the operator when measuring the divergence angles on the protractor. Additionally, it requires a lot of tedious work and about 45 minutes per plant. To make this process more repeatable and accurate I explored using 3D optical tomography as an alternative method to the divergometer.
The tomography software Photomodeler Scanner was the primary software used. I generated optical reference targets which are unique circular patterns that can be recognized by the software to determine the location of the camera between each photograph. Each plant was photographed six times from three different angles (Figure 3.2). The images were loaded into Photomodeler Scanner and a 3D triangular mesh model was generated. These meshes were edited in MeshLab to remove any extraneous object such as the base used to hold the plant in place and any background objects that were picked up by the software. Once the mesh was devoid of any objects besides the plant, the new mesh was exported. The clean mesh was then processed with a custom algorithm which identified the shoot vs siliques and measured divergence angles between siliques.
promotes stem cell-ness as well as its own negative regu- lator, through the CLV3 ligand and CLV1-CLV2-CRN receptors . In the 1990s, this genetic network was further shown to incorporate hormones, and notably auxin, which is thought to be the main inducer of organ positioning and emergence. More specifically, the PIN- FORMED 1 gene (PIN1) encodes an auxin efflux carrier and extensive literature (e.g., [4–6]) conclusively showed that organogenesis at the SAM requires PIN1-dependent auxin transport towards the site of primordium (i.e., incipient organ) initiation. Other hormones were shown to contribute to organogenesis, albeit to a lower degree (e.g., [7–10]). More recently, the structural elements of meristematic cells were analyzed and shown to consist of a stereotypical tissue-scale pattern of cortical micro- tubule arrays (and arguably cellulose deposition), with isotropic orientations in the central zone and circumfer- ential orientations in the peripheral zone [11, 12]; patterns of cell wall components and regulators were also identified . Among the structural components of plant cells, the plasma membrane has received very little attention at the shootmeristem. Yet, its position at the interface between the cell wall and the cytoplasm may make its composition a key coordinating feature for the meristem, at the nexus between biochemical and mechanical cues.
* To whom correspondence should be addressed. E-mail: email@example.com
Received 10 August 2010; Revised 4 November 2010; Accepted 30 November 2010
Plant microRNAs (miRNAs) play crucial regulatory roles in various developmental processes. In this study, we characterize the miRNA profile of the shootapicalmeristem (SAM) of an important legume crop, soybean, by integrating high-throughput sequencing data with miRNA microarray analysis. A total of 8423 non-redundant sRNAs were obtained from two libraries derived from micro-dissected SAM or mature leaf tissue. Sequence analysis allowed the identification of 32 conserved miRNA families as well as 8 putative novel miRNAs. Subsequent miRNA profiling with microarrays verified the expression of the majority of these conserved and novel miRNAs. It is noteworthy that several miRNAs* were expressed at a level similar to or higher than their corresponding mature miRNAs in SAM or mature leaf, suggesting a possible biological function for the star species. In situ hybridization analysis revealed a distinct spatial localization pattern for a conserved miRNA, miR166, and its star speciessuggesting that they serve different roles in regulating leaf development. Furthermore, localization studies showed that a novel soybean miRNA, miR4422a, was nuclear-localized. This study also indicated a novel expression pattern of miR390 in soybean. Our approach identified potential key regulators and provided vital spatial information towards understanding the regulatory circuits in the SAM of soybean during shoot development.
Plants continuously generate new tissues and organs through the activity of populations of undifferentiated stem cells, called meristems. Here, we discuss the so-called shootapical mer- istem (SAM), which generates all the aerial parts of the plant. It has been known for many years that auxin plays a central role in the functioning of this meristem. Auxin is not homogeneously distributed at the SAM and it is thought that this distribution is interpreted in terms of differential gene expression and patterned growth. In this context, auxin transporters of the PIN and AUX families, creating auxin maxima and minima, are crucial regulators. However, auxin transport is not the only factor involved. Auxin biosynthesis genes also show specific, patterned activi- ties, and local auxin synthesis appears to be essential for meristem function as well. In addition, auxin perception and signal transduction defining the competence of cells to react to auxin, add further complexity to the issue. To unravel this intricate signaling network at the SAM, systems biology approaches, involving not only molecular genetics but also live imaging and computational modeling, have become increasingly important.
Functional Diversification and Redundancy of AGO10 and Other AGOs in Arabidopsis
In addition to SAM maintenance, AGO10 also plays a critical role in organ polarity and vascular development. Although we have not examined these developmental processes in our study, it is likely that AGO10 regulates these biological events through its association with miR166/165. Of course, we have no reason to exclude the possibility that AGO10 might bind to other sRNAs to regulate their targets. Our sequencing results revealed that AGO10 does recruit a spectrum of miRNAs and numerous ta-siRNAs, although their relative ratios are very low. A previous study with Arabidopsis AGO4 suggests that a single AGO protein may function as a catalytic engine of RNA cleavage while it can also execute slicing-independent regulation of sRNA targets (Qi et al., 2006). Given that AGO10 does possess slicer activity, it will be intriguing to investigate whether or not this activity is required for regulation of sRNA targets other than HD-ZIP III family genes.
The floral transition is the process by which flowering plants switch from vegetative growth to the production of flowers. Consistent with the importance of this developmental transition, flowering is highly regulated through several genetic pathways, some of which respond to environmental cues. Arabidopsis thaliana flowers earlier under long-days (LD) of spring than under short-days (SD) of winter, and day-length, or photoperiod, is one of the most important environmental stimuli influencing the flowering response. Photoperiod is perceived in the leaves, while the floral transition occurs at the shootapicalmeristem (SAM). In LD, a genetic cascade is activated in the leaf vasculature, so that a key transcriptional regulator called CONSTANS activates the genes FLOWERING LOCUS T (FT) and its homolog TWIN SISTER OF FT (TSF). FT protein is then transported through the phloem, eventually reaching the SAM, where it triggers the floral transition. By forming a complex with FD, a bZIP transcription factor, FT activates target genes, such as SUPPRESSOR OF OVEREXPRESSION OF CO (SOC1), FRUITFULL (FUL) and later APETALA1 (AP1), all of which encode MADS-box transcription factors. However, the floral transition involves a dramatic transcriptional reprogramming of the shootmeristem, and a complete picture of the global changes in gene expression occurring specifically in the SAM is still missing. Therefore, in the first part of this work, SAMs were specifically collected by the use of laser microdissection from plants experiencing a shift from SD to LD. RNA isolated from the meristems was converted to cDNA and gene expression quantified through next-generation sequencing by RNA-seq. Genes were grouped according to those increased or reduced in expression, with a particular focus on novel genes that were up-regulated similarly to SOC1 or FUL. Among them, the expression of a selected set of genes was tested by in situ hybridisation on wild-type apices to confirm their activation at the SAM, and to uncover their spatial pattern of mRNA expression. Several novel genes were confirmed to be induced by transferring plants to LD and they showed specific spatial patterns of expression in various regions of the SAM. Moreover, apices of ft tsf double mutants were also hybridised, to reveal whether those genes are induced by the photoperiodic cascade downstream of FT/TSF. Surprisingly, while many genes were induced only in the presence of FT/TSF, similarly to SOC1, some of them still respond to photoperiod in the ft tsf double mutants, suggesting that additional unknown signals may play a role in response to inductive day-length independently of FT and TSF. Further preliminary studies on a set of these novel genes are described in this study.
2.4.4 Known SAM function genes and SAM variation
This study uncovered 23 candidate genes associated with SAM size and shape. Notably, our GWAS did not detect any SAM master regulatory genes previously
identified by mutational analyses, corroborating the results of previous QTL analyses of maize SAM morphology (Thompson et al. 2014, 2015). A successful GWAS ultimately links phenotypic variation with allelic polymorphisms. As such, our GWAS would fail to identify SAM master regulators if these genes were fixed in our population, perhaps due to strong purifying selective pressure for SAM function as observed in some species (Bauchet et al. 2014). However, our genotyping matrix includes ample polymorphisms within the coding sequences of multiple SAM master regulatory genes (Supplementary Data 6). For example, after filtering and quality control, 118 SNPs were identified in the SAM maintenance gene, knotted1 (kn1) (Kerstetter et al. 1997) and 12 SNPs were found in the SAM size regulator, aberrant phyllotaxy1 (abph1) (Jackson and Hake 1999), although SNPs in neither gene were significantly associated with SAM volume. Likewise, although 23 SNPs were identified in the leucine-rich repeat receptor-like, faciated ear2 (fea2) (Taguchi-Shiobara et al. 2001), significant associations were not detected between SAM volume and fea2 SNPs by GWAS. Loss of fea2, a putative CLAVATA2 ortholog, dramatically affects the shape and size of the maize inflorescence meristem (IM) (Taguchi-Shiobara et al. 2001), and natural variation in the regulation of fea2 was shown to underlie ear morphological variation between maize inbreds B73 and Mo17 (Bommert et al. 2013a).
The maize shootapicalmeristem (SAM) comprises a small pool of stem cells that generate all above-ground organs. Although mutational studies have identiﬁed genetic networks regulating SAM function, little is known about SAM morphological variation in natural populations. Here we report the use of high-throughput image processing to capture rich SAM size variation within a diverse maize inbred panel. We demonstrate correlations between seedling SAM size and agronomically important adult traits such as ﬂowering time, stem size and leaf node number. Combining SAM phenotypes with 1.2 million single nucleotide polymorphisms (SNPs) via genome-wide association study reveals unexpected SAM morphology candidate genes. Analyses of candidate genes implicated in hormone transport, cell division and cell size conﬁrm correlations between SAM morphology and trait-associated SNP alleles. Our data illustrate that the microscopic seedling SAM is predictive of adult phenotypes and that SAM morphometric variation is associated with genes not previously predicted to regulate SAM size.
Accepted for publication April 21, 2004
Shoot and floral meristem activity in higher plants is controlled by complex signaling networks consisting of positive and negative regulators. The Arabidopsis ULTRAPETALA1 (ULT1) gene has been shown to act as a negative regulator of meristem cell accumulation in inflorescence and floral meristems, as loss- of-function ult1 mutations cause inflorescence meristem enlargement, the production of extra flowers and floral organs, and a decrease in floral meristem determinacy. To investigate whether ULT1 functions in known meristem regulatory pathways, we generated double mutants between ult1 alleles and null alleles of the meristem-promoting genes SHOOTMERISTEMLESS (STM) and WUSCHEL (WUS). We found that, although the ult1 alleles have no detectable embryonic or vegetative phenotypes, ult1 mutations restored extensive organ-forming capability to stm null mutants after germination and increased leaf and floral organ production in stm partial loss-of-function mutants. Mutations in ULT1 also partially suppressed the wus shoot and floral meristem phenotypes. However, wus was epistatic to ult1 in the center of the flower, and WUS transcriptional repression was delayed in ult1 floral meristems. Our results show that during the majority of the Arabidopsis life cycle, ULT1 acts oppositely to STM and WUS in maintaining meristem activity and functions in a separate genetic pathway. However, ULT1 negatively regulates WUS to establish floral meristem determinacy, acting through the WUS-AG temporal feedback loop.
Nine MIR165/166 loci are present in the Arabidopsis genome, each of which has the potential to regulate any or all of its five class III HD-ZIP gene targets either by clearing the transcripts from specific cells or by regulating the level of transcript accumulation. To account for the observed expression of the HD-ZIP genes in combinatorial tissue- and stage-specific patterns, it seems reasonable to expect that the nine MIR loci will also have dynamic and differential transcription profiles. Further examination of the regulatory interactions between the various MIR165/166 family members, their class III HD-ZIP target genes, and meristem maintenance factors will reveal additional insights into the complex interplay between polar lateral organs and the shootapicalmeristem from which they derive.
Modern imaging approaches enable the acquisition of 3D and 4D datasets capturing plant organ development at cellular resolution. Computational analyses of these data enable the digitization and analysis of individual cells. In order to fully harness the information encoded within these datasets, annotation of the cell types within organs may be performed. This enables data points to be placed within the context of their position and identity, and for equivalent cell types to be compared between samples. The shootapicalmeristem (SAM) in plants is the apical stem cell niche from which all above ground organs are derived. We developed 3DCellAtlas Meristem which enables the complete cellular annotation of all cells within the SAM with up to 96% accuracy across all cell types in Arabidopsis and 99% accuracy in tomato SAMs. Successive layers of cells are identified along with the central stem cells, boundary regions, and layers within developing primordia. Geometric analyses provide insight into the morphogenetic pro‑ cess that occurs during these developmental processes. Coupling these digital analyses with reporter expression will enable multidimensional analyses to be performed at single cell resolution. This provides a rapid and robust means to perform comprehensive cellular annotation of plant SAMs and digital single cell analyses, including cell geometry and gene expression. This fills a key gap in our ability to analyse and understand complex multicellular biology in the apical plant stem cell niche and paves the way for digital cellular atlases and analyses.
Analysis of variance revealed that the interaction effects of genotypes, BAP and NAA were very highly significant (p< 0.001) for number of shoots/explant, shoot length and leaves/shoot. On MS media devoid of BAP and NAA, young shoots were developed from the primary shoot and showed shoot elongation in both genotypes after being cultured for a month (control, data was not taken). This might be due to the presence of methylene blue, which stimulates shoot growth and increasing survival [25, 26], and adenine hemisulfate enhanced shoot multiplication . B4906 gave the highest (16.88 ±0.54) shoots/explant with 5.94±0.17 cm average shoot length and 6.33±0.29 leaves/shoot on MS media with 1.5 mgl -1 BAP and 0.4 mgl -1 NAA (Table 1; Fig. 2). Whereas, Pr1013 produced maximum of 11.70 ±0.28 shoots/explant and 4.48±0.08 cm shoot length with 4.95±0.11 leaves/shoot on MS media fortified by 2 mgl -1 BAP and 0.5 mgl -1 NAA (Table1; Fig.3). This multiplication rate difference might be due to genotypic difference, which affects the frequency of shoot organogenesis , and also endogenous cytokinin and auxin concentration differences [29, 30]. The performance of each cultivar is expected to be different in in vitro culture as a field response regarding shoot number and shoot length as described by . This requires that novel or modified in vitro regeneration procedures must be developed for each genotype because of the significant variations in response to hormone combinations.
How organisms attain their specific shapes and modify their growth patterns in response to environmental and chemical signals has been the subject of many investigations. Plant cells are at high turgor pressure and are surrounded by a rigid yet flexible cell wall, which is the primary determinant of plant growth and morphogenesis. Cellulose microfibrils, synthesized by plasma membrane-localized cellulose synthase complexes, are major tension-bearing components of the cell wall that mediate directional growth. Despite advances in understanding the genetic and biophysical regulation of morphogenesis, direct studies of cellulose biosynthesis and its impact on morphogenesis of different cell and tissue types are largely lacking. In this study, we took advantage of mutants of three primary cellulose synthase ( CESA ) genes that are involved in primary wall cellulose synthesis. Using field emission scanning electron microscopy, live cell imaging and biophysical measurements, we aimed to understand how the primary wall CESA complex acts during shootapicalmeristem development. Our results indicate that cellulose biosynthesis impacts the mechanics and growth of the shootapicalmeristem.
ABSTRACT The shootapicalmeristem contains a pool of undifferentiated stem cells and controls initiation of all aerial plant organs. In maize (Zea mays), leaves are formed throughout vegetative development; on transition to ﬂoral development, the shootmeristem forms the tassel. Due to the regulated balance be- tween stem cell maintenance and organogenesis, the structure and morphology of the shootmeristem are constrained during vegetative development. Previous work identi ﬁed loci controlling meristem architecture in a recombinant inbred line population. The study presented here expanded on this by investigating shootapicalmeristem morphology across a diverse set of maize inbred lines. Crosses of these lines to common parents showed varying phenotypic expression in the F1, with some form of heterosis occasionally ob- served. An investigation of meristematic growth throughout vegetative development in diverse lines linked the timing of reproductive transition to ﬂowering time. Phenotypic correlations of meristem morphology with adult plant traits showed an association between the meristem and ﬂowering time, leaf shape, and yield traits, revealing links between the control and architecture of undifferentiated and differentiated plant organs. Finally, quantitative trait loci mapping was utilized to map the genetic architecture of these mer- istem traits in two divergent populations. Control of meristem architecture was mainly population-speciﬁc, with 15 total unique loci mapped across the two populations with only one locus identiﬁed in both populations.
(Haberlandt, 1902). Our study represents an early step towards realizing this potential. In vitro culture experiments support the idea that cell identity in plants is largely governed by positional cues mediated by specific hormones (Steward et al., 1964). We propose a model in which partition of cell identity within a callus on SIM is mediated through non-homogeneous distributions of auxin and cytokinin, which are initially broadly distributed and therefore induce broad CUC2 and WUS expression, respectively. The expression of these genes may further feed back on hormone synthesis, transport or perception, to enhance gradients of hormone signaling, which then alters CUC2 and WUS expression. This feedback could lead to self- organizing patterns observed during de novo shootmeristem initiation. If this is the case, the primary difference between shootmeristem initiation in planta and shootmeristem induction in culture is the initial distribution of auxin and cytokinin. Auxin and cytokinin distribution is tightly controlled at all stages during development in planta, whereas this distribution must be gradually reorganized from disrupted initial conditions during shoot induction in culture. In vivo imaging of this dynamic process during gene and hormone perturbations should test the validity of this model.
The establishment of the primary meristems through proliferation after germination is essential for plant post-embryonic development. Cytokinins have long been considered a key regulator of plant cell division. Here we show that cytokinins are essential for early seedling development of Arabidopsis. Loss of cytokinin perception leads to a complete failure of meristem establishment and growth arrest after germination. We also present evidence that cytokinin signaling is involved in activation of the homeobox gene STIMPY (STIP or WOX9) expression in meristematic tissues, which is essential for maintaining the meristematic fate. Cytokinin-independent STIP expression is able to partially compensate for the shootapicalmeristem growth defects in mutants that cannot sense cytokinin. These findings identify a new branch of the cytokinin signaling network, linking cytokinin to the process of meristem and seedling establishment.
Considering the importance of plastids to plant biology and their potential in enhancing cultivars of high socioeconomic importance, it is somewhat surprising that relatively limited research has been undertaken into their development during embryogenesis and germination; particularly into the differ- ences seen between the plastid dynamics of root and shootmeristem-derived tissues. As individual systems of gene expres- sion and development, these early differences could have pro- found impacts on general plant biology, crop improvement and biotechnology. Before further research can be performed, however, a detailed characterization of a suitable model system is fundamental.
However, at the twofold and higher cutoffs, CNGC5, AtKUP9 and ZIP1 were no longer detected as CPEGs.
Similarities among cell types based on expression profiles We performed the sample-wise hierarchical clustering of transcriptome to determine similarities in the gene expression profiles among cell types. Data from the ten SAM cell populations as well as from 16 cell populations of the Arabidopsis root were considered (Birnbaum et al., 2003; Brady et al., 2007). The distance matrix required for this step was generated from the distance- transformed Spearman correlation coefficients obtained from all pair- wise sample comparisons using the average among their normalized replicates (see supplementary material Table S6). The resulting clustering dendrogram (Fig. 4C) indicates that the expression profiles of adjacent cell types in the CZ and the PZ are more similar than those that are spatially distant. For example, FIL cell population share greater similarities with KAN1 cell population compared with CLV3 (CZ) cell population (Fig. 4C). Similarly, the two vascular cell types, the xylem and phloem, clustered together; however, they showed a lower degree of relatedness to stem cell population of the CZ.
While recent studies provided evidence of a function of TPL/TPR in BES1/BZR1-mediated control of cell elongation, it is at present unknown whether this family of co-repressors is also involved in the promotion of cell proliferation in response to BR signaling. Here, we show that mutation of the EAR domain in the protein encoded by bes1-D reverses both the organ boundary and QC defects of bes1-D overexpressors. Increased TPL gene dosage aggravates the organ fusion and QC cell division phenotype of bes1-D mutants, while overexpression of the protein encoded by tpl-1 largely overrides bes1-D effects. We show that TPL binds to conserved BRRE and G-box elements in the CUC3 and BRAVO promoters through complex formation with BES1, and that pTPL::TPL seedlings display similar organ fusion defects and increased QC division rates to bes1-D mutants. Together, these results unveil a pivotal role of the co-repressor TPL in BR-regulated expression in the root and shoot meristems, and demonstrate that this function is essential to organ boundary initiation and maintenance, and to the preservation of low QC cell division rates.
HD-Zip IIγ and HD-Zip ΙΙδ proteins contain an LxLxL type of EAR repression motif (Ciarbelli et al., 2008; Kagale et al., 2010) and behave as negative regulators of gene expression (Ohgishi et al., 2001; Sawa et al., 2002). All HD-Zip II promoters are significantly enriched for HD-Zip-binding sequences, suggesting that members of this family negatively regulate each other (Ciarbelli et al., 2008). Here, we demonstrate that HAT3 and ATHB4 exhibit synergistic interaction with HD-Zip IIγ proteins, which involves cross-regulation within the two subfamilies. The finding that ATHB2 became induced in the HAT3-ATHB4 expression domain in the apical part of the embryo, marking the incipient cotyledons, as well as in the SAM of hat3 athb4, and thus can at least partially compensate for HAT3 and ATHB4 function, indicates that HD-Zip IIγ and HD-Zip ΙΙδ proteins are to some extent functionally interchangeable. In agreement, athb2 loss-of-function mutations significantly enhance the hat3 athb4 phenotype. Almost all hat3 athb4 athb2 mutants display radialized cotyledons and, among these, more than one-third shows loss of bilateral symmetry.