Optimized genetic transformation of Zanthoxylum zanthoxyloides by Agrobacterium rhizogenes and the production of chelerythrine and skimmiamine in hairy root cultures

Zanthoxylum zanthoxyloides is an endangered African tree producing numerous bioactive substances including antileukemic and antisickling agents. Here, the potential of Z. zanthoxyloides hairy root cultures was tested for the production of bioactive substances with limited natural resources. The efficiency of Agrobacterium rhizogenes LBA9402‐mediated transformation of leaf material was evaluated using different techniques. An optimal transformation frequency of 77% was obtained after 11 days by inoculating A. rhizogenes directly onto the central vein of 14‐week‐old leaves followed by a co‐cultivation period of 3 days. Different treatments in immersion mode (manual wounding, acetosyringone, CaCl₂, ultrasonication) never exceeded these results. A maximum growth rate of 0.37 cm/day was determined during the exponential phase. Liquid chromatography‐diode array detection analysis showed the presence of skimmiamine, sesamine, chelerythrine, and chelerythrine derivatives in Z. zanthoxyloides hairy root lines. The maximum production of skimmiamine and chelerythrine in 28‐day‐old hairy root cultures was 45 ± 2 and 107 ± 4 mg/100 g dry weight, respectively. The present results highlight the potential of Z. zanthoxyloides hairy root cultures for the sustainable production of skimmiamine and chelerythrine.     

Dendrobium tamenglongense sp. nov. (Orchidaceae) from Manipur,India

Dendrobium tamenglongense R. Kishor, Y. N. Devi, H. B. Sharma, J. Tongbram & S. P. Vij sp. nov. from Manipur, India is described and illustrated as a new species. The new species differs from D. jaintianum in having larger and glabrous leaves with acute apices, green sepal apices, a 1–3‐flowered inflorescence, smaller and non‐fragrant flowers and a 3‐lobed labellum with fimbriate midlobe.     

Lasiolactols A and B Produced by the Grapevine Fungal Pathogen Lasiodiplodia mediterranea

A strain of Lasiodiplodia mediterranea, a fungus associated with grapevine decline in Sicily, produced several metabolites in liquid medium. Two new dimeric γ‐lactols, lasiolactols A and B ( 1 and 2 ), were characterized as (2S*,3S*,4R*,5R*,2′S*,3′S*,4′R*,5′R*)‐ and (2R*,3S*,4R*,5R*,2′R*,3′S*,4′R*,5′R*)‐(5‐(4‐hydroxymethyl‐3,5‐dimethyl‐tetrahydro‐furan‐2‐yloxy)‐2,4‐dimethyl‐tetrahydro‐furan‐3‐yl]‐methanols by IR, 1D‐ and 2D‐NMR, and HR‐ESI‐MS. Other four metabolites were identified as botryosphaeriodiplodin, (5R)‐5‐hydroxylasiodiplodin, (–)‐(1R,2R)‐jasmonic acid, and (–)‐(3S,4R,5R)‐4‐hydroxymethyl‐3,5‐dimethyldihydro‐2‐furanone ( 3  –  6 , resp.). The absolute configuration (R) at hydroxylated secondary C‐atom C(7) was also established for compound 3 . The compounds 1  –  3 , 5, and 6 , tested for their phytotoxic activities to grapevine cv. Inzolia leaves at different concentrations (0.125, 0.25, 0.5, and 1 mg/ml) were phytotoxic and compound 5 showed the highest toxicity. All metabolites did not show in vitro antifungal activity against four plant pathogens.     

Multiplex CRISPR/Cas9‐mediated metabolic engineering increases soya bean isoflavone content and resistance to soya bean mosaic virus

Isoflavonoids, which include a variety of secondary metabolites, are derived from the phenylpropanoid pathway and are distributed predominantly in leguminous plants. These compounds play a critical role in plant–environment interactions and are beneficial to human health. Isoflavone synthase (IFS) is a key enzyme in isoflavonoid synthesis and shares a common substrate with flavanone‐3‐hydroxylase (F3H) and flavone synthase II (FNS II). In this study, CRISPR/Cas9‐mediated multiplex gene‐editing technology was employed to simultaneously target GmF3H1, GmF3H2 and GmFNSII‐1 in soya bean hairy roots and plants. Various mutation types and frequencies were observed in hairy roots. Higher mutation efficiencies were found in the T₀ transgenic plants, with a triple gene mutation efficiency of 44.44%, and these results of targeted mutagenesis were stably inherited in the progeny. Metabolomic analysis of T₀ triple‐mutants leaves revealed significant improvement in isoflavone content. Compared with the wild type, the T₃ generation homozygous triple mutants had approximately twice the leaf isoflavone content, and the soya bean mosaic virus (SMV) coat protein content was significantly reduced by one‐third after infection with strain SC7, suggesting that increased isoflavone content enhanced the leaf resistance to SMV. The isoflavone content in the seeds of T₂ triple mutants was also significantly increased. This study provides not only materials for the improvement of soya bean isoflavone content and resistance to SMV but also a simple system to generate multiplex mutations in soya bean, which may be beneficial for further breeding and metabolic engineering.     

Resistance to Phytophthora pathogens is dependent on gene silencing pathways in plants

RNA silencing is one of the main defence mechanisms employed by plants to fight pathogens. p19 protein encoded by the tomato bushy stunt virus (TBSVp19) is known as a suppressor of RNA silencing via siRNA sequestration to prevent the assembly of RISC. To better understand the impact of TBSVp19 on silencing and its roles in Phytophthora pathogens, we used the transient expression assay in Nicotiana benthamiana and found that the leaves expressing TBSVp19 were more susceptible to Phytophthora parasitica. Furthermore, we demonstrated that TBSVp19‐mediated plant susceptibility in N. benthamiana is dependent on RNA‐dependent RNA polymerase 6 (RDR6). We also tested the role of RNA silencing in resistance of soybean hairy roots to Phytophthora. The lesion size induced by P. sojae on TBSVp19‐expressing soybean hairy roots was slightly, but significantly larger than GFP‐expressing soybean hairy roots. Finally, the Arabidopsis gene silencing mutants ago1‐27, zip‐1, sgs3‐11 and rdr6‐11 were also examined for their resistance to P. parasitica. The results clearly showed that resistance levels of the mutants were visibly reduced compared with the wild type. Taken together, these results suggest that the gene silencing system in plants is essential for resistance to Phytophthora pathogens.     

Isolation of the major chiral compounds from Bubonium graveolens essential oil by HPLC and absolute configuration determination by VCD

The chirality issues in the essential oils (EOs) of leaves and flowers from Bubonium graveolens were addressed by chiral high‐performance liquid chromatography (HPLC) with polarimetric detection and vibrational circular dichroism (VCD). The chemical compositions of the crude oils of three samples were established by gas chromatography / mass spectrometry (GC/MS). The well‐known cis ‐chrysanthenyl acetate ( 1 ), oxocyclonerolidol ( 2 ), and the recently disclosed cis ‐acetyloxychrysanthenyl acetate ( 3 ), the three major chiral compounds, were isolated by preparative HPLC. The naturally occurring oxocycloneroledol ( 2 ), mostly found in the leaf oil (49.4–55.6%), presents a (+) sign in the mobile phase during HPLC on a chiral stationary phase (CSP) with a Jasco polarimetric detection. The naturally occurring cis ‐chrysanthenyl acetate ( 1 ) and cis ‐acetyloxychrysanthenyl acetate ( 3 ), mostly found in the flower EO (35.9–74.9% and 10.0–34.3%, respectively), both present a (−) sign. HPLC on a CSP with polarimetric detection is an unprecedented approach to readily differentiate the flower and leaf EOs according to their chiral signature. The comparison of the experimental and calculated VCD spectra of pure isolated 1 , 2, and 3 provided their absolute configuration as being (1S ,5R ,6S )‐(−)‐2,7,7‐trimethylbicyclo[3.1.1]hept‐2‐en‐6‐yl acetate 1 , (2R ,6R )‐(+)‐6‐ethenyl‐2,6‐dimethyl‐2‐(4‐methylpent‐3‐en‐1‐yl)dihydro‐2H‐pyran‐3(4H)‐one) 2 and (1S ,5R ,6R ,7S )‐(−)‐7‐(acetyloxy)‐2,6‐dimethylbicyclo[3.1.1]hept‐2‐en‐6‐yl]methyl acetate 3 . Compounds 1 , 2, and 3 were already known in B. graveolens but this is the first report of the absolute configuration of (+)‐ 2 and (−)‐ 3 . The VCD chiral signatures of the crude oils were also recorded.     

840. ROSA HIRTULA

Rosa hirtula (Regel) Nakai, a species endemic to Japan, is illustrated. It occurs in central Honshū around Mt. Fuji and Hakone and vicinity. Rosa hirtula is similar to R. roxburghii Tratt., particularly to the single‐flowered form, f. normalis Rehder & E. H. Wils., and is distinguished by its tree habit, hairy rachis, and lanceolate to narrowly oblong leaflets with acuminate to acuminate‐acute apex, serrulate margins, and hairy midvein on the lower surface.     

Two phenological variants of Terminalia alata coexist in a dry dipterocarp forest

Two morphological variants of Terminalia alata (Combretaceae) differed in leaf flushing phenology and spatial distribution in a Cambodian deciduous forest. The hairy‐type trees displayed leaf exchange behavior in the middle of the dry season. The glabrous type flushed new leaves 3 months after the wet season started. The leafless period of the hairy type was estimated to be <1 month, whereas that of the glabrous type lasted more than 5 months. The landscape‐scale leaf exchange behavior was similar to that of the hairy type. The two types showed clear spatial separation. The hairy type was limited to flat areas with deep soils. The dominance of the glabrous type in hilly areas with shallow soils suggests that it is adapted to water‐limited environments. The abundance of the glabrous type in hilly areas and its unique leaf phenology probably influence the carbon, energy and water balance at the landscape level.     

Abelmoschus enbeepeegearense sp. nov. (Malvaceae), an endemic species of okra from Western Ghats,India

Abelmoschus enbeepeegearense J. John et al. is a new species occurring at low elevations in the Western Ghats of India, comprising Kerala, Karnataka and Tamil Nadu. The taxon is morphologically allied to A. moschatus subsp. moschatus, A. moschatus subsp. tuberosus and A. crinitus, but easily distinguishable by virtue of its orthotropic branching, 3–5‐angled leaves, glandular hairy plant body with whitish waxy secretions, glandular non‐setose epiclayx segmens which is more than eight in number and ovate hirsute fruits with a short mucro at the apex. It can be crossed with all three taxa with varying degree of success, but the hybrids are sterile. The material belonging to it was earlier identified as and placed under A. moschatus Medik. The taxon is described and illustrated with notes on its phenology, ecology and distribution. In additions, a key to all Abelmoschus taxa occurring in India is provided.     

Flavonoid content and antioxidant activity of Artemisia vulgaris L. “hairy” roots

We investigated the effect of Agrobacterium rhizogenes-mediated transformation on antioxidant activity of Artemisia vulgaris “hairy” roots. It appeared that transformation may increase flavonoid content as well as DPPH-scavenging activity and ability to reduce Fe³⁺ as compared to the non-transformed plants. Some “hairy” roots accumulated flavonoids up to 73.1?±?10.6?mg RE/g DW (while the amount of flavonoids in the leaves of non-transformed plants was up to 49.4?±?5.0?mg RE/g DW). DPPH-scavenging activity of some “hairy” root lines was 3–3.8 times higher than such one of the roots of the control plants. The Fe³⁺-reducing power of most transgenic root extracts exceeded such power of the extracts of the roots of the control plants. The decrease in SOD activity was found in the most “hairy” root lines compared to the control roots. The increase of flavonoid content correlated with the increase of ability of extracts to scavenge DPPH*- radical and Fe³⁺ - reducing power. No correlation between SOD activity of extracts and concentration of flavonoids was found (p?≥?0.2).Thus, transformation has led to the alteration in flavonoid accumulation and antioxidant activity in A. vulgaris “hairy” roots. Transgenic roots with high-antioxidant properties can be selected after A. rhizogenes-mediated transformation.     

银杏毛状根褐化机制的初步研究

该实验依据现有发根农杆菌侵染法,以银杏无菌苗叶片为外植体,对农杆菌诱导银杏毛状根的实验操作进行优化,并观察记录银杏毛状根继代生长情况,测定继代不同天数的毛状根组织内多酚含量、多酚合成相关酶基因和POD基因相对表达量,以探讨银杏毛状根褐化的生理机制。结果显示:(1)银杏农杆菌侵染最适预培养时间为3d、菌种为R1601、共培养基为White、侵染时间为30min,优化侵染操作后的银杏毛状根诱导率可达72.36%;银杏毛状根继代生长的最适IBA浓度为0.5mg/L。(2)银杏毛状根初始发根时颜色为乳白色,多在伤口处簇生,伸长生长时无向地性,不分叶正背面都能在伤口处诱导出根,且在除菌培养20d时毛状根长可达0.4～0.8cm;继代培养25d时毛状根为白色,继代35d时部分毛状根从根部至中部开始转黄,继代45d时大部分毛状根由白转黄,根部至中部开始有褐化出现。(3)继代35d时毛状根的PAL、C4 H、4CL和POD基因表达量分别为25d的68%、44%、51%、52%;继代45d时PAL、C4 H、4CL、POD基因表达量分别为35d的19%、75%、86%、105%。(4)继代35d、45d的银杏毛状根总酚类含量分别为17.14和13.78mg/g。研究发现,PAL基因表达量变化与其下游的两个关键酶基因(C4 H、4CL)表达量变化不一致,且PAL基因表达量的大幅降低并未对下游C4 H、4CL基因表达有明显影响,说明PAL基因表达量变化对其下游合成速率影响不大,可能不是银杏毛状根苯丙氨酸代谢途径的限速酶;而C4 H与4CL基因表达量变化趋势高度一致,说明C4H应是该途径中的限速酶之一;推测继代前期可能是由PPO作为关键酶参与毛状根褐化反应,导致后期毛状根生长受抑制,POD基因表达水平随之上升,POD参与褐化反应。     

Crystal structure of the effector AvrLm4–7 of Leptosphaeria maculans reveals insights into its translocation into plant cells and recognition by resistance proteins

The avirulence gene AvrLm4–7 of Leptosphaeria maculans, the causal agent of stem canker in Brassica napus (oilseed rape), confers a dual specificity of recognition by two resistance genes (Rlm4 and Rlm7) and is strongly involved in fungal fitness. In order to elucidate the biological function of AvrLm4–7 and understand the specificity of recognition by Rlm4 and Rlm7, the AvrLm4–7 protein was produced in Pichia pastoris and its crystal structure was determined. It revealed the presence of four disulfide bridges, but no close structural analogs could be identified. A short stretch of amino acids in the C terminus of the protein, (R/N)(Y/F)(R/S)E(F/W), was well‐conserved among AvrLm4–7 homologs. Loss of recognition of AvrLm4–7 by Rlm4 is caused by the mutation of a single glycine to an arginine residue located in a loop of the protein. Loss of recognition by Rlm7 is governed by more complex mutational patterns, including gene loss or drastic modifications of the protein structure. Three point mutations altered residues in the well‐conserved C–terminal motif or close to the glycine involved in Rlm4‐mediated recognition, resulting in the loss of Rlm7‐mediated recognition. Transient expression in Nicotiana benthamiana (tobacco) and particle bombardment experiments on leaves from oilseed rape suggested that AvrLm4–7 interacts with its cognate R proteins inside the plant cell, and can be translocated into plant cells in the absence of the pathogen. Translocation of AvrLm4–7 into oilseed rape leaves is likely to require the (R/N)(Y/F)(R/S)E(F/W) motif as well as an RAWG motif located in a nearby loop that together form a positively charged region.     

Memecylon trunciflorum sp. nov. (Melastomataceae–Olisbeoideae) from the Udzungwa Mountains,southern Tanzania

Described and illustrated is Memecylon trunciflorum R. D. Stone, an evidently localized endemic of the Udzungwa Mountains in southern Tanzania. The new species was previously confused with the vegetatively similar but distantly related M. erythranthum Gilg and M. semseii A. Fern. & R. Fern., from which it is distinguished by its anther connectives bearing a dorsal oil‐gland and by its ellipsoid to obovoid fruits (vs anther connective gland absent and fruits globose in M. erythranthum and M. semseii). The new species is placed in M. sect. Magnifoliata R. D. Stone together with M. magnifoliatum A. Fern. & R. Fern., from which it differs by its smaller leaves mostly 9.5–15.0 × 3.5–6.0 cm (vs 18–35 × 8–13 cm), transverse veins 8–18 pairs (vs 25–28 pairs), short‐pedunculate inflorescences with secondary axes well developed (vs peduncles and secondary axes absent), white flowers (vs bluish purple), and smaller fruits mostly 11.5–14.5 × 9–11 mm on longer fruiting pedicels 8.0–13.5 mm (vs fruits 17–20 × 12–14 mm on pedicels 5.0–7.5 mm). Despite its local endemism, Memecylon trunciflorum has been assessed as ‘Least Concern’ according to IUCN criteria, although this assessment is dependent on the continued safeguarding of the Udzungwa Mountains National Park.     

Sonication-assisted Agrobacterium rhizogenes-mediated transformation of Verbascum xanthophoeniceum Griseb. for bioactive metabolite accumulation

An efficient protocol for the establishment of transformed root culture of Verbascum xanthophoeniceum using sonication-assisted Agrobacterium rhizogenes-mediated transformation is reported. Only 10 days after the inoculation with A. rhizogenes ATCC 15834 and 45 s ultrasound exposure, hairy roots appeared on 75% of the Verbascum leaves. Ten hairy root lines were isolated, although only half of them were free of bacterial contamination and started growing when excised from mother explants. The transgenic nature of the most vigorously growing hairy root clones (VX1 and VX6) was confirmed by polymerase chain reaction. Under submerged cultivation both hairy root clones accumulated high biomass amounts (12.8 and 14.3 g L⁻¹, respectively) and significant amounts of bioactive phenylethanoid glycoside verbascoside (over 6-times more than in mother plant leaves). LC-APCI-MS analyses confirmed verbascoside accumulation in hairy root clones along with three other phenylethanoid glycosides (forsythoside B, leucosceptoside B and martynoside) and an iridoid glycoside aucubin. This is the first report on the induction of hairy roots of Verbascum plants.     

Production of transgenic Aralia elata regenerated from Agrobacterium rhizogenes-mediated transformed roots

Transgenic hairy roots were induced from petiole and root segments of in vitro plant Aralia elata, a medicinal woody shrub, after co-cultivation with A. rhizogenes ATCC 15834. The percentage of putative hairy root induction from root segments was higher (26.7%) than petiole explants (10.0%). Hairy roots showed active production of lateral roots with vigorous elongation. Transgenic plants were regenerated from hairy roots via somatic embryogenesis. These plants had wrinkled leaves, short petioles and numerous lateral hairy roots. The RT-PCR analysis showed the expression of rol A, B, C, D, aux 1 and 2 genes differed between the transgenic lines. Endogenous IAA level was higher in transgenic than non-transgenic plants. Conclusively, transgenic hairy roots were developed for first time in A. elata and the transgenic hairy root lines showed distinct morphological growth pattern and gene expression.     

Rubia austrozhejiangensis sp. nov. (Rubiaceae) from Zhejiang,China

Rubia austrozhejiangensis Z. P. Lei, Y. Y. Zhou & R. W. Wang, a new species of Rubiaceae from China, is described and illustrated. The new species is similar to R. ovatifolia Z. Ying Zhang and R. argyi (H. Lév. & Vaniot) H. Hara ex Lauener, but differs from the former in having stems and branches cylindrical, not quadrate‐angled, long‐ovate to ovate‐lanceolate leaf blades, many‐flowered inflorescence, and smaller mericarps, 3–4 mm in diameter. In R. ovatifolia, stems and branches are quadrate‐angled, leaf blades ovate, ovate‐cordate to rounded cordate, and the inflorescences are sparsely flowered. Compared to R. argyi, the new species has cylindrical, not quadrate‐angled stems and branches, leaf blades that are long‐ovate to ovate‐lanceolate, 3–5‐veined, and slightly reflexed corolla lobes. In R. argyi, stems and branches are quadrate‐angled or winged, the corolla lobes are spreading, and the mericarps are 5–7 mm in diameter.     

Rhododendron subroseum sp. nov. and R. denudatum var. glabriovarium var. nov. (Ericaceae) from the Guizhou Province,China

One new species, Rhododendron subroseum Xiang Chen & Jiayong Huang, and one new variety, R. denudatum var. glabriovarium Xiang Chen & Xun Chen (Ericaceae), from the Guizhou Province, China are described and illustrated. Rhododendron subroseum is most similar to R. morii from which it differs mainly by having glandular branchlets, a subcordate leaf blade base and a mucronate leaf blade apex, a less floriferous inflorescence, a campanulate to broadly‐campanulate corolla, and a glandular‐hairy style. Rhododendron denudatum var. glabriovarium differs from R. denudatum var. denudatum mainly by having a glabrous ovary. Both of the new taxa are quite rare and therefore their protection is highly desired.