Flavonoids as a class of important secondary metabolites are widely present in land plants, and chalcone isomerase (CHI) is the key rate-limiting enzyme that participates in catalyzing the stereospecific isomerization of chalcones to yield their corresponding flavanones. However, the phylogenetic dynamics and functional divergence of CHI family genes during the evolutionary path of green plants remains poorly understood. Here, a total of 122 CHI genes were identified by performing a genome-wide survey of 15 representative green plants from the most ancestral basal plant chlorophyte algae to higher angiosperm plants. Phylogenetic, orthologous groups (OG) classification, and genome structure analysis showed that the CHI family genes have evolved into four distinct types (types I–IV) containing eight OGs after gene duplication, and further studies indicated type III CHIs consist of three subfamilies (FAP1, FAP2, and FAP3). The phylogeny showed FAP3 CHIs as an ancestral out-group positioned on the outer layers of the main branch, followed by type IV CHIs, which are placed in an evolutionary intermediate between FAP3 CHIs and bona fide CHIs (including type I and type II). The results imply a potential intrinsic evolutionary connection between CHIs existing in the green plants. The amino acid substitutions occurring in several residues have potentially affected the functional divergence between CHI proteins. This is supported by the analysis of transcriptional divergence and cis-acting element analysis. Evolutionary dynamics analyses revealed that the differences in the total number of CHI family genes in each plant are primarily attributed to the lineage-specific expansion by natural selective forces. The current studies provide a deeper understanding of the phylogenetic relationships and functional diversification of CHI family genes in green plants, which will guide further investigation on molecular characteristics and biological functions of CHIs.

The plant hormones gibberellins (GAs) regulate plant growth and development and are closely related to the yield of cash crops. The GA oxidases (GAoxs), including the GA2ox, GA3ox, and GA20ox subfamilies, play pivotal roles in GAs’ biosynthesis and metabolism, but their classification and evolutionary pattern in Gramineae crops remain unclear. We thus conducted a comparative genomic study of GAox genes in six Gramineae representative crops, namely, Setaria italica (Si), Zea mays (Zm), Sorghum bicolor (Sb), Hordeum vulgare (Hv), Brachypodium distachyon (Bd), and Oryza sativa (Os). A total of 105 GAox genes were identified in these six crop genomes, belonging to the C₁₉-GA2ox, C₂₀-GA2ox, GA3ox, and GA20ox subfamilies. Based on orthogroup (OG) analysis, GAox genes were divided into nine OGs and the number of GAox genes in each of the OGs was similar among all tested crops, which indicated that GAox genes may have completed their family differentiations before the species differentiations of the tested species. The motif composition of GAox proteins showed that motifs 1, 2, 4, and 5, forming the 2OG-FeII_Oxy domain, were conserved in all identified GAox protein sequences, while motifs 11, 14, and 15 existed specifically in the GA20ox, C₁₉-GA2ox, and C₂₀-GA2ox protein sequences. Subsequently, the results of gene duplication events suggested that GAox genes mainly expanded in the form of WGD/SD and underwent purification selection and that maize had more GAox genes than other species due to its recent duplication events. The cis-acting elements analysis indicated that GAox genes may respond to growth and development, stress, hormones, and light signals. Moreover, the expression profiles of rice and maize showed that GAox genes were predominantly expressed in the panicles of the above two plants and the expression of several GAox genes was significantly induced by salt or cold stresses. In conclusion, our results provided further insight into GAox genes’ evolutionary differences among six representative Gramineae and highlighted GAox genes that may play a role in abiotic stress.

The cAMP-dependent protein kinase A, cGMP-dependent protein kinase G and phospholipid-dependent protein kinase C (AGC) perform various functions in plants, involving growth, immunity, apoptosis and stress response. AGC gene family is well described in Arabidopsis, however, limited information is provided about AGC genes in rice, an important cereal crop. This research studied the AGC gene family in the AA genome species: Oryza sativa ssp. japonica, Oryza sativa ssp. indica, Oryza nivara, Oryza rufipogon, Oryza glaberrima, Oryza meridionalis, Oryza barthii, Oryza glumaepatula and Oryza longistaminata were searched and classified into six subfamilies, and it was found that these species have similar numbers of members. The analysis of gene duplication and selection pressure indicated that the AGC gene family expanded mainly by segmental or whole genome duplication (WGD), with purifying selection during the long evolutionary period. RNA-seq analysis revealed that OsAGCs of subfamily V were specifically highly expressed in leaves, and the expression patterns of these genes were compared with that of photosynthesis-related genes using qRT-PCR, discovered that OsAGC9, OsAGC20, and OsAGC22 might participate in photosynthesis. These results provide an informative perspective for exploring the evolutionary of AGC gene family and its practical application in rice.

Effective panicle numbers (PNs) and Tiller numbers (TNs) are important traits affecting rice (Oryza sativa L.) architecture and grain yield. However, the molecular mechanisms underlying PN and TN heterosis remain unknown in rice. In addition, new PN- or TN-related genes need to be detected and discovered. In this study, in order to detect rice quantitative trait loci (QTLs) and the heterosis-related loci of PN or TN in rice, we developed a high generation recombinant inbred line (RIL) population from a cross of two elite cultivars, Luohui9 (Xian/Indica) and RPY geng (Geng/Japonica), and two testcross hybrid populations derived from the crosses of RILs and two cytoplasmic male sterile lines, YTA (Xian/Indica) and Z7A (Geng/Japonica). Finally, nine QTLs of PN across four seasons were identified, and two QTLs of TN in 191HB were mapped. Besides this, six heterosis-related QTLs of PN and five heterosis-related QTLs of TN were located. We found that heterosis-related QTLs of PN or TN covered multiple known genes, such as MOC1, TAC1 and OsETR2. Furthermore, homologous gene analysis identified one candidate gene of PN (LOC_10g25720). Together, these findings uncover multiple heterosis-related loci, and provide a new insight into the heterosis mechanism of PN and TN in rice.

The CYP76 subfamily, a member of the CYP superfamily, plays crucial roles in the biosynthesis of phytohormones in plants, involving biosynthesis of secondary metabolites, hormone signaling, and response to environmental stresses. Here, we conducted a genome-wide analysis of the CYP76 subfamily in seven AA genome species: Oryza sativa ssp. japonica, Oryza sativa ssp. indica, Oryza rufipogon, Oryza glaberrima, Oryza meridionalis, Oryza barthii, and Oryza glumaepatula. These were identified and classified into three groups, and it was found that Group 1 contained the largest number of members. Analysis of cis-acting elements revealed a large number of elements related to jasmonic acid and light response. The gene duplication analysis revealed that the CYP76 subfamily expanded mainly in SD/WGD and tandem forms and underwent strong purifying selection during evolution. Expression pattern analysis of OsCYP76s in various developmental stages revealed that the majority of OsCYP76s exhibit relatively restricted expression patterns in leaves and roots. We further analyzed the expression of CYP76s in O. sativa, japonica, and O. sativa, indica under cold, flooding, drought, and salt abiotic stresses by qRT-PCR. We found that OsCYP76-11 showed a huge increase in relative expression after drought and salt stresses. After flooding stress, OsiCYP76-4 showed a greater increase in expression compared to other genes. CYP76 in japonica and indica showed different response patterns to the same abiotic stresses, revealing functional divergence in the gene family during evolution; these may be the key genes responsible for the differences in tolerance to indica japonica. Our results provide valuable insights into the functional diversity and evolutionary history of the CYP76 subfamily and pave the way for the development of new strategies for improving stress tolerance and agronomic traits in rice.

Lenvatinib plus PD-1 inhibitor combination therapy has emerged as a frontline treatment for advanced hepatocellular carcinoma (HCC), but substantial interpatient variability in therapeutic responses underscores the urgent need for reliable predictive biomarkers. Here, we enrolled 86 HCC patients treated with lenvatinib plus tislelizumab and integrated digital spatial profiling (DSP), multiplex immunofluorescence (mIF), and immunohistochemistry (IHC) to dissect intratumoral heterogeneity and identify spatially resolved biomarkers associated with treatment outcomes. DSP analysis demonstrated distinct molecular features between epithelial cell-enriched (EP-enriched) and immune cell-enriched (IM-enriched) regions, effectively capturing spatial intratumoral heterogeneity in HCC. Furthermore, we found that FAP-α expression was significantly elevated in non-responders, predominantly localized to IM-enriched regions, and correlated with M2-type macrophage infiltration accompanied by suppressed T cell infiltration. Using an mIF validation cohort of 22 patients, we confirmed that FAP-α outperformed immune cell markers as a prognostic biomarker. IHC validation in 54 patients revealed FAP-α+ cell proportion as a robust predictor (sensitivity: 80%; specificity: 71.4%), with 85.7% of patients above the optimal threshold exhibiting treatment non-response. Upregulation of FAP-α during HCC progression and association with shorter overall survival were also observed by using public data analysis. Collectively, leveraging large clinical cohorts and cross-validation across multiple technologies, we systematically dissected the spatial molecular landscape of HCC and identified FAP-α as a clinically translatable biomarker for lenvatinib plus PD-1 inhibitor therapy, providing a rationale for clinical patient stratification and guiding personalized treatment strategies.