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 (N 2) 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.

Phytaspases are plant cell death-related proteases of the subtilisin-like protease family that possess an unusual 11 aspartate cleavage specificity. Although phytaspase activity is widespread in plants, phytaspase of Arabidopsis 12 thaliana (L.) Heynh. has escaped detection and identification thus far. Here, we show that a single gene (At4 13 g10540) out of 56 A. thaliana subtilisin-like protease genes encodes a phytaspase. The recombinant phytaspase was 14 overproduced in Nicotiana benthamiana Domin leaves, isolated, and its substrate specificity and properties were 15 characterised. At pH 5.5, at physiological mildly acidic reaction conditions, the Arabidopsis phytaspase was shown 16 to be strictly Asp-specific. The strongly preferred cleavage motifs of the enzyme out of a panel of synthetic peptide 17 substrates were YVAD and IETD, while the VEID-based substrate preferred by the tobacco and rice phytaspases 18 was almost completely resistant to hydrolysis. At neutral pH, however, the Arabidopsis phytaspase could hydrolyse 19 peptide substrates after two additional amino acid residues, His and Phe, in addition to Asp. This observation may 20 indicate that the repertoire of Arabidopsis phytaspase targets could possibly be regulated by the conditions of the 21 cellular environment. Similar to tobacco and rice phytaspases, the Arabidopsis enzyme was shown to accumulate in 22 the apoplast of epidermal leaf cells. However, in stomatal cells Arabidopsis phytaspase was observed inside the 23 cells, possibly co-localising with vacuole. Our study thus demonstrates that the Arabidopsis phytaspase possesses 24 both important similarities with and distinctions from the already known phytaspases, and is likely to be the most 25 divergent member of the phytaspase family. 26Additional keywords: apoplast, aspartate specificity, proteolysis, subtilisin-like protease. 27 N. V. Chichkova et al. 28 Identification and properties of A. thaliana phytaspase 29Although plant proteases of the phytaspase family are important contributors to stress-induced plant cell death, 30phytaspase of a classical model plant Arabidopsis thaliana has escaped identification thus far. We identified the 31Arabidopsis phytaspase-encoding gene and characterised the recombinant enzyme. Substrate specificity and 32

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.

3 hormaechei sp. improves the early vegetative growth and 4 the P and K uptake of okra (Abelmoschus esculentus) 5 seedling 6 Muhamad Aidilfitri Abstract 23 Limited information is available that seed biopriming by plant growth-promoting bacteria such 24 as those among Enterobacter spp. play a prominent role to enhance vegetative growth of plants.25 Contrary to Enterobacter cloacae, Enterobacter hormaechei is a less-studied counterpart 26 despite its vast potential in plant growth-promotion mainly through the inorganic phosphorus 27 (P) and potassium (K) solubilization abilities. To this end, 18 locally isolated bacterial pure 28 cultures screened and three strains showed high P-and K-solubilizing capabilities. Light 29 microscopy, biochemical tests and 16S rRNA gene sequencing revealed that strains 15a1 and 30 40a were closely related to Enterobacter hormaechei while strain 38 was closely related to 31 Enterobacter cloacae (Accession number: MN294583; MN294585; MN294584). All 32 Enterobacter spp. shared common plant growth-promoting traits, namely N 2 fixators, indole-33 3-acetic acid producers and siderophore producers. Gibberellic acid was only produced by 34 strain 38 and 40a, while exopolysaccharide formation was solely detected on agar containing 35 colonies of strain 38. Under in vitro germination assay of okra (Abelmoschus esculentus) seeds, 36 Enterobacter spp. significantly improved overall germination parameters and vigor index 37 (19.6%) of seedlings. The efficacy of root colonization of Enterobacter spp. on the pre-treated 38 seedling root tips was confirmed using Scanning Electron Microscopy (SEM). The pot 39 experiment of bioprimed seeds of okra seedling showed significant improvement of the plant 40 growth (> 28%) which corresponded to the increase of P and K uptakes (> 89%) as compared 41 to the uninoculated control plants. The leaf surface area and the SPAD chlorophyll index of 42 bioprimed plants were increased up to 29% and 9% respectively. This report revealed that the 43 under-explored species of P-and K-solubilizing Enterobacter hormaechei sp. with multiple 44 plant beneficial traits hold as a good potential sustainable approach for enhancement of soil 45 fertility and P and K uptakes of plants. 3 46