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A pectin glucuronyltransferase gene is essential for intercellular attachment in the plant meristem.

Intercellular attachment is an essential process in the morphogenesis of multicellular organisms. A unique mutant, nolac-H18 (nonorganogenic callus with loosely attached cells), generated by T-DNA transformation using leaf-disk cultures of haploid Nicotiana plumbaginifolia, lost the ability to form tight intercellular attachments and adventitious shoots. The gene tagged with T-DNA, named NpGUT1 (glucuronyltransferase 1), was similar to the gene for the catalytic domains of animal glucuronyltransferases and was expressed predominantly in shoot and root apical meristems. The transformation of NpGUT1 complemented the nolac-H18 mutation, and the expression of antisense NpGUT1 RNA produced crumbled shoots. The mutation caused defects in the glucuronic acid of rhamnogalacturonan II of pectin, which drastically reduced the formation of borate cross-linking of rhamnogalacturonan II. NpGUT1, which encodes a unique glucuronyltransferase, is a glycosyltransferase gene identified in pectin biosynthesis and is essential for intercellular attachment in plant meristems and tissues.

Amino Acid Sequence↗

Modeling the organization of the WUSCHEL expression domain in the shoot apical meristem.

MOTIVATION: The above-ground tissues of higher plants are generated from a small region of cells situated at the plant apex called the shoot apical meristem. An important genetic control circuit modulating the size of the Arabidopsis thaliana meristem is a feed-back network between the CLAVATA3 and WUSCHEL genes. Although the expression patterns for these genes do not overlap, WUSCHEL activity is both necessary and sufficient (when expressed ectopically) for the induction of CLAVATA3 expression. However, upregulation of CLAVATA3 in conjunction with the receptor kinase CLAVATA1 results in the downregulation of WUSCHEL. Despite much work, experimental data for this network are incomplete and additional hypotheses are needed to explain the spatial locations and dynamics of these expression domains. Predictive mathematical models describing the system should provide a useful tool for investigating and discriminating among possible hypotheses, by determining which hypotheses best explain observed gene expression dynamics. RESULTS: We are developing a method using in vivo live confocal microscopy to capture quantitative gene expression data and create templates for computational models. We present two models accounting for the organization of the WUSCHEL expression domain. Our preferred model uses a reaction-diffusion mechanism in which an activator induces WUSCHEL expression. This model is able to organize the WUSCHEL expression domain. In addition, the model predicts the dynamical reorganization seen in experiments where cells, including the WUSCHEL domain, are ablated, and it also predicts the spatial expansion of the WUSCHEL domain resulting from removal of the CLAVATA3 signal. AVAILABILITY: An extended description of the model framework and image processing algorithms can be found at http://www.computableplant.org, together with additional results and simulation movies. SUPPLEMENTARY INFORMATION: http://www.computableplant.org/ and alternatively for a direct link to the page, http://computableplant.ics.uci.edu/bti1036 can be accessed.

Arabidopsis↗

Fine mapping and characterization of linked quantitative trait loci involved in the transition of the maize apical meristem from vegetative to generative structures.

Quantitative trait locus (QTL) mapping has detected two linked QTL in the 8L chromosome arm segment introgressed from Gaspé Flint (a Northern Flint open-pollinated population) into the background of N28 (a Corn Belt Dent inbred line). Homozygous recombinant lines, with a variable length of the introgressed segment, confirmed the presence of the two previously identified, linked QTL. In the N28 background, Gaspé Flint QTL alleles at both loci induce a reduction in node number, height, and days to anthesis (pollen shed). Given the determinate growth pattern of maize, the phenotypic effects indicate that the two QTL are involved in the transition of the apical meristem from vegetative to generative structures. Relative to the effects of the two QTL in the background of N28, we distinguish two general developmental factors affecting the timing of pollen shed. The primary factor is the timing of the transition of the apical meristem. The second, derivative factor is the global extent of internode elongation. Having separated the two linked QTL, we have laid the foundation for the positional cloning of the QTL with a larger effect.

Analysis of Variance↗

Cytokinin levels in leaves, leaf exudate and shoot apical meristem of Arabidopsis thaliana during floral transition.

Understanding the complete picture of floral transition is still impaired by the fact that physiological studies mainly concern plant species whose genetics is poorly known, and vice versa. Arabidopsis thaliana has been successfully used to unravel signalling pathways by genetic and molecular approaches, but analyses are still required to determine the physiological signals involved in the control of floral transition. In this work, the putative role of cytokinins was investigated using vegetative plants of Arabidopsis (Columbia) induced to flower synchronously by a single 22 h long day. Cytokinins were analysed in leaf extracts, leaf phloem exudate and in the shoot apical meristem at different times during floral transition. It was found that, in both the leaf tissues and leaf exudate, isopentenyladenine forms of cytokinins increased from 16 h after the start of the long day. At 30 h, the shoot apical meristem of induced plants contained more isopentenyladenine and zeatin than vegetative controls. These cytokinin increases correlate well with the early events of floral transition.

Arabidopsis↗

Changes in gene expression during meristem activation processes in Solanum tuberosum with a focus on the regulation of an auxin response factor gene.

A suppression subtractive hybridization approach (SSH) was used to generate a cDNA library enriched in clones representing genes that are up-regulated in the potato tuber apical bud on dormancy release. The sequences of cDNAs representing 385 different genes were determined. This study focuses on the characterization of one of these cDNAs. On the basis of sequence similarity, the cDNA was identified as encoding a member of the auxin response factor family (ARF6). The expression pattern of potato ARF6 was determined by in situ hybridization. In apical tuber buds in the early stages of sprouting, relatively high levels of ARF6-specific transcripts were detected, especially in the peripheral zones of the tunica and corpus of the apical meristems. Expression was also detected in procambial and early vascular tissues, both subtending the meristem and in adjacent leaf primordia. By contrast, in dormant buds no expression of ARF6 could be detected. The expression pattern was also determined during the tuberization process; steady-state expression levels decreased c. 10-fold in the apical region as tuberization proceeded. In non-growing buds, exhibiting correlative inhibition, ARF6-specific transcript levels were relatively low, but rapidly increased when apical dominance was removed by excision of the apical bud. The effects of gibberellin and auxin on axillary bud growth and ARF6 expression are described.

Amino Acid Sequence↗

The rubber tree (Hevea brasiliensis Muell. Arg.) homologue of the LEAFY/FLORICAULA gene is preferentially expressed in both male and female floral meristems.

The rubber tree (Hevea brasiliensis Muell. Arg.) is an important source of natural rubber in tropical regions and, as with many woody species, shows a long juvenile phase. To understand the genetic and molecular mechanisms underlying the reproductive process in rubber trees, H. brasiliensis RRIM600 flower and inflorescence development have been characterized, the rubber tree FLORICAULA/LEAFY (FLO/LFY) orthologue, HbLFY, cloned, and its expression patterns were analysed during vegetative and reproductive development. The rubber tree, similar to other Euphorbiaceae species, produces lateral inflorescences containing male, female, and bisexual flowers. HbLFY is expressed in lateral meristems that give rise to inflorescences and in all flower meristems, consistent with a role in reproductive development. Complementation studies using Arabidopsis lfy mutants indicated that the biological function of LFY might be conserved among Brassicaceae and Euphorbiaceae species.

Arabidopsis↗

The co-ordination of cell division, differentiation and morphogenesis in the shoot apical meristem: a perspective.

Whether morphogenesis is cell division-driven or organismal-based has been a long-running debate in plant biology. This article is a summary of a series of experiments aimed at distinguishing these alternate views by local manipulation of parameters of cell division frequency, orientation, and growth within the shoot apical meristem. These data, put in the context of other investigations in this area, support an organismal view of plant morphogenesis and support the idea that the cell wall plays a key role in the mechanism by which this is achieved. At the same time, the data indicate that the intimate but variable relationship between cell growth and division within the organism means that cell proliferation can indirectly influence this process, leading to a context-dependent influence on morphogenesis. Finally, cell growth and proliferation are intimately related with the process of differentiation as cells exit the meristem. In the final part of the article the molecular mechanism by which these basic cellular parameters are intertwined is discussed.

Cell Differentiation↗

Root meristems in Medicago truncatula tissue culture arise from vascular-derived procambial-like cells in a process regulated by ethylene.

Leaf explants of Medicago truncatula were used to investigate the origins of auxin-induced root formation. On the application of auxin there is some callus formation (not the massive amount that occurs in response to auxin plus cytokinin) and roots appear shortly after the first visible callus. Histological examination reveals morphologically distinctive sheets of callus cells that emanate from the veins of the leaf explants and, within this cell type, root primordia are produced as well as some vascular tissue cells. What is suggested is that the vein-derived cells (VDCs) are procambial-like and function as pluripotent stem cells with a propensity to form root meristems or vascular tissues in response to added auxin. The development of root primordia from these pluripotent cells was clearly up-regulated by the use of the sickle (skl) mutant, which is a mutant impaired in ethylene signal transduction while the wild type and the sunn mutant, defective in auxin polar transport, produced similar numbers of roots. The skl mutant in generating many more roots concomitantly formed fewer vascular tissues. The root meristems differentiate similarly to normal roots producing a central cylinder of vascular tissue, which connects with the leaf explant veins. The VDCs appear to be derived from the cells of or near the phloem. The leaf observations suggest that a pool of stem cells exist in vascular tissue that, in combination with auxin and perhaps other factors, drive a diversity of plant development outcomes that is species specific. The way auxin interacts with other hormones is a key factor in determining the stem cell fate. The histological data in this study also assist in the interpretation of the molecular analysis of auxin-induced root formation in cultured leaves of M. truncatula.

Ethylenes↗

Nuclear import of the transcription factor SHOOT MERISTEMLESS depends on heterodimerization with BLH proteins expressed in discrete sub-domains of the shoot apical meristem of Arabidopsis thaliana.

The gene SHOOT MERISTEMLESS (STM) is required for the initiation and the maintenance of the shoot apical meristem (SAM) in Arabidopsis and encodes a MEINOX/three amino acid loop extension (TALE)-HD-type transcription factor. Translational fusions with the green fluorescent protein showed that STM is not nuclear by default. In a yeast two-hybrid screen performed with a meristem-enriched cDNA library, three interacting BLH (Bel1-like homeodomain) transcription factors were identified. According to bimolecular fluorescence complementation, STM is targeted into the nuclear compartment through heterodimerization with BLH partner proteins, which are expressed in distinct SAM domains from the center to the periphery. On a functional level, overexpression experiments in transgenic Arabidopsis plants suggest that individual heterodimers provide distinct contributions. These results contribute to our understanding of the STM transcription factor function in the SAM and also shed new light on the evolution of the TALE-HD super gene family in animal and plant lineages.

Active Transport, Cell Nucleus↗

NFL1, a Nicotiana tabacum LEAFY-like gene, controls meristem initiation and floral structure.

The Arabidopsis LEAFY (LFY) gene product induces cells of the shoot apical meristem to differentiate into floral primordia by acting as a master regulator of downstream floral homeotic genes. Tobacco, an allotetraploid, possesses two homologous genes, NFL1 and NFL2, which are 97% identical in amino acid sequence and share 73% amino acid sequence identity with LFY. In order to test whether the highly conserved tobacco orthologue, NFL1, shares functional identity with LFY, we created transgenic tobacco and Arabidopsis plants that constitutively express the NFL1 cDNA. Our results indicate that NFL1 plays a critical role in the allocation of meristematic cells that differentiate lateral structures such as leaves and branches, thereby determining the architecture of the wild-type tobacco shoot. NFL1 also regulates floral meristem development and does so through the control of cell proliferation as well as cell identity. Surprisingly, unlike ectopic LFY expression, which can act as a floral trigger, ectopic NFL1 expression does not promote severe precocious flowering in Nicotiana tabacum suggesting that variations in amino acid sequence among members of the LFY-like gene family have led to divergence in the functional roles of these genes.

Arabidopsis Proteins↗

Endogenous ascorbic acid modulates meristem reactivation in white spruce somatic embryos and affects thymidine and uridine metabolism.

Previously, we demonstrated that, in some cell lines, exogenous applications of ascorbic acid (ASC) enhance the conversion frequency of white spruce (Picea glauca (Moench) Voss) somatic embryos, by stimulating mitotic activity in the apical meristems. To examine this event in more detail, we investigated the effects of ASC on de novo, salvage and degradation pathways of pyrimidine metabolism by following the metabolic fate of (14)C-labeled orotic acid, thymidine, uridine and uracil in shoot and root poles of germinating embryos, after altering the cellular ASC content of the embryos. Alterations in endogenous ASC content did not affect the utilization of either orotic acid or uracil, but affected the metabolism of thymidine and uridine. Specifically, a lowering of endogenous ASC content by applications of lycorine (L), an inhibitor of the last enzyme of the ASC de novo biosynthetic pathway, resulted in a lower embryo conversion frequency, as well as a reduced percentage of thymidine and uridine incorporated into nucleotides and nucleic acids. The reduction in thymidine and uridine anabolism was mainly ascribed to the decreased activities of thymidine kinase (TRK) and uridine kinase (URK), the respective salvage enzymes of thymidine and uridine, measured in L-treated embryos. These effects were solely a result of a decrease in endogenous ASC content because applications of ascorbic acid plus lycorine (ASC + L) increased embryo conversion frequency, thymidine and uridine salvage activities, and TRK and URK activities to near control values. Inclusion of exogenous ASC in the germination medium did not affect the percentage of embryos able to convert to viable plantlets, although it increased thymidine and uridine utilization for nucleic acid synthesis in the shoot and root poles of the embryos. Taken together, these findings confirm that cellular ASC plays a key role in the reactivation of the apical meristems of germinating white spruce somatic embryos.

Ascorbic Acid↗

Cellular and molecular events in a newly organizing lateral root meristem.

Spontaneous or auxin-induced lateral root formation in radish and Arabidopis provides an efficient system in which to examine molecular and cellular events associated with the initiation of a new meristem. Subtracted cDNA libraries made at different times in lateral root initiation were used as a source of genes that are expressed differentially during this developmental process, and expression studies on a small gene family of ribosomal protein genes were conducted. From analysis of cell division patterns in pericycle cells the number of founder cells for lateral roots was established. By the use of in vitro growth assays lateral root formation was determined to be a two-stage process. First a primordium is formed, and subsequently a subset of primordial cells begins to function as the lateral root apical meristem. This mode of root development has implications for pattern formation in newly organizing organs.

Arabidopsis↗

KNOX homeodomain protein directly suppresses the expression of a gibberellin biosynthetic gene in the tobacco shoot apical meristem.

To identify genes targeted by the tobacco KNOX homeodomain protein, Nicotiana tabacum homeobox 15 (NTH15), we have generated an inducible system using the human glucocorticoid receptor. In this system, steroid treatment strictly induced NTH15 function and immediately suppressed the expression of a gibberellin (GA) biosynthetic gene encoding GA 20-oxidase (Ntc12) and also resulted in a decrease in bioactive GA levels. The repression of Ntc12 was observed even when indirect effects were blocked by cycloheximide. NTH15 mRNA was present in corpus cells of the shoot apical meristem (SAM), whereas Ntc12 mRNA was observed in leaf primordia and rib meristem but not in the corpus. Recombinant NTH15 protein strongly bound to a 5-bp dyadsymmetric sequence, GTGAC, in the first intron of Ntc12 in vitro. Mutation of this sequence in the Ntc12 gene abolished the NTH15-dependent suppression of Ntc12 in the corpus of the SAM. Our results indicate that NTH15 directly represses Ntc12 expression in the corpus of the wild-type SAM to maintain the indeterminate state of corpus cells. The suppression of NTH15 within cells at the flanks of the SAM permits GA biosynthesis, which promotes organized cell proliferation and consequently induces the determination of cell fate.

Cell Division↗

Mutations in two independent genes lead to suppression of the shoot apical meristem in maize.

The shoot apical meristem (SAM), initially formed during embryogenesis, gives rise to the aboveground portion of the maize (Zea mays) plant. The shootless phenotype (sml) described here is caused by disruption of SAM formation due to the synergistic interaction of mutations at two genetic loci. Seedlings must be homozygous for both sml (shootmeristemless), and the unlinked dgr (distorted growth) loci for a SAM-less phenotype to occur. Seedlings mutant only for sml are impaired in their morphogenesis to different extents, whereas the dgr mutation alone does not have a recognisable phenotype. Thus, dgr can be envisaged as being a dominant modifier of sml and the 12 (normal):3 (distorted growth):1 (shoot meristemless) segregation observed in the F(2) of the double heterozygote is the result of the interaction between the sml and dgr genes. Other segregation patterns were also observed in the F(2), suggesting instability of the dgr gene. Efforts to rescue mutant embryos by growth on media enriched with hormones have been unsuccessful so far. However, mutant roots grow normally on medium supplemented with kinetin at a concentration that suppresses wild-type root elongation, suggesting possible involvement of the mutant in the reception or transduction of the kinetin signal or transport of the hormone. The shootless mutant appears to be a valuable tool with which to investigate the organization of the shoot meristem in monocots as well as a means to assay the origins and relationships between organs such as the scutellum, the coleoptile, and leaves that are initiated during the embryogenic process.

Adenine↗

The Medicago species A2-type cyclin is auxin regulated and involved in meristem formation but dispensable for endoreduplication-associated developmental programs.

Phytohormones as well as temporal and spatial regulation of the cell cycle play a key role in plant development. Here, we investigated the function and regulation of an alfalfa (Medicago sativa) A2-type cyclin in three distinct root developmental programs: in primary and secondary root development, nodule development, and nematode-elicited gall formation. Using transgenic plants carrying the Medsa;cycA2;2 promoter-beta-glucuronidase gene fusion, in combination with other techniques, cycA2;2 expression was localized in meristems and proliferating cells in the lateral root and nodule primordia. Rapid induction of cycA2;2 by Nod factors demonstrated that this gene is implicated in cell cycle activation of differentiated cells developing to nodule primordia. Surprisingly, cycA2;2 was repressed in the endoreduplicating, division-arrested cells both during nodule development and formation of giant cells in nematode-induced galls, indicating that CycA2;2 was dispensable for S-phase in endoreduplication cycles. Overexpression of cycA2;2 in transgenic plants corresponded to wild type protein levels and had no apparent phenotype. In contrast, antisense expression of cycA2;2 halted regeneration of somatic embryos, suggesting a role for CycA2;2 in the formation or activity of apical meristems. Expression of cycA2;2 was up-regulated by auxins, as expected from the presence of auxin response elements in the promoter. Moreover, auxin also affected the spatial expression pattern of this cyclin by shifting the cycA2;2 expression from the phloem to the xylem poles.

Animals↗

The hpa1 mutant of Arabidopsis reveals a crucial role of histidine homeostasis in root meristem maintenance.

Histidine (His) is an essential ingredient for protein synthesis and is required by all living organisms. In higher plants, although there is considerable evidence that His is essential for plant growth and survival, there is very little information as to whether it plays any specific role in plant development. Here, we present evidence for such a role of this amino acid in root development in Arabidopsis (Arabidopsis thaliana) from the characterization of a novel Arabidopsis mutant, hpa1, which has a very short root system and carries a mutation in one of the two Arabidopsis histidinol-phosphate aminotransferase (HPA) genes, AtHPA1. We have established that AtHPA1 encodes a functional HPA and that its complete knockout is embryo lethal. Biochemical analysis shows that the mutation in hpa1 only resulted in a 30% reduction in free His content and had no significant impact on the total His content. It did not cause any known symptoms of His starvation. However, the mutant displayed a specific developmental defect in root meristem maintenance and was unable to sustain primary root growth 2 d after germination. We have demonstrated that the root meristem failure in the mutant is tightly linked to the reduction in free His content and could be rescued by either exogenous His supplementation or AtHPA1 overexpression. Our results therefore reveal an important role of His homeostasis in plant development.

Amino Acid Sequence↗

Short vegetative phase-like MADS-box genes inhibit floral meristem identity in barley.

Analysis of the functions of Short Vegetative Phase (SVP)-like MADS-box genes in barley (Hordeum vulgare) indicated a role in determining meristem identity. Three SVP-like genes are expressed in vegetative tissues of barley: Barley MADS1 (BM1), BM10, and Vegetative to Reproductive Transition gene 2. These genes are induced by cold but are repressed during floral development. Ectopic expression of BM1 inhibited spike development and caused floral reversion in barley, with florets at the base of the spike replaced by tillers. Head emergence was delayed in plants that ectopically express BM1, primarily by delayed development after the floral transition, but expression levels of the barley VRN1 gene (HvVRN1) were not affected. Ectopic expression of BM10 inhibited spike development and caused partial floral reversion, where florets at the base of the spike were replaced by inflorescence-like structures, but did not affect heading date. Floral reversion occurred more frequently when BM1 and BM10 ectopic expression lines were grown in short-day conditions. BM1 and BM10 also inhibited floral development and caused floral reversion when expressed in Arabidopsis (Arabidopsis thaliana). We conclude that SVP-like genes function to suppress floral meristem identity in winter cereals.

Arabidopsis↗

The 14-amino acid CLV3, CLE19, and CLE40 peptides trigger consumption of the root meristem in Arabidopsis through a CLAVATA2-dependent pathway.

CLAVATA3 (CLV3), CLV3/ESR19 (CLE19), and CLE40 belong to a family of 26 genes in Arabidopsis thaliana that encode putative peptide ligands with unknown identity. It has been shown previously that ectopic expression of any of these three genes leads to a consumption of the root meristem. Here, we show that in vitro application of synthetic 14-amino acid peptides, CLV3p, CLE19p, and CLE40p, corresponding to the conserved CLE motif, mimics the overexpression phenotype. The same result was observed when CLE19 protein was applied externally. Interestingly, clv2 failed to respond to the peptide treatment, suggesting that CLV2 is involved in the CLE peptide signaling. Crossing of the CLE19 overexpression line with clv mutants confirms the involvement of CLV2. Analyses using tissue-specific marker lines revealed that the peptide treatments led to a premature differentiation of the ground tissue daughter cells and misspecification of cell identity in the pericycle and endodermis layers. We propose that these 14-amino acid peptides represent the major active domain of the corresponding CLE proteins, which interact with or saturate an unknown cell identity-maintaining CLV2 receptor complex in roots, leading to consumption of the root meristem.

Amino Acid Sequence↗