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Item Reconstruction of gene innovation associated with major evolutionary transitions in the kingdom Fungi(BioMed Central Ltd, 2022-12) Wu B; Hao W; Cox MPBACKGROUND: Fungi exhibit astonishing diversity with multiple major phenotypic transitions over the kingdom's evolutionary history. As part of this process, fungi developed hyphae, adapted to land environments (terrestrialization), and innovated their sexual structures. These changes also helped fungi establish ecological relationships with other organisms (animals and plants), but the genomic basis of these changes remains largely unknown. RESULTS: By systematically analyzing 304 genomes from all major fungal groups, together with a broad range of eukaryotic outgroups, we have identified 188 novel orthogroups associated with major changes during the evolution of fungi. Functional annotations suggest that many of these orthogroups were involved in the formation of key trait innovations in extant fungi and are functionally connected. These innovations include components for cell wall formation, functioning of the spindle pole body, polarisome formation, hyphal growth, and mating group signaling. Innovation of mitochondria-localized proteins occurred widely during fungal transitions, indicating their previously unrecognized importance. We also find that prokaryote-derived horizontal gene transfer provided a small source of evolutionary novelty with such genes involved in key metabolic pathways. CONCLUSIONS: The overall picture is one of a relatively small number of novel genes appearing at major evolutionary transitions in the phylogeny of fungi, with most arising de novo and horizontal gene transfer providing only a small additional source of evolutionary novelty. Our findings contribute to an increasingly detailed portrait of the gene families that define fungal phyla and underpin core features of extant fungi.Item Exploring plant volatile-mediated interactions between native and introduced plants and insects(Springer Nature Limited, 2022-09-14) Effah E; Svendsen L; Barrett DP; Clavijo McCormick AIn invasion scenarios, native and introduced species co-occur creating new interactions and modifying existing ones. Many plant-plant and plant-insect interactions are mediated by volatile organic compounds (VOCs), however, these have seldom been studied in an invasion context. To fill this knowledge gap, we explored some interactions mediated by VOCs between native and introduced plants and insects in a New Zealand system. We investigated whether a native plant, Leptospermum scoparium (mānuka), changes its volatile profile when grown adjacent to two European introduced plants, Calluna vulgaris (heather) and Cytisus scoparius (Scotch broom), in a semi-field trial using potted plants without above- or below-ground physical contact. We also investigated the influence of plant cues on the host-searching behaviour of two beetles, the native Pyronota festiva (mānuka beetle), and the introduced biocontrol agent Lochmaea suturalis (heather beetle), by offering them their host-plant and non-host volatiles versus clean air, and their combination in a Y-tube olfactometer. As a follow-up, we performed preference/feeding tests in Petri dishes with fresh plant material. Results of the semi-field experiment show a significant reduction in green leaf volatiles, sesquiterpenes and total volatile emissions by mānuka plants neighbouring heather. In the Y-tube assays, the native beetle P. festiva performed poorly in discriminating between host and non-host plants based on plant volatile cues only. However, it performed relatively well in the Petri dish tests, where other cues (i.e., visual, gustatory or tactile) were present. In contrast, the introduced beetle L. suturalis showed high host-specificity in both Y-tube and Petri dish assays. This study illustrates the importance of VOCs in mediating interactions between introduced and native species, suggesting that invasive plants can disrupt native plants' communication and affect the host-searching behaviour of native insects. It also reinforces the relevance of regular host testing on introduced weed biocontrol agents to avoid unwanted host shifts or host-range expansion.Item Insect bioactive capabilities of Epichloë festucae var lolii AR48 infected Lolium perenne : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Manawatū, New Zealand(Massey University, 2018) Miller, Taryn AngelaAs the modern world expands and develops, new innovative methodologies for more efficient and environmentally friendly agricultural practices are required. Loss of crops through abiotic (e.g. drought) and biotic (e.g. herbivory) stresses has a major effect on the success of an agricultural industry. For animal production pasture crops are a key aspect of animal husbandry and directly affects yield and health. Symbiotic fungi belonging to the genus Epichloë form associations with cool season forage grasses and have been exploited as a new innovative method for insect pest management. Ryegrass infected with the asexual E. festucae var lolii strain AR48 has insect bioactivity against both the stem boring fly (SBF-Ceradontha australis) and cutworm moth caterpillar (CC -Agrotis ipsilion). The bioactive/s targeting both insects is currently unknown. The aim of this thesis was to identify the gene/s and/or bioactive/s present in AR48 infected ryegrass that have bioactivity against the SBF and/or CC. Two approaches were taken; the known insect bioactive secondary metabolite pathways in Epichloë were investigated in AR48 through bioinformatics and mass spectrometry, and the gene ‘makes caterpillars floppy’ (mcf), encoding an insect toxin like protein, was investigated through reverse genetics and insect bioactivity trials. A new indole diterpene compound (IDT) was identified in AR48 infected plant material and this compound was absent in other Epichloë strains that do not have SBF and CC bioactivity. The same mcf gene allele as that present in the E. typhina mcf model, previously identified as having CC bioactivity, is present and predicted to be functional in AR48. The other Epichloë strains also have mcf genes predicted to be functional, however the mcf allele is different to the bioactive E. typhina mcf model. Overall, this project was able to identify a new IDT compound with potential insect bioactivity as well as identify two Epichloë mcf gene alleles that potentially have differing insect bioactivities.Item Functional characterisation of constitutive expresser of pathogenesis-related genes 5 : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Molecular Biology at Massey University, Palmerston North, New Zealand(Massey University, 2017) Faisal, MuhammadAs reported previously, CPR5 negatively regulates the onset of leaf death, hypersensitive response, disease resistance and early leaf senescence. cpr5 plants contain aberrant trichomes and higher levels of ROS, SA and JA. Cell-cycle, JA/ET, ABA and sugar signalling are also affected in cpr5 plants. These results suggest that CPR5 is a master regulator of multiple processes. However, how CPR5 manages to exert pleiotropic effects is still poorly understood. The first objective of the current study was the purification of the CPR5 protein to solve its crystal structure. Extensive in silico analyses were carried out and the results showed that CPR5 is predicted to be a membrane protein with 4 or 5 transmembrane (TM) domains. Additionally, CPR5 contains intrinsically disordered regions (IDRs) at its N-terminus. Proteins containing IDRs and TM domains are often difficult to purify for crystallization studies. Therefore, the undesirable regions of CPR5 such as, IDR and TM domains were deleted and a set of 24 constructs were developed. Despite several efforts, none of the CPR5 recombinant proteins were isolated. In addition to predicting IDR and TM domains, in silico results also predicted three NLS-encoding clusters, casein kinase phosphorylation sites, multiple start codons, coiled-coil domains and glycine motifs. To find out the roles of these putative structural elements on CPR5 functions, firstly a CPR5 cDNA was synthesised and termed as SynCPR5. Subsequently, predicted sites or motifs were mutated in SynCPR5 through sitedirected mutagenesis and a set of 25 mutated CPR5 transgenes (cDNA constructs) were developed. Using a complementation strategy, all the constructs were transformed into cpr5- 2 plants. The results show that the complementation of cpr5-2 plants with SynCPR5, fully restored HR-like lesions, wildtype-like trichomes and leaves on SynCPR5 plants. Further physiological characterization such as, transcript abundance of SynCPR5, PR1, PR5 and PDF1.2, leaf area measurements and ploidy levels showed that CPR5 regulates some of its functions and phenotypes quantitatively as well as qualitatively. When compared with the wildtype, better growth (larger leaves) but enhanced disease susceptibility was found in metCPR5 transgenic lines (in which putative start codons were mutated), indicating that CPR5 regulates a balance between growth and resistance. Functional characterization of NLS mutants (nlsCPR5) showed that NLS-encoding clusters are important for CPR5 proper functions. However, current evidence is insufficient to relate their role in CPR5 localization. Moreover, in silico results show that putative NLS clusters are present in the region of CPR5 which were annotated as intrinsically disordered region (IDR). Similar phenotypes shown by both nlsCPR5 and Del63CPR5 (in which the first 63 amino acids of CPR5 including putative NLS were deleted), indicate that the putative NLS clusters could be part of IDR and may have dual functions. Loss-of-function phenotypes shown by coiled-coil domain mutants (ccdCPR5) reinforce the role of coiled-coil domains in CPR5 homo-dimerization. Moreover, in contrast to previous reports, the downregulation of PDF1.2 in the majority of CPR5 complementation lines proposes CPR5 to be a positive regulator of PDF1.2. Based on the results presented in the current study, putative CPR5 IDRs and coiled-coil domains are proposed to facilitate CPR5 dimerization in order to restrict the entry of deregulated cargos into the nucleus. Moreover, these results uncover a novel role of CPR5 in the regulation of balance between plant growth and resistance. Furthermore, this study, for the first time, reports evidence of the requirement of NLS clusters for CPR5 functions.Item The economic significance of the phytoextraction of nickel, cobalt and gold from metalliferous soils : a thesis in partial fulfilment of the requirements for the degree of Master of Science at Massey University(Massey University, 2000) Keeling, Scott MPhytoextraction of heavy metals is a relatively new technology that has potential applications for the remediation of many contaminated sites around the world. The technology has significant applications in the minerals industry for the treatment of low-grade ores and metalliferous mine waste. This study concerns the investigation of the potential to remove heavy metals, in particular nickel, cobalt and gold, from artificial and lateritic substrates. Four experiments comprise this study of the phytoextraction of nickel, cobalt and gold using both accumulator and non-accumulator species. Nickel and cobalt bioavailability was determined by ammonium acetate extraction for both artificial and laterite substrates. It was found that ammonium acetate extractability was predictive for nickel accumulation from a nickel-only artificial substrate. Cobalt bioavailability did not predict the accumulation response of either Alyssum bertolonii or Berkheya coddii grown of artificial substrates. The potential for phytoextraction of nickel and cobalt was investigated using the known nickel hyperaccumulators A. bertolonii and B. coddii, grown on artificially prepared substrates. The substrates were nickel-only (4 mg/kg to 1000 mg/kg), cobalt-only (4 mg/kg to 1000 mg/kg) and nickel-cobalt mixed (1:1 ratio, 4 mg/kg to 500 mg/kg) amendments of sulphates to commercial potting mix. Hyperaccumulation from nickel-only and cobalt-only substrates resulted in typical logarithmic metal uptake by both species. The cobalt-only substrates were phytotoxic to B. coddii above a concentration of 15-20 mg/kg. Phytotoxicity significantly reduced biomass production in B. coddii without effecting the bioaccumulation coefficient. No corresponding cobalt phytotoxicity was observed in A. bertolonii over the experimental range, although biomass production appears to favour substrate concentrations below 30 mg/kg. The bioavailability and hyperaccumulation of cobalt from the mixed nickel-cobalt substrates dramatically reduced the nickel accumulation potential of both species at substrate concentrations below 300 mg/kg. At higher substrate metal concentrations both species return to nickel dominant hyperaccumulation. Induced gold accumulation in B. coddii and Iberis intermedia was investigated using, sequential ammonium thiocyanate and ammonium thiosulphate chelation to, a 5 mg/kg gold artificial substrate. An attempt to determine gold bioavailability by ammonium thiocyanate and ammonium thiosulphate extraction was made on the substrate. It was found that neither chelator extraction could be correlated with plant accumulation induced by the same concentration of the reagent. Ammonium thiocyanate induction resulted in plant gold accumulation at or below the substrate concentration. Ammonium thiosulphate induced gold accumulation in I. Intermedia reached 48.8 mg/kg when treatment with a 1% solution. B. coddii accumulated 9.3 mg/kg gold for the same treatment. Five consignments of metalliferous lateritic materials from Western Australia were investigated. Three substrates originated from Project Murrin Murrin nickel and cobalt mine operated by Anaconda Nickel Ltd. and two substrates originated from Boddington Gold Mine operated by Worsley Alumina Ltd. Nickel and cobalt accumulation by A. bertolonii and B. coddii was found to be significantly lower than observed using artificial substrates. Nickel and cobalt bioavailability, determined by ammonium acetate extraction, failed to predict the accumulation responses from laterite substrates. This is attributed to elemental interference by, and possibly ammonium acetate chelation of, other mobile heavy metals in these substrates. A hypothesis deserved of further research. Hyperaccumulation of nickel was observed for both species on the Anaconda Nickel Ltd. SAP substrate only. Appreciable cobalt accumulation (≈90 mg/kg) was observed on the SAP substrate for both species and on the Boddington Gold Mine B5 substrate for B. coddii. Phytomining scenarios were determined for both species grown on the SAP substrate. A. bertolonii could produce 13 kg of nickel and 0.8 kg of cobalt per hectare with a value of US$ 163. B. coddii could produce 23.8 kg of nickel and 2.1 kg of cobalt per hectare at a value of US$ 319. These levels of production could be improved by fertilisation and/or substrate acidification. A preliminary investigation into induced gold accumulation from laterite substrates by I. Intermedia, A. longiflora, Brassica juncea and Limum usitatissimum was made using the acid biased chelator ammonium thiocyanate. It was found that an acidified amendment of ammonium thiocyanate greatly improved the phytoaccumulation of gold from the lateritic substrates. An amendment of 2M HC1 produced appreciable gold mobility and phytoaccumulation and indicates that gold solubility is the primary control on plant uptake. Analysis of various plant tissues indicated that Acacia longiflora stored significant gold in its roots compared to foliar components. All plant-substrate combinations indicated a trend towards increasing acidification and gold phytoaccumulation. No plant-substrate-treatment combination produced an economically viable phytomining scenario.Item Investigation of relationships through which biodynamic growing practices affect plant growth and nutrient composition : a thesis presented in partial fulfilment of the requirements of the degree of Master of Science in Soil Science at Massey University(Massey University, 2003) Bacchus, GillMore research attention has been paid to development of indicators of soil quality in relation to environmental sustainability than to food quality. Challenges in measuring and showing relationships between soil quality, food quality and human health are discussed. Comparative and long-term studies have shown that organic and biodynamic farming methods and use of composts and manures favourably affect soil quality, enhancing organic matter content and soil organism activity. However, measured effects on food quality have been lacking or inconsistent. Antioxidants, nitrates, trace elements, protein quality and ratios between element concentrations can be measured in assessing food quality. Many of these factors vary considerably with growing conditions and soil management techniques. Effects of factors such as water, light, soil type, variety and nutrient supply on lettuce growth, lettuce nutrient requirements, and recent research into effects of light on plant signalling and nitrogen metabolism arc reviewed and discussed. Recent research into factors influencing food quality has focussed on integration of growth and differentiation forces into "Vital quality" food. The biodynamic field-spray preparations 500 and 501 are used to balance effects of growth or "shade" forces from humus and fertilisers with the differentiating or "light' forces on plants. Literature indicates that the preparation 501 (silica-spray) appears to increase plant nutrient assimilation and production of more complex organic acids. Similar effects have been found for silica compounds applied to soil or nutrient solution. The main objective of the experimental work conducted for this thesis was to investigate whether relationships exist between soil management techniques and application of biodynamic sprays and plant product quality. Transplanted lettuces (cv. Canasta) were grown in a factorial designed field trial on Te Puke Series sandy loam with six treatments: control, soluble fertilisers (DAPCAN) and compost, each with, or without, biodynamic field-sprays 500 (twice) and 501 (3 times). High variability between plants within treatments and small differences between treatment means for most parameters measured prevented many statistically significant differences or relationships being found. Compost amendments appeared to enhance water and nutrient uptake during a dry season. However compost application at a rate to provide equivalent nitrogen to the soluble fertilisers resulted in high leaf concentrations of nitrates and potassium and low DM% and concentrations of sugars, antioxidants, calcium and magnesium. Plants in treatments given compost had highest yields; highest N, P and K concentrations at 28 days from transplanting (DAT); and highest K at 48 DAT. Plants in treatments given soluble fertilisers had highest Ca, Mg, Fe, Zn. and Cu concentrations and greater Ca: P and K: Ca + Mg ratios at 48 DAT. Application of biodynamic field-sprays appeared to have different effects on the plants in plots, depending upon whether they received compost or not. Plants in treatments given field-sprays but no compost had generally small head weight, greater dry matter % and root: shoot ratios at 28 DAT, and highest crude protein and Ferric reducing ability of plasma (FRAP) antioxidant concentration at 48 DAT. Plants in the biodynamic treatment, given compost and field-sprays, had highest P uptake between 28 and 48 DAT and highest fresh-weight at 48 DAT. Measurements of nitrate and sugar contents of leaf cell sap and amino acid concentrations in leaves yielded few, or no, significant differences between treatment means. Microbial activity measured by soil respiration ex situ at 28 DAT was highest in composted plots and lowest in sprayed plots. Measurement of AM fungi colonisation of roots gave inconclusive results. In a sensory evaluation, no significant differences in taste, bitterness, sweetness and preference ranking were found between lettuces from the different treatments. A greenhouse pot trial was undertaken to study the effects of the biodynamic silica spray in more detail. Lettuce transplants (cv. Cos Little Gem) were grown in the same soil and biodynamic compost as were used in the first trial and preparation 500 applied. Half the plants were sprayed 3 times with preparation 501. Measurements before and after the last spray time yielded insignificant differences in light absorption at most wavelengths, net photosynthesis and nitrate, sugar and amino acid concentration in leaves. Silica sprayed plants had higher rates of transpiration and stomatal conductance and higher estimated light absorption of blue and near infrared wavelengths 21/2 hours after spraying. Mainly inconclusive effects of treatments were due partly to the large natural plant to plant (within replication) variation. It was concluded that organic and biodynamic management of lettuces may result in some favourable quality attributes compared to soluble fertilisers but not necessarily all. Results are likely to be specific to particular climatic and soil conditions. It is recommended that further trials be carried out to evaluate influences of biodynamic practices on vegetable food quality in controlled, well-replicated conditions, to improve likelihood of showing statistical differences between treatments. Such trials are needed in a variety of soil, climatic and management conditions, to better understand how different conditions and their interactions affect food quality parameters. Relationships between biodynamic preparation application, soil biota populations and activity, plant metabolism and food product quality, particularly nitrogen assimilation into complex molecules such as essential amino acids, should be explored.Item Changes in inhibitor levels, stomatal aperture, and growth of Pisum sativum L. subjected to wilting stress cycles during different developmental stages : a thesis submitted in partial fulfilment of the requirements for the degree of Master of Horticultural Science at Massey University(Massey University, 1974) Ivey, Ian DavidPlants of Pisum Sativum L., grown under controlled environmental conditions, were subjected to wilting stress cycles at different developmental stages and analyzed for changes in inhibitor levels, stomatal aperture, water status, and effects on final yield. As leaf water potential decreased past a critical level, stomatal aperture decreased markedly and, at the same time, inhibitor levels increased rapidly. The naximum inhibitor levels attained, as determined by several different methods of assessment, approximately halved with each later wilting cycle, whilst the degree of stomatal closure was approximately the same for each cycle. During the recovery phase, plant water status recovered to normal 24 hours after rewatering. At this time inhibitor levels had decreased markedly and, in the later cycles, had apparently declined to normal levels. However stomatal aperture had only recovered slightly at this point in all cycles and by 4 days after rewatering stomata had generally regained normal apertures. Results of the final yield analysis were confounded somewhat by the shooting of basal buds, particularly on plants subjected to wilting cycles during the preflovrering and flowering stages. and a possible explanation for this lateral growth is discussed. However the pod swelling stage was more sensitive to water stress than other stages. Changes in inhibitor levels alone, did not appear to be related directly to stomatal responses or any sensitivity of particular growth stages. Some possible reasons for these observations are presented on the basis of evidence available in the literature.Item Coordinated transcriptional regulation between a reactive oxygen species-responsive gene network and the circadian clock in Arabidopsis thaliana : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Plant Biology at Massey University, Plant Biology, New Zealand(Massey University, 2012) Lai, Alvina GraceMost organisms have evolved endogenous biological clocks as internal timekeepers to fine-tune physiological processes to the external environment. Energetic cycles such as photosynthesis and glycolytic cycles are physiological processes that have been shown to be under clock control. This work sought to understand the mechanism of the synchrony between the circadian oscillator and products of energetic cycles. The fact that plants rely on photosynthesis for survival,and that photosynthesis relies on the sun, this would have meant that oxygen levels would have fluctuated across the day. A common by-product of oxygen metabolism and photosynthesis is the Reactive Oxygen Species(ROS). Evidence has proposed ROS as regulators of cellular signaling and plant development. However, if ROS levels are left unmanaged, it may cause oxidative stress in organisms, which could damage cellular components and disrupt normal mechanisms of cellular signaling. Therefore, it is advantageous for plants to be able to anticipate such periodic burst in ROS. My research investigates the role of the circadian clock in regulating ROS homeostasis in the model plant Arabidopsis thaliana. I found that ROS production and scavenging wax and wane in a periodic manner under diurnal and circadian conditions. Not only that, at the transcriptional level, ROS7 responsive genes exhibited time-of-day specific phases under diurnal and circadian conditions,suggesting the role of the circadian clock in ROS signaling. Mutations in the core-clock regulator, CIRCADIAN3 CLOCK3 ASSOCIATED3 1 (CCA1), affect both the transcriptional regulation of ROS genes and ROS homeostasis. Furthermore, mis- expressions of other clock genes such as EARLY3 FLOWERING3 33 (ELF3), LUX3 ARRHYTHMO3 (LUX) and TIMING3 OF3 CAB3 EXPRESSION3 13 (TOC1) also have profound effects on ROS signaling and homeostasis, thus suggesting a global clock effect on ROS networks. Taken together, CCA1 is proposed as a master regulator of ROS signaling where the response to oxidative stress is dependent on the time of CCA1 expression. Plants exhibit the strongest response at dawn, the time when CCA1 peaks. Moreover, CCA1 can associate to the Evening Element or CCA17Binding Site on promoters of ROS genes in vivo to coordinate transcription. A common feature of circadian clocks is the presence of multiple interlocked transcriptional feedback loops. It is shown here that the oscillator incorporates ROS as a component of the loop where ROS signals could feed back to affect circadian behavior by changing CCA1 and TOC1 transcription. The clock regulates a plethora of output pathways; particularly the transcription of an output gene FLAVIN3BINDING3KELCH3REPEAT3FHBOX31(FKF1) is affected by ROS signals. Temporal coordination of ROS signaling by CCA1 and the reciprocal control of circadian behavior by ROS revealed a mechanistic link of which plants match their physiology to the environment to confer fitness.Item Aminoacid metabolism in plants : the biosynthesis of [beta]-cyanoalanine and asparagine in lupins : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University(Massey University, 1970) Lever, Michaelβ-Cyanoalanine synthase in etiolated seedlings of Lupinus angustifolius is found mainly in the mitochondrial fraction of the cotyledons and stems. In seedlings developing at 25°C in the dark it reaches a maximal concentration after 5 days, at the same time as asparagine accumulation is most rapid and other physiological changes occur. However, maximal ability to assimilate HCN gas to asparagine develops after 3 to 4 days, before asparagine accumulation begins. A partial purification of lupin β-cyanoalanine synthase is described and its distinction from cysteine synthase confirmed. Carbon-14 labelled substrates were supplied to etiolated seedlings of lupin in order to identify precursors of asparagine. Four carbon acids related to the TCA cycle were readily converted to asparagine i n vivo but not in vitro; the carbon skeleton of aspartate is retained in asparagine. Comparison of the distribution of label in the carbon skeletons of aspartate and asparagine from plants supplied carboxyl labelled fumarate shows a separation of the aspartate pool for asparagine biosynthesis from most cell aspartate. Metobolites that could be expected to give rise to cyanide and β-cyanoalanine are relatively ineffective as asparagine precursors. Thus the β-cyanoalanine pathway is not of major importance in asparagine biosynthesis in lupins.Item Some effects of water stress and environment on soybean plants : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Botany at Massey University(Massey University, 1970) Beardsell, Michael FletcherAlthough a great deal of research has been carried out on the effects of water stress on plant processes, the influence of environmental conditions on plant response to water stress has received comparatively little attention. In this study the rates of CO2 exchange and transpiration and the leaf water status of whole soybean plants (Glycine max (L.) Merr. cv. Merit) were measured under contrasting sets of environmental conditions when (a) the plants were maintained under conditions of adequate soil water supply. (b) water stress was imposed by withholding water and, (c) when water stress was imposed and then relieved by rewatering. Light intensity and quality, atmospheric CO2 concentration, wind speed and daylength were all constant; the between-treatment variables were air temperature and vapour pressure deficit (VPD). Plants were grown under one of four environmental treatments in a growth cabinet and the experiments carried out under very similar conditions in a plant chamber with facilities for measuring CO2 exchange and transpiration. Details of this equipment are given. Under conditions of adequate soil water supply rates of photosynthesis were lower under low VPD than under high VPD conditions at the same temperature. The effect was particularly marked at low temperature (22.5°C). Between-treatment differences in photosynthetic rate appeared to be mainly attributable to differences in the magnitude of the mesophyll resistance to CO2 transfer. Transpiration rates were largely determined by the VPD, plants under high VPD treatments having the higher rates. At low VPD temperature had little effect on the rate of transpiration, but at high VPD plants under low temperature had lower rates of transpiration than plants under high temperature (27.5°C). Possible mechanisms whereby low temperatures may reduce transpiration under conditions of high VPD are discussed. When water stress was imposed the rates of photosynthesis and transpiration declined in parallel under all treatments at soil moisture tensions in excess of 0.2 atm. This suggested that both plant processes were subject to a common controlling mechanism, probably stomatal diffusion resistance. At soil moisture tensions below 0.2 atm. the rates of photosynthesis and transpiration were independent of the soil moisture status. Between 0.2 and 0.4 atm. tension they appeared to be determined by plant, soil and atmospheric factors. The relative rates of photosynthesis and transpiration were reduced to a greater extent at any tension between 0.2 and 0.4 atm. under high VPD than under low VPD conditions. Above 0.4 atm. soil moisture tension the rates of photosynthesis and transpiration became independent of the atmospheric conditions and it is suggested that transpiration was limited chiefly by the rate of movement of water into the root zone from the surrounding soil. Photosynthesis may have been limited by direct effects of dehydration on the biochemical components of the process at these severe stress levels. It was thus possible to distinguish three stages in the development of water stress, the significance and possible general application of which are discussed. Under high temperature/high VPD conditions the rates of photosynthesis and transpiration recovered simultaneously and to a very similar extent when stress was relieved by rewatering, the degree of recovery being inversely proportional to the soil moisture tension at the time of rewatering. Possible causes of the failure of the rates of photosynthesis and transpiration to recover to their original prestressed values are discussed. These results are discussed in relation to the findings of other workers, and suggestions for further research in this field made.