Murray R. Badger

Photoprotection in plants: a new light on photosystem II damage

Sunlight damages photosynthetic machinery, primarily photosystem II (PSII), and causes photoinhibition that can limit plant photosynthetic activity, growth and productivity. The extent of photoinhibition is associated with a balance between the rate of photodamage and its repair. Recent studies have shown that light absorption by the manganese cluster in the oxygen-evolving complex of PSII causes primary photodamage, whereas excess light absorbed by light-harvesting complexes acts to cause inhibition of the PSII repair process chiefly through the generation of reactive oxygen species. As we review here, PSII photodamage and the inhibition of repair are therefore alleviated by photoprotection mechanisms associated with avoiding light absorption by the manganese cluster and successfully consuming or dissipating the light energy absorbed by photosynthetic pigments, respectively.

Apoplastic Synthesis of Nitric Oxide by Plant Tissues

Nitric oxide (NO) is an important signaling molecule in animals and plants. In mammals, NO is produced from Arg by the enzyme NO synthase. In plants, NO synthesis from Arg using an NO synthase–type enzyme and from nitrite using nitrate reductase has been demonstrated previously. The data presented in this report strongly support the hypothesis that plant tissues also synthesize NO via the nonenzymatic reduction of apoplastic nitrite. As measured by mass spectrometry or an NO-reactive fluorescent probe, Hordeum vulgare (barley) aleurone layers produce NO rapidly when nitrite is added to the medium in which they are incubated. NO production requires an acid apoplast and is accompanied by a loss of nitrite from the medium. Phenolic compounds in the medium can increase the rate of NO production. The possible significance of apoplastic NO production for germinating grain and for plant roots is discussed.

d-Ribulose-1,5-bisphosphate carboxylase-oxygenase: Improved methods for the activation and assay of catalytic activities

Abstract Improved methods for the activation and assay of d -ribulose-1,5-bisphosphate carboxylase and oxygenase are described. The importance of fully activating the enzyme before starting either reaction is emphasized. The enzyme is activated by preincubation with 20 m m MgCl 2 and 10 m m NaHCO 3 , pH 8.6. To avoid inactivation effects, both assays are limited to a 1- or 2-min duration. The initial rate of carboxylation is determined by a modification of the conventional 14 CO 2 fixation method, while the initial rate of oxygenase activity is measured polarographically. Both assays are initiated by the addition of the fully activated enzyme to otherwise complete reaction mixtures. The use of a “CO 2 -free” oxygenase reaction mixture is emphasized along with the difficulties arising from the inevitable carryover of CO 2 into the reaction mixture with the activated enzyme.

Evolution and diversity of CO2 concentrating mechanisms in cyanobacteria

Cyanobacteria have developed an effective photosynthetic CO2 concentrating mechanism (CCM) for improving the efficiency of carboxylation by a relatively inefficient Rubisco. The development of this CCM was presumably in response to the decline in atmospheric CO2 levels and rising O2, both of which were triggered by the development of oxygenic photosynthesis by cyanobacteria themselves. In the past few years there has been a rapid expansion in our understanding of the mechanism and genes responsible for the CCM. In addition, there has been a recent expansion in the availability of complete cyanobacterial genomes, thus increasing our potential to examine questions regarding both the evolution and diversity of components of the CCM across cyanobacteria. This paper considers various CCM and photosynthesis gene components across eight cyanobacteria where significant genomic information is available. Significant conclusions from our analysis of the distribution of various genes indicated the following. Firstly, cyanobacteria have developed with two types of carboxysomes, and this is correlated with the form of Rubisco present. We have coined the terms α-cyanobacteria to refer to cyanobacteria containing Form 1A Rubisco and α-carboxysomes, and β-cyanobacteria having Form 1B Rubisco and β-carboxysomes. Secondly, there are two NDH-1 CO2 uptake systems distributed variably, withProchlorococcus marinus species appearing to lack this CO2 uptake system. There are at least two HCO3- transport systems distributed variably, with some α-cyanobacteria having an absence of systems identified in β-cyanobacteria. Finally, there are multiple forms of carbonic anhydrases (CAs), but with only β-carboxysomal CA having a clearly shown role at present. The α-cyanobacteria appear to lack a clearly identifiable carboxysomal CA. A pathway for the evolution of cyanobacterial CCMs is proposed. The acquisition of carboxysomes triggered by the rapid decline of atmospheric CO2 in the Phanerozoic is argued to be the initial step. This would then be followed by the development of NDH-1 CO2-uptake systems, followed by the development of low-and high-affinity HCO3- transporters. An intriguing question is, were carboxysomes developed first in cyanobacteria, or did they originate by the lateral transfer of pre-existing proteobacterial bacterial microcompartment genes? The potentially late evolution of the CCM genes in cyanobacteria argues for a polyphyletic and separate evolution of CCMs in cyanobacteria, algae, and higher plants.

Identification of a SulP-type bicarbonate transporter in marine cyanobacteria

Cyanobacteria possess a highly effective CO2-concentrating mechanism that elevates CO2 concentrations around the primary carboxylase, Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase). This CO2-concentrating mechanism incorporates light-dependent, active uptake systems for CO2 and HCO–3. Through mutant studies in a coastal marine cyanobacterium, Synechococcus sp. strain PCC7002, we identified bicA as a gene that encodes a class of HCO–3 transporter with relatively low transport affinity, but high flux rate. BicA is widely represented in genomes of oceanic cyanobacteria and belongs to a large family of eukaryotic and prokaryotic transporters presently annotated as sulfate transporters or permeases in many bacteria (SulP family). Further gain-of-function experiments in the freshwater cyanobacterium Synechococcus PCC7942 revealed that bicA expression alone is sufficient to confer a Na+-dependent, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{HCO}}_{3}^{-}\end{equation*}\end{document} uptake activity. We identified and characterized three cyanobacterial BicA transporters in this manner, including one from the ecologically important oceanic strain, Synechococcus WH8102. This study presents functional data concerning prokaryotic members of the SulP transporter family and represents a previously uncharacterized transport function for the family. The discovery of BicA has significant implications for understanding the important contribution of oceanic strains of cyanobacteria to global CO2 sequestration processes.

Multiple Rubisco forms in proteobacteria: their functional significance in relation to CO2 acquisition by the CBB cycle

Rubisco is the predominant enzymatic mechanism in the biosphere by which autotrophic bacteria, algae, and terrestrial plants fix CO(2) into organic biomass via the Calvin-Benson-Basham reductive pentose phosphate pathway. Rubisco is not a perfect catalyst, suffering from low turnover rates, a low affinity for its CO(2) substrate, and a competitive inhibition by O(2) as an alternative substrate. As a consequence of changing environmental conditions over the past 3.5 billion years, with decreasing CO(2) and increasing O(2) in the atmosphere, Rubisco has evolved into multiple enzymatic forms with a range of kinetic properties, as well as co-evolving with CO(2)-concentrating mechanisms to cope with the different environmental contexts in which it must operate. The most dramatic evidence of this is the occurrence of multiple forms of Rubisco within autotrophic proteobacteria, where Forms II, IC, IBc, IAc, and IAq can be found either singly or in multiple combinations within a particular bacterial genome. Over the past few years there has been increasing availability of genomic sequence data for bacteria and this has allowed us to gain more extensive insights into the functional significance of this diversification. This paper is focused on summarizing what is known about the diversity of Rubisco forms, their kinetic properties, development of bacterial CO(2)-concentrating mechanisms, and correlations with metabolic flexibility and inorganic carbon environments in which proteobacteria perform various types of obligate and facultative chemo- and photoautotrophic CO(2) fixation.

Novel gene products associated with NdhD3/D4‐containing NDH‐1 complexes are involved in photosynthetic CO2 hydration in the cyanobacterium, Synechococcus sp. PCC7942

Cyanobacteria possess light‐dependent CO2 uptake activity that results in the net hydration of CO2 to HCO3– and may involve a protein‐mediated carbonic anhydrase (CA)‐like activity. This process is vital for the survival of cyanobacteria and may be a contributing factor in the ecological success of this group of organisms. Here, via isolation of mutants of Synechococcus sp. PCC7942 that cannot grow under low‐CO2 conditions, we have identified two novel genes, chpX and chpY, that are involved in light‐dependent CO2 hydration and CO2 uptake reactions; co‐inactivation of both these genes abolished both activities. The function and mechanism of the CO2 uptake systems supported by each chp gene product differs, with each associated with functionally distinct NAD(P)H dehydrogenase (NDH‐1) complexes. The ChpX system has a low affinity for CO2 and is de‐pendent on photosystem I cyclic electron transport, whereas the inducible ChpY system has a high affinity for CO2 and is dependent on linear electron transport. We believe that ChpX and ChpY are involved in a unique, net hydration of CO2 to HCO3–, that is coupled electron flow within the NDH‐1 complex on the thylakoid membrane.

The relationship between steady-state gas exchange of bean leaves and the levels of carbon-reduction-cycle intermediates

The relationship between the gas-exchange characteristics of attached leaves of Phaseolus vulgaris L. and the pool sizes of several carbon-reduction-cycle intermediates was examined. After determining the rate of CO2 assimilation at known intercellular CO2 pressure, O2 pressure and light, the leaf was rapidly killed (<0.1 s) and the levels of ribulose-1,5-bisphosphate (RuBP), 3-phosphoglyceric acid (PGA), fructose-1,6-bisphosphate, fructose-6-phosphate, glucose-6-phosphate, glyceraldehyde-3-phosphate, and dihydroxyacetone phosphate were measured. In 210 mbar O2, photosynthesis appeared RuBP-saturated at low CO2 pressure and RuBP-limited at high CO2 pressure. In 21 mbar (2%) O2, the level of RuBP always appeared saturating. Very high levels of PGA and other phosphate-containing compounds were found with some conditions, especially under low oxygen.

A rapid, non-invasive procedure for quantitative assessment of drought survival using chlorophyll fluorescence

BackgroundAnalysis of survival is commonly used as a means of comparing the performance of plant lines under drought. However, the assessment of plant water status during such studies typically involves detachment to estimate water shock, imprecise methods of estimation or invasive measurements such as osmotic adjustment that influence or annul further evaluation of a specimens response to drought.ResultsThis article presents a procedure for rapid, inexpensive and non-invasive assessment of the survival of soil-grown plants during drought treatment. The changes in major photosynthetic parameters during increasing water deficit were monitored via chlorophyll fluorescence imaging and the selection of the maximum efficiency of photosystem II (Fv/Fm) parameter as the most straightforward and practical means of monitoring survival is described. The veracity of this technique is validated through application to a variety of Arabidopsis thaliana ecotypes and mutant lines with altered tolerance to drought or reduced photosynthetic efficiencies.ConclusionThe method presented here allows the acquisition of quantitative numerical estimates of Arabidopsis drought survival times that are amenable to statistical analysis. Furthermore, the required measurements can be obtained quickly and non-invasively using inexpensive equipment and with minimal expertise in chlorophyll fluorometry. This technique enables the rapid assessment and comparison of the relative viability of germplasm during drought, and may complement detailed physiological and water relations studies.

How does cyclic electron flow alleviate photoinhibition in Arabidopsis

Cyclic electron flow (CEF) around photosystem I has a role in avoiding photoinhibition of photosystem II (PSII), which occurs under conditions in which the rate of photodamage to PSII exceeds the rate of its repair. However, the molecular mechanism underlying how CEF contributes to photoprotection is not yet well understood. We examined the effect of impairment of CEF and thermal energy dissipation (qE) on photoinhibition using CEF (pgr5) and qE (npq1 and npq4) mutants of Arabidopsis (Arabidopsis thaliana) exposed to strong light. Impairment of CEF by mutation of pgr5 suppressed qE and accelerated photoinhibition. We found that impairment of qE, by mutations of pgr5, npq1, and npq4, caused inhibition of the repair of photodamaged PSII at the step of the de novo synthesis of the D1 protein. In the presence of the chloroplast protein synthesis inhibitor chloramphenicol, impairment of CEF, but not impairment of qE, accelerated photoinhibition, and a similar effect was obtained when leaves were infiltrated with the protonophore nigericin. These results suggest that CEF-dependent generation of ΔpH across the thylakoid membrane helps to alleviate photoinhibition by at least two different photoprotection mechanisms: one is linked to qE generation and prevents the inhibition of the repair of photodamaged PSII at the step of protein synthesis, and the other is independent of qE and suppresses photodamage to PSII.