Maize Root Apex Research Articles

Mn3 O4 Nanoparticles Alleviate ROS-Inhibited Root Apex Mitosis Activities to Improve Maize Drought Tolerance.

Poly (acrylic) acid coated Mn3O4 nanoparticles (PAA@Mn3 O4 nanoparticles (PMO, 11.02 nm, -28.93 mV)) are synthesized to investigate whether they can help to improve maize drought tolerance and the relevant mechanisms behind this. In planta experimental results show that under drought (15% PEG 6000, polyethylene glycol, mimicking drought stress, 96 h), compared with the control plants, 500 mg L-1 PMO (root application, 96 h) improves maize drought tolerance, showing an increase of root length (21.6%), shoot length (21.2%), fresh weight (7.8%) and total protein (67.2%) content. In addition, PMO significantly decreases the malondialdehyde (MDA) content by 74.7% in maize under drought, compared with the control group. Further, PMO treated maize root apex shows significantly increased mitotic index (MI, 35.5%), and decreased hydrogen peroxide (40.9%). Compared with the control under drought (15% PEG, 96 h), thr root apex of maize plants treated with PMO (500 mg L-1 , root application, 96 h) have significantly lower level of H2 O2 . Overall, the results show that PMO can alleviate drought-inhibited cell mitosis activities via maintaining ROS (reactive oxygen species) homeostasis. In this study, it is not only shown that PMO can be a good nano-regulator candidate to improve maize drought tolerance, but also that PMO has potential to modulate plant cell mitosis activities.

Transition zone cells reach G2 phase before initiating elongation in maize root apex.

ABSTRACTRoot elongation requires cell divisions in the meristematic zone and cell elongation in the elongation zone. The boundary between dividing and elongating cells is called the transition zone. In the meristem zone, initial cells are continuously dividing, but on the basal side of the meristem cells exit the meristem through the transition zone and enter in the elongation zone, where they stop division and rapidly elongate. Throughout this journey cells are accompanied by changes in cell cycle progression. Flow cytometry analysis showed that meristematic cells are in cycle, but exit when they enter the elongation zone. In addition, the percentage of cells in G2 phase (4C) strongly increased from the meristem to the elongation zone. However, we did not observe remarkable changes in the percentage of cells in cell cycle phases along the entire elongation zone. These results suggest that meristematic cells in maize root apex stop the cell cycle in G2 phase after leaving the meristem.

Nitric Oxide-Mediated Maize Root Apex Responses to Nitrate are Regulated by Auxin and Strigolactones.

Nitrate (NO3-) is a key element for crop production but its levels in agricultural soils are limited. Plants have developed mechanisms to cope with these NO3- fluctuations based on sensing nitrate at the root apex. Particularly, the transition zone (TZ) of root apex has been suggested as a signaling-response zone. This study dissects cellular and molecular mechanisms underlying NO3- resupply effects on primary root (PR) growth in maize, confirming nitric oxide (NO) as a putative modulator. Nitrate restoration induced PR elongation within the first 2 h, corresponding to a stimulation of cell elongation at the basal border of the TZ. Xyloglucans (XGs) immunolocalization together with Brefeldin A applications demonstrated that nitrate resupply induces XG accumulation. This effect was blocked by cPTIO (NO scavenger). Transcriptional analysis of ZmXET1 confirmed the stimulatory effect of nitrate on XGs accumulation in cells of the TZ. Immunolocalization analyses revealed a positive effect of nitrate resupply on auxin and PIN1 accumulation, but a transcriptional regulation of auxin biosynthesis/transport/signaling genes was excluded. Short-term nitrate treatment repressed the transcription of genes involved in strigolactones (SLs) biosynthesis and transport, mainly in the TZ. Enhancement of carotenoid cleavage dioxygenases (CCDs) transcription in presence of cPTIO indicated endogenous NO as a negative modulator of CCDs activity. Finally, treatment with the SLs-biosynthesis inhibitor (TIS108) restored the root growth in the nitrate-starved seedlings. Present report suggests that the NO-mediated root apex responses to nitrate are accomplished in cells of the TZ via integrative actions of auxin, NO and SLs.

Nitrate sensing by the maize root apex transition zone: a merged transcriptomic and proteomic survey.

Nitrate is an essential nutrient for plants, and crops depend on its availability for growth and development, but its presence in agricultural soils is far from stable. In order to overcome nitrate fluctuations in soil, plants have developed adaptive mechanisms allowing them to grow despite changes in external nitrate availability. Nitrate can act as both nutrient and signal, regulating global gene expression in plants, and the root tip has been proposed as the sensory organ. A set of genome-wide studies has demonstrated several nitrate-regulated genes in the roots of many plants, although only a few studies have been carried out on distinct root zones. To unravel new details of the transcriptomic and proteomic responses to nitrate availability in a major food crop, a double untargeted approach was conducted on a transition zone-enriched root portion of maize seedlings subjected to differing nitrate supplies. The results highlighted a complex transcriptomic and proteomic reprogramming that occurs in response to nitrate, emphasizing the role of this root zone in sensing and transducing nitrate signal. Our findings indicated a relationship of nitrate with biosynthesis and signalling of several phytohormones, such as auxin, strigolactones, and brassinosteroids. Moreover, the already hypothesized involvement of nitric oxide in the early response to nitrate was confirmed with the use of nitric oxide inhibitors. Our results also suggested that cytoskeleton activation and cell wall modification occurred in response to nitrate provision in the transition zone.

The electrical network of maize root apex is gravity dependent.

Investigations carried out on maize roots under microgravity and hypergravity revealed that gravity conditions have strong effects on the network of plant electrical activity. Both the duration of action potentials (APs) and their propagation velocities were significantly affected by gravity. Similarly to what was reported for animals, increased gravity forces speed-up APs and enhance synchronized electrical events also in plants. The root apex transition zone emerges as the most active, as well as the most sensitive, root region in this respect.

The carbohydrate distribution on maize root apex in early growth stages

The carbohydrate distribution on maize root apex in early growth stages

Nitric oxide modulates dynamic actin cytoskeleton and vesicle trafficking in a cell type-specific manner in root apices.

NO is an important regulatory molecule in eukaryotes. Much of its effect is ascribed to the action of NO as a signalling molecule. However, NO can also directly modify proteins thus affecting their activities. Although the signalling functions of NO are relatively well recognized in plants, very little is known about its potential influence on the structural integrity of plant cells. In this study, the reorganization of the actin cytoskeleton, and the recycling of wall polysaccharides in plants via the endocytic pathway in the presence of NO or NO-modulating substances were analysed. The actin cytoskeleton and endocytosis in maize (Zea mays) root apices were visualized with fluorescence immunocytochemistry. The organization of the actin cytoskeleton is modulated via NO levels and the extent of such modulation is cell-type specific. In endodermis cells, actin cables change their orientation from longitudinal to oblique and cellular cross-wall domains become actin-depleted/depolymerized. The reaction is reversible and depends on the type of NO donor. Actin-dependent vesicle trafficking is also affected. This was demonstrated through the analysis of recycled wall material transported to newly-formed cell plates and BFA compartments. Therefore, it is concluded that, in plant cells, NO affects the functioning of the actin cytoskeleton and actin-dependent processes. Mechanisms for the reorganization of the actin cytoskeleton are cell-type specific, and such rearrangements might selectively impinge on the functioning of various cellular domains. Thus, the dynamic actin cytoskeleton could be considered as a downstream effector of NO signalling in cells of root apices.

Actin Turnover-Mediated Gravity Response in Maize Root Apices

The dynamic actin cytoskeleton has been proposed to be linked to gravity sensing in plants but the mechanistic understanding of these processes remains unknown. We have performed detailed pharmacological analyses of the role of the dynamic actin cytoskeleton in gravibending of maize (Zea mays) root apices. Depolymerization of actin filaments with two drugs having different mode of their actions, cytochalasin D and latrunculin B, stimulated root gravibending. By contrast, drug-induced stimulation of actin polymerization and inhibition of actin turnover, using two different agents phalloidin and jasplakinolide, compromised the root gravibending. Importantly, all these actin drugs inhibited root growth to similar extent suggesting that high actin turnover is essential for the gravity-related growth responses rather than for the general growth processes. Both latrunculin B and cytochalasin D treatments inhibited root growth but restored gravibending of the decapped root apices, indicating that there is a strong potential for effective actin-mediated gravity sensing outside the cap. This elusive gravity sensing outside the root cap is dependent not only on the high rate of actin turnover but also on weakening of myosin activities, as general inhibition of myosin ATPases induced stimulation of gravibending of the decapped root apices. Collectively, these data provide evidence for the actin turnover-mediated gravity sensing outside the root cap.

Localization of aluminium in the maize root apex: can morin detect cell wall-bound aluminium?

Morin is a fluorochrome which forms a fluorescent complex with aluminium (Al) and is thus used to localize Al in plant tissues. However, reports about the cellular distribution of Al-apoplastic versus symplastic-based on morin staining are often conflicting. The objective of this work was to investigate whether Al localization with morin staining can show the proper cellular distribution of Al. Fresh root cross-sections were made from root apices of maize (cv. Lixis) treated with 25 muM Al for 6 h and stained with morin. Fluorescence microscopic investigation showed Al-morin fluorescence in the cytosol, but not in the cell wall. This is in contrast to the growing evidence which shows that Al mainly accumulates in the cell wall, especially bound to the pectin matrix. Therefore, in vitro analyses were carried out to study whether morin can form a fluorescent complex with Al, which is bound to pectin, cell wall, and other Al-binding ligands such as phosphate, galacturonate, DNA, and ATP. Compared with the control treatment without Al-binding ligands, fluorescence intensity was reduced by about 10-fold in the presence of pectin and isolated cell walls, but fairly unaffected in the presence of phosphate and galacturonate. Al associated with DNA and ATP also formed a fluorescent complex with morin. This implies that, although Al is mainly accumulated in the cell wall, it cannot be detected with morin as it is tightly bound to cell-wall pectin. Thus, morin staining should not be used to study the distribution of Al between cell compartments.

Actin-dependent fluid-phase endocytosis in inner cortex cells of maize root apices.

The fluorescent dye Lucifer Yellow (LY) is a well-known and widely-used marker for fluid-phase endocytosis. In this paper, both light and electron microscopy revealed that LY was internalized into transition zone cells of the inner cortex of intact maize root apices. The internalized LY was localized within tubulo-vesicular compartments invaginating from the plasma membrane at actomyosin-enriched pit-fields and individual plasmodesmata, as well as within adjacent small peripheral vacuoles. The internalization of LY was blocked by pretreating the roots with the F-actin depolymerizing drug latrunculin B, but not with the F-actin stabilizer jasplakinolide. F-actin enriched plasmodesmata and pit-fields of the inner cortex also contain abundant plant-specific unconventional class VIII myosin(s). In addition, 2,3 butanedione monoxime, a general inhibitor of myosin ATPases, partially inhibited the uptake of LY into cells of the inner cortex. Conversely, loss of microtubules did not inhibit fluid-phase endocytosis of LY into these cells. In conclusion, specialized actin- and myosin VIII-enriched membrane domains perform a tissue-specific form of fluid-phase endocytosis in maize root apices. The possible physiological relevance of this process is discussed.

Short‐term boron deprivation enhances levels of cytoskeletal proteins in maize, but not zucchini, root apices

By using indirect immunofluorescence microscopy and Western blot analysis, we have demonstrated increased levels of actin and tubulin proteins as well as an altered polymerization pattern of their cytoskeletal assemblies in maize, but not zucchini root apices, as a response to early boron deprivation. Northern blot analysis, however, did not show significant increases in the amount of steady‐state mRNAs of actin and tubulin. This finding indicates that these rapid cytoskeletal responses to boron removal are very likely regulated at translational/post‐translational levels. Interestingly, these increased levels of cytoskeletal proteins coincided well with a reduction in the water‐extractable, but not with the cell wall‐bound, fraction of boron. This implicates that free boric acid, or other more labile boron complexes, might be involved in the activation of cytoskeletal responses in maize root apices. In fact, our experimental approach revealed that maize was suffering from boron deprivation as early as zucchini. This was evidenced by its slightly reduced root elongation rate recorded within 3–5 h of boron deprivation. Importantly, however, maize roots can recover from this early inhibition indicating an effective adaptation mechanism. In contrast, zucchini roots apparently lack this boron‐deprivation response pathway and suffer extensively when exposed to boron‐free environment. This leads to the tentative working hypothesis for an adaptive mechanism of maize roots to boron deprivation by enhancing its cytoskeletal protein levels and altering their polymerization patterns in order to mechanically reinforce the cell periphery complex of their cells. This testable hypothesis requires further experimental verification.

Short-term boron deprivation inhibits endocytosis of cell wall pectins in meristematic cells of maize and wheat root apices.

By using immunofluorescence microscopy, we observed rapidly altered distribution patterns of cell wall pectins in meristematic cells of maize (Zea mays) and wheat (Triticum aestivum) root apices. This response was shown for homogalacturonan pectins characterized by a low level (up to 40%) of methylesterification and for rhamnogalacturonan II pectins cross-linked by a borate diol diester. Under boron deprivation, abundance of these pectins rapidly increased in cell walls, whereas their internalization was inhibited, as evidenced by a reduced and even blocked accumulation of these cell wall pectins within brefeldin A-induced compartments. In contrast, root cells of species sensitive to the boron deprivation, like zucchini (Cucurbita pepo) and alfalfa (Medicago sativa), do not internalize cell wall pectins into brefeldin A compartments and do not show accumulation of pectins in their cell walls under boron deprivation. For maize and wheat root apices, we favor an apoplastic target for the primary action of boron deprivation, which signals deeper into the cell via endocytosis-mediated pectin signaling along putative cell wall-plasma membrane-cytoskeleton continuum.

F-actin-dependent endocytosis of cell wall pectins in meristematic root cells. Insights from brefeldin A-induced compartments.

Brefeldin A (BFA) inhibits exocytosis but allows endocytosis, making it a valuable agent to identify molecules that recycle at cell peripheries. In plants, formation of large intracellular compartments in response to BFA treatment is a unique feature of some, but not all, cells. Here, we have analyzed assembly and distribution of BFA compartments in development- and tissue-specific contexts of growing maize (Zea mays) root apices. Surprisingly, these unique compartments formed only in meristematic cells of the root body. On the other hand, BFA compartments were absent from secretory cells of root cap periphery, metaxylem cells, and most elongating cells, all of which are active in exocytosis. We report that cell wall pectin epitopes counting rhamnogalacturonan II dimers cross-linked by borate diol diester, partially esterified (up to 40%) homogalacturonan pectins, and (1-->4)-beta-D-galactan side chains of rhamnogalacturonan I were internalized into BFA compartments. In contrast, Golgi-derived secretory (esterified up to 80%) homogalacturonan pectins localized to the cytoplasm in control cells and did not accumulate within characteristic BFA compartments. Latrunculin B-mediated depolymerization of F-actin inhibited internalization and accumulation of cell wall pectins within intracellular BFA compartments. Importantly, cold treatment and protoplasting prevented internalization of wall pectins into root cells upon BFA treatment. These observations suggest that cell wall pectins of meristematic maize root cells undergo rapid endocytosis in an F-actin-dependent manner.

Mastoparan alters subcellular distribution of profilin and remodels F-actin cytoskeleton in cells of maize root apices.

Indirect immunofluorescence localization of profilin in cells of maize root apices revealed that this abundant protein was present both in the cytoplasm and within nuclei. Nucleo-cytoplasmic partitioning of profilin exhibits tissue-specific and developmental features. Mastoparan-mediated activation of heterotrimeric G-proteins, presumably through triggering a phosphoinositide-signaling pathway based on phosphatidylinositol-4,5-bisphosphate (PIP(2)), induced relocalization of profilin from nuclei into the cytoplasm of root apex cells. In contrast, PIP(2) accumulated within nuclei of mastoparan-treated root cells. Intriguingly, cytoplasmic accumulation of profilin was associated with remodeling of F-actin arrays in root apex cells. Specifically, dense F-actin networks were dismantled and distinct actin patches became associated with the periphery of small vacuoles. On the other hand, disruption of F-actin with the G-actin sequestering agent latrunculin B does not affect the subcellular distribution of profilin or PIP(2). These data suggest that nuclear profilin can mediate a stimulus-response action on the actin cytoskeleton which is somehow linked to a phosphoinositide-signaling cascade.

A Polarity Crossroad in the Transition Growth Zone of Maize Root Apices: Cytoskeletal and Developmental Implications

Due to their simple and regular anatomy, root apices represent a unique model object for studying growth, polarity, and morphogenesis. This advantageous anatomy has been exploited to characterize the developmental changes that occur as root cells progress from their origin in the meristem up to their final nongrowing state at the proximal limit of the elongation region. A new growth region located between the apical meristem and the distal portion of the region of rapid cell elongation was discovered and designated as the ‘transition zone.’ Cells of this zone accomplish a developmental transition recently from cytoplasmically driven expansion to vacuome-driven elongation. Cells traversing the transition zone use cytoskeletal elements to regulate both growth polarity and the maintenance of cellular growth per se. Transition zone cells are also sensitive to diverse endogenous clues and exogenous factors such as auxin, ethylene, extracellular calcium, mechanical pressure, aluminum, and microorganisms. This high sensitivity of transition zone cells, which are not engaged in mitotic divisions, seems to be related to their specific cytoarchitecture whereby postmitotic nuclei occupy a central position within the cell, with their radial perinuclear microtubules extending to the cell periphery. Future studies are challenged to identify genes and proteins that determine the various sensory behaviors of cells in this transitional phase of development, and which, in turn, drive directed growth responses (tropisms) of root apices in response to diverse external stimuli.

A patch-clamp study on the physiology of aluminum toxicity and aluminum tolerance in maize. Identification and characterization of Al(3+)-induced anion channels.

The presence of Al(3+) in the rhizosphere induces citrate efflux from the root apex of the Al-tolerant maize (Zea mays) hybrid South American 3, consequently chelating and reducing the activity of toxic Al(3+) at the root surface. Because citrate is released from root apical cells as the deprotonated anion, we used the patch-clamp technique in protoplasts isolated from the terminal 5 mm of the root to study the plasma membrane ion transporters that could be involved in Al-tolerance and Al-toxicity responses. Acidification of the extracellular environment stimulated inward K(+) currents while inhibiting outward K(+) currents. Addition of extracellular Al(3+) inhibited the remaining K(+) outward currents, blocked the K(+) inward current, and caused the activation of an inward Cl(-) current (anion efflux). Studies with excised membrane patches revealed the existence of Al-dependent anion channels, which were highly selective for anions over cations. Our success in activating this channel with extracellular Al(3+) in membrane patches excised prior to any Al(3+) exposure indicates that the machinery required for Al(3+) activation of this channel, and consequently the whole root Al(3+) response, is localized to the root-cell plasma membrane. This Al(3+)-activated anion channel may also be permeable to organic acids, thus mediating the Al-tolerance response (i.e. Al-induced organic acid exudation) observed in intact maize root apices.

Tissue-specific subcellular immunolocalization of a myosin-like protein in maize root apices

Using a heterologous myosin antibody raised against the whole molecule of bovine muscle myosin, we have identified a myosin-like protein in maize. Immunoblots of subcellular fractions isolated from roots identified one distinct band at about 210 kDa in the microsomal protein fraction and one band at about 180 kDa in the soluble protein fraction. Indirect immunofluorescence was performed using maize root apex sections to reveal endocellular distributions of the myosin-like protein. Both diffuse and particulate labelling patterns were observed throughout the cytoplasm of all root cells. In mitotic cells, myosin-like protein was excluded from spindle regions. Amyloplast surfaces were labelled prominently in cells of the root cap statenchyma and in all root cortex cells. On the other hand, myosin-like protein was prominently enriched at cellular peripheries in cells of the pericycle and outer stele in the form of continuous peripheral labelling. From all root apex tissues, phloem elements showed the most abundant presence of myosinlike protein.

Effects of myosin ATPase inhibitor 2,3-butanedione 2-monoxime on distributions of myosins, F-actin, microtubules, and cortical endoplasmic reticulum in maize root apices.

2,3-Butanedione 2-monoxime (BDM) is a general inhibitor of myosin ATPases of eukaryotic cells, and its effects on animal and yeast cells are well described. Using immunofluorescence and electron microscopy, we have analyzed the impacts of BDM on distributions of plant myosins, actin filaments (AFs), microtubules (MTs), and cortical endoplasmic reticulum (ER) elements in various cell types of maize root apices. Treatment of growing maize roots with BDM altered the typical distribution patterns of unconventional plant myosin VIII and of putative maize homologue(s) of myosin II. This pharmacological agent also induced a broad range of impacts on AFs and on cortical ER elements associated with plasmodesmata and pit fields. BDM-mediated effects on the actomyosin cytoskeleton were especially pronounced in cells of the root transition zone. Additionally, BDM elicited distinct reactions in the MT cytoskeleton; endoplasmic MTs vanished in all cells of the transition zone and cortical MTs assembled in increased amounts preferentially at plasmodesmata and pit-fields. Our data indicate that AFs and MTs interact together via BDM-sensitive plant myosins, which can be considered as putative integrators of the plant cytoskeleton. Morphometric analysis revealed that cell growth was prominently inhibited in the transition zone and the apical part, but not the central part, of the elongation region. Obviously, myosin-based contractility of the actin cytoskeleton is essential for the developmental progression of root cells through the transition zone.

Genotypical differences in aluminum resistance of maize are expressed in the distal part of the transition zone. Is reduced basipetal auxin flow involved in inhibition of root elongation by aluminum?

Short-term Al treatment (90 microM Al at pH 4.5 for 1 h) of the distal transition zone (DTZ; 1-2 mm from the root tip), which does not contribute significantly to root elongation, inhibited root elongation in the main elongation zone (EZ; 2.5-5 mm from the root tip) to the same extent as treatment of the entire maize (Zea mays) root apex. Application of Al to the EZ had no effect on root elongation. Higher genotypical resistance to Al applied to the entire root apex, and specifically to the DTZ, was expressed by less inhibition of root elongation, Al accumulation, and Al-induced callose formation, primarily in the DTZ. A characteristic pH profile along the surface of the root apex with a maximum of pH 5.3 in the DTZ was demonstrated. Al application induced a substantial flattening of the pH profile moreso in the Al-sensitive than in the Al-resistant cultivar. Application of indole-3-acetic acid to the EZ but not to the meristematic zone significantly alleviated the inhibition of root elongation induced by the application of Al to the DTZ. Basipetal transport of exogenously applied [(3)H]indole-3-acetic acid to the meristematic zone was significantly inhibited by Al application to the DTZ in the Al-sensitive maize cv Lixis. Our results provide evidence that the primary mechanisms of genotypical differences in Al resistance are located within the DTZ, and suggest a signaling pathway in the root apex mediating the Al signal between the DTZ and the EZ through basipetal auxin transport.

Molecular cloning and characterisation of a maize cDNA for a homologue of the large subunit of the eukaryotic initiation factor 3 (eIF3).

In order to identify genes that are specifically expressed in distinct cell populations of the maize root apex, we have constructed PCR-directed cDNA libraries from microdissected populations of cells, and screened them by differential hybridisation. A meristem-specific cDNA was isolated and characterised. This cDNA, termed ZmeIF3A, encodes a protein homologous to the large subunit of the eukaryotic translation Initiation Factor 3 (eIF3), which is an essential multi-protein complex for the initiation of protein synthesis. The ZmeIF3A protein is most similar to the yeast homologue RPG1, lacking the repeated C-terminal domain characteristic of its mammalian counterparts. However, despite this similarity, it fails to replace the RPG1 protein in complementation experiments on yeast mutants. Analysis of gene expression in situ showed that the ZmeIF3A transcript is expressed in the region of the root meristem surrounding the central stele. ZmeIF3A mRNA is also expressed in the young root, the male inflorescence, and the developing cob and seed. In maize, ZmeIF3A is encoded by one or two genomic sequences. This is the first report on the isolation and characterisation of a cDNA from higher plants that encodes a product homologous to a component of the eIF3 complex.