The role of MicroRNAs in stress response in the resurrection plant Tripogon loliiformis

Research in this thesis focussed on the improvement of agricultural crops in increasing water use efficiency that impacts global crop productivity. The study identified key genetic regulatory mechanisms that the resurrection plant Tripogon loliiformis utilises to tolerate desiccation. Due to the conserved nature of the pathways involved, this information can be transferred for the enhancement of drought tolerance and water use efficiency in agricultural crops. Specifically this study used high throughput sequencing, microscopy and plant transformation to further the understanding of post-transcriptional regulatory mechanisms. It was shown that T. loliiformis uses microRNAs to regulate pro-survival autophagy pathways to tolerate desiccation.

Quorum sensing in the plant pathogen Erwinia carotovora subsp. carotovora

Erwinia carotovora subsp. carotovora (Ecc) is a Gram-negative enterobacterium that causes soft-rot in potato and other crops. The main virulence determinants, the extracellular plant cell wall -degrading enzymes (PCWDEs), lead to plant tissue maceration. In order to establish a successful infection the production of PCWDEs are controlled by a complex regulatory network, including both specific and global activators and repressors. One of the most important virulence regulation systems in Ecc is mediated by quorum sensing (QS), which is a population density -dependent cell-to-cell communication mechanism used by many Gram-negative bacteria. In these bacteria N-acylhomoserine lactones (AHSL), act as diffusible signaling molecules enabling communication between bacterial cells. The AHSLs are structurally diverse and differ in their acyl chain length. This gives the bacteria signaling specificity and enables the recognition and communication within its own species. In order to detect and respond to the AHSLs the bacteria use QS regulators, LuxR-type proteins. The aim of this study was to get a deeper understanding of the Ecc QS system. In the first part of the study we showed that even different strains of Ecc use different dialects and of physiological concentrations, only the cognate AHSL with the correct acyl chain is recognized as a signal that can switch on virulence genes. The molecular basis of the substrate specificity of the AHSL synthase ExpI was investigated in order to recognize the acyl chain length specificity determinants of distinct AHSL synthases. Several critical residues that define the size of the substrate-binding pocket were identified. We demonstrated that in the ExpISCC1 mutations M127T and F69L are sufficient to change the N-3-oxohexanoyl-L-homoserine lactone producing ExpISCC1 to an N-3-oxooctanoyl-L-homoserine lactone (3-oxo-C8-HSL) producing enzyme. In the second study the means of sensing specificity and response to the AHSL signaling molecule were investigated. We demonstrated that the AHSL receptor ExpR1 of Ecc strain SCC3193 has strict specificity for the cognate AHSL 3-oxo-C8-HSL. In addition we identified a second AHSL receptor ExpR2 with a novel property to sense AHSLs with different acyl chain lengths. In the absence of AHSLs ExpR1 and ExpR2 were found to act synergistically to repress the virulence gene expression. This repression was shown to be released by addition of AHSLs and appears to be largely mediated by the global negative regulator RsmA. In the third study random transposon mutagenesis was used to widen the knowledge of the Ecc QS regulon. Two new QS-controlled target genes, encoding a DNA-binding regulator Hor and a plant ferredoxin-like protein FerE, were identified. The QS control of the identified genes was executed by the QS regulators ExpR1 and ExpR2 and as expression of PCWDE genes mediated by the RsmA repressor. Hor was shown to contribute to bacterial virulence at least partly through its control of PCWDE production, while FerE was shown to contribute to oxidative stress tolerance and in planta fitness of the bacteria. In addition our results suggest that QS is central to the control of oxidative stress tolerance in Ecc. In conclusion, these results indicate that Ecc strain SCC3193 is able to react and respond both to the cognate AHSL signal and the signals produced by other bacterial species, in order to control a wide variety of functions in the plant pathogen Ecc.

Maintaining plant species richness by cattle grazing - mesic semi-natural grasslands as focal habitats

Semi-natural grasslands are the most important agricultural areas for biodiversity. The present study investigates the effects of traditional livestock grazing and mowing on plant species richness, the main emphasis being on cattle grazing in mesic semi-natural grasslands. The two reviews provide a thorough assessment of the multifaceted impacts and importance of grazing and mowing management to plant species richness. It is emphasized that livestock grazing and mowing have partially compensated the suppression of major natural disturbances by humans and mitigated the negative effects of eutrophication. This hypothesis has important consequences for nature conservation: A large proportion of European species originally adapted to natural disturbances may be at present dependent on livestock grazing and / or mowing. Furthermore, grazing and mowing are key management methods to mitigate effects of nutrient-enrichment. The species composition and richness in old (continuously grazed), new (grazing restarting 3-8 years ago) and abandoned (over 10 years) pastures differed consistently across a range of spatial scales, and was intermediate in new pastures compared to old and abandoned pastures. In mesic grasslands most plant species were shown to benefit from cattle grazing. Indicator species of biologically valuable grasslands and rare species were more abundant in grazed than in abandoned grasslands. Steep S-SW-facing slopes are the most suitable sites for many grassland plants and should be prioritized in grassland restoration. The proportion of species trait groups benefiting from grazing was higher in mesic semi-natural grasslands than in dry and wet grasslands. Consequently, species trait responses to grazing and the effectiveness of the natural factors limiting plant growth may be intimately linked High plant species richness of traditionally mowed and grazed areas is explained by numerous factors which operate on different spatial scales. Particularly important for maintaining large scale plant species richness are evolutionary and mitigation factors. Grazing and mowing cause a shift towards the conditions that have occurred during the evolutionary history of European plant species by modifying key ecological factors (nutrients, pH and light). The results of this Dissertation suggest that restoration of semi-natural grasslands by private farmers is potentially a useful method to manage biodiversity in the agricultural landscape. However, the quality of management is commonly improper, particularly due to financial constraints. For enhanced success of restoration, management regulations in the agri-environment scheme need to be defined more explicitly and the scheme should be revised to encourage management of biodiversity.

Effects of oxygen provision on the physiology of baker's yeast Saccharomyces cerevisiae

The availability of oxygen has a major effect on all organisms. The yeast Saccharomyces cerevisiae is able to adapt its metabolism for growth in different conditions of oxygen provision, and to grow even under complete lack of oxygen. Although the physiology of S. cerevisiae has mainly been studied under fully aerobic and anaerobic conditions, less is known of metabolism under oxygen-limited conditions and of the adaptation to changing conditions of oxygen provision. This study compared the physiology of S. cerevisiae in conditions of five levels of oxygen provision (0, 0.5, 1.0, 2.8 and 20.9% O2 in feed gas) by using measurements on metabolite, transcriptome and proteome levels. On the transcriptional level, the main differences were observed between the three level groups, 0, 0.5 2.8 and 20.9% O2 which led to fully fermentative, respiro-fermentative and fully respiratory modes of metabolism, respectively. However, proteome analysis suggested post-transcriptional regulation at the level of 0.5 O2. The analysis of metabolite and transcript levels of central carbon metabolism also suggested post-transcriptional regulation especially in glycolysis. Further, a global upregulation of genes related to respiratory pathways was observed in the oxygen-limited conditions and the same trend was seen in the proteome analysis and in the activities of enzymes of the TCA cycle. The responses of intracellular metabolites related to central carbon metabolism and transcriptional responses to change in oxygen availability were studied. As a response to sudden oxygen depletion, concentrations of the metabolites of central carbon metabolism responded faster than the corresponding levels of gene expression. In general, the genome-wide transcriptional responses to oxygen depletion were highly similar when two different initial conditions of oxygen provision (20.9 and 1.0% O2) were compared. The genes related to growth and cell proliferation were transiently downregulated whereas the genes related to protein degradation and phosphate uptake were transiently upregulated. In the cultures initially receiving 1.0% O2, a transient upregulation of genes related to fatty acid oxidation, peroxisomal biogenesis, response to oxidative stress and pentose phosphate pathway was observed. Additionally, this work analysed the effect of oxygen on transcription of genes belonging to the hexose transporter gene family. Although the specific glucose uptake rate was highest in fully anaerobic conditions, none of the hxt genes showed highest expression in anaerobic conditions. However, the expression of genes encoding the moderately low affinity transporters decreased with the decreasing oxygen level. Thus it was concluded that there is a relative increase in high affinity transport in anaerobic conditions supporting the high uptake rate.

The plant Banjauri (Vicoa indica) exhibits antifertility activity in adult female bonnet monkeys (Macaca radiata)

The antifertility activity of the plant Vicoa indica was tested in proven fertile bonnet monkeys. The dry powder of the whole plant was fed to the cycling monkeys on day 1 to 14 of menstrual cycle or day 9 to 14 of cycle or on day 2 to 5 after delivery and the fertility was evaluated in the following cycle in cycle fed monkey or after weaning the young one in the post-partum fed monkeys. Results indicated that while feeding in the post-partum monkeys did not confer any protection against pregnancy feeding during day 1 to 14 of cycle, protected from pregnancy. The monkeys did not become pregnant even after exposure to the proven fertile male monkeys for 13 ovulatory cycles while all the vehicle fed monkeys became pregnant within 3 cycles.

Plant regeneration from protoplasts of Capsicum annuum L cv. California Wonder

Plant regeneration from mesophyll protoplasts of pepper, Capsicum annuum L. cv. California Wonder has been demonstrated via shoot organogenesis, Protoplasts isolated from fully expanded leaves of 3-week-old axenic shoots when cultured in TM medium supplemented with 1 mgl(-1) NAA, 1 mgl(-1) 2, 4-D, 0.5 mgl(-1) BAP (CM 1) resulted in divisions with a frequency ranging from 20-25%. Antioxidant ascorbic acid and polyvinylpyrrolidone (PVP) in the medium and incubation in the dark helped overcome browning of protoplasts. Microcalli and macrocalli were formed in TM medium containing 2 mgl(-1) NAA and 0.5 mgl(-1) BAP (CM II) and MS gelled medium containing 2 mgl(-1) NAA and 0.5 mgl(-1) BAP (CM III), respectively, Regeneration of plantlets was possible via caulogenesis, Microshoots, 2-5 per callus appeared on MS gelled medium enriched with 0.5 mgl(-1) IAA, 2 mgl(-1) GA and 10 mgl(-1) BAP (CM IVc). Rooting of microshoots was obtained on half strength gelled medium containing 1 mgl(-1) NAA and 0.5 mgl(-1) BAP, Protoplasts isolated from cotyledons failed to divide and degenerated eventually.

Analysis of the amino acid sequences of plant Bowman-Birk inhibitors

Plant seeds contain a large number of protease inhibitors of animal, fungal, and bacterial origin. One of the well-studied families of these inhibitors is the Bowman-Birk family(BBI). The BBIs from dicotyledonous seeds are 8K, double-headed proteins. In contrast, the 8K inhibitors from monocotyledonous seeds are single headed. Monocots also have a 16K, double-headed inhibitor. We have determined the primary structure of a Bowman-Birk inhibitor from a dicot, horsegram, by sequential edman analysis of the intact protein and peptides derived from enzymatic and chemical cleavage. The 76-residue-long inhibitor is very similar to that ofMacrotyloma axillare. An analysis of this inhibitor along with 26 other Bowman-Birk inhibitor domains (MW 8K) available in the SWISSPROT databank revealed that the proteins from monocots and dicots belong to related but distinct families. Inhibitors from monocots show larger variation in sequence. Sequence comparison shows that a crucial disulphide which connects the amino and carboxy termini of the active site loop is lost in monocots. The loss of a reactive site in monocots seems to be correlated to this. However, it appears that this disulphide is not absolutely essential for retention of inhibitory function. Our analysis suggests that gene duplication leading to a 16K inhibitor in monocots has occurred, probably after the divergence of monocots and dicots, and also after the loss of second reactive site in monocots.

A simple method for the isolation of plant protoplasts

A simple protoplast isolation protocol that was designed to recover totipotent plant protoplasts with relative ease has been described. The key elements of the protocol are, tissue digestion at slightly elevated temperatures and use of protoplast-releasing enzymes that are stable and efficient at higher temperatures. Besides enzymes, the protoplast isolation cocktail consisted of an osmoticum (mannitol or MgSO4), and a protectant (CaCl2 2H2O), all dissolved in distilled water. The protocol has ensured reproducibility, higher yields and is gentle on protoplasts as the protoplasts obtained were amenable to cell wall regeneration and cell division. Plant regeneration was demonstrated forNicotiana tabacum cv. Thompson from protoplasts isolated by this method. Wall regeneration and cell division were obtained in other species. The merits of the protocol are, simple and easy-to-handle procedure, non-requirement of preconditioning of donor plant and explants, incubation without agitation, satisfactory yields, culturability of the protoplasts isolated and applicability of the protocol to a large number of species including mucilage-containing plants.

Plant guard cell anion channel SLAC1 regulates stomatal closure

Plants are rooted to their growth place; therefore it is important that they react adequately to changes in environmental conditions. Stomatal pores, which are formed of a pair of guard cells in leaf epidermis, regulate plant gas-exchange. Importantly, guard cells protect the plant from desiccation in drought conditions by reducing the aperture of the stomatal pore. They serve also as the first barrier against the major air pollutant ozone, but the behaviour of guard cells during ozone exposure has not been sufficiently addressed. Aperture of the stomatal pore is regulated by the influx and efflux of osmotically active ions via ion channels and transporters across the guard cell membrane, however the molecular identity of guard cell plasma membrane anion channel has remained unknown. In the frame of this study, guard cell behaviour during ozone exposure was studied using the newly constructed Arabidopsis whole-rosette gas-exchange system. Ozone induced a Rapid Transient Decrease (RTD) in stomatal conductance within 10 min from the start of exposure, which was followed by a recovery in the conductance within the next 40 min. The decrease in stomatal conductance was dependent on the applied ozone concentration. Three minutes of ozone exposure was sufficient to induce RTD and further ozone application during the closure-recovery process had no effect on RTD, demonstrating that the whole process is programmed within the first three minutes. To address the molecular components responsible for RTD, the ozone response was measured in 59 different Arabidopsis mutants involved in guard cell signalling. Four of the tested mutants slac1 (originally rcd3), ost1, abi1-1 and abi2-1 lacked RTD completely. As the ozone sensitive mutant slac1 lacked RTD, the next aim of this study was to identify and characterize SLAC1. SLAC1 was shown to be a central regulator in response to all major factors regulating guard cell aperture: CO2, light/darkness transitions, ozone, relative air humidity, ABA, NO, H2O2, and extracellular Ca2+. It encodes the first guard cell plasma membrane slow type anion channel to be identified at the molecular level. Interestingly, the rapid type anion conductance was intact in slac1 mutant plants. For activation, SLAC1 needs to be phosphorylated. Protein kinase OST1 was shown to phosphorylate several amino acids in the N-terminal tail of SLAC1, Ser120 was one of its main targets, which led to SLAC1 activation. The lack of RTD in type 2C protein phosphatase mutants abi1-1 and abi2-1, suggests that these proteins have a regulatory role in ozoneinduced activation of the slow type anion channel.

Meadow plant growth and competition under elevated ozone and carbon dioxide

The main aim of my thesis project was to assess the impact of elevated ozone (O3) and carbon dioxide (CO2) on the growth, competition and community of meadow plants in northern Europe. The thesis project consisted of three separate O3 and CO2 exposure experiments that were conducted as open-top-chamber (OTC) studies at Jokioinen, SW Finland, and a smaller-scale experiment with different availabilities of resources in greenhouses in Helsinki. The OTC experiments included a competition experiment with two- and three-wise interactions, a mesocosm-scale meadow community with a large number of species, and a pot experiment that assessed intraspecific differences of Centaurea jacea ecotypes. The studied lowland hay meadow proved to be an O3-sensitive biotope, as the O3 concentrations used (40-50 ppb) were moderate, and yet, six out of nine species (Campanula rotundifolia, Centaurea jacea, Fragaria vesca, Ranunculus acris, Trifolium medium, Vicia cracca) showed either significant reductions in biomass or reproductive development, visible O3 injury or any two as a response to elevated O3. The plant species and ecotypes exhibited large intra- and interspecific variation in their response to O3, but O3 and CO2 concentrations did not cause changes in their interspecific competition or in community composition. However, the largest O3-induced growth reductions were seen in the least abundant species (C. rotundifolia and F. vesca), which may indicate O3-induced suppression of weak competitors. The overall effects of CO2 were relatively small and mainly restricted to individual species and several measured variables. Based on the present studies, most of the deleterious effects of tropospheric O3 are not diminished by a moderate increase in CO2 under low N availability, and variation exists between different species and variables. The present study indicates that the growth of several herb species decreases with increasing atmospheric O3 concentrations, and that these changes may pose a threat to the biodiversity of meadows. Ozone-induced reductions in the total community biomass production and N pool are likely to have important consequences for the nutrient cycling of the ecosystem.