Secondary Cell Wall Research Articles

The walnut shell network: 3D visualisation of symplastic and apoplastic transport routes in sclerenchyma tissue

Main conclusionHigh symplastic connectivity via pits was linked to the lignification of the developing walnut shell. With maturation, this network lessened, whereas apoplastic intercellular space remained and became relevant for shell drying.The shell of the walnut (Juglans regia) sclerifies within several weeks. This fast secondary cell wall thickening and lignification of the shell tissue might need metabolites from the supporting husk tissue. To reveal the transport capacity of the walnut shell tissue and its connection to the husk, we visualised the symplastic and apoplastic transport routes during shell development by serial block face-SEM and 3D reconstruction. We found an extensive network of pit channels connecting the cells within the shell tissue, but even more towards the husk tissue. Each pit channel ended in a pit field, which was occupied by multiple plasmodesmata passing through the middle lamella. During shell development, secondary cell wall formation progressed towards the interior of the cell, leaving active pit channels open. In contrast, pit channels, which had no plasmodesmata connection to a neighbouring cell, got filled by cellulose layers from the inner cell wall lamellae. A comparison with other nut species showed that an extended network during sclerification seemed to be linked to high cell wall lignification and that the connectivity between cells got reduced with maturation. In contrast, intercellular spaces between cells remained unchanged during the entire sclerification process, allowing air and water to flow through the walnut shell tissue when mature. The connectivity between inner tissue and environment was essential during shell drying in the last month of nut development to avoid mould formation. The findings highlight how connectivity and transport work in developing walnut shell tissue and how finally in the mature state these structures influence shell mechanics, permeability, conservation and germination.

A flax mutant with impaired intrusive growth of phloem and xylem fibres demonstrates constitutive gravitropic response

Intrusive growth is a type of growth in which a cell exceeds the growth rate of its neighbours and intrudes between them, reaching a much greater length. This process provides plant fibres with their exceptional length. Fibres are the most abundant cell type in the mechanical tissues of plants. At the same time, the plant fibres are of fundamental importance for the production of textiles, paper, biocomposites, etc. Here we describe a mutant of flax (reduced fibre 1, rdf) in which intrusive growth of fibres is impaired in both phloem and xylem. In addition to the intrinsic differences in fibre length, the mutant is characterized by a constitutive gravitropic response, mechanical aberrations at the macro- and nanolevels, disruption of the cambium and uneven transition of xylem cells to secondary cell wall formation. Gelatinous cell walls in both phloem and xylem of mutant plants have disturbed structure and reduced elasticity. The existence of this mutant-control pair offers both prospects for finding the molecular players involved in triggering intrusive growth, cell wall thickening and for understanding the principles of plant mechanical tissue functioning.

LsNRL4 enhances photosynthesis and decreases leaf angles in lettuce.

SummaryLettuce (Lactuca sativa) is one of the most important vegetables worldwide and an ideal plant for producing protein drugs. Both well‐functioning chloroplasts that perform robust photosynthesis and small leaf angles that enable dense planting are essential for high yields. In this study, we used an F2 population derived from a cross between a lettuce cultivar with pale‐green leaves and large leaf angles to a cultivar with dark‐green leaves and small leaf angles to clone LsNRL4, which encodes an NPH3/RPT2‐Like (NRL) protein. Unlike other NRL proteins in lettuce, the LsNRL4 lacks the BTB domain. Knockout mutants engineered using CRISPR/Cas9 and transgenic lines overexpressing LsNRL4 verified that LsNRL4 contributes to chloroplast development, photosynthesis and leaf angle. The LsNRL4 gene was not present in the parent with pale‐green leaves and enlarged leaf angles. Loss of LsNRL4 results in the enlargement of chloroplasts, decreases in the amount of cellular space allocated to chloroplasts and defects in secondary cell wall biosynthesis in lamina joints. Overexpressing LsNRL4 significantly improved photosynthesis and decreased leaf angles. Indeed, the plant architecture of the overexpressing lines is ideal for dense planting. In summary, we identified a novel NRL gene that enhances photosynthesis and influences plant architecture. Our study provides new approaches for the breeding of lettuce that can be grown in dense planting in the open field or in modern plant factories. LsNRL4 homologues may also be used in other crops to increase photosynthesis and improve plant architecture.

Comparative genomic analysis of the COBRA genes in six Rosaceae species and expression analysis in Chinese white pear (Pyrus bretschneideri).

COBRA-Like (COBL) genes encode a glycosylphosphatidylinositol (GPI) anchoring protein unique to plants. In current study, 87 COBRA genes were identified in 6 Rosaceae species, including Pyrus bretschneideri (16 genes), Malus domestica (22 genes), Fragaria vesca (13 genes), Prunus mume (11 genes), Rubus occidentalis (13 genes) and Prunus avium (12 genes). We revealed the evolution of the COBRA gene in six Rosaceae species by phylogeny, gene structure, conservative sequence, hydrophobicity analysis, gene replication events and sliding window analysis. In addition, based on the analysis of expression patterns in pear fruit combined with bioinformatics, we identified PbCOBL12 and PbCOBL13 as potential genes regulating secondary cell wall (SCW) formation during pear stone cell development. This study aimed to understand the evolutionary relationship of the COBRA gene in Rosaceae species, clarify the potential function of COBRA in pear fruit development, and provide essential theoretical basis and gene resources for improving pear fruit quality through genetical modification mechanism.

Characterization, Expression Profiling, and Biochemical Analyses of the Cinnamoyl-CoA Reductase Gene Family for Lignin Synthesis in Alfalfa Plants.

Cinnamoyl-CoA reductase (CCR) is a pivotal enzyme in plant lignin synthesis, which has a role in plant secondary cell wall development and environmental stress defense. Alfalfa is a predominant legume forage with excellent quality, but the lignin content negatively affects fodder digestibility. Currently, there is limited information on CCR characteristics, gene expression, and its role in lignin metabolism in alfalfa. In this study, we identified 30 members in the CCR gene family of Medicago sativa. In addition, gene structure, conserved motif, and evolution analysis suggested MsCCR1–7 presumably functioned as CCR, while the 23 MsCCR-likes fell into three categories. The expression patterns of MsCCRs/MsCCR-likes suggested their role in plant development, response to environmental stresses, and phytohormone treatment. These results were consistent with the cis-elements in their promoters. Histochemical staining showed that lignin accumulation gradually deepened with the development, which was consistent with gene expression results. Furthermore, recombinant MsCCR1 and MsCCR-like1 were purified and the kinetic parameters were tested under four substrates. In addition, three-dimensional structure models of MsCCR1 and MsCCR-like1 proteins showed the difference in the substrate-binding motif H212(X)2K215R263. These results will be useful for further application for legume forage quality modification and biofuels industry engineering in the future.

Review: Tertiary cell wall of plant fibers as a source of inspiration in material design

The review summarizes the plant lifehacks on material design on the example of the impressive cellulose-enriched cell wall deposited by fibers of many plants. This specific cell wall type is called tertiary since it is deposited after the secondary cell wall and is very distinct in the machinery of formation and function. The basic principles of tertiary cell wall performance include: 1) original composition (two major players – cellulose microfibrils and the version of rhamnogalacturonan I that forms specific supramolecular structures); 2) original cell wall design with axial orientation of all cellulose microfibrils, pronounced lateral interactions between them and the presence of the entrapped rhamnogalacturonan I; 3) dynamic changes in cell wall supramolecular organization due to rhamnogalacturonan I modifications in muro in the course of fiber maturation; 4) the built-in sensors that trace the cell wall state; 5) incorporation of tertiary cell wall into the system with higher level of organization.

Salinity induces discontinuous protoxylem via a DELLA-dependent mechanism promoting salt tolerance in Arabidopsis seedlings.

Summary Salinity is detrimental to plants and developmental adjustments limiting salt uptake and transport is therefore important for acclimation to high salt. These parameters may be influenced by xylem morphology, however how plant root xylem development is affected by salt stress remains unclear.Using molecular and genetic techniques and detailed phenotypic analyses, we demonstrate that salt causes distinct effects on Arabidopsis seedling root xylem and reveal underlying molecular mechanisms.Salinity causes intermittent inhibition of protoxylem cell differentiation, generating protoxylem gaps, in Arabidopsis and several other eudicot seedlings. The extent of protoxylem gaps in seedlings positively correlates with salt tolerance. Reduced gibberellin signalling is required for protoxylem gap formation. Mutant analyses reveal that the xylem differentiation regulator VASCULAR RELATED NAC DOMAIN 6 (VND6), along with secondary cell wall producing and cell wall modifying enzymes, including EXPANSIN A1 (EXP1), are involved in protoxylem gap formation, in a DELLA‐dependent manner.Salt stress is likely to reduce levels of bioactive gibberellins, stabilising DELLAs, which in turn activates multiple factors modifying protoxylem differentiation. Salt stress impacts seedling survival and formation of protoxylem gaps may be a measure to enhance salt tolerance.

Transcriptional and metabolic changes associated with internode development and reduced cinnamyl alcohol dehydrogenase activity in sorghum.

The molecular mechanisms associated with secondary cell wall (SCW) deposition in sorghum remain largely uncharacterized. Here, we employed untargeted metabolomics and large-scale transcriptomics to correlate changes in SCW deposition with variation in global gene expression profiles and metabolite abundance along an elongating internode of sorghum, with a major focus on lignin and phenolic metabolism. To gain deeper insight into the metabolic and transcriptional changes associated with pathway perturbations, a bmr6 mutant [with reduced cinnamyl alcohol dehydrogenase (CAD) activity] was analyzed. In the wild type, internode development was accompanied by an increase in the content of oligolignols, p-hydroxybenzaldehyde, hydroxycinnamate esters, and flavonoid glucosides, including tricin derivatives. We further identified modules of genes whose expression pattern correlated with SCW deposition and the accumulation of these target metabolites. Reduced CAD activity resulted in the accumulation of hexosylated forms of hydroxycinnamates (and their derivatives), hydroxycinnamaldehydes, and benzenoids. The expression of genes belonging to one specific module in our co-expression analysis correlated with the differential accumulation of these compounds and contributed to explaining this metabolic phenotype. Metabolomics and transcriptomics data further suggested that CAD perturbation activates distinct detoxification routes in sorghum internodes. Our systems biology approach provides a landscape of the metabolic and transcriptional changes associated with internode development and with reduced CAD activity in sorghum.

Data mining of transcriptional biomarkers at different cotton fiber developmental stages.

Advancement of the gene expression study provides comprehensive information on pivotal genes at different cotton fiber development stages. For the betterment of cotton fiber yield and their quality, genetic improvement is a major target point for the cotton community. Therefore, various studies were carried out to understand the transcriptional machinery of fiber leading to the detailed integrative as well as innovative study. Through data mining and statistical approaches, we identified and validated the transcriptional biomarkers for staged specific differentiation of fiber. With the unique mapping read matrix of ~ 200 cotton transcriptome data and sequential statistical analysis, we identified several important genes that have a deciding and specific role in fiber cell commitment, initiation and elongation, or secondary cell wall synthesis stage. Based on the importance score and validation analysis, IQ domain 26, Aquaporin, Gibberellin regulated protein, methionine gamma lyase, alpha/beta hydrolases, and HAD-like superfamily have shown the specific and determining role for fiber developmental stages. These genes are represented as transcriptional biomarkers that provide a base for molecular characterization for cotton fiber development which will ultimately determine the high yield.

Engineering Curcumin Biosynthesis in Poplar Affects Lignification and Biomass Yield.

Lignocellulosic biomass is recalcitrant toward deconstruction into simple sugars mainly due to the presence of lignin. By engineering plants to partially replace traditional lignin monomers with alternative ones, lignin degradability and extractability can be enhanced. Previously, the alternative monomer curcumin has been successfully produced and incorporated into lignified cell walls of Arabidopsis by the heterologous expression of DIKETIDE-CoA SYNTHASE (DCS) and CURCUMIN SYNTHASE2 (CURS2). The resulting transgenic plants did not suffer from yield penalties and had an increased saccharification yield after alkaline pretreatment. Here, we translated this strategy into the bio-energy crop poplar. Via the heterologous expression of DCS and CURS2 under the control of the secondary cell wall CELLULOSE SYNTHASE A8-B promoter (ProCesA8-B), curcumin was also produced and incorporated into the lignified cell walls of poplar. ProCesA8-B:DCS_CURS2 transgenic poplars, however, suffered from shoot-tip necrosis and yield penalties. Compared to that of the wild-type (WT), the wood of transgenic poplars had 21% less cellulose, 28% more matrix polysaccharides, 23% more lignin and a significantly altered lignin composition. More specifically, ProCesA8-B:DCS_CURS2 lignin had a reduced syringyl/guaiacyl unit (S/G) ratio, an increased frequency of p-hydroxyphenyl (H) units, a decreased frequency of p-hydroxybenzoates and a higher fraction of phenylcoumaran units. Without, or with alkaline or hot water pretreatment, the saccharification efficiency of the transgenic lines was equal to that of the WT. These differences in (growth) phenotype illustrate that translational research in crops is essential to assess the value of an engineering strategy for applications. Further fine-tuning of this research strategy (e.g., by using more specific promoters or by translating this strategy to other crops such as maize) might lead to transgenic bio-energy crops with cell walls more amenable to deconstruction without settling in yield.

PtomtAPX is an autonomous lignification peroxidase during the earliest stage of secondary wall formation in Populus tomentosa Carr

At present, a cooperative process hypothesis is used to explain the supply of enzyme (class III peroxidases and/or laccases) and substrates during lignin polymerization. However, it remains elusive how xylem cells meet the needs of early lignin rapid polymerization during secondary cell wall formation. Here we provide evidence that a mitochondrial ascorbate peroxidase (PtomtAPX) is responsible for autonomous lignification during the earliest stage of secondary cell wall formation in Populus tomentosa. PtomtAPX was relocated to cell walls undergoing programmed cell death and catalysed lignin polymerization in vitro. Aberrant phenotypes were caused by altered PtomtAPX expression levels in P. tomentosa. These results reveal that PtomtAPX is crucial for catalysing lignin polymerization during the early stages of secondary cell wall formation and xylem development, and describe how xylem cells provide autonomous enzymes needed for lignin polymerization during rapid formation of the secondary cell wall by coupling with the programmed cell death process.

Overexpression of PeNAC122 gene promotes wood formation and tolerance to osmotic stress in poplars

Finding the adequate balance between wood formation and abiotic stress resistance is still an important challenge for industrial woody crops. In this study, PeNAC122, a member of the NAC transcription factor (TF) family highly expressed in xylem, was cloned from Populus euphratica. Tissue expression and β-glucuronidase (GUS) staining showed that PeNAC122 was exclusively expressed in phloem fiber and secondary xylem of stems. Subcellular and yeast transactivation assays confirmed that PeNAC122 protein existed in the nucleus and did not have transcriptional activation and inhibitory activity. Overexpression of PeNAC122 poplar lines exhibited reduced plant height, thickened xylem, and accumulated lignin content in stems, and also upregulates the expression of secondary cell wall biosynthetic genes. Moreover, overexpression of PeNAC122 lines displayed more tolerance to PEG6000-induced osmotic stress, with stronger photosynthetic performance, higher antioxidant enzyme activity, and less accumulation of reactive oxygen species in leaves, and higher expression levels of stress response genes DREB2A, RD29, and NCED3. These results indicate that PeNAC122 plays a crucial role in wood formation and abiotic stress tolerance, which, in addition to potential use in improving wood quality, provides further insight into the role of NAC family TFs in balancing wood development and abiotic stress resistance.

Tissue decay tested in modern Metasequoia leaves: Implications for early diagenesis of leaves in fossil Lagerstätten

Sedimentary deposits yielding extraordinarily-preserved fossils (known as Lagerstätten) may provide significant insights into the physiology and environments of ancient plants, particularly when the fossils represent their original characteristics with limited diagenetic modifications. To better understand molecular, isotopic, and morphological changes during the early stages of diagenesis, degradation experiments were conducted in two time series: 1) a laboratory decay series using fungi on leaves over the course of a month and 2) a natural decay series with leaves collected from different stages of leaf senescence and early diagenesis. Both experiments used modern leaves of the dawn redwood Metasequoia glyptostroboides, referred to as a “living fossil” due to the morphological stability of the genus Metasequoia over the past 100 million years. Both decay series demonstrate that microbial degradation of polysaccharides occurs on extremely short timescales and results in cell collapse, first in the exclusively cellulose-based primary cell walls and then much later in the lignin-strengthened secondary cell walls. Despite morphological and molecular changes, the stable carbon isotopic composition of bulk leaves and n-alkanes remained virtually unchanged. Together, these findings suggest that: 1) rapid burial and tissue stabilization is essential in the formation of Lagerstätte fossils, 2) polysaccharides play a key role in maintaining three-dimensional fossil leaf structures and thus polysaccharide preservation implies rapid burial and minimal microbial degradation, and 3) carbon isotope signals, including at the molecular level, altered little during diagenesis. Thus, interpretations of physiological and environmental signals from conifer leaves in Lagerstätten are not likely impacted by early diagenesis.

Rapid growth of Moso bamboo (Phyllostachys edulis): Cellular roadmaps, transcriptome dynamics, and environmental factors.

Moso bamboo (Phyllostachys edulis) shows remarkably rapid growth (114.5 cm/day), but the underlying biological mechanisms remain unclear. After examining more than 12,750 internodes from more than 510 culms from 17 Moso populations, we identified internode 18 as a representative internode for rapid growth. This internode includes a 2-cm cell division zone (DZ), a cell elongation zone up to 12 cm, and a secondary cell wall (SCW) thickening zone. These zones elongated 11.8 cm, produced approximately 570,000,000 cells, and deposited ∼28 mg g−1 dry weight (DW) lignin and ∼44 mg g−1 DW cellulose daily, far exceeding vegetative growth observed in other plants. We used anatomical, mathematical, physiological, and genomic data to characterize development and transcriptional networks during rapid growth in internode 18. Our results suggest that (1) gibberellin may directly trigger the rapid growth of Moso shoots, (2) decreased cytokinin and increased auxin accumulation may trigger cell DZ elongation, and (3) abscisic acid and mechanical pressure may stimulate rapid SCW thickening via MYB83L. We conclude that internode length involves a possible tradeoff mediated by mechanical pressure caused by rapid growth, possibly influenced by environmental temperature and regulated by genes related to cell division and elongation. Our results provide insight into the rapid growth of Moso bamboo.

Nanoscale Mechanism of Moisture-Induced Swelling in Wood Microfibril Bundles.

Understanding nanoscale moisture interactions is fundamental to most applications of wood, including cellulosic nanomaterials with tailored properties. By combining X-ray scattering experiments with molecular simulations and taking advantage of computed scattering, we studied the moisture-induced changes in cellulose microfibril bundles of softwood secondary cell walls. Our models reproduced the most important experimentally observed changes in diffraction peak locations and widths and gave new insights into their interpretation. We found that changes in the packing of microfibrils dominate at moisture contents above 10–15%, whereas deformations in cellulose crystallites take place closer to the dry state. Fibrillar aggregation is a significant source of drying-related changes in the interior of the microfibrils. Our results corroborate the fundamental role of nanoscale phenomena in the swelling behavior and properties of wood-based materials and promote their utilization in nanomaterials development. Simulation-assisted scattering analysis proved an efficient tool for advancing the nanoscale characterization of cellulosic materials.

PtrWOX13A Promotes Wood Formation and Bioactive Gibberellins Biosynthesis in Populus trichocarpa.

WUSCHEL-related homeobox (WOX) genes are plant-specific transcription factors (TFs) involved in multiple processes of plant development. However, there have hitherto no studies on the WOX TFs involved in secondary cell wall (SCW) formation been reported. In this study, we identified a Populus trichocarpa WOX gene, PtrWOX13A, which was predominantly expressed in SCW, and then characterized its functions through generating PtrWOX13A overexpression poplar transgenic lines; these lines exhibited not only significantly enhanced growth potential, but also remarkably increased SCW thicknesses, fiber lengths, and lignin and hemicellulose contents. However, no obvious change in cellulose content was observed. We revealed that PtrWOX13A directly activated its target genes through binding to two cis-elements, ATTGATTG and TTAATSS, in their promoter regions. The fact that PtrWOX13A responded to the exogenous GAs implies that it is responsive to GA homeostasis caused by GA inactivation and activation genes (e.g., PtrGA20ox4, PtrGA2ox1, and PtrGA3ox1), which were regulated by PtrWOX13A directly or indirectly. Since the master switch gene of SCW formation, PtrWND6A, and lignin biosynthesis regulator, MYB28, significantly increased in PtrWOX13A transgenic lines, we proposed that PtrWOX13A, as a higher hierarchy TF, participated in SCW formation through controlling the genes that are components of the known hierarchical transcription regulation network of poplar SCW formation, and simultaneously triggering a gibberellin-mediated signaling cascade. The discovery of PtrWOX13A predominantly expressed in SCW and its regulatory functions in the poplar wood formation has important implications for improving the wood quality of trees via genetic engineering.

Flexible and digestible wood caused by viral-induced alteration of cell wall composition

Woody plant material represents a vast renewable resource that has the potential to produce biofuels and other bio-based products with favorable net CO2 emissions.1,2 Its potential has been demonstrated in a recent study that generated novel structural materials from flexible moldable wood.3 Apple rubbery wood (ARW) disease is the result of a viral infection that causes woody stems to exhibit increased flexibility.4 Although ARW disease is associated with the presence of an RNA virus5 known as apple rubbery wood virus (ARWV), how the unique symptoms develop is unknown. We demonstrate that the symptoms of ARWV infections arise from reduced lignification within the secondary cell wall of xylem fibers and result in increased wood digestibility. In contrast, the mid-lamellae region and xylem ray cells are largely unaffected by the infection. Gene expression and proteomic data from symptomatic xylem clearly show the downregulation of phenylalanine ammonia lyase (PAL), the enzyme catalyzing the first committed step in the phenylpropanoid pathway leading to lignin biosynthesis. A large increase in soluble phenolics in symptomatic xylem, including the lignin precursor phenylalanine, is also consistent with PAL downregulation. ARWV infection results in the accumulation of many host-derived virus-activated small interfering RNAs (vasiRNAs). PAL-derived vasiRNAs are among the most abundant vasiRNAs in symptomatic xylem and are likely the cause of reduced PAL activity. Apparently, the mechanism used by the virus to alter lignin exhibits similarities to the RNAi strategy used to alter lignin in genetically modified trees to generate comparable improvements in wood properties.6-8.

A semi-thin section technique-based approach to quantify the xylem secondary cell wall deposition process

Summary Identification of wood formation, cell wall deposition, lignification, and the maturation process contribute to a better understanding of biomass accumulation processes. Traditional methods for studying xylem development are limited by dyeing effects and discrimination experience, are non-quantitative for the degree of cell wall deposition and lignification, or are unsuitable for broad-leaved trees. In this study, we integrated several already existing methods to improve the discriminative accuracy of the cell development stage and to quantitatively describe the cell wall deposition and lignification degree for both softwood and hardwood tree species. To do this, we collected tree microcores every 7–14 days during a growing season for two species, one conifer (Platycladus orientalis) and one broad-leaved tree (Acer truncatum), in the mountainous areas in Beijing, China. We tracked the xylem development using semi-thin section technology combined with polarization microscopy. This integrated approach allows a quantitative description of the xylem cell wall deposition and lignification process of both hardwood and softwood tree species. This approach can be applied to demonstrate the dynamic process between the cambium layer and the production of wood cells and to describe the cell wall deposition and lignification process of wood cells from generation to maturation. This approach has certain application prospects for exploring scientific issues related to wood formation and accumulation processes in forestry and ecological studies.

MiR395c Regulates Secondary Xylem Development Through Sulfate Metabolism in Poplar.

Secondary xylem development requires the coordination of multiple regulatory factors, including plant hormones, transcription factors, and microRNAs (miRNAs). MiR395 is an important regulator involved in sulfate metabolism, but its function in plant development is unclear. This study investigated the functions of miR395c in the secondary xylem development in Populus alba × P. glandulosa. MiR395c was highly expressed in the shoot apex and secondary xylem. The overexpression of miR395c resulted in an increase in both secondary xylem width and vessel dimension, as well as a decrease in the thickness of the secondary cell wall of the xylem fiber. Further analysis showed that miR395c inhibited biosynthesis of sulfate metabolic products by targeting ATPS genes, which led to the reduction of Abscisic acid (ABA) synthesis and down-regulation of MYB46 expression. Our results indicate that miR395c regulates the secondary xylem development process via sulfate metabolism in Populus.

Comprehensive analysis of the MYB transcription factor gene family in Morus alba

BackgroundThe V-myb myeloblastosis viral oncogene homolog (MYB) family of proteins is large, containing functionally diverse transcription factors. However, MYBs in Morus are still poorly annotated and a comprehensive functional analysis of these transcription factors is lacking.ResultsIn the present study, a genome-wide identification of MYBs in Morus alba was performed. In total 166 MaMYBs were identified, including 103 R2R3-MYBs and four 3R-MaMYBs. Comprehensive analyses, including the phylogenetic analysis with putative functional annotation, motif and structure analysis, gene structure organization, promoter analysis, chromosomal localization, and syntenic relationships of R2R3-MaMYBs and 3R-MaMYBs, provided primary characterization for these MaMYBs. R2R3-MaMYBs covered the subgroups reported for R2R3-MYBs in Arabidopsis and Populus, and had two Morus-specific subgroups, indicating the high retention of MYBs in Morus. Motif analysis revealed high conservative residues at the start and end of each helix and residues consisting of the third helix in R2 and R3 repeats. Thirteen intron/exon patterns (a–m) were summarized, and the intron/exon pattern of two introns with phase numbers of 0 and 2 was the prevalent pattern for R2R3-MaMYBs. Various cis-elements in promoter regions were identified, and were mainly related to light response, development, phytohormone response, and abiotic and biotic stress response and secondary metabolite production. Expression patterns of R2R3-MaMYBs in different organs showed that MaMYBs involved in secondary cell wall components and stress responsiveness were preferentially expressed in roots or stems. R2R3-MaMYBs involved in flavonoid biosynthesis and anthocyanin accumulation were identified and characterized based on functional annotation and correlation of their expression levels with anthocyanin contents.ConclusionBased on a comprehensive analysis, this work provided functional annotation for R2R3-MYBs and an informative reference for further functional dissection of MYBs in Morus.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12870-022-03626-5.