Abstract
Plant roots acquire nutrients and water from the surrounding soil and transport them upwards to the shoots. Before reaching the central vasculature of the root, nutrients and water radially cross the layers of epidermis, cortex and endodermis. The endodermis surrounds the central root cylinder, i.e. the stele, with the vascular tissues and forms physical, hydrophobic barriers including Casparian strips and suberin lamellae. These barriers make the endodermis act as a selective barrier to control the transport of nutrients from the soil to the vasculature and to prevent backflow of nutrients from the stele into the cortex. The endodermal barriers, especially suberin, are highly influenced by environmental conditions, including nutrient supply. However, the functional effects that the root barriers have on nutrient acquisition are still elusive.
The objective of this Ph.D. thesis was to investigate how barley (Hordeum vulgare) roots change their suberization in response to manganese (Mn) availability and how this affects Mn acquisition. An additional objective was to study the consequences of the Mn-induced changes in suberization for radial gradients in other plant nutrients across the root.
To analyze the functional effects of root barriers on nutrient acquisition, a method based on laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) for multi-element bio-imaging in roots of Arabidopsis (Arabidopsis thaliana L.) was developed. A major challenge was that the sample preparation in conventional protocols of microscopy causes leakage and/or rearrangement of diffusible ions during sample dehydration. A protocol that preserves ions in their native state by encapsulating fresh roots in paraffin before embedding them into a medium for cryostat sectioning was developed. This method was tested on a mutant of Arabidopsis unable to synthesize the metal chelator nicotianamine. The obtained results showed that the developed method allowed detailed studies of ion distribution in plant roots. To further explore tissue-specific ionomic analysis of barley roots, a method combining laser capture microdissection (LCM) with ICP-MS was developed. Root tissue samples were cut and collected by LCM followed by digestion and analysis by ICP-MS. With this method, the concentrations of magnesium, phosphorus, potassium and manganese in the stele and in the cortex of barley roots were studied, showing that these elements had substantially higher concentration in the stele than in the cortex, with the steepest gradient obtained for manganese. The developed analytical techniques were complemented by development of suberin staining protocols able to visualize changes in suberin across whole roots.
The obtained results show that suberization of the endodermis in the main axis of barley roots decreased in response to mild Mn deficiency but increased under strong Mn deficiency. Resupply of Mn to deficient plants reverted the suberin pattern to that of the roots of control plants. Multi-elemental bio-imaging and xylem analyses revealed that the reduced suberization in mildly Mn deficient plants promoted radial Mn transport across the endodermis at a greater distance from the root tip. Less suberin also favored the accumulation of calcium and sodium in the stele, but caused leakage of potassium from the stele to the cortex. On the contrary, with enhanced suberization, the accumulation of calcium and sodium decreased, whereas that of potassium increased. It is concluded that the suberization under Mn deficiency is not a linear process and that changes in suberization affect the ion homeostasis in barley roots.
The objective of this Ph.D. thesis was to investigate how barley (Hordeum vulgare) roots change their suberization in response to manganese (Mn) availability and how this affects Mn acquisition. An additional objective was to study the consequences of the Mn-induced changes in suberization for radial gradients in other plant nutrients across the root.
To analyze the functional effects of root barriers on nutrient acquisition, a method based on laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) for multi-element bio-imaging in roots of Arabidopsis (Arabidopsis thaliana L.) was developed. A major challenge was that the sample preparation in conventional protocols of microscopy causes leakage and/or rearrangement of diffusible ions during sample dehydration. A protocol that preserves ions in their native state by encapsulating fresh roots in paraffin before embedding them into a medium for cryostat sectioning was developed. This method was tested on a mutant of Arabidopsis unable to synthesize the metal chelator nicotianamine. The obtained results showed that the developed method allowed detailed studies of ion distribution in plant roots. To further explore tissue-specific ionomic analysis of barley roots, a method combining laser capture microdissection (LCM) with ICP-MS was developed. Root tissue samples were cut and collected by LCM followed by digestion and analysis by ICP-MS. With this method, the concentrations of magnesium, phosphorus, potassium and manganese in the stele and in the cortex of barley roots were studied, showing that these elements had substantially higher concentration in the stele than in the cortex, with the steepest gradient obtained for manganese. The developed analytical techniques were complemented by development of suberin staining protocols able to visualize changes in suberin across whole roots.
The obtained results show that suberization of the endodermis in the main axis of barley roots decreased in response to mild Mn deficiency but increased under strong Mn deficiency. Resupply of Mn to deficient plants reverted the suberin pattern to that of the roots of control plants. Multi-elemental bio-imaging and xylem analyses revealed that the reduced suberization in mildly Mn deficient plants promoted radial Mn transport across the endodermis at a greater distance from the root tip. Less suberin also favored the accumulation of calcium and sodium in the stele, but caused leakage of potassium from the stele to the cortex. On the contrary, with enhanced suberization, the accumulation of calcium and sodium decreased, whereas that of potassium increased. It is concluded that the suberization under Mn deficiency is not a linear process and that changes in suberization affect the ion homeostasis in barley roots.
| Original language | English |
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| Publisher | Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen |
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| Publication status | Published - 2019 |