Transcriptome analysis of apical meristem enriched bud samples for size dependent flowering commitment in Crocus sativus reveal role of sugar and auxin signalling.

BACKGROUND: Cultivation of Crocus sativus (saffron) faces challenges due to inconsistent flowering patterns and variations in yield. Flowering takes place in a graded way with smaller corms unable to produce flowers. Enhancing the productivity requires a comprehensive understanding of the underlying genetic mechanisms that govern this size-based flowering initiation and commitment. Therefore, samples enriched with non-flowering and flowering apical buds from small (< 6 g) and large (> 14 g) corms were sequenced. METHODS AND RESULTS: Apical bud enriched samples from small and large corms were collected immediately after dormancy break in July. RNA sequencing was performed using Illumina Novaseq 6000 to access the gene expression profiles associated with size dependent flowering. De novo transcriptome assembly and analysis using flowering committed buds from large corms at post-dormancy and their comparison with vegetative shoot primordia from small corms pointed out the major role of starch and sucrose metabolism, Auxin and ABA hormonal regulation. Many genes with known dual responses in flowering development and circadian rhythm like Flowering locus T and Cryptochrome 1 along with a transcript showing homology with small auxin upregulated RNA (SAUR) exhibited induced expression in flowering buds. Thorough prediction of Crocus sativus non-coding RNA repertoire has been carried out for the first time. Enolase was found to be acting as a major hub with protein-protein interaction analysis using Arabidopsis counterparts. CONCLUSION: Transcripts belong to key pathways including phenylpropanoid biosynthesis, hormone signaling and carbon metabolism were found significantly modulated. KEGG assessment and protein-protein interaction analysis confirm the expression data. Findings unravel the genetic determinants driving the size dependent flowering in Crocus sativus.

Transcription factor OsSHR2 regulates rice architecture and yield per plant in response to nitrogen.

MAIN CONCLUSION: Mutation of OsSHR2 adversely impacted root and shoot growth and impaired plant response to N conditions, further reducing the yield per plant. Nitrogen (N) is a crucial factor that regulates the plant architecture. There is still a lack of research on it. In our study, it was observed that the knockout of the SHORTROOT 2 (OsSHR2) which was induced by N deficiency, can significantly affect the regulation of plant architecture response to N in rice. Under N deficiency, the mutation of OsSHR2 significantly reduced root growth, and impaired the sensitivity of the root meristem length to N deficiency. The mutants were found to have approximately a 15% reduction in plant height compared to wild type. But mutants showed a significant increase in tillering at post-heading stage, approximately 26% more than the wild type, particularly in high N conditions. In addition, due to reduced seed setting rate and 1000-grain weight, mutant yield was significantly decreased by approximately 33% under low N fertilizer supply. The mutation also changed the distribution of N between the vegetative and reproductive organs. Our findings suggest that the transcription factor OsSHR2 plays a regulatory role in the response of plant architecture and yield per plant to N in rice.

Expression analyses of CUP-SHAPED COTYLEDON and SHOOT MERISTEMLESS in the one-leaf plant Monophyllaea glabra reveal neoteny evolution of shoot meristem.

The one-leaf plant Monophyllaea glabra exhibits a unique developmental manner in which only one cotyledon continues growing without producing new vegetative organs. This morphology is formed by specific meristems, the groove meristem (GM) and the basal meristem (BM), which are thought to be modified shoot apical meristem (SAM) and leaf meristem. In this study, we analysed the expression of the organ boundary gene CUP-SHAPED COTYLEDON (CUC) and the SAM maintenance gene SHOOT MERISTEMLESS (STM) orthologs by whole-mount in situ hybridisation. We found that CUCs did not show clear border patterns around GM and BM during the vegetative phase. Furthermore, double-colour detection analysis at the cellular level revealed that CUC and STM expression overlapped in the GM region during the vegetative phase. We also found that this overlap is dissolved in the reproductive phase when normal shoot organogenesis is observed. Since co-expression of these genes occurs during SAM initiation under embryogenesis in Arabidopsis, our results demonstrate that GM is a prolonged stage of pre-mature SAM. Therefore, we propose that neotenic meristems could be a novel plant trait acquired by one-leaf plants.

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.

An AGO10:miR165/6 module regulates meristem activity and xylem development in the Arabidopsis root.

The RNA-silencing effector ARGONAUTE10 influences cell fate in plant shoot and floral meristems. ARGONAUTE10 also accumulates in the root apical meristem (RAM), yet its function(s) therein remain elusive. Here, we show that ARGONAUTE10 is expressed in the root cell initials where it controls overall RAM activity and length. ARGONAUTE10 is also expressed in the stele, where post-transcriptional regulation confines it to the root tip's pro-vascular region. There, variations in ARGONAUTE10 levels modulate metaxylem-vs-protoxylem specification. Both ARGONAUTE10 functions entail its selective, high-affinity binding to mobile miR165/166 transcribed in the neighboring endodermis. ARGONAUTE10-bound miR165/166 is degraded, likely via SMALL-RNA-DEGRADING-NUCLEASES1/2, thus reducing miR165/166 ability to silence, via ARGONAUTE1, the transcripts of cell fate-influencing transcription factors. These include PHABULOSA (PHB), which controls meristem activity in the initials and xylem differentiation in the pro-vasculature. During early germination, PHB transcription increases while dynamic, spatially-restricted transcriptional and post-transcriptional mechanisms reduce and confine ARGONAUTE10 accumulation to the provascular cells surrounding the newly-forming xylem axis. Adequate miR165/166 concentrations are thereby channeled along the ARGONAUTE10-deficient yet ARGONAUTE1-proficient axis. Consequently, inversely-correlated miR165/166 and PHB gradients form preferentially along the axis despite ubiquitous PHB transcription and widespread miR165/166 delivery inside the whole vascular cylinder.

Golgi-localized APYRASE 1 is critical for Arabidopsis growth by affecting cell wall integrity under boron deficiency.

Many nucleoside triphosphate-diphosphohydrolases (NTPDases/APYRASEs, APYs) play a key role in modulating extracellular nucleotide levels. However, the Golgi-localized APYs, which help control glycosylation, have rarely been studied. Here, we identified AtAPY1, a gene encoding an NTPDase in the Golgi apparatus, which is required for cell wall integrity and plant growth under boron (B) limited availability. Loss of function in AtAPY1 hindered cell elongation and division in root tips while increasing the number of cortical cell layers, leading to swelling of the root tip and abundant root hairs under low B stress. Further, expression pattern analysis revealed that B deficiency significantly induced AtAPY1, especially in the root meristem and stele. Fluorescent-labeled AtAPY1-GFP localized to the Golgi stack. Biochemical analysis showed that AtAPY1 exhibited a preference of UDP and GDP hydrolysis activities. Consequently, the loss of function in AtAPY1 might disturb the homoeostasis of NMP-driven NDP-sugar transport, which was closely related to the synthesis of cell wall polysaccharides. Further, cell wall-composition analysis showed that pectin content increased and borate-dimerized RG-II decreased in apy1 mutants, along with a decrease in cellulose content. Eventually, altered polysaccharide characteristics presumably cause growth defects in apy1 mutants under B deficiency. Altogether, these data strongly support a novel role for AtAPY1 in mediating responses to low B availability by regulating cell wall integrity.

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.

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.

Allium cepa tests: Exploring bleomycin induced cyto-genotoxicity and altered cell cycle kinetics in root tips meristematic cells.

Bleomycin, commonly employed in treating Hodgkin's lymphoma and testicular cancer, is associated with significant pulmonary toxicity. While various studies have assessed the toxic impact of chemotherapeutic agents on aquatic and terrestrial environments, limited data exist on bleomycin's effects, especially concerning higher plants. To address this gap, we utilized the Allium cepa assays, renowned for evaluating chemical and biochemical agents' toxic effects, to investigate bleomycin's impact on the terrestrial ecosystem. Our study aimed to assess bleomycin's cyto-genotoxic effects on A. cepa root tip cells at minimal concentrations (10-40 µg mL-1) and varied exposure durations (2, 4, 6, and 24 h). Analysis of nuclear and mitotic abnormalities in bleomycin-treated A. cepa root tip cells, alongside an acridine orange-ethidium bromide double staining assay, illuminated its influence on cell viability. Additionally, agarose gel electrophoresis determined the drug's potential for DNA degradation, unveiling the underlying mechanisms of cyto-genotoxicity. Results also demonstrated a decline in the mitotic index with increased bleomycin concentrations and exposure time, elevated frequencies of various cyto-genotoxic abnormalities, including sticky chromosomes, chromatid breaks, laggards, bridges, polar deviations, nuclear lesions, and hyperchromasia. The study indicated the potential risks of bleomycin even at low concentrations and brief exposures, highlighting its severe adverse effects on genetic material of plant, potentially contributing to cell death. Consequently, this investigation unveils bleomycin's cyto-genotoxic effects on higher plant system, underscoring its threat to terrestrial ecosystems, particularly upon chronic and unmonitored exposure.

Leaf and shoot apical meristem transcriptomes of quinoa (Chenopodium quinoa Willd.) in response to photoperiod and plant development.

Understanding the regulation of flowering time is crucial for adaptation of crops to new environment. In this study, we examined the timing of floral transition and analysed transcriptomes in leaf and shoot apical meristems of photoperiod-sensitive and -insensitive quinoa accessions. Histological analysis showed that floral transition in quinoa initiates 2-3 weeks after sowing. We found four groups of differentially expressed genes in quinoa genome that responded to plant development and floral transition: (i) 222 genes responsive to photoperiod in leaves, (ii) 1812 genes differentially expressed between accessions under long-day conditions in leaves, (iii) 57 genes responding to developmental changes under short-day conditions in leaves and (iv) 911 genes responding to floral transition within the shoot apical meristem. Interestingly, among numerous candidate genes, two putative FT orthologs together with other genes (e.g. SOC1, COL, AP1) were previously reported as key regulators of flowering time in other species. Additionally, we used coexpression networks to associate novel transcripts to a putative biological process based on the annotated genes within the same coexpression cluster. The candidate genes in this study would benefit quinoa breeding by identifying and integrating their beneficial haplotypes in crossing programs to develop adapted cultivars to diverse environmental conditions.

The peptide GOLVEN10 alters root development and noduletaxis in Medicago truncatula.

The conservation of GOLVEN (GLV)/ROOT MERISTEM GROWTH FACTOR (RGF) peptide encoding genes across plant genomes capable of forming roots or root-like structures underscores their potential significance in the terrestrial adaptation of plants. This study investigates the function and role of GOLVEN peptide-coding genes in Medicago truncatula. Five out of fifteen GLV/RGF genes were notably upregulated during nodule organogenesis and were differentially responsive to nitrogen deficiency and auxin treatment. Specifically, the expression of MtGLV9 and MtGLV10 at nodule initiation sites was contingent upon the NODULE INCEPTION transcription factor. Overexpression of these five nodule-induced GLV genes in hairy roots of M. truncatula and application of their synthetic peptide analogues led to a decrease in nodule count by 25-50%. Uniquely, the GOLVEN10 peptide altered the positioning of the first formed lateral root and nodule on the primary root axis, an observation we term 'noduletaxis'; this decreased the length of the lateral organ formation zone on roots. Histological section of roots treated with synthetic GOLVEN10 peptide revealed an increased cell number within the root cortical cell layers without a corresponding increase in cell length, leading to an elongation of the root likely introducing a spatiotemporal delay in organ formation. At the transcription level, the GOLVEN10 peptide suppressed expression of microtubule-related genes and exerted its effects by changing expression of a large subset of Auxin responsive genes. These findings advance our understanding of the molecular mechanisms by which GOLVEN peptides modulate root morphology, nodule ontogeny, and interactions with key transcriptional pathways.

STAMENLESS1 activates SUPERWOMAN 1 and FLORAL ORGAN NUMBER 1 to control floral organ identities and meristem fate in rice.

Floral patterns are unique to rice and contribute significantly to its reproductive success. SL1 encodes a C2H2 transcription factor that plays a critical role in flower development in rice, but the molecular mechanism regulated by it remains poorly understood. Here, we describe interactions of the SL1 with floral homeotic genes, SPW1, and DL in specifying floral organ identities and floral meristem fate. First, the sl1 spw1 double mutant exhibited a stamen-to-pistil transition similar to that of sl1, spw1, suggesting that SL1 and SPW1 may located in the same pathway regulating stamen development. Expression analysis revealed that SL1 is located upstream of SPW1 to maintain its high level of expression and that SPW1, in turn, activates the B-class genes OsMADS2 and OsMADS4 to suppress DL expression indirectly. Secondly, sl1 dl displayed a severe loss of floral meristem determinacy and produced amorphous tissues in the third/fourth whorl. Expression analysis revealed that the meristem identity gene OSH1 was ectopically expressed in sl1 dl in the fourth whorl, suggesting that SL1 and DL synergistically terminate the floral meristem fate. Another meristem identity gene, FON1, was significantly decreased in expression in sl1 background mutants, suggesting that SL1 may directly activate its expression to regulate floral meristem fate. Finally, molecular evidence supported the direct genomic binding of SL1 to SPW1 and FON1 and the subsequent activation of their expression. In conclusion, we present a model to illustrate the roles of SL1, SPW1, and DL in floral organ specification and regulation of floral meristem fate in rice.

PHYTOCHROME A controls the DNA damage response and cell death tolerance within the Arabidopsis root meristem.

The DNA damage response avoids mutations into dividing cells. Here, we analysed the role of photoreceptors on the restriction of root growth imposed by genotoxic agents and its relationship with cell viability and performance of meristems. Comparison of root growth of Arabidopsis WT, phyA-211, phyB-9, and phyA-211phyB-9 double mutants unveiled a critical role for phytochrome A (PhyA) in protecting roots from genotoxic stress, regeneration and cell replenishment in the meristematic zone. PhyA was located on primary root tips, where it influences genes related to the repair of DNA, including ERF115 and RAD51. Interestingly, phyA-211 mutants treated with zeocin failed to induce the expression of the repressor of cell cycle MYB3R3, which correlated with expression of the mitotic cyclin CycB1, suggesting that PhyA is required for safeguarding the DNA integrity during cell division. Moreover, the growth of the primary roots of PhyA downstream component HY5 and root growth analyses in darkness suggest that cell viability and DNA damage responses within root meristems may act independently from light and photomorphogenesis. These data support novel roles for PhyA as a key player for stem cell niche maintenance and DNA damage responses, which are critical for proper root growth.

Transcriptome Analysis of Rice Root Tips Reveals Auxin, Gibberellin and Ethylene Signaling Underlying Nutritropism.

Nutritropism is a positive tropism toward nutrients in plant roots. An NH4+ gradient is a nutritropic stimulus in rice (Oryza sativa L.). When rice roots are exposed to an NH4+ gradient generated around nutrient sources, root tips bend toward and coil around the sources. The molecular mechanisms are largely unknown. Here, we analyzed the transcriptomes of the inside and outside of bending root tips exhibiting nutritropism to reveal nutritropic signal transduction. Tissues facing the nutrient sources (inside) and away (outside) were separately collected by laser microdissection. Principal component analysis revealed distinct transcriptome patterns between the two tissues. Annotations of 153 differentially expressed genes implied that auxin, gibberellin and ethylene signaling were activated differentially between the sides of the root tips under nutritropism. Exogenous application of transport and/or biosynthesis inhibitors of these phytohormones largely inhibited the nutritropism. Thus, signaling and de novo biosynthesis of the three phytohormones are necessary for nutritropism. Expression patterns of IAA genes implied that auxins accumulated more in the inside tissues, meaning that ammonium stimulus is transduced to auxin signaling in nutritropism similar to gravity stimulus in gravitropism. SAUR and expansin genes, which are known to control cell wall modification and to promote cell elongation in shoot gravitropism, were highly expressed in the inside tissues rather than the outside tissues, and our transcriptome data are unexplainable for differential elongation in root nutritropism.

Re-examining meristems through the lens of evo-devo.

The concept of the meristem was introduced in 1858 to characterize multicellular, formative, and proliferative tissues that give rise to the entire plant body, based on observations of vascular plants. Although its original definition did not encompass bryophytes, this concept has been used and continuously refined over the past 165 years to describe the diverse apices of all land plants. Here, we re-examine this matter in light of recent evo-devo research and show that, despite displaying high anatomical diversity, land plant meristems are unified by shared genetic control. We also propose a modular view of meristem function and highlight multiple evolutionary mechanisms that are likely to have contributed to the assembly and diversification of the varied meristems during the course of plant evolution.

Production of transgenic periclinal chimeras in pumpkin - a tool for revealing cell fates of L1 meristem.

Genetic engineering is commonly used to improve the agronomic traits of crops. However, genetic transformation in pumpkin remains a challenge. Conducting transformation trials, we accidentally created transgenic L1 periclinal chimeras in pumpkins. Using our modified Agrobacterium-mediated transformation, we generated transgenic L1 periclinal chimeras which have high value in research on development of the meristem. Fluorescence observations of transformed L1 cells enabled us to reveal cell fates. These L1 cells can develop into stomata, epidermal hairs, seed coat, and epidermis of the root, stem, leaf, flower, and fruit. These periclinal chimeras can be propagated vegetatively with minimal risk of transgene flow. This study offers new perspectives on development of the meristem and a promising technique for creating transgenic periclinal chimeras in plants.

The transcription factor HSFA7b controls thermomemory at the shoot apical meristem by regulating ethylene biosynthesis and signaling in Arabidopsis.

The shoot apical meristem (SAM) is responsible for overall shoot growth by generating all aboveground structures. Recent research has revealed that the SAM displays an autonomous heat stress (HS) memory of a previous non-lethal HS event. Considering the importance of the SAM for plant growth, it is essential to determine how its thermomemory is mechanistically controlled. Here, we report that HEAT SHOCK TRANSCRIPTION FACTOR A7b (HSFA7b) plays a crucial role in this process in Arabidopsis, as the absence of functional HSFA7b results in the temporal suppression of SAM activity after thermopriming. We found that HSFA7b directly regulates ethylene response at the SAM by binding to the promoter of the key ethylene signaling gene ETHYLENE-INSENSITIVE 3 to establish thermotolerance. Moreover, we demonstrated that HSFA7b regulates the expression of ETHYLENE OVERPRODUCER 1 (ETO1) and ETO1-LIKE 1, both of which encode ethylene biosynthesis repressors, thereby ensuring ethylene homeostasis at the SAM. Taken together, these results reveal a crucial and tissue-specific role for HSFA7b in thermomemory at the Arabidopsis SAM.

Hormonal and genetic control of pluripotency in bryophyte model systems.

Land plant meristems are reservoirs of pluripotent stem cells where new tissues emerge, grow and eventually differentiate into specific cell identities. Compared to algae, where cells are produced in two-dimensional tissues via tip or marginal growth, land plants have meristems that allow three-dimensional growth for successful exploration of the terrestrial environment. In land plants, meristem maintenance leads to indeterminate growth and the production of new meristems leads to branching or regeneration via reprogramming of wounded somatic cells. Emerging model systems in the haploid dominant and monophyletic bryophytes are allowing comparative analyses of meristem gene regulatory networks to address whether all plants use common or diverse programs to organise, maintain, and regenerate meristems. In this piece we aim to discuss recent advances in genetic and hormonal control of bryophyte meristems and possible convergence or discrepancies in an exciting and emerging field in plant biology.

R-SNARE protein YKT61 mediates root apical meristem cell division via BRASSINOSTEROID-INSENSITIVE1 recycling.

Root growth is sustained by cell division and differentiation of the root apical meristem (RAM), in which brassinosteroid (BR) signaling mediated via the dynamic targeting of BRASSINOSTEROID-INSENSITIVE1 (BRI1) plays complex roles. BRI1 is constitutively secreted to the plasma membrane (PM), internalized, and recycled or delivered into vacuoles, whose PM abundance is critical for BR signaling. Vesicle-target membrane fusion is regulated by heterotetrameric SNARE complexes. SNARE proteins have been implicated in BRI1 targeting, but how SNAREs affect RAM development is unclear. We report that Arabidopsis (Arabidopsis thaliana) YKT61, an atypical R-SNARE protein, is critical for BR-controlled RAM development through the dynamic targeting of BRI1. Functional loss of YKT61 is lethal for both male and female gametophytes. By using weak mutant alleles of YKT61, ykt61-partially complemented (ykt61-pc), we show that YKT61 knockdown results in a reduction of RAM length due to reduced cell division, similar to that in bri1-116. YKT61 physically interacts with BRI1 and is critical for the dynamic recycling of BRI1 to the PM. We further determine that YKT61 is critical for the dynamic biogenesis of vacuoles, for the maintenance of Golgi morphology, and for endocytosis, which may have a broad effect on development. Endomembrane compartments connected via vesicular machinery, such as SNAREs, influence nuclear-controlled cellular activities such as division and differentiation by affecting the dynamic targeting of membrane proteins, supporting a retro-signaling pathway from the endomembrane system to the nucleus.