ROLE OF CELLULASE IN AERENCHYMA DEVELOPMENT IN SUNFLOWER

A hypothesis that ethylene causes aerenchyma development in waterlogged plants through increased cellulase activity was tested with sunflower, Helianthus annuus L. Treatment with commercial cellulase induced aerenchyma development in sunflower stem sections. Some of the cellulase-treated cortical cells enlarged radially and some disintegrated, leading to intercellular space. Cell disintegration started with progressive plasmolysis and severe plasmolysis was associated with or was apparently followed by cell wall breakdown. Localized stem treatment of an intact sunflower with ethylene increased cellulase activity in that part of the stem. Localized stem treatment of an intact sunflower with a water jacket increased cellulase activity in that part of the stem. When the lower part of the sunflower was waterlogged, the cellulase activity in the waterlogged stem increased. Present and earlier results suggest that aerenchyma development is a plant adaptation to waterlogging conditions. The deficiency of oxygen in a waterlogged plant triggers the anaerobic stimulation of ethylene production, which causes an increase in cellulase activity leading to aerenchyma development and enhancing the transport of oxygen to the roots. It is proposed that there is competition between neighboring cortical cells for water after an increase in cell wall plasticity by the action of cellulase. The competition causes progressive plasmolysis and eventual disintegration of weaker cells.     

AERENCHYMA DEVELOPMENT IN WATERLOGGED PLANTS

Aerenchyma development in waterlogged Helianthus annuus, Lycopersicon esculentum, and Salix fragilis was studied. More than half of the root cortical tissue sometimes became an air cavity in willow roots which developed in water. There was no cortical aerenchyma in the terminal portion, but more advanced aerenchyma developed towards the base of the sunflower roots formed in water. Waterlogged sunflower and tomato plants developed lysigenous aerenchyma in the cortex of waterlogged stems within two days.     

Nitric oxide is essential for the development of aerenchyma in wheat roots under hypoxic stress

In response to flooding/waterlogging, plants develop various anatomical changes including the formation of lysigenous aerenchyma for the delivery of oxygen to roots. Under hypoxia, plants produce high levels of nitric oxide (NO) but the role of this molecule in plant‐adaptive response to hypoxia is not known. Here, we investigated whether ethylene‐induced aerenchyma requires hypoxia‐induced NO. Under hypoxic conditions, wheat roots produced NO apparently via nitrate reductase and scavenging of NO led to a marked reduction in aerenchyma formation. Interestingly, we found that hypoxically induced NO is important for induction of the ethylene biosynthetic genes encoding ACC synthase and ACC oxidase. Hypoxia‐induced NO accelerated production of reactive oxygen species, lipid peroxidation, and protein tyrosine nitration. Other events related to cell death such as increased conductivity, increased cellulase activity, DNA fragmentation, and cytoplasmic streaming occurred under hypoxia, and opposing effects were observed by scavenging NO. The NO scavenger cPTIO (2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide potassium salt) and ethylene biosynthetic inhibitor CoCl₂ both led to reduced induction of genes involved in signal transduction such as phospholipase C, G protein alpha subunit, calcium‐dependent protein kinase family genes CDPK, CDPK2, CDPK 4, Ca‐CAMK, inositol 1,4,5‐trisphosphate 5‐phosphatase 1, and protein kinase suggesting that hypoxically induced NO is essential for the development of aerenchyma.     

Ethylene-Induced Activation of Xylanase in Adventitious Roots of Maize as a Response to the Stress Effect of Root Submersion

Submersion of roots of ten-day-old maize (Zea maysL.) seedlings was accompanied by a decrease in pO₂and an increase in pCO₂of the medium adjacent to the roots. These changes stimulated ethylene evolution in intact plants. Enhanced biosynthesis of ethylene was accompanied by xylanase activation in adventitious roots. As a result, an enhanced formation of aerenchyma was observed in the cortex of adventitious roots. Therefore, these processes resulted in the development of a ventilation system by which O₂can reach the root system exposed to hypoxia. The volume of aerenchyma was assessed by the volume of gas cavities (porosity). In contrast to the main root, the growth of adventitious roots was not inhibited under these conditions. Enlargement of the stem base and increase in the number of aerenchymatous adventitious roots facilitated the oxygen supply to the submerged organs of the plants.     

MESOCOTYL ROOT FORMATION IN ECHINOCHLOA PHYLLOPOGON (POACEAE) IN RELATION TO ROOT ZONE AERATION

Echinochloa phyllopogon was grown hydroponically under four root zone gassing treatments to determine aeration effects on the growth and development of the plant root system. Although mesocotyl growth and the number of nodal roots were unaffected by the treatments, other aspects of plant growth were altered. Shoot growth was reduced by hypoxic (5 kPa partial pressure O₂ in nitrogen gas) and anoxic conditions (O₂ free nitrogen gas), but not by ethylene (0.1 ppm in air). Seminal root growth was unaffected by hypoxia or ethylene treatments, but was reduced under anoxia. Hypoxic environments stimulated the emergence of roots along the length of the mesocotyl when compared to aerobic controls; anoxic and ethylene treatments had no significant effects. Mesocotyl roots elongated from primordia that were produced de novo in response to the hypoxic treatment. Under hypoxic conditions, aerenchyma was present in the cortex of nodal roots and to a lesser extent in seminal roots, but mesocotyl roots were devoid of aerenchyma under these conditions. The results are compared with the literature concerning flooding and aeration effects on growth and development in other species.     

Transduction of an Ethylene Signal Is Required for Cell Death and Lysis in the Root Cortex of Maize during Aerenchyma Formation Induced by Hypoxia

Ethylene has been implicated in signaling cell death in the lysigenous formation of gas spaces (aerenchyma) in the cortex of adventitious roots of maize (Zea mays) subjected to hypoxia. Various antagonists that are known to modify particular steps in signal transduction in other plant systems were applied at low concentrations to normoxic and hypoxic roots of maize, and the effect on cell death (aerenchyma formation) and the increase in cellulase activity that precedes the appearance of cell degeneration were measured. Both cellulase activity and cell death were inhibited in hypoxic roots in the presence of antagonists of inositol phospholipids, Ca2+- calmodulin, and protein kinases. By contrast, there was a parallel promotion of cellulase activity and cell death in hypoxic and normoxic roots by contact with reagents that activate G-proteins, increase cytosolic Ca2+, or inhibit protein phosphatases. Most of these reagents had no effect on ethylene biosynthesis and did not arrest root extension. These results indicate that the transduction of an ethylene signal leading to an increase in intracellular Ca2+ is necessary for cell death and the resulting aerenchyma development in roots of maize subjected to hypoxia.     

Characteristics of Helianthus annuus Plant Cell Culture as a Producer of Immunologically Active Exopolysaccharides

The aim of this work was to determine relationships between the growth and biosynthesis of exopolysaccharides by sunflower (Helianthus annuus 1805) plant cell culture and the uptake of the main compounds from the nutrient medium through the study of their time courses. It was established that plant cell culture biosynthesized 12.8 g/L biomass and 4.3 g/L exopolysaccharides. The calculated maximum specific growth rate and doubling time were μ_max = 0.21 d^–1 and t_d = 3.31 d, respectively. The productivity of exopolysaccharides in the plant in vitro system was found to be 0.43 g/(L × d). Data regarding the physiology of Helianthus annuus 1805 plant cell culture are presented. On the basis of the achieved results it can be concluded that the plant cell suspension of Helianthus annuus 1805 is a good producer of exopolysaccharides which exhibit immunostimulating activity.     

ANAEROBIC ELEVATION OF ETHYLENE CONCENTRATION IN WATERLOGGED PLANTS

Anaerobic elevation of ethylene concentration in waterlogged and non-waterlogged Helianthus annuus L. and Lycopersicon esculentum Mill. was studied. A balloon method was devised to provide an anaerobic atmosphere around the intact sunflower stem. Anaerobic conditions were also produced by bubbling nitrogen into the floodwater. Ethylene concentration in the stem of waterlogged plants was higher when nitrogen was bubbled through the floodwater than when aerated, the effect being greater for the soil-grown plants than for the sand-cultured plants. Ethylene concentration in the stem of waterlogged plants was highest in the region exposed to anaerobiosis, and less with increasing distance or height on the non-waterlogged part of the stems. Intact sunflower stems increased their ethylene concentration in that part of the stem which was maintained in an oxygen-free atmosphere. The results suggest that enhanced ethylene production in waterlogged plants primarily occurs in the waterlogged part of roots and stems.     

Temperature Sensitivity of the Latent Phase in Ethylene-induced Elongation

The temperature sensitivity is reported for the latent period preceding ethylene-induced elongation in the adaxial half of the leaf petiole of Helianthus annuus. When intact plants were exposed to 10 μl of ethylene/l of air over the temperature range 18 to 35 C, the minimum latent time was 62 minutes at 28 C and the maximum was 132 minutes at 18 C. The temperature coefficient, Q₁₀, changed from 2.1 below 28 C, to 0.7 above. In 100 μl of ethylene/l of air, the latent time was reduced by 14% at 18 C, but was significantly increased at 28 and 38 C. These results show that the latent period in the elongation response of the petiole to ethylene cannot be reduced below about 60 minutes by raising either the leaf temperature or the atmospheric ethylene concentration.     

THE ORIGIN OF HELIANTHUS MULTIFLORUS

Heiser , CHARLES B., Jr . (Indiana U., Bloomington), and Dale M. Smith . The origin of Helianthus multiflorus. Amer. Jour. Bot. 47(10): 860–865. Illus. 1960.—The cultivated ornamental sunflower, Helianthus multiflorus L., has been known since 1591 when it was described from Europe by Tabernaemontanus. Both Gray and Bailey concluded that it was a variety of H. decapetalus. The plant is invariably sterile and Dod was first to suggest a hybrid origin from H. annuus and H. decapetalus. Helianthus multiflorus has been found to be triploid (2n = 51) and at meiosis generally shows 17 bivalents and 17 univalents. Morphologically, the plant is similar to H. decapetalus (n = 34) but differs from it in the more hispid stem, broader leaves and phyllaries, and other characters, all of which could have been derived from H. annuus (n = 17). The artificial hybrid between H. decapetalus and H. annuus, while not readily obtained, has been secured, and the hybrid is rather similar to H. multiflorus. It is concluded that this taxon most likely originated in Europe from spontaneous hybridization between H. decapetalus and H. annuus, following the introduction of these species from North America.     

Hormonally Induced Changes in the Stem and Petiole Anatomy and Cellulase Enzyme Patterns in Phaseolus vulgaris L

Time course changes were observed in petiole and stem anatomy and cellulase enzyme patterns in bean (Phaseolus vulgaris L.) explants when 10⁻⁵ or 10⁻²m indoleacetic acid in lanolin paste was applied to acropetal cut surfaces in the presence or absence of ethylene. Auxin (10⁻²m) in the presence of ethylene stimulated rapid ordered cell division and dedifferentiation, with ensuing lateral root formation. Auxin (10⁻⁵m) caused moderate cortical swelling, pit formation in pith parenchyma, and chloroplast development in certain cortical cells. Exogenous ethylene reduced cell division activity and caused cortical cell swelling and separation. Removal of endogenously generated ethylene by mercuric perchlorate resulted in less ordered cell division patterns and no lateral root formation. Auxin treatments enhanced formation of an active acidic pI cellulase, exogenous ethylene-stimulated formation of an active basic pI cellulase. The absence of basic pI cellulase activity by the removal of endogenously generated ethylene suggests a close dependence of basic pI cellulase activity on ethylene.     

The influence of oxygen deficiency on ethylene synthesis, 1-aminocyclopropane-1-carboxylic acid levels and aerenchyma formation in roots of Zea mays

The relationship between ethylene production, 1-aminocyclopropane-l-carboxylic acid (ACC) concentration and aerenchyma formation (ethylene-promoted cavitation of the cortex) was studied using nodal roots of maize (Zea mays L. cv. LG11) subjected to various O₂ treatments. Ethylene evolution was 7–8 fold faster in roots grown at 3 kPa O₂ than in those from aerated solution (21 kPa O₂), and transferring roots from aerated solution to 3 kPa O₂ enhanced ethylene synthesis within less than 2 h. Ethylene production and ACC accumulation were closely correlated in different zones of hypoxic roots, regardless of whether O₂ was furnished to the roots through aerenchyma or external solution. Both ethylene production and ACC concentrations (fresh weight basis) were more than 10-fold greater in the distal 0–10 mm than in the fully expanded zone of roots at 3 kPa O₂. Aerenchyma formation occurred in the apical 20 mm of these roots. Roots transferred from air to anoxia accumulated less than 0. 1 nmol ACC (mg protein)^-1 for the first 1.75 h; no ethylene was produced in this time. The subsequent rise in ACC levels shows that ACC can reach high concentrations even in the absence of O₂, presumably due to a de-repression of ACC synthase. The hypothesis was therefore tested that anoxia in the apical region of the root caused enhanced synthesis of ACC, which was transported to more mature regions (10–20 mm behind the apex), where ethylene could be produced and aerenchyma formation stimulated. Surprisingly, exposure of intact root tips to anoxia inhibited aerenchyma formation in the mature root axis. High osmotic pressures around the growing region or excision of apices had the same effect, demonstrating that a growing apex is required for high rates of aerenchyma formation in the adjacent tissue.     

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.     

Ethylene Biosynthesis during Aerenchyma Formation in Roots of Maize Subjected to Mechanical Impedance and Hypoxia

Germinated maize (Zea mays L.) seedlings were enclosed in modified triaxial cells in an artificial substrate and exposed to oxygen deficiency stress (4% oxygen, hypoxia) or to mechanical resistance to elongation growth (mechanical impedance) achieved by external pressure on the artificial substrate, or to both hypoxia and impedance simultaneously. Compared with controls, seedlings that received either hypoxia or mechanical impedance exhibited increased rates of ethylene evolution, greater activities of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, ACC oxidase, and cellulase, and more cell death and aerenchyma formation in the root cortex. Effects of hypoxia plus mechanical impedance were strongly synergistic on ethylene evolution and ACC synthase activity; cellulase activity, ACC oxidase activity, or aerenchyma formation did not exhibit this synergism. In addition, the lag between the onset of stress and increases in both ACC synthase activity and ethylene production was shortened by 2 to 3 h when mechanical impedance or impedance plus hypoxia was applied compared with hypoxia alone. The synergistic effects of hypoxia and mechanical impedance and the earlier responses to mechanical impedance than to hypoxia suggest that different mechanisms are involved in the promotive effects of these stresses on maize root ethylene biosynthesis.     

G proteins as regulators in ethylene-mediated hypoxia signaling

Waterlogging or flooding are frequently or constitutively encountered by many plant species. The resulting reduction in endogenous O₂ concentration poses a severe threat. Numerous adaptations at the anatomical, morphological and metabolic level help plants to either escape low oxygen conditions or to endure them. Formation of aerenchyma or rapid shoot elongation are escape responses, as is the formation of adventitious roots. The metabolic shift from aerobic respiration to anaerobic fermentation contributes to a basal energy supply at low oxygen conditions. Ethylene plays a central role in hypoxic stress signaling, and G proteins have been recognized as crucial signal transducers in various hypoxic signaling pathways. The programmed death of parenchyma cells that results in hypoxia-induced aerenchyma formation is an ethylene response. In maize, aerenchyma are induced in the absence of ethylene when G proteins are constitutively activated. Similarly, ethylene induced death of epidermal cells that cover adventitious roots at the stem node of rice is strictly dependent on heterotrimeric G protein activity. Knock down of the unique Gα gene RGA1 in rice prevents epidermal cell death. Finally, in Arabidopsis, induction of alcohol dehydrogenase with resulting increased plant survival relies on the balanced activities of a small Rop G protein and its deactivating protein RopGAP4. Identifying the general mechanisms of G protein signaling in hypoxia adaptation of plants is one of the tasks ahead.Key words: submergence, hypoxia, ethylene, G protein, reactive oxygen species, H₂O₂     

Activity of C<Subscript>4</Subscript> enzymes in C<Subscript>3</Subscript>-type herbaceous plants

The activity of enzymes characteristic for C₄-type photosynthesis was determined in different organs of two herbaceous plants: Reynoutria japonica Houtt. and Helianthus tuberosus L. The activity of phosphoenolpyruvate carboxylase (PEPC) was usually higher in the roots, some of the stem tissues and petioles in comparison to the leaf blades. The highest activity of malic enzymes (NAD-ME, NADP-ME) and phosphoenolpyruvate carboxykinase (PEPCK) was in the petioles and stem tissues of both plants and the lowest in the leaf blades and the pith of Helianthus tuberosus L.     

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     

Distinct mechanisms for aerenchyma formation in leaf sheaths of rice genotypes displaying a quiescence or escape strategy for flooding tolerance

Background and Aims

Rice is one of the few crops able to withstand periods of partial or even complete submergence. One of the adaptive traits of rice is the constitutive presence and further development of aerenchyma which enables oxygen to be transported to submerged organs. The development of lysigenous aerenchyma is promoted by ethylene accumulating within the submerged plant tissues, although other signalling mechanisms may also co-exist. In this study, aerenchyma development was analysed in two rice (Oryza sativa) varieties, ‘FR13A’ and ‘Arborio Precoce’, which show opposite traits in flooding response in terms of internode elongation and survival.

Methods

The growth and survival of rice varieties under submergence was investigated in the leaf sheath of ‘FR13A’ and ‘Arborio Precoce’. The possible involvement of ethylene and reactive oxygen species (ROS) was evaluated in relation to aerenchyma formation. Cell viability and DNA fragmentation were determined by FDA/FM4-64 staining and TUNEL assay, respectively. Ethylene production was monitored by gas chromatography and by analysing ACO gene expression. ROS production was measured by using Amplex Red assay kit and the fluorescent dye DCFH₂-DA. The expression of APX1 was also evaluated. AVG and DPI solutions were used to test the effect of inhibiting ethylene biosynthesis and ROS production, respectively.

Key Results

Both the varieties displayed constitutive lysigenous aerenchyma formation, which was further enhanced when submerged. ‘Arborio Precoce’, which is characterized by fast elongation when submerged, showed active ethylene biosynthetic machinery associated with increased aerenchymatous areas. ‘FR13A’, which harbours the Sub1A gene that limits growth during oxygen deprivation, did not show any increase in ethylene production after submersion but still displayed increased aerenchyma. Hydrogen peroxide levels increased in ‘FR13A’ but not in ‘Arborio Precoce’.

Conclusions

While ethylene controls aerenchyma formation in the fast-elongating ‘Arborio Precoce’ variety, in ‘FR13A’ ROS accumulation plays an important role.     

Phytoremediation potential of Helianthus annuus L in sewage-irrigated Indo-Gangetic alluvial soils

The study of phytoremediation potential of Helianthus annuus L was conducted in the sewage-irrigated Indo-Gangetic alluvial soils, India. Calcium @ 1.0% and Zn @ 40 ppm enhanced the yield of H. annuus L and minimized the toxicity of Cr in the investigated soils. The study indicated that H. annuus L is highly sensitive to Cr and Zn in terms of metallic pollution; and may be used as indicator plant. For Cr-phytoremediation, humic acid treatment @ 500 mL/acre induced the Cr-accumulation in roots (p < 0.007) and in shoots (p < 0.015), which was recorded 3.21 and 3.16 mg/kg in root and shoot of H. annuus L, respectively. We suggest that H. annuus L fulfils the necessary condition for efficiently increasing species bioaccumulation after soil treatment with humic acid in Cr-polluted sewage-irrigated soils through soil- plant rhizospheric processes.