AGAMOUS regulates various target genes via cell cycle-coupled H3K27me3 dilution in floral meristems and stamens.

The MADS domain transcription factor AGAMOUS (AG) regulates floral meristem termination by preventing maintenance of the histone modification lysine 27 of histone H3 (H3K27me3) along the KNUCKLES (KNU) coding sequence. At 2 d after AG binding, cell division has diluted the repressive mark H3K27me3, allowing activation of KNU transcription prior to floral meristem termination. However, how many other downstream genes are temporally regulated by this intrinsic epigenetic timer and what their functions are remain unknown. Here, we identify direct AG targets regulated through cell cycle-coupled H3K27me3 dilution in Arabidopsis thaliana. Expression of the targets KNU, AT HOOK MOTIF NUCLEAR LOCALIZED PROTEIN18 (AHL18), and PLATZ10 occurred later in plants with longer H3K27me3-marked regions. We established a mathematical model to predict timing of gene expression and manipulated temporal gene expression using the H3K27me3-marked del region from the KNU coding sequence. Increasing the number of del copies delayed and reduced KNU expression in a polycomb repressive complex 2- and cell cycle-dependent manner. Furthermore, AHL18 was specifically expressed in stamens and caused developmental defects when misexpressed. Finally, AHL18 bound to genes important for stamen growth. Our results suggest that AG controls the timing of expression of various target genes via cell cycle-coupled dilution of H3K27me3 for proper floral meristem termination and stamen development.

The Arabidopsis TETRATRICOPEPTIDE-REPEAT THIOREDOXIN-LIKE (TTL) family members are involved in root system formation via their interaction with cytoskeleton and cell wall remodeling.

Lateral roots (LR) are essential components of the plant edaphic interface; contributing to water and nutrient uptake, biotic and abiotic interactions, stress survival, and plant anchorage. We have identified the TETRATRICOPEPTIDE-REPEAT THIOREDOXIN-LIKE 3 (TTL3) gene as being related to LR emergence and later development. Loss of function of TTL3 leads to a reduced number of emerged LR due to delayed development of lateral root primordia (LRP). This trait is further enhanced in the triple mutant ttl1ttl3ttl4. TTL3 interacts with microtubules and endomembranes, and is known to participate in the brassinosteroid (BR) signaling pathway. Both ttl3 and ttl1ttl3ttl4 mutants are less sensitive to BR treatment in terms of LR formation and primary root growth. The ability of TTL3 to modulate biophysical properties of the cell wall was established under restrictive conditions of hyperosmotic stress and loss of root growth recovery, which was enhanced in ttl1ttl3ttl4. Timing and spatial distribution of TTL3 expression is consistent with its role in development of LRP before their emergence and subsequent growth of LR. TTL3 emerged as a component of the root system morphogenesis regulatory network.

Structural adaptations in plants from the humid equatorial Andes indicate a trade-off between hydraulic transport efficiency and safety.

PREMISE: Environmental gradients of mountains are reflected in traits that are common to high-elevation plants worldwide. Closely related species of Senecio from the equatorial Andes grow as broad-leaved climbers in montane forests, basal broad-leaved rosette herbs in azonal marshy habitats, and ascending, narrow-leaved subshrubs in high-elevation páramo. Habitat variation along the elevational gradient enables testing whether modifications in leaf and stem functional traits among species were driven by contrasting environmental conditions. METHODS: We used quantitative analyses to describe changes in morphological and anatomical traits of leaves and stems in 10 species from various habitats. We applied univariate (linear regression, hierarchical ANOVA) and multivariate (NMDS ordination, permutational MANOVA) techniques to examine the correlation of traits with the species' habitats and elevation. RESULTS: Species from the humid and frost-free montane forest develop xylem optimized for transport efficiency by increasing the internal diameter and length of the conduits. In contrast, páramo species are optimized toward hydraulic safety by producing narrower conduits and are more likely to prevent the risk of frost-induced cavitation. Moreover, species from the high-elevation páramo habitats present a set of water-transport-related anatomical traits of leaf lamina, allowing for efficient regulation of transpiration losses. CONCLUSIONS: Morphological and anatomical traits of leaves and stems in species of Senecio inhabiting montane forests and high-elevation páramo in the equatorial Andes demonstrate a trade-off between hydraulic safety and efficiency of water transport.

At-Hook Motif Nuclear Localised Protein 18 as a Novel Modulator of Root System Architecture.

The At-Hook Motif Nuclear Localized Protein (AHL) gene family encodes embryophyte-specific nuclear proteins with DNA binding activity. They modulate gene expression and affect various developmental processes in plants. We identify AHL18 (At3G60870) as a developmental modulator of root system architecture and growth. AHL18 is involved in regulation of the length of the proliferation domain and number of dividing cells in the root apical meristem and thereby, cell production. Both primary root growth and lateral root development respond according to AHL18 transcription level. The ahl18 knock-out plants show reduced root systems due to a shorter primary root and a lower number of lateral roots. This change results from a higher number of arrested and non-developing lateral root primordia (LRP) rather than from a decreased LRP initiation. The over-expression of AHL18 results in a more extensive root system, longer primary roots, and increased density of lateral root initiation events. AHL18 is thus involved in the formation of lateral roots at both LRP initiation and their later development. We conclude that AHL18 participates in modulation of root system architecture through regulation of root apical meristem activity, lateral root initiation and emergence; these correspond well with expression pattern of AHL18.

The role of the testa during the establishment of physical dormancy in the pea seed.

BACKGROUND: A water-impermeable testa acts as a barrier to a seed's imbibition, thereby imposing dormancy. The physical and functional properties of the macrosclereids are thought to be critical determinants of dormancy; however, the mechanisms underlying the maintenance of and release from dormancy in pea are not well understood. METHODS: Seeds of six pea accessions of contrasting dormancy type were tested for their ability to imbibe and the permeability of their testa was evaluated. Release from dormancy was monitored following temperature oscillation, lipid removal and drying. Histochemical and microscopic approaches were used to characterize the structure of the testa. KEY RESULTS: The strophiole was identified as representing the major site for the entry of water into non-dormant seeds, while water entry into dormant seeds was distributed rather than localized. The major barrier for water uptake in dormant seeds was the upper section of the macrosclereids, referred to as the 'light line'. Dormancy could be released by thermocycling, dehydration or chloroform treatment. Assays based on either periodic acid or ruthenium red were used to visualize penetration through the testa. Lipids were detected within a subcuticular waxy layer in both dormant and non-dormant seeds. The waxy layer and the light line both formed at the same time as the establishment of secondary cell walls at the tip of the macrosclereids. CONCLUSIONS: The light line was identified as the major barrier to water penetration in dormant seeds. Its outer border abuts a waxy subcuticular layer, which is consistent with the suggestion that the light line represents the interface between two distinct environments - the waxy subcuticular layer and the cellulose-rich secondary cell wall. The mechanistic basis of dormancy break includes changes in the testa's lipid layer, along with the mechanical disruption induced by oscillation in temperature and by a decreased moisture content of the embryo.

Arabidopsis thaliana plants lacking the ARP2/3 complex show defects in cell wall assembly and auxin distribution.

Background and Aim: The cytoskeleton plays an important role in the synthesis of plant cell walls. Both microtubules and actin cytoskeleton are known to be involved in the morphogenesis of plant cells through their role in cell wall building. The role of ARP2/3-nucleated actin cytoskeleton in the morphogenesis of cotyledon pavement cells has been described before. Seedlings of Arabidopsis mutants lacking a functional ARP2/3 complex display specific cell wall-associated defects. Methods: In three independent Arabidopsis mutant lines lacking subunits of the ARP2/3 complex, phenotypes associated with the loss of the complex were analysed throughout plant development. Organ size and anatomy, cell wall composition, and auxin distribution were investigated. Key Results: ARP2/3-related phenotype is associated with changes in cell wall composition, and the phenotype is manifested especially in mature tissues. Cell walls of mature plants contain less cellulose and a higher amount of homogalacturonan, and display changes in cell wall lignification. Vascular bundles of mutant inflorescence stems show a changed pattern of AUX1-YFP expression. Plants lacking a functional ARP2/3 complex have decreased basipetal auxin transport. Conclusions: The results suggest that the ARP2/3 complex has a morphogenetic function related to cell wall synthesis and auxin transport.

Casparian bands and suberin lamellae in exodermis of lateral roots: an important trait of roots system response to abiotic stress factors.

Background and Aims: Root absorptive characteristics rely on the presence of apoplastic barriers. However, little is known about the establishment of these barriers within a complex root system, particularly in a major portion of them - the lateral roots. In Zea mays L., the exodermis differentiates under the influence of growth conditions. Therefore, the species presents a suitable model to elucidate the cross-talk among environmental conditions, branching pattern and the maturation of barriers within a complex root system involved in the definition of the plant-soil interface. The study describes the extent to which lateral roots differentiate apoplastic barriers in response to changeable environmental conditions. Methods: The branching, permeability of the outer cell layers and differentiation of the endo- and exodermis were studied in primary roots and various laterals under different types of stress of agronomic importance (salinity, heavy metal toxicity, hypoxia, etc.). Histochemical methods, image analysis and apoplastic tracer assays were utilized. Key Results: The results show that the impact of growth conditions on the differentiation of both the endodermis and exodermis is modulated according to the type/diameter of the root. Fine laterals clearly represent that portion of a complex root system with a less advanced state of barrier differentiation, but with substantial ability to modify exodermis differentiation in response to environmental conditions. In addition, some degree of autonomy in exodermal establishment of Casparian bands (CBs) vs. suberin lamellae (SLs) was observed, as the absence of lignified exodermal CBs did not always fit with the lack of SLs. Conclusions: This study highlights the importance of lateral roots, and provides a first look into the developmental variations of apoplastic barriers within a complex root system. It emphasizes that branching and differentiation of barriers in fine laterals may substantially modulate the root system-rhizosphere interaction.

Towards Better Understanding of Pea Seed Dormancy Using Laser Desorption/Ionization Mass Spectrometry.

Seed coats of six pea genotypes contrasting in dormancy were studied by laser desorption/ionization mass spectrometry (LDI-MS). Multivariate statistical analysis discriminated dormant and non-dormant seeds in mature dry state. Separation between dormant and non-dormant types was observed despite important markers of particular dormant genotypes differ from each other. Normalized signals of long-chain hydroxylated fatty acids (HLFA) in dormant JI64 genotype seed coats were significantly higher than in other genotypes. These compounds seem to be important markers likely influencing JI64 seed imbibition and germination. HLFA importance was supported by study of recombinant inbred lines (JI64xJI92) contrasting in dormancy but similar in other seed properties. Furthemore HLFA distribution in seed coat was studied by mass spectrometry imaging. HLFA contents in strophiole and hilum are significantly lower compared to other parts indicating their role in water uptake. Results from LDI-MS experiments are useful in understanding (physical) dormancy (first phases of germination) mechanism and properties related to food processing technologies (e.g., seed treatment by cooking).

Potassium transporter KUP9 regulates plant response to K+ deficiency and affects carbohydrate allocation in A.thaliana.

Due to the essential roles of K+ in plants, its up to 10% share in plant dry matter, and its mostly low availability in soil, effective potassium management poses a significant challenge for the plant. To enable efficient uptake and allocation of K+, numerous transporters and channels have evolved. During the last two decades, efforts have been made to characterise these transport proteins in Arabidopsis thaliana using knock-out mutants. Several KT/HAK/KUP transporters have been assigned specific functions. In this work, we contribute to an understanding of the role of AtKUP9 in plant adaptation to low K+ availability. We found that in vitro, atkup9 has reduced lateral root growth under low-K conditions, and root apical meristem proliferation is reduced in lateral roots compared with the primary root. We also documented AtKUP9 expression in both roots and shoots and showed that AtKUP9 expression is modulated during plant ontogeny and as a result of K+ deprivation. Altered carbohydrate allocation was also documented in atkup9. Mutants exported more soluble saccharides from leaves under K+ rich conditions and, under K+ deficiency, atkup9 accumulated more soluble saccharides in the shoots. A possible role of AtKUP9 in these processes is discussed.

Lateral root development in the maize (Zea mays) lateral rootless1 mutant.

BACKGROUND AND AIMS: The maize lrt1 (lateral rootless1) mutant is impaired in its development of lateral roots during early post-embryonic development. The aim of this study was to characterize, in detail, the influences that the mutation exerts on lateral root initiation and the subsequent developments, as well as to describe the behaviour of the entire plant under variable environmental conditions. METHODS: Mutant lrt1 plants were cultivated under different conditions of hydroponics, and in between sheets of moist paper. Cleared whole mounts and anatomical sections were used in combination with both selected staining procedures and histochemical tests to follow root development. Root surface permeability tests and the biochemical quantification of lignin were performed to complement the structural data. KEY RESULTS: The data presented suggest a redefinition of lrt1 function in lateral roots as a promoter of later development; however, neither the complete absence of lateral roots nor the frequency of their initiation is linked to lrt1 function. The developmental effects of lrt1 are under strong environmental influences. Mutant primordia are affected in structure, growth and emergence; and the majority of primordia terminate their growth during this last step, or shortly thereafter. The lateral roots are impaired in the maintenance of the root apical meristem. The primary root shows disturbances in the organization of both epidermal and subepidermal layers. The lrt1-related cell-wall modifications include: lignification in peripheral layers, the deposition of polyphenolic substances and a higher activity of peroxidase. CONCLUSIONS: The present study provides novel insights into the function of the lrt1 gene in root system development. The lrt1 gene participates in the spatial distribution of initiation, but not in its frequency. Later, the development of lateral roots is strongly affected. The effect of the lrt1 mutation is not as obvious in the primary root, with no influences observed on the root apical meristem structure and maintenance; however, development of the epidermis and cortex are impaired.

Effect of Aluminosilicates' Particle Size Distribution on the Microstructural and Mechanical Properties of Metakaolinite-Based Geopolymers.

The present study focused on investigating the differences in properties between calcined and milled aluminosilicates with different particle size distributions. Two types of clay, i.e., kaolin and kaolinitic claystone, were subjected to calcination at 750 °C, and subsequent milling to obtain different fractions with distinct particle size distributions. These fractions were then combined with a potassium alkaline activator and quartz sand in a 50:50 weight ratio to form a geopolymer composite. The geopolymer binders were then characterized using a mercury intrusion porosimeter (MIP), scanning electron microscopy (SEM), and a rotary rheometer. Mechanical tests were conducted on the geopolymer composites prepared from aluminosilicates with varying particle size distributions. The findings indicated that aluminosilicates with a finer particle size distribution exhibited higher levels of dissolved aluminum (10,000 mg/kg) compared to samples with coarser particle size distributions (1000 mg/kg). Additionally, as the particle size distribution decreased, the dynamic viscosity of the geopolymer binders increased, while the average pore size decreased. Finally, the mechanical properties of the geopolymer composites derived from both tested aluminosilicates demonstrated a decline in performance as the mean particle size increased beyond 10 µm.

Effect of Different Types of Aluminosilicates on the Thermo-Mechanical Properties of Metakaolinite-Based Geopolymer Composites.

In this study, the effect of different types of aluminosilicates on the thermo-mechanical properties of metakaolinite-based geopolymer binders and composites was examined. The metakaolinite-based geopolymer binders and composites were produced from three different types of aluminosilicates (one metakaolin and two calcined claystones) and a potassium alkaline activator. Chamotte was added as a filler, amounting to 65% by volume, to create geopolymer composites. Geopolymer binders were characterized by X-ray diffraction, rotary rheometer and scanning electron microscopy. The mechanical properties, thermal dilatation and thermal conductivity were investigated on geopolymer composites with three different aluminosilicates before and after exposure to high temperatures (up to 1200 °C). The results showed that the geopolymer binders prepared from calcined claystones had a lower dynamic viscosity (787 and 588 mPa·s) compared to the geopolymer binders prepared from metakaolin (1090 mPa·s). Geopolymer composites based on metakaolin had lower shrinkage (0.6%) and higher refractoriness (1520 °C) than geopolymers from calcined claystones (0.9% and 1.5%, 1500 °C and 1470 °C). Geopolymers based on calcined kaolinitic claystones are a promising material with higher compressive (95.2 and 71.5 MPa) and flexural strength (12.4 and 10.7 MPa) compared to geopolymers based on metakaolin (compressive strength 57.7 MPa).

Apical meristem exhaustion during determinate primary root growth in the moots koom 1 mutant of Arabidopsis thaliana.

An indeterminate developmental program allows plant organs to grow continuously by maintaining functional meristems over time. The molecular mechanisms involved in the maintenance of the root apical meristem are not completely understood. We have identified a new Arabidopsis thaliana mutant named moots koom 1 (mko1) that showed complete root apical meristem exhaustion of the primary root by 9 days post-germination. MKO1 is essential for maintenance of root cell proliferation. In the mutant, cell division is uncoupled from cell growth in the region corresponding to the root apical meristem. We established the sequence of cellular events that lead to meristem exhaustion in this mutant. Interestingly, the SCR and WOX5 promoters were active in the mko1 quiescent center at all developmental stages. However, during meristem exhaustion, the mutant root tip showed defects in starch accumulation in the columella and changes in auxin response pattern. Therefore, contrary to many described mutants, the determinate growth in mko1 seedlings does not appear to be a consequence of incorrect establishment or affected maintenance of the quiescent center but rather of cell proliferation defects both in stem cell niche and in the rest of the apical meristem. Our results support a model whereby the MKO1 gene plays an important role in the maintenance of the root apical meristem proliferative capacity and indeterminate root growth, which apparently acts independently of the SCR/SHR and WOX5 regulatory pathways.

Arabinogalactan Proteins in Plant Roots - An Update on Possible Functions.

Responsiveness to environmental conditions and developmental plasticity of root systems are crucial determinants of plant fitness. These processes are interconnected at a cellular level with cell wall properties and cell surface signaling, which involve arabinogalactan proteins (AGPs) as essential components. AGPs are cell-wall localized glycoproteins, often GPI-anchored, which participate in root functions at many levels. They are involved in cell expansion and differentiation, regulation of root growth, interactions with other organisms, and environmental response. Due to the complexity of cell wall functional and regulatory networks, and despite the large amount of experimental data, the exact molecular mechanisms of AGP-action are still largely unknown. This dynamically evolving field of root biology is summarized in the present review.

Effect of Potassium Phosphate Content in Aluminosilicate Matrix on Mechanical Properties of Carbon Prepreg Composites.

Six matrices based on alkali-activated aluminosilicate with different amounts of potassium phosphate were prepared for the production of six-layer composite plates. The addition of potassium phosphate in the matrix was 2 wt%, 4 wt%, 6 wt%, 8 wt% and 10 wt% of its total weight. The matrix without the potassium phosphate was also prepared. The aim of this study was to determine whether this addition has an effect on the tensile strength or Young's modulus of composites at temperatures up to 800 °C. Changes in the thickness and weight of the samples after this temperature were also monitored. Carbon plain weave fabric was chosen for the preparation of the composites. The results show that under normal conditions, the addition of potassium phosphate has no significant effect on the mechanical properties; the highest measured tensile strengths were around 350 MPa. However, at temperatures of 600 °C and 800 °C the addition of potassium phosphate had a positive effect, with the tensile strength of the composites being up to 300% higher than the composites without the addition. The highest measured values of composites after one hour at 600 °C were higher than 100 MPa and after 1 h at 800 °C higher than 85 MPa.

Effect of K/Al Molar Ratio on the Thermo-Mechanical Properties of Metakaolinite-Based Geopolymer Composites.

A metakaolinite-based geopolymer binder was prepared by using calcined claystone as the main raw material and potassium as the alkaline activator. Chamotte was added (65 vol%) to form geopolymer composites. Potassium hydroxide (KOH) was used to adjust the molar ratio of K/Al and the effect of K/Al on thermo-mechanical properties of geopolymer composites was investigated. This study aimed to analyze the effect of K/Al ratio and exposure to high temperatures (up to 1200 °C) on the compressive and flexural strengths, phase composition, pore size distribution, and thermal dilatation. With an increasing K/Al ratio, the crystallization temperature of the new phases (leucite and kalsilite) decreased. Increasing content of K/Al led to a decline in the onset temperature of the major shrinkage. The average pore size slightly increased with increasing K/Al ratio at laboratory temperature. Mechanical properties of geopolymer composites showed degradation with the increase of the K/Al ratio. The exception was the local maximum at a K/Al ratio equal to one. The results showed that the compressive strength decreases with increasing temperature. For thermal applications above 600 °C, it is better to use samples with lower K/Al ratios (0.55 or 0.70).

Arabidopsis exocyst subunits SEC8 and EXO70A1 and exocyst interactor ROH1 are involved in the localized deposition of seed coat pectin.

• Polarized deposition of cell wall pectins is a key process in Arabidopsis thaliana myxospermous seed coat development. The exocyst, an octameric secretory vesicle tethering complex, has recently been shown to be involved in the regulation of cell polarity in plants. Here, we used the Arabidopsis seed coat to study the participation of the exocyst complex in polarized pectin delivery. • We characterized the amount of pectinaceous mucilage and seed coat structure in sec8 and exo70A1 exocyst mutants. Using a yeast two-hybrid screen, we identified a new interactor of the exocyst subunit Exo70A1, termed Roh1, a member of the DUF793 protein family. • T-DNA insertions in SEC8, EXO70A1 caused considerable deviations from normal seed coat development, in particular reduced pectin deposition and defects in the formation of the central columella of seed epidermal cells. A gain-of-function mutation of ROH1 also caused reduced pectin deposition. Interestingly, we observed a systematic difference in seed coat development between primary and secondary inflorescences in wild-type plants: siliques from secondary branches produced seeds with thicker seed coats. • The participation of exocyst subunits in mucilage deposition provides direct evidence for the role of the exocyst in polarized cell wall morphogenesis.

134Cs Uptake and Growth at Various Cs+ and K+ Levels in Arabidopsis AtKUP7 Mutants.

Radiocaesium is a pollutant with a high risk for the environment, agricultural production, and human health. It is mobile in ecosystems and can be taken up by plants via potassium transporters. In this study, we focused on the role of potassium transporter AtKUP7 of the KT/HAK/KUP family in Cs+ and K+ uptake by plants and in plant tolerance to caesium toxicity. We detected that Arabidopsiskup7 mutant accumulates significantly lower amounts of 134Cs in the root (86%) and in the shoot (69%) compared to the wild-type. On the other hand ability of the mutant to grow on media with toxic (100 and 200 µM) concentrations of Cs+ was not changed; moreover its growth was not impaired on low K+. We further investigated another mutant line in AtKUP7 and found that the growth phenotype of the kup7 mutants in K+ deficient conditions is much milder than previously published. Also, their accumulation of K+ in shoots is hindered only by severe potassium shortage.

Exodermis and Endodermis Respond to Nutrient Deficiency in Nutrient-Specific and Localized Manner.

The exodermis is a common apoplastic barrier of the outer root cortex, with high environmentally-driven plasticity and a protective function. This study focused on the trade-off between the protective advantages provided by the exodermis and its disadvantageous reduction of cortical membrane surface area accessible by apoplastic route, thus limiting nutrient acquisition from the rhizosphere. We analysed the effect of nutrient deficiency (N, P, K, Mg, Ca, K, Fe) on exodermal and endodermal differentiation in maize. To differentiate systemic and localized effects, nutrient deficiencies were applied in three different approaches: to the root system as a whole, locally to discrete parts, or on one side of a single root. Our study showed that the establishment of the exodermis was enhanced in low-N and low-P plants, but delayed in low-K plants. The split-root cultivation proved that the effect is non-systemic, but locally coordinated for individual roots. Within a single root, localized deficiencies didn't result in an evenly differentiated exodermis, in contrast to other stress factors. The maturation of the endodermis responded in a similar way. In conclusion, N, P, and K deficiencies strongly modulated exodermal differentiation. The response was nutrient specific and integrated local signals of current nutrient availability from the rhizosphere.

Selected Simple Methods of Plant Cell Wall Histochemistry and Staining for Light Microscopy.

Histochemical methods allow for identification and localization of various components within the tissue. Such information on the spatial heterogeneity is not available with biochemical methods. However, there is limitation of the specificity of such detection in context of complex tissue, which is important to consider, and interpretations of the results should regard suitable control treatments if possible. Such methods are valuable extension to specific optical and spectroscopic analytical methods. Here we present a set of selected simple methods of staining and histochemical tests with comments based on our laboratory experience.