Background: Asparagus officinalis L. is a worldwide cultivated vegetable enrichened in both nutrient and steroidal saponins with multiple pharmacological activities. The upstream biosynthetic pathway of steroidal saponins (USSP) for cholesterol (CHOL) synthesis has been studied, while the downstream pathway of steroidal saponins (DSSP) starting from cholesterol and its regulation in asparagus remains unknown. Results: Metabolomics, Illumina RNAseq, and PacBio IsoSeq strategies were applied to different organs of both cultivated green and purple asparagus to detect the steroidal metabolite profiles & contents and to screen their key genes for biosynthesis and regulation. The results showed that there is a total of 427 compounds, among which 18 steroids were detected with fluctuated concentrations in roots, spears and flowering twigs of two garden asparagus cultivars. The key genes of DSSP include; steroid-16-hydroxylase (S16H), steroid-22-hydroxylase (S22H) and steroid-22-oxidase-16-hydroxylase (S22O-16H), steroid-26-hydroxylase (S26H), steroid-3-β-glycosyltransferase (S3βGT) and furostanol glycoside 26-O-beta-glucosidases (F26GHs) which were correlated with the contents of major steroidal saponins were screened, and the transcriptional factors (TFs) co-expressing with the resulted from synthetic key genes, including zinc fingers (ZFs), MYBs and WRKYs family genes were also screened. Conclusions: Based on the detected steroidal chemical structures, profiles and contents which correlated to the expressions of screened synthetic and TFs genes, the full steroidal saponin synthetic pathway (SSP) of asparagus, including its key regulation networks was proposed for the first time.

Agrobacterium sp. ATCC31749 is an efficient curdlan producer at low pH and under nitrogen starvation. The helix-turn-helix transcriptional regulatory protein (crdR) essential for curdlan production has been analyzed, but whether crdR directly acts to cause expression of the curdlan biosynthesis operon (crdASC) is uncertain. To elucidate the molecular function of crdR in curdlan biosynthesis, we constructed a crdR knockout mutant along with pBQcrdR and pBQNcrdR vectors with crdR expression driven by a T5 promoter and crdR native promoter, respectively. Also, we constructed a pAG with the green fluorescent protein (GFP) gene driven by a curdlan biosynthetic operon promoter (crdP) to measure the effects of crdR expression on curdlan biosynthesis. Compared with wild-type (WT) strain biomass production, the biomass of the crdR knockout mutant was not significantly different in either exponential or stationary phases of growth. Mutant cells were non-capsulated and planktonic and produced significantly less curdlan. WT cells were curdlan-capsulated and aggregated in the stationery phase. pBQcrdR transformed to the WT strain had a 38% greater curdlan yield and pBQcrdR and pBQNcrdR transformed to the crdR mutant strain recovered 18% and 105% curdlan titers of the WT ATCC31749 strain, respectively. Consistent with its function of promoting curdlan biosynthesis, curdlan biosynthetic operon promoter (crdP) controlled GFP expression caused the transgenic strain to have higher GFP relative fluorescence in the WT strain, and no color change was observed with low GFP relative fluorescence in the crdR mutant strain as evidenced by fluorescent microscopy and spectrometric assay. q-RT-PCR revealed that crdR expression in the stationary phase was greater than in the exponential phase, and crdR overexpression in the WT strain increased crdA, crdS, and crdC expression. We also confirmed that purified crdR protein can specifically bind to the crd operon promoter region, and we inferred that crdR directly acts to cause expression of the curdlan biosynthesis operon (crdASC). CrdR is a positive transcriptional regulator of the crd operon for promoting curdlan biosynthesis in ATCC31749. The potential binding region of crdR is located within the −98 bp fragment upstream from the crdA start codon

Introduction: Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) is a key enzyme in the terpenoid biosynthetic pathway, playing a crucial role in plant stress responses. However, the HMGR gene family in Asparagus taliensis, a traditional Chinese medicinal herb with high steroidal saponin content and strong stress tolerance, remains poorly understood. This study investigates the stress response mechanisms of the HMGR gene family in A. taliensis under abiotic stress conditions.Methods: A comprehensive genome-wide analysis of the HMGR gene family in A. taliensis was conducted. The analysis included chromosomal localization, phylogenetic tree construction, linear analysis, gene structure characterization, motif distribution, cis-acting elements, and protein structure. Candidate AtaHMGR10 gene were overexpressed in Arabidopsis thaliana to analyze phenotypic changes under osmotic and salt stress, including seed germination rate and primary root length. Physiological parameters were also analyzed, and gene expression was validated using qPCR under drought, osmotic, and salt stress conditions.Results: A total of 18 HMGR gene family members were identified in A. taliensis. The functions and evolution of AtaHMGR genes are conserved. AtaHMGR10 was selected as a promising candidate due to its unique expression profile. Docking analysis revealed that AtaHMGR10 has conserved motifs for binding both HMG-CoA and NADPH/NADH, showing equal affinity for both. Overexpression of AtaHMGR10 in transgenic A. thaliana enhanced tolerance to abiotic stresses, as evidenced by higher germination rates, improved primary root length, increased chlorophyll and proline levels, enhanced peroxidase (POD) and catalase (CAT) activities, and reduced malondialdehyde (MDA) content compared to non-transgenic plants under stress conditions.Discussion: These findings highlight the role of AtaHMGR10 in enhancing plant stress tolerance, particularly in combating drought, osmotic, and salt stress. This understanding of its potential function provides avenues for improving crop resilience to abiotic stress through future gene modification.

Asparagus L., widely distributed in the old world is a genus under Asparagaceae, Asparagales. The species of the genus were mainly used as vegetables, traditional medicines as well as ornamental plants. However, the evolution and functions of mitochondrial (Mt) genomes (mitogenomes) remains largely unknown. In this study, the typical herbal medicine A. taliensis and ornamental plant A. setaceus were used to assemble and annotate the mitogenomes, and the resulting mitogenomes were further compared with published mitogenome of A. officinalis for the analysis of their functions in the context of domestication and adaptative evolution. The mitochondrial genomes of both A. taliensis and A. setaceus were assembled as complete circular ones. The phylogenetic trees based on conserved protein-coding genes of Mt genomes and whole chloroplast (Cp) genomes showed that, the phylogenetic relationship of the sampled 13 species of Asparagus L. were not exactly consistent. The collinear analyses between the nuclear (Nu) and Mt genomes confirmed the existence of mutual horizontal genes transfers (HGTs) between Nu and Mt genomes within these species. Based on RNAseq data, the Mt RNA editing were predicted and atp1 and ccmB RNA editing of A. taliensis were further confirmed by DNA sequencing. Simultaneously homologous search found 5 Nu coding gene families including pentatricopeptide-repeats (PPRs) involved in Mt RNA editing. Finally, the Mt genome variations, gene expressions and mutual HGTs between Nu and Mt were detected with correlation to the growth and developmental phenotypes respectively. The results suggest that, both Mt and Nu genomes co-evolved and maintained the Mt organella replication and energy production through TCA and oxidative phosphorylation . The assembled and annotated complete mitogenomes of both A. taliensis and A. setaceus provide valuable information for their phylogeny and concerted action of Nu and Mt genomes to maintain the energy production system of Asparagus L. in the context of domestication and adaptation to environmental niches. Mitochondrial genomes of A. taliensis and A. setaceus were assembled and annotated, and phylogenetic trees of 13 sampled species in the genus Asparagus based on both mitochondrial and chloroplast genomes showed that A. officinalis, A. taliensis and A. setaceus are represented species of Asparagus L. Independent horizontal gene transfers (HGTs) between the mitochondrial and nuclear genomes were detected with reduction in dioecious species, while Mt RNA editing rate was higher in dioecious species with consistency of their higher copies and higher expression levels of involved nuclear coding RNA editing enzyme genes. Detected different gene copies and expression levels of both pathways of tricarboxylic acid cycle(TCA) and oxidative phosphorylation genes indicates the different efficiency of citric acid accumulation and ATP synthesis due to adaptation and/or domestication among species of A. officinalis, A. taliensis and A. setaceus.

• 204 phenolics were detected with different accumulation in roots, stems and flowering twigs of both purple and green asparagus influences their respective nutritional values and environmental adaption. • A complete synthetic pathway of phenols and flavonoids with 97 synthetic and 17 regulatory gene in transcriptional isoforms level was proposed. • Alternative splicing and fusion of phenolic synthesis and regulatory genes involved in differential accumulation of phenolic substances in various organs of asparagus were found. • MYB_9 and bHLH_2/7 were predicted as major regulatory players for anthocyanin biosynthesis in purple asparagus.