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Chickpea (Cicer arietinum L.) seed is a potential source of protein ingredients with desirable nutritional and functional properties. Knowledge of molecular characteristics of a food protein is essential before a protein can gain widespread use as a food ingredient. The objectives of this study were to prepare chickpea proteins using different extraction methods and precipitation methods and to investigate molecular characteristics using polyacrylamide gel electrophoresis (PAGE; Native and SDS), reversed phase high performance liquid chromatography (RP-HPLC) and electrospray ionization mass spectrometry (ESI-MS) techniques. Proteins of ground chickpea seed were extracted with sodium hydroxide (NaOH) and with citric acid solutions and precipitated with addition of acid and by cryoprecipitation. The protein contents of the protein preparation ranged from 49% to 97%. The microstructures of chickpea protein isolates examined by scanning electron microscope (SEM) revealed the presence of starch grains in the cryoprecipitates from citric acid extraction but not in isoelectric precipitates. The globulins (legumins and vicilins), glutelins, and albumins from both citric acid and NaOH isolates were characterized by Native-PAGE. The cryoprecipitates contained mainly the globulin-rich proteins. With SDS-PAGE characterization, protein subunits were identified as follows: (i) legumin subunits: MW 40, 39, 26, 23, and 22 kDa, (ii) vicilin subunits: MW 50, 37, 33, 19, and 15 kDa, (iii) glutelin subunits: 58, 55, and 54 kDa, and (iv) albumin subunits: 10 kDa. Separation of fractions of isolated chickpea proteins by RP-HPLC showed that early eluting fractions (Rt 20-30 min) consisted of subunits of MW 6.5-31 kDa (SDS-PAGE). At elution time 30-36 min, the fractions obtained were composed mainly of mixtures of legumin and vicilin subunits (MW 14-45 kDa). The major subunits of chickpea protein fractions from both cryoprecipitates and isoelectric precipitates are legumin basic subunit (MW∼23 kDa) and vicilin-rich proteins (MW∼19, 17, 15 kDa). ESI-MS analysis of fractions separated by RP-HPLC showed MW ranging between 5.1 and 53.5 kDa. The subunits of MW 35366, 27626, 22864, 20531, 16092, and 15626 Da of fractions from ESI-MS corresponded to MW 35.3, 28.0, 24.1, 20.5, 16.1, and 15.3 kDa identified in SDS-PAGE. These fractions were identified as legumin-rich and vicilin-rich proteins.

Six different lots of soya beans (Glycine max (L.) Merr.) were examined. Seed hydroperoxide levels were highly correlated with viability, but not with moisture contents. It was proposed that moisture contents may exert a similar antioxidant effect at intermediate levels as has been observed in dry foods. Seeds treated with ferrous sulphate were significantly (S% level) invigorated. Furthermore, this treatment was observed to give rise to a reduction in the peroxide value of soya bean axes over the first hour of imbibition, an increase in 2,3,S-triphenyl-tetrazolium chloride reduction and protein synthesis, and a decline in electrolyte leakage. It was proposed that this was due to the antioxidant activity of the ferrous iron, leading to an attenuation of free-radical induced autoxidation. Ferrous sulphate treated seeds produced more aldehydes than untreated seeds. This result suggested that aldehydes may not be responsible for declining seed vigour. Hexanal, pentanal and butanal production from heated dry seeds was significantly correlated with seed germination, CVG and hydroperoxide levels. The thermal breakdown of the hydroperoxides was postulated to be the source of these compounds. A GC technique was developed using model systems of oxidized methyl oleate, linoleate, linolenate and soya bean bulk oil. The analysis of seed lipid oxidation products revealed marked differences in the proportions of the products compared to bulk and monolayer oxidation. The selective production of the 13-hydroperoxide implicated enzymatic or metalloprotein involvement. The implications of the results of this study with regard to the present theories of seed ageing were discussed. / Thesis (Ph.D.)-University of Natal, Durban, 1990.

Cowpea (Vigna unguiculata. L) is an important African crop. However, it is also an underutilized grain legume. Consequently, there is not enough research data on cowpea seed physiology. Whereas there is evidence of cowpea being a drought tolerant crop, there is no evidence to associate plant drought tolerance with seed quality in response to water stress. This study sought to understand the effect of production site and water stress on cowpea seed quality development with respect to germination capacity and vigour. Patterns of raffinose family of oligosaccharides (RFO) during seed development to mature dry stage were used to physiologically relate seed performance to water stress. The effect of water stress and exogenous ABA on the accumulation of stress LEA proteins (dehydrins) in relation to seed quality development and germination was investigated. RFOs are known for their roles in desiccation sensitivity but no studies have shown their significance in cowpeas. Seeds of six cowpea cultivars were produced at two distinct growth sites characterised by irrigated and dry land conditions. The seeds were assessed during six developmental stages, for water content, dry matter accumulation, and performance. Harvested seeds were then planted in a pot experiment under controlled conditions to examine the effect of water stress on seed quality development and data collected during three developmental stages. Harvested seeds from the pot experiment were subsequently analyzed for changes in RFO accumulation during development using gas chromatography. The seeds were also used to investigate the effect of water stress and ABA on the accumulation of stress LEA proteins (dehydrins) in relation to seed quality development in cowpea. In addition, this study evaluated the use of image analysis as a method that can be used to objectively determine seed coat colour variation in cowpea. Statistical variation in individual seed’s solute leakage for cowpea cultivars differing in seed coat colour and produced under different environmental conditions was explored and correlations were done between seed conductivity test with other aspects of seed performance during germination. Furthermore the results of the conductivity test were compared with accelerated aging test, in relation to seed performance. The study provided evidence that cowpea seed lots produced under different environmental, and possibly management conditions may not differ with respect to seed quality as determined by germination capacity and vigour. However, significant differences between sites with respect to seed maturation patterns determined by water content and dry matter accumulation were observed. Adverse maternal environmental effects on the subsequent performance of seeds in a drought tolerant crop may not necessarily lead to poor performance. Cultivar differences in response to simulated drought conditions at the whole plant and tissue level can be considerable and highly variable; however, these differences may not have adverse effects on the germination and vigour of the seeds. Drought avoidance mechanisms at the whole plant level in cowpea are quite efficient in allowing the species to adapt to simulated drought conditions. These mechanisms may allow the cowpea cultivars to maintain metabolism and restore conditions for their continued growth under water stress; and produce few seeds of high germination capacity and vigour. Stachyose was found to be the predominant member of the raffinose family of oligosaccharides in cowpea. It is suggested that stachyose accumulation could be used as an indicator of stress tolerance in cowpea. However, the relationship between RFO concentration and the acquisition of desiccation remained as a matter of speculation in the present study and is still generally inconclusive. There was no evidence to suggest the acquisition of maximum desiccation tolerance is associated with maximum seed vigour. It is suggested in cowpea, which is drought tolerant, that maximum vigour does not necessarily imply the acquisition of maximum desiccation tolerance; rather there is a minimum level of desiccation tolerance that is required for the development of optimal seed vigour. The use of an in vivo approach in the study of LEA function in cowpea enabled the accurate comparison of two different groups of LEA proteins in developing cowpea seeds under conditions of water stress and in relation to germination and vigour. Both group 1 LEA and group 2 LEA (dehydrin) were shown to increase in concentration in response to water stress. In addition group 1 LEA protein was observed to be relatively abundant in cowpea seeds. A maternal influence on LEA protein gene expression under conditions of water stress, which may induce dehydrin accumulation vii during the earlier stages of seed development, was implied by the observation that dehydrin-like proteins were induced after two weeks of development in cowpea plants subjected to stress during the vegetative phase. In addition, the exogenous application of ABA delayed radicle protrusion; this was associated with a delay in the disappearance of LEA proteins and is suggestive of a relationship between LEA protein accumulation and the acquisition of desiccation tolerance. The study has demonstrated that image analysis can objectively discriminate seed coat colour variation in cowpea. Dark coloured seeds in general performed better than light coloured seeds; however seed coat colour was not always associated with better performance. A newly developed Aging Stress Differential Index (ASDI) has been used in this study to demonstrate a link between seed coat colour and sensitivity to water stress. The ASDI correlated well with the observations relating stress tolerance to stachyose accumulation. The skewed distribution patterns in individual electrical conductivity and the presence of extreme values may have implications with respect to the suitability of using standard statistical analyses which compare mean values to evaluate such data. In addition variation in individual electrical conductivity may also be influenced by cultivar differences and the chemical composition of the seed coat. Therefore associations between seed coat colour and electrical conductivity as a measure of performance should be treated with caution. The AA test does reflect changes in seed vigour, however ranked electrical conductivity values after AA did not consistently reflect differences in seed performance between cultivars and sites, and they did not correlate well with other aspects of performance. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2007.

It has been suggested that lipid peroxidation is involved in the loss of seed vigour, although many attempts to examine the relationships between lipid peroxidation and seed vigour have proved equivocal. Studies were undertaken on seed lots of cabbage and soya bean to find evidence for peroxidation by the analysis of i) total and polar fatty acid levels; ii) lipid hydroperoxides; iii) volatile products produced on heating dry seeds; and iv) volatile products produced on imbibition. The loss of polyunsaturated fatty acids (PUFAs) detected in the dry seeds was clearly related to germinability in both soya bean and cabbage seeds. Furthermore, an increase in hydroperoxides was observed in both seed types. Although the relationship of the level of hydroperoxides to germinability was less clear than for the decline in the level of PUFAs, these results suggested that the loss of PUFAs was possibly due to evidence the peroxidation of the seed obtained from the heating lipid, indirect of the seeds suggesting that hydroperoxide breakdown may be necessary in order that the changes in PUFAs become apparent. In contrast to the poor relationship observed between germinability and hydroperoxide level, a marked relationship between hydroperoxide level and seed moisture content was observed in the cabbage seeds. This may be significant with regard to the observed relationship between storability and seed moisture content, although no such relationship was seen in the soya beans. Certain volatile compounds derived from dry heated seeds were related to seed vigour in both seed types and evidence suggests that the lipid hydroperoxides were the source of these compounds. Although the total volatiles counts evolved from imbibing cabbage seeds showed no quantitative relationship to seed vigour, one peak was noted which was clearly associated with the vigour of these seeds. The variability in the volatiles evolved from soya beans on imbibition, however, precluded the detection of any possible relationship between these and seed vigour. In both seed types, results suggest that the volatiles derived on imbibition were of a different source to those derived on heating. A marked increase in the level of hydroperoxides was observed in whole cabbage seeds and soya bean axes of low vigour over the first hour of imbibition. This may suggest that an exacerbation of damage on imbibition was associated with low vigour seeds. In contrast to this, in the seeds of high vigour, the increase in hydroperoxide levels was markedly less or rapidly reduced, suggesting the possible activity of repair mechanisms. Ferrous ions were shown to invigorate both seed types, particularly cabbage seeds. It is suggested that the invigorating effect of these compounds was due to the facilitation of repair, including hydroperoxide breakdown and the quenching of any free radicals. / Thesis (M.Sc.)-University of Natal, Durban, 1986.

The aim of the study was to determine the effect of day/night temperatures (22/l6°C, 2712l oC and 33/27°C) and phosphorus levels (0, 0.12 and 1.2g per 20 kg soil) on seedling establishment and seed viability during three stages of seed development (15, 22 and 33 days after flowering) for seed of normal and quality protein maize cultivars. Soluble carbohydrate accumulation and mineral element content were determined using environmental scanning electron microscopy (ESEM) in relation to seed phytate levels and seed germination capacity at different stages of development. Leaf emergence rate and plant height during seed development were significantly (P < 0.05) influenced by temperature and phosphorus nutrition. Phosphorus in seed is stored primarily in the form of phytic acid, also known as phytate. Accumulation of phytate takes place during maturation phase of seed development. Phosphorus nutrition and temperature also caused a.significant (P < 0.05) increase in seed germination at all stages of seed development. Furthermore, phosphorus nutrition and temperature influenced occurrence of soluble carbohydrates in seeds. Myo-inositol, the sugar alcohol that forms the basic structure of phytate, was increased by P nutrition and increasing growth temperature. Whereas, QPM maize was generally found to perform poorly than normal maize, with respect to phytate content, seed germination and seedling establishment, both cultivars displayed the same responses to phosphorus nutrition and temperature. In both cultivars, globoids, the sites of phytate synthesis and storage, were found only in the embryonic axis. Subsequently, there were significantly low levels of mineral elements (P, Mg and K) found in the endosperm, compared with embryonic axis. This finding suggested that the embryonic axis plays a major role in seed performance, through its effects emanating from phyate metabolism. Myo-inositol plays a role in membrane biogenesis during stress conditions such as temperature by maintaining the integrity of the cell wall and minimizes the leaching of cations essential during germination. Myo-inositol, although it occurs in small concentrations, could be used to indicate seed quality in maize, because its accumulation was found to be associated with enhanced phyate levels and better seed germination in a wide range of temperatures. Low vigour seeds are associated with high electrolyte leakage during imbibition. Mineral elements form a significant portion of the imbibition leachate, which causes seeds to lose nutrients for early seedling growth. This study provided evidence that phosphorus nutrition can alleviate poor seed vigour of maize by improving phytate levels. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2006.

Young developing fruits of nasturtium (Tropaeolum majus L.) accumulate large deposits of non-fucosylated "storage" XG in periplasmic spaces of cotyledon cells. The only XG that is clearly fucosylated in these fruits Is the structural fraction (approx. 1% total) integrated into growing primary walk. Storage XG can be fucosylated by a nasturtium transferase in vitro, but this does not happen in vivo, even as a transitory signal required for secretion which would subsequently be cleaved to produce mature non-fucosylated storage XG in the periplasmic space. The two fucosylated subunits that are formed in vitro are identical to those found in structural XG in vivo. A block appears to develop in the secretory machinery of young cotyledon cells resulting in extended galactosylation and diversion of XG traffic to the periplasm without fucosylation. The primary walls buried beneath accretions of storage XG eventually swell and lose cohesion, probably because they continue to extend without incorporating components like fucosylated XG that are needed for maintaining wall integrity.

In Scotland, bilberry is well known as a member of the dwarf shrub community on moorlands, which provide valuable habitats for wildlife. Recently it has been envisaged that bilberry populations could play an important role in improving the dwarf shrub vegetation in degraded moorland areas. This study was therefore undertaken to investigate the above-ground growth dynamics of bilberry with emphasis on responses to grazing by sheep and seedling establishment. The leaf production of bilberry was increased by higher nitrogen availability. High nitrogen supply also led to greater activation of dormant buds. Abortion of buds was less when nitrogen was continuously supplied. The reproductive capacity of bilberry was shown to be decreased by herbage removal. In contrast flowering was increased following supply of high nitrogen in glasshouse conditions. Almost all the flowers developed into fruits in the field, but a very low production of fruits was reported from bilberry plants that were grown in the glasshouse. Results of a series of laboratory experiments on seed production, germination, seed storage and evaluation of a moorland seed bank confirmed that the scanty seedling recruitment of bilberry was due to the limited production of 'germinable' seeds in berries and risks imposed by the environment after dispersal particularly when seeds are in the litter layer and/or soil. This could suggest that limited picking of bilberry fruits would not have a detrimental effect on establishment of bilberry populations in moorlands. Therefore, areas could be identified which were suitable for public access and berry exploitation in Scotland. The berry production was estimated approximately to be about 3220-9660 tones per year in this zone.

The present investigation can be divided into two main sections: the first dealing with the post-shedding embryogenesis of Encephalartos natalensis and the second concerned with the cell wall properties of immature and mature embryos of this species. Development of the embryo of E. natalensis from a rudimentary meristematic structure approximately 700 μm in length, extends over six months after the seed is shed from the strobilus. Throughout its development the embryo remains attached to a long suspensor. Differentiation of the shoot meristem flanked by two cotyledonary protuberances occurs over the first two months, during which peripheral tannin channels become apparent. Tannins, apparently elaborated by the endoplasmic reticulum, first accumulate in the large central vacuole and ultimately fill the channel. By the fourth month of development the root meristem is apparent and procambial tissue forming discrete vascular bundles can be discerned in the elongating cotyledons. Between four and six months, mucilage ducts differentiate, and, after six months when the seed becomes germinable, the embryo is characterised by cotyledons far longer than the axis. Shoot and root meristem cells remain ultrastructurally similar throughout embryo ontogeny, containing small vacuoles, many welldifferentiated mitochondria and ER profiles, abundant polysomes, plastids containing small starch deposits and Golgi bodies. Unusually however, Golgi bodies are infrequent in other cells including those elaborating mucilage which is accumulated in distended ER and apparently secreted into the duct lumen directly by ER-derived vesicles. The nonmeristematic cells accumulate massive starch deposits to the exclusion of any protein bodies, and only very sparse lipid, features which are considered in terms of the prolonged period of embryo development and the high atmospheric oxygen content of the Carboniferous Period, when cycads are suggested to have originated. With regard to plant cell walls, the present investigation employed immunofluorescence microscopy and immunocytochemistry to characterise the cell walls of immature and mature embryos of the recalcitrant-seeded E. natalensis to determine wall composition and potential changes with development. These techniques, together with cryo-scanning- and transmissionelectron microscopy (TEM) were used to analyse potential changes in the cell walls of mature embryos upon desiccation. Immature cell walls appeared to be composed of low- and high methyl esterified epitopes of pectin, rhamnogalacturonan-associated arabinan, and the hemicellulose xyloglucan, while partially-esterified epitopes of pectin appear to have a punctuate distribution in the wall. Arabinogalactan protein recognised by the LM2 antibody, along with rhamnogalacturonan-associated galactan and the hemicellulose xylan, were not positively localised using immunological probes, suggesting that the embryo of the current species does not possess these epitopes. Interestingly, mature embryos appeared to be identical to immature ones with respect to the cell wall components investigated, implying that these may not change during the protracted post-shedding embryogeny of this species. Analysis of the monosaccharide composition of the walls by gas liquid chromatography complemented the immuno-labelling work. However, there appeared to be abnormally high levels of glucose (Glc), which may indicate the presence of Glc-rich polymers not accounted for by the antibodies used in the current study. Preliminary Glc-normalised data revealed that there may be considerable quantities of arabinose polymers in the wall comparable to that found in desiccation tolerant plants. Drying appeared to induce some degree of cell wall folding in mature embryos, correlating with their possession of wall plasticisers such as arabinose polymers, but this was limited, due to the abundance of amyloplasts, which filled the cytoplasmic space. From the results of this study, it is proposed that the embryo cell walls of E. natalensis are constitutively prepared for the flexibility required during cell growth and expansion, which may facilitate the observed moderate cell wall folding in mature embryos upon drying. This, together with an abundant supply of amyloplasts in the cytomatrix may provide sufficient mechanical stabilisation during desiccation even though the seeds of this species are highly desiccation sensitive. Overall, this study has been a relatively comprehensive coverage of histological and ultrastructural aspects of embryogenesis in E. natalensis. This work will form a pivotal basis for future studies, which may ultimately lead to the successful germplasm cryopreservation and in vitro production on a commercial scale of these, and other, endangered cycad species. Furthermore, the work on cell walls in this investigation has provided improved comprehension of the responses of seed cell walls to dehydration. / Ph.D. University of KwaZulu-Natal, Durban 2013.

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