Potent inhibition of TCP transcription factors by miR319 ensures proper root growth in Arabidopsis.

KEY MESSAGE: Proper root growth depends on the clearance of TCP transcripts from the root apical meristem by microRNA miR319. The evolutionarily conserved microRNA miR319 regulates genes encoding TCP transcription factors in angiosperms. The miR319-TCP module controls cell proliferation and differentiation in leaves and other aerial organs. The current model sustains that miR319 quantitatively tunes TCP activity during leaf growth and development, ultimately affecting its size. In this work we studied how this module participates in Arabidopsis root development. We found that misregulation of TCP activity through impairment of miR319 binding decreased root meristem size and root length. Cellular and molecular analyses revealed that high TCP activity affects cell number and cyclin expression but not mature cell length, indicating that, in roots, unchecking the expression of miR319-regulated TCPs significantly affects cell proliferation. Conversely, tcp multiple mutants showed no obvious effect on root growth, but strong defects in leaf morphogenesis. Therefore, in contrast to the quantitative regulation of the TCPs by miR319 in leaves, our data suggest that miR319 clears TCP transcripts from root cells. Hence, we provide new insights into the functions of the miR319-TCP regulatory system in Arabidopsis development, highlighting a different modus operandi for its action mechanism in roots and shoots.

Dual expression and anatomy lines allow simultaneous visualization of gene expression and anatomy.

Studying the developmental genetics of plant organs requires following gene expression in specific tissues. To facilitate this, we have developed dual expression anatomy lines, which incorporate a red plasma membrane marker alongside a fluorescent reporter for a gene of interest in the same vector. Here, we adapted the GreenGate cloning vectors to create two destination vectors showing strong marking of cell membranes in either the whole root or specifically in the lateral roots. This system can also be used in both embryos and whole seedlings. As proof of concept, we follow both gene expression and anatomy in Arabidopsis (Arabidopsis thaliana) during lateral root organogenesis for a period of over 24 h. Coupled with the development of a flow cell and perfusion system, we follow changes in activity of the DII auxin sensor following application of auxin.

Proteolytic and Structural Changes in Rye and Triticale Roots under Aluminum Stress.

Proteolysis and structural adjustments are significant for defense against heavy metals. The purpose of this study was to evaluate whether the Al3+ stress alters protease activity and the anatomy of cereale roots. Azocaseinolytic and gelatinolytic measurements, transcript-level analysis of phytocystatins, and observations under microscopes were performed on the roots of Al3+-tolerant rye and tolerant and sensitive triticales exposed to Al3+. In rye and triticales, the azocaseinolytic activity was higher in treated roots. The gelatinolytic activity in the roots of rye was enhanced between 12 and 24 h in treated roots, and decreased at 48 h. The gelatinolytic activity in treated roots of tolerant triticale was the highest at 24 h and the lowest at 12 h, whereas in treated roots of sensitive triticale it was lowest at 12 h but was enhanced at 24 and 48 h. These changes were accompanied by increased transcript levels of phytocystatins in rye and triticale-treated roots. Light microscope analysis of rye roots revealed disintegration of rhizodermis in treated roots at 48 h and indicated the involvement of root border cells in rye defense against Al3+. The ultrastructural analysis showed vacuoles containing electron-dense precipitates. We postulate that proteolytic-antiproteolytic balance and structural acclimation reinforce the fine-tuning to Al3+.

Arabidopsis thaliana Myb59 Gene Is Involved in the Response to Heterodera schachtii Infestation, and Its Overexpression Disturbs Regular Development of Nematode-Induced Syncytia.

Transcription factors are proteins that directly bind to regulatory sequences of genes to modulate and adjust plants' responses to different stimuli including biotic and abiotic stresses. Sedentary plant parasitic nematodes, such as beet cyst nematode, Heterodera schachtii, have developed molecular tools to reprogram plant cell metabolism via the sophisticated manipulation of genes expression, to allow root invasion and the induction of a sequence of structural and physiological changes in plant tissues, leading to the formation of permanent feeding sites composed of modified plant cells (commonly called a syncytium). Here, we report on the AtMYB59 gene encoding putative MYB transcription factor that is downregulated in syncytia, as confirmed by RT-PCR and a promoter pMyb59::GUS activity assays. The constitutive overexpression of AtMYB59 led to the reduction in A. thaliana susceptibility, as indicated by decreased numbers of developed females, and to the disturbed development of nematode-induced syncytia. In contrast, mutant lines with a silenced expression of AtMYB59 were more susceptible to this parasite. The involvement of ABA in the modulation of AtMYB59 gene transcription appears feasible by several ABA-responsive cis regulatory elements, which were identified in silico in the gene promoter sequence, and experimental assays showed the induction of AtMYB59 transcription after ABA treatment. Based on these results, we suggest that AtMYB59 plays an important role in the successful parasitism of H. schachtii on A. thaliana roots.

New evidence of arsenic translocation and accumulation in Pteris vittata from real-time imaging using positron-emitting 74As tracer.

Pteris vittata is an arsenic (As) hyperaccumulator plant that accumulates a large amount of As into fronds and rhizomes (around 16,000 mg/kg in both after 16 weeks hydroponic cultivation with 30 mg/L arsenate). However, the sequence of long-distance transport of As in this hyperaccumulator plant is unclear. In this study, we used a positron-emitting tracer imaging system (PETIS) for the first time to obtain noninvasive serial images of As behavior in living plants with positron-emitting 74As-labeled tracer. We found that As kept accumulating in rhizomes as in fronds of P. vittata, whereas As was retained in roots of a non-accumulator plant Arabidopsis thaliana. Autoradiograph results of As distribution in P. vittata showed that with low As exposure, As was predominantly accumulated in young fronds and the midrib and rachis of mature fronds. Under high As exposure, As accumulation shifted from young fronds to mature fronds, especially in the margin of pinna, which resulted in necrotic symptoms, turning the marginal color to gray and then brown. Our results indicated that the function of rhizomes in P. vittata was As accumulation and the regulation of As translocation to the mature fronds to protect the young fronds under high As exposure.

Effects of graphene on morphology, microstructure and transcriptomic profiling of Pinus tabuliformis Carr. roots.

Graphene has shown great potential for improving growth of many plants, but its effect on woody plants remains essentially unstudied. In this work, Pinus tabuliformis Carr. bare-rooted seedlings grown outdoors in pots were irrigated with a graphene solution over a concentration range of 0-50 mg/L for six months. Graphene was found to stimulate root growth, with a maximal effect at 25 mg/L. We then investigated root microstructure and carried out transcript profiling of root materials treated with 0 and 25 mg/L graphene. Graphene treatment resulted in plasma-wall separation and destruction of membrane integrity in root cells. More than 50 thousand of differentially expressed genes (DEGs) were obtained by RNA sequencing, among which 6477 could be annotated using other plant databases. The GO enrichment analysis and KEGG pathway analysis of the annotated DEGs indicated that abiotic stress responses, which resemble salt stress, were induced by graphene treatment in roots, while responses to biotic stimuli were inhibited. Numerous metabolic processes and hormone signal transduction pathways were altered by the treatment. The growth promotion effects of graphene may be mediated by encouraging proline synthesis, and suppression of the expression of the auxin response gene SMALL AUXIN UP-REGULATED RNA 41 (SAUR41), PYL genes which encode ABA receptors, and GSK3 homologs.

A microscopic study on scattering in tissue section of Alternanthera philoxeroides under polarized light.

Like any other biological tissue, plant tissue also exhibits optical properties like refraction, transmission, absorption, coloration, scattering and so on. Several studies have been conducted using different parts of plants such as leaves, seedlings, roots, stems and so on, and their optical properties have been analyzed to study plant physiology, influence of environmental cues on plant metabolism, light propagation through plant parts and the like. Thus, it is essential to study in detail the optical properties of several plant parts to determine their structural relationship. In this backdrop, an experimental study was conducted to observe and analyze the optical properties of node and inter-nodal tissue cross-sections of the plant Alternanthera philoxeroides under a polarizing microscope constructed and standardized in the laboratory. The observed optical properties of the microscopic tissue sections have been then studied to determine a significant structural relationship between nodal and inter-nodal tissue arrangement patterns as a whole. Tissue sections that have undergone a sort of biological perturbation like loss of water (dried in air for 15 min) have also been studied to study the change in the pattern of tissue optical property when compared with that of normal plant-tissue cross-sections under a polarizing microscope. This type of biological perturbation was chosen for the study because water plays an important role in maintenance of the normal physiological processes in plants and most other forms of life.

Vacuolar occupancy is crucial for cell elongation and growth regardless of the underlying mechanism.

In the physiological range, the phytohormone auxin inhibits the growth of underground tissues. In the roots of Arabidopsis thaliana, cell size inhibition has been shown to be accompanied by auxin-mediated reduction of vacuole size. A tonoplast-localized protein family (Networked 4) with actin-binding capacity was demonstrated to modulate the compactness of the vacuole. Overexpression of NET4A led to smaller, more spherical and compact vacuoles, which occupied less cellular space compared to wild type. This reduction of vacuolar occupancy is similar to the observed auxin-induced decrease in occupancy, albeit there are enormous morphological differences. Here, we show that a net4a net4b double mutant and a NET4A overexpressor line are still sensitive to auxin-induced vacuolar constrictions. However, the overexpressor showed a partial auxin resistance accompanied by more compact vacuoles, thereby indicating an additional regulatory mechanism. Furthermore, we show that other NET superfamily members do not compensate for the loss of NET4A and NET4B expression on the transcriptional level. This leads us to hypothesize that regulation of vacuole size is a general mechanism to regulate cell expansion and that other players besides NET4 must participate in regulating the vacuole-cytoskeleton interface.

Osmotic stress-induced somatic embryo maturation of coffee Coffea arabica L., shoot and root apical meristems development and robustness.

Somatic embryogenesis (SE) is the most important plant biotechnology process for plant regeneration, propagation, genetic transformation and genome editing of coffee, Coffea arabica L. Somatic embryo (SEs) conversion to plantlets is the principal bottleneck for basic and applied use of this process. In this study we focus on the maturation of SEs of C. arabica var. Typica. SEs conversion to plantlet up to 95.9% was achieved under osmotic stress, using 9 g/L gelrite, as compared with only 39.34% in non-osmotic stress. Mature SEs induced in osmotic stress developed shoot and root apical meristems, while untreated SEs were unable to do it. C. arabica regenerated plants from osmotic stress were robust, with higher leaf and root area and internode length. To understand a possible regulatory mechanism, gene expression of key genes of C. arabica, homologous to sequences in the Arabidopsis thaliana genome, were analyzed. A set of two component system and cytokinin signaling-related coding genes (AHK1, AHK3, AHP4 and ARR1) which interact with WUSCHEL and WOX5 homedomains and morphogenic genes, BABY-BOOM, LEC1, FUS3 and AGL15, underwent significant changes during maturation of SEs of C. arabica var. Typica. This protocol is currently being applied in genetic transformation with high rate of success.

Three-dimensional reconstructions of haustoria in two parasitic plant species in the Orobanchaceae.

Parasitic plants infect other plants by forming haustoria, specialized multicellular organs consisting of several cell types, each of which has unique morphological features and physiological roles associated with parasitism. Understanding the spatial organization of cell types is, therefore, of great importance in elucidating the functions of haustoria. Here, we report a three-dimensional (3-D) reconstruction of haustoria from two Orobanchaceae species, the obligate parasite Striga hermonthica infecting rice (Oryza sativa) and the facultative parasite Phtheirospermum japonicum infecting Arabidopsis (Arabidopsis thaliana). In addition, field-emission scanning electron microscopy observation revealed the presence of various cell types in haustoria. Our images reveal the spatial arrangements of multiple cell types inside haustoria and their interaction with host roots. The 3-D internal structures of haustoria highlight differences between the two parasites, particularly at the xylem connection site with the host. Our study provides cellular and structural insights into haustoria of S. hermonthica and P. japonicum and lays the foundation for understanding haustorium function.

Cell-type action specificity of auxin on Arabidopsis root growth.

The plant hormone auxin plays a critical role in root growth and development; however, the contributions or specific roles of cell-type auxin signals in root growth and development are not well understood. Here, we mapped tissue and cell types that are important for auxin-mediated root growth and development by manipulating the local response and synthesis of auxin. Repressing auxin signaling in the epidermis, cortex, endodermis, pericycle or stele strongly inhibited root growth, with the largest effect observed in the endodermis. Enhancing auxin signaling in the epidermis, cortex, endodermis, pericycle or stele also caused reduced root growth, albeit to a lesser extent. Moreover, we established that root growth was inhibited by enhancement of auxin synthesis in specific cell types of the epidermis, cortex and endodermis, whereas increased auxin synthesis in the pericycle and stele had only minor effects on root growth. Our study thus establishes an association between cellular identity and cell type-specific auxin signaling that guides root growth and development.

Elucidating the morpho-physiological adaptations and molecular responses under long-term waterlogging stress in maize through gene expression analysis.

Waterlogging stress in maize is one of the emerging abiotic stresses in the current climate change scenario. To gain insights in transcriptional reprogramming during late hours of waterlogging stress under field conditions, we aimed to elucidate the transcriptional and anatomical changes in two contrasting maize inbreds viz. I110 (susceptible) and I172 (tolerant). Waterlogging stress reduced dry matter translocations from leaves and stems to ears, resulting in a lack of sink capacity and inadequate grain filling in I110, thus decreased the grain yield drastically. The development of aerenchyma cells within 48 h in I172 enabled hypoxia tolerance. The upregulation of alanine aminotransferase, ubiquitin activating enzyme E1, putative mitogen activated protein kinase and pyruvate kinase in I172 suggested that genes involved in protein degradation, signal transduction and carbon metabolism provided adaptive mechanisms during waterlogging. Overexpression of alcohol dehydrogenase, sucrose synthase, aspartate aminotransferase, NADP dependent malic enzyme and many miRNA targets in I110 indicated that more oxygen and energy consumption might have shortened plant survival during long-term waterlogging exposure. To the best of our knowledge, this is the first report of transcript profiling at late stage (24-96 h) of waterlogging stress under field conditions and provides new visions to understand the molecular basis of waterlogging tolerance in maize.

Cytokinesis in fra2 Arabidopsis thaliana p60-Katanin Mutant: Defects in Cell Plate/Daughter Wall Formation.

Cytokinesis is accomplished in higher plants by the phragmoplast, creating and conducting the cell plate to separate daughter nuclei by a new cell wall. The microtubule-severing enzyme p60-katanin plays an important role in the centrifugal expansion and timely disappearance of phragmoplast microtubules. Consequently, aberrant structure and delayed expansion rate of the phragmoplast have been reported to occur in p60-katanin mutants. Here, the consequences of p60-katanin malfunction in cell plate/daughter wall formation were investigated by transmission electron microscopy (TEM), in root cells of the fra2 Arabidopsis thaliana loss-of-function mutant. In addition, deviations in the chemical composition of cell plate/new cell wall were identified by immunolabeling and confocal microscopy. It was found that, apart from defective phragmoplast microtubule organization, cell plates/new cell walls also appeared faulty in structure, being unevenly thick and perforated by large gaps. In addition, demethylesterified homogalacturonans were prematurely present in fra2 cell plates, while callose content was significantly lower than in the wild type. Furthermore, KNOLLE syntaxin disappeared from newly formed cell walls in fra2 earlier than in the wild type. Taken together, these observations indicate that delayed cytokinesis, due to faulty phragmoplast organization and expansion, results in a loss of synchronization between cell plate growth and its chemical maturation.

Multiseriate cortical sclerenchyma enhance root penetration in compacted soils.

Mechanical impedance limits soil exploration and resource capture by plant roots. We examine the role of root anatomy in regulating plant adaptation to mechanical impedance and identify a root anatomical phene in maize (Zea mays) and wheat (Triticum aestivum) associated with penetration of hard soil: Multiseriate cortical sclerenchyma (MCS). We characterize this trait and evaluate the utility of MCS for root penetration in compacted soils. Roots with MCS had a greater cell wall-to-lumen ratio and a distinct UV emission spectrum in outer cortical cells. Genome-wide association mapping revealed that MCS is heritable and genetically controlled. We identified a candidate gene associated with MCS. Across all root classes and nodal positions, maize genotypes with MCS had 13% greater root lignin concentration compared to genotypes without MCS. Genotypes without MCS formed MCS upon exogenous ethylene exposure. Genotypes with MCS had greater lignin concentration and bending strength at the root tip. In controlled environments, MCS in maize and wheat was associated improved root tensile strength and increased penetration ability in compacted soils. Maize genotypes with MCS had root systems with 22% greater depth and 49% greater shoot biomass in compacted soils in the field compared to lines without MCS. Of the lines we assessed, MCS was present in 30 to 50% of modern maize, wheat, and barley cultivars but was absent in teosinte and wild and landrace accessions of wheat and barley. MCS merits investigation as a trait for improving plant performance in maize, wheat, and other grasses under edaphic stress.

Three-dimensional imaging reveals that positions of cyst nematode feeding sites relative to xylem vessels differ between susceptible and resistant wheat.

KEY MESSAGE: Resistance conferred by the Cre8 locus of wheat prevents cereal cyst nematode feeding sites from reaching and invading root metaxylem vessels. Cyst nematodes develop syncytial feeding sites within plant roots. The success of these sites is affected by host plant resistance. In wheat (Triticum aestivum L.), 'Cre' loci affect resistance against the cereal cyst nematode (CCN) Heterodera avenae. To investigate how one of these loci (Cre8, on chromosome 6B) confers resistance, CCN-infected root tissue from susceptible (-Cre8) and resistant (+Cre8) wheat plants was examined using confocal microscopy and laser ablation tomography. Confocal analysis of transverse sections showed that feeding sites in the roots of -Cre8 plants were always adjacent to metaxylem vessels, contained many intricate 'web-like' cell walls, and sometimes 'invaded' metaxylem vessels. In contrast, feeding sites in the roots of +Cre8 plants were usually not directly adjacent to metaxylem vessels, had few inner cell walls and did not 'invade' metaxylem vessels. Models based on data from laser ablation tomography confirmed these observations. Confocal analysis of longitudinal sections revealed that CCN-induced xylem modification that had previously been reported for susceptible (-Cre8) wheat plants is less extreme in resistant (+Cre8) plants. Application of a lignin-specific stain revealed that secondary thickening around xylem vessels in CCN-infected roots was greater in +Cre8 plants than in -Cre8 plants. Collectively, these results indicate that Cre8 resistance in wheat acts by preventing cyst nematode feeding sites from reaching and invading root metaxylem vessels.

Soil PAHs contamination effect on the cellular and subcellular organelle changes of Phragmites australis Cav.

The concentrations of ∑16 priority polycyclic aromatic hydrocarbons (PAHs) for soils, roots, and above-ground parts of reed (Phragmites australis Cav.) were determined on different monitoring plots located near the city of Kamensk-Shakhtinsky, southern Russia, where historically received industrial sewage and sludge. The total PAHs concentration in monitoring soil plots was significantly higher than those in the background site which situated at the distance of 2 km from the contamination source. Accordingly, the maximum accumulation was found for phenanthrene and chrysene among the 16 priority PAHs in most of the plant samples collected in the impact zone. The effects of PAHs' pollution on changes of Phragmites australis Cav. cellular and subcellular organelles in the studied monitoring sites were also determined using optical and electron microscopy, respectively. The obtained data showed that increasing of PAHs contamination negatively affected the ultrastructural changes of the studied plants. Phragmites australis Cav. showed a high level of adaptation to the effect of stressors by using tissue and cell levels. In general, the detected alterations under the PAHs effect were possibly connected to changes in biochemical and histochemical parameters as a response for reactive oxygen species and as a protective response against oxidative stress. The obtained results introduce innovative findings of cellular and subcellular changes in plants exposed to ∑16 priority PAHs as very persistent and toxic contaminants.

Multi-Walled Carbon Nanotubes Can Promote Brassica napus L. and Arabidopsis thaliana L. Root Hair Development through Nitric Oxide and Ethylene Pathways.

Here, we report that multi-walled carbon nanotubes (MWCNTs) can promote plant root hair growth in the species analyzed in this study; however, low and excessive concentrations of MWCNTs had no significant effect or even an inhibiting influence. Further results show that MWCNTs can enter rapeseed root cells. Meanwhile, nitrate reductase (NR)-dependent nitric oxide (NO) and ethylene syntheses, as well as root hair formation, were significantly stimulated by MWCNTs. Transcription of root hair growth-related genes were also modulated. The above responses were sensitive to the removal of endogenous NO or ethylene with a scavenger of NO or NO/ethylene synthesis inhibitors. Pharmacological and molecular evidence suggested that ethylene might act downstream of NR-dependent NO in MWCNTs-induced root hair morphogenesis. Genetic evidence in Arabidopsis further revealed that MWCNTs-triggered root hair growth was abolished in ethylene-insensitive mutants ein2-5 and ein3-1, and NR mutant nia1/2, but not in noa1 mutant. Further data placed NO synthesis linearly before ethylene production in root hair development triggered by MWCNTs. The above findings thus provide some insights into the molecular mechanism underlying MWCNTs control of root hair morphogenesis.

A non-classical route of efficient plant uptake verified with fluorescent nanoparticles and root adhesion forces investigated using AFM.

Classical plant uptake is limited to hydrophilic or water-dispersible material. Therefore, in order to test the uptake behaviour of hydrophobic particles, here, we tested the fate of hydrophobic particles (oleylamine coated Cu2-xSe NPs (CS@OA)) in comparison to hydrophilic particles (chitosan-coated Cu2-xSe NPs (CS@CH)) by treatment on the plant roots. Surprisingly, hydrophobic CS@OA NPs have been found to be ~ 1.3 times more efficient than hydrophilic CS@CH NPs in tomato plant root penetration. An atomic force microscopy (AFM) adhesion force experiment confirms that hydrophobic NPs experience non-spontaneous yet energetically favorable root trapping and penetration. Further, a relative difference in the hydrophobic vs. hydrophilic NPs movement from roots to shoots has been observed and found related to the change in protein corona as identified by two dimensional-polyacrylamide gel electrophoresis (2D-PAGE) analysis. Finally, the toxicity assays at the give concentration showed that Cu2-xSe NPs lead to non-significant toxicity as compared to control. This technology may find an advantage in fertilizer application.

Enhanced Cd accumulation by Graphene oxide (GO) under Cd stress in duckweed.

Effective phytoremediation by aquatic plant such as duckweed could be applied to solve Cd pollution. In the present study, the impact of Graphene oxide (GO) on the accumulation of Cd in duckweed has been studied. The response of duckweed was also investigated, concluding growth, Cd2+ flux, and gene expression response. Results showed that GO promoted the accumulation of Cd in duckweed. After 6 h of Cd enrichment in duckweed, Cd content was about 1.4 times that of the control group at fronds and 1.25 times that of the control group at roots, meanwhile, Cd content in the water system was 0.67 times that of the control group. The Cd2+ influx increased significantly. 4471 genes were up-regulated and 3230 genes were down-regulated significantly as duckweed treated with GO under Cd treatment. Moreover, phagosome pathway was downregulated, some key proteins: Stx7, Rab7 and Tubastatin B (TUBB) were significantly downregulated with GO addition under Cd stress. Scanning electron microscope (SEM) observation showed that GO and Cd were attached on the cell surface of duckweed as white crystal. GO could be applied in phytoremediation by duckweed of Cd in aquatic system.

Plant Cell Wall Changes in Common Wheat Roots as a Result of Their Interaction with Beneficial Fungi of Trichoderma.

Plant cell walls play an important role in shaping the defense strategies of plants. This research demonstrates the influence of two differentiators: the lifestyle and properties of the Trichoderma species on cell wall changes in common wheat seedlings. The methodologies used in this investigation include microscopy observations and immunodetection. In this study was shown that the plant cell wall was altered due to its interaction with Trichoderma. The accumulation of lignins and reorganization of pectin were observed. The immunocytochemistry indicated that low methyl-esterified pectins appeared in intercellular spaces. Moreover, it was found that the arabinogalactan protein epitope JIM14 can play a role in the interaction of wheat roots with both the tested Trichoderma strains. Nevertheless, we postulate that modifications, such as the appearance of lignins, rearrangement of low methyl-esterified pectins, and arabinogalactan proteins due to the interaction with Trichoderma show that tested strains can be potentially used in wheat seedlings protection to pathogens.