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.     

EFFECTS OF BORON DEFICIENCY ON MITOSIS AND INCORPORATION OF TRITIATED THYMIDINE INTO NUCLEI OF SUNFLOWER ROOT TIPS

Boron deprivation has multiple effects upon root growth within 6 hr after this essential micronutrient is withheld. Root elongation is inhibited and this response has been attributed to a cessation of mitosis and DNA synthesis. Our preliminary results using an autoradiographic analysis of sunflower roots labeled with [³H]-thymidine demonstrated no difference in label distribution between +/-B root tips. We found that mitosis in inhibited in -B roots but does not completely cease. Scintillation counting of whole root tips shows that boron-deficient roots up to 72 hr of treatment incorporate radioactive label at a level comparable to that of the controls. Because mitosis and presumably DNA synthesis are affected by prolonged boron deficiency, these results may be brought about by a change in membrane integrity or permeability. We propose that effects of boron deprivation on DNA synthesis and mitosis in sunflower are secondary and that primary events involve alterations in cellular membranes.     

Early effects of boron deficiency on indoleacetic Acid oxidase levels of squash root tips

The indoleacetic acid (IAA) oxidase activity of root tips of boron-sufficient, -deficient, recovering, and IAA-treated boron-sufficient squash plants (Cucurbita pepo L.) was determined. Apical and subapical root sections displayed an increase in IAA oxidase activity between 6 and 9 hours after boron was withheld, and after 24 hours the activity of the apical sections showed a 20-fold increase over +B controls. Root elongation of -B plants was inhibited before an increase in oxidase activity could be detected. Roots of plants subjected to 12 hours of -B treatment and then transferred to +B treatment for recovery regained normal elongation rates and oxidase activity within 18 to 20 hours. IAA treatment of +B plants increased IAA oxidase activity of apical and subapical root sections and also inhibited root elongation and caused symptoms similar to -B treatments.     

Effect of boron on cell elongation and division in squash roots

This work establishes that cessation of root elongation of intact squash (Cucurbita pepo L.) plants is an early result of boron deficiency. Root elongation is slowed by 6 hours and is virtually stopped as early as 24 hours after boron is first withheld from the nutrient solution. As root elongation ceased, cell elongation progressed distally into the region normally occupied by the apical meristem and eventually the meristem became indistinguishable. Differentiation was determined by use of an elongation index in which cell length was compared to cell width. This index ranged from a low of 0.8 in boron-sufficient root meristems to a high of 3 in root meristems grown in a boron-deficient nutrient solution for 98 hours. It is concluded that a continuous supply of boron is not essential for cell elongation but is required for maintenance of meristematic activity. Boron may act as a regulator of cell division in this tissue.     

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.     

Molecular specificity of 2-aminopurine in Saccharomyces cerevisiae

The rates of DNA, RNA and protein synthesis were investigated by incorporation of radioactive precursors into the excised root tips of V. faba. 2-h exposure to 0.1% caffeine resulted in inhibition of protein synthesis to about 60% of the control rate. RNA synthesis was reduced in the range of 20–30%. The same concentration of caffeine did not affect the rate of DNA synthesis even during 12-h incubation, but concentrations higher than 1% caused a significant decrease in [³H]thymidine incorporation.     

Phospholipid metabolism in roots and microsomes of sunflower seedlings: Inhibition of choline phosphotransferase activity by boron

The action of boron on phospholipid composition and synthesis in roots and microsomes from sunflower seedlings has been studied. The fatty acid composition and relative amounts of individual molecular species of phospholipids in roots and microsomes were very similar. In both the content of phospholipids was decreased and the relative levels of their component fatty acids changed by treatment with 50 ppm of boron. This concentration of boron in the culture medium was found to inhibit the in vivo [1-^14C] acetate incorporation into root lipids and that of [Me-¹⁴C] choline into phosphatidylcholine of root microsomes. Cytidine-5-diphospho (CDP)-[Me-¹⁴C] choline incorporation into phosphatidylcholine of isolated microsomes was also inhibited by 50 ppm of boron when present in the growth medium of seedlings. These results indicate that the decrease in phosphatidylcholine labelling from [¹⁴C] choline observed when root microsomes were treated with boron would be caused by a decrease in CDP-choline phosphotransferase activity.     

Effect of trifluralin on growth, morphology, and nucleic Acid synthesis

Roots and shoots of corn seedlings (Zea mays L. var. Dixie 18) germinated in trifluralin (α,α,α-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine) solutions are characterized by radial enlargement of the cortical cells and by multinucleate cells in the meristematic regions. Trifluralin inhibits elongation of Avena coleoptile sections at concentrations of 0.1 μm to 10 μm. Synthesis of DNA, RNA, and protein is suppressed in the root tips while no significant effect is noticeable in the shoots of corn germinated in trifluralin. A ³²P time-course study of 48, 72, and 96 hours utilizing phenol extraction and MAK column separation of corn root and shoot nucleic acids showed suppression of ³²P incorporation in the treated roots; however, the 72 and 96 hour treated shoots incorporated a much greater amount than the control with most of the increased incorporation found in the sRNA and DNA fractions. The increased activity in the DNA may be due to a high G-C type DNA. No selective suppression or enhancement of any particular RNA species was noticed in the treated plants.     

Protein synthesis in trifluralin-treated corn root tips

The effect of trifluralin (,,-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine) on protein synthesis in corn (Zea mays) root tips was determined. ¹⁴C-leucine uptake and subsequent incorporation into protein in intact and excised root tips was measured. Root tips were treated with and without 15 µM trifluralin for 2 hr; incorporation of ¹⁴C-leucine was observed during a 20-min interval. Total amino acid content in the soluble pool and protein hydrolysate was reduced in the excised tissue by the herbicide. Kinetic analysis showed trifluralin had no effect on endogenous leucine pool size nor on the rate of protein synthesis in intact tissue. Uptake was unaffected; however, in excised tissue uptake increased 100% over the control. While ¹⁴C-leucine content was greater in both the soluble pool and protein in treated, excised root tips, analysis showed the apparent increase in protein synthesis was in response to increased pool size.     

Compartmentation and Fluxes of Sugars in Roots of Phaseolus Vulgaris Under Phosphate Deficiency

The influence of phosphate deficiency on the sugar accumulation and sugar partitioning in the root cells of bean (Phaseolus vulgaris L.) was studied. Bean plants were cultured 17 - 19 d on a phosphate-sufficient and phosphate-deficient nutrient medium. Phosphate deficit in the growth medium resulted in increased sugar concentration for about 30 % in the apoplastic and cytoplasmic compartments as well as in the vacuoles of root cells. However, the distribution of sugars between apoplast and cytoplasm compartment and vacuole was not affected by decreased phosphate concentration. About 20 % of sugars were found in the apoplast and cytoplasm, about 80 % in the vacuole. Low phosphate concentration enhanced influx of exogenous ¹⁴C-sucrose into meristematic and elongation zones of root. The ¹⁴C-labelled sugar content in the root tips increased for about 60 % as compared to control plants. Phosphate deficiency increased also ¹⁴C-glucose uptake and content in the root tips. However, the amount of ¹⁴CO₂ liberated during respiration of P-deficient roots (after feeding with uniformly labelled ¹⁴C-glucose) was lower than ¹⁴CO₂ respired by control plants, thus a large part of accumulated sugars seems to be metabolically inactive. This revised version was published online in July 2006 with corrections to the Cover Date.     

Dynamics of growth,proliferation and differentiation of wheat root cells exposed to a high nickel concentration

The dynamics of root growth, proliferation of initial cells of the root cap, rhizodermis, and central metaxylem, as well as structural changes in the cells induced by a 72-h exposure to a high (0.1 mM) concentration of NiSO₄ were studied in 3-day-old wheat (Triticum aestivum L.) seedlings. In the roots of control plants, we observed a 12-h rhythm of changes in the length of the cells that completed elongating. Upon the treatment with nickel, this effect was negated, and a considerable reduction in the root length increment was observed in 12 h. In 24 h, root growth essentially ceased. Cell elongation was suppressed acropetally, and the cells, whose elongation was over, became shorter. In the meristem and apical part of the elongation zone, slow cell growth continued during the second and even third days. Autoradiography showed that the earliest effect of nickel on the processes of root morphogenesis observed in 6 h was a suppression of cell transition to DNA synthesis. The cells, where DNA synthesis has already started or which were in other stages of the cycle, continued to pass slowly through the cycle and completed it. Sister cells formed as a result of division subsequently left the cycle in the phase G1 and transited to dormancy. It was found that the main mechanism of cell proliferation cessation was the suppression of cell transition to DNA synthesis. In the cells elongating when exposed to nickel, tissue-specific changes in the nucleus structure were observed (chromatolysis in the rhizodermis and cortex, pycnosis in the endodermis, a disturbance of the nucleus structure in the central metaxylem). These disorders were only observed after cessation of elongation. Root incubation in 0.1 mM nickel solution did not affect the onset of cell differentiation in the xylem and metaphloem and shifted its beginning to the root tip. However, in 24 h the initiation and growth of root hairs were suppressed. It was concluded that tissue-specific nickel-induced changes in the nucleus structure in the elongating cells do not cause the cessation of root growth, although point to nickel toxic effect on the cells in the course of elongation.     

Effects of aluminium on nitrate uptake and assimilation

A study was conducted to examine the hypothesis that the effects of external Al on NO₃^? uptake and assimilation depend upon the concentration of Al present. Young soybean seedlings [Glycine max (L.) Merrill, cv. Essex], growing under moderate acidity stress at pH 4-2, were exposed to a range of {A1³⁺} in solution for 3d, and to labelled 99 atom %¹⁵NO₃^? during the final hour of Al exposure. Uptake of ¹⁵NO₃^?g^?1 root dry weight was increased by about 28% in the presence of Al at {A1³⁺} below 10 mmolm^?3, and NO₃^? uptake was decreased by about 12% when the {A1³⁺} increased to 44mmoln^?3. The stimulation phase closely paralleled stimulation of root elongation. At higher {A1³⁺}, the inhibition of root elongation was much more severe than that of NO₃^? uptake. There was no indication of a separate effect of Al on root ¹⁵NO₃^? reduction in situ, as the accumulation of reduced ¹⁵N in the root remained a similar percentage of ¹⁵NO₃^? uptake at all {A1³⁺}. At higher {A1³⁺}, the atom %¹⁵N enrichment of the insoluble reduced-N (protein) fraction of root tips increased. This suggests that the Al inhibition of root elongation did not result from disruption of the N supply to the root apex.     

Incorporation of nitrate nitrogen in rice seedlings transferred to anaerobic conditions

Summary Incorporation of¹⁵NO₃- into amino acids was studied in 3-day-old aerobic rice seedlings (with coleoptile and root) subjected for 24h to anaerobic conditions. The incorporation of¹⁵N into glutamate, glutamine and alanine accounted for 89% and 84% of total incorporation in coleoptile and root, respectively. These findings indicate that, after the primary incorporation of¹⁵N into glutamate and glutamine, the main fate of nitrate nitrogen in rice seedlings subjected to anoxia is alanine.     

Kinetics and relative significance of remobilized and current C and N incorporation in leaf and root growth zones of Lolium perenne after defoliation: assessment by 13C and 15N steady-state labelling

The contribution of pre-defoliation reserves and current assimilates to leaf and root growth was examined in Lolium perenne L. during regrowth after defoliation. Differential steady-state labelling with ¹³C (CO₂ with δ¹³C = -0.0281 and -0.0088) and ¹⁵N (NO₃^? with 1.0 and 0.368 atom percentage, i.e. δ¹⁵N = 1.742 and 0.0052, respectively) was applied for 2 weeks after defoliation. Rapidly growing tissues were isolated, i.e. the basal elongation and maturation zones of the most rapidly expanding leaves and young root tips, with a biomass turnover rate > 1 d^?1. C and N weights of the elongation zone showed a transient decline. The dry matter and C concentration in fresh biomass of leaf growth zones transiently decreased by up to 25% 2 d after defoliation, while the N concentration remained constant. This ‘dilution’ of growth zone C indicates a decreased net influx of carbohydrates relative to growth-related influx of water and N in expanding cells, immediately after defoliation. Recovery of the total C and N weights of the leaf elongation zone coincided with net incorporation of currently absorbed C and N, as shown by the kinetics of δ¹³C and atom percentage ¹⁵N in the growth zones after defoliation. C isotope discrimination (Δ¹³C) in leaf growth zones was about 23‰, 1–2‰ higher than the Δ in root tips. Δ¹⁵N in the leaf and root growth zones was 10±3‰. The leaf elongation zones (at 0–0.03 m from the tiller base) and the distant root tips (about 0.2 m from the base) exhibited similar kinetics of current C and N incorporation. The amount of pre-defoliation C and N in the growth zones, expressed as a fraction of total C and N, decreased from 1.0 to 0.5 at 3 (C) and 5 (N) d after defoliation, and to 0.1 at 5 (C) and 14 (N) d after defoliation. Thus, the dependence of growth zones on current assimilate supply was significant, and stronger for C than for N. The important roles of current assimilates (as compared to pre-defoliation reserves) and ‘dilution’ of dry matter in regrowth after defoliation are discussed in relation to the method of labelling and the functional and morphological heterogeneity of shoot tissues.     

Effects of rhizosphere oxygen concentration on root physiological characteristics and anatomical structure at the tillering stage of rice

Oxygen is essential for all aerobic organisms. Higher plants need oxygen to sustain metabolism and growth. After experiencing anaerobic stress for a period of time, most plant tissues will be damaged. This study examined the physiological characteristics and anatomical structures in the root tips of rice seedlings (cultivars Xiushui09 and Chunyou84) in response to different rhizosphere oxygen environments. The results showed that moderate oxygen (MO: 2.5–3.5 mg L⁻¹) increased the dry weight accumulation and formation of rice roots (including length, surface area, number of tips) in both genotypes. Compared to under normal conditions (NC), the magnitudes of increase in these four variables were 4.67, 66.40, 35.44 and 49.50% in Xiushui09 and 12.25, 15.59, 13.57 and 13.48% in Chunyou84, respectively. Low oxygen (LO: 0–1.0 mg L⁻¹) stress decreased the root surface area but increased root volume and average diameter. LO and high oxygen (HO: >6.8 ± 0.21 mg L⁻¹) stress each damaged the apical cells' ultrastructure, reduced the number of organelles, and increased electrical conductivity. Meanwhile, the root activity and respiration of rice seedlings decreased; the abscisic acid content increased compared to levels under NC. Under MO treatment, the cell membrane was not damaged, the root tip organelles were rich, the soluble protein content, root activity, respiration rate and gibberellic acid content increased compared to levels under NC; the magnitudes of these increases were 24.76, 36.00, 8.00 and 283.00% in Xiushui09 and 4.78, 40.00, 15.45 and 49.35% in Chunyou84, respectively. In conclusion, MO optimised rice root morphology and enhanced root physiological activity.     

樟树(Cinnamomum camphora)幼苗细根形态对氮磷添加和幼苗密度的响应

全球氮沉降对森林生态系统结构和功能的影响已成为现代生态学研究热点之一,我国华南地区氮沉降的增长引起了土壤酸化和磷限制加剧等一系列生态问题。密度制约着植物个体对环境资源的吸收利用,是自然界中十分重要的选择压力之一。因此研究樟树(Cinnamomum camphora)幼苗的细根形态对氮磷添加和密度的响应,有利于了解亚热带树木根系对氮沉降和磷添加与林分密度的响应过程和机制,并为全球变化背景下樟树林生态系统的管理提供依据。本研究以1年生樟树幼苗为试验材料,选择氯化铵(NH_4Cl)作为氮肥以模拟大气氮沉降,并且以二水合磷酸二氢钠(NaH_2PO_4·2H_2O)模拟磷添加,氮磷处理设置4个水平,即对照、施N、施P和施N+P;种植密度设置10、20、40和80株/m~2 4个水平。测定各处理樟树幼苗细根的根长、表面积、体积和根尖数,分析氮磷添加、密度和两者交互作用对樟树幼苗细根的影响。研究结果表明,与对照处理相比,N、P和N+P处理促进了幼苗细根长度、表面积、体积以及根尖数的增加。低密度条件下的N添加对幼苗根系形态的促进效果强于P添加。N+P处理对10、20、40株/m~2幼苗根系形态的促进效果最佳,而各处理对80株/m~2幼苗根系形态的促进效果均无显著性差异。随着种植密度的增大,幼苗细根长度、表面积、体积和根尖数均减少。樟树幼苗的细根长度、表面积、体积和根尖数在各密度间和不同氮磷添加处理间均有显著性差异,密度和氮磷处理间的交互作用对根系形态各指标均无显著影响。     

Relationship between intracellular pH and N metabolism in maize (Zea mays L.) roots

In vivo ³¹P nuclear magnetic resonance (NMR) was used to characterize the effect of the N form (NO₃ vs. NH₄) and the external pH (4, 6, and 8), on the intracellular pH of root tips (0–5 mm) and root segments (5–30 mm). Ammonium-grown root tips were the most sensitive to changes in the external pH. In vivo ¹⁵N NMR was used to characterize the pathway of primary ammonium assimilation in the ammonium-grown roots and to compare the activity of the apical and more-basal root parts. The kinetics of ¹⁵NH₄ ⁺ incorporation showed that primary assimilation in both root tips and root segments followed the glutamine synthetase (GS) pathway. In agreement with the reported gradient of GS along the seminal root of maize, incorporation of label into glutamine amide was more rapid in tips than in segments. It is suggested that this higher GS activity increases the endogenous proton production and thus contributes to the greater dependence of the cytoplasmic pH on the external pH in the ammonium-treated root tips.     

Incorporation of [C]Glucose into Cell Wall Polysaccharides of Cotton Roots: Effects of NaCl and CaCl(2)

Cotton (Gossypium hirsutum L. cv Acala SJ-2) seedlings were grown in nutrient solutions with four combinations of NaCl (0.1 and 150 millimolar) and CaCl₂ (1 and 10 millimolar) for 7 days, and then exposed to [¹⁴C]glucose for 5 hours. Uptake and incorporation of [¹⁴C]glucose into various cell wall fractions of the root tips were determined. At 1 millimolar Ca²⁺, treatment with 150 millimolar NaCl slightly stimulated uptake but considerably inhibited glucose incorporation into noncellulosic and cellulosic polysaccharides. Supplemental Ca²⁺ did not affect incorporation of glucose into the noncellulosic fraction (regardless of NaCl treatment) but completely alleviated the inhibitory effect of NaCl on glucose incorporation into cellulose. We suggest that high Na⁺ concentrations reduce synthesis of cellulose in cotton roots via disturbance of plasma membrane integrity and that supplemental Ca²⁺ counteracts this effect. The effects on cellulose biosynthesis are proposed to be related to Ca²⁺ displacement from the plasma membrane.     

Elongation and termination reactions of protein synthesis on maize root tip polyribosomes studied in a homologous cell-free system

We show that the control of gene expression at the level of elongation and termination of protein synthesis can be observed in vitro. Free cytoplasmic polyribosomes were isolated from maize (Zea mays) root tips, and translated in root tip extracts that had been fractionated with ammonium sulfate to contain elongation factors, and be depleted in initiation factors. The root tip extract performs elongation and termination reactions as efficiently as wheat germ extracts. The translation products of the maize system are the same as made in vivo. The dependence of these in vitro elongation and termination reactions on pH was determined. Total protein synthesis in this system exhibits an optimum at pH ~7.5. However, the pH dependence of rates of synthesis of individual proteins is not at all uniform; many polyribosomes become stalled when translated at low pH. These data were compared with the elongation and termination capacity of polyribosomes isolated from oxygenated and hypoxic root tips (tissue having, respectively, high and low cytoplasmic pH values). We observed an inverse relationship between the relative abundance of many specific translatable mRNAs in polyribosomes of hypoxic root tips, and the relative rates of elongation and termination reactions on the different mRNAs at low pH in vitro. These results suggest that changes in intracellular pH in hypoxic root tips can be sensed directly by the translational machinery and thereby selectively modulate gene expression.     

Root Growth Inhibition in Boron-Deficient or Aluminum-Stressed Squash May Be a Result of Impaired Ascorbate Metabolism

Although cessation of growth is the most apparent symptom of boron deficiency, the biochemical function of boron in growth processes is not well understood. We propose that the action of boron in root meristems is associated with ascorbate metabolism. Total inhibition of root growth in squash (Cucurbita pepo L.) plants transferred to boron-free medium coincided with a major decrease (up to 98%) in the ascorbate concentration of root apices. Under low-boron conditions, in which root growth was partially inhibited, ascorbate concentration declined in proportion to growth rate. The decline in ascorbate concentration in boron-deficient root tips was not related to ascorbate oxidation. Ascorbate added to the medium improved root growth in plants supplied with insufficient boron. Increasing concentrations of aluminum in the nutrient medium caused progressive inhibition of root growth and a parallel reduction in ascorbate concentration of root apices. Elevated boron levels improved root growth under toxic aluminum conditions and produced root apices with higher ascorbate concentrations. To our knowledge, this is the first report of a correlation between boron nutrition, ascorbate concentration in root apices, and growth. These findings show that root growth inhibition resulting from either boron deficiency or aluminum toxicity may be a consequence of disrupted ascorbate metabolism.