Absorption and Translocation of Zn Foliar Fertilisers
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Absorption and Translocation of Zn Foliar Fertilisers Cui Li Master of Science A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2018 School of Agriculture and Food Sciences Abstract Crop Zn deficiency is associated with low Zn availability in soils and is a widespread problem, also resulting in human Zn deficiency. Foliar Zn fertilisation has been shown to be an efficient approach for overcoming this problem. However, the mechanisms by which Zn moves across the leaf surface (penetration across the cuticle and epidermal cells and into the underlying tissues) and is subsequently translocated remain unclear, with this being the main focus of the present research. Specifically, using ZnSO4 and nano-ZnO, the roles of leaf cuticle, trichomes, and stomata in foliar Zn absorption were examined, with the distribution and the speciation of the absorbed Zn in the leaves determined. In addition, the influence of plant Zn status on foliar Zn absorption was also examined. The first experiment aimed to obtain a general understanding of the effects of leaf properties on the foliar absorption of Zn. Methyl jasmonate (MeJA) (0, 0.1, 0.5, 1, and 2.5 mM) was applied to leaves of soybean (Glycine max), sunflower (Helianthus annuus), and tomato (Solanum lycopersicum) to alter leaf properties. It was found for all three plant species that treating of MeJA caused substantial increases (1.3–3.5-times higher) in leaf trichome density, and the hydrophobicity of the adaxial leaf surface. The changes in stomatal density and leaf cuticle thickness varied with plant species and MeJA concentration. Based upon these results, the second experiment used 0 (control) and 1 mM MeJA treatments to examine the effects of changes in leaf properties on the absorption of foliar-applied Zn, Mn, and Fe, using synchrotron-based X-ray fluorescence microscopy (µ-XRF) to examine the distribution of elements in situ within hydrated leaves. Interestingly, foliar absorption of Zn, Mn, and Fe increased up to 3- to 5-fold in sunflower leaves treated with 1 mM MeJA, but decreased by 0.5- to 0.9-fold in leaves of tomato treated with 1 mM MeJA. It was found that these changes were related to the thickness of the cuticle and epidermal cell wall, suggesting that the cuticle is important for absorption of foliar-applied nutrients. However, subsequent translocation of the absorbed Zn, Mn, and Fe within the leaf tissues was limited (moving < 1.3 mm in 6 h) irrespective of the plant species and treatment. Of the three nutrients, Zn was translocated a greater distance within the vein than in the interveinal tissues (being translocated ca. two-fold further). The third experiment gave specific consideration to the role of trichomes in foliar Zn absorption. Using µ-XRF for the in situ analyses of nutrient distribution in leaves of soybean and tomato, it was found that upon the foliar-application of ZnSO4, Zn accumulated in some 1 non-glandular trichomes of soybean, but not in any trichomes of tomato. However, examining foliar Zn absorption in soybean near-isogenic lines (NILs) differing 10-fold in trichome density found that foliar Zn absorption was not related to leaf trichome density. Therefore, it is suggested that, for soybean and tomato, trichomes are not an important pathway for the movement of foliar-applied Zn across the leaf surface. However, as trichomes are highly diverse, depending upon the plant species and their properties, trichomes could potentially be important for foliar Zn absorption. Next, the importance of the cuticle, stomata, and trichomes in foliar Zn absorption was compared in sunflower – a species in which trichomes are known to play an important role in metal sequestration when supplied in the rooting environment. Analyses of intact hydrated leaves using µ-XRF showed that foliar-applied Zn accumulated rapidly (≤ 15 min) within non-glandular trichomes (NGTs), with the NGTs having the highest Zn concentrations within the leaf tissue. Examining cross sections of the leaf, it was confirmed that the NGTs of sunflower are indeed important for accumulation of the foliar applied Zn. Interestingly, Zn was found to accumulate rapidly in trichomes on both the adaxial and abaxial leaf surfaces for sunflower leaves, despite the Zn being only applied to the adaxial surface, with this translocation seemingly due to transport through bundle sheath extensions. For sunflower, not only was Zn found to accumulate rapidly in the NGTs, but using nanoscale secondary ion mass spectrometry (NanoSIMS), it was found that the cuticle also has a role in Zn foliar absorption, with Zn appearing to move across the cuticle before accumulating in the walls of the epidermal cells. In contrast, however, no marked accumulation of Zn was found within the stomatal cavity, indicating that movement of Zn through the stomata was not likely to be important. However, it is necessary to extend the findings of this study to other plant species and other forms of Zn fertilisers which have different chemical properties. A fifth experiment using sunflower examined the influence of the plant Zn status on foliar Zn absorption. It was found that the absorption of Zn in Zn deficient sunflower was 50-66% lower than for Zn sufficient sunflower. However, in situ µ-XRF analyses showed that the pattern of Zn absorption was similar for both plants, indicating the mechanisms of Zn foliar absorption in Zn sufficient and deficient sunflower were likely the same. Rather, Zn deficiency decreased the leaf trichome density and altered the chemical composition of the cuticle and epidermal layer, with these presumably being the main causes of the reduced Zn absorption in Zn deficient sunflower. In addition, in situ synchrotron-based X-ray absorption 2 spectroscopy (XAS) analyses showed that the Zn in the leaves following foliar application was mostly present as Zn phytate (37-53%) in Zn sufficient leaves, but as Zn phosphate (about 55%) in Zn deficient leaves. Across several of the experiments described above, the foliar absorption of ZnSO4 and nano- ZnO were compared. It was found that the absorption of Zn when supplied as ZnSO4 was ca. 10-fold higher than when supplied as nano-ZnO in soybean, and ca. 100-fold higher in sunflower after 6 h. Using µ-XRF analysis, it was found that their pattern of absorption did not differ between the two types of fertilisers. Furthermore, using XAS analysis, it was found that the speciation of the Zn within the leaf tissue following absorption was similar. These results suggest that the nano-ZnO was absorbed as soluble Zn ions rather than as intact nanoparticles, and nano fertilisers could potentially be used as a slow release foliar fertiliser provided they can be retained on the leaf surface during plant growth. Overall, the cuticle appears to be an important pathway for Zn foliar absorption, although depending upon the plant species and the properties of the trichomes, it is possible that trichomes are also important for foliar Zn absorption. Regardless of the plant species or the form of Zn supplied, the subsequent translocation of bulk Zn after its initial absorption was limited. The Zn status of sunflower plants was found to influence the extent of the absorption of foliar-applied Zn, with Zn deficiency leading to decreased absorption of foliar applied Zn. Compared with ZnSO4, the absorption of nano-ZnO was markedly lower despite their distribution and speciation within the leaf tissues being similar, suggesting that nano-ZnO was absorbed as soluble Zn rather than as intact nanoparticles. However, if nano-ZnO can be retained on the leaf surface, it would potentially have great value as a slow release fertiliser to provide sustained release. 3 Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, financial support and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my higher degree by research candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis and have sought permission from co- authors for any jointly authored works included in the thesis. 4 Publications during candidature 1. Li C, Wang P, Menzies NW, Lombi E, Kopittke PM (2018) Effects of methyl jasmonate on plant growth and leaf properties. Journal of Plant Nutrition and Soil Science 181: 409-418. 2. Li C, Wang P, Menzies NW, Lombi E, Kopittke PM (2017) Effects of changes in leaf properties mediated by methyl jasmonate (MeJA) on foliar absorption of Zn, Mn and Fe.