Editing Chloroplast (section)

== Chloroplast DNA ==

{{Main|Chloroplast DNA}}
{{See also|List of sequenced plastomes}}

Chloroplasts, like other endosymbiotic organelles, contain a [[genome]] separate from that in the cell [[Cell nucleus|nucleus]]. The existence of [[chloroplast DNA]] (cpDNA) was identified biochemically in 1959,<ref name="Stocking-1959" /> and confirmed by electron microscopy in 1962.<ref name="Plaut-1962" /> The discoveries that the chloroplast contains ribosomes<ref name="Lyttleton-1962" /> and performs protein synthesis<ref name="Heber, U.-1962" /> revealed that the chloroplast is genetically semi-autonomous. Chloroplast DNA was first sequenced in 1986.<ref name="UniHamburg">{{cite web|title=Chloroplasts and Other Plastids|url=http://www.biologie.uni-hamburg.de/b-online/e23/23a.htm|publisher=University of Hamburg|access-date=27 December 2012|url-status=dead|archive-url=https://web.archive.org/web/20120925191743/http://www.biologie.uni-hamburg.de/b-online/e23/23a.htm|archive-date=25 September 2012}}</ref> Since then, hundreds of chloroplast genomes from various species have been [[DNA sequencing|sequenced]], but they are mostly those of [[land plants]] and [[green algae]]—[[glaucophytes]], [[red algae]], and other algal groups are extremely underrepresented, potentially introducing some [[Selection bias|bias]] in views of "typical" chloroplast DNA structure and content.<ref name="Sandelius-2009">{{cite book |last=Sandelius |first=Anna Stina | name-list-style=vanc |title=The Chloroplast: Interactions with the Environment |year=2009 |publisher=Springer |isbn=978-3-540-68696-5 |page=18 |url=https://books.google.com/books?id=aQR__H2XBnUC&pg=PA18}}</ref>

{{Chloroplast DNA|caption='''Chloroplast DNA''' Interactive gene map of chloroplast DNA from ''[[Nicotiana tabacum]]''. Segments with labels on the inside are on the B strand of [[DNA]], segments with labels on the outside are on the A strand. Notches indicate [[introns]].}}

=== Molecular structure ===

With few exceptions, most chloroplasts have their entire chloroplast genome combined into a single large circular DNA molecule,<ref name="Sandelius-2009" /> typically 120,000–170,000 [[base pair]]s long<ref name="Dann-2002">{{cite book |last=Dann |first=Leighton |url=http://www.bioscience-explained.org/ENvol1_2/pdf/ctDNAEN.pdf |title=Bioscience—Explained |publisher=BIOSCIENCE EXPLAINED |year=2002 |location=Green DNA |archive-url=https://web.archive.org/web/20101214102105/http://www.bioscience-explained.org/ENvol1_2/pdf/ctDNAEN.pdf |archive-date=14 December 2010 |url-status=live |name-list-style=vanc}}</ref><ref name="Clegg-1994" /><ref name="Shaw-2007" /><ref name="Milo">{{cite web | vauthors=Milo R, Phillips R |url=http://book.bionumbers.org/how-large-are-chloroplasts/|title=Cell Biology by the Numbers: How large are chloroplasts?|website =book.bionumbers.org |access-date=7 February 2017}}</ref> and a mass of about 80–130&nbsp;million [[dalton (unit)|daltons]].<ref name="Burgess-1989b">{{cite book |last=Burgess |first=Jeremy | name-list-style=vanc |title=An introduction to plant cell development |year=1989 |publisher=Cambridge university press |location=Cambridge |isbn=0-521-31611-1 |page=62 |url=https://books.google.com/books?id=r808AAAAIAAJ&pg=PA62}}</ref> While chloroplast genomes can almost always be assembled into a circular map, the physical DNA molecules inside cells take on a variety of linear and branching forms.<ref name="Sandelius-2009" /><ref>{{Cite journal |last=Green |first=Beverley R. |date=28 April 2011 |title=Chloroplast genomes of photosynthetic eukaryotes |url=https://onlinelibrary.wiley.com/doi/10.1111/j.1365-313X.2011.04541.x |journal=The Plant Journal |language=en |volume=66 |issue=1 |pages=34–44 |doi=10.1111/j.1365-313X.2011.04541.x |pmid=21443621 |issn=0960-7412}}</ref> New chloroplasts may contain up to 100 copies of their genome,<ref name="Dann-2002" /> though the number of copies decreases to about 15–20 as the chloroplasts age.<ref name="PlantBiochem-2005">{{cite book|title=Plant Biochemistry |edition=3rd |year=2005|publisher=Academic Press|page=[https://archive.org/details/isbn_9788131200032/page/517 517]|url=https://archive.org/details/isbn_9788131200032|url-access=registration |quote=number of copies of ctDNA per chloroplast. |isbn=978-0-12-088391-2}}</ref>

Chloroplast DNA is usually condensed into [[nucleoid]]s, which can contain multiple copies of the chloroplast genome. Many nucleoids can be found in each chloroplast.<ref name="Burgess-1989b" /> In primitive [[red algae]], the chloroplast DNA nucleoids are clustered in the center of the chloroplast, while in green plants and [[green algae]], the nucleoids are dispersed throughout the [[stroma (fluid)|stroma]].<ref name="Kobayashi-2002" /> Chloroplast DNA is not associated with true [[histone]]s, proteins that are used to pack DNA molecules tightly in eukaryote nuclei.<ref name="Campbell-2009c" /> Though in [[red algae]], similar proteins tightly pack each chloroplast DNA ring in a [[nucleoid]].<ref name="Kobayashi-2002">{{cite journal | vauthors=Kobayashi T, Takahara M, Miyagishima SY, Kuroiwa H, Sasaki N, Ohta N, Matsuzaki M, Kuroiwa T | display-authors=6 | title=Detection and localization of a chloroplast-encoded HU-like protein that organizes chloroplast nucleoids | journal=The Plant Cell | volume=14 | issue=7 | pages=1579–89 | date=July 2002 | pmid=12119376 | pmc=150708 | doi=10.1105/tpc.002717 | bibcode=2002PlanC..14.1579K }}</ref>

Many chloroplast genomes contain two [[inverted repeat]]s, which separate a long single copy section (LSC) from a short single copy section (SSC).<ref name="Shaw-2007">{{cite journal | vauthors=Shaw J, Lickey EB, Schilling EE, Small RL | s2cid=30501148 | title=Comparison of whole chloroplast genome sequences to choose noncoding regions for phylogenetic studies in angiosperms: the tortoise and the hare III | journal=American Journal of Botany | volume=94 | issue=3 | pages=275–88 | date=March 2007 | pmid=21636401 | doi=10.3732/ajb.94.3.275 }}</ref> A given pair of inverted repeats are rarely identical, but they are always very similar to each other, apparently resulting from [[concerted evolution]].<ref name="Sandelius-2009" /> The inverted repeats vary wildly in length, ranging from 4,000 to 25,000 [[base pair]]s long each and containing as few as four or as many as over 150 genes.<ref name="Sandelius-2009" /> The inverted repeat regions are highly [[Conserved sequence|conserved]] in land plants, and accumulate few mutations.<ref name="Shaw-2007" /><ref name="Kolodner-1979">{{cite journal |vauthors=Kolodner R, Tewari KK |date=January 1979 |title=Inverted repeats in chloroplast DNA from higher plants |journal=Proceedings of the National Academy of Sciences of the United States of America |volume=76 |issue=1 |pages=41–5 |bibcode=1979PNAS...76...41K |doi=10.1073/pnas.76.1.41 |pmc=382872 |pmid=16592612 |doi-access=free}}</ref>

Similar inverted repeats exist in the genomes of cyanobacteria and the other two chloroplast lineages ([[glaucophyta]] and [[rhodophyceae]]), suggesting that they predate the chloroplast.<ref name="Sandelius-2009" /> Some chloroplast genomes have since lost<ref name="Kolodner-1979" /><ref name="Palmer-1982" /> or flipped the inverted repeats (making them [[direct repeat]]s).<ref name="Sandelius-2009" /> It is possible that the inverted repeats help stabilize the rest of the chloroplast genome, as chloroplast genomes which have lost some of the inverted repeat segments tend to get rearranged more.<ref name="Palmer-1982">{{cite journal | vauthors=Palmer JD, Thompson WF | title=Chloroplast DNA rearrangements are more frequent when a large inverted repeat sequence is lost | journal=Cell | volume=29 | issue=2 | pages=537–50 | date=June 1982 | pmid=6288261 | doi=10.1016/0092-8674(82)90170-2 | s2cid=11571695 }}</ref>

===DNA repair and replication===

In chloroplasts of the moss ''[[Physcomitrella patens]]'', the [[DNA mismatch repair]] protein Msh1 interacts with the [[homologous recombination|recombination]]al [[DNA repair|repair]] proteins [[RecA]] and RecG to maintain chloroplast [[genome]] stability.<ref name="Odahara-2017">{{cite journal | vauthors=Odahara M, Kishita Y, Sekine Y | title=MSH1 maintains organelle genome stability and genetically interacts with RECA and RECG in the moss Physcomitrella patens | journal=The Plant Journal | volume=91 | issue=3 | pages=455–465 | date=August 2017 | pmid=28407383 | doi=10.1111/tpj.13573 | doi-access=free }}</ref> In chloroplasts of the plant ''[[Arabidopsis thaliana]]'' the [[RecA]] protein maintains the integrity of the chloroplast's DNA by a process that likely involves the recombinational repair of [[DNA damage (naturally occurring)|DNA damage]].<ref name="Rowan-2010">{{cite journal | vauthors=Rowan BA, Oldenburg DJ, Bendich AJ | title=RecA maintains the integrity of chloroplast DNA molecules in Arabidopsis | journal=Journal of Experimental Botany | volume=61 | issue=10 | pages=2575–88 | date=June 2010 | pmid=20406785 | pmc=2882256 | doi=10.1093/jxb/erq088 }}</ref>

[[File:CpDNA Replication.png|thumb|upright=1.65|Chloroplast DNA replication via multiple [[D-loop]] mechanisms. Adapted from Krishnan NM, Rao BJ's paper "A comparative approach to elucidate chloroplast genome replication."]]
The mechanism for chloroplast DNA (cpDNA) replication has not been conclusively determined, but two main models have been proposed. Scientists have attempted to observe chloroplast replication via [[electron microscopy]] since the 1970s.<ref name="Krishnan-2009">{{cite journal | vauthors=Krishnan NM, Rao BJ | title=A comparative approach to elucidate chloroplast genome replication | journal=BMC Genomics | volume=10 | issue=237 | page=237 | date=May 2009 | pmid=19457260 | pmc=2695485 | doi=10.1186/1471-2164-10-237 | doi-access=free }}</ref><ref name="Heinhorst-1993">{{cite journal | vauthors=Heinhorst S, Cannon GC |title=DNA replication in chloroplasts|journal=Journal of Cell Science|date=1993|volume=104|pages=1–9|doi=10.1242/jcs.104.1.1|url=https://aquila.usm.edu/cgi/viewcontent.cgi?article=7560&context=fac_pubs}}</ref> The results of the microscopy experiments led to the idea that chloroplast DNA replicates using a double displacement loop (D-loop). As the D-loop moves through the circular DNA, it adopts a theta intermediary form, also known as a Cairns replication intermediate, and completes replication with a rolling circle mechanism.<ref name="Krishnan-2009"/><ref name="Bendich-2004">{{cite journal |vauthors=Bendich AJ |date=July 2004 |title=Circular chloroplast chromosomes: the grand illusion |journal=The Plant Cell |volume=16 |issue=7 |pages=1661–6 |doi=10.1105/tpc.160771 |pmc=514151 |pmid=15235123|bibcode=2004PlanC..16.1661B }}</ref> Transcription starts at specific points of origin. Multiple replication forks open up, allowing replication machinery to transcribe the DNA. As replication continues, the forks grow and eventually converge. The new cpDNA structures separate, creating daughter cpDNA chromosomes.

In addition to the early microscopy experiments, this model is also supported by the amounts of [[deamination]] seen in cpDNA.<ref name="Krishnan-2009"/> Deamination occurs when an amino group is lost and is a mutation that often results in base changes. When adenine is deaminated, it becomes [[hypoxanthine]]. Hypoxanthine can bind to cytosine, and when the XC base pair is replicated, it becomes a GC (thus, an A → G base change).<ref name=Biocyclopedia>{{cite web|title=Effect of chemical mutagens on nucleotide sequence|url=http://www.biocyclopedia.com/index/genetics/mutations_molecular_level_mechanism/effect_of_chemical_mutagens_on_nucleotide_sequence.php|website=Biocyclopedia|access-date=24 October 2015}}</ref>
[[File:Adenine Deaminates to Guanine.png|thumb|left|upright=1.35|Over time, base changes in the DNA sequence can arise from deamination mutations. When adenine is deaminated, it becomes hypoxanthine, which can pair with cytosine. During replication, the cytosine will pair with guanine, causing an A --> G base change.]]

In cpDNA, there are several A → G deamination gradients. DNA becomes susceptible to deamination events when it is single stranded. When replication forks form, the strand not being copied is single stranded, and thus at risk for A → G deamination. Therefore, gradients in deamination indicate that replication forks were most likely present and the direction that they initially opened (the highest gradient is most likely nearest the start site because it was single stranded for the longest amount of time).<ref name="Krishnan-2009"/> This mechanism is still the leading theory today; however, a second theory suggests that most cpDNA is actually linear and replicates through homologous recombination. It further contends that only a minority of the genetic material is kept in circular chromosomes while the rest is in branched, linear, or other complex structures.<ref name="Krishnan-2009"/><ref name="Bendich-2004"/>

One of competing model for cpDNA replication asserts that most cpDNA is linear and participates in [[homologous recombination]] and replication structures similar to the linear and circular DNA structures of [[bacteriophage T4]].<ref name="Bendich-2004"/><ref name="Bernstein-1973">{{cite journal | vauthors=Bernstein H, Bernstein C | title=Circular and branched circular concatenates as possible intermediates in bacteriophage T4 DNA replication | journal=Journal of Molecular Biology | volume=77 | issue=3 | pages=355–61 | date=July 1973 | pmid=4580243 | doi=10.1016/0022-2836(73)90443-9 }}</ref> It has been established that some plants have linear cpDNA, such as maize, and that more species still contain complex structures that scientists do not yet understand.<ref name="Bendich-2004"/> When the original experiments on cpDNA were performed, scientists did notice linear structures; however, they attributed these linear forms to broken circles.<ref name="Bendich-2004"/> If the branched and complex structures seen in cpDNA experiments are real and not artifacts of concatenated circular DNA or broken circles, then a D-loop mechanism of replication is insufficient to explain how those structures would replicate.<ref name="Bendich-2004"/> At the same time, homologous recombination does not expand the multiple A --> G gradients seen in plastomes.<ref name="Krishnan-2009"/> Because of the failure to explain the deamination gradient as well as the numerous plant species that have been shown to have circular cpDNA, the predominant theory continues to hold that most cpDNA is circular and most likely replicates via a D loop mechanism.

=== Gene content and protein synthesis ===

The ancestral cyanobacteria that led to chloroplasts probably had a genome that contained over 3000 genes, but only approximately 100 genes remain in contemporary chloroplast genomes.<ref name="Milo" /><ref name="McFadden-2001">{{cite journal | vauthors=McFadden GI | title=Chloroplast origin and integration | journal=Plant Physiology | volume=125 | issue=1 | pages=50–3 | date=January 2001 | pmid=11154294 | pmc=1539323 | doi=10.1104/pp.125.1.50 }}</ref><ref name="Clegg-1994">{{cite journal | vauthors=Clegg MT, Gaut BS, Learn GH, Morton BR | title=Rates and patterns of chloroplast DNA evolution | journal=Proceedings of the National Academy of Sciences of the United States of America | volume=91 | issue=15 | pages=6795–801 | date=July 1994 | pmid=8041699 | pmc=44285 | doi=10.1073/pnas.91.15.6795 | bibcode=1994PNAS...91.6795C | doi-access=free }}</ref> These genes code for a variety of things, mostly to do with the [[protein synthesis|protein pipeline]] and [[photosynthesis]]. As in [[prokaryotes]], genes in chloroplast DNA are organized into [[operons]].<ref name="McFadden-2001" /> Unlike [[prokaryotic]] DNA molecules, chloroplast DNA molecules contain [[introns]] (plant [[mitochondrial DNA]]s do too, but not human mtDNAs).<ref name="Alberts-2002b" />

Among land plants, the contents of the chloroplast genome are fairly similar.<ref name="Shaw-2007" />

==== Chloroplast genome reduction and gene transfer ====

Over time, many parts of the chloroplast genome were transferred to the [[nuclear genome]] of the host,<ref name="Dann-2002" /><ref name="Clegg-1994" /><ref>{{cite journal | vauthors=Huang CY, Ayliffe MA, Timmis JN | title=Direct measurement of the transfer rate of chloroplast DNA into the nucleus | journal=Nature | volume=422 | issue=6927 | pages=72–6 | date=March 2003 | pmid=12594458 | doi=10.1038/nature01435 | bibcode=2003Natur.422...72H | s2cid=4319507 }}</ref> a process called ''[[endosymbiotic gene transfer]]''. As a result, the chloroplast genome is heavily [[genome reduction|reduced]] compared to that of free-living cyanobacteria. Chloroplasts may contain 60–100 genes whereas cyanobacteria often have more than 1500 genes in their genome.<ref name="Martin-2002">{{cite journal | vauthors=Martin W, Rujan T, Richly E, Hansen A, Cornelsen S, Lins T, Leister D, Stoebe B, Hasegawa M, Penny D | display-authors=6 | title=Evolutionary analysis of Arabidopsis, cyanobacterial, and chloroplast genomes reveals plastid phylogeny and thousands of cyanobacterial genes in the nucleus | journal=Proceedings of the National Academy of Sciences of the United States of America | volume=99 | issue=19 | pages=12246–51 | date=September 2002 | pmid=12218172 | pmc=129430 | doi=10.1073/pnas.182432999 | bibcode=2002PNAS...9912246M | doi-access=free }}</ref> Recently, a plastid without a genome was found, demonstrating chloroplasts can lose their genome during endosymbiotic the gene transfer process.<ref>{{cite journal | vauthors=Smith DR, Lee RW | title=A plastid without a genome: evidence from the nonphotosynthetic green algal genus Polytomella | journal=Plant Physiology | volume=164 | issue=4 | pages=1812–9 | date=April 2014 | pmid=24563281 | pmc=3982744 | doi=10.1104/pp.113.233718 }}</ref>

Endosymbiotic gene transfer is how we know about the [[#Secondary and tertiary endosymbiosis|lost chloroplasts]] in many CASH lineages. Even if a chloroplast is eventually lost, the genes it donated to the former host's nucleus persist, providing evidence for the lost chloroplast's existence. For example, while [[diatoms]] (a [[heterokontophyte]]) now have a [[red algal derived chloroplast]], the presence of many [[green algal]] genes in the diatom nucleus provide evidence that the diatom ancestor had a [[green algal derived chloroplast]] at some point, which was subsequently replaced by the red chloroplast.<ref name="Moustafa-2009" />

In land plants, some 11–14% of the DNA in their nuclei can be traced back to the chloroplast,<ref name="Nowack-2011" /> up to 18% in ''[[Arabidopsis]]'', corresponding to about 4,500 protein-coding genes.<ref name="Archibald-2006">{{cite journal | vauthors=Archibald JM | title=Algal genomics: exploring the imprint of endosymbiosis | journal=Current Biology | volume=16 | issue=24 | pages=R1033-5 | date=December 2006 | pmid=17174910 | doi=10.1016/j.cub.2006.11.008 | doi-access=free | bibcode=2006CBio...16R1033A }}</ref> There have been a few recent transfers of genes from the chloroplast DNA to the nuclear genome in land plants.<ref name="Clegg-1994" />

Of the approximately 3000 proteins found in chloroplasts, some 95% of them are encoded by nuclear genes. Many of the chloroplast's protein complexes consist of subunits from both the chloroplast genome and the host's nuclear genome. As a result, [[protein synthesis]] must be coordinated between the chloroplast and the nucleus. The chloroplast is mostly under nuclear control, though chloroplasts can also give out signals regulating [[gene expression]] in the nucleus, called ''[[retrograde signaling]]''.<ref name="Koussevitzky-2007">{{cite journal|vauthors= Koussevitzky S, Nott A, Mockler TC, Hong F, Sachetto-Martins G, Surpin M, Lim J, Mittler R, Chory J|display-authors=6|title=Signals from chloroplasts converge to regulate nuclear gene expression|journal=Science|volume=316|issue=5825|pages=715–9|date=May 2007|pmid= 17395793|doi= 10.1126/science.1140516|bibcode= 2007Sci...316..715K|s2cid=245901639}}
* {{cite magazine |author=Bob Grant |date=1 April 2009 |title=Communicating with chloroplasts |magazine=The Scientist |url=https://www.the-scientist.com/hot-paper/communicating-with-chloroplasts-44253}}</ref> Recent research indicates that parts of the retrograde signaling network once considered characteristic for land plants emerged already in an algal progenitor,<ref>{{Cite journal |last1=de Vries |first1=Jan |last2=Curtis |first2=Bruce A. |last3=Gould |first3=Sven B. |last4=Archibald |first4=John M. |date=10 April 2018 |title=Embryophyte stress signaling evolved in the algal progenitors of land plants |journal=Proceedings of the National Academy of Sciences |language=en |volume=115 |issue=15 |pages=E3471–E3480 |doi=10.1073/pnas.1719230115 |issn=0027-8424 |pmc=5899452 |pmid=29581286 |bibcode=2018PNAS..115E3471D |doi-access=free }}</ref><ref>{{Cite journal |last1=Nishiyama |first1=Tomoaki |last2=Sakayama |first2=Hidetoshi |last3=de Vries |first3=Jan |last4=Buschmann |first4=Henrik |last5=Saint-Marcoux |first5=Denis |last6=Ullrich |first6=Kristian K. |last7=Haas |first7=Fabian B. |last8=Vanderstraeten |first8=Lisa |last9=Becker |first9=Dirk |last10=Lang |first10=Daniel |last11=Vosolsobě |first11=Stanislav |last12=Rombauts |first12=Stephane |last13=Wilhelmsson |first13=Per K.I. |last14=Janitza |first14=Philipp |last15=Kern |first15=Ramona |date=July 2018 |title=The Chara Genome: Secondary Complexity and Implications for Plant Terrestrialization |journal=Cell |language=en |volume=174 |issue=2 |pages=448–464.e24 |doi=10.1016/j.cell.2018.06.033|pmid=30007417 |s2cid=206569169 |doi-access=free }}</ref><ref>{{Cite journal |last1=Zhao |first1=Chenchen |last2=Wang |first2=Yuanyuan |last3=Chan |first3=Kai Xun |last4=Marchant |first4=D. Blaine |last5=Franks |first5=Peter J. |last6=Randall |first6=David |last7=Tee |first7=Estee E. |last8=Chen |first8=Guang |last9=Ramesh |first9=Sunita |last10=Phua |first10=Su Yin |last11=Zhang |first11=Ben |last12=Hills |first12=Adrian |last13=Dai |first13=Fei |last14=Xue |first14=Dawei |last15=Gilliham |first15=Matthew |date=12 March 2019 |title=Evolution of chloroplast retrograde signaling facilitates green plant adaptation to land |journal=Proceedings of the National Academy of Sciences |language=en |volume=116 |issue=11 |pages=5015–5020 |doi=10.1073/pnas.1812092116 |issn=0027-8424 |pmc=6421419 |pmid=30804180 |bibcode=2019PNAS..116.5015Z |doi-access=free }}</ref> integrating into co-expressed cohorts of genes in the closest algal relatives of land plants.<ref>{{Cite journal |last1=Dadras |first1=Armin |last2=Fürst-Jansen |first2=Janine M. R. |last3=Darienko |first3=Tatyana |last4=Krone |first4=Denis |last5=Scholz |first5=Patricia |last6=Sun |first6=Siqi |last7=Herrfurth |first7=Cornelia |last8=Rieseberg |first8=Tim P. |last9=Irisarri |first9=Iker |last10=Steinkamp |first10=Rasmus |last11=Hansen |first11=Maike |last12=Buschmann |first12=Henrik |last13=Valerius |first13=Oliver |last14=Braus |first14=Gerhard H. |last15=Hoecker |first15=Ute |date=28 August 2023 |title=Environmental gradients reveal stress hubs pre-dating plant terrestrialization |journal=Nature Plants |volume=9 |issue=9 |pages=1419–1438 |language=en |doi=10.1038/s41477-023-01491-0 |pmid=37640935 |pmc=10505561 |bibcode=2023NatPl...9.1419D |issn=2055-0278}}</ref>

==== Protein synthesis ====

{{See also|transcription (genetics)|Translation (biology)|label 1=Transcription|label 2=translation}}
Protein synthesis within chloroplasts relies on two [[RNA polymerase]]s. One is coded by the chloroplast DNA, the other is of [[cell nucleus|nuclear]] origin. The two RNA polymerases may recognize and bind to different kinds of [[promoter (genetics)|promoters]] within the chloroplast genome.<ref name="Hedtke-1997">{{cite journal | vauthors=Hedtke B, Börner T, Weihe A | title=Mitochondrial and chloroplast phage-type RNA polymerases in Arabidopsis | journal=Science | volume=277 | issue=5327 | pages=809–11 | date=August 1997 | pmid=9242608 | doi=10.1126/science.277.5327.809 }}</ref> The [[ribosome]]s in chloroplasts are similar to bacterial ribosomes.<ref name="Harris-1994">{{cite journal | vauthors=Harris EH, Boynton JE, Gillham NW | title=Chloroplast ribosomes and protein synthesis | journal=Microbiological Reviews | volume=58 | issue=4 | pages=700–54 | date=December 1994 | pmid=7854253 | pmc=372988 | doi=10.1128/MMBR.58.4.700-754.1994 }}</ref>

{{Expand section|Genome size differences between algae and land plants, chloroplast stuff coded by the nucleus|date=January 2013}}

=== Protein targeting and import ===

{{See also|Translation (biology)|label 1=Translation}}

Because so many chloroplast genes have been moved to the nucleus, many [[protein]]s that would originally have been [[Translation (biology)|translated]] in the chloroplast are now synthesized in the cytoplasm of the plant cell. These proteins must be directed back to the chloroplast, and imported through at least two chloroplast membranes.<ref name="Soll-2004">{{cite journal | vauthors=Soll J, Schleiff E | title=Protein import into chloroplasts | journal=Nature Reviews Molecular Cell Biology | volume=5 | issue=3 | pages=198–208 | date=March 2004 | pmid=14991000 | doi=10.1038/nrm1333 | s2cid=32453554 | url=http://nbn-resolving.de/urn:nbn:de:bvb:19-epub-3587-4 }}</ref>

Curiously, around half of the protein products of transferred genes aren't even targeted back to the chloroplast. Many became [[exaptations]], taking on new functions like participating in [[cell division]], [[protein routing]], and even [[disease resistance]]. A few chloroplast genes found new homes in the [[mitochondrial genome]]—most became nonfunctional [[pseudogenes]], though a few [[tRNA]] genes still work in the [[mitochondrion]].<ref name="Martin-2002" /> Some transferred chloroplast DNA protein products get directed to the [[secretory pathway]],<ref name="Martin-2002" /> though many [[#Secondary and tertiary endosymbiosis|secondary plastids]] are bounded by an outermost membrane derived from the host's [[cell membrane]], and therefore [[topologically]] outside of the cell because to reach the chloroplast from the [[cytosol]], the [[cell membrane]] must be crossed, which signifies entrance into the [[extracellular space]]. In those cases, chloroplast-targeted proteins do initially travel along the secretory pathway.<ref name="Keeling-2010" />

Because the cell acquiring a chloroplast [[#Primary endosymbiosis|already]] had [[Mitochondrion|mitochondria]] (and [[peroxisomes]], and a [[cell membrane]] for secretion), the new chloroplast host had to develop a unique [[protein targeting system]] to avoid having chloroplast proteins being sent to the wrong [[organelle]].<ref name="Soll-2004" />

{{plain image with caption|File:Tetrapeptide structural formulae.svg| The two ends of a polypeptide are called the [[N-terminus]], or ''amino end'', and the [[C-terminus]], or ''carboxyl end''.<ref name="Campbell-2009a">{{cite book | vauthors=Campbell NA, Reece JB, Urry LA, Cain ML, Wasserman, Minorsky PV, Jackson RB |title=Biology | edition=8th | year=2009 |publisher=Benjamin Cummings (Pearson) | page=340 | isbn=978-0-8053-6844-4 }}</ref> This [[polypeptide]] has four [[amino acids]] linked together. At the left is the [[N-terminus]], with its [[amino group|amino]] (H<sub>2</sub>'''N''') group in green. The blue [[C-terminus]], with its [[carboxyl group]] ('''C'''O<sub>2</sub>H) is at the right.|370px|right|bottom|triangle|#00aa15}}

In most, but not all cases, nuclear-encoded chloroplast proteins are [[Translation (biology)|translated]] with a ''[[cleavable transit peptide]]'' that's added to the N-terminus of the protein precursor. Sometimes the transit sequence is found on the C-terminus of the protein,<ref name="Lung-2012">{{cite journal | vauthors=Lung SC, Chuong SD | title=A transit peptide-like sorting signal at the C terminus directs the Bienertia sinuspersici preprotein receptor Toc159 to the chloroplast outer membrane | journal=The Plant Cell | volume=24 | issue=4 | pages=1560–78 | date=April 2012 | pmid=22517318 | pmc=3398564 | doi=10.1105/tpc.112.096248 | bibcode=2012PlanC..24.1560L }}</ref> or within the functional part of the protein.<ref name="Soll-2004" />

==== Transport proteins and membrane translocons ====

After a chloroplast [[polypeptide]] is synthesized on a [[ribosome]] in the [[cytosol]], an enzyme [[enzyme specificity|specific]] to chloroplast proteins<ref name="Waegemann-1996">{{cite journal | vauthors=Waegemann K, Soll J | title=Phosphorylation of the transit sequence of chloroplast precursor proteins | journal=The Journal of Biological Chemistry | volume=271 | issue=11 | pages=6545–54 | date=March 1996 | pmid=8626459 | doi=10.1074/jbc.271.11.6545 | doi-access=free }}</ref> [[phosphorylates]], or adds a [[phosphate group]] to many (but not all) of them in their transit sequences.<ref name="Soll-2004" />
Phosphorylation helps many proteins bind the polypeptide, keeping it from [[protein folding|folding]] prematurely.<ref name="Soll-2004" /> This is important because it prevents chloroplast proteins from assuming their active form and carrying out their chloroplast functions in the wrong place—the [[cytosol]].<ref name="May-2000" /><ref name="Jarvis-2001">{{cite journal | vauthors=Jarvis P, Soll J | title=Toc, Tic, and chloroplast protein import | journal=Biochimica et Biophysica Acta (BBA) - Molecular Cell Research | volume=1541 | issue=1–2 | pages=64–79 | date=December 2001 | pmid=11750663 | doi=10.1016/S0167-4889(01)00147-1 | doi-access=free }}</ref> At the same time, they have to keep just enough shape so that they can be recognized by the chloroplast.<ref name="May-2000">{{cite journal | vauthors=May T, Soll J | title=14-3-3 proteins form a guidance complex with chloroplast precursor proteins in plants | journal=The Plant Cell | volume=12 | issue=1 | pages=53–64 | date=January 2000 | pmid=10634907 | pmc=140214 | doi=10.1105/tpc.12.1.53 | bibcode=2000PlanC..12...53M }}</ref> These proteins also help the polypeptide get imported into the chloroplast.<ref name="Soll-2004" />

From here, chloroplast proteins bound for the stroma must pass through two protein complexes—the [[TOC complex]], or ''[[translocon|'''t'''ranslocon]] on the '''o'''uter '''c'''hloroplast membrane'', and the [[TIC translocon]], or '''''t'''ranslocon on the '''i'''nner '''c'''hloroplast membrane [[translocon]]''.<!--Yes, that's what the source says--><ref name="Soll-2004" /> Chloroplast polypeptide chains probably often travel through the two complexes at the same time, but the TIC complex can also retrieve preproteins lost in the [[chloroplast intermembrane space|intermembrane space]].<ref name="Soll-2004" />