Physiological response and transcriptome analyses of leguminous Indigofera bungeana Walp. to drought stress.

Objective: Indigofera bungeana is a shrub with high quality protein that has been widely utilized for forage grass in the semi-arid regions of China. This study aimed to enrich the currently available knowledge and clarify the detailed drought stress regulatory mechanisms in I. bungeana, and provide a theoretical foundation for the cultivation and resistance breeding of forage crops. Methods: This study evaluates the response mechanism to drought stress by exploiting multiple parameters and transcriptomic analyses of a 1-year-old seedlings of I. bungeana in a pot experiment. Results: Drought stress significantly caused physiological changes in I. bungeana. The antioxidant enzyme activities and osmoregulation substance content of I. bungeana showed an increase under drought. Moreover, 3,978 and 6,923 differentially expressed genes were approved by transcriptome in leaves and roots. The transcription factors, hormone signal transduction, carbohydrate metabolism of regulatory network were observed to have increased. In both tissues, genes related to plant hormone signaling transduction pathway might play a more pivotal role in drought tolerance. Transcription factors families like basic helix-loop-helix (bHLH), vian myeloblastosis viral oncogene homolog (MYB), basic leucine zipper (bZIP) and the metabolic pathway related-genes like serine/threonine-phosphatase 2C (PP2C), SNF1-related protein kinase 2 (SnRK2), indole-3-acetic acid (IAA), auxin (AUX28), small auxin up-regulated rna (SAUR), sucrose synthase (SUS), sucrosecarriers (SUC) were highlighted for future research about drought stress resistance in Indigofera bungeana. Conclusion: Our study posited I. bungeana mainly participate in various physiological and metabolic activities to response severe drought stress, by regulating the expression of the related genes in hormone signal transduction. These findings, which may be valuable for drought resistance breeding, and to clarify the drought stress regulatory mechanisms of I. bungeana and other plants.

Transcriptional regulation of proanthocyanidin biosynthesis pathway genes and transcription factors in Indigofera stachyodes Lindl. roots.

BACKGROUND: Proanthocyanidins (PAs) have always been considered as important medicinal value component. In order to gain insights into the PA biosynthesis regulatory network in I. stachyodes roots, we analyzed the transcriptome of the I. stachyodes in Leaf, Stem, RootI (one-year-old root), and RootII (two-year-old root). RESULTS: In this study, a total of 110,779 non-redundant unigenes were obtained, of which 63,863 could be functionally annotated. Simultaneously, 75 structural genes that regulate PA biosynthesis were identified, of these 6 structural genes (IsF3'H1, IsANR2, IsLAR2, IsUGT72L1-3, IsMATE2, IsMATE3) may play an important role in the synthesis of PAs in I. stachyodes roots. Furthermore, co-expression network analysis revealed that 34 IsMYBs, 18 IsbHLHs, 15 IsWRKYs, 9 IsMADSs, and 3 IsWIPs hub TFs are potential regulators for PA accumulation. Among them, IsMYB24 and IsMYB79 may be closely involved in the PA biosynthesis in I. stachyodes roots. CONCLUSIONS: The biosynthesis of PAs in I. stachyodes roots is mainly produced by the subsequent pathway of cyanidin. Our work provides new insights into the molecular pathways underlying PA accumulation and enhances our global understanding of transcriptome dynamics throughout different tissues.

Degradation of the Indospicine Toxin from Indigofera spicata by a Mixed Population of Rumen Bacteria.

The leguminous plant species, Indigofera linnaei and Indigofera spicata are distributed throughout the rangeland regions of Australia and the compound indospicine (L-2-amino-6-amidinohexanoic acid) found in these palatable forage plants acts as a hepatotoxin and can accumulate in the meat of ruminant livestock and wild camels. In this study, bovine rumen fluid was cultivated in an in vitro fermentation system provided with Indigofera spicata plant material and the ability of the resulting mixed microbial populations to degrade indospicine was determined using UPLC-MS/MS over a 14 day time period. The microbial populations of the fermentation system were determined using 16S rRNA gene amplicon sequencing and showed distinct, time-related changes occurring as the rumen-derived microbes adapted to the fermentation conditions and the nutritional substrates provided by the Indigofera plant material. Within eight days of commencement, indospicine was completely degraded by the microbes cultivated within the fermenter, forming the degradation products 2-aminopimelamic acid and 2-aminopimelic acid within a 24 h time period. The in vitro fermentation approach enabled the development of a specifically adapted, mixed microbial population which has the potential to be used as a rumen drench for reducing the toxic side-effects and toxin accumulation associated with ingestion of Indigofera plant material by grazing ruminant livestock.

Profiling the Philippine Blue: Liquid chromatography/mass spectrometry-based metabolomics study on Philippine Indigofera.

RATIONALE: High-throughput liquid chromatography/mass spectrometry (LC/MS) analysis presents an interesting platform for natural dyes research. A particular example is the assessment of the dynamic changes in fermentation mixtures of Philippine Indigofera, and in the investigation of commercially available indigo prepared using traditional and optimized methods. METHODS: Leaves from Indigofera tinctoria and Indigofera suffruticosa were subjected to methanolic extraction and aqueous fermentation for 48 h. Indigo powders prepared following 2-day and 15-day fermentation were also subjected to profiling using ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/QTOF-MS). MS2 spectra were annotated through a library search in the community-curated Global Natural Products Social Molecular Networking (GNPS). Spectra with no library hits in GNPS were annotated by analysis of their fragmentation pathways. RESULTS: UHPLC/MS-based detection and fragmentation analysis led to characterization of leucoindigo and the unreported tryptanthrin intermediate, 5a-hydroxy-5,5a-dihydroindolo[2,1-b]quinazoline-6,12-dione, in the fermentation extract of I. tinctoria leaves. Indigo-associated metabolites were absent in an Indigofera specimen in Laguna Province, which explained why it did not produce blue dye. Locally produced indigo was abundant in indigotin and indirubin, differentiated based on product ions with the corresponding predicted fragmentation pattern. The relative intensity of indigotin, however, decreased with the traditional process of extended fermentation to produce indigo. CONCLUSIONS: The study is the first to demonstrate simultaneous MS-based analysis of reaction intermediates, indigotin dye, side products, and catabolites on actively transforming fermentation extracts of I. tinctoria. New results include annotated mass spectra for leucoindigo, and for the unreported 5a-hydroxy-5,5a-dihydroindolo[2,1-b]quinazoline-6,12-dione, which is probably an intermediate in tryptranthrin synthesis. The proposed fragmentation schemes could guide the annotation of analogous compounds in complex mixtures.

Plasmodium chabaudi-infected mice spleen response to synthesized silver nanoparticles from Indigofera oblongifolia extract.

Malaria is a worldwide serious-threatening infectious disease caused by Plasmodium and the parasite resistance to antimalarial drugs has confirmed a significant obstacle to novel therapeutic antimalarial drugs. In this article, we assessed the antioxidant and anti-inflammatory activity of nanoparticles prepared from Indigofera oblongifolia extract (AgNPs) against the infection with Plasmodium chabaudi caused in mice spleen. AgNPs could significantly suppress the parasitaemia caused by the parasite to approximately 98% on day 7 postinfection with P. chabaudi and could improve the histopathological induced spleen damage. Also, AgNPs were able to increase the capsule thickness of the infected mice spleen. In addition, the AgNPs functioned as an antioxidant agent that affects the change in glutathione, nitric oxide and catalase levels in the spleen. Moreover spleen IL1ß, IL-6 and TNF-α-mRNA expression was regulated by AgNPs administration to the infected mice. These results indicated the anti-oxidant and the anti-inflammatory protective role of AgNPs against P. chabaudi-induced spleen injury.

[Advances in biosynthesis of indigo in plants].

Natural indigo, as one of the oldest dyes, is also a pivotal dye utilized in cotton fabrics today. A diversity of plants rich in indigo compounds belong to traditional Chinese herbal medicines. Indigo compounds have a variety of biological and pharmacological activities, including anticonvulsant, antibacterial, antifungal, antiviral and anticancer activities. A substantial progress in indigo biosynthesis has been made lately. This paper summarizes the value of indigo from the aspects of cultural history, biosynthetic pathways and the medicinal activities of its related derivatives involved in the pathways. In addition, the latest research advancements in indigo biosynthetic pathways is demonstrated in this paper, which would lay the theoretical foundation for the exploration and utilization of natural indigo.

Body Weight Gain, Nutrients Degradability and Fermentation Rumen Characteristics of Boerka Goat Supplemented Green Concentrate Pellets (GCP) Based on Indigofera zollingeriana.

BACKGROUND AND OBJECTIVE: Indigofera zollingeriana leguminous have been known widely to have a concentrate feed characteristic due to its high nutrient contents (crude protein, vitamin and some mineral) and its highly dry matter (DM) digestibility. This study aimed to identify the effects body weight gain, nutrients degradability, fermentation rumen characteristics and blood metabolite of Boerka goat supplemented green concentrate pellets (GCP)based on Indigofera zollingeriana. MATERIALS AND METHODS: Twenty four male Boer x Kacang crossbreeds with age of approximately male phase to 6 months and average initial body weight (BW) 13±0.5 kg were used in feeding and degestion trials. The study was assigned according to randomized block design with four dietary treatments and six goats were allocated to one of four treatments in randomised block design. The animals feed was offered chopped fresh Brachiaria humidicola (ad libitum) and feed treatments were offered daily at 4.0% body weight (BW). RESULTS: The digestibility increased in line with the increasing proportion of I. zollingeriana in the green concentrate pellets (GCP). The tannin content of GCP seemed to not significantly impacted on feed intake. The daily body weight gain and efficiency of feed utilization increased as the proportion of I. zollingeriana GCP increased. Increasing of the proportion C. calotyhrsus in GCP affected the concentration of ammonia (NH3) and VFA of the rumen liquids of goat. CONCLUSION: Green concentrate pellets composing 90% I. zollingeriana gave the best results in term of daily body weight gain, feed intake, nutrient degradability, efficiency of feed utilization, rumen fermentation in Boerka goats.

In Vitro Biodegradation of Hepatotoxic Indospicine in Indigofera spicata and Its Degradation Derivatives by Camel Foregut and Cattle Rumen Fluids.

The known accumulation of the hepatotoxin indospicine in tissues of camels and cattle grazing Indigofera pasture plants is unusual in that free amino acids would normally be expected to be degraded during the fermentation processes in these foregut fermenters. In this study, in vitro experiments were carried out to examine the degradability of indospicine of Indigofera spicata by camel and cattle foregut microbiota. In the first experiment, a 48 h in vitro incubation was carried out using foregut fluid samples that were collected from 15 feral camels and also a fistulated cow. Degradability of indospicine ranged between 97% and 99%, with the higher value of 99% for camels. A pooled sample of foregut fluids from three camels that were on a roughage diet was used in a second experiment to examine the time-dependent degradation of indospicine present in the plant materials. Results indicated that camels' foregut fluids have the ability to biodegrade ∼99% of the indospicine in I. spicata within 48 h of incubation and produced 2-aminopimelamic acid and 2-aminopimelic acid. The time-dependent degradation analysis showed rapid indospicine degradation (65 nmol/h) during the first 8-18 h of incubation followed by a slower degradation rate (12 nmol/h) between 18 and 48 h. Indospicine degradation products were also degraded toward the end of the experiment. The results of these in vitro degradation studies suggest that dietary indospicine may undergo extensive degradation in the foregut of the camel, resulting in trace levels after 48 h. The retention time for plant material in the camel foregut varies depending on feed quality, and the results of this study together with the observed accumulation of indospicine in camel tissues suggest that, although indospicine can be degraded by foregut fermentation, this degradation is not complete before the passage of the digesta into the intestine.

Accumulation, Persistence, and Effects of Indospicine Residues in Camels Fed Indigofera Plant.

Indospicine (l-2-amino-6-amidinohexanoic acid) is a natural hepatotoxin found in all parts of some Indigofera plants such as Indigofera linnaei and Indigofera spicata. Several studies have documented a susceptibility to this hepatotoxin in different species of animals, including cattle, sheep, dogs, and rats, which are associated with mild to severe liver disease after prolonged ingestion. However, there is little published data on the effects of this hepatotoxin in camels, even though Indigofera plants are known to be palatable to camels in central Australia. The secondary poisoning of dogs after prolonged dietary exposure to residual indospicine in camel muscle has raised additional food safety concerns. In this study, a feeding experiment was conducted to investigate the in vivo accumulation, excretion, distribution, and histopathological effects of dietary indospicine on camels. Six young camels (2-4 years old), weighing 270-390 kg, were fed daily a roughage diet consisting of Rhodes grass hay and lucerne chaff, supplemented with Indigofera and steam-flaked barley. Indigofera (I. spicata) was offered at 597 mg DM/kg body weight (bw)/day, designed to deliver 337 µg indospicine/kg bw/day, and fed for a period of 32 days. Blood and muscle biopsies were collected over the period of the study. Concentrations of indospicine in the plasma and muscle biopsy samples were quantitated by validated ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The highest concentrations in plasma (1.01 mg/L) and muscle (2.63 mg/kg fresh weight (fw)) were found at necropsy (day 33). Other tissues were also collected at necropsy, and analysis showed ubiquitous distribution of indospicine, with the highest indospicine accumulation detected in the pancreas (4.86 ± 0.56 mg/kg fw) and liver (3.60 ± 1.34 mg/kg fw), followed by the muscle, heart, and kidney. Histopathological examination of liver tissue showed multiple small foci of predominantly mononuclear inflammatory cells. After cessation of Indigofera intake, indospicine present in plasma in the remaining three camels had a longer terminal elimination half-life (18.6 days) than muscle (15.9 days), and both demonstrated monoexponential decreases.

Indigofera oblongifolia mitigates lead-acetate-induced kidney damage and apoptosis in a rat model.

This study was conducted to appraise the protective effect of Indigofera oblongifolia leaf extract on lead acetate (PbAc)-induced nephrotoxicity in rats. PbAc was intraperitoneally injected at a dose of 20 mg/kg body weight for 5 days, either alone or together with the methanol extract of I. oblongifolia (100 mg/kg). Kidney lead (Pb) concentration; oxidative stress markers including lipid peroxidation, nitrite/nitrate, and glutathione (GSH); and antioxidant enzyme activities, namely superoxide dismutase, catalase, GSH peroxidase, and GSH reductase were all determined. The PbAc injection elicited a marked elevation in Pb concentration, lipid peroxidation, and nitrite/nitrate, with a concomitant depletion in GSH content compared with the control and a remarkable decrease in antioxidant enzymes. Oxidant/antioxidant imbalance, Pb accumulation, and histological changes in the kidneys were successfully prevented by the pre-administration of I. oblongifolia extract. In addition, the elevated expression of proapoptotic protein, Bax, in the kidneys of the PbAc-injected rats was reduced as a result of I. oblongifolia pre-administration, while the hitherto reduced expression of the anti-apoptotic protein Bcl-2 was elevated. Based on the current findings, it can be concluded that I. oblongifolia successfully minimizes the deleterious effects in kidney function and histological coherence associated with nephrotoxicity by strengthening the antioxidant defense system, suppressing oxidative stress, and mitigating apoptosis.

Determination of hepatotoxic indospicine in Australian camel meat by ultra-performance liquid chromatography-tandem mass spectrometry.

Indospicine is a hepatotoxic amino acid found in Indigofera plant spp. and is unusual in that it is not incorporated into protein but accumulates as the free amino acid in the tissues (including muscle) of animals consuming these plants. Dogs are particularly sensitive to indospicine, and secondary poisoning of dogs has occurred from the ingestion of indospicine-contaminated horse meat and more recently camel meat. In central Australia, feral camels are known to consume native Indigofera species, but the prevalence of indospicine residues in their tissues has not previously been investigated. In this study, a method was developed and validated with the use of ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to determine the level of indospicine in camel meat samples using isotopically labeled indospicine as an internal standard. UPLC-MS/MS analysis showed that the method is reproducible, with high recovery efficiency and a quantitation limit of 0.1 mg/kg. Camel meat samples from the Simpson Desert were largely contaminated (≈50%) by indospicine with levels up to 3.73 mg/kg (fresh weight) determined. However, the majority of samples (95%) contained less than 1 mg/kg indospicine.

25th anniversary article: progress in chemistry and applications of functional indigos for organic electronics.

Indigo and its derivatives are dyes and pigments with a long and distinguished history in organic chemistry. Recently, applications of this 'old' structure as a functional organic building block for organic electronics applications have renewed interest in these molecules and their remarkable chemical and physical properties. Natural-origin indigos have been processed in fully bio-compatible field effect transistors, operating with ambipolar mobilities up to 0.5 cm(2) /Vs and air-stability. The synthetic derivative isoindigo has emerged as one of the most successful building-blocks for semiconducting polymers for plastic solar cells with efficiencies > 5%. Another isomer of indigo, epindolidione, has also been shown to be one of the best reported organic transistor materials in terms of mobility (∼2 cm(2) /Vs) and stability. This progress report aims to review very recent applications of indigoids in organic electronics, but especially to logically bridge together the hereto independent research directions on indigo, isoindigo, and other materials inspired by historical dye chemistry: a field which was the root of the development of modern chemistry in the first place.