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1.1. Description of the work performed since the beginning of the project

During this project, we have developed a morphology and chlorophyll fluorescence based screening platform using Arabidopsis seedlings grown on plate. This simple and fully automated non-invasive screening methodology allows to simultaneously monitoring stress-induced changes in photosynthetic yield and plant growth. By using oxidative (methyl viologen, hydrogen peroxide), and osmotic (polyethylene glycol, NaCl) stressors, we analyzed the stress performance of auxin-related lines which are mutants and overexpressors of genes involved in control points of auxin homeostasis including biosynthesis, transport, metabolism, and signaling. Through this method, we deciphered some components of the ROS-auxin regulatory network involved in plant stress adaptation. In this sense, for each selected line we could determine the specificity of the response that means, the type of stress to which the auxin-related line show improved or suboptimal effect on root or shoot growth and photosynthesis in comparison to wild type plants.
By using cell biology tools, the effect of ROS accumulation upon oxidative stress on the underlying mechanisms controlling auxin-dependent root apical meristem activity and lateral root development and on the subcellular trafficking mechanisms controlling PIN polarity and degradation was inferred. Our results contribute to the understanding of stress-induced root growth and root development retardation effect.

1.2. Description of the main results achieved so far

1.2.1. Uncovering ROS-auxin regulatory components involved in plant growth and photosynthesis regulation

First, we generated a transcriptional specific ROS-auxin crosstalk regulatory networks which served to pick up stress responsive and involved in auxin homeostasis. The real biological potential of the selected genes was tested through the selection of mutants or overexpressing lines and further functional assessments via a screening methodology in which root and shoot growth and changes in photosystem II photochemical efficiency (Fv/Fm), non-photochemical quenching (NPQ) and photochemical quenching are used to determine the plant stress response. In total, we have analyzed 50 auxin-related Arabidopsis lines. From them, lines with altered photosynthesis under control or stress growth conditions (6 lines); altered growth and photosynthesis (9 lines); or altered growth (4 lines) were selected. I general, lines affected in auxin biosynthesis or transport show lower levels of NPQ under stress, which measures a change in the efficiency of heat dissipation processes that protect the leaf from photoinhibition under unfavorable growth conditions. Therefore, changes in auxin levels and/or metabolism modulate NPQ photo protection mechanism rather than chloroplast photochemical processes. From 15 auxin signaling-related lines, only 4 were selected. Mutant for the auxin responsive protein IAA1 shows altered photosynthetic response and wild type effect on root and shoot growth upon stress, while mutants for IAA19 affect only plant growth. Moreover, under stress the absence of the auxin response factors ARF6 or ARF7impact on plant growth and photosynthesis, while arf9 mutants specifically affect stress-induced growth retardation.

1.2.2. Biochemical and Molecular characterization of selected lines.

Based on the screening results, we further characterized auxin transport PIN6-related mutants and overexpressors which showed decreased NPQ values under oxidative and NaCl stress conditions. Moreover, pin6-2 and 35S:PIN6 plants grown on soil display decreased CO2 fixation rates and lower stomatal closure after 11 days of watering with 150 mM NaCl. Interestingly, the stress-induced growth retardation effect in response to NaCl treatment is inhibited in mutants and overexpressors. In addition, changes on auxin transport and homeostasis in PIN6 mutants and overexpressors do not affect ROS accumulation in roots and shoots. Altered auxin homeostasis in pin6-2 and 35S:PIN6 plants is associated to altered rosette shape, and results in significantly improved water retention under salt stress treatments. Hence, our results reveal that PIN6-dependant auxin transport is an important regulators of morphological and physiological stress adaptation mechanisms, and provide molecular evidence for the interplay between auxin and photosynthesis.
Results obtained using YUC knockout mutants and overexpressors deciphered the specificity of the stress response in terms of growth and photosynthetic performance. We have started the characterization of YUC-related lines showing altered stress induced morphogenetic responses.

1.2.3: Dissection of SIMR responses on auxin transport and homeostasis.

We studied the effect of H2O2 treatment on the subcellular trafficking mechanisms controlling PIN polarity and degradation. Interestingly, we observed that the oxidative stress induced by H2O2 disassembles actin filaments (data not shown). In addition, we have characterized the effect of oxidative and osmotic stress on root apical meristem activity and on the development of lateral roots. Our results show that oxidative stress alters root system architecture by reducing lateral root initiation and accelerating lateral root development. In addition, oxidative stress alters PIN transporters abundance and auxin signaling in emerged primordia.

1.3. Conclusions

The results obtained in this project increase our poor understanding of how developmental, environmental and photosynthetic signals are integrated by cooperative modulation of ROS and auxin pathways. Moreover, target genes selected via the screening are new identified ROS-auxin components and represent an important step to shed light on the mechanisms by which plants adapt to stress.
Understanding the molecular bases of the observed alterations in growth and photosynthesis of the most stress-responsive auxin-related lines is an important breakthrough in the elucidation of ROS-auxin homeostasis convergence points and in its interaction with photosynthesis upon stress.

1.4. Potential impact of the results

Besides the effects on plant morphology, regulation of photosynthesis is also a part of plant stress adaptation. Reduced photosynthesis is in part responsible for some of the observed symptoms in stress-adapted plants such as growth retardation, reduced metabolism and reallocation of metabolic resources. Notwithstanding the large number of evidence showing interplay between ROS and auxin on photosynthesis, research on this topic has not been addressed. Owing to the vital importance of photosynthetic processes for plant growth and survival, the regulatory components at the ROS-auxin interface we have selected in this project constitute one of the first steps to uncover the contribution of auxin on photosynthesis.

We have deciphered the role of auxin-related genes during stress adaptation and their effect of shoot, root or photosynthesis. The generated date open new and promising perspectives to investigate the molecular mechanism by which the selected targets modulates growth or photosynthesis under specific environmental stresses and would lead to future tools to improve yields on crops.
In the light of a growing need for a more sustainable society, detailed understanding of plant responses to environmental hardships may result in novel strategies to improve yields of cultivated crops. Improved yields of cultivated crops, used for food and nonfood applications, are highly desirable since the European ambitions regarding the future use of renewable resources, such as biofuels or bioplastics, will require an important increase in total biomass production.