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 modification-dependent production of peptide hormones facilitates acquisition of pluripotency during leaf-to-callus transition in Arabidopsis.

Chromatin configuration is critical for establishing tissue identity and changes substantially during tissue identity transitions. The crucial scientific and agricultural technology of in vitro tissue culture exploits callus formation from diverse tissue explants and tissue regeneration via de novo organogenesis. We investigated the dynamic changes in H3ac and H3K4me3 histone modifications during leaf-to-callus transition in Arabidopsis thaliana. We analyzed changes in the global distribution of H3ac and H3K4me3 during the leaf-to-callus transition, focusing on transcriptionally active regions in calli relative to leaf explants, defined by increased accumulation of both H3ac and H3K4me3. Peptide signaling was particularly activated during callus formation; the peptide hormones RGF3, RGF8, PIP1 and PIPL3 were upregulated, promoting callus proliferation and conferring competence for de novo shoot organogenesis. The corresponding peptide receptors were also implicated in peptide-regulated callus proliferation and regeneration capacity. The effect of peptide hormones in plant regeneration is likely at least partly conserved in crop plants. Our results indicate that chromatin-dependent regulation of peptide hormone production not only stimulates callus proliferation but also establishes pluripotency, improving the overall efficiency of two-step regeneration in plant systems.

The HOS1-PIF4/5 module controls callus formation in Arabidopsis leaf explants.

A two-step plant regeneration has been widely exploited to genetic manipulation and genome engineering in plants. Despite technical importance, understanding of molecular mechanism underlying in vitro plant regeneration remains to be fully elucidated. Here, we found that the HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 1 (HOS1)-PHYTOCHROME INTERACTING FACTOR 4/5 (PIF4/5) module participates in callus formation. Consistent with the repressive role of HOS1 in PIF transcriptional activation activity, hos1-3 mutant leaf explants exhibited enhanced callus formation, whereas pif4-101 pif5-3 mutant leaf explants showed reduced callus size. The HOS1-PIF4/5 function would be largely dependent on auxin biosynthesis and signaling, which are essential for callus initiation and proliferation. Our findings suggest that the HOS1-PIF4/5 module plays a pivotal role in auxin-dependent callus formation in Arabidopsis.

Ectopic expression of WOX5 promotes cytokinin signaling and de novo shoot regeneration.

KEY MESSAGE: WOX5 has a potential in activating cytokinin signaling and shoot regeneration, in addition to its role in pluripotency acquisition. Thus, overexpression of WOX5 maximizes plant regeneration capacity during tissue culture. In vitro plant regeneration involves two steps: callus formation and de novo shoot organogenesis. The WUSCHEL-RELATED HOMEOBOX 5 (WOX5) homeodomain transcription factor is known to be mainly expressed during incubation on callus-inducing medium (CIM) and involved in pluripotency acquisition in callus, but whether WOX5 also affects de novo shoot regeneration on cytokinin-rich shoot-inducing medium (SIM) remains unknown. Based on the recent finding that WOX5 promotes cytokinin signaling, we hypothesized that ectopic expression of WOX5 beyond CIM would further enhance overall plant regeneration capacity, because intense cytokinin signaling is particularly required for shoot regeneration on SIM. Here, we found that overexpression of the WOX5 gene on SIM drastically promoted de novo shoot regeneration from callus with the repression of type-A ARABIDOPSIS RESPONSE REGULATOR (ARR) genes, negative regulators of cytokinin signaling. The enhanced shoot regeneration phenotypes were indeed dependent on cytokinin signaling, which were partially suppressed in the progeny derived from crossing WOX5-overexpressing plants with cytokinin-insensitive 35S:ARR7 plants. The function of WOX5 in enhancing cytokinin-dependent shoot regeneration is evolutionarily conserved, as conditional overexpression of OsWOX5 on SIM profoundly enhanced shoot regeneration in rice callus. Overall, our results provide the technical advance that maximizes in vitro plant regeneration by constitutively expressing WOX5, which unequivocally promotes both callus pluripotency and de novo shoot regeneration.

Dynamic changes in DNA methylation occur in TE regions and affect cell proliferation during leaf-to-callus transition in Arabidopsis.

Plant somatic cells can be reprogrammed into pluripotent cell mass, called callus, through a two-step in vitro tissue culture method. Incubation on callus-inducing medium triggers active cell proliferation to form a pluripotent callus. Notably, DNA methylation is implicated during callus formation, but a detailed molecular process regulated by DNA methylation remains to be fully elucidated. Here, we compared genome-wide DNA methylation profiles between leaf and callus tissues in Arabidopsis using whole-genome bisulphite-sequencing. Global distribution of DNA methylation showed that CHG methylation was increased, whereas CHH methylation was reduced especially around transposable element (TE) regions during the leaf-to-callus transition. We further analysed differentially expressed genes around differentially methylated TEs (DMTEs) during the leaf-to-callus transition and found that genes involved in cell cycle regulation were enriched and also constituted a coexpression gene network along with pluripotency regulators. In addition, a conserved DNA sequence analysis for upstream cis-elements led us to find a putative transcription factor associated with cell fate transition. CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) was newly identified as a regulator of plant regeneration, and consistently, the cca1lhy mutant displayed altered phenotypes in callus proliferation. Overall, these results suggest that DNA methylation coordinates cell cycle regulation during callus formation, and CCA1 may act as a key upstream coordinator at least in part in the processes.

Arabidopsis ATXR2 represses de novo shoot organogenesis in the transition from callus to shoot formation.

Plants exhibit high regenerative capacity, which is controlled by various genetic factors. Here, we report that ARABIDOPSIS TRITHORAX-RELATED 2 (ATXR2) controls de novo shoot organogenesis by regulating auxin-cytokinin interaction. The auxin-inducible ATXR2 Trithorax Group (TrxG) protein temporally interacts with the cytokinin-responsive type-B ARABIDOPSIS RESPONSE REGULATOR 1 (ARR1) at early stages of shoot regeneration. The ATXR2-ARR1 complex binds to and deposits the H3K36me3 mark in the promoters of a subset of type-A ARR genes, ARR5 and ARR7, thus activating their expression. Consequently, the ATXR2/ARR1-type-A ARR module transiently represses cytokinin signaling and thereby de novo shoot regeneration. The atxr2-1 mutant calli exhibit enhanced shoot regeneration with low expression of ARR5 and ARR7, which ultimately upregulates WUSCHEL (WUS) expression. Thus, ATXR2 regulates cytokinin signaling and prevents premature WUS activation to ensure proper cell fate transition, and the auxin-cytokinin interaction underlies the initial specification of shoot meristem in callus.

The DME demethylase regulates sporophyte gene expression, cell proliferation, differentiation, and meristem resurrection.

The flowering plant life cycle consists of alternating haploid (gametophyte) and diploid (sporophyte) generations, where the sporophytic generation begins with fertilization of haploid gametes. In Arabidopsis, genome-wide DNA demethylation is required for normal development, catalyzed by the DEMETER (DME) DNA demethylase in the gamete companion cells of male and female gametophytes. In the sporophyte, postembryonic growth and development are largely dependent on the activity of numerous stem cell niches, or meristems. Analyzing Arabidopsis plants homozygous for a loss-of-function dme-2 allele, we show that DME influences many aspects of sporophytic growth and development. dme-2 mutants exhibited delayed seed germination, variable root hair growth, aberrant cellular proliferation and differentiation followed by enhanced de novo shoot formation, dysregulation of root quiescence and stomatal precursor cells, and inflorescence meristem (IM) resurrection. We also show that sporophytic DME activity exerts a profound effect on the transcriptome of developing Arabidopsis plants, including discrete groups of regulatory genes that are misregulated in dme-2 mutant tissues, allowing us to potentially link phenotypes to changes in specific gene expression pathways. These results show that DME plays a key role in sporophytic development and suggest that DME-mediated active DNA demethylation may be involved in the maintenance of stem cell activities during the sporophytic life cycle in Arabidopsis.

JA-pretreated hypocotyl explants potentiate de novo shoot regeneration in Arabidopsis.

Plant regeneration involves critical checkpoints including pluripotency acquisition and de novo organogenesis. However, comprehensive understanding of the mechanisms that underlie plant regeneration remains limited. Here, we found that calli derived from jasmonate (JA)-pretreated hypocotyl explants exhibited increased rates of de novo shoot regeneration. In contrast, exogenous JA treatment during callus formation on CIM did not influence the plant regeneration process. The enhanced shoot regeneration was diminished in coi1-1 mutants, indicating that JA-pretreated explants potentiate shoot regeneration in a COI1-dependent manner. These results suggest that the JA-responsive COI1 protein likely contributes to plant regeneration efficiency via regulation of hormone-signaling crosstalk and/or cell proliferation.

ARABIDOPSIS TRITHORAX 4 Facilitates Shoot Identity Establishment during the Plant Regeneration Process.

Plant cells have a remarkable plasticity that allows cellular reprogramming from differentiated cells and subsequent tissue regeneration. Callus formation occurs from pericycle-like cells through a lateral root developmental pathway, and even aerial parts can also undergo the cell fate transition. Pluripotent calli are then subjected primarily to shoot regeneration in in vitro tissue culture. Successful completion of plant regeneration from aerial explants thus entails a two-step conversion of tissue identity. Here we show that a single chromatin modifier, ARABIDOPSIS TRITHORAX 4 (ATX4)/SET DOMAIN GROUP 16, is dynamically regulated during plant regeneration to address proper callus formation and shoot regeneration. The ATX4 protein massively activates shoot identity genes by conferring H3K4me3 deposition at the loci. ATX4-deficient mutants display strong silencing of shoot identity and thus enhanced callus formation. Subsequently, de novo shoot organogenesis from calli is impaired in atx4 mutants. These results indicate that a series of epigenetic reprogramming of tissue identity underlies plant regeneration, and molecular components defining tissue identity can be used as invaluable genetic sources for improving crop transformation efficiency.

JMJ30-mediated demethylation of H3K9me3 drives tissue identity changes to promote callus formation in Arabidopsis.

Plant somatic cells can be reprogrammed by in vitro tissue culture methods, and massive genome-wide chromatin remodeling occurs, particularly during callus formation. Since callus tissue resembles root primordium, conversion of tissue identity is essentially required when leaf explants are used. Consistent with the fact that the differentiation state is defined by chromatin structure, which permits limited gene profiles, epigenetic changes underlie cellular reprogramming for changes to tissue identity. Although a histone methylation process suppressing leaf identity during leaf-to-callus transition has been demonstrated, the epigenetic factor involved in activation of root identity remains elusive. Here, we report that JUMONJI C DOMAIN-CONTAINING PROTEIN 30 (JMJ30) stimulates callus formation by promoting expression of a subset of LATERAL ORGAN BOUNDARIES-DOMAIN (LBD) genes that establish root primordia. The JMJ30 protein binds to promoters of the LBD16 and LBD29 genes along with AUXIN RESPONSE FACTOR 7 (ARF7) and ARF19 and activates LBD expression. Consistently, the JMJ30-deficient mutant displays reduced callus formation with low LBD transcript levels. The ARF-JMJ30 complex catalyzes the removal of methyl groups from H3K9me3, especially at the LBD16 and LBD29 loci to activate their expression during leaf-to-callus transition. Moreover, the ARF-JMJ30 complex further recruits ARABIDOPSIS TRITHORAX-RELATED 2 (ATXR2), which promotes deposition of H3K36me3 at the LBD16 and LBD29 promoters, and the tripartite complex ensures stable LBD activation during callus formation. These results indicate that the coordinated epigenetic modifications promote callus formation by establishing root primordium identity.

ATXR2 as a core regulator of de novo root organogenesis.

Tissue identity is plastically regulated in plants, and chromatin modifiers/remodelers are main players of cell fate changes. Callus formation is an intriguing example of cell fate transition. Leaf explants can form callus tissues, which resemble lateral root primordium, on callus-inducing medium (CIM). We recently demonstrated that the ARABIDOPSIS TRITHORAX-RELATED 2 (ATXR2) protein, which deposits H3K36me3 at genomic level, regulates callus formation on CIM. Consistent with the role of ATXR2 in conferring root identity, lateral root formation was significantly reduced in atxr2-deficient mutants. Furthermore, atxr2 mutants also displayed defects in adventitious root formation from wounded leaf tissues on hormone-free medium. Our findings indicate that ATXR2 is a genuine regulator of de novo root organogenesis.

Arabidopsis ATXR2 deposits H3K36me3 at the promoters of LBD genes to facilitate cellular dedifferentiation.

Cellular dedifferentiation, the transition of differentiated somatic cells to pluripotent stem cells, ensures developmental plasticity and contributes to wound healing in plants. Wounding induces cells to form a mass of unorganized pluripotent cells called callus at the wound site. Explanted cells can also form callus tissues in vitro. Reversible cellular differentiation-dedifferentiation processes in higher eukaryotes are controlled mainly by chromatin modifications. We demonstrate that ARABIDOPSIS TRITHORAX-RELATED 2 (ATXR2), a histone lysine methyltransferase that promotes the accumulation of histone H3 proteins that are trimethylated on lysine 36 (H3K36me3) during callus formation, promotes early stages of cellular dedifferentiation through activation of LATERAL ORGAN BOUNDARIES DOMAIN (LBD) genes. The LBD genes of Arabidopsis thaliana are activated during cellular dedifferentiation to enhance the formation of callus. Leaf explants from Arabidopsis atxr2 mutants exhibited a reduced ability to form callus and a substantial reduction in LBD gene expression. ATXR2 bound to the promoters of LBD genes and was required for the deposition of H3K36me3 at these promoters. ATXR2 was recruited to LBD promoters by the transcription factors AUXIN RESPONSE FACTOR 7 (ARF7) and ARF19. Leaf explants from arf7-1arf19-2 double mutants were defective in callus formation and showed reduced H3K36me3 accumulation at LBD promoters. Genetic analysis provided further support that ARF7 and ARF19 were required for the ability of ATXR2 to promote the expression of LBD genes. These observations indicate that the ATXR2-ARF-LBD axis is key for the epigenetic regulation of callus formation in Arabidopsis.

RNA-Seq Analysis of the Arabidopsis Transcriptome in Pluripotent Calli.

Plant cells have a remarkable ability to induce pluripotent cell masses and regenerate whole plant organs under the appropriate culture conditions. Although the in vitro regeneration system is widely applied to manipulate agronomic traits, an understanding of the molecular mechanisms underlying callus formation is starting to emerge. Here, we performed genome-wide transcriptome profiling of wild-type leaves and leaf explant-derived calli for comparison and identified 10,405 differentially expressed genes (> two-fold change). In addition to the well-defined signaling pathways involved in callus formation, we uncovered additional biological processes that may contribute to robust cellular dedifferentiation. Particular emphasis is placed on molecular components involved in leaf development, circadian clock, stress and hormone signaling, carbohydrate metabolism, and chromatin organization. Genetic and pharmacological analyses further supported that homeostasis of clock activity and stress signaling is crucial for proper callus induction. In addition, gibberellic acid (GA) and brassinosteroid (BR) signaling also participates in intricate cellular reprogramming. Collectively, our findings indicate that multiple signaling pathways are intertwined to allow reversible transition of cellular differentiation and dedifferentiation.

Histone deacetylation-mediated cellular dedifferentiation in Arabidopsis.

Chromatin structure determines the accessibility of transcriptional regulators to target DNA and contributes to regulation of gene expression. Posttranslational modifications of core histone proteins underlie the reversible changes in chromatin structure. Epigenetic regulation is closely associated with cellular differentiation. Consistently, we found that histone deacetylation is required for callus formation from leaf explants in Arabidopsis . Treatment with trichostatin A (TSA) led to defective callus formation on callus-inducing medium (CIM). Gene expression profiling revealed that a subset of HDAC genes, including HISTONE DEACETYLASE 9 (HDA9), HD-TUINS PROTEIN 1 (HDT1), HDT2, HDT4, and SIRTUIN 1 (SRT1), was significantly up-regulated in calli. In support of this, genetic mutations of HDA9 or HDT1 showed reduced capability of callus formation, probably owing to their roles in regulating auxin and embryonic and meristematic developmental signaling. Taken together, our findings suggest that histone deacetylation is intimately associated with the leaf-to-callus transition, and multiple signaling pathways are controlled by means of histone modification during cellular dedifferentiation.

Evidence-based practice for pain management for cancer patients in an acute care setting.

The purpose of this study is to implement an evidence utilization project using an audit and feedback approach to improve cancer pain management. A three-phased audit and feedback approach was used. A 46-bed oncology nursing unit in the university's cancer centre was selected as a research site. Nursing records extracted from 137 patients (65 for the baseline assessment and 72 for the follow-up audit) were used to examine nurse compliance with four audit criteria derived from best practice guidelines related to the assessment and management of pain. We observed a significant improvement in compliance from baseline to follow-up for the following criteria: documenting the side effects of opioids (2-83%), use of a formalized pain assessment tool (22-75%), and providing education for pain assessment and management to patients and caregivers (0-47%). The audit and feedback method was applicable to the implementation of clinical practice guidelines for cancer pain management. Leadership from both administrative personnel and staff nurses working together contributes to the spread of an evidence-based practice culture in clinical settings. As it was conducted in a single oncology nursing unit and was implemented over a short period of time, the results should be carefully interpreted.