In vitro propagation of Indonesian stevia (Stevia rebaudiana) genotype using axenic nodal segments.

OBJECTIVE: The high industrial demand for Stevia cultivation (Stevia rebaudiana) has increased due to its high stevioside content derived from the leaves. However, the low germination rate makes the cultivation of the plant become the main obstacle. Therefore, an efficient cultivation technique is required. This present work aims to analyze the effect of five combinations of Kinetin (Kin) and benzyladenine (BA) on stevia micropropagation using nodal segment explants. RESULTS: The micropropagation of stevia was performed using Murashige and Skoog (MS) medium supplemented with BA and Kin. We analyzed different organogenesis and callogenesis responses. In addition, the number of shoots and root formed during in vitro culture were also observed. Our results demonstrated that all treatments with Kin, both alone and in combination with BA, resulted in the development of callus on all nodal segment explants. Explants treated in MS with 1 mg L-1 BA exhibited the best average of shoot number (36.27). In contrast, the treatment without PGR resulted in the best root formation (2.6). The overall results suggested that different combination of BA and Kin resulted in distinct organogenesis responses, where 1 mg L-1 of BA was potentially used for boosting the number of shoots in micropropagation of stevia accession Mini.

Plastid DNA Barcoding and RtActin cDNA Fragment Isolation of Reutealis Trisperma: A Promising Bioresource for Biodiesel Production.

Reutealis trisperma belonging to the family Euphorbiaceae is currently used for biodiesel production, and rapid development in plant-based biofuel production has led to its increasing demand. However, massive utilization of bio-industrial plants has led to conservation issues. Moreover, genetic information on R trisperma is still limited, which is crucial for developmental, physiological, and molecular studies. Studying gene expression is essential to explain plant physiological processes. Nonetheless, this technique requires sensitive and precise measurement of messenger RNA (mRNA). In addition, the presence of internal control genes is important to avoid bias. Therefore, collecting and preserving genetic data for R trisperma is indispensable. In this study, we aimed to evaluate the application of plastid loci, rbcL, and matK, to the DNA barcode of R trisperma for use in conservation programs. In addition, we isolated and cloned the RtActin (RtACT) gene fragment for use in gene expression studies. Sequence information was analyzed in silico by comparison with other Euphorbiaceae plants. For actin fragment isolation, reverse-transcription polymerase chain reaction was used. Molecular cloning of RtActin was performed using the pTA2 plasmid before sequencing. We successfully isolated and cloned 592 and 840 bp of RtrbcL and RtmatK fragment genes, respectively. The RtrbcL barcoding marker, rather than the RtmatK plastidial marker, provided discriminative molecular phylogenetic data for R Trisperma. We also isolated 986 bp of RtACT gene fragments. Our phylogenetic analysis demonstrated that R trisperma is closely related to the Vernicia fordii Actin gene (97% identity). Our results suggest that RtrbcL could be further developed and used as a barcoding marker for R trisperma. Moreover, the RtACT gene could be further investigated for use in gene expression studies of plant.

Parthenocarpic tomato mutants, iaa9-3 and iaa9-5, show plant adaptability and fruiting ability under heat-stress conditions.

Fruit set is one of the main problems that arise in tomato plants under heat-stress conditions, which disrupt pollen development, resulting in decreased pollen fertility. Parthenocarpic tomatoes can be used to increase plant productivity during failure of the fertilisation process under heat-stress conditions. The aim of this study were to identify the plant adaptability and fruiting capability of ?iaa9-3 and iaa9-5 tomato mutants under heat-stress conditions. The iaa9-3 and iaa9-5 and wild-type Micro-Tom (WT-MT) plants were cultivated under two temperature conditions: normal and heat-stress conditions during plant growth. The results showed that under the heat-stress condition, iaa9-3 and iaa9-5 showed delayed flowering time, increased number of flowers, and increased fruit set and produced normal-sized fruit. However, WT-MT cannot produce fruits under heat stress. The mutants can grow under heat-stress conditions, as indicated by the lower electrolyte leakage and H2O2 concentration and higher antioxidant activities compared with WT-MT under heat-stress conditions. These results suggest that iaa9-3 and iaa9-5 can be valuable genetic resources for the development of tomatoes in high-temperature environmental conditions.

Transcriptome dataset from Solanum lycopersicum L. cv. Micro-Tom; wild type and two mutants of INDOLE-ACETIC-ACID (SlIAA9) using long-reads sequencing oxford nanopore technologies.

OBJECTIVE: Tomatoes are the most widely consumed fruit vegetable and are relatively easy to cultivate. However, an increase in temperature causes some plants to respond with a decrease in fruit production. So, it is necessary to develop plants resistant to extreme temperature changes. The tomato cv. Micro-Tom has genetic variations in the gene of INDOLE-ACETIC-ACID, namely SlIAA9-3 and SlIAA9-5. However, the genetic information regarding the full-length transcript of the gene from this type of tomato plant is unknown. Therefore, this study aimed to determine the full-length transcript of the genes of these three types of tomatoes using long-reads sequencing technology from Oxford Nanopore. DATA DESCRIPTION: The total RNA from three types of Micro-Tom was isolated with the RNeasy PowerPlant Kit. Then, the RNA sequencing process used PCR-cDNA Barcoding kit - SQK-PCB109 and continued with the processing of raw reads based on the protocol from microbepore protocol ( https://github.com/felixgrunberger/microbepore ). The resulting raw reads were 578 374, 409 905, and 851 948 for wildtype, iaa9-3, and iaa9-5, respectively. After obtaining cleaned reads, each sample was mapped to the tomato reference genome (S. lycopersicum ITAG4.0) with the Minimap2 program. In particular, 965 genes were expressed only in the iaa9-3 mutant, and 2332 genes were expressed only in the iaa9-5 mutant. Whereas in the wild type, 1536 genes are specifically expressed. In cluster analysis using the heatmap analysis, separate groups were obtained between the wild type and the two mutants. This proves an overall difference in transcript levels between the wild type and the mutants.

SlIAA9 Mutation Maintains Photosynthetic Capabilities under Heat-Stress Conditions.

Tomato is one of the most widely consumed horticultural products. However, tomato is very sensitive to changes in temperature. Daily average temperatures above 32 °C severely reduced tomato plant growth, development, and productivity. Therefore, climate change-induced global warming is a major threat to future tomato production. Good photosynthetic capability under heat stress conditions is known to be a major sign of heat tolerance. Tomato INDOLE-ACETIC-ACID (SlIAA9) is a transcriptional repressor in auxin signaling. SlIAA9 mutation caused heightened endogenous auxin response and biosynthesis within plant tissues. In this study, we studied the photosynthetic capability of iaa9-3 and iaa9-5 mutants under heat-stress conditions. We discovered that both iaa9-3 and iaa9-5 could maintain their photosynthetic capability after 14 days of heat treatment (>40 °C), differing from Wild Type-Micro-Tom (WT-MT) tomato. Both iaa9 mutants had higher net photosynthetic rate, stomatal conductance, leaf total chlorophyll, leaf carotenoids, Fv/Fm value, and lower leaf MDA than WT-MT. These results suggested that the SlIAA9 mutation benefits plant adaptation to heat stress.

Anti-cancer potency by induced apoptosis by molecular docking P53, caspase, cyclin D1, cytotoxicity analysis and phagocytosis activity of trisindoline 1,3 and 4.

Cancer is one of the leading causes of death in the world. Efforts to find and develop cancer drugs from natural products continue with the exploration of trisindoline, a substance that is isolated from marine sponges Hyrtios altum. Trisindoline is an indole trimer alkaloid compound that has been successfully synthesized into trisindoline 1, 3 and 4. Trisindoline is cytotoxic in cell lines and in this study, trisindoline was able to induce apoptosis in the in silico and in vitro tests that were carried out. The in silico test was carried out through molecular docking using the Autodock Vina method and the Molecular Dynamics (MD) Simulation QM / MM AMBER. The target proteins used were protein p53 and caspase -9 which played a role in the apoptotic pathway and cyclin D1 which played a role in cell proliferation. Meanwhile, cytotoxicity analysis was carried out using the MTT method (3- (4,5-dimethyltiazol -2-yl) -2,5 -dipenyl tetrazolium bromide). Nevertheless, the ability of trisindoline to induce phagocytosis is still unrevealed. The phagocytosis assay was carried out by assessing the macrophage capacity and phagocytic index using the latex-beads model. The in silico results showed that the binding affinity values between the target protein Cdk-2 and the trisindoline 1, trisindoline 3 and trisindoline 4 ligands were -7.3 kcal / mol, -7.7 kcal / mol and -6.6 kcal / mol respectively. The binding affinity values between the target protein p53 and the trisindoline 1, trisindoline 3 and trisindoline 4 ligands were -7.5 kcal / mol, -7.4 kcal / mol and -7.5 kcal / mol respectively. The binding affinity values between the target protein caspase-9 and the trisindoline 1, trisindoline 3 and trisindoline 4 ligands were -7.5 kcal / mol, -7.1 kcal / mol and -7.2 kcal / mol respectively. The results of RMSD (Root Mean Square Deviation), RMSF (Root Mean Square Fluctuation), and hydrogen bonds in the MD (Molecular Dynamics) Simulation showed that Cdk-2 formed a protein complex with trisindoline 3, protein p53 with trisindoline 1 and caspase-9 with trisindoline 1. The cytotoxicity assay was carried out in the MCF-7 cell line and the IC50 value obtained for trisindoline 1 was 2.059 µM, for trisindoline 3 was 3.9759 ​​µM, for trisindoline 4 was 15.46 µM and for doxorubicin was 9.88 µM. Furthermore, the phagocytosis test was carried out using trisindoline 1, 3 and 4. Our results showed that 6.25 µg mL-1 of trisindoline 1 and trisindoline 3 were able to induce the phagocytosis capacity of macrophage cells of 38.34; whereas trisindoline 4 at a concentration of 50 µg mL-1 induces a phagocytosis capacity of 32.89. Trisindoline 1, 3 and 4 showed potentials of immunostimulants at low concentrations but showed potentials of immunosuppressants at high concentrations. The overall results demonstrated that trisindoline 1 and 3 are potential anti-cancer candidates capable of activating the apoptotic pathway.

Genetic diversity and growth responses of Indonesian tomato (Solanum lycopersicum L.) genotypes under lead stress.

Tomato (Solanum lycopersicum L.) is cultivated and consumed worldwide, including in Indonesia. It is used in the food, cosmetic, and pharmaceutical industries, due to its high content of carotenoid (lycopene) compounds that have antioxidant and anticancer activities. In Indonesia, although several cultivars of tomato are cultivated, including Opal, Permata, Mutiara, and Rewako, studies on their genetic information are limited. Unpredicted climate change as well as heavy metal contamination, especially Pb pollution, has threatened Indonesian food security. Therefore, our study aimed to analyze the genetic diversity of the four local tomatoes using random amplified polymorphic DNA (RAPD) markers and to determine the growth responses of several local tomato genotypes under Pb stress. In this study, morphological responses to Pb, including plant height and root length were observed. The RAPD analysis showed that Rewako and Permata were distinct, whereas Opal and Mutiara were closely related, possessing 81.8% similarity. Pb stress influenced plant height and root length in the four tomato genotypes, with each genotype exhibiting different morphological responses than others. However, the closely related Mutiara and Opal genotypes demonstrated similar responses to Pb stress to Permata and Rewako. Our study demonstrates that RAPD are sensitive and efficient for elucidating the genomic profile of the tomato genotypes. In addition, our results suggest that genetic variation among tomato genotypes might influence the morphological responses against Pb stress.

In Silico Analysis of Partial Fatty Acid Desaturase 2 cDNA From Reutealis trisperma (Blanco) Airy Shaw.

Reutealis trisperma oil is a new source for biodiesel production. The predominant fatty acids in this plant are stearic acid (9%), palmitic acid (10%), oleic acid (12%), linoleic acid (19%), and α-eleostearic acid (51%). The presence of polyunsaturated fatty acids (PUFAs), linoleic acid, and α-eleostearic acid decreases the oxidation stability of R. trisperma biodiesel. Although several studies have suggested that the fatty acid desaturase 2 (FAD2) enzyme is involved in the regulation of fatty acid desaturation, little is known about the genetic information of FAD2 in R. trisperma. The objectives of this study were to isolate, characterize, and determine the relationship between the R. trisperma FAD2 fragment and other Euphorbiaceae plants. cDNA fragments were isolated using reverse transcription polymerase chain reaction (PCR). The DNA sequence obtained by sequencing was used for further analysis. In silico analysis identified the fragment identity, subcellular localization, and phylogenetic construction of the R. trisperma FAD2 cDNA fragment and Euphorbiaceae. The results showed that a 923-bp partial sequence of R. trisperma FAD2 was successfully isolated. Based on in silico analysis, FAD2 was predicted to encode 260 amino acids, had a domain similarity with Omega-6 fatty acid desaturase, and was located in the endoplasmic reticulum membrane. The R. trisperma FAD2 fragment was more closely related to Vernicia fordii (HM755946.1).

An ethnobotanical study of medicinal plants used by the Tengger tribe in Ngadisari village, Indonesia.

The people of Tengger, Indonesia have used plants as traditional medicine for a long time. However, this local knowledge has not been well documented until recently. Our study aims to understand the utilization of plants in traditional medicine by the people of Tengger, who inhabit the Ngadisari village, Sukapura District, Probolinggo Regency, Indonesia. We conducted semi-structured and structured interviews with a total of 52 informants that represented 10% of the total family units in the village. The parameters observed in this study include species use value (SUV), family use value (FUV), plant part use (PPU), and the relative frequency of citation that was calculated based on fidelity level (FL). We successfully identified 30 species belonging to 28 genera and 20 families that have been used as a traditional medicine to treat 20 diseases. We clustered all the diseases into seven distinct categories. Among the recorded plant families, Poaceae and Zingiberaceae were the most abundant. Plant species within those families were used to treat internal medical diseases, respiratory-nose, ear, oral/dental, and throat problems. The plant species with the highest SUV was Foeniculum vulgare Mill. (1.01), whereas the Aloaceae family (0.86) had the highest FUV. Acorus calamus L. (80%) had the highest FL percentage. The leaves were identified as the most used plant part and decoction was the dominant mode of a medicinal preparation. Out of the plants and their uses documented in our study, 26.7% of the medicinal plants and 71.8% of the uses were novel. In conclusion, the diversity of medicinal plant uses in the Ngadisari village could contribute to the development of new plant-based drugs and improve the collective revenue of the local society.

Isolation and identification of cholestane and dihydropyrene from Calophyllum inophyllum.

The highest population of Calophyllum inophyllum is in Indonesia. One of the bioactive compound contained in C. inophyllum leave is cholestane. The extraction method was employed to obtain crude extract from these leaves. It was followed by identification of the bioactive compounds. The purpose of this research was to develop a process for isolating and identifying cholestane and dihydropyrene from methanolic extract of C. inophyllum leaves in high yield and purity. The effect of crude extract to non-polar solvent mass ratio and non-polar solvent types on the separation of the bioactive compounds were also systematically investigated. New compounds (trans-2-[2-(trifluoromethyl)phenyl]-10b,10c-dimethyl-10b, 10c-dihydropyrene and anti-4-aza-B-homo-5.alpha-cholestane-3-one) were also identified in C. inophyllum leaves. The successful separation was obtained by employing CS2 as the solvent and crude extract to CS2 mass ratio of 1/10 (g/g).

Data of root anatomical responses to periodic waterlogging stress of tobacco (Nicotiana tabacum) varieties.

The data of root anatomical structure and the formation of aerenchyma tissues of five varieties of tobacco under waterlogging stress were obtained by modified paraffin method. Each tobacco varieties performed distinct anatomical adaptation response, including changes of cortical tissue, stele diameter, xylem diameter and the formation of aerenchyma under periodic waterlogging stress.

Transcription profile data of phorbol esters biosynthetic genes during developmental stages in Jatropha curcas.

Jatropha curcas is currently known as an alternative source for biodiesel production. Beside its high free fatty acid content, J. curcas also contains typical diterpenoid-toxic compounds of Euphorbiaceae plant namely phorbol esters. This article present the transcription profile data of genes involved in the biosynthesis of phorbol esters at different developmental stages of leaves, fruit, and seed in Jatropha curcas. Transcriptional profiles were analyzed using reverse transcription-polymerase chain reaction (RT-PCR). We used two genes including GGPPS (Geranylgeranyl diphospate synthase), which is responsible for the formation of common diterpenoid precursor (GGPP) and CS (Casbene Synthase), which functions in the synthesis of casbene. Meanwhile, J. curcas Actin (ACT) was used as internal standard. We demonstrated dynamic of GGPPS and CS expression among different stage of development of leaves, fruit and seed in Jatropha.

Arabidopsis ERG28 tethers the sterol C4-demethylation complex to prevent accumulation of a biosynthetic intermediate that interferes with polar auxin transport.

Sterols are vital for cellular functions and eukaryotic development because of their essential role as membrane constituents. Sterol biosynthetic intermediates (SBIs) represent a potential reservoir of signaling molecules in mammals and fungi, but little is known about their functions in plants. SBIs are derived from the sterol C4-demethylation enzyme complex that is tethered to the membrane by Ergosterol biosynthetic protein28 (ERG28). Here, using nonlethal loss-of-function strategies focused on Arabidopsis thaliana ERG28, we found that the previously undetected SBI 4-carboxy-4-methyl-24-methylenecycloartanol (CMMC) inhibits polar auxin transport (PAT), a key mechanism by which the phytohormone auxin regulates several aspects of plant growth, including development and responses to environmental factors. The induced accumulation of CMMC in Arabidopsis erg28 plants was associated with diagnostic hallmarks of altered PAT, including the differentiation of pin-like inflorescence, loss of apical dominance, leaf fusion, and reduced root growth. PAT inhibition by CMMC occurs in a brassinosteroid-independent manner. The data presented show that ERG28 is required for PAT in plants. Furthermore, it is accumulation of an atypical SBI that may act to negatively regulate PAT in plants. Hence, the sterol pathway offers further prospects for mining new target molecules that could regulate plant development.

DOLICHOL PHOSPHATE MANNOSE SYNTHASE1 mediates the biogenesis of isoprenyl-linked glycans and influences development, stress response, and ammonium hypersensitivity in Arabidopsis.

The most abundant posttranslational modification in nature is the attachment of preassembled high-mannose-type glycans, which determines the fate and localization of the modified protein and modulates the biological functions of glycosylphosphatidylinositol-anchored and N-glycosylated proteins. In eukaryotes, all mannose residues attached to glycoproteins from the luminal side of the endoplasmic reticulum (ER) derive from the polyprenyl monosaccharide carrier, dolichol P-mannose (Dol-P-Man), which is flipped across the ER membrane to the lumen. We show that in plants, Dol-P-Man is synthesized when Dol-P-Man synthase1 (DPMS1), the catalytic core, interacts with two binding proteins, DPMS2 and DPMS3, that may serve as membrane anchors for DPMS1 or provide catalytic assistance. This configuration is reminiscent of that observed in mammals but is distinct from the single DPMS protein catalyzing Dol-P-Man biosynthesis in bakers' yeast and protozoan parasites. Overexpression of DPMS1 in Arabidopsis thaliana results in disorganized stem morphology and vascular bundle arrangements, wrinkled seed coat, and constitutive ER stress response. Loss-of-function mutations and RNA interference-mediated reduction of DPMS1 expression in Arabidopsis also caused a wrinkled seed coat phenotype and most remarkably enhanced hypersensitivity to ammonium that was manifested by extensive chlorosis and a strong reduction of root growth. Collectively, these data reveal a previously unsuspected role of the prenyl-linked carrier pathway for plant development and physiology that may help integrate several aspects of candidate susceptibility genes to ammonium stress.

Characterization of plant carotenoid cyclases as members of the flavoprotein family functioning with no net redox change.

The later steps of carotenoid biosynthesis involve the formation of cyclic carotenoids. The reaction is catalyzed by lycopene beta-cyclase (LCY-B), which converts lycopene into beta-carotene, and by capsanthin-capsorubin synthase (CCS), which is mainly dedicated to the synthesis of kappa-cyclic carotenoids (capsanthin and capsorubin) but also has LCY-B activity. Although the peptide sequences of plant LCY-Bs and CCS contain a putative dinucleotide-binding motif, it is believed that these two carotenoid cyclases proceed via protic activation and stabilization of resulting carbocation intermediates. Using pepper (Capsicum annuum) CCS as a prototypic carotenoid cyclase, we show that the monomeric protein contains one noncovalently bound flavin adenine dinucleotide (FAD) that is essential for enzyme activity only in the presence of NADPH, which functions as the FAD reductant. The reaction proceeds without transfer of hydrogen from the dinucleotide cofactors to beta-carotene or capsanthin. Using site-directed mutagenesis, amino acids potentially involved in the protic activation were identified. Substitutions of alanine, lysine, and arginine for glutamate-295 in the conserved 293-FLEET-297 motif of pepper CCS or LCY-B abolish the formation of beta-carotene and kappa-cyclic carotenoids. We also found that mutations of the equivalent glutamate-196 located in the 194-LIEDT-198 domain of structurally divergent bacterial LCY-B abolish the formation of beta-carotene. The data herein reveal plant carotenoid cyclases to be novel enzymes that combine characteristics of non-metal-assisted terpene cyclases with those attributes typically found in flavoenzymes that catalyze reactions, with no net redox, such as type 2 isopentenyl diphosphate isomerase. Thus, FAD in its reduced form could be implicated in the stabilization of the carbocation intermediate.