Root Tips Of Seedlings Research Articles

Proline accumulation in maize (Zea mays L.) primary roots at low water potentials. II. Metabolic source of increased proline deposition in the elongation zone

The proline (Pro) concentration increases greatly in the growing region of maize (Zea mays L.) primary roots at low water potentials (psiw), largely as a result of an increased net rate of Pro deposition. Labeled glutamate (Glu), ornithine (Orn), or Pro was supplied specifically to the root tip of intact seedlings in solution culture at high and low psiw to assess the relative importance of Pro synthesis, catabolism, utilization, and transport in root-tip Pro deposition. Labeling with [3H]Glu indicated that Pro synthesis from Glu did not increase substantially at low psiw and accounted for only a small fraction of the Pro deposition. Labeling with [14C]Orn showed that Pro synthesis from Orn also could not be a substantial contributor to Pro deposition. Labeling with [3H]Pro indicated that neither Pro catabolism nor utilization in the root tip was decreased at low psiw. Pro catabolism occurred at least as rapidly as Pro synthesis from Glu. There was, however, an increase in Pro uptake at low psiw, which suggests increased Pro transport. Taken together, the data indicate that increased transport of Pro to the root tip serves as the source of low-psiw-induced Pro accumulation. The possible significance of Pro catabolism in sustaining root growth at low psiw is also discussed.

Fluorescent Banding Pattern of Peach (Prunus persica (L.) Batsch) Chromosomes.

The chromomycin A3 (CMA) and 4'-6-diamidino-2-phenylindole (DAPI) chromosome banding patterns of peach (Prunus persica (L.) Batsch) (2n=16) were determined on root tips of open-pollinated seedlings of three cultivars : 'Ohatsumomo', 'Akatsuki', and 'Momo daigi tsukuba 4 gou' ('Tsukuba No.4'). Root tips were pretreated in 2mM 8-hydroxyquinoline at 10°C for 4hr ; the chromosome samples were prepared by the enzymatic maceration and air-drying method. CMA positive (+) and DAPI negative (-) bands were observed in the satellite position of two chromosomes, terminal position of two chromosomes, and proximal position of two chromosomes. Ten chromosomes did not have any CMA+/DAPI-band. The number and positions of CMA+/DAPI-bands were stable, and there was no difference among the seedlings from the three cultivars. No CMA-/DAPI+band was observed.

Phosphorus compartmentation in Pinus serotina Michx. (pond pine); observations from in vivo nuclear magnetic resonance spectroscopy

31P nuclear magnetic resonance (NMR) spectroscopy was used to estimate the amount of inorganic phosphate (Pi) present in the cytoplasm and vacuole of root tips and subapical root segments of pond pine (Pinus serotina Michx.). In root tips of seedlings grown with 100 mmol m–3P (HP) the cytoplasmic Pi content, on a root volume basis, was ≈ 1·5 μmol cm–3 and the vacuolar Pi content, on a root volume basis, was ≈ 3·4 μmol cm–3. In root tips from Pi starved seedlings the cytoplasmic Pi content, on a root volume basis, was ≈ 0·75 μmol cm–3; vacuolar Pi was too low to be reliably estimated. Similar results were obtained with subapical root segments; the Pi concentration in the cytoplasm was maintained at around 2 mol m–3 while that in the vacuole varied with Pi supply. This work demonstrates for the first time that quantitative measurements of the subcellular compartmentation of Pi can be made in young tissues of a woody species. The results indicate that cytoplasmic Pi levels are maintained across a range of external Pi supplies probably by withdrawing Pi stored in the vacuole.

Expression of NADH-dependent glutamate synthase protein in the epidermis and exodermis of rice roots in response to the supply of ammonium ions.

The mRNA and protein for NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) in root tips of rice (Oryza sativa L. cv. Sasanishiki) plants increases dramatically within 12 h of supplying a low concentration (> 0.05 mM) of ammonium ions (T. Yamaya et al., 1995, Plant Cell Physiol 36: 1197-1204). To identify the specific cells which are responsible for this rapid increase, the cellular localization of NADH-GOGAT protein was investigated immunocytologically with an affinity-purified anti-NADH-GOGAT immunoglobulin G. When root tips (> 1 mm) of rice seedlings which had been grown for 26 d in water were immuno-stained, signals for the NADH-GOGAT protein were detected in the central cylinder, in the apical meristem, and in the primordia of the secondary roots, Signals for ferredoxin-dependent GOGAT (Fd-GOGAT; EC 1.4.7.1) protein were also seen in the same three areas. When the roots were supplied with 1 mM ammonium ions for 24 h, there were strong signals for the NADH-GOGAT protein in two cell layers of the root surface, i.e. epidermis and exodermis, in addition to the cells giving signals in the absence of ammonium ions. The supply of ammonium ions was less effective on the profile of signals for Fd-GOGAT. Although the supply of ammonium ions had less effect on the expression of cytosolic glutamine synthetase (GS; EC 6.3.1.2), this enzyme was also found to be located in the epidermis and exodermis, as well as in the central cylinder and cortex. The results indicate that NADH-GOGAT, coupled to the cytosolic GS reaction, is probably important for the assimilation of ammonium ions in the two cell layers of the root surface.

Promoter trap markers differentiate structural and positional components of polar development in Arabidopsis.

To investigate mechanisms involved in establishing polar organization in Arabidopsis embryos and seedlings, we used promoter trapping to identify molecular markers (beta-glucuronidase fusion genes) expressed in spatially restricted patterns along the apical-basal axis. Three markers were identified that are expressed, respectively, in the embryonic and seedling root tip (POLARIS), cotyledons and shoot and root apices (EXORDIUM), and root cap (COLUMELLA). Each marker was crossed into the mutants hydra and emb30, which are defective in embryonic and seedling morphogenesis. All three markers were expressed in hydra mutants in patterns similar to those observed in phenotypically wild-type embryos and seedlings. In emb30 mutants, the EXORDIUM marker was expressed in cotyledons but not in the expected position of shoot and root meristems, and the marker COLUMELLA was not expressed at all, which is consistent with the view that the emb30 mutant, but not hydra, lacks shoot and root meristems. However, POLARIS was expressed in the basal part of hydra embryos lacking an embryonic root and in the basal parts of both hydra and emb30 seedlings. Expression of POLARIS is inducible by exogenous auxin and suppressed by cytokinin but is unaffected by inhibitors of polar auxin transport or cell division. We conclude that POLARIS differentiates positional aspects of polar development from structural aspects.

In‐vitro‐cultured subclover root can develop Fe‐deficiency stress response

The Fe‐deficiency stress response is induced in most plants under Fe‐deficient conditions, but whether the shoot and/or the root control development of the stress response is not known. The objectives of the present study were to determine whether in‐vitro‐cultured subclover roots can develop Fe‐deficiency stress response and to examine this approach as a possible screening technique for Fe‐deficiency resistance. One‐cm long root tips of subclover seedlings were cultured in modified White's medium without (‐Fe) or with (+Fe) 100 μM Fe3+EDTA. Root Fe3+ reduction and H+ release were evaluated. On the first day after transfer to the ‐Fe medium, the Fe‐deficiency‐resistant cultivar Koala (Trifolium brachycalycinum Katzn. and Morley) started to release H+, resulting in a decrease in pH of the culture medium, while the susceptible cultivar Karridale (T. subterraneum L.) did not release H+ until the second day. The H+‐release rate of the ‐Fe Koala was approximately twice as high as that of the ‐Fe Karridale for the first 4 days of ‐Fe treatment. Both Koala and Karridale reached their highest H+‐release rates on the fourth day after ‐Fe treatment initiation. The +Fe Koala released H+ after several days of culture, but the H+ release of the ‐Fe Koala was severalfold greater than that of the +Fe Koala. The implicit correlation between H+ release and Fe‐deficiency resistance was substantiated by using a series of subclover cultivars with a range of susceptibilities to Fe deficiency. The pH of the ‐Fe culture media of the series of cultivars was positively correlated to their Fe‐chlorosis scores reported in previous research. The results of the present study indicate that root itself has the full ability to develop Fe‐deficiency stress response and the response is dependent on the root Fe status. The results also suggest that root culture could be used as a simple and efficient alternative technique for screening germplasm for Fe‐deficiency resistance.

Intracellular pH and Proton-Transport in Barley Root Cells under Salt Stress: in Vivo 31P-NMR Study

Salt stress-induced changes of intracellular pH and in levels of phosphorous compounds were monitored in intact root tips of barley seedlings (Hordeum vulgare cv. Akashin-riki) by in vivo 31P-nuclear magnetic resonance (NMR) spectroscopy. Vacuolar alkalization was observed after treatment with both 300 and 500 mM NaCl. Much of the observed apparent alkalization of the cytoplasm was eliminated when the effect of Na+ ions on the titration curve was considered. Within 1 h after the initiation of salt stress, levels of glucose-6-phosphate and UDP-glucose decreased markedly, and such decreases might lead directly or indirectly to cell death. Simultaneous measurements of the external and intracellular pH revealed the promotion of external acidification and internal alkalization during salt stress. Possible mechanisms of Na+/H+ antiport at the tonoplast and the role of proton-pump in the plasma membrane are discussed.

Mycorrhizal colonization and lead distribution in root tissues of Norway spruce seedlings

AbstractNon‐inoculated spruce seedlings (Picea abies Karst.) and spruce seedlings colonized with Lactarius rufus Fr., Pisolithus tinctorius Coker & Couch or Paxillus involutus Fr. were grown for 35 or 37 weeks in a microcosm system on two types of natural forest humus differing in Pb content. Using X‐ray microanalysis, the distribution and content of Pb in the tissues of mycorrhizal and non‐mycorrhizal root tips were compared. No significant difference in the Pb contents of root cortex cell walls of non‐mycorrhizal and seedlings colonized by Lactarius rufus, Pisolithus tinctorius, or indigenous mycorrhizal fungi (mainly Tylospora sp.) was found. However, in root tips of seedlings colonized by Paxillus involutus, due to a higher binding capacity for cations, the Pb content in cell walls of the root cortex were higher than in non‐mycorrhizal roots. Pb contents in cell walls of the cortex of mycorrhizal and non‐mycorrhizal roots were 3 times higher in plants growing in humus with a high Pb content than in plants growing in humus with a low Pb content. It is concluded that increasing contents of Pb in the organic matter may lead to an increased loading of the apoplast with Pb. The mycobionts tested in this investigation did not exclude Pb from root tissues.

Aluminum stress and protein synthesis in near isogenic lines of Triticum aestivum differing in aluminum tolerance

Aluminum (Al) stress was examined in three lines of wheat (Triticum aestivum L.) by measuring root lengths, protein synthesis and protein accumulation in seedling root tips grown in a hydroponic system. An Al‐sensitive, recurrent wheat parent (cv. Katepwa) showed very little root growth in low Al concentrations. In contrast, an Al‐tolerant near isogenic line (Alikat) and Al‐tolerant donor (cv. Maringa) had much greater root growth. Segregation data from an F2 population (Katepwa × Alikat) showed that one major gene controlled Al tolerance based on root growth (X2= 0.651). All three lines showed an approximately 2‐fold increase in [35S]‐Met incorporation in root tips after 3 days in Al and a comparable increase in root‐tip dry weight. Maringa and Alikat root tips showed an increased total protein content while Katepwa root tips showed no increase in total protein content during the Al stress. Based on higher specific activities, insoluble proteins were preferentially translated in all three lines during Al stress. Proteinase activity in Katepwa root tips was 1.7‐fold higher during Al stress, with Maringa and Alikat showing no change in proteinase activity. The Al‐induced, increased proteinase activity in Katepwa appeared to inhibit soluble protein accumulation.

Interactive effects of Ca2+ and NaCl salinity on the ionic relations and proline accumulation in the primary root tip of Sorghum bicolor

The effects of supplemental Ca2+ supply and NaCl salinity on the ionic relations and levels of proline and other amino acids in the primary root of Sorghum bicolor (cv. Hegari) seedlings were investigated. Two days of exposure to 150 mM NaCl resulted in a 50‐fold increase in the proline level in the 0–10 mm root tips of seedlings supplied with 5.0 mM Ca2+, but only a 4‐fold increase in seedlings with 0.5 mM Ca2+. In contrast to the high levels of proline in the root tip, proline accumulation was only modest in the expanded tissues of the root. The enhancement of proline accumulation in the root tip of salinized seedlings with the Ca2+ supplement may be related to their more favorable tissue K to Na ratio. Thus, elevated Ca2+ may mitigate the NaCl‐induced inhibition of S. bicolor root growth via the maintenance of net K to Na selectivity and the enhancement of proline accumulation in the root tip.

Regulation of H+ Extrusion and Cytoplasmic pH in Maize Root Tips Acclimated to a Low-Oxygen Environment.

We tested the hypothesis that H+ extrusion contributes to cytoplasmic pH regulation and tolerance of anoxia in maize (Zea mays) root tips. We studied root tips of whole seedlings that were acclimated to a low-oxygen environment by pretreatment in 3% (v/v) O2. Acclimated root tips characteristically regulate cytoplasmic pH near neutrality and survive prolonged anoxia, whereas nonacclimated tips undergo severe cytoplasmic acidosis and die much more quickly. We show that the plasma membrane H+-ATPase can operate under anoxia and that net H+ extrusion increases when cytoplasmic pH falls. However, at an external pH near 6.0, H+ extrusion contributes little to cytoplasmic pH regulation. At more acidic external pH values, net H+ flux into root tips increases dramatically, leading to a decrease in cytoplasmic pH and reduced tolerance of anoxia. We present evidence that, under these conditions, H+ pumps are activated to partly offset acidosis due to H+ influx and, thereby, contribute to cytoplasmic pH regulation and tolerance of anoxia. The regulation of H+ extrusion under anoxia is discussed with respect to the acclimation response and mechanisms of intracellular pH regulation in aerobic plant cells.

Sugar levels regulate the expression of ribosomal protein genes encoding protein S28 and ubiquitin-fused protein S27a in maize primary root tips

In order to isolate glucose-starvation-repressed cDNAs in maize ( Zea mays L.) root tips, a cDNA library was constructed with poly(A) + mRNA from excised root tips of 3-day old maize seedlings. After differential screening of the library, we isolated two cDNAs named star1 and star2, which were identified by comparison with sequence data bases. star1 corresponds to a new member of the ubiquitin fusion gene family in maize coding for a monomer of ubiquitin in frame with ribosomal protein S27a (Ubi-S27a). star2 encodes the maize homologue of ribosomal protein S28. In maize root tips submitted to carbohydrate deprivation, the Ubi-S27a and S28 mRNA levels decrease very rapidly and become undetectable after 24 h of starvation. The effects of starvation on the Ubi-S27a and S28 mRNA expression can be reversed by re-feeding the root tips with glucose. The Ubi-S27a and S28 mRNA expression is most prevalent when root tips are incubated in the presence of high glucose concentrations and metabolizable sugars such as fructose and sucrose, but not in the presence of non-metabolizable sugars such as mannitol and l-glucose. Transcript levels of maize Ubi-S27a and S28 are preferentially abundant in dividing tissues of maize seedlings. In root tips incubated with cell division inhibitors, the decrease of Ubi-S27a and S28 mRNA steady-state levels parallels that of histone H4 mRNA, that was used as a marker of cell division. These results indicate that the regulation of the expression of the two ribosomal protein genes that we monitored, may be controlled by sugar levels in a nutrient supply- and growth rate-dependent manner.

Expression of an auxin-inducible promoter of tobacco in Arabidopsis thaliana

The expression of the auxin-inducible Nt103-1 gene of tobacco was studied in Arabidopsis thaliana. For this purpose we introduced a gene fusion between the promoter of the gene and the β-glucuronidase reporter gene (GUS) into Arabidopsis thaliana. The expression and location of GUS activity were studied histochemically in time and after incubation of seedlings on medium containing auxins or other compounds. The auxins 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), and 1-naphthylacetic acid (1-NAA) were able to induce GUS activity in the root tips of transgenic seedlings. The auxin transport inhibitor 2,3,5-triiodobenzoic acid was able to induce GUS activity not only in the root tip, but also in other parts of the root. Induction by the inactive auxin analog 3,5-dichlorophenoxyacetic acid was much weaker. Compounds like glutathione and the heavy metal CuSO4 were weak inducers. GUS activity observed after induction by glutathione was located in the transition zone. Salicylic acid and compounds increasing the concentration of hydrogen peroxide in the cell were also very well able to induce GUS activity in the roots. The possible involvement of hydrogen peroxide as a second messenger in the pathway leading to the induction of the Nt103-1 promoter is discussed.

Transcription of tobacco phytochrome-A genes initiates at multiple start sites and requires multiple cis-acting regulatory elements.

Promoter regions of the Nicotiana tabacum PHYA1 and PHYA2 genes display 89% sequence identity. Analysis of the 5' ends of both the PHYA1 and the PHYA2 transcript revealed multiple, distinct mRNA species, each differing in length and in abundance. The levels of the major PHYA1, PHYA2 transcripts were found to be auto-regulated by phytochrome. This auto-regulation was most efficient in 2-8-day old seedlings. Furthermore, we examined the expression pattern of the PHYA1-GUS reporter gene, containing a 4700 bp PHYA1 promoter fragment or its 5'-deletion derivatives, by GUS histochemistry and by RNase protection assays in transgenic tobacco plants. Our data indicate that the PHYA1 promoter contains three regions which are necessary for the maximum level and regulated expression. We show that a 264 bp promoter fragment contains a cis-regulatory element(s) responsible for expression in the root tips of transgenic seedlings. The major cis-regulatory elements required for high-level transcription and expression in other organs are located in separate regions of the PHYA1 promoter. These data indicate the contribution of multiple cis-regulatory elements for the maximum and regulated expression of tobacco genes coding for phytochrome A protein.

Effect of Chilling on Assimilatory Sulfate Reduction and Glutathione Synthesis in Maize

The contents of cysteine, γ-glutamylcysteine and glutathione, as well as the activity of enzymes of assimilatory sulfate reduction and glutathione synthesis in second leaves and root tips of maize seedlings ( Zea mays L. cv. LG 11) cultivated at low temperatures, were compared with controls grown at 25 °C. In vitro nitrate reductase (EC 1.6.6.1) activity was measured for comparison. Compared with the controls, adenosine 5'-phosphosulfate sulf otransf erase activity was 3.5- and 5.5-fold higher at 12 °C in second leaves and root tips, respectively. After 3 days of growth, the activity of y,-glutamylcysteine synthetase (EC 6.3.2.2) and glutathione synthetase (EC 6.3.2.3) in the second leaves was also significantly higher at 12 °C compared with 25 °C. Consistent with these enzyme activities, there was an increase in cysteine and glutathione contents both in second leaves and root tips after 3 days of growth at 12 °C. Nitrate reductase activity of second leaves and root tips was not affected by growth at 12 °C, indicating that the increase in adenosine 5'-phosphosulfate sulfotransferase activity and of the enzymes of glutathione synthesis was specific. Our results demonstrate that the increased activity of a key enzyme of assimilatory sulfate reduction and of the enzymes of glutathione synthesis contribute to the increased glutathione levels measured at 12 °C.

Nucleotide Levels Do Not Critically Determine Survival of Maize Root Tips Acclimated to a Low-Oxygen Environment.

We tested the hypothesis that ATP levels and energy charge determine the resistance of maize (Zea mays) root tips to anoxia. We focused on root tips of whole maize seedlings that had been acclimated to low O2 by exposure to an atmosphere of 3% (v/v) O2 in N2. Acclimated anoxic root tips characteristically have higher ATP levels and energy charge and survive longer under anoxia than nonacclimated tips. We poisoned intact, acclimated root tips with either fluoride or mannose, causing decreases in ATP and energy charge to values similar to or, in most cases, below those found in nonacclimated anoxic tips. With the exception of the highest fluoride concentration used, the poisoned, acclimated tips remained much more tolerant of anoxia than nonacclimated root tips. We conclude that high ATP and energy charge are not components critical for the survival of acclimated root tips during anoxia. The reduced nucleotide status in poisoned, acclimated root tips had little effect on cytoplasmic pH regulation during anoxia. This result indicates that in anoxic, acclimated root tips either cytoplasmic pH regulation is not dominated by ATP-dependent processes or these processes can continue in vivo largely independently of any changes in ATP levels in the physiological range. The role of glycolytic flux in survival under anoxia is discussed.

Metabolic Acclimation of Root Tips to Oxygen Deficiency

Survival of roots in O2 deficient media appears to depend on two completely opposing stratagems; the one involving cell survival, the other, cell death. In the apical zone of maize primary roots, the ability of cells to tolerate hypoxia or anoxia is paramount so that the meristem is not destroyed. Transcriptional and post-transcriptional regulation of protein synthesis ensures that enzymes of the glycolytic and ethanolic fermentation pathway are abundant in seedling root tips that have previously acclimated to low O2. By contrast, anaerobically shocked cells are much less able to tolerate anoxia and quickly succumb. Acclimation is associated with higher anaerobic respiration rates, greater energy metabolism and improved survival after transfer to anoxia.Hypoxia also sets in motion an entirely different set of responses in the zone close to the tip where cell elongation is ceasing, resulting in a change of root structure. Ethylene-dependent lysis of cortical cells is induced, these cells being selectively sacrificed to form aerenchyma and thereby improve the internal oxygenation of the root. The significance of the hypoxic induction of changes in the activities of various key enzymes associated with cell death and lysis is discussed.

Sorghum seedling root tip elemental contents after concomitant exposure to PCMBS and mineral nutrient solution

Abstract p‐Chloromercuribenzenesulfonic acid (PCMBS) (0, 0.01, 0.1, or 1 mM) was evaluated for influence on calcium (Ca), phosphorus (P), boron (B), zinc (Zn), manganese (Mn), iron (Fe), magnesium (Mg), and copper (Cu) uptake in 1‐hr by 1‐cm root tips of seedling Sorghum bicolor (L.) Moench, cv GP‐10, SC 283, Funk G522DR, and SC 574 from Hoagland and Arnon complete mineral nutrient solution. Calcium uptake was inhibited by PCMBS (1 mM) in all four cultivars but genetic differences in response were evident. Responses of the cultivars to PCMBS influence on the other elements uptake showed complete genetic diversity within each element measured and between cultivars within each element.

Karyotype analysis and optimization of mitotic index in Picea mariana (black spruce) preparations from seedling root tips and embryogenic cultures

The effect of hydroxyurea and colchicine solutions on mitotic index was investigated in embryogenic suspensions and in seedling root tips of Picea mariana. Hydroxyurea treatment and 0.6 per cent colchicine solution significantly increased the quantity of mitotic cells which were mostly in the metaphase stage. The highest mitotic index was observed in cells of embryogenic suspensions. The embryogenic lines were cytologically stable and their utilization for karyotype studies and technique development holds great potential. The arm ratios and relative chromosome length values showed the same trends between embryogenic cultures and seedling root tips and among the five black spruce populations studied. This study shows that the black spruce karyotype is asymmetric and semi-advanced, as in Larix and Abies. Three pairs of chromosomes are of the sm-type with submedian centromeres. One pair is median-submedian (msm), six pairs are m-type having median centromeres and two pairs are metacentric or M-type.

Sorghum seedling root tip elemental contents after concomitant exposure to PCMBS and mineral nutrient solution

Sorghum seedling root tip elemental contents after concomitant exposure to PCMBS and mineral nutrient solution