Efficient regeneration of protoplasts from Solanum lycopersicum cultivar Micro-Tom.

Protoplast regeneration has become a key platform for genetic and genome engineering. However, we lack reliable and reproducible methods for efficient protoplast regeneration for tomato (Solanum lycopersicum) cultivars. Here, we optimized cell and tissue culture methods for protoplast isolation, microcallus proliferation, shoot regeneration, and plantlet establishment of the tomato cultivar Micro-Tom. A thin layer of alginate was applied to protoplasts isolated from third to fourth true leaves and cultured at an optimal density of 1 × 105 protoplasts/ml. We determined the optimal culture media for protoplast proliferation, callus formation, de novo shoot regeneration, and root regeneration. Regenerated plantlets exhibited morphologically normal growth and sexual reproduction. The entire regeneration process, from protoplasts to flowering plants, was accomplished within 5 months. The optimized protoplast regeneration platform enables biotechnological applications, such as genome engineering, as well as basic research on plant regeneration in Solanaceae species.

Genomic overview of INA-induced NPR1 targeting and transcriptional cascades in Arabidopsis.

The phytohormone salicylic acid (SA) triggers transcriptional reprogramming that leads to SA-induced immunity in plants. NPR1 is an SA receptor and master transcriptional regulator in SA-triggered transcriptional reprogramming. Despite the indispensable role of NPR1, genome-wide direct targets of NPR1 specific to SA signaling have not been identified. Here, we report INA (functional SA analog)-specific genome-wide targets of Arabidopsis NPR1 in plants expressing GFP-fused NPR1 under its native promoter. Analyses of NPR1-dependently expressed direct NPR1 targets revealed that NPR1 primarily activates genes encoding transcription factors upon INA treatment, triggering transcriptional cascades required for INA-induced transcriptional reprogramming and immunity. We identified genome-wide targets of a histone acetyltransferase, HAC1, including hundreds of co-targets shared with NPR1, and showed that NPR1 and HAC1 regulate INA-induced histone acetylation and expression of a subset of the co-targets. Genomic NPR1 targeting was principally mediated by TGACG-motif binding protein (TGA) transcription factors. Furthermore, a group of NPR1 targets mostly encoding transcriptional regulators was already bound to NPR1 in the basal state and showed more rapid and robust induction than other NPR1 targets upon SA signaling. Thus, our study unveils genome-wide NPR1 targeting, its role in transcriptional reprogramming, and the cooperativity between NPR1, HAC1, and TGAs in INA-induced immunity.

REGENOMICS: A web-based application for plant REGENeration-associated transcriptOMICS analyses.

In plants, differentiated somatic cells exhibit an exceptional ability to regenerate new tissues, organs, or whole plants. Recent studies have unveiled core genetic components and pathways underlying cellular reprogramming and de novo tissue regeneration in plants. Although high-throughput analyses have led to key discoveries in plant regeneration, a comprehensive organization of large-scale data is needed to further enhance our understanding of plant regeneration. Here, we collected all currently available transcriptome datasets related to wounding responses, callus formation, de novo organogenesis, somatic embryogenesis, and protoplast regeneration to construct REGENOMICS, a web-based application for plant REGENeration-associated transcriptOMICS analyses. REGENOMICS supports single- and multi-query analyses of plant regeneration-related gene-expression dynamics, co-expression networks, gene-regulatory networks, and single-cell expression profiles. Furthermore, it enables user-friendly transcriptome-level analysis of REGENOMICS-deposited and user-submitted RNA-seq datasets. Overall, we demonstrate that REGENOMICS can serve as a key hub of plant regeneration transcriptome analysis and greatly enhance our understanding on gene-expression networks, new molecular interactions, and the crosstalk between genetic pathways underlying each mode of plant regeneration. The REGENOMICS web-based application is available at http://plantregeneration.snu.ac.kr.

Negative evidence on the transgenerational inheritance of defense priming in Arabidopsis thaliana.

Defense priming allows plants to enhance their immune responses to subsequent pathogen challenges. Recent reports suggested that acquired resistances in parental generation can be inherited into descendants. Although epigenetic mechanisms are plausible tools enabling the transmission of information or phenotypic traits induced by environmental cues across generations, the mechanism for the transgenerational inheritance of defense priming in plants has yet to be elucidated. With the initial aim to elucidate an epigenetic mechanism for the defense priming in plants, we reassessed the transgenerational inheritance of plant defense, however, could not observe any evidence supporting it. By using the same dipping method with previous reports, Arabidopsis was exposed repeatedly to Pseudomonas syringae pv tomato DC3000 (Pst DC3000) during vegetative or reproductive stages. Irrespective of the developmental stages of parental plants that received pathogen infection, the descendants did not exhibit primed resistance phenotypes, defense marker gene (PR1) expression, or elevated histone acetylation within PR1 chromatin. In assays using the pressure-infiltration method for infection, we obtained the same results as above. Thus, our results suggest that the previous observations on the transgenerational inheritance of defense priming in plants should be more extensively and carefully reassessed. [BMB Reports 2022; 55(7): 342-347].

Optimization of protoplast regeneration in the model plant Arabidopsis thaliana.

BACKGROUND: Plants have a remarkable reprogramming potential, which facilitates plant regeneration, especially from a single cell. Protoplasts have the ability to form a cell wall and undergo cell division, allowing whole plant regeneration. With the growing need for protoplast regeneration in genetic engineering and genome editing, fundamental studies that enhance our understanding of cell cycle re-entry, pluripotency acquisition, and de novo tissue regeneration are essential. To conduct these studies, a reproducible and efficient protoplast regeneration method using model plants is necessary. RESULTS: Here, we optimized cell and tissue culture methods for improving protoplast regeneration efficiency in Arabidopsis thaliana. Protoplasts were isolated from whole seedlings of four different Arabidopsis ecotypes including Columbia (Col-0), Wassilewskija (Ws-2), Nossen (No-0), and HR (HR-10). Among these ecotypes, Ws-2 showed the highest potential for protoplast regeneration. A modified thin alginate layer was applied to the protoplast culture at an optimal density of 1 × 106 protoplasts/mL. Following callus formation and de novo shoot regeneration, the regenerated inflorescence stems were used for de novo root organogenesis. The entire protoplast regeneration process was completed within 15 weeks. The in vitro regenerated plants were fertile and produced morphologically normal progenies. CONCLUSION: The cell and tissue culture system optimized in this study for protoplast regeneration is efficient and reproducible. This method of Arabidopsis protoplast regeneration can be used for fundamental studies on pluripotency establishment and de novo tissue regeneration.

De Novo Shoot Regeneration Controlled by HEN1 and TCP3/4 in Arabidopsis.

Plants have the ability to regenerate whole plant body parts, including shoots and roots, in vitro from callus derived from a variety of tissues. However, the underlying mechanisms for this de novo organogenesis, which is based on the totipotency of callus cells, are poorly understood. Here, we report that a microRNA (miRNA)-mediated posttranscriptional regulation plays an important role in de novo shoot regeneration. We found that mutations in HUA ENHANCER 1 (HEN1), a gene encoding a small RNA methyltransferase, cause cytokinin-related defects in de novo shoot regeneration. A hen1 mutation caused a large reduction in the miRNA319 (miR319) level and a subsequent increase in its known target (TCP3 and TCP4) transcript levels. TCP transcription factors redundantly inhibited shoot regeneration and directly activated the expression of a negative regulator of cytokinin response ARABIDOPSIS THALIANA RESPONSE REGULATOR 16 (ARR16). A tcp4 mutation at least partly rescued the shoot-regeneration defect and derepression of ARR16 in hen1. These findings demonstrate that the miR319-TCP3/4-ARR16 axis controls de novo shoot regeneration by modulating cytokinin responses.

Salicylic acid-induced transcriptional reprogramming by the HAC-NPR1-TGA histone acetyltransferase complex in Arabidopsis.

Plant immunity depends on massive expression of pathogenesis-related genes (PRs) whose transcription is de-repressed by pathogen-induced signals. Salicylic acid (SA) acts as a major signaling molecule in plant immunity and systemic acquired resistance triggered by bacterial or viral pathogens. SA signal results in the activation of the master immune regulator, Nonexpressor of pathogenesis-related genes 1 (NPR1), which is thought to be recruited by transcription factors such as TGAs to numerous downstream PRs. Despite its key role in SA-triggered immunity, the biochemical nature of the transcriptional coactivator function of NPR1 and the massive transcriptional reprogramming induced by it remain obscure. Here we demonstrate that the CBP/p300-family histone acetyltransferases, HACs and NPR1 are both essential to develop SA-triggered immunity and PR induction. Indeed HACs and NPR1 form a coactivator complex and are recruited to PR chromatin through TGAs upon SA signal, and finally the HAC-NPR1-TGA complex activates PR transcription by histone acetylation-mediated epigenetic reprogramming. Thus, our study reveals a molecular mechanism of NPR1-mediated transcriptional reprogramming and a key epigenetic aspect of the central immune system in plants.

Epigenetic reprogramming by histone acetyltransferase HAG1/AtGCN5 is required for pluripotency acquisition in Arabidopsis.

Shoot regeneration can be achieved in vitro through a two-step process involving the acquisition of pluripotency on callus-induction media (CIM) and the formation of shoots on shoot-induction media. Although the induction of root-meristem genes in callus has been noted recently, the mechanisms underlying their induction and their roles in de novo shoot regeneration remain unanswered. Here, we show that the histone acetyltransferase HAG1/AtGCN5 is essential for de novo shoot regeneration. In developing callus, it catalyzes histone acetylation at several root-meristem gene loci including WOX5, WOX14, SCR, PLT1, and PLT2, providing an epigenetic platform for their transcriptional activation. In turn, we demonstrate that the transcription factors encoded by these loci act as key potency factors conferring regeneration potential to callus and establishing competence for de novo shoot regeneration. Thus, our study uncovers key epigenetic and potency factors regulating plant-cell pluripotency. These factors might be useful in reprogramming lineage-specified plant cells to pluripotency.

Epigenetic control of juvenile-to-adult phase transition by the Arabidopsis SAGA-like complex.

During growth and development, plants undergo a series of phase transitions from the juvenile-to-adult vegetative phase to the reproductive phase. In Arabidopsis, vegetative phase transitions and flowering are regulated by SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) factors. SPL mRNAs are post-transcriptionally regulated by miR156 in an age-dependent manner; however, the role of other mechanisms in this process is not known. In this study, we demonstrate that the HAG1/GCN5- and PRZ1/ADA2b-containing SAGA-like histone acetyltransferase (HAT) complex directly controls the transcription of SPLs and determines the time for juvenile-to-adult phase transition. Thus, epigenetic control by the SAGA-like HAT complex determines the transcriptional output of SPLs, which might be a prerequisite for the subsequent post-transcriptional regulation by miR156. Importantly, this epigenetic control mechanism is also crucial for miR156-independent induction of SPLs and acceleration of phase transition by light and photoperiod or during post-embryonic growth.

Negative regulatory roles of DE-ETIOLATED1 in flowering time in Arabidopsis.

Arabidopsis flowers early under long days (LD) and late under short days (SD). The repressor of photomorphogenesis DE-ETIOLATED1 (DET1) delays flowering; det1-1 mutants flower early, especially under SD, but the molecular mechanism of DET1 regulation remains unknown. Here we examine the regulatory function of DET1 in repression of flowering. Under SD, the det1-1 mutation causes daytime expression of FKF1 and CO; however, their altered expression has only a small effect on early flowering in det1-1 mutants. Notably, DET1 interacts with GI and binding of GI to the FT promoter increases in det1-1 mutants, suggesting that DET1 mainly restricts GI function, directly promoting FT expression independent of CO expression. Moreover, DET1 interacts with MSI4/FVE, which epigenetically inhibits FLC expression, indicating that the lack of FLC expression in det1-1 mutants likely involves altered histone modifications at the FLC locus. These data demonstrate that DET1 acts in both photoperiod and autonomous pathways to inhibit expression of FT and SOC1. Consistent with this, the early flowering of det1-1 mutants disappears completely in the ft-1 soc1-2 double mutant background. Thus, we propose that DET1 is a strong repressor of flowering and has a pivotal role in maintaining photoperiod sensitivity in the regulation of flowering time.

Repression of flowering under a noninductive photoperiod by the HDA9-AGL19-FT module in Arabidopsis.

Posttranslational acetylation of histones is reversibly regulated by histone deacetylases (HDACs). Despite the evident significance of HDACs in Arabidopsis development, the biological roles and underlying molecular mechanisms of many HDACs are yet to be elucidated. By a reverse-genetic approach, we isolated an hda9 mutant and performed phenotypic analyses on it. In order to address the role of HDA9 in flowering, genetic, molecular, and biochemical approaches were employed. hda9 flowered early under noninductive short-day (SD) conditions and had increased expression of the floral integrator FLOWERING LOCUS T (FT) and the floral activator AGAMOUS-LIKE 19 (AGL19) compared with the wild-type. The hda9 mutation increased histone acetylation and RNA polymerase II occupancy at AGL19 but not at FT during active transcription, and the HDA9 protein directly targeted AGL19. AGL19 expression was higher under SD than under inductive long-day (LD) conditions, and an AGL19 overexpression caused a strong up-regulation of FT. A genetic analysis showed that an agl19 mutation is epistatic to the hda9 mutation, masking both the early flowering and the increased FT expression of hda9. Taken together, our data indicate that HDA9 prevents precocious flowering under SD conditions by curbing the hyperactivation of AGL19, an upstream activator of FT, through resetting the local chromatin environment.

Genetic identification of a novel locus, ACCELERATED FLOWERING 1 that controls chromatin modification associated with histone H3 lysine 27 trimethylation in Arabidopsis thaliana.

Flowering on time is a critically important for successful reproduction of plants. Here we report an early-flowering mutant in Arabidopsis thaliana, accelerated flowering 1-1D (afl1-1D) that exhibited pleiotropic developmental defects including semi-dwarfism, curly leaf, and increased branching. Genetic analysis showed that afl1-1D mutant is a single, dominant mutant. Chromosomal mapping indicates that AFL1 resides at the middle of chromosome 4, around which no known flowering-related genes have been characterized. Expression analysis and double mutant studies with late flowering mutants in various floral pathways indicated that elevated FT is responsible for the early-flowering of afl1-1D mutant. Interestingly, not only flowering-related genes, but also several floral homeotic genes were ectopically overexpressed in the afl1-1D mutants in both FT-dependent and -independent manner. The degree of histone H3 Lys27-trimethylation (H3K27me3) was reduced in several chromatin including FT, FLC, AG and SEP3 in the afl1-1D, suggesting that afl1-1D might be involved in chromatin modification. In support, double mutant analysis of afl1-1D and lhp1-4 revealed epistatic interaction between afl1-1D and lhp1-4 in regard to flowering control. Taken together, we propose that AFL1 regulate various aspect of development through chromatin modification, particularly associated with H3K27me3 in A. thaliana.

Transcription elongation factor Tcea3 regulates the pluripotent differentiation potential of mouse embryonic stem cells via the Lefty1-Nodal-Smad2 pathway.

Self-renewal and pluripotency are hallmark properties of pluripotent stem cells, including embryonic stem cells (ESCs) and iPS cells. Previous studies revealed the ESC-specific core transcription circuitry and showed that these core factors (e.g., Oct3/4, Sox2, and Nanog) regulate not only self-renewal but also pluripotent differentiation. However, it remains elusive how these two cell states are regulated and balanced during in vitro replication and differentiation. Here, we report that the transcription elongation factor Tcea3 is highly enriched in mouse ESCs (mESCs) and plays important roles in regulating the differentiation. Strikingly, altering Tcea3 expression in mESCs did not affect self-renewal under nondifferentiating condition; however, upon exposure to differentiating cues, its overexpression impaired in vitro differentiation capacity, and its knockdown biased differentiation toward mesodermal and endodermal fates. Furthermore, we identified Lefty1 as a downstream target of Tcea3 and showed that the Tcea3-Lefty1-Nodal-Smad2 pathway is an innate program critically regulating cell fate choices between self-replication and differentiation commitment. Together, we propose that Tcea3 critically regulates pluripotent differentiation of mESCs as a molecular rheostat of Nodal-Smad2/3 signaling.

Small RNA and transcriptome deep sequencing proffers insight into floral gene regulation in Rosa cultivars.

BACKGROUND: Roses (Rosa sp.), which belong to the family Rosaceae, are the most economically important ornamental plants--making up 30% of the floriculture market. However, given high demand for roses, rose breeding programs are limited in molecular resources which can greatly enhance and speed breeding efforts. A better understanding of important genes that contribute to important floral development and desired phenotypes will lead to improved rose cultivars. For this study, we analyzed rose miRNAs and the rose flower transcriptome in order to generate a database to expound upon current knowledge regarding regulation of important floral characteristics. A rose genetic database will enable comprehensive analysis of gene expression and regulation via miRNA among different Rosa cultivars. RESULTS: We produced more than 0.5 million reads from expressed sequences, totalling more than 110 million bp. From these, we generated 35,657, 31,434, 34,725, and 39,722 flower unigenes from Rosa hybrid: 'Vital', 'Maroussia', and 'Sympathy' and Rosa rugosa Thunb., respectively. The unigenes were assigned functional annotations, domains, metabolic pathways, Gene Ontology (GO) terms, Plant Ontology (PO) terms, and MIPS Functional Catalogue (FunCat) terms. Rose flower transcripts were compared with genes from whole genome sequences of Rosaceae members (apple, strawberry, and peach) and grape. We also produced approximately 40 million small RNA reads from flower tissue for Rosa, representing 267 unique miRNA tags. Among identified miRNAs, 25 of them were novel and 242 of them were conserved miRNAs. Statistical analyses of miRNA profiles revealed both shared and species-specific miRNAs, which presumably effect flower development and phenotypes. CONCLUSIONS: In this study, we constructed a Rose miRNA and transcriptome database, and we analyzed the miRNAs and transcriptome generated from the flower tissues of four Rosa cultivars. The database provides a comprehensive genetic resource which can be used to better understand rose flower development and to identify candidate genes for important phenotypes.

Rhythmic oscillation of histone acetylation and methylation at the Arabidopsis central clock loci.

Circadian clock genes are regulated by a transcriptional-translational feedback loop. In Arabidopsis, LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) transcripts are highly expressed in the morning. Translated LHY and CCA1 proteins repress the expression of TIMING OF CAB EXPRESSION 1 (TOC1), which peaks in the evening. TOC1 protein induces expression of LHY and CCA1, forming a negative feedback loop which is believed to constitute the oscillatory mechanism of the clock. The rhythmic oscillation of mouse clock genes mPERIOD 1 (mPER1) and mPER2 has been correlated with regular alteration of chromatin structure through histone acetylation/deacetylation. However, little is known about the relationship between the transcriptional activity of Arabidopsis clock genes and their chromatin status. Here, we report that histone H3 acetylation (H3Ac) and H3 lysine 4 tri-methylation (H3K4me3) levels at LHY, CCA1, and TOC1 are positively correlated with the rhythmic transcript levels of these genes, whereas H3K36me2 level shows a negative correlation. Thus, our study suggests rhythmic transcription of Arabidopsis clock genes might be regulated by rhythmic histone modification, and it provides a platform for future identification of clock-controlling histone modifiers.

Control of seed germination by light-induced histone arginine demethylation activity.

For optimal survival, various environmental and endogenous factors should be monitored to determine the appropriate timing for seed germination. Light is a major environmental factor affecting seed germination, which is perceived by phytochromes. The light-dependent activation of phytochrome B (PHYB) modulates abscisic acid and gibberellic acid signaling and metabolism. Thus far, several negative regulators of seed germination that act when PHYB is inactive have been reported. However, neither positive regulators of seed germination downstream of PHYB nor a direct mechanism for regulation of the hormone levels has been elucidated. Here, we show that the histone arginine demethylases, JMJ20 and JMJ22, act redundantly as positive regulators of seed germination. When PHYB is inactive, JMJ20/JMJ22 are directly repressed by the zinc-finger protein SOMNUS. However, upon PHYB activation, JMJ20/JMJ22 are derepressed, resulting in increased gibberellic acid levels through the removal of repressive histone arginine methylations at GA3ox1/GA3ox2, which in turn promotes seed germination.

HDA19 is required for the repression of salicylic acid biosynthesis and salicylic acid-mediated defense responses in Arabidopsis.

To cope with a lifetime of exposure to a variety of pathogens, plants have developed exquisite and refined defense mechanisms that vary depending on the type of attacking pathogen. Defense-associated transcriptional reprogramming is a central part of plant defense mechanisms. Chromatin modification has recently been shown to be another layer of regulation for plant defense mechanisms. Here, we show that the RPD3/HDA1-class histone deacetylase HDA19 is involved in the repression of salicylic acid (SA)-mediated defense responses in Arabidopsis. Loss of HDA19 activity increased SA content and increased the expression of a group of genes required for accumulation of SA as well as pathogenesis related (PR) genes, resulting in enhanced resistance to Pseudomonas syringae. We found that HDA19 directly associates with and deacetylates histones at the PR1 and PR2 promoters. Thus, our study shows that HDA19, by modifying chromatin to a repressive state, ensures low basal expression of defense genes, such as PR1, under unchallenged conditions, as well as their proper induction without overstimulation during defense responses to pathogen attacks. Thus, the role of HDA19 might be critical in preventing unnecessary activation and self-destructive overstimulation of defense responses, allowing successful growth and development.

Identification of regulators required for the reactivation of FLOWERING LOCUS C during Arabidopsis reproduction.

FLOWERING LOCUS C (FLC) is a central floral repressor for the determination of flowering time in Arabidopsis. FLC expression is reactivated upon fertilization and regulated during seed development to ensure the appropriate floral behavior; however, the molecular mechanism for this process is largely unknown. Here, we report the identification of crucial regulators for FLC reactivation during embryogenesis by analyzing FLC::GUS and endogenous FLC expression. We newly define that the full reactivation of FLC requires a FRIGIDA (FRI)-containing protein complex throughout embryogenesis. Mutations in EARLY FLOWERING 7 (ELF7) and VERNALIZATION INDEPENDENCE4 (VIP4) showed severe defects in the reactivation of FLC transcription, suggesting that both of the genes, Arabidopsis homologs of the members of the yeast RNA polymerase II-associated factor 1 (Paf1) complex, are indispensable for FLC reactivation. actin-related protein 6 (arp6), arabidopsis trithorax 1 (atx1), arabidopsis trithorax-related 7 (atxr7), and atx1 atxr7 double mutants also caused the downregulation of FLC during seed development, but the defects were less severe than those in mutants for the FRI- and Paf1-complexes. These results suggest that the ARP6-containing Swr1-complex and FLC-specific histone methyltransferases, ATX1 and ATXR7, have relatively partial roles in FLC reactivation. In contrast to the roles of the histone modifiers, factors in the DNA methylation pathway and biogenesis of small RNAs are not involved in FLC regulation during reproduction. Taken together, our results demonstrate that adjustment by select FLC activators is critical for the re-establishment of an FLC expression state after fertilization to ensure competence for optimal flowering in the next generation.