SHOOT MERISTEMLESS Research Articles

The pentatricopeptide repeat protein DG1 promotes the transition to bilateral symmetry during Arabidopsis embryogenesis through GUN1-mediated plastid signals.

During Arabidopsis embryogenesis, the transition of the embryo's symmetry from radial to bilateral between the globular and heart stage is a crucial event, involving the formation of cotyledon primordia and concurrently the establishment of a shoot apical meristem (SAM). However, a coherent framework of how this transition is achieved remains to be elucidated. In this study, we investigated the function of DELAYED GREENING 1 (DG1) in Arabidopsis embryogenesis using a newly identified dg1-3 mutant. The absence of chloroplast-localized DG1 in the mutants led to embryos being arrested at the globular or heart stage, accompanied by an expansion of WUSCHEL (WUS) and SHOOT MERISTEMLESS (STM) expression. This finding pinpoints the essential role of DG1 in regulating the transition to bilateral symmetry. Furthermore, we showed that this regulation of DG1 may not depend on its role in plastid RNA editing. Nevertheless, we demonstrated that the DG1 function in establishing bilateral symmetry is genetically mediated by GENOMES UNCOUPLED 1 (GUN1), which represses the transition process in dg1-3 embryos. Collectively, our results reveal that DG1 functionally antagonizes GUN1 to promote the transition of the Arabidopsis embryo's symmetry from radial to bilateral and highlight the role of plastid signals in regulating pattern formation during plant embryogenesis.

Expression analyses of CUP-SHAPED COTYLEDON and SHOOT MERISTEMLESS in the one-leaf plant Monophyllaea glabra reveal neoteny evolution of shoot meristem

The one-leaf plant Monophyllaea glabra exhibits a unique developmental manner in which only one cotyledon continues growing without producing new vegetative organs. This morphology is formed by specific meristems, the groove meristem (GM) and the basal meristem (BM), which are thought to be modified shoot apical meristem (SAM) and leaf meristem. In this study, we analysed the expression of the organ boundary gene CUP-SHAPED COTYLEDON (CUC) and the SAM maintenance gene SHOOT MERISTEMLESS (STM) orthologs by whole-mount in situ hybridisation. We found that CUCs did not show clear border patterns around GM and BM during the vegetative phase. Furthermore, double-colour detection analysis at the cellular level revealed that CUC and STM expression overlapped in the GM region during the vegetative phase. We also found that this overlap is dissolved in the reproductive phase when normal shoot organogenesis is observed. Since co-expression of these genes occurs during SAM initiation under embryogenesis in Arabidopsis, our results demonstrate that GM is a prolonged stage of pre-mature SAM. Therefore, we propose that neotenic meristems could be a novel plant trait acquired by one-leaf plants.

Dissecting the Roles of the Cytokinin Signaling Network: The Case of De Novo Shoot Apical Meristem Formation.

Numerous biotechnological applications require a fast and efficient clonal propagation of whole plants under controlled laboratory conditions. For most plant species, the de novo regeneration of shoots from the cuttings of various plant organs can be obtained on nutrient media supplemented with plant hormones, auxin and cytokinin. While auxin is needed during the early stages of the process that include the establishment of pluripotent primordia and the subsequent acquisition of organogenic competence, cytokinin-supplemented media are required to induce these primordia to differentiate into developing shoots. The perception of cytokinin through the receptor ARABIDOPSIS HISTIDINE KINASE4 (AHK4) is crucial for the activation of the two main regulators of the establishment and maintenance of shoot apical meristems (SAMs): SHOOTMERISTEMLESS (STM) and the WUSCHEL-CLAVATA3 (WUS-CLV3) regulatory circuit. In this review, we summarize the current knowledge of the roles of the cytokinin signaling cascade in the perception and transduction of signals that are crucial for the de novo establishment of SAMs and lead to the desired biotechnological output-adventitious shoot multiplication. We highlight the functional differences between individual members of the multigene families involved in cytokinin signal transduction, and demonstrate how complex genetic regulation can be achieved through functional specialization of individual gene family members.

Gibberellins regulate masculinization through the SpGAI-SpSTM module in dioecious spinach.

The sex of dioecious plants is mainly determined by genetic factors, but it can also be converted by environmental cues such as exogenous phytohormones. Gibberellic acids (GAs) are well-known inducers of flowering and sexual development, yet the pathway of gibberellin-induced sex conversion in dioecious spinach (Spinacia oleracea L.) remains elusive. Based on sex detection before and after GA3 application using T11A and SSR19 molecular markers, we confirmed and elevated the masculinization effect of GA on a single female plant through exogenous applications of GA3, showing complete conversion and functional stamens. Silencing of GIBBERELLIC ACID INSENSITIVE (SpGAI), a single DELLA family protein that is a central GA signaling repressor, results in similar masculinization. We also show that SpGAI can physically interact with the spinach KNOX transcription factor SHOOT MERISTEMLESS (SpSTM), which is a homolog of the flower meristem identity regulator STM in Arabidopsis. The silencing of SpSTM also masculinized female flowers in spinach. Furthermore, SpSTM could directly bind the intron of SpPI to repress SpPI expression in developing female flowers. Overall, our results suggest that GA induces a female masculinization process through the SpGAI-SpSTM-SpPI regulatory module in spinach. These insights may help to clarify the molecular mechanism underlying the sex conversion system in dioecious plants while also elucidating the physiological basis for the generation of unisexual flowers so as to establish dioecy in plants.

ABSCISIC ACID-INSENSITIVE 5-KIP-RELATED PROTEIN 1-SHOOT MERISTEMLESS modulates reproductive development of Arabidopsis.

Soil (or plant) water deficit accelerates plant reproduction. However, the underpinning molecular mechanisms remain unknown. By modulating cell division/number, ABSCISIC ACID-INSENSITIVE 5 (ABI5), a key bZIP (basic (region) leucine zippers) transcription factor, regulates both seed development and abiotic stress responses. The KIP-RELATED PROTEIN (KRP) cyclin-dependent kinases (CDKs) play an essential role in controlling cell division, and SHOOT MERISTEMLESS (STM) plays a key role in the specification of flower meristem identity. Here, our findings show that abscisic acid (ABA) signaling and/or metabolism in adjust reproductive outputs (such as rosette leaf number and open flower number) under water-deficient conditions in Arabidopsis (Arabidopsis thaliana) plants. Reproductive outputs increased under water-sufficient conditions but decreased under water-deficient conditions in the ABA signaling/metabolism mutants abscisic acid2-1 (aba2-1), aba2-11, abscisic acid insensitive3-1 (abi3-1), abi4-1, abi5-7, and abi5-8. Further, under water-deficient conditions, ABA induced-ABI5 directly bound to the promoter of KRP1, which encodes a CDK that plays an essential role in controlling cell division, and this binding subsequently activated KRP1 expression. In turn, KRP1 physically interacted with STM, which functions in the specification of flower meristem identity, promoting STM degradation. We further demonstrate that reproductive outputs are adjusted by the ABI5-KRP1-STM molecular module under water-deficient conditions. Together, our findings reveal the molecular mechanism by which ABA signaling and/or metabolism regulate reproductive development under water-deficient conditions. These findings provide insights that may help guide crop yield improvement under water deficiency.

Molecular Insights into the Accelerated Sprouting of and Apical Dominance Release in Potato Tubers Subjected to Post-Harvest Heat Stress.

Climate change-induced heat stress (HS) increasingly threatens potato (Solanum tuberosum L.) production by impacting tuberization and causing the premature sprouting of tubers grown during the hot season. However, the effects of post-harvest HS on tuber sprouting have yet to be explored. This study aims to investigate the effects of post-harvest HS on tuber sprouting and to explore the underlying transcriptomic changes in apical bud meristems. The results show that post-harvest HS facilitates potato tuber sprouting and negates apical dominance. A meticulous transcriptomic profiling of apical bud meristems unearthed a spectrum of differentially expressed genes (DEGs) activated in response to HS. During the heightened sprouting activity that occurred at 15-18 days of HS, the pathways associated with starch metabolism, photomorphogenesis, and circadian rhythm were predominantly suppressed, while those governing chromosome organization, steroid biosynthesis, and transcription factors were markedly enhanced. The critical DEGs encompassed the enzymes pivotal for starch metabolism, the genes central to gibberellin and brassinosteroid biosynthesis, and influential developmental transcription factors, such as SHORT VEGETATIVE PHASE, ASYMMETRIC LEAVES 1, SHOOT MERISTEMLESS, and MONOPTEROS. These findings suggest that HS orchestrates tuber sprouting through nuanced alterations in gene expression within the meristematic tissues, specifically influencing chromatin organization, hormonal biosynthesis pathways, and the transcription factors presiding over meristem fate determination. The present study provides novel insights into the intricate molecular mechanisms whereby post-harvest HS influences tuber sprouting. The findings have important implications for developing strategies to mitigate HS-induced tuber sprouting in the context of climate change.

HISTONE DEACETYLASE19 Controls Ovule Number Determination and Transmitting Tract Differentiation.

The gynoecium is critical for the reproduction of flowering plants as it contains the ovules and the tissues that foster pollen germination, growth, and guidance. These tissues, known as the reproductive tract (ReT), comprise the stigma, style, and transmitting tract (TT). The ReT and ovules originate from the carpel margin meristem (CMM) within the pistil. SHOOT MERISTEMLESS (STM) is a key transcription factor for meristem formation and maintenance. In all above-ground meristems, including the CMM, local STM downregulation is required for organ formation. However, how this downregulation is achieved in the CMM is unknown. Here, we have studied the role of HISTONE DEACETYLASE 19 (HDA19) in Arabidopsis (Arabidopsis thaliana) during ovule and ReT differentiation based on the observation that the hda19-3 mutant displays a reduced ovule number and fails to differentiate the TT properly. Fluorescence-activated cell sorting coupled with RNA-sequencing revealed that in the CMM of hda19-3 mutants, genes promoting organ development are downregulated while meristematic markers, including STM, are upregulated. HDA19 was essential to downregulate STM in the CMM, thereby allowing ovule formation and TT differentiation. STM is ectopically expressed in hda19-3 at intermediate stages of pistil development, and its downregulation by RNA interference alleviated the hda19-3 phenotype. Chromatin immunoprecipitation assays indicated that STM is a direct target of HDA19 during pistil development and that the transcription factor SEEDSTICK is also required to regulate STM via histone acetylation. Thus, we identified factors required for the downregulation of STM in the CMM, which is necessary for organogenesis and tissue differentiation.

Condensation of STM is critical for shoot meristem maintenance and salt tolerance in Arabidopsis

The shoot meristem generates the entire shoot system and is precisely maintained throughout the life cycle under various environmental challenges. In this study, we identified a prion-like domain (PrD) in the key shoot meristem regulator SHOOT MERISTEMLESS (STM), which distinguishes STM from other related KNOX1 proteins. We demonstrated that PrD stimulates STM to form nuclear condensates, which are required for maintaining the shoot meristem. STM nuclear condensate formation is stabilized by selected PrD-containing STM-interacting BELL proteins in vitro and in vivo. Moreover, condensation of STM promotes its interaction with the Mediator complex subunit MED8 and thereby enhances its transcriptional activity. Thus, condensate formation emerges as a novel regulatory mechanism of shoot meristem functions. Furthermore, we found that the formation of STM condensates is enhanced upon salt stress, which allows enhanced salt tolerance and increased shoot branching. Our findings highlight that the transcription factor partitioning plays an important role in cell fate determination and might also act as a tunable environmental acclimation mechanism.

ERF5 enhances protocorm-like body regeneration via enhancement of STM expression in Dendrobium orchid.

Orchid plants develop protocorms upon germination and produce protocorm-like structures called protocorm-like bodies (PLBs) from protocorms and somatic cells via tissue culture. Protocorm-like bodies have broad technical application potential in the orchid industry and their regeneration is a distinct developmental process in the plant kingdom. However, little is known about this unparalleled developmental program. In this study, we identified a PLB-abundant gene, ethylene response factor (ERF), and a transcription factor named DoERF5, and determined its important role in PLB regeneration in Dendrobium orchid. Overexpression of DoERF5 in Dendrobium greatly enhanced the PLB regeneration from PLB and stem explants, and upregulated the expression of WOUND-INDUCED DEDIFFERENTIATION (DoWIND) homologs and SHOOT MERISTEMLESS (DoSTM), as well as the genes involved in cytokinin biosynthesis (DoIPT) and the cytokinin response factors (DoARRs). However, silencing DoERF5 reduced the regeneration rate of PLBs, and downregulated the expression of DoWIND homologs, DoSTM and DoARRs. We demonstrated that DoERF5 is directly bound to the DoSTM promoter and regulates its expression. In addition, overexpression of DoSTM in Dendrobium orchid resulted in favorable regeneration of PLBs. Our results clarify that DoERF5 regulates the regeneration of PLB by enhancing DoSTM expression. Our findings provide new insights into how DoERF5 mediates PLB regeneration and offers technical potential in improving clonal propagation, preservation, and the bioengineering of orchids.

Meristem genes are essential for the vegetative reproduction of Kalanchoë pinnata.

Several Kalanchoë species reproduce asexually by forming plantlets in the leaf crenulations. Some species produce plantlets incessantly via somatic embryogenesis and organogenesis, whereas others exclusively develop plantlets after leaf detachment, presumably through organogenesis. SHOOT MERISTEMLESS (STM), which mediates SAM functions, appears to be involved in Kalanchoë plantlet formation, suggesting that meristem genes may be essential for plantlet formation. However, the genetic regulatory network for establishing and maintaining plantlet primordia in Kalanchoë remains elusive. Here, we showed that meristem genes were differentially expressed in the leaf crenulations of K. pinnata during plantlet development after leaf detachment. The regulatory interactions among these meristem genes are largely conserved in K. pinnata crenulations. Moreover, transgenic antisense (AS) plants with lower expression of these key meristem genes formed significantly fewer plantlets with some morphological defects, suggesting that the meristem genes play an important role in plantlet formation and development. Our research revealed that key meristem genetic pathways were co-opted to the leaf margin to facilitate the unique asexual reproduction mechanism in K. pinnata. This also highlights how evolutionary tinkering invents new structures such as epiphyllous buds and plantlets by rewiring pre-existing genetic pathways.

From the archives: Ear apical degeneration 1 is essential for maize ear development, SHOOT MERISTEMLESS regulates floral fate, and the role of profilin in pollen tube growth.

From the archives: Ear apical degeneration 1 is essential for maize ear development, SHOOT MERISTEMLESS regulates floral fate, and the role of profilin in pollen tube growth.

Novel R2R3-MYB Transcription Factor LiMYB75 Enhances Leaf Callus Regeneration Efficiency in Lagerstroemia indica

Lagerstroemia indica is an important woody ornamental plant worldwide. However, the application of many technologies, such as transgenic breeding and genome editing, has been severely hampered due to the lack of efficient calli induction and regeneration technology. Here, we discussed a reliable and efficient calli induction and regeneration protocol using whole-leaf explants. This protocol’s effectiveness for the calli induction and regeneration systems in crape myrtle were up to 70.33% and 44.33%, respectively. Next, an efficient and stable Agrobacterium-mediated genetic transformation system was created from leaf calli, and the green fluorescent protein (GFP) was able to detect up to 90% of its positive frequency. Meanwhile, two positive lines’ transfer DNA insertion sites and directions were identified using whole genome sequencing. LiMYB75, a novel R2R3-MYB transcription factor, was identified and transferred to the L. indica genome to enhance the leaf calli regeneration frequency. Surprisingly, overexpressing LiMYB75 increased the frequency of calli regeneration in the leaf by 1.27 times and the number of regenerated plantlets per callus by 4.00 times compared to the wild type, by regulating the expression levels of genes involved in callus formation, such as SHOOT MERISTEMLESS (STM). Overall, our findings revealed a simple, reliable, and highly efficient transformation approach and identified the desirable candidate gene LiMYB75, which improves L. indica’s calli regeneration efficiency.

Targeted mutagenesis of BnaSTM leads to abnormal shoot apex development and cotyledon petiole fusion at the seedling stage in Brassica napus L.

The Arabidopsis homeodomain transcription factor SHOOT MERISTEMLESS (STM) is crucial for shoot apical meristem (SAM) function, which cooperates with CLAVATA3 (CLV3)/WUSCHEL (WUS) feedback regulation loops to maintain the homeostasis of stem cells in SAM. STM also interacts with the boundary genes to regulate the tissue boundary formation. However, there are still few studies on the function of STM in Brassica napus, an important oil crop. There are two homologs of STM in B. napus (BnaA09g13310D and BnaC09g13580D). In the present study, CRISPR/Cas9 technology was employed to create the stable site-directed single and double mutants of the BnaSTM genes in B. napus. The absence of SAM could be observed only in the BnaSTM double mutants at the mature embryo of seed, indicating that the redundant roles of BnaA09.STM and BnaC09.STM are vital for regulating SAM development. However, different from Arabidopsis, the SAM gradually recovered on the third day after seed germination in Bnastm double mutants, resulting in delayed true leaves development but normal late vegetative and reproductive growth in B. napus. The Bnastm double mutant displayed a fused cotyledon petiole phenotype at the seedling stage, which was similar but not identical to the Atstm in Arabidopsis. Further, transcriptome analysis showed that targeted mutation of BnaSTM caused significant changes for genes involved in the SAM boundary formation (CUC2, CUC3, LBDs). In addition, Bnastm also caused significant changes of a sets of genes related to organogenesis. Our findings reveal that the BnaSTM plays an important yet distinct role during SAM maintenance as compared to Arabidopsis.

Arabidopsis thaliana SHOOT MERISTEMLESS Substitutes for Medicago truncatula SINGLE LEAFLET1 to Form Complex Leaves and Petals.

LEAFY plant-specific transcription factors, which are key regulators of flower meristem identity and floral patterning, also contribute to meristem activity. Notably, in some legumes, LFY orthologs such as Medicago truncatula SINGLE LEAFLET (SGL1) are essential in maintaining an undifferentiated and proliferating fate required for leaflet formation. This function contrasts with most other species, in which leaf dissection depends on the reactivation of KNOTTED-like class I homeobox genes (KNOXI). KNOXI and SGL1 genes appear to induce leaf complexity through conserved downstream genes such as the meristematic and boundary CUP-SHAPED COTYLEDON genes. Here, we compare in M. truncatula the function of SGL1 with that of the Arabidopsis thaliana KNOXI gene, SHOOT MERISTEMLESS (AtSTM). Our data show that AtSTM can substitute for SGL1 to form complex leaves when ectopically expressed in M. truncatula. The shared function between AtSTM and SGL1 extended to the major contribution of SGL1 during floral development as ectopic AtSTM expression could promote floral organ identity gene expression in sgl1 flowers and restore sepal shape and petal formation. Together, our work reveals a function for AtSTM in floral organ identity and a higher level of interchangeability between meristematic and floral identity functions for the AtSTM and SGL1 transcription factors than previously thought.

SHOOT MERISTEMLESS participates in the heterophylly of Hygrophila difformis (Acanthaceae).

In heterophyllous plants, leaf shape shows remarkable plasticity in response to environmental conditions. However, transgenic studies of heterophylly are lacking and the molecular mechanism remains unclear. Here, we cloned the KNOTTED1-LIKE HOMEOBOX family gene SHOOT MERISTEMLESS (STM) from the heterophyllous plant Hygrophila difformis (Acanthaceae). We used molecular, morphogenetic, and biochemical tools to explore its functions in heterophylly. HdSTM was detected in different organs of H. difformis, and its expression changed with environmental conditions. Heterologous, ectopic expression of HdSTM in Arabidopsis (Arabidopsis thaliana) increased leaf complexity and CUP-SHAPED COTYLEDON (CUC) transcript levels. However, overexpression of HdSTM in H. difformis did not induce the drastic leaf change in the terrestrial condition. Overexpression of HdSTM in H. difformis induced quick leaf variations in submergence, while knockdown of HdSTM led to disturbed leaf development and weakened heterophylly in H. difformis. HdCUC3 had the same spatiotemporal expression pattern as HdSTM. Biochemical analysis revealed a physical interaction between HdSTM and HdCUC3. Our results provide genetic evidence that HdSTM is involved in regulating heterophylly in H. difformis.

Overexpression of PdeGATA3 results in a dwarf phenotype in poplar by promoting the expression of PdeSTM and altering the content of gibberellins.

In previous studies, GA20 oxidase (GA20ox) has been identified to be an important enzyme in the biosynthesis of GA, and SHOOTMERISTEMLESS (STM) can repress the expression of GA20ox. In this study, the GATA transcription factor PdeGATA3 was identified in the poplar line NL895, and its overexpression (OE) transgenic lines showed a dwarf phenotype. RNA sequencing (RNA-Seq) analysis suggested that OE PdeGATA3 could promote the expression of PdeSTM and repress the expression of PdeGA20ox. Therefore, we hypothesized that PdeGATA3 would directly promote the expression of PdeSTM and that PdeSTM would repress the expression of PdeGA20ox. Four experiments, a dual-luciferase reporter assay, GUS transient coexpression assay, yeast one-hybrid assay and electrophoretic mobility shift assay, were conducted and verified that PdeGATA3 could promote the expression of PdeSTM by binding GATA-Boxes in its promoter. OE PdeSTM in poplar resulted in a dwarf phenotype and repressed the expression of PdeGA20ox. GA measurement of the OE PdeSTM and PdeGATA3 lines showed that GA3 and GA4 contents were significantly lower than those in the wild type (WT). Accordingly, we put forward a regulation model involving plant height regulation by PdeGATA3, PdeSTM and PdeGA20ox.

Comparative Transcriptome Analysis of Two Kalanchoë Species during Plantlet Formation.

Few species in the Kalanchoë genus form plantlets on their leaf margins as an asexual reproduction strategy. The limited molecular studies on plantlet formation show that an organogenesis ortholog, SHOOTMERISTEMLESS (STM) and embryogenesis genes, such as LEAFY COTYLEDON1 (LEC1) and FUSCA3 are recruited during plantlet formation. To understand the mechanisms of two Kalanchoë plantlet-forming species with different modes of plantlet formation, RNA-sequencing analysis was performed. Differentially expressed genes between the developmental stages were clustered in K. daigremontiana (Raym.-Hamet and H. Perrier) and K. pinnata (Lam. Pers.), respectively. Of these gene clusters, GO terms that may be involved in plantlet formation of both species, such as signaling, response to wounding, reproduction, regulation of hormone level, and response to karrikin were overrepresented. Compared with the common GO terms, there were more unique GO terms overrepresented during the plantlet formation of each species. A more in-depth investigation is required to understand how these pathways are participating in plantlet formation. Nonetheless, this transcriptome analysis is presented as a reliable basis for future studies on plantlet formation and development in two Kalanchoë plantlet-forming species.

SKI-INTERACTING PROTEIN interacts with SHOOT MERISTEMLESS to regulate shoot apical meristem formation.

The shoot apical meristem (SAM), which is formed during embryogenesis, generates leaves, stems, and floral organs during the plant life cycle. SAM development is controlled by SHOOT MERISTEMLESS (STM), a conserved Class I KNOX transcription factor that interacts with another subclass homeodomain protein, BELL, to form a heterodimer, which regulates gene expression at the transcriptional level in Arabidopsis (Arabidopsis thaliana). Meanwhile, SKI-INTERACTING PROTEIN (SKIP), a conserved protein in eukaryotes, works as both a splicing factor and as a transcriptional regulator in plants to control gene expression at the transcriptional and posttranscriptional levels by interacting with distinct partners. Here, we show that, similar to plants with a loss of function of STM, a loss of function of SKIP or the specific knockout of SKIP in the SAM region resulted in failed SAM development and the inability of the mutants to complete their life cycle. In comparison, Arabidopsis mutants that expressed SKIP specifically in the SAM region formed a normal SAM and were able to generate a shoot system, including leaves and floral organs. Further analysis confirmed that SKIP interacts with STM in planta and that SKIP and STM regulate the expression of a similar set of genes by binding to their promoters. In addition, STM also interacts with EARLY FLOWERING 7 (ELF7), a component of Polymerase-Associated Factor 1 complex, and mutation in ELF7 exhibits similar SAM defects to that of STM and SKIP. This work identifies a component of the STM transcriptional complex and reveals the mechanism underlying SKIP-mediated SAM formation in Arabidopsis.

The GATA factor HANABA TARANU promotes runner formation by regulating axillary bud initiation and outgrowth in cultivated strawberry.

A runner, as an elongated branch, develops from the axillary bud (AXB) in the leaf axil and is crucial for the clonal propagation of cultivated strawberry (Fragaria × ananassa Duch.). Runner formation occurs in at least two steps: AXB initiation and AXB outgrowth. HANABA TARANU (HAN ) encodes a GATA transcription factor that affects AXB initiation in Arabidopsis and promotes branching in grass species, but the underlying mechanism is largely unknown. Here, the function of a strawberry HAN homolog FaHAN in runner formation was characterized. FaHAN transcripts can be detected in the leaf axils. Overexpression (OE) of FaHAN increased the number of runners, mainly by enhancing AXB outgrowth, in strawberry. The expression of the strawberry homolog of BRANCHED1 , a key inhibitor of AXB outgrowth in many plant species, was significantly downregulated in the AXBs of FaHAN -OE lines, whereas the expression of the strawberry homolog of SHOOT MERISTEMLESS, a marker gene for AXB initiation in Arabidopsis, was upregulated. Moreover, several genes of gibberellin biosynthesis and cytokinin signaling pathways were activated, whereas the auxin response pathway genes were repressed. Further assays indicated that FaHAN could be directly activated by FaNAC2, the overexpression of which in strawberry also increased the number of runners. The silencing of FaNAC2 or FaHAN inhibited AXB initiation and led to a higher proportion of dormant AXBs, confirming their roles in the control of runner formation. Taken together, our results revealed a FaNAC2-FaHAN pathway in the control of runner formation and have provided a means to enhance the vegetative propagation of cultivated strawberry.

Overexpression of the AtWUSCHEL gene promotes somatic embryogenesis and lateral branch formation in birch (Betula platyphylla Suk.)

Birch (Betula platyphylla Suk.) is a deciduous tree with medicinal and ornamental value. During the process of genetic transformation, somatic embryos do not easily develop into transgenic plants, which is a limitation in genetic breeding. The Arabidopsis thaliana WUSCHEL (AtWUS) gene, which is a transcription factor, plays an important role in maintaining and regulating stem cell characteristics, which determines whether the stem cell population is differentiated. To explore methods for inducing somatic embryogenesis (SE) in birch, we overexpressed the AtWUS gene and transferred it to birch. The expression of AtWUS increased the SE rate from 101.4 to 717.1%. The expression of the AtWUS gene led to the downregulation of BpWUS gene expression in both calli and globular embryos as well as bud meristems. The expression of a few genes, i.e., BpLEC1 (LEAFY COTYLEDON 1), BpLEC2 (LEAFY COTYLEDON 2) and BpFUS3 (FUSCA 3), was upregulated during both embryogenesis and bud meristem development. However, BpABI3 (ABSCISIC ACID INSENSITIVE 3) gene expression was upregulated only in calli embryos, while BpSTM (SHOOT MERISTEMLESS) and BpCUC2 (CUP-SHAPED COTYLEDON 2) gene expression was upregulated only in bud meristems. This result indicated that overexpression of the AtWUS gene promoted SE by increasing the expression of SE-related genes. In conclusion, this study focused on the role of the AtWUS gene in birch SE and the molecular mechanism by which SE was promoted. This work indicates that overexpression of the WUSCHEL gene from Arabidopsis thaliana in birch can promote somatic embryogenesis and increase the development of lateral branches and buds.