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Some physiological effects of freezing and flooding were studied on winter barley (Hordeum vulgare L.). The effects of freezing on plants grown under field conditions were determined by comparing plants exposed to naturally-occurring freezing temperatures with plants protected from such temperatures by thermostatically-controlled soil heating cables. The results from field trials in 1982/83 and 1983/84 showed the effects of freezing to include: (i) A reduction in the establishment of late-sown plants due to soil heaving. (ii) A retardation of plant and apical development. (iii) Occasional transient reductions in above ground dry matter and leaf area. (iv) Variable effects upon certain yield components. Attributes not greatly affected by such temperatures included cold hardiness and yield on a per plant basis. Controlled environment studies showed that non-lethal freezing temperatures ranging from -4 to -10oC significantly reduced shoot photosynthesis and dark respiration, the greater reductions being in photosynthesis. Factors involved in these reductions include damage to leaves and roots, neither of which were critical for plant survival. When a conductivity method was used to estimate the damage to different plant parts resulting from extracellular freezing, leaf laminae were found to be more freezing tolerant than roots, and young laminae were more tolerant than older laminae. Some effects of flooding were also studied using controlled environments, and cultivar differences in flooding tolerance were found. This tolerance was investigated by comparing the response to flooding of a tolerant cultivar (Athene) with a sensitive one (Maris Otter). Flooding sensitivity was shown as wilting in the leaves of Maris Otter, the onset of wilting being associated with the leakage of electrolytes and accumulation of ethanol in the leaf laminae. The results of exogenous application of ethanol to leaves, suggest the basis of this `tolerance' is ethanol exclusion. The production of aerenchyma was not associated with flooding tolerance.

γ-Hydroxybutyrate (GHB) is an intermediate of γ-aminobutyrate (GABA) catabolism in plants subjected to abiotic stress and its formation is catalyzed by two NADPH-dependent glyoxylate/succinic semialdehyde reductases (GLYRs). GABA and/or GHB accumulation in ‘Honeycrisp’ and ‘Empire’ apple fruit stored under controlled atmosphere (CA) conditions (i.e., low temperature, low O2, elevated CO2) in the presence or absence of the ethylene-antagonist 1-methylcyclopropene, coincided with the onset of physiological injury, suggesting an association with cellular disruption. Salinity and chilling stresses differentially influenced the expression of GABA pathway genes and the levels of GHB among various GABA pathway mutants of Arabidopsis. Furthermore, the occurrence of GHB in glyr1/glyr2 double knockout mutants indicates the presence of an additional pathway for GHB production. Evidence for GHB oxidation was not detectable in cell-free leaf extracts, suggesting the existence of a novel enzyme for GHB turnover. / NSERC Alexander Graham Bell Canada Graduate Scholarship (CGS-M), Ontario Graduate Scholarship (OGS), NSERC, Ontario Apple Growers, Rohm & Haas, MITACS

Post-transcriptional gene silencing (PTGS) is a powerful mechanism that can be adapted to genetically modify crop plants. PTGS operates in many plant signaling pathways including those mediating stress responses. Given the small number of miRNAs known, research on the characterization of stress-related micro-RNA (miRNA) and their targets could provide the basis for engineering stress tolerant traits in crops. Indeed, several examples of miRNA mediated crop tolerance have been reported. In the research presented here, we aimed to analyze the role of small non-coding RNA (smRNA) pathways involved in plant stress. In particular, we focused on miRNA-mediated PTGS in phosphate (Pi) starvation. The analysis was split into two research projects. First, to identify potential miRNA targets we began by analyzing the response and recovery of coding and long non-coding RNAs (lncRNA) to Pi starvation in shoot and root. The results obtained were the first genome-wide description of the root’s Pi starvation response and recovery. We found that the root's response involved a widely different set of genes than that of the shoot. In the second research project, the results of the first project were correlated with the responses of miRNA and trans-acting small-interfering RNA (tasiRNA) during Pi starvation. Many miRNA circuits have been predicted before, however, tasiRNA circuits are not as well defined. Therefore, we made use of the double-stranded RNA-binding protein 4 (DRB4) smRNA libraries to enhance our prediction of tasiRNAs. Altogether, we provided evidence to support the following miRNA-mRNA pairs that may function in Pi starvation: IPS1:miR399:PHO2; miR399:RS4; miR399:NF-YA10; miR398:CSD1/2; miR2111:TPS11; miR164:NAC6; miR157:TMO7; miR157:PSB28; RPS2:miR169:IPS2; miR397:LAC2; TAS4:PAP1; NR1:PAP1; and Chr3_1967672:TMO7. In general, we found that non-miR399 related circuits were active only during the root’s recovery from Pi starvation. The functional roles of the genes targeted by these PTGS circuits suggested that the local root response to Pi starvation was influenced by the plant's systemic response pathways via PHR1-mediated PTGS. Finally, since many PTGS targets function to modulate concentrations of reactive oxygen species and sucrose, we hypothesized that the candidate PTGS circuits found in our research mediate a general stress recovery process by modulating metabolites involved in signaling pathways.

Some Arabidopsis lesion mimic mutants (LMM) show alterations in abiotic stress responses as well as pathogen resistance. cpr22 is a LMM which has a mutation in cyclic nucleotide-gated ion channels, is a typical LMM exhibiting elevated levels of salicylic acid (SA), spontaneous cell death, constitutive expression of defense genes, and enhanced resistance to various pathogens in an SA dependant manner. cpr22 defense responses are suppressed in high humidity and enhanced by low humidity. To investigate environmental effects, microarray analyses were conducted. Expression of several genes related to abscisic acid (ABA) signaling was altered and ABA levels increased in cpr22 after humidity shift. Furthermore, significant alterations in ABA-related phenotypes were observed. Double mutant analysis with nahG plants indicated that alterations in ABA signaling were attributable to elevated SA levels. These results suggest a negative effect of SA on ABA signaling/abiotic stress responses during the activation of defense responses.

Some Arabidopsis lesion mimic mutants (LMM) show alterations in abiotic stress responses as well as pathogen resistance. cpr22 is a LMM which has a mutation in cyclic nucleotide-gated ion channels, is a typical LMM exhibiting elevated levels of salicylic acid (SA), spontaneous cell death, constitutive expression of defense genes, and enhanced resistance to various pathogens in an SA dependant manner. cpr22 defense responses are suppressed in high humidity and enhanced by low humidity. To investigate environmental effects, microarray analyses were conducted. Expression of several genes related to abscisic acid (ABA) signaling was altered and ABA levels increased in cpr22 after humidity shift. Furthermore, significant alterations in ABA-related phenotypes were observed. Double mutant analysis with nahG plants indicated that alterations in ABA signaling were attributable to elevated SA levels. These results suggest a negative effect of SA on ABA signaling/abiotic stress responses during the activation of defense responses.

Proteases play a crucial role in plant defence mechanisms as well as acclimation to changing metabolic demands and environmental cues. Proteases regulate the development of a plant from germination through to senescence and plant death. In this thesis the role of proteases and their inhibitors in plant response to cold stress and CO2 enrichment were investigated. The activity and inhibition of cysteine proteases (CP), as well as degradation of their potential target proteins was investigated in transgenic tobacco plants expressing the rice cystatin, OC-I. Expression of OC-I caused a longer life span; delayed senescence; significant decrease in in vitro CP activity; a concurrent increase in protein content; and protection from chilling-induced decreases in photosynthesis. An initial proteomics study identified altered abundance of a cyclophilin, a histone, a peptidyl-prolyl cis-trans isomerase and two RuBisCO activase isoforms in OC-I expressing leaves. Immunogold labelling studies revealed that RuBisCO and OC-I is present in RuBisCO vesicular bodies (RVB) that appear to be important in RuBisCO degradation in leaves under optimal and stress conditions. Plants need to respond quickly to changes in the environment that cause changes in the demand for photosynthesis. In this study the effect of CO2 enrichment on photosynthesis-related genes and novel proteases and protease inhibitors regulated by CO2 enrichment and/or development, was investigated. Maize plants grown to maturity with CO2 enrichment showed significant changes in leaf chlorophyll and protein content, increased epidermal cell size, and decreased epidermal cell density. An increased stomatal index in leaves grown at high-CO2 indicates that leaves adjust their stomatal densities through changes in epidermal cell numbers rather than stomatal numbers. Photosynthesis and carbohydrate metabolism were not significantly affected. Developmental stage affected over 3000 transcripts between leaf ranks 3 and 12, while 142 and 90 transcripts were modified by high CO2 in the same leaf ranks respectively. Only 18 transcripts were affected by CO2 enrichment exclusively. Particularly, two novel CO2 -modulated serine protease inhibitors modulated by both sugars and pro-oxidants, were identified. Growth with high CO2 decreased oxidative damage to leaf proteins. / Thesis (PhD)--University of Pretoria, 2009. / Plant Science / unrestricted

1. Plant quality (bottom-up effects) and natural enemies (top-down effects) affect herbivore performance. Furthermore, plant quality can also influence the impact of natural enemies. 2. Lower plant quality through reduced irrigation increased the abundance of the cryptic species from the Bemisia tabaci complex [hereafter B. tabaci Middle East Asia Minor 1 (MEAM1)], but not its natural enemies on cotton. It was therefore predicted that lower plant quality would diminish the impact of natural enemies in regulating this herbivore. 3. Over three cotton seasons, plant quality was manipulated via differential irrigation and natural enemy abundance with insecticides. Life tables were used to evaluate the impact of these factors on mortality of immature B. tabaci (MEAM1) over nine generations. 4. Mortality of B. tabaci (MEAM1) was consistently affected by natural enemies but not by plant quality. This pattern was driven by high levels of sucking predation, which was the primary (key) factor associated with changes in immature mortality across all irrigation and natural enemy treatments. Dislodgement (chewing predation and weather) and parasitism contributed as key factors in some cases. Analyses also showed that elimination of sucking predation and dislodgement would have the greatest effect on overall mortality. 5. The top-down effects of natural enemies had dominant effects on populations of B. tabaci (MEAM1) relative to the bottom-up effects of plant quality. Effects were primarily due to native generalist arthropod predators and not more host-specific aphelinid parasitoids. The findings of this study demonstrate the important role of arthropod predators in population suppression and validate the importance of conservation biological control in this system for effective pest control.

Irrigated lands generate crop yields that are more than double those of rain-fed lands. Unfortunately, these systems are often heavily reliant on water supplies, which are diminishing globally. Alternative use of impaired quality waters for irrigation can reduce soil quality through secondary salinization, affecting plant health and yields. With salinization of agricultural lands increasing around the world, further understanding the impacts of this on crop production are required. The aim of this research is to assess the influence of soil salinity and nutrient stress on leaf photosynthetic pigments, gas exchange and biochemical photosynthetic parameters in wheat plants. The feasibility of estimating key photosynthetic pigments from in-situ leaf hyperspectral data is examined using vegetation indices, linear regression models and a random forest machine learning technique. Results showed that salinity stress presented a significant increase in the chlorophyll and carotenoid contents per leaf area, although the total pigment contents per plant was reduced as a consequence of lower production of leaf matter. While nutrient application enhanced the photosynthetic pigment content per leaf area, its interaction with salinity stress was found to be significant and varied with salinity level. A strong positive relationship was found between SPAD-502 measurements and leaf chlorophyll content and confirmed that SPAD-based retrieval of photosynthetic pigments can be undertaken with confidence irrespective of any prevailing stress in wheat plants. Photosynthetic parameters directly related to biomass accumulation (such as Vcmax, Jmax and gs) varied considerably with stress levels and growth stages, with high values of these parameters observed at low stress and in periods of more vigorous growth. Employing a random forest machine learning approach with all hyperspectral data as input features significantly improved the predictability and accuracy relative to the univariate linear regression model. However, using vegetation indices as direct predictors further improved the estimation accuracy and robustness of the random forest model. Overall, the findings from this research have implications for large scale estimation of vegetation photosynthetic traits from remotely sensed data, and offer a mechanism by which early detection of stress may be monitored, providing a means for enacting a timely crop management response.

Stomata are cellular valves in plants that play an essential role in the regulation of gas exchange and are distributed in the epidermis of aerial organs. In Arabidopsis thaliana, stomatal production and development are coordinated by the mitogen-activated protein kinase (MAPK) signalling pathway, which modulates a variety of other processes, including cell proliferation, regulation of cytokinesis, programed cell death, and response to abiotic and biotic stress. The environment also plays a role in stomatal development, by influencing the frequency at which stomata develop in leaves. This thesis presents an analysis of stomatal development in Arabidopsis mutants in two MAPK pathways: MEKK1-MKK1/MKK2-MPK4, and MAP3K17/18-MKK3. Obtained results demonstrate the effect of stress conditions on stomatal development and specify the involvement of analysed MAPK in stomatal patterning. First, both analysed pathways modulate stomatal patterning in Arabidopsis cotyledons. Second, plant growth-promoting bacteria tested enhance stomatal density and affect guard cell morphology. Third, the sucrose or mannitol treatment increases defects in stomatal patterning. Finally, salt stress or high temperature can suppress stomatal defects in mutants of the MEKK1-MKK1/MKK2-MPK4 pathway.