Cold acclimation induces rapid and dynamic changes in freeze tolerance mechanisms in the cryophile Deschampsia antarctica E. Desv.

The cryophilic Antarctic hair grass, Deschampsia antarctica E. Desv., one of two higher plants indigenous to Antarctica, represents a unique resource for the study of freeze tolerance mechanisms. We have previously characterized a multi-gene family in D. antarctica encoding ice recrystallization inhibition proteins (IRIPs) whose transcript levels are responsive to cold acclimation, and whose products confer ice recrystallization inhibition (RI) activity that can account for activity seen in cold acclimated plants. We used molecular and physiological analyses to investigate temporal responses of D. antarctica to cold acclimation and de-acclimation, and sub-zero acclimation. Quantitative profiling revealed that IRIP transcript levels significantly increased and decreased within hours of cold acclimation and de-acclimation, respectively, becoming up to 1000-fold more abundant in fully acclimated plants. Western analysis detected three major immuno-reactive bands whose pattern of accumulation mirrored that of transcript. These data correlated with the onset and decline of RI activity in acclimated and de-acclimated leaves. Plant survival-based testing revealed that cold acclimation enhanced freeze tolerance by 5 °C within 4 d, and that sub-zero acclimation conferred an additional 3 °C of tolerance. Thus, D. antarctica is highly responsive to temperature fluctuations, able to rapidly deploy IRIP based RI activity and enhance its freeze tolerance.

Ice recrystallization inhibition proteins (IRIPs) and freeze tolerance in the cryophilic Antarctic hair grass Deschampsia antarctica E. Desv.

Antarctic hair grass (Deschampsia antarctica E. Desv.), the only grass indigenous to Antarctica, has well-developed freezing tolerance, strongly induced by cold acclimation. Here, we show that in response to low temperatures, D. antarctica expresses potent recrystallization inhibition (RI) activity that, inhibits the growth of small ice crystals into potentially damaging large ones, is proteinaceous and localized to the apoplasm. A gene family from D. antarctica encoding putative homologs of an ice recrystallization inhibition protein (IRIP) has been isolated and characterized. IRIPs are apoplastically targeted proteins with two potential ice-binding motifs: 1-9 leucine-rich repeats (LRRs) and c. 16 'IRIP' repeats. IRIP genes appear to be confined to the grass subfamily Pooideae and their products, exhibit sequence similarity to phytosulphokine receptors and are predicted to adopt conformations with two ice-binding surfaces. D. antarctica IRIP (DaIRIP) transcript levels are greatly enhanced in leaf tissue following cold acclimation. Transgenic Arabidopsis thaliana expressing a DaIRIP has novel RI activity, and purified DaIRIP, when added back to extracts of leaves from non-acclimated D. antarctica, can reconstitute the activity found in acclimated plants. We propose that IRIP-mediated RI activity may contribute to the cryotolerance of D. antarctica, and thus to its unique ability to have colonized Antarctica.

Processing of the dual targeted precursor protein of glutathione reductase in mitochondria and chloroplasts.

Pea glutathione reductase (GR) is dually targeted to mitochondria and chloroplasts by means of an N-terminal signal peptide of 60 amino acid residues. After import, the signal peptide is cleaved off by the mitochondrial processing peptidase (MPP) in mitochondria and by the stromal processing peptidase (SPP) in chloroplasts. Here, we have investigated determinants for processing of the dual targeting signal peptide of GR by MPP and SPP to examine if there is separate or universal information recognised by both processing peptidases. Removal of 30 N-terminal amino acid residues of the signal peptide (GRDelta1-30) greatly stimulated processing activity by both MPP and SPP, whereas constructs with a deletion of an additional ten amino acid residues (GRDelta1-40) and deletion of 22 amino acid residues in the middle of the GR signal sequence (GRDelta30-52) could be cleaved by SPP but not by MPP. Numerous single mutations of amino acid residues in proximity of the cleavage site did not affect processing by SPP, whereas mutations within two amino acid residues on either side of the processing site had inhibitory effect on processing by MPP with a nearly complete inhibition for mutations at position -1. Mutation of positively charged residues in the C-terminal half of the GR targeting peptide inhibited processing by MPP but not by SPP. An inhibitory effect on SPP was detected only when double and triple mutations were introduced upstream of the cleavage site. These results indicate that: (i) recognition of processing site on a dual targeted GR precursor differs between MPP and SPP; (ii) the GR targeting signal has similar determinants for processing by MPP as signals targeting only to mitochondria; and (iii) processing by SPP shows a low level of sensitivity to single mutations on targeting peptide and likely involves recognition of the physiochemical properties of the sequence in the vicinity of cleavage rather than a requirement for specific amino acid residues.

A transcriptomic and proteomic characterization of the Arabidopsis mitochondrial protein import apparatus and its response to mitochondrial dysfunction.

Mitochondria import hundreds of cytosolically synthesized proteins via the mitochondrial protein import apparatus. Expression analysis in various organs of 19 components of the Arabidopsis mitochondrial protein import apparatus encoded by 31 genes showed that although many were present in small multigene families, often only one member was prominently expressed. This was supported by comparison of real-time reverse transcriptase-polymerase chain reaction and microarray experimental data with expressed sequence tag numbers and massive parallel signature sequence data. Mass spectrometric analysis of purified mitochondria identified 17 import components, their mitochondrial sub-compartment, and verified the presence of TIM8, TIM13, TIM17, TIM23, TIM44, TIM50, and METAXIN proteins for the first time, to our knowledge. Mass spectrometry-detected isoforms correlated with the most abundant gene transcript measured by expression data. Treatment of Arabidopsis cell culture with mitochondrial electron transport chain inhibitors rotenone and antimycin A resulted in a significant increase in transcript levels of import components, with a greater increase observed for the minor isoforms. The increase was observed 12 h after treatment, indicating that it was likely a secondary response. Microarray analysis of rotenone-treated cells indicated the up-regulation of gene sets involved in mitochondrial chaperone activity, protein degradation, respiratory chain assembly, and division. The rate of protein import into isolated mitochondria from rotenone-treated cells was halved, even though rotenone had no direct effect on protein import when added to mitochondria isolated from untreated cells. These findings suggest that transcription of import component genes is induced when mitochondrial function is limited and that minor gene isoforms display a greater response than the predominant isoforms.

A plant outer mitochondrial membrane protein with high amino acid sequence identity to a chloroplast protein import receptor.

We have identified a novel protein on the outer membrane of Arabidopsis thaliana mitochondria. This protein displays 67% sequence identity with the 64 kDa translocase of the outer envelope membrane of chloroplasts (Toc). A mitochondrial localisation for this protein was determined by (i). its presence in the proteome of highly purified Arabidopsis mitochondria, (ii). Western blot analysis with antibodies to Toc64 from pea that indicate its presence in Arabidopsis and pea mitochondria, (iii). green fluorescent protein fusion proteins that indicate an exclusive mitochondrial localisation for this protein, and (iv). expression profiles in various tissue types and during development that are more similar to translocase of the outer mitochondrial membrane components than to chloroplastic Toc components. Thus Arabidopsis mitochondria contain a protein with high sequence identity to a plastid protein import receptor.

Molecular definition of the ascorbate-glutathione cycle in Arabidopsis mitochondria reveals dual targeting of antioxidant defenses in plants.

Key components of the ascorbate-glutathione cycle in Arabidopsis cell organelles are encoded by single organellar targeted isoforms that are dual localized in the chloroplast stroma and the mitochondrion. We demonstrate the presence of the ascorbate-glutathione cycle in purified Arabidopsis mitochondria using enzymatic activity, proteomic and in vitro and in vivo subcellular targeting data that identify the gene products responsible. In vitro experiments using a dual import assay assessing mitochondrial and chloroplast imports simultaneously show dual targeting of ascorbate peroxidase, monodehydroascorbate reductase, and glutathione reductase gene products to mitochondria and chloroplasts, while a putative dehydroascorbate reductase protein is only imported into mitochondria. In vivo subcellular localization using green fluorescent protein fusion proteins show clear targeting of all gene products to mitochondria. Transcript levels show these genes are induced by oxidative chemical stresses targeted to chloroplasts and/or mitochondria and are elevated during photosynthetic operation in the light. Together these data present a model of an integrated ascorbate-glutathione antioxidant defense common to plastids and mitochondria that is linked at the level of the genome in Arabidopsis.

Dual targeting ability of targeting signals is dependent on the nature of the mature protein.

The targeting ability of three signals previously shown to support the import of passenger proteins into both mitochondria and chloroplasts was investigated with authentic mitochondrial or chloroplastic proteins. An in vitro dual import assay that maintained import specificity showed that the ability of dual signals to support mitochondrial and chloroplastic import depended on the nature of the passenger protein. All dual targeting signals supported import of their native mature protein as a passenger into both mitochondria and chloroplasts. However the glutathione reductase targeting signal only supported mitochondrial import with the mitochondrial protein alternative oxidase, and chloroplast import with the small subunit of ribulose-1,5-bisphosphate carboxylase / oxygenase. The Arabidopsis histidyl-tRNA synthetase targeting signal only supported mitochondrial import with the alternative oxidase as a passenger, but the small subunit of ribulose-1,5-bisphosphate carboxylase / oxygenase was imported into both mitochondria and chloroplasts. The Arabidopsis asparaginyl-tRNA synthetase supported import of alternative oxidase and the small subunit of ribulose-1,5-bisphosphate carboxylase / oxygenase into both mitochondria and chloroplasts. Analysis of the targeting signals of all known dual targeted proteins using targeting predictions indicates that most of them are more strongly predicted to be chloroplast-targeted. Secondary structure predictions indicate the ability of most dual targeted signals to form both α-helical and ß-sheet-type structures, a feature of mitochondrial and plastid targeting signals, respectively. Thus, it appears that a major determinant of dual targeting ability is the nature of the mature or passenger protein.

Characterization of the targeting signal of dual-targeted pea glutathione reductase.

We investigated the dual targeting signal of pea glutathione reductase (GR) that had been previously shown to be capable of targeting the passenger protein phosphinothricin acetyl transferase to mitochondria and chloroplasts in vivo. We confirmed that GR was imported into mitochondria and chloroplasts in vitro. Rupture of the outer mitochondrial membrane after the import assay indicated that GR was imported into both the intermembrane space and the matrix. Changing positive and hydrophobic residues in the targeting signal we investigated if dual targeting of GR was due to an overlapping or separate signal. Overall single mutations had a greater effect on mitochondrial import compared to chloroplasts, especially those on positive residues. Precursors containing both positive and hydrophobic residue mutations (double mutants) indicated that there might be some redundancy in targeting information for chloroplastic import as double mutants had a greater effect than predicted from the single mutants. Fusion of the targeting signal to the green fluorescent protein (GFP) followed by transient transformation indicated that this signal was only capable of targeting this passenger protein to plastids. Additionally, fusion of the complete coding sequence of GR to GFP also resulted in an exclusive chloroplastic localization. Mutations in the targeting signal that reduced import into plastids in vitro also displayed altered patterns of GFP localizations in vivo. These results indicate that some residues in the signal for dual localisation of GR play a role in both mitochondrial and chloroplastic import, and thus the signal is overlapping.

A novel in vitro system for simultaneous import of precursor proteins into mitochondria and chloroplasts.

Most chloroplast and mitochondrial precursor proteins are targeted specifically to either chloroplasts or mitochondria. However, there is a group of proteins that are dual targeted to both organelles. We have developed a novel in vitro system for simultaneous import of precursor proteins into mitochondria and chloroplasts (dual import system). The mitochondrial precursor of alternative oxidase, AOX was specifically targeted only to mitochondria. The chloroplastic precursor of small subunit of pea ribulose bisphosphate carboxylase/oxygenase, Rubisco, was mistargeted to pea mitochondria in a single import system, but was imported only into chloroplasts in the dual import system. The dual targeted glutathione reductase GR precursor was targeted to both mitochondria and chloroplasts in both systems. The GR pre-sequence could support import of the mature Rubisco protein into mitochondria and chloroplasts in the single import system but only into chloroplasts in the dual import system. Although the GR pre-sequence could support import of the mature portion of the mitochondrial FAd subunit of the ATP synthase into mitochondria and chloroplasts, mature AOX protein was only imported into mitochondria under the control of the GR pre-sequence in both systems. These results show that the novel dual import system is superior to the single import system as it abolishes mistargeting of chloroplast precursors into pea mitochondria observed in a single organelle import system. The results clearly show that although the GR pre-sequence has dual targeting ability, this ability is dependent on the nature of the mature protein.