ENHANCER OF SHOOT REGENERATION Research Articles

ESR2–HDA6 complex negatively regulates auxin biosynthesis to delay callus initiation in Arabidopsis leaf explants during tissue culture

Plants exhibit an astonishing ability to regulate organ regeneration upon wounding. Excision of leaf explants promotes the biosynthesis of indole-3-acetic acid (IAA), which is polar-transported to excised regions, where cell fate transition leads to root founder cell specification to induce de novo root regeneration. The regeneration capacity of plants has been utilized to develop in vitro tissue culture technologies. Here, we report that IAA accumulation near the wounded site of leaf explants is essential for callus formation on 2,4-dichlorophenoxyacetic acid (2,4-D)-rich callus-inducing medium (CIM). Notably, a high concentration of 2,4-D does not compensate for the action of IAA because of its limited efflux; rather, it lowers IAA biosynthesis via a negative feedback mechanism at an early stage of in vitro tissue culture, delaying callus initiation. The auxin negative feedback loop in CIM-cultured leaf explants is mediated by an auxin-inducible APETALA2 transcription factor, ENHANCER OF SHOOT REGENERATION 2 (ESR2), along with its interacting partner HISTONE DEACETYLASE 6 (HDA6). The ESR2–HDA6 complex binds directly to, and removes the H3ac mark from, the YUCCA1 (YUC1), YUC7, and YUC9 loci, consequently repressing auxin biosynthesis and inhibiting cell fate transition on 2,4-D-rich CIM. These findings indicate that negative feedback regulation of auxin biosynthesis by ESR2 and HDA6 interferes with proper cell fate transition and callus initiation.

ENHANCER OF SHOOT REGENERATION1 promotes de novo root organogenesis after wounding in Arabidopsis leaf explants.

Plants have an astonishing ability to regenerate new organs after wounding. Here, we report that the wound-inducible transcription factor ENHANCER OF SHOOT REGENERATION1 (ESR1) has a dual mode of action in activating ANTHRANILATE SYNTHASE ALPHA SUBUNIT1 (ASA1) expression to ensure auxin-dependent de novo root organogenesis locally at wound sites of Arabidopsis (Arabidopsis thaliana) leaf explants. In the first mode, ESR1 interacts with HISTONE DEACETYLASE6 (HDA6), and the ESR1-HDA6 complex directly binds to the JASMONATE-ZIM DOMAIN5 (JAZ5) locus, inhibiting JAZ5 expression through histone H3 deacetylation. As JAZ5 interferes with the action of ETHYLENE RESPONSE FACTOR109 (ERF109), the transcriptional repression of JAZ5 at the wound site allows ERF109 to activate ASA1 expression. In the second mode, the ESR1 transcriptional activator directly binds to the ASA1 promoter to enhance its expression. Overall, our findings indicate that the dual biochemical function of ESR1, which specifically occurs near wound sites of leaf explants, maximizes local auxin biosynthesis and de novo root organogenesis in Arabidopsis.

Histone deacetylation regulates de novo shoot regeneration.

During de novo plant organ regeneration, auxin induction mediates the formation of a pluripotent cell mass called callus, which regenerates shoots upon cytokinin induction. However, molecular mechanisms underlying transdifferentiation remain unknown. Here, we showed that the loss of HDA19, a histone deacetylase (HDAC) family gene, suppresses shoot regeneration. Treatment with an HDAC inhibitor revealed that the activity of this gene is essential for shoot regeneration. Further, we identified target genes whose expression was regulated through HDA19-mediated histone deacetylation during shoot induction and found that ENHANCER OF SHOOT REGENERATION 1 and CUP-SHAPED COTYLEDON 2 play important roles in shoot apical meristem formation. Histones at the loci of these genes were hyperacetylated and markedly upregulated in hda19. Transient ESR1 or CUC2 overexpression impaired shoot regeneration, as observed in hda19. Therefore, HDA19 mediates direct histone deacetylation of CUC2 and ESR1 loci to prevent their overexpression at the early stages of shoot regeneration.

NARROW AND DWARF LEAF 1, the Ortholog of Arabidopsis ENHANCER OF SHOOT REGENERATION1/DORNRÖSCHEN, Mediates Leaf Development and Maintenance of the Shoot Apical Meristem in Oryza sativa L.

The molecular basis for leaf development, a major focus in developmental biology, remains unclear in the monocotyledonous grass, rice (Oryza sativa). Here, we performed a mutant screen in rice and identified an AP2-type transcription factor family protein, NARROW AND DWARF LEAF1 (NDL1). NDL1 is the ortholog of Arabidopsis thaliana (subsequently called Arabidopsis) ENHANCER OF SHOOT REGENERATION1 (ESR1)/DORNRÖSCHEN (DRN) and mediates leaf development and maintenance of the shoot apical meristem (SAM). Loss of function of NDL1 results in bladeless leaves and SAMs that are flat, rather than dome-shaped, and lack cell proliferation activity. This loss of function also causes reduced auxin signaling. Moreover, as is the case with Arabidopsis ESR1/DRN, NDL1 plays crucial roles in shoot regeneration. Importantly, we found that NDL1 is not expressed in the SAM but is expressed in leaf primordia. We propose that NDL1 cell autonomously regulates leaf development, but non-cell autonomously regulates SAM maintenance in rice.

Physical state of the culture medium triggers shift in morphogenetic pattern from shoot bud formation to somatic embryo in Solanum khasianum.

Solanum khasianum is a rich source of steroidal alkaloids that are important secondary metabolites with enormous pharmaceutical uses. Development of plantlets from somatic tissues, under in vitro conditions, takes place both through adventitious shoot bud differentiation or somatic embryogenesis (SE) pathway. We observed that the physical state of medium, solid or liquid, determined the regenerant differentiation patterns from root segment explants in S. khasianum. In the solidified medium, the root segments developed adventitious shoot buds whereas somatic embryos were regenerated in the liquid medium. Varying gradients from liquid to solid medium were further used to confirm the effect of solidified condition on regeneration pathway. Histological analysis of developing shoot buds and somatic embryos was also performed to confirm their development and differentiation patterns. In order to further confirm the developmental pathways, qRT-PCR analysis of the marker genes of SE and shoot regeneration was also performed. While SOMATIC EMBRYOGENESIS RECEPTOR KINASE1 (SkSERK1) expression was significantly up-regulated during the early embryogenic stage, the LATE EMBRYOGENESIS ABUNDANT (SkLEA) proteinwas found to be highly expressed in the mature embryos. Expression of the HISTONE DEACETYLASE (HDA6), a repressor of SE related genes, was highly decreased during embryogenesis in the liquid culture. Furthermore, expression of the ENHANCER OF SHOOT REGENERATION (ESR) gene was comparatively increased during shoot regeneration in the culture usingsolid medium. Our results point out that the physical state of the medium in S. khasianum plays a decisive role in differentiation pattern which was independent of hormonal supplements.

A Gene Regulatory Network for Cellular Reprogramming in Plant Regeneration.

Wounding triggers organ regeneration in many plant species, and application of plant hormones, such as auxin and cytokinin, enhances their regenerative capacities in tissue culture. Recent studies have identified several key players mediating wound- and/or plant hormone-induced cellular reprogramming, but the global architecture of gene regulatory relationships underlying plant cellular reprogramming is still far from clear. In this study, we uncovered a gene regulatory network (GRN) associated with plant cellular reprogramming by using an enhanced yeast one-hybrid (eY1H) screen systematically to identify regulatory relationships between 252 transcription factors (TFs) and 48 promoters. Our network analyses suggest that wound- and/or hormone-invoked signals exhibit extensive cross-talk and regulate many common reprogramming-associated genes via multilayered regulatory cascades. Our data suggest that PLETHORA 3 (PLT3), ENHANCER OF SHOOT REGENERATION 1 (ESR1) and HEAT SHOCK FACTOR B 1 (HSFB1) act as critical nodes that have many overlapping targets and potentially connect upstream stimuli to downstream developmental decisions. Interestingly, a set of wound-inducible APETALA 2/ETHYLENE RESPONSE FACTORs (AP2/ERFs) appear to regulate these key genes, which, in turn, form feed-forward cascades that control downstream targets associated with callus formation and organ regeneration. In addition, we found another regulatory pathway, mediated by LATERAL ORGAN BOUNDARY/ASYMMETRIC LEAVES 2 (LOB/AS2) TFs, which probably plays a distinct but partially overlapping role alongside the AP2/ERFs in the putative gene regulatory cascades. Taken together, our findings provide the first global picture of the GRN governing plant cell reprogramming, which will serve as a valuable resource for future studies.

WIND1 Promotes Shoot Regeneration through Transcriptional Activation of ENHANCER OF SHOOT REGENERATION1 in Arabidopsis.

Many plant species display remarkable developmental plasticity and regenerate new organs after injury. Local signals produced by wounding are thought to trigger organ regeneration but molecular mechanisms underlying this control remain largely unknown. We previously identified an AP2/ERF transcription factor WOUND INDUCED DEDIFFERENTIATION1 (WIND1) as a central regulator of wound-induced cellular reprogramming in plants. In this study, we demonstrate that WIND1 promotes callus formation and shoot regeneration by upregulating the expression of the ENHANCER OF SHOOT REGENERATION1 (ESR1) gene, which encodes another AP2/ERF transcription factor in Arabidopsis thaliana The esr1 mutants are defective in callus formation and shoot regeneration; conversely, its overexpression promotes both of these processes, indicating that ESR1 functions as a critical driver of cellular reprogramming. Our data show that WIND1 directly binds the vascular system-specific and wound-responsive cis-element-like motifs within the ESR1 promoter and activates its expression. The expression of ESR1 is strongly reduced in WIND1-SRDX dominant repressors, and ectopic overexpression of ESR1 bypasses defects in callus formation and shoot regeneration in WIND1-SRDX plants, supporting the notion that ESR1 acts downstream of WIND1. Together, our findings uncover a key molecular pathway that links wound signaling to shoot regeneration in plants.

Arabidopsis ENHANCER OF SHOOT REGENERATION 2 and PINOID are involved in in vitro shoot regeneration

ENHANCER OF SHOOT REGENERATION 2 (ESR2), like ESR1, plays several critical roles during in vitro shoot regeneration. We investigated the genetic interaction between ESR2 and PINOID (PID) during shoot regeneration in this study. Both esr2 and pid mutations markedly decreased the e ciency of shoot regeneration from root segments at comparable levels (27% and 35% of shoot numbers on wild type, respectively); while esr2-2 pid double mutants dramatically decreased the e ciency of shoot regeneration (4.7% of shoot numbers on wild type). Our results demonstrated an additive or synergistic e ect of these two mutations on shoot regeneration. Expression of ESR2 in pid explants during shoot regeneration was abnormal a er day 5, although pid mutation did not a ect ESR2 expression until day 5. In conclusion, PID appears to be required for late development of the shoot apical meristem during shoot regeneration.

Arabidopsis ENHANCER OF SHOOT REGENERATION ( ESR) 1 and ESR2 regulate in vitro shoot regeneration and their expressions are differentially regulated

The Arabidopsis ENHANCER OF SHOOT REGENERATION ( ESR) 1 and ESR2 genes are thought to play critical roles in in vitro shoot regeneration. In this study, we investigated the functions and expression patterns of ESR1 and ESR2 during shoot regeneration by using their mutants and promoter-reporter systems. Shoot regeneration efficiencies of esr1 esr2 double mutants from hypocotyl explants decreased drastically; although the effects on shoot regeneration of the esr1 single mutation were much less marked than those of the esr2 single mutation, especially from root explants, their effects were additive. We found that ESR1 was initially expressed 1 day after transfer onto shoot-inducing medium (SIM), compared with 4 days for ESR2 expression. These results suggest that the functions of ESR1 and ESR2 in shoot regeneration are not redundant, even though they encode similar transcription factors and the ESR2 gene substituted with an ESR1 coding region complements the esr2 mutation. We also found that ESR1 expression was localized to a small number of cells in the lateral root meristem (LRM)-like structures, and the ESR1-expressing cells appeared to proliferate to form shoot apical meristem (SAM)-like structures. Thus, ESR1 may be involved in the change of LRM to SAM in tissue culture.

Identification of ENHANCER OF SHOOT REGENERATION 1-upregulated genes during in vitro shoot regeneration

The Arabidopsis ENHANCER OF SHOOT REGENERATION 1 (ESR1) is thought to be a key gene for commitment to in vitro shoot regeneration in tissue culture. ESR1 encodes a member of the ETHYLENE RESPONSIVE FACTOR (ERF) family of transcription factors. Here, we report identification of genes downstream of ESR1 during in vitro shoot regeneration. We previously demonstrated that the ESR1 protein functions as a transcriptional activator; in the present study, genes upregulated after induction of ESR1 overexpression were screened by microarray experiments. Seven genes, including CUP-SHAPED COTYLEDON 1 (CUC1), CLAVATA3/EMBRYO SURROUNDING REGION-RELATED PEPTIDE 2 (CLE2), and GCN5-related N-acetyltransferase 1 (GNAT1) were identified as ESR1-upregulated genes by screening. CUC1, CLE2, and GNAT1 were also upregulated by translocation of ESR1-ER (estrogen receptor) fusions to the nucleus in the presence of cycloheximide, suggesting that these genes are possibly the direct target of the ESR1 protein. Transcript levels of CUC1, CLE2, and GNAT1 as well as ESR1 increased during the early in vitro shoot regeneration process, although their time courses were not necessarily similar. Thus, these genes may function downstream of ESR1 and may be involved in the shoot differentiation process.

A domain containing the ESR motif in ENHANCER OF SHOOT REGENERATION 1 functions as a transactivation domain

The Arabidopsis ENHANCER OF SHOOT REGENERATION 1 (ESR1) gene is thought to regulate the initiation of in vitro shoot regeneration. ESR1 encodes a transcriptional activator belonging to the ETHYLENE-RESPONSIVE FACTOR family. In this study, we investigated the functions of the C-terminal domain of the ESR1 protein. Effector- reporter assays using the GAL4 DNA-binding domain fused with portions of the C-terminal domain revealed that the ESR motif, which is a short amino acid sequence commonly seen in the C-terminal domain of ESR1 and ESR2, functions as a transactivation domain. We also examined the effects of partial deletions of the C-terminal domain on the in vitro shoot regeneration-enhancing effects of ESR1 overexpression. The results revealed that the ESR motif and a region between the AP2/ERF domain and the ESR motif were required for the shoot regeneration-enhancing effects of ESR1. The region between the AP2/ERF domain and the ESR motif appeared to have an essential function other than nuclear localization, DNA-binding, and transactivation. Substitution of the ESR motif by the VP16 transactivation domain abolished the ability of ESR1 to enhance shoot regeneration, suggesting that the ESR motif not only has transactivation activity but also has other functions.

The Arabidopsis transcription factor ESR1 induces in vitro shoot regeneration through transcriptional activation

The Arabidopsis Enhancer of Shoot Regeneration 1 (ESR1) gene regulates initiation of in vitro shoot regeneration. In this study, we investigated the transcription-modulating potential of ESR1. ESR1 induced reporter gene expression when overexpressed transiently in Arabidopsis leaf cells. Experiments using a fusion protein with the GAL4 DNA-binding domain located a transactivating domain of ESR1 within the C-terminal region. A nuclear localization signal was also located within the AP2/ERF domain. These results demonstrated that ESR1 functions as a transcriptional activator. Furthermore, we examined whether transcriptional modulation by ESR1 affects the in vitro shoot regeneration efficiency. Overexpression of ESR1 fused with the VP16 transactivating domain enhanced in vitro shoot regeneration as well as overexpressed wild-type ESR1 did, while overexpression of ESR1 fused with a strong repression domain, SRDX, inhibited shoot regeneration. These results suggest that ESR1 induces shoot regeneration through its transactivating ability.

The AP2 transcription factors DORNRÖSCHEN and DORNRÖSCHEN-LIKE redundantly controlArabidopsisembryo patterning via interaction with PHAVOLUTA

DORNROSCHEN (DRN) (also known as ENHANCER OF SHOOT REGENERATION1; ESR1) and DRN-LIKE (DRNL; also known as ESR2) are two linked paralogues encoding AP2 domain-containing proteins. drn mutants show embryo cell patterning defects and, similarly to drnl mutants, disrupt cotyledon development at incomplete penetrance. drn drnl double mutants with weak or strong drnl alleles show more highly penetrant and extreme phenotypes, including a pin-like embryo without cotyledons, confirming a high degree of functional redundancy for the two genes in embryo patterning. Altered expression of PIN1::PIN1-GFP and DR5::GFP in drn mutant embryos places DRN upstream of auxin transport and response. A yeast two-hybrid screen with DRN followed by co-immunoprecipitation and bimolecular fluorescence complementation revealed PHAVOLUTA (PHV) to be a protein interaction partner in planta. drn phv double mutants show an increased penetrance of embryo cell division defects. DRNL can also interact with PHV and both DRN and DRNL can heterodimerise with additional members of the class III HD-ZIP family, PHABULOSA, REVOLUTA, CORONA and ATHB8. Interactions involve the PAS-like C-terminal regions of these proteins and the DRN/DRNL AP2 domain.