Applications of molecular markers in genetic analysis

Restriction fragment length polymorphism (RFLP) and microsatellite molecular markers were used to map two soybean nodulation genes, enod2 and leghemoglobin (lbc3). In addition, a high annealing temperature DNA amplification fingerprinting (DAF) method was developed for DNA fingerprinting of soybean cyst nematode (SCN), Mychorrizae, aphid, centipedegrass, and bermudagrass samples. Recombinant inbred lines (RILs) as well as an F\u3c sub \u3e2 segregating population of soybean Glycine max (L. Merr) facilitated the mapping of two expressed sequence tags (EST) involved in early nodulation and subsequent nitrogen fixation in soybean. For the early nodulin gene enodl2, the parents of RILs, Minsoy and Noirl, showed a polymorphism (5.5 vs. 5.9 kb) after EcoRV digestion. RFLP patterns of 42 RILs were analyzed using the MAPMAKER program linking enod2 to the seed coat color gene, I, with a distance of 11.1 cM on linkage group U3 of RIL map. Enodl2 and I are located close to Rhg4 , a soybean cyst nematode (SCN) resistance gene, and a locus for seed coat hardness. The molecular marker pAllO and seed coat color were used to integrate enod2 on an F2 segregating population (72 plants) generated from a cross between cultivar Bragg and G. soja (Sieb and Zucc), PI468.397. Enod2 was mapped in the same order as on the RIL map but 18.5 cM from the I locus. A microsatellite from the 5\u27 region of enod2B was mapped in the same position, demonstrating that enod2B and not enod2A was mapped. An RFLP for lbc3 (leghemoglobin) segregated independently from enod2 and the nts-l supemodulating locus suggesting that in soybean, symbiotically significant loci (including rj1, rj2, and rj6) are not clustered. To overcome potential problems caused by mismatch priming and secondary DNA structure and taking advantage of high primer-template ratios used in DAF reactions, annealing temperature of 55°C were used with single short arbitrary oligonucleotide as well as mini-hairpin primers to provide high resolution DNA profiles of soybean. Initially, high annealing temperatures for three arbitrary octamer primers in polymerase chain reaction (PGR) were tested for DNA fingerprinting of two soybean cultivars, Minsoy and Noirl. Fifteen PGR programs differing in levels of annealing temperature (47, 55, and 60°C), denaturation, annealing, and extension time (30, 60, and 120 second), and presence/ absence of extension step (+/- 72°C) were tested. The number of bands (amplification products) ranged from 7 (Program 10) to 51 (Program 3). The average ramping temperature for heating and cooling were calculated 1.42 and 1.27 sec/°C, respectively. Intensity of the silver-stained bands in a 10% polyacrylamide gel was high for the most PCR programs. Program 15, DAF-15, (95°C/30 sec, 55°C/120, and 72°C/30 sec) generated a complex DNA fingerprinting profiles for tested primers in Minsoy and Noirl. These profiles contained an average of 42 sharp and highly intense bands using both octamer primers 8-4 and 8-8 for DNA amplification. Using high annealing temperature increased stringency of primer-template annealing, avoided potential mismatching and hybrid molecule formation, and consequently improved reproducibility of DNA fingerprinting. Newly-developed high annealing temperature DAF was used successfully and detected markers linked to the enod2 gene and analyzed DNA fingerprinting of soybean cyst nematode (SCN), Mycorrhizae, aphid, centipedegrass, and bermudagrass samples. RFLP patterns of 41 homozygous F2 individuals for enod2 gene were set into two bulks of 26 and 15 with RFLP patterns identical to their parental patterns Bragg and G. soja, respectively. Screening of the bulks B and S with 31 primers resulted in detection of four polymorphic bands using primers HpC29 and HpC30 and DAF-15 program. Due to low number of polymorphic bands in the B and S bulks, sub-pools were generated and screened. B1 and SI sub-pools were tested with total 196 primers of which 32 were used for screening of sub-pools B3 versus S2. Primers Hp30, HpC22 and HpC30 generated 1, 1 and 4 polymorphic markers, respectively, in the B3 vs. S2. The major screening was focused on the B1 versus SI sub-pools which resulted in screening of 196 mini-hairpin and unstructured primers of which a set of 9 primers detected 20 polymorphic bands. Primer HpD25 generated polymorphic bands with 920B1, 320B1, 220S1, and 185B1 base pairs which were reliable and reproducible. These bands are promising bands for further analysis such as cloning and generating SCAR markers in the region of genome containing the enod2 gene. Key Words: nitrogen fixation, RFLP, recombinant inbred lines, integration mapping, annealing temperature, PCR, DNA fingerprinting, arbitrary primers, soybean. Abbreviations: RFLP, restriction fragment length polymorphism; SCN, soybean cyst nematode; RJLs, recombinant inbred lines; CHS, chalcone synthase; QTL, quantitative trait locus; lbc3, leghemoglobin gene; DAF, DNA amplification fingerprinting; PCR, polymerase chain reaction

Genetic and molecular analyses of host symbiotic genes and an in vitro regeneration system for Cicer arietinum L

Ineffectively nodulating plant mutants PM405B, PM638A, and PM796B were used in molecular/genetic analyses of root nodule formation in chickpea. To establish the mode of inheritance of the mutant nodule phenotype in chickpea mutant PM638A, reciprocal crosses were made between PM638A and wild-type ICC640. The F\sb2 segregation data fit a 3 mutants: 1 wild-type monohybrid phenotypic ratio, indicating that ineffective nodulation is due to a monogenic dominant, nuclear mutation, tentatively designated as Rn\sb7 . Segregation analysis of F\sb3 progeny confirmed this model. Early (ENod2) and late (Lb) nodulin cDNAs were used as heterologous probes to identify and study the expression of corresponding chickpea genes. ENod2- and Lb-homologous sequences were detected in the chickpea genome by Southern analysis. Northern analysis of root or nodule RNA extracted at different developmental stages indicated that chickpea ENod2 and Lb genes behave as early and late nodulin genes, respectively, and are expressed in a developmentally regulated nodule-specific manner. Comparison of nodulin gene expression in wild-type and ineffective nodules lead to the following conclusions (1) the rn\sb4 (PM405B) and Rn\sb7 (PM638A) mutations do not prevent the expression of ENod2 gene; (2) the rn\sb4 mutation eliminated detectible levels of Lb mRNA. Rn\sb7 mutation reduced levels of detectible Lb mRNA, and rn\sb5 (PM796B) mutation did not reduce Lb gene expression. (3) The symbiotic process in PM638A (nod\sp+fix\sp-) is blocked at a later developmental stage as compared to that in PM405B (nod\sp+fix\sp-). To develop an in vitro regeneration system for chickpea, immature cotyledons were cultured on B5 basal medium with various growth regulators. Non-morphogenic callus formed in response to various auxins previously reported to induce somatic embryogenesis on immature soybean cotyledons. However, different concentrations of zeatin induced formation of white cotyledon-like structures (CLS) at the proximal end of cotyledons. No morphogenesis, or occasional formation of fused, deformed CLS, was observed in response to kinetin or 6-benzyladenine (BA), respectively. Maximum frequency (64%) of explants forming CLS, was induced by 13.7 $\mu$M zeatin plus 0.2 $\mu$M indoleacetic acid. Shoots formed at the base of CLS, proliferated in medium with 4.4 $\mu$M BA or 46 $\mu$M kinetin, and required 4.9 $\mu$M indolebutyric acid or 5.4 $\mu$M naphthaleneacetic acid to produce roots

Nodulins in root nodule development

In de bodem zijn Rhizobium bacteriën in staat de wortels van vlinderbloemige planten (erwt, boon, klaver) te infecteren en aan te zetten tot de vorming van knolletjes. In die wortelknolletjes zijn de bacteriën in staat om stikstof uit de lucht te binden en om te zetten in ammonia. Met de ammonia kan de plant zich in belangrijke mate voorzien in haar stikstofbehoefte. Op hun beurt krijgen de bacteriën voedingsstoffen van de plant, zodat beide partners profiteren van deze symbiose.Een stikstofbindende wortelknol is een uiterst gespecialiseerd plante-orgaan, dat gevormd wordt in een aantal opeenvolgende stappen, waarin rhizobia de plant binnendringen, en deze aanzetten tot de vorming van een wortelknolstructuur. Uiteindelijk vullen de Rhizobium bacteriën ongeveer de helft van de cellen in de wortelknol, veranderen van vorm en beginnen vervolgens met de stikstofbinding. Gedurende dit proces wisselen plant en bacterie waarschijnlijk voortdurend signalen uit om het goede verloop van het proces te bewerkstelligen.Het onderzoek naar het mechanisme van wortelknolvorming en stikstofbinding op moleculair niveau heeft een hoge vlucht genomen. Zowel in de bacterie als in de plant zijn genen geïdentificeerd, die alleen in de wortelknol tot expressie komen. Rhizobium bacteriën bezitten naast hun chromosoom een zogenaamd sym plasmide, waarop de genen liggen die betrokken zijn bij de symbiose. Geïdentificeerd zijn de genen voor wortelknolvorming ( nod genen) en stikstofbinding ( nif en fix genen). Over de functie van de genproducten en de regulatie van de expressie van deze genen is veel bekend, maar wat met name de ( nod genen precies teweegbrengen is nog onduidelijk. Een twintig- tot dertigtal genen van de plant komen uitsluitend in de wortelknol tot expressie; dit zijn de zogenaamde noduline genen. Onderzoek naar de expressie van noduline genen in relatie tot de ontwikkeling van de wortelknol heeft laten zien dat er tenminste twee klassen noduline genen onderscheiden kunnen worden: vroege en late noduline genen. De vroege noduline genen komen ruim voor de stikstof-binding tot expressie, als het orgaan de wortelknol wordt aangelegd. Vroege nodulines spelen daarom waarschijnlijk een rol bij het vormen van de structuur van de wortelknol. Late noduline genen komen tot expressie rond het tijdstip dat de wortelknol met stikstofbinding begint. Het is dan ook aannemelijk dat late nodulines betrokken zijn bij het functioneren van de wortelknol. Mogelijk scheppen zij de voorwaarden voor stikstofbinding en het transport van gebonden stikstof. Omdat de late noduline genen min of meer tegelijkertijd tot expressie komen, is het waarschijnlijk dat deze genen op één en dezelfde wijze worden gereguleerd.Het merendeel van de noduline genen die tot nu toe geïdentificeerd zijn, behoort tot de klasse van late noduline genen. Het best bestudeerde late noduline is leghemoglobine, een myoglobine- achtig eiwit dat de zuurstofhuishouding in de wortelknol regelt. Van slechts een gering aantal van de overige nodulines is de functie in de wortelknol bekend. Ook over de manier waarop de plant ervoor zorgt dat noduline genen op het juiste moment en op de juiste plaats, dus alleen in de wortelknol, tot expressie komen, en over de rol van de Rhizobium bacteriën bij dit proces, is de kennis nog gering.Dit proefschrift beoogt een bijdrage te leveren aan de kennis over nodulines en noduline genexpressie. De beschreven experimenten hebben tot doel inzicht te krijgen in het mechanisme van de regulatie van noduline genexpressie. Vooral de rol van Rhizobium genen bij de inductie van noduline genexpressie staat daarbij centraal. Anderzijds komt ook de functie van vroege nodulines tijdens de vorming van een wortelknol ter sprake.Na een korte algemene inleiding over wortelknolvorming (hoofdstuk een) wordt in hoofdstuk twee een cDNA kloon beschreven die een vroeg noduline gen representeert. Deze cDNA kloon, pGmENOD2, is geïsoleerd uit een cDNA bank gemaakt tegen wortelknoIRNA van soja. Het ENOD2 DNA blijkt te coderen voor een noduline met een molecuulgewicht van 75.000, aangeduid met Ngm-75. De aminozuurvolgorde van dit noduline, afgeleid uit de DNA sequentie, laat zien dat Ngm-75 een zeer proline-rijk eiwit is, met een repeterend motief in zijn primaire structuur. Dit duidt erop dat Ngm-75 een structureel eiwit zou kunnen zijn. Het gen dat codeert voor Ngm-75 komt tot expressie in wortelknol-achtige structuren, die weliswaar door Rhizobium bacteriën geïnduceerd zijn, maar waarin geen bacteriën aangetroffen worden, zogenaamde 'lege' knollen. Ngm-75 lijkt daarom geen functie te hebben in het proces waarbij de Rhizobium bacteriën de plant binnendringen, maar eerder lijkt dit vroege noduline een bijdrage te leveren aan de vorming van de wortelknolstructuur.In hoofdstuk drie wordt de regulatie van de expressie van late noduline genen op het niveau van het DNA onder de loep genomen. Leghemoglobine cDNA kloons, geïsoleerd uit een cDNA bank gemaakt van wortelknoIRNA van de erwt, zijn gebruikt om een leghemoglobine gen te isoleren uit een genomische bank gemaakt van erwteDNA. Van dit leghemoglobine gen van de erwt is de DNA sequentie bepaald. Uit de analyse van deze sequentie blijkt dat het geïsoleerde gen volledig is en alle kenmerken bezit van een actief gen. De vergelijking van de promotergebied van het uit de erwt geïsoleerde leghemoglobine gen met de promotergebieden van soja leghemoglobine genen laat zien dat er overeenkomstige sequenties voorkomen. Omdat de overeenkomstige sequenties ook worden aangetroffen in de promotergebieden van andere late noduline genen, zijn deze sequenties wellicht betrokken bij de wortelknolspecifieke expressie van alle late noduline genen.In de hoofdstukken vier en vijf staat de communicatie tussen plant en bacterie centraal. Gepoogd wordt te achterhalen welke genen van Rhizobium betrokken zijn bij het aanschakelen van noduline genen. Hierbij is gebruik gemaakt van de mogelijkheden die de bacteriële genetica biedt om bacteriën te construeren die weliswaar een gedefiniëerd deel van het totale Rhizobium genoom missen, maar toch nog wortelknollen kunnen induceren. Deze studies zijn uitgevoerd met wikke ( Vicia sativa subsp. nigra ), omdat dit kleine vlinderbloemige plantje snel reageert op inoculatie met (genetisch veranderde) Rhizobium bacteriën. In hoofdstuk vier is de basis gelegd voor de analyse door het identificeren van de noduline genen van wikke. Uit wortelknollen werd RNA geïsoleerd, en in vitro vertaald in eiwitten, welke werden gescheiden op tweedimensionale polyacrylamide gels. Door het vergelijken van het aldus verkregen eiwitpatroon met dat van worteIRNA zijn vijftien noduline mRNAs geïdentificeerd, waaronder één vroeg noduline mRNA. Een tweede vroeg noduline mRNA van wikke is geïdentificeerd op Northern blots met behulp van de in hoofdstuk twee beschreven soja cDNA kloon pGmENOD2.De expressie van de noduline genen van wikke is vervolgens bestudeerd in wortelknollen geïnduceerd door een Rhizobium stam waarin het sym plasmide is vervangen door een plasmide met alleen 12 kb van het nod gebied. Alle noduline genen bleken tot expressie te komen. Kennelijk speelt de informatie op het sym plasmide buiten deze 12 kb nod gebied geen enkele rol bij de inductie van noduline genexpressie. In wortelknollen geïnduceerd door een Agrobacterium transconjugant, waarin het Ti plasmide is vervangen door dezelfde 12 kb van het nod gebied, bleken alleen de twee vroege noduline genen tot expressie te komen. Het nod gebied is dus het enige Rhizobium DNA dat betrokken lijkt te zijn bij de inductie van vroege noduline genexpressie. Hoewel dit resultaat tegelijkertijd suggereert dat het Rhizobium c hromosoom betrokken moet zijn bij de inductie van late noduline gen expressie, mag die conclusie niet zomaar getrokken worden, want cytologisch onderzoek laat zien dat de Agrobacterium transconjugant een afweerreactie van de plant oproept. Het is dus mogelijk dat de genen op het nod gebied weliswaar in staat zijn om late noduline genen aan te schakelen, maar dat de verdere ontwikkeling van de wortelknol al gestopt is door de tussenkomst van het afweermechanisme, vóórdat die late noduline genen aangeschakeld konden worden. Agrobacterium transconjuganten zijn dus niet bruikbaar om de rol van de nod genen bij de inductie van late noduline genexpressie te onderzoeken.In hoofdstuk vijf worden experimenten besproken die, zij het indirect, laten zien dat de nod genen inderdaad betrokken zijn bij de expressie van late noduline genen. Gezien het fenotype van mutaties in de diverse nod genen aanwezig in de 12 kb van het nod gebied zijn het waarschijnlijk de nodA , B en C genen, die één of meer signalen aan de plant geven, waardoor de expressie wordt geïnduceerd van de vroege en vervolgens mogelijk ook van de late noduline genen.Uit bovenstaande experimenten bleek dat er een correlatie bestaat tussen de ontwikkeling van een wortelknol, zoals die op microscopisch niveau gevolgd kan worden, en de expressie van noduline genen. De correlatie tussen de expressie van een bepaald noduline gen en het bereiken van een bepaald ontwikkelingsstadium biedt de mogelijkheid om te speculeren over de functie die dat noduline zou kunnen hebben. Op grond van de experimenten beschreven in de hoofdstukken vier en vijf kunnen vroege zowel als late noduline genen verder onderverdeeld worden in subklassen, die ieder correleren met een stap in de ontwikkeling van een wortelknol. Hoofdstuk zes, tenslotte, is een overzicht van de huidige kennis over nodulines, noduline genen en de regulatie van noduline genexpressie. De resultaten gepresenteerd in de hoofdstukken twee tot en met vi# worden in dit laatste hoofdstuk met deze kennis geïntegreerd.</p

The medicago truncatula genome and analysis of nodule-specific genes

The nitrogen-fixing plant Medicago truncatula is an important model system for identifying legume genes and determining their functions. With over 255 megabases of the genome, representing about 85% of the euchromatic regions, having been sequenced, my analysis reveals 50,540 predicted protein-encoding genes, 632 tRNA genes, 45 miRNA precursor candidates, and repetitive elements covering 11% of the sequence. ~ 50% predicted genes are supported by ESTs or TCs. About 40% of the predicted genes are intronless and there is evidence for 55% of them being expressed. A comparison of the Medicago truncatula, Oryza sativa, Arabidopsis thaliana, Lotus japonicus, Glycine max, and Populus trichocarpa genomes shows that the Medicago genome uniquely contains a high number of very short genes encoded by predicted genes with fewer than 99 nucleotides. The Gene Ontology (GO) annotation of the predicted genes showed that the nucleic acid binding domains are the most abundant domains in M.truncatula. The comparison between GO the annotation of M.truncatula, O. sativa, A.thaliana, L. japonicus, G. max, and P. trichocarpa reveals that all the six genomes have similar percentage of each of the major functional domains. The comparison of the top 40 Interpro domains in M. truncatula with the corresponding domains in the other five plants also indicates that most of the overrepresenting domains are overrepresenting in all the six genomes although some species-specific domains, such those for late nodulation, only are present in M. truncatula.The in silico analysis of the Medicago Gene Index 9.0 revealed that 191 genes only are expressed in root nodules, with100 of them similar to known GenBank sequences. Of the several gene familys, my analysis of 50 nodule-specific cysteine-rich peptides (NCR) indicates that they have a conserved signal peptide, a conserved cysteine motif, and a highly divergent remaining sequence. Many of the NCR genes are clustered while others are dispersed throughout the Medicago genome, suggesting that they have ungone a recent tandem or segmental gene duplication. A Ka/Ks analysis of NCR genes indicates that although some NCR genes underwent positive selection, others underwent purifying selection. That the NCR intron sequence is highly conserved suggests it may act as an enhancer for nodule-specific NCR gene expression in combination with the conserved upstream cis-acting motifs. The phylogenetic tree of both defensin and NCR genes reveals that after gene duplication, some of the defensin genes still remained defensins as is the case with the five medicago defensins, while the other duplicated defensin genes mutated such that they now seem to function in symbiosis as NCR genes.I also analyzed the three members of the glycine-rich peptide (GRP) gene family that are encoded on chromosome 2. These studies reveal that the GRP1 gene that is 5.2 Mb from the GRP2-GRP3 cluster arose from tandem gene duplication followed by either a deletion or an insertion from a common ancestor, an idea that is supported by sequence conservation in the signal peptide, the glycine-motif, and the 200bp upstream DNA sequence. In addition, my Ka/Ks analysis indicates that positive selection played an important role during GRP gene evolution. Furthermore, the Leghemoglobin (Lb) genes that originated from nonsymbiotic hemoglobins (Hb) seem to have undergone a purifying selection that has preserved their ability to function during oxygen transport. Finally, the nodule-specific genes all seem to contain one or both of two nodule-specific motifs (CTCCT and AAAGAT) in their promoter regions suggesting that nodule-specific gene expression likely is co-regulated and about 50% of the predicted nodule-specific genes are clustered and have corresponding EST, indicating that they are expressed in root nodules

The Identification, Functional Characterization and Phylogeny of the Nodulin-Like Anion Transporter (NLAT) Family in Plants

A cDNA was isolated from soybean (Glycine max) nodules that encodes a putative transporter (GmN70). GmN70 is expressed predominantly in mature nitrogenfixing root nodules. By western-blot and immunocytochemical analyses, GmN70 was localized to the symbiosome membrane of infected root nodule cells, suggesting a transport role in symbiosis. To investigate its transport function, cRNA encoding GmN70 was expressed in Xenopus laevis oocytes, and two-electrode voltage clamp analysis was performed. Ooctyes expressing GmN70 showed outward currents that are carried by anions with a selectivity of nitrate \u3e nitrite \u3e\u3e chloride. These currents showed little sensitivity to pH or the nature of the counter cation in the oocyte bath solution. One-half maximal currents were induced by nitrate concentrations between 1 to 3 mM. A global protein BLAST search for NLAT-related proteins revealed the presence of multiple homologs in all plant genomes sequenced with members segregating into 6 distinct monophyletic clades. Two genes from Arabidopsis clustering within the same clade 1 (chromosome nomenclature:At2g39210 and At2g28120) and shared a high amino acid sequence identity with GmN70 (respective 63.7% and 56.3%) and cluster in the same clade (Clade 1) as GmN70 and LjN70. Investigation of the transport properties of these two Arabidopsis NLAT-like genes (renamed AtNLAT1;1 [At2g39210] and AtNLAT1;2 [At2g28120] showed similar transport properties as GmN70 and LjN70 and transported the inorganic anions, nitrate, nitrite, and chloride. Expression analysis and sub-cellular localization of AtNLAT1;1 show it to be expressed predominately in the leaf on the plasma membrane. Its expression is regulated by a diverse set of biotic and abiotic stress signals including Pseudomonas syringae pv. tomato, salicylic acid, salinity, touch, and wounding suggesting a role in stress adaptation in plants. Consistent with these findings, transgenic Arabidopsis lines in which AtNLAT1;1 expression is knocked-down show an enhanced sensitivity to NaCl as well as a perturbed ionomic profile as revealed by inductively coupled plasma-mass spectroscopy (ICP-MS). Overall, the data presented in this body of work have provided insight into the biochemical and biophysical function and expression profiles of two members of the NLAT family, GmN70 and AtNLAT1;1

Early nodulin gene expression and the action of nod factors in Vicia sativa

Bacteria of the genera Rhizobium, Bradyrhizobium or Azorhizobium secrete lipo-oligosaccharide signal molecules, which play a pivotal role in the induction of early steps of root nodule formation on legumes. In these nodules the bacteria are hosted and a proper environment is provided for the bacteria to fix atmospheric nitrogen into ammonia, making the plant for its growth independent of nitrogen compounds in the soil.The goal of the research described in this thesis is to provide insight in the mechanism by which the Nod factors secreted by Rhizobium bacteria initiate root nodule formation. Such kind of study requires a plant-bacteria system in which both the morphological and molecular changes induced by the Nod factor can be examined. Working on the Nod factors secreted by Rhizobium leguminosarum bv. viciae the choice of the host plant is limited to the genera Pisum, Lathyrus, Lens and Vicia. We decided that Viciasativa (vetch) would be the most useful plant for our studies, since the plant is small and various morphological changes, like root hair deformation and the formation of nodule primordia, that are induced by the Nod factors can easily be observed (Chapter 1).Nod factors can induce the expression of early nodulin genes. These genes are expressed during different developmental stages of root nodule formation and the expression of these genes can therefore be used as molecular markers of root nodule development and Nod factor induced processes. To study Nod factor induced plant responses in V.sativa on the molecular level, the homologues of the early nodulin genes of pea (Pisum sativum) were isolated from V. sativa and their expression pattern was studied by in situ hybridization during root nodule development (Chapter 2).To obtain information about the pathway from Nod factor to early nodulin gene expression, the mechanisms controlling ENOD12 expression were studied. With a promoter analysis in transgenic V.hirsuta root nodules we have identified that the 200 bp immediately upstream of the transcription start are sufficient to induce nodule specific and Nod factor induced expression (Chapter 3). For the isolation of transcription factors involved in controlling ENOD12 expression an expression library was screened and a preliminary characterization of cDNA clones encoding polypeptides that bind to the PsENOD12 promoter is described in Chapter 4.In Chapter 5 early nodulin gene expression during Nod factor induced morphological changes, like root hair deformation and nodule primordia induction, was studied. Furthermore we examined whether RNA and protein synthesis are required for root hair deformation and for the activation of the early nodulin genes. These studies provided new insights about the mode of action of Nod factors.In Chapter 6 is discussed to what extent V. sativa is a suitable host plant to study the mode of action of Nod factors and in which way the studies reported in this thesis have contributed to elucidate the mechanism by which Nod factors induce a diversity of plant responses

Characterization of a potato Proteinase Inhibitor II gene whose expression in not wound-responsive

A potato Proteinase Inhibitor II gene (pin2T) was isolated and characterized both at the molecular level and the functional level. The open reading frame as well as the 5[superscript]\u27 and 3[superscript]\u27 flanking regions were sequenced. The coding region and 3[superscript]\u27 flanking region showed 86% and 87% identity with those of the previously isolated wound-inducible potato Proteinase Inhibitor IIK gene (pin2K) sequence, respectively. However, the 5[superscript]\u27 flanking region of the pin2T is highly homologous (91% identity) with that of pin2K from -767 to +29, relative to the transcription start site of the wound-inducible pin2K. The 5[superscript]\u27 flanking region of pin2T was linked to the reporter gene (chloramphenicol acetyl transferase, CAT; or [beta]-glucuronidase, GUS) coding sequences in constructions that contained the terminator from the wound-inducible pin2K gene. The chimeric genes were transferred to tobacco plants using Agrobacterium tumefaciens. The presence of the constructions in the transgenic tobacco plants was confirmed by polymerase chain reaction (PCR). Expression of CAT or GUS activities in transgenic plants driven by pin2T promoter indicated that pin2T is not a wound-inducible gene. GUS activities driven by promoter deletion mutants ruled out the presence of the silencer sequences in upstream portion of the pin2T promoter that differed from pin2K. Comparison of pin2T promoter sequence with that of the wound-inducible pin2K indicates that there are four small deletions which are located at -221 to -200, -263 to -254, -523 to -462, and -759 to -708 relative to the transcription start site of pin2K. When these deleted sequences are searched through Genebank, we identified one sequence that is found three times in pin2K and is completely deleted from pin2T. This sequence, 5[superscript]\u27-AGTAAA-3[superscript]\u27, is found in a wide variety of other wound-inducible genes but is not easily found in the published promoter sequences of the other plant genes. Nuclear proteins from unwounded and wounded potato leaves bound to the proximal promoter region, downstream of the 5[superscript]\u27-AGTAAA-3[superscript]\u27, of pin2T. A third deletion located at -523 to -462 relative to the transcription initiation site of pin2K, was moderately homologous (72% identity) with a putative sucrose-responsive element of sucrose-inducible genes. Deletion of this sequence was correlated with a loss of sucrose inducibility in the pin2T promoter. CAT activity driven by pin2T was not enhanced by sucrose, while sucrose enhances the expression of pin2K-CAT construct in transgenic tobacco plants. In addition to the sucrose effect on the chimeric gene expression, the effects of plant hormones, abscisic acid (ABA) and [alpha]-naphthalene acetic acid ([alpha]-NAA), were examined in transgenic tobacco leaves and calli, respectively. CAT activity driven by the non-wound-inducible pin2T promoter was not induced by ABA or derepressed in the absence of [alpha]-NAA as was the wound-inducible pin2K promoter

Transcriptional and Post-Transcriptional Regulation of Nodule-Specific Gene Expression in Soybean

Lateral roots and nodules are two important nutrient acquisition organs in soybean. The evolutionary origin of nodules from lateral roots has been highly hypothesized based on morphological similarities and genetic studies, but gene expression profiles during the formation of these organs have not been compared. In addition, the role of post-transcriptional gene regulation during nodule development has not been thoroughly explored. Bridging these knowledge gaps is crucial to develop genetic/biotechnological strategies to optimize nutrient acquisition and sustainable production of crops. To answer some of the outstanding questions about regulation of gene expression during nodule development, (i) global transcriptome analyses of lateral root and nodule were compared to identify organ-specific enrichment patterns of transcription factors and hormone signaling elements; (ii) small RNA and degradome/Parallel Analysis of RNA Ends (PARE) libraries were generated to identify miRNAs and their cleavage products respectively in nodule tissues; (iii) miRNA qPCR quantification methods were optimized; and (iv) the effect of misexpression of selected miRNAs on nodule development were evaluated. Analysis of transcriptome data showed very little overlap in transcription factor expression profiles between emerging nodules and emerging lateral roots. The expression profiles of certain key hormone biosynthesis and signaling genes were distinct between nodules and lateral roots. Interestingly, members of gene families associated with shoot axillary meristem formation were enriched in nodules, but not in lateral roots. Analysis of small RNA and PARE libraries resulted in the identification of 497 previously unknown miRNA precursors and validated 353 miRNA-target pairs. These and additional results suggested that inverse expression of miRNA and target is likely to be one of the mechanisms that direct nodule-specific gene expression. In addition, methods for miRNA quantification by qPCR were optimized, and a potential role for miR169 in regulating hormone homeostasis during nodule development was identified through functional assays. In summary, nodules might have adopted not only the developmental pathways of lateral roots, but also shoot axillary meristems. Furthermore, the inverse expression of miRNAs and their targets between nodules and adjacent root tissues might be a mechanism that spatially limits target gene expression to the nodule and/or root tissues