Local auxin biosynthesis promotes shoot patterning and stem cell differentiation in Arabidopsis shoot apex.

The shoot apical meristem (SAM) of higher plants comprises distinct functional zones. The central zone (CZ) is located at the meristem summit and harbors pluripotent stem cells. Stem cells undergo cell division within the CZ and give rise to descendants, which enter the peripheral zone (PZ) and become recruited into lateral organs. Stem cell daughters that are pushed underneath the CZ form rib meristem (RM). To unravel the mechanism of meristem development, it is essential to know how stem cells adopt distinct cell fates in the SAM. Here, we show that meristem patterning and floral organ primordia formation, besides auxin transport, are regulated by auxin biosynthesis mediated by two closely related genes of the TRYPTOPHAN AMINOTRANSFERASE family. In Arabidopsis SAM, TAA1 and TAR2 played a role in maintaining auxin responses and the identity of PZ cell types. In the absence of auxin biosynthesis and transport, the expression pattern of the marker genes linked to the patterning of the SAM is perturbed. Our results prove that local auxin biosynthesis, in concert with transport, controls the patterning of the SAM into the CZ, PZ and RM.

ELONGATED HYPOCOTYL 5 regulates BRUTUS and affects iron acquisition and homeostasis in Arabidopsis thaliana.

Iron (Fe) is an essential micronutrient for both plants and animals. Fe-limitation significantly reduces crop yield and adversely impacts on human nutrition. Owing to limited bioavailability of Fe in soil, plants have adapted different strategies that not only regulate Fe-uptake and homeostasis but also bring modifications in root system architecture to enhance survival. Understanding the molecular mechanism underlying the root growth responses will have critical implications for plant breeding. Fe-uptake is regulated by a cascade of basic helix-loop-helix (bHLH) transcription factors (TFs) in plants. In this study, we report that HY5 (Elongated Hypocotyl 5), a member of the basic leucine zipper (bZIP) family of TFs, plays an important role in the Fe-deficiency signaling pathway in Arabidopsis thaliana. The hy5 mutant failed to mount optimum Fe-deficiency responses, and displayed root growth defects under Fe-limitation. Our analysis revealed that the induction of the genes involved in Fe-uptake pathway (FIT-FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR, FRO2-FERRIC REDUCTION OXIDASE 2 and IRT1-IRON-REGULATED TRANSPORTER1) is reduced in the hy5 mutant as compared with the wild-type plants under Fe-deficiency. Moreover, we also found that the expression of coumarin biosynthesis genes is affected in the hy5 mutant under Fe-deficiency. Our results also showed that HY5 negatively regulates BRUTUS (BTS) and POPEYE (PYE). Chromatin immunoprecipitation followed by quantitative polymerase chain reaction revealed direct binding of HY5 to the promoters of BTS, FRO2 and PYE. Altogether, our results showed that HY5 plays an important role in the regulation of Fe-deficiency responses in Arabidopsis.

Antioxidant and Antibacterial Activities Evaluation, Phytochemical Characterisation of Rhizome from Angiopteris helferiana and Barks from Saurauia fasciculata in Nepal.

Ethnomedicinally, more than 2000 plants were found to be used in Nepal. Among them, the red colored rhizome of Angiopteris helferiana and the bark of Saurauia fasciculata have been used widely to treat muscle fatigue, bone pain, fever, postpartum hemorrhage, and thirst by healers in Kaski and Tanahun districts, Nepal. However, scientific evidence towards their traditional uses is lacking till December, 2023. Therefore, we report the phytochemicals, total phenolic content (TPC), total flavonoid content (TFC), total carbohydrate content (TCC), antioxidant and antibacterial activities of A. helferiana and S. fasciculata extracts. Phytochemical analysis indicated that A. helferiana and S. fasciculata extracts were potential sources of chemicals such as phenols, flavonoids, tannins, terpenoids, saponins, and carbohydrates. The TPC, TFC, and TCC of extracts were determined by using an ultraviolet visible spectrophotometer. Among the extracts tested, A. helferiana extracts showed the highest phenolic and carbohydrate contents of 208.33 ± 12.96 mg of gallic acid equivalent/g and 564.16 ± 2.92 mg of D-glucose equivalent/g of dry extract, respectively. Similarly, S. fasciculata revealed the highest flavonoid content of 30.35 ± 0.1 mg quercetin equivalent/g of dry extract. The extract of A. helferiana and S. fasciculata exhibited potent antioxidant activity by scavenging 2,2-diphenyl-1-picrylhydrazyl radicals with an IC50 of 25.9 µg/ml and 31.07 µg/ml, respectively. The antibacterial activity of the A. helferiana and S. fasciculata extract against Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli was determined using an agar-well diffusion protocol that revealed the potential antibacterial activity of A. helferiana against E. coli. The present study will help validate the traditional uses of A. helferiana rhizomes and S. fasciculata barks as a healing medicine and inspire the researcher towards further research, development, and formulation.

ELONGATED HYPOCOTYL5 Negatively Regulates DECREASE WAX BIOSYNTHESIS to Increase Survival during UV-B Stress.

Understanding how the distinct cell types of the shoot apical meristem (SAM) withstand ultraviolet radiation (UVR) stress can improve cultivation of plants in high-UVR environments. Here, we show that UV-B irradiation selectively kills epidermal and niche cells in the shoot apex. Plants harboring a mutation in DECREASE WAX BIOSYNTHESIS (DEWAX) are tolerant to UV-B. Our data show that DEWAX negatively regulates genes involved in anthocyanin biosynthesis. ELONGATED HYPOCOTYL5 (HY5) binds to the DEWAX promoter elements and represses its expression to promote the anthocyanin biosynthesis. The HY5-DEWAX regulatory network regulates anthocyanin content in Arabidopsis (Arabidopsis thaliana) and influences the survivability of plants under UV-B irradiation stress. Our cell sorting-based study of the epidermal cell layer transcriptome confirms that core UV-B stress signaling pathway genes are conserved and upregulated in response to UV-B irradiation of the SAM. Furthermore, we show that UV-B induces genes involved in shoot development and organ patterning. We propose that the HY5-DEWAX regulatory relationship is conserved; however, changes in the expression levels of these genes can determine anthocyanin content in planta and, hence, fitness under UV-B irradiation stress.

DNA-dependent homodimerization, sub-cellular partitioning, and protein destabilization control WUSCHEL levels and spatial patterning.

The homeodomain transcription factor WUSCHEL (WUS) promotes stem cell maintenance in inflorescence meristems of Arabidopsis thaliana WUS, which is synthesized in the rib meristem, migrates and accumulates at lower levels in adjacent cells. Maintenance of WUS protein levels and spatial patterning distribution is not well-understood. Here, we show that the last 63-aa stretch of WUS is necessary for maintaining different levels of WUS protein in the rib meristem and adjacent cells. The 63-aa region contains the following transcriptional regulatory domains: the acidic region, the WUS-box, which is conserved in WUS-related HOMEOBOX family members, and the ethylene-responsive element binding factor-associated amphiphilic repression (EAR-like) domain. Our analysis reveals that the opposing functions of WUS-box, which is required for nuclear retention, and EAR-like domain, which participates in nuclear export, are necessary to maintain higher nuclear levels of WUS in cells of the rib meristem and lower nuclear levels in adjacent cells. We also show that the N-terminal DNA binding domain, which is required for both DNA binding and homodimerization, along with the homodimerization sequence located in the central part of the protein, restricts WUS from spreading excessively and show that the homodimerization is critical for WUS function. Our analysis also reveals that a higher level of WUS outside the rib meristem leads to protein destabilization, suggesting a new tier of regulation in WUS protein regulation. Taken together our data show that processes that influence WUS protein levels and spatial distribution are highly coupled to its transcriptional activity.

Threshold-dependent transcriptional discrimination underlies stem cell homeostasis.

Transcriptional mechanisms that underlie the dose-dependent regulation of gene expression in animal development have been studied extensively. However, the mechanisms of dose-dependent transcriptional regulation in plant development have not been understood. In Arabidopsis shoot apical meristems, WUSCHEL (WUS), a stem cell-promoting transcription factor, accumulates at a higher level in the rib meristem and at a lower level in the central zone where it activates its own negative regulator, CLAVATA3 (CLV3). How WUS regulates CLV3 levels has not been understood. Here we show that WUS binds a group of cis-elements, cis- regulatory module, in the CLV3-regulatory region, with different affinities and conformations, consisting of monomers at lower concentration and as dimers at a higher level. By deleting cis elements, manipulating the WUS-binding affinity and the homodimerization threshold of cis elements, and manipulating WUS levels, we show that the same cis elements mediate both the activation and repression of CLV3 at lower and higher WUS levels, respectively. The concentration-dependent transcriptional discrimination provides a mechanistic framework to explain the regulation of CLV3 levels that is critical for stem cell homeostasis.

WUSCHEL protein movement mediates stem cell homeostasis in the Arabidopsis shoot apex.

WUSCHEL (WUS) is a homeodomain transcription factor produced in cells of the niche/organizing center (OC) of shoot apical meristems. WUS specifies stem cell fate and also restricts its own levels by activating a negative regulator, CLAVATA3 (CLV3), in adjacent cells of the central zone (CZ). Here we show that the WUS protein, after being synthesized in cells of the OC, migrates into the CZ, where it activates CLV3 transcription by binding to its promoter elements. Using a computational model, we show that maintenance of the WUS gradient is essential to regulate stem cell number. Migration of a stem cell-inducing transcription factor into adjacent cells to activate a negative regulator, thereby restricting its own accumulation, is a theme that is unique to plant stem cell niches.

A high-resolution gene expression map of the Arabidopsis shoot meristem stem cell niche.

The shoot apical meristem (SAM) acts as a reservoir for stem cells. The central zone (CZ) harbors stem cells. The stem cell progenitors differentiate in the adjacent peripheral zone and in the rib meristem located just beneath the CZ. The SAM is further divided into distinct clonal layers: the L1 epidermal, L2 sub-epidermal and L3 layers. Collectively, SAMs are complex structures that consist of cells of different clonal origins that are organized into functional domains. By employing fluorescence-activated cell sorting, we have generated gene expression profiles of ten cell populations that belong to different clonal layers as well as domains along the central and peripheral axis. Our work reveals that cells in distinct clonal layers exhibit greater diversity in gene expression and greater transcriptional complexity than clonally related cell types in the central and peripheral axis. Assessment of molecular functions and biological processes reveals that epidermal cells express genes involved in pathogen defense: the L2 layer cells express genes involved in DNA repair pathways and telomere maintenance, and the L3 layers express transcripts involved in ion balance and salt tolerance besides photosynthesis. Strikingly, the stem cell-enriched transcriptome comprises very few hormone-responsive transcripts. In addition to providing insights into the expression profiles of hundreds of transcripts, the data presented here will act as a resource for reverse genetic analysis and will be useful in deciphering molecular pathways involved in cell type specification and their functions.

Plant stem cell maintenance involves direct transcriptional repression of differentiation program.

In animal systems, master regulatory transcription factors (TFs) mediate stem cell maintenance through a direct transcriptional repression of differentiation promoting TFs. Whether similar mechanisms operate in plants is not known. In plants, shoot apical meristems serve as reservoirs of stem cells that provide cells for all above ground organs. WUSCHEL, a homeodomain TF produced in cells of the niche, migrates into adjacent cells where it specifies stem cells. Through high-resolution genomic analysis, we show that WUSCHEL represses a large number of genes that are expressed in differentiating cells including a group of differentiation promoting TFs involved in leaf development. We show that WUS directly binds to the regulatory regions of differentiation promoting TFs; KANADI1, KANADI2, ASYMMETRICLEAVES2 and YABBY3 to repress their expression. Predictions from a computational model, supported by live imaging, reveal that WUS-mediated repression prevents premature differentiation of stem cell progenitors, being part of a minimal regulatory network for meristem maintenance. Our work shows that direct transcriptional repression of differentiation promoting TFs is an evolutionarily conserved logic for stem cell regulation.

WUSCHEL mediates stem cell homeostasis by regulating stem cell number and patterns of cell division and differentiation of stem cell progenitors.

Plant stem cell populations, unlike their animal counterparts, do not use cell migration and oriented cell divisions to maintain their size, and therefore require a precise coordination between self-renewing divisions of stem cells, and rates of cell division and differentiation among stem cell progenitors. Shoot apical meristems (SAMs) of higher plants harbor a set of stem cells within the central zone (CZ) that divide infrequently. Stem cell daughters that are displaced towards the surrounding peripheral zone (PZ) divide at a faster rate and enter into differentiation at specific locations to form leaves or flowers. The relative ratios of cells in the CZ and the PZ are maintained, despite a constant displacement of cells from the CZ into the PZ, and subsequent allocation of cells within the PZ to form organ primordia. The mechanisms that mediate this homeostatic balance are not well understood. A homeodomain transcription factor WUSCHEL, expressed in the rib meristem (RM), located beneath the CZ, has been shown to provide nonautonomous cues for stem cell specification. By employing transient spatial manipulation and live imaging, we show that an elevated level of WUS not only induces expansion of the CZ, but also results in increased cell division rates in cells of the PZ; conversely, decreases in WUS level lead to a smaller CZ and are associated with a reduction in cell division rate. Moreover, low levels of WUS lead to enlarged organ primordia, by elevating the responsiveness of the PZ cells to the plant hormone auxin. This reveals a function of WUS in mediating the balance between differentiating and non-differentiating cells of the PZ. Regulation of stem cell numbers, growth and differentiation patterns by a single transcription factor forms a interconnected and self-correcting feedback loop to provide robustness to stem cell homeostasis in a dynamic cellular environment.

Gene expression map of the Arabidopsis shoot apical meristem stem cell niche.

Despite the central importance of stem cells in plant growth and development, the molecular signatures associated with them have not been revealed. Shoot apical meristems (SAMs) harbor a small set of stem cells located at the tip of each plant and they are surrounded by several million differentiating cells. This imposes a major limitation in isolating pure populations of stem cells for genomic analyses. We have developed a system to isolate pure populations of distinct cell types of the SAMs, including stem cells. We have used this system to profile gene expression from 4 different cell samples of SAMs. The cell sample-specific gene expression profiling has resulted in a high-resolution gene expression map to reveal gene expression networks specific to individual spatial domains of SAMs. We demonstrate that the cell sample-specific expression profiling is sensitive in identifying rare transcripts expressed in a few specific subsets of cells of SAMs. Our extensive RNA in situ analysis reveals that the expression map can be used as a predictive tool in analyzing the spatial expression patterns of genes and it has led to the identification of unique gene expression patterns within the SAMs. Furthermore, our work reveals an enrichment of DNA repair and chromatin modification pathways in stem cells suggesting that maintenance of genome stability and flexible chromatin may be crucial for stem cell function. The gene expression map should guide future reverse genetics experiments, high-resolution analyses of cell-cell communication networks and epigenetic modifications.

DETORQUEO, QUIRKY, and ZERZAUST represent novel components involved in organ development mediated by the receptor-like kinase STRUBBELIG in Arabidopsis thaliana.

Intercellular signaling plays an important role in controlling cellular behavior in apical meristems and developing organs in plants. One prominent example in Arabidopsis is the regulation of floral organ shape, ovule integument morphogenesis, the cell division plane, and root hair patterning by the leucine-rich repeat receptor-like kinase STRUBBELIG (SUB). Interestingly, kinase activity of SUB is not essential for its in vivo function, indicating that SUB may be an atypical or inactive receptor-like kinase. Since little is known about signaling by atypical receptor-like kinases, we used forward genetics to identify genes that potentially function in SUB-dependent processes and found recessive mutations in three genes that result in a sub-like phenotype. Plants with a defect in DETORQEO (DOQ), QUIRKY (QKY), and ZERZAUST (ZET) show corresponding defects in outer integument development, floral organ shape, and stem twisting. The mutants also show sub-like cellular defects in the floral meristem and in root hair patterning. Thus, SUB, DOQ, QKY, and ZET define the STRUBBELIG-LIKE MUTANT (SLM) class of genes. Molecular cloning of QKY identified a putative transmembrane protein carrying four C(2) domains, suggesting that QKY may function in membrane trafficking in a Ca(2+)-dependent fashion. Morphological analysis of single and all pair-wise double-mutant combinations indicated that SLM genes have overlapping, but also distinct, functions in plant organogenesis. This notion was supported by a systematic comparison of whole-genome transcript profiles during floral development, which molecularly defined common and distinct sets of affected processes in slm mutants. Further analysis indicated that many SLM-responsive genes have functions in cell wall biology, hormone signaling, and various stress responses. Taken together, our data suggest that DOQ, QKY, and ZET contribute to SUB-dependent organogenesis and shed light on the mechanisms, which are dependent on signaling through the atypical receptor-like kinase SUB.

Automated tracking of stem cell lineages of Arabidopsis shoot apex using local graph matching.

Shoot apical meristems (SAMs) of higher plants harbor stem-cell niches. The cells of the stem-cell niche are organized into spatial domains of distinct function and cell behaviors. A coordinated interplay between cell growth dynamics and changes in gene expression is critical to ensure stem-cell homeostasis and organ differentiation. Exploring the causal relationships between cell growth patterns and gene expression dynamics requires quantitative methods to analyze cell behaviors from time-lapse imagery. Although technical breakthroughs in live-imaging methods have revealed spatio-temporal dynamics of SAM-cell growth patterns, robust computational methods for cell segmentation and automated tracking of cells have not been developed. Here we present a local graph matching-based method for automated-tracking of cells and cell divisions of SAMs of Arabidopsis thaliana. The cells of the SAM are tightly clustered in space which poses a unique challenge in computing spatio-temporal correspondences of cells. The local graph-matching principle efficiently exploits the geometric structure and topology of the relative positions of cells in obtaining spatio-temporal correspondences. The tracker integrates information across multiple slices in which a cell may be properly imaged, thus providing robustness to cell tracking in noisy live-imaging datasets. By relying on the local geometry and topology, the method is able to track cells in areas of high curvature such as regions of primordial outgrowth. The cell tracker not only computes the correspondences of cells across spatio-temporal scale, but it also detects cell division events, and identifies daughter cells upon divisions, thus allowing automated estimation of cell lineages from images captured over a period of 72 h. The method presented here should enable quantitative analysis of cell growth patterns and thus facilitating the development of in silico models for SAM growth.

A cellular expression map of epidermal and subepidermal cell layer-enriched transcription factor genes integrated with the regulatory network in Arabidopsis shoot apical meristem.

Transcriptional control of gene expression is an exquisitely regulated process in both animals and plants. Transcription factors (TFs) and the regulatory networks that drive the expression of TF genes in epidermal and subepidermal cell layers in Arabidopsis are unexplored. Here, we identified 65 TF genes enriched in the epidermal and subepidermal cell layers of the shoot apical meristem (SAM). To determine the cell type specificity in different stages of Arabidopsis development, we made YFP based transcriptional fusion constructs by taking a 3-kb upstream noncoding region above the translation start site. Here, we report that for ~52% (22/42) TF genes, we detected transcription activity. TF genes derived from epidermis show uniform expression in early embryo development; however, in the late globular stage, their transcription activity is suppressed in the inner cell layers. Expression patterns linked to subepidermal cell layer identity were apparent in the postembryonic development. Potential upstream regulators that could modulate the activity of epidermal and subepidermal cell layer-enriched TF genes were identified using enhanced yeast-one-hybrid (eY1H) assay and validated. This study describes the activation of TF genes in epidermal and subepidermal cell layers in embryonic and postembryonic development of Arabidopsis shoot apex.

Inter-cell-layer signalling during Arabidopsis ovule development mediated by the receptor-like kinase STRUBBELIG.

Plant organs, such as ovules and flowers, arise through cellular events that are precisely co-ordinated between cells within and across clonally distinct cell layers. Receptor-like kinases are cell-surface receptors that perceive and relay intercellular information. In Arabidopsis the leucine-rich repeat receptor-like kinase STRUBBELIG (SUB) is required for integument initiation and outgrowth during ovule development, floral organ shape and the control of the cell division plane in the first subepidermal cell layer of floral meristems, among other functions. A major goal is to understand SUB-mediated signal transduction at the molecular level. Present evidence suggests that SUB affects neighbouring cells in a non-cell-autonomous fashion. In addition, our results indicate that SUB is an atypical, or kinase-dead, kinase. Forward genetics identified three genes, QUIRKY (QKY), ZERZAUST and DETORQUEO, that are thought to contribute to SUB-dependent signal transduction. QKY encodes a predicted membrane-bound protein with four cytoplasmic C(2) domains. By analogy to animal proteins with related domain topology, we speculate that QKY may be involved in Ca(2+)-dependent signalling and membrane trafficking. Studying SUB-dependent signalling will contribute to our understanding of how atypical kinases mediate signal transduction and how cells co-ordinate their behaviour to allow organs, such as ovules, to develop their three-dimensional architecture.

Labeling and Sorting of Arabidopsis SAM Cell Populations to Capture Their Transcriptome Profile.

In higher plants, the cells that form aboveground tissues and organs are derived from the shoot apical meristem (SAM). SAM is dynamic in nature and divided into central zone (CZ), peripheral zone (PZ), and rib meristem (RM). Stem cells reside in the CZ, and their progenitors differentiate to form lateral organs in PZ and stem tissue in RM. Besides zones, the SAM is also divided into distinct clonal cell layers that show patterned cell division. Here, we describe methods to tag and isolate cell types from both cell layers and zones of SAM in high purity using cell sorter. This method enable plant biologist in rapid isolation of desired cell types from SAM to record their transcriptome, epigenome, proteome, and metabolome. The information generated by this approach will elucidate the mechanism of stem cell self-renewal, differentiation, and organogenesis in SAM.

The Arabidopsis receptor-like kinase STRUBBELIG mediates inter-cell-layer signaling during floral development.

In plants important questions relate to the mechanisms that control signaling between the histogenic cell layers of apical meristems and developing organs. The Arabidopsis putative atypical leucine-rich repeat receptor-like kinase STRUBBELIG (SUB) regulates amongst others floral organ shape, the plane of cell division in cells of the first subepidermal cell layer of floral meristems, ovule integument morphogenesis, and root hair patterning. Reporter assays using a functional translational fusion between SUB and EGFP indicate that SUB expression is largely confined to interior tissues in young flowers, ovules, and roots. In contrast, SUB mRNA expression can be monitored in all cell layers of those tissues. Specifically, SUB protein is not detectable in cells that show a sub mutant phenotype. Rather, SUB is detected in directly neighbouring cells in flower and ovule primordia, or in cells that are separated from mutant cells by two cell diameters in the root. Inhibitor studies corroborate a posttranscriptional regulation of SUB. Phenotypic analysis of sub-1 plants expressing a SUB:EGFP gene under the control of tissue and epidermis-specific promoters support the notion that SUB-dependent signal transduction relies on the production of secondary intercellular signals. The combined results indicate that SUB acts in a non-cell-autonomous fashion, functions in a radial inside-out signaling process, and mediates cell morphogenesis and cell fate across clonally distinct cell layers in floral primordia, developing ovules, and root meristems.

Genome wide gene expression analyses of Arabidopsis shoot stem cell niche cell populations.

The shoot apical meristem (SAM) of higher plants harbors stem cells at their tips. In the SAM, stem cell niches maintain the pluripotent nature of these cell types in the central zone (CZ) and allow them to enter in to differentiation pathways either in the peripheral zone (PZ) or rib meristem (RM). Apart from functional zones, SAM is also subdivided in to distinct cell layers termed as; L1 / epidermal, L2 / subepidermal and L3 / corpus cell layer. Thus, SAM is a complex structure made up of multiple cell types having discrete cell identities. In a recent study, we employed the fluorescent activated cell sorting approach to isolate the cell population of functional zones and cell layers and identified the cell population expressed genes (CPEGs). The Gene Ontology (GO) analysis revealed cellular functions of the identified CPEGs. The cell layers of the SAM are involved in pathogen defense, cell differentiation and photosynthesis. We found many genes in SAM CEPGs that responded to hormone treatment. These observations in the future will help researchers working in the area of shoot apex biology to elucidate the gene regulatory network involved in cell and tissue specialization.

Gene expression analysis of shoot apical meristem cell types.

Shoot apical meristems (SAMs) of higher plants harbor a set of stem-cells and provide cells for the development of all the above-ground biomass of plants. Most of the important pattern formation events such as maintenance of stem-cell identity, specification and differentiation of leaf/flower primordia, and temporal control of the transition from vegetative to reproductive program are determined in SAMs. Genetic analysis has revealed molecular and hormonal pathways involved in stem-cell maintenance, organ differentiation, and flowering time. However, limited information is available as to how different pathways interact with each other to function as a network in specifying different cell types and their function. Deciphering gene networks that underlie cell fate transitions requires new approaches aimed at assaying genome-scale expression patterns of genes at a single cell-type resolution. Here we provide details of experimental methods involved in protoplasting of SAM cells, generating cell type-specific gene expression profiles, and analysis platforms for identifying and inferring gene networks.

WUSCHEL protein movement and stem cell homeostasis.

Stem cell maintenance is essential for growth and development of plants and animals. Similar to animal studies, transcription factors play a critical role in plant stem cell maintenance, however the regulatory logic is not well understood. Shoot apical meristems (SAMs) harbor a pool of pluoripotent stem cells and they provide cells for the development of all above-ground organs. Molecular genetic studies spanning more than a decade have revealed cell-cell communication logic underlying stem cell homeostasis. WUSCHEL (WUS), a homeodomain transcription factor expressed in cells of the organizing center specifies stem cells in overlying cells of the central zone (CZ) and also activates a negative regulator-CLAVATA3 (CLV3). CLV3, a small secreted peptide, binds to CLAVATA1 (CLV1) and also possibly to CLV1-related receptors to activate signaling which restricts WUS transcription. Though the CLV-WUS feedback network explains the cell-cell communication logic of stem cell maintenance, how WUS communicates with adjacent cells had remained elusive. In October 15 2011 issue of Genes and Development, we report that WUS protein synthesized in cells of organizing center migrates into adjacent cells via cell-cell movement and activates CLV3 transcription by directly binding to promoter elements.