Characterization of two CYP80 enzymes provides insights into aporphine alkaloid skeleton formation in Aristolochia contorta.

Aporphine alkaloids are a large group of natural compounds with extensive pharmaceutical application prospects. The biosynthesis of aporphine alkaloids has been paid attentions in the past decades. Here, we determined the contents of four 1-benzylisoquinoline alkaloids and five aporphine alkaloids in root, stem, leaf, and flower of Aristolochia contorta Bunge, which belongs to magnoliids. Two CYP80 enzymes were identified and characterized from A. contorta. Both of them catalyze the unusual C-C phenol coupling reactions and directly form the aporphine alkaloid skeleton. AcCYP80G7 catalyzed the formation of hexacyclic aporphine corytuberine. AcCYP80Q8 catalyzed the formation of pentacyclic proaporphine glaziovine. Kingdom-wide phylogenetic analysis of the CYP80 family suggested that CYP80 first appeared in Nymphaeales. The functional divergence of hydroxylation and C-C (or C-O) phenol coupling preceded the divergence of magnoliids and eudicots. Probable crucial residues of AcCYP80Q8 were selected through sequence alignment and molecular docking. Site-directed mutagenesis revealed two crucial residues E284 and Y106 for the catalytic reaction. Identification and characterization of two aporphine skeleton-forming enzymes provide insights into the biosynthesis of aporphine alkaloids.

The MYB Activator WHITE PETAL1 Associates with MtTT8 and MtWD40-1 to Regulate Carotenoid-Derived Flower Pigmentation in Medicago truncatula.

Carotenoids are a group of natural tetraterpenoid pigments with indispensable roles in the plant life cycle and the human diet. Although the carotenoid biosynthetic pathway has been well characterized, the regulatory mechanisms that control carotenoid metabolism, especially in floral organs, remain poorly understood. In this study, we identified an anthocyanin-related R2R3-MYB protein, WHITE PETAL1 (WP1), that plays a critical role in regulating floral carotenoid pigmentation in Medicago truncatula Carotenoid analyses showed that the yellow petals of the wild-type M. truncatula contained high concentrations of carotenoids that largely consisted of esterified lutein and that disruption of WP1 function via Tnt1 insertion led to substantially reduced lutein accumulation. WP1 mainly functions as a transcriptional activator and directly regulates the expression of carotenoid biosynthetic genes including MtLYCe and MtLYCb through its C-terminal acidic activation motif. Further molecular and genetic analyses revealed that WP1 physically interacts with MtTT8 and MtWD40-1 proteins and that this interaction facilitates WP1's function in the transcriptional activation of both carotenoid and anthocyanin biosynthetic genes. Our findings demonstrate the molecular mechanism of WP1-mediated regulation of floral carotenoid pigmentation and suggest that the conserved MYB-basic-helix-loop-helix-WD40 regulatory module functions in carotenoid biosynthesis in M. truncatula, with specificity imposed by the MYB partner.

ATP-Binding Cassette G Transporters SGE1 and MtABCG13 Control Stigma Exsertion.

Stigma exsertion is an important agricultural trait that facilitates the application of heterosis in crop breeding. Although several quantitative trait loci associated with stigma exsertion have been fine-mapped or cloned, the underlying genetic basis, particularly in legumes, remains unclear. In this study, we identified and characterized the exserted stigma mutant stigma exsertion1 (sge1) in the model legume Medicago truncatula The exserted stigma phenotype of sge1 is mainly caused by physical interaction between floral organs, in which normal petal and stamen elongation are inhibited due to flower cuticle defects. SGE1 encodes an ATP-binding cassette G (ABCG) transporter that plays a critical role in regulating floral cutin and wax secretion in M. truncatula SGE1 physically interacts with another half-size transporter, MtABCG13, to form a functional heterodimer. Mutation of MtABCG13 results in flower cuticle defects similar to those in sge1 as well as stigma exsertion, indicating that SGE1 and MtABCG13 are indispensable for flower cuticle secretion and collaboratively control stigma exsertion in M. truncatula Our findings reveal novel functions for ABCG transporters in determining stigma exsertion by affecting the physical interactions of floral organs, providing insight into the molecular mechanism underlying stigma exsertion in leguminous plants with complex zygomorphic flowers.

HEADLESS, a WUSCHEL homolog, uncovers novel aspects of shoot meristem regulation and leaf blade development in Medicago truncatula.

The formation and maintenance of the shoot apical meristem (SAM) are critical for plant development. However, the underlying molecular mechanism of regulating meristematic cell activity is poorly understood in the model legume Medicago truncatula. Using forward genetic approaches, we identified HEADLESS (HDL), a homolog of Arabidopsis WUSCHEL, required for SAM maintenance and leaf development in M. truncatula. Disruption of HDL led to disorganized specification and arrest of the SAM and axillary meristems, resulting in the hdl mutant being locked in the vegetative phase without apparent stem elongation. hdl mutant leaves are shorter in the proximal-distal axis due to reduced leaf length elongation, which resulted in a higher blade width/length ratio and altered leaf shape, uncovering novel phenotypes undescribed in the Arabidopsis wus mutant. HDL functions as a transcriptional repressor by recruiting MtTPL through its conserved WUS-box and EAR-like motif. Further genetic analysis revealed that HDL and STENOFOLIA (STF), a key regulator of M. truncatula lamina outgrowth, act independently in leaf development although HDL could recruit MtTPL in the same manner as STF does. Our results indicate that HDL has conserved and novel functions in regulating shoot meristems and leaf shape in M. truncatula, providing new avenues for understanding meristem biology and plant development.

AGAMOUS AND TERMINAL FLOWER controls floral organ identity and inflorescence development in Medicago truncatula.

Angiosperms integrate a multitude of endogenous and environmental signals to control floral development, thereby ensuring reproductive success. Here, we report the identification of AGAMOUS AND TERMINAL FLOWER (AGTFL), a novel regulator of floral development in Medicago truncatula. Mutation of AGTFL led to the transformation of carpels and stamens into numerous sepals and petals and altered primary inflorescence identity. AGTFL encodes a nucleus-localized protein containing a putative Myb/SANT-like DNA-binding domain and a PKc kinase domain. Molecular and genetic analyses revealed that AGTFL regulates the transcription of MtAGs and MtTFL1 to control floral organ identity and inflorescence development.

The Smi-miR858a-SmMYB module regulates tanshinone and phenolic acid biosynthesis in Salvia miltiorrhiza.

Tanshinones and phenolic acids are two major classes of bioactive compounds in Salvia miltiorrhiza. Revealing the regulatory mechanism of their biosynthesis is crucial for quality improvement of S. miltiorrhiza medicinal materials. Here we demonstrated that Smi-miR858a-Smi-miR858c, a miRNA family previously known to regulate flavonoid biosynthesis, also played critical regulatory roles in tanshinone and phenolic acid biosynthesis in S. miltiorrhiza. Overexpression of Smi-miR858a in S. miltiorrhiza plants caused significant growth retardation and tanshinone and phenolic acid reduction. Computational prediction and degradome and RNA-seq analyses revealed that Smi-miR858a could directly cleave the transcripts of SmMYB6, SmMYB97, SmMYB111, and SmMYB112. Yeast one-hybrid and transient transcriptional activity assays showed that Smi-miR858a-regulated SmMYBs, such as SmMYB6 and SmMYB112, could activate the expression of SmPAL1 and SmTAT1 involved in phenolic acid biosynthesis and SmCPS1 and SmKSL1 associated with tanshinone biosynthesis. In addition to directly activating the genes involved in bioactive compound biosynthesis pathways, SmMYB6, SmMYB97, and SmMYB112 could also activate SmAOC2, SmAOS4, and SmJMT2 involved in the biosynthesis of methyl jasmonate, a significant elicitor of plant secondary metabolism. The results suggest the existence of dual signaling pathways for the regulation of Smi-miR858a in bioactive compound biosynthesis in S. miltiorrhiza.

Functional Specialization of Duplicated AGAMOUS Homologs in Regulating Floral Organ Development of Medicago truncatula.

The C function gene AGAMOUS (AG) encodes for a MADS-box transcription factor required for floral organ identity and floral meristem (FM) determinacy in angiosperms. Unlike Arabidopsis, most legume plants possess two AG homologs arose by an ancient genome duplication event. Recently, two euAGAMOUS genes, MtAGa and MtAGb, were characterized and shown to fulfill the C function activity in the model legume Medicago truncatula. Here, we reported the isolation and characterization of a new mtaga allele by screening the Medicago Tnt1 insertion mutant collection. We found that MtAGa was not only required for controlling the stamen and carpel identity but also affected pod and seed development. Genetic analysis indicated that MtAGa and MtAGb redundantly control Medicago floral organ identity, but have minimal distinct functions in regulating stamen and carpel development in a dose-dependent manner. Interestingly, the stamens and carpels are mostly converted to numerous vexillum-like petals in the double mutant of mtaga mtagb, which is distinguished from Arabidopsis ag. Further qRT-PCR analysis in different mtag mutants revealed that MtAGa and MtAGb can repress the expression of putative A and B function genes as well as MtWUS, but promote putative D function genes expression in M. truncatula. In addition, we found that the abnormal dorsal petal phenotype observed in the mtaga mtagb double mutant is associated with the upregulation of CYCLOIDEA (CYC)-like TCP genes. Taken together, our data suggest that the redundant MtAGa and MtAGb genes of M. truncatula employ a conserved mechanism of action similar to Arabidopsis in determining floral organ identity and FM determinacy but may have evolved distinct function in regulating floral symmetry by coordinating with specific floral dorsoventral identity factors.

Generation of Wheat Transcription Factor FOX Rice Lines and Systematic Screening for Salt and Osmotic Stress Tolerance.

Transcription factors (TFs) play important roles in plant growth, development, and responses to environmental stress. In this study, we collected 1,455 full-length (FL) cDNAs of TFs, representing 45 families, from wheat and its relatives Triticum urartu, Aegilops speltoides, Aegilops tauschii, Triticum carthlicum, and Triticum aestivum. More than 15,000 T0 TF FOX (Full-length cDNA Over-eXpressing) rice lines were generated; of these, 10,496 lines set seeds. About 14.88% of the T0 plants showed obvious phenotypic changes. T1 lines (5,232 lines) were screened for salt and osmotic stress tolerance using 150 mM NaCl and 20% (v/v) PEG-4000, respectively. Among them, five lines (591, 746, 1647, 1812, and J4065) showed enhanced salt stress tolerance, five lines (591, 746, 898, 1078, and 1647) showed enhanced osmotic stress tolerance, and three lines (591, 746, and 1647) showed both salt and osmotic stress tolerance. Further analysis of the T-DNA flanking sequences showed that line 746 over-expressed TaEREB1, line 898 over-expressed TabZIPD, and lines 1812 and J4065 over-expressed TaOBF1a and TaOBF1b, respectively. The enhanced salt and osmotic stress tolerance of lines 898 and 1812 was confirmed by retransformation of the respective genes. Our results demonstrate that a heterologous FOX system may be used as an alternative genetic resource for the systematic functional analysis of the wheat genome.