Cotton CSLD3 restores cell elongation and cell wall integrity mainly by enhancing primary cellulose production in the Arabidopsis cesa6 mutant.

KEY MESSAGE: Overexpression of cotton cellulose synthase like D3 (GhCSLD3) gene partially rescued growth defect of atcesa6 mutant with restored cell elongation and cell wall integrity mainly by enhancing primary cellulose production. Among cellulose synthase like (CSL) family proteins, CSLDs share the highest sequence similarity to cellulose synthase (CESA) proteins. Although CSLD proteins have been implicated to participate in the synthesis of carbohydrate-based polymers (cellulose, pectins and hemicelluloses), and therefore plant cell wall formation, the exact biochemical function of CSLD proteins remains controversial and the function of the remaining CSLD genes in other species have not been determined. In this study, we attempted to illustrate the function of CSLD proteins by overexpressing Arabidopsis AtCSLD2, -3, -5 and cotton GhCSLD3 genes in the atcesa6 mutant, which has a background that is defective for primary cell wall cellulose synthesis in Arabidopsis. We found that GhCSLD3 overexpression partially rescued the growth defect of the atcesa6 mutant during early vegetative growth. Despite the atceas6 mutant having significantly reduced cellulose contents, the defected cell walls and lower dry mass, GhCSLD3 overexpression largely restored cell wall integrity (CWI) and improved the biomass yield. Our result suggests that overexpression of the GhCSLD protein enhances primary cell wall synthesis and compensates for the loss of CESAs, which is required for cellulose production, therefore rescuing defects in cell elongation and CWI.

Sucrose Synthase Enhances Hull Size and Grain Weight by Regulating Cell Division and Starch Accumulation in Transgenic Rice.

Grain size and weight are two important determinants of grain yield in rice. Although overexpression of sucrose synthase (SUS) genes has led to several improvements on cellulose and starch-based traits in transgenic crops, little is reported about SUS enhancement of hull size and grain weight in rice. In this study, we selected transgenic rice plants that overexpressed OsSUS1-6 genes driven with the maize Ubi promoter. Compared to the controls (wild type and empty vector line), all independent OsSUS homozygous transgenic lines exhibited considerably increased grain yield and grain weights. Using the representative OsSUS3 overexpressed transgenic plants, four independent homozygous lines showed much raised cell numbers for larger hull sizes, consistent with their enhanced primary cell wall cellulose biosynthesis and postponed secondary wall synthesis. Accordingly, the OsSUS3 transgenic lines contained much larger endosperm volume and higher starch levels than those of the controls in the mature grains, leading to increased brown grain weights by 15-19%. Hence, the results have demonstrated that OsSUS overexpression could significantly improve hull size and grain weight by dynamically regulating cell division and starch accumulation in the transgenic rice.

Cellulose Synthase Mutants Distinctively Affect Cell Growth and Cell Wall Integrity for Plant Biomass Production in Arabidopsis.

Cellulose is the most characteristic component of plant cell walls, and plays a central role in plant mechanical strength and morphogenesis. Despite the fact that cellulose synthase (CesA) mutants exhibit a reduction in cellulose level, much remains unknown about their impacts on cell growth (elongation and division) and cell wall integrity that fundamentally determine plant growth. Here, we examined three major types of AtCesA mutants (rsw1, an AtCesA1 mutant; prc1-1 and cesa6, AtCesA6-null mutants; and IRX3, an AtCesA7 mutant) and transgenic mutants that overexpressed AtCesA genes in the background of AtCesA6-null mutants. We found that AtCesA6-null mutants showed a reduced cell elongation of young seedlings with little impact on cell division, which consequently affected cell wall integrity and biomass yield of mature plants. In comparison, rsw1 seedlings exhibited a strong defect in both cell elongation and division at restrictive temperature, whereas the IRX3 mutant showed normal seedling growth. Analyses of transgenic mutants indicated that primary wall AtCesA2, AtCesA3, AtCesA5 and AtCesA9 genes played a partial role in restoration of seedling growth. However, co-overexpression of AtCesA2 and AtCesA5 in AtCesA6-null mutants could greatly enhance cell division and fully restore wall integrity, leading to a significant increase in secondary wall thickness and biomass production in mature plants. Hence, this study has demonstrated distinct functions of AtCesA genes in plant cell growth and cell wall deposition for biomass production, which helps to expalin our recent finding that only three AtCesA6-like genes, rather than other AtCesA genes of the AtCesA family, could greatly enhance biomass production in transgenic Arabidopsis plants.

Three AtCesA6-like members enhance biomass production by distinctively promoting cell growth in Arabidopsis.

Cellulose is an abundant biopolymer and a prominent constituent of plant cell walls. Cellulose is also a central component to plant morphogenesis and contributes the bulk of a plant's biomass. While cellulose synthase (CesA) genes were identified over two decades ago, genetic manipulation of this family to enhance cellulose production has remained difficult. In this study, we show that increasing the expression levels of the three primary cell wall AtCesA6-like genes (AtCesA2, AtCesA5, AtCesA6), but not AtCesA3, AtCesA9 or secondary cell wall AtCesA7, can promote the expression of major primary wall CesA genes to accelerate primary wall CesA complex (cellulose synthase complexes, CSCs) particle movement for acquiring long microfibrils and consequently increasing cellulose production in Arabidopsis transgenic lines, as compared with wild-type. The overexpression transgenic lines displayed changes in expression of genes related to cell growth and proliferation, perhaps explaining the enhanced growth of the transgenic seedlings. Notably, overexpression of the three AtCesA6-like genes also enhanced secondary cell wall deposition that led to improved mechanical strength and higher biomass production in transgenic mature plants. Hence, we propose that overexpression of certain AtCesA genes can provide a biotechnological approach to increase cellulose synthesis and biomass accumulation in transgenic plants.

Ectopic expression of a novel OsExtensin-like gene consistently enhances plant lodging resistance by regulating cell elongation and cell wall thickening in rice.

Plant lodging resistance is an important integrative agronomic trait of grain yield and quality in crops. Although extensin proteins are tightly associated with plant cell growth and cell wall construction, little has yet been reported about their impacts on plant lodging resistance. In this study, we isolated a novel extensin-like (OsEXTL) gene in rice, and selected transgenic rice plants that expressed OsEXTL under driven with two distinct promoters. Despite different OsEXTL expression levels, two-promoter-driven OsEXTL-transgenic plants, compared to a rice cultivar and an empty vector, exhibited significantly reduced cell elongation in stem internodes, leading to relatively shorter plant heights by 7%-10%. Meanwhile, the OsEXTL-transgenic plants showed remarkably thickened secondary cell walls with higher cellulose levels in the mature plants, resulting in significantly increased detectable mechanical strength (extension and pushing forces) in the mature transgenic plants. Due to reduced plant height and increased plant mechanical strength, the OsEXTL-transgenic plants were detected with largely enhanced lodging resistances in 3 years field experiments, compared to those of the rice cultivar ZH11. In addition, despite relatively short plant heights, the OsEXTL-transgenic plants maintain normal grain yields and biomass production, owing to their increased cellulose levels and thickened cell walls. Hence, this study demonstrates a largely improved lodging resistance in the OsEXTL-transgenic rice plants, and provides insights into novel extensin functions in plant cell growth and development, cell wall network construction and wall structural remodelling.

AtCSLD3 and GhCSLD3 mediate root growth and cell elongation downstream of the ethylene response pathway in Arabidopsis.

CSLD3, a gene of the cellulose synthase-like D family, affects root hair elongation, but its interactions with ethylene signaling and phosphate-starvation are poorly understood. Here, we aim to understand the role of CSLD3 in the context of the ethylene signaling and phosphate starvation pathways in Arabidopsis plant growth. Therefore, we performed a comparative analysis of the csld3-1 mutant, CSLD3-overexpressing lines, and ethylene-response mutants, such as the constitutive ethylene-response mutant i-ctr1. We found that CSLD3 overexpression enhanced root and hypocotyl growth by increasing cell elongation, and that the root growth was highly sensitive to ethylene treatment (1 µM ACC), in particular under phosphate starvation. However, the CSLD3-mediated hypocotyl elongation occurred independently of the ethylene signaling pathway. Notably, the typical induction of root hair and root elongation by ethylene and phosphate-starvation was completely abolished in the csld3-1 mutant. Furthermore, i-ctr1 csld3-1 double-mutants were hairless like the csld3-1 parent, confirming that CSLD3 acts downstream of the ethylene signaling pathway during root growth. Moreover, the CSLD3 levels positively correlated with cellulose levels, indicating a role of CSLD3 in cellulose synthesis, which may explain the observed growth effects. Our results establish how CSLD3 works in the context of the ethylene signaling and phosphate-starvation pathways during root hair growth, cell elongation, and cell wall biosynthesis.

A pqr2 mutant encodes a defective polyamine transporter and is negatively affected by ABA for paraquat resistance in Arabidopsis thaliana.

Despite the paraquat-resistant mutants that have been reported in plants, this study identified a novel A. thaliana mutant (pqr2) from an XVE inducible activation library based on its resistance to 2 µM paraquat. The pqr2 mutant exhibited a termination mutation in the exon of AT1G31830/PAR1/PQR2, encoded a polyamine uptake transporter AtPUT2/PAR1/PQR2. The PQR2 mutation could largely reduce superoxide accumulation and cell death in the pqr2 plants under paraquat treatment. Moreover, compared with wild type, the pqr2 mutant exhibited much reduced tolerance to putrescine, a classic polyamine compound, which confirmed that PQR2 encoded a defective polyamine transporter. Notably, co-treated with ABA and paraquat, both pqr2 mutant and wild type exhibited a lethal phenotype from seed germination, but the wild type like pqr2 mutant, could remain paraquat-resistance while co-treated with high dosage of Na2WO4, an ABA synthesis inhibitor. Gene expression analysis suggested that ABA signaling should widely regulate paraquat-responsive genes distinctively in wild type and pqr2 mutant. Hence, this study has for the first time reported about ABA negative effect on paraquat-resistance in A. thaliana, providing insight into the ABA signaling involved in the oxidative stress responses induced by paraquat in plants.

Chromosome-scale genome assembly of medicinal plant Tinospora sagittata (Oliv.) Gagnep. from the Menispermaceae family.

Tinospora sagittata (Oliv.) Gagnep. is an important medicinal tetraploid plant in the Menispermaceae family. Its tuber, Radix Tinosporae, used in traditional Chinese medicine, is rich in diterpenoids and benzylisoquinoline alkaloids (BIAs). To enhance our understanding of medicinal compounds' biosynthesis and Menispermaceae's evolution, we herein report assembling a high-quality chromosome-scale genome with both PacBio HiFi and Illumina sequencing technologies. PacBio Sequel II generated 2.5 million circular consensus sequencing (CCS) reads, and a hybrid assembly strategy with Illumina sequencing resulted in 4483 contigs. The assembled genome size was 2.33 Gb, consisting of 4070 scaffolds (N50 = 42.06 Mb), of which 92.05% were assigned to 26 pseudochromosomes. T. sagittata's chromosomal-scale genome assembly, the first species in Menispermaceae, aids Menispermaceae evolution and T. sagittata's secondary metabolites biosynthesis understanding.

An omics-based characterization of Wolfiporia cocos reveals three CYP450 members involved in the biosynthetic pathway of pachymic acid.

Wolfiporia cocos is a medicinal mushroom used in China. It biosynthesizes pachymic acid (PA), a main therapeutic triterpene associated with therapies. Nowadays, the unknown PA biosynthesis leads to difficulties in increasing its content in W. cocos. Herein, we report sequencing, assembling, and characterization of the genome and several transcriptomes of W. cocos. Sequence mining determined candidate genes that encode lanosterol synthase, sterol O-acyltransferase, and sterol C-24 methyltransferase likely involved in the steps from lanosterol to PA. Gene cluster analysis identified four CYP450 cDNAs likely involved in the biosynthesis of PA, namely WcCYP64-1, WcCYP64-2, WcCYP52, and WcCYP_FUM15, which were subjected to both overexpression and silencing in mycelia. The overexpression of each of WcCYP64-1, WcCYP52 and WcCYP_FUM15 increased the content of PA, 16α-hydroxytrametenolic acid, eburicoic acid, and tumulosic acid, while the silencing of each gene either significantly or slightly decreased the contents of these four compounds, indicating their involvement in the PA biosynthesis. In addition, different temperatures affected the expression of these genes and the formation of PA. By contrast, the overexpression and silencing of WcCYP64-2 did not alter the formation of these compounds. Taken together, these findings determine more potential steps in the biosynthetic pathway of PA for metabolic engineering.

Analysis of five rice 4-coumarate:coenzyme A ligase enzyme activity and stress response for potential roles in lignin and flavonoid biosynthesis in rice.

4-Coumarate:coenzyme A ligase (4CL) catalyzes the conversion of hydroxycinnamates into corresponding CoA esters for biosynthesis of flavonoids and lignin. In this study, five members of the 4CL gene family from rice were cloned and analyzed. Recombinant 4CL data revealed that 4-coumaric acid and ferulic acid were the two main substrates of 4CL (Os4CL1/3/4/5) for monolignol biosynthesis in rice. Os4CL2 was specifically expressed in the anther and was strongly activated by UV irradiation, suggesting its potential involvement in flavonoid formation. Moreover, bioinformatics analysis showed that the existence of valine residue at the substrate-binding pocket may mainly affect rice 4CL activities toward sinapic acid.

An integrative analysis of four CESA isoforms specific for fiber cellulose production between Gossypium hirsutum and Gossypium barbadense.

Cotton fiber is an excellent model system of cellulose biosynthesis; however, it has not been widely studied due to the lack of information about the cellulose synthase (CESA) family of genes in cotton. In this study, we initially identified six full-length CESA genes designated as GhCESA5-GhCESA10. Phylogenetic analysis and gene co-expression profiling revealed that CESA1, CESA2, CESA7, and CESA8 were the major isoforms for secondary cell wall biosynthesis, whereas CESA3, CESA5, CESA6, CESA9, and CESA10 should involve in primary cell wall formation for cotton fiber initiation and elongation. Using integrative analysis of gene expression patterns, CESA protein levels, and cellulose biosynthesis in vivo, we detected that CESA8 could play an enhancing role for rapid and massive cellulose accumulation in Gossypium hirsutum and Gossypium barbadense. We found that CESA2 displayed a major expression in non-fiber tissues and that CESA1, a housekeeping gene like, was predominantly expressed in all tissues. Further, a dynamic alteration was observed in cell wall composition and a significant discrepancy was observed between the cotton species during fiber elongation, suggesting that pectin accumulation and xyloglucan reduction might contribute to cell wall transition. In addition, we discussed that callose synthesis might be regulated in vivo for massive cellulose production during active secondary cell wall biosynthesis in cotton fibers.

A telomere-to-telomere reference genome provides genetic insight into the pentacyclic triterpenoid biosynthesis in Chaenomeles speciosa.

Chaenomeles speciosa (2n = 34), a medicinal and edible plant in the Rosaceae, is commonly used in traditional Chinese medicine. To date, the lack of genomic sequence and genetic studies has impeded efforts to improve its medicinal value. Herein, we report the use of an integrative approach involving PacBio HiFi (third-generation) sequencing and Hi-C scaffolding to assemble a high-quality telomere-to-telomere genome of C. speciosa. The genome comprised 650.4 Mb with a contig N50 of 35.5 Mb. Of these, 632.3 Mb were anchored to 17 pseudo-chromosomes, in which 12, 4, and 1 pseudo-chromosomes were represented by a single contig, two contigs, and four contigs, respectively. Eleven pseudo-chromosomes had telomere repeats at both ends, and four had telomere repeats at a single end. Repetitive sequences accounted for 49.5% of the genome, while a total of 45 515 protein-coding genes have been annotated. The genome size of C. speciosa was relatively similar to that of Malus domestica. Expanded or contracted gene families were identified and investigated for their association with different plant metabolisms or biological processes. In particular, functional annotation characterized gene families that were associated with the biosynthetic pathway of oleanolic and ursolic acids, two abundant pentacyclic triterpenoids in the fruits of C. speciosa. Taken together, this telomere-to-telomere and chromosome-level genome of C. speciosa not only provides a valuable resource to enhance understanding of the biosynthesis of medicinal compounds in tissues, but also promotes understanding of the evolution of the Rosaceae.

Distinct cellulose and callose accumulation for enhanced bioethanol production and biotic stress resistance in OsSUS3 transgenic rice.

Genetic modification of plant cell walls is an effective approach to reduce lignocellulose recalcitrance in biofuel production, but it may affect plant stress response. Hence, it remains a challenge to reduce biomass recalcitrance and simultaneously enhance stress resistance. In this study, the OsSUS3-transgenic plants exhibited increased cell wall polysaccharides deposition and reduced cellulose crystallinity and xylose/arabinose proportion of hemicellulose, resulting in largely enhanced biomass saccharification and bioethanol production. Additionally, strengthening of the cell wall also contributed to plant biotic resistance. Notably, the transgenic plants increased stress-induced callose accumulation, and promoted the activation of innate immunity, leading to greatly improved multiple resistances to the most destructive diseases and a major pest. Hence, this study demonstrates a significant improvement both in bioethanol production and biotic stress resistance by regulating dynamic carbon partitioning for cellulose and callose biosynthesis in OsSUS3-transgenic plants. Meanwhile, it also provides a potential strategy for plant cell wall modification.

Molecular characterization, chromosomal and expression patterns of three aquaglyceroporins (AQP3, 7, 9) from pig.

As one subgroup of aquaporin, aquaglyceroporin including AQP3, 7, 9, 10 facilitates glycerol transport as well as water transport. In this study, we cloned the full length coding sequences of porcine (Sus scrofa) AQP3, 7 and 9 and the genomic sequence of AQP3 including 6 exons and 5 introns. Additionally, as a first step toward understanding the regulatory mechanisms of AQP9 in pig, we cloned and analyzed the upstream genomic sequence of the ATG translation initiation codon and found two negative insulin response elements (TGTTTTC and TATTTTG.), glucocorticoid-responsive elements, several CCAAT enhancer binding protein (C/EBP) sites, hepatocyte nuclear factor (HNF) sites, and NF-kappaB sites in this region. Subsequently, semi-quantitative analysis showed that AQP3 selectively expressed in spleen, stomach, kidney and lung. AQP7 and AQP9 were ubiquitously detected in all tissues examined and highly expressed in adipose tissue and liver, respectively. Finally, both AQP3 and AQP7 were assigned to chromosome 10q while AQP9 was mapped to chromosome 1q. This is the first report of molecular characterization of aquaglyceroporin in pig, which provides basic observations useful for future assessing and characterizing the role of aquaglyceroporin.

Clenbuterol inhibits SREBP-1c expression by activating CREB1.

As a beta(2)-adrenergic agonist, clenbuterol decreases body fat, but the molecular mechanism underlying this process is unclear. In the present study, we treated 293T and L-02 cells with clenbuterol and found that clenbuterol downregulates SREBP-1c expression and upregulates CREB1 expression. Considering SREBP-1c has the function of regulating the transcription of several lipogenic enzymes, we considered that the downregulation of SREBP-1c is responsible for body fat reduction by clenbuterol. Many previous studies have found that clenbuterol markedly increases intracellular cAMP levels, therefore, we also investigated whether CREB1 is involved in this process. The data from our experiments indicate that CREB1 overexpression inhibits SREBP-1c transcription, and that this action is antagonized by CREB2, a competitive inhibitor of CREB1. Furthermore, since PPARs are able to repress SREBP-1c transcription, we investigated whether clenbuterol and CREB1 function via a pathway involving PPAR activation. However, our results showed that clenbuterol or CREB1 overexpression suppressed PPARs transcription in 293T and L-02 cells, which suggested that they impair SREBP-1c expression in other ways.

AtCesA8-driven OsSUS3 expression leads to largely enhanced biomass saccharification and lodging resistance by distinctively altering lignocellulose features in rice.

BACKGROUND: Biomass recalcitrance and plant lodging are two complex traits that tightly associate with plant cell wall structure and features. Although genetic modification of plant cell walls can potentially reduce recalcitrance for enhancing biomass saccharification, it remains a challenge to maintain a normal growth with enhanced biomass yield and lodging resistance in transgenic plants. Sucrose synthase (SUS) is a key enzyme to regulate carbon partitioning by providing UDP-glucose as substrate for cellulose and other polysaccharide biosynthesis. Although SUS transgenic plants have reportedly exhibited improvement on the cellulose and starch based traits, little is yet reported about SUS impacts on both biomass saccharification and lodging resistance. In this study, we selected the transgenic rice plants that expressed OsSUS3 genes when driven by the AtCesA8 promoter specific for promoting secondary cell wall cellulose synthesis in Arabidopsis. We examined biomass saccharification and lodging resistance in the transgenic plants and detected their cell wall structures and wall polymer features. RESULTS: During two-year field experiments, the selected AtCesA8::SUS3 transgenic plants maintained a normal growth with slightly increased biomass yields. The four independent transgenic lines exhibited much higher biomass enzymatic saccharification and bioethanol production under chemical pretreatments at P < 0.01 levels, compared with the controls of rice cultivar and empty vector transgenic line. Notably, all transgenic lines showed a consistently enhanced lodging resistance with the increasing extension and pushing forces. Correlation analysis suggested that the reduced cellulose crystallinity was a major factor for largely enhanced biomass saccharification and lodging resistance in transgenic rice plants. In addition, the cell wall thickenings with the increased cellulose and hemicelluloses levels should also contribute to plant lodging resistance. Hence, this study has proposed a mechanistic model that shows how OsSUS3 regulates cellulose and hemicelluloses biosyntheses resulting in reduced cellulose crystallinity and increased wall thickness, thereby leading to large improvements of both biomass saccharification and lodging resistance in transgenic rice plants. CONCLUSIONS: This study has demonstrated that the AtCesA8::SUS3 transgenic rice plants exhibited largely improved biomass saccharification and lodging resistance by reducing cellulose crystallinity and increasing cell wall thickness. It also suggests a powerful genetic approach for cell wall modification in bioenergy crops.

Resistin overexpression impaired glucose tolerance in hepatocytes.

Resistin is a 12.5-kDa cysteine-rich protein secreted from adipose tissue and is an important factor linking obesity with insulin resistance. Here, we investigated the effect of resistin on glucose tolerance in adult human hepatocytes (L-02 cells). In this study, resistin cDNA was transfected into L-02 cells, and glucose concentration and glucokinase activity were determined subsequently. The data indicated resistin impaired, insulin-stimulated glucose utilization, which implied liver was a target tissue of resistin. To understand its molecular mechanism, mRNA levels of key genes in glucose metabolism and insulin signaling pathway were analyzed. The results demonstrated resistin-stimulated expression of glucose-6-phosphatase (G6Pase), sterol regulatory element-binding protein 1c (SREBP1c) and suppressor of cytokine signaling 3 (SOCS-3), repressed expression of peroxisome proliferator-activated receptor gamma (PPARgamma) as well as insulin receptor substrate 2 (IRS-2). Given that glucokinase (GK) activity and glucose transporter 2 (GLUT2) expression were not altered, we presumed that resistin did not effect them. Moreover, resistin lowered mRNA levels of IRS-2 while stimulating SOCS-3 expression, which suggests it impairs glucose tolerance by blocking the insulin signal transduction pathway.

Proteomic profiling of cellulase-aid-extracted membrane proteins for functional identification of cellulose synthase complexes and their potential associated- components in cotton fibers.

Cotton fibers are an excellent model for understanding of cellulose biosynthesis in higher plants. In this study, we determined a high cellulose biosynthesis activity in vitro by optimizing biochemical reaction conditions in cotton fibers. By adding a commercial cellulase enzyme into fibers extraction process, we extracted markedly higher levels of GhCESA1 and GhCESA8 proteins and observed an increase in ß-1,4-glucan and ß-1,3-glucan products in vitro. LC-MS/MS analysis of anti-GhCESA8-immunoprecipitated proteins showed that 19 proteins could be found in three independent experiments including four CESAs (GhCESA1,2,7,8), five well-known non-CESA proteins, one callose synthase (CALS) and nine novel proteins. Notably, upon the cellulase treatment, four CESAs, one CALS and four novel proteins were measured at relatively higher levels by calculating total peptide counts and distinct peptide numbers, indicating that the cellulase-aid-extracted proteins most likely contribute to the increase in ß-glucan products in vitro. These results suggest that the cellulase treatment may aid to release active cellulose synthases complexes from growing glucan chains and make them more amenable to extraction. To our knowledge, it is the first time report about the functional identification of the potential proteins that were associated with plant cellulose and callose synthases complexes by using the cellulase-aided protein extraction.

Positive selection drives adaptive diversification of the 4-coumarate: CoA ligase (4CL) gene in angiosperms.

Lignin and flavonoids play a vital role in the adaption of plants to a terrestrial environment. 4-Coumarate: coenzyme A ligase (4CL) is a key enzyme of general phenylpropanoid metabolism which provides the precursors for both lignin and flavonoids biosynthesis. However, very little is known about how such essential enzymatic functions evolve and diversify. Here, we analyze 4CL sequence variation patterns in a phylogenetic framework to further identify the evolutionary forces that lead to functional divergence. The results reveal that lignin-biosynthetic 4CLs are under positive selection. The majority of the positively selected sites are located in the substrate-binding pocket and the catalytic center, indicating that nonsynonymous substitutions might contribute to the functional evolution of 4CLs for lignin biosynthesis. The evolution of 4CLs involved in flavonoid biosynthesis is constrained by purifying selection and maintains the ancestral role of the protein in response to biotic and abiotic factors. Overall, our results demonstrate that protein sequence evolution via positive selection is an important evolutionary force driving adaptive diversification in 4CL proteins in angiosperms. This diversification is associated with adaption to a terrestrial environment.