Catalase is well-known as an antioxidant dismutating H 2 O 2 to O 2 and H 2 O. However, catalases evolved when metabolism was largely sulfur-based, long before O 2 and reactive oxygen species (ROS) became abundant, suggesting catalase metabolizes reactive sulfide species (RSS). Here we examine catalase metabolism of H 2 S n , the sulfur analog of H 2 O 2 , hydrogen sulfide (H 2 S) and other sulfur-bearing molecules using H 2 S-specific amperometric electrodes and fluorophores to measure polysulfides (H 2 S n ; SSP4) and ROS (dichlorofluorescein, DCF). Catalase eliminated H 2 S n , but did not anaerobically generate H 2 S, the expected product of dismutation. Instead, catalase concentration- and oxygen-dependently metabolized H 2 S and in so doing acted as a sulfide oxidase with a P 50 of 20 mmHg. H 2 O 2 had little effect on catalase-mediated H 2 S metabolism but in the presence of the catalase inhibitor, sodium azide (Az), H 2 O 2 rapidly and efficiently expedited H 2 S metabolism in both normoxia and hypoxia suggesting H 2 O 2 is an effective electron acceptor in this reaction. Unexpectedly, catalase concentration-dependently generated H 2 S from dithiothreitol (DTT) in both normoxia and hypoxia, concomitantly oxidizing H 2 S in the presence of O 2 . H 2 S production from DTT was inhibited by carbon monoxide and augmented by NADPH suggesting that catalase heme-iron is the catalytic site and that NADPH provides reducing equivalents. Catalase also generated H 2 S from garlic oil, diallyltrisulfide, thioredoxin and sulfur dioxide, but not from sulfite, metabisulfite, carbonyl sulfide, cysteine, cystine, glutathione or oxidized glutathione. Oxidase activity was also present in catalase from Aspergillus niger . These results show that catalase can act as either a sulfide oxidase or sulfur reductase and they suggest that these activities likely played a prominent role in sulfur metabolism during evolution and may continue do so in modern cells as well. This also appears to be the first observation of catalase reductase activity independent of peroxide dismutation.

Catalase as a sulfide-sulfur oxido-reductase: An ancient (and modern?) regulator of reactive sulfur species (RSS)

Preface.- Section I.- 1. The Genus Chenopodium - A Potential Food Source E. de la Cruz Torres et al.- 2. Thin Cell Layer Technology in Micropropagation of Jatropha curcas L. D. Tan Nhut et al.- 3. New Achievements in Panax vietnamensis Research D. Tan Nhut et al.- Section II.- 4. Molecular Biology and Physiology of the Resurrection Glacial Relic Haberlea rhodopensis M. Benina et al.- 5. Cell Morphometry as Predictor of Protein Legume In Vitro Growth S.J. Ochatt.- 6. Application of TILLING for Orphan Crop Improvement K. Esfeld et al.- Section III.- 7. Neglected Oil Crop Biotechnology S. Tiwari, S. Kumar.- 8. Prospects for Quinoa (Chenopodium quinoa Willd.) Improvement through Biotechnology E.N. Jellen et al.- 9. Biotechnology of Eruca sativa Mill. S.M.H. Slater.- 10. Biotechnology of Stylosanthes A. Chandra.- 11. Biotechnology of Miscanthus S.J. Dalton.- Section IV.- 12. Tissue Culture and Genetic Transformation of Safflower (Carthamus tinctorius L.) M. Sujatha, S.D. Gupta.- 13. Plant Regeneration and Genetic Transformation in Jatropha M. Sujatha et al.- 14. Genetic Transformation of Bael (Aegle marmelos Corr.) R. Pati, M. Muthukumar.- 15. Genetic Transformation of Taro X. He et al.- 16. Bioprospecting and Genetic Engineering of Mangrove Genes to Enhance Salinity Tolerance in Crop Plants A.B. Das. Index.

Biotechnology of Neglected and Underutilized Crops

A large part of chemodiversity of plant triterpenes is due to the modification of their side chains. Reduction or isomerization of double bonds in the side chains is often an important step for the diversification of triterpenes, although the enzymes involved are not fully understood. Withanolides are a large group of structurally diverse C28 steroidal lactones derived from 24-methylenecholesterol. These compounds are found in the Indian medicinal plant Withania somnifera, also known as ashwagandha, and other members of the Solanaceae. The pathway for withanolide biosynthesis is unknown, preventing sustainable production via white biotechnology and downstream pharmaceutical usages. In the present study, based on genome and transcriptome data we have identified a key enzyme in the biosynthesis of withanolides: a DWF1 paralog encoding a sterol Δ24-isomerase (24ISO). 24ISO originated from DWF1 after two subsequent duplication events in Solanoideae plants. Withanolides and 24ISO appear only in the medicinal plants in the Solanoideae, not in crop plants such as potato and tomato, indicating negative selection during domestication. 24ISO is a unique isomerase enzyme evolved from a reductase and as such has maintained the FAD-binding oxidoreductase structure and requirement for NADPH. Using phylogenetic, metabolomic, and gene expression analysis in combination with heterologous expression and virus-induced gene silencing, we showed that 24ISO catalyzes the conversion of 24-methylenecholesterol to 24-methyldesmosterol. We propose that this catalytic step is the committing step in withanolide biosynthesis, opening up elucidation of the whole pathway and future larger-scale sustainable production of withanolides and related compounds with pharmacological properties.

https://www.pnas.org/content/pnas/115/34/E8096.full.pdf

Third DWF1 paralog in Solanaceae, sterol Δ24-isomerase, branches withanolide biosynthesis from the general phytosterol pathway

Jatropha curcas is a promising feedstock for biofuel production because its oil is highly suitable for processing bio-jet fuels and biodiesel. However, Jatropha exhibits a long juvenile stage in subtropical areas. miR172, a conserved small non-protein-coding RNA molecule with 21 nucleotides, regulates a wide range of developmental processes. To date, however, no studies have examined the function of miR172 in Jatropha. There are five miR172 precursors encoding two mature miR172s in Jatropha, which are expressed in all tissues, with the highest expression level in leaves, and the levels are up-regulated with age. Overexpression of JcmiR172a resulted in early flowering, abnormal flowers, and altered leaf morphology in transgenic Arabidopsis and Jatropha. The expression levels of miR172 target genes were down-regulated, and the flower identity genes were up-regulated in the JcmiR172a-overexpressing transgenic plants. Interestingly, we showed that JcmiR172 might be involved in regulation of stem vascular development through manipulating the expression of cellulose and lignin biosynthesis genes. Overexpression of JcmiR172a enhanced xylem development and reduced phloem and pith development. This study helped elucidate the functions of miR172 in perennial plants, a known age-related miRNA involved in the regulation of perennial plant phase change and organ development.

/pdf/mir172-regulates-both-vegetative-and-reproductive-1wr1iybg6v.pdf

miR172 Regulates both Vegetative and Reproductive Development in the Perennial Woody Plant Jatropha curcas.

Glycosylation of sterols, catalysed by sterol glycosyltransferases (SGTs), improves the sterol solubility, chemical stability and compartmentalization, and helps plants to adapt to environmental changes. The SGTs in medicinal plants are of particular interest for their role in the biosynthesis of pharmacologically active substances. WsSGTL1, a SGT isolated from Withania somnifera, was expressed and functionally characterized in transgenic tobacco plants. Transgenic WsSGTL1-Nt lines showed an adaptive mechanism through demonstrating late germination, stunted growth, yellowish-green leaves and enhanced antioxidant system. The reduced chlorophyll content and chlorophyll fluorescence with decreased photosynthetic parameters were observed in WsSGTL1-Nt plants. These changes could be due to the enhanced glycosylation by WsSGTL1, as no modulation in chlorophyll biogenesis-related genes was observed in transgenic lines as compared to wildtype (WT) plants. Enhanced accumulation of main sterols like, campesterol, stigmasterol and sitosterol in glycosylated form was observed in WsSGTL1-Nt plants. Apart from these, other secondary metabolites related to plant’s antioxidant system along with activities of antioxidant enzymes (SOD, CAT; two to fourfold) were enhanced in WsSGTL1-Nt as compared to WT. WsSGTL1-Nt plants showed significant resistance towards Spodoptera litura (biotic stress) with up to 27 % reduced larval weight as well as salt stress (abiotic stress) with improved survival capacity of leaf discs. The present study demonstrates that higher glycosylation of sterols and enhanced antioxidant system caused by expression of WsSGTL1 gene confers specific functions in plants to adapt under different environmental challenges.

WsSGTL1 Gene from Withania somnifera, Modulates Glycosylation Profile, Antioxidant System and Confers Biotic and Salt Stress Tolerance in Transgenic Tobacco

In the present study, Agrobacterium tumefaciens-mediated transformation protocol has been optimized for Jatropha curcas—an important biofuel plant using leaf segments (LS) and hypocotyl segments (HS) from in vitro-seedlings. Among different accessions used, ‘GJ-Ran-C2’ was selected as the best for transformation. A. tumefaciens strain EHA101, containing the binary vector pIG121Hm was used for transformation, along with the GUS reporter gene containing intron and CaMV 35S as promoter. Various parameters were optimized to get the highest transient transformation expression (TTE). Augmentin® was used in the medium to control A. tumefaciens. Selection of transgenic shoots was done in the presence of kanamycin. Integration of GUS gene was confirmed by PCR analysis, whereas the expression of GUS transgene was confirmed by RT-PCR, using actin gene as internal control. GUS histochemical analysis of explants just after cocultivation showed blue color in approximately all the LS and HS but stable transformation efficiency was 5% in case of LS, whereas 4% in HS.

Agrobacterium tumefaciens -mediated transformation protocol of Jatropha curcas L. using leaf and hypocotyl segments

Sterol glycosyltransferases (SGTs) belong to family 1 of glycosyltransferases (GTs) and are enzymes responsible for synthesis of sterol-glucosides (SGs) in many organisms. WsSGTL1 is a SGT of Withania somnifera that has been found associated with plasma membranes. However its biological function in W.somnifera is largely unknown. In the present study, we have demonstrated through RNAi silencing of WsSGTL1 gene that it performs glycosylation of withanolides and sterols resulting in glycowithanolides and glycosylated sterols respectively, and affects the growth and development of transgenic W.somnifera. For this, RNAi construct (pFGC1008-WsSGTL1) was made and genetic transformation was done by Agrobacterium tumefaciens. HPLC analysis depicts the reduction of withanoside V (the glycowithanolide of W.somnifera) and a large increase of withanolides (majorly withaferin A) content. Also, a significant decrease in level of glycosylated sterols has been observed. Hence, the obtained data provides an insight into the biological function of WsSGTL1 gene in W.somnifera.

https://www.tandfonline.com/doi/pdf/10.1080/15592324.2015.1078064

RNAi-mediated gene silencing of WsSGTL1 in W.somnifera affects growth and glycosylation pattern

Withania somnifera (L.) Dunal, commonly called Indian Ginseng (Ashwagandha), is an important medicinal plant having a number of chemotypes which vary in their regenerative potentiality. In the present study, regenerative potentiality of four chemotypes of W. somnifera was compared using leaf explants. The study correlated that the chemotype (NIMTLI-130) with high total phenolic content (TPC), high H 2 O 2 concentration and low proline content was less regenerative, whereas, the chemotype (NIMTLI-101) with lesser TPC, lower H 2 O 2 concentration and high proline content was more regenerative. NIMTLI-130 showed higher SOD, GPX and APX activity compared to NIMTLI-101, but less CAT activity as well as low proline content and a weak detoxification effect resulting in lower regeneration. The study revealed that the difference in in vitro shoot organogenesis of different chemotypes largely depends on the antioxidant protection system of the plant involving non-enzymatic compounds and activities of antioxidant enzymes.

/pdf/effect-of-antioxidant-protection-system-on-regeneration-4gogpzic7k.pdf

Syed Saema

Papers

Agrobacterium tumefaciens -mediated transformation protocol of Jatropha curcas L. using leaf and hypocotyl segments

RNAi-mediated gene silencing of WsSGTL1 in W.somnifera affects growth and glycosylation pattern

Effect of antioxidant protection system on regeneration potential of different chemotypes of Withania somnifera (L.) Dunal ― A comparative analysis