In order to improve our understanding on LEA proteins and their molecular functions in drought tolerance, the present work analyzes in the first place, the composition of LEA subproteomes from Arabidopsis seeds and maize embryos; second, three maize embryo LEA proteins from groups 1, 2, and 3 are analyzed in order to detect functional differences among them and finally, transgenic maize plants over-expressing group 5 “rab28” lea gene are characterized. The following results are presented: - Chapter 1. Proteomic approach to analyze the composition of LEA subproteomes from Arabidopsis seeds by mass spectrometry. The main objective was the development and isolation method to obtain enriched LEA populations from Arabidopsis seeds. LEA subproteomes were obtained using an extraction procedure that combines heat stability and acid solubility of LEA proteins. To identify the protein content, we followed two approaches: first, a classical 1D (SDSPAGE) gel-based procedure associated with MS analysis using an electrospray ionization source coupled on-line to liquid chromatography (LC-ESI-MSMS) and second, a gel-free protocol associated with an off-line HPLC and analysis via matrix assisted laser desorption/ionization (LC-MALDI-MSMS). - Chapter 2. Proteomic analysis of LEA proteins accumulated in maize mature seeds. Identification of LEA protein content by mass spectrometry and selection of three LEA proteins, Emb564, Rab17 and Mlg3, as representatives of groups 1, 2 and 3 for further study. Comparative functional analysis covering different aspects of maize Emb564, Rab17 and Mlg3 proteins, posttranslational modifications, subcellular localization and their properties in in-vitro and in- vivo assays. - Chapter 3. Characterization of transgenic maize plants expressing maize group 5 rab28 LEA gene under the ubiquitin promoter. Evaluation of Rab28 transcripts and protein levels, phenotype and stress tolerance traits of transgenic plants under drought stress. Investigation of the subcellular localization of transgenic and wild-type Rab28 protein using immunocytochemical approaches. / Las proteínas LEA, originalmente fueron descritas en las semillas de algodón; se acumulan en grandes cantidades en estructuras tolerantes a la desecación (semillas, polen) y en tejidos vegetativos sometidos a estrés abiótico, sequía, salinidad y frío. También se hallan en organismos anidrobióticos, en plantas de resurrección, algunos invertebrados y microorganismos. La presencia de proteínas LEA se correlaciona con la adquisición de tolerancia a la desecación. Desde un principio se les atribuyó un papel en las respuestas de las plantas en la adaptación al estrés (revisado en Bartels and Salamini 2001, Tunnacliffe 2007, Shih et al. 2010, Tunnacliffe 2010, Hand et al. 2011). Las proteínas LEA se clasifican en diversos grupos en función de dominios y secuencias de aminoácidos específicos (Wise 2010, Batagglia et al 2008, Bies-Ethève et al 2008). Los grupos 1, 2 y 3 son los más relevantes ya que abarcan la mayoría de las proteínas de la familia LEA. Una característica general de estas proteínas es su elevada hidrofilicidad, alto contenido de aminoácidos cargados y su falta de estructura en estado hidratado. A pesar de encontrarse mayoritariamente en forma de “random coil”, algunas adquieren un cierto grado de estructura durante la deshidratación o en la presencia de agentes promotores de α-hélices (Shih et al. 2010, Hand et al. 2011). A nivel celular se han hallado en todas las localizaciones, citosol, núcleo, nucleolo, mitocondria, cloroplasto, vacuola, retículo endoplásmico, peroxisoma y membrana plasmática, donde se supone ejercen su función protectora frente al estrés (Tunnacliffe and Wise 2007, Hundertmark and Hincha 2008). En relación a las modificaciones post-traduccionales, algunas se hallan fosforiladas (Jiang and Wang 2004; Plana et al. 1991, Heyen et al. 2002, Rohrig et al. 2006). Los efectos protectores de las varias proteínas LEA se han demostrado mediante ensayos in vitro y en aproximaciones transgénicas que han dado lugar a fenotipos resistentes a la sequía, sal y frío. Por lo general, se considera que estas proteínas contribuyen a la protección y a la estabilización de macromoléculas y estructuras celulares en las respuestas de adaptación al estrés en plantas; sin embargo, sus funciones específicas aún no han sido esclarecidas. A nivel molecular se ha propuesto que las funciones de las proteínas LEA pueden ser variadas: estabilización y renaturalización de proteínas, mantenimiento de membranas, en combinación, o no, con azúcares, tampones de hidratación (substitución de moléculas de agua), afinidad por iones y función antioxidante (Tunnacliffe and Wise 2007, Shih et al. 2010, Batagglia et al. 2008). Para finalizar, diremos que los objetivos principales de esta tesis consisten en ampliar los conocimientos sobre las proteínas LEA y sus funciones relativas a la tolerancia a la sequía. Los resultados están presentados en forma de capítulos.

Maize is susceptible to biotic and abiotic stresses. The most important abiotic stresses in Africa are drought and low soil fertility. Aflatoxin contamination is a potential problem in areas facing drought and low soil fertility. Three studies were conducted to evaluate maize germplasm for tolerance to stress. In the first study, fifteen maize inbred lines crossed in a diallel were evaluated under drought, low N stress, and well-watered conditions at six locations in three countries to estimate general (GCA) and specific combining ability (SCA), investigate genotype x environment interaction, and estimate genetic diversity and its relationship with grain yield and heterosis. GCA effects were not significant for grain yield across environments. Lines with good GCA effect for grain yield were P501 and CML258 across stresses. Lines CML339, CML341, and SPLC7-F had good GCA effects for anthesis silking interval across stresses. Additive genetic effects were more important for grain yield under drought and well-watered conditions. Heterosis estimates were highest in stress environments. Clustering based on genetic distance calculated using marker data from AFLP, RFLP, and SSRs grouped lines according to origin. Genetic distance was positively correlated with grain yield and specific combining ability. In the second study, synthetic hybrids were evaluated at seven locations in three countries to estimate GCA and SCA effects under low N stress and optimal conditions and investigate genotype x environment interaction. GCA effects were significant for all traits across low N stress and optimal conditions. The highest yielding synthetic hybrids involved synthetics developed from stress tolerant lines. Synthetics 99SADVIA-# and SYNA00F2 had good GCA for grain yield across low N stress conditions. Heterosis was highly correlated with grain yield. Optimal environments explained more variation than stress environments. The third study evaluated the agronomic performance and aflatoxin accumulation of single and three-way cross white maize hybrids at five locations in Texas. Inbreds CML343, Tx601W, and Tx110 showed positive GCA effects for grain yield. Significant GCA effects for reduced aflatoxin concentration were observed in lines CML269, CML270, and CML78 across locations. Differences in performance between single and three-way crosses hybrids were dependent mostly on the inbred lines.

The production of hydrogen (H2) and methane (CH4) related to olivine hydration (i.e. serpentinization) is considered a major contributor to abiotic hydrocarbon synthesis on Earth. Recent discoveries have highlighted the importance of low temperature (<100oC) serpentinization at continental peridotite outcrops. Such sites produce substantial fluxes of abiotic CH4 from gas seeps and/or springs. A limited number of studies in the southern hemisphere offer research on low temperature abiotic hydrocarbon synthesis in natural ultramafic environments, though large areas of exposed ophiolite are prevalent. This study assesses the origin and flux of CH4 and related water-rock interactions from a previously undiscovered site in the Dun Mountain Ophiolite Belt (DMOB), located at Red Hills, New Zealand. Methane emissions from a hyper-alkaline (pH >11.6) and reduced spring of calcium hydroxide (Ca2+-OH-) type waters near the Maitlands Fault were between 730 to 17,000 mg m 2day 1. The δ13C and δD values of CH4 emitting from this spring are consistent with CH4 of abiotic origin (δ13C: 32.7 ‰ VPDB, δD: 363 ‰ V SMOW). Hyper-alkaline fluids emitting from the spring are concentrated in dissolved CH4 (2.2 mg/L) and H2 (0.7 mg/L) and display δ13CCH4 signatures consistent with other sites worldwide. Extensive and localised carbonate precipitation occurs at the hyper-alkaline Ca-rich spring. Isotopic evaluation of carbonate nodules are kinetically fractionated with 13C and 18O depletions up to 30.8 ‰ and 9.3 ‰, respectively. This disequilibrium between the mineralogy and interacting fluids and gases represents a potential habitable environment for microorganisms. Porous, layered carbonates located on the outer edges of the hyper-alkaline spring are the result of atmospheric CO2 interaction with magnesium bicarbonate (Mg2+-HCO3) and Ca2+-OH- hyper-alkaline waters. The precipitation of these carbonates offers potential insight towards low temperature CO2 sequestration. Additionally, various forms of Fe-rich amorphous material precipitate in association with Mg2+-HCO3 type waters at the Red Hills. The identification of bacteria and diatoms within this material offers supporting information regarding microbial survival in metal-rich, reduced environments. This multidisciplinary study demonstrates the interconnected nature of geological, biological and atmospheric interactions in ultramafic environments at low temperature on Earth.

Recent studies have demonstrated the omnipresence of antibacterial agents in the aquatic environment due to high usage and widespread applications of these compounds in medicine and agriculture, raising concerns over proliferation of antibiotic-resistant bacteria and other adverse health effects. Tetracyclines (TCs) are among the most widely used antibiotics and their fate and transformation in the soil-water environment are not yet well understood. Based on TCs' strong tendency to interact with metals, their environmental fate and transport are expected to be greatly influenced by metal species commonly present in waters and soils and thus the focus of this study. The study results show that TCs are highly susceptible to oxidative transformation mediated by dissolved Mn(II) and Cu(II) ions and manganese dioxide under environmentally relevant conditions. The oxidative transformation can occur via different TC structural moieties and reaction pathways when different metal species are involved, leading to complicated product formation patterns. It was also found that Al oxide surfaces can promote the acid-catalyzed isomeration and dehydration of TCs. To better evaluate the surface reactions of Mn oxide with TCs and other compounds, a new kinetic model was successfully developed to describe the complex reaction kinetics based on the experimental results with TCs and three other classes of antibacterial agents. Overall, this work significantly advances the fundamental understanding of the reaction mechanisms of TC compounds and provides the knowledge basis for better risk assessment of these compounds in the environment.

Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: Sutherlandia frutescens (L.) R. Br., also regarded as Lessertia frutescens, is a leguminous, perennial shrub indigenous to South Africa. Extracts prepared from the leaves have traditionally been used for the treatment of various diseases. Reports have also indicated that S. frutescens provides certain health benefits to cancer and HIV/AIDS patients. Analysis of extracts indicated the presence of several compounds (bitter triterpenoid glycosides, several flavonoids, amino acids, small amounts of saponins (no alkaloids though), asparagine, Larginine, canavanine, gamma-aminobutyric acid (GABA) and pinitol) which contribute to the medicinal properties of this plant. The first part of this study involved testing the effect of six treatments (light, dark, soaking of seeds, physical scarification, chemical scarification and flaming of seeds) on the in vitro germination of Sutherlandia seeds to elucidate the factors which control seed germination. Those treatments which removed the seed coat were most successful for germination with physical scarification being the most efficient method, resulting in 98.6% of the seeds germinating after 21 days. Although the organogenesis of Sutherlandia explants (cotyledons and hypocotyls) in vitro were investigated (results not included in this thesis), omitting plant growth regulators (PGR) in the cultivation medium was best for shoot multiplication. However, this PGR-free system successfully provided a continuous supply of plant material for further studies. It would be possible to successfully adopt it for commercial production of plants to assist with cultivation of Sutherlandia as a field crop. Another advantage of this system is spontaneous rooting with 85% of the in vitro microshoots rooting in PGR-free medium. These rooted plants were acclimated in the glasshouse using vented lids to harden off the shoots and this method resulted in 100% survival of plants. The second part of this study investigated the induction of hairy root cultures of S. frutescens using Agrobacterium-mediated transformation. The efficiency of three Agrobacterium strains (A4T, LBA9402 and C58C1) to transform different S. frutescens explants (cotyledons and hypocotyls) was analyzed. All three strains were equally efficient at inducing hairy roots in both hypocotyls and cotyledons. However, transformation of S. frutescens was dependent on the type of explant used with the hypocotyls being more efficiently transformed than the cotyledons. Overall the transformation of both the hypocotyl (93%) and cotyledon (47%) was highest when the strain A4T was used. Four hairy root clones were selected and their cultivation in a liquid system was optimized by investigating their growth in four different types of media (Gamborg B5 (Gamborg et al., 1968), White’s (White, 1934; White, 1954), MS (Murashige and Skoog, 1962) and half strength MS medium). All the growth of hairy root clones was best in the B5 and MS medium, with White’s medium being the least effective cultivation medium. Molecular analysis of hairy roots was used to prove the transgenic status of these four putative transgenic clones. This was achieved using polymerase chain reaction (PCR) amplification of rol A (320 bp), B (780 bp) and C (600 bp) genes to determine the presence of the TL-DNA in the plant genome. During Southern hybridization a radioactively labeled rol A probe was used to determine the copy number of the rol A gene. The three rol genes were present in all four hairy root clones. The third part of this study focused on the effect of three abiotic stress factors (nitrogen availability, salinity and drought) on the synthesis of four metabolites (gamma-aminobutyric acid (GABA), asparagine, arginine and canavanine). The effect of nitrogen availability on metabolite synthesis and the morphology was determined using in vitro shoot cultures as well as the hairy root clone C58C1-g. Nitrogen availability studies were conducted by cultivating the microshoots or root tips on modified MS medium. The MS medium contained either the normal amount of nitrogen (1.9 g L-1 KNO3 and 1.65 g L-1 NH4NO3) in the MS medium (1x nitrogen), half the normal nitrogen concentration in MS medium (0.5x nitrogen) or twice the normal nitrogen concentration in MS medium (2x nitrogen). The arginine and asparagine levels in the roots and shoots and the canavanine level in the shoots were directly correlated with the amount of nitrogen in the medium (as the nitrogen level increased, the metabolite levels increased). The GABA level in the shoots was inversely correlated with the amount of nitrogen in the medium. Several reasons may explain these metabolic changes including the assimilation of extra nitrogen into asparagine, canavanine and arginine in the shoots. The reduced GABA levels may indicate the preferential flux of the free GABA into other nitrogen assimilatory pathways such as protein synthesis as well as its rapid utilization to replenish the tricarboxilic acid cycle intermediates. The effect of water (induced by including 3% (w/v) PEG in the medium) and salt stress (induced by including either 50 or 100 mM NaCl in the medium) was only investigated in the shoot cultures as the root cultures lacked the synthesis of canavanine. Water stress did not significantly alter the metabolite levels, but resulted in a significant decrease in the growth (fresh weight and total shoot length) and the rooting response of these microshoots. Salt stress only resulted in a significant increase in arginine levels with increasing salinity and also caused a reduction in the rooting and growth response. Lowered plant vigour may be the first visual sign of water stress. Addition of NaCl may lead to ion toxicity and requires osmotic adjustment resulting in changes at the metabolic level concomitant to physiological growth changes. Finally, the anti-bacterial activity and the phytochemistry of transgenic root cultures and untransformed in vitro and ex vitro plant material was examined. Only the extracts prepared from the wild harvested leaf material exhibited moderate anti-bacterial activity (1.25 mg ml-1) against all the bacteria (Escherichia coli, Klebsiella pneumoniae, Bacillus subtilis and Staphylococcus aureus) tested. Changes to the secondary metabolism of hairy roots were investigated using TLC and LC-MS analysis. Several of the compounds in the hairy root extracts were present in higher levels than in the control root extracts. Transformation also increased the complexity of the phytochemical pattern of the hairy roots, either due the synthesis of novel compounds or upregulated synthesis of existing metabolic pathways. The production of hairy roots and the establishment in a liquid system during this study was an important step towards upscaling these cultures to a bioreactor. In future these roots can assist in developing cultures which produce a high yield of the desired metabolites. / AFRIKAANSE OPSOMMING: Sutherlandia frutescens (L.) R. Br., ook bekend as Lessertia frutescens is ‘n peulagtige meerjarige struik, inheems tot Suid Afrika. Ekstrakte wat van die blare voorberei word, is tradisioneel gebruik vir die behandeling van verskeie siektes. Berigte het ook daarop gedui, dat S. frutescens sekere gesondheidsvoordele vir kanker en HIV/VIGS pasiënte inhou. ‘n Ontleding van die ekstrakte, dui op die teenwoordigheid van verskeie verbindings (bitter triterpenoïed glikosiede, verskeie flavonoïede, aminosure, klein hoeveelhede saponiene (alhoewel geen alkaloïede), asparagien, L-arginien, canavanien, gamma-aminobottersuur (GABS) en pinitol) wat tot die medisinale eienskappe van hierdie plant bydrae. Die eerste deel van die studie het die effek van ses behandelings (lig, donker, week van sade, fisiese skarifikasie, chemiese skarifikasie en die vlam van sade) op die in vitro ontkieming van Sutherlandia sade getoets met die doel om die faktore wat saadontkieming beheer, te identifiseer. Die beste behandeling vir saadontkieming was dié behandelings wat die saadhuid verwyder het. Die mees effektiewe metode van saadhuidverwydering was die fisiese skarifikasie van sade, wat gelei het tot ‘n 98.6% ontkieming van sade na 21 dae. Alhoewel in vitro organogenese van Sutherlandia eksplante (kotiel en hipokotiel) ondersoek was (resultate nie ingesluit in die tesis nie), was plant groei reguleerders (PGR) uitgesluit in die groeimedium om stingelvermeerdering te bevorder. Nie te min was die PGR-vrye sisteem suksesvol om ‘n voortdurende bron van plant material vir verder studies te verskaf. Dit sou egter moontlik wees om die PGR-vrye sisteem suksesvol te kon aanpas vir die kommersiële produksie van plante met die doel om Sutherlandia as ‘n landbougewas te bevorder. ‘n Verdere voordeel van dié sisteem, is die spontane wortelvorming, met 85% van die in vitro mikrostingels wat wortels in die PGR-vrye medium produseer het. Hierdie bewortelde plante was in die glashuis geakklimatiseer met behulp van geventileerde deksels (vir stingel afharding) en het tot ‘n 100% oorlewing gelei. Die tweede deel van die studie het die induksie van S. frutescens harige wortelkulture met behulp van Agrobacterium-bemiddelde transformasie ondersoek. Die effektiwiteit van drie Agrobacterium stamme (A4T, C58C1 en LBA9402) om verskillende S. frutescens eksplante (kotiel en hipokotiele) te transformeer, was geanaliseer. Al drie stamme was ewe effektief om harige wortels op beide hipokotiel en kotiele te induseer. S. frutescens transformasie blyk egter tog van die tipe eksplant afhanklik te wees, aangesien die hipokotiele meer effektief as die kotiele getransformeer kon word. Met inagneming van beide die hipokotiel (93%) en kotiel vii (47%), was transformasie optimaal met die gebruik van die A4T stam. Vier harige wortelklone was geselekteer en hulle produksie in ‘n vloeibare sisteem was geoptimiseer deur hulle groei in vier verskillende tipe media (Gamborg B5 (Gamborg et al., 1968), White’s (White, 1934; White, 1954), MS (Murashige and Skoog, 1962) en half-sterkte MS medium) te ondersoek. B5 en MS medium was beskou as die beste vir alle die harige wortelklone se groei, terwyl White’s medium die minste doeltreffende groeimedium was. Molekulêre analise van die harige wortels was gebruik ten einde die transgeniese status van die vier vermoedelike transgeniese klone te bewys. Dit was behaal deur polimerase kettingreaksie amplifisering (PKR) van die rol A, B en C gene ten einde die teenwoordigheid van die TL-DNS in die plant genoom aan te toon. Tydens Southern hibridisasie was ‘n radioaktief gemerkte peiler gebruik om die aantal rol A geen kopieë te bepaal. Die drie rol gene was teenwoordig in al vier harige wortelklone. Die derde deel van die studie het gefokus op die effek van drie abiotiese stress faktore (stikstof beskikbaarheid, sout- en droogte stres) op die produksie van vier metaboliete (GABS, asparagien, canavanien en arginien). Die effek van stikstof beskikbaarheid op die metaboliet produksie asook die morfologie was bestudeer deur gebruik te maak van in vitro mikrostingels asook die harige wortel kloon C58C1-g. Stikstof beskikbaarheidstudies was uitgevoer deur die mikrostingels of wortelpunte in ‘n gewysigde MS medium te groei. Die MS medium was aangepas om die normale hoeveelheid stikstof (1.9 g L-1 KNO3 en 1.65 g L-1 NH4NO3) in MS medium (1x stikstof), of die helfte van die normale stikstof konsentrasie (0.5x stikstof) of twee keer die normale stikstof konsentrasie in MS medium (2x stikstof) te bevat. Die arginien en asparagien vlakke in die wortels en stingels, asook die canavanien vlak in die stingels was positief gekorreleerd aan die stikstof konsentrasie in die medium. Die GABS vlak in die stingels was egter omgekeerd eweredig aan die stikstof konsentrasie in die medium. Verskeie redes kan aangevoer word om die metaboliet veranderinge te verduidelik, insluitende die assimilasie van addisionele stikstof in asparagien, canavanien en arginien in die stingels. Die verlaagde GABS vlakke kan dui op die voorkeur van vrye GABS vloei na ander stikstofassimilerende metaboliese paaie soos proteïen sintese, asook die snelle benutting van GABS ten einde die Trikarboksielsuursiklus intermediêre produkte aan te vul. Die effek van droogte (geïnduseer deur die byvoeging van 3% (m/v) PEG tot die medium) en sout stres (geïnduseer deur 50 of 100 mM NaCl byvoeging tot die medium) was slegs in die stingel kulture ondersoek weens die afwesigheid van canavanien produksie in die wortel kulture. Water stres het nie ‘n betekenisvolle verandering in die metaboliet vlakke meegebring nie, maar dit het wel tot ‘n beduidende afname in groei (vars massa en totale stingel lengte) en bewortelingsreaksie in die mikrostingels gelei. Sout stres het slegs tot ‘n betekenisvolle viii toename in arginien vlakke asook ‘n afname in die wortelvorming en groeireaksie tydens die toenemende sout vlakke gelei. ‘n Verlaging in plant groeikragtigheid mag ‘n eerste visuele teken van water stres wees. Die toevoeging van NaCl tot die medium kan tot ioontoksisiteit lei en plante reageer deur middel van osmotiese aanpassing wat tot veranderinge in die metaboliet vlakke asook veranderinge in fisiologiese groei, lei. Die finale deel van die studie het die anti-bakteriële aktiwiteit en die fitochemie van die transgeniese wortel kulture asook die ongetransformeerde in vitro en ex vitro plant materiaal ondersoek. Slegs die ekstrakte verkry vanaf blaar materiaal geoes uit die natuur, het matige anti-bakteriële aktiwiteit (1.25 mg ml-1) teen al die bakterië (Escherichia coli, Klebsiella pneumoniae, Bacillus subtilis en Staphylococcus aureus) wat ondersoek is, getoon. Aanpassings in die sekondêre metabolisme van die harige wortels is deur middel van dunlaag chromatografie (DLC) en vloeibare chromatografie-massa spektroskopiese (VC-MS) analise ondersoek. Verskeie verbindings was in hoër vlakke in die harige wortels teenwoordig, as in die kontrole wortel ekstrakte. Transformasie het ook die kompleksiteit van die harige wortels se fitochemiese patroon verhoog, moontlik weens die produksie van nuwe verbindings of weens die opregulasie van bestaande metaboliese paaie. Die produksie van harige wortels en die vestiging daarvan in ‘n vloeibare sisteem tydens hierdie studie word beskou as ‘n belangrike stap na die opskalering van die kulture na bioreaktore. Hierdie wortels kan toekomstig tot die ontwikkeling van kulture met ‘n hoë produksie van gewenste metaboliete lei.

All plants have a protection against the surrounding environment called a cuticle coating. When this cuticle coating is constructed it is believed that the family of protein called lipid transfer proteins (LTPs) is involved. The LTPs are small and cysteine rich. In Marchantia polymorpha the groups of LTPs called LTPd and LTPg can be found. 8 and 4 in each group respectively. In the genes of LTPd there is an intron placed downstream of the start codon. Firstly, a sequence database search was performed and LTPd2 and LTPd3 were chosen for further experiments in this study. Secondly, a control that the intron was present in the samples were done by preforming a PCR reaction of cDNA from isolated RNA taken from untreated Marchantia polymorpha. A gel electrophoresis of the product was also performed. Lastly, the amount of alternative splicing in LTPd2 and LTPd3 from Marchantia polymorpha after treated with cold and dehydration were studied using quantitative PCR. For the qPCR MpACT and the exon of respective gene were used as references. The ΔCt values and the expression fold (2ΔΔCt) calculated from the qPCR results showed that most of the transcript with introns preserved were upregulated after subjected to stress. Only the intron in MpLTPd2 and MpLTPd3 with MpACT as reference showed a small downregulation after the cold treatment. The intron in MpLTPd3 with MpLTPd3s exon as reference didn’t show any difference. None of the intron transcript in any of the genes on the other hand showed any significant difference in the alternative splicing. This could be because of small sample groups when the test was performed. In conclusion, there were no significant difference in intron expression between treated and control samples. Therefore, nothing can be said about the change in alternative splicing in MpLTPds after cold and dehydration treatments.

Du fait de sa croissance exceptionnelle combinée aux propriétés supérieures de son bois, l'Eucalyptus est devenu le feuillu le plus planté au monde et s'est imposé comme source de biomasse pour la production de papier et de biocarburants de seconde génération. Le bois est composé de parois secondaires lignifiées et sa formation est finement régulée par un réseau complexe, et globalement mal connu, de facteurs de transcription (FT). Les parois secondaires sont composées de 80% de polysaccharides, ciblés pour la plupart des bioproduits à haute valeur ajoutée, tandis que la lignine (20%) est responsable de la récalcitrance de la biomasse à la dégradation enzymatique mais augmente le potentiel énergétique du bois par combustion. Malgré son adaptabilité remarquable à différents sols et climats, la croissance de l'Eucalyptus varie fortement suivant ces facteurs. L'Eucalyptus est largement planté sur des sols lessivés dans les régions tropicales et subtropicales où les plantations industrielles font face à des épisodes de sécheresse de plus en plus fréquents, en combinaison avec des forts manques de nutriments, nécessitant de gros apports en fertilisants. Dans les région tempérées telles que l'Europe du Nord, la principale limitation à l'implantation de cet arbre dépourvu d'endodormance, est l'exposition au froid. Ces contraintes abiotiques sont aggravées par le changement climatique et leur impact sur la formation du bois et sa qualité restent peu documentés. Quelques données suggèrent que ces stress affectent le dépôt de la paroi secondaire ainsi que la structure du xylème. Cependant, ces résultats sont très hétérogènes entre différentes espèces et principalement focalisés sur des tissus différents du bois. La sélection de clones adaptés et le développement de pratiques culturales plus viables, sont essentiels pour améliorer la productivité et la qualité du bois, ce qui requiert une meilleure compréhension de la réponse des arbres au froid et au manque d'eau en interaction avec la nutrition. Dans le but de décrypter les régulations induites par le froid dans la différenciation du xylème, nous avons effectué une approche ciblée sur des Eucalyptus acclimatés au froid. Des analyses de biochimie, d'histochimie et de transcriptomique, ont révélé que le froid déclenche un dépôt de paroi secondaire précoce dans les cellules du xylème en développement, caractérisé par un fort dépôt de lignine. En parallèle, pour caractériser l'effet du manque d'eau combiné à différents régimes nutritifs, sur la formation et la qualité du bois, nous avons tiré profit d'un dispositif expérimental mis en place au champ avec un clone commercial d'Eucalyptus, soumis à une exclusion de pluie combinée à une fertilisation au potassium. Nous avons combiné des analyses globales du transcriptome et du métabolome, avec l'analyse des propriétés structurales et biochimiques du bois. L'approche intégrative de ces jeux de données a révélé que la fertilisation au potassium induit une répression de la biosynthèse de la paroi secondaire ainsi qu'une régulation de l'activité cambiale et la modification dans les propriétés du bois, avec une forte interaction avec l'exclusion d'eau. Ces deux approches ont permis l'identification de différents FT non caractérisés qui constituent des candidats prometteurs dans le contrôle de l'activité cambiale et du dépôt de paroi secondaire chez un ligneux. Leur caractérisation fonctionnelle chez le peuplier et l'Eucalyptus a révélé un nouveau régulateur clé de la biosynthèse de paroi secondaire, et plusieurs facteurs MYB potentiellement impliqués dans la balance entre formation de la paroi secondaire et croissance. / Due to its outstanding growth combined to superior wood properties, Eucalyptus genus has become the most planted hardwood on earth and emerged as the most appealing sources of renewable biomass feedstock for paper and second-generation biofuels. Wood is composed of lignified secondary cell walls (SCWs) and its formation is tightly regulated by a complex, partially unknown, transcription factors (TFs) network. SCWs are composed by 80% of polysaccharides targeted for most of value-added bioproducts, whereas lignin (20%) is responsible for biomass recalcitrance to enzymatic degradation but increase wood energetic potential for combustion. Despite its remarkable adaptability to various soils and climate environment Eucalyptus growth varies strongly according to these factors. Eucalyptus is extensively grown in highly weathered soils in tropical and subtropical regions where plantations are facing more frequent drought episodes in combination to nutrient starvation, requiring high amounts of expensive fertilizers. In temperate regions such as North of Europe, the main limitation for the expansion of this non-dormant tree is cold exposure, which reduces dramatically its growth. The effects of these stresses are emphasized in the actual context of climate change which induces sharp contrasting periods, and their impacts on wood formation and quality remain unknown. Scarce data from literature suggest that these stresses affect secondary cell wall (SCW) deposition as well as xylem cell patterning. However these results are highly heterogeneous among different species and mainly focused on non-woody tissues. The selection of adapted clones and the development of more sustainable cultural practices are crucial to improve wood productivity and quality, which require a better understanding of tree response to cold and water stress in interaction with nutrition. In order to unravel the regulation of xylem differentiation by low temperature, we performed a targeted approach on cold-acclimated Eucalyptus trees. By biochemical, histochemical and transcriptomic analyses, we revealed that low temperature trigger a precocious SCW deposition in developing xylem cells, characterized by a strong lignin deposition. In parallel, we aimed to characterize the effect of water stress combined to different mineral nutrition regimes, on wood formation and quality. To this end, we took advantage of an experimental design set up on field with a highly productive Eucalyptus commercial clone submitted to both rainfall exclusion combined to potassium fertilization. We combined large scale analyses of transcriptome and metabolome, with wood structural and biochemical properties analyses. The integrative approach with these datasets revealed that potassium fertilization induces a repression of SCW biosynthesis, together with regulation of cambial activity and modifications in wood properties, with a strong interaction with water exclusion. Both approaches allowed to point out several uncharacterized yet TFs which are highly promising candidates in the control of cambial activity and SCW deposition in a woody perennial. Characterization of their function in poplar and Eucalyptus revealed a new key regulator of SCW biosynthesis in wood, and several MYB TFs potentially involved in the trade-off between SCW biosynthesis and growth.

Doctor of Philosophy / Department of Agronomy / S.V. Krishna Jagadish / Sorghum (Sorghum bicolor (L.) Moench) is one of the hardiest crop to abiotic stresses compared with other grain crops. However early stage chilling, terminal heat and drought stress are three most damaging abiotic stresses that have limited sorghum productivity in the US Great plains and other locations having similar environmental conditions. Three studies were conducted with an overall goal aimed at increasing grain sorghum’s resilience to harsh climatic conditions. In the first study, four promising chilling stress tolerant sorghum advanced breeding lines, a known early stage chilling tolerant Chinese landrace (Shan Qui Red - SQR) and a susceptible US elite cultivar (RTx430) as checks were assessed for chilling tolerance during emergence and early growth under field and controlled environments. Aerial phenotyping using unmanned aircraft systems (UAS) fitted with multispectral camera was used to capture reflectance-based vegetation indices (NDVI and NDRE) in field experiments. Some advanced breeding lines with superior agronomic background also recorded significantly better emergence, seedling growth and vigor compared to SQR under chilling conditions. Aerial phenotyping indices from images taken between 30 and 60 days after emergence were consistently correlated with destructive measurements under early plantings, indicating their effectiveness in differentiating chilling responses. Second study was conducted to understand physiological mechanisms inducing heat stress resilience in sorghum during flowering. A diverse set of sorghum inbreds and selected hybrids were tested under greenhouse, growth chamber facilities and field conditions. A highly conserved early-morning-flowering mechanism was observed across all the inbreds and hybrids, with the peak anthesis wherein >90% of florets completed flowering within 30 min after dawn. The conserved response was consistent even under drought stress and heat stress exposure imposed at different times of the day. Our findings report a novel heat escaping early-morning-flowering mechanism effectively employed by sorghum to minimize heat stress impact at anthesis. Another experiment with sequential increase in daytime temperature treatments suggest heat stress induced loss in pollen viability to be a key factor resulting in reduced seed-set and grain yield. The findings suggest heat stress could have a greater impact on post-pollen germination processes such as fertilization, embryo formation and development. We identified a heat tolerant genotype “Macia” which appears to be a promising donor for developing improved heat tolerant sorghum hybrids. In the third study, a bi-parental recombinant inbred lines (RILs) mapping population developed from elite post flowering drought susceptible cultivar (RTx430) and a known drought tolerant cultivar (SC35) were evaluated under wide spectrum of environments and moisture conditions. Several novel and major QTL for grain yield, panicle neck diameter, effective quantum yield of photosystem II and chlorophyll content were identified. The genomic regions and the candidate genes within these regions can potentially help in improving source and sink dynamics in sorghum under diverse environments. The findings from these studies will complement ongoing efforts in developing future sorghum with enhanced resilience to different abiotic stresses that continue to limit sorghum productivity.

Seed addition experiment was conducted on the moisture gradient with aim to demonstrate that many species not accounted by current measures of species pools in a site should be included because they are in fact able to establish successfully if biotic filter is removed. Transplant experiment was established to compare the sensitivity of seedling recruitment and survival of transplants in the field. Recruitment of seedlings in field was observed and compared with germination of seeds under different chilling treatments in laboratory.

O desempenho de materiais compósitos plástico-madeira ou ―wood plastic composites‖(WPC) requerem uma avaliação eficiente de sua resistência ao envelhecimento natural ao longo do tempo e contra sua biodeterioração por micro-organismos. Neste sentido, o objetivo deste estudo foi investigar os mecanismos de degradação abiótica e biótica de compósitos termoplásticos com fibras vegetais, a fim de determinar seu comportamento, do ponto de vista da estabilidade, nas condições em que serão usados. A aplicação proposta para estes materiais é sua utilização em ambientes externos, com o propósito de substituir a madeira, em artefatos para construção civil, como portas, janelas, pisos, decks e divisórias. Além disso, a eficácia da utilização do agente de acoplamento (AC) na durabilidade dos compósitos envelhecidos foi investigada. Para a produção dos compósitos foi utilizada a proporção polímero-madeira de 70/30 m/m, sendo que a matriz polimérica foi constituída de uma blenda de polipropileno-copolímero de etileno acetato de vinila (PP-EVA) pós-consumo provenientes de tampas de garrafa, e as madeiras usadas na forma de serragem, utilizando as espécies de eucalipto e pinus. O polipropileno graftizado com anidrido maleico foi usado como AC na proporção de 3 % m/m. As misturas foram processadas por extrusão e os compósitos moldados por compressão térmica e injeção. No teste de degradação por fungos foram utilizados quatro espécies de fungos basidiomicetos, Trametes villosa, Trametes versicolor, Pycnoporus sanguineus e Fuscoporia ferrea, todos de podridão branca. Na degradação abiótica, foram avaliadas as propriedades físicas, mecânicas, térmicas, reológicas, químicas e morfológicas dos compósitos nos diferentes estágios e formas de degradação. Para a degradação biótica, foram realizados ensaios de biodegradação em solo simulado (respirometria) e inoculação com fungos (deterioração fúngica). Os resultados mostraram que as condições climáticas afetaram diretamente as características dos compósitos avaliados, causando mudanças na cor e em sua viscosidade, com o aumento do índice de fluidez dos materiais e também alterações em suas estruturas químicas, com aumento do índice de carbonila, indicando a ocorrência de foto-oxidação das amostras. Em relação aos ensaios de biodegradabilidade, houve uma pequena perda de massa nas amostras avaliadas e formação de biofilmes nas superfícies dos compósitos, verificados por microscopia eletrônica de varredura (MEV). A deterioração fúngica ocorre especialmente na superfície das amostras. O fungo Fuscoporia ferrea foi o mais efetivo na deterioração dos compósitos, com maior perda de massa e até mesmo o surgimento de estruturas de reprodução após o período de incubação, porém não penetrou na parte interna dos materiais. Por respirometria também foi verificado um aumento na geração de CO2 ao longo do período avaliado, indicando que há um início de metabolização das amostras por micro-organismos incubadas no solo, principalmente nas amostras expostas a nove meses de intemperismo natural. A presença das fibras protegeu a matriz de PP-EVA da degradação na face não exposta diretamente ao sol, o que é interessante para aplicações que visam longa vida útil, porém a utilização de aditivos para preservação contra radiação UV, oxigênio e micro-organismos se tornam necessárias para otimização e maior durabilidade destes materiais em estudos futuros. / Performance tests on "wood plastic composites" (WPC) require efficient evaluation of their resistance to natural aging over time and against its biodegradation by microorganisms. In this sense, the objective of this study was to investigate the abiotic and biotic degradation mechanisms of thermoplastic composites with vegetable fibers, in order to determine its behavior from the point of view of stability under conditions that will be used. The proposed application for these materials is their use outdoors, in order to replace the wood, in building construction, such as doors, windows, floors, decks and bulkheads. In addition, the effective use of the coupling agent (CA) in durability of aged composites was investigated. For the production of composite was used wood-polymer ratio of 70/30 w/w, wherein the polymeric matrix is comprised of a blend of polypropylene and ethylene-vinyl acetate copolymer (PP-EVA) from post-consumer caps bottle, and the woods used in the form of sawdust, using the eucalyptus and pine species. The polypropylene grafted with maleic anhydride was used as coupling agent in the proportion of 3% w/w. The mixtures were processed by extrusion and composites molded by injection and thermal compression. In fungal degradation tests were used four species of basidiomycete fungi, Trametes villosa, Trametes versicolor, Pycnoporus sanguineus and Fuscoporia ferrea, all white rot fungi. In abiotic degradation, physical, thermal, mechanical, rheological, chemical and morphological properties of composites at different stages of degradation and shapes were evaluated. For biotic degradation, biodegradation tests were performed on soil (respirometry) and inoculated with fungi (fungal deterioration). The results showed that the climatic conditions directly affect the characteristics of the evaluated composites, causing changes in color and its viscosity with increasing melt flow index of the materials and also changes in their chemical structures with increased carbonyl index of samples exposed to natural weathering, thus indicating a photo-oxidation of the samples. Regarding the degradation tests, there was a small weight loss in the evaluated samples, and growth of microorganisms on the surface of composites, evidenced by scanning electron microscopy (SEM). The fungal deterioration occurs especially on the surface. The fungus Fuscoporia ferrea was the most effective in composites deterioration, with greater weight loss and even the emergence of reproductive structures after the incubation period, but did not penetrate inside the material. By respirometry also can be seen an increase in the generation of CO2 over the months, indicating a beginning of metabolism of the samples incubated by microorganisms in the soil, especially in samples exposed to 9 months of natural weathering. The presence of fibers protected the PP-EVA matrix of degradation in the face not exposed to direct sunlight, which is interesting for applications that aim to long life, but the use of additives for preservation against UV radiation, oxygen and microorganisms become necessary for optimization and durability of these materials in future studies.