Phosphorus (P) is an essential macronutrient, critical for plant structure and function. However, excessive soil P fertilization practices have severely altered the global P biogeochemical cycle, as 85% of all mined rock P is used in fertilizer production. This has led to the current “phosphorus paradox”, where increased demand for a non-renewable resource threatens future supplies, while the run-off of labile P from soils is causing major environmental pollution through eutrophication. More sustainable, targeted applications of P fertilizers are required to conserve this limited resource and to minimize adverse environmental effects.
Foliar P fertilization is one alternative strategy that could be applied in-season to meet plant demand when required, or when environmental conditions are favourable. While foliar-applied P has been studied in a range of crops and shown high absorption, this has not always been translated into increased grain yield or plant production. This may be because P absorption pathways across the leaf surface remain unclear, although they are critically important for understanding the mechanisms for penetration and successful assimilation into leaf tissue.
The aim of this thesis was to develop and apply bioimaging techniques for visualizing foliar-applied P absorption and translocation in barley. The first experiment involved testing vanadate (VO43-), an analogous ion to phosphate (PO43-), as a potential tracer for foliar-applied P absorption using high resolution laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) scans of leaf cross-sections. Additionally, a chlorophyll a fluorescence assay was developed using non-photochemical quenching (NPQ) as a proxy for the visualization of physiologically latent leaf P-deficiency, via an Imaging-PAM system. This study showed that vanadate was an appropriate and highly useful foliar-applied phosphate tracer, where fiber cells overlying leaf veins were identified as major zones for phosphate and vanadate accumulation, with indications of subsequent penetration through the underlying bundle sheath extension. Synchrotron X-ray Absorption Near Edge Structure spectroscopy showed the vanadate was limited in its use as a tracer for phosphate translocation, due to reduction processes occurring inside the leaf tissue. Furthermore, the NPQ assay showed that foliar-applied P was able to restore P functionality in treated P-deficient leaves within 24 h. The assay also revealed that absorbed foliar-applied P was rapidly translocated to newly emerging leaves within 7 days, while the P sprayed leaves reverted back to P-deficiency within the same period.
The second experiment applied both LA-ICP-MS elemental scans and Imaging-PAM chlorophyll a fluorescence assays to compare the absorption of foliar-applied Mn and P in nutrient deficient plants at two growth stages. Mn-deficiency led to the thinning of the cuticle and epidermal cell wall, as well as reduced proportions of lipids, and increased stomatal and trichome densities in older plants. Foliar-applied Mn was absorbed and either partially or fully restored Mn functionality within 6 h. In contrast, P-deficiency led to severe decreases in plant biomass production but did not highly alter leaf surface properties, aside from increasing the cuticle and epidermal cell wall thickness. No restoration of P functionality in P-deficient leaves was measured within the 6 h application time. Furthermore, LA-ICP-MS scans showed that fiber cells and trichomes were important structures for P absorption via the use of vanadate as a phosphate tracer. Mn accumulation was visible in some adaxial epidermal cells and throughout the mesophyll.
Finally, the third experimental chapter examined the relationship between trichome density and the thickness of the cuticle and epidermal cell wall with foliar-applied P absorption and investigated whether plant P status quantitatively affected foliar-applied P absorption and translocation. Four Australian barley cultivars (Flagship, Gairdner, Macquarie, Westminster) were grown hydroponically under P-sufficient and P-deficient conditions and 32P spiked solutions were applied to the adaxial epidermis for 48 h and for 7 days. Cultivars varied in trichome density. P-deficiency increased trichome density in Macquarie and decreased trichome density in Flagship. Foliar-applied P absorption was high amongst all four cultivars, 7 days after application. P-deficient plants absorbed significantly less foliar-applied P, although this could not be attributed to specific changes in leaf structure. Autoradiographs showed that the P-deficient cultivars translocated most of the absorbed foliar-applied P to the newly emerging leaf, while P-sufficient plants showed translocation to both older leaves and emerging shoots.
Overall, the bioimaging techniques developed and applied in this study revealed the importance of fiber cells and trichomes for foliar-applied P absorption in barley. They also indicated that absorption could restore P-deficient leaf P functionality, but that absorption pathways and the ability to restore nutrient functionality differed for P and Mn. It was also shown that foliar-applied P absorption was high amongst the cultivars examined, although the exact mechanisms of penetration remained elusive despite previous indications of the importance of trichomes and fiber cells. While much research is required to fully understand absorption mechanisms, foliar P applications show promise as a potential strategy for improving the sustainability of P fertilization. It is hoped that the bioimaging techniques and information produced by this research will help optimize foliar-applied P fertilization formulations and practices to suit plant physiological processes.