The availability of complete genomic sequences and technologies that allow comprehensive analysis of global expression profiles of messenger RNA have greatly expanded our ability to monitor the internal state of a cell. Yet biological systems ultimately need to be explained in terms of the activity, regulation and modification of proteins--and the ubiquitous occurrence of post-transcriptional regulation makes mRNA an imperfect proxy for such information. To facilitate global protein analyses, we have created a Saccharomyces cerevisiae fusion library where each open reading frame is tagged with a high-affinity epitope and expressed from its natural chromosomal location. Through immunodetection of the common tag, we obtain a census of proteins expressed during log-phase growth and measurements of their absolute levels. We find that about 80% of the proteome is expressed during normal growth conditions, and, using additional sequence information, we systematically identify misannotated genes. The abundance of proteins ranges from fewer than 50 to more than 10(6) molecules per cell. Many of these molecules, including essential proteins and most transcription factors, are present at levels that are not readily detectable by other proteomic techniques nor predictable by mRNA levels or codon bias measurements.

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Global analysis of protein expression in yeast

This research is part of an effort that the ICT (Institute of Tropical Cultivation) has been doing for several years tending to develop superior genotypes of cocoa (Theobroma cacao L.). That is why this study aims to find tolerant or moderately tolerant cocoa genotypes and accessions to water stress with resistance to pests and diseases and high production and industrial quality. Twenty genotypes of cocoa seedlings were investigated, during the period of 6 months, in a soil with sandy-loam texture under nursery conditions, of controlled irrigation. A split plot design was used, with 40 treatments and 3 repetitions. In addition, daily data of the micro climatic characteristics (T °, HR) were taken, in which different indicators of variable were evaluated such as the stomatal conductance (CE) that is greatly influenced by the T ° and HR. The results obtained indicate that the genotypes that showed moderate tolerance to water stress were UNG - 77, UNG - 53, ICT - 1281 and ICT - 1112; the non-tolerant ones were PAS - 93, CEPEC - 2002, ICT - 2142, ICT - 1092, CP - 2005 - C10, TSH - 1188, CCN - 51, IMC - 67, PH - 17, AYP - 15, ICS - 6, BN - 34, ICT - 1506, PAS - 91, PH - 990 and ICS - 1. Under optimal conditions, the most outstanding genotype was ICS-1, both in plant height, number of leaves, and stomatal conductance, this being proof that this genotype develops excellently and stands out if it has the right conditions and water availability.

Stomatal conductance and photosynthesis

Theory is developed to explain the carbon isotopic composition of plants. It is shown how diffusion of gaseous CO2 can significantly affect carbon isotopic discrimination. The effects on discrimination by diffusion and carboxylation are integrated, yielding a simple relationship between discrimination and the ratio of the intercellular and atmospheric partial pressures of CO2. The effects of dark respiration and photorespiration are also considered, and it is suggested that they have relatively little effect on discrimination other than via their effects on intercellular p(CO2). It is also suggested that various environmental factors such as light, temperature, salinity and drought will also have effects via changes in intercellular p(CO2). A simple method is suggested for assessing water use efficiencies in the field.

On the Relationship Between Carbon Isotope Discrimination and the Intercellular Carbon Dioxide Concentration in Leaves

Na+ Tolerance and Na+ Transport in Higher Plants

Drought is a major threat to agricultural production. Plants synthesize the hormone abscisic acid (ABA) in response to drought, triggering a signalling cascade in guard cells that results in stomatal closure, thus reducing water loss. ABA triggers an increase in cytosolic calcium in guard cells ([Ca2+]cyt) that has been proposed to include Ca2+ influx across the plasma membrane. However, direct recordings of Ca2+ currents have been limited and the upstream activation mechanisms of plasma membrane Ca2+ channels remain unknown. Here we report activation of Ca2+-permeable channels in the plasma membrane of Arabidopsis guard cells by hydrogen peroxide. The H2O2-activated Ca2+ channels mediate both influx of Ca2+ in protoplasts and increases in [Ca2+]cyt in intact guard cells. ABA induces the production of H2O2 in guard cells. If H2O2 production is blocked, ABA-induced closure of stomata is inhibited. Moreover, activation of Ca2+ channels by H2O2 and ABA- and H2O2-induced stomatal closing are disrupted in the recessive ABA-insensitive mutant gca2. These data indicate that ABA-induced H2O2 production and the H2O2-activated Ca2+ channels are important mechanisms for ABA-induced stomatal closing.

Calcium channels activated by hydrogen peroxide mediate abscisic acid signalling in guard cells

Using the patch-clamp technique we discovered that the voltage dependent anion channels in the plasma membrane of guard cells are activated by a rise in cytoplasmic Ca2+ in the presence of nucleotides. Upon activation, these anion channels catalyse anion currents 10-20 times higher than in the inactivated state, thus shifting the plasma membrane from a K+ conducting state to an anion conducting state. Prolonged stimulation by depolarizing voltages results in the inactivation of the anion current (t1/2 = 10-12 s). We suggest that activation of the anion channel by Ca2+ and nucleotides is a key event in the regulation of salt efflux from guard cells during stomatal closure.

Ca2+ and nucleotide dependent regulation of voltage dependent anion channels in the plasma membrane of guard cells.

When stomata of Vicia faba opened (from a stomatal aperture of about 2 micrometers to one of 12 micrometers) the solute content of the guard cells increased by 4.8 x 10(-12) osmoles per stoma. During the same time an average of 4.0 x 10(-12) gram equivalents of K(+) were transported into each pair of guard cells. This amount of K(+), if associated with dibasic anions, is sufficient to produce the changes in guard cell volume and osmotic pressure associated with stomatal opening. Analysis of Cl, P, and S showed that these elements were not transported in significant amounts during stomatal opening. This finding suggests that the anions balancing K(+) were predominantly organic. K(+) was specifically required because no other elements, likely to be present as cations, were found to accumulate in appreciable quantities in guard cells of open stomata.

Stomatal opening quantitatively related to potassium transport: evidence from electron probe analysis.

Mature leaves of Phaseolus vulgaris L (red kidney bean), Xanthium strumarium L (cocklebur), and Gossypium hirsutum L (cotton) were used to study accumulation of abscisic acid (ABA) during water stress The water status of individual, detached leaves was monitored while the leaves slowly wilted, and samples were cut from the leaves as they lost water The leaf sections were incubated at their respecitive water contents to allow ABA to build up or not At least 8 h were required for a new steady-state level of ABA to be established The samples from any one leaf covered a range of known water potentials (ψ), osmotic pressures (π), and turgor pressures (p) The π and p values were calculated from “pressure-volume curves”, using a pressure bomb to measure the water potentials Decreasing water potential had little effect on ABA levels in leaves at high turgor Sensitivity of the production of ABA to changes in ψ progressively increased as turgor approached zero At p=1 bar, ABA content averaged 4 times the level found in fully turgid samples Below p=1 bar, ABA content increased sharply to as much as 40 times the level found in unstressed samples ABA levels rose steeply at different water potentials for different leaves, according to the ψ at which turgor became zero These differences were caused by the different osmotic pressures of the leaves that were used; ψ must cqual -π for turgor to be zero Leaves vary in π, not only among species, but also between plants of one and the same species depending on the growing conditions A difference of 6 bars (calculated at ψ=0) was found between the osmotic pressures of leaves from two groups of G hirsutum plants; one group had previously experienced periodic water stress, and the other group had never been stressed When individual leaves were subsequently wilted, the leaves from stress-conditioned plants required a lower water potential in order to accumulate ABA than did leaves from previously unstressed plants On the basis of these results we suggest that turgor is the critical parameter of plant water relations which controls ABA production in water-stressed leaves

Correlation between loss of turgor and accumulation of abscisic acid in detached leaves.

The occurrence of O2-insensitive photosynthesis at high quantum flux and moderate temperature in Spinacia oleracea was characterized by analytical gas exchange measurements on intact leaves In addition photosynthetic metabolite pools were measured in leaves which had been rapidly frozen under defined gas conditions Upon switching to low O2 in O2-insensitive conditions the ATP/ADP ratio fell dramatically within one minute The P-glycerate pool increased over the same time Ribulose bisphosphate initially declined, then increased and exceeded the pool size measured in air The pools of hexose monophosphates and UDPglucose were higher at a partial pressure of O2 of 21 millibars than at 210 millibars These results are consistent with the hypothesis that the rate of sucrose synthesis limited the overall rate of assimilation under O2-insensitive conditions

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Limitation of Photosynthesis by Carbon Metabolism: II. O2-Insensitive CO2 Uptake Results from Limitation Of Triose Phosphate Utilization

Flux response curves were determined at 16 wavelengths of light for the conductance for water vapor of the lower epidermis of detached leaves of Xanthium strumarium L An action spectrum of stomatal opening resulted in which blue light (wavelengths between 430 and 460 nanometers) was nearly ten times more effective than red light (wavelengths between 630 and 680 nanometers) in producing a conductance of 15 centimoles per square meter per second Stomata responded only slightly to green light An action spectrum of stomatal responses to red light corresponded to that of CO2 assimilation; the inhibitors of photosynthetic electron transport, cyanazine (2-chloro-4[1-cyano-1-methylethylamino]-6-ethylamino-s-triazine) and 3-(3,4-dichlorophenyl)-1,1-dimethylurea, eliminated the response to red light This indicates that light absorption by chlorophyll is the cause of stomatal sensitivity to red light Determination of flux response curves on leaves in the normal position (upper epidermis facing the light) or in the inverted position (lower epidermis facing the light) led to the conclusion that the photoreceptors for blue as well as for red light are located on or near the surfaces of the leaves; presumably they are in the guard cells themselves

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Klaus Raschke

Papers

Ca2+ and nucleotide dependent regulation of voltage dependent anion channels in the plasma membrane of guard cells.

Stomatal opening quantitatively related to potassium transport: evidence from electron probe analysis.

Correlation between loss of turgor and accumulation of abscisic acid in detached leaves.

Limitation of Photosynthesis by Carbon Metabolism: II. O2-Insensitive CO2 Uptake Results from Limitation Of Triose Phosphate Utilization

Effect of Light Quality on Stomatal Opening in Leaves of Xanthium strumarium L.