Hormonal and gravitropic specificity in the regulation of growth and cell wall synthesis in pulvini and internodes from shoots of Avena sativa L. (oat)

Segments can be cut from the peduncular-1 internode of oat (Avena sativa L.) shoots so as to contain the graviresponsive leaf-sheath pulvinus and gibberellin-sensitive internodal tissue. Incorporation of [14C]glucose was used to monitor cell wall synthesis in these two tissues as affected by gravistimulus, indoleacetic acid (IAA), gibberellic acid (GA3), and fusicoccin (FC). Pulvinar cell wall synthesis was promoted by IAA and FC (both within about 1 h), as well as by gravistimulus (starting between 3 and 6 h), whereas GA3 had no effect on nongravistimulated pulvini. In contrast, GA3 and FC promoted internodal cell wall synthesis (initiated between 1 and 2 h), whereas IAA and gravistimulus caused a decrease in internodal uptake. FC preferentially promoted incorporation into the matrix component of the wall in both tissues. Gravistimulus failed to increase responsiveness of pulvinar tissue to IAA, whereas GA3 partially overcame gravistimulus-promoted incorporation into pulvinar cell wall, probably because of preferential movement of label into the rapidly elongating internode. The results demonstrate that these eight stimulus/tissue combinations can be examined easily in an isolated 10-mm stem segment, providing new opportunities for the comparative study of tissue- and stimulus-specific events in gene regulation and signal transduction in agronomically important cereals.     

Changes in the ultrastructure of cell walls, cellulose synthesis, and glucan synthase activity from gravistimulated pulvini of oat (Avena sativa)

Ultrastructural analyses of the cell walls from top and bottom halves of gravistimulated pulvini from oat leaves show a decrease in the density of material within the cell walls from the lower halves of pulvini after 24 h of gravistimulation. Assays of cellulose synthesis with a 14C-sucrose pulse-chase experiment indicate no difference in the amount of new cellulose synthesized in top compared with bottom halves of gravistimulated pulvini. The highest rate of cellulose synthesis occurs with 12-24 h of gravistimulation. Treatment of graviresponding pulvini with 2,6-dichlorobenzonitrile (DCBN) had only a minor effect on segment gravitropic curvature. We also found that there is no difference in the activities of either glucan synthase I or glucan synthase II in top halves as compared with bottom halves of gravistimulated pulvini. We conclude that the graviresponse in oat stems is not driven by new cell wall synthesis but, rather, by changes in cell wall plasticity and osmotic potential.     

A role for inositol 1,4,5-trisphosphate in gravitropic signaling and the retention of cold-perceived gravistimulation of oat shoot pulvini

Plants sense positional changes relative to the gravity vector. To date, the signaling processes by which the perception of a gravistimulus is linked to the initiation of differential growth are poorly defined. We have investigated the role of inositol 1,4,5-trisphosphate (InsP(3)) in the gravitropic response of oat (Avena sativa) shoot pulvini. Within 15 s of gravistimulation, InsP(3) levels increased 3-fold over vertical controls in upper and lower pulvinus halves and fluctuated in both pulvinus halves over the first minutes. Between 10 and 30 min of gravistimulation, InsP(3) levels in the lower pulvinus half increased 3-fold over the upper. Changes in InsP(3) were confined to the pulvinus and were not detected in internodal tissue, highlighting the importance of the pulvinus for both graviperception and response. Inhibition of phospholipase C blocked the long-term increase in InsP(3), and reduced gravitropic bending by 65%. Short-term changes in InsP(3) were unimpaired by the inhibitor. Gravitropic bending of oat plants is inhibited at 4 degrees C; however, the plants retain the information of a positional change and respond at room temperature. Both short- and long-term changes in InsP(3) were present at 4 degrees C. We propose a role for InsP(3) in the establishment of tissue polarity during the gravitropic response of oat pulvini. InsP(3) may be involved in the retention of cold-perceived gravistimulation by providing positional information in the pulvini prior to the redistribution of auxin.     

Changes in starch and inositol 1,4,5-trisphosphate levels and auxin transport are interrelated in graviresponding oat (Avena sativa) shoots

This study was conducted to unravel a mechanism for the gravitropic curvature response in oat (Avena sativa) shoot pulvini. For this purpose, we examined the downward movement of starch-filled chloroplast gravisensors, differential changes in inositol 1,4,5-trisphosphate (IP(3)) levels, transport of indole-3-acetic acid (IAA) and gravitropic curvature. Upon gravistimulation, the ratio for IAA levels in lower halves versus those in upper halves (L/U) increased from 1.0 at 0 h and reached a maximum value of 1.45 at 8 h. When shoots were grown in the dark for 10 d, to deplete starch in the chloroplast, the gravity-induced L/U of IAA was reduced to 1.0. N-naphthylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid (TIBA), both auxin transport inhibitors, significantly reduced the amount of gravitropic curvature and gravity-induced lateral IAA transport, but did not reduce the gravity-induced late change in the L/U ratio of IP(3) levels. U73122, a specific phospholipase C (PLC) inhibitor, decreased gravity-induced curvature. Because U73122 reduced the ratio of L/U of IAA imposed by gravistimulation, it is clear that IAA transport is correlated with changes in IP(3) levels upon gravistimulation. These results indicate that gravistimulation-induced differential lateral IAA transport may result from the onset of graviperception in the chloroplast gravisensors coupled with gravity-induced asymmetric changes in IP(3) levels in oat shoot pulvini.     

Factors inducing regeneration response in oat (Avena sativa L.) anther culture

The efficiency of embryogenesis of anther culture was compared using four cultivars of oat (Avena sativa L.): ‘Akt’, ‘Bingo’, ‘Bajka’, and ‘Chwat’. Despite the high resistance of oat to the process of androgenesis, all tested cultivars produced embryo-like structures and only two of them, ‘Akt’ and ‘Chwat’, produced fertile doubled haploid plants. A strong cultivar dependency was observed during induction of androgenesis. Further, cold pretreatment together with high temperature shock enhanced the efficiency of this technique. The highest number of embryo-like structures and haploid plants was obtained from cv. ‘Chwat’ (3.6% and 0.8%, respectively). Embryo-like structure formation also depended on the distance from the base of the flag leaf to the penultimate leaf of the panicle. Most of them were observed on anthers harvested from panicles of which the distance from the base of the flag leaf to the penultimate leaf was less than 4 cm. The presence of the induction medium supplemented with different plant growth regulators was essential for the induction of embryo-like structures but did not increase the production of haploid plants and doubled haploid lines. The highest number of embryo-like structures and plants was obtained on W14 medium with the addition of 2.0 mg/dm³ 2,4-dichlorophenoxyacetic acid and 0.5 mg/dm³ kinetin (2.7%). The low haploid plant regeneration rate (from 0.03 to 0.05%) still limits the practical application of anther culture for the production of doubled haploid lines in oat.

     

Transferability and utility of white oat (Avena sativa) microsatellite markers for genetic studies in black oat (Avena strigosa)

Preservation and use of wild oat species germplasm are essential for further improvement of cultivated oats. We analyzed the transferability and utility of cultivated (white) oat Avena sativa (AACCDD genome) microsatellite markers for genetic studies of black oat A. strigosa (A(s)A(s) genome) genotypes. The DNA of each black oat genotype was extracted from young leaves and amplified by PCR using 24 microsatellite primers developed from white oat. The PCR products were separated on 3% agarose gel. Eighteen microsatellite primer pairs amplified consistent products and 15 of these were polymorphic in A. strigosa, demonstrating a high degree of transferability. Microsatellite primer pairs AM3, AM4, AM21, AM23, AM30, and AM35 consistently amplified alleles only in A. sativa, which indicates that they are putative loci for either the C or D genomes of Avena. Using the data generated by the 15 polymorphic primer pairs, it was possible to separate 40 genotypes of the 44 that we studied. The four genotypes that could not be separated are probably replicates. We conclude that A. sativa microsatellites have a high transferability index and are a valuable resource for genetic studies and characterization of A. strigosa genotypes.     

Synthesis of proteins during the development of the first leaf of oat (Avena sativa)

Distal segments of the first leaf of intact oat ( Avena sativa L. cv. Victory) plants at different stages of development were labelled with ³⁵S-methionine. During 12-h labelling periods similar amounts of label were retained in the segments, but incorporation into protein decreased from 28% in 7-day- to 3% in 27-day-old plants. Two-dimensional polyacrlamide gel electrophoretic analysis of the proteins revealed that even at a late stage of senescence a great many proteins are still being synthesized. Synthesis of ribulosebisphosphate carboxylase and other chloroplast-associated proteins declined more rapidly than general protein synthesis. With increasing leaf age, two sets of proteins with a relatively high molecular weight around 67 kDa and isoelectric points between 6.5 and 6.8 became the most prominently synthesized proteins. Since these proteins were hardly visible upon silver-staining, they seem to be subject to rapid turnover. An involvement of these proteins in senescence might explain the requirement of protein synthesis for senescence to proceed.     

Changes in phosphorylation of 50 and 53 kDa soluble proteins in graviresponding oat (Avena sativa) shoots

The present work indicates that phosphorylation of a 50 kDa soluble protein is involved in the gravitropic response in graviresponsive pulvini of oat (Avena sativa) stems. This 50 kDa protein shows a differential pattern of phosphorylation between lower and upper halves of pulvini both in vivo and in vitro. The differential phosphorylation of this protein is detected only when stem segments are gravistimulated for short and long time periods. The differential phosphorylation of the 50 kDa protein occurs as early as 5 min after the initiation of gravistimulation. This corresponds closely to the presentation time of 5.2 min. This differential phosphorylation pattern was changed by treatments with cycloheximide, implying that a newly-synthesized protein is involved in the differential phosphorylation during the gravitropic response. An autophosphorylation experiment shows that the 50 kDa protein has kinase activity. The phosphorylation patterns of a 53 kDa protein were similar to those of the 50 kDa protein, but were only expressed in vitro. These findings indicate that the differential phosphorylation of the 50 (and 53 kDa) soluble proteins in graviresponding oat shoots may be an important component of the gravity signal transduction pathway.     

Aluminum resistance mechanisms in oat (Avena sativa L.)

Background and aims

Enhanced aluminum (Al) resistance has been observed in dicots over-expressing enzymes involved in organic acid synthesis; however, this approach for improving Al resistance has not been investigated in monocots. Among the cereals, oat (Avena sativa L.) is considered to be Al resistant, but the basis of resistance is not known.

Methods

A hydroponic assay and hematoxylin staining for Al accumulation in roots were used to evaluate Al resistance in 15 oat cultivars. Malate and citrate release from roots was measured over a 24?h period. A malate dehydrogenase gene, neMDH, from alfalfa (Medicago sativa L.) was used to transform oat.

Results

Oat seedlings were highly resistant to Al, as a concentration of 325?μM AlK(SO₄)₂ was needed to cause a 50% decrease in root growth. Most oat cultivars tested are naturally resistant to high concentrations of Al and effectively excluded Al from roots. Al-dependent release of malate and Al-independent release of citrate was observed. Al resistance was enhanced in a transgenic oat line with the highest accumulation of neMDH protein. However, overall root growth of this line was reduced and expression of neMDH in transgenic oat did not enhance malate secretion.

Conclusions

Release of malate from oat roots was associated with Al resistance, which suggests that malate plays a role in Al resistance of oat. Over-expression of alfalfa neMDH enhanced Al resistance in some lines but was not effective alone for crop improvement.     

Endogenous phytohormone profile during oat (Avena sativa L.) haploid embryo development

The development of embryos requires interaction of many endogenous hormones. The aim of the study was to determine which endogenous phytohormones are involved in the process of oat (Avena sativa L.) haploidization. Oat haploids were obtained by wide crossing with Zea mays L. The hormonal profiles of the ovaries with (OE) and without developed embryo (OWE) were compared. Phytohormone contents were measured by UHPLC coupled with mass spectrometer. The total content of indole-3-acetic acid (IAA), trans-zeatin (tZ), and kinetin (KN) in OE was significantly higher than in OWE. 4-Chloroindole-3-acetic acid was detected only in OWE. There were no differences between OE and OWE in the content of gibberellins (GA₁, GA₃, GA₄, GA₆, GA₇) and stress hormones (abscisic, salicylic, jasmonic acids). Content of endogenous KN was highly negatively correlated with the percentage of haploid embryos, germinated haploid embryos, haploid plants on MS (in vitro), haploid plants in soil (ex vitro), and doubled haploid lines. The tZ content negatively correlated with the frequency of haploid embryo formation, germination, and haploid plants obtained in vitro, as opposed to GA₁, which correlated positively. A positive correlation was found between IAA and tZ in OE, whereas in OWE it was a negative correlation. The profiles of phytohormones in OE and OWE were determined; however, their mode of action needs to be clarified.

     

Isolation and structure of peptides from oat (Avena sativa) seedlings

The level of proteolytic activity in tissues of oat seedlings was characterized under acidic conditions, and the number and content of the main components in low-molecular-mass fractions of the extract were determined. The structures of the majority of predominant peptide components isolated from the extract were studied. The use of a database of protein structures helped suggest possible structures of protein precursors of the peptides isolated. Detailed information on a plant peptidome was obtained for the first time.     

Ultrastructural investigations and EDX-analyses of Al-treated oat (Avena sativa) roots

Seedlings of Avena sativa were precultivated hydroponically at pH 4.1 and subsequently treated with solutions containing 0 or 400 µM Al. Electron microscopic and X-ray microanalytic (EDXA) investigations were carried out on root tips after 1 to 10 days of treatment. Root growth and mitotic activity decreased rapidly upon application of Al. The meristematic tissues of Al-treated roots showed enhanced vacuolation. The cells, however, remained intact after a longer period of Al-treatment and no alterations in ultrastructure (for example of the nucleus) were visible. The EDX analyses of bulk frozen hydrated tissues showed that Al was predominantly localised in the walls of the outermost cortex cells. One day after onset of Al-stress no intracellular Al was detectable. Even after 10 days with continuous Al-stress, only small amounts of the absorbed Al were localised within the cells. These results suggest that the plasma membrane is a very effective barrier for Al in oats. It is improbable that impairments of cytoplasmatic functions are primary effects of Al-intoxication.     

Cell wall and enzyme changes during the graviresponse of the leaf-sheath pulvinus of oat (Avena sativa)

The graviresponse of the leaf-sheath pulvinus of oat (Avena sativa) involves an asymmetric growth response accompanied by several asymmetric processes, including degradation of starch and cell wall synthesis. To understand further the cellular and biochemical events associated with the graviresponse, changes in cell walls and their constituents and the activities of related enzymes were investigated in excised pulvini. Asymmetric increases in dry weight with relatively symmetric increases in wall weight accompanied the graviresponse. Starch degradation could not account for increases in wall weight. However, a strong asymmetry in invertase activity indicated that hydrolysis of exogenous sucrose could contribute significantly to the increases in wall and dry weights. Most cell wall components increased proportionately during the graviresponse. However, beta-D-glucan did not increase symmetrically, but rather increased in proportion in lower halves of gravistimulated pulvini. This change resulted from an increase in glucan synthase activity in lower halves. The asymmetry of beta-D-glucan content arose too slowly to account for initiation of the graviresponse. A similar pattern in change in wall extensibility was also observed. Since beta-D-glucan was the only wall component to change, it is hypothesized that this change is the basis for the change in wall extensibility. Since wall extensibility changed too slowly to account for growth initiation, it is postulated that asymmetric changes in osmotic solutes act as the driving factor for growth promotion in the graviresponse, while wall extensibility acts as a limiting factor during growth.     

Changes in cuticular transpiration rate and cuticular lipids of oat (Avena sativa) seedlings induced by water stress

Two cultivars of oat ( Avena sativa L. cvs Pendek and Stormogul II) were exposed to short periods of water-deficit stress on five consecutive days. The plants responded to the stress by decreasing their cuticular transpiration rate. After two stress periods the cuticular transpiration rate was reduced by 30% for Pendek and by 47% for Stormogul II, and after another three stress periods by 30% and 20%, respectively. These reductions were correlated neither to changes in the total amount of what is generally called epicuticular lipids, nor to changes in any of the major individual constituents of the epicuticular lipids (alkanes, free and esterified fatty acids or free primary alcohols). After removal of the epicuticular lipids the long chain free primary alcohols of the leaves were extracted and determined. The amount of these presumably intracuticular alcohols increased after stress and changed to shorter chain length. From these results it is concluded that the intra- as well as the epicuticular lipids must be taken into consideration when discussing leaf surface lipids as protecting agents against water loss.     

Kinetics of mitotic rhythmic activity in oat (Avena sativa) coleoptiles following irradiation

The analysis of different intensity (10-200 Gy) irradiation influence on rhythmical processes in mitotic activity of oats coleoptiles was carries out. Obtained results indicate that irradiation disorders phase and amplitude of the cell division rhythm in investigated material. This disturbance, as judged by doze dependence, is associated with unknown molecular structures.     

Purification and properties of apoplastic amylase from oat (Avena sativa) seedlings

The protein fraction extracted with a high ionic strength buffer from the cell wall preparation of oat ( Avena sativa L.) coleoptiles and first leaves contained starch-degrading (amylase) activity. The activity of apoplastic amylase in the coleoptiles and first leaves continued to increase in parallel with organ growth. One of the apoplastic amylases recovered from shoot cell wall preparations was purified by sequential ion exchange and gel filtration chromatography, and the catalytic properties of the enzyme were analysed. The purified enzyme gave a single 25 kDa protein band on SDS-PAGE. The enzyme exhibited maximum activity at pH 5.0 against maltooligosaccharides. The purified enzyme hydrolysed soluble starch and maltooligosaccharides larger than tetraose at maltose unit, but did not hydrolyse β -limit dextrin or p -nitrophenyl- α - d -glucopyranoside. These results as well as the findings that the molecular size and the catalytic properties of the purified enzyme are different from those of known amylases obtained from Gramineae caryopses suggest that this enzyme is a novel type of β -amylase present in cell walls of vigorously elongating Gramineae shoot organs.     

Barley yellow dwarf virus: effects on carbohydrate metabolism in oat (Avena sativa) during cold hardening

Barley yellow dwarf virus (BYDV) causes significant losses in yield and in overwintering ability of winter cereals. Mechanisms by which the physiology of plants is affected by the virus are not clear. To see how carbohydrates in the crown of winter cereals were affected by BYDV, fructan isomers of degree of polymerization (DP) 3–5, fructan DP>6 and the simple sugars, glucose, fructose and sucrose, were measured before and during cold hardening in three oat ( Avena sativa L.) cultivars, 'Wintok', 'Coast Black' and 'Fulghum'. On a fresh weight basis fructan DP>6 decreased by 50% in infected 'Wintok' and 'Coast Black' and by 25% in 'Fulghum'. Two DP3, one DP4 and one DP5 isomer were significantly higher than non-infected controls. The percentages of simple sugars in infected crowns were significantly higher than controls in all three cultivars in every week except the first week of hardening. Crude enzyme extracts from BYDV infected plants incubated with sucrose suggested higher invertase and lower sucrose-sucrosyl transferase activity. When incubated with 1-kestose and neokestin, no significant difference was found in fructose fructosyl transferase or in hydrolase activity. The activity of unidentified enzymes catalysing the synthesis of larger (DP>5) fructan was altered by BYDV. The decrease of carbohydrates in the crown induced indirectly by BYDV may alter the plant's capacity to regenerate tillers in the spring. The ability of plants to prevent or tolerate carbohydrate fluctuations induced by BYDV infection may be an important genetically regulated characteristic for developing virus-resistant cultivars.     

Auxin,gibberellins and the gravitropic response of grass leaf sheath pulvini

The role of hormones in mediating tropic responses has been a central question in plant biology. Another key issue concerns how interactions between hormones regulate plant responses. In the September 2007 issue of Physiologia Plantarum, we published a paper relevant to both these questions.¹ This paper focuses on gravitropism in the barley leaf sheath pulvinus. The results support the Cholodny-Went theory on hormones and tropic responses, and highlight how an environmental factor (gravity) appears to first affect auxin content and consequently that of bioactive gibberellins (GAs). It appears that while GAs do not actually trigger the gravitropic bending of barley pulvini, they do act to magnify the bending response.Key words: auxin, gibberellin, Cholodny-Went theory, barley, pulvinus, gravitropism, mutant     

Isolation and characterization of novel microsatellite markers for Avena sativa (Poaceae) (oat)

? Premise of the study: A new set of microsatellite primers was developed for Avena sativa and characterized to assess the level of genetic diversity among cultivars and wild genotypes. ? Methods and Results: Using an enrichment genomic library, 14 simple sequence repeat markers were identified. The loci of these markers were characterized and found to be polymorphic in size among 48 genotypes of oat from diverse geographical locations. The number of alleles per locus ranged from two to eight, while the observed heterozygosity ranged from 0.031 to 0.75. ? Conclusions: These newly identified microsatellite markers will facilitate genetic diversity studies, fingerprinting, and genetic mapping of oat. Moreover, these new primers for A. sativa will aid future studies of polyploidy and hybridization in other species in this genus.