Physiological Basis for Genotypic Variation in Tolerance to and Recovery from Pre-flowering Drought in Peanut.

Drought during the pre-flowering stage can increase yield of peanut. There is limited information on genotypic variation for tolerance to and recovery from pre-flowering drought (PFD) and more importantly the physiological traits underlying genotypic variation. The objectives of this study were to determine the effects of moisture stress during the pre-flowering phase on pod yield and to understand some of the physiological responses underlying genotypic variation in response to and recovery from PFD. A glasshouse and field experiments were conducted at Khon Kaen University, Thailand. The glasshouse experiment was a randomized complete block design consisting of two watering regimes, i.e. fully-irrigated control and 1/3 available soil water from emergence to 40 days after emergence followed by adequate water supply, and 12 peanut genotypes. The field experiment was a split-plot design with two watering regimes as main-plots, and 12 peanut genotypes as sub-plots. Measurements of N-2 fixation, leaf area (LA) were made in both experiments. In addition, root growth was measured in the glasshouse experiment. Imposition of PFD followed by recovery resulted in an average increase in yield of 24 % (range from 10 % to 57 %) and 12 % (range from 2 % to 51 %) in the field and glasshouse experiments, respectively. Significant genotypic variation for N-2 fixation, LA and root growth was also observed after recovery. The study revealed that recovery growth following release of PFD had a stronger influence on final yield than tolerance to water deficits during the PFD. A combination of N-2 fixation, LA and root growth accounted for a major portion of the genotypic variation in yield (r = 0.68-0.93) suggesting that these traits could be used as selection criteria for identifying genotypes with rapid recovery from PFD. A combined analysis of glasshouse and field experiments showed that LA and N-2 fixation during the recovery had low genotype x environment interaction indicating potential for using these traits for selecting genotypes in peanut improvement programs.

Of tolerance in mice and men : studies in APECED and Aire

Autoimmune diseases affect 5 % of the population and come in many forms, such as diabetes, rheumatoid arthritis and MS. However, how and why autoimmune diseases arise are not yet fully resolved. In this thesis, the onset of autoimmunity was investigated using both patient samples and a mouse model of autoimmunity. Autoimmune diseases are usually complex, due to a number of different causative genes and environmental factors. However, a few monogenic autoimmune diseases have been described, which are caused by mutations in only one gene per disease. One of such disease is called APECED (autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy) and is enriched in the Finnish population. The causative gene behind APECED is named AIRE from AutoImmune REgulator. How malfunction of just one gene product can cause the multitude of disease components found in APECED is not yet resolved. This thesis sought out to find out more about the functions of AIRE, in order to reveal why APECED and other autoimmune diseases arise and what goes wrong? Usually, immune cells are taught to distinguish between self and non-self during their development. That way, immune cells can fight off bacteria and microbes while leaving the tissues and organs of the host organism itself unharmed. In APECED, the development of immune cells called αβ T cells is incomplete. The cells are not able to fully distinguish between self and non-self. This leads to autodestruction of self tissues and autoimmune disease. One of the achievements of this thesis was the finding that the development of another set of T cells called γδ T cells is not affected by AIRE in mice or in men. Instead, we found that another type of immune cell important in tolerance, called the dendritic cell is defective in APECED patients and is not able to respond to microbial stimulus in a normal fashion. Finally, we studied Aire-deficient mice and found that autoantibodies expressed in the mice were not targeted against the same molecules as those found in APECED patients. This indicates differences in the autoimmune pathology in mice and men. More work is still required before we understand the mechanisms of tolerance and autoimmunity well enough to be able to cure APECED, let alone the more complex autoimmune diseases. Yet altogether, the findings of this thesis work bring us one step closer to finding out why and how APECED and common autoimmune diseases arise.

Early growth and adult health: focus on blood pressure, glucose tolerance status and body composition

Theory of developmental origins of adult health and disease proposes that experiences during critical periods of early development may have consequences on health throughout a lifespan. Thesis studies aimed to characterize associations between early growth and some components of the metabolic syndrome cluster. Participants belong to two epidemiological cohorts with data on birth measurements and, for the younger cohort, on serial recordings of weight and height during childhood. They were born as singletons between 1924-33 and 1934-44 in the Helsinki University Central Hospital, and 500 and 2003 of them, respectively, attended clinical studies at the age of 65-75 and 56-70 years, respectively. In the 65-75 year old men and women, the well-known inverse relationship between birth weight and systolic blood pressure (SBP) was confined to people who had established hypertension. Among them a 1-kg increase in birth weight was associated with a 6.4-mmHg (95% CI: 1.0 to 11.9) decrease in SBP. This relationship was further confined to people with the prevailing Pro12Pro polymorphism of the peroxisome proliferator-activated receptor-γ2 (PPARγ2) gene. People with low birth weight were more likely to receive angiotensin-converting enzyme inhibitors/angiotensin-receptor blockers (ACEI/ARB, p=0.03), and, again, this relationship was confined to the carriers of the Pro12Pro (p=0.01 for interaction). These results suggest that the inverse association between birth weight and systolic BP becomes focused in hypertensive people because pathological features of BP regulation, associated with slow fetal growth, become self-perpetuating in adult life. Insulin resistance of the Pro12Pro carriers with low birth weight may interact with the renin-angiotensin system leading to raised BP levels. Habitual physical activity protected men and women who were small at birth, and thus at increased risk for the development of type 2 diabetes, against glucose intolerance more strongly. Among subjects with birth weight ≤3000 g, the odds ratio (OR) for glucose intolerance was 5.2 (95% CI: 2.1 to 13) in those who exercised less than 3 times per week compared to regular exercisers; in those who scored their exercise light compared with moderate exercisers (defined as comparable to brisk walking) the OR was 3.5 (1.5 to 8.2). In the 56-70 year old men a 1 kg increase in birth weight corresponded to a 4.1 kg (95% CI: 3.1 to 5.1) and in women to a 2.9 kg (2.1 to 3.6) increase in adult lean mass. Rapid gain in body mass index (BMI), i.e. crossing from an original BMI percentile to a higher one, before the age of 2 years increased adult lean mass index (LMI, lean mass/height squared) without excess fat accumulation whereas rapid gain in BMI during later childhood, despite the concurrent rise in LMI, resulted in a relatively higher increase in adult body fat mass. These findings illustrate how genes, the environment and their interactions, early growth patterns, and adult lifestyle modify adult health risks which originate from early life.

Traffic tolerance of warm season turfgrasses under Community Sportsfield Conditions

The four-year Horticulture Australia (HAL) project is the first scientific study within Australia to assess simulated and actual wear studies of warm-season turfgrasses suitable for sportfield use. The study has allowed researchers and turf professionals to compare traffic (wear and compaction) tolerance and turf management requirements (e.g. mowing) of the current dominant varieties.

DAQ00152 Herbicide Tolerance Screening in Northern Region (Phase IV)

The previous projects (phase I - III) highlighted that northern region wheat and barley cultivars differ considerably in their sensitivity to herbicides. The new project will focus on increased screening of advanced breeding lines and new cultivars lines to commonly used herbicides, for barley, chickpea and wheat. Studies on impact of environment on herbicide x genotype responses will also be undertaken with the national team. The new information will be added to the existing information package on herbicide tolerance. Thus, adverse impacts of herbicides on productivity in northern region will be reduced, as growers and agronomists will select safer herbicides for their sown variety, or select more tolerant varieties for their important herbicides.

Herbicide tolerance of barley and wheat cultivars in NR (phase III)

During the past 10 years, this project tested 23 barley and 51 wheat varieties with 19 and 34 registered herbicides, respectively. It concentrated on new varieties and herbicides. The research highlighted that Northern Region (NR) wheat and barley varieties differed considerably in their sensitivity to these herbicides. Overall, 9 per cent of wheat variety x herbicide combinations and 6 per cent of barley variety x herbicide combinations had significant yield losses (3 to 38%) from herbicides at recommended rates and crop stages. In addition, 21 to 23 per cent had significant yield losses from herbicides at double rates, indicating a narrow margin of crop safety.

Identifying the Function of Sorghum's Drought Tolerance Stay-Green QTL

The goal of this research is to understand the function of allelic variation of genes underpinning the stay-green drought adaptation trait in sorghum in order to enhance yield in water-limited environments. Stay-green, a delayed leaf senescence phenotype in sorghum, is primarily an emergent consequence of the improved balance between the supply and demand of water. Positional and functional fine-mapping of candidate genes associated with stay-green in sorghum is the focus of an international research partnership between Australian (UQ/DAFFQ) and US (Texas A&M University) scientists. Stay-green was initially mapped to four chromosomal regions (Stg1, Stg2, Stg3, and Stg4) by a number of research groups in the US and Australia. Physiological dissection of near-isolines containing single introgressions of Stg QTL (Stg1-4) indicate that these QTL reduce water demand before flowering by constricting the size of the canopy, thereby increasing water availability during grain filling and, ultimately, grain yield. Stg and root angle QTL are also co-located and, together with crop water use data, suggest the role of roots in the stay-green phenomenon. Candidate genes have been identified in Stg1-4, including genes from the PIN family of auxin efflux carriers in Stg1 and Stg2, with 10 of 11 PIN genes in sorghum co-locating with Stg QTL. Modified gene expression in some of these PIN candidates in the stay-green compared with the senescent types has been found in preliminary RNA expression profiling studies. Further proof-of-function studies are underway, including comparative genomics, SNP analysis to assess diversity at candidate genes, reverse genetics and transformation.

Evaluation of potential candidate genes involved in salinity tolerance in striped catfish (Pangasianodon hypophthalmus) using an RNA-Seq approach

Increasing salinity levels in freshwater and coastal environments caused by sea level rise linked to climate change is now recognized to be a major factor that can impact fish growth negatively, especially for freshwater teleost species. Striped catfish (Pangasianodon hypophthalmus) is an important freshwater teleost that is now widely farmed across the Mekong River Delta in Vietnam. Understanding the basis for tolerance and adaptation to raised environmental salinity conditions can assist the regional culture industry to mitigate predicted impacts of climate change across this region. Attempt of next generation sequencing using the ion proton platform results in more than 174 million raw reads from three tissue libraries (gill, kidney and intestine). Reads were filtered and de novo assembled using a variety of assemblers and then clustered together to generate a combined reference transcriptome. Downstream analysis resulted in a final reference transcriptome that contained 60,585 transcripts with an N50 of 683 bp. This resource was further annotated using a variety of bioinformatics databases, followed by differential gene expression analysis that resulted in 3062 transcripts that were differentially expressed in catfish samples raised under two experimental conditions (0 and 15 ppt). A number of transcripts with a potential role in salinity tolerance were then classified into six different functional gene categories based on their gene ontology assignments. These included; energy metabolism, ion transportation, detoxification, signal transduction, structural organization and detoxification. Finally, we combined the data on functional salinity tolerance genes into a hypothetical schematic model that attempted to describe potential relationships and interactions among target genes to explain the molecular pathways that control adaptive salinity responses in P. hypophthalmus. Our results indicate that P. hypophthalmus exhibit predictable plastic regulatory responses to elevated salinity by means of characteristic gene expression patterns, providing numerous candidate genes for future investigations.

Field evaluation of tolerance to Tobacco streak virus in sunflower germplasm, and observations of seasonal disease spread

Strong statistical evidence was found for differences in tolerance to natural infections of Tobacco streak virus (TSV) in sunflower hybrids. Data from 470 plots involving 23 different sunflower hybrids tested in multiple trials over 5 years in Australia were analysed. Using a Bayesian Hierarchical Logistic Regression model for analysis provided: (i) a rigorous method for investigating the relative effects of hybrid, seasonal rainfall and proximity to inoculum source on the incidence of severe TSV disease; (ii) a natural method for estimating the probability distributions of disease incidence in different hybrids under historical rainfall conditions; and (iii) a method for undertaking all pairwise comparisons of disease incidence between hybrids whilst controlling the familywise error rate without any drastic reduction in statistical power. The tolerance identified in field trials was effective against the main TSV strain associated with disease outbreaks, TSV-parthenium. Glasshouse tests indicate this tolerance to also be effective against the other TSV strain found in central Queensland, TSV-crownbeard. The use of tolerant germplasm is critical to minimise the risk of TSV epidemics in sunflower in this region. We found strong statistical evidence that rainfall during the early growing months of March and April had a negative effect on the incidence of severe infection with greatly reduced disease incidence in years that had high rainfall during this period.

Development of salinity tolerance in rice by constitutive-overexpression of genes involved in the regulation of programmed cell death

Environmental factors contribute to over 70% of crop yield losses worldwide. Of these drought and salinity are the most significant causes of crop yield reduction. Rice is an important staple crop that feeds more than half of the world’s population. However among the agronomically important cereals rice is the most sensitive to salinity. In the present study we show that exogenous expression of anti-apoptotic genes from diverse origins, AtBAG4 (Arabidopsis), Hsp70 (Citrus tristeza virus) and p35 (Baculovirus), significantly improves salinity tolerance in rice at the whole plant level. Physiological, biochemical and agronomical analyses of transgenic rice expressing each of the anti-apoptotic genes subjected to salinity treatment demonstrated traits associated with tolerant varieties including, improved photosynthesis, membrane integrity, ion and ROS maintenance systems, growth rate, and yield components. Moreover, FTIR analysis showed that the chemical composition of salinity-treated transgenic plants is reminiscent of non-treated, unstressed controls. In contrast, wild type and vector control plants displayed hallmark features of stress, including pectin degradation upon subjection to salinity treatment. Interestingly, despite their diverse origins, transgenic plants expressing the anti-apoptotic genes assessed in this study displayed similar physiological and biochemical characteristics during salinity treatment thus providing further evidence that cell death pathways are conserved across broad evolutionary kingdoms. Our results reveal that anti-apoptotic genes facilitate maintenance of metabolic activity at the whole plant level to create favorable conditions for cellular survival. It is these conditions that are crucial and conducive to the plants ability to tolerate/adapt to extreme environments.

Tripogon loliiformis elicits a rapid physiological and structural response to dehydration for desiccation tolerance

Resurrection plants can withstand extreme dehydration to an air-dry state and then recover upon receiving water. Tripogon loliiformis (F.Muell.) C.E.Hubb. is a largely uncharacterised native Australian desiccation-tolerant grass that resurrects from the desiccated state within 72 h. Using a combination of structural and physiological techniques the structural and physiological features that enable T. loliiformis to tolerate desiccation were investigated. These features include: - (i) a myriad of structural changes such as leaf folding, cell wall folding and vacuole fragmentation that mitigate desiccation stress; - (ii) potential role of sclerenchymatous tissue within leaf folding and radiation protection; - (iii) retention of ~70% chlorophyll in the desiccated state; - (iv) early response of photosynthesis to dehydration by 50% reduction and ceasing completely at 80 and 70% relative water content, respectively; - (v) a sharp increase in electrolyte leakage during dehydration, and; - (vi) confirmation of membrane integrity throughout desiccation and rehydration. Taken together, these results demonstrate that T. loliiformis implements a range of structural and physiological mechanisms that minimise mechanical, oxidative and irradiation stress. These results provide powerful insights into tolerance mechanisms for potential utilisation in the enhancement of stress-tolerance in crop plants.

Mechanical properties and damage tolerance of Y2SiO5

Y2SiO5 has potential applications as functional-structural ceramic and environmental/thermal barrier coating material. As an important grain-boundary phase in the sintered Si3N4, it also influences the mechanical and dielectric performances of the host material. In this paper, we present the mechanical properties of Y2SiO5 including elastic moduli, hardness, strength and fracture toughness, and try to understand the mechanical features from the viewpoint of crystal structure. Y2SiO5 has low shear modulus, low hardness, as well as high capacity for dispersing mechanical damage energy and for resisting crack penetration. Particularly, it can be machined by cemented carbides tools. The crystal structure characteristics of Y2SiO5 suggest the low-energy weakly bonded atomic planes crossed only by the easily breaking Y-O bonds as well as the rotatable rigid SiO4 tetrahedra are the origins of low shear deformation, good damage tolerance and good machinability of this material. TEM observations also demonstrate that the mechanical damage energy was dispersed in the form of the micro-cleavages, stacking faults and twins along these weakly bonded atomic planes, which allows the "microscale-plasticity" for Y2SiO5.

Cow's milk allergy and the development of tolerance

Aims: To gain insight on the immunological processes behind cow’s milk allergy (CMA) and the development of oral tolerance. To furthermore investigate the associations of HLA II and filaggrin genotypes with humoral responses to early oral antigens. Methods: The study population was from a cohort of 6209 healthy, full-term infants who in a double-blind randomized trial received supplementary feeding at maternity hospitals (mean duration 4 days): cow’s milk (CM) formula, extensively hydrolyzed whey formula or donor breast milk. Infants who developed CM associated symptoms that subsided during elimination diet (n=223) underwent an open oral CM challenge (at mean age 7 months). The challenge was negative in 112, and in 111 it confirmed CMA, which was IgE-mediated in 83. Patients with CMA were followed until recovery, and 94 of them participated in a follow-up study at age 8-9 years. We investigated serum samples at diagnosis (mean age 7 months, n=111), one year later (19 months, n=101) and at follow-up (8.6 years, n=85). At follow-up, also 76 children randomly selected from the original cohort and without CM associated symptoms were included. We measured CM specific IgE levels with UniCAP (Phadia, Uppsala, Sweden), and β-lactoglobulin, α-casein and ovalbumin specific IgA, IgG1, IgG4 and IgG levels with enzyme-linked immunosorbent assay in sera. We applied a microarray based immunoassay to measure the binding of IgE, IgG4 and IgA serum antibodies to sequential epitopes derived from five major CM proteins at the three time points in 11 patients with active IgE-mediated CMA at age 8-9 years and in 12 patients who had recovered from IgE-mediated CMA by age 3 years. We used bioinformatic methods to analyze the microarray data. We studied T cell expression profile in peripheral blood mononuclear cell (PBMC) samples from 57 children aged 5-12 years (median 8.3): 16 with active CMA, 20 who had recovered from CMA by age 3 years, 21 non-atopic control subjects. Following in vitro β-lactoglobulin stimulation, we measured the mRNA expression in PBMCs of 12 T-cell markers (T-bet, GATA-3, IFN-γ, CTLA4, IL-10, IL-16, TGF-β, FOXP3, Nfat-C2, TIM3, TIM4, STIM-1) with quantitative real time polymerase chain reaction, and the protein expression of CD4, CD25, CD127, FoxP3 with flow cytometry. To optimally distinguish the three study groups, we performed artificial neural networks with exhaustive search for all marker combinations. For genetic associations with specific humoral responses, we analyzed 14 HLA class II haplotypes, the PTPN22 1858 SNP (R620W allele) and 5 known filaggrin null mutations from blood samples of 87 patients with CMA and 76 control subjects (age 8.0-9.3 years). Results: High IgG and IgG4 levels to β-lactoglobulin and α-casein were associated with the HLA (DR15)-DQB1*0602 haplotype in patients with CMA, but not in control subjects. Conversely, (DR1/10)-DQB1*0501 was associated with lower IgG and IgG4 levels to these CM antigens, and to ovalbumin, most significantly among control subjects. Infants with IgE-mediated CMA had lower β -lactoglobulin and α-casein specific IgG1, IgG4 and IgG levels (p<0.05) at diagnosis than infants with non-IgE-mediated CMA or control subjects. When CMA persisted beyond age 8 years, CM specific IgE levels were higher at all three time points investigated and IgE epitope binding pattern remained stable (p<0.001) compared with recovery from CMA by age 3 years. Patients with persisting CMA at 8-9 years had lower serum IgA levels to β-lactoglobulin at diagnosis (p=0.01), and lower IgG4 levels to β-lactoglobulin (p=0.04) and α-casein (p=0.05) at follow-up compared with patients who recovered by age 3 years. In early recovery, signal of IgG4 epitope binding increased while that of IgE decreased over time, and binding patterns of IgE and IgG4 overlapped. In T cell expression profile in response to β –lactoglobulin, the combination of markers FoxP3, Nfat-C2, IL-16, GATA-3 distinguished patients with persisting CMA most accurately from patients who had become tolerant and from non-atopic subjects. FoxP3 expression at both RNA and protein level was higher in children with CMA compared with non-atopic children. Conclusions: Genetic factors (the HLA II genotype) are associated with humoral responses to early food allergens. High CM specific IgE levels predict persistence of CMA. Development of tolerance is associated with higher specific IgA and IgG4 levels and lower specific IgE levels, with decreased CM epitope binding by IgE and concurrent increase in corresponding epitope binding by IgG4. Both Th2 and Treg pathways are activated upon CM antigen stimulation in patients with CMA. In the clinical management of CMA, HLA II or filaggrin genotyping are not applicable, whereas the measurement of CM specific antibodies may assist in estimating the prognosis.

Role of an RNA polymerase interacting protein, MsRbpA, from Mycobacterium smegmatis in phenotypic tolerance to rifampicin

Rifampicin and its derivatives are at the forefront of the current standard chemotherapeutic regimen for active tuberculosis; they act by inhibiting the transcription activity of prokaryotic RNA polymerase. Rifampicin is believed to interact with the beta subunit of RNA polymerase. However, it has been observed that protein-protein interactions with RNA polymerase core enzyme lead to its reduced susceptibility to rifampicin. This mechanism became more diversified with the discovery of RbpA, a novel RNA polymerase-binding protein, in Streptomyces coelicolor that could mitigate the effect of rifampicin on RNA polymerase activity. MsRbpA is a homologue of RbpA in Mycobacterium smegmatis. On deciphering the role of MsRbpA in M. smegmatis we found that it interacts with RNA polymerase and increases the rifampicin tolerance levels, both in vitro and in vivo. It interacts with the beta subunit of RNA polymerase. However, it was found to be incapable of rescuing rifampicin-resistant RNA polymerases in the presence of rifampicin at the respective IC50.