Transcriptome analysis reveals important regulatory factors for condensed tannins synthesis in acorn.

Condensed tannins are widely present in the fruits and seeds of plants and effectively prevent them from being eaten by animals before maturity due to their astringent taste. In addition, condensed tannins are a natural compound with strong antioxidant properties and significant antibacterial effects. Four samples of mature and near-mature Quercus fabri acorns, with the highest and lowest condensed tannin content, were used for genome-based transcriptome sequencing. The KEGG enrichment analysis revealed that the differentially expressed genes (DEGs) were highly enriched in phenylpropanoid biosynthesis and starch and sucrose metabolism. Given that the phenylpropanoid biosynthesis pathway is a crucial step in the synthesis of condensed tannins, we screened for significantly differentially expressed transcription factors and structural genes from the transcriptome data of this pathway and found that the expression levels of four MADS-box, PAL, and 4CL genes were significantly increased in acorns with high condensed tannin content. The quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) experiment further validated this result. In addition, yeast one-hybrid assay confirmed that three MADS-box transcription factors could bind the promoter of the 4CL gene, thereby regulating gene expression levels. This study utilized transcriptome sequencing to discover new important regulatory factors that can regulate the synthesis of acorn condensed tannins, providing new evidence for MADS-box transcription factors to regulate the synthesis of secondary metabolites in fruits.

AP3 promotes the synthesis of condensed tannin in fruit by positively regulating ANR expression.

Condensed tannins are often found in fruits and nuts and have an astringent flavor. The synthesis pathway of condensed tannins is already clear, but few related regulatory factors have been explored. Previous studies about MADS-box transcription factors have mainly focused on the regulation of floral organ development. Recent studies have shown that MADS-box are also involved in fruit development, maturation, and quality. The fruit of Quercus fabri is rich in starch and nutrients in its kernel but is difficult to eat directly because of its high condensed tannin content. This study identified and functionally characterized the MADS-box transcription factor QfAP3 in Q. fabri. Functional analysis based on overexpression in Micro-Tom showed that QfAP3 promoted condensed tannin synthesis. By analyzing the expression trend of key genes in the condensed tannin synthesis pathway in Micro-Tom plants, we found that the expression trend of ANR was consistent with that of QfAP3, and QfAP3 could bind to the promoter of ANR and positively regulate it. This study has discovered new functions of MADS-box transcription factors in fruit quality formation, developed new regulatory factors for the synthesis pathway of condensed tannin, and provided a biotechnological method that can effectively reduce astringency in fruit.

LcERF19, an AP2/ERF transcription factor from Litsea cubeba, positively regulates geranial and neral biosynthesis.

The APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factors (TFs) are involved in the regulation of specialized terpenoid biosynthesis. However, the AP2/ERF TFs in Litsea cubeba have not been characterized and their role in the biosynthesis of terpenoids is unknown. Here, 174 LcAP2/ERF TFs were identified in L. cubeba and categorized into four subfamilies: 27 AP2, 7 RAV, 1 Soloist, and 139 ERF. Transcriptomic and qRT-PCR assays both showed that the expression levels of LcERF19 were similar to that of terpene synthase LcTPS42 in the pericarp, which is related to the synthesis of geranial and neral in L. cubeba. LcERF19 was further shown to encode a nuclear-localized protein and its expression was strongly induced by jasmonate. Yeast one-hybrid and dual-luciferase assays showed that LcERF19 associated with GCC box elements of the LcTPS42 promoter and promoted its activity. Transient overexpression of LcERF19 in L. cubeba and overexpression of LcERF19 in tomato resulted in a significant increase in geranial and neral. Our findings show that LcERF19 enhances geranial and neral biosynthesis through activation of LcTPS42 expression, which provides a strategy to improve the flavor of tomato and other fruits.

Effects of Drought Stress and Rehydration on Physiological and Biochemical Properties of Four Oak Species in China.

Quercus fabri Hance, Quercus serrata Thunb, Quercus acutissima Carruth, and Quercus variabilis BL are four Chinese oak species commonly used for forestation. To ensure the survival of seedlings, we first need to understand the differences in drought resistance of the four oak species at the seedling stage, and comprehensively evaluate their drought resistance capabilities. The four oak seedlings were divided into drought-rewatering treatment group and well watered samples (control group). For the seedlings of the drought-rewatering treatment group, drought stress lasting 31 days was used, and then re-watering for 5 days. The water parameters, osmotic solutes content, antioxidant enzyme activity and photosynthesis parameters of the seedlings in the two groups were measured every 5 days. Compared with the control group, the relative water content, water potential, net photosynthetic rate, transpiration rate, and stomatal conductance levels of the four oaks all showed a downward trend under continuous drought stress, and showed an upward trend after rehydration. The soluble protein, soluble sugar, proline, peroxidase, superoxide dismutase and catalase content of the four oaks increased first and then decreased under drought stress, and then increased after rehydration. The content of glycine betaine and malondialdehyde continued to increase, and gradually decreased after rehydration. The weight of each index was calculated by principal component analysis, and then the comprehensive evaluation of each index was carried out through the membership function method. The drought resistance levels of the four oak species were as follows: Q. serrata > Q. fabri > Q. variabilis > Q. acutissima.

Comprehensive identification of bHLH transcription factors in Litsea cubeba reveals candidate gene involved in the monoterpene biosynthesis pathway.

Litsea cubeba (Lour.) Person, an economically important aromatic plant producing essential oils, has lemon-like fragrance and 96.44-98.44% monoterpene contents. bHLH transcription factor plays an important role in plant secondary metabolism and terpene biosynthesis. In this study, we used bioinformatics to identify bHLH transcription factors in L. cubeba, 173 bHLH genes were identified from L. cubeba and divided these into 26 subfamilies based on phylogenetic analysis. The majority of bHLHs in each subfamily shared comparable structures and motifs. While LcbHLHs were unevenly distributed across 12 chromosomes, 10 tandem repeats were discovered. Expression profiles of bHLH genes in different tissues demonstrated that LcbHLH78 is a potential candidate gene for regulating monoterpene biosynthesis. LcbHLH78 and the terpene synthase LcTPS42 showed comparable expression patterns in various tissues and fruit development stages of L. cubeba. Subcellular localization analysis revealed that LcbHLH78 protein localizes to the nucleus, consistent with a transcription factor function. Importantly, transient overexpression of LcbHLH78 increased geraniol and linalol contents. Our research demonstrates that LcbHLH78 enhances terpenoid biosynthesis. This finding will be beneficial for improving the quality of L. cubeba and provides helpful insights for further research into the control mechanism of LcbHLH genes over terpenoid biosynthesis.

Genetic diversity and population structure of Quercus fabri Hance in China revealed by genotyping-by-sequencing.

Analysis of genetic diversity and population structure among Quercus fabri populations is essential for the conservation and utilization of Q. fabri resources. Here, the genetic diversity and structure of 158 individuals from 13 natural populations of Quercus fabri in China were analyzed using genotyping-by-sequencing (GBS). A total of 459,564 high-quality single nucleotide polymorphisms (SNPs) were obtained after filtration for subsequent analysis. Genetic structure analysis revealed that these individuals can be clustered into two groups and the structure can be explained mainly by the geographic barrier, showed gene introgression from coastal to inland areas and high mountains could significantly hinder the mutual introgression of genes. Genetic diversity analysis indicated that the individual differences within groups are greater than the differences between the two groups. These results will help us better understand the genetic backgrounds of Q. fabri.