Photorespiration is complemented by cyclic electron flow and the alternative oxidase pathway to optimize photosynthesis and protect against abiotic stress

Photosynthesis Research - Optimization of photosynthetic performance and protection against abiotic stress are essential to sustain plant growth. Photorespiratory metabolism can help plants to...     

The flexibility of metabolic interactions between chloroplasts and mitochondria in Nicotiana tabacum leaf

To examine the effect of mitochondrial function on photosynthesis, wild-type and transgenic Nicotiana tabacum with varying amounts of alternative oxidase (AOX) were treated with different respiratory inhibitors. Initially, each inhibitor increased the reduction state of the chloroplast electron transport chain, most severely in AOX knockdowns and least severely in AOX overexpressors. This indicated that the mitochondrion was a necessary sink for photo-generated reductant, contributing to the ‘P700 oxidation capacity’ of photosystem I. Initially, the Complex III inhibitor myxothiazol and the mitochondrial ATP synthase inhibitor oligomycin caused an increase in photosystem II regulated non-photochemical quenching not evident with the Complex III inhibitor antimycin A (AA). This indicated that the increased quenching depended upon AA-sensitive cyclic electron transport (CET). Following 12 h with oligomycin, the reduction state of the chloroplast electron transport chain recovered in all plant lines. Recovery was associated with large increases in the protein amount of chloroplast ATP synthase and mitochondrial uncoupling protein. This increased the capacity for photophosphorylation in the absence of oxidative phosphorylation and enabled the mitochondrion to act again as a sink for photo-generated reductant. Comparing the AA and myxothiazol treatments at 12 h showed that CET optimized photosystem I quantum yield, depending upon the P700 oxidation capacity. When this capacity was too high, CET drew electrons away from other sinks, moderating the P700⁺ amount. When P700 oxidation capacity was too low, CET acted as an electron overflow, moderating the amount of reduced P700. This study reveals flexible chloroplast–mitochondrion interactions able to overcome lesions in energy metabolism.     

Adenylate ratios in the cytosol,chloroplasts and mitochondria of barley leaf protoplasts during photosynthesis at different carbon dioxide concentrations

Barley (Hordeum vulgare) protoplasts were incubated in darkness and in the light at saturating and limiting CO₂ concentrations. The protoplasts were fractioned by a membrane filtration technique which allows quenching of the metabolism by acidification within about 0.1 s and the ATP/ADP ratios in the cytasol, chloroplasts and mitochondria were determined. It is concluded that the cytosolic ATP/ADP ratio is considerably higher during photosynthesis at limiting CO₂ (which is the normal situation for a C₃ plant in air) compared to photosynthesis at saturating CO₂ or darkness.     

AtPAP2 is a tail-anchored protein in the outer membrane of chloroplasts and mitochondria

To date, Arabidopsis purple acid phosphatase 2 (AtPAP2) is the only known plant protein that is dual-targeted to chloroplasts and mitochondria by a C-terminal targeting signal. Using in vitro organelle import and green fluorescence protein (GFP) localization assays, we showed that AtPAP2 is located on, but not imported across the outer membrane (OM) of chloroplasts and mitochondria and exposed its N-terminal enzymatic domain to the cytosol. It was also found that a short stretch of 30 amino acids (a.a.) at the C-terminal region (a.a. 615-644) that contains a stretch of 18 hydrophobic residues, a WYAK motif and 8 hydrophilic residues is sufficient for dual-targeting. Mutation of WYAK to WYAE had no effect on dual-targeting ability suggesting that the charge within this flanking region alone is not an important determinant for dual-targeting.        

Fine‐tuned regulation of the K+/H+ antiporter KEA3 is required to optimize photosynthesis during induction

KEA3 is a thylakoid membrane localized K⁺/H⁺ antiporter that regulates photosynthesis by modulating two components of proton motive force (pmf), the proton gradient (?pH) and the electric potential (?ψ). We identified a mutant allele of KEA3, disturbed proton gradient regulation (dpgr) based on its reduced non‐photochemical quenching (NPQ) in artificial (CO₂‐free with low O₂) air. This phenotype was enhanced in the mutant backgrounds of PSI cyclic electron transport (pgr5 and crr2‐1). In ambient air, reduced NPQ was observed during induction of photosynthesis in dpgr, the phenotype that was enhanced after overnight dark adaptation. In contrast, the knockout allele of kea3‐1 exhibited a high‐NPQ phenotype during steady state in ambient air. Consistent with this kea3‐1 phenotype in ambient air, the membrane topology of KEA3 indicated a proton efflux from the thylakoid lumen to the stroma. The dpgr heterozygotes showed a semidominant and dominant phenotype in artificial and ambient air, respectively. In dpgr, the protein level of KEA3 was unaffected but the downregulation of its activity was probably disturbed. Our findings suggest that fine regulation of KEA3 activity is necessary for optimizing photosynthesis.     

Method to protect a targeted amino acid residue during random mutagenesis

To generate a random mutant library that is free from mutation at a particular amino acid residue, we replace the codon of interest with a detachable, short DNA sequence containing a BsaXI recognition site. After PCR mutagenesis, this sequence is removed and intramolecular ligation of the sequences flanking the insert regenerates the gene. The three-base cohesive ends for ligation correspond to the codon for the targeted residue and any sequences with mutations at this site will fail to ligate. As a result, only the variants that are free from mutation at this site are in the proper reading frame. In a random library of C₃₀ carotenoid synthase CrtM, this method was used to exclude readily accessible mutations at position F26, which confer C₄₀ synthase function. This enabled us to identify two additional mutations, W38C and E180G, which confer the same phenotype but are present in the random library at much lower frequencies.     

Critical review: incorporating the arrangement of mitochondria and chloroplasts into models of photosynthesis and carbon isotope discrimination

Photosynthesis Research - The arrangement of mitochondria and chloroplasts, together with the relative resistances of cell wall and chloroplast, determine the path of diffusion out of the leaf for...     

Stomatal conductance is a key parameter to assess limitations to photosynthesis and growth potential in barley genotypes

Fourteen genotypes of barley were compared for response to salinity by monitoring the parameters gas exchange and chlorophyll fluorescence. We present relationships between stomatal conductance (gs) gas exchange chlorophyll fluorescence parameters and aboveground dry matter (AGDM). We found that genetic variability provided a continuum of data for gs across control and saline conditions. We used this continuum of gs values to test the overall relationships between gs and net photosynthesis (A), leaf internal CO2 concentration (Ci), actual quantum yield of PSII electron transport (PhiPSII), relative electron yield over net CO2 assimilation rate (ETR/A), and AGDM. The relationship between gs and A was highly significant (P < 0.0001) for both control and saline treatments, while correlations between gs and Ci, and Ci and A were significant only under control conditions. Unexpectedly, we found positive correlations between gs and PhiPSII (P < 0.0001) for both conditions. A comparison between relationships of gs and A, and gs and PhiPSII seemed to indicate a possible acclimation to salinity at the chloroplastic level. Finally, the relationships between gs and ETR/A were exceptionally strong for both growing conditions (P < 0.0001) indicating that, as gs values were negatively affected in barley by genetics and salinity as main or interactive effects, there was a progressive increase in photorespiration in barley. Overall, we found that stomatal conductance was a key parameter in the study of barley responses to limiting situations for photosynthesis. We also found a strong relationship between AGDM and gs regardless of growing conditions and genotypes. For breeding evaluations to select barley genotypes for salinity tolerance, it may be possible to replace all measurements of gas exchange and chlorophyll fluorescence by the simple use of a porometer.     

Arabidopsis thaliana GLN2-encoded glutamine synthetase is dual targeted to leaf mitochondria and chloroplasts

In higher plants, photorespiratory Gly oxidation in leaf mitochondria yields ammonium in large amounts. Mitochondrial ammonium must somehow be recovered as glutamate in chloroplasts. As the first step in that recovery, we report glutamine synthetase (GS) activity in highly purified Arabidopsis thaliana mitochondria isolated from light-adapted leaf tissue. Leaf mitochondrial GS activity is further induced in response to either physiological CO(2) limitation or transient darkness. Historically, whether mitochondria are fully competent for oxidative phosphorylation in actively photorespiring leaves has remained uncertain. Here, we report that light-adapted, intact, leaf mitochondria supplied with Gly as sole energy source are fully competent for oxidative phosphorylation. Purified intact mitochondria efficiently use Gly oxidation (as sole energy, NH(3), and CO(2) source) to drive conversion of l-Orn to l-citrulline, an ATP-dependent process. An A. thaliana genome-wide search for nuclear gene(s) encoding mitochondrial GS activity yielded a single candidate, GLN2. Stably transgenic A. thaliana ecotype Columbia plants expressing a p35S::GLN2::green fluorescent protein (GFP) chimeric reporter were constructed. When observed by laser scanning confocal microscopy, leaf mesophyll and epidermal tissue of transgenic plants showed punctate GFP fluorescence that colocalized with mitochondria. In immunoblot experiments, a 41-kD chimeric GLN2::GFP protein was present in both leaf mitochondria and chloroplasts of these stably transgenic plants. Therefore, the GLN2 gene product, heretofore labeled plastidic GS-2, functions in both leaf mitochondria and chloroplasts to faciliate ammonium recovery during photorespiration.     

RNA editing gives a new meaning to the genetic information in mitochondria and chloroplasts

RNA editing in plant mitochondria and chloroplasts alters mRNA sequences to code for different proteins than the DNA. Most of these C-to-U transitions occur in open reading frames, but a few are observed in intron sequences. Influences of the nuclear genome on editing patterns suggest that cytoplasmic factors participate in this process.     

Priming for JA-dependent defenses using hexanoic acid is an effective mechanism to protect Arabidopsis against B. cinerea

Soil drench treatments with hexanoic acid can effectively protect Arabidopsis plants against Botrytis cinerea through a mechanism based on a stronger and faster accumulation of JA-dependent defenses.Plants impaired in ethylene, salicylic acid, abscisic acid or glutathion pathways showed intact protection by hexanoic acid upon B. cinerea infection. Accordingly, no significant changes in the SA marker gene PR-1 in either the SA or ABA hormone balance were observed in the infected and treated plants. In contrast, the JA signaling pathway showed dramatic changes after hexanoic acid treatment, mainly when the pathogen was present. The impaired JA mutants, jin1-2 and jar1, were unable to display hexanoic acid priming against the necrotroph. In addition, hexanoic acid-treated plants infected with B. cinerea showed priming in the expression of the PDF1.2, PR-4 and VSP1 genes implicated in the JA pathways. Moreover, JA and OPDA levels were primed at early stages by hexanoic acid. Treatments also stimulated increased callose accumulation in response to the pathogen. Although callose accumulation has proved an effective IR mechanism against B. cinerea, it is apparently not essential to express hexanoic acid-induced resistance (HxAc-IR) because the mutant pmr4.1 (callose synthesis defective mutant) is protected by treatment.We recently described how hexanoic acid treatments can protect tomato plants against B. cinerea by stimulating ABA-dependent callose deposition and by priming OPDA and JA-Ile production. We clearly demonstrate here that Hx-IR is a dependent plant species, since this acid protects Arabidopsis plants against the same necrotroph by priming JA-dependent defenses without enhancing callose accumulation.     

From extracellular to intracellular: the establishment of mitochondria and chloroplasts.

Paracoccus and Rhodopseudomonas are unusual among bacteria in having a majority of the biochemical features of mitochondria; blue-green algae have many of the features of chloroplasts. The theory of serial endosymbiosis proposes that a primitive eukaryote successively took up bacteria and blue-green algae to yield mitochondria and chloroplasts respectively. Possible characteristics of transitional forms are indicated both by the primitive amoeba, Pelomyxa, which lacks mitochondria but contains a permanent population of endosymbiotic bacteria, and by several anomalous eukaryotic algae, e.g. Cyanophora, which contain cyanelles instead of chloroplasts. Blue-green algae appear to be obvious precursors of red algal chloroplasts but the ancestry of other chloroplasts is less certain, though the epizoic symbiont, Prochloron, may resemble the ancestral green algal chloroplast. We speculate that the chloroplasts of the remaining algae may have been a eukaryotic origin. The evolution or organelles from endosymbiotic precursors would involve their integration with the host cell biochemically, structurally and numerically.     

Rubisco activase is a key regulator of non-steady-state photosynthesis at any leaf temperature and, to a lesser extent, of steady-state photosynthesis at high temperature

The role of Rubisco activase in steady-state and non-steady-state photosynthesis was analyzed in wild-type (Oryza sativa) and transgenic rice that expressed different amounts of Rubisco activase. Below 25°C, the Rubisco activation state and steady-state photosynthesis were only affected when Rubisco activase was reduced by more than 70%. However, at 40°C, smaller reductions in Rubisco activase content were linked to a reduced Rubisco activation state and steady-state photosynthesis. As a result, overexpression of maize Rubisco activase in rice did not lead to an increase of the Rubisco activation state, nor to an increase in photosynthetic rate below 25°C, but had a small stimulatory effect at 40°C. On the other hand, the rate at which photosynthesis approached the steady state following an increase in light intensity was rapid in Rubisco activase-overexpressing plants, intermediate in the wild-type, and slowest in antisense plants at any leaf temperature. In Rubisco activase-overexpressing plants, Rubisco activation state at low light was maintained at higher levels than in the wild-type. Thus, rapid regulation by Rubisco activase following an increase in light intensity and/or maintenance of a high Rubisco activation state at low light would result in a rapid increase in Rubisco activation state and photosynthetic rate following an increase in light intensity. It is concluded that Rubisco activase plays an important role in the regulation of non-steady-state photosynthesis at any leaf temperature and, to a lesser extent, of steady-state photosynthesis at high temperature.     

Locating gaps in amino acid sequences to optimize the homology between two proteins

A method for optimally locating gaps in the amino acid sequences of homologous proteins is presented. The method involves three steps: (1) demonstration that the sequences are indeed homologous, (2) location of regions where the homologous pairing is reasonably certain, and (3) location of gaps between these regions so as to minimize the total number of mutations required to account for the differences between the two sequences. The major virtues of this procedure are that the assertion of homology does not depend upon the prior introduction of gaps and that a genetic rather than a chemical test is the basis for asserting a genetic relationship.This project received support from grants from NSF (GB-7486) and NIH (NB 04545-06).     

Yeast Nej1 is a key participant in the initial end binding and final ligation steps of nonhomologous end joining

In Saccharomyces cerevisiae, the key components of the nonhomologous end joining (NHEJ) pathway that repairs DNA double-strand breaks (DSBs) are yeast Ku (yKu), Mre11-Rad50-Xrs2, Dnl4-Lif1, and Nej1. Here, we examined the role of Nej1 in NHEJ by a combination of molecular genetic and biochemical approaches. As expected, the recruitment of Nej1 to in vivo DSBs is dependent upon yKu. Surprisingly, Nej1 is required for the stable binding of yKu to in vivo DSBs, in addition to Dnl4-Lif1. Thus, Nej1 and Dnl4-Lif1 are independently recruited by yKu to in vivo DSBs, forming a stable ternary complex that channels DSBs into the NHEJ pathway. In accord with these results, purified Nej1 interacts with yKu and preferentially binds to DNA ends bound by yKu. Furthermore, the binding of a mixture of Nej1 and Dnl4-Lif1 to DNA ends bound by yKu is greater than the sum of the binding of the individual proteins, indicating that pairwise interactions among yKu, Nej1, and Dnl4-Lif1 contribute to complex assembly at DNA ends. Nej1 stimulates intermolecular ligation by Dnl4-Lif1, but, more interestingly, the addition of Nej1 results in more than one intermolecular ligation per Dnl4 molecule. Thus, Nej1 not only plays an important role in determining repair pathway choice by participating in the initial NHEJ complex formed at DSBs but also contributes to the reactivation of Dnl4-Lif1 after repair is complete, thereby increasing the capacity of the NHEJ repair pathway.     

Inability of the mutagen-blocking agent oleic acid to protect against colon carcinogenesis in the rat

Oleic acid has been found in cooked beef and human feces. It has also been found to protect against mutagenesis as measured by the Salmonella microsomal assay test. Addition of oleic acid to the diet of rats treated with 1,2-dimethylhydrazine, a colon carcinogen, failed to protect the rats against the induction of colon cancer.