Boby Varghese

Desiccation-induced changes in lipid peroxidation, superoxide level and antioxidant enzymes activity in neem [Azadirachta indica A.Juss] seeds

The freshly harvested mature neem seeds (42.2 % seed moisture content) with 100 % viability deteriorate when naturally desiccated to below 10.9 %. The desiccation-induced loss of viability was closely associated with over accumulation of superoxide anion and lipid peroxidation products both in the embryonic axes and cotyledons. The levels of superoxide anion and lipid peroxidation products were higher in axes compared to cotyledons. Superoxide dismutase activity was not much affected, both in the axes and cotyledons of 100 % viable seeds during desiccation from 42.2 % to 10.9 % seed moisture content. Steep rise in its activity was observed during drying below lowest safe moisture content (LSMC). Activities of catalase and peroxidase exhibited substantially higher levels in the 100 % viable seeds dehydrated up to LSMC. Their activities declined sharply in seeds with water content below LSMC. Impairment of catalase and peroxidase activities possibly lead to enhanced accumulation of reactive oxygen species. The accumulation of superoxide anion, lipid peroxidation and differential expression of superoxide dismutase and catalse/peroxidase activities in response to desiccation (below LSMC) is discussed to explain the intermediate storage physiology of neem seeds.

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.

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. dregeanas 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.

Examining the utility of hyperspectral remote sensing and partial least squares to predict plant stress responses to sulphur dioxide pollution: a case study of Trichilia dregeana Sond.

Abstract The use of air quality monitoring stations is expensive, with pollution data being either unavailable or inaccessible. Hence, effects of atmospheric sulphur dioxide (SO2) levels on biomarkers related to environmental stress were investigated for Trichilia dregeana tree leaves, in order to assess their bioindicator potential. Leaves were sampled randomly from trees at three industrial sites within the South Durban Basin, and an ex situ control, across two seasons (n = 28, per season). Ground-level SO2 concentrations were measured daily and ranged between 1 and 25 ppb. There were significant (p < 0.001) differences across sites and seasons for leaf area and leaf chlorophyll content. Partial least squares regression (PLSR) was used to quantify the relationship between biomarkers and hyperspectral data. For leaf chlorophyll content and leaf area, r2 values ranged from 0.325–0.475 to 0.429–0.586, with root mean square error of prediction (RMSEP) ranging between 8.75–8.98 and 9.20–12.52. The variable importance in projection (VIP) method was utilized and significant hyperspectral wavebands were identified, within the red-edge region, at 552 and 704 nm for spring, and at 552 and 708 nm for summer. Notably, PLSR was able to relate hyperspectral data-sets to both biomarkers, showing promise in identifying stress in T. dregeana leaves. However, the interaction between leaf chlorophyll content and leaf area suggests that a simultaneous prediction of these biomarkers may be more suitable.

Photo-oxidation modulates green fern spore longevity during dry storage

Desiccation tolerance and longevity of plant propagules in the dry state have significant implications for biotechnological applications. In this study fern spores were used as a unicellular model to characterize some of the mechanisms of ageing during dry storage of plant propagules (at relative humidity ca. 15%). More specifically, we compared the potential relationships among indicators of photo-oxidative stress and spore viability during dry storage between green (chlorophyllous) spores of Todea barbara and non-green spores of Christella dentata. Green spores stored under the light aged faster than those stored in the dark, and faster than light- and dark-stored non-green spores of C. dentata. This rapid ageing in light-stored green spores was associated with significantly lower antioxidant activity (relative to time zero and dark-stored spores) during storage, and a burst of hydrogen peroxide during the latter stages of storage, which was not a feature of dark-stored spores. We attribute these signs of enhanced oxidative-stress mediated ageing in light-stored spores to photo-oxidative processes, similar to those described in other homoiochlorophyllous organisms. Additionally, high antioxidant activity and low levels of reactive oxygen species in green spores compared with non-green spores suggests differing mechanisms of coping with life in the dry state.

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.