Effects of Ce^(3+) on improvement of spectral characteristics and function of chloroplasts damaged by linolenic acid in spinach

Linolenic acid has great effects on the structure and function of chloroplast. We studied the effects of Ce3＋ on the improvement of chloroplast spectral characteristics and oxygen evolution damaged by linolenic acid in spinach. Results showed that Ce3＋ could decrease the light absorption increased by linolenic acid and promote the distribution of excitation energy to PS II and alleviate the decrease of PS Ⅱ fluo- rescence yield caused by linolenic acid. The linolenic acid treatments in various concentrations reduced the oxygen-evolving rate of chloroplasts, but the rate was accelerated since adding Ce3＋.

Exogenous GSH protects tomatoes against salt stress by modulating photosystem Ⅱ efficiency, absorbed light allocation and H2O2-scavenging system in chloroplasts

The effects of exogenous GSH（reduced glutathione）on photosynthetic characteristics,photosystem Ⅱ efficiency,absorbed light energy allocation and the H2O2-scavenging system in chloroplasts of salt-stressed tomato（Solanum lycopersicum L.）seedlings were studied using hydroponic experiments in a greenhouse.Application of exogenous GSH ameliorated saline-induced growth inhibition,the disturbed balance of Na＋ and Cl- ions and Na＋/K＋ ratios,and the reduction of the net photosynthetic rate（Pn）.GSH also increased the maximal photochemical efficiency of PSⅡ（Fv/Fm）,the electron transport rate（ETR）,the photochemical quenching coefficient（qP）,and the non-photochemical quenching coefficient（NPQ）.In addition,GSH application increased the photochemical quantum yield（Y（Ⅱ））and relative deviation from full balance between the photosystems（β/α-1）and decreased the PSⅡ excitation pressure（1-qP）and quantum yield of non-regulated energy dissipation（Y（NO））in leaves of salt-stressed tomatoes without BSO（L-buthionine-sulfoximine,an inhibitor of key GSH synthesis enzymeγ-glutamylcysteine synthetase）or with BSO.Further,the addition of GSH depressed the accumulation of H2O2 and malondialdehyde（MDA）,induced the redistribution of absorbed light energy in PSⅡ reaction centers,and improved the endogenous GSH content,GSH/GSSH ratio and activities of H2O2-scavenging enzymes（including superoxidase dismutase（SOD）,catalase（CAT）,peroxidase（POD）and key enzymes in the AsA-GSH cycle and Grx system）in the chloroplasts of salt-stressed plants with or without BSO.Therefore,GSH application alleviates inhibition of salt-induced growth and photosynthesis mainly by overcoming stomatal limitations,improving the PSⅡ efficiency,and balancing the uneven distribution of light energy to reduce the risk of ROS generation and to mediate chloroplast redox homeostasis and the antioxidant defense system to protect the chloroplasts from oxidative damage.Thus,GSH may be used as a potential tool for alleviating salt stress in tomato plants.

Isolation of Chloroplasts from Marine Microalga Isochrysis galbana Parke for Their Lipid Composition Analysis

Marine microalga Isochrysis galbana is an important feed species with a high nutritional value.Different from other uni-cellular algae,its cell contains two chloroplasts which are the major sites for lipid synthesis.Here,we optimized a chloroplast isola-tion approach suitable for the isolation of I.galbana chloroplasts and determined the purity and integrity of the isolated chloroplasts through microscopic observations and enzyme activity assay.The chloroplast lipids were analyzed with a ultrahigh-performance li-quid chromatography-Q Exactive Orbitrap-mass spectrometry.This newly developed isolation approach is simple and reliable to isolate chloroplasts with high integrity and purity.The average yield of intact chloroplasts was 15.3%±0.1%.Glycolipids and acyl-glycerols were the main chloroplast lipids.Glycolipids accounted for 56.6%of chloroplast lipid.Digalactosyldiacylglycerol(DGDG),monogalactosyldiacylglycerol(MGDG)and sulfoquinovosyldiacylglycerol(SQDG)were the main glyceroglycolipids.The fatty acyl R1/R2 were mostly 18:4/16:1,18:3/16:1 and 18:4/18:5 in DGDGs,14:0/18:4,18:4/18:5,18:4/18:4 and 18:3/18:4 in MGDGs and 16:0/14:0,16:0/18:3,and 18:4/18:3 in SQDGs.In addition,diacylglycerol(DAG)was the most abundant acylglycerols;the content of 22:6/18:4-DAG was the highest.There was a little amount of glycosphingolipid(GSL)in chloroplast.Digalactosylmonoglyceride(DGMG),monogalactosylmonoglyceride(MGMG),sulfoquinovosylmonoacylglycerol(SQMG),monoglyceride(MAG),phospholi-pids(PLs),ceramide(Cer)and betaine lipids were nearly undetectable in chloroplast.The fatty acid proportions of DGDGs,MGDGs,SQDGs,DAGs,triglycerides(TAGs)and GSLs were either higher or lower than or similar to those of whole-cell.Collectively,our isolation approach is applicable to many aspects of chloroplast biology,and may offer a reference for the isolation of chloroplasts from other marine microalgae.

Phylogenomic analysis of transcriptomic sequences of mitochondria and chloroplasts for marine red algae(Rhodophyta)in China

The chloroplast and mitochondrion of red algae （Phylum Rhodophyta） may have originated from different endosymbiosis. In this study, we carried out phylogenomic analysis to distinguish their evolutionary lin-eages by using red algal RNA-seq datasets of the 1 000 Plants （1KP） Project and publicly available complete genomes of mitochondria and chloroplasts of Rhodophyta. We have found that red algae were divided into three clades of orders, Florideophyceae, Bangiophyceae and Cyanidiophyceae. Taxonomy resolution for Class Florideophyceae showed that Order Gigartinales was close to Order Halymeniales, while Order Graci-lariales was in a clade of Order Ceramials. We confirmed Prionitis divaricata （Family Halymeniaceae） was closely related to the clade of Order Gracilariales, rather than to genus Grateloupia of Order Halymeniales as reported before. Furthermore, we found both mitochondrial and chloroplastic genes in Rhodophyta under negative selection （Ka/Ks〈1）, suggesting that red algae, as one primitive group of eukaryotic algae, might share joint evolutionary history with these two organelles for a long time, although we identified some dif-ferences in their phylogenetic trees. Our analysis provided the basic phylogenetic relationships of red algae, and demonstrated their potential ability to study endosymbiotic events.

Phylogenomic analysis of transcriptomic sequences of mitochondria and chloroplasts of essential brown algae(Phaeophyceae)in China

The chloroplast and mitochondrion of brown algae （Class Phaeophyceae of Phylum Ochrophyta） may have originated from different endosymbiosis. In this study, we carried out phylogenomic analysis to distinguish their evolutionary lineages by using algal RNA-seq datasets of the 1 000 Plants （1KP） Project and publicly available complete genomes of mitochondria and chloroplasts of Kingdom Chromista. We have found that there is a split between Class Phaeophyceae of Phylum Ochrophyta and the others （Phylum Cryptophyta and Haptophyta） in Kingdom Chromista, and identified more diversity in chloroplast genes than mitochondrial ones in their phylogenetic trees. Taxonomy resolution for Class Phaeophyceae showed that it was divided into Laminariales-Ectocarpales clade and Fucales clade, and phylogenetic positions of Kjellmaniella crassi-folia, Hizikia fusifrome and Ishige okamurai were confirmed. Our analysis provided the basic phylogenetic relationships of Chromista algae, and demonstrated their potential ability to study endosymbiotic events.

Efficient transcription of the larvicidal <i>cry</i>4<i>Ba</i>gene from <i>Bacillus thuringiensis</i>in transgenic chloroplasts of the green algal <i>Chlamydomonas reinhardtii</i>

Unicellular micro-alga Chlamydomonas reinhardtii has been recognized as a promising host for expressing recombinant proteins albeit its limited utility due to low levels of heterologous protein expression. Here, transcription of the 3.4-kb mosquito-larvicidal cry4Ba gene from Bacillus thuringiensis in transgenic C. reinhardtii chloroplasts under control of the promoter and 5’-untranslated region of photosynthetic psbA gene was accomplished. Inverted repeats in chloroplast genomes of the host strain with deleted endogenous psbA genes were selected as recombination targets. Two transformant lines were obtained by dual-phenotypic screening via exhibition of resistance to spectinomycin and restoration of photosynthetic activity. Stable and site-specific integration of intact cry4Ba and psbA genes into chloroplast genomes found in both transgenic lines implied homoplasmy of organelle populations. Achievement in cotranscription of cry4Ba and psbA transgenes revealed by RT-PCR and Northern blot analyses demonstrates the sufficiency of this system’s transcription machinery, offering the further innovation for insecticidal protein production.

Chloroplasts prevent precocious flowering through a GOLDEN2-LIKE–B-BOX DOMAIN PROTEIN module

The timing of flowering is tightly controlled by signals that integrate environmental and endogenous cues.Sugars produced by carbon fixation in the chloroplast are a crucial endogenous cue for floral initiation.Chloroplasts also convey information directly to the nucleus through retrograde signaling to control plant growth and development.Here,we show that mutants defective in chlorophyll biosynthesis and chloroplast development flowered early,especially under long-day conditions,although low sugar accumulation was seen in some mutants.Plants treated with the bleaching herbicide norflurazon also flowered early,suggesting that chloroplasts have a role in floral repression.Among retrograde signaling mutants,the golden2-like 1(glk1)glk2 double mutants showed early flowering under long-day conditions.This early flowering was completely suppressed by constans(co)and flowering locus t(ft)mutations.Leaf vascular-specific knockdown of both GLK1 and GLK2 phenocopied the glk1 glk2 mutants.GLK1 and GLK2 repress flowering by directly activating the expression of B-BOX DOMAIN PROTEIN 14(BBX14),BBX15,and BBX16 via CCAATC cis-elements in the BBX genes.BBX14/15/16 physically interact with CO in the nucleus,and expression of BBXs hampered CO-mediated FT transcription.Simultaneous knockdown of BBX14/15/16 by artificial miRNA(35S::amiR-BBX14/15/16)caused early flowering with increased FT transcript levels,whereas BBX overexpression caused late flowering.Flowering of glk1/2 and 35S::amiR-BBX14/15/16 plants was insensitive to norflurazon treatment.Taking these observations together,we propose that the GLK1/2-BBX14/15/16 module provides a novel mechanism explaining how the chloroplast represses flowering to balance plant growth and reproductive development.

Maize ZmFDR3 localized in chloroplasts is involved in iron transport

Iron is an essential nutrient for plant metabolism such that Fe-limited plants display chlorosis and suffer from reduced photosynthetic efficiency. Differential display previously identified genes whose expression was elevated in Fe-deficient maize roots. Here,we describe the functional characterization of one of the genes identified in the screen,ZmFDR3 (Zea maize Fe-deficiency-related). Heterologous functional complementation assays using a yeast iron uptake mutant showed that ZmFDR3 functions in iron transport. ZmFDR3 contains a domain found in FliN-proteins of the type III secretion system and is predicted to localize to the thylakoid of plastids. Fluorescence immunocytochemistry showed that ZmFDR3 is localized in the plastids of roots,stems and leaves,with high expression found in guard cell chloroplasts. Transgenic tobacco expressing a 35S-ZmFDR3 construct contains elevated iron content,displays well arranged thylakoid membranes and has photosynthetic indices that are higher than those of the wild type. Together,these results suggest that ZmFDR3 functions in chloroplast iron transport.

Ion Channels in Plant Bioenergetic Organelles, Chloroplasts and Mitochondria： From Molecular Identification to Function

Recent technical advances in electrophysiological measurements, organelle-targeted fluorescence imaging, and organelle proteomics have pushed the research of ion transport a step forward in the case of the plant bioenergetic organelles, chloroplasts and mitochondria, leading to the molecular identification and functional characterization of several ion transport systems in recent years. Here we focus on channels that mediate relatively high-rate ion and water flux and summarize the current knowledge in this field, focusing on targeting mechanisms, proteomics, electrophysiology, and physiological function. In addition, since chloroplasts evolved from a cyanobacterial ancestor, we give an overview of the information available about cyanobacterial ion channels and discuss the evolutionary origin of chloroplast channels. The recent molecular identification of some of these ion channels allowed their physiological functions to be studied using genetically modified Arabidopsis plants and cyanobacteria. The view is emerging that alteration of chloroplast and mitochondrial ion homeostasis leads to organelle dysfunction, which in turn significantly affects the energy metabolism of the whole organism. Clear-cut identification of genes encoding for chan- nels in these organelles, however, remains a major challenge in this rapidly developing field. Multiple stra- tegies including bioinformatics, cell biology, electrophysiology, use of organelle-targeted ion-sensitive probes, genetics, and identification of signals eliciting specific ion fluxes across organelle membranes should provide a better understanding of the physiological role of organellar channels and their contribution to signaling pathways in plants in the future.

On the Impact of Precursor Unfolding during Protein Import into Chloroplasts

Protein translocation across membranes is a fundamental cellular process. The majority of the proteins of organelles such as mitochondria and chloroplasts is synthesized in the cytosol and subsequently imported in a posttranslational manner. The precursor proteins have to be unfolded at least for translocation, but it has also been assumed that they are unfolded during transport to the organelle in the cytosol. Unfolding is governed by chaperones and the translocon itself. At the same time, chaperones provide the energy for the import process. The energetic properties of the chloroplast translocon were studied by import of the Ig-like module of the muscle protein titin fused to the transit peptide of the chloroplast targeted oxygen evolving complex subunit of 33 kDa （OE33）. Our results suggest that p（OE33）titin is folded prior to import and that translocation is initiated by unfolding after having bound to the translocon at the chloroplast surface. Using a set of stabilizing and destabilizing mutants of titin previously analyzed by atomic force microscopy and as passenger for mitochondrial translocation, we studied the unfolding force provided by the chloroplast translocon. Based on these results, a model for translocation is discussed.

Molecular Mechanism of the Specificity of Protein Import into Chloroplasts and Mitochondria in Plant Cells

Plants possess both types of endosymbiotic organelles, chloroplasts and mitochondria. Transit peptides and presequences function as signal sequences for specific import into chloroplasts and mitochondria, respectively. However, how these highly similar signal sequences confer the protein import specificity remains elusive. Here, we show that mitochondrial- or chloroplast-specific import involves two distinct steps, specificity determination and translocation across envelopes, which are mediated by the N-terminal regions and functionally interchangeable C-terminal regions, respectively, of transit peptides and presequences. A domain harboring multiple-arginine and hydrophobic sequence motifs in the N-terminal regions of presequences was identified as the mitochondrial specificity factor. The presence of this domain and the absence of arginine residues in the N-terminal regions of otherwise common targeting signals confers specificity of protein import into mitochondria and chloroplasts, respectively. AtToc159, a chloroplast import receptor, also contributes to determining chloroplast import specificity. We propose that common ancestral sequences were functionalized into mitochondrial- and chloroplast-specific signal sequences by the presence and absence, respectively, of multiple-arginine and hydrophobic sequence motifs in the N-terminal region.

A Golden2-like transcription factor, BnGLK1a, improves chloroplast development, photosynthesis, and seed weight in rapeseed

Enhancing photosynthetic efficiency is a major goal for improving crop yields under agricultural field conditions and is associated with chloroplast biosynthesis and development.In this study,we demonstrate that Golden2-like 1a(BnGLK1a)plays an important role in regulating chloroplast development and photosynthetic efficiency.Overexpressing BnGLK1a resulted in significant increases in chlorophyll content,the number of thylakoid membrane layers and photosynthetic efficiency in Brassica napus,while knocking down BnGLK1a transcript levels through RNA interference(RNAi)had the opposite effects.A yeast two-hybrid screen revealed that BnGLK1a interacts with the abscisic acid receptor PYRABACTIN RESISTANCE 1-LIKE 1-2(BnPYL1-2)and CONSTITUTIVE PHOTOMORPHOGENIC 9 SIGNALOSOME 5A subunit(BnCSN5A),which play essential roles in regulating chloroplast development and photosynthesis.Consistent with this,BnGLK1a-RNAi lines of B.napus display hypersensitivity to the abscisic acid(ABA)response.Importantly,overexpression of BnGLK1a resulted in a 10%increase in thousand-seed weight,whereas seeds from BnGLK1a-RNAi lines were 16%lighter than wild type.We propose that BnGLK1a could be a potential target in breeding for improving rapeseed productivity.Our results not only provide insights into the mechanisms of BnGLK1a function,but also offer a potential approach for improving the productivity of Brassica species.

Synergistic effects of carbon cycle metabolism and photosynthesis in Chinese cabbage under salt stress

Chinese cabbage(Brassica rapa ssp. pekinensis) has a long cultivation history and is one of the vegetable crops with the largest cultivation area in China. However, salt stress severely damages photosynthesis and hormone metabolism, nutritional balances, and results in ion toxicity in plants. To better understand the mechanisms of salt-induced growth inhibition in Chinese cabbage, RNA-seq and physiological index determination were conducted to explore the impacts of salt stress on carbon cycle metabolism and photosynthesis in Chinese cabbage. Here, we found that the number of thylakoids and grana lamellae and the content of starch granules and chlorophyll in the leaves of Chinese cabbage under salt stress showed a time-dependent response, first increasing and then decreasing. Chinese cabbage increased the transcript levels of genes related to the photosynthetic apparatus and carbon metabolism under salt stress, probably in an attempt to alleviate damage to the photosynthetic system and enhance CO_(2) fixation and energy metabolism. The transcription of genes related to starch and sucrose synthesis and degradation were also enhanced;this might have been an attempt to maintain intracellular osmotic pressure by increasing soluble sugar concentrations. Soluble sugars could also be used as potential reactive oxygen species(ROS) scavengers, in concert with peroxidase(POD)enzymes, to eliminate ROS that accumulate during metabolic processes. Our study characterizes the synergistic response network of carbon metabolism and photosynthesis under salt stress.

Across two phylogeographic breaks: Quaternary evolutionary history of a mountain aspen (Populus rotundifolia) in the Hengduan Mountains

Biogeographical barriers to gene flow are central to plant phylogeography.In East Asia,plant distribution is greatly influenced by two phylogeographic breaks,the Mekong-Salween Divide and Tanaka-Kaiyong Line,however,few studies have investigated how these barriers affect the genetic diversity of species that are distributed across both.Here we used 14 microsatellite loci and four chloroplast DNA fragments to examine genetic diversity and distribution patterns of 49 populations of Populus rotundifolia,a species that spans both the Mekong-Salween Divide and the Tanaka-Kaiyong Line in southwestern China.Demographic and migration hypotheses were tested using coalescent-based approaches.Limited historical gene flow was observed between the western and eastern groups of P.rotundifolia,but substantial flow occurred across both the Mekong-Salween Divide and Tanaka-Kaiyong Line,manifesting in clear admixture and high genetic diversity in the central group.Wind-borne pollen and seeds may have facilitated the dispersal of P.rotundifolia following prevalent northwest winds in the spring.We also found that the Hengduan Mountains,where multiple genetic barriers were detected,acted on the whole as a barrier between the western and eastern groups of P.rotundifolia.Ecological niche modeling suggested that P.rotundifolia has undergone range expansion since the last glacial maximum,and demographic reconstruction indicated an earlier population expansion around 600 Ka.The phylogeographic pattern of P.rotundifolia reflects the interplay of biological traits,wind patterns,barriers,niche differentiation,and Quaternary climate history.This study emphasizes the need for multiple lines of evidence in understanding the Quaternary evolution of plants in topographically complex areas.

A mutation in the promoter of the yellow stripe-like transporter gene in cucumber results in a yellow cotyledon phenotype

Leaf color mutants in higher plants are considered to be ideal materials for studying the chlorophyll biosynthesis,photosynthesis mechanism and chloroplast development.Herein,we identified a spontaneous mutant,yc412,in cultivated cucumber that exhibited yellow cotyledons.The yellow-lethal mutant was diagnosed with an abnormal chloroplast ultrastructure,and reduced photosynthetic capacity and pigment content.Through bulked segregant analysis-based whole-genome sequencing and fine genetic mapping,we narrowed the yellow cotyledons (yc) locus to a 96.8 kb interval on chromosome 3.By resequencing and molecular cloning,we showed that Csyc is a potential candidate gene,which encodes a yellow stripe-like (YSL) transporter.The T to C mutation in the promoter region of Csyc caused the yellow cotyledon phenotype in yc412.Compared to YZU027A (WT),the expression of Csyc was significantly downregulated in the cotyledons of yc412.Silencing of Csyc in cucumber via virus-induced gene silencing resulted in chlorotic leaves,mainly by suppressing the chlorophyll content.Furthermore,a comparative transcriptome analysis revealed that chloroplast-related genes and chlorophyll biosynthesis genes were significantly downregulated in yc412 cotyledons.Our results provide new insights into the molecular function of the YSL transporter in plant chloroplast development and chlorophyll synthesis.

Na_(2)CO_(3)-responsive Photosynthetic and ROS Scavenging Mechanisms in Chloroplasts of Alkaligrass Revealed by Phosphoproteomics

Alkali-salinity exerts severe osmotic,ionic,and high-p H stresses to plants.To understand the alkali-salinity responsive mechanisms underlying photosynthetic modulation and reactive oxygen species(ROS)homeostasis,physiological and diverse quantitative proteomics analyses of alkaligrass(Puccinellia tenuiflora)under Na_(2)CO_(3)stress were conducted.In addition,Western blot,real-time PCR,and transgenic techniques were applied to validate the proteomic results and test the functions of the Na_(2)CO_(3)-responsive proteins.A total of 104 and 102 Na_(2)CO_(3)-responsive proteins were identified in leaves and chloroplasts,respectively.In addition,84 Na_(2)CO_(3)-responsive phosphoproteins were identified,including 56 new phosphorylation sites in 56 phosphoproteins from chloroplasts,which are crucial for the regulation of photosynthesis,ion transport,signal transduction,and energy homeostasis.A full-length Pt FBA encoding an alkaligrass chloroplastic fructosebisphosphate aldolase(FBA)was overexpressed in wild-type cells of cyanobacterium Synechocystis sp.Strain PCC 6803,leading to enhanced Na_(2)CO_(3)tolerance.All these results indicate that thermal dissipation,state transition,cyclic electron transport,photorespiration,repair of photosystem(PS)Ⅱ,PSI activity,and ROS homeostasis were altered in response to Na_(2)CO_(3)stress,which help to improve our understanding of the Na_(2)CO_(3)-responsive mechanisms in halophytes.

Determinants of the Specif icity of Protein Targeting to Chloroplasts or Mitochondria

Chloroplasts and mitochondria are both thought to have arisen through primary endosymbiosis (Kutschera and Niklas, 2005). As each organelle evolved, most of its genome was transferred to the host nucleus resulting in the vast majority of its proteome being nuclear encoded and synthesized on cytosolic ribosomes (Shi and Theg, 2013).

Normal Structure and Function of Endothecium Chloroplasts Maintained by ZmMs33-Mediated Lipid Biosynthesis in Tapetal Cells Are Critical for Anther Development in Maize

Genic male sterility(GMS)is critical for heterosis utilization and hybrid seed production.Although GMS mutants and genes have been studied extensively in plants,it has remained unclear whether chloroplast-associated photosynthetic and metabolic activities are involved in the regulation of anther development.In this study,we characterized the function of ZmMs33/ZmGPAT6,which encodes a member of the glycerol-3-phosphate acyltransferase(GPAT)family that catalyzes the first step of the glycerolipid synthetic pathway.We found that normal structure and function of endothecium(En)chloroplasts maintained by ZmMs33-mediated lipid biosynthesis in tapetal cells are crucial for maize anther development.ZmMs33 is expressed mainly in the tapetum at early anther developmental stages and critical for cell proliferation and expansion at late stages.Chloroplasts in En cells of wild-type anthers function as starch storage sites before stage 10 but as photosynthetic factories since stage 10 to enable starch metabolism and carbohydrate supply.Loss of ZmMs33 function inhibits the biosynthesis of glycolipids and phospholipids,which are major components of En chloroplast membranes,and disrupts the development and function of En chloroplasts,resulting in the formation of abnormal En chloroplasts containing numerous starch granules.Further analyses reveal that starch synthesis during the day and starch degradation at night are greatly suppressed in the mutant anthers,leading to carbon starvation and low energy status,as evidenced by low trehalose-6-phosphate content and a reduced ATP/AMP ratio.The energy sensor and inducer of autophagy,SnRK1,was activated to induce early and excessive autophagy,premature PCD,and metabolic reprogramming in tapetal cells,finally arresting the elongation and development of mutant anthers.Taken together,our results not only show that ZmMs33 is required for normal structure and function of En chloroplasts but also reveal that starch metabolism and photosynthetic activities of En chloroplasts at different developmental stages are essential for normal anther development.These findings provide novel insights for understanding how lipid biosynthesis in the tapetum,the structure and function of En chloroplasts,and energy and substance metabolism are coordinated to maintain maize anther development.

Dual Targeting to Mitochondria and Chloroplasts： Characterization of Thr-tRNA Synthetase Targeting Peptide

There is a group of proteins that are encoded by a single gene, expressed as a single precursor protein and dually targeted to both mitochondria and chloroplasts using an ambiguous targeting peptide. Sequence analysis of 43 dual targeted proteins in comparison with 385 mitochondrial proteins and 567 chloroplast proteins ofArabidopsis thaliana revealed an overall significant increase in phenylalanines, leucines, and serines and a decrease in acidic amino acids and glycine in dual targeting peptides （dTPs）. The N-terminal portion of dTPs has significantly more serines than mTPs. The number of arginines is similar to those in mTPs, but almost twice as high as those in cTPs. We have investigated targeting determinants of the dual targeting peptide of Thr-tRNA synthetase （ThrRS-dTP） studying organellar import of N- and C-terminal deletion constructs of ThrRS-dTP coupled to GFR These results show that the 23 amino acid long N-terminal portion of ThrRS-dTP is crucial but not sufficient for the organellar import. The C-terminal deletions revealed that the shortest peptide that was capable of conferring dual targeting was 60 amino acids long. We have purified the ThrRS- dTP（2-60） to homogeneity after its expression as a fusion construct with GST followed by CNBr cleavage and ion exchange chromatography. The purified ThrRS-dTP（2-60） inhibited import of pF1β into mitochondria and of pSSU into chloroplasts at μM concentrations showing that dual and organelle-specific proteins use the same organellar import pathways. Furthermore, the CD spectra of ThrRS-dTP（2-60） indicated that the peptide has the propensity for forming α-helical structure in membrane mimetic environments; however, the membrane charge was not important for the amount of induced helical structure. This is the first study in which a dual targeting peptide has been purified and investigated by biochemical and biophysical means.

Essentials of Proteolytic Machineries in Chloroplasts

Plastids are unique organelles that can alter their structure and function in response to environmental and developmental stimuli. Chloroplasts are one type of plastid and are the sites for various metabolic pro- cesses, including photosynthesis. For optimal photosynthetic activity, the chloroplast proteome must be properly shaped and maintained through regulated proteolysis and protein quality control mechanisms. Enzymatic functions and activities are conferred by protein maturation processes involving consecutive proteolytic reactions. Protein abundances are optimized by the balanced protein synthesis and degrada- tion, which is depending on the metabolic status. Malfunctioning proteins are promptly degraded. Twenty chloroplast proteolytic machineries have been characterized to date. Specifically, processing peptidases and energy-driven processive proteases are the major players in chloroplast proteome biogenesis, remod- eling, and maintenance. Recently identified putative proteases are potential regulators of photosynthetic functions. Here we provide an updated, comprehensive overview of chloroplast protein degradation ma- chineries and discuss their importance for photosynthesis. Wherever possible, we also provide structural insights into chloroplast proteases that implement regulated proteolysis of substrate proteins/peptides.