Tessa Pocock

Cold-regulated cereal chloroplast late embryogenesis abundant-like proteins. Molecular characterization and functional analyses.

Cold acclimation and freezing tolerance are the result of complex interaction between low temperature, light, and photosystem II (PSII) excitation pressure. Previous results have shown that expression of the Wcs19 gene is correlated with PSII excitation pressure measured in vivo as the relative reduction state of PSII. Using cDNA library screening and data mining, we have identified three different groups of proteins, late embryogenesis abundant (LEA) 3-L1, LEA3-L2, and LEA3-L3, sharing identities with WCS19. These groups represent a new class of proteins in cereals related to group 3 LEA proteins. They share important characteristics such as a sorting signal that is predicted to target them to either the chloroplast or mitochondria and a C-terminal sequence that may be involved in oligomerization. The results of subcellular fractionation, immunolocalization by electron microscopy and the analyses of target sequences within the Wcs19 gene are consistent with the localization of WCS19 within the chloroplast stroma of wheat (Triticum aestivum) and rye (Secale cereale). Western analysis showed that the accumulation of chloroplastic LEA3-L2 proteins is correlated with the capacity of different wheat and rye cultivars to develop freezing tolerance. Arabidopsis was transformed with the Wcs19 gene and the transgenic plants showed a significant increase in their freezing tolerance. This increase was only evident in cold-acclimated plants. The putative function of this protein in the enhancement of freezing tolerance is discussed.

IDENTIFICATION OF A PSYCHROPHILIC GREEN ALGA FROM LAKE BONNEY ANTARCTICA: CHLAMYDOMONAS RAUDENSIS ETTL. (UWO 241) CHLOROPHYCEAE1

An unusual psychrophilic green alga was isolated from the deepest portion of the photic zone (<0.1% of incident PAR) at a depth of 17 m in the permanently ice‐covered lake, Lake Bonney, Antarctica. Here we identify and report the first detailed morphological and molecular examination of this Antarctic green alga, which we refer to as strain UWO 241. To determine the taxonomic identity, UWO 241 was examined using LM and TEM and partial sequences of the small subunit (SSU), internal transcribed spacer (ITS) 1 and ITS2 regions (including the 5.8S) of the ribosomal operon. These data were compared with those of previously described taxa. We identified UWO 241 as a strain of Chlamydomonas raudensis Ettl (SAG 49.72). Chlamydomonas raudensis is closely related to C. noctigama Korshikov (UTEX 2289) as well as foraminifer symbionts such as C. hedleyi Lee, Crockett, Hagen et Stone (ATCC 50216). In addition, its morphology, pigment complement, and phototactic response to temperature are reported. Chlamydomonas raudensis (UWO 241) contains relatively high levels of lutein and low chl a/b ratios (1.6±0.15), and the phototactic response was temperature dependent. The Antarctic isolate (UWO 241) included the typical photosynthetic pigments found in all chl a/b containing green algae. It possesses a small eyespot and, interestingly, was positively phototactic only at higher nonpermissive growth temperatures. Comparison of SSU and ITS rDNA sequences confirms the identification of the strain UWO 241 as C. raudensis Ettl and contradicts the previous designation as C. subcaudata Wille.

The Antarctic psychrophile, Chlamydomonas raudensis Ettl (UWO241) (Chlorophyceae, Chlorophyta), exhibits a limited capacity to photoacclimate to red light

The psychrophilic Antarctic alga, Chlamydomonas raudensis Ettl (UWO241), grows under an extreme environment of low temperature and low irradiance of a limited spectral quality (blue‐green). We investigated the ability of C. raudensis to acclimate to long‐term imbalances in excitation caused by light quality through adjustments in photosystem stoichiometry. Log‐phase cultures of C. raudensis and C. reinhardtii grown under white light were shifted to either blue or red light for 12 h. Previously, we reported that C. raudensis lacks the ability to redistribute light energy via the short‐term mechanism of state transitions. However, similar to the model of mesophilic alga, C. reinhardtii, the psychrophile retained the capacity for long‐term adjustment in energy distribution between PSI and PSII by modulating the levels of PSI reaction center polypeptides, PsaA/PsaB, with minimal changes in the content of the PSII polypeptide, D1, in response to changes in light quality. The functional consequences of the modulation in PSI/PSII stoichiometry in the psychrophile were distinct from those observed in C. reinhardtii. Exposure of C. raudensis to red light caused 1) an inhibition of growth and photosynthetic rates, 2) an increased reduction state of the intersystem plastoquinone pool with concomitant increases in nonphotochemical quenching, 3) an uncoupling of the major light‐harvesting complex from the PSII core, and 4) differential thylakoid protein phosphorylation profiles compared with C. reinhardtii. We conclude that the characteristic low levels of PSI relative to PSII set the limit in the capacity of C. raudensis to photoacclimate to an environment enriched in red light.

The Determination and Quantification of Photosynthetic Pigments by Reverse Phase High-Performance Liquid Chromatography, Thin-Layer Chromatography, and Spectrophotometry

Chorophylls and carotenoids are functionally important pigment molecules in photosynthetic organisms. Methods for the determination of chlorophylls a and b, beta-carotene, neoxanthin, and the pigments that are involved in photoprotective cycles such as the xanthophylls are discussed. These cycles involve the reversible de-epoxidation of violaxanthin into antheraxanthin and zeaxanthin, as well as the reversible de-epoxidation of lutein-5,6-epoxide into lutein. This chapter describes pigment extraction procedures from higher plants and green algae. Methods for the determination and quantification using high-performance liquid chromatograpy (HPLC) are described as well as methods for the separation and purification of pigments for use as standards using thin-layer chromatography (TLC). In addition, several spectrophotometric methods for the quantification of chlorophylls a and b are described.

Photoprotection of Photosystem II: Reaction Center Quenching Versus Antenna Quenching

Understanding the role of the xanthophyll cycle and elucidating the mechanisms of antenna quenching through the non-photochemical dissipation of excess absorbed energy in the photoprotection of the photochemical apparatus continues to be a major focus of photosynthetic research. In addition to antenna quenching, there is evidence for the non-photochemical dissipation of excess energy through the PS II reaction center. Hence, this photoprotective mechanism is called reaction center quenching. One technique to assess reaction center quenching is photosynthetic thermoluminescence. This technique represents a simple but powerful probe of PS II photochemistry that measures the light emitted due to the reversal of PS II charge separation through the thermally-dependent recombination of the negative charges stabilized on Q− A and Q− B on the acceptor side of PS II with the positive charges accumulated in the S2- and S3-states of the oxygen evolving complex. Changes in the temperature maxima for photosynthetic thermoluminescence may reflect changes in redox potentials of recombining species within PS II reaction centers. Exposure of Synechococcussp. PCC 7942, Pinus sylvestrisL., Arabidopsis thaliana, and Chlamydomonas reinhardtii to either lowtemperatures or to high light induces a significant downshift in the temperature maxima for S2Q− B and S3Q− B recombinations relative to S2Q− A and S3Q− A recombinations. These shifts in recombination temperatures are indicative of lower activation energy for the S2Q− B redox pair recombination and a narrowing of the free energy gap betweenQAandQB electron acceptors. This, in turn, is associated with a decrease in the overall thermoluminescence emission. We propose that environmental factors such as high light and low temperature result in an increased population of reduced QA (Q− A), that is, increased excitation pressure, facilitating non-radiative P680+Q− A radical pair recombination within the PS II reaction center. The underlying molecular mechanisms regulating reaction center quenching appear to be species dependent. We conclude that reaction center quenching and antenna quenching are complementary mechanisms that may function to photoprotect PS II to different extents in vivo depending on the species as well as the environmental conditions to which the organism is exposed.

Effects of growth under UVA radiation on CO2 assimilation, carbon partitioning, PSII photochemistry and resistance to UVB radiation in Brassica napus cv. Topas

The effects of growth of Brassica napus L. cv. Topas under PAR or PAR+UVA radiation was assessed with respect to sensitivity to subsequent exposure to UVB radiation. Despite the fact that growth under PAR+UVA induced minimal effects in photosystem II (PSII) photochemistry, growth under PAR+UVA inhibited the accumulation of the photosynthetic end products, sucrose and starch. This was associated with a decreased capacity for ribulose 1,5-bisphosphate (RuBP) regeneration, a decreased capacity for light- and CO 2 -saturated rates of CO2 assimilation, a decrease in the apparent quantum yield for CO2 assimilation, an over-reduction of chloroplast stroma, an increased susceptibility to the feedback effects on photosynthesis and a stimulation of glycolysis compared to controls grown under PAR. Subsequent exposure to UVB decreased the maximum Rubisco activity in leaves of both PAR- and PAR+UVA-grown plants. However, the decrease in the capacity for CO2 assimilation in PAR-grown plants exposed to UVB did not appear to be associated with limitations at the level of PSII linear electron transport, but rather with a decreased capacity for sucrose biosynthesis, limited triose-P utilization and a decreased capacity for RuBP regeneration. In contrast, the decreased capacity for CO 2 assimilation in PAR+UVA-grown plants exposed to UVB was associated with an inhibition of PSII photochemistry and a decreased supply of ATP. Thus, growth under UVA radiation appears to induce feedback-limited photosynthesis and does not enhance resistance of Brassica napus to UVB radiation.

Diphenyl carbazide restores electron transport in isolated, illuminated chloroplasts after electron transport from water has been eliminated by mild heat treatment

Freshly isolated, illuminated chloroplasts oxidize water and transfer the resulting electrons through the photosynthetic electron transport chains in their thylakoid membranes to the artificial electron acceptor, dichlorophenol indophenol (DCPIP). As a consequence, DCPIP is reduced and the decline in absorbance over time can be used to measure the rate of electron transfer. When gently heated, chloroplasts lose the capacity to oxidize water and the transfer of electrons to DCPIP is eliminated. Electron transport through chloroplasts to DCPIP is restored in the presence of the artificial electron donor diphenylcarbazide (DPC). If students gain experience with the DCPIP photoreduction assay and are given information on normal chloroplast function, they should be able to predict the behavior of heat‐treated chloroplasts in a variety of experimental conditions. A number of such predictions are outlined and tested. The experiments can all be conducted with a limited repertoire of equipment and easily prepared solutions. Consequently, this work is well suited to an investigative study in which each student group, in consultation with instructors, can make and test its own prediction. The ways in which changing different variables can affect the quality of the experimental results is emphasized. Additional studies, on measurements of rates of oxygen evolution and emitted chlorophyll fluorescence, are briefly described to support the inferences that heat‐treated chloroplasts do not oxidize water and that the vectorial transfer of electrons through them to DCPIP is identical to that in untreated chloroplasts.