Chloroplast Engineering: Fundamental Insights and Its Application in Amelioration of Environmental Stress.

Chloroplasts are specialized organelle that are responsible for converting light energy to chemical energy, thereby driving the carbon dioxide fixation. Apart from photosynthesis, chloroplast is the site for essential cellular processes that determine the plant adaptation to changing environment. Owing to the presence of their own expression system, it provides an optimum platform for engineering valued traits as well as site for synthesis of bio-compounds. Advancements in technology have further enhanced the scope of using chloroplast as a multifaceted tool for the biotechnologist to develop stress-tolerant plants and ameliorate environmental stress. Focusing on chloroplast biotechnology, this review discusses the advances in chloroplast engineering and its application in enhancing plant adaptation and resistance to environmental stress and the development of new bioproducts and processes. This is accomplished through analysis of its biogenesis and physiological processes, highlighting the chloroplast engineering and recent developments in chloroplast biotechnology. In the first part of the review, the evolution and principles of structural organization and physiology of chloroplast are discussed. In the second part, the chief methods and mechanisms involved in chloroplast transformation are analyzed. The last part represents an updated analysis of the application of chloroplast engineering in crop improvement and bioproduction of industrial and health compounds.

Comparison of hypotensive properties of dexmedetomidine versus clonidine for induced hypotension during functional endoscopic sinus surgery: A randomised, double-blind interventional study.

BACKGROUND AND AIMS: Excessive bleeding is a major concern in functional endoscopic sinus surgery (FESS) under general anaesthesia; this can be decreased by various hypotensive agents. This study was conducted to compare the hypotensive effectiveness and haemodynamic stability of dexmedetomidine and clonidine in patients undergoing elective FESS. METHODS: In this prospective double-blinded interventional study, 70 adult patients of either sex, 20-50 years of age, posted for elective FESS were randomly assigned to two groups. Group A received a loading dose of intravenous (IV) dexmedetomidine 1 µg/kg, followed by infusion of 1 µg/kg/h, and group B received a loading dose of IV clonidine 2 µg/kg, followed by 1 µg/kg/h infusion. Surgical field quality, emergence time, sedation score, visual analogue score, recovery profile and haemodynamic parameters were recorded. Statistical analysis was done by Student's unpaired t-test to evaluate the significance of normally distributed variables, whereas Mann-Whitney test and Chi-square test were used for ordinal data and categorical variables and proportions, respectively. RESULTS: In both the groups, target mean arterial pressure (MAP) of 65-70 mmHg and improved surgical field quality were achieved. MAP and heart rate (HR) were statistically significantly lower in the dexmedetomidine group with a longer duration of post-operative analgesia (P = 0.001). None of the groups showed any statistically significant adverse effects. CONCLUSIONS: Both dexmedetomidine and clonidine can be used for controlled hypotension to improve surgical field quality in FESS. Dexmedetomidine provides more haemodynamic stability and an additional benefit of post-operative analgesia and conscious sedation.

Oral teratoma in a neonate: A case report of anesthetic challenge.

Congenital teratoma of oral cavity in a neonate is a rare condition associated with compromised airway and challenges anesthesiologist in airway management. In this report, we describe a scenario of neonate with multiple oral teratoma, cleft palate, and bifid tongue who presented with respiratory distress for surgical excision of mass. The compromised airway can be successfully managed by appropriate prior planning and effective communication between anesthesiologist and surgical team.

Membrane-Specific Targeting of Tail-Anchored Proteins SECE1 and SECE2 Within Chloroplasts.

Membrane proteins that are imported into chloroplasts must be accurately targeted in order to maintain the identity and function of the highly differentiated internal membranes. Relatively little is known about the targeting information or pathways that direct proteins with transmembrane domains to either the inner envelope or thylakoids. In this study, we focused on a structurally simple class of membrane proteins, the tail-anchored proteins, which have stroma-exposed amino-terminal domains and a single transmembrane domain within 30 amino acids of the carboxy-terminus. SECE1 and SECE2 are essential tail-anchored proteins that function as components of the dual SEC translocases in chloroplasts. SECE1 localizes to the thylakoids, while SECE2 localizes to the inner envelope. We have used transient expression in Arabidopsis leaf protoplasts and confocal microscopy in combination with a domain-swapping strategy to identify regions that contain important targeting determinants. We show that membrane-specific targeting depends on features of the transmembrane domains and the short C-terminal tails. We probed the contributions of these regions to targeting processes further through site-directed mutagenesis. We show that thylakoid targeting still occurs when changes are made to the tail of SECE1, but changing residues in the tail of SECE2 abolishes inner envelope targeting. Finally, we discuss possible parallels between sorting of tail-anchored proteins in the stroma and in the cytosol.

The integration of chloroplast protein targeting with plant developmental and stress responses.

The plastids, including chloroplasts, are a group of interrelated organelles that confer photoautotrophic growth and the unique metabolic capabilities that are characteristic of plant systems. Plastid biogenesis relies on the expression, import, and assembly of thousands of nuclear encoded preproteins. Plastid proteomes undergo rapid remodeling in response to developmental and environmental signals to generate functionally distinct plastid types in specific cells and tissues. In this review, we will highlight the central role of the plastid protein import system in regulating and coordinating the import of functionally related sets of preproteins that are required for plastid-type transitions and maintenance.

Sorting of SEC translocase SCY components to different membranes in chloroplasts.

Membrane proteins that are imported into chloroplasts must be accurately routed in order to establish and maintain the highly differentiated membranes characteristic of these organelles. Little is known about the targeting information or pathways involved, especially in the case of proteins with multiple transmembrane domains. We have studied targeting of the SCY components of the two SEC translocases in chloroplasts. SCY1 and SCY2 share a similar, highly conserved structure with 10 transmembrane domains, but are targeted to different membranes: the thylakoids and inner envelope, respectively. We used protoplast transfections and a confocal microscopy imaging assay in combination with a domain-swapping approach to investigate sorting pathways and identify important targeting elements in these proteins. We show that the N-terminal region of SCY1 contains targeting determinants that allow SCY1 to be recruited to the signal-recognition particle pathway. In addition, substituting the N-terminal region of SCY1 for the N-terminal region of SCY2 causes SCY2 to be displaced out of the inner envelope. The region of SCY2 that contains transmembrane domains 3 and 4 is necessary for localization to the inner envelope and may serve as a membrane anchor, enhancing the integration of other transmembrane domains via either stop-transfer or post-import mechanisms.

Chaperone-assisted Post-translational Transport of Plastidic Type I Signal Peptidase 1.

Type I signal peptidase (SPase I) is an integral membrane Ser/Lys protease with one or two transmembrane domains (TMDs), cleaving transport signals off translocated precursor proteins. The catalytic domain of SPase I folds to form a hydrophobic surface and inserts into the lipid bilayers at the trans-side of the membrane. In bacteria, SPase I is targeted co-translationally, and the catalytic domain remains unfolded until it reaches the periplasm. By contrast, SPases I in eukaryotes are targeted post-translationally, requiring an alternative strategy to prevent premature folding. Here we demonstrate that two distinct stromal components are involved in post-translational transport of plastidic SPase I 1 (Plsp1) from Arabidopsis thaliana, which contains a single TMD. During import into isolated chloroplasts, Plsp1 was targeted to the membrane via a soluble intermediate in an ATP hydrolysis-dependent manner. Insertion of Plsp1 into isolated chloroplast membranes, by contrast, was found to occur by two distinct mechanisms. The first mechanism requires ATP hydrolysis and the protein conducting channel cpSecY1 and was strongly enhanced by exogenously added cpSecA1. The second mechanism was independent of nucleoside triphosphates and proteinaceous components but with a high frequency of mis-orientation. This unassisted insertion was inhibited by urea and stroma extract. During import-chase assays using intact chloroplasts, Plsp1 was incorporated into a soluble 700-kDa complex that co-migrated with the Cpn60 complex before inserting into the membrane. The TMD within Plsp1 was required for the cpSecA1-dependent insertion but was dispensable for association with the 700-kDa complex and also for unassisted membrane insertion. These results indicate cooperation of Cpn60 and cpSecA1 for proper membrane insertion of Plsp1 by cpSecY1.

The Sec2 translocase of the chloroplast inner envelope contains a unique and dedicated SECE2 component.

Biogenesis of chloroplasts involves a series of protein trafficking events. Nuclear-encoded proteins are imported into the organelle, and then trafficked to various chloroplast locations by systems that are directly homologous to bacterial systems. Although the thylakoid-based systems have been studied extensively, much less is known about the systems that reside and function in the inner envelope membrane. One such system, the Sec2 system, is homologous to both the thylakoid-based Sec1 system and bacterial Sec systems, and may mediate both integration and translocation across the inner envelope. At a minimum, this system is expected to include three components, but only two, SCY2 and SECA2, have been identified in Arabidopsis. Bioinformatics and protein modeling were used to identify the protein encoded by At4g38490 as a candidate for the missing component (SECE2). Cellular localization, biochemistry, protein interaction assays in yeast, and co-immunoprecipitation experiments were used to establish that this protein is an integral membrane protein of the inner envelope, and specifically interacts with the SCY2 component in vivo. Sequence analyses indicated that SECE2 proteins are found in a variety of plants, and differ from the thylakoid SECE1 proteins in a stroma-exposed helical domain, which may contribute to their specificity. Finally, a genetic analysis indicated that SECE2 plays an essential role in plant growth and development.

The MADS-Domain Factors AGAMOUS-LIKE15 and AGAMOUS-LIKE18, along with SHORT VEGETATIVE PHASE and AGAMOUS-LIKE24, Are Necessary to Block Floral Gene Expression during the Vegetative Phase.

Multiple factors, including the MADS-domain proteins AGAMOUS-LIKE15 (AGL15) and AGL18, contribute to the regulation of the transition from vegetative to reproductive growth. AGL15 and AGL18 were previously shown to act redundantly as floral repressors and upstream of FLOWERING LOCUS T (FT) in Arabidopsis (Arabidopsis thaliana). A series of genetic and molecular experiments, primarily focused on AGL15, was performed to more clearly define their role. agl15 agl18 mutations fail to suppress ft mutations but show additive interactions with short vegetative phase (svp) mutations in ft and suppressor of constans1 (soc1) backgrounds. Chromatin immunoprecipitation analyses with AGL15-specific antibodies indicate that AGL15 binds directly to the FT locus at sites that partially overlap those bound by SVP and FLOWERING LOCUS C. In addition, expression of AGL15 in the phloem effectively restores wild-type flowering times in agl15 agl18 mutants. When agl15 agl18 mutations are combined with agl24 svp mutations, the plants show upward curling of rosette and cauline leaves, in addition to early flowering. The change in leaf morphology is associated with elevated levels of FT and ectopic expression of SEPALLATA3 (SEP3), leading to ectopic expression of floral genes. Leaf curling is suppressed by sep3 and ft mutations and enhanced by soc1 mutations. Thus, AGL15 and AGL18, along with SVP and AGL24, are necessary to block initiation of floral programs in vegetative organs.

Isolation of leaf peroxisomes from Arabidopsis for organelle proteome analyses.

The isolation of cell organelles from model organisms in high purity is important for biochemical analyses of single proteins, entire metabolic pathways, and protein complexes and is absolutely essential for organelle proteome analyses. The efficient enrichment of nearly all cell organelles is more difficult from Arabidopsis as compared to traditional model plants and especially challenging for peroxisomes. Leaf peroxisomes are generally very instable in aqueous solution due to the presence of a single membrane and (para-)crystalline inclusions in the matrix. Leaf peroxisomes from Arabidopsis are particularly fragile and, moreover, strongly physically adhere to chloroplasts and mitochondria for largely unknown reasons. Here, we provide a detailed protocol for the isolation of Arabidopsis leaf peroxisomes by Percoll followed by sucrose density gradient centrifugation that yields high purity suitable for proteome analyses. Diverse enzymatic and immuno-biochemical methods are summarized to assess purity and intactness.