Sucrose Utilization via Invertase and Sucrose Synthase with Respect to Accumulation of Cellulose and Callose Synthesis in Wheat Roots under Oxygen Deficiency

The sucrose cleavage by sucrose synthase (SuSy) and neutral invertase was studied in wheat roots (Triticum aestivum L.) subjected to hypoxia or anoxia for 4 days. By in situ activity staining, increased SuSy activity was observed in the tip region and stele of root axes while the activity of invertase decreased. Cellulose content significantly increased in hypoxically treated roots. The cellulose deposition was correlated with regions of high SuSy activity, being mainly located in the pericycle and endodermis. Invertase activity was distributed along the root without clear difference between cortex and stele. Under root hypoxia, a significant increase in the structural carbohydrates, callose and especially cellulose, was shown. Increasing levels of soluble carbohydrates were partially used to synthesize cellulose for secondary wall thickening and callose to counteract the tissue injury following low-oxygen stress. Under strict anoxia, the roots were much more injured but sustained a high level of cellulose and callose while the soluble carbohydrates almost disappeared.     

Hypoxia induces anoxia tolerance in roots and shoots of lupine seedlings

Lupine seedlings were exposed to 4 kPa partial pressure oxygen (hypoxically pretreated) for 18 hours before treatment with strictly anaerobic conditions (anoxia). Seedlings previously exposed to hypoxia were more tolerant than the controls (not hypoxically pretreated) to anoxic stress in both roots and shoots. Hypoxic pretreatment induced roots and shoots survival in anoxia. Improved viability of roots, following hypoxic pretreatment, was associated with increased activity of ADH. In nonacclimated roots and shots significant increase in LDH activity occurd during the first hours under anoxia but the in vitro activity of LDH was two orders of magnitude lower than that of ADH. The results are discussed in relation to the ability of lupine seedlings to survive anoxia.     

Tolerance of crop plants to oxygen deficiency stress: fermentative activity and photosynthetic capacity of entire seedlings under hypoxia and anoxia

The study investigates the reactions of rice, wheat and maize to anoxia (plants without access to oxygen) and hypoxia (roots with very limited access to oxygen). We studied the adaptations of these intact crop plants because they are known to differ widely in their tolerance to oxygen deficiency. In hypoxia, there was an accumulation of sugars, especially in wheat and maize, although both flood-sensitive species significantly increased the activities of fermentative and glycolytic enzymes, clearly more than in rice. In rice, avoiding an oxygen limitation due to the effective aeration system (30% of root cross-sectional area) may have accounted for only a minor metabolic reaction to hypoxia. In anoxia, maize and wheat quickly lost viability and nearly all photosynthetic capacity, while most rice leaves stayed turgid and green, losing only 50% of the photosynthetic capacity. A strong metabolic arrest under anoxia was obvious for the sucrolytic, glycolytic and fermentative enzymes in all tested species, but was most pronounced in rice. Of the 14 enzymes studied, rice showed the lowest activity increase in hypoxia for 11 enzymes, and the strongest activity decrease in anoxia for 8 enzymes. However, rice was able even under anoxia to keep a 1/4 of the ATP level of the aerated control, while it was at the detection limit in maize and wheat. It appears that in anoxic rice, the switch to metabolic dormancy and maintenance of basic shoot meristems diminishes the needs for energy and substrate. Additionally, rice already has lower sugar demand under hypoxia, and sugar supply appears to be sustained under anoxia by a functioning anaerobic amylase and by the photosynthetically active shoot.     

Hormonal Status in Wheat and Rice Seedlings under Anoxia

A comparative analysis of the effects of anoxia on growth, fresh weight gain, and phytohormones in wheat (Triticum aestivum L.) and rice (Oryza sativa L.) seedlings was performed. In both plant species, a total cessation of root growth occurred during the initial hours of anoxia. In an anaerobic environment, the fresh weight of wheat seedlings decreased. An increase in the shoot length and weight under the stress conditions was found only in rice seedlings. During the initial hours of anoxia, the level of free ABA in wheat and rice tissues increased manifold, and the accumulation of a free ABA form occurred at the expense of the hydrolysis of its bound forms. The IAA content in plant tissues also increased. In wheat, the accumulation of IAA was short, but in rice, a high hormone level was retained during the entire experiment, and, as a result, its concentration exceeded that of ABA. A level of cytokinins in the tissues of both plant species was affected by anoxia to a lesser extent than that of other phytohormones. This level somewhat decreased under anoxia similarly to the level in darkness under aeration. It is suggested that IAA accumulation in hypoxia-tolerant rice seedlings under anoxia favors maintenance of shoot growth and simultaneous inhibition of root growth. At the same time, in the hypoxia-sensitive wheat, an increase in the ABA level resulted in growth cessation.     

Antioxidant responses of shoots and roots of lentil to NaCl-salinity stress

The effect of salt stress (100 mM and 200 mM NaCl) on antioxidant responses in shoots and roots of 14-day-old lentil (Lens culinaris M.) seedlings was investigated. Salt stress caused a significant decrease in length, wet-dry weight and an increase in proline content of both shoot and root tissues. In leaf tissues, high salinity treatment resulted in a 4.4 fold increase in H₂O₂ content which was accompanied by a significant level of lipid peroxidation and an increase in electrolyte leakage. Root tissues were less affected with respect to these parameters. Leaf tissue extracts exhibited four activity bands, of which two were identified as Cu/Zn-SOD and others as Fe-SOD and Mn-SOD. Fe-SOD activity was missing in root extracts. In both tissues Cu/Zn-SOD activity comprised 70–75% of total SOD activity. Salt stress did not cause a significant increase in total SOD activity of leaf tissues but a significant enhancement (88%) was observed in roots mainly due to an enhancement in Cu/ZnSOD isoforms. Compared to leaf tissues a significantly higher constitutive ascorbate peroxidase (APX) and glutathion reductase (GR) activity was observed in root tissues. Upon salt stress no significant change in the activity of APX, catalase (CAT) and GR was observed in root tissues but a higher APX activity was present when compared to leaf tissues. On the other hand, in leaf tissues, with the exception of CAT, salt stress caused significant enhancement in the activity of other antioxidant enzymes. These results suggested that, root tissues of lentil are protected better from NaCl stress induced oxidative damage due to enhanced total SOD activity together with a higher level of APX activity under salinity stress. To our knowledge this is the first report describing antioxidant enzyme activities in lentil.     

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.     

海滨滨麦叶片和根对不同厚度沙埋的生理响应差异分析

以烟台海岸抗风沙植物滨麦为研究材料,通过对不同厚度沙埋下其叶片和根部抗氧化酶活力(超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT))、丙二醛(MDA)含量和渗透调节物含量变化的分析,探讨了叶片和根部对沙埋生理响应的差异。试验按滨麦成株株高(约40 cm)对其进行了轻度沙埋(在株高1/4处)、中度沙埋(2/4处)和重度沙埋(3/4处)。在沙埋第6天,分别测定了不同厚度沙埋处理下,植株各段叶片和根抗氧化酶活力、MDA和渗透调节物含量。结果表明,轻度和中度沙埋均加速植株生长。与对照相比,经轻度、中度沙埋处理6 d,叶片平均MDA含量增加,在重度沙埋下降低。不同厚度沙埋6 d,叶片平均SOD活力和脯氨酸含量增加,而CAT活力、可溶性糖和可溶性蛋白质含量下降。但不同厚度沙埋均使沙上叶片MDA、脯氨酸、可溶性蛋白质含量和SOD和CAT活力增加,尤其是叶片顶部增加最为明显,使沙下叶片MDA、可溶性糖、可溶性蛋白质含量和CAT活力下降,导致同株沙上和沙下叶片MDA、脯氨酸、可溶性糖、可溶性蛋白质含量和SOD和CAT活力差异显著(P0.05)。与叶片相比,根中MDA、可溶性蛋白质含量和SOD和CAT活力较低,而POD活力和可溶性糖含量较高并与叶片差异显著(P0.05)。不同厚度沙埋6 d,滨麦根中MDA和可溶性蛋白质含量变化较小,可溶性糖含量和CAT、POD、SOD活力略有降低。研究表明,滨麦根和叶片对不同厚度沙埋的生理响应不同。沙埋直接作用于叶片并诱发叶内氧自由基积累,但叶片通过快速激活的抗氧化酶保护系统(CAT、SOD)维持氧自由基代谢平衡,以及渗透调节物(脯氨酸、可溶性糖)的积累维护细胞水分代谢平衡,并满足能量的需求和快速生长。但在不同厚度沙埋下,由于根系不受沙埋直接影响而生理变化较小,并且还维持较低的膜脂过氧化水平,这可能是根能维持正常的吸水输水功能并在沙埋处理过程中和沙埋后地上叶片快速生长摆脱沙埋的重要物质基础。     

Improved procedure for detection of superoxide dismutase isoforms in potato,Solanum tuberosum L.

Superoxide dismutase (SOD) in-gel activity assay with selective inhibitors (KCN and H₂O₂) is one of the most commonly used methods for identification of SOD isoform types, i.e., FeSOD, MnSOD or Cu/ZnSOD, and evaluation of oxidative stress response in plants. However, there are potential pitfalls that surround this assay, such as problem to detect isoforms with low activity, comigration of SOD isoforms or application of inappropriate inhibitor concentration. We propose an improved method based on the combination of in-gel analysis of SOD activity and native-PAGE immunoblotting for identification of isoforms and determination of SOD isoenzyme activity pattern in potato. Depending on cultivar and growing conditions, one MnSOD, 3 FeSOD and 5–6 Cu/ZnSOD isoforms were identified in potato leaves. The most important qualitative difference between ex vitro- and in vitro-grown plants was the presence of additional FeSOD and Cu/ZnSOD isoforms in plantlets grown in vitro. Compared with results of in-gel activity assay with selective inhibitors, new method allowed accurate identification of comigrating FeSOD and Cu/ZnSOD isoforms and two protein bands of ambiguous identities. Potato SODs were also characterized by SDS-PAGE immunoblotting and single MnSOD (23.6 kDa), three Cu/ZnSOD polypeptides (17.9, 17 and 16.3 kDa) and single FeSOD (25.1 kDa) polypeptide were detected in leaves of four examined cultivars. The difference in the number of FeSOD and Cu/ZnSOD isoforms/polypeptides between native-PAGE and SDS-PAGE immunoblots suggests that SOD proteins may have undergone post-translational modifications affecting protein mobility or existence of isoforms that differ from each other in total protein charge, but not in molecular weight.     

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.     

Response of the ascorbate-glutathione cycle to re-aeration following hypoxia in lupine roots.

The response of the enzymes and metabolites of the ascorbate-glutathione pathway to oxidative stress caused by re-aeration following hypoxia was studied in roots of hydroponically grown lupine (Lupinus luteus L. cv. Juno) seedlings. Lupine roots were deprived of oxygen by subjecting them to hypoxia for 48 and 72 h and then re-aerated for up to 4 h. An increased content of total ascorbate was observed in lupine roots immediately after hypoxia, whereas total glutathione level decreased. However, a significant increase in the reduced forms of both metabolites was found directly after hypoxia. Re-admission of oxygen caused the decrease of the ratios of reduced to oxidized forms of ascorbate and glutathione, indicating oxidative stress. While monodehydroascorbate reductase (MDHAR, EC 1.6.5.4) activity remained unaltered during re-aeration the increase in activities of ascorbate peroxidase (APX, EC 1.11.1.11) and glutathione reductase (GR, EC 1.6.4.2) was observed 30 min after transfer from hypoxic condition. Dehydroascorbate reductase (DHAR, EC 1.8.5.1) activity approached the control level during a whole re-aeration period. Native gel electrophoresis combined with specific activity staining revealed seven isoforms of APX, five isoforms of GR and three different proteins with DHA reductase activity in roots extracts. However, immediately after hypoxic treatment APX-5 isoform and GR-1 isoform were not observed in roots. This experimental system was also used to investigate superoxide anion level in roots utilizing the superoxide anion-specific indicator dihydroethidium (DHE). Intense DHE-derived fluorescence was found in re-aerated root tips as compared to control roots, indicating that re-aeration induced superoxide anion production in hypoxically pretreated roots.     

Regulation of root anaerobiosis and carbon translocation by light and root aeration in Isoetes alpinus

The activity of alcohol dehydrogenase (ADH) was measured in corms and roots of the submerged freshwater macrophyte Isoetes alpinus Kirk. growing in situ, and related to its capacity for internal oxygen transport and to carbohydrate translocation. ADH activity was present in roots but not corms at uniform activity (0.15–0.35 × 10^?6 mol g^?1 fresh weight s^?1) over the entire plant depth range (3–7 m depth), and was intermediate to that developed in excised roots after 1‐week exposure to either dissolved oxygen at air‐saturation or to anoxia. Responses of photosynthesis and root oxygen release to light intensity confirmed that shoot‐to‐root oxygen transport saturated at similar light intensities to photosynthetic oxygen evolution, but was positive in the dark and at irradiances below the compensation point for photosynthesis, due to contributions to transport by oxygen diffusion from the external medium. Transport of ¹⁴C‐labelled photo‐assimilates to roots nevertheless ceased when intact plants were exposed to a combination of leaf darkness and root external anoxia, even when high ¹⁴C concentrations were present in shoots, but remained high when the roots were provided with external oxygen. The lack of any control over permeability of the root surface to gases in this species suggested that ADH activity and reduced translocation is most likely caused by development of hypoxic tissues in the apical tissue. These results suggest that reductions in ambient light intensity may have indirect effects on I. alpinus viability by increasing the degree of root hypoxia and impairing carbon partitioning.     

Internal oxygen transport inRumex species and its significance for respiration under hypoxic conditions

Rumex thyrsiflorus, Rumex crispus andRumex maritimus show a differential flood-tolerance in the river ecosystem in the Netherlands.R. thyrsiflorus occurs at high-elevated habitats and is flood-intolerant, the other two species occur at lower-elevated habitats and are flood-tolerant. We compared their respiratory activity under aerobic and anaerobic conditions in the root environment and quantified the internal gas transport. The results indicate that aerial oxygen can be used for root respiration in both aerobically and anaerobically grown plants. The amount of oxygen used via internal aeration increased with decreasing oxygen concentration in the root environment. Aerobically grown plants ofR. maritimus andR. crispus already showed a high internal aeration, but there was a significant increase in internal oxygen transport in anaerobic plants, where new, aerenchymatous roots had formed. This indicates the functional significance of new root formation for respiration in these species upon hypoxia. After two weeks of anaerobiosis, more than 50% of the total respiration of the roots of young plants ofR. maritumus and 40% of roots of young plants ofR. crispus was due to internal aeration at low oxygen concentrations in the root environment. InR. maritimus both young and old plants performed in this way, inR. crispus only young plants, whileR. thyrsiflorus showed some internal aeration, but this was hardly detectable. These differences can be explained on the basis of a different morphology and concomitant diffusive resistance of both root and shoot system. In experiments with different submergence levels of the shoot, the amount of internal aeration was positively correlated to the total leaf area protruding above the water surface inR. maritimus. This indicates a functional significance of the petiole and leaf elongation response upon total submergence of this species.     

Effects of metalaxyl enantiomers stress on root activity and leaf antioxidant enzyme activities in tobacco seedlings

The objective of this experiment was to study the effects of metalaxyl enantiomers on the activity of roots and antioxidative enzymes in tobacco seedlings. Water culture experiment was conducted to analyze the effects of different concentrations of metalaxyl enantiomers (30 and 10 mg L^?1) on root activity and leaf superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities and malondialdehyde (MDA) content of tobacco seedlings. The results showed that metalaxyl significantly inhibited root activity and significantly improved leaf SOD, POD, and CAT activities and MDA content. A better physiological response in tobacco seedlings was observed at 30 mg L^?1 than at 10 mg L^?1 metalaxyl. The stereoselectivity for different enantiomers had no obvious effect on root activity and the leaf POD activity, but it affected significantly the SOD and CAT activities and MDA content. The SOD activity was promoted more by R‐enantiomer than by S‐enantiomer at 30 mg L^?1 metalaxyl, and the same effect was observed on CAT activity from the beginning to the end of the stress period. The MDA content under the stress by R‐enantiomer was higher than that under the stress by S‐enantiomer at 10 mg L^?1 metalaxyl.     

Effect of drought on biomass, protein content, lipid peroxidation and antioxidant enzymes in two sesame cultivars

The effects of drought on growth, protein content, lipid peroxidation, superoxide dismutase (SOD), peroxidase (POX), catalase (CAT) and polyphenol oxidase (PPO) were studied in leaves and roots of Sesamum indicum L. cvs. Darab 14 and Yekta. Four weeks after sowing, plants were grown under soil moisture corresponding to 100, 75, 50 and 25 % field capacity for next four weeks. Fresh and dry masses, and total protein content in leaves and roots decreased obviously under drought. However, several new proteins appeared and content of some proteins was affected. Measurement of malondialdehyde content in leaves and roots showed that lipid peroxidation was lower in Yekta than in Darab 14. Severe stress increased SOD, POX, CAT and PPO activities in leaves and roots, especially in Yekta. According to the present study Yekta is more resistant to drought than Darab 14.     

The role of nitrate reduction in the anoxic metabolism of roots II. Anoxic metabolism of tobacco roots with or without nitrate reductase activity

The effects of root anoxia on a tobacco (Nicotiana tabacum) wild type (WT) and a transformant (LNR-H) lacking root nitrate reductase were compared. LNR-H plants were visibly more sensitive to oxygen deprivation than WT, showing rapid and heavy wilting symptoms. LNR-H roots also produced substantially more ethanol and lactate than WT roots under anoxia, and their sugar and sugar-P content, as well as their ATP levels, remained higher. The fermentation rates of WT and LNR-H roots were unaffected by sugar feeding and the higher fermentation rate in the LNR-H roots was associated with a greater acidification of the cytoplasm under anoxia. From these observations it is concluded: (i) that the absence of NR activity in the LNR-H roots does not necessarily limit NADH recycling; and (ii) that nitrate reduction in the WT roots results in a more acidifying metabolism. It is the higher metabolic rate in the LNR-H roots that leads to the greater cytoplasmic acidification under anoxia despite the absence of a contribution from the metabolism of nitrate. Competition for NADH cannot explain this difference in metabolic rate, and it remains unclear why the NR-free LNR-H, and tungstate-treated WT roots, had much higher fermentation rates than WT roots. The difference in anaerobic metabolism could still be due to the presence or absence of nitrate reductase and the possibility that this could occur through the production of nitric oxide is discussed.     

Second messenger and cAMP-dependent protein kinase responses to dehydration and anoxia stresses in frogs

The effects of whole body dehydration (up to 40% of total body water lost) or anoxia exposure (up to 2 days under N₂ gas) at 5 °C on tissue levels of adenosine 3′–5′ cyclic monophosphate (cAMP) and the percentage of cAMP-dependent protein kinase present as the free catalytic subunit (PKAc), as well as the levels of the protein kinase C (PKC) second messenger, inositol 1,4,5-trisphosphate (IP₃), were assessed in two anurans, the freeze-tolerant wood frog, Rana sylvatica, and the freeze-intolerant leopard frog, Rana pipiens. Dehydration of wood frogs resulted in a rapid elevation of liver cAMP and PKAc; cAMP was 3.4-fold greater than control values in animals that had lost 5% of total body water, whereas PKAc was elevated threefold in 20% dehydrated frogs. These results indicate protein kinase A mediation of the liver glycogenolysis and hyperglycemia that is induced by dehydration in this species. Skeletal muscle PKAc content also rose with dehydration but neither cAMP nor PKAc was affected by dehydration in leopard frog tissues. Anoxia exposure had different effects on signal transduction systems. PKAc was elevated after 1 h anoxia in R. sylvatica brain and was sustained over time but the enzyme was unaffected in other organs; by contrast, R. pipiens showed variable responses by PKAc to anoxia in three organs. Both species showed rapid (within 30 min) and large (3 to 7.8-fold) increases in IP₃ in liver of anoxic frogs that decreased slowly with continued anoxia. IP₃ also increased quickly in heart of anoxia-exposed wood frogs. This suggests that PKC may mediate various metabolic adjustments that promote hypoxia/anoxia resistance such as coordinating metabolic rate depression. A progressive rise in liver IP₃ during dehydration in wood frogs (reaching fourfold higher than controls in 40% dehydrated animals) may also mediate similar hypoxia resistance adaptations under this stress since anurans experience progressive hypoxia due to increased blood viscosity when water loss reaches high values. The patterns of second messenger and PKAc changes in wood frog liver during dehydration closely parallel the changes seen in these same parameters during natural freezing suggesting that the freeze tolerance of selected terrestrially hibernating anurans may have evolved out of various anuran mechanisms of dehydration resistance. Accepted: 2 January 1997     

Functional linkage between N acquisition strategies and aeration capacities of hydrophytes for efficient oxygen consumption in roots

We evaluated the specific strategies of hydrophytes for root O₂ consumption in relation to N acquisition and investigated whether the strategies varied depending on the aeration capacity. Aeration capacity of roots is an important factor for determining hypoxia tolerance in plants. However, some hydrophytes possessing quite different aeration capacities often co‐occur in wetlands, suggesting that root O₂ consumption also strongly affects hypoxia tolerance. We cultivated Phragmites australis with high aeration capacity and Zizania latifolia with low aeration capacity in hypoxic conditions with NH or NO treatment and compared the growth, N uptake, N assimilation and root respiration between the two species. In Z. latifolia grown with NH treatment, high N uptake activity and restrained root growth led to sufficient N acquisition and decrease in whole‐root respiration rate. These characteristics consequently compensated for the low aeration capacity. In contrast, in P. australis, low N uptake activity was compensated by active root growth, but the whole‐root respiration rate was high. This high root respiration rate was allowed by the high aeration capacity. The O₂ consumption‐related traits of hydrophyte roots were closely correlated with N acquisition strategies, which consequently led to a compensational relationship with the root aeration capacity. It is likely that this functional linkage plays an important role as a core mechanism in the adaptation of plants to hypoxic soils.