De novo organ regeneration is an excellent biological system for the study of fundamental questions regarding stem cell initiation, cell fate determination, and hormone signaling. Despite the general belief that auxin and cytokinin responses interact to regulate de novo organ regeneration, the molecular mechanisms underlying such a cross talk are little understood. Here, we show that spatiotemporal biosynthesis and polar transport resulted in local auxin distribution in Arabidopsis (Arabidopsis thaliana), which in turn determined the cytokinin response during de novo shoot regeneration. Genetic and pharmacological interference of auxin distribution disrupted the cytokinin response and ATP/ADP ISOPENTENYLTRANSFERASE5 (AtIPT5) expression, affecting stem cell initiation and meristem formation. Transcriptomic data suggested that AUXIN RESPONSE FACTOR3 (ARF3) mediated the auxin response during de novo organ regeneration. Indeed, mutations in ARF3 caused ectopic cytokinin biosynthesis via the misexpression of AtIPT5, and this disrupted organ regeneration. We further showed that ARF3 directly bound to the promoter of AtIPT5 and negatively regulated AtIPT5 expression. The results from this study thus revealed an auxin-cytokinin cross talk mechanism involving distinct intermediate signaling components required for de novo stem cell initiation and shed new light on the mechanisms of organogenesis in planta.

ORCID IDs: 0000-0003-3103-8629 (W.J.M.); 0000-0002-3129-5206 (X.S.Z.)Plants are known for their capacity to regenerate the whole body through de novo formation of apical meristems from a mass of proliferating cells named callus. Exogenous cytokinin and auxin determine cell fate for the establishment of the stem cell niche, which is the vital step of shoot regeneration, but the underlying mechanisms remain unclear. Here, we show that type-B ARABIDOPSIS RESPONSE REGULATORs (ARRs), critical components of cytokinin signaling, activate the transcription of WUSCHEL (WUS), which encodes a key regulator for maintaining stem cells. In parallel, type-B ARRs inhibit auxin accumulation by repressing the expression of YUCCAs, which encode a key enzyme for auxin biosynthesis, indirectly promoting WUS induction. Both pathways are essential for de novo regeneration of the shoot stem cell niche. In addition, the dual regulation of type-B ARRs on WUS transcription is required for the maintenance of the shoot apical meristem in planta. Thus, our results reveal a long-standing missing link between cytokinin signaling and WUS regulator, and the findings provide critical information for understanding cell fate specification.

Plants have a profound capacity to regenerate organs from differentiated somatic tissues, based on which propagating plants in vitro was made possible. Beside its use in biotechnology, in vitro shoot regeneration is also an important system to study de novo organogenesis. Phytohormones and transcription factor WUSCHEL (WUS) play critical roles in this process but whether and how epigenetic modifications are involved is unknown. Here, we report that epigenetic marks of DNA methylation and histone modifications regulate de novo shoot regeneration of Arabidopsis through modulating WUS expression and auxin signaling. First, functional loss of key epigenetic genes—including METHYLTRANSFERASE1 (MET1) encoding for DNA methyltransferase, KRYPTONITE (KYP) for the histone 3 lysine 9 (H3K9) methyltransferase, JMJ14 for the histone 3 lysine 4 (H3K4) demethylase, and HAC1 for the histone acetyltransferase—resulted in altered WUS expression and developmental rates of regenerated shoots in vitro. Second, we showed that regulatory regions of WUS were developmentally regulated by both DNA methylation and histone modifications through bisulfite sequencing and chromatin immunoprecipitation. Third, DNA methylation in the regulatory regions of WUS was lost in the met1 mutant, thus leading to increased WUS expression and its localization. Fourth, we did a genome-wide transcriptional analysis and found out that some of differentially expressed genes between wild type and met1 were involved in signal transduction of the phytohormone auxin. We verified that the increased expression of AUXIN RESPONSE FACTOR3 (ARF3) in met1 indeed was due to DNA demethylation, suggesting DNA methylation regulates de novo shoot regeneration by modulating auxin signaling. We propose that DNA methylation and histone modifications regulate de novo shoot regeneration by modulating WUS expression and auxin signaling. The study demonstrates that, although molecular components involved in organogenesis are divergently evolved in plants and animals, epigenetic modifications play an evolutionarily convergent role in this process.

Seed development includes an early stage of endosperm proliferation and a late stage of embryo growth at the expense of the endosperm in Arabidopsis thaliana. Abscisic acid (ABA) has known functions during late seed development, but its roles in early seed development remain elusive. In this study, we report that ABA-deficient mutants produced seeds with increased size, mass, and embryo cell number but delayed endosperm cellularization. ABSCISIC ACID DEFICIENT2 (ABA2) encodes a unique short-chain dehydrogenase/reductase that functions in ABA biosynthesis, and its expression pattern overlaps that of SHORT HYPOCOTYL UNDER BLUE1 (SHB1) during seed development. SHB1 RNA accumulation was significantly upregulated in the aba2-1 mutant and was downregulated by the application of exogenous ABA. Furthermore, RNA accumulation of the basic/region leucine zipper transcription factor ABSCISIC ACID-INSENSITIVE5 (ABI5), involved in ABA signaling, was decreased in aba2-1. Consistent with this, seed size was also increased in abi5. We further show that ABI5 directly binds to two discrete regions in the SHB1 promoter. Our results suggest that ABA negatively regulates SHB1 expression, at least in part, through the action of its downstream signaling component ABI5. Our findings provide insights into the molecular mechanisms by which ABA regulates early seed development.

Plant mature cells have the capability to reverse their state of differentiation and produce new organs under cultured conditions. Two phases, dedifferentiation and redifferentiation, are commonly characterized during in vitro organogenesis. In these processes, cells undergo fate switch several times regulated by both extrinsic and intrinsic factors, which are associated with reentry to the cell cycle, the balance between euchromatin and heterochromatin, reprogramming of gene expression, and so forth. This short article reviews the advances in the mechanism of organ regeneration from plant somatic cells in molecular, genomic and epigenetic aspects, aiming to provide important information on the mechanism underlying cell fate switch during in vitro plant organogenesis.

Contents Summary 1334 I. Introduction 1334 II. Regeneration-initial cell: the origin of regeneration 1335 III. Acquiring regeneration competency: the essential intermediate step for hormone-induced regeneration 1335 IV. Hormonal induction of stem cell regulators: the program for de novo establishment of apical meristems 1337 V. Conclusions and perspectives 1337 Acknowledgements 1338 Author contributions 1338 References 1338 SUMMARY: High cellular plasticity confers remarkable regeneration capacity to plants. Based on the activity of stem cells and their regulators, higher plants are capable of regenerating new individuals. De novo organogenesis exemplifies the regeneration of the whole plant body and is exploited widely in agriculture and biotechnology. In this Tansley insight article, we summarize recent advances that facilitate our understanding of the molecular mechanisms underlying de novo organogenesis. According to our current knowledge, this process can be divided into three steps, including activation of regeneration-initial cells, acquisition of competency and de novo establishment of apical meristems. The functions of stem cells and their regulators are critical to de novo organogenesis, whereas auxin and cytokinin act as triggers and linkers between different steps.

Inflorescence regeneration in vitro provides a simplified approach for the study of inflorescence development. In this study, high frequency of regenerated inflorescences was established using Arabidopsis stage-10 pistil as the explants on the inducing medium containing the 2 mg/L zeatin and 0.01 mg/L indole-3-acetic acid. TERMINAL FLOWER 1 (TFL1) expression was detected in callus at 6 days after transferred to inducing medium, and LEAFY (LFY) expression was detectable subsequently, suggesting that both genes play important roles as they function on inflorescence development in the plant. To investigate the formation of the stem cell organizing center, we examined the WUSCHEL (WUS) and CLAVATA3 (CLV3) expression within callus during inflorescence regeneration. WUS signals start to accumulate on callus at 4 days after induction, and then, the CLV3 signals are induced on callus at 5 days on the inflorescence-inducing medium. The expression domain of WUS is below that of CLV3, indicating that the patterns of the organizing center and stem cell formation are similar to that in zygotic and somatic embryogenesis. However, more cells of the organizing center were observed within callus than pro-embryo, suggesting that inflorescence differentiation requires more cells of the organizing center. Furthermore, it was found that the WUS expression is controlled by the ratio of cytokinin with auxin. The results suggest that other factors besides WUS and CLV3 are required for inflorescence regeneration.

The MADS box genes are essential for floral development of Arabidopsis because they regulate flowering time and the development of floral organs. In this study, a MADS box gene, TaMADS1, was isolated and characterized from wheat (Triticum aestivum L.). The analysis of amino acid sequences and phylogenetic tree suggested that the TaMADS1 gene might be a SEPALLATA (SEP)-like gene. RNA hybridization indicated that the TaMADS1 transcripts began to accumulate in spikelets, and then, in floret primordia and floral organ primordia in wheat. In the later stage of floret development, the TaMADS1 transcripts accumulated in four whorls of young floral organs. The ectopic expression of TaMADS1 in Arabidopsis caused early flowering and altered the development of all floral organs. Further studies demonstrated that the early flowering phenotype in transgenic plants could be correlated with the upregulation of some flowering time genes and flower meristem identity genes. Our results suggest that TaMADS1 could be a putative SEP-like gene, and has diverse roles in flower development.