Physiological, morphological and molecular acclimation of Populus spp. to high salinity
by Shayla Sharmin
Date of Examination:2022-02-17
Date of issue:2022-03-25
Advisor:Prof. Dr. Andrea Polle
Referee:Prof. Dr. Andrea Polle
Referee:PD Dr. Thomas Teichmann
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EnglishSalinization of terrestrial land is a global environmental problem, causing loss in soil fertility. Cultivation of salt tolerant species counteracts soil degradation and ensures productive utilization of salt-affected lands. But the tolerance mechanisms of salt-tolerant plants are not entirely understood. Salt tolerance of a plant involves several adaptations at the physiological, morphological and molecular levels. Morphological adaptation via increased tissue thickness and succulence is one of the strategies followed by halophytes (salt tolerant plant) under salinity. Succulent tissues possess larger cell with higher water content. However, little is known about morphological acclimation processes to high salinity in non-halophytic plants such as the salt-tolerant Populus euphratica. Under salinity, the gradual buildup of salt ions in the plant cells causes toxicity and leads the plant to death. In addition, salt ions can greatly affect the uptake and supply of macronutrients like potassium, calcium and magnesium. However, it is not completely known how the root to shoot translocation of macronutrients is affected by sodium under salinity. In addition, it would be interesting to check whether the reduction in translocation processes can reduce salt accumulation in leaves under salinity or not. In this thesis, I studied the acclimation and tolerance mechanisms in a salt-tolerant, P. euphratica and in a salt-susceptible poplar species, Populus x canescens. I addressed the following goals: (i) to characterize root morphology and plant gas exchange under salinity and describe the underlying molecular regulation, (ii) to investigate whether root thickening under salinity contributes to enhanced salt tolerance of P. euphratica, and (iii) to dissect the influence of the transpirational pull and salinity on the distribution of nutrients in the plant. To characterize the root morphology of P. euphratica under high salinity, I adapted the plants to increased salinity by gradually increasing NaCl concentrations from 50 mM to 150 mM and investigated the changes in root morphology after short- (2 days) and long-term (12 days) high salt (150 mM NaCl) exposure. Gas exchange was measured during the salt exposure period in salt-stressed plants and controls (grown without NaCl) to observe responses of plant under salinity. The molecular regulation induced by short- and long-term salt exposure was studied by transcriptome analysis of roots and leaves. To investigate the impact of auxin in root morphology under salinity, transgenic P. euphratica plants transformed with auxin-inducible promoter reporter GH3::GUS construct were used. With increasing NaCl concentration in the medium from 50 mM to 150 mM, gas exchange significantly declined in stressed plants. However, the stressed plants showed an improved CO2 assimilation rate after 11 days of exposure to 150 mM NaCl by consuming sub-stomatal CO2 more efficiently. After long-term salt exposure, main roots showed two-times and lateral roots 1.5 times greater diameters than control roots. Longitudinal sections revealed that an increased number of cortex cells was responsible for the increased thickness of main roots under stress. The dry-to-fresh mass ratio in long-term stressed main and lateral roots did not differ from controls indicating no changes in water content in thick roots compared to control thin roots. In contrast, increased dry-to-fresh mass ratio in leaves after short- and long-term salt exposure suggested that water content decreased in leaves under salinity. Na content increased significantly in main roots and leaves, but not in lateral roots, after long-term salt exposure. Among macronutrients, K and Ca decreased significantly in main and lateral roots under salinity, but nutrient levels were maintained in the leaves. The transgenic plants containing GH3::GUS construct did not show strong noticeable changes in the GUS staining in main and lateral roots after long-term salt exposure. This observation suggested that auxin has no apparent role in the alterations of root morphology under salinity. The salt-induced changes in the regulation of genes for cell wall organization and biogenesis were studied by transcriptome analyses. Pectin methylesterase, expansin, expansin-like, cellulose synthase, cellulose synthase-like, xyloglucan endotransglucosylase/hydrolase, fasciclin-like arabinogalactan, MYB and NAC-type transcription factors were the most abundant differentially expressed gene families. The salt-induced regulation of cell wall associated genes was less affected in leaves than in roots suggesting no stimulation for alteration in leaf morphology. The upregulation of genes involved in the synthesis of cell wall polysaccharides and cell wall extension in the main roots after short-term salt exposure indicated that the reactions to modify cell wall were activated. However, most of these genes were downregulated in thick main roots after long-term salt exposure. The increase in lateral root thickness was likely a result of up- and down-regulation of these cell wall associated genes at both times. Therefore, this study shows that P. euphratica adjusted root morphology by increasing root diameter under high salinity. Increased root thickness in response to salinity was likely induced by salt accumulation in the tissue and regulated by cell wall modifying genes to manage ion toxicity through increased volume for salt deposition. To investigate the second goal, I studied the contribution of physiological root plasticity of P. euphratica to cope with saline conditions and explored the underlying transcriptional regulation. To distinguish between Na+ present in roots and newly taken up Na+, 22Na+ was used as a tracer. Both Na+ uptake from and extrusion into the external solution was strongly reduced in salt-acclimated thick roots compared with non-acclimated thin roots. Transcriptome analyses showed high expression of genes required for the control of Na+ levels (Na+/H+ antiporters: SOS1, NHX, NhaD and ATPases) in both salt-acclimated and non-acclimated roots. Significant increases in transcript abundances were found initially (after 2 days of 150 mM NaCl application) for two NHX family members and many genes with potential functions in Ca2+ signaling. But these responses disappeared in thick, salt-acclimated roots and the majority of differentially expressed genes related to Ca2+ signaling were downregulated. Transcriptional upregulation was observed for stress signaling via NADPH oxidases, irrespective of short- or long-term salt exposure. Thick roots showed higher K+ retention under salt stress than thin roots, presumably as the result of transcriptional downregulation of K+ transporters and non-selective cation channels. Thick roots contained elevated P concentrations, which corresponded to enhanced transcript levels of P transporters. In conclusion, acclimated thick roots have a high ability to control Na+ levels without additional transcriptional activation of the SOS pathway. They are relatively resistant toward changes in salt levels and maintain a favorable nutrient balance. Water flow through the xylem is driven by transpiration and this is thought to be the major driving force for the translocation process. To investigate the third goal, I used the salt-sensitive hybrid poplar, Populus × canescens. The phytohormone Abscisic acid (ABA) is known to regulate stomatal openings and consequently affects the transpiration rate. To differentiate between the effects of transpiration pull from those of salt, we compared salt-stressed, ABA treated, and combined salt- and ABA treated poplars (P. × canescens) with untreated controls. The root content of macronutrients like K+, Ca2+ and Mg2+ was reduced in response to salinity. Decreased Ca2+ levels in roots corresponded to decreased Ca2+ levels in leaves. The same was observed for Mg2+ levels in roots and leaves. In contrast, K+ levels in leaves increased under salt stress, although root K+ levels were reduced. However, reduced transpiration in response to salinity did not decrease Na+ accumulation in the leaves. During ABA treatment, leaf Ca2+ and leaf Mg2+ levels decreased comparably to the salt treatment, while leaf K+ level was unaffected, although stomatal conductance was reduced by salt or ABA treatment. Thus, these results suggest that Ca2+ and Mg2+ levels in leaves were predominantly affected by the transpirational pull, while loading and retention of K+ in leaves are enhanced in response to salt stress, but independently of the transpirational pull. In summary, the salt-tolerant poplar P. euphratica employs a morphological adaptation mechanism in response to high salinity by developing root thickening. Increased root thickness in P. euphratica is induced by salt. An increase in the number of cortex cells which leads to enlarged diameter of first order main roots in response to high salinity is likely a strategy to control ion toxicity via increasing the volume for salt deposition. Moreover, salt-induced thick roots contribute to the outstanding salt tolerance of P. euphratica by controlling Na+ influx from and efflux into the outside environment as well as enhancing K+ retention ability in response to high salinity. Besides, this species is able to maintain the levels of macronutrients K+, Ca2+ and Mg2+ in the leaves, even though root contents of K+ and Ca2+ is negatively affected due to salinity. The salt-induced thick roots might be an evolutionary adaptation of P. euphratica to salinity, which is associated with constant activation rather than over-expression of stress regulatory pathways. Thus, P. euphratica has an improved strategy to maintain a favourable nutrient balance, to control ion toxicity and to continue gas exchange efficiently under high salinity.
Keywords: Populus; Salinity; Morphology; Root; NaCl