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Abstract WRKY transcription factors are pivotal regulators in various aspects of plant biology, including growth, development, secondary metabolic biosynthesis, and responses to both biotic and abiotic stresses. The legume plant Grona styracifolia is widely utilized for its medicinal properties in treating urinary calculi and combating SARS-CoV-2, owing to its bioactive component schaftoside. However, the regulatory function of GsWRKYs in schaftoside biosynthesis within G. styracifolia remains elusive. In the G. styracifolia genome, we identified a total of 102 GsWRKYs, classified phylogenetically into Group I (18), II (68), and III (16). Genomic analysis revealed an uneven distribution of GsWRKYs on chromosomes (Chr), with prevalence on Chr 1, followed by Chr 2, 3, 5, and 6. Among the 82 duplicated GsWRKYs, comprising 12, 54, and 16 members in Group I, II, and III respectively, 11 GsWRKYs were tandemly duplicated genes located across Chr 2 (2), Chr 5 (7), and Chr 9 (2). Weighted gene co-expression network analysis unveiled that 2 Group I (GsWRKY44 and GsWRKY95) and 14 Group II GsWRKYs, including two pairs of segmentally duplicated Group II GsWRKYs associated with thermomorphogenesis, exhibited coexpression with Grona styracifolia C-glycosyltransferases (GsCGT), a gene encoding a C-glucosyltransferase involved in schaftoside biosynthesis. Furthermore, GsWRKY95 demonstrated coexpression with other schaftoside biosynthetic genes. Dual-luciferase and yeast one-hybrid assays provided additional evidence that GsWRKY95 binds to the W-box of GsCGT, activating its expression. In addition, GsWRKY95- and GsCGT-coexpressing G r o na styracifolia chalcone synthase (GsCHSs), along with 11 pairs of segmentally duplicated Group II GsWRKYs, responded to both abiotic and biotic stresses. Notably, certain GsWRKYs were identified as regulators specific to schaftoside biosynthesis in stems, roots, and leaves. These findings suggest that duplication events, particularly in segmentally duplicated Group II GsWRKYs, play a pivotal role in orchestrating the hierarchical regulation of schaftoside biosynthesis. Overall, our results establish a foundation for genetically enhancing G. styracifolia to abundantly produce schaftoside, thereby contributing to its medicinal efficacy.

Abstract Grona styracifolia is a photophilous legume that contains abundant flavonoids with multiple pharmacological activities, which is used to cure urethral and biliary calculus in China for thousands of years. The authentication of the rate-limiting enzymes involved in the flavonoids biosynthesis pathway enabled a better understanding of the molecular aspect of quality formation and modulation of this medicinal herb. In this study, the chemical distribution characteristics and content of flavonoids in different tissues of Grona styracifolia were analyzed using ultraperormance liquid chromatography coupled with Q-TOF mass spectrometry and showed that active flavonoids were primarily synthesized and stored in the leaves. Subsequently, RNA sequencing (RNA-seq)-based transcriptome profiling of the different tissues revealed that the flavonoids biosynthesis in the leaves was the most active. Meanwhile, 27 full-length transcripts inferred encoding vital enzymes involved in the flavonoids biosynthesis were preliminarily excavated. Finally, four CHSs, four CHIs, and one FNSII were successfully characterized by heterologous expression, which involved in three rate-limiting steps of the flavonoid biosynthetic pathway. In conclusion, these results laid a foundation for further investigation of the molecular mechanism of the biosynthesis and modulation of active flavonoids in Grona styracifolia.

Grona styracifolia is a photophilous legume that contains abundant flavonoids with multiple pharmacological activities, which is used to cure urethral and biliary calculus in China for thousands of years. The authentication of the rate-limiting enzymes involved in the flavonoids biosynthesis pathway enabled a better understanding of the molecular aspect of quality formation and modulation of this medicinal herb. In this study, the chemical distribution characteristics and content of flavonoids in different tissues of Grona styracifolia were analyzed using ultraperormance liquid chromatography coupled with Q-TOF mass spectrometry and showed that active flavonoids were primarily synthesized and stored in the leaves. Subsequently, RNA sequencing (RNA-seq)-based transcriptome profiling of the different tissues revealed that the flavonoids biosynthesis in the leaves was the most active. Meanwhile, 27 full-length transcripts inferred encoding vital enzymes involved in the flavonoids biosynthesis were preliminarily excavated. Finally, four CHSs, four CHIs, and one FNSII were successfully characterized by heterologous expression, which involved in three rate-limiting steps of the flavonoid biosynthetic pathway. In conclusion, these results laid a foundation for further investigation of the molecular mechanism of the biosynthesis and modulation of active flavonoids in Grona styracifolia., (© 2023. The Author(s).)