WUSCHEL-related Homeobox Research Articles

Comprehensive study of rice YABBY gene family: evolution, expression and interacting proteins analysis.

As plant-specific transcription regulators, YABBYs are involved in plant growth, development and stress responses. However, little information is available about genome-wide screening and identification of OsYABBY-interacting proteins. In this study, phylogenetic relationship, gene structure, protein structure and gene expression profile of eight OsYABBYs were carried out, all of which indicated that OsYABBYs were involved in different developmental processes and had functional differentiation. More importantly, PPI (protein-protein interaction) analysis and molecular docking simulation predicted that WUSCHEL-related homeobox (WOX) proteins might be interacting proteins of OsYABBYs. Yeast two-hybrid (Y2H) and luciferase complementation imaging assays (LCI) further confirmed that OsYABBYs (except for OsYABBY7) could interact with OsWOX3A in vitro and in vivo. In addition, OsYABBY3 and OsYABBY5 also could interact with OsWUS. Taken together, our results provided valuable information for further elucidating OsYABBYs regulation mechanism in improving rice performance.

FRUCTOSE INSENSITIVE1 regulates stem cell function in Arabidopsis in response to fructose signalling.

Stem cell function in different meristems of Arabidopsis is mainly defined by WUSCHEL (WUS)/WUSCHEL-RELATED HOMEOBOX (WOX) family of proteins. Sugars have also been demonstrated to play pivotal roles in stem cell function and development of plants. As a cytosolic fructose 1,6-bisphosphatase, FRUCTOSE INSENSITIVE1 (FINS1) has been demonstrated to regulate plant growth in response to fructose signalling. However, it remains to be elucidated how stem cell function is regulated in response to fructose signalling. Our work showed that FINS1 interacts with WUS/WOX protein as a complex, which further binds to the promoter of WUS/WOX and regulates its expression in response to fructose signalling. FINS1 might act as a bifunctional factor that promotes WUS/WOX expression in the presence of low concentrations of fructose, and represses WUS/WOX expression in the presence of high concentrations of fructose. Therefore, FINS1 regulates stem cell function in response to fructose signalling during the growth and development of Arabidopsis.

Identification and Expression Profile of CLE41/44-PXY-WOX Genes in Adult Trees Pinus sylvestris L. Trunk Tissues during Cambial Activity

WUSCHEL (WUS)-related homeobox (WOX) protein family members play important roles in the maintenance and proliferation of the stem cells in the cambium, the lateral meristem that forms all the wood structural elements. Most studies have examined the function of these genes in angiosperms, and very little was known about coniferous trees. Pine is one of the most critical forest-forming conifers globally, and in this research, we studied the distribution of WOX4, WOX13, and WOXG genes expression in Pinus sylvestris L. trunk tissues. Further, we considered the role of TDIF(CLE41/44)/TDR(PXY) signaling in regulating Scots pine cambial activity. The distribution of CLE41/44-PXY-WOXs gene expression in Scots pine trunk tissues was studied: (1) depending on the stage of ontogenesis (the first group of objects); and (2) depending on the stage of cambial growth (the second group of objects). The first group of objects is lingonberry pine forests of different ages (30-, 80-, and 180-year-old stands) in the middle taiga subzone. At the time of selection, all the trees of the studied groups were at the same seasonal stage of development: the formation of late phloem and early xylem was occurring in the trunk. The second group of objects is 40-year-old pine trees that were selected growing in the forest seed orchard. We took the trunk tissue samples on 27 May 2022, 21 June 2022, and 21 July 2022. We have indicated the spatial separation expressed of PsCLE41/44 and PsPXY in pine trunk tissues. PsCLE41/44 was differentially expressed in Fraction 1, including phloem cells and cambial zone. Maximum expression of the PsPXY gene occurred in Fraction 2, including differentiating xylem cells. The maximum expression of the PsCLE41/44 gene occurred on 27 May, when the number of cells in the cambial zone was the highest, and then it decreased to almost zero. The PsPXY gene transcript level increased from May to the end of July. We found that the highest transcript level of the PsWOX4 gene was during the period of active cell proliferation in the cambial zone, and also in the trees with the cambial age 63 years, which were characterized by the largest number of cell layers in the cambial zone. In this study, we have examined the expression profiles of genes belonging to the ancient clade (PsWOXG and PsWOX13) in stem tissues in Scots pine for the first time. We found that, in contrast to PsWOX4 (high expression that was observed during the period of active formation of early tracheids), the expression of genes of the ancient clade of the WOX genes was observed during the period of decreased cambial activity in the second half of the growing season. We found that PsWOX13 expression was shifted to Fraction 1 in most cases and increased from the phloem side, while PsWOXG expression was not clearly bound to a certain fraction. Based on the data, the role of the CLE41/44-PXY-WOX signaling module in regulating P. sylvestris cambial growth is discussed.

Genome-Wide Analysis of WOX Multigene Family in Sunflower (Helianthus annuus L.).

The WUSCHEL-related homeobox (WOX) is a family of specific transcription factors involved in plant development and response to stress, characterized by the presence of a homeodomain. This study represents the first comprehensive characterization of the WOX family in a member of the Asteraceae family, the sunflower (H. annuus L.). Overall, we identified 18 putative HaWOX genes divided by phylogenetic analysis in three major clades (i.e., ancient, intermediate, and WUS). These genes showed conserved structural and functional motifs. Moreover, HaWOX has homogeneously distributed on H. annuus chromosomes. In particular, 10 genes originated after whole segment duplication events, underpinning a possible evolution of this family along with the sunflower genome. In addition, gene expression analysis evidenced a specific pattern of regulation of the putative 18 HaWOX during embryo growth and in ovule and inflorescence meristem differentiation, suggesting a pivotal role for this multigenic family in sunflower development. The results obtained in this work improved the understanding of the WOX multigenic family, providing a resource for future study on functional analysis in an economically valuable species such as sunflower.

Transcription factor WOX11 regulates protein translation via ribosome protein acetylation in rice roots.

WUSCHEL-related Homeobox 11 regulates protein translation via lysine acetylation of ribosome protein mediated by ROS in rice root meristem.

Isolation and Expression of Transcription Factors Involved in Somatic Embryo Development by Transcriptome Analysis of Embryogenic Callus of Thuja koraiensis

Thuja koraiensis Nakai (Cupressaceae) is an endangered and ecologically important conifer endemic to Korea. Previously, we established a protocol for micropropagation in T. koraiensis, which involved somatic embryogenesis from embryogenic callus of T. koraiensis. However, the molecular mechanisms underlying somatic embryogenesis remain unclear. Herein, we performed transcriptomic analysis to identify somatic embryogenesis-related genes of T. koraiensis via Illumina RNA sequencing. We conducted de novo transcriptome assembly using a Trinity assembler, which produced 274,077 transcript contigs clustered into 205,843 transcripts (unigenes), with an average length of 825 base pairs. Of all the unigenes, 14.69%, 18.62%, and 7.4% had homologs in the Gene Ontology, NCBI Non-redundant Protein, and NCBI Nucleotide databases, respectively. Among these mRNA sequences, expression of putative embryogenesis-associated transcription factors, namely BABYBOOM (BBM), WUSCHEL-RELATED HOMEOBOX (WOX), and SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK), was analyzed during somatic embryogenesis. RT-PCR analysis revealed that TkBBM, TkWOX, and TkSERK were highly expressed in embryogenic callus and seedling roots, whereas their expression was reduced in seedling leaves. Our findings provide new insights into the roles of BBM, WOX, and SERK in somatic embryogenesis. Our results may serve as a reference for comparative transcriptome analysis in related species and further aid functional genomics studies in conifers.

Overexpression of Liriodenron WOX5 in Arabidopsis Leads to Ectopic Flower Formation and Altered Root Morphology

Roots are essential for plant growth, and studies on root-related genes, exemplified by WUSCHEL-RELATED HOMEOBOX5 (WOX5), have mainly concentrated on model organisms with less emphasis on the function of these genes in woody plants. Here, we report that overexpression of the WOX5 gene from Liriodendron hybrid (LhWOX5) in Arabidopsis leads to significant morphological changes in both the aerial and subterranean organs. In the Arabidopsis aerial parts, overexpression of LhWOX5 results in the production of ectopic floral meristems and leaves, possibly via the ectopic activation of CLV3 and LFY. In addition, in the Arabidopsis root, overexpression of LhWOX5 alters root apical meristem morphology, leading to a curled and shortened primary root. Importantly, these abnormal phenotypes in the aerial and subterranean organs caused by constitutive ectopic expression of LhWOX5 mimic the observed phenotypes when overexpressing AtWUS and AtWOX5 in Arabidopsis, respectively. Taken together, we propose that the LhWOX5 gene, originating from the Magnoliaceae plant Liriodendron, is a functional homolog of the AtWUS gene from Arabidopsis, while showing the highest degree of sequence similarity with its ortholog, AtWOX5. Our study provides insight into the potential role of LhWOX5 in the development of both the shoot and root.

Identification of WUSCHEL-related homeobox (WOX) gene family members and determination of their expression profiles during somatic embryogenesis in Phoebe bournei.

WUSCHEL-related homeobox (WOX) transcription factor (TF)-encoding genes play crucial roles during embryo development. The function of WOX genes in embryonic development has been thoroughly studied in Arabidopsis thaliana, but little is known about their function in woody species, especially Phoebe bournei, an endemic and endangered species in China. In the present study, a total of 15 WOX genes were identified in P. bournei, and phylogenetic analysis resulted in their assignment to three typical clades: an ancient clade, an intermediate clade, and a modern/WUS clade. The gene structure and sequence characteristics and the physicochemical properties of WOX proteins were also analyzed. Promoter prediction indicated that WOX genes are likely involved in plant growth and development and hormone responses. Subsequently, we evaluated the expression patterns of WOX genes in response to auxin (IAA), abscisic acid (ABA), and methyl jasmonate (MeJA) treatments. According to tissue-specific expression patterns, we screened nine WOX genes that were present in embryonic calli and that might participate in the somatic embryogenesis (SE) of P. bournei. Furthermore, the expression profiles of these nine WOX genes during three phases of embryogenic calli development and three phases of somatic embryo development, namely, spheroid embryogenesis, immature cotyledon-producing embryogenesis and mature cotyledon-producing embryogenesis, were monitored. Overall, we systematically analyzed the expression patterns of WOX genes in P. bournei during SE, the information of which provides a basis for further elucidating the molecular mechanism through which WOX TFs function in P. bournei embryo development.

Expression dynamics of WOX homeodomain transcription factors during somatic embryogenesis in Liriodendron hybrids.

The relict woody plant genus Liriodendron contains two endangered species, Liriodendron chinense and Liriodendron tulipifera. Understanding the molecular mechanisms involved in early embryo development is important for horticultural and ecological research, particularly for the development of improved somatic embryogenesis systems. However, the specific molecular processes underlying embryogenesis in these species remain largely unexplored. To address this, we investigated expression of the WOX ( WUSCHEL-related homeobox) gene family of transcription factors throughout somatic embryogenesis. We confirmed expression of eight out of 11 novel candidate LcWOX genes in L.chinense using qRT-PCR and examined spatiotemporal expression patterns of the expressed genes using stable reporter lines that had been transformed with different LcWOX promoters driving GUS expression. We observed embryo developmental stages and expression patterns that broadly correlated with those reported for Arabidopsis somatic embryogenesis. LcWUS was weakly expressed during the transition stage and was predominantly restricted to the apical meristem. LcWOX5 was specifically expressed in the root meristem and restricted to the cotyledons thereafter, and LcWOX4 expression was restricted to the vascular tissue of cotyledonary embryos. In contrast, LcWOX9 was expressed in the embryonic callus and the entire embryonic cell mass, then became restricted to the basal cells, indicating a potential role in regulating embryonic maintenance. Our findings provide insights into spatiotemporally specific WOX transcription and shed new light on potential functions of WOX genes during Liriodendron somatic embryogenesis.

Genome-wide identification of WOX family members in nine Rosaceae species and a functional analysis of MdWOX13-1 in drought resistance

WUSCHEL-related homeobox (WOX) transcription factors (TFs) are important in plant development processes and evolutionary novelties. In this study, a genome-wide comprehensive analysis of WOX genes from nine Rosaceae species was carried out, and their potential roles in Malus were subsequently investigated. 125 WOXs in 9 Rosaceae species were identified and classified into three clades, i.e., the ancient, intermediate, and WUS clades. Prunus. domestica contained the most intra-genomic collinearity among the nine Rosaceae species. Additionally, the cis-elements in WOX gene family members were compared and classified into three categories, including phytohormone-responsive, plant growth and development, and abiotic and biotic stresses. Overexpression (OE) of MdWOX13–1 also increased the callus weight and enhanced ROS scavenging against drought stress. Furthermore, via yeast-one hybrid assay and LUC analyses, MdWOX13–1 could directly bind to the MdMnSOD promoter. Therefore, our results will facilitate further study of the WOX genes’ function in the Rosaceae family.

Induction of Somatic Embryogenesis in Plants: Different Players and Focus on WUSCHEL and WUS-RELATED HOMEOBOX (WOX) Transcription Factors.

In plants, other cells can express totipotency in addition to the zygote, thus resulting in embryo differentiation; this appears evident in apomictic and epiphyllous plants. According to Haberlandt's theory, all plant cells can regenerate a complete plant if the nucleus and the membrane system are intact. In fact, under in vitro conditions, ectopic embryos and adventitious shoots can develop from many organs of the mature plant body. We are beginning to understand how determination processes are regulated and how cell specialization occurs. However, we still need to unravel the mechanisms whereby a cell interprets its position, decides its fate, and communicates it to others. The induction of somatic embryogenesis might be based on a plant growth regulator signal (auxin) to determine an appropriate cellular environment and other factors, including stress and ectopic expression of embryo or meristem identity transcription factors (TFs). Still, we are far from having a complete view of the regulatory genes, their target genes, and their action hierarchy. As in animals, epigenetic reprogramming also plays an essential role in re-establishing the competence of differentiated cells to undergo somatic embryogenesis. Herein, we describe the functions of WUSCHEL-RELATED HOMEOBOX (WOX) transcription factors in regulating the differentiation-dedifferentiation cell process and in the developmental phase of in vitro regenerated adventitious structures.

Constructing the maize inflorescence regulatory network by using efficient tsCUT&Tag assay

Maize ear development determines the crop yield, and many important transcription factors (TFs) have been identified to function in this process. However, their transcriptional regulation mechanisms are still elusive. In this study, we generated the genome-wide DNA binding sites for 8 TFs which are known to function or highly expressed in inflorescence by applying the tsCUT&Tag method in maize leaf protoplast. We exposed a regulatory grid of 4 WUSCHEL-related homeobox (WOX) genes and revealed their potential regulatory mechanisms. In addition, a hierarchical regulation network for the determinacy and specification of maize inflorescence meristems were also constructed using the binding profiles of a floral development gene INDETERMINATE FLORAL APEX1 (IFA1) and 3 MADS-box genes. Our study provides an in-depth understanding and new insights of the regulatory mechanisms during maize inflorescence development.

<i>MtWOX2</i> gene in somatic embryogenesis of <i>Medicago truncatula</i>

Somatic embryogenesis (SE) is one of the plant regeneration pathways. This process is very similar to zygotic embryogenesis, but embryos develop not from zygote, but from vegetative tissues. This process is widely used in biotechnology for plant transformation and propagation. Somatic embryos can derive directly from the vegetative tissues or through the formation of callus. The search for SE stimulators is very important for plant biotechnology.
 Several genes were reported to be the regulators of this process, among them the WOX (WUSCHEL-RELATED HOMEOBOX) family members are presented. These genes encode homeodomain-containing transcription factors, participating in different developmental processes. WOX2 is known for its participation in zygotic embryogenesis. It is expressed in the zygote and later in the apical domain of the embryo. We study the role of this gene in the somatic embryogenesis. Overexpression of MtWOX2 in some cases leads to the development of embryogenic calli with increased size. We performed transcriptome analysis of Medicago truncatula calli with overexpression of this gene compared to the calli overexpressing GUS.
 It was shown that MtWOX2 overexpression led to the changes in expression levels of genes, enriched with several GO pathways, including groups related to oxidative stress and ROS formation, response to toxic substance and auxin-activated signaling pathway. Among differentially expressed genes there are members of several TF families, e.g. MADS-box, BHLH, MYB, bZIP and others. These genes may regulate embryogenic callus development. Together, these results can be used for the search of new morphogenic regulators applicable for plant transformation.
 The research was supported by RFBR grant 20-016-00124.

The <i>MtWOX</i> and <i>MtCLE</i> genes in the regulation of <i>Medicago truncatula</i> somatic embryogenesis

Plant somatic cells can be reprogrammed into totipotent embryonic cells that are able to form differentiated embryos in a process called somatic embryogenesis (SE). SE can occur naturally in various plant species and it is widely used for clonal propagation, transformation and regeneration of different crops. This process is regulated by hormone treatment and many proteins, among which WUSCHEL-related homeobox (WOX) transcription factors are believed to play crucial roles. The WOX family is involved in the regulation of a wide range of key developmental programs in different plant organs and tissues. CLE peptides are well-known hormonal regulators of plant development. WOX and CLE genes can be related to each other through feedback regulatory loops.
 Our previous studies have shown that MtWOX9-1 stimulates SE in Medicago truncatula [1] and overexpression of the MtWOX9-1 gene increases the expression level of the MtCLE08, MtCLE16, MtCLE18 genes in SE. In this study, we examine the overexpression effect of MtCLE08, 16, and 18 on the expression level of MtWOX9-1 gene and a number of other MtWOX genes, which were shown to change expression in SE according to the transcriptomic data. No significant impact of MtCLE overexpression on any MtWOX gene under study was found.
 Our findings could be a helpful point for searching and studying new morphogenetic regulators controlling SE and could have a positive impact on plant biotechnology in improving the transformation and regeneration capacity for other legumes.
 The research was supported by grant from the Russian Foundation for Basic Research No. 20-016-00124.

Genome-Wide Identification and Expression Profile Analysis of the WUSCHEL-Related Homeobox (WOX) Genes in Woodland Strawberry (Fragaria vesca)

The WUSCHEL-related homeobox (WOX) is a unique transcription factor family belonging to plants. Its members play important roles in regulation of plant stem cell division and differentiation or dynamic balance of the organ development. In this study, 16 Fragaria vesca WOX members (FvWOXs) were identified in the woodland strawberry (Fragaria vesca) genome. According to the phylogenetic analysis, FvWOX proteins can be classified into three clades: the ancient clade, the WUS clade and the intermediate clade. The gene structure and protein motif analysis showed that FvWOXs are highly conserved to each other in their respective clades. Chromosome location analysis revealed that FvWOXs are unevenly distributed in the Fragaria vesca chromosomes. Expression analysis showed that FvWOX genes are widely expressed in various tissues of Fragaria vesca. Moreover, when treated with 6-benzylaminopurine (6-BA) or gibberellin (GA3), expression level changes of the genes highly expressed in crowns were detected at 48 h after treatment. The subcellular localization of FvWUSb was determined in tobacco leaves, indicating that the protein is located in the nucleus. This study enlarged our recognition about the WUSCHEL-related homeobox genes in Fragaria vesca, and laid a foundation for further researches about this gene family in strawberry.

Genome-wide analysis of the WOX gene family and the role of EjWUSa in regulating flowering in loquat (Eriobotrya japonica).

The WUSCHEL (WUS)-related homeobox (WOX) gene family plays a crucial role in stem cell maintenance, apical meristem formation, embryonic development, and various other developmental processes. However, the identification and function of WOX genes have not been reported in perennial loquat. In this study, 18 EjWOX genes were identified in the loquat genome. Chromosomal localization analysis showed that 18 EjWOX genes were located on 12 of 17 chromosomes. Gene structure analysis showed that all EjWOX genes contain introns, of which 11 EjWOX genes contain untranslated regions. There are 8 pairs of segmental duplication genes and 0 pairs of tandem duplication genes in the loquat WOX family, suggesting that segmental duplications might be the main reason for the expansion of the loquat WOX family. A WOX transcription factor gene named EjWUSa was isolated from loquat. The EjWUSa protein was localized in the nucleus. Protein interactions between EjWUSa with EjWUSa and EjSTM were verified. Compared with wild-type Arabidopsis thaliana, the 35S::EjWUSa transgenic Arabidopsis showed early flowering. Our study provides an important basis for further research on the function of EjWOX genes and facilitates the molecular breeding of loquat early-flowering varieties.

Auxin-Induced WUSCHEL-RELATED HOMEOBOX13 Mediates Asymmetric Activity of Callus Formation upon Cutting.

Plants have the regenerative ability to reconnect cut organs, which is physiologically important to survive severe tissue damage. The ability to reconnect organs is utilized as grafting to combine two different individuals. Callus formation at the graft junction facilitates organ attachment and vascular reconnection. While it is well documented that local wounding signals provoke callus formation, how callus formation is differentially regulated at each cut end remains elusive. Here, we report that callus formation activity is asymmetrical between the top and bottom cut ends and is regulated by differential auxin accumulation. Gene expression analyses revealed that cellular auxin response is preferentially upregulated in the top part of the graft. Disruption of polar auxin transport inhibited callus formation from the top, while external application of auxin was sufficient to induce callus formation from the bottom, suggesting that asymmetric auxin accumulation is responsible for active callus formation from the top end. We further found that the expression of a key regulator of callus formation, WUSCHEL-RELATED HOMEOBOX 13 (WOX13), is induced by auxin. The ectopic callus formation from the bottom end, which is triggered by locally supplemented auxin, requires WOX13 function, demonstrating that WOX13 plays a pivotal role in auxin-dependent callus formation. The asymmetric WOX13 expression is observed both in grafted petioles and incised inflorescence stems, underscoring the generality of our findings. We propose that efficient organ reconnection is achieved by a combination of local wounding stimuli and disrupted long-distance signaling.

RsCLE22a regulates taproot growth through an auxin signaling-related pathway in radish (Raphanus sativus L.).

CLAVATA3/EMBRYO SURROUNDING REGION-related (CLE) peptides are a class of small molecules involved in plant growth and development. Although radish (Raphanus sativus) is an important root vegetable crop worldwide, the functions of CLE peptides in its taproot formation remain elusive. Here, a total of 48 RsCLE genes were identified from the radish genome. RNA in situ hybridization showed that RsCLE22a gene was highly expressed in the vascular cambium. Overexpression of RsCLE22a inhibited root growth by impairing stem cell proliferation in Arabidopsis, and radish plants with exogenous supplementation of RsCLE22 peptide (CLE22p) showed a similar phenotype. The vascular cambial activity was increased in RsCLE22a-silenced plants. Transcriptome analysis revealed that CLE22p altered the expression of several genes involved in meristem development and hormone signal transduction in radish. Immunolocalization results showed that CLE22p increased auxin accumulation in vascular cambium. Yeast one-hybrid and dual-luciferase assays showed that the WUSCHEL-RELATED HOMEOBOX 4 (RsWOX4) binds to RsCLE22a promoter and activates its transcription. The expression level of RsWOX4 was related to vascular cambial activity and was regulated by auxin. Furthermore, a RsCLE22a-RsWOX4 module is proposed to regulate taproot vascular cambium activity through an auxin signaling-related pathway in radish. These findings provide novel insights into the regulation of root growth in a horticultural crop.

Characterization of WOX genes revealed drought tolerance, callus induction, and tissue regeneration in Gossypium hirsutum.

Cotton is an important natural fiber crop; its seeds are the main oil source. Abiotic stresses cause a significant decline in its production. The WUSCHEL-related Homeobox (WOX) genes have been involved in plant growth, development, and stress responses. However, the functions of WOX genes are less known in cotton. This study identified 39, 40, 21, and 20 WOX genes in Gossypium hirsutum, Gossypium barbadense, Gossypium arboreum, and Gossypium raimondii, respectively. All the WOX genes in four cotton species could be classified into three clades, which is consistent with previous research. The gene structure and conserved domain of all WOX genes were analyzed. The expressions of WOX genes in germinating hypocotyls and callus were characterized, and it was found that most genes were up-regulated. One candidate gene Gh_ A01G127500 was selected to perform the virus-induced gene silencing (VIGS) experiment, and it was found that the growth of the silenced plant (pCLCrVA: GhWOX4_A01) was significantly inhibited compared with the wild type. In the silenced plant, there is an increase in antioxidant activities and a decrease in oxidant activities compared with the control plant. In physiological analysis, the relative electrolyte leakage level and the excised leaf water loss of the infected plant were increased. Still, both the relative leaf water content and the chlorophyll content were decreased. This study proved that WOX genes play important roles in drought stress and callus induction, but more work must be performed to address the molecular functions of WOX genes.

Genome-wide identification and expression profiling of WUSCHEL-related homeobox (WOX) genes confer their roles in somatic embryogenesis, growth and abiotic stresses in banana.

The online version contains supplementary material available at 10.1007/s13205-022-03387-w.