Freezing tolerance in alpine plants as assessed by the FDA-staining method

We established a method employing fluorescein diacetate (FDA)-staining with a laser scan microscope for evaluation of the freezing tolerance of leaf mesophyll cells, and applied it to assessments of freezing tolerance in leaves of nine alpine plants, Arcterica nana, Cassiope lycopodioides, Diapensia lapponica, Empetrum nigrum, Loiseleuria procumbens, Phyllodoce nipponica, Rhododendron aureum, Schizocodon soldanelloides, and Vaccinium vitis-idaea, which naturally occur on Mt. Iwo (36°00′N, 138°22′W, 2760 m a.s.l.) in 2001. The results obtained with the FDA-staining method were strongly correlated with those of the electrolyte-leakage test, suggesting that this method is highly reliable. Leaf mesophyll cells in all these plants collected in early September survived after freezing at -5°C, but did not survive below -30°C. The survival ratio at subzero temperatures, ranging from -5 to -16°C, varied among the species. On the other hand, all the mesophyll cells of the plants collected in the middle of November survived freezing at -30°C. However, the survival ratio decreased after freezing at -80°C in Phyllodoce nipponica and Empetrum nigrum. Using the FDA-staining method, we were able to confirm the increase of the freezing tolerance of alpine plants from September to November. Advantages of the new method are discussed

A new application of the SFDA-staining method to assessment of the freezing tolerance in leaves of alpine plants

For the first time, this study used 5- (6-) sulfofluorescein diacetate (SFDA), a fluorescent product in plant cells converted by esterase activity to fluorescein-5- (and 6-) sulfonic acid (FSA), to assess the freezing tolerance of leaf cells. We were able to readily distinguish living and dead cells, and detect differences in freezing tolerance among five alpine plants using the SFDA-staining method. We also compared this method with two conventional methods, the electrolyte leakage test and fluorescein diacetate (FDA) staining method. The electrolyte leakage test often over- or underestimated freezing injury. With the uninjured control samples, the FDA-staining method failed to stain all leaf cells, while the SFDA-staining method stained almost 100%. From these results, we concluded that SFDA-staining is a more convenient, accurate and reproducible method for analyses of freezing tolerance

Thermal acclimation of photosynthesis: on the importance of adjusting our definitions and accounting for thermal acclimation of respiration

Abstract While interest in photosynthetic thermal acclimation has been stimulated by climate warming, comparing results across studies requires consistent terminology. We identify five types of photosynthetic adjustments in warming experiments: photosynthesis as measured at the high growth temperature, the growth temperature, and the thermal optimum; the photosynthetic thermal optimum; and leaf-level photosynthetic capacity. Adjustments of any one of these variables need not mean a concurrent adjustment in others, which may resolve apparently contradictory results in papers using different indicators of photosynthetic acclimation. We argue that photosynthetic thermal acclimation (i.e., that benefits a plant in its new growth environment) should include adjustments of both the photosynthetic thermal optimum (T opt ) and photosynthetic rates at the growth temperature (A growth ), a combination termed constructive adjustment. However, many species show reduced photosynthesis when grown at elevated temperatures, despite adjustment of some photosynthetic variables, a phenomenon we term detractive adjustment. An analysis of 70 studies on 103 species shows that adjustment of T opt and A growth are more common than adjustment of other photosynthetic variables, but only half of the data demonstrate constructive adjustment. No systematic differences in these patterns were found between different plant functional groups. We also discuss the importance of thermal acclimation of respiration for net photosynthesis measurements, as respiratory temperature acclimation can generate apparent acclimation of photosynthetic processes, even if photosynthesis is unaltered. We show that while dark respiration is often used to estimate light respiration, the ratio of light to dark respiration shifts in a non-predictable manner with a change in leaf temperature

Growth and Accumulation of Secondary Metabolites in Perilla as Affected by Photosynthetic Photon Flux Density and Electrical Conductivity of the Nutrient Solution

The global demand for medicinal plants is increasing. The quality of plants grown outdoors, however, is difficult to control. Myriad environmental factors influence plant growth and directly impact biosynthetic pathways, thus affecting the secondary metabolism of bioactive compounds. Plant factories use artificial lighting to increase the quality of medicinal plants and stabilize production. Photosynthetic photon flux density (PPFD) and electrical conductivity (EC) of nutrient solutions are two important factors that substantially influence perilla (Perilla frutescens, Labiatae) plant growth and quality. To identify suitable levels of PPFD and EC for perilla plants grown in a plant factory, the growth, photosynthesis, and accumulation of secondary metabolites in red and green perilla plants were measured at PPFD values of 100, 200, and 300 μmol m-2 s-1 in nutrient solutions with EC values of 1.0, 2.0, and 3.0 dS m-1. The results showed significant interactive effects between PPFD and EC for both the fresh and dry weights of green perilla, but not for red perilla. The fresh and dry weights of shoots and leafy areas were affected more by EC than by PPFD in green perilla, whereas they were affected more by PPFD than by EC in red perilla. Leaf net photosynthetic rates were increased as PPFD increased in both perilla varieties, regardless of EC. The perillaldehyde concentration (mg g-1) in red perilla was unaffected by the treatments, but accumulation in plants (mg per plant) was significantly enhanced as the weight of dry leaves increased. Perillaldehyde concentrations in green perilla showed significant differences between combinations of the highest PPFD with the highest EC and the lowest PPFD with the lowest EC. Rosmarinic acid concentration (mg g-1) was increased in a combination of low EC and high PPFD conditions. Optimal cultivation conditions of red and green perilla in plant factory will be discussed in terms of plant growth and contents of medicinal ingredients

Antisense reductions in the PsbO protein of photosystem II leads to decreased quantum yield but similar maximal photosynthetic rates

Photosystem (PS) II is the multisubunit complex which uses light energy to split water, providing the reducing equivalents needed for photosynthesis. The complex is susceptible to damage from environmental stresses such as excess excitation energy and high temperature. This research investigated the in vivo photosynthetic consequences of impairments to PSII in Arabidopsis thaliana (ecotype Columbia) expressing an antisense construct to the PsbO proteins of PSII. Transgenic lines were obtained with between 25 and 60% of wild-type (WT) total PsbO protein content, with the PsbO1 isoform being more strongly reduced than PsbO2. These changes coincided with a decrease in functional PSII content. Low PsbO (less than 50% WT) plants grew more slowly and had lower chlorophyll content per leaf area. There was no change in content per unit area of cytochrome b6f, ATP synthase, or Rubisco, whereas PSI decreased in proportion to the reduction in chlorophyll content. The irradiance response of photosynthetic oxygen evolution showed that low PsbO plants had a reduced quantum yield, but matched the oxygen evolution rates of WT plants at saturating irradiance. It is suggested that these plants had a smaller pool of PSII centres, which are inefficiently connected to antenna pigments resulting in reduced photochemical efficiency.This work was supported by an Australian Postgraduate Award to SAD, the Australian Research Council Centre of Excellence in Plant Energy Biology (MRB), and grants from the Australian Research Council (WSC)

Overexpression of BUNDLE SHEATH DEFECTIVE 2 improves the efficiency of photosynthesis and growth in Arabidopsis

Bundle Sheath Defective 2, BSD2, is a stroma-targeted protein initially identified as a factor required for the biogenesis of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) in maize. Plants and algae universally have a homologous gene for BSD2 and its deficiency causes a RuBisCO-less phenotype. As RuBisCO can be the rate-limiting step in CO2 assimilation, the overexpression of BSD2 might improve photosynthesis and productivity through the accumulation of RuBisCO. To examine this hypothesis, we produced BSD2 overexpression lines in Arabidopsis. Compared with wild type, the BSD2 overexpression lines BSD2ox-2 and BSD2ox-3 expressed 4.8-fold and 8.8-fold higher BSD2 mRNA, respectively, whereas the empty-vector (EV) harbouring plants had a comparable expression level. The overexpression lines showed a significantly higher CO2 assimilation rate per available CO2 and productivity than EV plants. The maximum carboxylation rate per total catalytic site was accelerated in the overexpression lines, while the number of total catalytic sites and RuBisCO content were unaffected. We then isolated recombinant BSD2 (rBSD2) from E. coli and found that rBSD2 reduces disulfide bonds using reductants present in vivo, for example glutathione, and that rBSD2 has the ability to reactivate RuBisCO that has been inactivated by oxidants. Furthermore, 15% of RuBisCO freshly isolated from leaves of EV was oxidatively inactivated, as compared with 0% in BSD2-overexpression lines, suggesting that the overexpression of BSD2 maintains RuBisCO to be in the reduced active form in vivo. Our results demonstrated that the overexpression of BSD2 improves photosynthetic efficiency in Arabidopsis and we conclude that it is involved in mediating RuBisCO activation.This work was supported in part by a JSPS KAKENHIGrant Number 26450081 (HS), 16H06552 (WY), A-STEP from theJapan Science and Technology Agency (HS), the Ministry of Edu-cation, Culture, Sports, Science and Technology (MEXT) as part ofJoint Research Program implemented at the Institute of PlantScience and Resources, Okayama University in Japan (HS), grantsfrom the Advanced Low Carbon Technology Research and Devel-opment Program from the Japan Science and Technology Agency(ST, TK, and HS), and the Join Usage/Research Center, Institute ofPlant Science and Resources, Okayama University (HS), and theJapan Society for the Promotion of Science under the Japan-UKResearch Cooperative Program from the Ministry of Education, Culture, Sports, Science and Technology of Japan (TK). JT is sup-ported by Research Fellowships for Young Scientists from JSPS,and FAB is supported by the Australian Government through theAustralian Research Council Centre of Excellence for TranslationalPhotosynthesis (CE1401000015)

Effect of Rubisco Activase Deficiency on the Temperature Response of CO2 Assimilation Rate and Rubisco Activation State: Insights from Transgenic Tobacco with Reduced Amounts of Rubisco Activase1[W][OA]

The activation of Rubisco in vivo requires the presence of the regulatory protein Rubisco activase. To elucidate its role in maintaining CO2 assimilation rate at high temperature, we examined the temperature response of CO2 assimilation rate at 380 μL L−1 CO2 concentration (A380) and Rubisco activation state in wild-type and transgenic tobacco (Nicotiana tabacum) with reduced Rubisco activase content grown at either 20°C or 30°C. Analyses of gas exchange and chlorophyll fluorescence showed that in the wild type, A380 was limited by ribulose 1,5-bisphosphate regeneration at lower temperatures, whereas at higher temperatures, A380 was limited by ribulose 1,5-bisphosphate carboxylation irrespective of growth temperatures. Growth temperature induced modest differences in Rubisco activation state that declined with measuring temperature, from mean values of 76% at 15°C to 63% at 40°C in wild-type plants. At measuring temperatures of 25°C and below, an 80% reduction in Rubisco activase content was required before Rubisco activation state was decreased. Above 35°C, Rubisco activation state decreased slightly with more modest decreases in Rubisco activase content, but the extent of the reductions in Rubisco activation state were small, such that a 55% reduction in Rubisco activase content did not alter the temperature sensitivity of Rubisco activation and had no effect on in vivo catalytic turnover rates of Rubisco. There was a strong correlation between Rubisco activase content and Rubisco activation state once Rubisco activase content was less that 20% of wild type at all measuring temperatures. We conclude that reduction in Rubisco activase content does not lead to an increase in the temperature sensitivity of Rubisco activation state in tobacco

Quantification of Rubisco activase content in leaf extracts

Rubisco activase functions to promote and maintain the catalytic activity of Rubisco. Studies with the activase-lacking Arabidopsis rca mutant (Salvucci et al. Photosynth Res 7:193-201, 1985; Salvucci et al. Plant Physiol 80:655-659, 1986), antisense activase tobacco, Arabidopsis and Flaveria bidentis plants (Mate et al. Plant Physiol 102:1119-1128, 1993; Eckardt et al. Plant Physiol 113:575-586, 1997; von Caemmerer et al. Plant Physiol 137:747-755, 2005) have shown that photosynthesis at atmospheric levels of CO2 is severely impaired when plants lack activase because Rubisco becomes sequestered in an inactive form. Activase protein has been detected in all plant species, including C3 and C4 plants and green algae (Salvucci et al. Plant Physiol 84:930-936, 1987). Rubisco activase is essential in all these photosynthetic organisms for photosynthesis and plant growth. The physiological importance of Rubisco activase is reinforced by recent studies indicating that it plays a role in the response of photosynthesis to temperature. In this chapter, we describe how to extract and quantify Rubisco activase content in leaf