Isatis tinctoria (woad)

Dyer's woad being invasive. Photo by Steve Dewey, Utah State University, bugwood.org

Background

(For a somewhat less than scientific consideration of this species, please read the Flights of Whimsy post.)

For well over 1,000 years, Isatis tinctoria was valued as the source of the brilliantly blue dye, indigo. Its importance declined about 150 years or so ago with the perfection of artificial dyes, but now is experiencing a bit of a resurgence. With increasing interest in “alternative crops” and natural products, I. tinctoria has “recently” been touted as a “new crop” for Europe, especially suitable in marginal areas.[1] This effort has been supported by the European Commission with the idea of replacing synthetic, petroleum based sources with natural, renewable ones. Based on historical cultivation and crop breeding, there are a number of genotypes with significantly different chemical characteristics. But woad is more than a source of blue dye, and has promise as a source of phytopharmacologicals; it’s secondary chemistry is in many ways unique.


Isatis tinctoria is in the Isatideae tribe of the Brassicaceae. Close relatives are the Brassiceae, Sysimbrieae and Thelypodioeae, while the Eutremeae (see E. salsugineum) are somewhat more remotely related. It’s 2n chromosome number is 14, but it is commonly autopolyploid (2n=28).[2] There are a number of subspecies and synonyms, including I. indigotica. (See BrassiBase). It should be noted here that I. indogotica has sometimes been used as a comparison species, but regardless of the final relationship, there are numerous opportunities for metabolome, transcriptome and genome comparisons that could shed light on the complexities of secondary metabolism.

I. tinctoria is native to the steppes and deserts of the Caucasus region, central Asia and Siberia. It tolerates serpentine soils with very high Mg/Ca ratios and high levels of metals, especially Ni and Zn, although it is not restricted to that ecosystem.[3] In the last 80 years, it has also established itself as an undesirable invasive in semiarid regions of the Western US.[4,5] It does well on rocky, oligotrophic soils with low water retention capacity and is often found in rugged, inaccessible terrain. Its persistence in range lands depends in part on low disturbance levels, as expected for “stress tolerant” plants in general.[6]

The root system of I. tinctoria undoubtedly contributes to its ability to persist in poor soils. It has a thick taproot capable of penetrating at least 1 m into the soil, with lateral roots concentrated in the top 30 cm. This allows the plants to compete well for soil moisture and nutrients. Recently, it has also drawn attention to this species as a possible “nitrogen catch crop”; in some agricultural soils, it showed high root densities even at 1.6-2.4 m, and the ability to reduce NO3-N under barley. The useful effect of this is to reduce leaching losses and protect groundwater quality.[7] Even at this, however, other plants (e.g fodder radish) may be better in many soils.[8]

Life history and ecologyI. tinctoria is generally a monocarpic (semelparous) biennial, producing a rosette in the first season, overwintering as dormant buds on the root crown, and flowering/fruiting in the second growing season. It propagates only by seed. Vernalization is required for the transition to flowering. Under less favorable conditions, however, the rosette stage can persist for multiple years until flowering is possible. And, to complete the possibilities, it can also be a winter annual, although vernalization is still required for flowering.[4,5] In any case, midsummer droughts and high temperatures can result in mortality as high as 75%.

Under natural conditions, the success of germination and seed longevity in the seed bank depend on whether or not the seed (one, sometimes two, per silique) is released from the fruit or remains encased. Free seeds germinate quite well; seeds in fruits, much less so. Seedling growth is also better from free seeds, possibly because of an unidentified water-soluble and leachable chemical inhibitor present in the fruit. Tied up with all this, seed longevity is short for free seeds, but at least 10 years for seeds still in the womb.[4]

To date, woad has not been subjected to any formal breeding effort and expresses a variety of phenotypes. There are, for example, genotypes that show upright or decumbent habits, smooth, pubescent, glaucous, shiny, serrated or smooth edged leaves, and a wide range of indigo precursor production between plants.[9] The effects of growth conditions, particularly irradiance levels, are significant, but have not been fully elucidated.[10]

As a crop in its own right, the effects of sowing date, plant density, nitrogen fertilization, irrigation rate and seedling transplanting also influence whole life cycle indigo production.[11] Genotype (e.g. I. tinctoria vs. I. indigotica) influences the propensity of the plants to regenerate after cutting, I. indigotica doing so poorly. Variation on this characteristic are known in other Brassicaceae species, e.g. Arabidopsis thaliana[12]

For more on growing, harvesting, processing, chemistry, synthesis and use of indigo, see reference [11]. For the description of a pilot project directed at large scale commercial production of indigo from woad, see reference [13]. Interestingly, neither N nor water availability nor planting density has a direct effect on indigo content per unit leaf even though plant growth and total leaf production are sensitive to those factors.

Metabolomics

Woad metabolomics – Interest in the metabolome of Isatis has been different from interest in other species considered on this site. The main focus in the literature and in practice has not been on stress-related changes in woad’s sugars, amino acids etc., but on secondary metabolites. Using dichloromethane extraction (supplemented with MeOH extraction for polar metabolites), Mohn et al.[14] identified more than 65 compounds found in the plants, including alkaloids, flavonoids, fatty acids, porphyrins, lignans, carotenoids, glucosinolates and cyclohexenones. Some of the compounds they identified are common to higher plants, some are more restricted to Isatis tinctoria and its close relatives.  One of the compounds – an indole alkaloid – was new to the literature.

Clearly, indigo is one important metabolite and understanding the factors controlling its production and quality may be important if it is to be genetically manipulated and exploited as a green alternative to the synthetic dye.[11] Interestingly, indigo is not synthesized directly by plants. Instead, it is a product derived from one of two secondary metabolites. The first, indoxyl, occurs mainly in the form of the glucoside, indican (indoxyl-β-D-glucoside). This is found in most indigo-producing plants.

Isatin B

However, in Isatis spp., the ester, isatan B (indoxyl-5- ketogluconate), rather than the glucoside is the major precursor.[15] Once the carbohydrate moiety is cleaved from the indoxyl group, two of the resulting molecules combine oxidatively to produce an indigo molecule. This combination occurs spontaneously in aerobic conditions and indigo precipitates from solution. 

Spontaneous indigo precipitation will also occur on broken or cut – or chewed – edges of leaves. This is a major potential problem in growing woad for indigo production since recovery of the dye once precipitated is difficult or impossible. This, of course, raises the question of what the role of indigo is in nature, not only in woad but in other species. It is tempting to speculate that it may be involved in some kind of defense against herbivory.

Indican and Isatin B synthesis – While the production of indigo itself is understood, the production of the precursors is not. In bacteria that produce indigo, tryptophan is considered the precursor,[16] but that doesn’t seem to be the case in higher plants; labelled tryptophan does not appear as labelling in indigo.[9] At present, there doesn’t seem to be a great rush to fill this gap, partly because it may not have immediate practical consequences in industrial indigo production, and partly because there seems to be more interest in engineering/improving microbial systems for indigo production which may prove to be less “messy” and just as “green” as using plants.[16] Of course, the genomic basis for indigo production is also unknown, and elucidation may well require figuring out the role of “unknown” and “orphan” genes, a task that seems to be on the back burner for many (even most) plant species.

Beyond indigo – Initially, two factors were responsible for the interest in the Isatis metabolome[14], and indigo production was not one of them. The first was that for centuries, woad was used as a medicinal plant in two distinct and unrelated cultures, i.e. in Europe and China. The second was that in both areas, its use was particularly targeted at ailments involving inflammatory processes. Indeed, both root and leaf preparations remain important herbal drugs in traditional Chinese medicine for such diseases.

As Matthius Hamburger noted in 2002, “the number of medicinal plants and phytopharmaceuticals with reasonably proven clinical efficacy in inflammatory and rheumatic diseases is small.”[17] Woad is one. Despite the large sales of putative medicinal plants as dietary supplements, the amount actually known about their active principles, their modes of action and their efficacy is often limited. The consequences of this for acceptance of medicinal plants by both consumers and regulators are significant.

This is a problem with solving. Inflammatory diseases affect very large numbers of people and anti-inflamitories, especially non-steroidal anti-inflammatory drugs (NSAIDs), are consumed in huge quantities. Unfortunately, extended use of these drugs can have a number of well-documented adverse effects, including gastrointestinal problems, increased danger of heart attack and stroke, renal failure, photosensitivity, and, for infants exposed to the drugs in utero, birth defects. Hence, if there were an alternative, rational, phytopharmacological approach with a better benefit/risk ratio, it could be useful.

Tryptanthrin – This is a weakly basic alkaloid, bright yellow in crystalline form. Its name derives from the observation that it is produced by the yeast Candida lipolytica when grown in L-tryptophan-containing medium.[18]

Tryptanthrin

Like indigo, it is synthesized by a diverse collection of plants, as well as by some fungi, bacteria and at least two mammals (Asian elephants – Elephas maximus and the bat Saccopteryx bilineata). It can also be synthesized chemically, and is a good prospect for chemical modifications to modify activity. As an herbal medicine, tryptanthrin has antiviral, antibacterial, antimycobacterial, antifungal, antiparasitic, and antineoplastic activities.[17,18] As an anti-inflammatory, tryptanthrin has been shown to be a potent COX-2 inhibitor, as well as 5-LOX, iNOS, serotonin and histamine release and human leucocytic elastase.[17] COX-2 and 5-LOX are key targets for NSAIDs.

The most potent activities were found in dichloromethane (DCM) or supercritical CO2 extracts, but maximal activity was found only when the complete extract was used instead of the purified tryptanthrin. Interestingly, tryptanthrin has no obvious structural resemblance with any of the synthetic COX-2 inhibitors in clinical use.

From the standpoint of woad as both a source of anti-inflammatories and as a genetically variable eXtreme plant, 67 Isatis samples – representative of the genetic diversity of the species – were screened for tryptanthrin content.  The genotypes originated from Morocco to Western Europe to East Asia. The variability was very large, covering almost a 30-fold concentration difference.[17]

Subsequent investigation of the effects of growth conditions, time of harvest and treatment of harvested material.[19] Post-harvest treatments of shock-freezing in LN2/freeze drying, air drying and drying at 40˚C showed very large effects – the freezing treatment yielded virtually no tryptanthrin while the 40˚ drying yielded the highest. This study also showed that breeding for phenotype and tryptanthrin concentration were also possible.  Given that, it is worthwhile to examine the current state of transcriptomic and genomic resources for the species.

Genomics/Transcriptomics

The Isatis tinctoria transcriptome – As noted earlier, I. tinctoria has 14 chromosomes (2n) but has usually been considered as an autopolyploid.[2] The reference transcriptome of the sub- or related-species, I. indigotica has been sequenced and reported using a mixed leaf and root sample.[20] Of approximately 33K unigenes, 84% had a hit in at least one protein or nucleotide database; 8 were found that were associated with the biosynthesis of indole and its derivatives. The authors considered the results to be a valuable first step in identifying genes that function in the synthesis of indigo, tryptanthrin and other indole alkaloids.

In addition, a few smaller transcriptome projects have been deposited to GenBank, including one associated with hairy roots and methyl jasmonate treatment leading to the production of anti-viral substances, transcriptome characterizations of autopolyploidization, medicinal compound production and the effects of nitrogen supply on the transcriptomics of alkaloid production. The full listing is here and depending on when this is read, additional bioprojects may have been added.

Genome resources – The reference genome for Isatis tinctoria has been sequenced by JGI as part of a broader project on the top 20 Brassicaceae. Although the release date is given as 2014-08-05, there appears, as yet, to be no assembled genome available and no publications related to the project. Raw and QC checked raw sequences are available for download from the JGI Portal or through the Isatis tinctoria BioProject at NCBI.

PubMed resources

The following is a partial list of publications on Isatis tinctoria/indigotica focussed on studies emphasizing a molecular aspect, but not limited to those on indigo or tryptanthrin. The list was generated by a search of PubMed; additional publications not indexed there are, of course, not included.  Abstracts and/or full text versions can be accessed by clicking doi, PMID or PMC numbers.

  1. Jie, C, Luo, Z, Chen, H, Wang, M, Yan, C, Mao, ZF et al. (2017) Indirubin, a bisindole alkaloid from Isatis indigotica, reduces H1N1 susceptibility in stressed mice by regulating MAVS signaling. Oncotarget 8:105615-105629. doi: 10.18632/oncotarget.22350. PubMed PMID:29285277 PubMed Central PMC5739664.
  2. Meng, L, Guo, Q, Liu, Y, Shi, J (2017) 8,4'-Oxyneolignane glucosides from an aqueous extract of "ban lan gen" (Isatis indigotica root) and their absolute configurations. Acta Pharm Sin B 7:638-646. doi: 10.1016/j.apsb.2017.09.006. PubMed PMID:29159023 PubMed Central PMC5687312.
  3. Ma, YQ, Li, DZ, Zhang, L, Li, Q, Yao, JW, Ma, Z et al. (2017) Ectopic expression of IiFUL isolated from Isatis indigotica could change the reproductive growth of Arabidopsis thaliana. Plant Physiol. Biochem. 121:140-152. doi: 10.1016/j.plaphy.2017.10.014. PubMed PMID:29102902 .
  4. He, L, Fan, F, Hou, X, Wu, H, Wang, J, Xu, H et al. (2017) 4(3H)-Quinazolone regulates innate immune signaling upon respiratory syncytial virus infection by moderately inhibiting the RIG-1 pathway in RAW264.7 cell. Int. Immunopharmacol. 52:245-252. doi: 10.1016/j.intimp.2017.09.010. PubMed PMID:28957692 .
  5. Nguyen, TK, Jamali, A, Grand, E, Morreel, K, Marcelo, P, Gontier, E et al. (2017) Phenylpropanoid profiling reveals a class of hydroxycinnamoyl glucaric acid conjugates in Isatis tinctoria leaves. Phytochemistry 144:127-140. doi: 10.1016/j.phytochem.2017.09.007. PubMed PMID:28930667 .
  6. Ma, R, Xiao, Y, Lv, Z, Tan, H, Chen, R, Li, Q et al. (2017) AP2/ERF Transcription Factor, Ii049, Positively Regulates Lignan Biosynthesis in Isatis indigotica through Activating Salicylic Acid Signaling and Lignan/Lignin Pathway Genes. Front Plant Sci 8:1361. doi: 10.3389/fpls.2017.01361. PubMed PMID:28824690 PubMed Central PMC5543283.
  7. Li, T, Wang, J, Lu, M, Zhang, T, Qu, X, Wang, Z et al. (2017) Selection and Validation of Appropriate Reference Genes for qRT-PCR Analysis in Isatis indigotica Fort. Front Plant Sci 8:1139. doi: 10.3389/fpls.2017.01139. PubMed PMID:28702046 PubMed Central PMC5487591.
  8. Sukenik, N, Iluz, D, Amar, Z, Varvak, A, Workman, V, Shamir, O et al. (2017) Early evidence (late 2nd millennium BCE) of plant-based dyeing of textiles from Timna, Israel. PLoS ONE 12:e0179014. doi: 10.1371/journal.pone.0179014. PubMed PMID:28658314 PubMed Central PMC5489155.
  9. Miceli, N, Filocamo, A, Ragusa, S, Cacciola, F, Dugo, P, Mondello, L et al. (2017) Chemical Characterization and Biological Activities of Phenolic-Rich Fraction from Cauline Leaves of Isatis tinctoria L. (Brassicaceae) Growing in Sicily, Italy. Chem. Biodivers. 14:. doi: 10.1002/cbdv.201700073. PubMed PMID:28622440 .
  10. Meng, L, Guo, Q, Liu, Y, Chen, M, Li, Y, Jiang, J et al. (2017) Indole alkaloid sulfonic acids from an aqueous extract of Isatis indigotica roots and their antiviral activity. Acta Pharm Sin B 7:334-341. doi: 10.1016/j.apsb.2017.04.003. PubMed PMID:28540170 PubMed Central PMC5430897.
  11. Meng, LJ, Guo, QL, Xu, CB, Zhu, CG, Liu, YF, Chen, MH et al. (2017) Diglycosidic indole alkaloid derivatives from an aqueous extract of Isatis indigotica roots. J Asian Nat Prod Res 19:529-540. doi: 10.1080/10286020.2017.1320547. PubMed PMID:28475367 .
  12. Kang, L, Li, P, Wang, A, Ge, X, Li, Z (2017) A Novel Cytoplasmic Male Sterility in Brassica napus (inap CMS) with Carpelloid Stamens via Protoplast Fusion with Chinese Woad. Front Plant Sci 8:529. doi: 10.3389/fpls.2017.00529. PubMed PMID:28428799 PubMed Central PMC5382163.
  13. Zhou, J, Liu, J, Lin, D, Gao, G, Wang, H, Guo, J et al. (2017) Boiling-induced nanoparticles and their constitutive proteins from Isatis indigotica Fort. root decoction: Purification and identification. J Tradit Complement Med 7:178-187. doi: 10.1016/j.jtcme.2016.08.007. PubMed PMID:28417088 PubMed Central PMC5388084.
  14. Liu, Y, Chen, M, Guo, Q, Li, Y, Jiang, J, Shi, J et al. (2017) Aromatic compounds from an aqueous extract of "ban lan gen" and their antiviral activities. Acta Pharm Sin B 7:179-184. doi: 10.1016/j.apsb.2016.09.004. PubMed PMID:28303224 PubMed Central PMC5343108.
  15. Liu, W, Liu, C, Liu, L, You, Y, Jiang, J, Zhou, Z et al. (2017) Simultaneous decolorization of sulfonated azo dyes and reduction of hexavalent chromium under high salt condition by a newly isolated salt-tolerant strain Bacillus circulans BWL1061. Ecotoxicol. Environ. Saf. 141:9-16. doi: 10.1016/j.ecoenv.2017.03.005. PubMed PMID:28284151 .
  16. Zhou, B, Li, J, Liang, X, Yang, Z, Jiang, Z (2017) Transcriptome profiling of influenza A virus-infected lung epithelial (A549) cells with lariciresinol-4-β-D-glucopyranoside treatment. PLoS ONE 12:e0173058. doi: 10.1371/journal.pone.0173058. PubMed PMID:28273165 PubMed Central PMC5342222.
  17. Milanović, V, Osimani, A, Taccari, M, Garofalo, C, Butta, A, Clementi, F et al. (2017) Insight into the bacterial diversity of fermentation woad dye vats as revealed by PCR-DGGE and pyrosequencing. J. Ind. Microbiol. Biotechnol. 44:997-1004. doi: 10.1007/s10295-017-1921-4. PubMed PMID:28246965 .
  18. Tsai, TY, Livneh, H, Hung, TH, Lin, IH, Lu, MC, Yeh, CC et al. (2017) Associations between prescribed Chinese herbal medicine and risk of hepatocellular carcinoma in patients with chronic hepatitis B: a nationwide population-based cohort study. BMJ Open 7:e014571. doi: 10.1136/bmjopen-2016-014571. PubMed PMID:28122837 PubMed Central PMC5278254.
  19. Ma, RF, Liu, QZ, Xiao, Y, Zhang, L, Li, Q, Yin, J et al. (2016) The phenylalanine ammonia-lyase gene family in Isatis indigotica Fort.: molecular cloning, characterization, and expression analysis. Chin J Nat Med 14:801-812. doi: 10.1016/S1875-5364(16)30097-8. PubMed PMID:27914524 .
  20. Bektaş, İ, Karaman, Ş, Dıraz, E, Çelik, M (2016) The role of natural indigo dye in alleviation of genotoxicity of sodium dithionite as a reducing agent. Cytotechnology 68:2245-2255. doi: 10.1007/s10616-016-0018-7. PubMed PMID:27757710 PubMed Central PMC5101296.
Search PubMed

 

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