Assessing Tolerance to Heavy-Metal Stress in Arabidopsis thaliana Seedlings

The deposited book chapter is a post-print version and has been submitted to peer review.The deposited book chapter version contains attached the supplementary materials within the pdf.This publication hasn't any creative commons license associated.The deposited book chapter is part of the book series: "Environmental Responses in Plants: Methods and Protocols" (pp.197-208) published by Springer.Heavy-metal soil contamination is one of the major abiotic stress factors that, by negatively affecting plant growth and development, severely limit agricultural productivity worldwide. Plants have evolved various tolerance and detoxification strategies in order to cope with heavy-metal toxicity while ensuring adequate supply of essential micronutrients at the whole-plant as well as cellular levels. Genetic studies in the model plant Arabidopsis thaliana have been instrumental in elucidating such mechanisms. The root assay constitutes a very powerful and simple method to assess heavy-metal stress tolerance in Arabidopsis seedlings. It allows the simultaneous determination of all the standard growth parameters affected by heavy-metal stress (primary root elongation, lateral root development, shoot biomass, and chlorophyll content) in a single experiment. Additionally, this protocol emphasizes the tips and tricks that become particularly useful when quantifying subtle alterations in tolerance to a given heavy-metal stress, when simultaneously pursuing a large number of plant lines, or when testing sensitivity to a wide range of heavy metals for a single line.Fundação para a Ciência e a Tecnologia grants: (EXPL/AGR-PRO/1013/2013, SFRH/BPD/44640/2008); GREEN-it "Bioresources for Sustainability": (UID/Multi/04551/2013).info:eu-repo/semantics/publishedVersio

Restoration of photosystem II photochemistry and carbon assimilation and related changes in chlorophyll and protein contents during the rehydration of desiccated Xerophyta scabrida leaves

Recovery of photosynthesis in rehydrating desiccated leaves of the poikilochlorophyllous desiccation-tolerant plant Xerophyta scabrida was investigated. Detached leaves were remoistened under 12 h light/dark cycles for 96 h. Water, chlorophyll (Chl), and protein contents, Chl fluorescence, photosynthesis–CO2 concentration response, and the amount and activity of Rubisco were measured at intervals during the rehydration period. Leaf relative water contents reached 87% in 12 h and full turgor in 96 h. Chl synthesis was slower before than after 24 h, and Chla:Chlb ratios changed from 0.13 to 2.6 in 48 h. The maximum quantum efficiency recovered faster during rehydration than the photosystem II operating efficiency and the efficiency factor, which is known to depend mainly on the use of the electron transport chain products. From 24 h to 96 h of rehydration, net carbon fixation was Rubisco limited, rather than electron transport limited. Total Rubisco activity increased during rehydration more than the Rubisco protein content. Desiccated leaves contained, in a close to functional state, more than half the amount of the Rubisco protein present in rehydrated leaves. The results suggest that in X. scabrida leaves Rubisco adopts a special, protective conformation and recovers its activity during rehydration through modifications in redox status

Label-free chemically specific imaging in planta with stimulated Raman scattering microscopy.

The growing world population puts ever-increasing demands on the agricultural and agrochemical industries to increase agricultural yields. This can only be achieved by investing in fundamental plant and agrochemical research and in the development of improved analytical tools to support research in these areas. There is currently a lack of analytical tools that provide noninvasive structural and chemical analysis of plant tissues at the cellular scale. Imaging techniques such as coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) microscopy provide label-free chemically specific image contrast based on vibrational spectroscopy. Over the past decade, these techniques have been shown to offer clear advantages for a vast range of biomedical research applications. The intrinsic vibrational contrast provides label-free quantitative functional analysis, it does not suffer from photobleaching, and it allows near real-time imaging in 3D with submicrometer spatial resolution. However, due to the susceptibility of current detection schemes to optical absorption and fluorescence from pigments (such as chlorophyll), the plant science and agrochemical research communities have not been able to benefit from these techniques and their application in plant research has remained virtually unexplored. In this paper, we explore the effect of chlorophyll fluorescence and absorption in CARS and SRS microscopy. We show that with the latter it is possible to use phase-sensitive detection to separate the vibrational signal from the (electronic) absorption processes. Finally, we demonstrate the potential of SRS for a range of in planta applications by presenting in situ chemical analysis of plant cell wall components, epicuticular waxes, and the deposition of agrochemical formulations onto the leaf surface