This thesis was undertaken to determine if there is any interrelationship between the two terminal enzymes of the haem biosynthetic pathway, protoporphyrinogen oxidase (PPO) and ferrochelatase, with particular reference to porphyria variegata (PV) and erythropoietic protoporphyria (EPP)• It has previously been found that both enzymes were deficient in PV and EPP, there being a qualitative difference in so far as ferrochelatase deficiency is concerned. / IT2018

Master of Science / Department of Agronomy / Kassim Al-Khatib / Field experiments were conducted near Manhattan, KS in 2005 and 2006 and Sabetha, KS in 2005 to determine the efficacy of S-metolachlor tank mixed with fomesafen on common waterhemp in soybean. Preemergence treatments included S-metolachlor + fomesafen at 0.91 + 0.22, 1.21 + 0.28, 1.52 + 0.36, and 1.82 + 0.43 kg ha-1 and S-metolachlor + metribuzin at 0.55 + 0.14 kg ha-1. These treatments were applied alone or followed by a postemergence glyphosate application at 0.88 kg ha-1. Ratings were taken 2, 4 and 8 weeks after treatment. The study showed that S-metolachlor + fomesafen gave excellent early season control of common waterhemp at both Sabetha and Manhattan. S-metolachlor + fomesafen at the 1.52+0.36 kg ha-1 rate gave greater weed control than S-metolachlor + metribuzin. A separate study was conducted to determine the competitiveness and fitness of a protox-resistant common waterhemp biotype. Protox-resistant and protox-susceptible biotypes of common waterhemp were grown under noncompetitive and competitive arrangements in the greenhouse. In the noncompetitive study a single plant of both biotypes was planted in 15-cm-diam pots. Photosynthesis, leaf area, and plant biomass were measured 10, 20, 30, and 40 day after transplanting (DATP). In general, photosynthesis rate and plant biomass was similar between biotypes. However, the protox-resistant biotype had higher leaf area then the susceptible biotype at 20, 30, and 40 DATP. Under competitive conditions, a replacement series study, photosynthesis, leaf area, plant height, and plant biomass were measured 7, 14, 21, and 28 DATP. In general protox-resistant and –susceptible common waterhemp values were similar 28 DATP. Relative crowding coefficient values 28 DATP were 0.86, 0.89, 1.09, and 1.13 for photosynthesis, leaf area, plant height, and plant biomass, respectively. Suggesting, protox resistance did not change the ability of common waterhemp to grow normally under competitive conditions.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Sugarcane is a tropical perennial grass species belonging to the Poaceae (true grasses) family. Mature sugarcane is comprised mostly of sugarcane stalks, which accumulate high amounts of sucrose, a fact that has led to its wide cultivation of sugarcane for sucrose production. Sugar yields from sugarcane have been improved in the past by either creating transgenic sugarcane or through using traditional breeding methods. Increasing sugar yields in sugarcane is still of interest and new cisgenic strategies are being considered to alleviate consumer concerns over transgenic plants. This thesis consists of two parts. The first was aimed at understanding the relation between trehalose-6-phosphate (T6P) synthesis and sucrose accumulation in sugarcane. In this study the E. coli genes involved in trehalose synthesis, otsA and otsB, were overexpressed in sugarcane in order to observe their effects on soluble sugar accumulation. Nine otsA and two otsB overexpressing lines were created, confirmed by gDNA insertion PCRs, sq-RT-PCR and immuno detection of encoded enzymes. Preliminary measurements of soluble sugars showed that four out of the nine otsA lines had significantly decreased and one line significantly increased sucrose concentrations. Correlating sq-RT-PCR results with soluble sugar measurements suggest that trehalose-6-phosphate synthase (TPS) expression affects sucrose levels in sugarcane, but further research of TPS activity is required before a conclusion can be reached. Further analysis of mature cane material in regard to relevant enzyme levels, carbohydrate levels and gene expression should contribute to more conclusive results. Three novel sugarcane TPS encoding sequences were isolated and proven to be functional through complementation of the growth defect in tps1Δ yeast grown on glucose as a carbon source. Sugarcane TPS isoforms named SoTPSa, SoTPSb and SoTPSc, were isolated by successful application of 5‟ RACE alongside standard PCR using primers based on other monocotyledonous TPS sequences. The encoded SoTPSa contains a 25 amino acid insertion within the partial TPP domain. The encoded SoTPSc contains a 126 amino acid long N terminal truncation, which removes one of the thirteen amino acids found within the active site of the TPS domain. Future characterization of the encoded enzymes will determine the effects of these modifications on TPS activity. The second part of this thesis describes initial efforts made in attempting to develop a cisgenic in vitro selectable marker system for sugarcane, S. officinarum callus, which uses a diphenylether type (DPE) herbicide as a selection agent and a sugarcane protoporphyrinogen oxidase (PPO) gene as a selection marker. Firstly the plastid targeted PPO from tobacco (NtPPO-1) was isolated and mutagenized, to mimic the double mutated Arabidopsis PPO, used by Li et al., (2003) in maize. However, sugarcane calli transformed with the double mutated NtPPO-1 and grown on media containing fomesafen herbicide, were incapable of regenerating. Future efforts will utilize a N-terminal sequence that is targeted to the plastid organelle, so as to ensure translocation of the enzyme to that subcellular location. Also, random mutations were induced in the NtPPO-1 gene to screen for mutations that confer DPE herbicide resistance, however this work is currently on hold until a heme deficient E. coli can be obtained. Secondly, attempts were made to isolate a putative sugarcane plastid targeted PPO gene, so as to eventually use this in developing a cisgenic strategy. 5‟ RACE was successful in revealing additional nucleotide sequence adding 1006 bp to the already known partial sugarcane PPO sequence. However the fragment isolated was still a partial sequence. / AFRIKAANSE OPSOMMING: Suikerriet is 'n tropiese meerjarige gras spesie wat deel is van die Poaceae (ware grasse) familie. Volwasse suikerriet bestaan hoofsaaklik uit suikerrietstamme, wat hoë hoeveelhede sukrose akkumuleer, 'n feit wat gelei het tot die wye verbouing van suikerriet vir sukrose produksie. In die verlede is suikeropbrengste vanuit suikerriet verbeter deur die skep van transgeniese suikerriet óf die gebruik van tradisionele teelmetodes. Toenemende suiker opbrengste in suikerriet is steeds van belang en nuwe cisgeniese strategieë word oorweeg om verbruikerskommer oor transgeniese plante te akkommodeer. Hierdie tesis bestaan uit twee dele. Die eerste deel is daarop gemik om die begrip van die verhouding tussen trehalose-6-fosfaat (T6P) sintese en sukrose ophoping in suikerriet te verstaan. In hierdie studie is die E. coli gene wat betrokke is in trehalose sintese, otsA en otsB, ooruitgedruk in suikerriet ten einde die uitwerking daarvan in die opgaar van oplosbare suiker te bestudeer. Nege otsA en twee otsB verhoogte uitdrukkings lyne is geskep, bevestig deur gDNA bygevoegde PKR, sq-RT-PKR en immuno opsporing van geïnkripteerde ensieme. Voorlopige metings van oplosbare suikers toon dat vier van die nege otsA lyne ʼn beduidende afname in sukrose vlakke en een lyn „n beduidende toegeneem in sukrose vlakke getoon het. Korrelerende sq-RT-PKR resultate met oplosbare suikermetings dui daarop dat trehalose-6- fosfaat sintese (TPS) geenuitdrukking sukrose vlakke sal affekteer, maar verdere navorsing van TPS aktiwiteit is nodig voordat 'n gevolgtrekking gemaak kan word. Verdere ontleding van volwasse riet materiaal met betrekking tot relevante ensiem vlakke, koolhidrate vlakke en geenuitdrukking, behoort by te dra tot meer volledige resultate. In hierdie studie is drie nuwe suikerriet TPS gene geïsoleer en dit is bewys as funksioneel deur die komplimentering van die groeidefek van tps1Δ gis, gegroei op glukose as 'n koolstof bron. Suikerriet TPS isoforme, genoem SoTPSa, SoTPSb en SoTPSc, is geïsoleer deur die suksesvolle toepassing van 5 'RACE, in kombinasie met standaard PKR, deur van spesiaal ontwerpte primers, gebaseer op ander eensaadlobbige TPS gene, gebruik te maak. Die gekodeerde SoTPSa bevat 'n 25 aminosuur invoeging binne-in die gedeeltelike TPP domein. Die gekodeerde SoTPSc bevat 'n 126 aminosuur lange N terminaal afkapping, wat een van die dertien aminosure binne die aktiewe terrein van die TPS domein verwyder. Toekomstige karakterisering van hierdie geïnkripteerde ensiemes sal die effek van hierdie veranderinge op TPS aktiwiteit bepaal. Die tweede deel van hierdie tesis beskryf die aanvanklike probeerslae wat gemaak is in 'n poging om „n cisgeniese in vitro selekteerbare merker vir suikerriet, S. officinarum kallus te ontwikkel. Hierin word gebruik gemaak van 'n difenylether tipe (DPE) onkruiddoder as 'n seleksie agent, en 'n suikerriet protoporphyrinogen oksidase (PPO) geen as 'n seleksie merker. In 'n poging om dit te bewerkstellig is daar eerstens plastied geteikende PPO van tabak (NtPPO-1) geïsoleer en geteikende mutagenese suksesvol daarop uitgevoer. Mutasies wat geinduseer is, is gegrond op die dubbele gemuteerde Arabidopsis PPO, wat gebruik was in mielies deur Li et al., (2003). Alhoewel die suikerriet kallus getransformeer is met die dubbele gemuteerde NtPPO-1 konstruk en geselekteer is op media wat fomesafen onkruiddoder bevat, was die kallus nie in staat om te regenereer nie. In toekomstige pogings sal probeer word om 'n N-terminale volgorde, geteiken op „n plastied organel, te benut sodat translokasie van die ensiem aan die plastied organel verseker kan word. So ook is toevallige mutasies veroorsaak in die NtPPO-1 gene om te soek vir nuwe mutasies wat DPE onkruiddoderweerstand verleen, maar hierdie werk is tans gestop totdat 'n heem gebrekkige E. coli mutant verkry kan word. Tweedens, is pogings aangewend om 'n vermeende suikerriet plastied geteikende PPO gene te isoleer, om uiteindelik te gebruik in die ontwikkeling van 'n cisgeniese strategie in suikeriet. 5 'RACE was suksesvol in die onthulling van bykomende nukleotiede volgorde deur 1006 bp by te voeg by die reeds bekende gedeeltelike suikerriet PPO fragment. Nie teenstaande is die fragment wat nuut geïsoleer is, steeds slegs 'n gedeeltelike volgorde volgens vergelykings met ander bekende plant PPO gene.

<p>Tall waterhemp management in agronomic crops continues to be an increasing problem due to widespread resistance to herbicides, including protoporphyrinogen oxidase (PPO)-inhibitors. With limited effective postemergence herbicides, especially in soybeans, research to further understand the selection of PPO-resistant (PPO-R) tall waterhemp and identification of new herbicide resistance mechanisms is crucial for improving weed management decisions in order to slow selection for herbicide resistance and prolong the effectiveness of PPO-inhibiting herbicides.</p> <p> Previous research has shown that soil-applied applications of PPO-inhibiting herbicides can increase the frequency of the PPO resistance trait (∆G210) in surviving tall waterhemp plants, even when applied in combination at the same ratio with the very long chain fatty acid inhibitor (VLCFA), <i>s-</i>metolachlor. Field experiments were conducted to determine if selection for tall waterhemp resistant individuals to PPO-inhibitors could be reduced when the soil residual activity of <i>s</i>-metolachlor persisted longer than the PPO-inhibitor herbicide. The frequency of ∆G210 in surviving individual plants increased as the fomesafen rate increased, but was independent of the rate of <i>s</i>-metolachlor. Additionally, heterozygosity of ∆G210 in surviving individuals did not change with any rate or combination of fomesafen and <i>s</i>-metolachlor. However, saflufenacil, standard PPO-inhibitor with relatively short soil residual activity, applied alone increased the number of homozygous PPO-R tall waterhemp by 15% compared to the high rate of <i>s</i>-metolachlor and the combination of saflufenacil and <i>s</i>-metolachlor. Furthermore, this research demonstrated that end of season control of tall waterhemp plays a more vital role in delaying a large-scale shift towards herbicide resistance through reduced seed production. This can be achieved through the combination of multiple effective herbicide sites of action, including soil residual PPO-inhibitors. Tall waterhemp control and density were greatest with the high rates of fomesafen plus <i>s</i>-metolachlor, which resulted in the lowest number of PPO-R tall waterhemp that survived herbicide treatment at the end of season.</p> <p> Prior to the research conducted in this thesis, the only known resistance mechanism to PPO-inhibiting herbicides in tall waterhemp has been the ∆G210 target site mutation. A previously developed TaqMan assay used to determine the presence or absence of the ∆G210 mutation has allowed accurate, high throughput screening of this mutation. However, suspected PPO-R tall waterhemp do not always receive positive confirmation indicating the presence of an alternative resistance mechanism. Identification of additional resistance mechanisms can provide valuable insight in regards to resistance to PPO-inhibiting herbicides as well as cross resistance to other herbicide modes of action, which can lead to improved tall waterhemp management decisions. Of 148 tall waterhemp populations collected across the Midwestern U.S., 84% of the populations sampled contained at least one PPO-R biotype with the ∆G210 mutation, although several individual plants across the Midwest U.S. exhibited phenotypic resistance to fomesafen that could not be explained by ∆G210. The percentage of PPO-R tall waterhemp without ∆G210 was 19, 5, 2, 1, and 2% for Iowa, Illinois, Indiana, Minnesota, and Missouri, respectively. Following the initial greenhouse screening, subsequent tall waterhemp populations were selected that exhibited low-, mid-, and high-level resistance to fomesafen that resulted in resistance ratios from 0.6 to 17X in response to fomesafen. This research documents the variability in fomesafen response to multiple tall waterhemp populations in addition to revealing the presence of additional resistance mechanism(s), other than the previously known ∆G210 mutation that has been the benchmark for resistance to PPO-inhibiting herbicides in tall waterhemp.</p> <p> Lastly, greenhouse and lab experiments were conducted to investigate the role of antioxidant enzymes with PPO-R tall waterhemp via ∆G210. The objectives of this research were to determine if the variability in resistance ratios for PPO-R tall waterhemp documented in greenhouse and field scenarios could be due to an enhanced antioxidant enzyme pathway. Basal levels of antioxidant enzymes in PPO-S populations were not different from PPO-R populations when pooled together by respective phenotype. However, enzyme activity of tall waterhemp populations varied at the individual level, but independent of the ∆G210 mutation. This indicates that an inherent enhanced antioxidant enzyme pathway does not cause the variability in fomesafen response in tall waterhemp. With the exception of glutathione reductase, antioxidant enzyme activity following fomesafen application was generally the same for PPO-R and PPO-S populations by increasing, decreasing, or remaining unchanged. Glutathione reductase activity in PPO-S populations decreased compared to PPO-R populations from 9 to 36 HAT. By 36 HAT, all antioxidant enzyme activity for PPO-S populations was lower compared to PPO-R populations most likely a consequence of more lipid peroxidation. This research shows that antioxidant enzyme activity correlated with fomesafen application and documents the variability observed within tall waterhemp populations with and without the ∆G210 mutation. </p>

Canola is a cool-season, oilseed crop grown throughout Europe, Canada, and the Northern Great Plains region of the United States. The expansion of canola production into new growing regions, such as the Southern Plains region, has resulted in new production challenges. The Southern Plains region cultivates canola as a winter annual compared to a spring annual for the Northern Great Plains and Canada. Given the difference in climate and weed spectrum, region-specific weed management systems need to be developed. Agronomic practices can affect seed oil content, protein content, and fatty acid composition, however the effect of herbicides on these and other characteristic of canola are unknown. Therefore, experiments were conducted in 2010 and 2011 to evaluate a broad spectrum of herbicides for potential use in South Texas canola production with respect to crop injury, effects on canola seed oil content, fatty acid composition, weed control, biomass yield, and forage quality. Visual crop injury at 42 DAE was unacceptable for saflufenacil at both 0.12 and 0.06 kg ai ha-1 and ethalfluralin at 1.05 kg ai ha-1. Trifluralin at 1.12 and 0.56 kg ai ha-1, S-metolachlor at 2.14 and 1.07 kg ai ha-1, pyroxasulfone at 0.24 and 0.12 kg ai ha-1, and pendimethalin at 0.8 kg ai ha-1 had lowest visual injury of all treatments. Fluroxypyr applied EPOST caused severe injury at both 0.21 and 0.11 kg ae ha-1. All other EPOST treatments did not cause any visible injury. Seed oil content was not affected by the herbicides evaluated. Fatty acid composition, specifically stearic acid, oleic acid, linolenic acid, and oleic to linolenic acid ratio, was affected by herbicide treatments. This research found that protoporphyrinogen oxidase (PPG oxidase) inhibitor herbicides, such as carfentrazone-ethyl and saflufenacil, negatively affect canola oil quality. Biomass yield was improved for all herbicide treatments except pendimethalin PRE when compared to the untreated plots. Crude protein content of canola forage was not affected by herbicide treatment. Digestible dry matter appeared to be reduced by treatments that included an EPOST application of sethoxydim. The research shows that pendimethalin and S-metolachlor may be suitable for canola production in South Texas based on low crop injury and effective weed control. Neither pendimethalin nor S-metolachlor is currently labeled for use in canola. The herbicides trifluralin, ethalfluralin, quizalofop P-ethyl, ethametsulfuron-methyl, sethoxydim, glyphosate, clethodim, and clopyralid are currently labeled for use in canola and were confirmed suitable for canola production in South Texas. Carfentrazone-ethyl is currently labeled for use in canola but the effects on oil quality should be considered.

ABSTRACT The porphyrias are a diverse group of metabolic disorders arising from diminished activity of enzymes in the heme biosynthetic pathway. They can present with acute neurovisceral symptoms, cutaneous symptoms, or both. The complexity of these disorders is demonstrated by the fact that some acute porphyria patients with the underlying genetic defect(s) are latent and asymptomatic while others present with severe symptoms. This indicates that there is at least one other risk factor required in addition to the genetic defect for symptom manifestation. A systematic review of the heme biosynthetic pathway highlighted the involvement of a number of micronutrient cofactors. An exhaustive review of the medical literature uncovered numerous reports of micronutrient deficiencies in the porphyrias as well as successful case reports of treatments with micronutrients. Many micronutrient deficiencies present with symptoms similar to those in porphyria, in particular vitamin B6. It is hypothesized that a vitamin B6 deficiency and related micronutrient deficiencies may play a major role in the pathogenesis of the acute porphyrias. In order to further investigate the porphyrias, a computational model of the heme biosynthetic pathway was developed based on kinetic parameters derived from a careful analysis of the literature. This model demonstrated aspects of normal heme biosynthesis and illustrated some of the disordered biochemistry of acute intermittent porphyria (AIP). The testing of this model highlighted the modifications necessary to develop a more comprehensive model with the potential to investigated hypotheses of the disordered biochemistry of the porphyrias as well as the discovery of new methods of treatment and symptom control. It is concluded that vitamin B6 deficiency might be the risk factor necessary in conjunction with the genetic defect to trigger porphyria symptoms.