Zhi-Fang Lin

Enhanced sensitivity of Arabidopsis anthocyanin mutants to photooxidation: a study with fluorescence imaging

Chlorophyll fluorescence imaging and antioxidative capability in detached leaves of the wild-type Arabidopsis thaliana ecotype Landsberg erecta (Ler) and in three mutants deficient in anthocyanin biosynthesis (tt3, tt4 and tt3tt4) were investigated under photooxidation stress induced by methyl viologen (5 μm) in the light. In comparison with the wild-type (WT) plant, photooxidation resulted in significant decreases in the contents of total phenolics and flavonoid, total antioxidative capability and chlorophyll fluorescence parameters (Fv/Fm, qP, ΦPSII, NPQ and ETR) as determined by chlorophyll fluorescence imaging, and in an increase in cell-membrane leakiness in the three anthocyanin mutants. The sequence of sensitivity to photooxidation in the leaves of the four phenotypes were tt3tt4 (deficient in both chalcone synthase locus (CHS) and dihydroflavonol 4-reductase locus (DFR)) > tt4 (deficient in CHS) > tt3 (deficient in DFR) > WT. The results demonstrate that anthocyanins might, along with other antioxidants, protect the photosynthetic apparatus against photooxidative damage. An interesting phenomenon was observed over the 270 min of the photooxidative treatment, that is, fluorescence imaging revealed that qP, ΦPSII and ETR appeared in three phases (fall → partial recovery → rapid fall). This was considered to be a modulation of reversible deactivation in PSII to cope with the moderate oxidative stress in the first two stages of short-term treatment (<150 min), followed finally by damage to PSII under severe oxidative stress with prolonged treatment.

Photosynthetic Characteristics of Two New Chlorophyll b-Less Rice Mutants

Two yellow rice mutants VG28-1 and VG30-5 were obtained during the tissue culture process from a rice plant (cv. Zhonghua No.11 japonica) with inserted maize Ds transposon element. Absorption spectra and pigment composition showed that two mutants had no chlorophyll (Chl) b and lower Chl a content in comparison to the wild type (WT). Net photosynthetic rate (PN), total electron transport rate (JF), photochemical quenching (qp), quantum yield of PS2 dependent non-cyclic electron transport (ΦPS2), fraction of Prate, and leaf area were lower but Fv/Fm and apparent quantum yield (AQY) remained at similar levels as in the WT plant. Xanthophyll cycle pool size (V+A+Z) on a Chl basis, and de-epoxidation state were enhanced in the mutants. The mutants had larger amounts of soluble protein and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO), especially the small subunit of RuBPCO, than WT. The characteristics of two rice mutants differed somewhat from the other common Chl b-less mutants originating from mutagenic agent treatments.

The antioxidative function of lutein: electron spin resonance studies and chemical detection

In the present study, both electron spin resonance (ESR) and chemical detection confirmed that lutein [extracted from alfalfa (Medicago sativa L.)], the most abundant xanthophyll in thylakoids of chloroplasts, could serve as an antioxidant to scavenge reactive oxygen species (ROS) in vitro. Lutein exhibited a greater capacity for scavenging hydroxyl (OH·) and superoxide (O2·-) radicals than β-carotene at the same concentration, whereas the opposite trend was observed in the capacity for scavenging singlet oxygen (1O2). The capacity of lutein for scavenging ROS from high to low is OH· > O2·- > 1O2. We hypothesise that lutein plays an important photoprotective role in scavenging O2·- and OH· under severe stress. This hypothesis is consistent with our previous report that the lut2 (lutein-deficient) Arabidopsis mutant is more susceptible to damage than the npq1 (lutein-replete but violaxanthin de-epoxidase-deficient) Arabidopsis mutant under severe stress during exposure to high light intensity at low temperature (Peng and Gilmore 2003).

Antioxidant enzymes regulate reactive oxygen species during pod elongation in Pisum sativum and Brassica chinensis.

Previous research has focused on the involvement of reactive oxygen species (ROS) in cell wall loosening and cell extension in plant vegetative growth, but few studies have investigated ROS functions specifically in plant reproductive organs. In this study, ROS levels and antioxidant enzyme activities were assessed in Pisum sativum and Brassica chinensis pods at five developmental stages. In juvenile pods, the high levels of O2 .− and.OH indicates that they had functions in cell wall loosening and cell elongation. In later developmental stages, high levels of.OH were also related to increases in cell wall thickness in lignified tissues. Throughout pod development, most of the O2 .− was detected on plasma membranes of parenchyma cells and outer epidermis cells of the mesocarp, while most of the H2O2 was detected on plasma membranes of most cells throughout the mesocarp. This suggests that these sites are presumably the locations of ROS generation. The antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) apparently contributed to ROS accumulation in pod wall tissues. Furthermore, specifically SOD and POD were found to be associated with pod growth through the regulation of ROS generation and transformation. Throughout pod development, O2 .− decreases were associated with increased SOD activity, while changes in H2O2 accumulation were associated with changes in CAT and POD activities. Additionally, high POD activity may contribute to the generation of.OH in the early development of pods. It is concluded that the ROS are produced in different sites of plasma membranes with the regulation of antioxidant enzymes, and that substantial ROS generation and accumulation are evident in cell elongation and cell wall loosening in pod wall cells.

In Situ Localisation of Superoxide Generated in Leaves of Alocasia macrorrhiza (L.) Shott under Various Stresses

Leaf discs of Alocasia macrorrhiza were treated with various stress factors, including two photo-oxidants, methyl viologen (MV) or riboflavin (RB); three pollutants, sodium bisulphite (NaHSO3), or the heavy metals lead or cadmium; or an osmotic medium, polyethylene glycol 6000. The in situ localisation sites for O2− generation were identified using specific dye nitro blue tetrazolium as a probe. The level of superoxide production was determined by scanning the blue-stained formazan area and was defined as the percentage of pixels from the stained portion versus the total number of pixels in the entire leaf disc area. All stress factors induced the generation of O2− in a time- or concentration-dependent pattern. Although superoxide production also was enhanced by longer time periods in untreated discs (control), the degree to which this occurred was less than that measured in leaves treated with either MV or RB. Generation sites were primarily found in the chloroplasts of stomatal guard cells and in the plasma membrane of the epidermis and mesophyll cells, indicating that they were most responsive to stress conditions. Nevertheless, the site of O2− generation varied among these stress factors.

Lead and cadmium induced alterations of cellular functions in leaves of Alocasia macrorrhiza L. Schott

Alocasia macrorrhiza is a fast growing and propagating herbaceous species commonly found in South China. To determine its physiological responses to Pb and Cd stresses, the biochemical, histochemical and cytochemical changes under PbAC2 and CdCl2 phytotoxicity were detected using leaf discs as an experimental model. After leaf discs were infiltrated in different concentrations of PbAC2 and CdCl2 solutions (0, 50, 100, 150, 200 microM) for 72 h, the formation of reactive oxygen species (H2O2 and O2-) in plant tissue were found to be exaggerated together with elevated OH concentration and cell death. Changes in chlorophyll fluorescence (Fv/Fm, PhiPSII, qP and NPQ) imaging colours/areas of leaf discs indicated decreased photosystem II functions by both heavy metal treatments and positive reactions of antioxidants under Pb2+ stress. Results showed that fluorescent detection of hydroxylated terephthlate using terephthalic acid as OH trap is a simple, yet valuable and specific method for monitoring OH generation in plant tissue under heavy metal stresses. As compared with Cd2+, Pb2+ was found to be less toxic, indicating that A. macrorrhiza tissue might have a potential tolerance to Pb.

Characteristics of sun- and shade-adapted populations of an endangered plant Primulina tabacum Hance

Primulina tabacum Hance is an endangered perennial herb distributed in calcium-rich and nitrogen-limited soil of the karst limestone areas in southern China. The morphological, ultrastructural, and physiological traits were determined for P. tabacum populations growing in three different environment conditions: twilight zone of a cave (site TZ, extremely low light intensity), at a cave entrance (site EZ, low light intensity), and in an open area (site OA, high light intensity). At site OA, P. tabacum plants were exposed to high light (635 μmol m−2 s−1 of mean daily photosynthetically active radiation) with drought stress, and expressed traits to minimize light capture and water loss. Compared to plants at sites EZ and TZ, those at site OA had thicker leaves with higher densities of stomata and pubescence, higher palisade/spongy ratio, higher light-saturated rate of net photosynthetic rate (Pmax), higher biomass, higher non-photochemical quenching coefficient (NPQ), and higher light saturation point (LSP) but fewer grana per chloroplast and less thylakoid stacking per granum. In contrast, P. tabacum growing at the cave vicinities: EZ (mean daily irradiance 59 μmol m−2 s−1) and TZ (mean daily irradiance 11 μmol m−2 s−1) showed typical shade-adapted characteristics for optimum light capture. The presence of sun- and shade-adapted characteristics indicates that P. tabacum has different strategies to cope with different environments but whether these strategies reflect genetic selection or phenological plasticity is yet to be determined. Such variability in physiological and morphological traits is important for the survival of P. tabacum in heterogeneous light conditions.

Enhancement of susceptivity to photoinhibition and photooxidation in rice chlorophyll b-less mutants

Two rice chlorophyll (Chl) b-less mutants (VG28-1, VG30-5) and the respective wild type (WT) plant (cv. Zhonghua No. 11) were analyzed for the changes in Chl fluorescence parameters, xanthophyll cycle pool, and its de-epoxidation state under exposure to strong irradiance, SI (1 700 µmol m−2 s−1). We also examined alterations in the chloroplast ultrastructure of the mutants induced by methyl viologen (MV) photooxidation. During HI (0–3.5 h), the photoinactivation of photosystem 2 (PS2) appeared earlier and more severely in Chl b-less mutants than in the WT. The decreases in maximal photochemical efficiency of PS2 in the dark (Fv/Fm), quantum efficiency of PS2 electron transport (ΦPS2), photochemical quenching (qP), as well as rate of photochemistry (Prate), and the increases in de-epoxidation state (DES) and rate of thermal dissipation of excitation energy (Drate) were significantly greater in Chl b-mutants compared with the WT plant. A relatively larger xanthophyll pool and 78–83 % conversion of violaxanthin into antheraxanthin and zeaxanthin in the mutants after 3.5 h of HI was accompanied with a high ratio of inactive/total PS2 (0.55–0.73) and high 1–qP (0.57–0.68) which showed that the activities of the xanthophyll cycle were probably insufficient to protect the photosynthetic apparatus against photoinhibition. No apparent difference of chloroplast ultrastructure was found between Chl b-less mutants and WT plants grown under low, LI (180 µmol m−2 s−1) and high, HI (700 µmol m−2 s−1) irradiance. However, swollen chloroplasts and slight dilation of thylakoids occurred in both mutants and the WT grown under LI followed by MV treatment. These typical symptoms of photooxidative damage were aggravated as plants were exposed to HI. Distorted and loose scattered thylakoids were observed in particular in the Chl b-less mutants. A greater extent of photoinhibition and photooxidation in these mutants indicated that the susceptibility to HI and oxidative stresses was enhanced in the photosynthetic apparatus without Chl b most likely as a consequence of a smaller antenna size.

Alteration of Components of Chlorophyll-Protein Complexes and Distribution of Excitation Energy Between the Two Photosystems in Two New Rice Chlorophyll b-less mutants

Two new yellow rice chlorophyll (Chl) b-less (lack) mutants VG28-1 and VG30-5 differ from the other known Chl b-less mutants with larger amounts of soluble protein and ribulose-1,5-bisphosphate carboxylase/oxygenase small sub-unit and smaller amounts of Chl a. We investigated the altered features of Chl-protein complexes and excitation energy distribution in these two mutants, as compared with wild type (WT) rice cv. Zhonghua 11 by using native mild green gel electrophoresis and SDS-PAGE, and 77 K Chl fluorescence in the presence of Mg2+. WT rice revealed five pigment-protein bands and fourteen polypeptides in thylakoid membranes. Two Chl b-less mutants showed only CPI and CPa pigment bands, and contained no 25 and 26 kDa polypeptides, reduced amounts of the 21 kDa polypeptide, but increased quantities of 32, 33, 56, 66, and 19 kDa polypeptides. The enhanced absorption of CPI and CPa and the higher Chl fluorescence emission ratio of F685/F720 were also observed in these mutants. This suggested that the reduction or loss of the antenna LHC1 and LHC2 was compensated by an increment in core component and the capacity to harvest photon energy of photosystem (PS) 1 and PS2, as well as in the fraction of excitation energy distributed to PS2 in the two mutants. 77 K Chl fluorescence spectra of thylakoid membranes showed that the PS1 fluorescence emission was shifted from 730 nm in WT rice to 720 nm in the mutants. The regulation of Mg2+ to excitation energy distribution between the two photosystems was complicated. 10 mM Mg2+ did not affect noticeably the F685/F730 emission ratio of WT thylakoid membranes, but increased the ratio of F685/F720 in the two mutants due to a reduced emission at 685 nm as compared to that at 720 nm.

Effect of light intensity on partitioning of photosynthetic electron transport to photorespiration in four subtropical forest plants.

Photosynthetic rate (Pn) and the partitioning of noncyclic photosynthetic electron transport to photorespiration (JO) in seedlings of four subtropical woody plants growing at three light intensities were studied in the summer time by measurements of chlorophyll fluorescence and CO2 exchange. ExceptSchima superba, an upper canopy tree species, the tree speciesCastanopsis fissa and two understory shrubsPsychotria rubra, Ardisia quinquegona had the highestPn at 36% of sunlight intensity. The total photosynthetic electron transport rate (JF) and the ratio ofJO/JF were elevated in leaves under full sunlight.JO/JF ratio reached 0.5–0.6 and coincided with the increasing of oxygenation rate of Rubisco (VO), the activity of glycolate oxidase and photorespiration rate at full sunlight. It is suggested that an increasing partitioning proportion of photosynthetic electron transport to photorespiration might be one of the protective regulation mechanisms in forest plant under strong summer light and high temperature conditions.