Isolation of intact and pure chloroplasts from leaves of Arabidopsis thaliana plants acclimated to low irradiance for studies on Rubisco regulation

A protocol is presented for low-cost and fast isolation of intact and pure chloroplasts from leaves of plants acclimated to low irradiance. The protocol is based on a differential centrifugation of cleared leaf homogenate and omits a centrifugation on Percoll gradient step. The intactness and purity of the chloroplasts isolated from leaves of low irradiance-acclimated plants by using this protocol (confirmed by phase contrast microscopy as well as enzymatic and immunological approaches) allows plausible studies on low irradiance-dependent Rubisco regulation

Post-translational Modifications in Regulation of Chloroplast Function: Recent Advances

Post-translational modifications (PTMs) of proteins enable fast modulation of protein function in response to metabolic and environmental changes. Phosphorylation is known to play a major role in regulating distribution of light energy between the Photosystems (PS) I and II (state transitions) and in PSII repair cycle. In addition, thioredoxin-mediated redox regulation of Calvin cycle enzymes has been shown to determine the efficiency of carbon assimilation. Besides these well characterized modifications, recent methodological progress has enabled identification of numerous other types of PTMs in various plant compartments, including chloroplasts. To date, at least N-terminal and Lys acetylation, Lys methylation, Tyr nitration and S-nitrosylation, glutathionylation, sumoylation and glycosylation of chloroplast proteins have been described. These modifications impact DNA replication, control transcriptional efficiency, regulate translational machinery and affect metabolic activities within the chloroplast. Moreover, light reactions of photosynthesis as well as carbon assimilation are regulated at multiple levels by a number of PTMs. It is likely that future studies will reveal new metabolic pathways to be regulated by PTMs as well as detailed molecular mechanisms of PTM-mediated regulation

Gel-based proteomic map of Arabidopsis thaliana root plastids and mitochondria

Background Non-photosynthetic plastids of plants are known to be involved in a range of metabolic and biosynthetic reactions, even if they have been difficult to study due to their small size and lack of color. The morphology of root plastids is heterogeneous and also the plastid size, density and subcellular distribution varies depending on the cell type and developmental stage, and therefore the functional features have remained obscure. Although the root plastid proteome is likely to reveal specific functional features,Arabidopsis thalianaroot plastid proteome has not been studied to date. Results In the present study, we separated Arabidopsis root protein fraction enriched with plastids and mitochondria by 2D-PAGE and identified 84 plastid-targeted and 77 mitochondrion-targeted proteins using LC-MS/MS. The most prevalent root plastid protein categories represented amino acid biosynthesis, carbohydrate metabolism and lipid biosynthesis pathways, while the enzymes involved in starch and sucrose metabolism were not detected. Mitochondrion-targeted proteins were classified mainly into the energetics category. Conclusions This is the first study presenting gel-based map ofArabidopsis thalianaroot plastid and mitochondrial proteome. Our findings suggest that Arabidopsis root plastids have broad biosynthetic capacity, and that they do not play a major role in a long-term storage of carbohydrates. The proteomic map provides a tool for further studies to compare changes in the proteome, e.g. in response to environmental cues, and emphasizes the role of root plastids in nitrogen and sulfur metabolism as well as in amino acid and fatty acid biosynthesis. The results enable taking a first step towards an integrated view of root plastid/mitochondrial proteome and metabolic functions inArabidopsis thalianaroots

Characterization of the complete chloroplast genome of Pinus uliginosa (Neumann) from the Pinus mugo complex

Taxonomic status of endangered peat-bog pine, Pinus uliginosa (Neumann) classified within the Pinus mugo complex, still remains to be elucidated. Here we present a complete chloroplast genome of P. uliginosa, to aid resolve its complex systematical position. The total genome size was 119,877 bp in length and contained a total of 112 genes, including 73 protein-coding genes, 35 tRNAs, and four rRNAs. The most of genes occur as a single copy. Five tRNA genes were duplicated from two to four times. Eighteen genes contain one intron, with a single gene containing two introns. No large inverted repeats were identified. The overall G + C content of P. uliginosa chloroplast genome is 38.5%

Genetic portrait of polyamine transporters in barley: insights in the regulation of leaf senescence

Nitrogen (N) is one of the most expensive nutrients to supply, therefore, improving the efficiency of N use is essential to reduce the cost of commercial fertilization in plant production. Since cells cannot store reduced N as NH3 or NH4+, polyamines (PAs), the low molecular weight aliphatic nitrogenous bases, are important N storage compounds in plants. Manipulating polyamines may provide a method to increase nitrogen remobilization efficiency. Homeostasis of PAs is maintained by intricate multiple feedback mechanisms at the level of biosynthesis, catabolism, efflux, and uptake. The molecular characterization of the PA uptake transporter (PUT) in most crop plants remains largely unknown, and knowledge of polyamine exporters in plants is lacking. Bi-directional amino acid transporters (BATs) have been recently suggested as possible PAs exporters for Arabidopsis and rice, however, detailed characterization of these genes in crops is missing. This report describes the first systematic study to comprehensively analyze PA transporters in barley (Hordeum vulgare, Hv), specifically the PUT and BAT gene families. Here, seven PUTs (HvPUT1-7) and six BATs (HvBAT1-6) genes were identified as PA transporters in the barley genome and the detailed characterization of these HvPUT and HvBAT genes and proteins is provided. Homology modeling of all studied PA transporters provided 3D structures prediction of the proteins of interest with high accuracy. Moreover, molecular docking studies provided insights into the PA-binding pockets of HvPUTs and HvBATs facilitating improved understanding of the mechanisms and interactions involved in HvPUT/HvBAT-mediated transport of PAs. We also examined the physiochemical characteristics of PA transporters and discuss the function of PA transporters in barley development, and how they help barley respond to stress, with a particular emphasis on leaf senescence. Insights gained here could lead to improved barley production via modulation of polyamine homeostasis

Root-type ferredoxin-NADP(+) oxidoreductase isoforms in Arabidopsis thaliana : Expression patterns, location and stress responses

In Arabidopsis, two leaf-type ferredoxin-NADP(+) oxidoreductase (LFNR) isoforms function in photosynthetic electron flow in reduction of NADP(+), while two root-type FNR (RFNR) isoforms catalyse reduction of ferredoxin in non-photosynthetic plastids. As the key to understanding, the function of RFNRs might lie in their spatial and temporal distribution in different plant tissues and cell types, we examined expression of RFNR1 and RFNR2 genes using beta-glucuronidase (GUS) reporter lines and investigated accumulation of distinct RFNR isoforms using a GFP approach and Western blotting upon various stresses. We show that while RFNR1 promoter is active in leaf veins, root tips and in the stele of roots, RFNR2 promoter activity is present in leaf tips and root stele, epidermis and cortex. RFNR1 protein accumulates as a soluble protein within the plastids of root stele cells, while RFNR2 is mainly present in the outer root layers. Ozone treatment of plants enhanced accumulation of RFNR1, whereas low temperature treatment specifically affected RFNR2 accumulation in roots. We further discuss the physiological roles of RFNR1 and RFNR2 based on characterization of rfnr1 and rfnr2 knock-out plants and show that although the function of these proteins is partly redundant, the RFNR proteins are essential for plant development and survival.Peer reviewe

Root-type ferredoxin-NADP(+) oxidoreductase isoforms in Arabidopsis thaliana: Expression patterns, location and stress responses

In Arabidopsis, two leaf-type ferredoxin-NADP(+) oxidoreductase (LFNR) isoforms function in photosynthetic electron flow in reduction of NADP(+), while two root-type FNR (RFNR) isoforms catalyse reduction of ferredoxin in non-photosynthetic plastids. As the key to understanding, the function of RFNRs might lie in their spatial and temporal distribution in different plant tissues and cell types, we examined expression of RFNR1 and RFNR2 genes using beta-glucuronidase (GUS) reporter lines and investigated accumulation of distinct RFNR isoforms using a GFP approach and Western blotting upon various stresses. We show that while RFNR1 promoter is active in leaf veins, root tips and in the stele of roots, RFNR2 promoter activity is present in leaf tips and root stele, epidermis and cortex. RFNR1 protein accumulates as a soluble protein within the plastids of root stele cells, while RFNR2 is mainly present in the outer root layers. Ozone treatment of plants enhanced accumulation of RFNR1, whereas low temperature treatment specifically affected RFNR2 accumulation in roots. We further discuss the physiological roles of RFNR1 and RFNR2 based on characterization of rfnr1 and rfnr2 knock-out plants and show that although the function of these proteins is partly redundant, the RFNR proteins are essential for plant development and survival.</p

Regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in response to the exposure of moderate irradiance-grown Arabidopsis thaliana plants to low irradiance

Wydział BiologiiRubisco jest dwufunkcyjnym enzymem, zaangażowanym w fazę ciemną fotosyntezy oraz w cykl fotorespiracyjny. U roślin wyższych dojrzała cząstka enzymu zbudowana jest z ośmiu dużych (LS) oraz ośmiu małych (SS) podjednostek. Rubisco podlega precyzyjnej regulacji w trakcie rozwoju ontogenetycznego roślin oraz pod wpływem zmian warunków środowiskowych. Mechanizmy regulacyjne mogą oddziaływać zarówno poprzez zmiany zawartości białka, jak i wpływać na aktywność enzymatyczną Rubisco. W pracy doktorskiej wykorzystano rośliny A. thaliana, które po hodowli w warunkach kontaktu ze światłem o umiarkowanym natężeniu poddawane były ekspozycji na światło o niskim natężeniu. W takim układzie doświadczalnym w ciągu 24 h dochodziło do silnego spadku zawartości LS Rubisco, któremu towarzyszyło masowe pojawianie się agregatów białkowych z udziałem LS- prawodopodobnie na skutek modyfikacji oksydacyjnych LS wywoływanych przez 1O2. W trakcie ekspozycji na nowe warunki świetlne dochodziło także do redukcji aktywności karboksylacyjnej Rubisco. Przywrócenie kontaktu roślin ze świtałem o umiarkowanym natężeniu prowadziło do zaniku agregatów, prawie całkowitego odtworzenia początkowej zawartości LS- Rubisco, a także przywrócenia wyjściowej aktywności karboksylacyjnej enzymu. Prawdopodobny sens fizjologiczny obserwowanych zmian zawartości i aktywności karboksylacyjnej Rubisco polega na przejściowym i odwracalnym wyeliminowaniu określonej puli cząstek Rubisco, które stają się funkcjonalnie nadmiarowe w warunkach kontaktu ze światłem o niskim natężeniu.Rubisco is a bifunctional enzyme that catalyzes two competing reactions, namely photosynthetic CO2 assimilation and photorespiratory carbon oxidation. In terrestrial plants Rubisco exists as a holoenzyme composed of eight large (LS) and eight small subunits (SS). Rubisco is highly regulated in response to fluctuations in the environment both by the changes in amount and enzymatic activity. However, no complex data are available on Rubisco regulatory mechanisms triggered in plants which are submitted to moderate–low irradiance shift. In present study, moderate irradiance-grown A. thaliana plants were exposed to low irradiance conditions. The moderate– low irradiance shift for a single photoperiod caused the exclusion of a certain pool of Rubisco under altered conditions owing to oxidative modifications resulting in the formation of protein aggregates involving Rubisco-LS. As a result, Rubisco carboxylase activity was reduced. The results of the determination of reactive oxygen species indicated that a moderate-low irradiance transition had stimulated 1O2 accumulation, and probably the Rubisco oxidative modifications leading to formation of aggregates encompassing Rubisco-LS were triggered by 1O2. When moderate irradiance regime was resumed, the majority of Rubisco-LS containing aggregates tended to be resolubilized, and this allowed Rubisco carboxylation activities to be largely recovered

PEP444c encoded within the MIR444c gene regulates microRNA444c accumulation in barley

Abstract MicroRNAs are small, noncoding RNA molecules that regulate the expression of their target genes. The MIR444 gene family is present exclusively in monocotyledons, and microRNAs444 from this family have been shown to target certain MADS-box transcription factors in rice and barley. We identified three barley MIR444 (MIR444a/b/c) genes and comprehensively characterised their structure and the processing pattern of the primary transcripts (pri-miRNAs444). Pri-microRNAs444 undergo extensive alternative splicing, generating functional and nonfunctional pri-miRNA444 isoforms. We show that barley pri-miRNAs444 contain numerous open reading frames (ORFs) whose transcripts associate with ribosomes. Using specific antibodies, we provide evidence that selected ORFs encoding PEP444a within MIR444a and PEP444c within MIR444c are expressed in barley plants. Moreover, we demonstrate that CRISPR-associated endonuclease 9 (Cas9)-mediated mutagenesis of the PEP444c-encoding sequence results in a decreased level of PEP444 transcript in barley shoots and roots and a 5-fold reduced level of mature microRNA444c in roots. Our observations suggest that PEP444c encoded by the MIR444c gene is involved in microRNA444c biogenesis in barley