Gene expression trajectories during male and female reproductive development in balsam poplar (Populus balsamifera L.).

Plant reproductive development from the first appearance of reproductively committed axes through to floral maturation requires massive and rapid remarshalling of gene expression. In dioecious species such as poplar this is further complicated by divergent male and female developmental programs. We used seven time points in male and female balsam poplar (Populus balsamifera L.) buds and catkins representing the full annual flowering cycle, to elucidate the effects of time and sex on gene expression during reproductive development. Time (developmental stage) is dominant in patterning gene expression with the effect of sex nested within this. Here, we find (1) evidence for five successive waves of alterations to the chromatin landscape which may be important in setting the overall reproductive trajectory, regardless of sex. (2) Each individual developmental stage is further characterized by marked sex-differential gene expression. (3) Consistent sexually differentiated gene expression regardless of developmental stage reveal candidates for high-level regulators of sex and include the female-specific poplar ARR17 homologue. There is also consistent male-biased expression of the MADS-box genes PISTILLATA and APETALA3. Our work provides insights into expression trajectories shaping reproductive development, its potential underlying mechanisms, and sex-specific translation of the genome information into reproductive structures in balsam poplar.

A signal cascade originated from epidermis defines apical-basal patterning of Arabidopsis shoot apical meristems.

In multicellular organisms, a long-standing question is how spatial patterns of distinct cell types are initiated and maintained during continuous cell division and proliferation. Along the vertical axis of plant shoot apical meristems (SAMs), stem cells are located at the top while cells specifying the stem cells are located more basally, forming a robust apical-basal pattern. We previously found that in Arabidopsis SAMs, the HAIRY MERISTEM (HAM) family transcription factors form a concentration gradient from the epidermis to the interior cell layers, and this gradient is essential for the stem cell specification and the apical-basal patterning of the SAMs. Here, we uncover that epidermis specific transcription factors, ARABIDOPSIS THALIANA MERISTEM LAYER 1 (ATML1) and its close homolog, define the concentration gradient of HAM in the SAM through activating a group of microRNAs. This study provides a molecular framework linking the epidermis-derived signal to the stem cell homeostasis in plants.

Gene regulatory network controlling carpel number variation in cucumber.

The WUSCHEL-CLAVATA3 pathway genes play an essential role in shoot apical meristem maintenance and floral organ development, and under intense selection during crop domestication. The carpel number is an important fruit trait that affects fruit shape, size and internal quality in cucumber, but the molecular mechanism remains elusive. Here, we found that CsCLV3 expression was negatively correlated with carpel number in cucumber cultivars. CsCLV3-RNAi led to increased number of petals and carpels, whereas overexpression of CsWUS resulted in more sepals, petals and carpels, suggesting that CsCLV3 and CsWUS function as a negative and a positive regulator for carpel number variation, respectively. Biochemical analyses indicated that CsWUS directly bound to the promoter of CsCLV3 and activated its expression. Overexpression of CsFUL1A , a FRUITFULL-like MADS-box gene, resulted in more petals and carpels. CsFUL1A can directly bind to the CsWUS promoter to stimulate its expression. Furthermore, we found that auxin participated in carpel number variation in cucumber through interaction of CsARF14 with CsWUS. Therefore, we have identified a gene regulatory pathway involving CsCLV3, CsWUS, CsFUL1A and CsARF14 in determining carpel number variation in an important vegetable crop - cucumber.

POLTERGEIST and POLTERGEIST-LIKE1 are essential for the maintenance of post-embryonic shoot and root apical meristems as revealed by a partial loss-of-function mutant allele of pll1 in Arabidopsis.

BACKGROUND: POLTERGEIST (POL) and POL-LIKE1 (PLL1) encoding related protein phosphatase 2Cs are essential for the establishment of both shoot and root meristems during embryogenesis. As the strong pol pll1 are seedling-lethal due to the lack of hypocotyl vasculature, the roles of POL/PLL1 for the post-embryonic development is difficult to be assessed. OBJECTIVE: To prepare a weak pol pll1 double mutant that are able to produce post-embryonic organs. METHODS: Several T-DNA insertion mutants of pll1 were crossed to pol-6 for the preparation of weak pol pll1. To understand the epistatic interactions between POL/PLL1 and CLAVATAs, the phenotypes of clvs pol pll1 were assessed and the expression patterns of stem cell markers were examined in pol pll1. POLpro:PLL1-GFP expression was examined during the embryogenesis with confocal microscopy. RESULTS: We isolated a pll1-3 (S544N) allele and prepared a weak pol-6 pll1-3. About 5% of pol-6 pll1-3 seedlings continued the post-embryonic development displaying short roots with reduced root meristem, wuschel-like adventitious phyllotaxis, and defective flowers lacking carpel. The clv1, clv2, and clv3 phenotypes led by enlarged shoot meristems were almost completely suppressed in the pol-6 pll1-3. POL/PLL1 were required for the indeterminate floral organ development displayed by agamous. PLL1-GFP asymmetrically localized in the shootward sides of columella cells and increased the size of distal root meristem region by enhancing the WUS-RELATED HOMEOBOX 5 expression suggesting that PLL1 might provide the stem cells and progenies with proper positional information for the asymmetric cell divisions. CONCLUSION: Together, POL/PLL1 are required for the maintenance of stem cell pools for the post-embryonic development in Arabidopsis.

Genes involved in the deformations of the shoot apical meristem in somatic embryos of Capsicum chinense Jacq.

Somatic embryos (SE) of habanero pepper (Capsicum chinense Jacq.) represent persistent deformations in the shoot apical meristem (SAM), which inhibits their capacity to form organs and subsequently plants. In dicotyledonous plants, SAM is formed in the apex, between cotyledons and it plays a central role in postembryonic shoot organ formation. Based on the previous knowledge on the role of some families of gene in the formation, organization and maintenance of the SAM, the expression patterns of WUS, WOX2, NAM, STM, PIN1 and PIN7 genes were analysed, which would allow us to elucidate the possible implication of these genes in SAM deformations in the SE of C. chinense. The results show that the expression patterns of STM and PIN1 in the SE were completely opposite to the respective expression pattern obtained in zygotic embryos (ZE). Moreover, NAM and PIN7 showed an over accumulation of transcripts in SE, compared with ZE. This is the first time in the genus Capsicum that alterations in the expression pattern of key genes of the SE development are reported, as well as its possible implication in the persistent deformations of the SAM.

BELL1-like homeobox genes regulate inflorescence architecture and meristem maintenance in rice.

Inflorescence architecture is diverse in angiosperms, and is mainly determined by the arrangement of the branches and flowers, known as phyllotaxy. In rice (Oryza sativa), the main inflorescence axis, called the rachis, generates primary branches in a spiral phyllotaxy, and flowers (spikelets) are formed on these branches. Here, we have studied a classical mutant, named verticillate rachis (ri), which produces branches in a partially whorled phyllotaxy. Gene isolation revealed that RI encodes a BELL1-type homeodomain transcription factor, similar to Arabidopsis PENNYWISE/BELLRINGER/REPLUMLESS, and is expressed in the specific regions within the inflorescence and branch meristems where their descendant meristems would soon initiate. Genetic combination of an ri homozygote and a mutant allele of RI-LIKE1 (RIL1) (designated ri ril1/+ plant), a close paralog of RI, enhanced the ri inflorescence phenotype, including the abnormalities in branch phyllotaxy and rachis internode patterning. During early inflorescence development, the timing and arrangement of primary branch meristem (pBM) initiation were disturbed in both ri and ri ril1/+ plants. These findings suggest that RI and RIL1 were involved in regulating the phyllotactic pattern of the pBMs to form normal inflorescences. In addition, both RI and RIL1 seem to be involved in meristem maintenance, because the ri ril1 double-mutant failed to establish or maintain the shoot apical meristem during embryogenesis.

Local Auxin Biosynthesis Is a Key Regulator of Plant Development.

Auxin is a major phytohormone that controls numerous aspects of plant development and coordinates plant responses to the environment. Morphogenic gradients of auxin govern cell fate decisions and underlie plant phenotypic plasticity. Polar auxin transport plays a central role in auxin maxima generation. The discovery of the exquisite spatiotemporal expression patterns of auxin biosynthesis genes of the WEI8/TAR and YUC families suggested that local auxin production may contribute to the formation of auxin maxima. Herein, we systematically addressed the role of local auxin biosynthesis in plant development and responses to the stress phytohormone ethylene by manipulating spatiotemporal patterns of WEI8. Our study revealed that local auxin biosynthesis and transport act synergistically and are individually dispensable for root meristem maintenance. In contrast, flower fertility and root responses to ethylene require local auxin production that cannot be fully compensated for by transport in the generation of morphogenic auxin maxima.

Cytokinin signalling regulates organ identity via the AHK4 receptor in Arabidopsis.

Mutual interactions of the phytohormones, cytokinins and auxin determine root or shoot identity during postembryonic de novo organogenesis in plants. However, our understanding of the role of hormonal metabolism and perception during early stages of cell fate reprogramming is still elusive. Here we show that auxin activates root formation, whereas cytokinins mediate early loss of the root identity, primordia disorganisation and initiation of shoot development. Exogenous and endogenous cytokinins influence the initiation of newly formed organs, as well as the pace of organ development. The process of de novo shoot apical meristem establishment is accompanied by accumulation of endogenous cytokinins, differential regulation of genes for individual cytokinin receptors, strong activation of AHK4-mediated signalling and induction of the shoot-specific homeodomain regulator WUSCHEL. The last is associated with upregulation of isopentenyladenine-type cytokinins, revealing higher shoot-forming potential when compared with trans-zeatin. Moreover, AHK4-controlled cytokinin signalling negatively regulates the root stem cell organiser WUSCHEL RELATED HOMEOBOX 5 in the root quiescent centre. We propose an important role for endogenous cytokinin biosynthesis and AHK4-mediated cytokinin signalling in the control of de novo-induced organ identity.

Epidermal expression of a sterol biosynthesis gene regulates root growth by a non-cell-autonomous mechanism in Arabidopsis.

The epidermis is hypothesized to play a signalling role during plant development. One class of mutants showing defects in signal transduction and radial patterning are those in sterol biosynthesis. The expectation is that living cells require sterols, but it is not clear that all cell types express sterol biosynthesis genes. The HYDRA1 (HYD1) gene of Arabidopsis encodes sterol Δ8-Δ7 isomerase, and although hyd1 seedlings are defective in radial patterning across several tissues, we show that the HYD1 gene is expressed most strongly in the root epidermis. Transgenic activation of HYD1 transcription in the epidermis of hyd1 null mutants reveals a major role in root patterning and growth. HYD1 expression in the vascular tissues and root meristem, though not endodermis or pericycle, also leads to some phenotypic rescue. Phenotypic rescue is associated with rescued patterning of the PIN1 and PIN2 auxin efflux carriers. The importance of the epidermis in controlling root growth and development is proposed to be, in part, due to its role as a site for sterol biosynthesis, and auxin is a candidate for the non-cell-autonomous signal.

Control of Endogenous Auxin Levels in Plant Root Development.

In this review, we summarize the different biosynthesis-related pathways that contribute to the regulation of endogenous auxin in plants. We demonstrate that all known genes involved in auxin biosynthesis also have a role in root formation, from the initiation of a root meristem during embryogenesis to the generation of a functional root system with a primary root, secondary lateral root branches and adventitious roots. Furthermore, the versatile adaptation of root development in response to environmental challenges is mediated by both local and distant control of auxin biosynthesis. In conclusion, auxin homeostasis mediated by spatial and temporal regulation of auxin biosynthesis plays a central role in determining root architecture.

The Roles of the Sole Activator-Type Auxin Response Factor in Pattern Formation of Marchantia polymorpha.

Cell division patterning is important to determine body shape in plants. Nuclear auxin signaling mediated by AUXIN RESPONSE FACTOR (ARF) transcription factors affects plant growth and development through regulation of cell division, elongation and differentiation. The evolutionary origin of the ARF-mediated pathway dates back to at least the common ancestor of bryophytes and other land plants. The liverwort Marchantia polymorpha has three phylogenetically distinct ARFs: MpARF1, the sole 'activator' ARF; and MpARF2 and MpARF3, two 'repressor' ARFs. Genetic screens for auxin-resistant mutants revealed that loss of MpARF1 function conferred auxin insensitivity. Mparf1 mutants showed reduced auxin-inducible gene expression and various developmental defects, including thallus twisting and gemma malformation. We further investigated the role of MpARF1 in gemma development, which is traceable at the cellular level. In wild-type plants, a gemma initial first undergoes several transverse divisions to generate a single-celled stalk and a gemma proper, followed by rather synchronous longitudinal divisions in the latter. Mparf1 mutants often contained multicelled stalks and showed defects in the execution and timing of the longitudinal divisions. While wild-type gemmae finally generate two meristem notches, Mparf1 gemmae displayed various numbers of ectopic meristems. These results suggest that MpARF1 regulates formative cell divisions and axis formation through auxin responses. The mechanism for activator ARF regulation of pattern formation may be shared in land plants and therefore important for the general acquisition of three-dimensional body plans.

Redox Changes During the Cell Cycle in the Embryonic Root Meristem of Arabidopsis thaliana.

AIMS: The aim of this study was to characterize redox changes in the nuclei and cytosol occurring during the mitotic cell cycle in the embryonic roots of germinating Arabidopsis seedlings, and to determine how redox cycling was modified in mutants with a decreased capacity for ascorbate synthesis. RESULTS: Using an in vivo reduction-oxidation (redox) reporter (roGFP2), we show that transient oxidation of the cytosol and the nuclei occurred at G1 in the synchronized dividing cells of the Arabidopsis root apical meristem, with reduction at G2 and mitosis. This redox cycle was absent from low ascorbate mutants in which nuclei were significantly more oxidized than controls. The cell cycle-dependent increase in nuclear size was impaired in the ascorbate-deficient mutants, which had fewer cells per unit area in the root proliferation zone. The transcript profile of the dry seeds and size of the imbibed seeds was strongly influenced by low ascorbate but germination, dormancy release and seed aging characteristics were unaffected. INNOVATION: These data demonstrate the presence of a redox cycle within the plant cell cycle and that the redox state of the nuclei is an important factor in cell cycle progression. CONCLUSIONS: Controlled oxidation is a key feature of the early stages of the plant cell cycle. However, sustained mild oxidation restricts nuclear functions and impairs progression through the cell cycle leading to fewer cells in the root apical meristem. Antioxid. Redox Signal. 27, 1505-1519.

TOPLESS mediates brassinosteroid control of shoot boundaries and root meristem development in Arabidopsis thaliana.

The transcription factor BRI1-EMS-SUPRESSOR 1 (BES1) is a master regulator of brassinosteroid (BR)-regulated gene expression. BES1 together with BRASSINAZOLE-RESISTANT 1 (BZR1) drive activated or repressed expression of several genes, and have a prominent role in negative regulation of BR synthesis. Here, we report that BES1 interaction with TOPLESS (TPL), via its ERF-associated amphiphilic repression (EAR) motif, is essential for BES1-mediated control of organ boundary formation in the shoot apical meristem and the regulation of quiescent center (QC) cell division in roots. We show that TPL binds via BES1 to the promoters of the CUC3 and BRAVO targets and suppresses their expression. Ectopic expression of TPL leads to similar organ boundary defects and alterations in QC cell division rate to the bes1-d mutation, while bes1-d defects are suppressed by the dominant interfering protein encoded by tpl-1, with these effects respectively correlating with changes in CUC3 and BRAVO expression. Together, our data unveil a pivotal role of the co-repressor TPL in the shoot and root meristems, which relies on its interaction with BES1 and regulation of BES1 target gene expression.

A Molecular Framework for the Embryonic Initiation of Shoot Meristem Stem Cells.

The establishment of pluripotent stem cells is a key event during plant and animal embryogenesis, but the underlying mechanisms remain enigmatic. We show that in the flowering plant Arabidopsis thaliana, expression of the shoot meristem stem cell marker CLV3 becomes detectable in transition stage embryos. Surprisingly, the key regulator of stem cell homeostasis WUSCHEL (WUS) is expressed but dispensable for stem cell initiation. Rather, the WUS paralog WOX2, a regulator of embryo patterning initiated in the zygote, functions in this process by shielding stem cell progenitors from differentiation. WOX2 upregulates HD-ZIP III transcription factors required for shoot identity and balances cytokinin versus auxin hormone pathways, revealing that classical plantlet regeneration procedures recapitulate the natural induction mechanism. Our findings link transcriptional regulation of early embryo patterning to hormonal control of stem cell initiation and suggest that similar strategies have evolved in plant and animal stem cell formation.

Regulatory function of homeodomain-leucine zipper (HD-ZIP) family proteins during embryogenesis.

Homeodomain-leucine zipper proteins (HD-ZIPs) form a plant-specific family of transcription factors functioning as homo- or heterodimers. Certain members of all four classes of this family are involved in embryogenesis, the focus of this review. They support auxin biosynthesis, transport and response, which are in turn essential for the apical-basal patterning of the embryo, radicle formation and outgrowth of the cotyledons. They transcriptionally regulate meristem regulators to maintain the shoot apical meristem once it is initiated. Some members are specific to the protoderm, the outermost layer of the embryo, and play a role in shoot apical meristem function. Within classes, homeodomain-leucine zippers tend to act redundantly during embryo development, and there are many examples of regulation within and between classes of homeodomain-leucine zippers. This indicates a complex network of regulation that awaits future experiments to uncover.

ZRF1 Chromatin Regulators Have Polycomb Silencing and Independent Roles in Development.

Histone H2A monoubiquitination (H2Aub1), catalyzed by Polycomb-Repressive Complex1 (PRC1), is a key epigenetic mark in Polycomb silencing. However, little is known about how H2Aub1 is read to exert downstream physiological functions. The animal ZUOTIN-RELATED FACTOR1 (ZRF1) has been reported to bind H2Aub1 to promote or repress the expression of varied target genes. Here, we show that the Arabidopsis (Arabidopsis thaliana) ZRF1 homologs, AtZRF1a and AtZRF1b, are key regulators of multiple processes during plant growth and development. Loss of function of both AtZRF1a and AtZRF1b in atzrf1a atzrf1b mutants causes seed germination delay, small plant size, abnormal meristem activity, abnormal flower development, as well as gametophyte transmission and embryogenesis defects. Some of these defects overlap with those described previously in the PRC1-defective mutants atbmi1a atbmi1b and atring1a atring1b, but others are specific to atzrf1a atzrf1b In line with this, 4,519 genes (representing more than 14% of all genes) within the Arabidopsis genome are found differentially expressed in atzrf1a atzrf1b seedlings, and among them, 114 genes are commonly up-regulated in atring1a atring1b and atbmi1a atbmi1b Finally, we show that in both atzrf1a atzrf1b and atbmi1a atbmi1b seedlings, the seed developmental genes ABSCISIC ACID INSENSITIVE3, CRUCIFERIN3, and CHOTTO1 are derepressed, in association with the reduced levels of H2Aub1 and histone H3 lysine-27 trimethylation (H3K27me3). Collectively, our results indicate that AtZRF1a/b play both PRC1-related and PRC1-unrelated functions in regulating plant growth and development and that AtZRF1a/b promote H2Aub1 and H3K27me3 deposition in gene suppression. Our work provides novel insight into the mechanisms of function of this family of evolutionarily conserved chromatin regulators.

Identification of three LRR-RKs involved in perception of root meristem growth factor in Arabidopsis.

A peptide hormone, root meristem growth factor (RGF), regulates root meristem development through the PLETHORA (PLT) stem cell transcription factor pathway, but it remains to be uncovered how extracellular RGF signals are transduced to the nucleus. Here we identified, using a combination of a custom-made receptor kinase (RK) expression library and exhaustive photoaffinity labeling, three leucine-rich repeat RKs (LRR-RKs) that directly interact with RGF peptides in Arabidopsis These three LRR-RKs, which we named RGFR1, RGFR2, and RGFR3, are expressed in root tissues including the proximal meristem, the elongation zone, and the differentiation zone. The triple rgfr mutant was insensitive to externally applied RGF peptide and displayed a short root phenotype accompanied by a considerable decrease in meristematic cell number. In addition, PLT1 and PLT2 protein gradients, observed as a gradual gradient decreasing toward the elongation zone from the stem cell area in wild type, steeply declined at the root tip in the triple mutant. Because RGF peptides have been shown to create a diffusion-based concentration gradient extending from the stem cell area, our results strongly suggest that RGFRs mediate the transformation of an RGF peptide gradient into a PLT protein gradient in the proximal meristem, thereby acting as key regulators of root meristem development.

Expression and genomic integration of transgenes after Agrobacterium-mediated transformation of mature barley embryos.

Mature embryos in tissue cultures are advantageous because of their abundance and rapid germination, which reduces genomic instability problems. In this study, 2-day-old isolated mature barley embryos were infected with 2 Agrobacterium hypervirulent strains (AGL1 and EHA105), followed by a 3-day period of co-cultivation in the presence of L-cystein amino acid. Chimeric expression of the b-glucuronidase gene (gusA) directed by a viral promoter of strawberry vein banding virus was observed in coleoptile epidermal cells and seminal roots in 5-day-old germinated seedlings. In addition to varying infectivity patterns in different strains, there was a higher ratio of transient b-glucuronidase expression in developing coleoptiles than in embryonic roots, indicating the high competency of shoot apical meristem cells in the mature embryo. A total of 548 explants were transformed and 156 plants developed to maturity on G418 media after 18-25 days. We detected transgenes in 74% of the screened plant leaves by polymerase chain reaction, and 49% of these expressed neomycin phosphotransferase II gene following AGL1 transformation. Ten randomly selected T0 transformants were analyzed using thermal asymmetric interlaced polymerase chain reaction and 24 fragments ranged between 200-600 base pairs were sequenced. Three of the sequences flanked with transferred-DNA showed high similarity to coding regions of the barley genome, including alpha tubulin5, homeobox 1, and mitochondrial 16S genes. We observed 70-200-base pair filler sequences only in the coding regions of barley in this study.

Plant development. Genetic control of distal stem cell fate within root and embryonic meristems.

The root meristem consists of populations of distal and proximal stem cells and an organizing center known as the quiescent center. During embryogenesis, initiation of the root meristem occurs when an asymmetric cell division of the hypophysis forms the distal stem cells and quiescent center. We have identified NO TRANSMITTING TRACT (NTT) and two closely related paralogs as being required for the initiation of the root meristem. All three genes are expressed in the hypophysis, and their expression is dependent on the auxin-signaling pathway. Expression of these genes is necessary for distal stem cell fate within the root meristem, whereas misexpression is sufficient to transform other stem cell populations to a distal stem cell fate in both the embryo and mature roots.

O cell, where art thou? The mechanisms of shoot meristem patterning.

Plants develop postembryonically from pools of continuously active stem cells embedded in specialized tissues called meristems, which are located at the growing points of shoot and root. How these stem cells are established, maintained and guided towards differentiation within the highly dynamic shoot apical meristem is only beginning to emerge. At the core of the complex regulatory system are spatially distinct subdomains within the shoot apex, in which cells carry out defined functions, despite highly similar phenotypes. Spatial and temporal control of these domains appears to rely on an elaborate network of phytohormone signaling, transcriptional loops and intercellular trafficking of key regulators. In this review, we aim at summarizing and connecting the mechanisms underlying the spatial organization of the shoot apical meristem and the sequence of molecular events occurring during the life of a shoot cell, from its birth towards its differentiation.