Plastid-expressed AdDjSKI enhances photosystem II stability, delays leaf senescence, and increases fruit yield in tomato plants under heat stress.

Heat stress substantially reduces tomato (Solanum lycopersicum) growth and yield globally, thereby jeopardizing food security. DnaJ proteins, constituents of the heat shock protein system, protect cells from diverse environmental stresses as HSP-70 molecular co-chaperones. In this study, we demonstrated that AdDjSKI, a serine-rich DnaJ III protein induced by pathogens, plays an important role in stabilizing photosystem II (PSII) in response to heat stress. Our results revealed that transplastomic tomato plants expressing the AdDjSKI gene exhibited increased levels of total soluble proteins, improved growth and chlorophyll content, reduced malondialdehyde (MDA) accumulation, and diminished PSII photoinhibition under elevated temperatures when compared with wild-type (WT) plants. Intriguingly, these transplastomic plants maintained higher levels of D1 protein under elevated temperatures compared with the WT plants, suggesting that overexpression of AdDjSKI in plastids is crucial for PSII protection, likely due to its chaperone activity. Furthermore, the transplastomic plants displayed lower accumulation of superoxide radical (O2 •─) and H2O2, in comparison with the WT plants, plausibly attributed to higher superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities. This also coincides with an enhanced expression of corresponding genes, including SlCuZnSOD, SlFeSOD, SlAPX2, and SltAPX, under heat stress. Taken together, our findings reveal that chloroplastic expression of AdDjSKI in tomatoes plays a critical role in fruit yield, primarily through a combination of delayed senescence and stabilizing PSII under heat stress.

Enhancing drought tolerance in chilli pepper through AdDjSKI-mediated modulation of ABA sensitivity, photosynthetic preservation, and ROS scavenging.

Drought stress threatens the productivity of numerous crops, including chilli pepper (Capsicum annuum). DnaJ proteins are known to play a protective role against a wide range of abiotic stresses. This study investigates the regulatory mechanism of the chloroplast-targeted chaperone protein AdDjSKI, derived from wild peanut (Arachis diogoi), in enhancing drought tolerance in chilli peppers. Overexpressing AdDjSKI in chilli plants increased chlorophyll content, reflected in the maximal photochemical efficiency of photosystem II (PSII) (Fv/Fm) compared with untransformed control (UC) plants. This enhancement coincided with the upregulated expression of PSII-related genes. Our subsequent investigations revealed that transgenic chilli pepper plants expressing AdDjSKI showed reduced accumulation of superoxide and hydrogen peroxide and, consequently, lower malondialdehyde levels and decreased relative electrolyte leakage percentage compared with UC plants. The mitigation of ROS-mediated oxidative damage was facilitated by heightened activities of antioxidant enzymes, including superoxide dismutase, catalase, ascorbate peroxidase, and peroxidase, coinciding with the upregulation of the expression of associated antioxidant genes. Additionally, our observations revealed that the ectopic expression of the AdDjSKI protein in chilli pepper plants resulted in diminished ABA sensitivity, consequently promoting seed germination in comparison with UC plants under different concentrations of ABA. All of these collectively contributed to enhancing drought tolerance in transgenic chilli plants with improved root systems when compared with UC plants. Overall, our study highlights AdDjSKI as a promising biotechnological solution for enhancing drought tolerance in chilli peppers, addressing the growing global demand for this economically valuable crop.

CRISPR/Cas9 based genome editing of Phytoene desaturase (PDS) gene in chilli pepper (Capsicum annuum L.).

An effective CRISPR/Cas9 reagent delivery system has been developed in a commercially significant crop, the chilli pepper using a construct harboring two distinct gRNAs targeting exons 14 and 15 of the Phytoene desaturase (CaPDS) gene, whose loss-of-function mutation causes a photo-bleaching phenotype and impairs the biosynthesis of carotenoids. The construct carrying two sgRNAs was observed to create visible albino phenotypes in cotyledons regenerating on a medium containing 80 mg/L kanamycin, and plants regenerated therefrom after biolistic-mediated transfer of CRISPR/Cas9 reagents into chilli pepper cells. Analysis of CRISPR/Cas9 genome-editing events, including kanamycin screening of mutants and assessing homozygosity using the T7 endonuclease assay (T7E1), revealed 62.5 % of transformed plants exhibited successful editing at the target region and displayed both albino and mosaic phenotypes. Interestingly, the sequence analysis showed that insertions and substitutions were present in all the plant lines in the targeted CaPDS region. The detected mutations were mostly 12- to 24-bp deletions that disrupted the exon-intron junction, along with base substitutions and the insertion of 1-bp at the protospacer adjacent motif (PAM) region of the target site. The reduction in essential photosynthetic pigments (chlorophyll a, chlorophyll b and carotenoid) in knockout chilli pepper lines provided further evidence that the CaPDS gene had been functionally disrupted. In this present study, we report that the biolistic delivery of CRISPR/Cas9 reagents into chilli peppers is very effective and produces multiple mutation events in a short span of time.

Chloroplast Genome Engineering: A Plausible Approach to Combat Chili Thrips and Other Agronomic Insect Pests of Crops.

The world population's growing demand for food is expected to increase dramatically by 2050. The agronomic productivity for food is severely affected due to biotic and abiotic constraints. At a global level, insect pests alone account for ~20% loss in crop yield every year. Deployment of noxious chemical pesticides to control insect pests always has a threatening effect on human health and environmental sustainability. Consequently, this necessitates for the establishment of innovative, environmentally friendly, cost-effective, and alternative means to mitigate insect pest management strategies. According to a recent study, using chloroplasts engineered with double-strand RNA (dsRNA) is novel successful combinatorial strategy deployed to effectively control the most vexing pest, the western flower thrips (WFT: Frankliniella occidentalis). Such biotechnological avenues allowed us to recapitulate the recent progress of research methods, such as RNAi, CRISPR/Cas, mini chromosomes, and RNA-binding proteins with plastid engineering for a plausible approach to effectively mitigate agronomic insect pests. We further discussed the significance of the maternal inheritance of the chloroplast, which is the major advantage of chloroplast genome engineering.

Woodfordia fruticosa (L.) Kurz: in vitro biotechnological interventions and perspectives.

Woodfordia fruticosa (L.) Kurz is a woody medicinal shrub (Lythraceae) commonly known as the "fire flame bush." W. fruticosa plant parts either alone or whole plant have a long history of recommended use in the Indian medicine systems of Ayurveda, Unani, and Siddha (AUS). This plant is prominently known for its pharmacological properties, viz., antimicrobial, anti-inflammatory, anti-peptic ulcer, hepatoprotective, immunomodulatory, antitumor, cardioprotective, analgesic, and wound healing activities. Its important phyto-constituents, woodfordin C, woodfordin I, oenothein B, and isoschimacoalin-A, exhibit in vitro or in vivo physiological activities beneficial to human health. As the plant is a rich storehouse of phyto-constituents, it is indiscriminately used in its wild habitats. Moreover, due to very poor seed viability and difficult-to-root qualities, it is placed under IUCN list of endangered plant species. For W. fruticosa, biomass production or to its conservation by in vitro regeneration is the best feasible alternative. Till date, only few important in vitro regeneration methods are reported in W. fruticosa. ISSR molecular markers based clonal fidelity and Agrobacterium-mediated transformation has been demonstrated, indicating that W. fruticosa is amenable to genetic manipulation and genome editing studies. This review presents concise summary of updated reports on W. fruticosa phyto-constituents and their biological activities, while a critical appraisal of biotechnological interventions, shortcomings, and factors influencing such potential areas success was presented. The unexplored gaps addressed here are relevant for W. fruticosa scientific innovations yet to come. In this paper, for the first time, we have presented a simple and reproducible protocol for synthetic seed production in W. fruticosa. KEY POINTS: • Critical and updated records on W. fruticosa phytochemistry and its activities • In vitro propagation and elicitation of secondary metabolites in W. fruticosa • Key bottlenecks, in vitro flowering, value addition, and outlook in W. fruticosa.

An Efficient Method of Mitochondrial DNA Isolation from Vigna radiata for Genomic Studies.

Isolation of mitochondrial DNA from root tissues of mung bean (Vigna radiata) is quite tedious, complex, and often results in low yield. Hence here we show a simple, rapid, and improved protocol for isolation of mitochondrial DNA from root tissues of hydroponically grown mung bean plants. This method involves purification of mitochondria and subsequent isolation of DNA from obtained purified mitochondria. For this purpose, mitochondria were isolated using a discontinuous Percoll gradient centrifugation followed by RNase I treatment to the isolated DNA to remove any traces of RNA contamination. The mitochondrial DNA was isolated from mitochondrial samples by commonly used CTAB method. The specificity of isolated mitochondrial DNA was confirmed using mtDNA-specific genes (NAD1 and COX3). ß-Actin primer was used to check the nuclear DNA contamination. PCR amplification was observed in mtDNA specific genes NAD1 and COX3 except nuclear encoded ß-actin gene suggesting that mitochondrial DNA was not contaminated by nuclear DNA.

An Overview of Important Enzymes Involved in Nitrogen Assimilation of Plants.

Nitrogen (N) is a macro-nutrient that is essential for growth development and resistance against biotic and abiotic stresses of plants. Nitrogen is a constituent of amino acids, proteins, nucleic acids, chlorophyll, and various primary and secondary metabolites. The atmosphere contains huge amounts of nitrogen but it cannot be taken up directly by plants. Plants can take up nitrogen in the form of nitrate, ammonium, urea, nitrite, or a combination of all these forms. In addition, in various leguminous rhizobia, bacteria can convert atmospheric nitrogen to ammonia and supply it to the plants. The form of nitrogen nutrition is also important in plant growth and resistance against pathogens. Nitrogen content has an important function in crop yield. Nitrogen deficiency can cause reduced root growth, change in root architecture, reduced plant biomass, and reduced photosynthesis. Hence, understanding the function and regulation of N metabolism is important. Several enzymes and intermediates are involved in nitrogen assimilation. Here we provide an overview of the important enzymes such as nitrate reductase, nitrite reductase, glutamine synthase, GOGAT, glutamate dehydrogenase, and alanine aminotransferase that are involved in nitrogen metabolism.

Measurement of Nitrate Reductase Activity in Tomato (Solanum lycopersicum L.) Leaves Under Different Conditions.

Nitrogen is one of the crucial macronutrients essential for plant growth, development, and survival under stress conditions. Depending on cellular requirement, plants can absorb nitrogen mainly in multiple forms such as nitrate (NO3-) or ammonium (NH4+) or combination of both via efficient and highly regulated transport systems in roots. In addition, nitrogen-fixing symbiotic bacteria can fix atmospheric nitrogen in to NH4+ via highly regulated complex enzyme system and supply to the roots in nodules of several species of leguminous plants. If NO3- is a primary source, it is transported from roots and then it is rapidly converted to nitrite (NO2-) by nitrate reductase (NR) (EC 1.6.6.1) which is a critical and very important enzyme for this conversion. This key reaction is mediated by transfer of two electrons from NAD(P)H to NO3-. This occurs via the three redox centers comprised of two prosthetic groups (FAD and heme) and a MoCo cofactor. NR activity is greatly influenced by factors such as developmental stage and various stress conditions such as hypoxia, salinity and pathogen infection etc. In addition, light/dark dynamics plays crucial role in modulating NR activity. NR activity can be easily detected by measuring the conversion of NO3- to NO2- under optimized conditions. Here, we describe a detailed protocol for measuring relative NR enzyme activity of tomato crude extracts. This protocol offers an efficient and straightforward procedure to compare the NR activity of various plants under different conditions.

Expression Analysis of Important Genes Involved in Nitrogen Metabolism Under Hypoxia.

Hypoxia or anoxia condition can occurs during flooding or waterlogging and can cause intense damage to the plants. Since oxygen is important for active operation of electron transport chain in mitochondria to generate energy production (ATP) any drop in oxygen can cause an energy crisis during flooding/waterlogging. To cope with this energy crisis plants have developed various anatomical, physiological, and biochemical adaptations. Perception of signals and induction of genes are required for initiation of these adaptive responses. Various genes involved in nitrogen, carbon, and fermentative metabolism play a role in hypoxic tolerance. Regulation of genes involved in nitrogen metabolism also plays a role under hypoxia. Hence in this present chapter we describe the expression of nitrate reductase-1 (NIA1), nitrate reductase-2 (NIA2), and glutamine synthetase-1 (GLN-1) during hypoxia in Arabidopsis.

Polyamine Induction in Postharvest Banana Fruits in Response to NO Donor SNP Occurs via l-Arginine Mediated Pathway and Not via Competitive Diversion of S-Adenosyl-l-Methionine.

Nitric oxide (NO) is known to antagonize ethylene by various mechanisms; one of such mechanisms is reducing ethylene levels by competitive action on S-adenosyl-L-methionine (SAM)-a common precursor for both ethylene and polyamines (PAs) biosynthesis. In order to investigate whether this mechanism of SAM pool diversion by NO occur towards PAs biosynthesis in banana, we studied the effect of NO on alterations in the levels of PAs, which in turn modulate ethylene levels during ripening. In response to NO donor sodium nitroprusside (SNP) treatment, all three major PAs viz. putrescine, spermidine and spermine were induced in control as well as ethylene pre-treated banana fruits. However, the gene expression studies in two popular banana varieties of diverse genomes, Nanjanagudu rasabale (NR; AAB genome) and Cavendish (CAV; AAA genome) revealed the downregulation of SAM decarboxylase, an intermediate gene involved in ethylene and PA pathway after the fifth day of NO donor SNP treatment, suggesting that ethylene and PA pathways do not compete for SAM. Interestingly, arginine decarboxylase belonging to arginine-mediated route of PA biosynthesis was upregulated several folds in response to the SNP treatment. These observations revealed that NO induces PAs via l-arginine-mediated route and not via diversion of SAM pool.

Current approaches to measure nitric oxide in plants.

Nitric oxide (NO) is now established as an important signalling molecule in plants where it influences growth, development, and responses to stress. Despite extensive research, the most appropriate methods to measure and localize these signalling radicals are debated and still need investigation. Many confounding factors such as the presence of other reactive intermediates, scavenging enzymes, and compartmentation influence how accurately each can be measured. Further, these signalling radicals have short half-lives ranging from seconds to minutes based on the cellular redox condition. Hence, it is necessary to use sensitive and specific methods in order to understand the contribution of each signalling molecule to various biological processes. In this review, we summarize the current knowledge on NO measurement in plant samples, via various methods. We also discuss advantages, limitations, and wider applications of each method.

Alternative oxidase is an important player in the regulation of nitric oxide levels under normoxic and hypoxic conditions in plants.

Plant mitochondria possess two different pathways for electron transport from ubiquinol: the cytochrome pathway and the alternative oxidase (AOX) pathway. The AOX pathway plays an important role in stress tolerance and is induced by various metabolites and signals. Previously, several lines of evidence indicated that the AOX pathway prevents overproduction of superoxide and other reactive oxygen species. More recent evidence suggests that AOX also plays a role in regulation of nitric oxide (NO) production and signalling. The AOX pathway is induced under low phosphate, hypoxia, pathogen infections, and elicitor treatments. The induction of AOX under aerobic conditions in response to various stresses can reduce electron transfer through complexes III and IV and thus prevents the leakage of electrons to nitrite and the subsequent accumulation of NO. Excess NO under various stresses can inhibit complex IV; thus, the AOX pathway minimizes nitrite-dependent NO synthesis that would arise from enhanced electron leakage in the cytochrome pathway. By preventing NO generation, AOX can reduce peroxynitrite formation and tyrosine nitration. In contrast to its function under normoxia, AOX has a specific role under hypoxia, where AOX can facilitate nitrite-dependent NO production. This reaction drives the phytoglobin-NO cycle to increase energy efficiency under hypoxia.

Efficient in vitro direct shoot organogenesis from seedling derived split node explants of maize (Zea mays L.).

Maize is one of the important cereal crops around the world. An efficient and reproducible regeneration protocol via direct organogenesis has been established using split nodes as ex-plants derived from 7 to 10 day old in vitro grown seedlings. Surface sterilized maize seeds were germinated on MS medium lacking plant growth regulators. Nodal sections of 7-10 day old seedlings were isolated, split longitudinally into two halves and cultured on regeneration medium containing different concentrations of 6-benzyladenine (2.20, 4.40, 6.60, 8.80, 11.0 and 13.2 µM) or kinetin (2.32, 4.65, 6.97, 9.29, 11.6 and 13.9 µM). Inclusion of 8.80 µM BA into MS supplemented medium triggered a high frequency of regeneration response from split node explants with a maximum number of shoots (12.0 ± 1.15) and the highest shoot length (3.0 ± 0.73) was obtained directly (without an intervening callus phase) within 4 weeks of culture. Further shoot elongation was achieved on medium containing 4.40 µM BA. The elongated micro shoots were rooted on MS medium fortified with 1.97 µM indole-3-butyric acid. The regenerated plantlets with roots were successfully hardened on earthen pots after proper acclimatization under greenhouse conditions. This new efficient regeneration method provides a solid foundation for genetic manipulation of maize for biotic and abiotic stresses and to enhance the nutritional values.

Agrobacterium tumefaciens - Mediated transformation of Woodfordia fruticosa (L.) Kurz.

In the present study, a protocol for Agrobacterium tumefaciens-mediated transformation has been optimized for Woodfordia fruticosa (L.) Kurz. Precultured axenic leaf segments were co-cultivated with A. tumefaciens strain LBA4404 harboring the binary plasmid pCAMBIA1301 with ß-glucuronidase (uidA) containing intron as the reporter gene and hygromycin phosphotransferase (hpt) as a selectable marker gene. After 3 days of co-cultivation, leaf segments were cultured on MS medium containing Thidiazuron (TDZ 4.54 µM) and Indole-3-acetic acid IAA (1.14 µM) + 20 mg/l hygromycin + 200 mg/l cefotaxime (PTSM1) for 4 weeks (includes a single subculture onto the same medium at a 2 week interval). They were subsequently cultured for 3 weeks on MS medium containing Thidiazuron (TDZ 4.54 µM) and Indole-3-acetic acid IAA (1.14 µM) + 25 mg/l hygromycin + 100 mg/l cefotaxime (PTSM2) medium for further development and shoot elongation. The hygromycin resistant shoots were rooted on a rooting medium (PTRM) containing half strength MS medium + 4.90 µM IBA + 25 mg/l hygromycin. A highest transformation efficiency of 44.5% with a mean number of 2.6 transgenic shoots per explant was achieved. Successful transformation was confirmed by the histochemical GUS activity of the regenerated shoots, PCR and RT-PCR analysis using respective primers. Southern blot analysis revealed that the hpt gene integrated into the genome of transgenic W. fruticosa. Establishment of genetic transformation protocol may facilitate the improvement of this medicinal plant in terms of enhancement of secondary metabolites.