Soja, uma cultura conhecida por sua importância econômica e nutricional, tem sido objeto de vários estudos que avaliam o impacto e as respostas efetivas das plantas aos estresses abióticos. O estresse salino é um dos principais estresses ambientais e afeta negativamente o crescimento e o rendimento das culturas, incluindo a soja. A edição de RNA é um processo pelo qual as sequências de nucleotídeos podem ser alteradas, revertendo mutações que podem mudar as sequências de proteínas para manter suas funções conservadas. As proteínas pentatricopeptide repeat (PPRs) são trans-elementos de edição caracterizados por reconhecer cis-elementos específicos de RNA e realizar a reação de edição. Vários estudos descreveram estes trans-elementos e seus sítios de edição cognatos, mas nem todas as proteínas que compõem o complexo de edição foram identificadas. A perda de eventos de edição de plastídios, resultante de mutações em fatores de edição de RNA ou através de interferência por estresse, leva a alterações de desenvolvimento, de fisiologia e da fotossíntese. O objetivo do presente trabalho é caracterizar os sítios de edição e os fatores associados à edição de RNA em Glycine max e a influência de estresses abióticos no processo de edição de RNA em cloroplastos. No capítulo 1, um método é apresentado para triar a edição de RNA de cloroplasto usando bibliotecas públicas de sRNAs de Arabidopsis, soja e arroz. Entre os sítios de edição previstos, 40,57, 34,78 e 25,31% foram confirmados utilizando sRNAs de Arabidopsis, soja e arroz, respectivamente. A análise de SNPs revelou alterações de C-to-U de 58,2, 43,9 e 37,5% nas respectivas espécies e identificou conhecidas e possíveis novas edições de RNA de adenosina para inosina (A-to-I) em tRNAs. O método e os dados revelam o potencial do uso de sRNA como uma fonte confiável para identificar novos e confirmar sítios de edição conhecidos. No capítulo 2, o processo de edição de RNA foi avaliado em cloroplastos de plantas de soja sob estresse salino. A abordagem de bioinformática utilizando bibliotecas de sRNAs e mRNAs foi empregada para detectar sítios específicos que mostram diferenças na taxa de edição. RT-qPCR foi usado para medir a taxa de edição nos sítios selecionados. Observamos diferenças nas taxas de edição nos transcritos dos genes ndhA, ndhB, rps14 e rps16 ao comparar os dados das bibliotecas controle e das tratadas com NaCl. Os ensaios de RT-qPCR 9 demonstraram um aumento na edição dos genes selecionados. Esses aumentos podem ser uma resposta para manter a homeostase das funções das proteínas do cloroplasto em resposta ao estresse salino. No capítulo 3, para identificar os fatores relacionados aos sítios de edição analisados, sondas biotiniladas de RNA foram projetadas com base nos sítios de edição de RNA de plastídio de soja para realizar um isolamento proteico específico do fator de edição. Proteínas que interagiram com as sondas foram isoladas através da ligação das sondas à biotina e foram identificadas utilizando espectrometria de massa. Entre os peptídeos detectados, cinco corresponderam a proteínas PPR. A comparação dos genes de Arabidopsis com as proteínas PPR da soja permitiu a identificação dos homólogos mais próximos. O presente estudo representa a primeira identificação do conjunto de sítios de edição de RNA, de fatores associados aos sítios de edição de RNA e a caracterização dos efeitos do estresse abiótico na edição de RNA em Glycine max. / Soybean, a crop known by its economic and nutritional importance, has been the subject of several studies that assess the impact and the effective plant responses to abiotic stresses. Salt stress is one of the main environmental stresses and negatively impacts crop growth and yield. RNA editing is a process whereby nucleotide sequences can be altered, reverting mutations that could change protein sequences to maintain their conserved functions. Pentatricopeptide repeat proteins are editing trans-elements characterized by recognize specific RNA cis-elements and perform the editing reaction. Several studies have described these trans-elements and their cognate editing sites, but not all proteins that compose the editing complex were identified. The loss of plastid editing events, resulting from mutations in RNA editing factors or through stress interference, leads to developmental, physiological and photosynthetic alterations. The aim of the present work is to characterize the editing sites and factors associated with RNA editing in Glycine max and the influence of abiotic stresses on the process of RNA editing in chloroplasts. In chapter 1, a method is presented to screen chloroplast RNA editing using public sRNA libraries from Arabidopsis, soybean and rice. Among the predicted editing sites, 40.57, 34.78, and 25.31% were confirmed using sRNAs from Arabidopsis, soybean and rice, respectively. SNP analysis revealed 58.2, 43.9, and 37.5% new C-to-U changes in the respective species and identified known and new putative adenosine to inosine (A-to-I) RNA editing in tRNAs. The method and data reveal the potential of sRNA as a reliable source to identify new and confirm known editing sites. In chapter 2, RNA editing process was evaluated in the chloroplast of soybean plants under salt stress. Bioinformatics approach using sRNA and mRNA libraries was employed to detect specific sites showing differences in editing efficiency. RT-qPCR was used to measure editing efficiency at selected sites. We observed differences in ndhA, ndhB, rps14 and rps16 editing rates between control and salt-treated libraries. RT-qPCR assays demonstrated an increase in editing efficiency of selected genes. These increases can be a response to keep the homeostasis of chloroplast protein functions in response to NaCl stress. In chapter 3, to identify the trans-acting factors of editing sites analyzed, we have designed RNA biotinylated probes based in soybean plastid RNA editing sites to perform 11 specific isolation of proteins associated to editosomes. Proteins that interacted with the probes were isolated by binding the probes to biotin and were identified using mass spectrometry. Among the detected peptides, five corresponded to PPR proteins. Comparison of Arabidopsis genes to the soybean PPR proteins allow identification of the closest related homologs. The present study represents the first identification of RNA editing sites set, associated factors to RNA editing sites and characterization of effects from abiotic stress in RNA editing in Glycine max.

Patch-clamp recordings in whole-cell configuration were made from ventrolateral medulla neurons of brainstem slices from 8-12-day-old rats. 5- Hydroxytryptamine (3-30 μM) concentration-dependently suppressed excitatory and inhibitory postsynaptic currents evoked by focal stimulation. An augmentation of inhibitory synaptic currents by 5-hydroxytryptamine was noted in a small number of neurons. 5-Hydroxytryptamine depressed synaptic currents with or without causing a significant change in holding currents and membrane conductances; the inward or outward currents induced by exogenously applied glutamate or GABA/glycine were also not significantly changed by 5- hydroxytryptamine. In paired-pulse paradigms designed to evaluate a presynaptic site of action, 5-hydroxytryptamine suppressed synaptic currents but enhanced the paired-pulse facilitation. 5-Hydroxytryptamine reduced the frequency of miniature excitatory postsynaptic currents without significantly affecting the amplitude. 5-Carboxamidotryptamine, 8-hydroxy-2(di-n- propylamino)tetralin, sumatriptan and N-(3-trifluoromethylphenyl)piperazine which exhibit 5-hydroxytryptamine1 receptor agonist activity, depressed synaptic currents with different potencies, with 5-carboxamidotryptamine being the most potent. The non-selective 5-hydroxytryptamine1 receptor antagonist pindolol attenuated the presynaptic effect of 5-hydroxytryptamine, whereas the 5-hydroxytryptamine(1A) antagonist pindobind-5- hydroxytryptamine(1A) and 5-hydroxytryptamine2 receptor antagonist ketanserin were ineffective. Our results indicate that 5-hydroxytryptamine suppressed synaptic transmission in ventrolateral medulla neurons by activating presynaptic 5-hydroxytryptamine1 receptors, probably the 5- hydroxytryptamine(1B)/5-hydroxytryptamine(1D) subtype. In addition, 5- hydroxytryptamine augmented inhibitory synaptic currents in a small number of neurons the site and mechanism of this potentiating action are not known.

Thesis(Ph.D.(Botany)) --University of Limpopo,2019 / Genetic transformation offers great opportunities for rapidly introducing, selecting or inducing desired characteristics in various leguminous plants for breeding purposes. But, this technique remains aloof for soybean improvement due to challenges such as genotype specificity, inefficient regeneration protocols and the rapid loss of viability in seeds required to develop explants. However, the rate of seed deterioration and its influence on in-vitro plant genetic transformation differs according to the age, storage duration and moisture content of the seeds used. The moisture status of the seeds is usually high during harvesting and deterioration (loss of viability) starts to occur when seeds are stored under ambient conditions for long periods. This seed deterioration also results in a phenomenon called “recalcitrance”, which is predominantly realised in soybean. In the present study, selected soybean genotypes were analysed for: (i) the efficiency of germination using seeds stored for 0, 3, 6 and 9-months under ambient conditions (ii) the effect of seed storage on in-vitro multiple shoot induction, (iii) the competency of the selected soybean genotypes on callus induction and Agrobacterium-mediated genetic transformation and (iv) the evaluation of protein profiles of the genotypes following co-cultivation of cotyledonary node explants with A. tumefaciens. The results obtained in this study showed that, seed stored for more than 3-months had reduced rates of germination, seedling development and in-vitro shoot multiplication. In particular, seed stored for 9-months showed a significant drop in seed germination, and less than 50% overall seed germination (Dundee-42%, LS678- 49%, TGx140-2F-44% and TGx1835-10E-48%) except for LS677 and Peking with 52 and 55%, respectively. The efficiency of multiple shoot induction also decreased with the prolonged seed storage, with all genotypes recording overall decline from about 96% to 40% regeneration efficiency over this period. The mean number of induced shoots decreased from more than 10.5 to 4.2 shoots per explant, for each genotype. The results obtained clearly indicated that efficient in-vitro shoot induction depended largely on seed storage duration, viability and significantly differed according to genotype. Following the evaluation for callus induction and Agrobacterium-mediated genetic transformation frequencies, the results indicated that the responses were genotype specific. This trend was consecutively observed in all soybean cultivars used (LS677, LS678, Dundee, Peking, TGx1740-2F and TGx1835-10E). Furthermore, the responses of the genotypes were also dependent on the culture media composition,especially, plant growth regulators and antibiotics. Amongst the cultivars used, Peking demonstrated the highest callus induction capacity (more than 70%) on MS-A and the mean number of shoots induced (1.65) using cotyledonary explants co-cultivated with Agrobacterium. This was followed by LS677 (1.42 shoots), LS678 (1.40 shoots), Dundee (1.30 shoots), TGx1835-10E (0.80 shoots) and TGx1740-2F (0.75 shoots), respectively. These genotypes also demonstrated low yields of proteins, extracted using a TCA buffer, and separated by means of two-dimensional polyacrylamide gel electrophoresis. The one-dimensional and two-dimensional profiles of proteins extracted from explants infected with Agrobacterium differed significantly to those expressed without co-cultivation of cotyledonary nodes with bacteria. These observations suggested that, the infection and co-cultivation of explants with Agrobacterium may have caused the expression of new proteins. Newly expressed proteins could also be found to either promote or inhibit transgene integration and expression on the cotyledonary node explants transformed with Agrobacterium tumefaciens for trait improvement. This study has clearly demonstrated that soybean production is confronted with a myriad of stress factors, including seed storage and quality problems due to unfavourable storage duration and weather conditions, amongst others. Thus, soybean seeds used for germination, callus induction, multiple shoot induction and genetic improvement should be harvested at R8 stage after reaching physiological maturity (with 20-35% seed moisture content) to avoid any mechanical damage, shattering or loss of seed viability. / National Research Foundation

FFifteen to thirty-five percent of the population in the United States experience tinnitus, a subjective "ringing in the ears". Up to 10% of tinnitus patients report their symptoms are severe and disabling. Tinnitus was induced in FBN rats using 116 dB (SPL) unilateral octave-band sound exposures centered at 16 kHz for one hour in an anesthetized preparation. Rats were assessed behaviorally by an operant conditioning paradigm as well as a gap detection method to verify the development of tinnitus. Both young (7 mos.) and aged (30 mos.) sound exposed rats showed significant elevated auditory brainstem-evoked response (ABR) thresholds for clix and all tested frequencies immediately after the sound exposure. Eighty days post-exposure, ABR thresholds for the young exposed rats were significantly close to the initial young control values while aged exposed rats showed residual thresholds shifts relative to aged controls. Sixteen weeks following sound exposure, young exposed rats showed significantly reduced gap detection at 24 and 32 kHz, suggestive of high frequency tinnitus. Aged exposed animals showed significant tinnitus-related behavioral changes near 10 kHz by both behavior methods. Message and protein levels of &alpha1-3 glycine receptor subunits (GlyRs), gephyrin, BDNF and its receptor TrkB were assessed in dorsal cochlear nucleus (DCN) fusiform cells 4 months post exposure utilizing quantitative in situ hybridization and immunocytochemistry. Young exposed rats showed significant decreases of GlyR &alpha1 protein at middle and high frequency regions in DCN unlike the contrasting increase of their message levels. Aged exposed rats showed higher &alpha1 subunit protein levels in the same high and middle DCN frequency regions. The GlyR anchoring protein, gephyrin, was significantly increased in both young and aged exposed rats, suggesting an intracellular receptor trafficking change following acoustic trauma. BDNF and TrkB were also increased over fusiform cells in both young and aged exposed rats. [3H] strychnine binding was used to evaluate DCN GlyR pharmacology and function following sound exposure. The age-related decrease in GlyR α1 protein was reflected in the significant age-related down-regulation of GlyR (Bmax). Tinnitus-related changes in GlyR &alpha1 protein level was reflected in the decline of the GlyR (Bmax) in young exposed rats and up-regulated GlyRs in aged exposed animals. The GlyRs in DCN of young exposed animals also demonstrated an increase in affinity, further suggesting a post-exposure receptor composition change. These findings suggest that both aging and/or sound exposure/tinnitus are associated with GlyR changes capable of altering alter the output of the DCN. Detailed characterization of these GlyR modifications could advance the development of novel selective drugs for tinnitus and age-related hearing loss.

The understanding of plant defense response in plant-pathogen interaction provides useful information to combat the disease. Soybean cyst nematode (SCN), Heterodera glycines, and charcoal rot causing fungus, Macrophomina phaseolina, are major pathogens of soybean, Glycine max, and infestation with SCN has found to aggravate the fungal disease severity. The gene expression analysis in resistant and susceptible interactions between H. glycines and G. max has identified some candidate resistance genes and signaling pathways but they are yet to be fully characterized. This dissertation aims to characterize one such gene, Conserved Oligomeric Golgi (COG) complex. The COG complex is important in structural and functional integrity of Golgi complex among eukaryotes, but very little is understood about its role in plants. This study demonstrated a defensive role of the COG complex in G. max against H. glycines. The transgenic plants for overexpression of COG genes, which were originally susceptible to the pathogen, showed reduced parasitism. In contrast, the RNA interference (RNAi) in originally resistant soybean lines showed a marked increase in parasitism. Further, these COG genes were found to be inducible by harpin elicitor molecules. In another study, the already proven resistance genes (NDR1, NPR1, EDS1 and TGA2) against H. glycines were investigated against the fungal pathogen. The transgenic plants for overexpression of these genes showed reduced disease severity, while the RNAi resulted increased severity compared to control lines. In addition, the H. glycines parasitism study and the candidate gene expression analysis in M. Phaseolina susceptible and moderately resistant G. max indicate that there are some cross communications between the defense processes of G. max to H. glycines and M. phaseolina.

No description available.

No description available.

This research was conducted to analyze the genetics of soybean mosaic virus (SMV) resistance in soybean [Glycine max (L.) Merr.] and to determine allelic relationships of SMV resistance genes and their interactions with SMV strain groups. In the first part of this study, the inheritance of SMV resistance in OX670 and 'Harosoy' was studied to determine the source and identity of the resistance gene/genes in OX670. Other researchers reported that OX670 possesses a single gene at a locus independent of Rsv1 and assigned the gene symbol Rsv2. Rsv2 was presumably derived from the cultivar 'Raiden'. However, later work showed that Raiden contains a single resistance gene at the Rsv1 locus, raising the possibility that the resistance gene in OX670 was not from Raiden. Harosoy and its derivatives make up much of the remaining pedigree of OX670. Results from crosses of OX670 with susceptible cultivars indicate that it contains two independent genes for SMV resistance. One is allelic to the Rsv1 locus, expresses resistance to SMV-G1 and G7 and is derived from Raiden. The other is allelic to the Rsv3 locus, expresses resistance to SMV-G7 but susceptibility to SMV-G1 and is derived from Harosoy. Therefore the Rsv2 locus does not appear to exist in OX670 or its ancestors. The presence of Rsv1 and Rsv3 makes OX670 resistant to all SMV strains from G1 through G7. The second study was conducted to investigate the inheritance and allelomorphic relationships of resistance gene(s) in 'Tousan 140' and 'Hourei', which were reported to carry single independent resistance genes when inoculated with the Japanese SMV strain C. Both of these lines exhibit resistance to strains SMV-G1 through G7. This inheritance study shows that Tousan 140 and Hourei each possess two resistance genes. One of the genes in Hourei confers resistance to SMV-G1 and G7 strains; the other gene confers susceptibility to SMV-G1 but resistance to SMV-G7. Allelism tests indicate that one of the genes in both Hourei and Tousan 140 is allelic to Rsv1, and the other is allelic to Rsv3. The two genes in Tousan 140 were separated into individual lines, R1 and R2. R1, most probably containing Rsv1, exhibited resistance to SMV-G1 through G3 but was susceptible to SMV-G5 through G7. Line R2, most likely possesses Rsv3 gene, was susceptible to SMV-G1 through G3 but resistant to SMV-G5 through G7. Therefore, presence of these two genes makes Tousan 140 resistant to SMV-G1 through G7. The objective of the third study was to investigate inheritance and allelomorphic relationships of SMV resistance in PI88788. PI88788 exhibits resistance to SMV-G1 through G7. Genetic analysis of our data reveals that SMV resistance in PI88788 is conferred by a single gene at a locus tentatively labeled 'Rsv4'. Expression of this gene in the homozygous state decreased accumulation rate and prevented vascular movement of SMV. In the heterozygous state vascular movement of the SMV was delayed but not prevented. / Ph. D.

Commercially available leaf area index (LAI) meters are tools that can be used in making insect management decisions. However, proper technique must be determined for LAI estimation, and accuracy must be validated for the meters. Full-season soybean require LAI values of at least 3.5 to 4.0 by early to mid-reproductive developmental stages to achieve maximum yield potential, but the relationship between double-crop soybean LAI and yield is unknown. This research (1) evaluated minimum plot size requirements for mechanically defoliated soybean experiments using the LAI-2000 Plant Canopy Analyzer, (2) compared LAI estimates among LAI-2000 detector types which respond to different wavelengths of light, (3) compared LAI-2000 estimates with directly determined LAI values for 0, 33, 66, and 100% mechanical defoliation levels, (4) used linear and non-linear models to describe the response of full-season and double-crop soybean yields to reductions in LAI through mechanical defoliation, and (5) evaluated the response of double-crop soybean yields to reductions in LAI through insect defoliation. The minimum plot size for obtaining accurate LAI estimates of defoliated canopies in soybean with 91 cm row centers is four rows by 2 m, with an additional 1 m at the ends of the two middle rows also defoliated. The wide-blue detector, which is found in newer LAI-2000 units and responds to wavelengths of light from 360 to 460 nm, gave higher LAI estimates than the narrow-blue detector, which responds to light from 400 to 490 nm. The unit with the narrow-blue detector gave estimates equal to directly determined LAI in two of three years for 0, 33, and 66% defoliation levels, while the units with the wide-blue detectors gave estimates higher than directly determined LAI in the two years that they were studied, except for a few accurate 33% defoliation estimates. Therefore, the LAI-2000 usually provides reasonable estimates of LAI. Yield decreased linearly with LAI when LAI values were below 3.5 to 4.0 by developmental stages R4 to R5 in both full-season and double-crop soybean. Usually, there was no relationship between yield and LAI at LAI values greater than 4.0. There was an average yield reduction of 820 ± 262 kg ha⁻¹ for each unit decrease in LAI below the critical 3.5 to 4.0 level; maximum yields ranged from 1909 to 3797 kg ha⁻¹. Insect defoliators did not defoliate double-crop soybean plots to LAI levels less than 4.0, and there was no yield difference between insect-defoliated and control plots. Therefore, double-crop soybean that maintains LAI values above the 3.5 to 4.0 critical level during mid-reproductive developmental stages is capable of tolerating defoliating pest / Ph. D.

Phytic acid, also known as myo-inositol hexakisphosphate or Ins(1,2,3,4,5,6)P6, is the major storage form of phosphorus in plant seeds. Phytic acid is poorly digested by non-ruminant animals such as swine and poultry, and it chelates mineral cations including calcium, iron, zinc, and potassium, classifying it as an anti-nutrient. The excretion of unutilized phytic acid in manure translates to an excess amount of phosphorus runoff that can lead to eutrophication of lakes and ponds. Understanding the phytic acid biosynthetic pathway will allow for the development of low phytic acid (lpa) soybeans by the down-regulation of specific genes. The goal of this research was to elucidate the pathway(s) for phytic acid biosynthesis in soybean (Glycine max). We have isolated several myo-inositol phosphate kinase genes in soybean as possible candidates for steps in the biosynthetic pathway. We have characterized the genes for four myo-inositol(1,3,4)P3 5/6-kinases (GmItpk1-4), one myo-inositol(1,4,5)P3 6/3/5-kinase (GmIpk2), and one myo-inositol(1,3,4,5,6)P5 2-kinase (GmIpk1). We have examined expression in developing seeds and other tissues by Northern blot analysis and quantitative RT-PCR. We have expressed all six genes as tagged fusion proteins in E. coli, and verified enzyme activity on the proposed substrates. For each enzyme, we have conducted biochemical characterization to determine enzyme kinetics and substrate specificities. We have verified in vivo activity of GmIpk2 and GmIpk1 by complementing yeast mutants in the respective genes. Our studies indicate the likelihood that three of the genes may be involved in phytic acid biosynthesis: GmItpk3, GmIpk2 and GmIpk1. For future work, to more fully understand the contribution of each kinase gene to phytic acid biosynthesis, an RNA interference approach will be employed. The gene sequences identified in this study will be used to construct silencing vectors for use in future transformation of soybean embryogenic cultures to determine the effects of down-regulation on myo-inositol phosphate profiles. / Ph. D.