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Transgenerational epigenetic regulation of heat stress response

Final Report Summary - TRANS-EPIGEN (Transgenerational epigenetic regulation of heat stress response)

Abiotic stresses, mainly high temperature, are among the most limiting factors of crop performance and limit their geographical distribution in Europe and worldwide. Although work on temperature tolerance goes back for many years back, little is known about the underlying molecular mechanisms. Before joining Dr. Grossniklaus’ Lab in Switzerland, Afif Hedhly initiated his work on heat stress effects during the reproductive phase of plants in his former Spanish host laboratories (Prof. Herrero’s group, EEAD-CSIC, Zaragoza, and Prof. Hormaza’s group, EELM-CSIC, Malaga) using temperate, subtropical, and tropical fruit trees. Results obtained pointed to phenotypic plasticity, probably mediated by epigenetic changes, as a key player in the plant’s response to changes in environmental conditions (Hedhly et al. 2009, Hedhly 2011). Research on fruit trees is, however, hindered by scarce knowledge at the molecular level, the single yearly flowering, and the long generation time. Thus, using short-cycled and well-characterized model species, such as Arabidopsis thaliana, as a bridge to identify genes potentially suitable to improve plant performance, is a valuable alternative.
This project was carried out to characterize the effect of heat stress on reproductive development of the model species Arabidopsis thaliana and its long lasting epigenetic effects. The demarcation of key developmental stages, during which abiotic stresses have long-lasting and/or transgenerational effects in plants is, however, poorly understood and the underlying molecular mechanisms are currently unknown. Thus, before undertaking the molecular characterization of these long-lasting epigenetic effects, we first had to characterize reproductive development in Arabidopsis thaliana and analyse the effects of heat stress.

The first important step was to characterize male and female development under control conditions from the meristematic stage to flower opening, along the main inflorescence once the first flower opened. At this stage there are around 32 buds, spanning all flower developmental stages, and we needed both a sensitive and quantitative technique. Light microscopy of cleared, whole-mount flowers was found the most promising. Furthermore, to judge the potential of this technique to characterise not only wild-type ovules (Schneitz et al. 1995) and stamen development (here) in a wild-type background but also any failure during the reproductive process, we took some uncharacterised male sterile lines available in the lab and are successfully characterising them.

Second, using different light and confocal microscopy techniques together with the whole-mount tissue clearing procedure, we characterized the effect of temperature on reproductive development. The effect of heat stress was equally characterized during all flower developmental stages, and a cartography of heat stress effects on reproductive development has been established. Heat stress affected all developmental stages but anther wall layer formation and the meiosis were found to be the most sensitive stages, leading to pollen grains of different ploidy levels, unbalanced chromosome numbers, and pollen sterility.

Transient starch accumulation within the flower represents an energy reservoir to sustain flower development, and any failure in carbohydrate metabolism is known to results in either male or female sterility. Despite the detailed knowledge of the molecular mechanisms of starch metabolism within autotrophic cells, and despite the importance of starch accumulation in heterotrophic cells for human consumption, starch metabolism in heterotrophic organs is not completely understood. Although Arabidopsis thaliana does not accumulate starch in its seeds, the molecular knowledge in this model species places it as an ideal system to characterize starch metabolism in heterotrophic organs, such as the flower and embryo, and to analyse how it reacts to heat stress. In this work we characterised for the first time starch dynamics in Arabidopsis thaliana during all developmental stages of stamen, carpel, and early embryo development both under control and heat stress conditions. Our approach allowed us to find not only a more complex picture of starch dynamics during stamen development (more waves of amylogenesis/amylolysis starch than previously reported) under control conditions but, interestingly, a starch excess phenotype under heat stress. To find out which genes mediate starch metabolism during stamen and early embryo development, and to identify those which are regulated by heat stress, these results are being complemented, amongst others, with a microarray analysis of genes known to play a role in starch metabolism in autotrophic organs and all their homologues.

One interesting effect of heat stress during ovule development is the formation of multiple megaspore mother cell (MMC)-like cells with the eventual development of more than one embryo sac. Additional nucellar sporophytic cells will initiate female gametophyte formation and form supernumerary embryo sacs in a way very similar to apospory, which occurs in various natural apomicts. Apomicts reproduce asexually through seeds without meiosis and fertilization. Recent analysis of some mutants defective in AGO9-dependent small RNA silencing (Olmedo-Monfil et al 2010) showed a similar phenotype, and established that MMC specification is under epigenetic control. To see whether heat stress is affecting this pathway, we initiated a molecular analysis of candidate genes in the pathway and of potential epigenetic changes during development. Apomixis has a tremendous potential for agricultural applications and it was a surprise that our research on the possible epigenetic effects of stress would converge on the longstanding interests in apomixis in the Grossniklaus’ laboratory.
An increasing body of evidence indicates that global warming is taking place and that this will have important effects in the next decades. While the reasons behind global warming are still controversial, its adverse consequences are clear and of increasing concern worldwide. Observed changes in phenotypic traits of different plant and animal species indicate that natural populations are responding to this change. In agricultural systems, a reduction in yield in different species and at different latitudes has been recorded. However, actual mechanisms underpinning these consequences are not well demarcated. This work has been undertaken to add some knowledge on the physiological and molecular mechanisms underlying immediate and long-lasting effects of heat stress on reproductive development. The work is still underway, financed by running projects in Dr. Grossniklaus’ lab, to publish the already advanced drafts and to answer further questions that arose during the realization of the project. My stay in Dr. Grossniklaus’ lab is undoubtedly important for reaching professional maturity, for performing independently, and for working in a multidisciplinary and inspiring atmosphere.

Hedhly A 
2011. Flowering sensitivity to temperature fluctuation. Environmental and Experimental Botany 74: 9-16.
Hedhly A, Hormaza JI, Herrero M 2009. Global warming and sexual plant reproduction. Trends in Plant Sciences 14, 30-36.
Olmedo-Monfil V, Durán-Figueroa N, Arteaga-Vázquez M, Demesa-Arévalo E, Autran D, Grimanelli D, Slotkin RK, Martienssen RA, Vielle-Calzada JP (2010) Control of female gamete formation by a small RNA pathway in Arabidopsis. Nature 464:628–632.
Schneitz K, Hülskamp M., Pruitt, R.E.1995. Wild-type ovule development in Arabidopsis thaliana: a light microscope study of cleared whole-mount tissue. The Plant Journal 7:731-749