Autoradiographic Examination of Meristems of Intact Boron-deficient Squash Roots Treated with Tritiated Thymidine

Intact roots of boron-sufficient squash (Cucurbita pepo L.) plants, plants entering boron deficiency, and plants recovering from boron deficiency were exposed to tritiated thymidine at the end of the treatment period to label the replicating DNA of root tip cells. Using histological sections, autoradiographs of intact root meristems were prepared. The labeling pattern in +B root tips revealed the presence of a well defined quiescent center. The ability of root tip cells to incorporate label is correlated with the total root elongation during the −B treatment period. A greater amount of total root elongation during boron deficiency and recovery reflects the fact that root tip cells have retained their ability to synthesize DNA and enter mitosis for a longer time. In roots recovering from boron deficiency, cells of the quiescent center were seen to play no part in the recovery process in roots treated for as long as 20 hours in a −B nutrient solution. They were inactive before, during, and after the −B treatment. Cessation of mitosis occurs as early as 6.5 hours after boron is withheld from the nutrient solution while DNA synthesis can occur for as long as 20 hours after withholding boron. It was concluded that boron is essential for continued DNA synthesis and mitotic activity. The absence of boron results in the cessation of mitosis and DNA synthesis within 20 hours from the time boron is withheld.     

Morphogenesis in Selaginella. III. Meristem determination and cell differentiation.

Selaginella willdenovii Baker is a prostrate vascular cryptogam with a dorsiventral stem. At each major branching of the stem tip a dorsal and a ventral angle meristem are formed. The ventral meristem becomes determined as a root and the dorsal meristem as a shoot. Indoleacetic acid (IAA) is transported basipetally in the stem and has been found to be the regulatory agent for meristem determination both in vitro and in vivo.Growth measurements of intact plants indicated that the sequence of development for each stem unit is frond expansion, internodal elongation, ventral meristem growth as a root, and dorsal meristem growth as a shoot. The principal experimental findings of this study are as follows. Triiodobenzoic acid (TIBA), an inhibitor of auxin transport alters the normal pattern of development in intact plants, causing ventral meristems to develop as shoots and dorsal meristems to develop precociously. Dorsal meristems grown in sterile culture on an auxin-free medium develop as shoots, but in the presence of IAA develop as roots. Meristems transferred after excision from auxin-free to plus-auxin medium on successive days showed an increasing tendency to develop as shoots, with more than 50% doing so by day 5. The mitotic index is low at the time of excision of the meristem, rises to a peak on day 5 and then declines.     

Metabolic Requirement of Cucurbita pepo for Boron

Lateral roots of intact summer squash seedlings (Cucurbita pepo L.) were used to quantify the effects of boron deficiency on DNA synthesis, protein synthesis, and respiration. The temporal relationship between changes in these metabolic activities and the cessation of root elongation caused by boron deprivation was determined. Transferring 5-day-old squash seedlings to a hydroponic culture medium without boron for 6 hours resulted in a 62% reduction in net root elongation and a 30% decrease in the incorporation of [³H]thymidine into DNA by root tips (apical 5-millimeter segments). At this time, root tips from both boron-deficient and boron-sufficient plants exhibited nearly identical rates of incorporation of [¹⁴C]leucine into protein and respiration as measured by O₂ consumption. After an additional 6 hours of boron deprivation, root elongation had nearly ceased. Concomitantly, DNA synthesis in root apices was 66% less than in the boron-sufficient control plants and protein synthesis was reduced 43%. O₂ consumption remained the same for both treatments. The decline and eventual cessation of root elongation correlated temporally with the decrease in DNA synthesis, but preceded changes in protein synthesis and respiration. These results suggest that boron is required for continued DNA synthesis and cell division in root meristems.     

Nutrient Sensing in Plant Meristems

Plants need nutrient to grow and plant cells need nutrient to divide. The meristems are the factories and cells that are left behind will expand and differentiate. However, meristems are not simple homogenous entities; cells in different parts of the meristem do different things. Positional cues operate that can fate cells into different tissue domains. However, founder/stem cells persist in specific locations within the meristem e.g. the quiescent centre of root apical meristem (RAM) and the lower half of the central zone of the shoot apical meristem (SAM). Given the complexity of meristems, do their cells simply respond to a diffusing gradient of photosynthate? This in turn begs the question, why do stem cell populations tend to have longer cell cycles than their immediate descendants given that like all other cells they are directly in the path of diffusing nutrient? In this review, we have examined the extent to which nutrient sensing might be operating in meristems. The scene is set for sugar sensing, the plant cell cycle, SAMs and RAMs. Special emphasis is given to the metabolic regulator, SnRK1 (SNF1-related protein kinase 1), hexokinase and the trehalose pathway in relation to sugar sensing. The unique plant cell cycle gene, cylin-dependent kinase B1;1 may have evolved to be particularly responsive to sugar signalling pathways. Also, the homeobox gene, STIMPY, emerges strongly as a link between sugar sensing, plant cell proliferation and development. Flowering can be influenced by sucrose and glucose levels and both meristem identity and organ identity genes could well be differentially sensitive to sucrose and glucose signals. We also describe how meristems deal with extra photosynthate as a result of exposure to elevated CO₂. What we review are numerous instances of how developmental processes can be affected by sugars/nutrients. However, given the scarcity of knowledge we are unable to provide uncontested links between nutrient sensing and specific activities in meristems.     

microRNA profiling of root tissues and root forming explant cultures in Medicago truncatula

Plant root architecture is regulated by the initiation and modulation of cell division in regions containing pluripotent stem cells known as meristems. In roots, meristems are formed early in embryogenesis, in the case of the root apical meristem (RAM), and during organogenesis at the site of lateral root or, in legumes, nodule formation. Root meristems can also be generated in vitro from leaf explants cultures supplemented with auxin. microRNAs (miRNAs) have emerged as regulators of many key biological functions in plants including root development. To identify key miRNAs involved in root meristem formation in Medicago truncatula, we used deep sequencing to compare miRNA populations. Comparisons were made between: (1) the root tip (RT), containing the RAM and the elongation zone (EZ) tissue and (2) root forming callus (RFC) and non-root forming callus (NRFC). We identified 83 previously reported miRNAs, 24 new to M. truncatula, in 44 families. For the first time in M. truncatula, members of conserved miRNA families miR165, miR181 and miR397 were found. Bioinformatic analysis identified 38 potential novel miRNAs. Selected miRNAs and targets were validated using Taqman miRNA assays and 5′ RACE. Many miRNAs were differentially expressed between tissues, particularly RFC and NRFC. Target prediction revealed a number of miRNAs to target genes previously shown to be differentially expressed between RT and EZ or RFC and NRFC and important in root development. Additionally, we predict the miRNA/target relationships for miR397 and miR160 to be conserved in M. truncatula. Amongst the predictions, were AUXIN RESPONSE FACTOR 10, targeted by miR160 and a LACCASE-like gene, targeted by miR397, both are miRNA/target pairings conserved in other species.     

Short term cultivation of isolated barley roots and their mitotic activity

Roots were excised from barley embryos cultivated in the complete liquid nutrient solution and cultivated in the same nutrient solution separately. The excised roots continued their growth but a progressive decrease in the growth rate was observed. There was a considerable short-term drop of the mitotic activity immediately after excision, which was followed by a compensatory increase and then equilibrium was reached 12 h after excision. During the next at least three days, the mitotic index of isolated barley roots varied between 5–6.5%, which is slightly lower than the mitotic index of the root meristems of isolated barley embryos under identical conditions. The mitotic cycle index of isolated barley roots and the size of the root meristem later decreased gradually.     

Carbohydrate metabolism in leaf meristems of tall fescue : I. Relationship to genetically altered leaf elongation rates

The physiological bases for genetic differences in leaf growth rates were examined in two genotypes of tall fescue (Festuca arundinacea Schreb.) selected for a 50% difference in leaf elongation rate. Genotypes had similar dark respiration rates and concentrations of carbohydrate fractions in the leaf meristem and in each daily growth segment above the meristem. Dark respiration rates and concentrations of nonreducing sugars, fructans, and takadiastase-soluble carbohydrates were highest in leaf intercalary meristems and declined acropetally with tissue age. Concentrations of reducing sugars were 1.0% of dry weight in leaf meristems, 3.7% of dry weight in tissue adjacent to the meristem, then decreased progressively with distance from the meristem. Glucose, fructose, and myo-inositol comprised over 90% of the monosaccharides present in leaf meristems. Soluble protein concentration was 9.7 milligrams per gram fresh weight in leaf meristems, 5.5 milligrams per gram in tissues immediately above the meristem and, thereafter, increased linearly with distance from the meristem.

Leaf meristems of the genotype exhibiting rapid leaf elongation contained 30% more soluble protein than those of the genotype selected for slow leaf elongation. The 4-fold difference in size of the leaf meristem appeared to be more important in influencing leaf elongation than were other characteristics examined.

     

Synthesis, salvage, and catabolism of uridine nucleotides in boron-deficient squash roots

Previous work has provided evidence that plants may require boron to maintain adequate levels of pyrimidine nucleotides, suggesting that the state of boron deficiency may actually be one of pyrimidine starvation. Since the availability of pyrimidine nucleotides is influenced by their rates of synthesis, salvage, and catabolism, we compared these activities in the terminal 3 centimeters of roots excised from boron-deficient and -sufficient squash plants (Cucurbita pepo L.). Transferring 5-day-old squash plants to a boron-deficient nutrient solution resulted in cessation of root elongation within 18 hours. However, withholding boron for up to 30 hours did not result in either impaired de novo pyrimidine biosynthesis or a change in the sensitivity of the de novo pathway to regulation by end product inhibition. Boron deprivation had no significant effect on pyrimidine salvage or catabolism. These results provide evidence that boron-deficient plants are not starved for uridine nucleotides collectively. Whether a particular pyrimidine nucleotide or derivative is limiting during boron deprivation remains to be examined.     

Rubidium Uptake and Boron Deficiency in Vicia faba L

Removal of boron from the nutrient solution reduced root elongationwithin 24 h in V. faba seedlings. This was accompanied by areduction in rubidium uptake per plant as well as an altereddistribution within the root. Although the uptake by the terminalcentimetre was reduced in boron deficiency, there was littleeffect on older tissue. The proportion of absorbed Rb transportedto the shoot, efflux rates, and other uptake characteristicswere similar for deficient and normal tissue. The reduced uptakeis considered to be a secondary effect associated with the requirementsof boron for root elongation and metabolism.     

The Root Apex of Arabidopsis thaliana Consists of Four Distinct Zones of Growth Activities: Meristematic Zone,Transition Zone,Fast Elongation Zone and Growth Terminating Zone

In the growing apex of Arabidopsis thaliana primary roots, cells proceed through four distinct phases of cellular activities. These zones and their boundaries can be well defined based on their characteristic cellular activities. The meristematic zone comprises, and is limited to, all cells that undergo mitotic divisions. Detailed in vivo analysis of transgenic lines reveals that, in the Columbia-0 ecotype, the meristem stretches up to 200 µm away from the junction between root and root cap (RCJ). In the transition zone, 200 to about 520 µm away from the RCJ, cells undergo physiological changes as they prepare for their fast elongation. Upon entering the transition zone, they progressively develop a central vacuole, polarize the cytoskeleton and remodel their cell walls. Cells grow slowly during this transition: it takes ten hours to triplicate cell length from 8.5 to about 35 µm in the trichoblast cell files. In the fast elongation zone, which covers the zone from 520 to about 850 µm from the RCJ, cell length quadruplicates to about 140 µm in only two hours. This is accompanied by drastic and specific cell wall alterations. Finally, root hairs fully develop in the growth terminating zone, where root cells undergo a minor elongation to reach their mature lengths.Key words: Arabidopsis, cytoskeleton, development, differentiation zone, elongation zone, growth, growth terminating zone, meristem, root apex, transition zone     

Comparative anatomy of embryogenesis in three species of Podostemaceae and evolution of the loss of embryonic shoot and root meristems

During embryogenesis in angiosperms, the embryonic shoot and root meristems are created at opposite poles of the embryo, establishing a vertical body plan. However, the aquatic eudicot family Podostemaceae exhibits an unusual horizontal body plan, which is attributed to the loss of embryonic shoot and root meristems. To infer the embryogenetic changes responsible for the loss of these meristems, we examined the embryogenesis of three podostemads with different meristem characters, that is, Terniopsis brevis with distinct shoot and root meristems, Zeylanidium lichenoides with reduced shoot and no root meristems, and Hydrobryum japonicum with no shoot and no root meristems. In T. brevis, as in other eudicots, the putative organizing center (OC) and L1 layer (=the epidermal cell layer) arose to generate a distinct shoot meristem initial, and the hypophysis formed the putative quiescent center (QC) of a root meristem. Z. lichenoides had a morphologically unrecognizable shoot meristem, because a distinct L1 layer did not develop, whereas the putative OC precursor arose normally. In H. japonicum, the vertical divisions of the apical cells of eight-cell embryo prevented putative OC initiation. In Z. lichenoides and H. japonicum, the putative QC failed to initiate because the hypophysis repeated longitudinal divisions during early embryogenesis. Based on their phylogenetic relationships, we infer that the conventional embryonic shoot meristem was lost in Podostemaceae via two steps, that is, the loss of a distinct L1 layer and the loss of the OC, whereas the loss of the embryonic root meristem occurred once by misspecification of the hypophysis.     

ELONGATA3 is required for shoot meristem cell cycle progression in Arabidopsis thaliana seedlings

A key feature of the development of a higher plant is the continuous formation of new organs from the meristems. Originally patterned during embryogenesis, the meristems must activate cell division de novo at the time of germination, in order to initiate post-embryonic development. In a mutagenesis screen aimed at finding new players in early seedling cell division control, we identified ELONGATA3 (ELO3) as a key regulator of meristem cell cycle activation in Arabidopsis. Our results show that plants carrying a hypomorphic allele of ELO3 fail to activate cell division in the meristems following germination, which leads to seedling growth arrest and lethality. Further analyses suggest that this is due to a failure in DNA replication, followed by cell cycle arrest, in the meristematic tissue. Interestingly, the meristem cell cycle arrest in elo3 mutants, but not the later leaf developmental defects that have been linked to the loss of ELO3 activities, can be relieved by the addition of metabolic sugars in the growth medium. This finding points to a new role by which carbohydrate availability promotes meristem growth. Furthermore, growth arrested elo3 mutants suffer a partial loss of shoot meristem identity, which provides further evidence that cell cycle activities can influence the control of tissue identity.     

Root growth, developmental changes in the apex, and hydraulic conductivity for Opuntia ficus-indica during drought

Developmental changes in the root apex and accompanying changes in lateral root growth and root hydraulic conductivity were examined for Opuntia ficus-indica (L.) Miller during rapid drying, as occurs for roots near the soil surface, and more gradual drying, as occurs in deeper soil layers. During 7 d of rapid drying (in containers with a 3-cm depth of vermiculite), the rate of root growth decreased sharply and most root apices died; such a determinate pattern of root growth was not due to meristem exhaustion but rather to meristem mortality after 3 d of drying. The length of the meristem, the duration of the cell division cycle, and the length of the elongation zone were unchanged during rapid drying. During 14 d of gradual drying (in containers with a 6-cm depth of vermiculite), root mortality was relatively low; the length of the elongation zone decreased by 70%, the number of meristematic cells decreased 30%, and the duration of the cell cycle increased by 36%. Root hydraulic conductivity ( L _P) decreased to one half during both drying treatments; L _P was restored by 2 d of rewetting owing to the emergence of lateral roots following rapid drying and to renewed apical elongation following gradual drying. Thus, in response to drought, the apical meristems of roots of O. ficus-indica near the surface die, whereas deeper in the substrate cell division and elongation in root apices continue. Water uptake in response to rainfall in the field can be enhanced by lateral root proliferation near the soil surface and additionally by resumption of apical growth for deeper roots.     

Embryo-patterning genes and reinforcement cues determine cell fate in theArabidopsis thalianaroot

The majority of plant organs arise from groups of continuously dividing cells, the meristems. Little is known about mechanisms of cell specification in meristems. Within theArabidopsisroot meristem, the fate of every cell can be predicted accurately, and the origin of these cells during the formation of the embryonic root primordium is known. Laser ablations reveal that, despite the regularity in cell lineage, position remains important to reinforce cell specification. Genetic analysis has revealed that many genes involved in the specification of the main cell types in the root act early, during embryogenesis, and an important question is whether the same or other genes are involved in the reinforcement of specification. Sub-specification of cell types, as exemplified by epidermal root hair cell specification, involves two pathways, one of which may act to reinforce earlier patterning events mediated by the other.     

Analysis of cell cycle in root meristems

We have analyzed the cell proliferation in a meristem assuming a single file model for root architecture. The meristem file appears to be built up by two clearly separated zones: the first going from the initial cell to the middle of the meristem and the second from the middle to the meristem boundary. The first half of the meristem shows an exponential age distribution for the cell population. In contrast, in the second half of the meristem, the cell kinetics of cycling cells strongly disagree with exponential kinetics and due to the compensation between the observed deviations in both halves, cell supply in the file meristem is in line with linear kinetics. However, we proposed that exponential kinetic equations offer a suitable approach to problems of cell cycle compartments and population age distributions in real meristems, where non-cycling cells cannot be identified inside the meristem, whether we consider the meristem as a whole or study a “window” inside it. Nevertheless, for more exact kinetic analysis, when estimating the proliferative fraction, the width of the “window” and its location along the axis must carefully be taken into account.     

Elevation in the Sucrose Content of the Shoot Apical Meristem of Sinapis alba at Floral Evocation

Nanogram tissue samples from apical meristems of Sinapis alba were assayed for sucrose, total soluble hexosyl equivalents ( glucose and fructose plus hexoses from sucrose hydrolysis), and total soluble glucosyl equivalents ( glucose plus glucose from sucrose hydrolysis). On dry weight basis, sucrose concentration increased by more than 50% within 10 hours after the start of either a long photoperiod or a short photoperiod displaced by 10 hours in the 24-hour cycle (`displaced short day'). (These treatments induce flower initiation) Glucose and fructose concentrations were close to zero in vegetative meristems and remained low compared to sucrose in meristems of induced plants. Within a single meristem, the peripheral and the central zones had similar concentrations of sucrose. Our results indicate that an early physiological event in floral transition is the accumulation of sucrose in the meristem.     

A molecular framework for the inhibition of Arabidopsis root growth in response to boron toxicity

Boron is an essential micronutrient for plants and is taken up in the form of boric acid (BA). Despite this, a high BA concentration is toxic for the plants, inhibiting root growth and is thus a significant problem in semi-arid areas in the world. In this work, we report the molecular basis for the inhibition of root growth caused by boron. We show that application of BA reduces the size of root meristems, correlating with the inhibition of root growth. The decrease in meristem size is caused by a reduction of cell division. Mitotic cell number significantly decreases and the expression level of key core cell cycle regulators is modulated. The modulation of the cell cycle does not appear to act through cytokinin and auxin signalling. A global expression analysis reveals that boron toxicity induces the expression of genes related with abscisic acid (ABA) signalling, ABA response and cell wall modifications, and represses genes that code for water transporters. These results suggest that boron toxicity produces a reduction of water and BA uptake, triggering a hydric stress response that produces root growth inhibition.     

Dependence of Root Cell Growth and Division on Root Diameter

Primary roots of 98 species from different families of monocotyledonous and dicotyledonous plants and adventitious roots obtained from bulbs and rhizomes of 24 monocot species were studied. Root growth rate, root diameter, length of the meristem and elongation zones, number of meristematic cells in a file of cortical cells, and length of fully elongated cells were evaluated in each species after the onset of steady growth. The mitotic cycle duration and relative cell elongation rate were calculated. In all species, the meristem length was approximately equal to two root diameters. When comparing different species, the rate of root growth increased with a larger root diameter. This was due to an increase in the number of meristematic cells in a row and, to a lesser degree, to a greater length of fully elongated cells. The duration of the mitotic cycle and the relative cell elongation rate did not correlate with the root diameter. It is suggested that the meristem size depends on the level of nutrient inflow from upper tissues, and is thereby controlled during further growth.     

The effect of butyrate on the cell cycle in root meristems of Pisum sativum

Sodium butyrate at 5 mM in aerated White's medium reduced the mitotic index in root meristems of seedlings of Pisum sativum to < 1% after 12 h. This effect was lessened as the butyrate concentrations were lowered. The fraction of the root meristem nuclei in G2 increased to ~ 70% after 12 h in butyrate. After 12 h exposure to butyrate, seedlings transferred lo medium without butyrate gradually re-established their normal root meristem mitotic pattern, with a burst of mitosis at 10 h after the transfer. Even a brief exposure to butyrate inhibited DNA synthesis, and nuclei released from butyrate exposure were still unable to resume normal DNA synthesis even after 12 h. This information suggests that butyrate halts progression through the cell cycle by arresting meristem nuclei in G2 and inhibiting DNA synthesis.     

ACTIVITY OF MARGINAL AND PLATE MERISTEMS DURING LEAF DEVELOPMENT OF XANTHIUM PENNSYLVANICUM

The mitotic and biosynthetic activities of the marginal and plate meristems were studied during the entire course of leaf development of Xanthium pennsylvanicum. In contrast to statements in the literature, marginal meristem activity is long in duration, as assayed by the mitotic counts and H³-thymidine incorporation. This me istem is active 23 days. The plate meristem is active for an additional 3 days after cessation of cell division in the marginal meristem, but the total duration of its mitotic activity is also approximately 23 days. Numerous periclinal cell divisions of the plate meristem form additional cell layers and contribute to the growth of the lamina in thickness. Incorporation of H³-thymidine increased during the course of leaf development. Cells between plastochronic ages 0 and 2.0 incorporated more of the radioisotopic precursor than those of younger leaf primordia. The uptake and incorporation of H³-thymidine into nuclear DNA was more sluggish during the early stages of development than in the more expanded leaves. No DNA synthesis was demonstrated after cessation of cell division in the leaf lamina. Metabolic or endomitotic DNA synthesis after leaf plastochron index (LPI) 3.0 seems improbable. No significant differences in the incorporation of H³-thymidine could be demonstrated between the marginal and plate meristems. This would indicate no distinct biosynthetic differences between the two meristems. The definitions of the marginal and plate meristems of Xanthium leaves were formulated in view of the above findings.