The phytohormone jasmonic acid (JA) is vital in plant defense and development. Although biosynthesis of JA and activation of JAresponsive gene expression by the bioactive form JA-isoleucine have been well-studied, knowledge on JA metabolism is incomplete. In particular, the enzyme that hydroxylates JA to 12-OH-JA, an inactive form of JA that accumulates after wounding and pathogen attack, is unknown. Here, we report the identification of four paralogous 2-oxoglutarate/Fe(II)-dependent oxygenases in Arabidopsis thaliana as JA hydroxylases and show that they down-regulate JA-dependent responses. Because they are induced by JA we named them JASMONATE-INDUCED OXYGENASES (JOXs). Concurrent mutation of the four genes in a quadruple Arabidopsis mutant resulted in increased defense gene expression and increased resistance to the necrotrophic fungus Botrytis cinerea and the caterpillar Mamestra brassicae. In addition, root and shoot growth of the plants was inhibited. Metabolite analysis of leaves showed that loss of function of the four JOX enzymes resulted in overaccumulation of JA and in reduced turnover of JA into 12-OH-JA. Transformation of the quadruple mutant with each JOX gene strongly reduced JA levels, demonstrating that all four JOXs inactivate JA in plants. The in vitro catalysis of 12-OH-JA from JA by recombinant enzyme could be confirmed for three JOXs. The identification of the enzymes responsible for hydroxylation of JA reveals a missing step in JA metabolism, which is important for the inactivation of the hormone and subsequent down-regulation of JA-dependent defenses.jasmonic acid | 2OG oxygenases | 12-OH-JA | plant defense

The phytohormone jasmonic acid (JA) is vital in plant defense and development. Although biosynthesis of JA and activation of JA-responsive gene expression by the bioactive form JAisoleucine (JA-Ile) have been well-studied, knowledge on JA metabolism is incomplete. In particular, the enzyme that hydroxylates JA to 12-OH-JA, an inactive form of JA that accumulates after wounding and pathogen attack, is unknown. Here, we report the identification of four paralogous 2-oxoglutarate/Fe(II)-dependent oxygenases in Arabidopsis thaliana as JA hydroxylases and show that they down-regulate JA-dependent responses. As they are induced by JA we named them JASMONATE-INDUCED OXYGENASEs (JOXs). Concurrent mutation of the four genes in a quadruple Arabidopsis mutant resulted in increased defense gene expression and increased resistance to the necrotrophic fungus Botrytis cinerea and the caterpillar Mamestra brassicae. In addition, root and shoot growth of the plants was inhibited. Metabolite analysis of leaves showed that loss of function of the four JOX enzymes resulted in over-accumulation of JA and in reduced turnover of JA into 12-OH-JA. Transformation of the quadruple mutant with each JOX gene strongly reduced JA levels, demonstrating that all four JOXs inactivate JA in plants. The in vitro catalysis of 12-OH-JA from JA by recombinant enzyme could be confirmed for three JOXs. The identification of the enzymes responsible for hydroxylation of JA reveals a missing step in JA metabolism, which is important for the inactivation of the hormone and subsequent down-regulation of JA-dependent defenses. SIGNIFICANCE STATEMENTIn plants, the hormone jasmonic acid (JA) is synthesized in response to attack by pathogens and herbivores, leading to activation of defense responses. Rapidly following JA accumulation, the hormone is metabolized, presumably to prevent inhibitive effects of high JA levels on growth and development. The enzymes that directly inactivate JA were so far unknown. Here, we identify four jasmonate-induced oxygenases (JOXs) in Arabidopsis that hydroxylate jasmonic acid to form inactive 12-OH-JA. A mutant that no longer produces the four enzymes hyperaccumulates JA, exhibits reduced growth, and is highly resistant to attackers that are sensitive to JA-dependent defense. The JOX enzymes thus play an important role in determining the amplitude and duration of JA responses to balance the growth-defense tradeoff.peer-reviewed)

The TCP-type transcription factors BRANCHED1 and BRANCHED2 shape plant architecture by suppressing bud outgrowth, with BRANCHED2 only playing a minor role in Arabidopsis. Here, we investigated the function of orthologs of these genes in the model tree Populus. We used CRISPR/Cas9 to generate loss-of-function mutants of previously identified Populus BRANCHED1-1 and BRANCHED2-1 candidate genes. BRANCHED1-1 mutants exhibited strongly enhanced bud outgrowth. BRANCHED2-1 mutants had an extreme bud outgrowth phenotype and possessed two ectopic leaves at each node. While BRANCHED1 function is conserved in poplar, BRANCHED2, in contrast to its Arabidopsis counterpart, plays an even more critical role in bud outgrowth regulation. In addition, we identified a new, not yet reported association of this gene to leaf development.

Abstract. Pratami MP, Fendiyanto MH, Satrio RD, Widana IDKK, Nikmah IA, Sari NIP, Awwanah M, Farah N, Darmadi D. 2021. Potential of invasive alien species Clidemia hirta as antibacterial against Salmonella typhi and Staphylococcus aureus. Biodiversitas 22: 3363-3369. Clidemia hirta D. Don is an invasive alien species (IAS) that is a threat to biodiversity in tropical country particularly Indonesia, and remains underutilized to date. Conversely, prevalence of typhus in Indonesia is generally higher every year. Thus, the aim of this study was to detect phytochemicals in ethanolic and aqueous extracts of C. hirta and their antibacterial activity against Salmonella typhi and Staphylococcus aureus. The research methods included sample identification, generating the simplicia, determination of water content, extraction, phytochemical screening tests, and antibacterial activity tests. Identification was made based on morphological characteristics. The water content in the dried powder of simplicia was 12.26 ± 0.39%. Phytochemical results showed that 70% ethanol extract of C. hirta contained flavonoids, saponins, tannins, and triterpenoids compounds. In addition, aqueous extract of C. hirta showed positive results on flavonoids, saponins, tannins, and steroids tests. Antibacterial activity results showed that ethanolic extracts of C. hirta inhibited S. typhi and S. aureus at all concentrations, while aqueous extract inhibited bacterial growth in only 12.5% ??and 25% concentrations. These findings indicate that C. hirta has antibacterial activity that inhibits S. typhi and S. aureus. This information can be used for adding preliminary data to metabolite interest researchers, i.e., biologists and biotechnologists in the future.

BRI1 BRASSINOSTEROID INSENSITIVE 1 CaCl2 Calcium chloride CalS Callose Synthase CBB Coomassie Brilliant Blue CBM Carbohydrate-binding module CC coiled-coil CC-NBS-LRR Coiled-coil nucleotide binding site leucine rich repeat cDNA complementary DNA CDPK/CPK CALCIUM DEPENDENT PROTEIN KINASE CEBiP CHITIN ELICITOR BINDING PROTEIN CERK1 CHITIN ELICITOR RECEPTOR LIKE KINASE CesA3 cellulose synthase subunit CLSM Confocal laser scanning microscopy cm centimeter CML CALMODULIN LIKE CO Chitoologisaccharide(s) Col-0 Columbia-0 Co-IP co-immunoprecipitation Com. Complementation CTAB Cetyl Trimethyl Ammonium Bromide CTP CHLOROPLAST-TARGETED PROTEIN DEPC Diethyl pyrocarbonate DAMP damage-associated molecular pattern ddH2O double-distilled water