CHAPTER A plants phenotype is the result of thee major factors: its genotype {@ll the genes, or alleles, that determine the plant’ traits), the pat tem of epigenetic modifications of its DNA, and the environment in vuhich it lives. In Chapter 1 we reviewed the fundamental structure and function of DNA, its packaging into chramosomnes, and the two major phases of gene expression: transcription and translation. In this, chapter we will discuss haw the camposition of the genome beyond its genes influences the physiology and evolution of the organism First we will look at the organization of the nuclear genome and the non-gene elements it contains. Then we will turn to the cytoplasmic genomes, which are contained inside the mitochondris and plastids Next we will discuss the cellular mechinery needed to trenscribe and translate the genes into functional proteins, and we will see how gene expression is regulated both transcriptionally and posttranscription- ally. We will then introduce some of the tools used to study cene function, and we will frish up with a discussion of the use of genetic engineering in research and agriculture Nuclear Genome Organization As discussed in Chapter 1. the nuclear genome contains most of the genes required for the plant’s physiological functions, The first plant genome to be fully sequenced, in 2000, was that of a small dicotyledonous angiosperm called thale cress, or Armbidopsis thaliana36 CHAPTER 2 ‘The genome of A. thalta de up of only about 15 rillion base pairs (157 Mbp), which aro distributed over five chromosomes. (By contrast. the genorne of the lily Evi yrinca, the plant species with the largest known, genome, containe 2s many a5 88,000 Mbp.) Within its nuclear genome, A. fhaliana holds some 27.400 protein: coding genes and elmest another 5000 geres thatare lther pseudogenes (nonfunctional genos) ox parts of transpo: sons (mobile LINA elements that we will discuss later in this chapter), In addition to these genes, move than, 1300 genes that produce ner~protein-coding RNAs (acRNAs) are known in A. thaliana, Many of these RNAS are prob- ably involved in gene segulation, Both transposons and ncRNAs ane described in more dolail later in thie chapter ‘The plant genome, however, corsists of much more than genes, In this section we will lake a look at the chemical ‘makeup of the genome, then see how certain zegione of the genome correspond to specific functions, ‘The nuclear genome is packaged into chromatin The nuclear genome consists af DNA molecules that are wrapped mound histone proteins to forin beadlike stv tures called nucleosomes (Gee Chapter 1), DNA and his tones, together with other proteins that bina to the DNA are refered fo 98 chromatin (so0 Figure 110), Two typoe of chromatin can be distinguished: euchvovmtin and het crocvannti. Ustorcally these two types were diffe ated based on thei appeerance in light microscopy when slainedl with specific dyes, Heterochromatin s sual more tightly packaged and thus appears datkor than the less condensed euchromatin. Mos! gones that ar actively transcribed ina plant ae located within the euchromatic rogions of a chromo some, while many genes located in heterochromatic regions are not tran- seribed—these genes are inactive, or silent. Compared with euchromatin, hetetochometin is relatively gene pact: Hoicrochomatic regions include the centromeres, several so-called knobs, and the regions immediately adjacent to tho telomeces, oF chromosome enc, xaovn as the subtelomeric regions (Gil ial. 2008, Heterochromatictnacturasoften con sistot highly zepetitive DNA sequences, © tandem repeats blocks of nucleatice motifs of about 150 to 160 bp that are repeated aver and over. A second cla of repeats isthe dispersed repeats. One type of dispersed repeat is known a simple sequence repeats (SSK) ot mice FIGURE 2.1 nucleolar o Chromasomal lancmatle, including cont row shows the ten chre common lines ere sh complementary t certain chramesemal lance rescent dyes andl *be seen as green dorsner the mdale af the chromosomes, the nucleo- otidles that are repeated huncireds or even thousands of times. Another dominant group of dispersed repeats found: in heterochromatin is the “jumping gones,” or Fanspecons (Heslop-Harvison and Scamidl 2007), Centromeres, telomeres, and nucleolar arganizers contain repetitive sequences The most prominentstractiral lands are centromeres, tolonieres, and nucleolar organizers, These regions contain repetitive DNA sequences that can be made visible by Muareseent in situ: hybridization (FISH) (Kato etal. 2005), a technique that uses fluorescently labeled molecular probes—nsually fragments of DNA—that bind. specifically to the secqience to bo idantified (FIGURE 2.1). Centromeres are constriction of the chromosomes where sister chromatids achore to cach other and where spindle fibers aitach during cell division. The attachment of fibers to the centromere is mediated by the kinetcchare, a pro tein complex surrounding tho centromere (see Chapter 1), Centromeres consist of highly repetitive DNA regions and inactive transposable elements (Jiang et al. 2003; Ma etal 2007). Telomeras cre sequences located atthe ends of each chromesome. Telomeres act as caps on the chromosome ends end prevent loss of DNA during DNA replication, ‘The RNA molecules that make up ribosomes (tRNA) are transcribed irom nucleolar organizer (NO) regions Because ribosomes are composed mostly of RNA, and. because many ribosomes are needed for translation, itis not surprising thatINOs contain hundreds of repeated cap- jes of each FRNA gene, Depending on the plant species, ‘one or several nucleolar organizers are prevent within the ‘kson chromosemes eres, talomeres, end , can be used to identify individval cwromosomes. macoma pais 4 differant inbred! lino of mal wer fete, fom A186 to 873). DNA sequences (prcbeq) rks vor lala with fluo- sto chromosome preperatiors, The cearomeres rosatellites. These repeats consist of sequence motifs as short as two nucle lar erganize” a a largo” graen arcs on chromosome , and the telomeres as faint tec cots, most Clearly vsinle 2 the top of chromosomes 2-4, The larger blue areas are specific haterochromatic regions: From Kato ota. 2008genome (maize bas one, on chromosome 6: see Figure 2.1). Due to thelr repetitive nature and their high GC content, NOs can be ewen through a light microscope (alter stain ing) and thus can serve as ehromosome-spectfie markers. ‘These markers can be used to map phenotypic traits to specific chromosomal segions, Despite its repetitivenature, 1DNA (DNA that encades cRINA) is actively transcribed. ‘The rDNA of the nucleolar organizer, along with proteins that transcribe the :DNA and process the :RNA primary transcripts for assembly into ribosomes, forms a prominent spuclear structure called the nucleolus (see Figure 1.9. Transposons are mobile sequences within the genome (One dominant type of sepetitive DNA within the heter- luomatic regions of the genome isthe transposon. Transpo- sons, or hansposable elements, are also known as “ump ing genes” beceuse some of themhave the ability to insert a copy of themselves in a nev location within the genome. “There are two large classes of transposons: the retroele- sents or retroirar'sposons (Class 1), and the DNA trans posons (Class 2). These two classes are distinguished by their mode of replication and insertion into new location (FIGURE 2.2), Retrotransposons make an RNA copy of themselves, which is then reverse-transcribed into DNA before itis inserted elsewhere in the genome (see Figure 2.28), Because they do not normally leave their original location, but rather generate additional copies of them selves, active retrotransposons tend to multiply within the genome (Bickbushand Malik 2002}, DNA transposons, by contrast, move from one position to another using a ci-and-paste mechanism catalyzed by an enzyme that fs encoded within the transpason sequence. This enzyme, tansposase, splices out the fransposon and inserts it ols ‘wherein the genome, in mest cases Keeping the total trans- poson copy number the same (see Figure 2.2B) ‘Transposition inio a genc can result in mutations. Ifa transposon lands within a ceding region, the gene may be inactivated. Insertion of a transposon close to a gene can also ater that gene's expression pattern. For example, the transposon may disrupt the gene's normal regulatory ele- ments, preventing transcription of since transposons often , Accidental ‘formation of 2N gametes 2. Fusion of normal haploid gommoter — ‘ond 8 can lead to the formation of an interspecies hybrid. These hybrds ‘an be viable but ere usualy stele they undorge pantensou genome | cupliestion they can give rise to allowetapibids 3, Accidental of diploid gametex ean bscta an Suto or allotetraplie 4. Lass of duplicated genes over evolutionary time can rest in slow tlploidastion, in which polyploide Fevert back to 2 cipleld state FIGURE 2.8 Continuum in tho evolution of polyploid ‘species. Diploids can give rise to autapclypioids or al lopolyplcids by the machariems outlinad in Figure 2.5, Polyploids can rever: 1 diploidy via the gradual loss of thet was absorbed by another prokaryotic organism, Over time, this original endosymbiont evalved into an organelle that wasno longer ableto live on its own. The host ell, along vith its endosymbiant, gave rise toa lineage of cells that sete eble to use oxygen In aereblc metabotisn; these cells, in turn, eventually gave rise toall animal cells, Plant cells, according to this theory. arose when a second endosymibio- sis event took place. This time, mitochondrion-containing call engulfed a photcsynthetic cyanobacterium, which over time evolved inside the cell into the chloroplast Two main fines of evidence are often cited in support of the endosymbiotie theory. First, both mitochondria and chloroplasts are enclosed by an outer and an inner ‘membrane, and the inner membrane is continuous with additional membrane-bound compartments inside the Diploid ancestor 2 5) fae Diploid spaces 88 Normal fermation OF IN gametes spit. Accidentsl fornation of 2N gametes me oe fpoma Diplo duplicated genes over evolutionary time. Light purge un derlying ovals represent the nuclei of a species; colored cirelas inside nuclei represent entre genomes. (Mer Comnai 2005) organelle, This observation is consistent with the idea that eagulfinent of the original aerobic or photosynthetic cell through invagination of the plaaraa membrane of the prokaryotic hest cell lefta double membrane around the new organelle. Second, both organellar genomes show sequence similarity to prokaryotic genomes, The oxganel- lat genomes, like those of prokaryates, aro notenclosed in a nuclear envelope and are called nucleoids Organellar genomes consist mostly of ‘near chromosomes Plastid genomes generally range in size from about 120 10 160 Kilobeses (kbp) and encode predouninantly genes that are neoded for photosyntiesis and the expression of44 CHAPTER 2 ) FIGURE 2.9_The complex stucture of mitochondrial ‘and plastid genomes, Each photagraph shows a single: branched DNA molecule conteining multisle connected cergancllar gonomos, (A) Micrograph of athiaiuen staines mitochonctial ONA from the liverwor: Marchantia poly morpha, (@) Line drawing of tho same molecule. The 106 kb scale bar shows the sze of one genome, (Cl i crograph of muktigenomis chloroplast DNA fram reaize stretched out in agarose. The 10 um scale bar fs equiva- lent to 30 kb of DNA sequence, which is roughly 20% of the total genome length (154 kb). (A, 8 fiom Older bburg and Bendich 1996; C trom Oldenburg and Bendich 2004) plastid geres. The mitochondrial genome is much more variable in size than the plestid genome. Plant mito- chondrial genomes range between approximately 180 and nearly 3000 kp—much larger than mitochondrial sgenomes of animals or fungi, many of which aze only 15 10 50 kbp. Plant mitochondrial DNA contains genes that encode proteins needed in the electron transport chain, ot thatare involved in providing co-factors for eleceon trans- port. Additionally, plané mitochondrial DNA carries genes for proteins requited for the organelles own gene expres: sion, such as ribosomal proteins, (RNAS, avi sRNAS. In both organelles many genes required for proper chloro plast or mitochondrial funetion aze no longer encodedt in the orgonellar genome itself but aver evolutionsry time have been transferred to the nucleus of moclezn plants. ‘These proteins are synthesized in the cytoplasm and then imported into the organelle For many years organellar chromosomes had been thought to containa genome-sized DNA molecale incirewe la form, similar io the circular placmida of bacteria, Recent data, however, show that mast of the DNA in both plant mitochondria and chloroplasts is foun in linear molecules that may contain more than one copy of the genome (FIG- URE 2.9). These copies are conneried fo ane another in hreacilo-tal orientation and the chromosomal DNA mol cules can be highly branched, resembling a bush or tree (Oldenburg anc Bendich 1998, 2004), unlike the simplex structures of linear nuclear chromesonies. While nuclear chromosomes are of constant sizo generation after gen efation, the chromosome size in mitochondria and chloro» plests can vary. Nonetheless, each o1garellar chromosone contains at least one complete genome. @ 5 © ‘ie ioum Orgenollar genetics do not obey Mendlian laws The genetics of organellar goncs are governed by vo princ ples that cistinguish them from Mendelian genetics, First, both nitochoncirla and plestids generally show unl- parental inheritance, which means that sexual offspring (via pollen and eggs) only inherit organelles from one parent. Among the gymiosperms, the conifers normally inhorit their plastids from the patemnal pazent. For angio: sperms the general rule is that the plastids come from the maternal parent. Hovrever there area few angiesperms in which plastids are inherited cither bipazentally or pater: nally. Mitochondrial inheritance is usually matecral inthe najity of planis butagainta few exceptions can be found; for example seme types of corifors, such as the eypeesses, show paternal inheritance of mitochondria In plants in whieh plastids are inheritec| maternally, th paternal plastids are exeluded as a consequence of the way. the male gemetophyte (pollen) is formed. Pollen formation inwelves twomitpticdivisionsof the micrygamewophyte. The first division results in a vegetative cell, which gives rise to the polten tude, arid a much smaller generative cel, which willlater produce the two sperm cells. During the frst div sion, plastids areexcludod from the generative cell and thus the male germ line, Consequently the zygote gets all ts plas Aids from the egg in ihe iemale megagemetophi—te, resulting ‘inmalormal inheritance of plastid gones (Miogensen 1696). the mechanism for paternal inheritance of plastids in come conifers, such as Douglas fir (Psewdotsusee meusiesi) lor Chinese pine (Pins tabulaeformis), is slightly more com- plicated and less wll understood. In many gymnosperms ‘he second mitotiedi¥ision, which peoctuces the two specm() (8) White sector nucle, occurs just as the pollen tube enters the female gametophyte. Paternal cytoplasm rich in plastids and, ‘some mitochondria clings to the sperm nucleus that enters, the egg cell AL the time of fevtlization, the ezg nucleus is surrouncled by maternal mitochondria but the maternal plastids are located in the periphery of the egg cell. After fasion of the sperm and egg, nuclei and formation of the zygote, cellularization follows in such a Fashion that the new cell walls essentially exclude all the maternal plastids, ancl most of the paternal mitochondia from the einbrye (Mogensen 1996; Guo et al. 2905} “The second major feature af mganellar inheritance is the fact that both chlowoplastsand mitochondsia can segsegate vegetatively. This means that a vogotalive col fas opposed, toa gamete) can give rise to another vegetative cell via znitosis that is genetically different. For example, during, mitosis, one daughter cell may receive plastids with one typeof genome, while other plastids with different genetic information, perhaps containing one or more mutetions, ae by chance distributed into the other dauighter cell, Veg lative segregation, which is alsoreterted toas sorting-out, can result in the formation of phenoty pically different sec tors within a tissue (FIGURE 2.10). The prosence of such, sectors in leaves may result in what horticulturists often, refer to as variegation (see Figuze 1631 for an example), Leaf variegation can be caused by mutations in nuclear, rnitechondrial, or chloroplast genes, Now that wo have looked at the organization of nuclear and cytoplasmic genomes in plants, we will turn to the structure of the nuclear genome and how it influences the ‘expression of the genes it contains, The basic mechanisms GENOME ORGANIZATION AND GENE EXPRESSION 45 ‘een sector prod regation ewcell with al wild ype Normal plastid laste Now cel is 2 swith more \S of id type and a rrotant pests } 2 seu Mata pli Newell ltt with all rian plastids FIGURE 2.10 Vegatative seqragation can lead to var iegation (4) Cell cvision in acall with 5oth normal (9) Allgreen (green) anc mutant (whitel chloroplasts can by chance ‘result in offspring with only mutant orgenelles.(@) Cells chat contain exclusively white chloroplasts pro- duce only whita chloroplast, leading t9 a white se tor. (©) Secio’s in which no céll arses thet contains only white chleroplasts stay green throughout. Varia gation can also be causec ky mutations in mizachon of gene transcription will be reviewed frst, followed by a description of the transcriptional regulation of gene expression Transcriptional Regulation of Nuclear Gene Expression ‘The path from gene to protein is a multistep process cata- lyzeel by many enzymes (FIGURE 2.11). Each step is subject toregulation by the plant to control the amount of protein that is produced! by each gene, Regulation of the first step, transcription, determines when and whether an mRNA is. ade. This level of regulation, which is referred to as tran- scriptional regulation, incluces the control of transcrip tion initiation, maintenance, and termination. The next Jovdl in the regulation of gene expression, Jnown as post- transcriptional regulation, occurs after transcription. This level, which will be covered later in this chapter, includes controls on mRNA stability, translation efficiency, and degradation. Finelly, protein stability (posttranslationsl regulation) plays an important sole in the overell activity of a gene or its product. RNA polymerase Il binds to the promoter region of. most protein-coding genes Gere transcription is facililated by an enzyme celled RNA, polymerase, which binds o the DNA tobe transcribed and makes an mRNA transcript complementary to the DNA. sequence. There afe several types of RNA polymerase.4& CHAPTER 2 Trarsgipton AUG (vartatiral Trasavonai —) farenere Satste Sopate | } Gnas. brie | == taxon [ERGA| on | f=» |S “Transeription | PremRNA “Transcription + capping and polyadenylation \-Teanseriptionsl regulation 6 TT AAA, mana | Nucteus Processing of pracureor < ) Cytopiasm Transport out of riedeuet stone sttrorscatonal | regulation feloned poiperrices Protein trodieation { | Postranslaional Se Fequlation (Protein mediation i Shastabiny) 255 proteasome FIGURE 2.11 Gens exprestion in aukeryotes, RNA poly capped and polyedenylsted pre-mRNA is then spiced merase Il binds to the promoters of genes that encade proteins. Unlice prokaryotic ganes, eukaryotic ganes are nict clustered in opeions, and each is divided ‘io introns and exons. Transcription from the template strand ora ceeds in the 2’ to & direction at the wranseription start site, and the growing RNA chain extends ene nuceotide ata time inthe §’ 20 direction. Tranelesien beging with the fist AUG encoding methionine, asin prokaryotes, and ende with a stop codon, The pre-mRNA tranzerps is frst “capped” by the adcition of 7-methylguanylete (m7) to tho 5! end, The Zend ie shoranee! sightly by cleavage ata specific site, and a poly-A tall added. The INA polymerase lis the polymerase thet transcribes mest protein-coding gers The region of the gene that binds RNA polymerase is called the promoter (FIGURE 2.124). We can divide the siructure of the eukaryotic promater into two parts: the core promoter or minimam promoter, consisting of the minimum upstream sequence required for gone expres bya splicecsome complex, ard the introns ere removed. ‘Tho mature mRNA exits the nucleus through the nuclear pperes and initiates translation on tibosomes in the cyto £0. As each ribosome progresses toward the 2 and of the mRNA, new ribosomes attach at she 5” end and begin translating, leading to the formation of palysames, Aer transletion some proteins are modified by acicition of chemical groups 20 the protein chain, The released poly peptides have characteristic halves, which are eegulat- ed by the ubiqutin pathway and 2 laege proteclytic cam plox calod sho 265 proteaseme (eee Figure 2.18) sion, aud the regulatory sequences, which control the activity of the care promoter Before transcription of a gene can start, several steps have to occur to allow the RNA polymerase to gain access to the gene’s nucleotide sequence. Recall that nuclear DNA is wrapped around histones, forming beadlike nucleosomes. As will be discussed in more detail in theGene regulatory proteins latory prove 100 cox “20 CAAT box Proximal contzcl elernents o Activating reguletery protein aserbly j Binding ste or Inhibiting reculatony GENOME ORGANIZATION AND GENE EXPRESSION 47 General, RNA polymeraca t tansciption,/ gas, / Grex 25 ‘TATA box Trarecrintion ere ore promoter ProonRNA | Leen RNA polymerase Il and seneral transcription factors, —_ CAAT b FIGURE 2.12 Crganizetion and regulation of atypical eu- karyotic gene. (A) Features of a typical eukeryolic cove promoter and the proteins that regulate gene expression. RNA polymerase ils situated at the TATA box in associa tion with the general transcriotion factors about 25 bp Upsteaam of the teansoription start site. Two cis-acting regulatory sequences that enhance the activiy of RINA polymerase ll are tha CAAT bew ane! the GC box, located {st ebout 80 ard 100 bp upstream, respectively, of the: ext section, the histones are subject to modifications, and only if those modifications are favorable to transcription, vyall RNA polymerase be able to bind to the DNA. Further. more, to be functional, the RNA pelymerases of eukary~ cles require additional proteins called general transerip- tion factors to position the polymerase at the transcription startsite, These general transcription factors, wagether with the RNA polymerase, make upa large, multisubunit tran. scription initiation complex (see Figure 212A). Trarserip: tion is initiated when the final transcription fector 40 join the complex phosphorylates the RNA polymerase, The RNA polymerase then separates from the initiation com= plexand proceeds along the antisense, or complementary, strand of the DNA in the 3 to 5 direction In addition to RNA polymerase and the general tcan- scription factors, most gones, especially those that play important roles in development, requise specifi trarserip- {uon factors (also often called gene regulatory proteins) for x TATA box transeription start site. The CAAT box and GC bos bind gene regulatory proteins. (6) Regulation of tenscription by distal regulatory sequences and transacting factors. The trans-acting factors can ect in cencert with the distal regulatory sequences to which they are Dound to activate twanseripticn by making direct physical contact with the transcription ination complex. The details of this pro~ ‘cess are til not wall understood. RNA polymerase to become active, These regulatory pre- teins ind to the DNA and become part ofthe transcription, initiation complex ‘An: examnple of a typical RNA polymerase fl promoter is shown schematically in Figure 212. The core pro- moter for genes transcribed hy RNA polymerase Tl typi cally entends about 100 bp upstream of the transcription initiation site and includes several sequence elements referred io as proximal promoter sequences. About 25 0 35 bp upstream of the transcription initiation site is @ short sequence called the TATA box, consisting af the sequence TATAAALA). The TATA box playsa crucial role in fran= scription because i! serves es the site of assembly of the ‘transcription initiation complex discussed above, In addition to the TATA box, the core promoters of cukaryotes also contain two additionel regulatory, sequences the CAAT bax and the GC box (see Figure 2.128), These two Sequences are binding sites for speciic48 CHAPTER 2 transcription factors. The DNA sequences themselves are termed cis-acting sequences, since they are adjacent (as), to the trans are regulating, The tcant- scription factors that bind to the cis-acting sequences ate also called trans-acting factors, since the genes that encode them are located elsewhere in the genome. Numerous other cis-acting saquiences lorated farther upstream of the proximal promoter sequences can exert cither positive or negative control over cukaryetic promot ers. These soquonces, termed distal regulatory sequences, are usually located within 1000 bp of the transcription in tiation site, The positively ecting transcription factors that bind to these sites are called activators, while those that Inhibit transcription are called repressors. In adcition to having regulatory sequences within the promoter itself eukaryotic genes can be regulated by contrel elements located tens of thousands of base pairs away from the tran: scription initiation site. Distently located positive regulatory sequences are caller enhancers, Enhancers may be located either upstrezm or downstream from the promoter How do all the traneeription factors thetbind to the es acting sequences regulate transcription? During the forma- ton of the initlation complex, the DNA between the core prometer and the most distally located regulatory sequences Joops out in such @ way as to allow all oF the transcription iption units @ i, a ee. NET Se metnyation NEE IC a Ce eae tesine Wether ® Hse ] 3 a Mone methyl Trine FIGURE 2.13 (A) The 2deition of a methyl gioup to the CS inoytosne is associated with transcriptional inectivity. B) The emino ac lysine [K, which occurs at several posi- sions in histones, can be mono-, ch, er zimathylated by histone methyltransferase (HT). (C) Histones ean ber modeled to sctivate gene transcription (top) of to repre factors bound to thatsegmentof DNA to make physical con- tact with the initiation complex (FIGURE 2.128). Threngh this physical contact, each transcription factor exerts its con- trol, either positive or negative, ever transcription. Epigenetic moditications holp determine gene activity As mnentioned in the previous section, transcription can be initiated only if the DNA is accessible to the RNA poly- merase and other required binding proteins. In order to make the DNA accessible, its packaging has t0 be "loos- ened,” a process mediated by covslent modifications of both DNA and histones, Because these modifications can change a gene's behavior without changing the DNA sequence of the gene itself, they are referred fo a8 epige netic modifications (from the Greek word epi, meaning “over” or “on top of”) ‘One commen type of DNA modification is the methyla- tion of cytosine residues (FIGURE 2.13). DINA sequences thataze frequently methylated in plentsazeCG, CHG, and CHIH (where H can be any nucleotide except guanine). In conliesi, cytosine methylation in mammals occurs mostly on CG. Cytosine methylation is eatalyzest by one of sev- eral methyltzansferases, whereas DNA demethylation is Amino acid chain of hetone ai S32 stone a Histon @ - nc Tce es vn antes ee aie 1 Vole 3 3 Dimethyl That ie ‘nine it foottom), Activation Is associated with acetylation by. Histone acetylransfersces (HATs) and methylation by HMT 2¢ lysine restive H3K4. These mocifcations promote ATP- dependent chreratin remacieling and! stimulate tanserip= on. Repression of transcription achieved by mata ‘ton of H3K? and deacetylation by histone deacetyiases,catalyzed by glycosylases that replace mothyleytesine with unmethylated cytosine. Epigenetic modifications can also cecur on histones, which, together with the DNA wrapped around them, make up the nucleasomes, Each histone has a “tail,” which is mace up of the first part of the hisione's ermine acid chain and protrudes outward from tho nucleosome, [Histone modifications eccur on these tals, usually within the outermost 40 or so amino acids. These modifications ‘can influence the conformation of the nucleosomes and thereby gene activity in the associated DNA. (© ATP-dependent chromatin remoceling protein GENOME ORGANIZATION AND GENE EXPRESSION 49 One of the histone modifications that influences gene activity is methylation, expecially at specific lysine resi- dues (single-letteramino acid abbreviation K) m the tailot histone type HB. Those residues are K4, K9, K27, and K36, counting from the outermost amino acielinersrd to the cen ter of he histonecoll. One, two, or three meth! groups can, beaded to a single lysine (FIGURE 2.138), Histones dim cthylated at position HBK4 are generally associated with active genes, whereas dimethylation at position H3K9 is often associated with inactive genes and elements, atch as silent transposons, Methyl gronpsare removed hy histone 1A polymerase land nstiption factors Activating histone nod fiations (@.g., acetyl groupe or netted HBR) Histone ceacetylase Histone, sudebiome GENE REPRESSION H9K9 histone rmethytirarateras Histone acetytransterase Deectiveting histone moaitications| (e.g. dimethyiated K3k9)50 CHAPTER 2 demethylases in mantmels and yeast (Bannister and Kou- zzacides 2005), and candidate genes with a similar function have been identified in plants (Choi et al. 2007). ‘Another form of modification that occurs on the histone tail is acetylation, which is catalyzed by enzymes called histone acetyliransfereses (HATS). Generally, acetylated histones aro associated with genes that aro activ scribed. Histone deacetylases (HDACs) can reverse this activation by removing acetyl gious, Poth mothylationand acetylation change the architecture of the chromatin complex, which may result in condense UUont or relaxation of the chiomatin. These changes oceur when mulliprotein chromatin remodeling complexes bind. to the medified histones. Using the energy released by ATP Iydrolysis to drive the reaction, these complexes open up, tho chromatin by displacing the nucleosomes in the ares slightly toward the 5 or 3’ dinection of the remodeling cos plex, The resulting gap between nucleosomes is nov wide enough for RNA polymerase to bind andl initiage transerip= tion (FIGURE 2.13C). Altematively, histone moditications, nay present novel binding sites for egulatory proteins thal affoct gone activity Scientists are only beginning to under stand the effects of specific chemical modifications on each of the first 49 or s0 amino acids of the histone tails. The entioly of histone modifications on a specific nucleosome is sometimes called the “hisione code” te underline the strong link between nucleosome constitution and gene activity tron Posttranscriptional Regulation of Nuclear Gene Expression Immediately after transcription, the resulting RNAS are. processed: their introns are removed by splicing, and 5” caps end 3’ poly tallsareadded. The transcriptsare then, exported to the cytoplasm for translation. ‘An organism often produces mRNA in response to 9 specific situation. In order to remain useful 2s a specific, response to a specific situation, individual mRNAS must havea finite lifetime. For example, in order to core with a ‘ansient environmental stess, a plant may need to briefly produce specific enaymes. After the stress subsides, it ‘would be wasteful maybe even detrimental, to continue to produce those enzymes. Thus mRNA production, activity, and stability are all regulated (Groen 1993). We diseussed the regulation of transcription (mRNA production) in the. previous section. Now we turn to mechanismisof posttran- seriptional regulation (roguletion of activity and stability) RNA stability can be influenced by cis-elements ‘One mechanism by which mRNA stability is regulated depends on the presence of certain sequences within the mRNA melecule itself, called efs-clements—an unfortu- nate choice of terms, since the same term is used for the. DNA regions that influence transcriptional sctivity, These cis-elements can be bound by RNA-binding proteins, which may either stabilize the mRNA or promote its deg- redation by nucleases (Hollams et al. 2002). Depending on. the types of cis-clements present, the stability of e given mRNA molecale can vary widely Noncoding RNAs regulate mRNA activity via the RNA interference (RNAi) pathway Another mechanism for regulating mRNA stability is the RNA interference (RNAi) pathway. This pathway involves several types of small RNA molecules that do not code for proteinaand are thusealied noncoding RNAS (reRNAs), The RNAi patineay has important roles in gene regulation and genomic defense, The RNAi pathway is aso! of collar ceactions to the presence of double-stranded RNA (dsRNA) molecules. Recall that mRNA is usually a single-stranded molecule (RNA), In plant cells, dsRNAs usually eccuras a result of one of three types of events: 1. The presence of microRNAs (miRNAs), which are involved in normal developmental precesses (FIGURE 214A) 2. The prociuction of short interfering RNAs (iIRNAs), which silence certain genes (FIGURE 2.1 48) 3. The introcluction of forsign RNAs, either by viral infection or via transiozmetion by a foveiga gene (FIGURE 2.14¢) Regardless of how the dsRNAs are produced, the cell _mounts the KNVAi response. The dsRNAs are chopped up, oF diced into small, 21- to 2-nucleotide RNAs, which bind to complementary single-stranded RNAs (e.g, mRNAs) from endogenous genes, viruses, of transgenes and pro- mote their degradation or trarslational inhibition. In some cases, the RNAi pathway can also lead to gene silencing or haterochromatization of endogenous DN.or introduced, foreign genes, To esploce RNAi inmre detail, we will first take a look al events leading to dsRNA accumiilation in the cell. Next we will discuss the molecular components and, downstream events of the RNAI process IMlcroRNAS REGULATE MANY DEVELOPMENTAL GENES, POSTTRANSCRIPTIONALLY MicroRNA (miRNAs) are involved in many develapmental processes, suchas le nd fower éevelopment, cel division, and the orientation {golmity) of plant organs (Kidner and Martienssen 2005) Al miRNAs ariso froin RNA poly:norase Il-medisted tran- scription ofa pecificlocus hat encodes primary MIRNA transcript (a pri-miRNA), which can vary in length from hhunaireds to thousand of nucleotides, This primary tran- sept is capped atthe 5" en! and polyadenslated atthe 3 end and ferme adouble-stianded stem, whosebase-paired armsbordera single-stranded loop. Next, the prismikNas are processed into presmiRNAs, which are vswally 70 to(9) NieraRIA pathway RNA pol GENOME ORGANIZATION AND GENE EXPRESSION 51 Tanarpion 4 Prien, a ETS | DCL, HYLT Nucleus Nuclear membrane ytoplasm EXPORTING, HASTY“ 5. miRNA-bound RISC can inhib {tarslation of a target mma, FIGURE 2.14 _RINAi pathways in plants. (A) MicroRNAS (miRNAs) ara part of rany genetic pathways during plant development. (8) Short interfering RNAS (SRNAS) are raquiraa t maintain hetarachronratin and to sense un 50 nucleotides long in animals, but can be up to several hundred nucleotides long in plants. In plants, pri-miRNAS are converted into miRNAs inside the ancleus by the pro. teins DICER-LIKE 1 (DCLI) andl HYPONASTIC LEAVES 1 (HYL1), both of which ae involved in processing the primary transcripts Aler processing, the mature miRNAs are ready to be used in KINA (soe Figure 2.14), SHORT INTERFERING RIVAS ORIGINATE FROM REPETITIVE. DNA Mature short interfering RNAS (SIRNAS) are struc turally and functionally similar to miRNAs and also leael 3. Transcription n.the nucleus by RNA dolymerase yields. aselFfolding remiRA, 2. The folded pri-miRNA is processed by Bett and HVUt inte 21- to 24 nucleotige fragirents 43. The Fragments atociate with AGO, forming the FIC complex 4. The RISC complex is exported 70 zne ‘itcpiasm by the proteins EXPORTINS 6. alternatively, RISC can cUt she target FHRNA and in tiae te degradation used genes. The siRNA pathway involving RARP2 fs illus- trated here, (C) Plant cells can mount an RNAi response: to infection by viruses. to the initiation of RNAi. However, siRNAs differ from AuIRNAs in the way they are generated. SIRNAS can be produced in two ways. Fist, they can arise from the tra scription of opposing promoters that practice mRNA on opposite strands and can thereby generate two fully or partially complementary single-stranded RNA (RNA) riolecules that can subsequently form. one dcuile-siranded molecule. The second way siRNAs can be generated is fiom ssRNAs that ae converted into dsRNAs by a special lass of RNA-dependent RNA polymerases (RARPS) [2° Figure2.14B). Note that transcription of siRNAS i accoun-52 CHAPTER 2 {8} Short interfering RNA pathway uceus RNA pot Iv uchromatin Heterochromatin nae Presinna renee + Ss 1. sits are transcribed fram ropeat ragions in heterochromatin by RNA polymerase IV (pel V7), 2. The pre siRNA is amplified bby RaR2, yialding lang deRNAS that are processed by DCD. Mature siRN 3, The fragments azociate with ACO, forming the FISC complex. Methylaxes and chromatin mosifans 4 siRNA-bound RISC can recruit methyleses and chromatin remodeling complexes (CMTS, BRM 3, DAD, anc KYA) Methyl — groups aaTRK, 1a. lternatively dela can alke bo rade ciroetly by tanseription fron ‘opposing promoters pew pew 22. Double sanded RMAC preduced by this patwey fre processed by DCLIIZ, C226 ag 5. Chromatin eo bythe sata to No transcription madoling comployas aro quiet apeat region: in tha genome, where they affect DNA and histone metryltion, plished by RNA polymerase IV, not RNA polymerase, as, jin miRNAs (Ramachandran and Chen 2008), Endogenous siRNAs are transcribed from chromo- somal regions thet otherwise do not scom to support much transcription: repetitive DNA, transposons, and, centiomeric regions. Indeed, siRNAs originating from ‘repeat regions are sometimes seferred to as repeat-ass0~ ciated silencing RNAs (rasiKNAS}, As you will see in the next section, this may not be coinciclentak it appears that the formation of siRNAs and the induction of RNAiacti- ally cause these regions to become heterochromatic and largely teanscriptionally silent. Once a double-steandedl RNA is produced, either by transcription of inverted repeats or by RARP2, itis cut into 21- to 24-nvcleotide RNA duplexes by members of the DICER protein family (see Figure 2148} Apart from these siRNAs of endogenous origin, exog- enous RNAs can algo trigger the formation of siRNAs ‘Sources for such exogenous RNAs include artificially mtro- duced transgenes and vital RNA. Inboth cases, RURPS and DICER-LIKE protéins are involved in producing matuze SiRNAS (soe Figure 214C).GENOME ORGANIZATION AND GENE EXPRESSION 53 (©) RNA’ resporve pathivay te virus infection usc((“AG re COS) = = eu toa oct — = 3 on Nucleus Viral RIA ip yplesm RIA virus @ 1A sina that injects RNA, SRN virus can immediately trigger « response medisted by DICER-LIKE (DCL) proteins 2. Viruses that injac sft ‘form dahivA as port oftheir ownal life ce Extracellular space DOWNSTREAM EVENTS OF THE RNAI PATHWAY INVOLVE ‘THE FORMATION OF AN RNAINDUCED SILENCING COM PLEX For miRNAs, siRNAs, and RNAs of exogenous origin, the end zesull of the RNAI process is similar: the inoetivation or silencing of their complementary mRNAs cor DNA sequences, After 21-to 24-nucleotide miRNAS or siRNAs have been formed by the DICER-LIKE proteins, cone strand of the short RNA duplex associates with a ribo: nuclease complex called the RNA-induced silencing com- plex (RISC) (sce Figure 2.14). In both animals and plants, RISC contains at least onecatalytic ARGONAUTE (AGO), protein. In some cases, KISC can recruit additional proteins to the complex. In Arabidopsis, ten different members of ‘the AGO gene family are Inown. The miRNAthat binds to AGO guides RISC 0 complementary mRNA. Upon bind ing to RISC, the target mRNA is cleaved by AGO, and the resulting fragments are released into the cytoplasm, where they are further degraded. KISC-bound mRNA molecules may also be translationally inhibited by RISC. targets viral RNA Tor degradation Is converted by DCL to viral i8NA, SANA SESS 4 Plant rakes convert rel si binds to RISC and thus fal deRNA, whatavoe tesouwes, YERARP fi ——- ro eSRNA, aus Plant DNA, Seliyic acid ——S Plant defense response © onavins sien sates ete pat seca Soest Rieciponer te sven poses 2. Viral attack inauces general plant defense Fesponses va the signaling malecila tlleylc act. RISC-bound sikNAs have an additional function: they facilitate methylation of DNA end associeted histones at sexquences complementary to the siRNA. Although RISC probably doesn't interact directly with DNA methyiases or histone methylases, the siRNA somehow guides those modification enzymes to the genomic sequence to be silenced. The chrcimatin structure is then “remodeled” in © an ATP-requiring reaction and subsequently metiylated, resulting in tighter condensation and heternchromatiza- tion of the DNA region involved (see Figure 2.13C) (Chap- man and Carrington 2007). SMALL RNAS AND RNAI COMBAT VIRAL INFECTION. In addition to the processing of miRNAs and endogenous SIRNAS, plants have aiso adapted the RNAi pathway as «type of moleculer inunune response against infection by viruses (Mlotshwa et al. 2008). The genomic structures of plant viruses are Guite diverse. Some viruses inject comble- stranded! DNA, othérs use single-or double-stranded RNA.54 CHAPTER 2 Nevertheless, each virus produces dsRNA at some point in its life cycle. Replication of RNA viruses in the host cell requires the formation of a dsRNA intermediate in the evte- plasm, Double-stranded DNA viruses, om the other hand, ‘flon produce deRNA hy transcription of overlapping open reading franes on opposite strands of thelr DNA. Whether from an RNA or a DNA virus, the deRNA is produced in the host cell’s nucleus. The plant’s DCL proteins recognize the dsRNA molecules and Initiate the RNAi pathway, which eventually leade to the destruction (of viral RNA. In the process of cutting invasive RNA into 21- to 2k-nucleotive siRNAs, the plant generates a pool of “memory” melecales that can travel vie the plasmodes- ‘mata throughout the entire plant body, effectively immu- izing the plant before the virus ean spread. COSUPPRESSION IS A GENE SILENCING PHENOMENON, MEDIATED BY RNA One of the first experiments leading, to the discovery of RNA involved an unexpacted plant response to the introduction of transgenes. In the early 190s, Richard Jorgensen and his colleagues were work ing with the petunia gene for chalcone syntaase, a key enzyme in the pathway that produces purple pigment molecules in pelunia flowers. When they insected a highly active copy of the gene into petunia plants. they expected to see a deepen- ing of the purple color in the flowers of the offspring Jo their suxprise, the flowers ranged in petal color from dark purple (as expected) to completely white (as if chalcone synthase avels had gone dows instead of up). This phe- nomenon—decreased expression of a gene when extra copies are introduced—was termed cosuppression. (Nith our present understand: ing of RNAI, we now know that in some calls, overexpression of chalcone synthase lriggered an RNA-dependent RNA polymerase to pro: duce dsRNA molecules, Which initiated the RNAV response. This response eventually led {0 postiranscriptional silencing and to methy: Ue Posttranslational regulation determines the life span of proteins As we have seen, mRNA stability pleys an important role in the ability of a gene lo producea functional peotein, We turn next to the stabilily of proteins and the mechanisms ‘hat regulate a protein's lie span, A prowin, once synthe- sized, has finite lifetime in the cell, ranging {rom a fow minutes to several hoursor even longer. Thus steady-state levels of cellular enzymes reflect an equilibrium between the synthesis and the degradation of these proteins, knows as tumover. In both plant and animal cells, there are two distinct pathways of protein tusnover, one in specialized Iytic vacuoles (called lysosemes in animal cells) and the other in the cytoplasm (see also Chapter 1) ‘The cytoplasinic pathway of protein tamover involves the ATP-dependent formation of a covalent bond between the protein that is to be degraded and a small, 76-amino acid polypeptide called ubiquitin, Addition of one or many molecales of ubiquitin toa protein is called ubiquit ‘mato, Ubiguitination marks a protein for destruction by a large, ATP- ‘of ubiquitin to a target protein, oe an neatas ‘mile times. + } Pol i J ebiguicnation oe oa, lation of both the introduced and the endog: enous copies of the chalcone synthase gore. Injerestingly, posttranscriptional silencing did not occur in all cells. The cells in which gene silencing took placo gave rise to white sectors, explaining why some of the transgenic petu: nia plants had variegated purple and white flowers. In summary, RNAi is a process in which ASRNA eliclts a posttranscriptional response thatleads to the silencing of specific tanscripts miRNAs aid in the regulation of genes—often developmental genes—while SIRNAS help to Leap hetercchromatin transcriptionally inactive oractas a molecular immune response against 4. The ubiqutinated protein is ‘hus targeted to the 295, proteescire, where itis degraded, FIGURE 2.15. Generalized diagram of the cytoplasmic pathway of protein degradation, Peptidesof the shortlived proteins in eukaryoticeells are degraded via the ubiquitin pathway (Lam 1997), Ubiquitination is initicted when the ubiquitin-activate jing enzyme (F1) catalyzes the ATP-dependent adenyiyla- tionof the C terminus of ubiquitin. The acenylylated ubig- ultin is then transionted to a eysteine residue om a second, enzyme. the ubiquitin-conjugating enzyme (E2). Pratsins destined for degradation ate bound by a third type of pro- fein, a ubiquitin ligese (B3), The E2-ubiquitin complex, then transfers its ubiquitin ta lysine residue of the protein ‘bound (0 B3. This process can occur multiple timesto form polymer of ubiquitin, The ubiquitinated protein is then targeted to the proteascme for degradation. As we now know, there are a multitude of proteinspe- cific ubiquitin ligases that regulate the tumover of specific target proteins (see Chapter 14) We will diseuss:an exaraple ‘of this pathway in more detail when we cover developmen ‘talzegulation by the plant hormene auxin in Chapter 19. Tools for Studying Gene Function Individuals that contain specific changes in their DNA sequence are called mutants. The analysis of mmutants an extremely powerful tool that ean help scientists infer the function of a gene or map ils location cn the chromosomes, Tn this section we will Lscuss how invutants are generated, and how thoy can be used in genetic analysis. We will also discuss some modern biatechnological tools that allow. researchers fo study or manipulate the expression of genes, Mutant analysis can help to elucidate gene function Throughout this book we will discuss the gencsand genetic pathways involved in physiological functions at length, often refering to certain types of mutants that allowed researchors to understand the genes and pathways under Giscussion. Why is a mutant gene a more powerful tool for elucidating gene function than the nermmal, wild-iype gene on its own? ‘The use of mutants for gene identification relies on the bili y to distinguish a mutant from a normal individ, 90 the change in the mutant’s nucleotice sequence must rest, in an altered phenotype. Ifa mutant can be restored to the rnommal phenotype with a wild-type version of a candidate _gene, the tsearcher knows thata mutation in that gene was responsible for conferring the originally observed mutant phenotype. This method is called complementation. For ‘example, assume thata plant with a single-gene mutation shovis.adelay in producing flowers compared with the Wild type. fave ean determine the sequence and location of the gene resporsibsle, we will presumably lean somethinigabout he mechanisins involved in floral development. Let’s now assume that a researcher ig able to find a gene in the mutent ‘genome that differs from the wild-type gene in its DNA sequence. Ifthe researcher Is able to show thar transferring. the wild-type geneinto the mutant restores the normel phe GENOME ORGANIZATION AND GENE EXPRESSION 55 notype, wocan be reasonably certain that the candidale gene playa role inthe initiation of flowering, athe 1929s, HJ. Muller and L.J. Stadler Independently oxperimented with the effects of X-rays on the stability bf chiomosomes in flies and in barley, respectively. Both researchers reposted heritable changes in the treated o4gas- isms. In the following years, other techniques for inducing, mutations were developed. These techniques include the use of ultraviolet or fast-neutran radiation and of muta- genic chemicals. For example, treatment with the chemi- cal etylmethanesulfonate (EMS) causes the addition of anetlyl group to a nucleotide, usually guanine, Ethylated guanine pairs with thymine instead of cytosine. The cell's DNA repair machinery then replaces the ethylated gua- ine with adenine, causing a permanent mutation froin G/C to A/T at that site, Mutagenesis with cither radia- ‘ion or chemicals induces nucleotide changes randomly heoughout the genome. ‘Thoreare several ways to map a mutation toils chromo- some and ultimately clone the affected gene. 1WEB TOPIC 2.1 explains a method called map-based cloning, which aises crosses between a mutant and a wild-type plant and genetic analysis of the offspring to narrow down the loca- tion of the matation to a short segment of the chromosome, which is then sequenced. “Another method of mutagenesis is the random insertion of transposons into genes. This technique involves cress Ing the plant of interest with a plant carrying an active ‘ansposon and screening their offspring for mutant phe- notype: caused by random insertion of the transposon into new locations. Because the sequonce of the transpe- son is known, these mutations are “lagged” thus the DNA sequences adjacent to the transposon can be readily found and analyzed todentify the mutated gene. This techniqa= 's called transposon tagging and is explained in detail in WEE TOPIC 2.2. Molecular techniques can measure the activity of genes Oncea gene oF interest bas been identified, scientists are usually interested in where andl when the gene is expressed. For example, a gene may be expressed only in ceproduc- live tissues, or only in vegetative ues. Likewise, a gene may encode general cell functions (so-called housekeeping Junctions) and be expressed continuously. or t may encode special functions and be expressecl only In response to a certain stimulus, such as a hormene or an environmental cue. In the pest, transcriptional analysis (the determination of the amount of mRNA produced from a gere ata given time) has boon performed mairly on single genes. Tools developed for this type of analysis include Northera blot- ling, reverse transcription polymerase chain reaction (PCR), and in situ hybridization, You ean find applications of each of these technique’ throughout this back. 4 more recently developed technigis, called microarray or gene chip tech=56 CHAPTER 2 nology, makes use of the information that has come cut of massive genome sequencing projects. All microarray techniques vse a solid support, such as a glass slide, amto which DNA sequences fare sported that ace representative of single genes of a givon species. Such arays can hold thousands cf spots, which can all be investigated together in a single experiment, greatly increasing the through put of gene analysis aver the classic methocls men- toned earler. RIVA extracted from a given tissue is first transfoemed by reverse transcription intoa more stable DNA copy (a cDNA) of each RNA molecule represented in the extract (FIGURE 2.16). The cDNA mixtureis then labeled witha fluerescent dye toallow lis visualization Iter ia the procedure, Aftor labeling, the cDINA mixture is applied to the microarray. Ench single stranded eDNA binds to (hybridizes swith) its corresponding (complementary) DNA spot on the artay, which represents Hie gene that pro: duced the corresponding mRNA in the original tis- sue extract. For example, if mRNA from gene X is present in the tissue extract, its eDNA will bind to the spot on the array representing gene X. If gene Y, on the other hand, was not expressed at the time of tissue sampling, there will be no cDNA te bind to the DNA spot on the array representing gane Y, ‘and that spot will stay blank, After hybridization, the microarray is seanned using « laser beam thatcan detect the fluorescent label applied to the eLNA. In tour example, the spot for gene X will ight up in the Iacer scan, whereas the spot for gene ¥ will rot There are several types of microarray techniques. While some types analyze twomRNA sampkes—for exarnple, from a treated! ane from a contol plant—on a singlearray (using “two-color labeling” see Figure 2.16), other types compare two samples using bw separate arvays. Since ils original application to the analysis of gene expression, microarray technology has been adapied to many other uses as well, rang: ing from diagnosing the genotypes af individuals in 4 population to determining the epigenetic status of ‘genes oF intergenic regions Gene fusions can introduce reporter genes The identification of a gene containing a mutation provides information about that gene's lecation ir the genome and about the eifect of its altered func tion on the plant’s phenotype. Psom the sequence FIGURE 2.16 Two.celorlabeling ie 9 microaray ‘technique thar can be used 10 compare gene ex: pression in different individuals or under diferent conditions, Droughtstressed pant 1. An experimental plant and a contol plant are . keptin identical conditions, \ excapt that the teatmant plant receives ne water 2. After harvest, RBA it ‘extracted from each plant, Fevetse transcribed 19

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