Characterising the Gene Expression, Enzymatic Activity and Subcellular Localisation of Arabidopsis thaliana Metacaspase 5 (AtMCA-IIb).

Metacaspases are a class of proteases found in plants that have gained attention in recent years due to their involvement in programmed cell death (PCD) and other essential cellular processes. Although structurally homologous to caspases found in animals, metacaspases have distinct properties and functions. There are nine metacaspase genes in the Arabidopsis thaliana genome; these can be type I or type II, and working out the function of each member of the gene family is challenging. In this study, we report the characterisation of one Arabidopsis type II metacaspase, metacaspase-5 (AtMC5; AtMCA-IIb). We detected the expression of AtMC5 only under specific conditions with a strong upregulation by ER stress and oxidative stress at a narrow 6 h time point. Recombinant AtMC5 was successfully purified from E. coli, with the recombinant AtMC5 working optimally at pH 7, using an optimised reaction buffer containing 10 mM calcium chloride together with 15% sucrose. Like other metacaspases, AtMC5 cleaved after arginine residue and demonstrated a substrate preference towards VRPR. Additionally, AtMC5-RFP was shown to be localised in the cytosol and nucleus of transfected cells. We found no evidence of a strong link between AtMC5 and PCD, and the data provide additional insights into the function of metacaspases in plants and will aid in future research toward further understanding their mode of action.

Okra Growth, Yield and Rhizosphere Microbiome Responses to the Encapsulated Bioinoculant Application under Reduced Fertilization Regime.

There is limited evidence that Enterobacter hormaechei can improve plant physiology and yield through soil phosphate (P) and potassium (K) amelioration. This study unraveled the effect of different soil inoculation methods i.e., free-cell and encapsulated (alginate bead containing sugar-protein hydrolysate and molasses) E. hormaechei 40a with different rates of PK-fertilization on okra P and K uptake, and soil rhizosphere bacterial community. The results revealed that 3HB (half-dose PK-fertilizer + encapsulated strain 40a) had the highest soil available P (SAP) and K (SAK), as well as P and K uptake for all plant organs, followed by 3F (full-dose PK-fertilizer), 3HI (half-dose PK-fertilizer + free-cell strain 40a), and 3H (half-dose PK-fertilizer), and improved yield by up to 75.6%. Both inoculated and full-dose fertilizer treatments produced larger pods (>15 cm) compared to 3H. We discovered increased bacterial richness and diversity in both 3HB and 3HI samples compared to uninoculated treatments. Both 3HB and 3F treatments were positively correlated with the increasing abundance of Acidobacteriales, Burkholderia caballeronia paraburkholderia, Gemmataceae, and Sphingomonas along with the SAP and SAK. The plant-beneficial effect of one-time 3HB treatment on okra growth and yield was comparable to biweekly inoculation in 3HI, suggesting a new cost-effective farming approach in precision agriculture.

Bioprospecting microwave-alkaline hydrolysate cocktail of defatted soybean meal and jackfruit peel biomass as carrier additive of molasses-alginate-bead biofertilizer.

The extraction of soluble hydrolysate protein and sugar from a biomass cocktail of defatted soybean meal (DSM) and jackfruit peel (JP) was examined using microwave-alkaline hydrolysis by varying the NaOH concentrations (0.04-0.11 M) and residence times (2-11 min). Based on the central composite design, the optimized parameters were achieved at 0.084 M NaOH concentration (100 mL), for 8.7 min at 300 W microwave power level to obtain the highest protein (5.31 mg/mL) and sugar concentrations (8.07 mg/mL) with > 75% recovery. Both raw and detoxified hydrolysate (using activated carbon) were correspondingly biocompatible with Enterobacter hormaechei strain 40a (P > 0.05) resulting in maximal cell counts of > 10 log CFU/mL. The optimized hydrolysate was prepared as an additive in molasses-alginate bead encapsulation of strain 40a. Further evaluation on phosphate and potassium solubilization performance of the encapsulated strain 40a exhibited comparable results with those of free cell counterpart (P > 0.05). The DSM-JP hydrolysate cocktail holds potential as a carrier additive of encapsulated-cell bead biofertilizers in order to sustain bacterial cell quality and consequently improve crop growth and productivity.

Response Surface Optimization of Extraction Conditions and In Vitro Antioxidant and Antidiabetic Evaluation of an Under-Valued Medicinal Weed, Mimosa pudica.

Mimosa pudica Linn is a well-known perennial herb and is traditionally used in ayurvedic medicine for the treatment of various illnesses. Despite its abundance in nature, the therapeutic potential of this invasive weed is deemed to be underappreciated in Malaysia. Previous studies have found an abundance of bioactive compounds associated with potent antioxidant properties in all parts of the plant. However, the optimum parameters required for the extraction of antioxidant compounds are still unknown. Therefore, the present study aimed to optimize the solvent extraction parameters of M. pudica using response surface methodology to enrich the accumulation of antioxidant compounds in the extracts. The effects of the optimized M. pudica extracts were then evaluated on the cell viability and glucose uptake ability in a 3T3-L1 adipocyte cell line. The highest total phenolic (91.98 mg of gallic acid equivalent per g of the dry extract) and total flavonoid content (606.31 mg of quercetin equivalent per g of the dry extract) were recorded when using 100% ethanol that was five-fold and three-fold higher, respectively, as compared to using 50% ethanol. The extract concentration required to achieve 50% of antioxidant activity (IC50 value) was 42.0 µg/mL using 100% ethanol as compared to 975.03 µg/mL using 50% ethanol. The results indicated that the use of 100% ethanol solvent had the greatest impact on the accumulation of antioxidant compounds in the extract (p < 0.05). Cell viability assay revealed that all extract concentration treatments recorded a viability level of above 50%. Glucose uptake assay using 2-NBDG analog showed that the cells treated with 50 µg/mL extract combined with insulin were five-fold higher than the control group. Given the high antioxidant and antidiabetic properties of this plant, M. pudica can be easily highlighted as a plant subject of interest, which warrants further investigation for nutraceutical prospects.

Seed biopriming with P- and K-solubilizing Enterobacter hormaechei sp. improves the early vegetative growth and the P and K uptake of okra (Abelmoschus esculentus) seedling.

Limited information is available that seed biopriming with plant growth-promoting Enterobacter spp. play a prominent role to enhance vegetative growth of plants. Contrary to Enterobacter cloacae, Enterobacter hormaechei is a less-studied counterpart despite its vast potential in plant growth-promotion mainly through the inorganic phosphorus (P) and potassium (K) solubilization abilities. To this end, 18 locally isolated bacterial pure cultures were screened and three strains showed high P- and K-solubilizing capabilities. Light microscopy, biochemical tests and 16S rRNA gene sequencing revealed that strains 15a1 and 40a were closely related to Enterobacter hormaechei while strain 38 was closely related to Enterobacter cloacae (Accession number: MN294583; MN294585; MN294584). All Enterobacter spp. shared common plant growth-promoting traits, namely nitrogen (N2) fixation, indole-3-acetic acid production and siderophore production. The strains 38 and 40a were able to produce gibberellic acid, while only strain 38 was able to secrete exopolysaccharide on agar. Under in vitro germination assay of okra (Abelmoschus esculentus) seeds, Enterobacter spp. significantly improved overall germination parameters and vigor index (19.6%) of seedlings. The efficacy of root colonization of Enterobacter spp. on the pre-treated seedling root tips was confirmed using Scanning Electron Microscopy (SEM). The pot experiment of bioprimed seeds of okra seedling showed significant improvement of the plant growth (> 28%) which corresponded to the increase of P and K uptakes (> 89%) as compared to the uninoculated control plants. The leaf surface area and the SPAD chlorophyll index of bioprimed plants were increased by up to 29% and 9% respectively. This report revealed that the under-explored species of P- and K-solubilizing Enterobacter hormaechei sp. with multiple plant beneficial traits presents a great potential sustainable approach for enhancement of soil fertility and P and K uptakes of plants.

Arabidopsis thaliana phytaspase: identification and peculiar properties.

Phytaspases are plant cell death-related proteases of the subtilisin-like protease family that possess an unusual aspartate cleavage specificity. Although phytaspase activity is widespread in plants, phytaspase of Arabidopsis thaliana (L.) Heynh. has escaped detection and identification thus far. Here, we show that a single gene (At4 g10540) out of 56 A. thaliana subtilisin-like protease genes encodes a phytaspase. The recombinant phytaspase was overproduced in Nicotiana benthamiana Domin leaves, isolated, and its substrate specificity and properties were characterised. At pH 5.5, at physiological mildly acidic reaction conditions, the Arabidopsis phytaspase was shown to be strictly Asp-specific. The strongly preferred cleavage motifs of the enzyme out of a panel of synthetic peptide substrates were YVAD and IETD, while the VEID-based substrate preferred by the tobacco and rice phytaspases was almost completely resistant to hydrolysis. At neutral pH, however, the Arabidopsis phytaspase could hydrolyse peptide substrates after two additional amino acid residues, His and Phe, in addition to Asp. This observation may indicate that the repertoire of Arabidopsis phytaspase targets could possibly be regulated by the conditions of the cellular environment. Similar to tobacco and rice phytaspases, the Arabidopsis enzyme was shown to accumulate in the apoplast of epidermal leaf cells. However, in stomatal cells Arabidopsis phytaspase was observed inside the cells, possibly co-localising with vacuole. Our study thus demonstrates that the Arabidopsis phytaspase possesses both important similarities with and distinctions from the already known phytaspases, and is likely to be the most divergent member of the phytaspase family.