The shoot apical meristem of vascular plants generates all the aboveground organs. During this process, the structure and function of the meristem are maintained by a group of regulatory genes, among which the WUSCHEL (WUS)-CLAVATA3 (CLV3) module plays the core role. To date, all of the insights into shoot meristem homeostasis have been derived from studies on herbaceous plants. The mechanism by which the shoot meristem is maintained in trees remains unknown. In this study, we analyzed the functions of the poplar genes PagWUS and PagCLV3, homologs of Arabidopsis WUS and CLV3, respectively, in the maintenance and regeneration of the shoot meristem. Our results reveal both conserved and divergent functions compared to those of their orthologs in herbaceous species. Similar to their herbaceous counterparts, PagWUS and PagCLV3 are specifically expressed in the organizing center and stem cells, respectively, and form a feedback loop that regulates shoot meristem maintenance. Overexpression of PagWUS promoted shoot regeneration. Compared with herbaceous species, poplar possesses a much larger stem cell niche. The function of the PagWUS-PagCLV3 module is consistent with the developmental characteristics of perennial trees in that it regulates the cessation of the shoot meristem and mediates the proper pattern of secondary growth. Disruption of PagCLV3 enhanced shoot regeneration capacity. Our results shed light on shoot meristem regulation in trees and pave the way for understanding the mechanisms of meristem activity and plant development.

In the root apical meristem, the stem cell niche (SCN) comprises a mitotically inactive quiescent center (QC) and adjacent, mitotically active stem cells that divide to form root tissues. Auxin dynamics are essential for specification and maintenance of the root SCN; however, the underlying mechanisms remain to be explored. Here, we report that Arabidopsis (Arabidopsis thaliana) AUXIN RESPONSE FACTOR3 (ARF3), ARF3-INTERACTING PROTEIN1/2 (AIP1/2), and SIN3-ASSOCIATED POLYPEPTIDE OF 18 KDA (SAP18) form a protein complex that specifies root SCN cell fate in response to auxin level. In cells proximal to the QC, the ARF3-AIP1/2-SAP18 complex bound the WOX5 promoter and restricted WOX5 expression to the QC by decreasing H3 histone acetylation, thereby maintaining the SCN. Disrupting the ARF3-AIP1/2-SAP18 complex via mutation or exposure to excessive amounts of auxin resulted in proximal and lateral expansion of WOX5 expression and inhibited root elongation by repressing cell division. During de novo specification of the SCN in lateral root primordia or regenerating root tips, accumulated auxin caused the ARF3-AIP1/2-SAP18 complex to dissociate, allowing the induction of WOX5 expression. In the reestablished meristem, the ARF3-AIP1/2-SAP18 complex confines WOX5 expression to the newly formed QC. Our findings provide insights into the roles of auxin dynamics in determining root SCN.

Somatic cells of higher plants possess the remarkable ability to regenerate new individuals via reestablishing apical meristems. Reconstitution of shoot meristem is the vital process and is required for application of plant biotechnology. Under in vitro culture condition, shoot meristem can be formed directly or indirectly, depending on the absence or presence of callus as the intermediate status. However, the difference of regulatory mechanisms between the two regeneration types remains unknown. In this study, we established a bi-directional system in which shoots regenerated directly from lateral root primordia (LRP) and indirectly from hypocotyl-derived callus simultaneously. The results based on this system revealed that regulation of WOX11 expression represents the difference between the two regeneration types in two aspects. Firstly, number of founder cells expressing WOX11 is tightly associated with regeneration types. Relatively more founder cells gave rise to callus and produce larger meristem, whereas less founder cells produce LRP that regenerate smaller meristem. Secondly, non-CG DNA methylation specifically regulated WOX11 transcription in LRP and promoted direct shoot regeneration, but had no influence on indirect regeneration. The results provide new insights for understanding the regulatory mechanisms of cell fate transition during de novo organogenesis.

KEY MESSAGE:ARF4-regulated shoot regeneration through competing with ARF5 for the interaction with IAA12. Plant possess the ability to regenerate shoot meristem and subsequent the whole individual. This process is the foundation for in vitro propagation and genetic engineering and provides a system for studying fundamental biological questions, such as hormonal signaling. Auxin response factor (ARF) family transcription factors are critical components of auxin signaling pathway that regulate the transcription of target genes. To date, the mechanisms underlying the functions of class-B ARFs which act as transcription repressors remains unclear. In this study, we found that ARF4, the transcriptional repressor, was involved in regulating shoot regeneration. ARF4 interacted with auxin/Indole-3-Acetic-Acid12 (IAA12). The expression signals of ARF4 displayed a dynamic pattern similar with those of ARF5 and IAA12 during shoot meristem formation. Enhanced expression of IAA12 compromised the shoot regeneration capacity. Induced expression of ARF4 complemented the regeneration phenotype of IAA12-overexpression but did not rescued the defects in the arf5 mutant, mp-S319. Further analysis revealed that ARF4 competed with ARF5 for the interaction with IAA12. The results indicate that ARF4-regulated shoot regeneration through cooperating with ARF5 and IAA12. Our findings provided new information for deciphering the function of class-B ARFs.

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.

Pluripotent stem cells (PSCs) are self-renewable cells with the potential to differentiate into all the cell types within an organism. PSCs exist transiently in early-stage mammalian embryos during ontogeny and are maintained in apical meristems of higher plants throughout postembryonic development. Through proper in vitro culture, somatic cells of both mammals and plants can be reprogrammed to generate induced PSCs (iPSCs). Recent studies have deciphered mechanisms underlying pluripotency gene activation and cell fate transition during plant iPSC generation. Here, we compare these mechanisms with those of their animal counterparts in the hope that this may trigger mutual learning of researchers from both fields, leading to advances and independent breakthroughs in this important area.

Plants are known for their capacity to regenerate organs, such as shoot, root and floral organs. Recently, a number of studies contributed to understanding the mechanisms of shoot and root regeneration. However, the mechanisms underlying floral organ regeneration are largely unknown. In this study, we established a carpel regeneration system in which two types of carpels were induced by exogenous cytokinin. For type I, all the floral organs in the regenerated inflorescence were transformed into carpels. For type II, carpels were generated directly from callus. The transcript level of AGAMOUS (AG), the carpel identity gene, was up-regulated during carpel induction. The expression signals of AG were detected in the initiating carpel primordia and regenerating carpels, and co-localized with those of two Type-B ARABIDOPSIS RESPONSE REGULATORs (ARRs), ARR1 and ARR10. Repression of either AG or type-B ARRs reduced carpel regeneration. Binding analyses showed that ARR1 and ARR10 directly bound to transcriptional regulatory regions of AG and positively regulated its expression. In addition, the expression of type-B ARRs overlapped with that of AG in the floral primordia in planta. Defects in type-B ARRs reduced the number of carpels. The results indicate that type-B ARRs control carpel regeneration through activating AG expression. Our results provide new information for understanding the mechanism of carpel formation.

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.

The remarkable developmental plasticity of plants greatly relies on the regulation of stem cell niches in two distal meristems, namely the shoot apical meristem (SAM) and the root apical meristem (RAM). Plants also have a remarkable capability to regenerate new stem cell niches in vitro and in vivo. These distinct features confer plants with an ability to survive and propagate successfully under ever-changing environmental conditions. Studies in the model flowering plant Arabidopsis have revealed that plant hormones, including auxin, cytokinin, and peptides, play key roles in the maintenance of stem cell niches in apical meristems and the de novo regeneration of a new SAM and RAM, which sheds a new light on the signals and molecular mechanisms underlying maintenance of plant stem cell niches and regeneration capacity.

Wen Jing Meng, Zhi Juan Cheng, Ya Lin Sang, Miao Miao Zhang, Xiao Fei Rong, Zhi Wei 6 Wang, Ying Ying Tang and Xian Sheng Zhang* 7 State Key Laboratory of Crop Biology, College of Life Sciences, College of Forestry, 8 Shandong Agricultural University, Taian, Shandong 271018, China. 9 1 These authors contributed equally to this work. 10 *Correspondence to: zhangxs@sdau.edu.cn 11 12 Short title: Cytokinin and Auxin Re-specify Stem Cell Niche 13 14 One-sentence summary: Type-B ARRs specify the shoot stem cell niche by directly 15 activating WUS transcription and repressing the expression of YUCs that indirectly promote 16 WUS induction. 17 18 The author responsible for distribution of materials integral to the findings presented in this 19 article in accordance with the policy described in the Instructions for Authors 20 (www.plantcell.org) is: Xian Sheng Zhang (zhangxs@sdau.edu.cn). 21 22 ABSTRACT 23 Plants are known for their capacity to regenerate the whole body through de novo 24 formation of apical meristems from a mass of proliferating cells named callus. Exogenous 25 cytokinin and auxin determine cell fate for the establishment of the stem cell niche, which is 26 the vital step of shoot regeneration, but the underlying mechanisms remain unclear. Here we 27 show that type-B ARABIDOPSIS RESPONSE REGULATORs (ARRs), critical components 28 of cytokinin signaling, activate the transcription of WUSCHEL (WUS), which encodes a key 29 regulator for maintaining stem cells. In parallel, type-B ARRs inhibit auxin accumulation by 30 repressing the expression of YUCCAs, which encode a key enzyme for auxin biosynthesis, 31 indirectly promoting WUS induction. Both pathways are essential for de novo regeneration of 32 the shoot stem cell niche. In addition, the dual regulation of type-B ARRs on WUS 33 transcription is required for the maintenance of the shoot apical meristem in planta. Thus, our 34 results reveal a long-standing missing link between cytokinin signaling and WUS regulator, 35 and the findings provide critical information for understanding cell fate specification. 36

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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.

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.

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.

Plant somatic cells have the capability to switch their cell fates from differentiated to undifferentiated status under proper culture conditions, which is designated as totipotency. As a result, plant cells can easily regenerate new tissues or organs from a wide variety of explants. However, the mechanism by which plant cells have such remarkable regeneration ability is still largely unknown. In this study, we used a set of meristem-specific marker genes to analyze the patterns of stem cell differentiation in the processes of somatic embryogenesis as well as shoot or root organogenesis in vitro . Our studies furnish preliminary and important information on the patterns of the de novo stem cell differentiation during various types of in vitro organogenesis.

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.

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.

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.