Effects of water saving and nitrogen reduction on the yield, quality, water and nitrogen use efficiency of Isatis indigotica in Hexi Oasis

Isatis indigotica planting is the backbone of the medicinal industry in Hexi Oasis, Gansu. In order to solve the problems insufficient water resources and excessive application of nitrogen fertilizer in this area, this paper explored the irrigation and nitrogen levels that can meet the multiple goals of Isatis indigotica. The two-factor split-plot field experiment (2018‒2019) was conducted in Minle County, Gansu Province, China, which contains 9 treatments. There were three levels of irrigation water: W1(low), W2(medium), and W3(high). The soil moisture contents were 60–70%, 70–80%, and 80–90% of the field water-holding capacity, respectively. The nitrogen application rate was classified into three levels, N1(low), N2(medium) and N3(high), which were 150, 200 and 250 kg N/ha, respectively. The standard local irrigation water amount and nitrogen application rate corresponded to W3N3. The results showed that the yield of Isatis indigotica increased first and then decreased with the increase of irrigation amount and nitrogen application rate, the yield of W2N2 is 12.2–17.1% higher than that of W1N1, the yield of W3N3 was 12.1–17.5% lower than that of W2N2. Saving water and reducing nitrogen can improve the quality of Isatis indigotica, compared with W3N3, the indigo, indirubin, (R,S)-epigoitrin and polysaccharides of W2N2 increased by 4.5–5.9%, 2.7–3.1%, 5.2–6.0%, and 1.8–2.1%, respectively. With the increase of nitrogen application rate, the water use efficiency (WUE) first increased and then decreased, as the irrigation volume increases, WUE decreases. Compared with W3N3, the WUE of W2N2 increased by 24.3–27.2%. With the increase of water input, the nitrogen fertilizer use efficiency (NUE) first increased and then decreased, as the nitrogen application rate increases, NUE decreases. Compared with W3N3, the NUE of W2W2 increased by 31.8–34.5%. Therefore, W2N2 can improve quality and increase water and nitrogen utilization efficiency on the basis of ensuring yield.

Identification of key gene networks controlling vernalization development characteristics of Isatis indigotica by full-length transcriptomes and gene expression profiles

Isatis indigotica Fort., as a common Chinese medicinal raw material, will lose its medicinal value if it blooms early, so it is highly valuable to clarify the induction mechanism of the vernalization of I. indigotica at low temperature. In this study, the concentrations of soluble sugar, proline, glutathione and zeatin in two germplasms of I. indigotica with different degrees of low temperature tolerance (Y1 and Y2) were determined at 10 days, 20 days and 30 days of low-temperature treatment, and the full-length transcriptome of 24 samples was sequenced by Nanopore sequencing with Oxford Nanopore Technologies (ONT). After that, the data of transcripts involved in the vernalization of I. indigotica at low temperature were obtained, and these transcripts were identified using weighted gene co-expression network analysis (WGCNA). The results revealed the massive accumulation of soluble sugar and proline in Y1 and Y2 after low temperature induction. A total of 18,385 new transcripts, 6168 transcription factors and 470 lncRNAs were obtained. Differential expression analysis showed that gibberellin, flavonoids, fatty acids and some processes related to low temperature response were significantly enriched. Eight key transcripts were identified by WGCNA, among which ONT.14640.1, ONT.9119.1, ONT.13080.2 and ONT.16007.1 encodes a flavonoid transporter, 9-cis-epoxycarotenoid dioxygenase 3 (NCED3), growth factor gene and L-aspartate oxidase in plants, respectively. It indicated that secondary metabolites such as hormones and flavonoids play an important role in the vernalization of I. indigotica. qRT-PCR proved the reliability of transcriptome results. These results provide important insights on the low-temperature vernalization of I. indigotica, and provide a research basis for analyzing the vernalization mechanism of I. indigotica.

Polysaccharide Nanoparticles from Isatis indigotica Fort. Root Decoction: Diversity, Cytotoxicity, and Antiviral Activity

It has been revealed that numerous nanoparticles are formed during the boiling preparation of traditional Chinese medical decoctions and culinary soups. They may possess physiological effects different from those of constituent components and are worth paying attention to but are barely noticed and investigated as of yet. In this study, six groups of nanoparticles, whose size ranged from 57 to 300 nm, were successfully isolated from the decoction of Isatis indigotica Fort. root, according to their particle size by the means of size-exclusive chromatography. All of the obtained nanoparticles have a high content of polysaccharides, which distinguishes them from the disclosed BLG protein nanoparticles. They also have high similarities in other compositions, surface charge, and stimuli responses. However, four out of these six nanoparticles (F2, F3, F4, and F5) exhibited significant antiviral activity against influenza virus H1N1, and their antiviral activities and cytotoxicity towards MDCK cells varied with their sizes. It suggested that the antiviral efficacy of BLG decoction could also be from its nanoparticles besides its well-known antiviral phytochemicals. It also implied that the biological effects of these polysaccharide nanoparticles, including cytotoxicity and antiviral activity, may be correlative with the physicochemical properties, especially the particle size.

Isatis indigotica: from (ethno) botany, biochemistry to synthetic biology

Isatis indigotica Fort. (Chinese woad) is a species with an ancient and well-documented history as an indigo dye and medicinal plant. It is often confused with Isatis tinctoria L. (European woad), a medicinal plant in Europe. Here, the differences between I. indigotica and I. tinctoria are systematically described. The usage development history, clinical applications and pharmacological activities, and chemical components of I. indigotica are also summarized. Lignans, indole alkaloids, and their corresponding derivatives have been identified as the major active ingredients of I. indigotica and are associated with anti-viral, anti-inflammatory, anti-cancer, and other health-promoting activities. Notable progress has been made in understanding the biosynthetic pathway and regulation mechanism of lignans and indole alkaloids in I. indigotica, the results from which should facilitate the process of targeted metabolic engineering or synthetic biology. Moreover, multiple biotechnology methods such as polyploid breeding and genetic engineering have been used with I. indigotica to result in, for example, greater yields, higher levels of bioactive component accumulation, and enhanced stress tolerance to salt, drought, and insects. Some issues require additional analyses, and suggestions for future research on I. indigotica are also discussed.

Integrative analyses of targeted metabolome and transcriptome of Isatidis Radix autotetraploids highlighted key polyploidization-responsive regulators

Background Isatidis Radix, the root of Isatis indigotica Fort. (Chinese woad) can produce a variety of efficacious compound with medicinal properties. The tetraploid I. indigotica plants exhibit superior phenotypic traits, such as greater yield, higher bioactive compounds accumulation and enhanced stress tolerance. In this study, a comparative transcriptomic and metabolomic study on Isatidis Radix autotetraploid and its progenitor was performed. Results Through the targeted metabolic profiling, 283 metabolites were identified in Isatidis Radix, and 70 polyploidization-altered metabolites were obtained. Moreover, the production of lignans was significantly increased post polyploidization, which implied that polyploidization-modulated changes in lignan biosynthesis. Regarding the transcriptomic shift, 2065 differentially expressed genes (DEGs) were identified as being polyploidy-responsive genes, and the polyploidization-altered DEGs were enriched in phenylpropanoid biosynthesis and plant hormone signal transduction. The further integrative analysis of polyploidy-responsive metabolome and transcriptome showed that 1584 DEGs were highly correlated with the 70 polyploidization-altered metabolites, and the transcriptional factors TFs-lignans network highlighted 10 polyploidy-altered TFs and 17 fluctuated phenylpropanoid pathway compounds. Conclusions These results collectively indicated that polyploidization contributed to the high content of active compounds in autotetraploid roots, and the gene–lignan pathway network analysis highlighted polyploidy–responsive key functional genes and regulators.

First report of southern root-knot nematode, Meloidogyne incognita, infecting Isatis indigotica Fortune in Anhui Province, China

Isatis indigotica Fortune, widely cultivated in China, is an important Chinese herbal medicine, mainly used to treat cold and fever. In October 2020, galls (Fig. 1), as many as 65 per root, were observed on the roots of I. indigotica in Taihe, Anhui Province, China (117°21'19.5"N, 32°57'59.5"E), and samples were taken. The infected plants were weak, and the leaves are wilting. Second-stage juveniles (J2s) were dissected from the egg masses released by females. Excretory pores of females were located nearby median bulb (Fig. 2A). The dorsal arch of the perineal pattern (n = 10) of the female was elliptical, and the dorsal arch was relatively high with smooth to wavy lines (Fig. 2B). Morphometrics of females (n=10): body length (L) = 595.5 ± 24.0 (570.0-620.5) μm, body width (W)= 350.5 ± 30.0 (320.0-390.5) μm, stylet length = 13.6 ± 0.7 (12.1-15.4) μm (Fig. 2A), and the distance from dorsal esophageal gland orifice to base of stylet (DGO) = 3.5 ± 0.2 (2.8-4.0) μm (Fig. 2B). J2s (n = 20) had the following characteristics: L = 383.2 ± 12.5 (337-430) μm (Fig. 2C), a = 22.0 ± 1.1 (20.3-24.4) μm, c = 8.4 ± 0.5 (7.5-10.5) μm, stylet length = 12.4 ± 1.5 (10.1-14.6) μm, DGO = 2.9 ± 0.6 (2.0-3.6) μm (Fig. 2D), tail length = 39.5 ±3.4 (32.0-48.5) μm and hyaline tail terminus = 10.5 ± 0.5 (9.5-11.2) μm (Fig.1E). There were four lines on the lateral field of J2s (Fig. 2F). Females and J2s obtained from galls had uniform morphological and molecular characteristics were confirmed to be Meloidogyne incognita. Live J2s were detected in all soil samples with a mean of 120 ± 15 J2s/100 ml of soil. Five 4-week-old I. indigotica plantlets, grown in pots (500cm3) with sterilized soil were inoculated with 1000 J2s from egg masses per pot and5 non-inoculated pots were used as control. Plants were well maintained under 25 ± 3°C in the greenhouse. Three plants were gently removed from the pots 30 days after inoculation, and an average of 50 galls per root was observed on the roots, and the resulting nematode reproduction factors (RF = final egg density ÷ 1,000, initial egg density) of 3.2, suggested that I. indigotica is a good host for M. incognita (Mojtahedi, 1988). There were no significant differences in main measurements and morphological characteristics between the Taihe population of M. incognita and that represented in "CIH descriptions of plant-parasitic nematodes" (Orton Williams, 1973). DNA was extracted from 5 single J2s, and ITS and 18S rDNA gene was amplified using the primer pair 18S/26S and 18s1.2a/18sr2b (Bernard et al. 2010; Vrain et al. 1992). The sequence of 18S rDNA (MW875892) was submitted to GenBank. Comparisons showed a sequence identity of greater than 99.8% for Meloidogyne incognita (MF177719.1). The rDNA sequences of M. incognita, M. hapla, M. javanica and M. arenaria are so homologous that rDNA-based differentiation is difficult. The SCAR primers can successfully distinguish M. incognita, M. hapla, M. javanica and M. arenaria. Five species-specific primer sets (Finc/Rinc; MORF-F/MTHIS-R; Jmv-F/Jmv-R; Far/Rar and Fjav/Rjav, Stanton et al. 1997; Wishart et al. 2002; Zijlstra et al. 2000) were used to species-specifically distinguish within the genus. The results (＋, ＋, －, －, －) proved that the Taihe population belonging to M. incognita. To our knowledge, this is the first report of M. incognita parasitizing I. indigotica. This finding may be important to medicinal plant industry, since M. incognita is one of the most harmful nematode pests in the world and would cause severe damage to I. indigotica.

Effects of water and nitrogen coupling on the photosynthetic characteristics, yield, and quality of Isatis indigotica

Isatis indigotica is a commercial medicinal crop that is widely cultivated with high water and nutrient application, in the arid areas of northwest China. Rational irrigation and nitrogen application are key factors for successful crop management. The objective of this study was to determine the effect of water and nitrogen coupling on the photosynthetic characteristics, yield, and quality of Isatis indigotica produced in northwestern China. Field trials were conducted for 2 consecutive years at an irrigation test station. Data on photosynthetic parameters, yield, and quality were collected from individual Isatis indigotica for each treatment during 2018–2019. The application of nitrogen significantly increased photosynthetic rates and yield under the same irrigation conditions. However, the yields were reduced in the excess water treatments (W3N1 and W3N2) and in the excess nitrogen treatments (W1N3, W2N3, and W3N3) in contrast to the optimum W2N2 treatment. Moreover, the quality indicators of the W2N2 treatment decreased compared with CK, which was due to water stress and more photoassimilates being available to the roots, but the effective quality index value could be effectively improved by greatly increasing the yield.