Identification of genes expressed in maize root cortical cells during lysigenous aerenchyma formation using laser microdissection and microarray analyses

? To adapt to waterlogging in soil, some gramineous plants, such as maize (Zea mays), form lysigenous aerenchyma in the root cortex. Ethylene, which is accumulated during waterlogging, promotes aerenchyma formation. However, the molecular mechanism of aerenchyma formation is not understood. ? The aim of this study was to identify aerenchyma formation-associated genes expressed in maize roots as a basis for understanding the molecular mechanism of aerenchyma formation. Maize plants were grown under waterlogged conditions, with or without pretreatment with an ethylene perception inhibitor 1-methylcyclopropene (1-MCP), or under aerobic conditions. Cortical cells were isolated by laser microdissection and their mRNA levels were examined with a microarray. ? The microarray analysis revealed 575 genes in the cortical cells, whose expression was either up-regulated or down-regulated under waterlogged conditions and whose induction or repression was suppressed by pretreatment with 1-MCP. ? The differentially expressed genes included genes related to the generation or scavenging of reactive oxygen species, Ca(2+) signaling, and cell wall loosening and degradation. The results of this study should lead to a better understanding of the mechanism of root lysigenous aerenchyma formation.     

Aerenchyma formation and porosity in root of a mangrove plant,<Emphasis Type="Italic"> Sonneratia alba</Emphasis> (Lythraceae)

Aerenchyma gas spaces are important for plants that grow in flooded and anaerobic sites or habitats, because these gas spaces provide an internal pathway for oxygen transport. The objective of this study is to characterize the development of aerenchyma gas spaces and observe the porosity in roots of Sonneratia alba. Tissue at different developmental stages was collected from four root types, i.e. cable root, pneumatophore, feeding root and anchor root, of S. alba. In S. alba, gas space is schizogenously produced in all root types, and increases in volume from the root meristem to mature root tissues. The aerenchyma formation takes place immediately, or 3–5 mm behind the root apex. At first, cortical cells are relatively round in cross sections (near the root apex); they then become two kinds of cells, rounded and armed, which combine together, forming intercellular spaces behind the root apex. The average dimensions of cortical cells increased more than 1.3 times in the vertical direction and over 3.3 times in the horizontal direction. At maturity, aerenchyma gas spaces are long tuberous structures without diaphragms and with numerous small pores on the lateral walls. Within the aerenchyma, many sclereids grow intrusively. Root porosity in all root types ranged from 0–60%. Pneumatophores and cable roots had the highest aerenchyma area (50–60%).     

Formation and function of secondary aerenchyma in hypocotyl, roots and nodules of soybean (Glycine max) under flooded conditions

Flooding is a major problem in many areas of the world and soybean is susceptible to the stress. Understanding the morphological mechanisms of flooding tolerance is important for developing flood-tolerant genotypes. We investigated secondary aerenchyma formation and function in soybean (Glycine max) seedlings grown under flooded conditions. Secondary aerenchyma, a white and spongy tissue, was formed in the hypocotyl, tap root, adventitious roots and root nodules after 3 weeks of flooding. Under irrigated conditions aerenchyma development was either absent or rare and phellem was formed in the hypocotyl, tap root, adventitious roots and root nodules. Secondary meristem partially appeared at the outer parts of the interfascicular cambium and girdled the stele, and then cells differentiated to construct secondary aerenchyma in the flooded hypocotyl. These morphological changes proceeded for 4 days after the initiation of the flooding. After 14 days of treatment, porosity exceeded 30% in flooded hypocotyl with well-developed secondary aerenchyma, while it was below 10% in hypocotyl of irrigated plants that had no aerenchyma. When Vaseline was applied to the hypocotyl of plants from a flooded treatment to prevent the entry of atmospheric oxygen into secondary aerenchyma, plant growth, especially that of roots, was sharply inhibited. Thus secondary aerenchyma might be an adaptive response to flooding.     

Construction of intraspecific linkage maps, detection of a chromosome inversion, and mapping of QTL for constitutive root aerenchyma formation in the teosinte Zea nicaraguensis

The teosinte Zea nicaraguensis, a wild relative of maize, possesses a flooding tolerance-related trait: the formation of constitutive root aerenchyma under drained (non-flooded) soil conditions. A previous study suggested that the degree of constitutive aerenchyma formation varies within Z. nicaraguensis. The objectives of this study were to construct linkage maps, to determine the marker order in a region of chromosome 4 in which recombination between maize and Z. nicaraguensis is suppressed, and to identify quantitative trait loci (QTL) controlling constitutive root aerenchyma formation in two segregating populations of Z. nicaraguensis. A total of 236 simple sequence repeat (SSR) markers were screened for polymorphism in an S1 population of Z. nicaraguensis. Seventy-one polymorphic SSR markers were assigned to 10 chromosomes, and a linkage map was constructed covering 793.5 cM. In the S1 map, a paracentric inversion was detected on the long arm of chromosome 4; this rearrangement was confirmed in an S1 linkage map of a different Z. nicaraguensis accession. Composite interval mapping analysis in 96 S1 plants revealed QTL for aerenchyma formation on chromosomes 1 (bins 1.06–1.07) and 7 (bin 7.01), explaining 17 and 12% of the total phenotypic variance, respectively. The QTL on chromosome 1 was verified by using 156 S2 plants. Near-isogenic lines exhibiting the presence or absence of the aerenchyma QTL have been developed that should be useful for genetic and physiological analyses of root aerenchyma formation.     

Inhibition by silver ions of gas space (aerenchyma) formation in adventitious roots of Zea mays L. subjected to exogenous ethylene or to oxygen deficiency

We have studied the role of ethylene in accelerating the lytic formation of gas spaces (aerenchyma) in the cortex of adventitious roots of maize (Zea mays L.) growing in poorly aerated conditions. Such roots had previously been shown to contain increased concentrations of ethylene. Ten day-old maize plants bearing seminal roots and one whorl of emerging adventitious roots were grown in nutrient solution bubbled with air, ethylene in air (0.1 to 5.0 l l^–1), or allowed to become oxygen-deficient in nonaerated (but not completely anaerobic) solution. Additions of 0.1 l l^–1 ethylene or more promoted the formation of aerenchyma, with lysis of up to 47% of the cortical cells. The effects of non-aeration were similar to those of exogenous ethylene. When silver ions, an ethylene antagonist, were present at low, non-toxic concentrations (circa 0.6 M), aerenchyma formation was prevented in ethylene treated roots and in those exposed to oxygen deficiency. Silver ions also blocked the inhibiting effect of exogenous ethylene on root extension. By contrast, the suppression of aerenchyma formation by silver ions under oxygendeficient conditions was associated with a retardation of root extension, indicating the importance of aerenchyma for root growth in poorly aerated media. Rates of production of ethylene by excised roots were stimulated by a previous non-aeration treatment. The effectiveness of Ag⁺ in inhibiting equally the action on cortical cells of exogenous ethylene and of non-aeration, supports the view that gas space (aerenchyma) formation in adventitious roots adpted to oxygendeficient environments is mediated by increased concentrations of endogenous ethylene. The possibility that extra ethylene could arise from increased biosynthesis of a precursor in root tissues with a restricted oxygen supply is discussed.     

Stimulation of ethylene production and gas-space (aerenchyma) formation in adventitious roots of Zea mays L. by small partial pressures of oxygen

Adventitious roots of two to four-weekold intact plants of Zea mays L. (cv. LG11) were shorter but less dense after extending into stagnant, non-aerated nutrient solution than into solution continuously aerated with air. Dissolved oxygen in the non-aerated solutions decreased from 21 kPa to 3–9 kPa within 24 h. When oxygen partial pressures similar to those found in non-aerated solutions (3, 5 and 12 kPa) were applied for 7 d to root systems growing in vigorously bubbled solutions, the volume of gas-space in the cortex (aerenchyma) was increased several fold. This stimulation of aerenchyma was associated with faster ethylene production by 45-mm-long apical root segments. When ethylene production by roots exposed to 5 kPa oxygen was inhibited by aminoethoxyvinylglycine (AVG) dissolved in the nutrient solution, aerenchyma formation was also retarded. The effect of AVG was reversible by concomitant applications of 1-aminocyclopropane-1-carboxylic acid, an immediate precursor of ethylene. Addition of silver nitrate, an inhibitor of ethylene action, to the nutrient solution also prevented the development of aerenchyma in roots given 5 kPa oxygen. Treating roots with only 1 kPa oxygen stimulated ethylene production but failed to promote gas-space formation. These severely oxygen-deficient roots seemed insensitive to the ethylene produced since a supplement of exogeneous ethylene that promoted aerenchyma development in nutrient solution aerated with air (21 kPa oxygen) failed to do so in nutrient solution supplied with 1 kPa oxygen. Both ethylene production and aerenchyma formation were almost completely halted when roots were exposed to nutrient solutions devoid of oxygen. Thus both processes require oxygen and are stimulated by oxygen-deficient surroundings in the 3-to 12-kPa range of oxygen partial pressures when compared with rates observed in air (21 kPa oxygen).Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine     

Variation for Root Aerenchyma Formation in Flooded and Non-Flooded Maize and Teosinte Seedlings

Morphological and anatomical factors such as aerenchyma formation in roots and the development of adventitious roots are considered to be amongst the most important developmental characteristics affecting flooding tolerance. In this study we investigated the lengths of adventitious roots and their capacity to form aerenchyma in three- and four-week-old seedlings of two maize (Zea mays ssp. mays, Linn.) inbred accessions, B64 and Na4, and one teosinte, Z. nicaraguensis Iltis & Benz (Poaceae), with and without a flooding treatment. Three weeks after sowing and following a seven day flooding treatment, both maize and teosinte seedlings formed aerenchyma in the cortex of the adventitious roots of the first three nodes. The degree of aerenchyma formation in the three genotypes increased with a second week of flooding treatment. In drained soil, the two maize accessions failed to form aerenchyma. In Z. nicaraguensis, aerenchyma developed in roots located at the first two nodes three weeks after sowing. In the fourth week, aerenchyma developed in roots of the third node, with a subsequent increase in aerenchyma in the second node roots. In a second experiment, we investigated the capacity of aerenchyma to develop in drained soil. An additional three teosinte species and 15 maize inbred lines, among them a set of flooding-tolerant maize lines, were evaluated. Evaluations indicate that accessions of Z. luxurians (Durieu & Asch. Bird) and two maize inbreds, B55 and Mo20W, form aerenchyma when not flooded. These materials may be useful genetic resources for the development of flooding-tolerant maize accessions.     

Aerenchyma tissue development and gas-pathway structure in root of Avicennia marina (Forsk.) Vierh.

The aerenchyma differentiation in cable roots, pneumatophores, anchor roots, and feeding roots of the mangrove plant, Avicennia marina (Verbenaceae) was analyzed using a light microscope and scanning electron microscope. In all types, cortex cells were arranged in longitudinal columns extending from the endodermis to the epidermis. No cells in the cortex had intercellular spaces at the root tip (0–150 m), and aerenchyma started developing at 200 m from the root apex. The aerenchyma formation was due to cell separation (schizogeny) rather than cell lysis. The cell separation occurred between the longitudinal cell columns, forming long intercellular spaces along the root axis. During aerenchyma formation, the cortex cells enlarged longitudinally by 1.8–3.9 times and widened horizontally by 2.2–2.9 times. As a result, the aerenchyma had a pronounced tubular structure that was radially long, elliptical or oval in cross section and that ran parallel to the root axis. The tube had tapering ends, as did vessel elements, although there were no perforated plates. The interconnection between neighboring tubes was made by abundant small pores or canals that were schizogenous intercellular spaces between the wall cells. All aerenchyma tubes in the root were interconnected by these small pores serving as a gas pathway.     

Progressive Cortical Senescence and Formation of Lysigenous Gas Space (Aerenchyma) Distinguished by Nuclear Staining in Adventitious Roots of Zea mays

The pattern of loss of nuclear integrity in the epidermis andcortex of maize adventitious roots was examined during (1) non-pathogeniccortical senescence associated with root ageing, and (2) lysigenousaerenchyma formation, to determine whether these phenomena arerelated. Nuclear integrity was estimated by counting the percentageof cells with nuclei detectable by acridine orange fluorescence. In roots of both soil-grown (90 d) and solution-grown (19 d)plants, nuclei were lost progressively, from the epidermis andfrom successively deeper cortical cell layers, with increasingdistance behind the root tips; this occurred irrespective ofthe degree of aeration in solution culture, and independentlyof aerenchyma formation. Aerenchyma developed in soil-grownplants and in sub-ambient oxygen concentrations (<5 kPa partialpressure) in solution culture. It started to form in the middlecortex and coincided with a marked loss of nuclear stainingin the inner cortex, especially in the innermost cortical celllayer next to the endodermis, but not in the remaining cellsof the middle cortex. Two distinct patterns of nuclear deletionfrom the cortex were thus demonstrated; they occurred independentlybut simultaneously in some conditions. These findings are discussed in relation to mechanisms of celldeath, and the metabolic status of root cortical cells participatingin ion transport to the xylem. Zea mays L., maize, roots, aerenchyma, cell death, nuclei     

Water in aerenchyma spaces in roots. A fast diffusion path for solutes

During a study of the diffusivity of sulphorhodamine G in the cortical apoplast of maize roots widely discrepant rates were found between different samples. In roots which had developed large aerenchyma spaces, the diffusion in some regions was very fast, indistinguishable from the rate in water. In other regions the rate was as much as 100 times slower. Examination of frozen intact roots with the cryo-scanning electron microscope showed the presence of liquid filling some of the aerenchyma spaces, while other spaces of the same root contained air. X-ray microanalysis of the liquid (for oxygen) showed that the liquid was water with few detectable ions. Similar liquid was present in small intercellular spaces within the spoke-like radial files of cells between the large spaces, or between remnants of collapsed cell walls at the edges of the large spaces. It is proposed that regions of roots with high diffusivity are those in which some of the aerenchyma spaces are filled with water. In seeking the origin of this liquid, the progress of aerenchyma formation could be followed in the frozen tissues. The first change observed in a group of contiguous cells was a loss of vacuolar solutes and of cell turgor. Next the walls broke apart and collapsed back onto the surrounding turgid cells leaving a volume of ion-poor liquid. The liquid was probably not that found in some aerenchyma spaces of the mature roots, because the final stage of space formation was a loss of the liquid, leaving an air filled cavity surrounded by a composite lining formed from the collapsed walls of the broken cells. It is likely that the liquid in the spaces of mature aerenchyma is exuded from the remaining living cortical cells at times when the root turgor is high. This would be consistent with several recent studies which have shown periodic exudation of water from the surface of turgid roots. The spasmodic occurrence of root cortex tissue with enhanced diffusivity would have important implications for the transport of nutrient ions across the root.Abbreviations CSEM cryo-scanning electron microscope - EDX energy dispersive X-ray microanalysis - SR-G sulphorhodamine G     

Roots of willow (Salix viminalis L.) show marked tolerance to oxygen shortage in flooded soils and in solution culture

Responses to soil flooding and oxygen shortage were studied in field, glasshouse and controlled environment conditions. Established stools ofSalix viminalis L., were compared at five field sites in close proximity but with contrasting water table levels and flooding intensities during the preceding winter. There was no marked effect of site on shoot extension rate, time to half maximum length or final length attained. When rooted cuttings were waterlogged for 4 weeks in a glasshouse, soil redox potentials quickly decreased to below zero. Shoot extension was slowed after a delay of 20 d, while, in the upper 100 mm of soil, formation and outgrowth of unbranched adventitious roots with enhanced aerenchyma development was promoted after 7 d. At depths of 100–200 mm and 200–300 mm, extension by existing root axes was halted by soil flooding, while adventitious roots from above failed to penetrate these deeper zones. After 4 weeks waterlogging, all arrested root tips recommenced elongation when the soil was drained; their extension rates exceeding those of roots that were well-drained throughout. Growth in fresh mass was also stimulated. The additional aerenchyma found in adventitious roots in the upper 100 mm of soil may have been ethylene regulated since gas space development was inhibited by silver nitrate, an ethylene action inhibitor. The effectiveness of aerenchyma was tested by blocking the entry of atmospheric oxygen into plants with lanolin applied to lenticels of woody shoots of plants grown in solution culture. Root extension was halved, while shoot growth remained unaffected. H Lambers Section editor     

Enhanced formation of aerenchyma and induction of a barrier to radial oxygen loss in adventitious roots of Zea nicaraguensis contribute to its waterlogging tolerance as compared with maize (Zea mays ssp. mays)

Enhancement of oxygen transport from shoot to root tip by the formation of aerenchyma and also a barrier to radial oxygen loss (ROL) in roots is common in waterlogging‐tolerant plants. Zea nicaraguensis (teosinte), a wild relative of maize (Zea mays ssp. mays), grows in waterlogged soils. We investigated the formation of aerenchyma and ROL barrier induction in roots of Z. nicaraguensis, in comparison with roots of maize (inbred line Mi29), in a pot soil system and in hydroponics. Furthermore, depositions of suberin in the exodermis/hypodermis and lignin in the epidermis of adventitious roots of Z. nicaraguensis and maize grown in aerated or stagnant deoxygenated nutrient solution were studied. Growth of maize was more adversely affected by low oxygen in the root zone (waterlogged soil or stagnant deoxygenated nutrient solution) compared with Z. nicaraguensis. In stagnant deoxygenated solution, Z. nicaraguensis was superior to maize in transporting oxygen from shoot base to root tip due to formation of larger aerenchyma and a stronger barrier to ROL in adventitious roots. The relationships between the ROL barrier formation and suberin and lignin depositions in roots are discussed. The ROL barrier, in addition to aerenchyma, would contribute to the waterlogging tolerance of Z. nicaraguensis.     

Larger adenylate energy charge and ATP/ADP ratios in aerenchymatous roots of Zea mays in anaerobic media as a consequence of improved internal oxygen transport

Internal transport of O₂ from the aerial tissues along the adventitious roots of intact maize plants was estimated by measuring the concentrations of adenine nucleotides in various zones along the root under an oxygen-free atmosphere. Young maize plants were grown in nutrient solution under conditions that either stimulated or prevented the formation of a lysigenous aerenchyma, and the roots (up to 210 mm long) were then exposed to an anaerobic (oxygen-free) nutrient solution. Aerenchymatous roots showed higher values than non-aerenchymatous ones for ATP content, adenylate energy charge and ATP/ADP ratios. We conclude that the lysigenous cortical gas spaces help maintain a high respiration rate in the tissues along the root, and in the apical zone, by improving internal transport of oxygen over distances of at least 210 mm. This contrasted sharply with the low energy status (poor O₂ transport) in non-aerenchymatous roots.Abbreviation AEC adenylate energy charge     

穿叶眼子菜茎叶结构及其通气组织发育解剖学研究

通气组织(aerenchyma)是植物薄壁组织内一些气室或腔隙的集合,对于水生及湿地植物体内的气体运输至关重要。该实验以沉水植物穿叶眼子菜为材料,利用石蜡切片技术,通过对茎的纵切面及横切面结构进行观察,从时间和空间上分析其茎、叶通气组织的发生过程。结果表明:(1)穿叶眼子菜的茎结构包括表皮、皮层及维管柱,通气组织发达,存在于内皮层与表皮之间;茎通气组织由距茎尖约0.6mm处开始形成,并成熟于约2.4mm处。(2)穿叶眼子菜的叶由表皮、皮层薄壁细胞及维管柱组成,其通气组织形成于靠近茎尖的第2~3片新生叶且仅形成于主叶脉。(3)穿叶眼子菜的茎和叶通气组织的发育过程相似,起初为排列致密的细胞团,然后由皮层细胞的分裂产生小的细胞间隙,随后的腔隙膨大过程涉及细胞的生长分裂及细胞降解,最终形成发达的通气组织。(4)穿叶眼子菜的通气组织发育过程可划分为实心期、形成期、膨大期、成熟期四个时期;不同时期茎通气组织的发达程度差异很大,实心期、形成期、膨大期和成熟期的孔隙度分别为0.54%、10.90%、27.61%和57.58%;但节处通气组织不发达,成熟期的节处孔隙度仅为3.62%。     

Ethylene‐dependent aerenchyma formation in adventitious roots is regulated differently in rice and maize

In roots of gramineous plants, lysigenous aerenchyma is created by the death and lysis of cortical cells. Rice (Oryza sativa) constitutively forms aerenchyma under aerobic conditions, and its formation is further induced under oxygen‐deficient conditions. However, maize (Zea mays) develops aerenchyma only under oxygen‐deficient conditions. Ethylene is involved in lysigenous aerenchyma formation. Here, we investigated how ethylene‐dependent aerenchyma formation is differently regulated between rice and maize. For this purpose, in rice, we used the reduced culm number1 (rcn1) mutant, in which ethylene biosynthesis is suppressed. Ethylene is converted from 1‐aminocyclopropane‐1‐carboxylic acid (ACC) by the action of ACC oxidase (ACO). We found that OsACO5 was highly expressed in the wild type, but not in rcn1, under aerobic conditions, suggesting that OsACO5 contributes to aerenchyma formation in aerated rice roots. By contrast, the ACO genes in maize roots were weakly expressed under aerobic conditions, and thus ACC treatment did not effectively induce ethylene production or aerenchyma formation, unlike in rice. Aerenchyma formation in rice roots after the initiation of oxygen‐deficient conditions was faster and greater than that in maize. These results suggest that the difference in aerenchyma formation in rice and maize is due to their different mechanisms for regulating ethylene biosynthesis.     

Aerenchyma Formed Under Phosphorus Deficiency Contributes to the Reduced Root Hydraulic Conductivity in Maize Roots

Root hydraulic conductivity has been shown to decrease under phosphorus （P） deficiency. This study Investigated how the formation of aerenchyma is related to this change. Root anatomy, as well as root hydraulic conductivity was studied In maize （Zea mays L.） roots under different phosphorus nutrition conditions. Plant roots under P stress showed enhanced degradation of cortical cells and the aerenchyma formation was associated with their reduced root hydraulic conductivity, supporting our hypothesis that air spaces that form in the cortex of phosphorusstressed roots Impede the radial transport of water in a root cylinder. Further evidence came from the variation In aerenchyma formation due to genotypic differences. Five maize inbred lines with different porosity in their root cortex showed a significant negative correlation with their root hydraulic conductivity. Shoot relative water content was also found lower In P-deficient maize plants than that in P-sufficient ones when such treatment was prolonged enough, suggesting a limitation of water transport due to lowered root hydraulic conductivity of P-deficient plants.     

Aerenchyma formation in roots of maize during sulphate starvation

Young maize ( Zea mays L., Poaceae) plants were grown in a complete, well-oxygenated nutrient solution and then deprived of their external source of sulphate. This treatment induced the formation of aerenchyma in roots. In addition to the effect of sulphate starvation on root anatomy, the presence and location of superoxide anions and hydrogen peroxide, and changes in calcium and pH were examined. By day 6 of sulphate deprivation, aerenchyma started to form in the roots of plants and the first aerenchymatous spaces were apparent in the middle of the cortex. S-starvation also induced thickening of the cell walls of the endodermis. Active oxygen species appeared in groups of intact mid-cortex cells. Formation of superoxide anion and hydrogen peroxide was found in degenerating cells of the mid-cortex. Very few nuclei in the cortex of S-starved roots fluoresced, being shrunken and near to the cell wall. By day 12 of S-deprivation, a fully developed aerenchyma was apparent and there were only a few 'chains' of cells bridging hypodermis to endodermis and stele of roots. Cell walls of endodermis of S-starved roots increased 68% in thickness. Intensive fluorescence in the cell walls of the endodermal, hypodermal and to a lesser extent of epidermal cells was observed due to the formation of active oxygen species, while there was no fluorescence in the cortical cells. There was a higher Ca concentration in the cells walls of the endodermis and epidermis, compared to the rest of the S-starved root tissues. A higher pH was observed, mainly in the cell walls of the hypodermis and to a lesser extent in the cell walls of the endodermis. Superoxide anion and hydrogen peroxide was found in degenerating cells of the root cortex. There was no fluorescence of nuclei in the cortex of S-starved roots.     

Cultivar variation in the effect of chlorsulfuron in depressing the uptake of copper in wheat

The application of herbicides has induced symptoms of nutrient deficiencies under some circumstances. This glasshouse study examined the effect of chlorsulfuron on the uptake and utilization of copper (Cu) in four cultivars of wheat plants (Triticum aestivum L. cvs. Kulin, Cranbrook, Gamenya and Bodallin) on a Cu-responsive soil. Application of chlorsulfuron depressed the concentration of Cu in wheat plants receiving either inadequate or adequate Cu. In plants with inadequate Cu supply, chlorsulfuron increased the severity of Cu deficiency. Shoot weight was markedly decreased by chlorsulfuron at all levels of Cu, through decreasing the number of tillers and the elongation of leaves. This decreased growth of shoots occurred prior to the effect on Cu concentration in tissues. The retranslocation of Cu in old tissues over time was unaffected by chlorsulfuron. In all wheat cultivars, the decreased growth of shoots were correlated with the concentration of Cu in the youngest fully emerged leaf blade with critical levels of 1.6−1.7 at day 25 and 0.9−1.0 μg g⁻¹ d. wt. at day 60. The application of chlorsulfuron tended to increase the critical level at day 25 but not at day 60. In addition, Kulin seems to be most, and Cranbrook least, sensitive to chlorsulfuron. This sensitivity was associated with the sensitivity of the cultivars to Cu deficiency. It is suggested that chlorsulfuron application induces Cu deficiency in wheat plants mainly due to effects on the uptake of Cu. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of Chilling on DNA Endoreplication in Root Cortex Cells and Root Hairs of Soybean Seedlings

Relative nuclear DNA contents in cortex parenchyma cells in root segments of 3- and 7-d-old soybean seedlings grown at 25 °C and in plants grown for 3 d at 25 °C, and then for 4 d at 10 °C, were determined with cytophotometry. Measurements revealed that in each variant the cortex cell nuclei with DNA content between 2C and 8C were in all the examined segments and nuclei with 8C – 16C DNA appeared in higher parts of roots. However, in chilled plant cells the number of 8C – 16C DNA nuclei was very low. Therefore, chilling inhibited endoreplication in comparison with plants grown at 25 °C for 7 d, and even reduced endopolyploidy level as compared to the initial seedlings, i.e. 3-d-old plants. DNA contents in root hairs grown at 25 °C (control) and in root hairs emerged at 10 °C were also determined. In controls 4C – 8C DNA nuclei predominated while in chilled plants an additional population of 2C – 4C DNA appeared. Thus a reduction of DNA synthesis was brought about by low temperature. The occurrence of an intermediate DNA contents besides those with full endoreplication cycles suggests the possibility of differential DNA replication. This suggestion seems to be supported by the lack of ³H-thymidine incorporation into root hair nuclei at the examined developmental stage both in control and chilled root hairs. The same number, but larger, chromocentric lumps in polyploid cortex cell nuclei of higher root zones, in comparison to meristematic nuclei, suggests that endoreduplication process occurred. This revised version was published online in July 2006 with corrections to the Cover Date.