Heterologous expression of bacterial dehydrin gene in Arabidopsis thaliana promotes abiotic stress tolerance.

Salinity, low temperature, and drought are major environmental factors in agriculture leading to reduced crop yield. Dehydrins (DHNs) are induced transcriptionally during cellular dehydration and accumulate in different tissues during abiotic stresses. Here we isolated and characterized a bacterial gene BG757 in Arabidopsis, encoding a putative dehydrin type protein. ABA induces the expression of various dehydrins in plants, therefore, to elucidate the potential role, ABA sensitivity was examined in Arabidopsis transgenic lines expressing BG757. Interestingly, BG757-expressing plants showed hypersensitivity towards NaCl and ABA during seed germination. In addition to germination, BG757-expressing plants also showed root growth retardation in the presence of ABA and NaCl when compared with wild type (WT), suggesting that BG757 positively regulate salt stress and ABA response. Furthermore, BG757-expressing plants showed significant drought tolerance compared with WT. Consistent with drought tolerance, expression levels of stress inducible genes (DREB2A, RD22, RD26, LEA7 and SOS1) were strongly upregulated in transgenic plants compared with WT. All together these results suggest that heterologous expression of bacterial gene, BG757 in plants promotes resistance to environmental stresses. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01358-w.

Delpinium uncinatum mediated green synthesis of AgNPs and its antioxidant, enzyme inhibitory, cytotoxic and antimicrobial potentials.

Green synthesis of nanoparticles is becoming a method of choice for biological research due to its environmentally benign outcomes, stability and ease of synthesis. In this study, silver nanoparticles (AgNPs) were synthesized using stem (S-AgNPs), root (R-AgNPs) and mixture of stem and root (RS-AgNPs) of Delphinium uncinatum. The synthesized nanoparticles were characterized by standardized techniques and evaluated for their antioxidant, enzyme inhibition, cytotoxic and antimicrobial potentials. The AgNPs exhibited efficient antioxidant activities and considerable enzyme inhibition potential against alpha amylase, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes. S-AgNPs showed strong cytotoxicity against human hepato-cellular carcinoma cells (HepG2) and high enzyme inhibitory effect (IC50 values 27.5µg/ml for AChE and 22.60 µg/ml for BChE) compared to R-AgNPs and RS-AgNPs. RS-AgNPs showed significant inhibition of Klebsiella pneumoniae and Aspergillus flavus and exhibited higher biocompatibility (<2% hemolysis) in human red blood cells hemolytic assays. The present study showed that biologically synthesized AgNPs using the extract of various parts of D. uncinatum have strong antioxidant and cytotoxic potentials.

Genome-wide in silico identification and characterization of the stress associated protein (SAP) gene family encoding A20/AN1 zinc-finger proteins in potato (Solanum tuberosum L.).

Stress associated proteins (SAPs) in plants have a key role in providing tolerance to multiple abiotic stresses. SAP gene family in Solanum tuberosum has not been fully studied before. This study identified 17 StSAP genes in S. tuberosum which code for A20/AN1 zinc-finger proteins. All the genes were distributed on ten different chromosomes and six segmental duplication events were identified. The SAPs in S. tuberosum and its orthologs in Arabidopsis thaliana were classified into six groups through the phylogenetic analysis. Introns across StSAP genes were identified in four genes. The promotor study of the StSAP genes showed different hormone and stress-related cis-elements that could potentially have a role in environmental stress response. The expression of StSAP genes in response to heat, mannitol, and salt were analyzed through in silico transcriptomic analysis. This study could potentially help in further understanding the functions of SAP genes in S. tuberosum.

Evaluation of analgesic, antiamnesic and antidiarrheal potentials of Medicago denticulata extract using animal model.

The analgesic, antidiarrheal, and neuro-pharmacological potentials of Medicago denticulata leaves extract were screened in animal models. Potential analgesic response was noted (*P < 0.05, **P < 0.01, ***P < 0.001) in formalin, acetic acid and heat-induced pain models in a dose-dependent manner. Maximum activity by means of writhing inhibition was documented for Medicago denticulata at 300 mg/kg that was found to be 71.79% (17.43 ± 1.31). In first phase, the Medicago denticulata at a dose of 150 and 300 mg/kg showed analgesic activity and reduced the pain by 54.18% (18.39 ± 1.67) and 62.90% (14.89 ± 1.56), respectively. In second phase, the Medicago denticulata at a dose of 150 and 300 mg/kg showed analgesic activity and reduced the pain by 69.48% (19.78 ± 1.44) and 70.89% (18.86 ± 1.58), respectively. In hot plate method, the Medicago denticulata at a dose of 150 and 300 mg/kg showed the maximum response of 61.16% (8.47 ± 1.23) and 67.39% (10.09 ± 1.04), respectively at 60 min. Scopolamine significantly reduces spontaneous alteration in Y-maze model for antiamnesic activity. Medicago denticulata significantly increased the discrimination index in a dose-dependent manner using novel object recognition test (NORT) model. Exploration time in sec for the novel object was increased significantly (P < 0.001) by donepezil decreased for familiar one with a discrimination index (DI) of 62.18%. Medicago denticulata significantly increased the discrimination index by 60.86% and 57.24% at 300 and 150 mg/kg b.w, respectively. The lowest DI of 53.80% at 75 mg/kg was observed in comparison to the amnesic group. The Medicago denticulata significant decreased the elevated levels of acetylcholinesterase (AChE) and malondialdehyde (MDA and enhancing level of acetylcholine (ACh), superoxide dismutase (SOD) and catalase (CAT) acting as an antioxidant agent. Medicago denticulata reduced the total number of diarrheal feces to lesser extent at dose-dependent manner. From the study results, it is suggested that the Medicago denticulata extract possess good analgesic and antiamnesic activity however the antidiarrheal effects of plant were negligible. In the current study, the traditional use of the plant as a source of medicine has been validated.

Therapeutic role of Typha elephantina leaves aqueous extract in paracetamol intoxicated rabbits.

Present study is aimed to investigate the hepatoprotective and hematopoietic effect of Typha elephantina leaves aqueous (T.E.AQ), extract in paracetamol (PCM) intoxicated rabbits. Experimental animals were divided into various groups. The blood was taken on day 7th (W1=Week 1), day 14th (W2 = week 2) and day 21st (W3 = week 3) of treatments and was analyzed for all hematological and serum biochemical markers. PCM administration caused marked increase in the levels of serum biochemical and hematological parameters. The leaves of T.E.AQ extract at dose rate 300mg/kg body weight significantly (P<0.05) reduced the elevated levels of serum biochemical and hematological indices towards normal values on third week (day 21st) of treatment while treatment in the first two weeks revealed non-significant effects even at all doses of extract. The levels of glutathione (GSH) and radical scavenging activity (RSA) were reduced and thiobarbituric acid reactive substances (TBARS) levels was high in the PCM feed animals. Administration of (T.E.AQ) extract at high dose (300mg/kg) significantly regulated and normalized these antioxidant values. The antioxidant capacity of (TE.AQ) extract, showed increase inhibition against various extract concentrations on the basis of percent scavenging of (DPPH) free radical. The histological sections of liver further supported the hepatoprotective activity of extract.

Iron-doped zinc oxide nanoparticles-triggered elicitation of important phenolic compounds in cell cultures of Fagonia indica.

The callus cultures of Fagonia indica could prove as factories for the production of important phytochemicals when triggered through different types of stress. In this study, we initiated callus cultures from healthy stem explants in the presence of iron-doped zinc oxide nanoparticles (Fe-ZnO-NPs). We performed experiments with the callus cultures of F. indica to determine the impact of Fe-ZnO-NPs in concentrations (15.62-250 µg/mL) on biomass accumulation, production of important phenolic and flavonoids, and antioxidative potential. Our results showed that maximum callus biomass [Fresh weight (FW) = 13.6 g and Dry weight (DW) = 0.58 ± 0.01] was produced on day 40 when the media was supplemented with 250 µg/mL Fe-ZnO-NPs. Similarly, maximum total phenolic content (268.36 µg GAE/g of DW) was observed in 40 days old callus added with 125 µg/mL Fe-ZnO-NPs. Maximum total flavonoid content (78.56 µg QE/g of DW) was recorded in 20 days old callus grown in 62.5 µg/mL Fe-ZnO-NPs containing media. Maximum total antioxidant capacity (390.74 µg AAE/g of DW) was recorded in 40 days old callus with 125 µg/mL Fe-ZnO-NPs treated cultures, respectively. Similarly, the highest free radical scavenging activity (93.02%) was observed in callus derived from media having 15.62 µg/mL Fe-ZnO-NPs. The antioxidant potential was observed to have positive correlation with TPC (r = 0.44). HPLC analysis showed that Fe-ZnO-NPs produced compounds (e.g., Epigallocatechin gallate) that were either absent or in lesser quantities in the control group. These results showed that Fe-ZnO-NPs elicitors could increase the biomass and activate secondary metabolism in F. indica cells. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11240-021-02123-1.

Comparative analysis of the effects of chemically and biologically synthesized silver nanoparticles on biomass accumulation and secondary metabolism in callus cultures of Fagonia indica.

Biotechnological strategies are needed to produce larger quantities of biomass and phytochemicals. In this study, callus cultures of Fagonia indica were elicited with different concentrations of chemically and biologically synthesized silver nanoparticles (chem- and bioAgNPs) to compare their effects on biomass, total phenolic content (TPC), total flavonoid content (TFC) and antioxidant activity of the extracts from callus. The results revealed that bioAgNPs being more biocompatible produced the highest biomass initially on day 10 (FW = 4.2152 ± 0.13 g; DW = 0.18527 ± 0.01 g) and day 20 (FW = 7.6558 ± 0.10 g; DW = 0.3489 ± 0.01 g) when supplemented in media as 62.5 µg/mL and 250 µg/mL, respectively. Initially, the highest TPC (319.32 ± 8.28 µg GAE/g of DW) was recorded on day 20 in chemAgNPs (31.25 µg/mL) induced callus as compared to TPC = 302.85 ± 3.002 µg GAE/g of DW in bioAgNPs-induced callus. Compared to the highest values of TFC (108.15 ± 2.10 µg QE/g of DW) produced in 15.6 µg/mL chemAgNPs-induced callus on day 20, TFC produced in bioAgNPs (62.5 µg/mL) was 168.61 ± 3.17 µg GAE/g of DW on day 10. Similarly, chemAgNPs-induced callus (62.5 µg/mL) showed the highest free radical scavenging activity (FRSA) i.e. 87.18% on day 20 while bioAgNPs (125 µg/mL) showed 81.69% FRSA on day 20 compared to highest among control callus (63.98% on day 40). The highest total antioxidant capacity of chemAgNPs-(125 µg/mL) induced callus was 330.42 ± 13.65 µg AAE/g of DW on day 20 compared to bioAgNPs-(62.5 µg/mL) induced callus (312.96 ± 1.73 µg AAE/g of DW) on day 10. Conclusively, bioAgNPs are potent elicitors of callus cultures of F. indica.

Dynamin-Like Proteins Are Potentially Involved in Membrane Dynamics within Chloroplasts and Cyanobacteria.

Dynamin-like proteins (DLPs) are a family of membrane-active proteins with low sequence identity. The proteins operate in different organelles in eukaryotic cells, where they trigger vesicle formation, membrane fusion, or organelle division. As discussed here, representatives of this protein family have also been identified in chloroplasts and DLPs are very common in cyanobacteria. Since cyanobacteria and chloroplasts, an organelle of bacterial origin, have similar internal membrane systems, we suggest that DLPs are involved in membrane dynamics in cyanobacteria and chloroplasts. Here, we discuss the features and activities of DLPs with a focus on their potential presence and activity in chloroplasts and cyanobacteria.

Understanding Plastid Vesicle Transport - Could it Provide Benefit for Human Medicine?

BACKGROUND: In plants, vesicle transport occurs in the secretory pathway in the cytosol, between the membranes of different compartments. Several protein components have been identified to be involved in the process and their functions were characterized. Both cargos and other molecules (such as hormones) have been shown to use vesicle transport, although the major constituents of vesicles are lipids which are transferred from donor to acceptor membranes. In humans, malfunction of the cytosolic vesicle transport system leads to different diseases. METHOD: To better understand and ultimately cure these human diseases, studying other model systems such as yeast can be beneficial. Plants with their cytosolic vesicle transport system could serve as another model system. However, this review focuses on plant vesicles not present in the cytosol but in the chloroplasts, where lipids produced in the surrounding envelope are transported through the aqueous stroma to the thylakoid membranes. Although chloroplast vesicles have found both biochemical and ultrastructural support, only two proteins have been characterized as components of the pathway. However, using bioinformatics a number of other proteins have been suggested as homologs to the cytosolic system. RESULTS & CONCLUSION: Based on these findings vesicles of chloroplasts are likely most similar to the vesicles trafficking from ER to Golgi, or may even be unique, but important experimental support is yet lacking. In this review, proposed vesicle transport components in chloroplasts are presented, and their possible future implementation for human medicine is discussed.

Gene expression pattern for putative chloroplast localized COPII related proteins with emphasis on Rab related proteins.

Vesicle transport occurs in the cytosol through COPI, COPII and a clathrin coated vesicle system for transport of lipids and proteins to different subcellular compartments. All three systems consist of several different protein components to maintain a functional transport. In chloroplasts photosynthesis takes place in thylakoids. Thylakoids contain a large amount of lipids and proteins but none of these components are produced there. Transport of lipids occurs from the envelope membrane where they are produced and through the aqueous stroma before being directed to the thylakoids. Nuclear encoded proteins use distinct pathways for entering thylakoids after import into chloroplasts. Transport of lipids through stroma requires either lipid transfer proteins, association between the envelope and the thylakoid membrane, or a vesicle transport system similar to the cytosolic one. No evidence exists for lipid transfer proteins in chloroplasts, nor for a consistent association between the envelope and the thylakoid membrane. However, vesicle transport has support from e.g., biochemical and genetics data as well as transelectron microscopy data. Moreover, a recent bioinformatics study revealed putatively COPII related proteins to be chloroplast localized in Arabidopsis and thus function in vesicle transport in chloroplasts. Here we present gene expression profiles of these putatively COPII related chloroplast localized proteins using Genevestigator (https://www.genevestigator.com/gv/) with special emphasis on Rab related proteins since they represent several stage of vesicle transport e.g., uncoating, tethering and fusion.

A novel chloroplast localized Rab GTPase protein CPRabA5e is involved in stress, development, thylakoid biogenesis and vesicle transport in Arabidopsis.

A novel Rab GTPase protein in Arabidopsis thaliana, CPRabA5e (CP = chloroplast localized) is located in chloroplasts and has a role in transport. Transient expression of CPRabA5e:EGFP fusion protein in tobacco (Nicotiana tabacum) leaves, and immunoblotting using Arabidopsis showed localization of CPRabA5e in chloroplasts (stroma and thylakoids). Ypt31/32 in the yeast Saccharomyces cerevisiae are involved in regulating vesicle transport, and CPRabA5e a close homolog of Ypt31/32, restores the growth of the ypt31Δ ypt32(ts) mutant at 37 °C in yeast complementation. Knockout mutants of CPRabA5e displayed delayed seed germination and growth arrest during oxidative stress. Ultrastructural studies revealed that after preincubation at 4 °C mutant chloroplasts contained larger plastoglobules, lower grana, and more vesicles close to the envelopes compared to wild type, and vesicle formation being enhanced under oxidative stress. This indicated altered thylakoid development and organization of the mutants. A yeast-two-hybrid screen with CPRabA5e as bait revealed 13 interacting partner proteins, mainly located in thylakoids and plastoglobules. These proteins are known or predicted to be involved in development, stress responses, and photosynthesis related processes, consistent with the stress phenotypes observed. The results observed suggest a role of CPRabA5e in transport to and from thylakoids, similar to cytosolic Rab proteins involved in vesicle transport.

Evolutionary, molecular and genetic analyses of Tic22 homologues in Arabidopsis thaliana chloroplasts.

The Tic22 protein was previously identified in pea as a putative component of the chloroplast protein import apparatus. It is a peripheral protein of the inner envelope membrane, residing in the intermembrane space. In Arabidopsis, there are two Tic22 homologues, termed atTic22-III and atTic22-IV, both of which are predicted to localize in chloroplasts. These two proteins defined clades that are conserved in all land plants, which appear to have evolved at a similar rates since their separation >400 million years ago, suggesting functional conservation. The atTIC22-IV gene was expressed several-fold more highly than atTIC22-III, but the genes exhibited similar expression profiles and were expressed throughout development. Knockout mutants lacking atTic22-IV were visibly normal, whereas those lacking atTic22-III exhibited moderate chlorosis. Double mutants lacking both isoforms were more strongly chlorotic, particularly during early development, but were viable and fertile. Double-mutant chloroplasts were small and under-developed relative to those in wild type, and displayed inefficient import of precursor proteins. The data indicate that the two Tic22 isoforms act redundantly in chloroplast protein import, and that their function is non-essential but nonetheless required for normal chloroplast biogenesis, particularly during early plant development.

New putative chloroplast vesicle transport components and cargo proteins revealed using a bioinformatics approach: an Arabidopsis model.

Proteins and lipids are known to be transported to targeted cytosolic compartments in vesicles. A similar system in chloroplasts is suggested to transfer lipids from the inner envelope to the thylakoids. However, little is known about both possible cargo proteins and the proteins required to build a functional vesicle transport system in chloroplasts. A few components have been suggested, but only one (CPSAR1) has a verified location in chloroplast vesicles. This protein is localized in the donor membrane (envelope) and vesicles, but not in the target membrane (thylakoids) suggesting it plays a similar role to a cytosolic homologue, Sar1, in the secretory pathway. Thus, we hypothesized that there may be more similarities, in addition to lipid transport, between the vesicle transport systems in the cytosol and chloroplast, i.e. similar vesicle transport components, possible cargo proteins and receptors. Therefore, using a bioinformatics approach we searched for putative chloroplast components in the model plant Arabidopsis thaliana, corresponding mainly to components of the cytosolic vesicle transport system that may act in coordination with previously proposed COPII chloroplast homologues. We found several additional possible components, supporting the notion of a fully functional vesicle transport system in chloroplasts. Moreover, we found motifs in thylakoid-located proteins similar to those of COPII vesicle cargo proteins, supporting the hypothesis that chloroplast vesicles may transport thylakoid proteins from the envelope to the thylakoid membrane. Several putative cargo proteins are involved in photosynthesis, thus we propose the existence of a novel thylakoid protein pathway that is important for construction and maintenance of the photosynthetic machinery.

Genes co-expressed with CPSAR1 identified using ATTED-II.

Identification of interacting proteins will help to investigate further the relationship between CPSAR1 and the vesicle transport system or the ribosomes. Thus, we adopted a bioinformatic approach, using the publicly available Arabidopsis thaliana trans-factor and cis-element prediction database, ATTED-II (http://atted.jp/), to identify putative protein interactors. The proteins directly linked to CPSAR1 were almost exclusively nucleus encoded and several were involved in protein synthesis of which three were thylakoid localized. The list of putative interacting proteins does not exclude any of the previous proposed actions of CPSAR1 but encourage more detailed examination of the role of CPSAR1.