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Dirigent proteins (DIRs) are a new class of proteins that were identified during the 8–8′ lignan biosynthetic pathway and involves the formation of (+) or (−)-pinoresinol through stereoselective coupling from E-coniferyl alcohol. These proteins are known to play a vital role in the development and stress response in plants. Various studies have reported the functional and structural characterization of dirigent gene family in different plants using in silico approaches. Here, we have summarized the importance of dirigent proteins in plants and their role in plant stress tolerance by analyzing the genome-wide analysis including gene structure, mapping of chromosomes, phylogenetic evolution, conserved motifs, gene structure, and gene duplications in important plants. Overall, this review would help to compare and clarify the molecular and evolutionary characteristics of dirigent gene family in different plants. [ABSTRACT FROM AUTHOR]

Abstract Background Dirigent (DIR) proteins mediate regioselectivity and stereoselectivity during lignan biosynthesis and are also involved in lignin, gossypol and pterocarpan biosynthesis. This gene family plays a vital role in enhancing stress resistance and in secondary cell-wall development, but systematical understanding is lacking in cotton. Results In this study, 107 GbDIRs and 107 GhDIRs were identified in Gossypium barbadense and Gossypium hirsutum, respectively. Most of these genes have a classical gene structure without intron and encode proteins containing a signal peptide. Phylogenetic analysis showed that cotton DIR genes were classified into four distinct subfamilies (a, b/d, e, and f). Of these groups, DIR-a and DIR-e were evolutionarily conserved, and segmental and tandem duplications contributed equally to their formation. In contrast, DIR-b/d mainly expanded by recent tandem duplications, accompanying with a number of gene clusters. With the rapid evolution, DIR-b/d-III was a Gossypium-specific clade involved in atropselective synthesis of gossypol. RNA-seq data highlighted GhDIRs in response to Verticillium dahliae infection and suggested that DIR gene family could confer Verticillium wilt resistance. We also identified candidate DIR genes related to fiber development in G. barbadense and G. hirsutum and revealed their differential expression. To further determine the involvement of DIR genes in fiber development, we overexpressed a fiber length-related gene GbDIR78 in Arabidopsis and validated its function in trichomes and hypocotyls. Conclusions These findings contribute novel insights towards the evolution of DIR gene family and provide valuable information for further understanding the roles of DIR genes in cotton fiber development as well as in stress responses.

Several biotic (bacterial and viral pathogenesis) and abiotic stress factors like salt, drought, cold, and extreme temperatures significantly reduce crop productivity and grain quality throughout the world. MicroRNAs (miRNAs) are small (~22 nucleotides) non-coding endogenous RNA molecules which negatively regulate gene expression at the post-transcriptional level either by degrading the target protein-coding mRNA genes or suppressing translation in plants. Dirigent (DIR) gene protein plays a crucial role as they are involved to dictate the stereochemistry of a compound synthesized by other enzymes as well as in lignifications against biotic and abiotic stress. In plants, several miRNAs, as well as their targets, are known to regulate stress response but systematic identification of the same is limited. The present work has been designed for in silico identification of miRNAs against a total of sixty-one DIR genes in Oryza sativa Indica followed by target prediction of identified miRNAs through the computational approach and thereafter validation of potential miRNAs in rice genotypes. We systematically identified 3 miRNA and their respective DIR specific target gene in Oryza sativa Indica. The expression of these three miRNAs and their respective DIR specific targets were validated in rice seedlings subjected to five different abiotic stress conditions (heavy metal, high temperature, low temperature, salinity and drought) by quantitative Real-Time PCR (qRT-PCR). Expression analysis indicated that miRNA under stress conditions regulates the gene expression of the DIR gene in rice. To the best of our knowledge this is this is the first report in any organism showing the expression of ath-miRf10317-akr, and osamiRf10761-akr miRNAs in response to various abiotic stresses.

Background: Dirigent (DIR) proteins mediate regioselectivity and stereoselectivity during lignan biosynthesis and are also involved in lignin, gossypol and pterocarpan biosynthesis. This gene family plays a vital role in enhancing stress resistance and in secondary cell-wall development, but systematical understanding is lacking in cotton. Results: In this study, 107 GbDIRs and 107 GhDIRs were identified in Gossypium barbadense and Gossypium hirsutum, respectively. Most of these genes have a classical gene structure without intron and encode proteins containing a signal peptide. Phylogenetic analysis showed that cotton DIR genes were classified into four distinct subfamilies (a, b/d, e, and f). Of these groups, DIR-a and DIR-e were evolutionarily conserved, and segmental and tandem duplications contributed equally to their formation. In contrast, DIR-b/d mainly expanded by recent tandem duplications, accompanying with a number of gene clusters. With the rapid evolution, DIR-b/d-III was a Gossypium-specific clade involved in atropselective synthesis of gossypol. RNA-seq data highlighted GhDIRs in response to Verticillium dahliae infection and suggested that DIR gene family could confer Verticillium wilt resistance. We also identified candidate DIR genes related to fiber development in G. barbadense and G. hirsutum and revealed their differential expression. To further determine the involvement of DIR genes in fiber development, we overexpressed a fiber length-related gene GbDIR78 in Arabidopsis and validated its function in trichomes and hypocotyls. Conclusions: These findings contribute novel insights towards the evolution of DIR gene family and provide valuable information for further understanding the roles of DIR genes in cotton fiber development as well as in stress responses. [ABSTRACT FROM AUTHOR]

Two western red cedar pinoresinol‐lariciresinol reductase (PLR) homologues were studied to determine their enantioselective, substrate versatility, and kinetic properties. PLRs are downstream of dirigent protein engendered, coniferyl alcohol derived, stereoselective coupling to afford entry into the 8‐ and 8′‐linked furofuran lignan, pinoresinol. Our investigations showed that each PLR homolog can enantiospecifically metabolize different furofuran lignans with modified aromatic ring substituents, but where phenolic groups at both C4/C4′ are essential for catalysis. These results are consistent with quinone methide intermediate formation in the PLR active site. Site‐directed mutagenesis and kinetic measurements provided additional insight into factors affecting enantioselectivity and kinetic properties. From these data, PLRs can be envisaged to allow for the biotechnological potential of generation of various lignan skeleta, that could be differentially "decorated" on their aromatic ring substituents, via the action of upstream dirigent proteins. [ABSTRACT FROM AUTHOR]

Pea phytoalexins (-)-maackiain and (+)-pisatin have opposite C6a/C11a configurations, but biosynthetically how this occurs is unknown. Pea dirigent-protein (DP) PsPTS2 generates 7,2'-dihydroxy-4',5'-methylenedioxyisoflav-3-ene (DMDIF), and stereoselectivity toward four possible 7,2'-dihydroxy-4',5'-methylenedioxyisoflavan-4-ol (DMDI) stereoisomers was investigated. Stereoisomer configurations were determined using NMR spectroscopy, electronic circular dichroism, and molecular orbital analyses. PsPTS2 efficiently converted cis-(3R,4R)-DMDI into DMDIF 20-fold faster than the trans-(3R,4S)-isomer. The 4R-configured substrate's near β-axial OH orientation significantly enhanced its leaving group abilities in generating A-ring mono-quinone methide (QM), whereas 4S-isomer's α-equatorial-OH was a poorer leaving group. Docking simulations indicated that the 4R-configured β-axial OH was closest to Asp 51 , whereas 4S-isomer's α-equatorial OH was further away. Neither cis-(3S,4S)- nor trans-(3S,4R)-DMDIs were substrates, even with the former having C3/C4 stereochemistry as in (+)-pisatin. PsPTS2 used cis-(3R,4R)-7,2'-dihydroxy-4'-methoxyisoflavan-4-ol [cis-(3R,4R)-DMI] and C3/C4 stereoisomers to give 2',7-dihydroxy-4'-methoxyisoflav-3-ene (DMIF). DP homologs may exist in licorice (Glycyrrhiza pallidiflora) and tree legume Bolusanthus speciosus, as DMIF occurs in both species. PsPTS1 utilized cis-(3R,4R)-DMDI to give (-)-maackiain 2200-fold more efficiently than with cis-(3R,4R)-DMI to give (-)-medicarpin. PsPTS1 also slowly converted trans-(3S,4R)-DMDI into (+)-maackiain, reflecting the better 4R configured OH leaving group. PsPTS2 and PsPTS1 provisionally provide the means to enable differing C6a and C11a configurations in (+)-pisatin and (-)-maackiain, via identical DP-engendered mono-QM bound intermediate generation, which PsPTS2 either re-aromatizes to give DMDIF or PsPTS1 intramolecularly cyclizes to afford (-)-maackiain. Substrate docking simulations using PsPTS2 and PsPTS1 indicate cis-(3R,4R)-DMDI binds in the anti-configuration in PsPTS2 to afford DMDIF, and the syn-configuration in PsPTS1 to give maackiain., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Key message: Identification of DIR encoding genes in flax genome. Analysis of phylogeny, gene/protein structures and evolution. Identification of new conserved motifs linked to biochemical functions. Investigation of spatio-temporal gene expression and response to stress.Abstract: Dirigent proteins (DIRs) were discovered during 8-8′ lignan biosynthesis studies, through identification of stereoselective coupling to afford either (+)- or (−)-pinoresinols from E-coniferyl alcohol. DIRs are also involved or potentially involved in terpenoid, allyl/propenyl phenol lignan, pterocarpan and lignin biosynthesis. DIRs have very large multigene families in different vascular plants including flax, with most still of unknown function. DIR studies typically focus on a small subset of genes and identification of biochemical/physiological functions. Herein, a genome-wide analysis and characterization of the predicted flax DIR 44-membered multigene family was performed, this species being a rich natural grain source of 8-8′ linked secoisolariciresinol-derived lignan oligomers. All predicted DIR sequences, including their promoters, were analyzed together with their public gene expression datasets. Expression patterns of selected DIRs were examined using qPCR, as well as through clustering analysis of DIR gene expression. These analyses further implicated roles for specific DIRs in (−)-pinoresinol formation in seed-coats, as well as (+)-pinoresinol in vegetative organs and/or specific responses to stress. Phylogeny and gene expression analysis segregated flax DIRs into six distinct clusters with new cluster-specific motifs identified. We propose that these findings can serve as a foundation to further systematically determine functions of DIRs, i.e. other than those already known in lignan biosynthesis in flax and other species. Given the differential expression profiles and inducibility of the flax DIR family, we provisionally propose that some DIR genes of unknown function could be involved in different aspects of secondary cell wall biosynthesis and plant defense. [ABSTRACT FROM AUTHOR]

Abiotic constraints are a source of concern in agriculture, because they can have a strong impact on plant growth and development, thereby affecting crop yield. The response of plants to abiotic constraints varies depending on the type of stress, on the species and on the organs. Although many studies have addressed different aspects of the plant response to abiotic stresses, only a handful has focused on the role of the cell wall. A targeted approach has been used here to study the expression of cell wall-related genes in different organs of alfalfa plants subjected for four days to three different abiotic stress treatments, namely salt, cold and heat stress. Genes involved in different steps of cell wall formation (cellulose biosynthesis, monolignol biosynthesis and polymerization) have been analyzed in different organs of Medicago sativa L. Prior to this analysis, an in silico classification of dirigent/dirigent-like proteins and class III peroxidases has been performed in Medicago truncatula and M. sativa. The final goal of this study is to infer and compare the expression patterns of cell wall-related genes in response to different abiotic stressors in the organs of an important legume crop. [ABSTRACT FROM AUTHOR]

Aquatic plant transition to land, and subsequent terrestrial plant species diversification, was accompanied by the emergence and massive elaboration of plant phenol chemo-diversity. Concomitantly, dirigent protein (DP) and dirigent-like protein subfamilies, derived from large multigene families, emerged and became extensively diversified. DP biochemical functions as gateway entry points into new and diverse plant phenol skeletal types then markedly expanded. DPs have at least eight non-uniformly distributed subfamilies, with different DP subfamily members of known biochemical/physiological function now implicated as gateway entries to lignan, lignin, aromatic diterpenoid, pterocarpan and isoflavene pathways. While some other DP subfamily members have jacalin domains, both these and indeed the majority of DPs throughout the plant kingdom await discovery of their biochemical roles. Methods and approaches were developed to discover DP biochemical function as gateway entry points to distinct plant phenol skeletal types in land plants. Various DP 3D X-ray structural determinations enabled structure-based comparative sequence analysis and modeling to understand similarities and differences among the different DP subfamilies. We consider that the core DP β-barrel fold and associated characteristics are likely common to all DPs, with several residues conserved and nearly invariant. There is also considerable variation in residue composition and topography of the putative substrate binding pockets, as well as substantial differences in several loops, such as the β1-β2 loop. All DPs likely bind and stabilize quinone methide intermediates, while guiding distinctive regio- and/or stereo-chemical entry into Nature's chemo-diverse land plant phenol metabolic classes., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Abstract: The compatible interaction between Erysiphe necator and Vitis vinifera induces significant alterations in the host transcriptome, affecting essentially those genes involved in signalling and secondary metabolite biosynthetic pathways. The precise transcriptomic changes vary from the early events to later stages of infection. In the present work, suppressive subtraction hybridization (SSH) was used to identify several differentially expressed transcripts in symptomatic and asymptomatic leaves from powdery mildew infected grapevines following a long term interaction. The detected transcripts show little or no correlation with similar expression studies concerning the early stages of infection which suggests distinct host responses occur before and after the infection is established. The transcription level of thirteen genes was assessed through qRT-PCR using appropriately selected and validated normalization genes. With one exception, all these genes underwent moderate levels of differential transcription, with log2-fold change values ranging from −2.65 to 4.36. The exception, a dirigent-like (DIR) protein, was upregulated over 180 fold in symptomatic leaves, suggesting an important role for stereochemical selectivity in the compatible interaction E. necator–V. vinifera. DIR copy number was determined in the genome of three grapevine cultivars exhibiting high (Carignan), moderate (Fernão Pires) and low (Touriga Nacional) sensitivity to E. necator. It was found to be a two-copy gene in all cultivars analyzed. Further analysis involving DIR metabolic neighbourhood transcripts was performed. The possible physiological significance of the detected DIR upregulation is discussed. [Copyright &y& Elsevier]

Abstract: Analysis of expressed sequence tags (ESTs) and full-length (FL)cDNAs from species of spruce (Picea spp.) revealed a family of 35 unique dirigent proteins (DIR) and DIR-like proteins. Phylogenetic analysis indicates the spruce DIR and DIR-like genes cluster into three distinct subfamilies, DIR-a, DIR-b/d, and DIR-f, of a larger plant DIR and DIR-like gene family. Gene-specific primers were designed for 31 unique spruce DIR family genes, and closely related isoforms, and used to evaluate patterns of constitutive expression, as well as responses to herbivory by stem-boring insects (i.e., white pine weevil, Pissodes strobi) in bark tissue and defoliating insects (i.e., western spruce budworm, Choristoneura occidentalis) in green apical shoots. Furthermore, meta-analysis of microarray gene expression data obtained from a series of independent experiments using the same 16.7K cDNA array platform identified several distinct expression clusters of the spruce DIR transcriptome closely matching phylogenetic clusters of sequence similarity. Members of the DIR-a family, which also contains functionally characterized DIR from other plant species, are most prominent for their induced response to feeding by weevils on Sitka spruce bark. [Copyright &y& Elsevier]

Conifers are resistant to attack from a large number of potential herbivores or pathogens. Previous molecular and biochemical characterization of selected conifer defence systems support a model of multigenic, constitutive and induced defences that act on invading insects via physical, chemical, biochemical or ecological (multitrophic) mechanisms. However, the genomic foundation of the complex defence and resistance mechanisms of conifers is largely unknown. As part of a genomics strategy to characterize inducible defences and possible resistance mechanisms of conifers against insect herbivory, we developed a cDNA microarray building upon a new spruce ( Picea spp.) expressed sequence tag resource. This first-generation spruce cDNA microarray contains 9720 cDNA elements representing c. 5500 unique genes. We used this array to monitor gene expression in Sitka spruce ( Picea sitchensis) bark in response to herbivory by white pine weevils ( Pissodes strobi, Curculionidae) or wounding, and in young shoot tips in response to western spruce budworm ( Choristoneura occidentalis, Lepidopterae) feeding. Weevils are stem-boring insects that feed on phloem, while budworms are foliage feeding larvae that consume needles and young shoot tips. Both insect species and wounding treatment caused substantial changes of the host plant transcriptome detected in each case by differential gene expression of several thousand array elements at 1 or 2 d after the onset of treatment. Overall, there was considerable overlap among differentially expressed gene sets from these three stress treatments. Functional classification of the induced transcripts revealed genes with roles in general plant defence, octadecanoid and ethylene signalling, transport, secondary metabolism, and transcriptional regulation. Several genes involved in primary metabolic processes such as photosynthesis were down-regulated upon insect feeding or wounding, fitting with the concept of dynamic resource allocation in plant defence. Refined expression analysis using gene-specific primers and real-time PCR for selected transcripts was in agreement with microarray results for most genes tested. This study provides the first large-scale survey of insect-induced defence transcripts in a gymnosperm and provides a platform for functional investigation of plant–insect interactions in spruce. Induction of spruce genes of octadecanoid and ethylene signalling, terpenoid biosynthesis, and phenolic secondary metabolism are discussed in more detail. [ABSTRACT FROM AUTHOR]

This review describes discoveries from this laboratory on monolignol, allylphenol and hydroxycinnamic acid coupling, and downstream metabolic conversions, affording various lignan skeleta. Stereoselective 8-8′ coupling (dirigent protein-mediated) of coniferyl alcohol to afford (+)-pinoresinol is comprehensively discussed, as is our current mechanistic/kinetic understanding of the protein’s radical-radical binding, orientation and coupling properties, and insights gained for other coupling modes, e.g. affording (−)-pinoresinol. In a species dependent manner, (+)- or (−)-pinoresinols can also undergo enantiospecific reductions, catalyzed by various bifunctional pinoresinol-lariciresinol reductases (PLR), to afford lariciresinol and then secoisolariciresinol. With X-ray structures giving a molecular basis for differing PLR enantiospecificities, comparisons are made herein to the X-ray structure of the related enzyme, phenylcoumaran benzylic ether reductase, capable of 8-5′ linked lignan regiospecific reductions. Properties of the enantiospecific secoisolariciresinol dehydrogenase (also discovered in our laboratory and generating 8-8′ linked matairesinol) are summarized, as are both in situ hybridization and immunolocalization of lignan pathway mRNA/proteins in vascular tissues. This entire 8-8′ pathway thus overall affords secoisolariciresinol and matairesinol, viewed as cancer preventative agent precursors, as well as intermediates to cancer treating substances, such as podophyllotoxin derivatives. Another emphasis is placed on allylphenol/hydroxycinnamic acid coupling and associated downstream metabolism, e.g. affording the antiviral creosote bush lignan, nordihydroguaiaretic acid (NDGA), and the fern lignans, blechnic/brainic acids. Regiospecific 8-8′ allylphenol coupling is described, as is characterization of the first enantiospecific membrane-bound polyphenol oxidase, (+)-larreatricin hydroxylase, involved in NDGA formation. Specific [13C]-labeling also indicated that Blechnum lignans arise from stereoselective 8-2′ hydroxycinnamic acid coupling. Abbreviations: CD – circular dichroism; e.e. – enantiomeric excess; DP – dirigent protein; ESI-MS – electrospray ionization mass spectrometry; MALDI -TOF – matrix assisted laser desorption ionization-time of flight; MALLS – multiangle laser light scattering; PLR – pinoresinol lariciresinol reductase; SDH – secoisolariciresinol dehydrogenase. [ABSTRACT FROM AUTHOR]

The discovery of a nine-member multigene dirigent family involved in control of monolignol radical–radical coupling in the ancient gymnosperm, western red cedar, suggested that a complex multidimensional network had evolved to regulate such processes in vascular plants. Accordingly, in this study, the corresponding promoter regions for each dirigent multigene member were obtained by genome-walking, with Arabidopsis being subsequently transformed to express each promoter fused to the β-glucuronidase (GUS) reporter gene. It was found that each component gene of the proposed network is apparently differentially expressed in individual tissues, organs and cells at all stages of plant growth and development. The data so obtained thus further support the hypothesis that a sophisticated monolignol radical–radical coupling network exists in plants which has been highly conserved throughout vascular plant evolution. [Copyright &y& Elsevier]

Plant dirigent proteins (DIRs) control the stereoselectivity of the monolignol coniferyl alcohol radical coupling. The main mechanistic hypothesis on this chemo- and stereoselective reaction invokes a binding of coniferyl alcohol radical substrates in the dirigent protein active site so that only one enantiomeric form can be produced. We have studied the influence of the Arabidopsis thaliana AtDIR6 protein on the transient coniferyl alcohol radical by EPR. Herein, we show that AtDIR6 stabilizes coniferyl alcohol radicals prior to directing their coupling towards the formation of (-)-pinoresinol., (© 2020 Wiley-VCH GmbH.)