Chloroplast-encoded Gene Research Articles

Temporal dynamics of chloroplast biogenesis revealed initiation of photosynthesis-related gene expression and protein complexes during alfalfa seed germination

The chloroplast biogenesis occurs in cotyledon during alfalfa seed germination before true leaf formation, and is extremely important for the followed plant development and growth. In this study, we conducted a simulation of alfalfa seed germination in the soil by using tin foil and focused on 10 pivotal time points of chloroplast biogenesis in cotyledons before and after light exposure, which showed significant differences in multispectral images, and covered the whole process of chloroplast biogenesis from proplastid, etioplast to mature chloroplast. We revealed three phases that referred to the programmed involvements of photosynthesis promotion, ultrastructure maturity, transcriptomic expression, and protein complex construction, and observed distinct transcriptional expressions of genes from nuclear and chloroplast genomes. In phase I at dark germination before light exposure, chloroplast-encoded genes showed up-regulated expressions together with the importation of chloroplast proteins. In phase II for the first day after light exposure, nuclear-encoded genes’ expressions were initiated at 2 h after light exposure (E2h), followed by swift assembly of chloroplast thylakoid membrane protein complexes, and roaring Fv/Fm and contents of chlorophyll a, chlorophyll b and carotenoid. The initiation at E2h was pronounced by the observation of gradual accumulation of single lamella, and facilitated the formation of granum stacks (thylakoid) at E8h in phase II. In phase III from the second day after light exposure, chloroplast became gradually complete with the fully established photosynthetic capacity. Altogether, our results layed a theoretical foundation for enhancing potential photosynthetic efficiency in alfalfa and related species.

Common environmental stress responses in a model marine diatom.

Marine planktonic diatoms are among the most important contributors to phytoplankton blooms and marine net primary production. Their ecological success has been attributed to their ability to rapidly respond to changing environmental conditions. Here, we report common molecular mechanisms used by the model marine diatom Thalassiosira pseudonana to respond to 10 diverse environmental stressors using RNA-Seq analysis. We identify a specific subset of 1076 genes that are differentially expressed in response to stressors that induce an imbalance between energy or resource supply and metabolic capacity, which we termed the diatom environmental stress response (d-ESR). The d-ESR is primarily composed of genes that maintain proteome homeostasis and primary metabolism. Photosynthesis is strongly regulated in response to environmental stressors but chloroplast-encoded genes were predominantly upregulated while the nuclear-encoded genes were mostly downregulated in response to low light and high temperature. In aggregate, these results provide insight into the molecular mechanisms used by diatoms to respond to a range of environmental perturbations and the unique role of the chloroplast in managing environmental stress in diatoms. This study facilitates our understanding of the molecular mechanisms underpinning the ecological success of diatoms in the ocean.

A splicing site change between exon 5 and 6 of the nuclear-encoded chloroplast-localized HvYGL8 gene results in reduced chlorophyll content and plant height in barley.

The chloroplast is an important cellular organelle and metabolic hub, which is not only responsible for plant photosynthesis but is also involved in the de novo biosynthesis of pigments, fatty acids, and hormone metabolisms. Several genes that are responsible for rice leaf color variations have been reported to be directly or indirectly involved in chlorophyll biosynthesis and chloroplast development, whereas a few genes have been functionally confirmed to be responsible for leaf color changes in barley at the molecular level. In this study, we obtained a yellow leaf and dwarf ygl8 mutant from the progeny of Morex (a variety of barley) seeds treated with EMS. We performed bulked-segregant analysis (BSA) and RNA-seq analysis and targeted a UMP kinase encoding gene, YGL8, which generated a splicing site change between exon 5 and 6 of YGL8 due to a G to A single-nucleotide transition in the 5th exon/intron junction in the ygl8 mutant. The splicing site change between exon 5 and 6 of YGL8 had no effects on chloroplast subcellular localization but resulted in an additional loop in the UMP kinase domain, which might disturb the access of the substrates. On one hand, the splicing site change between exon 5 and 6 of YGL8 downregulated the transcriptional expression of chloroplast-encoded genes and chlorophyll-biosynthesis-related genes in a temperature-dependent manner in the ygl8 mutant. On the other hand, the downregulation of bioactive GA-biosynthesis-related GA20ox genes and cell-wall-cellulose-biosynthesis-related CesA genes was also observed in the ygl8 mutant, which led to a reduction in plant height. Our study will facilitate the understanding of the regulation of leaf color and plant height in barley.

The effects of fullerene on photosynthetic apparatus, chloroplast-encoded gene expression, and nitrogen assimilation in Zea mays under cobalt stress.

Carbon nanostructures, such as the water-soluble fullerene (FLN) derivatives, are considered perspective agents for agriculture. FLN can be a novel nano-agent modulating plant response against stress conditions. However, the mechanism underlying the impacts of FLN on plants in agroecosystems remains unclear. Zea mays was exposed to exogenous C60 -FLN applications (FLN1: 100; FLN2: 250; and FLN3: 500 mg L-1 ) with/without cobalt stress (Co, 300 μM) for 3 days (d). In the maize chloroplasts, Co stress disrupted the photosynthetic efficiency and the expression of genes related to the photosystems (psaA and psbA). FLNs effectively improved the efficiency and photochemical reaction of photosystems. Co stress induced the accumulation of reactive oxygen species (ROS) as confirmed by ROS-specific fluorescence in guard cells. Co stress increased only chloroplastic superoxide dismutase (SOD) and peroxidase (POX). Stress triggered oxidative damages in maize chloroplasts, measured as an increase in TBARS content. In Co-stressed seedlings exposed to FLN1 and FLN2 exposures, the hydrogen peroxide (H2 O2 ) was scavenged through the nonenzymes/enzymes-related to the AsA-GSH cycle by preserving ascorbate (AsA) conversion, as well as GSH/GSSG and glutathione (GSH) redox state. Also, the alleviation effect of FLN3 against stress could be attributed to increased glutathione S-transferase (GST) activity and AsA regeneration. FLN applications reversed the inhibitory effects of Co stress on nitrogen assimilation. In maize chloroplasts, FLN increased the activities of nitrate reductase (NR), glutamate dehydrogenase (GDH), nitrite reductase (NiR), and glutamine synthetase (GS), which provided conversion of inorganic nitrogen (N) into organic N. The ammonium (NH4 + ) toxicity was removed via GS and GDH but not glutamate synthase (GOGAT). The increased NAD-GDH (deaminating) and NADH-GDH (aminating) activities indicated that GDH was needed more for NH4 + detoxification. Therefore, FLN exposure to Co-stressed maize plants might play a role in N metabolism regarding the partitioning of N assimilates. Exogenous FLN conceivably removed Co toxicity by improving the expressions of genes related to reaction center proteins of photosystems, increasing the level of enzymes related to the defense system, and improving the N assimilation in maize chloroplasts.

Molecular Characterization of Caulerpa racemosa (Caulerpales, Chlorophyta) from Indonesia Based on the Plastid tufA Gene

The green seaweed Caulerpa racemosa is a seaweed of high prospect that is being given emphasis by the Indonesian government. However, C. racemosa in Indonesia may include multiple species level-entity exhibiting morphological overlap and require molecular analysis for species identification. Molecular documentation of species richness of indigenous populations of C. racemosa is essential to underpin cultivar development and conservation of the species to avoid overharvesting. The present study aimed to determine the genetic diversity of C. racemosa and document the haplotype network of the specimens from four different locations (Bintan Island, Jepara, Takalar and Osi Island) using the chloroplast tufA gene. Twenty individuals from four areas were collected and amplified with the chloroplast-encoded gene tufA for species identification against publicly available data. The identification of C. racemosa based on the tufA gene showed that the species found in four locations were C. cylindracea (previously C. racemosa var. cylindracea), C. macra (previously C. racemosa var. macra), C. racemosa, and C. oligophylla (previously C. racemosa var. lamourouxii). This study records the existence of C. cylindracea in Takalar and Jepara, Indonesia for the first time. The most diverse C. racemosa species was in Osi Island, where the exploitation of this seaweed is very low. In contrast, the lowest number of C. racemosa varieties were found in Takalar, where exploitation is very high. There were only minor light variations of Caulerpa species in the tufA gene in four different sites with only four haplotypes found, and each haplotype corresponded to another species.

Integrative Taxonomic, Ecological and Genotyping Study of Charophyte Populations from the Egyptian Western-Desert Oases and Sinai Peninsula.

Present-day information available on the charophyte macroalgae in Egypt, including their phylogenetic affinities, remains largely incomplete. In this study, nine charophyte populations were collected from different aquatic biotopes across the Egyptian Western-Desert Oases and Sinai Peninsula. All populations were investigated using an integrative polyphasic approach including phylogenetic analyses inferred from the chloroplast-encoded gene (rbcL) and the internal transcribed spacer (ITS1) regions, in parallel with morphotaxonomic assignment, ultrastructure of the oospore walls, and autecology. The specimens identified belonged to the genera Chara, Nitella, and Tolypella, with predominance of the first genus to which five species were assigned though they presented some interesting aberrant taxonomic features: C. aspera, C. contraria, C. globata, C. tomentosa, and C. vulgaris. Based on our integrative study, the globally rare species C. globata was reported for the second time for the whole African continent. The genus Nitella was only represented by N. flagellifera, and based on the available literature, it is a new record for North Africa. Noteworthy, an interesting Tolypella sp., morphologically very similar to T. glomerata, was collected and characterized and finally designated with the working name ‘Tolypella sp. PBA–1704 from a desert, freshwater wetland’, mainly based on its concatenated rbcL+ITS1 phylogenetic position. This study not only improved our understanding on the diversity, biogeography and autecological preferences of charophytes in Egypt, but it also broadened our knowledge on this vulnerable algal group in North Africa, emphasizing the need of more in-depth research work in the future, particularly in the less–impacted desert habitats.

Are Cyanobacteria an Ancestor of Chloroplasts or Just One of the Gene Donors for Plants and Algae?

Chloroplasts of plants and algae are currently believed to originate from a cyanobacterial endosymbiont, mainly based on the shared proteins involved in the oxygenic photosynthesis and gene expression system. The phylogenetic relationship between the chloroplast and cyanobacterial genomes was important evidence for the notion that chloroplasts originated from cyanobacterial endosymbiosis. However, studies in the post-genomic era revealed that various substances (glycolipids, peptidoglycan, etc.) shared by cyanobacteria and chloroplasts are synthesized by different pathways or phylogenetically unrelated enzymes. Membranes and genomes are essential components of a cell (or an organelle), but the origins of these turned out to be different. Besides, phylogenetic trees of chloroplast-encoded genes suggest an alternative possibility that chloroplast genes could be acquired from at least three different lineages of cyanobacteria. We have to seriously examine that the chloroplast genome might be chimeric due to various independent gene flows from cyanobacteria. Chloroplast formation could be more complex than a single event of cyanobacterial endosymbiosis. I present the “host-directed chloroplast formation” hypothesis, in which the eukaryotic host cell that had acquired glycolipid synthesis genes as an adaptation to phosphate limitation facilitated chloroplast formation by providing glycolipid-based membranes (pre-adaptation). The origins of the membranes and the genome could be different, and the origin of the genome could be complex.

A valine residue deletion in ZmSig2A, a sigma factor, accounts for a revertible leaf-color mutation in maize

A nuclear-encoded sigma (σ) factor is essential for the transcriptional regulation of plant chloroplast-encoded genes. Five putative maize σ factors have been identified by database searches, but their functions are unknown. We report a maize leaf color mutant etiolated/albino leaf 1 (eal1) that was derived from space mutation breeding. The eal1 mutant displays etiolated or albino leaves that then gradually turn to normal green at the seedling stage. The changes in eal1 leaf color are associated with changes in photosynthetic pigment content and chloroplast development. Map-based cloning revealed that a single amino-acid deletion changing Val480-Val481-Val482 to Val480-Val481, in the C-terminal domain σ4 of the putative σ factor ZmSig2A, is responsible for the eal1 mutation. In comparison with the expression level of the wild-type (WT) allele ZmSig2A+ in WT plants, much higher expression of the mutant allele ZmSig2AΔV in eal1 plants was detected before the eal1 plants turned to normal green. ZmSig2A shows the highest similarity to rice OsSig2A and Arabidopsis SIG2. Ectopic expression of ZmSig2A+ or ZmSig2AΔV driven by the cauliflower mosaic virus 35S promoter rescued the pale green leaf of the sig2 mutant, but ectopic expression of ZmSig2AΔV driven by the SIG2 promoter did not. We propose that the Val deletion generated a new weak allele of ZmSig2A that cannot completely abolish the ZmSig2A function. Some genes involved in chloroplast development and photosynthesis-associated nuclear genes showed significant expression differences between eal1 and WT plants. We conclude that ZmSig2A encoding a σ factor is essential for maize chloroplast development. The eal1 mutant with a weak allele of ZmSig2A represents a valuable genetic resource for investigating the regulation of ZmSig2A-mediated chloroplast development in maize. The eal1 mutation may be useful as a marker for early identification and elimination of false hybrids or transgene transmission in the application of genetic male sterility to commercial hybrid seed production.

A procedure to introduce point mutations into the Rubisco large subunit gene in wild-type plants.

Photosynthetic inefficiencies limit the productivity and sustainability of crop production and the resilience of agriculture to future societal and environmental challenges. Rubisco is a key target for improvement as it plays a central role in carbon fixation during photosynthesis and is remarkably inefficient. Introduction of mutations to the chloroplast-encoded Rubisco large subunit rbcL is of particular interest for improving the catalytic activity and efficiency of the enzyme. However, manipulation of rbcL is hampered by its location in the plastome, with many species recalcitrant to plastome transformation, and by the plastid's efficient repair system, which can prevent effective maintenance of mutations introduced with homologous recombination. Here we present a system where the introduction of a number of silent mutations into rbcL within the model plant Nicotiana tabacum facilitates simplified screening via additional restriction enzyme sites. This system was used to successfully generate a range of transplastomic lines from wild-type N. tabacum with stable point mutations within rbcL in 40% of the transformants, allowing assessment of the effect of these mutations on Rubisco assembly and activity. With further optimization the approach offers a viable way forward for mutagenic testing of Rubisco function in planta within tobacco and modification of rbcL in other crops where chloroplast transformation is feasible. The transformation strategy could also be applied to introduce point mutations in other chloroplast-encoded genes.

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 thaliana PAP 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.

Gene Loss and Evolution of the Plastome.

Chloroplasts are unique organelles within the plant cells and are responsible for sustaining life forms on the earth due to their ability to conduct photosynthesis. Multiple functional genes within the chloroplast are responsible for a variety of metabolic processes that occur in the chloroplast. Considering its fundamental role in sustaining life on the earth, it is important to identify the level of diversity present in the chloroplast genome, what genes and genomic content have been lost, what genes have been transferred to the nuclear genome, duplication events, and the overall origin and evolution of the chloroplast genome. Our analysis of 2511 chloroplast genomes indicated that the genome size and number of coding DNA sequences (CDS) in the chloroplasts genome of algae are higher relative to other lineages. Approximately 10.31% of the examined species have lost the inverted repeats (IR) in the chloroplast genome that span across all the lineages. Genome-wide analyses revealed the loss of the Rbcl gene in parasitic and heterotrophic plants occurred approximately 56 Ma ago. PsaM, Psb30, ChlB, ChlL, ChlN, and Rpl21 were found to be characteristic signature genes of the chloroplast genome of algae, bryophytes, pteridophytes, and gymnosperms; however, none of these genes were found in the angiosperm or magnoliid lineage which appeared to have lost them approximately 203–156 Ma ago. A variety of chloroplast-encoded genes were lost across different species lineages throughout the evolutionary process. The Rpl20 gene, however, was found to be the most stable and intact gene in the chloroplast genome and was not lost in any of the analyzed species, suggesting that it is a signature gene of the plastome. Our evolutionary analysis indicated that chloroplast genomes evolved from multiple common ancestors ~1293 Ma ago and have undergone vivid recombination events across different taxonomic lineages.

Disruption of a Rice Chloroplast-Targeted Gene OsHMBPP Causes a Seedling-Lethal Albino Phenotype

BackgroundChloroplast development is coordinately regulated by plastid- and nuclear-encoding genes. Although many regulators have been reported to be involved in chloroplast development, new factors remain to be identified, given the complexity of this process.ResultsIn this study, we characterized a rice mutant lethal albinic seedling 1(las1)form of a 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (OsHMBPP) that was targeted to the chloroplasts. The LAS1 mutation caused the albino lethal phenotype in seedlings. Transmission electron microscopy indicated that las1 were defective in early chloroplast development. LAS1 is preferentially expressed in leaves, implying its role in controlling chloroplast development. The expression levels of many chloroplast-encoded genes were altered significantly in las1. The expression levels of nuclear-encoded gene involved in Chl biosynthesis were also decreased in las1. We further investigated plastidic RNA editing in las1 and found that the edit efficiency of four chloroplast genes were markly altered. Compared with WT, las1 exhibited defective in biogenesis of chloroplast ribosomes.ConclusionsOur results show that LAS1/OsHMBPP plays an essential role in the early chloroplast development in rice.

Gertia stigmatica gen. et sp. nov. (Kareniaceae, Dinophyceae), a New Marine Unarmored Dinoflagellate Possessing the Peridinin-type Chloroplast with an Eyespot

Marine unarmored dinoflagellates in the family Kareniaceae are known to possess chloroplasts of haptophyte origin, which contain fucoxanthin and its derivatives as major carotenoids, and lack peridinin. In the present study, the first species with the peridinin-type chloroplast in this family, Gertia stigmatica gen. et sp. nov., is described on the basis of ultrastructure, photosynthetic pigment composition, and molecular phylogeny inferred from nucleus- and chloroplast-encoded genes. Cells of G. stigmatica were small and harboring a chloroplast with an eyespot and two pyrenoids. The apical structure complex was straight, similar to Karenia and Karlodinium. Under transmission electron microscopy, the chloroplast was surrounded by two membranes, and the eyespot was composed of a single layer of osmiophilic globules (eyespot type A); this was never previously reported from the Kareniaceae. High performance liquid chromatography demonstrated the chloroplast contains peridinin, and neither fucoxanthin nor 19'-acyloxyfucoxanthins was identified. A phylogeny based on nucleus-encoded rDNAs suggested a position of G. stigmatica in the Kareniaceae, but not clustered within the previously described genera, i.e., Karenia, Karlodinium and Takayama. A phylogeny of chloroplast-encoded psbA, psbC and psbD indicated the chloroplast is of peridinin-type typical of dinoflagellates, but the most related species remains unclear.

Microscopy and phylogeny of Pyramimonas tatianae sp. nov. (Pyramimonadales, Chlorophyta), a scaly quadriflagellate from Golden Horn Bay (eastern Russia) and formal description of Pyramimonadophyceae classis nova

ABSTRACT Nearly two decades ago a scaly quadriflagellate culture was established from a sample collected in Golden Horn Bay, eastern Russia. Here we present a comparative analysis of pheno- and genotypic characters and show that the isolate did not match any existing species of Pyramimonas and is therefore described as P. tatianae sp. nov. The species was probably identified as P. aff. cordata in previous studies on material from the same area. Based on an ultrastructural account of the cell and its external body scales it was found to belong to the subgenus Vestigifera. This was supported by a phylogenetic analysis using the chloroplast-encoded rbcL gene. The cell dimensions of P. tatianae were 6–7 µm long and 5–6 µm wide and it thus represented one of the smaller species of the genus. The cup-shaped chloroplast was divided into four lobes reaching from the middle to the anterior part of the cell. A single posterior eyespot was observed adjacent to an excentric pyrenoid. The ultrastructure of the body and flagellar scales were illustrated from material prepared for whole mounts and thin sections. The new species was compared with P. cordata and P. mitra, two other vestigiferans with which it shares some morphological features. The sequence divergence between P. tatianae and the most closely related species, P. mitra, was 2.5%. The phylogeny of Vestigifera revealed two lineages, one comprising cold-water species and the cosmopolitan P. orientalis, and the other species from temperate-subtropical waters. The tree topology suggested a southbound dispersal route. This was further supported by all antarctic Pyramimonas species having encystment stages as part of their life cycle. It was therefore probable that their ancestors also were capable of producing cysts allowing transportation over great distances. A formal description of the class Pyramimonadophyceae comprising both extant and extinct species was also provided.

The tomato WV gene encoding a thioredoxin protein is essential for chloroplast development at low temperature and high light intensity

BackgroundChloroplast biogenesis, a complex process in higher plants, is the key to photoautotrophic growth in plants. White virescent (wv) mutants have been used to unfold the molecular mechanisms underlying the regulation of chloroplast development and chloroplast gene expression in plants. However, most of genes controlling white virescent phenotype still remain unknown.ResultsIn this study, we identified a temperature- and light intensity-sensitive mutant, named as wv. The content of chlorophyll was dramatically decreased in the immature leaves of wv mutant under the conditions of low temperature and high-light intensity. TEM observation showed that the chloroplasts in the young leaves of wv mutant lacked an organized thylakoid membrane, whereas crescent-shaped chloroplasts with well-developed stromal and stacked grana thylakoids in the mature leaves were developed. Immunoblot analyses suggested that proteins of photosynthetic complexes were decreased substantially in wv mutants. Based on map-based cloning and transgenic analysis, we determined that the wv phenotype was caused by single base mutation in the first intron of WV gene, which encoded a thioredoxin protein with 365 amino acids. qRT-PCR analysis revealed that the expression of WV gene was significantly down-regulated in wv mutant. In addition, knockdown of WV gene through RNAi also resulted in white virescent young leaves, suggesting that the mutation possibly blocks the differentiation of chloroplasts through inhibiting the expression of WV gene. Furthermore, the expression of WV peaked in apical buds and gradually decreased along with the developmental stage, which was consistent with the wv mutant phenotype. Expression analysis of chloroplast-encoded genes by qRT-PCR showed that the wv mutation affected the expression pattern of chloroplast-encoded PEP dependent genes.ConclusionOur results suggested that wv mutant was sensitive to low temperature and light intensity. WV gene was essential for chloroplast differentiation. A single base mutation in the first intron resulted in down-regulation of WV gene expression, which inhibited the expression of chloroplast-encoded genes, thereby blocking chloroplast formation and chlorophyll synthesis.

Pyrimidine Salvage: Physiological Functions and Interaction with Chloroplast Biogenesis.

The synthesis of pyrimidine nucleotides, an essential process in every organism, is accomplished by de novo synthesis or by salvaging pyrimdines from e.g. nucleic acid turnover. Here, we identify two Arabidopsis (Arabidopsis thaliana) uridine/cytidine kinases, UCK1 and UCK2, which are located in the cytosol and are responsible for the majority of pyrimidine salvage activity in vivo. In addition, the chloroplast has an active uracil salvage pathway. Uracil phosphoribosyltransferase (UPP) catalyzes the initial step in this pathway and is required for the establishment of photosynthesis, as revealed by analysis of upp mutants. The upp knockout mutants are unable to grow photoautotrophically, and knockdown mutants exhibit a variegated phenotype, with leaves that have chlorotic pale areas. Moreover, the upp mutants did not show altered expression of chloroplast-encoded genes, but transcript accumulation of the LIGHT HARVESTING COMPLEX B nuclear genes LHCB1.2 and LHCB2.3 was markedly reduced. An active UPP homolog from Escherichia coli failed to complement the upp mutant phenotype when targeted to the chloroplast, suggesting that the catalytic function of UPP is not the important factor for the chloroplast phenotype. Indeed, the expression of catalytically inactive Arabidopsis UPP, generated by introduction of point mutations, did complement the upp chloroplast phenotype. These results suggest that UPP has a vital function in chloroplast biogenesis unrelated to its catalytic activity and driven by a moonlighting function.

Comparative transcriptome profiling of genes and pathways involved in leaf-patterning of Clivia miniata var. variegata

Clivia miniata var. variegata (Cmvv) typically possesses yellow- and green-striped leaves. The striped plant not only has a high ornamental value but also be suitable for photosynthesis and chloroplast development research. Our previous study had revealed that yellow stripes (YSs) of Cmvv leaves contain chlorophyll-less ineffective chloroplasts. However, mechanism of Cmvv variegation is yet to be investigated. In the study, transcriptomes of both the YSs and green stripes (GSs) from single Cmvv leaves were compared using high-throughput sequencing. A total of 688 differential expression genes (DEGs) were identified based on biological replications. The qRT-PCR results indicated that transcriptome profiles accurately reflected global transcriptome differences between YSs and GSs. Subcellular localization analysis suggested that 56 DEG proteins were targeted to chloroplasts, and might be involved in anterograde signaling and leaf patterning. Moreover, the DEGs were mostly enriched in photosynthesis and plant-pathogen interaction KEGG pathways. Meanwhile, there should be coordination interaction between the two pathways. Seven of the eight DEGs involved in photosynthesis KEGG pathway were chloroplast-encoded genes and distributed among different cistrons of chloroplast DNA (cpDNA) large single copy regions (LSC) which are more prone to mutation. It was proposed that the YSs were caused by mutation(s) in cpDNA LSC. Thus, when the primary zygote of Cmvv was chimeric in LSC, leaf might be yellow- and green-striped. The study would give new insights into plant variegation and offers candidate genes to guide future research attempting to breed variegated plants.

WSL5, a pentatricopeptide repeat protein, is essential for chloroplast biogenesis in rice under cold stress.

Chloroplasts play an essential role in plant growth and development, and cold conditions affect chloroplast development. Although many genes or regulators involved in chloroplast biogenesis and development have been isolated and characterized, many other components affecting chloroplast biogenesis under cold conditions have not been characterized. Here, we report the functional characterization of a white stripe leaf 5 (wsl5) mutant in rice. The mutant develops white-striped leaves during early leaf development and is albinic when planted under cold stress. Genetic and molecular analysis revealed that WSL5 encodes a novel chloroplast-targeted pentatricopeptide repeat protein. RNA sequencing analysis showed that expression of nuclear-encoded photosynthetic genes in the mutant was significantly repressed, and expression of many chloroplast-encoded genes was also significantly changed. Notably, the wsl5 mutation causes defects in editing of rpl2 and atpA, and splicing of rpl2 and rps12. wsl5 was impaired in chloroplast ribosome biogenesis under cold stress. We propose that the WSL5 allele is required for normal chloroplast development in maintaining retrograde signaling from plastids to the nucleus under cold stress.

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.

WHITE PANICLE1, a Val-tRNA Synthetase Regulating Chloroplast Ribosome Biogenesis in Rice, Is Essential for Early Chloroplast Development.

Chloroplasts and mitochondria contain their own genomes and transcriptional and translational systems. Establishing these genetic systems is essential for plant growth and development. Here we characterized a mutant form of a Val-tRNA synthetase (OsValRS2) from Oryza sativa that is targeted to both chloroplasts and mitochondria. A single base change in OsValRS2 caused virescent to albino phenotypes in seedlings and white panicles at heading. We therefore named this mutant white panicle 1 (wp1). Chlorophyll autofluorescence observations and transmission electron microscopy analyses indicated that wp1 mutants are defective in early chloroplast development. RNA-seq analysis revealed that expression of nuclear-encoded photosynthetic genes is significantly repressed, while expression of many chloroplast-encoded genes also changed significantly in wp1 mutants. Western-blot analyses of chloroplast-encoded proteins showed that chloroplast protein levels were reduced in wp1 mutants, although mRNA levels of some genes were higher in wp1 than in wild type. We found that wp1 was impaired in chloroplast ribosome biogenesis. Taken together, our results show that OsValRS2 plays an essential role in chloroplast development and regulating chloroplast ribosome biogenesis.