Contrasting modes of regulation of PS II light utilization with changing irradiance in normal and psbS mutant leaves of Arabidopsis thaliana

Complementary techniques of chlorophyll a fluorescence, steady state CO₂ exchange, and O₂ release during a multiple turnover flash were applied to compare responses to irradiance for leaves of wild type and psbS mutants. The latter included variants in which the psbS gene was deleted (npq4-1) or possessed a single point mutation (npq4-9). Nonphotochemical quenching (NPQ) was reduced by up to 80 and 50%, respectively, in these lines at high irradiance. Analysis of changes in steady-state fluorescence yields and quantum yield of linear electron transport in the context of the reversible radical pair model of Photosystem II (PS II) indicated that NPQ occurs by nonradiative deactivation of chlorophyll singlet states in normal leaves. In contrast, application of the same criteria together with the observed irreversibility of NPQ and decline in density of functional PS II reaction centers following excessive illumination indicated a change in reaction center properties for the psbS deletion phenotype (Npq4-1^–). Specifically, PS II reaction centers in Npq4-1^– convert to a photochemically inactive, yet strongly quenching, form in intense light. The possibility of formation of a carotenoid or chlorophyll cation quencher in the reaction center is discussed. Results for the point mutant phenotype (Npq4-9^–) were intermediate to those of wild-type and Npq4-1^–. Furthermore, wild-type leaves exhibited a significant reversible increase in the PS II in vivo rate constant for photochemistry (k_P0) in saturating compared to limiting light. Changes in k_P0 could not be accounted for in terms of a classic phosphorylation-dependent (state transition) mechanism. Changes in k_P0 may arise from alternate pigment—protein conformations that alter the way excitons equilibrate among PS II chromophores. The lack of similar irradiance-dependent changes in k_P0 for the psbS mutants suggests a role for the PS II-S protein in the regulation of exciton distribution.This revised version was published online in October 2005 with corrections to the Cover Date.     

PAM fluorometer based on medium-frequency pulsed Xe-flash measuring light: A highly sensitive new tool in basic and applied photosynthesis research

A newly developed modulation fluorometer is described which employs repetitive 1 s Xe-flashes for excitation light. Similar to the standard PAM Chlorophyll Fluorometer, which uses 1 s LED pulses for measuring light, the integrated measuring light intensity is sufficiently low to monitor the dark-fluorescence level, F_o. The maximal fluorescence yield, F_m, can be determined with high selectivity upon application of a saturating light pulse. The Xe-PAM displays exceptionally high sensitivity, enabling quenching analysis at chlorophyll concentrations as low as 1 g/l, thus allowing to assess photosynthesis of phytoplankton in natural waters like lakes, rivers and oceans. Due to high flexibility in the choice of excitation and emission wavelengths, this system also provides the experimental basis for a thorough study of fluorescence and photosynthesis properties of various algae classes with differing antenna organisation. By appropriate modifications, the instrument may as well be used to measure with great sensitivity and selectivity other types of fluorescence (e.g. NADPH-fluorescence), as well as light-scattering and absorbance changes.     

The metal ion transporter IRT1 is necessary for iron homeostasis and efficient photosynthesis in Arabidopsis thaliana

The mutants irt1-1 and irt1-2 of Arabidopsis thaliana were identified among a collection of T-DNA-tagged lines on the basis of a decrease in the effective quantum yield of photosystem II. The mutations responsible interfere with expression of IRT1, a nuclear gene that encodes the metal ion transporter IRT1. In irt1 mutants, photosensitivity and chlorophyll fluorescence parameters, as well as abundance and composition of the photosynthetic apparatus, are significantly altered. Additional effects of the mutation under greenhouse conditions, including chlorosis and a drastic reduction in growth rate and fertility, are compatible with a deficiency in iron transport. Propagation of irt1 plants on media supplemented with additional quantities of iron salts restores almost all aspects of wild-type behaviour. The irt2-1 mutant, which carries an En insertion in the highly homologous IRT2 gene of Arabidopsis thaliana, was identified by reverse genetics and shows no symptoms of iron deficiency. This, together with the finding that irt1-1 can be complemented by 35S::IRT1 but not by 35S::IRT2, demonstrates that, although the products of the two genes are closely related, only AtIRT1 is required for iron homeostasis under physiological conditions.     

A non-destructive selection method for resistance to fusicoccin in Arabidopsis thaliana

A mutagenised population of seeds of Arabidopsis thaliana was allowed to germinate in the presence of the positively charged aminoglycoside hygromycin (4 μg/ml) and the fungal toxin fusicoccin (5×10^–6 m). This hygromycin concentration, which is non-toxic by itself, becomes toxic when used together with fusicoccin, which stimulates cation uptake. Seeds that had germinated after 3–5 days and produced seedlings with green cotyledons were potentially resistant to fusicoccin and were therefore transferred into sterile Magenta vessels containing 1/2-strength Murashige and Skoog medium. This selection procedure is non-destructive, i.e. it allows the recovery of viable seedlings and their growth into adult plants thus permitting direct physiological characterisation. Received: 16 February 1998 / Revision received: 11 August 1998 / Accepted: 13 August 1998     

NaCI胁迫对盐芥和拟南芥光合作用的影响

本研究检测了与盐芥(Ghellungiella halophila)和拟南芥(Arabidopsis thaliana)光合作用相关的叶绿素、净光合速率(photosynthetic rate,Pn)、气孔导度(stomatal conductance,Gs)、胞间隙CO2浓度以及叶绿素荧光参数等指标,观察到随着NaCl浓度逐渐增加,盐芥的叶绿素a／b值(Chl a／Chl b)、类胡萝卜素／总叶绿素值(Car／Chl)显著高于拟南芥,且二比值变化幅度较小并保持较高水平。盐胁迫下拟南芥净光合速率下降、气孔导度下降和胞间CO2浓度减小。气孔因素是引起拟南芥光合能力下降的主要因素。叶绿素荧光参数的变化表明,50-200 mmol·L-1NaCl降低拟南芥叶绿体对光能的吸收能力,而且降低叶绿体的光化学活性,使电子传递速率和光能转化效率大幅度下降,造成光能转化为化学能的过程受阻,进一步加剧了光合放氧和碳同化能力的降低。而50-200 mmol·L-1NaCl胁迫没有使盐芥的光合作用受到不良影响。     

Thioredoxins m are major players in the multifaceted light-adaptive response in Arabidopsis thaliana

Thioredoxins (TRXs) are well-known redox signalling players, which carry out post-translational modifications in target proteins. Chloroplast TRXs are divided into different types and have central roles in light energy uptake and the regulation of primary metabolism. The isoforms TRX m1, m2, and m4 from Arabidopsis thaliana are considered functionally related. Knowing their key position in the hub of plant metabolism, we hypothesized that the impairment of the TRX m signalling would not only have harmful consequences on chloroplast metabolism but also at different levels of plant development. To uncover the physiological and developmental processes that depend on TRX m signalling, we carried out a comprehensive study of Arabidopsis single, double, and triple mutants defective in the TRX m1, m2, and m4 proteins. As light and redox signalling are closely linked, we investigated the response to high light (HL) of the plants that are gradually compromised in TRX m signalling. We provide experimental evidence relating the lack of TRX m and the appearance of novel phenotypic features concerning mesophyll structure, stomata biogenesis, and stomatal conductance. We also report new data indicating that the isoforms of TRX m fine-tune the response to HL, including the accumulation of the protective pigment anthocyanin. These results reveal novel signalling functions for the TRX m and underline their importance for plant growth and fulfilment of the acclimation/response to HL conditions.     

A Putative Chloroplast Thylakoid Metalloprotease VIRESCENT3 Regulates Chloroplast Development in Arabidopsis thaliana

The chloroplast is the site of photosynthesis and many other essential plant metabolic processes, and chloroplast development is an integral part of plant growth and development. Mutants defective in chloroplast development can display various color phenotypes including the intriguing virescence phenotype, which shows yellow/white coloration at the leaf base and greening toward the leaf tip. Through large scale genetic screens, we identified a series of new virescent mutants including virescent3-1 (vir3-1), vir4-1, and vir5-1 in Arabidopsis thaliana. We showed that VIR3 encodes a putative chloroplast metalloprotease by map-based cloning. Through site-directed mutagenesis, we showed that the conserved histidine 235 residue in the zinc binding motif HEAGH of VIR3 is indispensable for VIR3 accumulation in the chloroplast. The chloroplast localization of VIR3 was confirmed by the transient expression of VIR3-GFP in leaf protoplasts. Furthermore, taking advantage of transgenic lines expressing VIR3-FLAG, we demonstrated that VIR3 is an intrinsic thylakoid membrane protein that mainly resides in the stromal lamellae. Moreover, topology analysis using transgenic lines expressing a dual epitope-tagged VIR3 indicated that both the N and C termini of VIR3 are located in the stroma, and the catalytic domain of VIR3 is probably facing the stroma. Blue native gel analysis indicated that VIR3 is likely present as a monomer or part of a small complex in the thylakoid membrane. This work not only implicates VIR3 as a new factor involved in early chloroplast development but also provides more insight into the roles of chloroplast proteases in chloroplast biogenesis.     

Different strategies of Cd tolerance and accumulation in Arabidopsis halleri and Arabidopsis arenosa

Pseudometallophytes are commonly used to study the evolution of metal tolerance and accumulation traits in plants. Within the Arabidopsis genus, the adaptation of Arabidopsis halleri to metalliferous soils has been widely studied, which is not the case for the closely related species Arabidopsis arenosa. We performed an in-depth physiological comparison between the A. halleri and A. arenosa populations from the same polluted site, together with the geographically close non-metallicolous (NM) populations of both species. The ionomes, growth, photosynthetic parameters and pigment content were characterized in the plants that were growing on their native site and in a hydroponic culture under Cd treatments. In situ, the metallicolous (M) populations of both species hyperaccumulated Cd and Zn. The NM population of A. halleri hyperaccumulated Cd and Zn while the NM A. arenosa did not. In the hydroponic experiments, the NM populations of both species accumulated more Cd in their shoots than the M populations. Our research suggests that the two Arabidopsis species evolved different strategies of adaptation to extreme metallic environments that involve fine regulation of metal homeostasis, adjustment of the photosynthetic apparatus and accumulation of flavonols and anthocyanins.     

东北主要树种光合作用可行的离体测定方法

树木光合作用的测定常因植株高大而难以开展, 其中离体测定是解决途径之一。但离体测定的方法及其可靠性因树种而异。选取东北东部温带森林中特性各异的7种主要树种: 针叶树(红松(Pinus koraiensis)、长白落叶松(Larix olgensis))、散孔材(白桦(Betula platyphylla)、胡桃楸(Juglans mandshurica))和环孔材(水曲柳(Fraxinus mandshurica)、黄榆(Ulmus macrocarpa)、蒙古栎(Quercus mongolica)), 首先采用光合速率恢复到光合诱导前稳定值90%的时间(T₉₀)长短和叶片蒸腾速率(E)的大小评估离体叶片水分供应状况及其光合活力, 以此确定较优的离体测定方案; 同时, 观测离体叶片的光合活力稳定时间; 最后通过比较原位测定和采用所确定的较优离体方案测定的各树种叶片气体交换参数, 论证采用离体测定光合作用的可靠性。结果表明: 除蒙古栎外的6个树种的离体叶片均具有较高、较稳定的水分供应和光合活力。离体枝条或复叶插入水中, 环剥去除切口处3 cm左右的韧皮部和剩余叶片的方法, 是这6个温带树种叶片光合能力的较优离体测定方法。6个树种叶片的T₉₀受树木特性的影响而差异显著(p < 0.05), 其中环孔材树种的T₉₀显著高于散孔材和针叶树种。6个树种离体叶片在1 h内均有较高、较稳定的水分供应和光合活力。在此期间离体所测得的绝大多数叶片的气体交换参数与其原位测定值之间的差异不显著。该研究提出了可行的树木叶片光合作用的离体测定方案, 适用于蒙古栎以外的其他6个温带树种。     

A simple method for the addition of rotenone in Arabidopsis thaliana leaves

A simple and reproducible method for the treatment of Arabidopsis thaliana leaves with rotenone is presented. Rosette leaves were incubated with rotenone and Triton X-100 for at least 15 h. Treated leaves showed increased expression of COX19 and BCS1a, 2 genes known to be induced in Arabidopsis cell cultures after rotenone treatment. Moreover, rotenone/Triton X-100 incubated leaves presented an inhibition of oxygen uptake. The simplicity of the procedure shows this methodology is useful for studying the effect of the addition of rotenone to a photosynthetic tissue in situ.     

Suppression of host photosynthesis by the parasitic plant Rhinanthus minor

BACKGROUND AND AIMS: Parasitism is well understood to have wide-ranging deleterious effects on host performance in species thus far characterized. Photosynthetic performance reductions have been noted in the Striga-Zea mays association; however, no such information exists for facultative hemiparasitic plants and their hosts, nor are the effects of host species understood. METHODS: Chlorophyll fluorimetry was used to study the effects of parasitism by the hemiparasite Rhinanthus minor on the grass Phleum bertolinii and the forb Plantago lanceolata, and the effects of host species on the photosynthetic apparatus of R. minor. KEY RESULTS: Parasitism by Rhinanthus led to a significant decrease in the host, and total (host + parasite) biomass in Phleum; however, in Plantago, no significant repression of growth was noted. Maximum quantum yield (F(v)/F(m)) was reduced in parasitized Plantago, relative to control plants, but not in Phleum. F(v)/F(m) was significantly lower in R. minor parasitizing Phleum than Plantago, suggesting Phleum to be a superior host to Plantago for R. minor. Steady-state quantum yield (Phi(PSII)) was significantly depressed in parasitized Phleum, but only at low irradiances in Plantago. Phi(PSII) was very low for R. minor grown on Plantago, but not Phleum. CONCLUSIONS: Shown here is the first evidence of the suppression of host photosynthesis by a facultative hemiparasitic plant, which has significant effects on total biomass production. Host identity is a significant factor in parasite success, with the forb Plantago lanceolata exhibiting apparent chemical as well as previously identified physical defences to parasitism. It is proposed that the electron transport rate (as denoted by Phi(PSII)) represents the limiting factor for biomass accumulation in this system, and that Plantago is able to suppress the growth of Rhinanthus by suppressing the electron transport rate.     

Mutants for photosystem I subunit D of Arabidopsis thaliana: effects on photosynthesis, photosystem I stability and expression of nuclear genes for chloroplast functions

In Arabidopsis thaliana, the D-subunit of photosystem I (PSI-D) is encoded by two functional genes, PsaD1 and PsaD2, which are highly homologous. Knock-out alleles for each of the loci have been identified by a combination of forward and reverse genetics. The double mutant psad1-1 psad2-1 is seedling-lethal, high-chlorophyll-fluorescent and deficient for all tested PSI subunits, indicating that PSI-D is essential for photosynthesis. In addition, psad1-1 psad2-1 plants show a defect in the accumulation of thylakoid multiprotein complexes other than PSI. Of the single-gene mutations, psad2 plants behave like wild-type (WT) plants, whereas psad1-1 markedly affects the accumulation of PsaD mRNA and protein, and photosynthetic electron flow. Additional effects of the psad1-1 mutation include a decrease in growth rate under greenhouse conditions and downregulation of the mRNA expression of most genes involved in the light phase of photosynthesis. In the same mutant, a marked decrease in the levels of PSI and PSII polypeptides is evident, as well as a light-green leaf coloration and increased photosensitivity. Increased dosage of PsaD2 in the psad1-1 background restores the WT phenotype, indicating that PSI-D1 and PSI-D2 have redundant functions.     

A high-throughput method for quantifying growth of phytopathogenic bacteria in Arabidopsis thaliana

Measuring the growth of pathogenic bacteria in leaves is a mainstay of plant pathology studies. We have made significant improvements to standard methods that will not only increase the throughput but also reduce the space limitations. Additionally, the method described here is as accurate as the standard method. Briefly, we infected leaves by dipping whole seedlings of Arabidopsis into a bacterial solution containing a surfactant. After harvest, the seedlings were then simply shaken in buffer. The resulting bacterial solutions were diluted in microtitre plates and spotted onto agar plates. Colony-forming units were then counted 40 h after plating. Therefore, we have eliminated most of the labour-intensive steps involved in measuring the growth of bacteria in Arabidopsis, and describe a method that could be automated. The assay is sensitive enough to detect small differences between pathogens or ecotypes.     

A replication stress-induced synchronization method for Arabidopsis thaliana root meristems

Synchronized cell cultures are an indispensable tool for the identification and understanding of key regulators of the cell cycle. Nevertheless, the use of cell cultures has its disadvantages, because it represents an artificial system that does not completely mimic the endogenous conditions that occur in organized meristems. Here, we present a new and easy method for Arabidopsis thaliana root tip synchronization by hydroxyurea treatment. A major advantage of the method is the possibility of investigating available Arabidopsis cell‐cycle mutants without the need to generate cell cultures. As a proof of concept, the effects of over‐expression of a dominant negative allele of the B‐type cyclin‐dependent kinase CDKB1;1 gene on cell‐cycle progression were tested. The previously observed prolonged G₂ phase was confirmed, but was found to be compensated for by a reduced G₁ phase. Furthermore, altered S‐phase kinetics indicated a functional role for CDKB1;1 during the replication process.     

A simple and rapid method for visualization of male meiotic chromosomes in Arabidopsis thaliana

Meiotic chromosomes are of basic interest to the geneticist and cell biologist who study their behavior. A rapid and highly repeatable method for visualization of meiotic chromosomes is useful. Here we describe a fast staining protocol for Arabidopsis male meiotic chromosomes. Meiocytes were squashed into a labeling buffer, the chromosome morphology could be analyzed using fluorescence without any additional treatment.     

Theory and practice of a portable photosynthesis instrument

Abstract. Theory and practice of non-steady-state portable photosynthesis instruments (LI-6000 and 6200, LI-COR Inc., Nebraska, U.S.A.) are presented. Mass balance equations for the time dependence of H₂O and CO₂ mol fractions within the leaf chamber were used to describe instrument function. Measurements for each run were fitted to an exponential function to estimate average rates of CO₂ assimilation and transpiration during the measurement period. Stomatal conductances and intercellular CO₂ mol fractions were also computed. Linear data analysis used in the LI-6200 produced similar results for assimilation rates, stomatal conductances and intercellular CO₂ concentrations compared to a more rigorous nonlinear analysis, provided humidity within the chamber was kept constant during the measurement period. Instrument performance for CO₂ fluxes was confirmed by injecting pure CO₂ at steady rates from a microsyringe into the chamber. Miniature evaporimeters were designed to check H₂O flux measurements. Significant discrepancies were observed between LI-6200 estimates of H₂O fluxes and direct measurement and errors were attributed to adsorption desorption of water vapour on chamber walls or to leaks. The leaf chamber should be stored at humidities and temperatures similar to those during measurement conditions for maximum reliability of results.