Phytohormones as Regulators of Mitochondrial Gene Expression in Arabidopsis thaliana.

The coordination of activities between nuclei and organelles in plant cells involves information exchange, in which phytohormones may play essential roles. Therefore, the dissection of the mechanisms of hormone-related integration between phytohormones and mitochondria is an important and challenging task. Here, we found that inputs from multiple hormones may cause changes in the transcript accumulation of mitochondrial-encoded genes and nuclear genes encoding mitochondrial (mt) proteins. In particular, treatments with exogenous hormones induced changes in the GUS expression in the reporter line possessing a 5'-deletion fragment of the RPOTmp promoter. These changes corresponded in part to the up- or downregulation of RPOTmp in wild-type plants, which affects the transcription of mt-encoded genes, implying that the promoter fragment of the RPOTmp gene is functionally involved in the responses to IAA (indole-3-acetic acid), ACC (1-aminocyclopropane-1-carboxylic acid), and ABA (abscisic acid). Hormone-dependent modulations in the expression of mt-encoded genes can also be mediated through mitochondrial transcription termination factors 15, 17, and 18 of the mTERF family and genes for tetratricopeptide repeat proteins that are coexpressed with mTERF genes, in addition to SWIB5 encoding a mitochondrial SWI/SNF (nucleosome remodeling) complex B protein. These genes specifically respond to hormone treatment, displaying both negative and positive regulation in a context-dependent manner. According to bioinformatic resources, their promoter region possesses putative cis-acting elements involved in responses to phytohormones. Alternatively, the hormone-related transcriptional activity of these genes may be modulated indirectly, which is especially relevant for brassinosteroids (BS). In general, the results of this study indicate that hormones are essential mediators that are able to cause alterations in the transcript accumulation of mt-related nuclear genes, which, in turn, trigger the expression of mt genes.

Effects of drought stress memory on the accumulation of stress-protective compounds in naturally grown pine and spruce.

Forest trees are subjected to multiple stressors during their long lifetime and therefore require effective and finely regulated stress-protective systems. Stressors can induce protective systems either directly or with the involvement of stress memory mechanisms. Stress memory has only begun to be uncovered in model plants and is unexplored in coniferous species. Therefore, we studied the possible role of stress memory in the regulation of the accumulation of stress-protective compounds (heat shock proteins, dehydrins, proline) in the needles of naturally grown Scots pine and Norway spruce trees subjected to the subsequent action of long-term (multiyear) and short-term (seasonal) water shortages. Although the water deficit was relatively mild, it significantly influenced the pattern of expression of stress memory-related heat shock factor (HSF) and SWI/SNF genes, indicating the formation of stress memory in both species. In spruce, dehydrin accumulation was increased by water shortage in a manner compatible with Type II stress memory. The accumulation of HSP40 in spruce needles was positively influenced by long-term water shortage, but this increase was unlikely to be of biological importance due to the concomitant decrease in HSP70, HSP90 and HSP101 accumulation. Finally, proline accumulation was negatively influenced by short-term water deficit in spruce. In pine, no one protective compound accumulated in response to water stress. Taken together, the results indicate that the accumulation of stress-protective compounds was generally independent of stress memory effects both in pine and in spruce.

Independent Responses of Photosynthesis and Plant Morphology to Alterations of PIF Proteins and Light-Dependent MicroRNA Contents in Arabidopsis thaliana pif Mutants Grown under Lights of Different Spectral Compositions.

The effects of the quality of light on the content of phytochrome interacting factors (PIFs) such as PIF3, PIF4 and PIF5, as well as the expression of various light-dependent microRNAs, in adult Arabidopsis thaliana pif mutant plants (pif4, pif5, pif3pif5, pif4pif5, pif3pif4pif5) were studied. We demonstrate that under blue light, the pif4 mutant had maximal expression of most of the studied microRNAs (miR163, miR319, miR398, miR408, miR833) when the PIF4 protein in plants was reduced. This finding indicates that the PIF4 protein is involved in the downregulation of this group of microRNAs. This assumption is additionally confirmed by the fact that under the RL spectrum in pif5 mutants, practically the same miRNAs decrease expression against the background of an increase in the amount of PIF4 protein. Unlike the WT and other mutants, the pif4 mutant responded to the BL spectrum not only by activating the expression of light-dependent miRNAs, but also by a significant increase in the expression of transcription factors and key light signalling genes. These molecular reactions do not affect the activity of photosynthesis but may be involved in the formation of a light quality-dependent phenotype.

The role of PAP4/FSD3 and PAP9/FSD2 in heat stress responses of chloroplast genes.

Chloroplasts' mechanisms of adaptation to elevated temperatures are largely determined by the gene expression of the plastid transcription apparatus. Gene disruption of iron-containing superoxide dismutase PAP4/FSD3 and PAP9/FSD2, which are parts of the DNA-RNA polymerase complex of plastids, contributed to a decrease in resistance to oxidative stress caused by the prolonged action of elevated temperatures (5 days, 30 °C). Under heat stress conditions, pap4/fsd3 and pap9/fsd2 mutants showed a decline in chlorophyll content and photosynthesis level, as measured by photosynthetic parameters, and a different amplitude of HSP gene response to heat stress. The expression of nuclear- and plastid-encoded photosynthesis genes and corresponding proteins was strongly inhibited in the mutants as compared with wild-type plants and was further suppressed or displayed no additional changes at 30 °C. NEP-dependent plastid genes, as well as NEP genes RPOTp and RPOTmp, were also downregulated in the mutants by high temperature or remained stable, unlike in wild-type seedlings where these genes were strongly upregulated. The results obtained correspond to the concept of the complex effect of various forms of reactive oxygen species under all types of stresses, including heat stress, and confirm the hypothesis of a new regulatory function in plastid transcription acquired by enzymatic proteins during evolution.

Quantitative analysis of differential dehydrin regulation in pine and spruce seedlings under water deficit.

Dehydrins are well-known components of plant responses to different stresses that cause dehydration, including drought, freezing, salinity, etc. In conifers, the dehydrin gene family is very large, implying that the members of this family have important physiological functions in conifer stress tolerance. However, dehydrin gene expression displays a wide range of responses to stress, from thousand-fold increased expression to decreased expression, and it is generally unknown how regulatory systems are connected at the mRNA and protein levels. Therefore, we studied these aspects of dehydrin regulation in Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies (L.) H. Karst) seedlings under polyethylene glycol 6000-induced osmotic stress ranging from relatively low (culture medium water potential of -0.15 MPa) to very high (-1.0 MPa) intensities. In pine, the major dehydrin protein was Dhn1 in both the roots and needles, and in spruce, two isoforms of the Dhn4 protein were the major dehydrins; both of these proteins are AESK-type dehydrins. The genes encoding these major proteins were highly expressed even under control conditions; surprisingly, we also observed several highly expressed dehydrin genes that were not abundantly translated. Under osmotic stress, the most prominent expression changes were observed for the dehydrin genes with low basal expression levels, whereas highly expressed genes generally demonstrated rather modest changes in expression. We report proposed constitutive physiological functions of the AESK-type dehydrins in Pinaceae plants.

Cytokinin-Regulated Expression of Arabidopsis thaliana PAP Genes and Its Implication for the Expression of Chloroplast-Encoded Genes.

Cytokinins (CKs) are known to regulate the biogenesis of chloroplasts under changing environmental conditions and at different stages of plant ontogenesis. However, the underlying mechanisms are still poorly understood. Apparently, the mechanisms can be duplicated in several ways, including the influence of nuclear genes that determine the expression of plastome through the two-component CK regulatory circuit. In this study, we evaluated the role of cytokinins and CK signaling pathway on the expression of nuclear genes for plastid RNA polymerase-associated proteins (PAPs). Cytokinin induced the expression of all twelve Arabidopsis thalianaPAP genes irrespective of their functions via canonical CK signaling pathway but this regulation might be indirect taking into consideration their different functions and versatile structure of promoter regions. The disruption of PAP genes contributed to the abolishment of positive CK effect on the accumulation of the chloroplast gene transcripts and transcripts of the nuclear genes for plastid transcription machinery as can be judged from the analysis of pap1 and pap6 mutants. However, the CK regulatory circuit in the mutants remained practically unperturbed. Knock-out of PAP genes resulted in cytokinin overproduction as a consequence of the strong up-regulation of the genes for CK synthesis.

Melatonin modifies the expression of the genes for nuclear- and plastid-encoded chloroplast proteins in detached Arabidopsis leaves exposed to photooxidative stress.

Melatonin, a potent regulator during plant development and stress responses, affects diverse plastid-related processes. However, its role in the regulation of plastid gene expression is largely unknown. In this study, exogenous melatonin was shown to reduce the negative influence of excess light by increasing the efficiency of the photosystems and rearranging the expression of chloroplast- and nuclear-encoded genes in detached Arabidopsis leaves. The positive effects of melatonin predominantly occurred at lower concentrations, while high doses had an inhibitory effect. The impact of melatonin was not straightforward. It mainly influenced the expression of the genes encoding the chloroplast transcription machinery and housekeeping genes involved in maintaining transcriptional activity and the functional state of chloroplasts. Despite the fact that melatonin treatment improved photosynthetic parameters, the levels of photosynthesis gene transcripts and photosynthetic proteins remained practically unaltered suggesting that melatonin impact on photosynthetic apparatus which would allow the balancing of chloroplast functions with stress responses is highly complicated.

Role of the PB-loop in ApcE and phycobilisome core function in cyanobacterium Synechocystis sp. PCC 6803.

The phycobilisome (PBS) is a giant highly-structured pigment-protein antenna of cyanobacteria and red algae. PBS is composed of the phycobiliproteins and several linker polypeptides. The large core-membrane linker protein (LCM or ApcE) influences many features and functions of PBS and consists of several domains including the chromophorylated PB-domain. Being homologous to the phycobiliprotein α-subunits this domain includes a so-called PB-loop insertion whose functions are still unknown. We have created the photoautotrophic mutant strain of the cyanobacterium Synechocystis sp. PCC 6803 with lacking PB-loop. Using various spectral techniques we have demonstrated that this mutation does not destroy the PBS integrity and the internal PBS excitation energy transfer pathways. At the same time, the deletion of the PB-loop leads to the decrease of connectivity between the PBS and thylakoid membrane and to the compensatory increase of the relative photosystem II content. Mutation provokes the violation of the thylakoid membranes arrangement, the inability to perform state transitions, and diminishing of the OCP-dependent non-photochemical PBS quenching. In essence, even such a minute mutation of the PBS polypeptide component, like the PB-loop deletion, becomes important for the concerted function of the photosynthetic apparatus.

Differential impact of heat stress on the expression of chloroplast-encoded genes.

Heat shock is one of the major abiotic factors that causes severe retardation in plant growth and development. To dissect the principal effects of hyperthermia on chloroplast gene expression, we studied the temporal dynamics of transcript accumulation for chloroplast-encoded genes in Arabidopsis thaliana and genes for the chloroplast transcription machinery against a background of changes in physiological parameters. A marked reduction in the transcript amounts of the majority of the genes at the early phases of heat shock (HS) was followed by a return to the baseline levels of rbcL and the housekeeping genes clpP, accD, rps14 and rrn16. The decline in the mRNA levels of trnE (for tRNAglu) and the PSI genes psaA and psaB was opposed by the transient increase in the transcript accumulation of ndhF and the PSII genes psbA, psbD, and psbN and their subsequent reduction with the development of stress. However, the up-regulation of PSII genes in response to elevated temperature was absent in the heat stress-sensitive mutants abi1 and abi2 with the impaired degradation of D2 protein. The expression of rpoA and rpoB, which encode subunits of PEP, was strongly down-regulated throughout the duration of the heat treatment. In addition, heat stress-induced PEP deficiency caused the compensatory up-regulation of the genes for the nuclear-encoded RNA polymerases RPOTp and RPOTmp, the PEP-associated proteins PAP6 and PAP8, the Ser/Thr protein kinase cPCK2, and the stress-inducible sigma factor gene SIG5. Thus, heat stress differentially modulates the transcript accumulation of plastid-encoded genes in A. thaliana at least in part via the expression of HS-responsive nuclear genes for the plastid transcription machinery.

Distinctive types of ATP-dependent Clp proteases in cyanobacteria.

Cyanobacteria are the only prokaryotes that perform oxygenic photosynthesis and are thought to be ancestors to plant chloroplasts. Like chloroplasts, cyanobacteria possess a diverse array of proteolytic enzymes, with one of the most prominent being the ATP-dependent Ser-type Clp protease. The model Clp protease in Escherichia coli consists of a single ClpP proteolytic core flanked on one or both ends by a HSP100 chaperone partner. In comparison, cyanobacteria have multiple ClpP paralogs plus a ClpP variant (ClpR), which lacks the catalytic triad typical of Ser-type proteases. In this study, we reveal that two distinct soluble Clp proteases exist in the unicellular cyanobacterium Synechococcus elongatus. Each protease consists of a unique proteolytic core comprised of two separate Clp subunits, one with ClpP1 and ClpP2, the other with ClpP3 and ClpR. Each core also associates with a particular HSP100 chaperone partner, ClpC in the case of the ClpP3/R core, and ClpX for the ClpP1/P2 core. The two adaptor proteins, ClpS1 and ClpS2 also interact with the ClpC chaperone protein, likely increasing the range of protein substrates targeted by the Clp protease in cyanobacteria. We also reveal the possible existence of a third Clp protease in Synechococcus, one which associates with the internal membrane network. Altogether, we show that presence of several distinctive Clp proteases in cyanobacteria, a feature which contrasts from that in most other organisms.

The gene complement for proteolysis in the cyanobacterium Synechocystis sp. PCC 6803 and Arabidopsis thaliana chloroplasts.

A set of 62 genes that encode the entire peptidase complement of Synechocystis sp. PCC 6803 has been identified in the genome database of that cyanobacterium. Sequence comparisons with the Arabidopsis genome uncovered the presumably homologous chloroplast components inherited from their cyanobacterial ancestor. A systematic gene disruption approach was chosen to individually inactivate, by customary transformation strategies, the majority of the cyanobacterial genes encoding peptidase subunits that are related to chloroplast enzymes. This allowed classification of the peptidases that are required for cell viability or are involved in specific stress responses. The comparative analysis between Synechocystis and Arabidopsis chloroplast peptidases showed that: (1) homologous enzymes that arose by gene duplications in cyanobacteria are functionally diverse and frequently do not complement each other, (2) the chloroplast appears to house a number of distinct peptidase polypeptide chains of cyanobacterial origin (49) which is comparable with a cyanobacterial cell (62) and (3) the peptidase complement in plastids results from a combination of the loss of some cyanobacterial peptidases and the gain or diversification of subclasses of peptidases. This reorganization in the pattern of proteolytic enzymes may reflect distinct environmental and physiological changes between prokaryotic and organellar systems.