Regulation of gene expression by SnRK1 kinases and miRNAs during the plant stress response

Dissertation presented to obtain the Ph.D degree in Plant Physiology

Acetylation of cell wall is required for structural integrity of the leaf surface and exerts a global impact on plant stress responses.

The epidermis on leaves protects plants from pathogen invasion and provides a waterproof barrier. It consists of a layer of cells that is surrounded by thick cell walls, which are partially impregnated by highly hydrophobic cuticular components. We show that the Arabidopsis T-DNA insertion mutants of REDUCED WALL ACETYLATION 2 (rwa2), previously identified as having reduced O-acetylation of both pectins and hemicelluloses, exhibit pleiotrophic phenotype on the leaf surface. The cuticle layer appeared diffused and was significantly thicker and underneath cell wall layer was interspersed with electron-dense deposits. A large number of trichomes were collapsed and surface permeability of the leaves was enhanced in rwa2 as compared to the wild type. A massive reprogramming of the transcriptome was observed in rwa2 as compared to the wild type, including a coordinated up-regulation of genes involved in responses to abiotic stress, particularly detoxification of reactive oxygen species and defense against microbial pathogens (e.g., lipid transfer proteins, peroxidases). In accordance, peroxidase activities were found to be elevated in rwa2 as compared to the wild type. These results indicate that cell wall acetylation is essential for maintaining the structural integrity of leaf epidermis, and that reduction of cell wall acetylation leads to global stress responses in Arabidopsis.

Localization of eIF4A-III in the nucleolus and splicing speckles is an indicator of plant stress

The mechanisms of long-term adaptation to low oxygen environment are quite well studied, but little is known about the sensing of oxygen shortage, the signal transduction and the short-term effects of hypoxia in plant cells. We have found that an RNA helicase eIF4A-III, a putative component of the Exon Junction Complex, rapidly changes its pattern of localisation in the plant nucleus under hypoxic conditions. In normal cell growth conditions GFP- eIF4A-III was mainly nucleoplasmic, but in hypoxia stress conditions it moved to the nucleolus and splicing speckles. This transition occurred within 15-20 min in Arabidopsis culture cells and seedling root cells, but took more than 2 h in tobacco BY-2 culture cells. Inhibition of respiration, transcription or phosphorylation in cells and ethanol treatment had similar effects to hypoxia. The most likely consequence is that a certain mRNA population will remain bound to the eIF4A-III and other mRNA processing proteins, rather than being transported from the nucleus to the cytoplasm, and thus its translation will be suspended.

Toward a hierarchical concept of plant stress

A number of attempts have been made to obtain a clear definition of biological stress. However, in spite of the efforts, some controversies on the concept of plant stress remain. The current versions are centered either on the cause (stress factor) or on the effect (stress response) of environmental stress. The objective of this study was to contribute to the definition of stress, using a hierarchical approach. Thus, we have performed an analysis of the most usual stress concepts and tested the relevance of considering different observation scales in a study on plant response to water deficit. Seedlings of Eucalyptus grandis were grown in vitro at water potentials ranging from -0.16 to -0.6 MPa, and evaluated according to growth and biochemical parameters. Data were analyzed through principal component analysis (PCA), which pointed to a hierarchical organization in plant responses to environmental disturbances. Growth parameters (height and dry weight) are more sensitive to water deficit than biochemical ones (sugars, proline, and protein), suggesting that higher hierarchical levels were more sensitive to environmental constraints than lower hierarchical ones. We suggest that before considering an environmental fluctuation as stressful, it is necessary to take into account different levels of plant response, and that the evaluation of the effects of environmental disturbances on an organism depends on the observation scale being used. Hence, a more appropriate stress concept should consider the hierarchical organization of the biological systems, not only for a more adequate theoretical approach, but also for the improvement of practical studies on plants under stress.

Family business: Multiple members of major phytohormone classes orchestrate plant stress responses

Low-molecular-weight compounds such as jasmonic, abscisic and salicylic acids are commonly thought to be regulators of plant stress responses. However, it is becoming clear that these molecules, often referred to as phytohormones, are only a part of bigger groups of compounds with biological activity. We propose that the concept of "hormone families" may help to better understand plant physiological responses by taking into account not only the alleged main regulators, but also their precursors, conjugates and catabolites. Novel approaches to profile potentially active compounds in plants are discussed.

The natural plant stress elicitor cis-jasmone causes cultivar-dependent reduction in growth of the stink bug, Euschistus heros and associated changes in flavonoid concentrations in soybean, Glycine max.

To test the hypothesis that the plant stress related elicitor cis-jasmone (cJ) provides protection in soybean pods against the seed-sucking stink bug pest, Euschistus heros, the growth of E. heros on cJ-treated pods was investigated using three soybean cultivars differing in insect susceptibility, i.e. BRS 134 (susceptible), IAC 100 (resistant) and Dowling (resistant). E. heros showed reduced weight gain when fed cJ-treated Dowling, whereas no effect on weight gain was observed when fed other treated cultivars. Using analysis of variance, a three factor (cultivar x treatment x time) interaction was observed with concentrations of the flavonoid glycosides daidzin and genistin, and their corresponding aglycones, daidzein and genistein. There were increases in genistein and genistin concentrations in cJ-treated Dowling at 144 and 120 h post treatment, respectively. Higher concentrations of malonyldaidzin and malonylgenistin in Dowling, compared to BRS 134 and IAC 100, were observed independently of time, the highest concentrations being observed in cJ-treated seeds. Levels of glycitin and malonylglycitin were higher in BRS 134 and IAC 100 compared to Dowling. Canonical variate analysis indicated daidzein (in the first two canonical variates) and genistein (in the first only) as important discriminatory variables. These results suggest that cJ treatment leads to an increase in the levels of potentially defensive isoflavonoids in immature soybean seeds, but the negative effect upon E. heros performance is cultivar-dependent.

Effects of vermicomposted spent mushroom compost on growing medium characteristics, plant growth, yield and abiotic stress tolerance

Treatment of agricultural biodegradable wastes and by-products can be carried out using composting or vermicomposting, or a combination of both treatment methods, to create a growing medium amendment suitable for horticultural use. When compared to traditional compost-maturation, vermicompost-maturation resulted in a more mature growing medium amendment i.e. lower C/N and pH, with increased nutrient content and improved plant growth response, increasing lettuce shoot fresh and dry weight by an average of 15% and 14%, respectively. Vermicomposted horse manure compost was used as a growing medium amendment for lettuce and was found to significantly increase lettuce shoot and root growth, and chlorophyll content. When used as a growing medium amendment for tomato fruit production, vermicomposted spent mushroom compost increased shoot growth and marketable yield, and reduced blossom end rot in two independent studies. Vermicompost addition to peat-based growing media increased marketable yield by an average of 21%. Vermicompost also improved tomato fruit quality parameters such as acidity and sweetness. Fruit sweetness, as measured using Brix value, was significantly increased in fruits grown with 10% or 20% vermicompost addition by 0.2 in truss one and 0.3 in truss two. Fruit acidity (% citric acid) was significantly increased in plants grown with vermicompost by an average of 0.65% in truss one and 0.68% in truss two. These changes in fruit chemical parameters resulted in a higher tomato fruit overall acceptability rating as determined by a consumer acceptance panel. When incorporated into soil, vermicomposted spent mushroom compost increased plant growth and reduced plant stress under conditions of cold stress, but not salinity or heat stress. The addition of 20% vermicompost to cold-stressed plants increased plant growth by an average of 30% and increased chlorophyll fluorescence by an average of 21%. Compared to peat-based growing medium, vermicompost had consistently higher nutrient content, pH, electrical conductivity and bulk density, and when added to a peat-based growing medium, vermicomposted spent mushroom compost altered the microbial community. Vermicompost amendment increased the microbial activity of the growing medium when incorporated initially, and this increased microbial activity was observed for up to four months after incorporation when plants were grown in it. Vermicomposting was shown to be a suitable treatment method for agricultural biodegradable wastes and by-products, with the resulting vermicompost having suitable physical, chemical and biological properties, and resulting in increased plant growth, marketable yield and yield quality, when used as an amendment in peat-based growing medium.

Evidence for a stress hypothesis: hemiparasitism effect on the colonization of Alchornea castaneaefolia A. Juss.uphorbiaceae) by Galling Insects

Stressed plants are generally more attacked by galling insects. In this study we investigated the relationship between population abundance and species richness of galling insects on the tree Alchornea castaneaefolia A. JUSS. (Euphorbiaceae), submited to stress induced by the hemiparasite Psittacanthus sp. (Loranthaceae) in the Amazon, Brazil. Branches of A. castaneaefolia attacked by the hemiparasite were more heavily infested by galling insects than non-attacked branches. The field observations partially corroborate the hypothesis that there would be an optimal level of host-plant stress for galling insect establishment.

Potato crop growth as affected by nitrogen and plant density

Growth and development variables and dry matter characteristics were studied for cultivar Snowden of potato (Solanum tuberosum L.) to evaluate nitrogen and plant density influence. Disregarding ending of season plant stress, the average number of actives haulms per plant was five and it was not affected by plant spacing. However, seasonal and final number of active haulms per plant were increased at 200 kg/ha of nitrogen. Maximum stem elongation was reached quickly with double density and had the tendency to keep constant at the highest and lowest nitrogen levels after 70 days after planting. Specific stem mass defined as mass per unit stem length was established as an indirect measure of stem thickness and load capacity. Specific leaf mass position in plant was higher at upper stem leaves, increased as plant density increased and did not vary markedly over time throughout the season. The rate of leaf appearance increased drastically due to more branching caused by high nitrogen level, and increased above ground dry matter per plant. Canopy growth and development influenced main tuber yield components. The number of active tubers per haulm decreased after 60 days after planting showing that tuberization is reversible. Tuber growth functions were established allowing the estimate of dry biomass partitioning coefficients for each plant organ.

Influence of host plant heterogeneity on the distribution of a birch aphid

1. The spatial and temporal abundance of the aphid Euceraphis betulae was investigated in relation to heterogeneity in host plant ( Betula pendula) vigour and pathogenic stress. The performance of aphids feeding on vigorous and stressed foliage was also examined. 2. The plant stress and plant vigour hypotheses have been suggested as opposing ways in which foliage quality influences herbivore abundance. In many plants, however, vigorous growing foliage co-exists with stressed or damaged foliage. 3. There was a negative correlation between branch growth ( vigour) and branch stress ( leaf chlorosis), with the most vigorous branches displaying little or no stress, and the most stressed branches achieving poor growth. There was a similar negative correlation between vigour and stress at the level of individual trees, which themselves represented a continuum in quality. 4. At the beginning of the season, E. betulae were intermittently more abundant on vigorous branches than on branches destined to become stressed, but aphids became significantly more abundant on stressed branches later in the season, when symptoms of stress became apparent. Similar patterns of aphid abundance were seen on vigorous and stressed trees in the following year. 5. Euceraphis betulae performance was generally enhanced when feeding on naturally stressed B. pendula leaves, but there was some evidence for elevated potential reproduction when feeding on vigorous leaves too. 6. Overall, plant stress probably influences E. betulae distribution more than plant vigour, but the temporal and spatial variability in plant quality suggests that plant vigour could play a role in aphid distribution early in the season.

Photoacoustics as a tool for the diagnosis of radicular stress: Measurements in eucalyptus seedlings

In reforesting companies (cellulose industry), eucalyptus is usually cultivated in small plastic containers (50 mL). As seedlings remain for about 120 days in these containers-until transplantation-their roots become space restricted, with consequent limitations in water and nutrient absorption. These restrictions may lead to plant stress, decreasing productivity. In this work, we used the photoacoustic technique to evaluate the photosynthetic activity of Eucalyptus grandis, E. urophylla and E. urograndis seedlings subjected to this limited space availability, seeking a correlation with morphological parameters and fluorescence measurements in these seedlings. Photoacoustic, fluorescence, and morphological analysis were conducted every 15 days, from 45 to 120 days after sowing. Fluorescence and photosynthetic rate were evaluated in vivo and in situ, the latter one using the open photoacoustic technique. Data show that root dry matter diminished markedly at 90 and 120 days after sowing; this behavior showed a high correlation with the gas exchange component of the photoacoustic signal, as well as with the fluorescence ratio Fv/Fm. These results indicate that the soil volume of the container becomes insufficient for the roots after 90 days, probably leading to a nutritional deficiency in plants, which explains the decrease observed in the photosynthetic rate of seedlings. (C) 2003 American Institute of Physics.

The Role of Phytochrome in Stress Tolerance

It is well-documented that phytochromes can control plant growth and development from germination to flowering. Additionally, these photoreceptors have been shown to modulate both biotic and abiotic stress. This has led to a series of studies exploring the molecular and biochemical basis by which phytochromes modulate stresses, such as salinity, drought, high light or herbivory. Evidence for a role of phytrochromes in plant stress tolerance is explored and reviewed.

Edge effect and phenology in Erythroxylum tortuosum (Erythroxylaceae), a typical plant of the Brazilian Cerrado

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Overexpression of UCP1 in tobacco induces mitochondrial biogenesis and amplifies a broad stress response

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Biochemical changes during the induction of mineral stress in plants of bell pepper cultivated in fertigation system

The greenhouse production associated with the fertigation management, have established in Brazil as economical alternative for several horticultural species. With this strategy this study had as aim to evaluate possible impacts in the metabolism of plants of bell pepper (Capsicum annuum L.; cv Elisa) in response to the increase of mineral concentration in the soil. During the experiments, the some nutrient concentrations were altered, to obtain high values of electric conductivity (EC) in the soil solution. The EC values commonly observed in the traditional fertigation system were adopted, as control. It was also verified the possibility of reduction of the mineral stress impact by the application of organic matter in the soil. Parameters of the antioxidative response system, as the superoxide dismutase (SOD) and catalase enzyme activities besides the proline content were evaluated to measure the extension of the saline stress and their effects on the plants. The increase of EC of the soil induced to the increase of the proline concentration and the SOD activity. Unexpectedly, it was verified that the saline stress inhibited the activity of the enzyme catalase. It was also concluded that the monitoring of EC of the soil is an indispensable tool to reach success in the fertigation system and that the study of the activity of the enzymes of the antioxidative response system, and the proline contents can be assumed as indicators in of the levels of stress in bell pepper plants (Capsicum annuum L.; cv Elisa).