The plant hormone abscisic acid (ABA) is able to regulate the expression of ABA-responsive genes via signaling transduction, and thus plays an important role in regulating plant responses to abiotic stresses. Hence, characterization of unknown ABA response genes may enable us to identify novel regulators of ABA and abiotic stress responses. By using RT-PCR analysis, we found that the expression levels of ABA-induced Serine-rich Repressor 1 (ASR1)and ASR2, two closely related unknown function genes, were increased in response to ABA treatment. Amino acid sequence analyses show that ASR1 contains an L×L×L motif and both ASR1 and ASR2 are enriched in serine. Transfection assays in Arabidopsis leaf protoplasts show that ASR1 and ASR2 were predominantly localized in the nucleus and were able to repress the expression of the reporter gene. The roles of ASRs in regulating ABA responses were examined by generating transgenic Arabidopsis plants over-expressing ASR1 and ASR2, respectively, and CRISPR/Cas9 gene-edited single and double mutants for ASR1 and ASR2. In both the seed germination and cotyledon greening assays, ABA sensitivity remained largely unchanged in the over-expression transgenic plants and the single mutants of ASR1 and ASR2, but greatly increased ABA sensitivity was observed in the asr1 asr2 double mutants. In root elongation assays, however, decreased ABA sensitivity was observed in the 35S:ASR1 and 35S:ASR2 transgenic plants, whereas increased ABA sensitivity was observed in the asr1 and asr2 single mutants, and ABA sensitivity was further increased in the asr1 asr2 double mutants. Transcriptome analysis show that the differentially expressed genes (DEGs) down-regulated in the 35S:ASR1 transgenic plant seedlings, but up-regulated in the asr1 asr2 double mutant seedlings were highly enriched in processes including responses to plant hormones and stress stimuli. Taken together, our results show that ASR1 and ASR2 are closely related ABA response genes, ASR1 and ASR2 are serine-rich novel transcription repressors, and they negatively regulate ABA responses in Arabidopsis in a redundant manner.

Floral coloration in Freesia hybrida is predominantly attributed to anthocyanins, with glycosylation playing a critical role in their stability and diversity. This study investigates the molecular mechanisms underlying color variation between F. hybrida cultivars, focusing on anthocyanin 5-O-glucosyltransferases (An5GTs). HPLC analysis revealed that ‘Pink Passion’ petals accumulate 3,5-O-diglucosidic anthocyanins, absent in ‘Red River^®’ and ‘Ambiance’. RNA-seq identified seven candidate Fh5GT genes, with phylogenetic and subcellular localization analyses confirming their classification as cytosolic glycosyltransferases. Expression profiling highlighted elevated transcript levels of Fh5GT1, Fh5GT3, and Fh5GT7 in ‘Pink Passion’, correlating with its di-glucosidic anthocyanin accumulation. In vitro enzymatic assays demonstrated that Fh5GT3 and Fh5GT7 preferentially glucosylate 3-O-monoglucosidic anthocyanins to form stable 3,5-O-diglucosides, with minimal activity on anthocyanidins to generate 5-O-glucosidic anthocyanins. Heterologous expression of Fh5GT3 and Fh5GT7 in Arabidopsis complemented anthocyanin deficiency in 5gt mutants, restoring pigmentation. These findings elucidate the potential role of 5GTs in modulating floral color diversity through anthocyanin modification, providing insights for targeted breeding strategies to enhance ornamental traits in horticultural species.

Floral scent is an essential and genetically complex trait in herbaceous peonies (Paeonia lactiflora Pall.); however, specific genes related to metabolic and regulatory networks remain scantily studied. Our study integrated metabolite profiling and RNA-sequencing to screen floral scent biosynthetic genes. Hence, the major molecules identified by headspace collection combined with cultivar-specific GC-MS analysis were geraniol, β-caryophyllene, 2-phenylethanol (2-PE), citronellol, and 1,8-cineole. Genes related to terpenoids and 2-PE biosynthesis were identified after the assembly and annotation of the P. lactiflora transcriptomes. Eight angiosperm-specific terpene synthases (TPSs) from the TPS-a and TPS-b clades, as well as enzymes linked to 2-PE synthesis such as aromatic amino acid decarboxylase (AADC), phenylacetaldehyde reductase (PAR), and geranial reductase (GER) were identified. The biochemical analysis of the enzymes encoded by PlPAR1 and PlGER1 generated 2-PE from phenylacetaldehyde (PAld). The pairwise alignment of AADC1 reveals a splice variant lacking a 124 bp fragment, thus highlighting the possible role of alternative splicing in modulating floral scent composition. This study offers insights into the molecular-level biosynthesis of terpenoids and 2-PE in Peonia taxa, and provides the basis for the functional characterization, breeding, and bioengineering of prospective candidate genes for the production of floral volatiles in the Paeonia genus.

EAR (Ethylene-responsive element binding factor-associated Amphiphilic Repression) motif-containing transcription repressors have been shown to regulate plant growth and development, and plant responses to plant hormones and environmental stresses including biotic and abiotic stresses. However, the functions of most EAR-motif-containing proteins remain largely uncharacterized. The plant hormone abscisic acid (ABA) also plays important roles in regulating plant responses to abiotic stresses via activation/repression of ABA-responsive genes. We report here the identification and functional characterization of two ABA-responsive EAR motif-containing protein genes, AtEAU1 (Arabidopsis thaliana EAR motif-containing ABAUp-regulated 1) and AtEAU2. Quantitative RT-PCR results show that the expressions of AtEAU1 and AtEAU2 were increased by ABA treatment, and were decreased in the ABA biosynthesis mutant aba1-5. Assays in transfected Arabidopsis protoplasts show that both AtEAU1 and AtEAU2 were specifically localized in the nucleus, and when recruited to the promoter region of the reporter gene by a fused DNA binding domain, repressed reporter gene expression. By using T-DNA insertion mutants and a gene-edited transgene-free mutant generated by CRISPR/Cas9 gene editing, we performed ABA sensitivity assays, and found that ABA sensitivity in the both ateau1 and ateau2 single mutants was increased in seedling greening assays. ABA sensitivity in the ateau1 ateau2 double mutants was also increased, but was largely similar to the ateau1 single mutants. On the other hand, all the mutants showed a wild type response to ABA in root elongation assays. Quantitative RT-PCR results show that the expression level of PYL4, an ABA receptor gene was increased, whereas that of ABI2, a PP2C gene was decreased in the ateau1 and ateau1 single, and the ateau1 ateau2 double mutants. In summary, our results suggest that AtEAU1 and AtEAU2 are ABA-response genes, and AtEAU1 and AtEAU2 are novel EAR motif-containing transcription repressors that negatively regulate ABA responses in Arabidopsis, likely by regulating the expression of some ABA signaling key regulator genes.