A dual-function transcription factor, SlJAF13, promotes anthocyanin biosynthesis in tomato.

Unlike modern tomato (Solanum lycopersicum) cultivars, cv. LA1996 harbors the dominant Aft allele, which is associated with anthocyanin synthesis in tomato fruit peel. However, the control of Aft anthocyanin biosynthesis remains unclear. Here, we used ethyl methanesulfonate-induced and CRISPR/Cas9-mediated mutation of LA1996 to show, respectively, that two class IIIf basic helix-loop-helix (bHLH) transcription factors, SlJAF13 and SlAN1, are involved in the control of anthocyanin synthesis. These transcription factors are key components of the MYB-bHLH-WD40 (MBW) complex, which positively regulates anthocyanin synthesis. Molecular and genetic analyses showed that SlJAF13 functions as an upstream activation factor of SlAN1 by binding directly to the G-Box motif of its promoter region. On the other hand, SlJAZ2, a JA signaling repressor, interferes with formation of the MBW complex to suppress anthocyanin synthesis by directly binding these two bHLH components. Unexpectedly, the transcript level of SlJAZ2 was in turn repressed in a SlJAF13-dependent manner. Mechanistically, SlJAF13 interacts with SlMYC2, inhibiting SlMYC2 activation of SlJAZ2 transcription, thus constituting a negative feedback loop governing anthocyanin accumulation. Taken together, our findings support a sophisticated regulatory network, in which SlJAF13 acts as an upstream dual-function regulator that fine tunes anthocyanin biosynthesis in tomato.

BrLETM2 Protein Modulates Anthocyanin Accumulation by Promoting ROS Production in Turnip (Brassica rapa subsp. rapa).

In 'Tsuda' turnip, the swollen root peel accumulates anthocyanin pigments in a light-dependent manner, but the mechanism is unclear. Here, mutant g120w which accumulated extremely low levels of anthocyanin after light exposure was identified. Segregation analysis showed that the anthocyanin-deficient phenotype was controlled by a single recessive gene. By using bulked-segregant analysis sequencing and CAPS marker-based genetic mapping analyses, a 21.6-kb region on chromosome A07 was mapped, in which a calcium-binding EF hand family protein named BrLETM2 was identified as the causal gene. RNA sequencing analysis showed that differentially expressed genes (DEGs) between wild type and g120w in light-exposed swollen root peels were enriched in anthocyanin biosynthetic process and reactive oxygen species (ROS) biosynthetic process GO term. Furthermore, nitroblue tetrazolium (NBT) staining showed that the ROS level decreased in g120w mutant. Anthocyanins induced by UV-A were abolished by the pre-treatment of seedlings with DPI (an inhibitor of nicotinamide adenine nucleoside phosphorylase (NADPH) oxidase) and decreased in g120w mutant. These results indicate that BrLETM2 modulates ROS signaling to promote anthocyanin accumulation in turnip under UV-A and provides new insight into the mechanism of how ROS and light regulate anthocyanin production.

A single amino acid substitution in the R2R3 conserved domain of the BrPAP1a transcription factor impairs anthocyanin production in turnip (Brassica rapa subsp. rapa).

The purple pigmentation in the epidermis of swollen roots of 'Tsuda' turnip (Brassica rapa subsp. rapa) is induced by light, providing a good system to investigate the genetic mechanism of light-dependent anthocyanin biosynthesis in B. rapa. Here, we identified the R2R3 MYB transcription factor gene PRODUCTION OF ANTHOCYANIN PIGMENT1 (BrPAP1a) as the critical gene in the anthocyanin-defective mutant w68. A nucleotide mutation in the turn region of the R3 domain was screened, which caused an amino acid substitution from glycine to serine (G94S). Functional analysis showed that the interaction of BrPAP1a with two bHLH factors ENHANCER OF GLABRA 3 (BrEGL3) and TRANSPARENT TESTA 8 (BrTT8) were impaired by the mutation. Expression of BrTT8 was activated by BrPAP1a and enhanced by MYB-bHLH-WDR (MBW) complexes, but blocked by the mutation. Furthermore, BrPAP1a directly bound the MYB-recognizing element (MRE) in the BrTT8 promoter, while the G94S substitution caused a loss of DNA-binding activity. Our findings indicate that G94 is required for protein interaction with BrTT8 and BrEGL3 and DNA-binding of BrPAP1a to activate BrTT8 expression, which leads to anthocyanin biosynthesis. Collectively, our data indicate the importance of the highly conserved amino acids within R2R3 MYB proteins in regulating anthocyanin biosynthesis and could aid programs to increase anthocyanins in turnip roots.

Highly efficient production of diverse rare ginsenosides using combinatorial biotechnology.

The rare ginsenosides are recognized as the functionalized molecules after the oral administration of Panax ginseng and its products. The sources of rare ginsenosides are extremely limited because of low ginsenoside contents in wild plants, hindering their application in functional foods and drugs. We developed an effective combinatorial biotechnology approach including tissue culture, immobilization, and hydrolyzation methods. Rh2 and nine other rare ginsenosides were produced by methyl jasmonate-induced culture of adventitious roots in a 10 L bioreactor associated with enzymatic hydrolysis using six ß-glycosidases and their combination with yields ranging from 5.54 to 32.66 mg L-1 . The yield of Rh2 was furthermore increased by 7% by using immobilized BglPm and Bgp1 in optimized pH and temperature conditions, with the highest yield reaching 51.17 mg L-1 (17.06% of protopanaxadiol-type ginsenosides mixture). Our combinatorial biotechnology method provides a highly efficient approach to acquiring diverse rare ginsenosides, replacing direct extraction from Panax plants, and can also be used to supplement yeast cell factories.