Best known as licorice, Glycyrrhiza Linn., a genus of herbaceous perennial legume, has been used as a traditional herbal medicine in Asia and a flavoring agent for tobacco and food industry in Europe and America. Abiotic stresses and hormonal treatments can significantly impact the development and metabolism of secondary metabolites in Glycyrrhiza. To better understand the biosynthesis of the trace-amount bioactive compounds, we first screened for the suitable reference genes for quantitative real-time reverse transcription PCR (qRT-PCR) analysis in Glycyrrhiza. The expression profiles of 14 candidate reference genes, including Actin1 (ACT), Clathrin complex AP1 (CAC), Cyclophilin (CYP), Heat-shock protein 40 (DNAJ), Dehydration responsive element binding gene (DREB), Translation elongation factor1 (EF1), Ras related protein (RAN), Translation initiation factor (TIF1), β-Tubulin (TUB), Ubiquitin-conjugating enzyme E2 (UBC2), ATP binding-box transpoter 2 (ABCC2), COP9 signal compex subunit 3 (COPS3), Citrate synthase (CS), and R3H domain protein 2 (R3HDM2) from two congeneric species, Glycyrrhiza uralensis F. and Glycyrrhiza inflata B., were examined under abiotic stresses (osmotic and salinity) and hormonal treatments (Abscisic acid (ABA) and methyl jasmonic acid (MeJA)) using a panel of software, including geNorm, NormFinder, BestKeeper, and Delta CT. The overall stability, however, was provided by RefFinder, a comprehensive ranking system integrating inputs from all four algorithms. In G. uralensis, the most stable reference genes under osmotic stress, salt stress, ABA treatment, and MeJA treatment were TIF1, DNAJ, CS, and ABCC2 for leaves and DNAJ, DREB, CAC, and CAC for roots, respectively. In comparison, the top ranked genes were TUB, CAC, UBC2, and RAN for leaves and TIF1, ABCC2, CAC, and UBC2 for roots, respectively, under stress and hormonal treatments in G. inflata. ACT and TIF1, on the other hand, were the least stable genes under the most experimental conditions in the two congeneric species. Finally, our survey of the reference genes in legume shows that EF, ACT, UBC2, and TUB were the top choices for the abiotic stresses while EF, UBC2, CAC, and ABCC2 were recommended for the hormonal treatments in Leguminosae. Our combined results provide reliable normalizers for accurate gene quantifications in Glycyrrhiza species, which will allow us to exploit its medicinal potential in general and antiviral activities in particular.

ABSCISIC ACID (ABA) INSENSITIVE5 (ABI5), the key regulator of abscisic acid (ABA) signaling pathway, plays a fundamental role in seed germination and postgerminative development. However, the detailed molecular mechanism underlying the repression function of ABI5 in these processes remains to be elucidated. In this study, we demonstrate that the conserved eukaryotic WD40 repeat protein RACK1 is a novel negative regulator of ABI5 in Arabidopsis. The RACK1 loss-of-function mutant is hypersensitive to ABA, while this phenotype was rescued by the mutation of ABI5. Moreover, overexpression of RACK1 suppresses ABI5 transcriptional activation activity for ABI5-targeted genes. RACK1 could also physically interact with ABI5 and facilitate its degradation. Furthermore, we found that RACK1 and the two substrate receptors for CUL4-based E3 ligases (DWA1 and DWA2) function together to mediate the turnover of ABI5, thereby efficiently turning down ABA signaling for seed germination and postgerminative growth. On the other hand, a series of molecular analyses demonstrated that ABI5 could bind with the promoter of RACK1 to repress its expression. Collectively, our findings suggest that RACK1 and ABI5 might form a feedback loop to regulate the homeostasis of ABA signaling for acute seed germination and early plant development.

Intracellular Ca ²⁺ controls various cellular functions, and local Ca ²⁺ dynamics are tightly regulated upon environmental cues. How cellular Ca ²⁺ balance is maintained during stress remains unresolved. Here, we identify an intracellular phosphoregulatory module comprising calcium-dependent protein kinase (CPK)–ER-type Ca ²⁺ -ATPases 1 (ECA1) that integrates [Ca ²⁺ ] _cyt flux with abscisic acid (ABA) homeostasis to modulate root growth under osmotic stress. Pharmacological and genetic disruption of ECA1-mediated Ca ²⁺ efflux triggered enhanced [Ca ²⁺ ] _cyt transients and [ABA] _cyt , exacerbating root growth hypersensitivity to osmotic stress. Biochemical assays reveal that CPK2/6/11 directly bind and phosphorylate ECA1, enhancing its Ca ²⁺ -pumping activity to restore cytosolic Ca ²⁺ equilibrium and subsequently prevent ABA _cyt overaccumulation. Genetic analysis shows that eca1cpk11 phenocopied eca1 hypersensitivity, while triple and quadruple mutants amplified osmotic sensitivity, demonstrating that CPKs and ECA1 function as a cooperative phosphoregulatory hub regulating Ca ²⁺ signaling and root growth. Our work resolves a critical gap by elucidating how plants couple ionic and hormonal messengers to balance plant growth and stress resilience.

Glycyrrhiza inflata Batalin is a medicinal licorice species that has been widely used by humans for centuries. Licochalcone A (LCA) is a characteristic flavonoid that accumulates in G. inflata roots with high economical value. However, the biosynthetic pathway and regulatory network of its accumulation remain largely unknown. Here we found that a histone deacetylase (HDAC) inhibitor nicotinamide (NIC) could enhance the accumulation of LCA and total flavonoids in G. inflata seedlings. GiSRT2, a NIC-targeted HDAC was functionally analyzed and its RNAi transgenic hairy roots accumulated much more LCA and total flavonoids than its OE lines and the controls, indicating a negative regulatory role of GiSRT2 in the accumulation of LCA and total flavonoids. Co-analysis of transcriptome and metabolome of RNAi-GiSRT2 lines revealed potential mechanisms in this process. An O-methyltransferase gene, GiLMT1 was up-regulated in RNAi-GiSRT2 lines and the encoded enzyme catalyzed an intermediate step in LCA biosynthesis pathway. Transgenic hairy roots of GiLMT1 proved that GiLMT1 is required for LCA accumulation. Together, this work highlights the critical role of GiSRT2 in the regulation of flavonoid biosynthesis and identifies GiLMT1 as a candidate gene for the biosynthesis of LCA with synthetic biology approaches.

Glycyrrhiza, a genus of perennial medicinal herbs, has been traditionally used to treat human diseases, including respiratory disorders. Functional analysis of genes involved in the synthesis, accumulation, and degradation of bioactive compounds in these medicinal plants requires accurate measurement of their expression profiles. Reverse transcription quantitative real-time PCR (RT-qPCR) is a primary tool, which requires stably expressed reference genes to serve as the internal references to normalize the target gene expression. In this study, the stability of 14 candidate reference genes from the two congeneric species G. uralensis and G. inflata, including ACT, CAC, CYP, DNAJ, DREB, EF1, RAN, TIF1, TUB, UBC2, ABCC2, COPS3, CS, R3HDM2, were evaluated across different tissues and throughout various developmental stages. More importantly, we investigated the impact of interactions between tissue and developmental stage on the performance of candidate reference genes. Four algorithms, including geNorm, NormFinder, BestKeeper, and Delta Ct, were used to analyze the expression stability and RefFinder, a comprehensive software, provided the final recommendation. Based on previous research and our preliminary data, we hypothesized that internal references for spatio-temporal gene expression are different from the reference genes suited for individual factors. In G. uralensis, the top three most stable reference genes across different tissues were R3HDM2, CAC and TUB, while CAC, CYP and ABCC2 were most suited for different developmental stages. CAC is the only candidate recommended for both biotic factors, which is reflected in the stability ranking for the spatio (tissue)-temporal (developmental stage) interactions (CAC, R3HDM2 and DNAJ). Similarly, in G. inflata, COPS3, R3HDM2 and DREB were selected for tissues, while RAN, COPS3 and CS were recommended for developmental stages. For the tissue-developmental stage interactions, COPS3, DREB and ABCC2 were the most suited reference genes. In both species, only one of the top three candidates was shared between the individual factors and their interactions, specifically, CAC in G. uralensis and COPS3 in G. inflata, which supports our overarching hypothesis. In summary, spatio-temporal selection of reference genes not only lays the foundation for functional genomics research in Glycyrrhiza, but also facilitates these traditional medicinal herbs to reach/maximize their pharmaceutical potential.

Abscisic acid (ABA) is a crucial phytohormone that functions as a master regulator of plant growth and development, as well as responses to diverse abiotic stresses, by integrating environmental cues with developmental programs. The transcription factor ABA INSENSITIVE 5 (ABI5) functions at the central hub of the ABA signaling pathway and mediates the expression of its target genes. Emerging evidence reveals extensive crosstalk between calcium-dependent protein kinases (CPKs)-mediated calcium signaling and the ABA-ABI5 cascade, enabling plants to balance growth and stress responses. However, the molecular mechanisms underlying the interactions between CPKs and ABA-ABI5 signaling are still elusive. In this study, we revealed that CPK2 enhances sensitivity to ABA during both seed germination and seedling root growth by promoting ABA-induced ABI5 expression and increasing ABA-mediated ABI5 stability. Compared to the wildtype (Col-0), the CPK2-OE line exhibited the highest sensitivity to ABA in both seed germination and root growth, while the cpk2abi5-7 double mutant showed the least sensitivity. The single mutants cpk2 and abi5-7, as well as the abi5-7CPK2-OE2 line, displayed intermediate phenotypes, suggesting that CPK2 acts upstream of ABI5. Biochemical and molecular biological studies revealed that CPK2 physically interacts with ABI5 and directly phosphorylates it at Ser42, Ser145, and Thr201. Moreover, both ABA-induced ABI5 expression and protein accumulation were significantly reduced in cpk2 mutants upon ABA treatment. Taken together, these findings provide compelling evidence that CPK2 exacerbates the ABA inhibition of seed germination and root growth by enhancing both the expression and stability of ABI5, thereby reinforcing stress adaptation during early plant development.