HD-Zip II transcription factors control distal stem cell fate in Arabidopsis roots by linking auxin signaling to the FEZ/SOMBRERO pathway.

In multicellular organisms, specialized tissues are generated by specific populations of stem cells through cycles of asymmetric cell divisions, where one daughter undergoes differentiation and the other maintains proliferative properties. In Arabidopsis thaliana roots, the columella - a gravity-sensing tissue that protects and defines the position of the stem cell niche - represents a typical example of a tissue whose organization is exclusively determined by the balance between proliferation and differentiation. The columella derives from a single layer of stem cells through a binary cell fate switch that is precisely controlled by multiple, independent regulatory inputs. Here, we show that the HD-Zip II transcription factors (TFs) HAT3, ATHB4 and AHTB2 redundantly regulate columella stem cell fate and patterning in the Arabidopsis root. The HD-Zip II TFs promote columella stem cell proliferation by acting as effectors of the FEZ/SMB circuit and, at the same time, by interfering with auxin signaling to counteract hormone-induced differentiation. Overall, our work shows that HD-Zip II TFs connect two opposing parallel inputs to fine-tune the balance between proliferation and differentiation in columella stem cells.

Neighbour-induced changes in root exudation patterns of buckwheat results in altered root architecture of redroot pigweed.

Roots are crucial in plant adaptation through the exudation of various compounds which are influenced and modified by environmental factors. Buckwheat root exudate and root system response to neighbouring plants (buckwheat or redroot pigweed) and how these exudates affect redroot pigweed was investigated. Characterising root exudates in plant-plant interactions presents challenges, therefore a split-root system which enabled the application of differential treatments to parts of a single root system and non-destructive sampling was developed. Non-targeted metabolome profiling revealed that neighbour presence and identity induces systemic changes. Buckwheat and redroot pigweed neighbour presence upregulated 64 and 46 metabolites, respectively, with an overlap of only 7 metabolites. Root morphology analysis showed that, while the presence of redroot pigweed decreased the number of root tips in buckwheat, buckwheat decreased total root length and volume, surface area, number of root tips, and forks of redroot pigweed. Treatment with exudates (from the roots of buckwheat and redroot pigweed closely interacting) on redroot pigweed decreased the total root length and number of forks of redroot pigweed seedlings when compared to controls. These findings provide understanding of how plants modify their root exudate composition in the presence of neighbours and how this impacts each other's root systems.

Investigation of multidirectional toxicity induced by high-dose molybdenum exposure with Allium test.

In this study, the multifaceted toxicity induced by high doses of the essential trace element molybdenum in Allium cepa L. was investigated. Germination, root elongation, weight gain, mitotic index (MI), micronucleus (MN), chromosomal abnormalities (CAs), Comet assay, malondialdehyde (MDA), proline, superoxide dismutase (SOD), catalase (CAT) and anatomical parameters were used as biomarkers of toxicity. In addition, detailed correlation and PCA analyzes were performed for all parameters discussed. On the other hand, this study focused on the development of a two hidden layer deep neural network (DNN) using Matlab. Four experimental groups were designed: control group bulbs were germinated in tap water and application group bulbs were germinated with 1000, 2000 and 4000 mg/L doses of molybdenum for 72 h. After germination, root tips were collected and prepared for analysis. As a result, molybdenum exposure caused a dose-dependent decrease (p < 0.05) in the investigated physiological parameter values, and an increase (p < 0.05) in the cytogenetic (except MI) and biochemical parameter values. Molybdenum exposure induced different types of CAs and various anatomical damages in root meristem cells. Comet assay results showed that the severity of DNA damage increased depending on the increasing molybdenum dose. Detailed correlation and PCA analysis results determined significant positive and negative interactions between the investigated parameters and confirmed the relationships of these parameters with molybdenum doses. It has been found that the DNN model is in close agreement with the actual data showing the accuracy of the predictions. MAE, MAPE, RMSE and R2 were used to evaluate the effectiveness of the DNN model. Collective analysis of these metrics showed that the DNN model performed well. As a result, it has been determined once again that high doses of molybdenum cause multiple toxicity in A. cepa and the Allium test is a reliable universal test for determining this toxicity. Therefore, periodic measurement of molybdenum levels in agricultural soils should be the first priority in preventing molybdenum toxicity.

Genome-wide identification of GRF transcription factors and their expression profile in stem meristem of broomcorn millet (Panicum miliaceum L.).

To understand the genome-wide information of the GRF family genes in broomcorn millet and their expression profile in the vegetative meristems, bioinformatic methods and transcriptome sequencing were used to analyze the characteristics, physical and chemical properties, phylogenetic relationship, chromosome distribution, gene structure, cis-acting elements and expression profile in stem meristem for the GRF family members. The results showed that the GRF gene family of millet contains 21 members, and the PmGRF gene is unevenly distributed on 12 chromosomes. The lengths of PmGRF proteins vary from 224 to 618 amino acids, and the isoelectric points are between 4.93-9.69. Each member of the family has 1-4 introns and 2-5 exons. The protein PmGRF13 is localized in both the nucleus and chloroplast, and the rest PmGRF proteins are located in the nucleus. Phylogenetic analysis showed that the 21 GRF genes were divided into 4 subfamilies (A,B,C and D) in broomcorn millet. The analysis of cis-acting elements showed that there were many cis-acting elements involved in light response, hormone response, drought induction, low temperature response and other environmental stress responses in the 2000 bp sequence upstream of the GRF genes. Transcriptome sequencing and qRT-PCR analyses showed that the expression levels of PmGRF3 and PmGRF12 in the dwarf variety Zhang778 were significantly higher than those of the tall variety Longmi12 in the internode and node meristems at the jointing stage, while the expression patterns of PmGRF4, PmGRF16 and PmGRF21 were reverse. In addition, the expression levels of PmGRF2 and PmGRF5 in the internode of Zhang778 were significantly higher than Longmi12. The other GRF genes were not or insignificantly expressed. These results indicated that seven genes, PmGRF2, PmGRF3, PmGRF4, PmGRF5, PmGRF12, PmGRF16 and PmGRF21, were related to the formation of plant height in broomcorn millet.

Cytogenetic Bioindication in Root Meristems for Vitality Assessment of Trees.

Our method describes how to collect forest tree root tips in the field, to store them for transfer to the lab, to pretreat root tips in order to arrest cells in metaphase, fix root tips to preserve specific morphological organizations, to stain fixed root tips by Feulgen's Reaction in order to increase contrast, and to prepare the root meristem for analyzing mitotic stages and chromosomal aberrations via light microscopy. We further describe how to classify chromosomal abnormalities and quantify them via aberration indices.

Root Hair Imaging Using Confocal Microscopy.

Plant genetics plays a key role in determining root hair initiation and development. A complex network of genetic interactions therefore closely monitors and influences root hair phenotype and morphology. The significance of these genes can be studied by employing, for instance, loss-of-function mutants, overexpression plant lines, and fluorescently labeled constructs. Confocal laser scanning microscopy is a great tool to visually observe and document these morphological features. This chapter elaborates the techniques involved in handling of microscopic setup to acquire images displaying root hair distribution along the fully elongated zone of Arabidopsis thaliana roots. Additionally, we illustrate an approach to visualize early fate determination of epidermal cells in the root apical meristem, by describing a method for imaging YFP tagged transgenic plant lines.

Immunohistochemical Detection of the Wall Components on the Example of Shoot Apical Meristem of Fagopyrum esculentum and Fagopyrum tataricum.

Immunohistochemistry is a method that allows the detection of individual components of cell walls in an extremely precise way at the level of a single cell and wall domains. The cell wall antibodies detect specific epitopes of pectins, arabinogalactan proteins (AGP), hemicelluloses, and extensins. The presented method visualization of the selected pectic and AGP epitopes using antibodies directed to wall components is described. The method of the analysis of the chemical composition of the wall is present on the example of the shoot apical meristems of Fagopurum esculentum and Fagopyrum tataricum. Recommended protocols for immunostaining and examination on fluorescence microscopy level are presented.

Dissecting the Roles of the Cytokinin Signaling Network: The Case of De Novo Shoot Apical Meristem Formation.

Numerous biotechnological applications require a fast and efficient clonal propagation of whole plants under controlled laboratory conditions. For most plant species, the de novo regeneration of shoots from the cuttings of various plant organs can be obtained on nutrient media supplemented with plant hormones, auxin and cytokinin. While auxin is needed during the early stages of the process that include the establishment of pluripotent primordia and the subsequent acquisition of organogenic competence, cytokinin-supplemented media are required to induce these primordia to differentiate into developing shoots. The perception of cytokinin through the receptor ARABIDOPSIS HISTIDINE KINASE4 (AHK4) is crucial for the activation of the two main regulators of the establishment and maintenance of shoot apical meristems (SAMs): SHOOTMERISTEMLESS (STM) and the WUSCHEL-CLAVATA3 (WUS-CLV3) regulatory circuit. In this review, we summarize the current knowledge of the roles of the cytokinin signaling cascade in the perception and transduction of signals that are crucial for the de novo establishment of SAMs and lead to the desired biotechnological output-adventitious shoot multiplication. We highlight the functional differences between individual members of the multigene families involved in cytokinin signal transduction, and demonstrate how complex genetic regulation can be achieved through functional specialization of individual gene family members.

Oxidative replication stress induced by long-term exposure to hydroxyurea in root meristem cells of Vicia faba.

KEY MESSAGE: Low concentrations of hydroxyurea, an inhibitor of DNA replication, induced oxidative and replicative stress in root apical meristem (RAM) cells of Vicia faba. Plant cells are constantly exposed to low-level endogenous stress factors that can affect DNA replication and lead to DNA damage. Long-term treatments of Vicia faba root apical meristems (RAMs) with HU leads to the appearance of atypical cells with intranuclear asynchrony. This rare form of abnormality was manifested by a gradual condensation of chromatin, from interphase to mitosis (so-called IM cells). Moreover, HU-treated root cells revealed abnormal chromosome structure, persisting DNA replication, and elevated levels of intracellular hydrogen peroxide (H2O2) and superoxide anion (O2∙-). Immunocytochemical studies have shown an increased number of fluorescent foci of H3 histones acetylated at lysine 56 (H3K56Ac; canonically connected with the DNA replication process). We show that continuous 3-day exposure to low concentrations (0.75 mM) of hydroxyurea (HU; an inhibitor of DNA replication) induces cellular response to reactive oxygen species and to DNA replication stress conditions.

Cyanobacterial elicitor enhances the biomass of Mentha piperita L. and improves the production of high-value rosmarinic acid under in vitro culture of apical meristem.

BACKGROUND: Rosmarinic acid (RA), like other phenolic compounds, is sources of antioxidants and anti-inflammatory agents in medicinal plants. In vitro culture of plants can improve the medicinal plants' metabolite profile and phenolic compound quantity. To date, various methods have been proposed to increase this medicinal metabolite in plants, among which the use of bioelicitors can be mentioned. In the present study, a native isolate of heterocystous cyanobacteria, Nostoc spongiaeforme var. tenue ISB65, was used to stimulate the production of biomass and content of RA in Mentha piperita L. (peppermint) grown in vitro from apical meristem. Mentha piperita L. explants were inoculated in half strength Murashige and Skoog (1/2 MS) medium containing cyanobacterial lysate (CL). After 50 days of culturing, the growth indices, the content of photosynthetic pigments, and RA in control and treated plants were measured. RESULTS: CL inoculation resulted in a significant enhancement in the vegetative growth indices of peppermint, including root and shoot length, plant biomass and leaf number. The content of photosynthetic pigments also increased in cyanobacteria-treated plants. Inoculation with CL increased the RA content by 2.3-fold, meaning that the plants treated with CL had the highest RA content (7.68 mg. g- 1 dry weight) compared to the control (3.42 mg. g- 1 dry weight). Additionally, HPLC analysis revealed the presence of several auxins in CL. CONCLUSIONS: The presence of auxins and the chemical content of CL such as K+ and Ca2+, as regulators of metabolic pathways and molecular activities of cells, may be responsible for the enhanced growth and phenolic compounds of plants under tissue culture conditions. An improvement in RA content in the tissue culture of medicinal plants treated with CL was reported for the first time in this investigation.

Control of phloem unloading and root development.

Root growth and development need proper carbon partitioning between sources and sinks. Photosynthesis products are unloaded from the phloem and enter the root meristem cell by cell. While sugar transporters play a major role in phloem loading, phloem unloading occurs via the plasmodesmata in growing root tips. The aperture and permeability of plasmodesmata strongly influence symplastic unloading. Recent research has dissected the symplastic path for phloem unloading and identified several genes that regulate phloem unloading in the root. Callose turnover and membrane lipid composition alter the shape of plasmodesmata, allowing fine-tuning to adapt phloem unloading to the environmental and developmental conditions. Unloaded sugars act both as an energy supply and as signals to coordinate root growth and development. Increased knowledge of how phloem unloading is regulated enhances our understanding of carbon allocation in plants. In the future, it may be possible to modulate carbon allocation between sources and sinks in a manner that would contribute to increased plant biomass and carbon fixation.

Unraveling the molecular mechanisms governing axillary meristem initiation in plants.

MAIN CONCLUSION: Axillary meristems (AMs) located in the leaf axils determine the number of shoots or tillers eventually formed, thus contributing significantly to the plant architecture and crop yields. The study of AM initiation is unavoidable and beneficial for crop productivity. Shoot branching is an undoubted determinant of plant architecture and thus greatly impacts crop yield due to the panicle-bearing traits of tillers. The emergence of the AM is essential for the incipient bud formation, and then the bud is dormant or outgrowth immediately to form a branch or tiller. While numerous reviews have focused on plant branching and tillering development networks, fewer specifically address AM initiation and its regulatory mechanisms. This review synthesizes the significant advancements in the genetic and hormonal factors governing AM initiation, with a primary focus on studies conducted in Arabidopsis (Arabidopsis thaliana L.) and rice (Oryza sativa L.). In particular, by elaborating on critical genes like LATERAL SUPPRESSOR (LAS), which specifically regulates AM initiation and the networks in which they are involved, we attempt to unify the cascades through which they are positioned. We concentrate on clarifying the precise mutual regulation between shoot apical meristem (SAM) and AM-related factors. Additionally, we examine challenges in elucidating AM formation mechanisms alongside opportunities provided by emerging omics approaches to identify AM-specific genes. By expanding our comprehension of the genetic and hormonal regulation of AM development, we can develop strategies to optimize crop production and address global food challenges effectively.

BnaA4.BOR2 contributes the tolerance of rapeseed to boron deficiency by improving the transport of boron from root to shoot.

Boron (B) is essential for plant growth. However, the molecular mechanism of B transport in rapeseed (Brassica napus L.) is unknown well. Here, we report that B transporter BnaA4.BOR2 is involved in the transport of B from root to shoot and its distribution in shoot cell wall and flower in rapeseed. The results of GUS staining and in-situ PCR analysis showed that BnaA4.BOR2 is mainly expressed in cortex and endodermis of root tip meristem zone and endodermis of mature zone. BnaA4.BOR2 was mainly localized in plasma membrane and showed B transport activity in yeast. Overexpression of Bna4.BOR2 could rescue the phenotype of Arabidopsis mutant bor2-2 under low-B condition. Furthermore, knockout of BnaA4.BOR2 could significantly enhance the sensitivity of rapeseed mutants to B deficiency, including inhibition of root elongation and biomass decrease of roots and shoots. The B concentration in xylem sap of BnaA4.BOR2 mutants was significantly decreased under B deficiency, which resulted in significantly lower B concentrations in shoot cell wall at seedling stage and flower organ at reproductive stage compared to that of wild-type QY10. The growth of BnaA4.BOR2 mutants were severely inhibited, exhibiting a typical B-deficient phenotype of "flowering without seed setting", leading to a sharp decrease in seed yield in B deficient soil. Taken together, these results indicate that BnaA4.BOR2 is critical for rapeseed growth and seed yield production under low B level, which is mainly expressed in cortex and endodermis, and contributed to the transport of B from roots to shoots and its distribution in shoot.

LONESOME HIGHWAY-TARGET OF MONOPTEROS5 transcription factor complex promotes a predifferentiation state for xylem vessel differentiation in the root apical meristem by inducing the expression of VASCULAR-RELATED NAC-DOMAIN genes.

In Arabidopsis thaliana, heterodimers comprising two bHLH family proteins, LONESOME HIGHWAY (LHW) and TARGET OF MONOPTEROS5 (TMO5) or its homolog TMO5-LIKE 1 (T5L1) control vascular development in the root apical meristem (RAM). The LHW-TMO5/T5L1 complex regulates vascular cell proliferation, vascular pattern organization, and xylem vessel differentiation; however, the mechanism of preparation for xylem vessel differentiation in the RAM remains elusive. We examined the relationship between LHW-T5L1 and VASCULAR-RELATED NAC-DOMAIN (VND) genes, which are key regulators of vessel differentiation, using reverse genetics approaches. LHW-T5L1 upregulated the expression of VND1, VND2, VND3, VND6, and VND7 but not that of other VNDs. The expression of VND1-VND3 in the RAM was decreased in lhw. In vnd1 vnd2 vnd3 triple loss-of-function mutant roots, metaxylem differentiation was delayed, and VND6 and VND7 expression was reduced. Furthermore, transcriptome analysis of VND1-overexpressing cells revealed that VND1 upregulates genes involved in the synthesis of secondary cell wall components. These results suggest that LHW-T5L1 upregulates VND1-VND3 at the early stages of vascular development in the RAM, and VNDs promote a predifferentiation state for xylem vessels by triggering low levels of VND6 and VND7 as well as genes for the synthesis of secondary cell wall materials.

Polarity establishment in the plant zygote at a glance.

The complex structures of multicellular organisms originate from a unicellular zygote. In most angiosperms, including Arabidopsis thaliana, the zygote is distinctly polar and divides asymmetrically to produce an apical cell, which generates the aboveground part of the plant body, and a basal cell, which generates the root tip and extraembryonic suspensor. Thus, zygote polarity is pivotal for establishing the apical-basal axis running from the shoot apex to the root tip of the plant body. The molecular mechanisms and spatiotemporal dynamics behind zygote polarization remain elusive. However, advances in live-cell imaging of plant zygotes have recently made significant insights possible. In this Cell Science at a Glance article and the accompanying poster, we summarize our understanding of the early steps in apical-basal axis formation in Arabidopsis, with a focus on de novo transcriptional activation after fertilization and the intracellular dynamics leading to the first asymmetric division of the zygote.

Decoding the leaf apical meristem of Guarea glabra Vahl (Meliaceae): insight into the evolution of indeterminate pinnate leaves.

In seed plants, growth of shoots and roots is indeterminate, while leaves are typically determinate organs that cease to grow after a certain developmental stage. This is due to the characteristics of the leaf meristem, where cell proliferation activity is retained only for a limited period. However, several plants exhibit indeterminacy in their leaves, exemplified by the pinnate compound leaves of Guarea and Chisocheton genera in the Meliaceae family. In these plants, the leaf meristem at the tip of the leaf retains meristematic activity and produces leaflets over years, resulting in a single leaf that resembles a twig. The molecular mechanism underlying the indeterminate leaf meristem of these plants has not been examined. In this research, we used Guarea glabra as a model to investigate the development of indeterminate pinnate leaves. Transcriptome analyses revealed that the gene expression profile in leaf apex tissue differed from that in the shoot apex. However, a class 1 KNOTTED-LIKE HOMEOBOX (KNOX1) gene which is lost in Brassicaceae was highly expressed in both tissues. We established an in situ hybridisation system for this species using Technovit 9100 to analyse the spatial expression patterns of genes. We revealed that the leaf meristematic region of G. glabra expresses KNOX1, LEAFY and ANGUSTIFORIA3 simultaneously, suggesting the involvement of these genes in the indeterminacy of the leaf meristem.

In situ seasonal patterns of root auxin concentrations and meristem length in an arctic sedge.

Seasonal dynamics of root growth play an important role in large-scale ecosystem processes; they are largely governed by growth regulatory compounds and influenced by environmental conditions. Yet, our knowledge about physiological drivers of root growth is mostly limited to laboratory-based studies on model plant species. We sampled root tips of Eriophorum vaginatum and analyzed their auxin concentrations and meristem lengths biweekly over a growing season in situ in a subarctic peatland, both in surface soil and at the permafrost thawfront. Auxin concentrations were almost five times higher in surface than in thawfront soils and increased over the season, especially at the thawfront. Surprisingly, meristem length showed an opposite pattern and was almost double in thawfront compared with surface soils. Meristem length increased from peak to late season in the surface soils but decreased at the thawfront. Our study of in situ seasonal dynamics in root physiological parameters illustrates the potential for physiological methods to be applied in ecological studies and emphasizes the importance of in situ measurements. The strong effect of root location and the unexpected opposite patterns of meristem length and auxin concentrations likely show that auxin actively governs root growth to ensure a high potential for nutrient uptake at the thawfront.

Do DEEPER ROOTING 1 Homologs Regulate the Lateral Root Slope Angle in Cucumber (Cucumis sativus)?

The architecture of the root system is fundamental to plant productivity. The rate of root growth, the density of lateral roots, and the spatial structure of lateral and adventitious roots determine the developmental plasticity of the root system in response to changes in environmental conditions. One of the genes involved in the regulation of the slope angle of lateral roots is DEEPER ROOTING 1 (DRO1). Its orthologs and paralogs have been identified in rice, Arabidopsis, and several other species. However, nothing is known about the formation of the slope angle of lateral roots in species with the initiation of lateral root primordia within the parental root meristem. To address this knowledge gap, we identified orthologs and paralogs of the DRO1 gene in cucumber (Cucumis sativus) using a phylogenetic analysis of IGT protein family members. Differences in the transcriptional response of CsDRO1, CsDRO1-LIKE1 (CsDRO1L1), and CsDRO1-LIKE2 (CsDRO1L2) to exogenous auxin were analyzed. The results showed that only CsDRO1L1 is auxin-responsive. An analysis of promoter-reporter fusions demonstrated that the CsDRO1, CsDRO1L1, and CsDRO1L2 genes were expressed in the meristem in cell files of the central cylinder, endodermis, and cortex; the three genes displayed different expression patterns in cucumber roots with only partial overlap. A knockout of individual CsDRO1, CsDRO1L1, and CsDRO1L2 genes was performed via CRISPR/Cas9 gene editing. Our study suggests that the knockout of individual genes does not affect the slope angle formation during lateral root primordia development in the cucumber parental root.

Light-sensitive short hypocotyl genes confer symbiotic nodule identity in the legume Medicago truncatula.

Legumes produce specialized root nodules that are distinct from lateral roots in morphology and function, with nodules intracellularly hosting nitrogen-fixing bacteria. We have previously shown that a lateral root program underpins nodule initiation, but there must be additional developmental regulators that confer nodule identity. Here, we show two members of the LIGHT-SENSITIVE SHORT HYPOCOTYL (LSH) transcription factor family, predominantly known to define shoot meristem complexity and organ boundaries, function as regulators of nodule organ identity. In parallel to the root initiation program, LSH1/LSH2 recruit a program into the root cortex that mediates the divergence into nodules, in particular with cell divisions in the mid-cortex. This includes regulation of auxin and cytokinin, promotion of NODULE ROOT1/2 and Nuclear Factor YA1, and suppression of the lateral root program. A principal outcome of LSH1/LSH2 function is the production of cells able to accommodate nitrogen-fixing bacteria, a key feature unique to nodules.

Genetic control of thermomorphogenesis in tomato inflorescences.

Understanding how plants alter their development and architecture in response to ambient temperature is crucial for breeding resilient crops. Here, we identify the quantitative trait locus qMULTIPLE INFLORESCENCE BRANCH 2 (qMIB2), which modulates inflorescence branching in response to high ambient temperature in tomato (Solanum lycopersicum). The non-functional mib2 allele may have been selected in large-fruited varieties to ensure larger and more uniform fruits under varying temperatures. MIB2 gene encodes a homolog of the Arabidopsis thaliana transcription factor SPATULA; its expression is induced in meristems at high temperature. MIB2 directly binds to the promoter of its downstream gene CONSTANS-Like1 (SlCOL1) by recognizing the conserved G-box motif to activate SlCOL1 expression in reproductive meristems. Overexpressing SlCOL1 rescue the reduced inflorescence branching of mib2, suggesting how the MIB2-SlCOL1 module helps tomato inflorescences adapt to high temperature. Our findings reveal the molecular mechanism underlying inflorescence thermomorphogenesis and provide a target for breeding climate-resilient crops.