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Plants produce specialized metabolites to protect themselves from biotic enemies. Members of the Solanaceae family accumulate phenylpropanoid-polyamine conjugates (PPCs) in response to attackers while also maintaining a chemical barrier of steroidal glycoalkaloids (SGAs). Across the plant kingdom, biosynthesis of such defense compounds is promoted by jasmonate signaling in which clade IIIe basic helix-loop-helix (bHLH) transcription factors play a central role. By characterizing hairy root mutants obtained through Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated protein 9 (CRISPR-Cas9) genome editing, we show that the tomato clade IIIe bHLH transcription factors, MYC1 and MYC2, redundantly control jasmonate-inducible PPC and SGA production, and are also essential for constitutive SGA biosynthesis. Double myc1 myc2 loss-of-function tomato hairy roots displayed suppressed constitutive expression of SGA biosynthesis genes, and severely reduced levels of the main tomato SGAs alpha-tomatine and dehydrotomatine. In contrast, basal expression of genes involved in PPC biosynthesis was not affected. CRISPR-Cas9(VQR) genome editing of a specific cis-regulatory element, targeted by MYC1/2, in the promoter of a SGA precursor biosynthesis gene led to decreased constitutive expression of this gene, but did not affect its jasmonate inducibility. Our results demonstrate that clade IIIe bHLH transcriptional regulators have evolved under the control of distinct regulatory cues to specifically steer constitutive and stress-inducible specialized metabolism.

Faba bean (Vicia faba L.) is a widely adapted and high-yielding legume cultivated for its protein-rich seeds(1). However, the seeds accumulate the pyrimidine glucosides vicine and convicine, which can cause haemolytic anaemia (favism) in 400 million genetically predisposed individuals(2). Here, we use gene-to-metabolite correlations, gene mapping and genetic complementation to identify VC1 as a key enzyme in vicine and convicine biosynthesis. We demonstrate that VC1 has GTP cyclohydrolase II activity and that the purine GTP is a precursor of both vicine and convicine. Finally, we show that cultivars with low vicine and convicine levels carry an inactivating insertion in the coding sequence of VC1. Our results reveal an unexpected, purine rather than pyrimidine, biosynthetic origin for vicine and convicine and pave the way for the development of faba bean cultivars that are free of these anti-nutrients.

The development of genetic transformation methods is critical for enabling the thorough characterization of an organism and is a key step in exploiting any species as a platform for synthetic biology and metabolic engineering approaches. In this work we describe the development of an Agrobacterium rhizogenes-mediated hairy root transformation protocol for the crop and medicinal legume fenugreek (Trigonella foenum-graecum). Fenugreek has a rich and diverse content in bioactive specialised metabolites, notably diosgenin, which is a common precursor for synthetic human hormone production. This makes fenugreek a prime target for identification and engineering of specific biosynthetic pathways for the production of triterpene and steroidal saponins, phenolics, and galactomanans. Through this transformation protocol, we identified a suitable promoter for robust transgene expression in fenugreek. Finally, we establish the proof of principle for the utility of the fenugreek system for metabolic engineering programs, by heterologous expression of known triterpene saponin biosynthesis regulators from the related legume Medicago truncatula in fenugreek hairy roots.

Anthriscus sylvestris (L.) Hoffm. (Apiaceae) is a common wild plant that accumulates the lignan deoxypodophyllotoxin. Deoxypodophyllotoxin can be hydroxylated at the C-7 position in recombinant organisms yielding podophyllotoxin, which is used as a semi-synthetic precursor for the anticancer drugs, etoposide phosphate and teniposide. As in vitro regeneration of A. sylvestris has not yet been reported, development of a regeneration protocol for A. sylvestris would be useful as a micropropagation tool and for metabolic engineering of the plant. Calli were induced from hypocotyl explants and transferred to shoot induction medium containing zeatin riboside. Regenerated shoots were obtained within 6 mo and were transferred onto growth regulator-free root induction medium containing 1% sucrose. Regenerated plants transferred to soil and acclimatized in a greenhouse. Plants were transferred to the field with a 100% survival rate. Regenerated plants flowered and were fully fertile. This is the first report of complete regeneration of A. sylvestris via shoot organogenesis from callus.

The Casuarinaceae family is a group of 96 species of trees and shrubs that are tolerant to adverse soil and climatic conditions. In the field, Casuarinaceae bears nitrogen-fixing root nodules (so called actinorhizal nodules) resulting from infection by the soil actinomycete Frankia. The association between Casuarina and Frankia is of tremendous ecological importance in tropical and subtropical areas where these trees contribute to land stabilization and soil reclamation. During differentiation of the actinorhizal nodule, a set of genes called actinorhizal nodulins is activated in the developing nodule. Understanding the molecular basis of actinorhizal nodule ontogenesis requires molecular tools such as genomics together with gene transfer technologies for functional analysis of symbiotic genes. Using the biological vectors Agrobacterium rhizogenes and A. tumefaciens, gene transfer into the two species Allocasuarina verticillata and Casuarina glauca has been successful. Transgenic Casuarinaceae plants proved to be valuable tools for exploring the molecular mechanisms resulting from the infection process of actinorhizal plants by Frankia.

Granule-bound starch synthase [GBSS; EC 24.1.21] determines the presence of amylose in reserve starches. Potato plants were transformed to produce antisense RNA from a gene construct containing a full-length granule-bound starch synthase cDNA in reverse orientation, fused between the cauliflower mosaic virus 35S promoter and the nopaline synthase terminator. The construct was integrated into the potato genome by Agrobacterium rhizogenes-mediated transformation. Inhibition of GBSS activity in potato tuber starch was found to vary from 70% to 100%. In those cases where total suppression of GBSS activity was found both GBSS protein and amylose were absent, giving rise to tubers containing amylose-free starch. The variable response of the transformed plants indicates that position effects on the integrated sequences might be important. The results clearly demonstrate that in tubers of potato plants which constitutively synthesize antisense RNA the starch composition is altered.

Large seeded grain legumes such as the common bean (Phaseolus vulgaris) and cowpea (Vigna unguiculata) are very important crops with seeds that are major protein source for people in developing countries, but their yields and improvement lag behind the economically more important cereals. For research purposes, genetic transformation is a powerful tool to obtain valuable information on gene expression and putative gene functions. In addition, through genetic transformation, candidate genes can be evaluated for their potential in agricultural biotechnology applications, such as resistance against biotic stresses. However, it remains difficult to stable genetically transform large seeded grain legumes such as Phaseolus and cowpea using Agrobacterium tumefaciens. In this paper a system is described to obtain so-called transgenic composite plants from P. vulgaris. These have a transgenic root system, obtained through Agrobacterium rhizogenes transformation of de-rooted seedlings. Their potentials for studies on important processes in the root system will be discussed.

Callus and hairy root cultures of Linum leonii were established. The genetic transformation in hairy roots was proven by PCR analysis, which showed integration of rol A and rol C genes into the plant genome. Calli and hairy roots accumulate the arylnaphthalene lignan justicidin B as a major constituent. Hairy roots produce 5-fold higher yields of justicidin B (10.8 mg g(-1) DW) compared to calli. Justicidin B shows strong cytotoxicity on the chronic myeloid leukemia LAMA-8 and K-562 cell lines and on the chronic lymphoid leukemia SKW-3 cell line with IC50 values of 1.11, 6.08, and 1.62 mu M, respectively. Apoptotic properties of justicidin B are reported for the first time.

Agrobacterium rhizogenes-mediated introduction of the wild-type allele of the gene encoding granule-bound starch synthase (GBSS) into the amylose-free starch mutant amf of potato leads to restoration of GBSS activity and amylose synthesis, which demonstrates that Amf is the structural gene for GBSS. Amylose was found in columella cells of root tips, in stomatal guard cells, tubers, and pollen, while in the control experiments using only vector DNA. these tissues remained amylose free. This confirms the fact that, in potato. GBSS is the only enzyme responsible for the presence of amylose. accumulating in all starch-containing tissues. Amylose-containing transformants showed no positive correlation between GBSS activity and amylose content, which confirms that the former is not the sole regulating factor in amylose metabolism.