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.

GIF1 controls ear inflorescence architecture and floral development by regulating key genes in hormone biosynthesis and meristem determinacy in maize.

BACKGROUND: Inflorescence architecture and floral development in flowering plants are determined by genetic control of meristem identity, determinacy, and maintenance. The ear inflorescence meristem in maize (Zea mays) initiates short branch meristems called spikelet pair meristems, thus unlike the tassel inflorescence, the ears lack long branches. Maize growth-regulating factor (GRF)-interacting factor1 (GIF1) regulates branching and size of meristems in the tassel inflorescence by binding to Unbranched3. However, the regulatory pathway of gif1 in ear meristems is relatively unknown. RESULT: In this study, we found that loss-of-function gif1 mutants had highly branched ears, and these extra branches repeatedly produce more branches and florets with unfused carpels and an indeterminate floral apex. In addition, GIF1 interacted in vivo with nine GRFs, subunits of the SWI/SNF chromatin-remodeling complex, and hormone biosynthesis-related proteins. Furthermore, key meristem-determinacy gene RAMOSA2 (RA2) and CLAVATA signaling-related gene CLV3/ENDOSPERM SURROUNDING REGION (ESR) 4a (CLE4a) were directly bound and regulated by GIF1 in the ear inflorescence. CONCLUSIONS: Our findings suggest that GIF1 working together with GRFs recruits SWI/SNF chromatin-remodeling ATPases to influence DNA accessibility in the regions that contain genes involved in hormone biosynthesis, meristem identity and determinacy, thus driving the fate of axillary meristems and floral organ primordia in the ear-inflorescence of maize.

A conserved superlocus regulates above- and belowground root initiation.

Plants continuously form new organs in different developmental contexts in response to environmental cues. Underground lateral roots initiate from prepatterned cells in the main root, but cells can also bypass the root-shoot trajectory separation and generate shoot-borne roots through an unknown mechanism. We mapped tomato (Solanum lycopersicum) shoot-borne root development at single-cell resolution and showed that these roots initiate from phloem-associated cells through a unique transition state. This state requires the activity of a transcription factor that we named SHOOTBORNE ROOTLESS (SBRL). Evolutionary analysis reveals that SBRL's function and cis regulation are conserved in angiosperms and that it arose as an ancient duplication, with paralogs controlling wound-induced and lateral root initiation. We propose that the activation of a common transition state by context-specific regulators underlies the plasticity of plant root systems.

COBL9 and COBL7 synergistically regulate root hair tip growth via controlling apical cellulose deposition.

Root hairs are cylindrical extensions of root epidermal cells that are important for the acquisition of water and minerals, interactions between plant and microbes. The deposition of cell wall materials in the tip enables root hairs to maintain elongation constantly. To date, our knowledge of the regulators that connect the architecture of cell wall and the root hair development remains very limited. Here, we demonstrated that COBL9 and COBL7, two genes of COBRA-Like family in Arabidopsis as well as their counterparts in rice, OsBC1L1 and OsBC1L8, regulate root hair growth. Single mutant cobl9, double mutants cobl7 cobl9 and double mutants osbc1l1 osbc1l8 all displayed prematurely terminated root hair elongation, though at varying levels. COBL7-YFP and COBL9-YFP accumulate prominently in the growing tips of newly emerged root hairs. Furthermore, cobl9, cobl7 cobl9 and osbc1l1 osbc1l8 mutants were defective in the enrichment of cellulose in the tips of the growing root hairs. We also discovered that overexpression of COBL9 could promote root hair elongation and salinity tolerance. Taken together, these results provide compelling evidence that the polarized COBL7 and COBL9 in the tip of the emerging root hairs have conserved roles in regulating root hair development and stress adaptation in dicots and monocots.

Efficient micropropagation of Thunbergia coccinea Wall. and genetic homogeneity assessment through RAPD and ISSR markers.

Thunbergia coccinea Wall. ex D. Don being a rare, ornamental and medicinal plant of India, is needed to propagate for conserving the germplasm and analyzing its phytochemical compounds in the future. A reliable protocol for direct in vitro propagation using nodal shoot meristem of T. coccinea as explant was standardized. The highest number of shoots per explant (22.17 ± 0.54) with maximum shoot length (2.36 ± 0.28) in cm was obtained in Murashige and Skoog (MS) medium supplemented with 9.70 µM of 6-furfurylaminopurine (Kinetin) and 0.053 µM of α-naphthaleneacetic acid (NAA) combination, among all the different plant growth regulators (PGR's) and concentrations tested. The aforesaid PGR's combination was optimum for axillary shoot bud induction and multiplication in T. coccinea. The best rooting was observed on the half-strength MS medium fortified with 2.68 µM NAA with the highest number of roots per shoot (3.75 ± 0.12) and maximum length (5.22 ± 0.32) in cm. All the in vitro raised plantlets were acclimatized in sterile sand and soil mixture (1:1) with a survival rate of 70% on earthen pots under greenhouse conditions. PCR-based RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter-Simple Sequence Repeat) molecular markers were employed to determine the genetic homogeneity amongst the plantlets. Twelve (12) RAPD and nine (9) ISSR primers developed a total of 104 and 91 scorable bands, respectively. The band profiles of micropropagated plantlets were monomorphic to the mother, donor in vivo plant, and similarity values varied from 0.9542-1.000. The dendrogram generated through UPGMA (unweighted pair group method with arithmetic mean) showed 99% similarities amongst all tested plants confirming the genetic uniformity of in vitro raised plants.

Gynoecium and fruit development in Arabidopsis.

Flowering plants produce flowers and one of the most complex floral structures is the pistil or the gynoecium. All the floral organs differentiate from the floral meristem. Various reviews exist on molecular mechanisms controlling reproductive development, but most focus on a short time window and there has been no recent review on the complete developmental time frame of gynoecium and fruit formation. Here, we highlight recent discoveries, including the players, interactions and mechanisms that govern gynoecium and fruit development in Arabidopsis. We also present the currently known gene regulatory networks from gynoecium initiation until fruit maturation.

WUSCHEL-related homeobox family genes in rice control lateral root primordium size.

The development of a plastic root system is essential for stable crop production under variable environments. Rice plants have two types of lateral roots (LRs): S-type (short and thin) and L-type (long, thick, and capable of further branching). LR types are determined at the primordium stage, with a larger primordium size in L-types than S-types. Despite the importance of LR types for rice adaptability to variable water conditions, molecular mechanisms underlying the primordium size control of LRs are unknown. Here, we show that two WUSCHEL-related homeobox (WOX) genes have opposing roles in controlling LR primordium (LRP) size in rice. Root tip excision on seminal roots induced L-type LR formation with wider primordia formed from an early developmental stage. QHB/OsWOX5 was isolated as a causative gene of a mutant that is defective in S-type LR formation but produces more L-type LRs than wild-type (WT) plants following root tip excision. A transcriptome analysis revealed that OsWOX10 is highly up-regulated in L-type LRPs. OsWOX10 overexpression in LRPs increased the LR diameter in an expression-dependent manner. Conversely, the mutation in OsWOX10 decreased the L-type LR diameter under mild drought conditions. The qhb mutants had higher OsWOX10 expression than WT after root tip excision. A yeast one-hybrid assay revealed that the transcriptional repressive activity of QHB was lost in qhb mutants. An electrophoresis mobility shift assay revealed that OsWOX10 is a potential target of QHB. These data suggest that QHB represses LR diameter increase, repressing OsWOX10 Our findings could help improve root system plasticity under variable environments.

Recruitment of an ancient branching program to suppress carpel development in maize flowers.

Carpels in maize undergo programmed cell death in half of the flowers initiated in ears and in all flowers in tassels. The HD-ZIP I transcription factor gene GRASSY TILLERS1 (GT1) is one of only a few genes known to regulate this process. To identify additional regulators of carpel suppression, we performed a gt1 enhancer screen and found a genetic interaction between gt1 and ramosa3 (ra3). RA3 is a classic inflorescence meristem determinacy gene that encodes a trehalose-6-phosphate (T6P) phosphatase (TPP). Dissection of floral development revealed that ra3 single mutants have partially derepressed carpels, whereas gt1;ra3 double mutants have completely derepressed carpels. Surprisingly, gt1 suppresses ra3 inflorescence branching, revealing a role for gt1 in meristem determinacy. Supporting these genetic interactions, GT1 and RA3 proteins colocalize to carpel nuclei in developing flowers. Global expression profiling revealed common genes misregulated in single and double mutant flowers, as well as in derepressed gt1 axillary meristems. Indeed, we found that ra3 enhances gt1 vegetative branching, similar to the roles for the trehalose pathway and GT1 homologs in the eudicots. This functional conservation over ∼160 million years of evolution reveals ancient roles for GT1-like genes and the trehalose pathway in regulating axillary meristem suppression, later recruited to mediate carpel suppression. Our findings expose hidden pleiotropy of classic maize genes and show how an ancient developmental program was redeployed to sculpt floral form.

Inhibition of ribosome biogenesis by actinomycin D affects Arabidopsis root development.

Actinomycin D has been reported to selectively inhibit rRNA synthesis and ribosome biogenesis, induce G2 checkpoint of cell cycle arrest in HeLa cells. In Arabidopsis, actinomycin D was also used as agent to preferentially inhibit the ribosome biosynthesis and ribosomal function. However, the function of actinomycin D on Arabidopsis root development remains to be elucidated. In this study, we exposed Arabidopsis seedlings to actinomycin D with the aim of evaluating the effects of ribosome biogenesis on root development. The results demonstrated that actinomycin D inhibited Arabidopsis root growth by reduced meristematic activity in a dose dependent manner. Exposure to actinomycin D decreased the expression of WOX5 and key stem cell niche-defining transcription factors SHR and PLT1, thus the loss function of QC identity and stem cell niche maintenance. In addition, dead cells were observed after actinomycin D treatment in root stele initials and DNA damage response was constitutively activated. Collectively, we propose that ribosome biogenesis plays key role in primary root growth through maintenance of root stem cell niche and DNA damage response in Arabidopsis.

Plasticity of bud outgrowth varies at cauline and rosette nodes in Arabidopsis thaliana.

Shoot branching is a complex mechanism in which secondary shoots grow from buds that are initiated from meristems established in leaf axils. The model plant Arabidopsis (Arabidopsis thaliana) has a rosette leaf growth pattern in the vegetative stage. After flowering initiation, the main stem elongates with the top leaf primordia developing into cauline leaves. Meristems in Arabidopsis initiate in the axils of rosette or cauline leaves, giving rise to rosette or cauline buds, respectively. Plasticity in the process of shoot branching is regulated by resource and nutrient availability as well as by plant hormones. However, few studies have attempted to test whether cauline and rosette branching are subject to the same plasticity. Here, we addressed this question by phenotyping cauline and rosette branching in three Arabidopsis ecotypes and several Arabidopsis mutants with varied shoot architectures. Our results showed no negative correlation between cauline and rosette branch numbers in Arabidopsis, demonstrating that there is no tradeoff between cauline and rosette bud outgrowth. Through investigation of the altered branching pattern of flowering pathway mutants and Arabidopsis ecotypes grown in various photoperiods and light regimes, we further elucidated that the number of cauline branches is closely related to flowering time. The number of rosette branches has an enormous plasticity compared with cauline branches and is influenced by genetic background, flowering time, light intensity, and temperature. Our data reveal different levels of plasticity in the regulation of branching at rosette and cauline nodes, and promote a framework for future branching analyses.

Strigo-D2-a bio-sensor for monitoring spatio-temporal strigolactone signaling patterns in intact plants.

Strigolactones (SLs) are a class of plant hormones that mediate biotic interactions and modulate developmental programs in response to endogenous and exogenous stimuli. However, a comprehensive view on the spatio-temporal pattern of SL signaling has not been established, and tools for a systematic in planta analysis do not exist. Here, we present Strigo-D2, a genetically encoded ratiometric SL signaling sensor that enables the examination of SL signaling distribution at cellular resolution and is capable of rapid response to altered SL levels in intact Arabidopsis (Arabidopsis thaliana) plants. By monitoring the abundance of a truncated and fluorescently labeled SUPPRESSOR OF MAX2 1-LIKE 6 (SMXL6) protein, a proteolytic target of the SL signaling machinery, we show that all cell types investigated have the capacity to respond to changes in SL levels but with very different dynamics. In particular, SL signaling is pronounced in vascular cells but low in guard cells and the meristematic region of the root. We also show that other hormones leave Strigo-D2 activity unchanged, indicating that initial SL signaling steps work in isolation from other hormonal signaling pathways. The specificity and spatio-temporal resolution of Strigo-D2 underline the value of the sensor for monitoring SL signaling in a broad range of biological contexts with highly instructive analytical depth.

Early sensing of phosphate deprivation triggers the formation of extra root cap cell layers via SOMBRERO through a process antagonized by auxin signaling.

KEY MESSAGE: The role of the root cap in the plant response to phosphate deprivation has been scarcely investigated. Here we describe early structural, physiological and molecular changes prior to the determinate growth program of the primary roots under low Pi and unveil a critical function of the transcription factor SOMBRERO in low Pi sensing. Mineral nutrient distribution in the soil is uneven and roots efficiently adapt to improve uptake and assimilation of sparingly available resources. Phosphate (Pi) accumulates in the upper layers and thus short and branched root systems proliferate to better exploit organic and inorganic Pi patches. Here we report an early adaptive response of the Arabidopsis primary root that precedes the entrance of the meristem into the determinate developmental program that is a hallmark of the low Pi sensing mechanism. In wild-type seedlings transferred to low Pi medium, the quiescent center domain in primary root tips increases as an early response, as revealed by WOX5:GFP expression and this correlates with a thicker root tip with extra root cap cell layers. The halted primary root growth in WT seedlings could be reversed upon transfer to medium supplemented with 250 µM Pi. Mutant and gene expression analysis indicates that auxin signaling negatively affects the cellular re-specification at the root tip and enabled identification of the transcription factor SOMBRERO as a critical element that orchestrates both the formation of extra root cap layers and primary root growth under Pi scarcity. Moreover, we provide evidence that low Pi-induced root thickening or the loss-of-function of SOMBRERO is associated with expression of phosphate transporters at the root tip. Our data uncover a developmental window where the root tip senses deprivation of a critical macronutrient to improve adaptation and surveillance.

Tissue-specific transcriptomics reveal functional differences in floral development.

Flowers are produced by floral meristems, groups of stem cells that give rise to floral organs. In grasses, including the major cereal crops, flowers (florets) are contained in spikelets, which contain one to many florets, depending on the species. Importantly, not all grass florets are developmentally equivalent, and one or more florets are often sterile or abort in each spikelet. Members of the Andropogoneae tribe, including maize (Zea mays), produce spikelets with two florets; the upper and lower florets are usually dimorphic, and the lower floret is greatly reduced compared to the upper floret. In maize ears, early development appears identical in both florets but the lower floret ultimately aborts. To gain insight into the functional differences between florets with different fates, we used laser capture microdissection coupled with RNA-sequencing to globally examine gene expression in upper and lower floral meristems in maize. Differentially expressed genes were involved in hormone regulation, cell wall, sugar, and energy homeostasis. Furthermore, cell wall modifications and sugar accumulation differed between the upper and lower florets. Finally, we identified a boundary domain between upper and lower florets, which we hypothesize is important for floral meristem activity. We propose a model in which growth is suppressed in the lower floret by limiting sugar availability and upregulating genes involved in growth repression. This growth repression module may also regulate floret fertility in other grasses and potentially be modulated to engineer more productive cereal crops.

Glutaredoxins regulate maize inflorescence meristem development via redox control of TGA transcriptional activity.

Glutaredoxins (GRXs) are small oxidoreductases that can modify target protein activities through control of the redox (reduction/oxidation) state by reducing or glutathionylating disulfide bridges. Although CC-type GRXs are plant specific and play important roles in many processes, the mechanisms by which they modulate the activity of target proteins in vivo are unknown. In this study, we show that a maize CC-type GRX, MALE STERILE CONVERTED ANTHER1 (MSCA1), acts redundantly with two paralogues, ZmGRX2 and ZmGRX5, to modify the redox state and the activity of its putative target, the TGA transcription factor FASCIATED EAR4 (FEA4) that acts as a negative regulator of inflorescence meristem development. We used CRISPR-Cas9 to create a GRX triple knockout, resulting in severe suppression of meristem, ear and tassel growth and reduced plant height. We further show that GRXs regulate the redox state, DNA accessibility and transcriptional activities of FEA4, which acts downstream of MSCA1 and its paralogues to control inflorescence development. Our findings reveal the function of GRXs in meristem development, and also provide direct evidence for GRX-mediated redox modification of target proteins in plants.

Flower meristem maintenance by TILLERS ABSENT 1 is essential for ovule development in rice.

Plant development depends on the activity of pluripotent stem cells in meristems, such as the shoot apical meristem and the flower meristem. In Arabidopsis thaliana, WUSCHEL (WUS) is essential for stem cell homeostasis in meristems and integument differentiation in ovule development. In rice (Oryza sativa), the WUS ortholog TILLERS ABSENT 1 (TAB1) promotes stem cell fate in axillary meristem development, but its function is unrelated to shoot apical meristem maintenance in vegetative development. In this study, we examined the role of TAB1 in flower development. The ovule, which originates directly from the flower meristem, failed to differentiate in tab1 mutants, suggesting that TAB1 is required for ovule formation. Expression of a stem cell marker was completely absent in the flower meristem at the ovule initiation stage, indicating that TAB1 is essential for stem cell maintenance in the 'final' flower meristem. The ovule defect in tab1 was partially rescued by floral organ number 2 mutation, which causes overproliferation of stem cells. Collectively, it is likely that TAB1 promotes ovule formation by maintaining stem cells at a later stage of flower development.

The Pellicle-Another Strategy of the Root Apex Protection against Mechanical Stress?

In grasses, the apical part of the root is covered by a two-layered deposit of extracellular material, the pellicle, which together with the outer periclinal wall of protodermal cells forms the three-layered epidermal surface. In this study, the effect of mechanical stress on the pellicle was examined. An experiment was performed, in which maize roots were grown in narrow diameter plastic tubes with conical endings for 24 h. Two groups of experimental roots were included in the analysis: stressed (S) roots, whose tips did not grow out of the tubes, and recovering (R) roots, whose apices grew out of the tube. Control (C) roots grew freely between the layers of moist filter paper. Scanning electron microscopy and confocal microscopy analysis revealed microdamage in all the layers of the epidermal surface of S roots, however, protodermal cells in the meristematic zone remained viable. The outermost pellicle layer was twice as thick as in C roots. In R roots, large areas of dead cells were observed between the meristematic zone and the transition zone. The pellicle was defective with a discontinuous and irregular outermost layer. In the meristematic zone the pellicle was undamaged and the protodermal cells were intact. The results lead to the conclusion that the pellicle may prevent damage to protodermal cells, thus protecting the root apical meristem from the negative effects of mechano-stress.

Evolution of RGF/GLV/CLEL Peptide Hormones and Their Roles in Land Plant Growth and Regulation.

Rooting is a key innovation during plant terrestrialization. RGFs/GLVs/CLELs are a family of secreted peptides, playing key roles in root stem cell niche maintenance and pattern formation. The origin of this peptide family is not well characterized. RGFs and their receptor genes, RGIs, were investigated comprehensively using phylogenetic and genetic analyses. We identified 203 RGF genes from 24 plant species, representing a variety of land plant lineages. We found that the RGF genes originate from land plants and expand via multiple duplication events. The lineage-specific RGF duplicates are retained due to their regulatory divergence, while a majority of RGFs experienced strong purifying selection in most land plants. Functional analysis indicated that RGFs and their receptor genes, RGIs, isolated from liverwort, tomato, and maize possess similar biological functions with their counterparts from Arabidopsis in root development. RGFs and RGIs are likely coevolved in land plants. Our studies shed light on the origin and functional conservation of this important peptide family in plant root development.

The role of auxin and sugar signaling in dominance inhibition of inflorescence growth by fruit load.

Dominance inhibition of shoot growth by fruit load is a major factor that regulates shoot architecture and limits yield in agriculture and horticulture crops. In annual plants, the inhibition of inflorescence growth by fruit load occurs at a late stage of inflorescence development termed the end of flowering transition. Physiological studies show this transition is mediated by production and export of auxin from developing fruits in close proximity to the inflorescence apex. In the meristem, cessation of inflorescence growth is controlled in part by the age-dependent pathway, which regulates the timing of arrest. Here, we show the end of flowering transition is a two-step process in Arabidopsis (Arabidopsis thaliana). The first stage is characterized by a cessation of inflorescence growth, while immature fruit continues to develop. At this stage, dominance inhibition of inflorescence growth by fruit load is associated with a selective dampening of auxin transport in the apical region of the stem. Subsequently, an increase in auxin response in the vascular tissues of the apical stem where developing fruits are attached marks the second stage for the end of flowering transition. Similar to the vegetative and floral transition, the end of flowering transition is associated with a change in sugar signaling and metabolism in the inflorescence apex. Taken together, our results suggest that during the end of flowering transition, dominance inhibition of inflorescence shoot growth by fruit load is mediated by auxin and sugar signaling.

CLAVATA3 mediated simultaneous control of transcriptional and post-translational processes provides robustness to the WUSCHEL gradient.

Regulation of the homeodomain transcription factor WUSCHEL concentration is critical for stem cell homeostasis in Arabidopsis shoot apical meristems. WUSCHEL regulates the transcription of CLAVATA3 through a concentration-dependent activation-repression switch. CLAVATA3, a secreted peptide, activates receptor kinase signaling to repress WUSCHEL transcription. Considering the revised regulation, CLAVATA3 mediated repression of WUSCHEL transcription alone will lead to an unstable system. Here we show that CLAVATA3 signaling regulates nuclear-cytoplasmic partitioning of WUSCHEL to control nuclear levels and its diffusion into adjacent cells. Our work also reveals that WUSCHEL directly interacts with EXPORTINS via EAR-like domain which is also required for destabilizing WUSCHEL in the cytoplasm. We develop a combined experimental and computational modeling approach that integrates CLAVATA3-mediated transcriptional repression of WUSCHEL and post-translational control of nuclear levels with the WUSCHEL concentration-dependent regulation of CLAVATA3. We show that the dual control by the same signal forms a seamless connection between de novo WUSCHEL synthesis and sub-cellular partitioning in providing robustness to the WUSCHEL gradient.