WUS Genes Research Articles

Comparative Transcriptome Analysis between Embryogenic and Non-Embryogenic Callus of Davidia involucrata

Davidia involucrata Baill. (D. involucrata), a rare and endangered wild plant, is native to China and is globally recognized as an ornamental tree species. However, D. involucrata exhibits inherent biological characteristics that contribute to its low reproductive efficiency. To address this challenge, somatic embryogenesis, a biotechnological method, offers numerous advantages, including enhanced reproductive efficiency, a large reproductive coefficient, and a complete structural composition. Consequently, somatic embryogenesis holds significant value in the propagation and genetic improvement of this particular tree species. In a previous study, we utilized immature zygotic embryos of D. involucrata as explants and induced somatic embryogenesis from embryogenic callus, thereby establishing a rapid propagation and plant regeneration scheme. In this study, we utilized Illumina RNA sequencing to compare the transcriptomes of the embryogenic callus (EC) and non-embryogenic callus (NEC) of D. involucrata. The analysis revealed 131,109 unigenes assembled from EC and NEC, and 12,806 differentially expressed genes (DEGs) were identified. To verify the authenticity of the transcriptome sequencing results, qRT-PCR was performed and 16 DEGs were screened, with the stable reference gene UBQ being selected. Our analysis focused on genes related to plant growth regulators and somatic embryogenesis, such as the Aux, IAA, ARF, GH3, AHP, ARR, CYCD, BBM, WUS, GRF, SERK, and WOX gene families. We found that certain genes in these families were significantly upregulated in EC induction compared to NEC, indicating that they play crucial roles in D. involucrata cell proliferation, differentiation, and cell totipotency. These results offer new insights into the role of these gene families in EC, and may guide efforts to improve the somatic embryo induction, culture conditions, and genetic transformation efficiency of D. involucrata.

MiR172 Regulates WUS during Somatic Embryogenesis in Arabidopsis via AP2.

In plants, the embryogenic transition of somatic cells requires the reprogramming of the cell transcriptome, which is under the control of genetic and epigenetic factors. Correspondingly, the extensive modulation of genes encoding transcription factors and miRNAs has been indicated as controlling the induction of somatic embryogenesis in Arabidopsis and other plants. Among the MIRNAs that have a differential expression during somatic embryogenesis, members of the MIRNA172 gene family have been identified, which implies a role of miR172 in controlling the embryogenic transition in Arabidopsis. In the present study, we found a disturbed expression of both MIRNA172 and candidate miR172-target genes, including AP2, TOE1, TOE2, TOE3, SMZ and SNZ, that negatively affected the embryogenic response of transgenic explants. Next, we examined the role of AP2 in the miR172-mediated mechanism that controls the embryogenic response. We found some evidence that by controlling AP2, miR172 might repress the WUS that has an important function in embryogenic induction. We showed that the mechanism of the miR172-AP2-controlled repression of WUS involves histone acetylation. We observed the upregulation of the WUS transcripts in an embryogenic culture that was overexpressing AP2 and treated with trichostatin A (TSA), which is an inhibitor of HDAC histone deacetylases. The increased expression of the WUS gene in the embryogenic culture of the hdac mutants further confirmed the role of histone acetylation in WUS control during somatic embryogenesis. A chromatin-immunoprecipitation analysis provided evidence about the contribution of HDA6/19-mediated histone deacetylation to AP2-controlled WUS repression during embryogenic induction. The upstream regulatory elements of the miR172-AP2-WUS pathway might involve the miR156-controlled SPL9/SPL10, which control the level of mature miR172 in an embryogenic culture.

Silencing of SlMYB55 affects plant flowering and enhances tolerance to drought and salt stress in tomato

The transcription factors of the MYB family are involved in plant growth and development and responses to biotic and abiotic stresses. Here, we isolated the R2R3-MYB transcription factor gene SlMYB55 and found that it is responsive to abscisic acid (ABA), drought, and salt stress. Notably, the expression levels of multiple stress-related and inflorescence and flowering time-related genes were changed in SlMYB55-RNAi plants compared to wild-type plants. Transient tobacco expression experiments indicated that SlMYB55 directly targets the WUS and 4CL genes to regulate the development of inflorescence and flavonoid biosynthesis. Yeast two-hybrid experiments showed that the SlMYB55 protein interacts with the MADS-box family protein MBP21. Based on these results, we concluded that SlMYB55 affects the biosynthesis of ABA, regulates drought and salt responses through ABA-mediated signal transduction pathways, and directly or indirectly affects the expression of genes related to drought and salt response, flowering time, sepal size and inflorescence, thereby regulating stress tolerance and flower development. In summary, this study identified essential roles for SlMYB55 in regulating drought and salt tolerance and flower development.

WUSCHEL Overexpression Promotes Callogenesis and Somatic Embryogenesis in Medicago truncatula Gaertn.

The induction of plant somatic embryogenesis is often a limiting step for plant multiplication and genetic manipulation in numerous crops. It depends on multiple signaling developmental processes involving phytohormones and the induction of specific genes. The WUSCHEL gene (WUS) is required for the production of plant embryogenic stem cells. To explore a different approach to induce somatic embryogenesis, we have investigated the effect of the heterologous Arabidopsis WUS gene overexpression under the control of the jasmonate responsive vsp1 promoter on the morphogenic responses of Medicago truncatula explants. WUS expression in leaf explants increased callogenesis and embryogenesis in the absence of growth regulators. Similarly, WUS expression enhanced the embryogenic potential of hairy root fragments. The WUS gene represents thus a promising tool to develop plant growth regulator-free regeneration systems or to improve regeneration and transformation efficiency in recalcitrant crops.

A peak in global DNA methylation is a key step to initiate the somatic embryogenesis of coconut palm (Cocos nucifera L).

DNA methylation, morphogenesis and gene expression during the somatic embryogenesis of Coconut are affected by 5-Azacytidine pretreatments, indicating that DNA methylation is an important factor throughout this process. Somatic embryogenesis (SE) is a process that can aid in the production of elite Cocos nucifera palms. It has been well established that epigenetic mechanisms are regulators of cell differentiation programs; however, their role in the coconut somatic embryogenesis has not yet been addressed. To this end, the morphogenetic changes, the global DNA methylation and the expression profiles of the SE-related genes and DNA methyltransferases genes were evaluated during the SE process, with and without the presence of 5-Azacytidine (AzaC). The results show that three days of pretreatments with 15µM and 20µM of AzaC significantly increased early somatic embryo formation (four- and tenfold, respectively). A clear peak of the global percentage of DNA methylation (approximately 13%) was determined at the beginning of the culture, followed by a re-establishing stage and a steady increase thereafter; in all cases, the levels of DNA methylation were lower after the pretreatments with AzaC. Additionally, the expression profiles of the SERK, WUS, BBM and LEC genes are modulated during the SE process and the pretreatments with AzaC affect the expression profiles of these genes, even at early stages. Furthermore, increased levels of expression were observed for the genes encoding for DNA methyltransferases (MET, CMT and DRM) at early and late stages of SE, indicating that DNA methylation is an important factor throughout the SE.

Genes involved in the deformations of the shoot apical meristem in somatic embryos of Capsicum chinense Jacq.

Somatic embryos (SE) of habanero pepper (Capsicum chinense Jacq.) represent persistent deformations in the shoot apical meristem (SAM), which inhibits their capacity to form organs and subsequently plants. In dicotyledonous plants, SAM is formed in the apex, between cotyledons and it plays a central role in postembryonic shoot organ formation. Based on the previous knowledge on the role of some families of gene in the formation, organization and maintenance of the SAM, the expression patterns of WUS, WOX2, NAM, STM, PIN1 and PIN7 genes were analysed, which would allow us to elucidate the possible implication of these genes in SAM deformations in the SE of C. chinense. The results show that the expression patterns of STM and PIN1 in the SE were completely opposite to the respective expression pattern obtained in zygotic embryos (ZE). Moreover, NAM and PIN7 showed an over accumulation of transcripts in SE, compared with ZE. This is the first time in the genus Capsicum that alterations in the expression pattern of key genes of the SE development are reported, as well as its possible implication in the persistent deformations of the SAM.

Selectable marker independent transformation of recalcitrant maize inbred B73 and sorghum P898012 mediated by morphogenic regulators BABY BOOM and WUSCHEL2

Key messageDiscriminatory co-expression of maizeBBMandWUStranscriptional factor genes promoted somatic embryogenesis and efficientAgrobacterium-mediated transformation of recalcitrant maize inbred B73 and sorghum P898012 genotypes without use of a selectable marker gene.The use of morphogenic regulators to overcome barriers in plant transformation is a revolutionary breakthrough for basic plant science and crop applications. Current standard plant transformation systems are bottlenecks for genetic, genomic, and crop improvement studies. We investigated the differential use of co-expression of maize transcription factors BABY BOOM and WUSCHEL2 coupled with a desiccation inducible CRE/lox excision system to enable regeneration of stable transgenic recalcitrant maize inbred B73 and sorghum P898012 without a chemical selectable marker. The PHP78891 expression cassette contains CRE driven by the drought inducible maize RAB17M promoter with lox P sites which bracket the CRE, WUS, and BBM genes. A constitutive maize UBIM promoter directs a ZsGreen GFP expression cassette as a reporter outside of the excision sites and provides transient, transgenic, and developmental analysis. This was coupled with evidence for molecular integration and analysis of stable integration and desiccation inducible CRE-mediated excision. Agrobacterium-mediated transgenic introduction of this vector showed transient expression of GFP and induced somatic embryogenesis in maize B73 and sorghum P898012 explants. Subjection to desiccation stress in tissue culture enabled the excision of CRE, WUS, and BBM, leaving the UBIM::GFP cassette and allowing subsequent plant regeneration and GFP expression analysis. Stable GFP expression was observed in the early and late somatic embryos, young shoots, vegetative plant organs, and pollen. Transgene integration and expression of GFP positive T0 plants were also analyzed using PCR and Southern blots. Progeny segregation analysis of primary events confirmed correlation between functional GFP expression and presence of the GFP transgene in T1 plants generated from self pollinations, indicating good transgene inheritance. This study confirms and extends the use of morphogenic regulators to overcome transformation barriers.

English

In order to determine the role of polyamines in the formation and development of the somatic embryos of Capsicum chinense, the effect of different concentrations (0, 0.01, 0.1, and 1.0 mM) of Putrescine, Spermidine and Spermine on the efficiency and morphology of the embryos was evaluated. The results show that none of the three polyamines evaluated had a significant effect on the number of embryos formed, except Spermidine (1 mM), which caused a significant reduction in their numbers, in comparison with the control treatment. However, the most noteworthy result was observed in the treatment containing 0.1 mM of Spermine. The embryos developed in this treatment showed harmonic apex-radicle development, pale-green coloration and the formation of two tiny cotyledonary leaves. Real-time PCR was used to analyze the differential expression of the WUS, WOX1 and WOX3 genes in somatic embryos treated with Spermine and untreated, including the zygotic embryo. The transcript levels of the genes analyzed were found to differ significantly between both types of embryos (somatic and zygotic), with the zygotic embryos presenting a higher level of transcripts; however, compared to the untreated somatic embryos, the somatic embryos treated with Spermine showed an increase in the transcript levels of the three genes analyzed (WUS, WOX1 and WOX3); the WOX1 gene in particular presented an accumulation pattern similar to that of the zygotic embryo of the species. Key words: Somatic embryos, zygotic embryos, polyamines, transcript patterns and morphology.

Pleiotropic effect of the fas5 mutation on the shoot development of Arabidopsis thaliana

The paper described a new mutation that causes the development of multiple meristematic foci as part of shoot apical meristem, which can give rise to new stem axes or cause stem fasciation. The wus-1 mutation represses development of additional apical meristem in fas5 mutant, indicating to the sequential action of the genes in the formation of the shoot apical meristem and FAS5 gene participation in spatial restriction of the WUS gene expression. This function gene FAS5 performs independently of other negative regulators of WUS gene--namely genes CLV, as demonstrated by additive phenotype of double mutants fas5 clv2-1 and fas5 clv3-2. Besides the effect on the development of the shoot apical meristem fas5 mutation causes a change in the shape and number of leaves, accelerates the plant transition to the reproductive stage and leads to the development of cell neoplasms on the stem (buds, stigmatic tissues and ovule-like structures). The mutation also causes changes in apical meristems and leaf cell morphology indicating the activation in cells of DNA endoreduplication. Pleiotropic effect of the fas5 mutation on different stages of ontogeny and different organs suggests that the FAS5 gene plays a complex regulatory role at all stages of the A. thaliana shoot development, and affects many direct or indirect target genes.

In Silicon Cloning and Bioinformatics Analysis of the Raphanus Sativus WUS Gene

The complete coding sequence ofRaphanus sativusWUS gene was obtained by usingArabidopsis thalianagene se-quence (NM_127349) as a probe in silico cloning. Then the hydrophilicity, secondary structure and advanced structure of WUS protein inRaphanus sativusare analyzed by using bioinformatics methods. The results show that the cDNA was 1298 bp, with no intron, contains an open reading frame of 936bp, encoding 312aa protein. The protein coded byRaphanus sativusgene showed 74% similarity toArabidopsis thaliana.

Over‐expression of WOX1 Leads to Defects in Meristem Development and Polyamine Homeostasis in ArabidopsisF

In plants, the meristem has to maintain a separate population of pluripotent cells that serve two main tasks, i.e., self-maintenance and organ initiation, which are separated spatially in meristem. Prior to our study, WUS and WUS-like WOX genes had been reported as essential for the development of the SAM. In this study, the consequences of gain of WOX1 function are described. Here we report the identification of an Arabidopsis gain-of-function mutant wox1-D, in which the expression level of the WOX1 (WUSCHEL HOMEOBOX 1) was elevated and subtle defects in meristem development were observed. The wox1-D mutant phenotype is dwarfed and slightly bushy, with a smaller shoot apex. The wox1-D mutant also produced small and dark green leaves, and exhibited a failure in anther dehiscence and male sterility. Molecular evidences showed that the transcription of the stem cell marker gene CLV3 was down-regulated in the meristem of wox1-D but accumulated in the other regions, i.e., in the root-hypocotyl junction and at the sites for lateral root initiation. The fact that the organ size and cell size in leaves of wox1-D are smaller than those in wild type suggests that cell expansion is possibly affected in order to have partially retarded the development of lateral organs, possibly through alteration of CLV3 expression pattern in the meristem. An S-adenosylmethionine decarboxylase (SAMDC) protein, SAMDC1, was found able to interact with WOX1 by yeast two-hybrid and pull-down assays in vitro. HPLC analysis revealed a significant reduction of polyamine content in wox1-D. Our results suggest that WOX1 plays an important role in meristem development in Arabidopsis, possibly via regulation of SAMDC activity and polyamine homeostasis, and/or by regulating CLV3 expression.

Interaction between the PINOID/ABRUPTUS gene with the AGAMOUS gene negatively regulating stem cells proliferetion in the Arabidopsis thaliana floral meristem

Complementary interaction between the AGAMOUS and PINOID/ABRUPTUS genes was revealed. In double mutant abr ag-1, there is a significant increasing proliferation of cells of the floral meristem and formation of branching shoot-like flowers. These data indicate the participation of the PID/ABR gene in limitation of stem cells proliferation in the floral meristem. The revealed increase of transcription level of WUS gene in flowers of the abr mutant, as well as distortion of auxin distribution, allow us to suggest that the PID/ABR gene controls auxin transport and participates in positioning of expression domains for the WUS gene.

PgWUS expression during somatic embryo development in a Panax ginseng 2c3 cell culture expressing the rolC oncogene

It is well known that embryogenesis in plants is regulated by the WOX family of transcription factors (W US homeobox); the WUS gene is the founding member of this family. Panax ginseng is one of the most valuable traditional Chinese medicinal herbs, and its reproduction must be maintained to fulfill production demands. A ginseng embryogenic cell culture 2c3 was previously obtained as a result of the transfer of a bacterial oncogene, rolC. This cell line has been used as a model system for studying plant somatic embryogenesis. Several genes that are homologous to the plant WUS genes have been shown to be expressed in the embryogenic 2c3 cell culture; no sequences homologous to WOX genes have been detected. We show that the expression levels of PgWUSII-6, PgWUSII-5, and PgWUSII-8 are significantly higher in the early developmental stages of somatic embryogenesis compared to vector-transformed cells. During late stages of embryogenesis, PgWUSI-3, PgWUSI-7, and PgWUSII-5 expression is activated. We represent the results suggesting that the initiation of P. ginseng somatic embryogenesis may correlate with significant activation of PgWUS expression.

Somatic embryogenesis in the Panax ginseng cell culture induced by the rolC oncogene is associated with increased expression of WUS and SERK genes

The embryogenic cell culture 2c3 was previously obtained by the transfer of the rolC gene from Agrobacterium rhizogenes into ginseng callus cells. It was found by us that the expression of SERK and WUS genes in the embryogenic culture 2c3 is increased. These genes are known to be responsible for the initial stimulus to the development of embryogenesis in plants. Taking into consideration earlier data, we suppose that the development of somatic embryos in the ginseng cell culture 2c3 is associated with changes in the work of the calcium signaling system and with the activation of expression of SERK and WUS genes.

A systems approach to morphogenesis in Arabidopsis thaliana: II. Modeling the regulation of shoot apical meristem structure

Two models of the mechanism maintaining a zonal structure in the shoot apical meristem (SAM) were built based on the analysis of experimental data on the interactions between CLV1, CLV2, CLV3, and WUS genes and the concepts of their role in this mechanism. The first model, a simple one-dimensional model with two morphogens, which is a variant of Wolpert’s French flag model [1], describes the regulation of zone distribution along the SAM vertical axis. Despite a number of simplifications, this model has stationary solutions with biologically meaningful interpretation. The simplifying assumptions were successively abandoned in constructing a two-dimensional model of the mechanism underlying the regulation of SAM structure. This model provides a better understanding of the distributed system that regulates the SAM structure, and allows more detailed formalization of the modern concepts and experimental data concerning this mechanism.

Activation of the WUS Gene Induces Ectopic Initiation of Floral Meristems on Mature Stem Surface in Arabidopsis thaliana

Activation of the WUS Gene Induces Ectopic Initiation of Floral Meristems on Mature Stem Surface in Arabidopsis thaliana

Activation of the WUS gene induces ectopic initiation of floral meristems on mature stem surface in Arabidopsis thaliana

A gain-of-function Arabidopsis mutant was identified via activation tagging genetic screening. The mutant exhibited clustered ectopic floral buds on the surface of inflorescence stems. The mutant was designated as sef for stem ectopic flowers. Our detailed studies indicate that the ectopic flower meristems are initiated from the differentiated cortex cells. Inverse PCR and sequence analysis indicated that the enhancer-containing T-DNA from the activation tagging construct, SKI015, was inserted upstream of the previously cloned WUS gene encoding a homeodomain protein. Studies from RT-PCR, RNA in situ hybridization and transgenic plant analysis further confirmed that the phenotypes of sef are caused by the overexpression of WUS. Our results suggest that overexpression of WUS could trigger the cell pluripotence and reestablish a new meristem in cortex. The type of new meristems caused by WUS overexpression was dependent upon the developmental and physiological stages of a plant. With the help of some undefined factors in the reproductive organs the new meristems differentiated into floral buds. In a vegetative growth plant, however, only the new vegetative buds can be initiated upon the overexpression of WUS. These studies provide new insights of WUS on flower development.