Allantoin: A Potential Compound for the Mitigation of Adverse Effects of Abiotic Stresses in Plants.

Stress-induced alterations vary with the species of plants, the intensity and duration of the exposure, and stressors availability in nature or soil. Purine catabolism acts as an inherent defensive mechanism against various abiotic stresses and plays a pivotal role in the stress acclimatisation of plants. The intermediate metabolite of purine catabolism, allantoin, compensates for soil nitrogen deficiency due to the low carbon/nitrogen ratio, thereby maintaining nitrogen homeostasis and supporting plant growth and development. Allantoin accounts for 90% of the total nitrogenous compound in legumes, while it contributes only 15% in non-leguminous plants. Moreover, studies on a variety of plant species have reported the differential accumulation of allantoin in response to abiotic stresses, endowing allantoin as a stress modulator. Allantoin functions as signalling molecule to stimulate stress-responsive genes (P5CS; pyrroline-5-carboxylase synthase) and ROS (reactive oxygen species) scavenging enzymes (antioxidant). Moreover, it regulates cross-talk between the abscisic acid and jasmonic acid pathway, and maintains ion homeostasis by increasing the accumulation of putrescine and/or spermine, consequently enhancing the tolerance against stress conditions. Further, key enzymes of purine catabolism (xanthine dehydrogenase and allantoinase) have also been explored by constructing various knockdown/knockout mutant lines to decipher their impact on ROS-mediated oxidative injury in plants. Thus, it is established that allantoin serves as a regulatory signalling metabolite in stress protection, and therefore a lower accumulation of allantoin also reduces plant stress tolerance mechanisms. This review gives an account of metabolic regulation and the possible contribution of allantoin as a photo protectant, osmoprotectant, and nitrogen recycler to reduce abiotic-stress-induced impacts on plants.

Cathodic Water Enhances Seedling Emergence and Growth of Controlled Deteriorated Orthodox Seeds.

All orthodox seeds eventually deteriorate during storage, a well-known problem in seed banking. Here we used a greenhouse study to test if priming deteriorated seeds with cathodic water can improve the emergence and subsequent seedling growth of three South African tree species, Bolusanthus speciosus, Combretum erythrophyllum and Erythrina caffra. Other priming solutions investigated were calcium magnesium (CaMg) solution and deionized water. In the present study, seeds were subjected to an artificial deterioration by increasing their water content to 14% and keeping them at 40 °C and 100% RH until they had lost 50% of their germination under laboratory conditions. Fresh and deteriorated seeds were primed with cathodic water, CaMg solution and deionized water, with non-primed fresh and deteriorated seeds as controls. Controlled deterioration significantly reduced total emergence and the biomass and photosynthetic parameters of the resulting seedlings. In one species (Bolusanthus speciosus), priming the deteriorated seeds with cathodic water significantly improved emergence parameters. However, in all species cathodic water significantly improved the total biomasses and other growth parameters of the seedlings derived from deteriorated seeds. Priming with CaMg solution and deionized water had little effect on emergence and while improving the growth of seedlings derived from deteriorated seeds, they were less effective than cathodic water. In fresh seeds, priming with all solutions resulted in small improvements in some parameters. Controlled deterioration of fresh seeds reduced the membrane stability index (MSI) in two of the three species and in all species increased the levels of the lipid oxidation products MDA and 4-HNE. Priming deteriorated seeds with cathodic water increased the MSI and reduced the MDA contents in all species and the 4-HNE content in one species. Other priming solutions were generally less effective in ameliorating oxidative stress. Results suggest that the strong antioxidative properties of cathodic water can explain its ability to ameliorate deterioration. In conclusion, the present study shows that priming with cathodic water is an effective way of invigorating deteriorated orthodox seeds and that it may have considerable potential in orthodox seed conservation.

Effects of Inorganic Salt Solutions on Vigour, Viability, Oxidative Metabolism and Germination Enzymes in Aged Cabbage and Lettuce Seeds.

This study assessed the potential of pre-hydration treatment with aqueous solutions (electrolysed [cathodic water; CW] and non-electrolysed) prepared from four different inorganic ion combinations: 1 mM CaCl2, 1 µm CaCl2 and 1 mM MgCl2 (CaMg, hereafter), 1 mM MgCl2 and 1 mM NaCl to invigorate controlled deteriorated (CDd) Brassicaoleracea (cabbage) and Lactucasativa (lettuce) seeds by assessing germination, vigour and biochemical markers (electrolyte leakage, lipid peroxidation products, protein carbonylation, and defence and germination associated enzymes) of oxidative stress. Additionally, the possible effects of pH of electrolysed CaMg and NaCl solutions were assessed. The inorganic salt solutions were applied to fresh seeds and seeds deteriorated to 75% viability (P75), 50% viability (P50) and 25% viability (P25); deionised water served as control. The pre-hydration treatment did not enhance normal seedling production in cabbage. However, Ca-containing and CW hydration treatments (CaCl2 CW, CaMg and CaMg CW [6.5], MgCl2 CW, NaCl CW and NaCl CW [6.5]) promoted normal seedling production of CDd lettuce seeds, while seedling vigour was enhanced by CaMg, CaMg CW (6.5), NaCl CW and NaCl CW (6.5) in CDd cabbage seeds, and CaCl2, CaCl2 CW, CaMg, CaMg CW (6.5), MgCl2 CW, NaCl CW and NaCl CW (6.5) in CDd lettuce seeds. The supplementation of Ca, a component of the ionised solutes, and/or the reducing potential of CW contributed to increased normal seedling production in lettuce seeds irrespective of the pH of treatment solutions or degree of deterioration. Overall, the pre-hydration treatments enhanced endogenous antioxidants leading to reduced levels of electrolyte leakage, lipid peroxidation, protein carbonylation, and enhanced germination enzyme activities in lettuce seeds. The study concluded that pre-hydration with selected inorganic salt solutions can invigorate debilitated lettuce seeds.

Influence of Cathodic Water Invigoration on the Emergence and Subsequent Growth of Controlled Deteriorated Pea and Pumpkin Seeds.

The quality of seeds in gene banks gradually deteriorates during long-term storage, which is probably, at least in part, a result of the progressive development of oxidative stress. Here, we report a greenhouse study that was carried out to test whether a novel approach of seed invigoration using priming with cathodic water (cathodic portion of an electrolysed calcium magnesium solution) could improve seedling emergence and growth in two deteriorated crop seeds. Fresh seeds of Pisum sativum and Cucurbita pepo were subjected to controlled deterioration to 50% viability at 14% seed moisture content (fresh weight basis), 40 °C and 100% relative humidity. The deteriorated seeds were thereafter primed with cathodic water, calcium magnesium solution and deionized water. In addition, to study the mechanism of the impacts of invigoration, the effects of such priming on the lipid peroxidation products malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) and on the reactive oxygen species (ROS) scavenging enzymes superoxide dismutase and catalase were also determined in the fresh and deteriorated seeds. All priming treatments improved seed emergence parameters, subsequent seedling photosynthesis and growth relative to the unprimed seeds. In general, cathodic water was most effective at invigorating deteriorated seeds. Analysis of the lipid peroxidation products and antioxidant enzyme activities in invigorated seeds provided support for the hypothesis that the effectiveness of cathodic water in invigoration of debilitated orthodox seeds in general and of pea and pumpkin seeds in particular derive from its ability to act as an antioxidant.

Zygotic embryo cell wall responses to drying in three gymnosperm species differing in seed desiccation sensitivity.

Plant cell walls (CWs) are dynamic in that they can change conformation during ontogeny and in response to various stresses. Though seeds are the main propagatory units of higher plants, little is known of the conformational responses of zygotic embryo CWs to drying. This study employed cryo-scanning electron microscopy to compare the effects of desiccation on zygotic embryo CW morphology across three gymnosperm species that were shown here to differ in seed desiccation sensitivity: Podocarpus henkelii (highly desiccation-sensitive), Podocarpus falcatus (moderately desiccation-sensitive), and Pinus elliottii (desiccation-tolerant). Fresh/imbibed (i.e. fresh Podocarpus at shedding and imbibed Pi. elliottii) embryos showed polyhedral cells with regular walls, typical of turgid cells with an intact plasmalemma. Upon desiccation to c. 0.05 g g-1 (dry mass basis), CWs assumed an undulating conformation, the severity of which appeared to depend on the amount and type of dry matter accumulated. After desiccation, intercellular spaces between cortical cells in all species were comparably enlarged relative to those of fresh/imbibed embryos. After rehydration, meristematic and cotyledonary CWs of P. henkelii and meristematic CWs of P. falcatus remained slightly undulated, suggestive of plasmalemma and/or CW damage, while those of Pi. elliottii returned to their original conformation. Cell areas in dried-rehydrated P. henkelii root meristem and cotyledon were also significantly lower than those from fresh embryos, suggesting incomplete recovery, even though embryo water contents were comparable between the two states. Electrolyte leakage measurements suggest that the two desiccation-sensitive species incurred significant plasmalemma damage relative to the tolerant species upon desiccation, in agreement with the CW abnormalities observed in these species after rehydration. Immunocytochemistry studies revealed that of the four CW epitopes common to embryos of all three species, an increase in arabinan (LM6) upon desiccation and rehydration in desiccation-tolerant Pi. elliottii was the only difference, although this was not statistically significant. Seed desiccation sensitivity in species like P. henkelii and P. falcatus may therefore be partly based on the inability of the plasmalemma and consequently CWs of dried embryos to regain their original conformation following rehydration.

Uncovering the basis of viability loss in desiccation sensitive Trichilia dregeana seeds using differential quantitative protein expression profiling by iTRAQ.

Recalcitrant seeds, unlike orthodox types, are desiccation sensitive and hence, cannot be stored using conventional seed storage methods In this study, relative changes of protein expression in T. dregeana seeds during desiccation and hydrated storage (a short- to medium-term storage method) were analysed to understand the basis of their desiccation- and storage-induced viability loss. Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) were used to compare (selected) protein expression levels across fresh, partially dehydrated and stored seeds. A total of 114 proteins were significantly differentially expressed in embryonic axes of fresh seeds and those seeds exposed to dehydration and hydrated storage (which exposed seeds to a mild dehydration stress). Proteins involved in protein synthesis were up-regulated in stored and dehydrated seeds, possibly in response to dehydration-induced repair processes and/or germinative development. A range of proteins related to antioxidant protection were variably up- and down-regulated in stored and dehydrated seeds, respectively. Additionally, a class I heat shock protein was down-regulated in dehydrated and stored seeds; no late embryogenesis abundant proteins were identified in both stored and dehydrated seeds; and storage and dehydration up-regulated proteins involved in the provision of energy for cell survival. The results suggest that dehydration- and storage-induced viability loss in recalcitrant seeds may be based on proteomic changes that lead to cellular redox imbalance and increased cell energy demands. This, together with the absence/down-regulation of proteins associated with desiccation tolerance in plant tissues may form part of the proteomic footprint for desiccation sensitivity in seeds.

The utility of biochemical, physiological and morphological biomarkers of leaf sulfate levels in establishing Brachylaena discolor leaves as a bioindicator of SO2 pollution.

The negative impacts of air pollution have made monitoring of air quality increasingly important, especially in heavily industrialized areas such as the South Durban Basin (SDB), in South Africa. Bioindicators such as trees can complement conventional air quality monitoring and be used to prioritise vulnerable areas. This study assessed the utility of Brachylaena discolor DC. tree leaves as a bioindicator of sulfur dioxide pollution. This involved correlating ground level sulfur dioxide concentrations ([SO2]) with B. discolor leaf sulfate concentrations ([sulfate]) at three industrial (treatment) sites within the SBD and control site at which [SO2] was significantly lower for all four seasons. Based on this significant positive correlation between the aforementioned (p = 0.005; ρ = 0.667) leaf sulfate levels were related to various biochemical (intracellular hydrogen peroxide production, total aqueous [TAA] and enzymic antioxidants [superoxide dismutase and catalase], lipid peroxidation [LPO] and electrolyte leakage), physiological (leaf chlorophyll fluorescence [LCF] and relative chlorophyll content) and morphological (leaf area [LA]) biomarkers of stress measured on leaves from trees at the treatment and control sites. Annual [SO2] and leaf [sulfate] at the treatments were significantly (p < 0.05) higher than the control and high by global standards. TAA, LPO, electrolyte leakage, LCF, and LA were significantly (p < 0.05) correlated with seasonal [sulfate]. Except for superoxide dismutase, catalase and relative chlorophyll content all other biomarkers could differentiate between the treatment sites and the control. However, TAA, electrolyte leakage and LA were the only biomarkers sensitive enough to reflect differences in annual [sulfate] across the treatment sites; these should be used to establish B. discolor leaves as a bioindicator of SO2 pollution.

Effects of simulated acid rain on germination, seedling growth and oxidative metabolism of recalcitrant-seeded Trichilia dregeana grown in its natural seed bank.

Increased air pollution in a number of developing African countries, together with the reports of vegetation damage typically associated with acid precipitation in commercial forests in South Africa, has raised concerns over the potential impacts of acid rain on natural vegetation in these countries. Recalcitrant (i.e. desiccation sensitive) seeds of many indigenous African species, e.g. must germinate shortly after shedding and hence, may not be able to avoid exposure to acid rain in polluted areas. This study investigated the effects of simulated acid rain (rainwater with pH adjusted to pH 3.0 and 4.5 with 70:30, H2 SO4 :HNO3 ) on germination, seedling growth and oxidative metabolism in a recalcitrant-seeded African tree species Trichilia dregeana Sond., growing in its natural seed bank. The results suggest that acid rain did not compromise T. dregeana seed germination and seedling establishment significantly, relative to the control (non-acidified rainwater). However, pH 3.0 treated seedlings exhibited signs of stress typically associated with acid rain: leaf tip necrosis, abnormal bilobed leaf tips, leaf necrotic spots and chlorosis, reduced leaf chlorophyll concentration, increased stomatal density and indications of oxidative stress. This may explain why total and root biomass of pH 3.0 treated seedlings were significantly lower than the control. Acid rain also induced changes in the species composition and relative abundance of the different life forms emerging from T. dregeana's natural seed bank and in this way could indirectly impact on T. dregeana seedling establishment success.

Uneven drying of zygotic embryos and embryonic axes of recalcitrant seeds: challenges and considerations for cryopreservation.

Cryopreservation is the most promising option for the long-term germplasm conservation of recalcitrant-seeded species. However, the variable post-cryo success achieved with the excised zygotic explants traditionally used for cryopreservation has been a concern for some time. Differential drying rates amongst explants of different species, uneven drying amongst explants within a batch of seeds and uneven drying across tissues within individual embryos could be contributory factors to this variable success and these phenomena form the foci of the present study. Using zygotic explants from a range of recalcitrant-seeded species, which included sub-tropical dicotyledonous trees and sub-tropical monocotyledonous geophytes, the study showed that embryo morphology and anatomy are critical determinants of the drying characteristics of the different tissues composing the explant and hence, post-cryo survival. The results suggest that the rates of drying of explants to water contents (WCs) in the theoretically optimal range for successful cryopreservation are species-specific, and that more rapid drying rates may promote post-cryo survival. However, the large variation in WC amongst individual explants in bulk samples challenges the selection of the theoretically optimum WC for cryopreservation. As a consequence of differential drying rates across the different tissues composing explants, either lethal ice crystal damage or desiccation damage may sometimes be likely in tissues responsible for the onwards development of the embryo. Drying times for cryopreservation of such explants should, therefore, be selected on the basis of WC of segments containing root or shoot meristem, rather than embryo bulk WC. Drying intensity and duration also interact with explant morphology and embryo/axis size and anatomy to bring about - or preclude - post-cryo survival.

Cryo-tolerance of zygotic embryos from recalcitrant seeds in relation to oxidative stress--a case study on two amaryllid species.

Oxidative stress is a major component of cryoinjury in plant tissues. This study investigated the ability of recalcitrant (i.e. desiccation sensitive) Amaryllis belladonna L. and Haemanthus montanus Baker zygotic embryos to survive cryopreservation, in relation to oxidative stress. The study also investigated whether glycerol cryoprotection promoted embryo post-cryo survival by protecting enzymic antioxidant activities. Zygotic embryos excised from hydrated stored seeds were subjected to various combinations of rapid dehydration (to < or >0.4 g g⁻¹ [dmb]), cryoprotection (with sucrose or glycerol), and cooling (either rapidly or slowly), and were thereafter assessed for viability, extracellular superoxide (·O2⁻) production, lipid peroxidation (TBARS) and antioxidant enzyme activities. Short-term hydrated storage of whole seeds was accompanied by ·O2⁻ production and lipid peroxidation, but ·O2⁻ levels were lower than in dehydrated and cooled embryos and viability was 100%, possibly associated with the high activities of certain antioxidant enzymes. Partial dehydration and cryoprotection (in H. montanus only) increased ·O2⁻ production (especially in cryoprotected-dried embryos) and was associated with some viability loss, but this was not correlated with enhanced lipid peroxidation. Cooling was generally accompanied by the greatest increase in ·O2⁻ production, and with a decline in viability. In A. belladonna only, post-cryo TBARS levels were generally higher than for fresh and pre-conditioned embryos. Partial dehydration and cooling decreased antioxidant activities, but these were consistently less severe in glycerol cryoprotected-dried, as opposed to non-cryoprotected-dried embryos. Post-cryo viability retention for glycerol cryoprotected-dried embryos was significantly higher than for non-cryoprotected-dried embryos, possibly facilitated by relatively low post-drying TBARS levels and high post-drying and post-rewarming activities of some antioxidant enzymes in the former. Pre-conditioning treatments such as glycerol cryoprotection, when used in combination with partial drying, may enhance post-cryo viability retention in recalcitrant zygotic embryos by protecting the activities of certain antioxidant enzymes during pre-conditioning for, and after retrieval from, cryostorage.

Differential drying rates of recalcitrant Trichilia dregeana embryonic axes: a study of survival and oxidative stress metabolism.

Studies to elucidate the biochemical basis of survival of excised embryonic axes (EAs) of recalcitrant seeds of Trichilia dregeana at different drying rates revealed significant differences between slow and rapid drying. Rapid drying allowed these EAs to survive dehydration to much lower water contents (WCs; ca. 0.31 g g⁻¹ dry mass basis with 73% germination) compared with slow drying, where 90% of the EAs lost viability at a WC of ca. 0.79 g g⁻¹. In EAs slowly dried within seeds, the levels of hydroxyl radical (three- to fivefold at WCs > 0.5 g g⁻¹) and lipid peroxidation (50% at similar WC) were significantly higher compared with those dried rapidly to comparable WCs. When EAs were dried slowly, enzymic antioxidant levels were not sustained and declined significantly with prolonged storage. In contrast, sustained activity of enzymic antioxidants was detected in rapidly dried EAs even at relatively low WCs. Furthermore, the greater decline in glutathione (GSH)/GSH disulphide ratio in EAs slowly dried within seeds compared with rapidly dried EAs and a shift in GSH redox potential to relatively more positive values in the EAs slowly dried within seeds was correlated with considerable viability loss. It is apparent from this study that greater retention of viability to lower WCs in rapidly dried EAs from recalcitrant seeds may at least be partly explained by the retention of functional antioxidant status. It is also suggested that the reduction of viability in rapidly dried EAs at very low WCs appears to be a non-oxidative process.

Oxidative metabolism-related changes in cryogenically stored neem (Azadirachta indica A. Juss) seeds.

The seeds of Azadirachta indica were successfully cryopreserved for 12 months with 45% survival following drying to 0.16 g H(2)O g(-1) dry mass (DM). Highest survival (94-96%) was recorded during the first month of cryostorage. Subsequent cryopreservation up to 12 months resulted in decreasing germination. Post-thawing pre-heat treatment enhanced the recovery marginally in seeds cryopreserved from 3 to 12 months. Viability of cryostored seeds was negatively correlated with leachate conductivity and accumulation of thiobarbituric acid reactive substances (TBRS) estimated in cotyledons and axes. Leachate conductivity of imbibed seeds was low during the first month of cryostorage but increased gradually with the duration of cryostorage to a maximum after 12 months. TBRS accumulation was gradual throughout cryostorage. Relatively low amounts of active oxygen species (AOS) detected during the first month of cryostorage were closely associated with very high activities of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) in seeds (cotyledons and axes). Marked accumulation of AOS from 3 to 12 months was associated with decrease in antioxidant enzyme activity.