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Tissue-specific reactive oxygen species signalling and ionic homeostasis in Chenopodium quinoa and Spinacia oleracea in the context of salinity stress tolerance


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Tanveer, M ORCID: 0000-0003-4560-6439 2020 , 'Tissue-specific reactive oxygen species signalling and ionic homeostasis in Chenopodium quinoa and Spinacia oleracea in the context of salinity stress tolerance', PhD thesis, University of Tasmania.

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Alongside with the drought, soil salinity is one the major environmental constraints that reduces crop growth and yield worldwide. According to recent reports, salinity stress is costing agricultural sector over US $27.3 billion per annum in lost revenues; which also aggravates the global food security. Improving salinity tolerance is a challenging task that requires understanding key physiological traits in naturally salt tolerant plant species. Halophytes are salt loving plants and they have very diverse array of physiological and biochemical mechanisms to encounter salinity stress. Therefore, understanding the fine-print of salinity-induced constraints on plant growth and/or salinity tolerance mechanisms in salt-tolerant halophytes is of a key importance for enhancing salinity stress tolerance in salt sensitive crop plants.
The extent of salinity tolerance depends considerably on crop species and families. Thus, comparing different plant species from the same family or genus can provide much better understanding of physiological mechanisms conferring differential salinity tolerance. In this work, we have selected Chenopodiaceae family, one of most important subfamilies of Amaranthaceae family that contains numerous plant species, including halophtytic (e.g. Chenopodium quinoa, Chenopodium album, Atriplex lentiformis) and glycophytic species (e.g. Spinacia oleracea (spinach). Amongst different plant species, we have selected Chenopodium quinoa (quinoa hereafter) as a dicotyledonous halophytic plant species and Spinacia oleracea (spinach hereafter) as glycophytic plants species. Some previous and basic reports showed that Chenopodiaceae family showed a considerable intra- and inter-specific variation at the whole plant level under saline conditions. However, to the best of our knowledge, no much specific details on the cellular mechanisms conferring this intra- and inter-specific variability in the context of salinity tolerance are available in the literature.
Salinity stress is very complex abiotic stress that induces cytosolic toxicity and oxidative damage by causing ROS production. Under saline conditions, the production of different ROS such as hydrogen peroxide (H\(_2\)O\(_2\)), superoxide radical (O\(_2\)\(^-\)) and hydroxyl radical (•OH) at different sites in cells causes significant damages to nucleic acids, proteins, and lipids. Elevated ROS levels also cause major disturbance to plant ionic homeostasis. At the same time, at low concentrations ROS can act as signalling molecules to control various physiological processes such as cell growth, pollen development, hormonal control, stress signalling and transduction, and ion transport across the plasma membrane.
Until now very few studies have been published showing the role of different types of ROS in regulating ion transport at tissue levels. Therefore, in order to understand above tissue specific and intra- and inter-specific variability in salinity tolerance, set of physiological, electrophysiological and confocal imaging experiments were conducted to answer some of these specific questions:
I. How does K\(^+\) retention pattern (a key determinant of the salinity tissue tolerance mechanism) in different tissues (root and leaf mesophyll) differ between halophyte and glycophyte species?
II. Can plants (especially halophytic plants) employ other ROS such as •OH and O\(_2\)\(^-\) to shape Ca\(^{2+}\) flux signatures?
III. Is there any specific Ca\(^{2+}\) or ROS signatures involved in early salt sensing in halophytes?
IV. How do halophytes avoid Na\(^+\) cytosolic toxicity and enhance K\(^+\) retention ability?
V. How can halophytes retain more K\(^+\)? Do they spend much energy (H\(^+\)-ATPase activity) to retain K\(^+\) as compared with spinach?
VI. What could be possible players behind reduced K\(^+\)-efflux from leaf mesophyll and roots in quinoa in relation to acclimation?
This work showed that salinity application arrested plant growth in a highly tissue- and treatment-specific manner and was more severe in glycophytic spinach plants however quinoa was able to withstand salinity stress and produced relative higher plant biomass even at sea level saline conditions (500mM NaCl). Analysis of shoot and xylem sap Na\(^+\) and K\(^+\) contents have revealed the key factor determining differential salinity tolerance between quinoa and spinach species was shoot K\(^+\) (not Na\(^+\)) content and kinetics of xylem ion loading suggested that quinoa species actively load and used Na\(^+\) for osmotic adjustment in shoot to avoid energy expensive synthesis of organic osmolytes. To further gain insight into such whole-plant observations, kinetics of K\(^+\), H\(^+\), Ca\(^{2+}\) flux responses from leaf mesophyll in were measured using non-invasive ion flux measuring MIFE technique in response to salinity stress and H\(_2\)O\(_2\) stress. Moreover, laser microscope confocal imaging technique was used to measure the cytosolic and vacuolar intensities of the fluorescent signals of K\(^+\), Na\(^+\) and Ca\(^{2+}\) from different root zones in response to salinity stress and H\(_2\)O\(_2\)2 stress.
It was also observed that mesophyll cells in glycophytic spinach lost 2 to 6-fold more K\(^+\) compared with its halophytic quinoa counterpart. Treatment with NaCl resulted in significant increase in a transient H\(^+\)-efflux in the leaf mesophyll in spinach, suggesting that spinach spent more ATP to up-regulate H\(^+\)-ATPase activity while quinoa avoid this mechanism to use same energy in defence system, consistent with recently suggested concept of the 'metabolic switch‘.
Among root zones, NaCl- and ROS- induced K\(^+\)-efflux was more pronounced in the root apex while mature zone showed relatively higher K\(^+\) retention, especially in quinoa. This differential sensitivity between different root zones was specifically originated from a 10-folds difference in K\(^+\)-efflux between the mature zone and the apical region (much poorer in the root apex) of the root. Three factors behind this poor K\(^+\) retention ability were: (1) an intrinsically lower H\(^+\)-ATPase activity in the root apex; (2) greater salt-induced membrane depolarization and (3) a higher ROS production under NaCl and a larger density of ROS-activated cation currents in root. Moreover ROS (H\(_2\)O\(_2\)) production was increased with time in all root zones in both species and accompanied with cytosolic Ca\(^{2+}\) elevation in quinoa, suggesting that quinoa (halophytic species) used ROS as a signalling moiety in stress adaptation. Elevation in cytosolic Ca\(^{2+}\) reduced cytosolic Na\(^+\), possibly by SOS1 pathway. Both species showed tremendous K\(^+\)-efflux in response to •OH and O\(_2\)\(^-\) radical but Ca\(^{2+}\)\) flux patterns revealed different results. In response to O\(_2\)\(^-\), a net Ca\(^{2+}\)-efflux was observed while in response to •OH, a net Ca\(^{2+}\)-influx was noted, suggesting halophytes may use ROS specific Ca\(^{2+}\) signatures to activate stress adaptation process. Moreover, halophytes (or at least quinoa) may use Ca\(^{2+}\)-efflux system to restore basal cytosolic Ca\(^{2+}\) level upon O\(_2\)\(^-\) treatment. Under long term salinity conditions, quinoa grown under higher NaCl level (300mM NaCl) showed much reduced responses to external H\(_2\)O\(_2\), suggesting desensitization of K\(^+\)-permeable ion channels to ROS. Moreover, quinoa showed strongest Ca\(^{2+}\) flux response to H\(_2\)O\(_2\) during acclimation, suggesting the important role of ROS-induced cytosolic Ca\(^{2+}\) elevation in stress signalling and adaptive cascade. Spinach was less efficient in doing so, thus showed massive K\(^+\)-efflux and reduced K\(^+\) retention ability.
In conclusion, results from current work showed that NaCl and ROS stress induced massive K\(^+\)\(^+\) loss. This loss was highly tissue-specific and more pronounced in glycophytic spinach plants. Several mechanisms were highlighted in this work behind such response in quinoa (i) higher vacuolar Na\(^+\) sequestration ability in roots, thus reduced Na\(^+\) cytosolic toxicity. (ii) It can employ H\(_2\)O\(_2\) to activate stress signalling cascades, (iii) higher K\(^+\) retention in leaf mesophyll was strongly correlated with plant biomass, SPAD and stomatal conductance, (iv) The results obtained indicated a major difference in distribution of energy between "metabolic" and "defence" pools. Future research should focus on the difference and mechanisms of the regulation of H\(^+\)-ATPase activity between halophytes and glycophytes, and the role of ROS in this process, (V) during acclimation, quinoa showed relatively more Na\(^+\) accumulation (based on coroNa green fluorescence signal) than spinach. Thus, it will be of a significant importance to reveal the contribution of numerous components (e.g. tonoplastic NHX, or FV/SV channels) towards vacuolar Na\(^+\) sequestration. One of most important discoveries in this study was the identification of the electrophysiological role of ROS specific Ca\(^{2+}\) signatures in the regulation of K\(^+\) homeostasis and stress adaptation. The use of some techniques such patch clamp and CRISPR/CAS 9 will help to reveal the molecular identity of different ion transporters in quinoa and spinach in response to different ROS especially to •OH and O\(_2\)\(^-\) radical at transcriptional and post transcriptional levels, to further understand the molecular basis of the observed physiological salinity tolerance mechanisms.

Item Type: Thesis - PhD
Authors/Creators:Tanveer, M
Keywords: Salinity tolerance; ROS signaling; Ionic Homeostasis; Halophytes; Tissue specificity
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