Biology subjects

Dhaubhadel, S.

Publications and source records attributed to Dhaubhadel, S..

5 recordsLinked to original sources

bioRxiv2026

Nucleoporin1 maintains male germ unit organization and transport in Arabidopsis pollen tubes, likely through shaping nuclear morphology

The male germ unit (MGU) in Arabidopsis pollen is comprised of one vegetative nucleus (VN) and two sperm nuclei (SN). It is evolutionarily specialized to deliver immotile sperm nuclei to an ovule for fertilization. Despite some progress in research on MGU, its organization and transport remain only partially understood. Here, we identified Nucleoporin1/136 as a new player in the structural organization and positioning of MGU in pollen tubes. We and others have previously reported the reduced fertility of nup1-1 plants; however, the mechanism remains unknown. In this work, we further examined the role of NUP1 in fertility using two mutant alleles, nup1-1 and nup1-2-/+. The reciprocal crosses between the nup1 mutants and the Col-0 wild type indicate that the nup1 mutant pollen is defective. To study the effect of a complete NUP1 knockout on pollen, we generated a transgenic line that produces pollen with and without NUP1 expression. This work led to the surprising discovery that the NUP1 protein is inherited from the pollen mother cell to the daughter cell during microgametophyte development. Subsequent in vitro experiments showed that NUP1 is required for pollen germination and pollen tube elongation. Further microscopic studies demonstrated that NUP1 is highly expressed in VN and essential for maintaining nuclear shape and size. We also demonstrated that NUP1 is required for proper MGU organization and transport, likely through maintaining VN morphology. Notably, our finding of nuclear morphology-mediated regulation of MGU may also explain the mechanistic details underlying the defective MGU movement in previously reported mutants such as kaku4, wit, and wip, which have abnormal nuclear morphology.

plant biology↗

bioRxiv2026

Multi-Substrate Specificity of Isoflavone hydroxylases (GmIFH) Drive Isoflavonoid Diversification in Soybean

Isoflavone hydroxylases (IFHs, CYP81E) convert isoflavone aglycones into their respective hydroxylated intermediates, which direct legume isoflavones into specialized defense pathways. In soybean, their functions have been studied mostly in the context of the daidzein-derived glyceollin biosynthesis. Here we combine metabolomics-guided feature mining, phylogenetic analysis, heterologous enzymology, structural elucidation, and in planta metabolite validation to determine the functional landscape of the soybean IFH family. Analysis of a soybean isoflavonoid-enriched metabolomic dataset revealed unidentified hydroxyisoflavone features that co-accumulated with glyceollins, indicating branch chemistry that is not well-recognized. The systematic characterization of the repertoire of soybean CYP81E has demonstrated that 9 out of 11 GmIFHs are catalytically active and collectively span both 2'- and 3'- hydroxylation of the major soybean isoflavone aglycones. Among them, GmIFH9A showed broad substrate scope and regioselectivity, yielding canonical and previously unknown hydroxylated isoflavone products. NMR and LC-MS/MS were used to identify and validate the hydroxylated isoflavone products as 2'-hydroxyglycitein and 2'-hydroxyformononetin, whose presence was also confirmed in soybean roots, thus confirming two of the hidden soybean isoflavonoid network metabolites. Kinetic studies also indicated that, although the majority of GmIFHs prefer daidzein and genistein as substrates, a few isoforms are active towards methoxylated isoflavones as well, indicating functional divergence in this expanded family. Our findings collectively redefine soybean IFHs as a multi-functional enzyme module that expands the hydroxyisoflavone chemical space and reveals new biosynthetic entry points beyond canonical glyceollin pathway.

biochemistry↗

bioRxiv2025

Identification and Functional Characterization of Isoflavone Synthase Gene Family in Pea (Pisum sativum): The Entry Point to Pisatin Biosynthesis

Isoflavone synthase (IFS), a cytochrome P450 monooxygenase of the CYP93C subfamily, catalyzes the conversion of flavanones into isoflavones, the first committed step in the biosynthesis of isoflavonoid phytoalexins. In pea (Pisum sativum L.), the phytoalexin pisatin plays a pivotal role in defense against pathogens. However, the molecular basis underlying IFS function in pea remains poorly understood. In this study, we performed a comprehensive genome-wide identification and characterization of IFS genes in pea. Three IFS candidates, PsIFS7A, PsIFS7B, and PsIFS7C, were identified that reside on chromosome 7, each harboring all conserved cytochrome P450 signature motifs. PsIFS genes exhibited predominant expression in root tissue, with transcript levels induced rapidly upon Aphanomyces euteiches infection. Enzymatic assays confirmed their catalytic activity in converting the flavanones naringenin and liquiritigenin into the isoflavones genistein and daidzein, respectively, both in vitro and in planta systems. Furthermore, all three PsIFS genes were found in close proximity to quantitative trait loci (QTL) associated with Aphanomyces root rot resistance. Together, these findings provide novel insights into the IFS gene family in pea and lay a foundation for metabolic engineering or molecular breeding strategies to enhance disease resistance through targeted modulation of pisatin biosynthesis.

biochemistry↗

bioRxiv2025

Investigating postharvest seed coat darkening in common beans: a molecular perspective beyond the major P gene

Many market classes of common beans (Phaseolus vulgaris) have a significant reduction in crop value due to the postharvest darkening of the seed coat. Seed coat darkening is caused by an elevated accumulation and oxidation of proanthocyanidins (PAs). In common bean, the major color gene P encodes for a bHLH protein with its Psdallele controlling the postharvest slow darkening seed coat trait. In the present investigation, we determined that P, PvMYB3A, and PvWD9 are the essential components that form a protein complex and regulate PA biosynthesis in pinto beans. P does not bind with the PA biosynthetic gene Anthocyanin Reductase (PvANR) promoter but regulates its expression by interacting with PvMYB3A, which directly binds to the PvANR promoter. PvWD9 is an essential member of the core protein complex, which requires one or more additional plant components in order to interact with its partner proteins P and PvMYB3A and create a functioning complex. The Psd isoform affects the accumulation of PA by functioning in a similar manner to its isoform P, albeit at a lower efficiency. Understanding the regulation of PA biosynthesis in common beans helps to explain variances in seed coat color and issues associated with darkening after harvest. HighlightColor and pattern of the common bean seed coat are important traits in bean breeding, which is determined by level of proanthocyanidins (PA). This study provides experimental evidence for the regulation of PA biosynthesis in common beans by the P-PvMYB3A-PvWD9 complex together with some yet unknown associated protein(s). In the slow darkening beans, substitution of P with its slow dakening isoform Psd reduces the target biosynthetic gene expression inflencing PA production.

molecular biology↗

bioRxiv2024

Discovery of the missing cytochrome P450 monooxygenase cyclases that conclude glyceollin biosynthesis in soybean

Glyceollins are isoflavonoid-derived metabolites produced by soybean that hold great promise in improving human and animal health due to their antimicrobial, and other medicinal properties. They play important roles in agriculture by defending soybean against one of its most destructive pathogens, Phytophthora sojae. Longstanding research efforts have focused on improving accessibility to glyceollins, yet chemical synthesis remains uneconomical. The fact that some of the key genes involved in the final step of glyceollin biosynthesis have not been identified, engineering the accumulation of these important compounds in microbes is not yet possible. Although the activity of a P450 cyclase was inferred to catalyze the final committed step in glyceollin biosynthesis forty years ago, the enzyme in question has never been conclusively identified. This study reports, for the first time, the identification of three cytochrome P450 monooxygenase cyclases that catalyze the final steps of glyceollin biosynthesis. Utilizing P. sojae-soybean transcriptome data, along with genome mining tools and co-expression network analysis, we have identified 16 candidate glyceollin synthases (GmGS). Heterologous expression of these candidate genes in yeast, coupled with in vitro enzyme assays, enabled us to discover three enzymes capable of producing two glyceollin isomers. GmGS11A and GmGS11B catalyzed the conversion of glyceollidin to glyceollin I, whereas GmGS13A converted glyceocarpin to glyceollin III. The functionality of these candidates was further confirmed in planta through gene silencing and overexpression in soybean hairy roots. This groundbreaking study not only contributes to the understanding of glyceollin biosynthesis, but also demonstrates a new synthetic biology strategy that could potentially be scaled up to produce valuable molecules for crop and disease management.

biochemistry↗