Estelle Giraud

The Absence of ALTERNATIVE OXIDASE1a in Arabidopsis Results in Acute Sensitivity to Combined Light and Drought Stress

Treatment of Arabidopsis (Arabidopsis thaliana) alternative oxidase1a (aox1a) mutant plants with moderate light under drought conditions resulted in a phenotypic difference compared with ecotype Columbia (Col-0), as evidenced by a 10-fold increase in the accumulation of anthocyanins in leaves, alterations in photosynthetic efficiency, and increased superoxide radical and reduced root growth at the early stages of seedling growth. Analysis of metabolite profiles revealed significant changes upon treatment in aox1a plants typical of combined stress treatments, and these were less pronounced or absent in Col-0 plants. These changes were accompanied by alteration in the abundance of a variety of transcripts during the stress treatment, providing a molecular fingerprint for the stress-induced phenotype of aox1a plants. Transcripts encoding proteins involved in the synthesis of anthocyanins, transcription factors, chloroplastic and mitochondrial components, cell wall synthesis, and sucrose and starch metabolism changed, indicating that effects were not confined to mitochondria, where the AOX1a protein is located. Microarray and quantitative reverse transcription-polymerase chain reaction analysis revealed that transcripts typically induced upon stress treatment or involved in antioxidant defense systems, especially chloroplast-located antioxidant defense components, had altered basal levels in untreated aox1a plants, suggesting a significant change in the basal equilibrium of signaling pathways that regulate these components. Taken together, these results indicate that aox1a plants have a greatly altered stress response even when mitochondria or the mitochondrial electron transport chain are not the primary target of the stress and that AOX1a plays a broad role in determining the normal redox balance in the cell.

Evidence for a SAL1-PAP Chloroplast Retrograde Pathway That Functions in Drought and High Light Signaling in Arabidopsis

This work describes a chloroplast-to-nucleus retrograde signaling pathway involving SAL1, a chloroplast and mitochondrial enzyme that degrades the phosphonucleotide 3′-phosphoadenosine 5′-phosphate (PAP). In the absence of SAL1 or in response to drought and high light, PAP accumulates and can move between the chloroplast and the nucleus, where it inhibits exoribonucleases, thereby inducing stress-responsive genes. Compartmentation of the eukaryotic cell requires a complex set of subcellular messages, including multiple retrograde signals from the chloroplast and mitochondria to the nucleus, to regulate gene expression. Here, we propose that one such signal is a phosphonucleotide (3′-phosphoadenosine 5′-phosphate [PAP]), which accumulates in Arabidopsis thaliana in response to drought and high light (HL) stress and that the enzyme SAL1 regulates its levels by dephosphorylating PAP to AMP. SAL1 accumulates in chloroplasts and mitochondria but not in the cytosol. sal1 mutants accumulate 20-fold more PAP without a marked change in inositol phosphate levels, demonstrating that PAP is a primary in vivo substrate. Significantly, transgenic targeting of SAL1 to either the nucleus or chloroplast of sal1 mutants lowers the total PAP levels and expression of the HL-inducible ASCORBATE PEROXIDASE2 gene. This indicates that PAP must be able to move between cellular compartments. The mode of action for PAP could be inhibition of 5′ to 3′ exoribonucleases (XRNs), as SAL1 and the nuclear XRNs modulate the expression of a similar subset of HL and drought-inducible genes, sal1 mutants accumulate XRN substrates, and PAP can inhibit yeast (Saccharomyces cerevisiae) XRNs. We propose a SAL1-PAP retrograde pathway that can alter nuclear gene expression during HL and drought stress.

The Transcription Factor ABI4 Is a Regulator of Mitochondrial Retrograde Expression of ALTERNATIVE OXIDASE1a

Plant cells integrate signals from external sources and from organelles to regulate gene expression, referred to as anterograde and retrograde signaling, respectively. Functional characterization of the promoter of ALTERNATIVE OXIDASE1a (AOX1a) from Arabidopsis (Arabidopsis thaliana), a marker for mitochondrial retrograde response, was carried out by testing the ability of the AOX1a promoter to drive expression of the reporter gene GUS. This approach identified a strong repressor element, designated the B element, that was necessary for an increased promoter activity in response to the mitochondrial complex I inhibitor rotenone. This element overlaps with a previously identified potential binding site for the transcription factor ABSCISIC ACID INSENSITIVE4 (ABI4). AOX1a promoter activity was fully derepressed in abi4 mutants and was unresponsive to rotenone. Furthermore, deletion of the B element of the AOX1a promoter resulted in increased GUS staining activity compared to the wild-type promoter in transgenic plants. Binding of the ABI4 transcription factor to this region of the AOX1a promoter was demonstrated by electromobility shift and yeast one-hybrid assays. Analysis of transcript abundance for AOX1a in abi4 mutant lines revealed significantly increased levels of AOX1a mRNA that could not be further induced by rotenone, consistent with the role of ABI4 as a repressor that is derepressed in response to rotenone. These results show that ABI4 plays a central role in mediating mitochondrial retrograde signals to induce the expression of AOX1a. Furthermore, they provide a molecular link between mitochondrial and chloroplast retrograde signaling, as ABI4 has been previously shown to act downstream of at least two chloroplast retrograde signaling pathways.

Alternative oxidase : a target and regulator of stress responses

The alternative oxidase (AOX) is found in all plants examined to date, fungi and lower invertebrates. We propose that AOX is not only part of the stress response in plants, but it also plays a central role in defining the stress response. Three lines of evidence support this proposal: (1) The absence of AOX leads to an alteration of stress defences in normal and stress conditions, (2) the expression of AOX is triggered by a variety of signals indicating that it is a common response and (3) AOX acts as a buffer that determines the threshold for the induction of programmed cell death. Therefore, AOX is not only one of many components involved in the defence response, its activity or lack of activity leads to a radical alteration of the defence equilibrium at a cellular level and thus it plays a central role in programming the stress response. This programming role of AOX can be achieved directly by its ability to suppress the induction of reactive oxygen species (ROS) and indirectly by causing changes in the energy status of cells owing to the non-phosphorylating nature of the alternative respiratory pathway. The latter is likely achieved in combination with a variety of alternative NAD(P)H dehydrogenases, that are co-regulated with AOX. Additionally, we explore the possible function of AOX as a component of the stress response beyond the plant frontier.

Physiological and Transcriptome Analysis of Iron and Phosphorus Interaction in Rice Seedlings

The antagonistic interaction between iron (Fe) and phosphorus (P) has been noted in the area of plant nutrition. To understand the physiology and molecular mechanisms of this interaction, we studied the growth performance, nutrient concentration, and gene expression profiles of root and shoot segments derived from 10-d-old rice (Oryza sativa) seedlings under four different nutrient conditions: (1) full strength of Fe and P (+Fe+P); (2) full strength of P and no Fe (−Fe+P); (3) full strength of Fe and no P (+Fe−P); and (4) without both Fe and P (−Fe−P). While removal of Fe in the growth medium resulted in very low shoot and root Fe concentrations, the chlorotic symptoms and retarded seedling growth were only observed on seedlings grown in the presence of P. Microarray data showed that in roots, 7,628 transcripts were significantly changed in abundance in the absence of Fe alone. Interestingly, many of these changes were reversed if P was also absent (−Fe−P), with only approximately 15% overlapping with –Fe alone (–Fe+P). Analysis of the soluble Fe concentration in rice seedling shoots showed that P deficiency resulted in significantly increased Fe availability within the plants. The soluble Fe concentration under –Fe–P conditions was similar to that under +Fe+P conditions. These results provide evidence that the presence of P can affect Fe availability and in turn can influence the regulation of Fe-responsive genes.

Protein transport in organelles: Dual targeting of proteins to mitochondria and chloroplasts

As many as fifty proteins have now been experimentally demonstrated to be targeted to both mitochondria and plastids, a phenomenon referred to as dual targeting. Although the first reported case of dual targeting of a protein was reported in 1995, there is still little understanding of the mechanism of dual targeting and any similarities or differences with respect to the targeting of location‐specific proteins. This minireview summarizes dual targeting in terms of signals, passenger proteins, receptors, regulation, why proteins may need to be dual targeted and the future challenges that remain in this area.

Defining the Mitochondrial Stress Response in Arabidopsis thaliana

To obtain a global overview of how mitochondria respond to stress, we aimed to define the plant mitochondrial stress response (MSR). By combining a set of 1196 Arabidopsis thaliana genes that putatively encode mitochondrial proteins with 16 microarray experiments on stress-related conditions, 45 nuclear encoded genes were defined as widely stress-responsive. Using green fluorescent protein (GFP) fusion assays, the mitochondrial targeting of a large number of these proteins was tested, confirming in total 26 proteins as mitochondrially targeted. Several of these proteins were observed to be dual targeted to mitochondria and plastids, including the small heat shock proteins sHSP23.5 and sHSP23.6. In addition to the well defined stress components of mitochondria, such as alternative oxidases, nicotinamide adenine dinucleotide (NAD(P)H) dehydrogenases, and heat shock proteins, a variety of other proteins, many with unknown function, were identified. The mitochondrial carrier protein family was over-represented in the stress-responsive genes, suggesting that stress induces altered needs for metabolite transport across the mitochondrial inner membrane. Although the genes encoding many of these proteins contain common cis-acting regulatory elements, it was apparent that a number of distinct regulatory processes or signals likely triggered the MSR. Therefore, these genes provide new model systems to study mitochondrial retrograde regulation, in addition to the widely used alternative oxidase model. Additionally, as changes in proteins responsive to stress did not correlate well with changes at a transcript level, it suggests that post-transcriptional mechanisms also play an important role in defining the MSR.

TCP transcription factors link the regulation of genes encoding mitochondrial proteins with the circadian clock in Arabidopsis thaliana.

The TCP family of transcription factors and site II promoter elements that they bind in Arabidopsis link the regulation of gene expression for mitochondrial proteins with a variety of circadian clock components to provide specific time-of-day expression for a variety of genes. Diurnal regulation of transcripts encoding proteins located in mitochondria, plastids, and peroxisomes is important for adaptation of organelle biogenesis and metabolism to meet cellular requirements. We show this regulation is related to diurnal changes in promoter activities and the presence of specific cis-acting regulatory elements in the proximal promoter region [TGGGC(C/T)], previously defined as site II elements, and leads to diurnal changes in organelle protein abundances. These site II elements can act both as activators or repressors of transcription, depending on the night/day period and on the number and arrangement of site II elements in the promoter tested. These elements bind to the TCP family of transcriptions factors in Arabidopsis thaliana, which nearly all display distinct diurnal patterns of cycling transcript abundance. TCP2, TCP3, TCP11, and TCP15 were found to interact with different components of the core circadian clock in both yeast two-hybrid and direct protein–protein interaction assays, and tcp11 and tcp15 mutant plants showed altered transcript profiles for a number of core clock components, including LATE ELONGATED HYPOCOTYL1 and PSEUDO RESPONSE REGULATOR5. Thus, site II elements in the promoter regions of genes encoding mitochondrial, plastid, and peroxisomal proteins provide a direct mechanism for the coordination of expression for genes involved in a variety of organellar functions, including energy metabolism, with the time-of-day specific needs of the organism.

A Membrane-Bound NAC Transcription Factor, ANAC017, Mediates Mitochondrial Retrograde Signaling in Arabidopsis

This work identifies a biological role for ANAC017 as an integral cellular component in mitochondrial retrograde signaling and a high-level transcriptional regulator that is necessary for H2O2-mediated primary stress responses in plants. Plants require daily coordinated regulation of energy metabolism for optimal growth and survival and therefore need to integrate cellular responses with both mitochondrial and plastid retrograde signaling. Using a forward genetic screen to characterize regulators of alternative oxidase1a (rao) mutants, we identified RAO2/Arabidopsis NAC domain-containing protein17 (ANAC017) as a direct positive regulator of AOX1a. RAO2/ANAC017 is targeted to connections and junctions in the endoplasmic reticulum (ER) and F-actin via a C-terminal transmembrane (TM) domain. A consensus rhomboid protease cleavage site is present in ANAC017 just prior to the predicted TM domain. Furthermore, addition of the rhomboid protease inhibitor N-p-Tosyl-l-Phe chloromethyl abolishes the induction of AOX1a upon antimycin A treatment. Simultaneous fluorescent tagging of ANAC017 with N-terminal red fluorescent protein (RFP) and C-terminal green fluorescent protein (GFP) revealed that the N-terminal RFP domain migrated into the nucleus, while the C-terminal GFP tag remained in the ER. Genome-wide analysis of the transcriptional network regulated by RAO2/ANAC017 under stress treatment revealed that RAO2/ANAC017 function was necessary for >85% of the changes observed as a primary response to cytosolic hydrogen peroxide (H2O2), but only ∼33% of transcriptional changes observed in response to antimycin A treatment. Plants with mutated rao2/anac017 were more stress sensitive, whereas a gain-of-function mutation resulted in plants that had lower cellular levels of H2O2 under untreated conditions.

Identification of Regulatory Pathways Controlling Gene Expression of Stress-Responsive Mitochondrial Proteins in Arabidopsis

In this study we analyzed transcript abundance and promoters of genes encoding mitochondrial proteins to identify signaling pathways that regulate stress-induced gene expression. We used Arabidopsis (Arabidopsis thaliana) alternative oxidase AOX1a, external NADP H-dehydrogenase NDB2, and two additional highly stress-responsive genes, At2g21640 and BCS1. As a starting point, the promoter region of AOX1a was analyzed and functional analysis identified 10 cis-acting regulatory elements (CAREs), which played a role in response to treatment with H2O2, rotenone, or both. Six of these elements were also functional in the NDB2 promoter. The promoter region of At2g21640, previously defined as a hallmark of oxidative stress, shared two functional CAREs with AOX1a and was responsive to treatment with H2O2 but not rotenone. Microarray analysis further supported that signaling pathways induced by H2O2 and rotenone are not identical. The promoter of BCS1 was not responsive to H2O2 or rotenone, but highly responsive to salicylic acid (SA), whereas the promoters of AOX1a and NDB2 were unresponsive to SA. Analysis of transcript abundance of these genes in a variety of defense signaling mutants confirmed that BCS1 expression is regulated in a different manner compared to AOX1a, NDB2, and At2g21640. These mutants also revealed a pathway associated with programmed cell death that regulated AOX1a in a manner distinct from the other genes. Thus, at least three distinctive pathways regulate mitochondrial stress response at a transcriptional level, an SA-dependent pathway represented by BCS1, a second pathway that represents a convergence point for signals generated by H2O2 and rotenone on multiple CAREs, some of which are shared between responsive genes, and a third pathway that acts via EDS1 and PAD4 regulating only AOX1a. Furthermore, posttranscriptional regulation accounts for changes in transcript abundance by SA treatment for some genes.