Calcium-Regulated in Vivo Protein Phosphorylation in Zea mays L. Root Tips

Calcium dependent protein phosphorylation was studied in corn (Zea mays L.) root tips. Prior to in vivo protein phosphorylation experiments, the effect of calcium, ethyleneglycol-bis-(β-aminoethyl ether)-N-N′-tetraacetic acid (EGTA) and calcium ionophore (A-23187) on phosphorus uptake was studied. Calcium increased phosphorus uptake, whereas EGTA and A-23187 decreased it. Consequently, phosphorus concentration in the media was adjusted so as to attain similar uptake in different treatments. Phosphoproteins were analyzed by two-dimensional gel electrophoresis. Distinct changes in phosphorylation were observed following altered calcium levels. Calcium depletion in root tips with EGTA and A-23187 decreased protein phosphorylation. However, replenishment of calcium following EGTA and ionophore pretreatment enhanced phosphorylation of proteins. Preloading of the root tips with ³²P in the presence of EGTA and A-23187 followed by a ten minute calcium treatment, resulted in increased phosphorylation indicating the involvement of calcium, calcium and calmodulin-dependent protein kinases. Calmodulin antagonist W-7 was effective in inhibiting calcium-promoted phosphorylation. These studies suggest a physiological role for calcium-dependent phosphorylation in calcium-mediated processes in plants.     

Nucleotide Metabolism in Salt-Stressed Zea mays L. Root Tips: I. Adenine and Uridine Nucleotides

Corn plants (Zea mays L. cv Pioneer 3906) were grown in a glass house on control and saline nutrient solutions, in winter and summer. There were two saline treatments, both with osmotic potential = −0.4 megapascal but with different Ca²⁺/Na⁺ ratios: 0.03 and 0.73. Root tips and shoot meristems (culm tissue) of 26 day-old plants were analyzed for nucleotides to ascertain if there were correlations between nucleotide pool size and the reduced growth on saline cultures. Several other cell components also were determined. Plants grown in winter were only half as large as those grown in summer mainly because of the lower light intensity and lower temperature. But the relative yield reduction on salt treatment compared to the control was similar in winter and summer. The two different salt treatments caused similar yield reductions. Neither salt treatment affected nucleotide pools in culm tissue, with the possible exception of UDPG in winter. In the case of root tips, salt treatment had little or no effect on nucleotide pool sizes in winter when many already seemed near a critical minimum, but in summer it reduced several pools including ATP, total adenine nucleotide, UTP, total uridine nucleotide, and UDP-glucose. The reductions were greatest on the salt treatment with low Ca²⁺/Na⁺. There was no simple correlation between the effects of salt stress on growth and on nucleotide pool size. The nucleotide pools of culm tissue indicated that in some respects this tissue was effectively insulated from the salt stress. Roots that were in direct contact with the saline solution indicated significant reductions in nucleotide pools only in the summer whereas growth was reduced both summer and winter. It is possible that the nucleotide concentrations of root cells in winter were already near a critical minimum so that nucleotide synthesis and growth were tightly linked. Significant reductions in nucleotide pools that would be expected to affect growth were more evident in summer when pools were larger and growth was more rapid. But even where ATP and total adenine nucleotides were reduced, the ratio of ATP:ADP and the adenylate energy charge remained unchanged indicating an active adenylate kinase that had access to most of the adenine nucleotide pools, and possible catabolism of excess AMP.     

Hypoxic Induction of Anoxia Tolerance in Root Tips of Zea mays

When root tips of fully aerobic, intact maize (Zea mays L.) seedlings are made anaerobic, viability normally is only 24 hours or less at 25°C. We find that viability can be extended to at least 96 hours if seedlings are given a hypoxic pretreatment for 18 hours by sparging the solution with 4% O₂ in nitrogen (v/v) before anoxia. Fully aerobic root tips (sparged with 40% O₂) had very low alcohol dehydrogenase (ADH) activity (per gram root fresh weight), and the level remained low under anoxia. In hypoxically pretreated roots, however, high levels of ADH activity were induced, and activity rose further during the initial 24 hours of anoxia, and then remained high at about 20 times that of controls in 40% O₂. ADH activity in roots in solution sparged with air (21% O₂) was about three times that in 40% O₂. Improved viability of hypoxically pretreated root tips was associated with maintenance of a high energy metabolism (ATP concentration, total adenylates, and adenylate energy charge). Roots that were not pretreated lost 94% of the total adenylates and ATP at 24 hours of anoxia. The relation between induced ADH activity, energy metabolism, and improved anoxia-tolerance in acclimated maize root tips is discussed.     

Root Growth Inhibitors from Root Cap and Root Meristem of Zea mays L.

A micro-assay based on the growth inhibition of root segmentsof the seminal roots of Zea mays has been used to investigatethe root-growth-inhibiting substances in root caps and meristemsrespectively of the roots of Zea mays. This micro-assay is sensitiveto 50 pg of IAA or less. Paper chromatography of the acid fractionof methanolic extracts shows the presence of one main inhibitorin root caps and a different main inhibitor in root meristems.Neither is IAA, whose presence in meristems is sometimes indicatedby small inhibitions (or stimulations) at the characteristicRf of IAA. A Commelina leaf-epidermis assay shows the presenceof one stomata-closing ABA-like substance in root caps and onein meristems, one corresponding in Rf to the main root-growthinhibitor from the root cap. The implications of these findingsfor the geotropic responses of roots is briefly discussed.     

The Movement and Physiological Effect of Indoleacetic Acid Following Point Applications to Root Tips of Zea mays

Pinpoint applications of labeled and non-labeled indoleacetic acid (IAA) on resin beads were made, without injury, to vertical roots of intact seedlings of Zea mays. Points of application were at the extreme tip of the root, 0.5, 2 and 5 mm from the root tip. The movement of label and bending of the roots was recorded. Radiolabel was found to move basipetally from the extreme tip and 0.5 mm applications to a similar extent, reaching 8 mm from the tip. The level of label in the growing zone after 4 h was 10% of that found in the extreme tip. Movement from 2 and 5 mm applications was equal in both directions. Higher amounts of non-labeled IAA caused bending towards the point of application if applied at 0.5 or 2 mm but not at 5 mm from the tip. It is proposed that any endogenous IAA in the root cap could move to the growing zone and cause a unilateral inhibition of growth, provided that it was in the same transport pool as the exogenously applied IAA.     

Suitability of the Osmotic Shock Procedure for the Analysis of Membrane Transport in Root Tips of Zea mays L.

Root tips of Zea mays L. previously subjected to osmotic shockwere studied as to uptake and efflux of glucose and uptake ofphosphate, as well as release of protein and other Lowry-positivesubstances. The osmotic shock procedure applied was similarto that frequently used in order to release periplasmic proteinsfrom bacteria. After treatment with hypertonic solutions of sorbitol (>0?3M) the uptake of phosphate and glucose is reduced and proteinis released into the medium. All osmotic treatments leadingto a significant reduction of either glucose or phosphate uptakeare accompanied by a strongly increased release of glucose andLowry-positive substances. Damage of plasma membrane barrierfunction can be directly observed by the uptake of Evans Blue(Gaff and Okong'o-Ogola, 1971) into the cells involving mainlyolder vacuolated rhizodermal cells. The occurrence and extent of the alterations of membrane semipermeabilityare strongly dependent on the duration of exposure to and theconcentration of the sorbitol solution used. The velocity ofthe osmotic transitions (from hypotonic to hypertonic and viceversa) is of minor importance. Sensibility of cells to osmotic stress increases with differentiationand vacuolation. Conditions for a selective reduction of phosphateuptake seem to occur when only meristem and near tip regionssubjected to a mild osmotic shock are used.     

Metabolism of Proline, Glutamate, and Ornithine in Proline Mutant Root Tips of Zea mays (L.)

In excised pro_1-1 mutant and corresponding normal type roots of Zea mays L. the uptake and interconversion of [¹⁴C]proline, [¹⁴C]glutamic acid, [¹⁴C]glutamine, and [¹⁴C]ornithine and their utilization for protein synthesis was measured with the intention of finding an explanation for the proline requirement of the mutant. Uptake of these four amino acids, with the exception of proline, was the same in mutant and normal roots, but utilization differed. Higher than normal utilization rates for proline and glutamic acid were noted in mutant roots leading to increased CO₂ production, free amino acid interconversion, and protein synthesis. Proline was synthesized from either glutamic acid (or glutamine) or ornithine in both mutant and normal roots; it did not accumulate but rather was used for protein synthesis. Ornithine was not a good precursor for proline in either system, but was preferentially converted to arginine and glutamine, particularly in mutant roots. The pro_1-1 mutant was thus not deficient in its ability to make proline. Based on these findings, and on the fact that ornithine, arginine, glutamic acid and aspartic acid are elevated as free amino acids in mutant roots, it is suggested that in the pro_1-1 mutant proline catabolism prevails over proline synthesis.     

Aluminum Exclusion Mechanism in Root Tips of Maize (Zea mays L.): Lysigeny of Aluminum Hyperaccumulator Cells

Abstract: Ultrastructural studies, together with X-ray microanalytical, immunocytochemical and cytochemical analysis performed in root tips of Al-resistant (C-525 M) and Al-sensitive (Adour 250, HS 7777 and BR 201 F) maize plants ( Zea mays L.), after 96 h exposure to 20 μM Al, showed qualitatively similar results in the four cultivars.
Al was identified in electron-opaque precipitates, which were insoluble even in EDTA chelate. They also contained an elevated proportion of P and also of K and Ca, some traces of Mn, Fe and Zn and sometimes of Mg. This elemental composition is similar to that described for phytin (Al-phytin), and the precipitates were localized in the two principal extraplasmatic compartments: cell walls and vacuoles. Al-phytin was detected in swollen areas of cell walls in membraneous concentric configurations, resembling myelin figures, probably rich in phosphatidyl inositol, which also intervene in the vacuolar internalization of Al-phytin and are similar to a peculiar form of endocytosis (not previously described). Abnormal apoplastic protuberances containing abundant electron-opaque Al-phytin deposits, agglutinated by callose (immunocytochemically identified), were shown in cortex cells with high mitotic activity (around 1 - 1.5 mm from cap root). Al-hyperaccumulator cells parallel to the root axis were correlated with longitudinal lysigenous intercellular spaces after cell death and dissolution (lysigeny). Indicators of activated lysigeny, as low levels of Al and callose (in agreement with other authors) and high levels of phosphoinositides, can mark Al-resistant genotypes, contrary to Al-sensitive genotypes, probably derived from a partially activated or even inactivated lysigeny.
The lysigeny of Al hyperaccumulator cells constitutes new ultrastructural evidence of an Al exclusion mechanism, supporting biochemical results reported by other investigators.     

Biosynthesis of Indole-3-Acetic Acid from L-Tryptophan in Coleoptile Tips of Maize (Zea mays L.)

Coleoptile tips (about 2.5 mm in length) were excised from 3-day-olddark-adapted maize (Zea mays L.) seedlings and incubated indarkness in potassium phosphate buffer that contained ¹⁴C-L-tryptophan(Trp). Subsequent analysis by gas chromatography-mass spectrometryindicated that a significant portion of endogenous indole-3-aceticacid (IAA) had been labeled with ¹⁴C. About 8% of the IAA thatdiffused from the tissue into the medium during incubation from0.5 to 2 h was labeled, and 12% of the IAA extracted from thetissue after a 2-h incubation was labeled. On the other hand,30% of the Trp extracted from the tissue after a 2-h incubationwas ¹⁴C-Trp, which was more than those determined for IAA. Sincethe experiments were carried out under the non-steady-stateconditions in which the tissue content of ¹⁴C-Trp increasedwith time, and since the extracted Trp included the ¹⁴C-Trpin the apoplastic space, it seemed that synthesis de novo fromTrp was the major means by which IAA was produced in the coleoptiletip. The conversion of Trp to IAA was not detected in sub-apicalsegments (5–7.5 mm from the top) that were incubated similarly,an indication that synthesis of IAA occurs specifically in thetip region. When intact seedlings were irradiated with a pulseof red light 2 h before excision of tips and the applicationof ¹⁴C-Trp, the amounts of extractable and diffusible IAA werereduced by 40–60% without a change in the rate of ¹⁴Cincorporation. This result indicated that the production ofIAA from Trp is controlled by a red-light signal. (Received May 15, 1995; Accepted September 1, 1995)     

Root Growth, Secondary Root Formation and Root Gravitropism in Carotenoid-deficient Seedlings of Zea mays L.

The effect of ABA on root growth, secondary-root formation androot gravitropism in seedlings of Zea mays was investigatedby using Fluridone-treated seedlings and a viviparous mutant,both of which lack carotenoids and ABA. Primary roots of seedlingsgrown in the presence of Fluridone grew significantly slowerthan those of control (i.e. untreated) roots. Elongation ofFluridone-treated roots was inhibited significantly by the exogenousapplication of 1 mM ABA. Exogenous application of 1 µMand 1 nM ABA had either no effect or only a slight stimulatoryeffect on root elongation, depending on the method of application.The absence of ABA in Fluridone-treated plants was not an importantfactor in secondary-root formation in seedlings less than 9–10d old. However, ABA may suppress secondary-root formation inolder seedlings, since 11-d-old control seedlings had significantlyfewer secondary roots than Fluridone-treated seedlings. Rootsof Fluridone-treated and control seedlings were graviresponsive.Similar data were obtained for vp-9 mutants of Z. mays, whichare phenotypically identical to Fluridone-treated seedlings.These results indicate that ABA is necessary for neither secondary-rootformation nor for positive gravitropism by primary roots. Zea mays, gravitropism, carotenoid-deficient, Fluridone, root growth, vp-9 mutant     

Exit from the Mitotic Cycle in Root Meristems of Zea mays L.

The choice between two modes of exit from the mitotic cycleat the margins of meristems has been made easier by surveyingthe range of the numbers of cell contacts between contiguousfiles in root apices of Zea mays L. The range shows that somecells must go out of cycle while others remain in cycle forat least three further generations. The view that cycling endsby a fall in the proliferative fraction is supported by theexistence of pulse-labelled telophases in the proximal regionof the menstem. These are most likely due to acceleration ofthe mitotic cycle which has to be contrasted with decelerationof the overall rate of cell proliferation. The work is discussedin relation to patterns of cycling in the different tissuesof the apex. mitotic cycle, cell size, meristem, proliferative fraction, Zea mays L, maize     

The Effects of Mechanical Impedance to Growth on the Levels of ABA and IAA in Root Tips of Zea mays L.

Extraction and analytical methods have been refined and newones devised to allow precise determinations by GC-EC of thelevels of abscisic acid (ABA) and indol-3ylacetic acid (IAA)in samples of maize root tips as small as 1.0 g fr. wt. Seminalroots of 5-d-old maize seedlings grown in normal (bulk density1200 kg m^–3) and compacted (bulk density 1600 kg m^–3)sand/garden loam mixtures have been examined. Seminal rootsfrom compacted soil had an average length of about 40% of thatof control roots and were much thicker. The ABA levels in 10mm tips of impeded roots (c. 25–35 ng g^–1 fr.wt.)did not differ significantly from those of normal root tipson both a fresh and dry weight basis. The levels in 0–1mm tips were approximately double those in the remaining 1–10mm zones. IAA levels were increased by about 3 times in impededroots (176.3 as compared with 52.4 ng g^–1 fr.wt) and itis concluded that this response is likely to be the main causeof the morphological and growth changes brought about by soilcompaction.     

Abscisic Acid Biosynthesis in Isolated Embryos of Zea mays L

Previous labeling experiments with ¹⁸O₂ have supported the hypothesis that stress-induced abscisic acid (ABA) is synthesized through an indirect pathway involving an oxygenated carotenoid (xanthophyll) as a precursor. To investigate ABA formation under nonstress conditions, an ¹⁸O₂ labeling experiment was conducted with isolated embryos from in vitro grown maize (Zea mays L.) kernels. Of the ABA produced during the incubation in ¹⁸O₂, three-fourths contained a single ¹⁸O atom located in the carboxyl group. Approximately one-fourth of the ABA synthesized during the experiment contained two ¹⁸O atoms. These results suggest that ABA synthesized in maize embryos under nonstress conditions also proceeds via the indirect pathway, requiring a xanthophyll precursor. It was also found that the newly synthesized ABA was preferentially released into the surrounding medium.     

The Proportion of Cells that Divide in Root Meristems of Zea mays L.

The proportion of cells that divide in four regions of the rootmeristem of Zea mays has been determined by an analysis of itscells pulse-labelled with tritiated thymidine. In the quiescent centre less than half of the cells divide andthe fastest of these (less than half of them) have a mitoticcycle duration of about 40 h at 23 °C compared with a cell-doublingtime of 230 h for the region. In the cap initials 80–90per cent of the cells divide and about 80 per cent of thesedivide once in 10 h. In the stele about 80 per cent of cellsdivide near the quiescent centre and all divide at 200 µmfrom the quesecent centre. The fast cells divide every 14 hin both regions, but the cell-doubling time increases from 18to 25 h near the quiescent centre. The root cap is completely replaced by its initials every dayand 10 000 cells are sloughed off. The rest of the meristemadds about 170 000 cells to the root every day. These figures are discussed in relation to the role of the quiescentcentre and the control of cell division.     

Leaf vasculature in Zea mays L.

The vascular system of the Zea mays L. leaf consists of longitudinal strands interconnected by transverse bundles. In any given transverse section the longitudinal strands may be divided into three types of bundle according to size and structure: small, intermediate, large. Virtually all of the longitudinal strands intergrade structurally however, from one bundle type to another as they descend the leaf. For example, all of the strands having large-bundle anatomy appear distally as small bundles, which intergrade into intermediates and then large bundles as they descend the leaf. Only the large bundles and the intermediates that arise midway between them extend basipetally into the sheath and stem. Most of the remaining longitudinal strands of the blade do not enter the sheath but fuse with other strands above and in the region of the blade joint. Despite the marked decrease in number of longitudinal bundles at the base of the blade, both the total and mean cross-sectional areas of sieve tubes and tracheary elements increase as the bundles continuing into the sheath increase in size. Linear relationships exist between leaf width and total bundle number, and between cross-sectional area of vascular bundles and both total and mean cross-sectional areas of sieve tubes and tracheary elements.     

Somatic embryogenesis in Zea mays L.

Summary Combining ability studies with respect to such green fodder quality characteristics as oxalic acid, calcium, sodium, potassium and green fodder yield were carried out in a 12 × 12 diallel cross set in pearl millet (Pennisetum typhoides (Burm) S. & H.). With regard to differential expression of gene effects, studies for quality traits were carried out in different seasons and on different plant parts. The relative proportions of general and specific combining variances indicated the preponderance of non-additive genetic variance. Parents possessing desirable fodder quality characteristics were identified on the basis of combining ability and per se performance, and selection criterion for crosses was discussed. It was recommended that leaf portion should be biochemically analysed and manipulated in an environment when the genes are expressed.Part of the Ph. D. dissertation submitted to the Punjab Agricultural University by the senior author in partial fulfilment of the requirements for the degree     

Effect of Exogenous Abscisic Acid on Proline Dehydrogenase Activity in Maize (Zea mays L.)

Plant responses to drought stress include proline and abscisic acid (ABA) accumulation. Proline dehydrogenase (PDH) (EC 1.4.3) is the first enzyme in the proline oxidation pathway, and its activity has been shown to decline in response to water stress (PJ Rayapati, CR Stewart [1991] Plant Physiol 95: 787-791). In this investigation, we determined whether ABA treatment affects PDH activity in a manner similar to drought stress in maize (Zea mays L.) seedlings. Four exogenous ABA treatments (0, 11, 33, and 100 micromolar ABA) were applied to well-watered maize seedlings. Mitochondria were isolated and PDH was solubilized using Nonidet P-40. PDH activity was measured by the reduction of iodonitrotetrazolium violet under proline-dependent conditions. There was no effect of ABA on PDH activity at 33 and 100 micromolar ABA, but there was a 38% decline at 11 micromolar. This decline was less than the 69% reduction in activity under drought stress. Endogenous ABA determinations and plant growth rate showed that ABA entered the plant and was affecting metabolic processes. ABA treatments had a small effect on shoot and root proline concentration, whereas drought stress caused a 220% increase in root tissues. We conclude that ABA is not part of the pathway linking drought stress and decreased PDH activity.     

The Ultrastructure of the Regenerating Root Cap of Zea mays L

Removal of the cap from the primary root of Zea mays activatescell division in the quiescent centre. It is the descendentsof these cells that eventually regenerate a new cap—aprocess that is complete in about 4 days at 23 °C. The ultrastructureof the cells of the regenerating cap was examined at daily intervals.During the first day after decapping the dictyosomes in theexposed outer layer of cells change from a relatively quiescentstate to one where they are secreting a polysaccharide slimewhich accumulates between the plasmalemma and the outer cellwall. Amyloplasts grow in size and appear to divide, and theendoplasmic reticulum proliferates. Many different cytoplasmicfeatures that are normally characteristic of cells in distinctlocations within the undisturbed cap occur, at first, all togetherwithin the few cells that are the source of the new regeneratingtissue. Regeneration of a normal structure in the new cap isachieved by progressive changes in the structures of the cellorganelles, apparently in response to the position that thecells containing them occupy within the growing cellular ensembleat the root apex. Zea mays, regeneration, root cap, ultrastructure     

Internode length in Zea mays L.

[¹³C, ³H]Gibberellin A₂₀ (GA₂₀) has been fed to seedlings of normal (tall) and dwarf-5 and dwarf-1 mutants of maize (Zea mays L.). The metabolites from these feeds were identified by combined gas chromatography-mass spectrometry. [¹³C, ³H]Gibberellin A₂₀ was metabolized to [¹³C, ³H]GA₂₉-catabolite and [¹³C, ³H]GA₁ by the normal, and to [¹³C, ³H]GA₂₉ and [¹³C, ³H]GA₁ by the dwarf-5 mutant. In the dwarf-1 mutant, [¹³C, ³H]GA₂₀ was metabolized to [¹³C, ³H]GA₂₉ and [¹³C, ³H]GA₂₉-catabolite; no evidence was found for the metabolism of [¹³C, ³H]GA₂₀ to [¹³C, ³H]GA₁. [¹³C, ³H]Gibberellin A₈ was not found in any of the feeds. In all feeds no dilution of ¹³C in recovered [¹³C, ³H]GA₂₀ was observed. Also in the dwarf-5 mutant, the [¹³C]label in the metabolites was apparently undiluted by endogenous [¹³C]GAs. However, dilution of the [¹³C]label in metabolites from [¹³C, ³H]GA₂₀ was observed in normal and dwarf-1 seedlings. The results from the feeding studies provide evidence that the dwarf-1 mutation of maize blocks the conversion of GA₂₀ to GA₁.Abbreviations GA_n gibberellin A_n - GC-MS combined gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - RP reverse phase     

Enhancement of Anaerobic Respiration in Root Tips of Zea mays following Low-Oxygen (Hypoxic) Acclimation

Root tips (10-millimeter length) were excised from hypoxically pretreated (HPT, 4% [v/v] oxygen at 25°C for 16 hours) or nonhypoxically pretreated (NHPT, 40% [v/v] oxygen) maize (Zea mays) plants, and their rates of respiration were compared by respirometry under aerobic and anaerobic conditions with exogenous glucose. The respiratory quotient under aerobic conditions with 50 millimolar glucose was approximately 1.0, which is consistent with glucose or other hexose sugars being utilized as the predominant carbon source in glycolysis. Under strictly anaerobic conditions (anoxia), glycolysis was accelerated appreciably in both HPT and NHPT root tips, but the rate of anaerobic respiration quickly declined in NHPT roots. [U-¹⁴C]Glucose supplied under anaerobic conditions was taken up and respired by HPT root tips up to five times more rapidly than by NHPT roots. When anaerobic ethanol production was measured with excised root tips in 50 millimolar glucose, HPT tissues consistently produced ethanol more rapidly than NHPT tissues. These data suggest that a period of low oxygen partial pressure is necessary to permit adequate acclimation of the root tip of maize to subsequent anoxia, resulting in more rapid rates of fermentation and generation of ATP.