Insights on desiccation tolerance of the lichen photobiont trebouxia sp. pl. in both thalline and isolated ones

Abstract

The study of desiccation tolerance of lichens, and of their phycobionts in particular, has been mainly focused on the antioxidant system that protects the cell against photo-oxidative stress produced during dehydration and rehydration. However, desiccation tolerance cannot be achieved in lichen phycobionts with antioxidant activity alone. The main objective of the thesis was to expand the knowledge of the poorly understood desiccation tolerant mechanisms. The first chapter presents an introduction to the subject of the thesis, based on the available background information, and explains the rationale of the thesis. The aim of the chapter 2 was to establish the optimal growing conditions for the isolated phycobiont Trebouxia erici Ahmadjian (SAG 32.85 = UTEX 911) and to determine how these working procedures could modify the results of further experiments. We concluded that cellulose-acetate discs for agar cultures should be inoculated with 14-day-old liquid cultures, and growth under irradiance of 30 mol m-2 s-1 PAR at 20 ºC. Experiments should be carried out with 21day-old algal discs. The experiments carried out in third chapter were designed on one hand, to determine the grade of desiccation tolerance of the isolated lichen phycobiont T. erici, and on the other hand, we studied the response of some protective mechanisms. The results showed that both long desiccations and rapid drying produced greater membrane damage and lower recovery of metabolic and photosynthetic activity than after brief desiccations and slow drying, respectively. Photosynthesis was never recovered totally neither after rapid nor slow drying. Classical mechanisms involved in the protection of the cell against oxidative stress were present in hydrated algae although were not enhanced during dehydration/rehydration. Likewise, in T. erici, dehydrins were also constitutively expressed. However, a slow drying time was required for activating the conformational change in the photosystem II which protect against photoinhibition in desiccation state. In the fourth chapter we decided to carry out proteomic and genetic expression analyses of the changes associated with desiccation and rehydration in the isolated phycobiont Trebouxia erici, in order to find out other proteins that may be involved on desiccation tolerance mechanisms of phycobionts. Proteomic analysis showed that desiccation caused up-regulation of around 19 proteins and down-regulation of 43 proteins. Among the proteins up-regulated during drying were found: a putative cation transport protein; a protein with an F-box domain; a cell division cycle 48 protein; -tubulin protein; a 90 kDa Heat shock protein. We observed that five Hsp90 and two -tubulin genes were activated during dehydration and mRNA was accumulated until the cell was completely dried. In the fifth chapter we investigated the role of the NO in the biochemical interaction between lichen symbionts and whether the NO may increase the endurance of lichenized phycobionts under desiccation. Rehydration of the lichen R. farinacea caused the release of NO together with a high production of ROS. The amount of NO detected decreased significantly with the addition of c-PTIO. In the isolated phycobionts, scavenging of the self produced NO caused a decrease in the recovery of photosynthetic activity after dehydration, probably due to the higher levels of photo-oxidative stress. The aim of the work presented in the sixth chapter was to determine the effect of cell ageing in desiccation resistance of T. erici. Phycobiont capacity to recover photosynthesis activity after desiccation was inversely related with cell ageing, suggesting that desiccation tolerance is age-dependent. The results presented in this thesis demonstrate that desiccation tolerance of T. erici is achieved by a complex system of constitutive and induced mechanisms. Moreover, some factors such as drying rate, desiccation duration, lichenization and ageing may alter the recovery capacity of T. erici after desiccation.

El estudio de la tolerancia a la desecación de los líquenes, y de sus fotobiontes en particular, ha sido principalmente enfocado hacia los sistemas antioxidantes que protegen a la célula frente al estrés fotoxidativo. Sin embargo, la tolerancia a la desecación no puede ser alcanzada solamente con sistemas antioxidantes. Por esta razón, el objetivo principal de esta tesis fue profundizar en el conocimiento de los mecanismos de tolerancia a la desecación en fotobiontes liquénicos. El fotobionte liquénico Trebouxia erici Ahmadjian mostró ser tolerante a la desecación incluso cuando es cultivado separado de su compañero micobionte. Sin embargo, la resistencia a la desecación dependió de la velocidad de deshidratación, del tiempo de desecación y del envejecimiento del cultivo. Tasas de deshidratación altas, largos periodos de desecación y cultivos viejos redujeron la capacidad de recuperación. Esto es debido a que la tolerancia a la desecación del fotobionte se consigue por un lado mediante mecanismos de protección celular que son constitutivamente expresados como, enzimas antioxidantes, xantofilas o proteínas LEA, junto con la activación Mientras que por otro, existen mecanismos que se activan durante la deshidratación, como son de la síntesis de proteínas implicadas en el transporte, la protección de membranas y proteínas, el citoesqueleto, el ciclo celular y el marcaje y la degradación de proteínas. Aunque los fotobiontes liquénicas tienen sus propios mecanismos para resistir la desecación, en la relación simbióntica la tolerancia a la desecación podría ser aumentada por otros mecanismos. Nuestros estudios con el liquen Ramalina farinacea Ach. y fotobiontes aislados mostraron que el gas bioactivo oxido de nitrógeno (NO) es liberado principalmente por el hongo durante la rehidratación del talo, el cual podría tener un papel importante en la maquinaria antioxidante del fotobionte durante las primeras fases de la rehidratación.