves for biolistic transformation. Young leaves of potato plants grown under aseptic conditions are arranged to cover the surface of a standard petri dish. (b) Exposure of biolistically bombarded leaf explants to a spectinomycincontaining regeneration medium. (c) Selection of primary transplastomic lines. After 11 weeks of selection on spectinomycin-containing medium, the leaf explants are swollen and largely bleached owing to inhibition of chloroplast protein biosynthesis. The arrow points to a putative transplastomic clone that is resistant to spectinomycin and regenerates into plantlets. (d ) Additional regeneration round under spectinomycin selection to purify the transplastomic line to homoplasmy. Leaflets from regenerating plantlets of the previous regeneration round were exposed to a spectinomycin-containing plant regeneration medium and regenerated again into shoots (picture taken after 11 weeks). (e) Additional regeneration round initiated from stem sections. The efficiency of regeneration and purification to homoplasmy from stem explants is similar to that from leaf explants (picture taken after 11 weeks). ( f ) Growth of homoplasmic transplastomic plants under aseptic conditions on a synthetic medium. Note the development of microtubers from the roots.

eliminated. In seed plants, the homoplasmic transplastomic state is typically achieved after two or three additional rounds of plant regeneration under selection (i.e., in the presence of spectinomycin if aadA was used as the selectable marker gene) (15, 116). If the gene or mutation to be introduced into the plastid genome is not absolutely linked to the selectable marker gene (Figure 4b,c), extended periods of heteroplasmy bear the risk of gene conversion between the two genome types (89). Gene conversion can eliminate the unlinked mutation so that, in the end, genomes harboring only the selectable marker gene but not the desired mutation or transgene are obtained, even though initially the desired change was there. It is therefore of the utmost importance to (a) pass the transplastomic lines through the additional selection cycles quickly to bring them to the stable homoplasmic state as fast as possible, and (b) analyze many regenerants in each regeneration round (dozens or sometimes hundreds) by Southern blotting and/or amplification by polymerase chain reaction (PCR) and sequencing. The latter is particularly important to be able to act quickly upon seeing the first signs of gene conversion (by checking many more regenerants or by going back to the previous regeneration round). www.annualreviews.org • Plastid Genetic Engineering

Gene conversion: a nonreciprocal transfer of genetic information in which one DNA sequence replaces a homologous DNA sequence such that the two sequences become identical

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Left flank

Right flank

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Transformation vector Left flank

M

aadARight flank

c

Transformation vector I Left flank

M

Right flank

Transformation vector II Target gene

Target gene


ptDNA

M

aadA

Target gene

Target gene

Right flank

ptDNA

ptDNA

aadA

Left flank aadA

Target gene

M Target gene aadA

Transformed ptDNA

Transformed ptDNA Cotransformed ptDNA

Knockout • Gene disruption • Gene deletion (replacement)

Site-directed mutagenesis

Cotransformation

• Introduction of point mutations, insertions/deletions, etc.

• Gene knockout • Site-directed mutagenesis

Figure 4 Strategies for modifying endogenous genes in the chloroplast genome [plastid DNA (ptDNA)]. (a) Construction of a gene knockout by disruption of the reading frame with the selectable marker gene cassette (aadA; red box). Alternatively, the target gene ( green box) can be excised from a cloned ptDNA fragment and replaced with the marker cassette. “Left flank” and “right flank” denote flanking regions of homology in which homologous recombination can take place (blue boxes). Possible recombination events (double crossovers) leading to successful plastid transformation are indicated by dashed arrows. (b) Introduction of mutations into a plastid gene by site-directed mutagenesis. Note that recombination in the region between the mutation (M) and the aadA gene ( gray dashed arrow) results in uncoupling of the two genetic changes and, hence, produces transplastomic lines that harbor the selectable marker gene but not the desired mutation in the target gene. The frequency of appearance of such transplastomic lines depends on the distance of the mutation from the aadA marker relative to the size of the left flank. (c) Introduction of mutations into a plastid gene by cotransformation. If the target gene is embedded in a complex operon, it may not be possible to insert the aadA marker in close proximity without interfering with the expression of other genes in the operon. In these cases, the aadA gene can be incorporated into a separate transformation plasmid (transformation vector II) and targeted to a neutral region elsewhere in the genome. Cointegration of the aadA gene and the (unselected) mutation in the target gene (supplied on transformation vector I) will occur in 5–20% of the transplastomic clones (95).

A common error in the analysis of transplastomic lines is to mistake promiscuous plastid DNA in the nucleus or the mitochondrion for heteroplasmy. Weak wild-type-like hybridization signals in DNA gel blot analyses or wild-type-like bands in PCR assays that persist over the regeneration rounds often come from plastid DNA transferred to the nuclear or mitochondrial genome. In these cases, homoplasmy can be verified by Southern blots with purified plastid DNA and/or by crosses and segregation assays that demonstrate a lack of phenotypic segregation in the next generation (73, 96).

Cotransformation Particle bombardment allows the simultaneous transformation of cells with multiple vectors. Although one might think that plastid transformation is so inefficient that the recipient chloroplast 220

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usually takes up only a single plasmid molecule, successful cotransformation with two plasmids has been demonstrated in both Chlamydomonas and tobacco (31, 91). Importantly, recovery of cotransformants does not require double selection for both transformation plasmids, thus allowing targeting of the selectable marker gene to one region of the plastid genome while introducing a genetic change (or another transgene) into a totally different region of the genome (Figure 4c). The frequency of cointegration of the unselected transgene or mutation is somewhat variable. Cotransformation frequencies between 5% and 20% have been reported in tobacco (31, 95), and it seems possible that mutations entailing negative phenotypic consequences result in low frequencies. Cotransformation approaches are particularly useful if defined changes are to be introduced into large, complex operons, where linkage to the selectable marker gene is not possible or bears the risk of interfering with transcription and/or RNA processing (95) (Figure 4c). Surprisingly, it is also possible to cotransform the nucleus and the plastid at the same time (58). If particles are simultaneously coated with a vector for nuclear transformation and a vector for plastid transformation, doubly transformed (transgenic and transplastomic) cells can be recovered. This type of cotransformation seems to require the presence of both plasmids on the same particle, because mixing particles that were individually coated with the two plasmids did not result in plastid-nucleus cotransformation. These findings have interesting implications regarding the mechanisms of biolistic DNA delivery and DNA uptake by the target compartment (58).

Episome: a DNA element that is not incorporated into the genome and can replicate autonomously

Transformable Species and Bottlenecks in Extending the Species Range After the initial success with plastid transformation in Chlamydomonas and tobacco, progress with extending the species range of the technology has been disappointingly slow. Table 2 lists species for which plastid transformation has been reproducibly obtained and confirmed by at least two independent reports. More than 25 years after the first report of plastid transformation in Chlamydomonas, the list of transformable species is still very short. Why is that so, and what are the bottlenecks in adapting chloroplast transformation protocols for new species? Plastid transformation is dependent on (a) a robust method of DNA delivery into the chloroplast, (b) the presence of an active homologous recombination machinery in the plastid, and (c) the availability of highly efficient selection and regeneration protocols for transplastomic cells. Although switching to a new species may require some optimization of the parameters of the biolistic procedure (especially adjustment of the particle velocity to the hardness and thickness of the cell wall and/or the leaf cuticle to penetrate), given the universality of particle gun–mediated transformation, it is generally assumed that the efficiency of DNA delivery does not pose a serious bottleneck. Homologous recombination is known to be very efficient in Chlamydomonas and seed plant plastids, but there is some uncertainty about its activity in other taxa. For example, in the unicellular flagellate Euglena gracilis, chloroplast transformation could be achieved, but the transforming DNA did not integrate into the resident genome and, hence, could only be maintained episomally by antibiotic selection (46). At least in seed plants, where biolistic bombardment requires little species-specific optimization (or is already established for transformation of the nuclear genome) and homologous recombination activity in the plastid is unlikely to be limiting, the efficiency of the tissue culture, selection, and regeneration procedures is considered the most serious bottleneck to plastid transformation. In addition to being easy to grow in tissue culture, tobacco has the great advantage of remaining somatically diploid, theoretically allowing plant regeneration from every single cell. Most other plants undergo somatic endopolyploidization or irreversible cell differentiation during leaf development, making it very difficult to select transplastomic cell lines and/or regenerate fertile www.annualreviews.org • Plastid Genetic Engineering

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Callus: an unorganized growing and dividing mass of plant cells whose formation in vitro can be induced by treatment of tissue explants with phytohormones Cybrid: a eukaryotic cell produced by the fusion of a whole cell with an enucleated cell (cytoplast)

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transplastomic plants (157). Therefore, success with plastid transformation in seed plants has been limited largely to species for which facile cell and tissue culture systems are available (Table 2). As described above, spectinomycin resistance conferred by chimeric aadA genes presently represents by far the most efficient selection principle for transplastomic cell lines (Table 1). Unfortunately, not all plant and algal species are equally sensitive to spectinomycin, and some species are even entirely insensitive to the drug. For example, a point mutation that is known to confer spectinomycin resistance in tobacco is naturally present in the plastid 16S rRNA genes of all cereals. Other inhibitors of plastid translation, such as streptomycin and kanamycin, do not provide good alternatives, because they do not sufficiently strongly inhibit callus growth in the dark (104). This is unfortunate, because the most efficient (nuclear) transformation protocols for cereals rely on bombardment of callus propagated in the dark, and selection of transgenic cell lines is conducted in the dark (to prevent terminal cell differentiation). Thus, although biolistic nuclear transformation is routine in many cereals, transformation of the plastid genome is likely to require novel selectable markers and/or the development of entirely new tissue culture and regeneration protocols. In conclusion, similar to plant nuclear transformation, there is no universal protocol for plastid transformation. Development of a plastid transformation protocol for a new species represents a significant challenge that involves tedious optimization work, especially with respect to the tissue culture and selection procedures involved. Unfortunately, this optimization work is exceedingly laborious and time consuming and is based largely on the trial-and-error principle.

Transfer of Transgenic Plastids Between Species An alternative to developing a plastid transformation protocol for a new species would be to transfer the transgenic chloroplasts from an easy-to-transform species into cells of a nontransformable species. A technically challenging way to achieve this involves generating cybrids (cytoplasmic hybrids) by using protoplast fusion techniques. To transfer transgenic chloroplasts from a transformable into a recalcitrant species, protoplasts must be prepared from both species. The nuclear genome in the protoplasts of the transformable species then needs to be destroyed (for example, by X-ray or γ-ray irradiation). Heterologous fusion of protoplasts from the two species produces cells with the nuclear genome of the recalcitrant species and the chloroplast (and mitochondrial) genomes of both species. Subsequent plant regeneration from fused protoplasts in the presence of the selection agent that kills nontransgenic chloroplasts gives rise to plants harboring the nuclear genome of the recalcitrant species and the chloroplast genome of the transformable species. Although proof-of-concept studies have demonstrated that this approach works (97, 135, 156), it is technically demanding and limited in applicability, because for species recalcitrant to plastid transformation, sufficiently efficient protoplast isolation, fusion, and regeneration protocols are usually not available. The recent discovery that chloroplast DNA moves from cell to cell in tissue grafts (162) opened up the exciting possibility of exploiting this process for the transfer of plastid transgenes between species. Compared with protoplast fusion and regeneration techniques, plant regeneration from excised graft sites is fast and simple (in that it requires only explant exposure to a selective regeneration medium) and is therefore available for many more plant species. The movement of plastid DNA across graft sites was initially demonstrated for two tobacco cultivars (162) and was subsequently shown to also occur between different species (164, 173). Transfer of DNA between grafted plants is an asexual process and, thus, represents a form of horizontal gene transfer (lateral gene transfer). Analysis of the transferred DNA sequences revealed that entire genomes and presumably entire organelles are transferred, qualifying this process as horizontal genome transfer 222

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(65, 162, 164). Because grafting is not restricted by species boundaries, it can potentially be used to transfer transgenic plastids even between rather distantly related species. However, the tight coevolution between the plastid genome and the nuclear genome makes it unlikely that plastids will function properly when combined with an alien nucleus from a distantly related species (72, 150). Therefore, the transfer of transformed plastid genomes between plants will likely remain restricted to the movement from readily transformable model cultivars used in research to elite cultivars grown commercially, or from a transformable species into a related recalcitrant species (from the same genus or family). Because the molecular determinants of plastome-genome incompatibilities are still largely unknown, the functionality of a plastid genome transferred into a new host cell cannot be reliably predicted and thus needs to be determined experimentally (72).

RNA editing: a posttranscriptional RNA processing step leading to the alteration of specific nucleotides in an mRNA molecule (in chloroplasts of seed plants by C-to-U conversion)


THE TOOLBOX FOR PLASTID GENOME ENGINEERING Over the years, the community has assembled a large toolbox for plastid genome engineering, especially in the two model species Chlamydomonas and tobacco. The following sections briefly review the parts and molecular tools that are relevant to the construction of transformation plasmids.

Vector Backbones Because integration of foreign DNA into the chloroplast genome relies on homologous recombination, there are no universal vectors for plastid transformation. The flanking regions required for targeting (Figure 4) must have sufficiently high sequence homology to the resident plastid genome to allow efficient homologous recombination. In the plastid genomes of seed plants, gene content and gene order are well conserved, and the nucleotide substitution rate is 3–4 times lower than in the nuclear genomes (49). This high degree of genome conservation usually allows the use of plastid transformation vectors for closely related species (88, 146), avoiding the need to construct species-specific vectors. However, a note of caution needs to be sounded here: RNA editing patterns can differ even between closely related species (85), and heterologous RNA editing sites often remain unprocessed when introduced into another species (22, 150). It is, therefore, advisable to carefully check the sequences of the flanks before using a vector for plastid transformation in a heterologous species. Numerous vector systems have been developed for plastid transformation in both Chlamydomonas and tobacco (e.g., 11, 78, 146, 192). Several reviews have discussed frequently used vectors and considerations for vector choice and vector design (e.g., 17, 110), and I refer interested readers to these articles for more information.

Promoters and Untranslated Regions A typical expression cassette for plastid transgenes consists of a promoter and a 5 untranslated region (UTR) upstream of the coding region and a 3 UTR downstream. Promoters recognized by the plastid-encoded RNA polymerase are of the bacterial type and usually confer significantly higher expression levels than the phage-type promoters recognized by the nucleus-encoded RNA polymerase (74). The 5 UTR contains the ribosome-binding site, also referred to as the ShineDalgarno sequence. It engages in complementary base pairing with the 3 end of the 16S ribosomal RNA and thereby mediates translation initiation. It is important to know that, as in bacteria, the spacing between the Shine-Dalgarno sequence and the initiation codon is absolutely critical to the efficiency of translation initiation in plastids (36, 47, 59). The 3 UTR confers transcript stability, typically by folding into a stable stem-loop-type RNA secondary structure (165). www.annualreviews.org • Plastid Genetic Engineering

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A large number of plastid promoters and UTRs conferring different strengths of transgene expression have been described (e.g., 56, 78, 160, 172), including some that confer extraordinarily high transgene expression levels. Owing to the prevalence of translational regulation in plastids (53), the choice of the 5 UTR is of particular importance. Interestingly, the 5 UTR from gene 10 of bacteriophage T7 proved to be superior to all plastid 5 UTRs and confers extreme transgene expression levels of up to 70% of the total soluble protein in tobacco (98, 132, 186). For reasons that are not entirely clear, the protein accumulation levels attainable in Chlamydomonas chloroplasts are much lower: Expression levels in the 1–5% range are reached only in rare cases and upon use of nonphotosynthetic mutant strains (117). An important general consideration in the choice of expression elements is that the repeated use of identical elements should be avoided, especially if they are larger than 200–300 base pairs. Using several copies of the same element in a directly repeated orientation is particularly dangerous because homologous recombination between them induces deletions (84), and indirectly repeated copies can cause inversions in the genome (also referred to as flip-flop recombination) (144). A recent comprehensive review article has summarized all factors known to affect the level of plastid transgene expression and some resulting general guidelines for vector design (17).


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Reporter Genes Several reporter genes suitable for monitoring gene expression (e.g., in promoter-reporter gene fusions) have been adapted for expression in chloroplasts of Chlamydomonas and seed plants. These include β-glucuronidase (14, 159), luciferases (118, 122), and the green fluorescent protein (GFP) and its derivatives with modified fluorescence properties (28, 142, 155). Especially in seed plants, GFP has quickly displaced all other reporters owing to the high expression levels attainable with both GFP and GFP fusion proteins and the low background fluorescence in most species (29, 126, 155). Selected applications that have provided new insights into the mechanisms and regulation of plastid gene expression are discussed below.

Operon Expression Plastid genes are arranged in gene clusters that are cotranscribed and, by analogy to bacteria, are called operons (Figures 1 and 2). The molecular mechanisms of operon expression in plastids are considerably more complex than in bacteria. Plastid operons are often transcribed from multiple promoters (including additional operon-internal promoters) and require extra processing steps, such as intron splicing and mRNA editing. Moreover, in contrast to bacteria, polycistronic primary transcripts often undergo posttranscriptional cleavage into monocistronic (or oligocistronic) units. A growing body of evidence suggests that, in many cases, intercistronic cleavage is functionally important to ensure efficient translation (47, 80). The reasons for this are not entirely clear, but aberrant RNA secondary structure formation (80) and a striking 5 -to-3 decline in the efficiency at which the individual cistrons of a polycistronic RNA are translated (47) may be involved. Although the requirement for intercistronic processing seems to be sequence context dependent (in that some operons are less dependent on it than others), it is advisable to take processing into consideration when constructing synthetic operons (109). Intercistronic processing can be induced by small sequence elements that fold into stem-loop-type RNA secondary structures and can be derived from processing sites in endogenous chloroplast operons. One such element, dubbed the intercistronic expression element, functions in all heterologous sequence contexts tested so far and, moreover, is short enough to be usable in multiple copies within the same synthetic operon without inducing unwanted homologous recombination (109, 149, 190). 224

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Inducible and Repressible Gene Expression A substantial number of genes encoded in the plastid genome are essential and, therefore, are not amenable to functional analysis by gene knockout (48, 149) (see below). Therefore, an inducible knockdown system would be a valuable tool for chloroplast reverse genetics. Also, the high-level expression of some transgenes (e.g., genes encoding hydrophobic proteins) has deleterious effects on plastid functions, including photosynthesis (76, 175, 189), which makes a robust system for inducible transgene expression highly desirable. Although many genes in the plastid genome respond to light at the level of transcription and/or translation, and the expression of some genes is turned off efficiently in the dark, the importance of light as an energy source and trigger of plant developmental processes prohibits the use of light as a stimulus for inducible or repressible (trans)gene expression in the chloroplast. Unfortunately, no other exogenous or endogenous cues (e.g., environmental factors or metabolites) are known that would regulate plastid genes qualitatively in an on/off manner. Therefore, inducible systems for plastids need to be constructed from heterologous components. Initial attempts to build such systems placed the inducible component in the nucleus, where efficient tools for inducible gene expression are available. A T7 RNA polymerase gene driven by an inducible promoter in the nucleus confers inducible expression in tobacco plastids if the protein is targeted to the plastid compartment by a suitable transit peptide and the target (trans)gene in the plastid genome is placed under the control of the T7 promoter (108, 119). A conceptually similar inducible system was developed for Chlamydomonas chloroplasts. Here, the nuclear transgene encodes a chloroplast protein that specifically binds to the 5 UTR of the psbD mRNA and is required for stable mRNA accumulation. Through the use of an inducible expression system in the nucleus, (trans)genes controlled by the psbD 5 UTR can be switched on or off at will by adding or removing the chemical inducer (140, 167). A more ambitious goal is to develop a chloroplast-only inducible expression system. This would have the advantage of not requiring nuclear transgenes that can outcross in field-grown plants and, therefore, represent a potential biosafety concern. The development of chloroplast-only systems has been attempted in tobacco, and two such systems have been described. One is based on the lac repressor (LacI) from Escherichia coli and isopropyl β-D-1-thiogalactopyranoside (IPTG) as a chemical inducer of transcription (125); the other relies on a synthetic riboswitch that is responsive to theophylline, a simple plant-derived secondary metabolite (179). Presently, both systems are somewhat limited in their induction range and are not nearly as efficient as inducible expression systems in bacteria and in the nuclei of eukaryotes.

Riboswitch: an RNA sensor that regulates gene expression (positively or negatively, transcriptionally or translationally) in response to the binding of a small molecule, typically a metabolite Chromoplast: a plastid type specialized in the storage of (colored) carotenoids; it occurs, for example, in flowers and fruits Amyloplast: a colorless plastid type specialized in the storage of starch; it occurs, for example, in roots and tubers

Expression in Nongreen Tissues The chloroplast genome is highly expressed in photosynthetic tissues, and although there are only approximately 100 proteins encoded in the plastid DNA (Figures 1 and 2), their contribution to the total protein content of green leaves can amount to more than 50%. Because most of the highly expressed plastid genes encode components of the photosynthetic apparatus (thylakoid membrane proteins and the large subunit of RuBisCO), the demand for plastid gene expression capacity in nonphotosynthetic tissues is dramatically lower. Consequently, the gene expression machinery is much less active in nongreen plastid types, such as chromoplasts and amyloplasts (86, 178). For a long time, it was thought that this problem was impossible to overcome and that high-level expression of plastid transgenes could be achieved only in photosynthetically active chloroplasts. However, recent genome-wide analyses of plastid gene expression at the transcriptional and translational levels (transcriptomics and translatomics) identified a small number of plastid genes that www.annualreviews.org • Plastid Genetic Engineering

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Site-specific recombination: a type of genetic exchange that involves specialized recombination enzymes and specific DNA sequences (recognition sequences)

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remain expressed at the RNA level or at the level of protein synthesis in chromoplasts of tomato fruits and amyloplasts of potato tubers (86, 178). This finding has facilitated the design of chimeric expression elements in which a plastid promoter from a gene that is still actively transcribed in nonphotosynthetic plastid types (but may be downregulated at the translational level) is combined with a 5 UTR from an mRNA that is associated with translating ribosomes (polysomes) in nongreen plastids (18). This strategy was highly successful and resulted in the identification of promoter-UTR combinations that dramatically improved transgene expression levels in roots, tubers, and fruits (29, 177, 188), with the best-performing constructs reaching 1% of the total fruit protein in tomato.

Marker Excision


For some commercial applications, it may be desirable to remove the selectable marker gene from the plastid genome after transformation and attainment of homoplasmy. Also, because of the superior performance of the aadA marker, its posttransformation removal can be useful to facilitate its repeated use in supertransformation experiments (i.e., transformation of an already transplastomic plant). Several techniques for marker gene elimination have been developed for both Chlamydomonas and tobacco. These methods (a) take advantage of the endogenous homologous recombination activity of the plastid to mediate marker gene deletion; (b) utilize site-specific recombination systems, such as the Cre/loxP system, to induce marker gene excision; or (c) employ cotransformation and genome segregation approaches (38, 62, 84, 92, 185). Methodological details and specific advantages and disadvantages of each technique have been reviewed recently (43, 112).

PLASTID TRANSFORMATION IN BASIC RESEARCH The availability of a transformation technology has revolutionized nearly all areas of chloroplast research. It facilitated the in vivo analysis of processes in gene expression that previously could be studied only in vitro or not at all. Moreover, it made possible new approaches in functional genomics and opened up an entirely new field: experimental genome evolution. The sections below briefly review selected areas of basic research that have greatly benefited from this technology. Rather than attempting to give a complete account of what has been done, I focus here on general approaches and principles.

Reverse Genetics Because mutations in plastid genomes can be neither easily induced nor mapped, the possibility of using chloroplast transformation to make specific changes in plastid genes and open reading frames (Figure 4) was particularly exciting. Over the years, nearly all plastid genes have been targeted in Chlamydomonas and/or tobacco plastids (for a complete list, see 149). This work has provided a wealth of new information about plastid gene functions and structure-function relationships in chloroplast protein complexes (e.g., photosystems and ribosomes). One of the unexpected discoveries resulting from the reverse genetic analysis of conserved open reading frames was the identification of a small group of plastid genes that encode photosystem assembly factors (26, 96, 148). Their subsequent in vivo tagging facilitated the isolation of additional (nucleus-encoded) components of the photosystem assembly machinery (2, 136) and thereby provided a novel entry point into the challenging problem of photosystem biogenesis. Reverse genetic approaches in

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chloroplasts also furthered our understanding of the molecular mechanisms of gene expression, for example, by elucidating the contributions of wobbling and superwobbling to the reading of the genetic code (3, 4, 143). Figure 4 illustrates common strategies in reverse genetics. Gene inactivation is most easily achieved by insertion of the selectable marker cassette (to disrupt the reading frame) or replacement of the target gene with the selection marker (Figure 4a). More subtle changes (e.g., point mutations and small insertions or deletions) are introduced by integrating the selectable marker cassette into an adjacent intergenic spacer that, ideally, provides a neutral insertion site (Figure 4b). Because the plastid genome is rather gene dense, it is not always possible to identify such a neutral insertion site where the selectable marker gene does not interfere with the expression of neighboring genes. In this case, a cotransformation strategy can be employed (Figure 4c). Cotransformation also represents the most suitable strategy for gene knockout if the target gene is embedded in a large operon displaying a complex expression pattern (95). Systematic reverse genetic analyses in the plastid genomes of Chlamydomonas and tobacco revealed several essential genes. These genes encode components of the gene expression machinery (e.g., most ribosomal proteins and tRNAs) (1, 144) but also a few other functions (e.g., an essential protease subunit and the only plastid-encoded protein involved in fatty acid biosynthesis) (94, 114, 153). The essentiality of a plastid gene is revealed by the inability to purify transplastomic knockout lines to homoplasmy. This is evidenced by stable heteroplasmy in the presence of antibiotic selection, a situation also referred to as balancing selection, and by rapid loss of the transplastome in the absence of selection (48). The functional analysis of essential plastid genes requires the identification of hypomorphic mutations (which cause only a partial loss of function; Figure 4b), methods for inducible gene repression (see above) (140), or conditional knockout approaches (e.g., by inducible gene excision with a site-specific recombinase) (99). The availability of alternative selectable markers (Table 1) and the development of marker recycling techniques (see above) also allow the construction of double or triple knockouts and the introduction of mutations in multiple plastid genes. Double-knockout approaches are particularly useful to probe the functions of two nonessential components of a multiprotein complex in order to reveal possible molecular interactions or synergistic effects. Recently, double-knockout analysis has been applied to plastid genes encoding nonessential ribosomal proteins (63, 145). This work uncovered a striking case of synthetic lethality in that the combined knockout of two nonessential ribosomal protein genes (rpl33 and rps15) resulted in loss of autotrophic growth (55).

Synthetic lethality: a condition in which a combination of mutations in two or more genes leads to the death of a cell or organism, whereas each individual mutation does not

In Vivo Analysis of Gene Expression Investigations into the mechanisms and regulation of gene expression require faithful experimental systems that contain all relevant components and accurately reproduce the functional properties of the molecular machinery involved. Because the validity of in vitro studies is often questioned, and in vitro systems are not even available for some steps in gene expression, plastid transformation quickly became widely used as a tool for the in vivo analysis of gene expression and its regulation at all levels. Transgenic approaches are particularly useful to identify and dissect cis-acting elements involved in gene expression at the DNA, RNA, or protein level. Cis-acting elements constitute sequence determinants for transcription, RNA metabolism, translation, and protein stability. They can be studied systematically, for example, by serial deletions, stepwise terminal truncations, or scanning point mutageneses. To this end, the candidate cis-elements are typically placed into a heterologous sequence context and often additionally tethered to a reporter gene, such as


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uidA (encoding β-glucuronidase) or gfp (encoding GFP). Systematic dissection of cis-elements in vivo using plastid transformation has been done for nearly all steps in gene expression, including transcription (by analyzing promoter architecture) (5, 93), RNA processing and RNA stability (124, 190), RNA editing (19, 20, 34, 77), translation (60, 79, 129, 191), and protein half-life (7). These in vivo approaches also provided some limited information about trans-acting factors recognizing these cis-elements. For example, they revealed the existence of site-specific, compartment-specific, and species-specific RNA editing factors that are encoded in the nuclear genome (21, 22, 33, 168). However, the molecular identification of the trans-acting factors involved in plastid gene expression and its regulation requires combining transplastomic technologies with biochemical and/or genetic approaches. The latter would greatly benefit from progress with plastid transformation in Arabidopsis thaliana. A workable plastid transformation protocol for this model plant would allow transplastomic approaches to be combined with the power of Arabidopsis nuclear genetics, for example, by conducting mutant screens in transplastomic lines expressing fusions of plastid expression elements with reporter genes.


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Experimental Evolution The acquisition of the cyanobacterial endosymbiont that marked the origin of photosynthetic eukaryotes was followed by the large-scale migration of genes from the genome of the endosymbiont to the nuclear genome of the host cell. This process, also known as endosymbiotic gene transfer (EGT), presumably has been active for more than a billion years and is thought to be largely responsible for the dramatic reduction in size and coding capacity that plastid genomes have experienced (Figures 1 and 2). Circumstantial phylogenetic evidence and the presence of apparently recently transferred plastid sequences in the nucleus (so-called promiscuous DNA) have suggested that gene transfer from the plastid genome to the nucleus is still ongoing (9, 121). The possibility of placing antibiotic resistance genes into the plastid genome and developing rigorous selection schemes to visualize their migration to the nucleus enabled experimental evolution approaches to study EGT in real time. Chloroplast transformation with a kanamycin resistance gene fused to a nuclear promoter produced transplastomic lines that are sensitive to kanamycin, because the nuclear (eukaryotic-type) promoter is not recognized by the prokaryotic-type transcription machinery of the plastid. Subsequent selection for kanamycin resistance identified events in which the kanamycin cassette had migrated to the nucleus, where the promoter is recognized by RNA polymerase II. These proof-of-concept studies provided direct experimental proof that EGT is still active and, moreover, revealed an astoundingly high rate of plastid-to-nucleus gene transfer (81, 163). Molecular analyses of integration events in the nucleus determined the sizes of the transferred plastid sequences (82) and provided evidence of frequent instability of the nuclear loci resulting from EGT (152). Moreover, refined genetic screens yielded new insights into the molecular mechanisms of EGT, including the demonstration that direct DNA-mediated gene transfer (rather than RNA/cDNA-mediated transfer) represents the prevailing transfer pathway (64) and the identification of molecular events that convert transferred plastid genes into functional nuclear genes (161). Another evolutionary process that could be reconstructed experimentally with the help of transplastomic approaches is organelle capture (chloroplast capture), a puzzling evolutionary phenomenon in which organelle genomes are apparently transferred between species. Whereas previously only cytoplasmic substitution following an introgression event had been considered as a capture mechanism, the discovery of plastid transfer across graft junctions provides a straightforward asexual mechanism (by horizontal genome transfer; see above) that might explain at least some cases of organelle capture (162, 164, 173). 228

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PLASTID TRANSFORMATION IN PLANT BIOTECHNOLOGY To plant biotechnologists, the plastid genome provides an attractive site for the integration of transgenes. Although plastid transformation is technically more challenging than nuclear transformation, accommodation of the transgene in the plastid genome offers several notable advantages. These lie in (a) the unique precision of the genetic engineering process in plastids resulting from the highly efficient homologous recombination system; (b) the absence from plastids of epigenetic transgene silencing mechanisms that interfere with transgene expression and/or durable expression over generations; (c) the possibility of stacking multiple transgenes in synthetic operons (see above); (d ) the extraordinarily high expression levels attainable, especially in seed plant plastids; and (e) the increased biosafety resulting from maternal inheritance of the plastid genome in most crops. The latter greatly reduces the probability of outcrossing of transgenes by pollination (147, 171). In view of these advantages, many biotechnological applications of plastid transformation have been explored over the years. Below, I summarize progress in some of the most intensely researched areas of plastid biotechnology.

Engineering Resistances The high transgene expression levels obtainable by transgene expression from the plastid genome make transplastomic technology an attractive choice in resistance engineering, especially in cases where the level of resistance is directly correlated to the expression level of the resistance protein. This is, for example, the case with insect resistance conferred by insecticidal proteins from Bacillus thuringiensis (Bt toxins) and herbicide resistance conferred by expression of herbicide-insensitive metabolic enzymes (44, 120, 186). Most of the resistances engineered into chloroplasts so far are based on transgenes that had been successfully used in nuclear transformation before. For example, herbicide resistances were obtained to glyphosate (by expression of glyphosate-insensitive 5-enolpyruvylshikimate-3-phosphate synthases) (186); glufosinate (by expression of the detoxifying enzyme phosphinothricin acetyltransferase) (84, 111); sulcotrione and isoxaflutole (by overexpression of 4-hydroxyphenylpyruvate dioxygenase) (50); imidazolinone, sulfonylurea, and pyrimidinylcarboxylates (by expression of insensitive acetolactate synthases) (154); and D-amino acids (by expression of D-amino acid oxidases) (66). In addition to the often very high protein accumulation levels, the main advantage of resistance gene expression from the plastid genome lies in the increased transgene containment provided by maternal chloroplast inheritance. Extreme expression levels have been reached in some studies, for example, with Bt toxin genes expressed in tobacco chloroplasts (44). However, in one reported case, high-level Bt protein accumulation in transgenic plastids resulted in a growth phenotype (32), suggesting that the expression level of the resistance gene needs to be carefully optimized in order to provide sufficient protection without incurring a yield penalty.

Metabolic Engineering A number of studies have been performed to evaluate the potential of transplastomic technology for metabolic pathway engineering, with the goal of increasing the nutritional value of crop plants or exploiting plants as production factories for metabolites of commercial interest. Because the chloroplast harbors a large number of biosynthetic pathways (and is often referred to as the biosynthetic center of the plant cell), many biochemical pathways in plants are amenable to engineering via chloroplast transformation. An important restriction is that plastid-produced enzymes stay put and cannot be exported from the organelle. Thus, metabolic pathway engineering www.annualreviews.org • Plastid Genetic Engineering

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through plastid transformation requires the presence of an accessible metabolite pool within the chloroplast. The feasibility of metabolic engineering in transgenic plastids has been demonstrated for several nutritionally important biochemical pathways, including carotenoid biosynthesis (6, 75, 184) and fatty acid biosynthesis (39, 113). The possibility of transgene stacking in synthetic operons arguably represents the greatest attraction of the transplastomic technology for metabolic pathway engineering (149). Because initial attempts to express native bacterial operons directly in plastids have met with limited success, operons are usually reengineered to optimize them for efficient expression from the chloroplast genome. Common adaptations include replacements of UTRs and intercistronic spacers, codon usage adaptation, and incorporation of intercistronic processing elements (190) (see above). Two general strategies for building plastid operons have proven successful: extension of endogenous plastid operons by additional genes (25, 78) and construction of fully synthetic operons by using rational design principles (109). An operon extension strategy was applied to optimize the production of the renewable and biodegradable plastic polyhydroxybutyrate in tobacco chloroplasts by coexpression of three bacterial enzymes: β-ketothiolase, acetoacetylCoA reductase, and polyhydroxybutyrate synthase (25). Principles of synthetic operon design were worked out using the vitamin E (tocopherol) biosynthetic pathway as an example and the three pathway enzymes homogentisate phytyltransferase, tocopherol cyclase, and γ-tocopherol methyltransferase (109). Although in both these cases only three transgenes were combined in an operon, there is every reason to believe that much larger operons can be constructed according to similar principles.

Molecular Farming Molecular farming represents a growing area of biotechnology that aims to harness the huge potential of plants as inexpensive factories for the large-scale production of recombinant pharmaceutical proteins and industrial enzymes. Motivated by the appealing concept of edible vaccines and in view of the very high foreign protein accumulation levels attainable in transgenic chloroplasts, much of the initial work on molecular farming in plastids focused on the expression of antigens for subunit vaccines (40, 61, 67, 123, 175, 189). Indeed, a number of viral and bacterial antigens could be expressed to high levels and proved to be immunogenic when tested in animal models using different immunization routes, including, in some cases, oral immunization (8, 35, 42, 71, 103). However, despite many promising expression studies and encouraging results from animal tests, no chloroplast-produced vaccine has entered the clinic yet. More recently, several other therapeutic proteins were successfully expressed in transgenic plastids. These include, for example, antibody fragments for passive immunization and/or diagnostics (102), a blood coagulation factor potentially applicable in hemophilia treatment (182), and several endolysins (132, 133). The latter are lytic proteins encoded in the genomes of bacteriophages that infect and eventually kill pathogenic bacteria. Endolysins are necessary and sufficient to induce the lysis of a bacterial cell and, therefore, hold great promise as future next-generation antibiotics (24). Many of the efforts to produce industrial proteins in plastids have focused on biofuel enzymes. The surging interest in biomass as a renewable energy source has created a great demand for large quantities of cheap enzymes that catalyze the efficient degradation of lignocellulosic matter into fermentable sugars. Representatives of almost all known classes of enzymes involved in cell wall degradation have been tested in transplastomic expression studies, including various endo- and exocellulases, glucosidases, xylanases, pectate lyases, and cutinases (90, 137, 180, 181, 187). Many of these enzymes could be expressed to high levels, including some enzymes from 230

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thermophilic biomass-degrading microorganisms. At the industrial scale, thermophilic enzymes offer the advantage of allowing enzymatic processing of plant biomass at elevated temperatures, thus protecting the released sugars from unwanted consumption by contaminating microbes. From the many (successful and unsuccessful) attempts to express pharmaceutical and industrial proteins in plastids, some important lessons have been learned. With very few exceptions (184), there is little evidence for problems with RNA accumulation being causally responsible for the failure to express a foreign protein to reasonably high levels. Instead, in most cases, protein stability appears to be the factor that limits transgene expression (12, 13, 45, 57). Unfortunately, very little is known about the determinants of protein (in)stability in plastids (7, 45), making it currently impossible to predict the expression potential of transgenes in plastids. If a protein turns out to be unstable in the chloroplast stroma, a possible alternative would be targeting it to the thylakoid lumen, where a different set of proteases resides (10, 102, 174). In addition, careful evaluation of the physicochemical properties of the protein to be expressed is highly recommended. Proteins harboring hydrophobic domains that tend to associate with plastid membranes often lead to problems with transgene expression and cause deleterious phenotypic effects (by interfering with thylakoid biogenesis or function) (76). Concerning posttranslational protein modifications, it seems clear that disulfide bonds are usually faithfully formed in chloroplasts (10, 158). However, for recombinant proteins requiring other posttranslational modifications (e.g., glycosylation or specific phosphorylation patterns), the chloroplast may not be the best site of production. Finally, there is a striking difference between seed plants and Chlamydomonas in the efficiency of transgene expression and the attainable expression levels: As mentioned above, for reasons that are not entirely clear, the protein accumulation levels achieved in Chlamydomonas chloroplasts are often much lower than those obtained in seed plant plastids. Although recently some progress has been made with the production of pharmaceutical proteins in Chlamydomonas chloroplasts, the expression levels reached so far do not nearly approach those regularly obtained in seed plant chloroplasts. Moreover, maximum expression rates seem to require knockout of photosynthesis, thus losing one of the greatest attractions of using plant cells as production hosts of recombinant proteins (117, 141).

SUMMARY AND OUTLOOK Over the past two decades, chloroplast transformation technology has become a mainstay of molecular and genetic research on plastids. In addition, numerous proof-of-concept applications in metabolic engineering, molecular farming, and resistance engineering have impressively demonstrated the great potential of plastid genome engineering in biotechnology. However, although exciting strides have been made in developing new tools for plastid genome engineering and efficient transgene expression, progress in expanding the species range of the technology has remained slow. The vast majority of transplastomic research is still done with the two best-established model systems: Chlamydomonas and tobacco. From many fruitless efforts made in the past, the community has come to realize that developing workable plastid transformation protocols for new species represents a daunting task and requires long-term investments in tedious optimization work directed toward the improvement of transformation protocols, tissue culture procedures, and selection conditions. Both the academic and the industrial sectors seem to largely shy away from making these investments. Unfortunately, in the past, the field has also suffered from the publication of several misleading reports that made exaggerated claims that turned out to be irreproducible. These cases included false claims about transformation protocols for new species and alternative marker genes for selection of transplastomic cells. Some but not all of these faulty papers have been retracted. www.annualreviews.org • Plastid Genetic Engineering

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Therefore, any newcomer to the field is advised to study the literature very carefully and, in case of doubt, consult with two or three recognized experts before investing in laborious and time-consuming transformation projects that use nonstandard markers or species. Despite many promising proof-of-concept applications, commercial products from transgenic plastids have not yet entered the market, and presently no transplastomic plants are grown commercially in the field or in greenhouses. Nonetheless, the unique attractions of transplastomic technology make a persuasive case for continued investments in technology development. In addition to protocols for major crop species (especially cereals), improved tools for inducible expression and repression of plastid (trans)genes as well as tissue-specific and developmentally regulated expression systems rank high on the wish list of researchers in the field. The emerging field of plant synthetic biology will also benefit greatly from the continued expansion of the toolbox for plastid genome engineering (149). Because of its small genome size and the unique precision with which the genome can be manipulated, the chloroplast lends itself to the exploration of synthetic biology applications, including the design of synthetic genomes, the large-scale engineering of novel metabolic pathways into plants, and the radical redesign of metabolic networks, the photosynthetic apparatus, and perhaps even the entire genetic system of the plastid.


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SUMMARY POINTS 1. Stable transformation of the plastid genome is routinely possible in the green alga Chlamydomonas reinhardtii and a few species of seed plants, but still challenging or not yet possible in many model species and crops. 2. Integration of foreign DNA into the plastid genome occurs exclusively by homologous recombination. 3. Particle gun–mediated (biolistic) transformation is the most efficient method for introducing DNA into the plastid compartment. 4. A large toolbox for plastid genome engineering is available, including tools for cotransformation, selectable marker gene removal, efficient transgene expression in nongreen tissues, and multigene engineering with synthetic operons. 5. Transgenic plastids can be horizontally transferred between related species. 6. Chloroplast transformation allows the study of all steps in plastid gene expression in vivo and the functional analysis of plastid genes by reverse genetics. 7. High transgene expression levels, transgene stacking in operons, and increased transgene containment (due to maternal plastid inheritance in most species) make plastid genome transformation a highly attractive technology in metabolic pathway engineering, resistance engineering, and molecular farming.

DISCLOSURE STATEMENT The author is not aware of any affiliations, memberships, funding, or financial holdings that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS I thank Claudia Hasse for photography and Dr. Stephanie Ruf (both from the Max-Planck-Institut fur ¨ Molekulare Pflanzenphysiologie) for discussion, help with artwork, and critical reading of the 232

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manuscript. I apologize to colleagues whose work could not be discussed because of space constraints. Work on plastid transformation in my laboratory is supported by grants from the European Union (EU-FP7 DISCO 613513 and COST Actions FA0804 and FA1006), the Deutsche Forschungsgemeinschaft (FOR 2092 and BO 1482/17-1), the Human Frontiers Science Program (RGP0005/2013), and the Max Planck Society.


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Annual Review of Plant Biology Volume 66, 2015


From the Concept of Totipotency to Biofortified Cereals Ingo Potrykus p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1 The Structure of Photosystem II and the Mechanism of Water Oxidation in Photosynthesis Jian-Ren Shen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p23 The Plastid Terminal Oxidase: Its Elusive Function Points to Multiple Contributions to Plastid Physiology Wojciech J. Nawrocki, Nicolas J. Tourasse, Antoine Taly, Fabrice Rappaport, and Francis-André Wollman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p49 Protein Maturation and Proteolysis in Plant Plastids, Mitochondria, and Peroxisomes Klaas J. van Wijk p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p75 United in Diversity: Mechanosensitive Ion Channels in Plants Eric S. Hamilton, Angela M. Schlegel, and Elizabeth S. Haswell p p p p p p p p p p p p p p p p p p p p p p p p 113 The Evolution of Plant Secretory Structures and Emergence of Terpenoid Chemical Diversity Bernd Markus Lange p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 139 Strigolactones, a Novel Carotenoid-Derived Plant Hormone Salim Al-Babili and Harro J. Bouwmeester p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 161 Moving Toward a Comprehensive Map of Central Plant Metabolism Ronan Sulpice and Peter C. McKeown p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 187 Engineering Plastid Genomes: Methods, Tools, and Applications in Basic Research and Biotechnology Ralph Bock p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 211 RNA-Directed DNA Methylation: The Evolution of a Complex Epigenetic Pathway in Flowering Plants Marjori A. Matzke, Tatsuo Kanno, and Antonius J.M. Matzke p p p p p p p p p p p p p p p p p p p p p p p p p 243 The Polycomb Group Protein Regulatory Network Iva Mozgova and Lars Hennig p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 269

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The Molecular Biology of Meiosis in Plants Raphaël Mercier, Christine Mézard, Eric Jenczewski, Nicolas Macaisne, and Mathilde Grelon p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 297 Genome Evolution in Maize: From Genomes Back to Genes James C. Schnable p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 329 Oxygen Sensing and Signaling Joost T. van Dongen and Francesco Licausi p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 345 Diverse Stomatal Signaling and the Signal Integration Mechanism Yoshiyuki Murata, Izumi C. Mori, and Shintaro Munemasa p p p p p p p p p p p p p p p p p p p p p p p p p p p p 369 Annu. Rev. Plant Biol. 2015.66:211-241. Downloaded from www.annualreviews.org Access provided by Columbia University on 07/17/17. For personal use only.

The Mechanism and Key Molecules Involved in Pollen Tube Guidance Tetsuya Higashiyama and Hidenori Takeuchi p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 393 Signaling to Actin Stochastic Dynamics Jiejie Li, Laurent Blanchoin, and Christopher J. Staiger p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 415 Photoperiodic Flowering: Time Measurement Mechanisms in Leaves Young Hun Song, Jae Sung Shim, Hannah A. Kinmonth-Schultz, and Takato Imaizumi p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 441 Brachypodium distachyon and Setaria viridis: Model Genetic Systems for the Grasses Thomas P. Brutnell, Jeffrey L. Bennetzen, and John P. Vogel p p p p p p p p p p p p p p p p p p p p p p p p p p p 465 Effector-Triggered Immunity: From Pathogen Perception to Robust Defense Haitao Cui, Kenichi Tsuda, and Jane E. Parker p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 487 Fungal Effectors and Plant Susceptibility Libera Lo Presti, Daniel Lanver, Gabriel Schweizer, Shigeyuki Tanaka, Liang Liang, Marie Tollot, Alga Zuccaro, Stefanie Reissmann, and Regine Kahmann p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 513 Responses of Temperate Forest Productivity to Insect and Pathogen Disturbances Charles E. Flower and Miquel A. Gonzalez-Meler p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 547 Plant Adaptation to Acid Soils: The Molecular Basis for Crop Aluminum Resistance Leon V. Kochian, Miguel A. Pineros, ˜ Jiping Liu, and Jurandir V. Magalhaes p p p p p p p p p 571 Terrestrial Ecosystems in a Changing Environment: A Dominant Role for Water Carl J. Bernacchi and Andy VanLoocke p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 599

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Contents

Plastid genomics in horticultural species: importance and applications for plant population genetics, evolution, and biotechnology.

Chloroplast genomes: diversity, evolution, and applications in genetic engineering.

Nanofat grafting: basic research and clinical applications.

Construction Biotechnology: a new area of biotechnological research and applications.

Engineering synergy in biotechnology.

Effective extraction and assembly methods for simultaneously obtaining plastid and mitochondrial genomes.

Frontiers in biomedical engineering and biotechnology.

Targeting thyroid cancer microenvironment: basic research and clinical applications.

HGF Receptor and Its Ligand: Multitask Tools with Applications from Basic Research to Therapy.

Plant genetics: Plastid genomes - lost in translation?

Qualitative research in CKD: an overview of methods and applications.

Editorial: Latest methods and advances in biotechnology.

MIPs as Tools in Environmental Biotechnology.

Applications of biotechnology in nutrition.

Neurostimulation in Alzheimer's disease: from basic research to clinical applications.

Aquaporins in Salivary Glands: From Basic Research to Clinical Applications.

Editorial: Applications of STEM (Science, Technology, Engineering and Mathematics) Tools in Microbiology of Infectious Diseases.

Evolutionary Dynamics of Cryptophyte Plastid Genomes.

Analytical methods in biotechnology: introduction.

Environmental equity research: review with focus on outdoor air pollution research methods and analytic tools.

Present status of genetic engineering and biotechnology in South Korea.

Tissue engineering and regenerative medicine in basic research: a year in review of 2014.

The extracellular matrix: Structure, composition, age-related differences, tools for analysis and applications for tissue engineering.

Membrane transporter engineering in industrial biotechnology and whole cell biocatalysis.