Meristematic Layer Research Articles

The identification and analysis of meristematic mutations within the apple tree that developed the RubyMac sport mutation

BackgroundUnderstanding the molecular basis of sport mutations in fruit trees has the potential to accelerate generation of improved cultivars.ResultsFor this, we analyzed the genome of the apple tree that developed the RubyMac phenotype through a sport mutation that led to the characteristic fruit coloring of this variety. Overall, we found 46 somatic mutations that distinguished the mutant and wild-type branches of the tree. In addition, we found 54 somatic gene conversions (i.e., loss-of-heterozygosity mutations) that also distinguished the two parts of the tree. Approximately 20% of the mutations were specific to individual cell lineages, suggesting that they originated from the corresponding meristematic layers. Interestingly, the de novo mutations were enriched for GC = > AT transitions while the gene conversions showed the opposite bias for AT = > GC transitions, suggesting that GC-biased gene conversions have the potential to counteract the AT-bias of de novo mutations. By comparing the gene expression patterns in fruit skins from mutant and wild-type branches, we found 56 differentially expressed genes including 18 involved in anthocyanin biosynthesis. While none of the differently expressed genes harbored a somatic mutation, we found that some of them in regions of the genome that were recently associated with natural variation in fruit coloration.ConclusionOur analysis revealed insights in the characteristics of somatic change, which not only included de novo mutations but also gene conversions. Some of these somatic changes displayed strong candidate mutations for the change in fruit coloration in RubyMac.

The vast majority of somatic mutations in plants are layer-specific

BackgroundPlant meristems are structured organs consisting of distinct layers of stem cells, which differentiate into new plant tissue. Mutations in meristematic layers can propagate into large sectors of the plant. However, the characteristics of meristematic mutations remain unclear, limiting our understanding of the genetic basis of somaclonal phenotypic variation.ResultsHere, we analyse the frequency and distribution of somatic mutations in an apricot tree. We separately sequence the epidermis (developing from meristem layer 1) and the flesh (developing from meristem layer 2) of several fruits sampled across the entire tree. We find that most somatic mutations (> 90%) are specific to individual layers. Interestingly, layer 1 shows a higher mutation load than layer 2, implying different mutational dynamics between the layers. The distribution of somatic mutations follows the branching of the tree. This suggests that somatic mutations are propagated to developing branches through axillary meristems. In turn, this leads us to the unexpected observation that the genomes of layer 1 of distant branches are more similar to each other than to the genomes of layer 2 of the same branches. Finally, using single-cell RNA sequencing, we demonstrate that layer-specific mutations were only transcribed in the cells of the respective layers and can form the genetic basis of somaclonal phenotypic variation.ConclusionsHere, we analyse the frequency and distribution of somatic mutations with meristematic origin. Our observations on the layer specificity of somatic mutations outline how they are distributed, how they propagate, and how they can impact clonally propagated crops.

Cell layer-specific expression of the homeotic MADS-box transcription factor PhDEF contributes to modular petal morphogenesis in petunia.

Floral homeotic MADS-box transcription factors ensure the correct morphogenesis of floral organs, which are organized in different cell layers deriving from distinct meristematic layers. How cells from these distinct layers acquire their respective identities and coordinate their growth to ensure normal floral organ morphogenesis is unresolved. Here, we studied petunia (Petunia × hybrida) petals that form a limb and tube through congenital fusion. We identified petunia mutants (periclinal chimeras) expressing the B-class MADS-box gene DEFICIENS in the petal epidermis or in the petal mesophyll, called wico and star, respectively. Strikingly, wico flowers form a strongly reduced tube while their limbs are almost normal, while star flowers form a normal tube but greatly reduced and unpigmented limbs, showing that petunia petal morphogenesis is highly modular. These mutants highlight the layer-specific roles of PhDEF during petal development. We explored the link between PhDEF and petal pigmentation, a well-characterized limb epidermal trait. The anthocyanin biosynthesis pathway was strongly downregulated in star petals, including its major regulator ANTHOCYANIN2 (AN2). We established that PhDEF directly binds to the AN2 terminator in vitro and in vivo, suggesting that PhDEF might regulate AN2 expression and therefore petal epidermis pigmentation. Altogether, we show that cell layer-specific homeotic activity in petunia petals differently impacts tube and limb development, revealing the relative importance of the different cell layers in the modular architecture of petunia petals.

Chimeras in Merlot grapevine revealed by phased assembly

Chimerism is the phenomenon when several genotypes coexist in a single individual. Used to understand plant ontogenesis they also have been valorised through new cultivar breeding. Viticulture has been taking economic advantage out of chimeras when the variant induced an important modification of wine type such as berry skin colour. Crucial agronomic characters may also be impacted by chimeras that aren’t identified yet. Periclinal chimera where the variant has entirely colonised a cell layer is the most stable and can be propagated through cuttings. In grapevine, leaves are derived from both meristem layers, L1 and L2. However, lateral roots are formed from the L2 cell layer only. Thus, comparing DNA sequences of roots and leaves allows chimera detection. In this study we used new generation Hifi long reads sequencing, recent bioinformatics tools and trio-binning with parental sequences to detect periclinal chimeras on ‘Merlot’ grapevine cultivar. Sequencing of cv. ‘Magdeleine Noire des Charentes’ and ‘Cabernet Franc’, the parents of cv. ‘Merlot’, allowed haplotype resolved assembly. Pseudomolecules were built with a total of 33 to 47 contigs and in few occasions a unique contig for one chromosome. This high resolution allowed haplotype comparison. Annotation was transferred from PN40024 VCost.v3 to all pseudomolecules. After strong selection of variants, 51 and 53 ‘Merlot’ specific periclinal chimeras were found on the Merlot-haplotype-CF and Merlot-haplotype-MG respectively, 9 and 7 been located in a coding region. A subset of positions was analysed using Molecular Inversion Probes (MIPseq) and 69% were unambiguously validated, 25% are doubtful because of technological noise or weak depth and 6% invalidated. These results open new perspectives on chimera detection as an important resource to improve cultivars through clonal selection or breeding.

The lipid-binding START domain regulates the dimerization of ATML1 via modulating the ZIP motif activity in Arabidopsis thaliana.

In Arabidopsis thaliana, the epidermis is the outermost cell layer composed of many specialized types of epidermal cells, such as pavement cells, trichomes, and guard cells. The homeodomain-leucine zipper (HD-ZIP) class Ⅳ transcription factors (TFs), which are unique to the plant kingdom, have been recognized as key regulators of epidermis development. Unlike animal HD proteins, which can bind to DNA as monomers, plant HD-ZIP class Ⅳ TFs bind to DNA as dimers, although little is known about the regulation of their dimerization process. Here, we show that the homodimerization of ARABIDOPSIS THALIANA MERISTEM LAYER 1 (ATML1) - HD-ZIP class Ⅳ TF that is required for protoderm development - is regulated by the lipid-binding steroidogenic acute regulatory protein-related lipid transfer (START) domain. We found that ATML1 forms homodimer through interaction via its ZIP motif in yeast and plant cells, although the interaction is abolished by generating a mutation into the lipid-binding START domain to disrupt the lipid-binding ability. These results suggest that lipidic ligands function as key regulators of protoderm development via modulating the dimerization of ATML1.

Leaf Epidermis: The Ambiguous Symplastic Domain.

The ability to develop secondary (post-cytokinetic) plasmodesmata (PD) is an important evolutionary advantage that helps in creating symplastic domains within the plant body. Developmental regulation of secondary PD formation is not completely understood. In flowering plants, secondary PD occur exclusively between cells from different lineages, e.g., at the L1/L2 interface within shoot apices, or between leaf epidermis (L1-derivative), and mesophyll (L2-derivative). However, the highest numbers of secondary PD occur in the minor veins of leaf between bundle sheath cells and phloem companion cells in a group of plant species designated “symplastic” phloem loaders, as opposed to “apoplastic” loaders. This poses a question of whether secondary PD formation is upregulated in general in symplastic loaders. Distribution of PD in leaves and in shoot apices of two symplastic phloem loaders, Alonsoa meridionalis and Asarina barclaiana, was compared with that in two apoplastic loaders, Solanum tuberosum (potato) and Hordeum vulgare (barley), using immunolabeling of the PD-specific proteins and transmission electron microscopy (TEM), respectively. Single-cell sampling was performed to correlate sugar allocation between leaf epidermis and mesophyll to PD abundance. Although the distribution of PD in the leaf lamina (except within the vascular tissues) and in the meristem layers was similar in all species examined, far fewer PD were found at the epidermis/epidermis and mesophyll/epidermis boundaries in apoplastic loaders compared to symplastic loaders. In the latter, the leaf epidermis accumulated sugar, suggesting sugar import from the mesophyll via PD. Thus, leaf epidermis and mesophyll might represent a single symplastic domain in Alonsoa meridionalis and Asarina barclaiana.

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.

TERMINAL FLOWER1 Functions as a Mobile Transcriptional Cofactor in the Shoot Apical Meristem.

The floral transition is a critical step in the life cycle of flowering plants, and several mechanisms control this finely orchestrated process. TERMINAL FLOWER1 (TFL1) is a floral repressor and close relative of the florigen, FLOWERING LOCUS T (FT). During the floral transition, TFL1 expression is up-regulated in the inflorescence apex to maintain the indeterminate growth of the shoot apical meristem (SAM). Both TFL1 and FT are mobile proteins, but they move in different ways. FT moves from the leaves to the SAM, while TFL1 appears to move within the SAM. The importance of TFL1 movement for its function in the regulation of flowering time and shoot indeterminacy and its molecular function are still largely unclear. Our results using Arabidopsis (Arabidopsis thaliana) indicate that TFL1 moves from its place of expression in the center of the SAM to the meristem layer L1 and that the movement in the SAM is required for the regulation of the floral transition. Chromatin immunoprecipitation sequencing and RNA sequencing demonstrated that TFL1 functions as a cotranscription factor that associates with and regulates the expression of hundreds of genes. These newly identified direct TFL1 targets provide the possibility to discover new roles for TFL1 in the regulation of floral transition and inflorescence development.

Morphological and Structural Characters of Trichomes on Various Organs of Rosa roxburghii

Rosa roxburghii Tratt (Rosaceae) of various organ surfaces are widely existing trichomes. Certain varieties have fruits that are thickly covered with macroscopic trichomes. R. roxburghii Tratt (RR) and R. roxburghii Tratt. f. esetosa Ku (RRE) are important commercial horticultural crops in China because of their nutritional and medicinal values. RRE is generally considered a smooth-fruit variant that arose from RR. Despite their economic importance, the morphological and anatomic features of organ trichomes have not been explored in detail for these two rose germplasms. In this research, we investigated the distribution, morphology, and structure of trichomes distributed on the stem, pedicel, fruit, sepal, and marginal lobule sepals (MLS) of RR as well as RRE. This was accomplished using scanning electron microscopy (SEM). There are various shapes of trichomes distributed on the surfaces of stems, pedicels, fruits, and sepals of the two germplasms. Binate prickles arose on the stem nodes in both germplasms, but acicular trichomes, papillary trichomes, and ribbon trichomes were present only on the surfaces of pedicels in RR. Likewise, flagelliform trichomes were present only on the surfaces of pedicels in RRE. Furthermore, a transection of stems shows that thorns in the two germplasms are composed of epidermis, meristematic layer, and parenchyma cells. The trichome epidermis and meristematic layer in stems of RR are composed of round cells, whereas RRE exhibits square cells in the same layers. Trichomes on the fruit of RR were macroscopic and of single flagelliform and acicular shape. RRE exhibited polymorphic trichomes of flagelliform, triangular, capitate glandular, and elliptic glandular shapes on the pericarp. On the surfaces of RR sepals, there are thick macroscopic acicular trichomes. In contrast, RRE sepals presented flagelliform trichomes and capitate glandular trichomes. It is interesting that no trichomes were found on the surfaces of the MLS in the two germplasms; however, stomata were densely packed on the MLS of RRE when compared with RR. For RR, the trichomes on both sepal and fruit are composed of an epidermis layer and parenchyma cells; however, the epidermis cells of sepal trichomes are polygon-shaped, in contrast to the round epidermis cells in fruit. These results suggest that the two rose germplasms are good candidates for understanding the trichome ontogeny in the genus and for further breeding of the smooth organ trait in this rose species.

HAIRY MERISTEM with WUSCHEL confines CLAVATA3 expression to the outer apical meristem layers.

The control of the location and activity of stem cells depends on spatial regulation of gene activities in the stem cell niche. Using computational and experimental approaches, we have tested and found support for a hypothesis for gene interactions that specify the Arabidopsis apical stem cell population. The hypothesis explains how the WUSCHEL gene product, synthesized basally in the meristem, induces CLAVATA3-expressing stem cells in the meristem apex but, paradoxically, not in the basal domain where WUSCHEL itself is expressed. The answer involves the activity of the small family of HAIRY MERISTEM genes, which prevent the activation of CLAVATA3 and which are expressed basally in the shoot meristem.

A Rice Glutamyl-tRNA Synthetase Modulates Early Anther Cell Division and Patterning.

Aminoacyl-tRNA synthetases (aaRSs) have housekeeping roles in protein synthesis, but little is known about how these aaRSs are involved in organ development. Here, we report that a rice (Oryza sativa) glutamyl-tRNA synthetase (OsERS1) maintains proper somatic cell organization and limits the overproliferation of male germ cells during early anther development. The expression of OsERS1 is specifically detectable in meristematic layer 2-derived cells of the early anther, and osers1 anthers exhibit overproliferation and disorganization of layer 2-derived cells, producing fused lobes and extra germ cells in early anthers. The conserved biochemical function of OsERS1 in ligating glutamate to tRNAGlu is enhanced by its cofactor aaRS OsARC. Furthermore, metabolomics profiling revealed that OsERS1 is an important node for multiple metabolic pathways, indicated by the accumulation of amino acids and tricarboxylic acid cycle components in osers1 anthers. Notably, the anther defects of the osers1 mutant are causally associated with the abnormal accumulation of hydrogen peroxide, which can reconstitute the osers1 phenotype when applied to wild-type anthers. Collectively, these findings demonstrate how aaRSs affect male organ development in plants, likely through protein synthesis, metabolic homeostasis, and redox status.

The Arabidopsis thaliana NGATHA1 transcription factor acts as a promoter of a general differentiation program and a carpel identity factor

The Arabidopsis thaliana NGATHA (NGA) family is a four-membered subclade of the B3-type transcription factor superfamily, and plays an essential role in the style development. NGAs also act as negative regulators of cell proliferation in leaf and petal growth. The notion that NGAs might act as promoters of a general differentiation program has been proposed to explain the involvement of NGAs in these seemingly discrete functions. Here, we tested this notion by activating NGA1 transcription in the expression domains of Arabidopsis thaliana Meristem Layer 1, AINTEGUMENTA, SHOOTMERISTEMLESS, CLAVATA3 (CLV3), and WUSCHEL (WUS), where cells remain mostly undifferentiated and pluripotent. We found that those specific cells expressing NGA1 lost their meristematic competence, becoming terminally differentiated. We also found that the inflorescence meristems of the plants expressing NGA1 in the CLV3 domain failed to develop floral meristems and differentiated into a terminal carpeloid cylinder without any other floral organs; similarly, the floral meristems of the plants expressing NGA1 in the WUS domain all developed into carpeloid cylinders with no other floral organs. Our results not only support the notion that NGA1 seems to be a promoter of a general differentiation program, but also raise the possibility that NGA1 may act as a carpel identity factor.

HISTOLOGICAL ANALYSIS OF In Vitro CULTURED COCONUT ENDOSPERM

Coconut is a very important plant for the livelihood of people in tropical countries. It is also used as an icon of tropical region. Coconut fruit is very heavy and can cause injuries if the fruit falls down and hits somebody who happens to be underneath a coconut tree. In order to avoid the accident,  the coconut fruits have to be regularly cut off. Coconut tree originated from in vitro cultured endosperm is a triploid plant that produces seedless fruit (without endocarp). Coconut fruit without endocarp is not heavy. The objective of this study was to investigate plant regeneration of fresh and in vitro cultured coconut endosperms. The fresh and developed in vitro cultured coconut endosperms were observed using histological analysis. Solid endosperm of seven month-old postanthesis coconut from “Samoan Dwarf” cultivar was freshly picked up and cultured in vitro on modified Branton & Blake formula. Histological study of fresh coconut endosperm showed that the endosperm consisted of parenchyma cells, which were relatively uniform in shape and size, with some nuclei consisted of 1 – 5 nucleoli. Three month-old calli of in vitro grown coconut endosperm in semi solid media showed that its cells varied in shape and size, characterized by high nucleus to cytoplasm ratio, high starch, protein and lipid contents which underwent many divisions. Seven month-old calli of in vitro grown coconut endosperm in liquid media showed embryogenic cells which resembled proembryos. Fourteen month-old bud-like structure of coconut endosperm in semi solid media showed a meristematic layer, tunica-corpus structure, cortex-like region and tracheids of xylem. These results indicated that the bud-like structure was an early stage of shoot bud formation in coconut endosperm. This is the first report of early stage of shoot bud formation occurring on coconut endosperm cultured in vitro.

The AP2-type transcription factors DORNRÖSCHEN and DORNRÖSCHEN-LIKE promote G1/S transition

The paralogous genes DORNRÖSCHEN (DRN) and DORNRÖSCHEN-LIKE (DRNL) encode AP2-type transcription factors that are expressed and act cell-autonomously in the central stem-cell zone or lateral organ founder cells (LOFCs) in the peripheral zone of the Arabidopsis shoot meristem (SAM), but their molecular contribution is unknown. Here, we show using the Arabidopsis thaliana MERISTEM LAYER 1 promoter that DRN and DRNL share a common function in cell cycle progression and potentially provide local competence for G1-S transitions in the SAM. Analysis of double transgenic DRN::erGFP and DRNL::erCERULEAN promoter fusion lines suggests that the trajectory of this cellular competence starts with DRN activity in the central stem-cell zone and extends locally via DRNL activity into groups of founder cells at the IM or FM periphery. Our data support the scenario that after gene duplication, DRN and DRNL acquired different transcription domains within the shoot meristem, but retained protein function that affects cell cycle progression, either centrally in stem cells or peripherally in primordial founder cells, a finding that is of general relevance for meristem function.

The phenotype of the CRINKLY4 deletion mutant of Physcomitrella patens suggests a broad role in developmental regulation in early land plants.

Deletion of the ancestral gene of the land plant multigene family of receptor like kinase CR4 in Physcomitrella patens demonstrates involvement in developmental control of gametophytic and sporophytic organs. The CRINKLY4 (CR4) family of receptor kinases in angiosperms consists of three clades, one including CR4, the CR4-related CCR1 and CCR2, a second including CCR3 and CCR4 family members, and a third and more distant clade. In addition to crinkly leaves in maize, which gave rise to the mutant gene name, CR4 is implicated in ovule, embryo, flower and root development in Arabidopsis thaliana. In root tips of the same species the module including a CLAVATA3/ESR-related protein, an Arabidopsis CR4, a CLAVATA1 and a WUSCHEL-related homeobox 5 (CLE40-ACR4-CLV1-WOX5) is implicated in meristem cell regulation. In embryos and shoots, CR4 acts together with A. thaliana MERISTEM LAYER 1 and PROTODERMAL FACTOR 2 to promote A. thaliana epidermis differentiation. Phylogenetic analysis has demonstrated that early land plants, e.g. mosses carry a single ancestral CR4 gene, together with genes encoding the other members of the CLE40-ACR4-CLV1-WOX5 signaling module. Here we show that CR4 serves as a broad regulator of morphogenesis both in gametophyte phyllids, archegonia and in sporophyte epidermis of the moss Physcomitrella patens. The phenotype of the CR4 deletion mutant in moss provides insight into the role of the ancestral CR4 gene as a regulator of development in early land plants.

Induction of somatic embryogenesis in explants of shoot cultures established from adult Eucalyptus globulus and E. saligna × E. maidenii trees.

A reproducible procedure for induction of somatic embryogenesis (SE) from adult trees of Eucalyptus globulus Labill. and the hybrid E. saligna Smith × E. maidenii has been developed for the first time. Somatic embryos were obtained from both shoot apex and leaf explants of all three genotypes evaluated, although embryogenic frequencies were significantly influenced by the species/genotype, auxin and explant type. Picloram was more efficient for somatic embryo induction than naphthaleneacetic acid (NAA), with the highest frequency of induction being obtained in Murashige and Skoog medium containing 40 µM picloram and 40 mg l(-1) gum Arabic, in which 64% of the shoot apex explants and 68.8% of the leaf explants yielded somatic embryos. The embryogenic response of the hybrid was higher than that of the E. globulus, especially when NAA was used. The cultures initiated on picloram-containing medium consisted of nodular embryogenic structures surrounded by a mucilaginous coating layer that emerged from a watery callus developed from the initial explants. Cotyledonary somatic embryos were differentiated after subculture of these nodular embryogenic structures on a medium lacking plant growth regulators. Histological analysis confirmed the bipolar organization of the somatic embryos, with shoot and root meristems and closed procambial tissue that bifurcated into small cotyledons. The root pole was more differentiated than the shoot pole, which appeared to be formed by a few meristematic layers. Maintenance of the embryogenic lines by secondary SE was attained by subculturing individual cotyledonary embryos or small clusters of globular and torpedo embryos on medium with 16.11 µM NAA at 4- to 5-week intervals. Somatic embryos converted into plantlets after being transferred to liquid germination medium although plant regeneration remained poor.

Proper selection of [formula omitted] controls in simulated microgravity research as illustrated with clinorotated plant cell suspension cultures

Understanding the physical and biological effects of the absence of gravity is necessary to conduct operations on space environments. It has been previously shown that the microgravity environment induces the dissociation of cell proliferation from cell growth in young seedling root meristems, but this source material is limited to few cells in each row of meristematic layers. Plant cell cultures, composed by a large and homogeneous population of proliferating cells, are an ideal model to study the effects of altered gravity on cellular mechanisms regulating cell proliferation and associated cell growth. Cell suspension cultures of Arabidopsis thaliana cell line (MM2d) were exposed to 2D-clinorotation in a pipette clinostat for 3.5 or 14 h, respectively, and were then processed either by quick freezing, to be used in flow cytometry, or by chemical fixation, for microscopy techniques. After long-term clinorotation, the proportion of cells in G1 phase was increased and the nucleolus area, as revealed by immunofluorescence staining with anti-nucleolin, was decreased. Despite the compatibility of these results with those obtained in real microgravity on seedling meristems, we provide a technical discussion in the context of clinorotation and proper 1 g controls with respect to suspension cultures. Standard 1 g procedure of sustaining the cell suspension is achieved by continuously shaking. Thus, we compare the mechanical forces acting on cells in clinorotated samples, in a control static sample and in the standard 1 g conditions of suspension cultures in order to define the conditions of a complete and reliable experiment in simulated microgravity with corresponding 1 g controls.

The over-expression of two transcription factors, ABS5/bHLH30 and ABS7/MYB101, leads to upwardly curly leaves.

Proper leaf development is essential for plant growth and development, and leaf morphogenesis is under the control of intricate networks of genetic and environmental cues. We are interested in dissecting these regulatory circuits genetically and report here the isolation of two Arabidopsis dominant mutants, abnormal shoot5-1D (abs5-1D) and abs7-1D identified through activation tagging screens. Both abs5-1D and abs7-1D display an intriguing upwardly curly leaf phenotype. Molecular cloning showed that the elevated expression of a bHLH transcription factor ABS5/T5L1/bHLH30 or a MYB transcription factor ABS7/MYB101 is the cause for the abnormal leaf phenotypes found in abs5-1D or abs7-1D, respectively. Protoplast transient expression assays confirmed that both ABS5/T5L1 and ABS7/MYB101 are targeted to the nucleus. Interestingly, the expression domains of auxin response reporter DR5::GUS were abnormal in leaves of abs5-1D and ABS5/T5L1 over-expression lines. Moreover, cotyledon venation analysis showed that more areoles and free-ending veins are formed in abs5-1D. We found that the epidermis-specific expressions of ABS5/T5L1 or ABS7/MYB101 driven by the Arabidopsis Meristem Layer 1 promoter (PAtML1) were sufficient to recapitulate the curly leaf phenotype of abs5-1D or abs7-1D. In addition, PAtML1::ABS5 lines exhibited similar changes in DR5::GUS expression patterns as those found in 35S-driven ABS5/T5L1 over-expression lines. Our work demonstrated that enhanced expressions of two transcription factors, ABS5/T5L1 and ABS7/MYB101, are able to alter leaf lamina development and reinforce the notion that leaf epidermis plays critical roles in regulating plant organ morphogenesis.

Post-Embryonic Induction of ATML1-SRDX Alters the Morphology of Seedlings

Arabidopsis thaliana MERISTEM LAYER 1 (ATML1), an HD-ZIP class IV homeobox gene, is one of the key regulators promoting epidermal cell differentiation in Arabidopsis thaliana. We recently showed that ATML1 was able to confer an ectopic shoot epidermis cell fate to non-epidermal tissues of seedlings, suggesting that ATML1 is a master regulator of epidermal cell fate. To further assess the roles of ATML1 and its homologs in epidermal cell differentiation, I generated transgenic plants expressing ATML1 fused with a transcriptional repressor sequence (ATML1-SRDX). Estradiol-induced expression of ATML1-SRDX in the seedlings decreased transcript levels of several epidermis-related genes. Moreover, these transgenic plants exhibited phenotypes such as increased permeability to a hydrophilic dye and fusion of leaves and cotyledons, which are reminiscent of epidermis and/or cuticle-deficient mutants. Epidermal cell morphology was severely affected in the strong lines: filamentous protrusions were formed on the surface of the cotyledons. Marker gene analyses showed that these protrusions did not have epidermis, mesophyll, root hair, or trichome cell identity, suggesting that post-embryonic expression of ATML1-SRDX was sufficient to alter cell identity in pre-existing protodermal cells of the cotyledons. Taken together, these results suggest that ATML1 and/or its target genes are not only necessary for the initial specification of epidermal cell fate but also may be necessary for the maintenance of epidermal cells in later stages.

THE PRODUCTION OF CAULIFLOWER MICROSHOOTS USING CURD MERISTEMATIC TISSUES AND HYPOCOTYL-DERIVED CALLUS

The capacity for microshoot production from cauliflower was investigated applying two different protocols. In the first, cauliflower curd meristematic tissue was used as a source of explants. The meristematic layer was shaved off and the clusters produced were homogenised using a commercial blender. In terms of its effect on the number and viability of microshoots, the use of 30 s blending duration treatment was found to be optimal between several treatments tested in the range 15 to 120 s. Explants were cultivated in agitated S23 (MS (Murashige and Skoog, 1962) + 3% sucrose) liquid media supplemented with different combinations of plant growth regulators. The use of 2 mg/L kinetin and 1 mg/L IBA gave the optimal results in terms of the number and viability of microshoots. The second protocol was designed to investigate the regeneration potential of hypocotyl explants of cauliflower via callus culture. The callus tissue was initiated from hypocotyl explants in callus induction medium (CIM), which consisted of S23 supplemented with 2,4-D at 1 mg/L and kinetin at 1.5 mg/L. The highest number of shoots was obtained after 28 days from sub-cultured hypocotyl derived callus on S23 basal media containing 0.5 mg/L of kinetin. This study demonstrated the ability of producing microshoots using various parts of cauliflower through both callus and without callus formation, which can be useful in the later applications of cauliflower tissue culture such as the production of artificial seeds.