MSH1 Is a Plant Organellar DNA Binding and Thylakoid Protein under Precise Spatial Regulation to Alter Development.

As metabolic centers, plant organelles participate in maintenance, defense, and signaling. MSH1 is a plant-specific protein involved in organellar genome stability in mitochondria and plastids. Plastid depletion of MSH1 causes heritable, non-genetic changes in development and DNA methylation. We investigated the msh1 phenotype using hemi-complementation mutants and transgene-null segregants from RNAi suppression lines to sub-compartmentalize MSH1 effects. We show that MSH1 expression is spatially regulated, specifically localizing to plastids within the epidermis and vascular parenchyma. The protein binds DNA and localizes to plastid and mitochondrial nucleoids, but fractionation and protein-protein interactions data indicate that MSH1 also associates with the thylakoid membrane. Plastid MSH1 depletion results in variegation, abiotic stress tolerance, variable growth rate, and delayed maturity. Depletion from mitochondria results in 7%-10% of plants altered in leaf morphology, heat tolerance, and mitochondrial genome stability. MSH1 does not localize within the nucleus directly, but plastid depletion produces non-genetic changes in flowering time, maturation, and growth rate that are heritable independent of MSH1. MSH1 depletion alters non-photoactive redox behavior in plastids and a sub-set of mitochondrially altered lines. Ectopic expression produces deleterious effects, underlining its strict expression control. Unraveling the complexity of the MSH1 effect offers insight into triggers of plant-specific, transgenerational adaptation behaviors.

Pyrophosphate-dependent fructose-6-phosphate 1-phosphotransferase induction and attenuation of Hsp gene expression during endosperm modification in quality protein maize.

Quality Protein Maize (QPM) is a hard-endosperm version of the high-lysine opaque2 (o2) maize (Zea mays) mutant, but the genes involved in modification of the soft o2 endosperm are largely unknown. Pyrophosphate-dependent fructose-6-phosphate 1-phosphotransferase (PFP) catalyzes the ATP-independent conversion of fructose-6-phosphate to fructose-1,6-bisphosphate in glycolysis. We found a large increase in transcript and protein levels of the α-regulatory subunit of PFP (PFPα) in QPM endosperm. In vitro enzyme assays showed a significant increase in forward PFP activity in developing endosperm extracts of QPM relative to the wild type and o2. An expressed retrogene version of PFPα of unknown function that was not up-regulated in QPM was also identified. The elevated expression levels of a number of ATP-requiring heat shock proteins (Hsps) in o2 endosperm are ameliorated in QPM. PFPα is also coinduced with Hsps in maize roots in response to heat, cold, and the unfolded protein response stresses. We propose that reduced ATP availability resulting from the generalized Hsp response in addition to the reduction of pyruvate, orthophosphate dikinase activity in o2 endosperm is compensated in part by increased PFP activity in QPM.

MutS HOMOLOG1 is a nucleoid protein that alters mitochondrial and plastid properties and plant response to high light.

Mitochondrial-plastid interdependence within the plant cell is presumed to be essential, but measurable demonstration of this intimate interaction is difficult. At the level of cellular metabolism, several biosynthetic pathways involve both mitochondrial- and plastid-localized steps. However, at an environmental response level, it is not clear how the two organelles intersect in programmed cellular responses. Here, we provide evidence, using genetic perturbation of the MutS Homolog1 (MSH1) nuclear gene in five plant species, that MSH1 functions within the mitochondrion and plastid to influence organellar genome behavior and plant growth patterns. The mitochondrial form of the protein participates in DNA recombination surveillance, with disruption of the gene resulting in enhanced mitochondrial genome recombination at numerous repeated sequences. The plastid-localized form of the protein interacts with the plastid genome and influences genome stability and plastid development, with its disruption leading to variegation of the plant. These developmental changes include altered patterns of nuclear gene expression. Consistency of plastid and mitochondrial response across both monocot and dicot species indicate that the dual-functioning nature of MSH1 is well conserved. Variegated tissues show changes in redox status together with enhanced plant survival and reproduction under photooxidative light conditions, evidence that the plastid changes triggered in this study comprise an adaptive response to naturally occurring light stress.

Expression of the rice CDPK-7 in sorghum: molecular and phenotypic analyses.

Sorghum (Sorghum bicolor (L.) Moench) is an important source for food, feed, and possesses many agronomic attributes attractive for a biofuels feedstock. A warm season crop originating from the semi-arid tropics, sorghum is relatively susceptible to both cold and freezing stress. Enhancing the ability of sorghum to tolerate cold and freezing offers a route to expand the acreage for production, and provides a potential drought avoidance strategy during flowering, an important parameter for protection of yield. Targeted perturbation of the signal transduction pathway, that is triggered by exposure to abiotic stress in plants, has been demonstrated in model systems as an avenue to augment tolerance. Calcium-dependent protein kinases (CDPKs) are key players in a plant's response to environmental assaults. To test the impact of modulating CDPK activity in sorghum as a means to enhanced abiotic stress tolerance, we introduced a constitutively expressed rice CDPK-7 (OsCDPK-7) gene construct. Sorghum transformants carrying this cassette, were not improved in cold or salt stress under the conditions tested. However, a lesion mimic phenotype and up-regulation of a number of pathogen related proteins, along with transcripts linked to photosynthesis were observed. These results demonstrate that modulating the Ca signaling cascade in planta via unregulated enhanced CDPK activity can lead to off-type effects likely due to the broadly integrated nature of these enzymes in signaling.

Proteomic analysis of atrazine exposure in Drosophila melanogaster (Diptera: Drosophilidae).

Atrazine is a widely used herbicide that has been reported to induce the activity of certain detoxification enzymes and to affect insecticide toxicity in organisms experiencing simultaneous exposure to both atrazine and insecticides. In this study, the effects of atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine) exposure on protein expression in male and female Drosophila melanogaster adults in both microsomal and cytosolic cell fractions was investigated by 2-dimensional gel electrophoresis. Differentially expressed proteins (vs. controls) were identified using matrix assisted laser desorption-time (MALDI-TOF) of flight mass spectrometry (MS). We identified a total of 28 proteins associated with energy production including glycolysis and mitochondrial respiration as differentially expressed and nine proteins associated with detoxification and response to oxidative stress. Most of these proteins were expressed in one sex or the other but not in both. Surprisingly, the only proteins associated with detoxification were identified as glutathione transferases. No cytochrome P450s were identified which have previously been shown to be responsive to atrazine exposure in D. melanogaster and proposed to be associated with insecticide/atrazine interactions. Results of this investigation support the role of atrazine in affecting mitochondrial electron transport and oxidative stress. However, the role of atrazine in pesticide interactions remains uncertain.

Rubisco activase and wheat productivity under heat-stress conditions.

Rubisco activase (RCA) constrains the photosynthetic potential of plants at high temperatures (heat stress). Endogenous levels of RCA could serve as an important determinant of plant productivity under heat-stress conditions. Thus, in this study, the possible relationship between expression levels of RCA and plant yield in 11 European cultivars of winter wheat following prolonged exposure to heat stress was investigated. In addition, the effect of a short-term heat stress on RCA expression in four genotypes of wheat, five genotypes of maize, and one genotype of Arabidopsis thaliana was examined. Immunoblots prepared from leaf protein extracts from control plants showed three RCA cross-reacting bands in wheat and two RCA cross-reacting bands in maize and Arabidopsis. The molecular mass of the observed bands was in the range between 40 kDa and 46 kDa. Heat stress affected RCA expression in a few genotypes of wheat and maize but not in Arabidopsis. In wheat, heat stress slightly modulated the relative amounts of RCA in some cultivars. In maize, heat stress did not seem to affect the existing RCA isoforms (40 kDa and 43 kDa) but induced the accumulation of a new putative RCA of 45-46 kDa. The new putative 45-46 kDa RCA was not seen in a genotype of maize (ZPL 389) that has been shown to display an exceptional sensitivity to heat stress. A significant, positive, linear correlation was found between the expression of wheat 45-46 kDa RCA and plant productivity under heat-stress conditions. Results support the hypothesis that endogenous levels of RCA could play an important role in plant productivity under supraoptimal temperature conditions.

A type III effector ADP-ribosylates RNA-binding proteins and quells plant immunity.

The bacterial plant pathogen Pseudomonas syringae injects effector proteins into host cells through a type III protein secretion system to cause disease. The enzymatic activities of most of P. syringae effectors and their targets remain obscure. Here we show that the type III effector HopU1 is a mono-ADP-ribosyltransferase (ADP-RT). HopU1 suppresses plant innate immunity in a manner dependent on its ADP-RT active site. The HopU1 substrates in Arabidopsis thaliana extracts were RNA-binding proteins that possess RNA-recognition motifs (RRMs). A. thaliana knockout lines defective in the glycine-rich RNA-binding protein GRP7 (also known as AtGRP7), a HopU1 substrate, were more susceptible than wild-type plants to P. syringae. The ADP-ribosylation of GRP7 by HopU1 required two arginines within the RRM, indicating that this modification may interfere with GRP7's ability to bind RNA. Our results suggest a pathogenic strategy where the ADP-ribosylation of RNA-binding proteins quells host immunity by affecting RNA metabolism and the plant defence transcriptome.

Biochemical and molecular characterization of photosystem I deficiency in the NCS6 mitochondrial mutant of maize.

Interorganellar signaling interactions are poorly understood. The maize non-chromosomal stripe (NCS) mutants provide models to study the requirement of mitochondrial function for chloroplast biogenesis and photosynthesis. Previous work suggested that the NCS6 mitochondrial mutation, a cytochrome oxidase subunit 2 (cox2) deletion, is associated with a malfunction of Photosystem I (PSI) in defective chloroplasts of mutant leaf sectors (Gu et al., 1993). We have now quantified the reductions of photosynthetic rates and PSI activity in the NCS6 defective stripes. Major reductions of the plastid-coded PsaC and nucleus-coded PsaD and PsaE PSI subunits and of their corresponding mRNAs are seen in mutant sectors; however, although the psaA/B mRNA is greatly reduced, levels of PsaA and PsaB (the core proteins of PSI) are only slightly decreased. Levels of the PsaL subunit and its mRNA appear to be unchanged. Tested subunits of other thylakoid membrane complexes--PSII, Cyt b6/f, and ATP synthase, have minor (or no) reductions within mutant sectors. The results suggest that specific signaling pathways sense the dysfunction of the mitochondrial electron transport chain and respond to down-regulate particular PSI mRNAs, leading to decreased PSI accumulation in the chloroplast. The reductions of both nucleus and plastid encoded components indicate that complex interorganellar signaling pathways may be involved.

Mitochondrial respiratory deficiencies signal up-regulation of genes for heat shock proteins.

The consequences of mitochondrial dysfunction are not limited to the development of oxidative stress or initiation of apoptosis but can result in the establishment of stress tolerance. Using maize mitochondrial mutants, we show that permanent mitochondrial deficiencies trigger novel Ca(2+)-independent signaling pathways, leading to constitutive expression of genes for molecular chaperones, heat shock proteins (HSPs) of different classes. The signaling to activate hsp genes appears to originate from a reduced mitochondrial transmembrane potential. Upon depolarization of mitochondrial membranes in transient assays, gene induction for mitochondrial HSPs occurred more rapidly than that for cytosolic HSPs. We also demonstrate that in the nematode Caenorhabditis elegans transcription of hsp genes can be induced by RNA interference of nuclear respiratory genes. In both organisms, activation of hsp genes in response to mitochondrial impairment is distinct from their responses to heat shock and is not associated with oxidative stress. Thus, mitochondria-to-nucleus signaling to express a hsp gene network is apparently a widespread retrograde mechanism to facilitate cell defense and survival.

Alternative NAD(P)H dehydrogenases of plant mitochondria.

Plant mitochondria have a highly branched electron transport chain that provides great flexibility for oxidation of cytosolic and matrix NAD(P)H. In addition to the universal electron transport chain found in many organisms, plants have alternative NAD(P)H dehydrogenases in the first part of the chain and a second oxidase, the alternative oxidase, in the latter part. The alternative activities are nonproton pumping and allow for NAD(P)H oxidation with varying levels of energy conservation. This provides a mechanism for plants to, for example, remove excess reducing power and balance the redox poise of the cell. This review presents our current understanding of the alternative NAD(P)H dehydrogenases present in plant mitochondria.

Differential expression of alternative oxidase genes in maize mitochondrial mutants.

We have examined the expression of three alternative oxidase (aox) genes in two types of maize mitochondrial mutants. Nonchromosomal stripe (NCS) mutants carry mitochondrial DNA deletions that affect subunits of respiratory complexes and show constitutively defective growth. Cytoplasmic male-sterile (CMS) mutants have mitochondrial DNA rearrangements, but they are impaired for mitochondrial function only during anther development. In contrast to normal plants, which have very low levels of AOX, NCS mutants exhibit high expression of aox genes in all nonphotosynthetic tissues tested. The expression pattern is specific for each type of mitochondrial lesion: the NADH dehydrogenase-defective NCS2 mutant has high expression of aox2, whereas the cytochrome oxidase-defective NCS6 mutant predominantly expresses aox3. Similarly, aox2 and aox3 can be induced differentially in normal maize seedlings by specific inhibitors of these two respiratory complexes. Translation-defective NCS4 plants show induction of both aox2 and aox3. AOX2 and AOX3 proteins differ in their ability to be regulated by reversible dimerization. CMS mutants show relatively high levels of aox2 mRNAs in young tassels but none in ear shoots. Significant expression of aox1 is detected only in NCS and CMS tassels. The induction pattern of maize aox genes could serve as a selective marker for diverse mitochondrial defects.