Triterpenoids are a diverse group of secondary metabolites that are associated with a variety of biological activities. Oleanolic acid, ursolic acid and betulinic acid are common triterpenoids in plants with diverse biological activities, including antifungal, antibacterial, anti-human immunodeficiency virus (HIV) and/or antitumor activities. In the present study, using the gene co-expression analysis tool of Medicago truncatula, we found a strong correlation between CYP716A12 and β-amyrin synthase (bAS), which encodes the enzyme responsible for the initial cyclization of 2,3-oxidosqualene to β-amyrin (the basic structural backbone of most triterpenoid saponins). Through an in vitro assay, we identified CYP716A12 as a β-amyrin 28-oxidase able to modify β-amyrin to oleanolic acid (through erythrodiol and, possibly, oleanolic aldehyde). We also confirmed its activity in vivo, by expressing CYP716A12 in transgenic yeast that endogenously produce β-amyrin. In addition, CYP716A12 was evaluated for its potential α-amyrin- and lupeol-oxidizing activities. Interestingly, CYP716A12 was able to generate ursolic acid (through uvaol and, possibly, ursolic aldehyde) and betulinic acid (through betulin). Hence, CYP716A12 was characterized as a multifunctional enzyme with β-amyrin 28-oxidase, α-amyrin 28-oxidase and lupeol 28-oxidase activities. We also identified homologs of CYP716A12 in grape (CYP716A15 and CYP716A17) that are involved in triterpenoid biosynthesis, which indicates the highly conserved functionality of the CYP716A subfamily among plants. These findings will be useful in the heterologous production of pharmacologically and industrially important triterpenoids, including oleanolic acid, ursolic acid and betulinic acid.

Potatoes (Solanum tuberosum) contain a-solanine and a-chaconine, two well-known toxic steroidal glycoalkaloids (SGAs). Sprouts and green tubers accumulate especially high levels of SGAs. Although SGAs were proposed to be biosynthesized from cholesterol, the biosynthetic pathway for plant cholesterol is poorly understood. Here, we identify sterol side chain reductase 2 (SSR2) from potato as a key enzyme in the biosynthesis of cholesterol and related SGAs. Using in vitro enzyme activity assays, we determined that potato SSR2 (St SSR2) reduces desmosterol and cycloartenol to cholesterol and cycloartanol, respectively. These reduction steps are branch points in the biosynthetic pathways between C-24 alkylsterols and cholesterol in potato. Similar enzymatic results were also obtained from tomato SSR2. St SSR2-silenced potatoes or St SSR2-disrupted potato generated by targeted genome editing had significantly lower levels of cholesterol and SGAs without affecting plant growth. Our results suggest that St SSR2 is a promising target gene for breeding potatoes with low SGA levels.

ABSTRACT␤-Glucan elicitor (GE), released from the cell wall of the phytopathogenic fungus Phytophthora megasperma by soybean glucanases, causes defense reactions in soybean. A GE-binding protein (GEBP) was purified from the membrane fraction of soybean root cells, and its cDNA was isolated. Expression of the cDNA clone in tobacco suspension cultured cells and in Escherichia coli conferred GE-binding activity to both. An antibody against the recombinant protein was found to inhibit the GE binding with the soybean cotyledon membrane fraction as well as the resulting accumulation of phytoalexin. Immunolocalization assays indicated that the GEBPs are located in the plasma membrane of root cells. These results suggest that the cDNA encodes a GE receptor and may mediate the signaling of the elicitor.Plants defend themselves from infection by invasive phytopathogenic fungi by a combination of constitutive as well as induced defenses such as phytoalexin accumulation; the hypersensitive reaction; and the production of chitinase, glucanase, and polygalacturonase inhibitor (1). Certain defense reactions are elicited by compounds referred to as elicitors, such as oligosaccharides, proteins, and glycoproteins released from fungal and plant cell walls (reviewed in refs. 2-4).

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Solanine and a-chaconine, steroidal glycoalkaloids (SGAs) found in potato (Solanum tuberosum), are among the best-known secondary metabolites in food crops. At low concentrations in potato tubers, SGAs are distasteful; however, at high concentrations, SGAs are harmful to humans and animals. Here, we show that POTATO GLYCOALKALOID BIOSYNTHESIS1 (PGA1) and PGA2, two genes that encode cytochrome P450 monooxygenases (CYP72A208 and CYP72A188), are involved in the SGA biosynthetic pathway, respectively. The knockdown plants of either PGA1 or PGA2 contained very little SGA, yet vegetative growth and tuber production were not affected. Analyzing metabolites that accumulated in the plants and produced by in vitro enzyme assays revealed that PGA1 and PGA2 catalyzed the 26-and 22-hydroxylation steps, respectively, in the SGA biosynthetic pathway. The PGA-knockdown plants had two unique phenotypic characteristics: The plants were sterile and tubers of these knockdown plants did not sprout during storage. Functional analyses of PGA1 and PGA2 have provided clues for controlling both potato glycoalkaloid biosynthesis and tuber sprouting, two traits that can significantly impact potato breeding and the industry.

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CRISPR/Cas9 is a programmable nuclease composed of the Cas9 protein and a guide RNA (gRNA) molecule. To create a mutant potato, a powerful genome-editing system was required because potato has a tetraploid genome. The translational enhancer dMac3, consisting of a portion of the OsMac3 mRNA 5′-untranslated region, greatly enhanced the production of the protein encoded in the downstream ORF. To enrich the amount of Cas9, we applied the dMac3 translational enhancer to the Cas9 expression system with multiple gRNA genes. CRISPR/Cas9 systems targeting the potato granule-bound starch synthase I (GBSSI) gene examined the frequency of mutant alleles in transgenic potato plants. The efficiency of the targeted mutagenesis strongly increased when the dMac3-installed Cas9 was used. In this case, the ratio of transformants containing four mutant alleles reached approximately 25% when estimated by CAPS analysis. The mutants that exhibited targeted mutagenesis in the GBSSI gene showed characteristics of low amylose starch in their tubers. This result suggests that our system may facilitate genome-editing events in polyploid plants.

The yeast vacuolar proton-translocating ATPase is a member of the third class of H(+)-pumping ATPase. A family of this type of H(+)-ATPase is now known to be ubiquitously distributed in eukaryotic vacuo-lysosomal organelles and archaebacteria. Nine VMA genes that are indispensable for expression of the enzyme activity have been cloned and characterized in the yeast Saccharomyces cerevisiae. This review summarizes currently available information on the VMA genes and cell biological functions of the VMA gene products.