Isatis tinctoria (woad)

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

(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 thaliana12

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.

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 metabolome14, 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.

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.

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. Zang, Y, Miao, Y, Wu, T, Cheng, Z (2020) Development of a thin-layer chromatography bioautographic assay for neuraminidase inhibitors hyphenated with electrostatic field induced spray ionisation-mass spectrometry for identification of active Isatis indigotica root compounds. J Chromatogr A :461597. doi: 10.1016/j.chroma.2020.461597. PubMed PMID:33250163 .
  2. Zeng, Q, Luo, C, Cho, J, Lai, D, Shen, X, Zhang, X et al. (2021) Tryptanthrin exerts anti-breast cancer effects both in vitro and in vivo through modulating the inflammatory tumor microenvironment. Acta Pharm 71:245-266. doi: 10.2478/acph-2021-0020. PubMed PMID:33151167 .
  3. Guo, Q, Li, Z, Shen, L, Xiao, Y, Cheng, Z (2020) Quantitative 1 H nuclear magnetic resonance (qHNMR) methods for accurate purity determination of glucosinolates isolated from Isatis indigotica roots. Phytochem Anal :. doi: 10.1002/pca.3003. PubMed PMID:33128329 .
  4. Jia, H, Wang, X, Shi, C, Guo, J, Ma, P, Ren, X et al. (2020) Hydrogen sulfide decreases Cd translocation from root to shoot through increasing Cd accumulation in cell wall and decreasing Cd2+ influx in Isatis indigotica. Plant Physiol Biochem 155:605-612. doi: 10.1016/j.plaphy.2020.08.033. PubMed PMID:32846396 .
  5. Zhang, ZJ, Morris-Natschke, SL, Cheng, YY, Lee, KH, Li, RT (2020) Development of anti-influenza agents from natural products. Med Res Rev 40:2290-2338. doi: 10.1002/med.21707. PubMed PMID:32677056 .
  6. Li, D, Oku, N, Shinozaki, Y, Kurokawa, Y, Igarashi, Y (2020) 4-Hydroxy-3-methyl-2(1H)-quinolone, originally discovered from a Brassicaceae plant, produced by a soil bacterium of the genus Burkholderia sp.: determination of a preferred tautomer and antioxidant activity. Beilstein J Org Chem 16:1489-1494. doi: 10.3762/bjoc.16.124. PubMed PMID:32647550 PubMed Central PMC7323622.
  7. Zhang, D, Sun, Y, Shi, Y, Wu, X, Jia, Q, Chen, K et al. (2020) Four new indole alkaloids from the roots of Isatis tinctoria. Nat Prod Res :1-7. doi: 10.1080/14786419.2020.1779716. PubMed PMID:32567364 .
  8. Pu, ZQ, Ma, YY, Lu, MX, Ma, YQ, Xu, ZQ (2020) Cloning of a SEPALLATA4-like gene (IiSEP4) in Isatis indigotica Fortune and characterization of its function in Arabidopsis thaliana. Plant Physiol Biochem 154:229-237. doi: 10.1016/j.plaphy.2020.05.031. PubMed PMID:32563851 .
  9. Guo, Q, Li, D, Xu, C, Zhu, C, Guo, Y, Yu, H et al. (2020) Indole alkaloid glycosides with a 1'-(phenyl)ethyl unit from Isatis indigotica leaves. Acta Pharm Sin B 10:895-902. doi: 10.1016/j.apsb.2019.09.001. PubMed PMID:32528835 PubMed Central PMC7280145.
  10. Zhang, D, Sun, Y, Chen, Z, Jia, Q, Zhu, W, Chen, K et al. (2020) Bisindole alkaloids with nitric oxide inhibitory activities from an alcohol extract of the Isatis indigotica roots. Fitoterapia 146:104654. doi: 10.1016/j.fitote.2020.104654. PubMed PMID:32502502 .
  11. Liang, X, Huang, Y, Pan, X, Hao, Y, Chen, X, Jiang, H et al. (2020) Erucic acid from Isatis indigotica Fort. suppresses influenza A virus replication and inflammation in vitro and in vivo through modulation of NF-κB and p38 MAPK pathway. J Pharm Anal 10:130-146. doi: 10.1016/j.jpha.2019.09.005. PubMed PMID:32373385 PubMed Central PMC7192973.
  12. Lotts, T, Kabrodt, K, Hummel, J, Binder, D, Schellenberg, I, Ständer, S et al. (2020) Isatis tinctoria L.-derived Petroleum Ether Extract Mediates Anti-inflammatory Effects via Inhibition of Interleukin-6, Interleukin-33 and Mast Cell Degranulation. Acta Derm Venereol 100:adv00131. doi: 10.2340/00015555-3476. PubMed PMID:32250439 .
  13. Qin, M, Wang, J, Zhang, T, Hu, X, Liu, R, Gao, T et al. (2020) Genome-Wide Identification and Analysis on YUCCA Gene Family in Isatis indigotica Fort. and IiYUCCA6-1 Functional Exploration. Int J Mol Sci 21:. doi: 10.3390/ijms21062188. PubMed PMID:32235744 PubMed Central PMC7139497.
  14. Wang, T, Wang, X, Zhuo, Y, Si, C, Yang, L, Meng, L et al. (2020) Antiviral activity of a polysaccharide from Radix Isatidis (Isatis indigotica Fortune) against hepatitis B virus (HBV) in vitro via activation of JAK/STAT signal pathway. J Ethnopharmacol 257:112782. doi: 10.1016/j.jep.2020.112782. PubMed PMID:32217096 .
  15. Zhang, D, Ruan, D, Li, J, Chen, Z, Zhu, W, Guo, F et al. (2020) Four undescribed sulfur-containing indole alkaloids with nitric oxide inhibitory activities from Isatis tinctoria L. roots. Phytochemistry 174:112337. doi: 10.1016/j.phytochem.2020.112337. PubMed PMID:32163787 .
  16. Lu, MX, Li, DZ, Pu, ZQ, Ma, YQ, Huang, X, Xu, ZQ et al. (2020) Ectopic expression of IiSHP2 from Isatis indigotica Fortune, a PLE-lineage MADS-box gene, influences leaf, floral organ and silique morphology in Arabidopsis thaliana. Physiol Mol Biol Plants 26:379-389. doi: 10.1007/s12298-019-00745-6. PubMed PMID:32158142 PubMed Central PMC7036382.
  17. Speranza, J, Miceli, N, Taviano, MF, Ragusa, S, Kwiecień, I, Szopa, A et al. (2020) Isatis tinctoria L. (Woad): A Review of its Botany, Ethnobotanical Uses, Phytochemistry, Biological Activities, and Biotechnological Studies. Plants (Basel) 9:. doi: 10.3390/plants9030298. PubMed PMID:32121532 PubMed Central PMC7154893.
  18. Xi, YF, Lou, LL, Han, FY, Liu, SF, Yao, GD, Lin, B et al. (2020) Four pairs of alkaloid enantiomers from Isatis indigotica Fortune Ex Land with neuroprotective effects against H2O2-induced SH-SY5Y cell injury. Bioorg Chem 96:103650. doi: 10.1016/j.bioorg.2020.103650. PubMed PMID:32044515 .
  19. Kang, M, Wu, H, Yang, Q, Huang, L, Hu, Q, Ma, T et al. (2020) A chromosome-scale genome assembly of Isatis indigotica, an important medicinal plant used in traditional Chinese medicine: An Isatis genome. Hortic Res 7:18. doi: 10.1038/s41438-020-0240-5. PubMed PMID:32025321 PubMed Central PMC6994597.
  20. Xu, C, Xin, Y, Chen, M, Ba, M, Guo, Q, Zhu, C et al. (2020) Discovery, synthesis, and optimization of an N-alkoxy indolylacetamide against HIV-1 carrying NNRTI-resistant mutations from the Isatis indigotica root. Eur J Med Chem 189:112071. doi: 10.1016/j.ejmech.2020.112071. PubMed PMID:32004936 PubMed Central PMC7111291.
Search PubMed

Bibliography for the backstory

1.
Spataro G, Negri V. Adaptability and variation in Isatis tinctoria L.: a new crop for Europe. Euphytica. 2007;163(1):89-102. doi: 101007/s10681-007-9604-2
2.
Lysak MA, Koch MA. Phylogeny, genome and karyotype evolution of crucifers (Brassicaceae). In: Schmidt R, Bancroft I, eds. Genetics and Genomics of the Brassicaceae. New York, NY: Springer New York; 2010:1-31. http://www.springerlink.com/index/10.1007/978-1-4419-7118-0_1.
3.
Tomović GM, Mihailović NL, Tumi AF, Gajić BA, Mišljenović TD, Niketić MS. Trace Metals in Soils and Several Brassicaceae Plant Species from Serpentine Sites of Serbia. Archives of Environmental Protection. 2014;39(4):1-21. http://www.degruyter.com/view/j/aep.2013.39.issue-4/aep-2013-0039/aep-2013-0039.xml.
4.
Jacobs J, Pokorny M. Ecology and Management of Dyer’s Woad (Isatis tinctoria L.). USDA Publications. https://www.nrcs.usda.gov/Internet/FSE_PLANTMATERIALS/publications/mtpmctn13107.pdf. Published 2008. Accessed November 13, 2017.
5.
Pokorny ML, Krueger-Mangold JM. Evaluating Montana’s Dyer’s Woad (Isatis tinctoria) Cooperative Eradication Project. Weed technol. 2017;21(01):262-269. https://www.cambridge.org/core/product/identifier/S0890037X00019825/type/journal_article.
6.
Grime JP. Evidence for the Existence of Three Primary Strategies in Plants and Its Relevance to Ecological and Evolutionary Theory. The American Naturalist. 2017;111(982):1169-1194. doi: 101086/283244
7.
Thorup-Kristensen K, Rasmussen CR. Identifying new deep-rooted plant species suitable as undersown nitrogen catch crops. Journal of Soil and Water Conservation. 2015;70(6):399-409. http://www.jswconline.org/cgi/doi/10.2489/jswc.70.6.399.
8.
Munkholm LJ, Hansen EM. Catch crop biomass production, nitrogen uptake and root development under different tillage systems. Soil Use & Management. 2012;28(4):517-529. http://doi.wiley.com/10.1111/sum.12001.
9.
Gilbert KG, Cooke DT. Dyes from plants: Past usage, present understanding and potential. Plant Growth Regulation. 2001;34:57-69. http://link.springer.com/10.1023/A:1013374618870.
10.
Stoker KG, Cooke DT, Hill DJ. Influence of light on natural indigo production from woad (Isatis tinctoria). Plant Growth Regulation. 1998;25(3):181-185. doi: 101023/a:1006042331385
11.
Sales E, Kanhonou R, Baixauli C, et al. Sowing date, transplanting, plant density and nitrogen fertilization affect indigo production from Isatis species in a Mediterranean region of Spain. Industrial Crops and Products. 2006;23(1):29-39. doi: 10.1016/j.indcrop.2005.03002
12.
Siddappaji MH, Scholes DR, Bohn M, Paige KN. Overcompensation in Response to Herbivory in Arabidopsis thaliana: The Role of Glucose-6-Phosphate Dehydrogenase and the Oxidative Pentose-Phosphate Pathway. G. 2013;195(2):589-598. doi: 10.1534/genetics.113154351
13.
Stoker KG, Cooke DT, Hill DJ. An Improved Method for the Large-Scale Processing of Woad (Isatis tinctoria) for Possible Commercial Production of Woad Indigo. Journal of Agricultural Engineering Research. 1998;71(4):315-320. doi: 10.1006/jaer.19980329
14.
Mohn T, Plitzko I, Hamburger M. A comprehensive metabolite profiling of Isatis tinctoria leaf extracts. Phytochemistry. 2009;70(7):924-934. http://dx.doi.org/10.1016/j.phytochem.2009.04.019.
15.
Gilbert KG. Dyes. In: Encyclopedia of Applied Plant Sciences. Elsevier; 2017:368-373. doi: 10.1016/b978-0-12-394807-600069-1
16.
Royo JL, Moreno-Ruiz E, Cebolla A, Santero E. Stable long-term indigo production by overexpression of dioxygenase genes using a chromosomal integrated cascade expression circuit. Journal of Biotechnology. 2004;116(2):113-124. doi: 10.1016/j.jbiotec.2004.10006
17.
Hamburger M. Isatis tinctoria – From the rediscovery of an ancient medicinal plant towards a novel anti-inflammatory phytopharmaceutical. Phytochemistry Reviews. 2002;1(3):333-344. doi: 101023/a:1026095608691
18.
The chemistry of tryptanthrin and its derivatives. Arkivoc. 2012;2012(1):546. doi: 10.3998/ark.5550190.0013113
19.
Oberthür C, Hamburger M. Tryptanthrin Content inIsatis tinctoriaLeaves – A Comparative Study of Selected Strains and Post-Harvest Treatments. P. 2004;70(7):642-645. doi: 101055/s-2004-827188
20.
Tang X, Xiao Y, Lv T, et al. High-Throughput Sequencing and De Novo Assembly of the Isatis indigotica Transcriptome. Devireddy lax, ed. PLoS ONE. 2014;9(9):e102963. doi: 10.1371/journal.pone0102963