Comparative analysis of molecular and physiological responses of two canola genotypes to drought stress

>Magister Scientiae - MScFood security has always been one of the priority concerns in Africa, and it is mostly threatened by drought stress due to climate change. Drought-induced stress is one of the serious limiting factors of plant production, and it is known to impose oxidative stress as a consequence of excessive reactive oxygen species (ROS) accumulation that lead to lipid peroxidation, which is manifested as increased cell death. Hence, this study investigated the influence of drought stress on two contrasting canola genotypes (Agamax and Garnet), by monitoring their physiological and molecular changes. The results showed that the plant growth and biomass of both genotypes were significantly affected by drought stress as a consequence of excessive ROS accumulation (manifested as H₂O₂ and OH· content). However, under drought stress conditions, the reduction in biomass and shoot length was more pronounced in the Garnet genotype when compared to that of the Agamax genotype. This was further supported by the increase in lipid peroxidation and cell death, which were shown to be significantly higher in the Garnet genotype when compared to the Agamax genotype under drought stress. ROS scavenging ability which prevents oxidative stress and ultimately ROS-induced cellular damage. Hence, given the higher levels of antioxidant activity coupled with the reduction in ROS accumulation that was observed in the Agamax genotype, we suggest that the Agamax genotype might be slightly less susceptible to drought stress, when compared to the Garnet genotype. Furthermore, understanding the proteomic responses of these two contrasting genotypes that showed a marked difference in response to drought stress might help in unlocking complex biological networks of proteins underlying drought stress tolerance. Hence we use two-dimensional (2D) gel electrophoresis coupled with Matrix assisted laser desorption/ionisation-time of flight/time of flight tandem mass spectrometry (MALDI TOFTOF MS) analysis for this part of the study, in order to detect and analyze those differentially expressed proteins or proteins whose abundance levels were influenced as a consequence of drought stress. To gain additional insight into the leaf proteomes of the two canola genotypes, a protamine sulphate precipitation (PSP) method was used to remove RuBisCo and confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. A total of 55 well resolved protein spots were selected for mass spectrometry analysis of which 31 (56%) were positively identified using the selective criteria analysis (SCA). All positively identified proteins were then classified into functional categories including protein folding (3%), photosynthetic (29%), detoxification and protection (20%), and energy related proteins whereas 16% could not be classified into any functional category. Apart from spot 32 (Fe superoxide dismutase) and spot 34 (chloroplast beta-carbonic anhydrase), no further significant difference in protein expression/abundance was observed for all the identified proteins for both genotypes in response to drought stress. Both proteins (spots 32 and 34) have been shown to contain antioxidant activity properties which suggest that they might play a crucial role in improving drought stress tolerance in canola plants

The role of p-coumaric acid on physiological and biochemical response of chia seedling under salt stress

Philosophiae Doctor - PhDThe role of phenolic acids in mitigating salt stress tolerance have been well documented. However, there are contradicting reports on the effect of exogenously applied phenolic acids on the growth and development of various plants species. A general trend was observed where phenolic acids were shown to inhibit plant growth and development, with the exception of a few documented cases. One of these such cases is presented in this thesis. This study investigates the role of exogenously applied p-coumaric acid (p-CA) on physio-biochemical and molecular responses of chia seedlings under salt stress. This study is divided into three parts. Part one (Chapter 3) focuses on the impact of exogenous p-coumaric acid on the growth and development of chia seedlings. In this section, chia seedlings were supplemented with exogenous p-CA and the various biochemical and plant growth parameters were measured. The results showed that exogenous p-CA enhanced the growth of chia seedlings. An increase in chlorophyll, proline and superoxide oxide contents were also observed in the p-CA treatment relative to the control. We suggested that the increase in chia seedling growth could possibly be via the activation of reactive oxygen species-signalling pathway involving O2− under the control of proline accumulation (Chapter 3). Given the allopathy, nature of p-coumaric acid it is noteworthy that the response observed in this study may be species dependent, as contrasting responses have been reported in other plant species. Part two (Chapter 4) of this study investigates the influence of piperonylic acid (an inhibitor of endogenous p-coumaric acid) on the growth and development of chia seedlings. In trying to illustrate whether p-CA does play a regulatory role in enhancing pseudocereal plant growth, we treated chia seedlings with the irreversible inhibitor of C4H enzyme, to inhibit the biosynthesis of endogenous p-CA. In this section, chia seedlings were treated with piperonylic acid and changes in plant growth, ROS-induced oxidative damage, p-CA content and antioxidant capacity was monitored. Inhibition of endogenous p-CA restricted chia seedling growth by enhancing ROS-induced oxidative damage as seen for increased levels of superoxide, hydrogen peroxide and the extent of lipid peroxidation. Although an increase in antioxidant activity was observed in response to piperonylic acid, this increase was not sufficient to scavenge the ROS molecules to prevent oxidative damage and ultimate cellular death manifested as reduced plant growth. The results presented in this section support our hypothesis that p-CA play an important regulatory role in enhancing chia seedling growth and development as shown in Chapter 3. Part three (Chapter 5) seeks to identify and functionally characterise p-coumaric acid induced putative protein biomarkers under salt stress conditions in chia seedlings. Previous studies have shown that p-CA reversing the negative effect caused by NaCl-induced salt stress. While these studies were able to demonstrate the involvement of p-CA in promoting plant growth under salt stress conditions, they focussed primarily on the physiological aspect, which lacks in-depth biochemical and molecular analysis (ionomic and proteomic data) which could help in detecting the genes/proteins involved in salt stress tolerance mechanisms. A comparative ionomics and proteomic study was conducted, with the aim of elucidating the pivotal roles of essential macro elements and/or key protein markers involved in p-CA induced salt stress tolerance in chia seedlings. With the exception of Na, all the other macro elements were decreased in the salt treatment. Contrary to what was observed for the salt treatment most of the macro elements were increased in the p-CA treatment. However, the addition of exogenous p-CA to salt stressed seedlings showed an increase in essential macro elements such as Mg and Ca which have been shown to play a key role in plant growth and development. In the proteomic analysis we identified 907 proteins associated with shoots across all treatments. Interestingly, only eight proteins were conserved amongst all treatments. A total of 79 proteins were unique to the p-CA, 26 to the combination treatment (NaCl + p-CA) and only two proteins were unique to the salt stress treatment. The unique proteins identified in each of the treatments were functionally characterised to various subcellular compartments and biological processes. Most of the positively identified proteins were localised to the chloroplast and plays key roles in photosynthesis, transportation, stress responses and signal transduction pathways. Moreover, the protein biomarkers identified in this study (especially in the p-CA treatment) are putative candidates for genetic improvement of salt stress tolerance in plants

Piperonylic acid alters growth, mineral content accumulation and reactive oxygen species-scavenging capacity in chia seedlings

p-Coumaric acid synthesis in plants involves the conversion of phenylalanine to trans-cinnamic acid via phenylalanine ammonia-lyase (PAL), which is then hydroxylated at the para-position under the action of trans-cinnamic acid 4-hydroxylase. Alternatively, some PAL enzymes accept tyrosine as an alternative substrate and convert tyrosine directly to p-coumaric acid without the intermediary of trans-cinnamic acid. In recent years, the contrasting roles of p-coumaric acid in regulating the growth and development of plants have been well-documented. To understand the contribution of trans-cinnamic acid 4-hydroxylase activity in p-coumaric acid-mediated plant growth, mineral content accumulation and the regulation of reactive oxygen species (ROS), we investigated the effect of piperonylic acid (a trans-cinnamic acid 4-hydroxylase inhibitor) on plant growth, essential macroelements, osmolyte content, ROS-induced oxidative damage, antioxidant enzyme activities and phytohormone levels in chia seedlings. Piperonylic acid restricted chia seedling growth by reducing shoot length, fresh weight, leaf area measurements and p-coumaric acid content. Apart from sodium, piperonylic acid signifcantly reduced the accumulation of other essential macroelements (such as K, P, Ca and Mg) relative to the untreated control. Enhanced proline, superoxide, hydrogen peroxide and malondialdehyde contents were observed. The inhibition of trans-cinnamic acid 4-hydroxylase activity signifcantly increased the enzymatic activities of ROS-scavenging enzymes such as superoxide dismutase, ascorbate peroxidase, catalase and guaiacol peroxidase. In addition, piperonylic acid caused a reduction in indole-3-acetic acid and salicylic acid content. In conclusion, the reduction in chia seedling growth in response to piperonylic acid may be attributed to a reduction in p-coumaric acid content coupled with elevated ROS-induced oxidative damage, and restricted mineral and phytohormone (indole-3-acetic acid and salicylic) levels

Exogenous p-coumaric acid improves salvia hispanica l. Seedling shoot growth

p-Coumaric acid (p-CA) belongs to a family of natural esters of hydroxycinnamic acid compounds that have been shown to modulate plant growth and metabolism. In this study, we investigated the effect of exogenous p-CA on plant growth, reactive oxygen species (ROS)-induced oxidative damage, photosynthetic metabolism, osmolyte content and changes in superoxide dismutase (SOD) enzymatic activity

Biocontrol Potential of Bacillus subtilis and Bacillus tequilensis against Four Fusarium Species

The use of biological control agents as opposed to synthetic agrochemicals to control plant pathogens has gained momentum, considering their numerous advantages. The aim of this study is to investigate the biocontrol potential of plant bacterial isolates against Fusarium oxysporum, Fusarium proliferatum, Fusarium culmorum, and Fusarium verticillioides. Isolation, identification, characterization, and in vitro biocontrol antagonistic assays of these isolates against Fusarium species were carried out following standard protocols. The bacterial endophytes were isolated from Glycine max. L leaves (B1), Brassica napus. L seeds (B2), Vigna unguiculata seeds (B3), and Glycine max. L seeds (B4). The bacterial isolates were identified using 16S rRNA PCR sequencing. A phylogenetic analysis shows that the bacterial isolates are closely related to Bacillus subtilis (B1) and Bacillus tequilensis (B2–B4), with an identity score above 98%. All the bacterial isolates produced a significant amount (p < 0.05) of indole acetic acid (IAA), siderophores, and protease activity. In vitro antagonistic assays of these isolates show a significant (p F. culmorum > F. verticillioides > F. oxysporum, compared to the control. The results suggest that these bacterial isolates are good biocontrol candidates against the selected Fusarium specie

Exogenous p-Coumaric Acid Improves Salvia hispanica L. Seedling Shoot Growth

p-Coumaric acid (p-CA) belongs to a family of natural esters of hydroxycinnamic acid compounds that have been shown to modulate plant growth and metabolism. In this study, we investigated the effect of exogenous p-CA on plant growth, reactive oxygen species (ROS)-induced oxidative damage, photosynthetic metabolism, osmolyte content and changes in superoxide dismutase (SOD) enzymatic activity. Exogenous p-CA improved Salvia hispanica (chia) growth by significantly enhancing shoot length, fresh and dry weights coupled with augmented levels of total chlorophyll and carotenoid contents. Furthermore, p-CA also triggered an induction in proline, glycine betaine (GB) and superoxide (O2∙&minus;) levels while no changes were observed for hydrogen peroxide (H2O2) and downstream malondialdehyde (MDA) content. Also, no change in SOD activity was observed in the p-CA treatment relative to the control. Therefore, the results suggest that exogenous p-CA improves chia seedling growth possibly via activation of a ROS-signalling pathway involving O2∙&minus; under the control of proline accumulation

Combined Application of Filter Cake and Macadamia Husk Compost Affects Soil Fertility and Plant Mineral Content of Orange-Fleshed Sweet Potatoes

A greenhouse pot experiment was conducted to investigate the influence of the combined application of filter cake and macadamia husk compost (FC+MHC) on the soil fertility and dry matter partitioning of Beauregard and 199062.1 cultivars of orange-fleshed sweet potato. The effects of the two organic wastes on the mineral nutrients in the leaves and the storage roots of the 199062.1 cultivar were also investigated. In addition to FC+MHC, four other treatments—filter cake only (FC), macadamia husk compost only (MHC), inorganic fertilizer only (IF), a combination of filter cake and inorganic fertilizer (FC+IF), a combination of macadamia husk compost and inorganic fertilizer (MHC+IF), and a control (CONT)—were included in the investigation for the purpose of comparison. To achieve this, 1 kg of compost was homogenized with 20 kg of soil and filled into graduated 25 L buckets. The experimental design was completely randomized. The plants were grown for 4 months. The results indicated that all treatments altered the soil fertility positively. There were indications that both filter cake and macadamia husk compost inhibited the absorption of iron (Fe), copper (Cu), and aluminum (Al). Also, zinc (Zn) and phosphorus (P) deficiencies in the initial soil were corrected after the application of the organic wastes. In terms of yield, FC+MHC was better than all other treatments. The outcome of this study will no doubt greatly benefit the resource-poor farmers of Northern KwaZulu-Natal who are involved in the production of orange-fleshed sweet potatoes

Biocontrol Potential of Bacillus subtilis and Bacillus tequilensis against Four Fusarium Species

The use of biological control agents as opposed to synthetic agrochemicals to control plant pathogens has gained momentum, considering their numerous advantages. The aim of this study is to investigate the biocontrol potential of plant bacterial isolates against Fusarium oxysporum, Fusarium proliferatum, Fusarium culmorum, and Fusarium verticillioides. Isolation, identification, characterization, and in vitro biocontrol antagonistic assays of these isolates against Fusarium species were carried out following standard protocols. The bacterial endophytes were isolated from Glycine max. L leaves (B1), Brassica napus. L seeds (B2), Vigna unguiculata seeds (B3), and Glycine max. L seeds (B4). The bacterial isolates were identified using 16S rRNA PCR sequencing. A phylogenetic analysis shows that the bacterial isolates are closely related to Bacillus subtilis (B1) and Bacillus tequilensis (B2&ndash;B4), with an identity score above 98%. All the bacterial isolates produced a significant amount (p &lt; 0.05) of indole acetic acid (IAA), siderophores, and protease activity. In vitro antagonistic assays of these isolates show a significant (p &lt; 0.05) growth inhibition of the fungal mycelia in the following order: F. proliferatum &gt; F. culmorum &gt; F. verticillioides &gt; F. oxysporum, compared to the control. The results suggest that these bacterial isolates are good biocontrol candidates against the selected Fusarium species

Methylglyoxal improves zirconium stress tolerance in Raphanus sativus seedling shoots by restricting zirconium uptake, reducing oxidative damage, and upregulating glyoxalase I

Abstract Raphanus sativus also known as radish is a member of the Brassicaceae family which is mainly cultivated for human and animal consumption. R. sativus growth and development is negatively affected by heavy metal stress. The metal zirconium (Zr) have toxic effects on plants and tolerance to the metal could be regulated by known signaling molecules such as methylglyoxal (MG). Therefore, in this study we investigated whether the application of the signaling molecule MG could improve the Zr tolerance of R. sativus at the seedling stage. We measured the following: seed germination, dry weight, cotyledon abscission (%), cell viability, chlorophyll content, malondialdehyde (MDA) content, conjugated diene (CD) content, hydrogen peroxide (H2O2) content, superoxide (O2 •− ) content, MG content, hydroxyl radical (·OH) concentration, ascorbate peroxidase (APX) activity, superoxide dismutase (SOD) activity, glyoxalase I (Gly I) activity, Zr content and translocation factor. Under Zr stress, exogenous MG increased the seed germination percentage, shoot dry weight, cotyledon abscission, cell viability and chlorophyll content. Exogenous MG also led to a decrease in MDA, CD, H2O2, O2 •− , MG and ·OH, under Zr stress in the shoots. Furthermore, MG application led to an increase in the enzymatic activities of APX, SOD and Gly I as well as in the complete blocking of cotyledon abscission under Zr stress. MG treatment decreased the uptake of Zr in the roots and shoots. Zr treatment decreased the translocation factor of the Zr from roots to shoots and MG treatment decreased the translocation factor of Zr even more significantly compared to the Zr only treatment. Our results indicate that MG treatment can improve R. sativus seedling growth under Zr stress through the activation of antioxidant enzymes and Gly I through reactive oxygen species and MG signaling, inhibiting cotyledon abscission through H2O2 signaling and immobilizing Zr translocation