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Plant cuticles are composed of wax and cutin, and evolved in the land plants as a hydrophobic boundary that reduces water loss from the plant epidermis. The expanding maize adult leaf displays a dynamic, proximodistal gradient of cuticle development, from the leaf base to the tip. Laser microdissection RNA Sequencing (LM-RNAseq) was performed along this proximodistal gradient, and complementary network analyses identified potential regulators of cuticle biosynthesis and deposition. Correlations between cuticle development and cell wall biosynthesis processes were identified, as well as evidence of roles for auxin and brassinosteroids. In addition, our network analyses suggested a previously undescribed function for PHYTOCHROME-mediated light signaling during cuticular wax deposition. Genetic analyses reveal that the phyB1 phyB2 double mutant of maize exhibits abnormal cuticle composition, supporting predictions of our coexpression analyses. Reverse genetic analyses also show that phy mutants of the moss Physcomitrella patens exhibit abnormal cuticle composition, suggesting a role for light-stimulated development of cuticular waxes during plant evolution.

Although extensive prior work has characterized cuticle composition, function, ultrastructure and development in many plant species, much remains to be learned about how these features are interrelated. Moreover, very little is known about the adult maize leaf cuticle in spite of its significance for agronomically important traits in this major crop. We analyzed cuticle composition, ultrastructure, and permeability along the developmental gradient of partially expanded adult maize leaves to probe the relationships between these features. The water barrier property is acquired at the cessation of cell expansion. Wax types and chain lengths accumulate asynchronously along the developmental gradient, while overall wax load does not vary. Cutin begins to accumulate prior to establishment of the water barrier and continues thereafter. Ultrastructurally, pavement cell cuticles consist of an epicuticular layer, a thin cuticle proper that acquires an inner, osmiophilic layer during development, and no cuticular layer. Cuticular waxes of the adult maize leaf are dominated by alkanes and wax esters localized mainly in the epicuticular layer. Establishment of the water barrier coincides with a switch from alkanes to esters as the major wax type, and the emergence of an osmiophilic (likely cutin-rich) layer of the cuticle proper.Higlight statementChemical, ultrastructural and functional analysis of cuticle development in partially expanded adult maize leaves revealed important roles for wax esters and an osmiophilic, likely cutin-rich, layer in protection from dehydration.

A comprehensive knowledge of proteomic states is essential for understanding biological systems. Using mass spectrometry, we mapped an atlas of developing maize seed proteotypes comprising 14,165 proteins and 18,405 phosphopeptides (from 4,511 proteins), quantified across eight tissues. We found that many of the most abundant proteins are not associated with detectable levels of their mRNAs, and we provide evidence for three potential explanations: transport of proteins between tissues; diurnal, out-of-phase accumulation of mRNAs and cognate proteins; and differential lifetimes of mRNAs compared with proteins. Likewise, many of the most abundant mRNAs were not associated with detectable levels of their proteins. Across the entire dataset, protein abundance was poorly correlated with mRNA levels and was largely independent of phosphorylation status. Comparisons between proteotypes revealed the quantitative contribution of specific proteins and phosphorylation events to the spatially and temporally regulated starch and oil biosynthetic pathways. Reconstruction of signaling networks established associations of proteins and phosphoproteins with distinct biological processes acting during seed development. Additionally, a protein kinase substrate network was reconstructed, enabling the identification of 762 potential substrates of specific protein kinases. Finally, examination of 694 transcription factors revealed remarkable constraints on patterns of expression and phosphorylation within transcription factor families. These results provide a resource for understanding seed development in a crop that is the foundation of modern agriculture.

Previous work has shown that the 0.02-0.05% of adult mouse bone marrow cells that bear the cell surface phenotype Thy-1loLin-Sca-1+ are enriched 1000- to 2000-fold for hematopoietic stem-cell activity in a variety of assays. When 50-100 cells of this phenotype are injected into an irradiated animal, they can permanently repopulate the entire hematopoietic system. In the present study, limiting-dilution and single-cell experiments were used to address the question of how individual Thy-1loLin-Sca-1+ stem cells contribute to repopulation of the hematopoietic system following irradiation. We calculated that 1 of 13 Thy-1loLin-Sca-1+ cells formed a clone comprising greater than 1% of peripheral white blood cells 3-7 weeks after injection. The majority of these clones included both lymphoid and myeloid lineages. Approximately one-third of the clones continued to produce new blood cells for 9 weeks or more, but the remainder disappeared earlier, including many that were multilineage. Thus, while the majority of Thy-1loLin-Sca-1+ bone marrow cells whose progeny are detected in the in vivo repopulation assay are pluripotential, only a subset undergo long-term self-renewal in vivo. Repopulation appears to be oligoclonal when limiting numbers of Thy-1loLin-Sca-1+ cells are injected. However, the number of clones contributing to hematopoiesis increases in proportion to the number of Thy-1loLin-Sca-1+ cells injected, bringing into question the notion that steady-state hematopoiesis in normal individuals is oligoclonal.

Summary Background In premitotic plant cells, the future division plane is predicted by a cortical ring of microtubules and F-actin called the preprophase band (PPB). The PPB persists throughout prophase, but is disassembled upon nuclear-envelope breakdown as the mitotic spindle forms. Following nuclear division, a cytokinetic phragmoplast forms between the daughter nuclei and expands laterally to attach the new cell wall at the former PPB site. A variety of observations suggest that expanding phragmoplasts are actively guided to the former PPB site, but little is known about how plant cells "remember" this site after PPB disassembly. Results In premitotic plant cells, Arabidopsis TANGLED fused to YFP (AtTAN::YFP) colocalizes at the future division plane with PPBs. Strikingly, cortical AtTAN::YFP rings persist after PPB disassembly, marking the division plane throughout mitosis and cytokinesis. The AtTAN::YFP ring is relatively broad during preprophase/prophase and mitosis; narrows to become a sharper, more punctate ring during cytokinesis; and then rapidly disassembles upon completion of cytokinesis. The initial recruitment of AtTAN::YFP to the division plane requires microtubules and the kinesins POK1 and POK2, but subsequent maintenance of AtTAN::YFP rings appears to be microtubule independent. Consistent with the localization data, analysis of Arabidopsis tan mutants shows that AtTAN plays a role in guidance of expanding phragmoplasts to the former PPB site. Conclusions AtTAN is implicated as a component of a cortical guidance cue that remains behind when the PPB is disassembled and directs the expanding phragmoplast to the former PPB site during cytokinesis.

SummaryIn plant cells, the plane of division is anticipated at the onset of mitosis by the presence of a preprophase band (PPB) of microtubules and F-actin at a cortical site that circumscribes the nucleus. During cytokinesis, the microtubule- and F-actin-based phragmoplast facilitates construction of a new cell wall and is guided to the forecast division site. Proper execution of this process is essential for establishing the cellular framework of plant tissues. The microtubule binding protein TANGLED1 (TAN1) of maize is a key player in the determination of division planes [1–3]. Lack of TAN1 leads to misguided phragmoplasts and mispositioned cell walls in maize. In a yeast two-hybrid screen for TAN1-interacting proteins, a pair of related kinesins was identified that shares significant sequence homology with two kinesin-12 genes in Arabidopsis thaliana (A. thaliana): PHRAGMOPLAST ORIENTING KINESIN 1 and 2 (POK1, POK2). POK1 and POK2 are expressed in tissues enriched for dividing cells. The phenotype of pok1;pok2 double mutants strongly resembles that of maize tan1 mutants, characterized by misoriented mitotic cytoskeletal arrays and misplaced cell walls. We propose that POK1 and POK2 participate in the spatial control of cytokinesis, perhaps via an interaction with the A. thaliana TAN1 homolog, ATN.

Plant cell shapes are defined by their surrounding walls, but microtubules and F-actin both play critical roles in cell morphogenesis by guiding the deposition of wall materials in expanding cells [1–7]. Leaf epidermal cells have lobed shapes, which are thought to arise through a microtubule-dependent pattern of locally polarized growth [8–13]. We have isolated a recessive mutation, brk1, which blocks the formation of epidermal cell lobes in the maize leaf. Mutant epidermal cells expand to the same extent as wild-type cells but fail to establish polar growth sites from which lobes arise. In expanding brk1 epidermal cells, microtubule organization differs little from that in wild-type, but localized enrichments of cortical F-actin seen at the tips of emerging lobes in wild-type cells fail to form. These observations suggest a critical role for F-actin in lobe formation and together with additional effects of brk1 on the morphogenesis of stomata and hairs suggest that Brk1 promotes multiple, actin-dependent cell polarization events in the developing leaf epidermis. The Brk1 gene encodes a novel, 8 kD protein that is highly conserved in plants and animals, suggesting that BRK1-related proteins may function in actin-dependent aspects of cell polarization in a wide spectrum of eukaryotic organisms.

Cytokinesis in plant cells involves a microtubule-containing structure, the phragmoplast, which guides the formation of new cell walls. Recent studies have identified kinesin-like proteins that appear to play a variety of roles in plant cytokinesis.

Background A new disease class of syndromes, described as linkeropathies, which are derived from defects in the glycosaminoglycan-linker region as well as glycosaminoglycan-side chains of proteoglycans is increasingly being recognized as a cause of human disease. Proteoglycans are an essential component of the extracellular matrix. Defects in the enzymatic process of proteoglycan synthesis broadly occur due to the incorrect addition of side chains. Previously, homozygous missense variants within the B3GAT3 gene encoding beta 1,3 glucuronyltransferase 3(GlcAT-I) responsible for the biosynthesis of glycosaminoglycans have been described in 7 individuals. Case presentation In this study, a 4-year-old patient with a severe phenotype of osteoporosis, hypotonia, joint laxity, fractures, scoliosis, biscuspid aortic valve and myopia was referred for next generation sequencing after extensive negative clinical testing. Whole exome sequencing was performed on the proband and his unaffected parents to identify the molecular basis of his disease. Sequencing revealed compound heterozygous variants in B3GAT3: c.1A > G (p.Met1?) and c.671 T > A (p.L224Q). Clinical and in vitro functional studies were then completed to verify the pathogenicity of the genotype and further characterize the functional basis of the patient’s disease demonstrating the patient had a decrease both in the protein level of B3GAT3 and in the glucuronyltransferase activity when compared to control samples. Independent in vitro assessment of each variant confirmed the B3GAT3: c.1A > G (p.Met1?) variant is functionally null and the c.671 T > A (p.L224Q) missense variant has significantly reduced glucuronyltransferase activity (~3% of control). Conclusions This is the first report of a patient with compound heterozygosity for a null variant in trans with a missense in B3GAT3 resulting in a severe phenotype, expanding both the genotypic and phenotypic spectrum of B3GAT3-related disease. Electronic supplementary material The online version of this article (doi:10.1186/s12881-016-0344-9) contains supplementary material, which is available to authorized users.