Regulation of respiratory metabolism in germinating seeds

Germination and early seedling growth are critical periods in the life cycle of plants. Starting from a quiescent dry state in the seed, embryos and seedlings need to maintain an efficient heterotrophic metabolism and cope with often stressful conditions, in order to rapidly reach autotrophy and start competing for nutriments and space. These processes are almost entirely dependent on mitochondrial respiration, which provides cellular energy as well as a metabolic platform involved in the conversion of seed reserves into building blocks for growth metabolism. It is therefore no surprise that seed mitochondria exhibit unusual properties in respect of desiccation and temperature tolerance. Stress proteins such as LEA (late embryogenesis abundant) proteins and sHSPs (small heat shock proteins) are involved in the protection of mitochondria in the dry state, and likely contribute to their thermal tolerance during germination. In many cases, fast germination increases the chances of successful emergence and establishment of seedlings, and this requires an efficient energy metabolism. Oxygen availability for respiration can be a challenge because of limiting oxygen diffusion rates in large seeds and/or within soils. It appears that, at least in legume seeds, mitochondria are able to self-adjust their oxygen consumption with the support of nitric oxide (NO) metabolism. This allows maximal energy production to be achieved under hypoxic conditions, without subjecting tissues to deleterious anoxia. In the context of ongoing and future climate change, it is of general importance to understand how mitochondrial functions have evolved to maintain energy homeostasis in organisms and tissues exposed to extreme environmental conditions such as desiccation. Such traits could offer interesting targets for plant adaptation and improvement

Integrative analysis of acquired thermotolerance in developmentally arrested Arabidopsis seedlings. Implication of energy metabolism

In the context of climate change, the increased frequency and intensity of heat waves will likely have a negative impact on plant physiology, due to the structural destabilization of proteins and membranes caused by high temperatures. As part of this thesis, we developed and characterized an original experimental setup in which Arabidopsis thaliana seedlings are arrested in their development because of mineral starvation. These seedlings exhibit a high metabolic plasticity, especially for energy metabolism, which allows them to survive in a steady state for weeks. Then, we performed an integrative analysis of the processes that allow these seedlings to survive an otherwise lethal heat stress (43°C, 2 h), thanks to a priming treatment at a nonlethal temperature (38°C, 2 h). Priming protects the energy metabolism and permits the recovery of organelle dynamics after stress. At the transcriptional level, primed seedlings overexpress many chaperone proteins and genes involved in photosynthesis, and in the regulation of the expression of mitochondrial and plastidial genomes. At the protein level, the accumulation of HSPs and other stress proteins favour seedling recovery, whereas in the absence of acclimation, heat shock provokes the decrease of ribosomal proteins and the accumulation of proteins implicated in protein degradation. This study highlights the relevance of multi-scale analysis to decipher mechanisms of stress response in plants

Simple system using natural mineral water for high-throughput phenotyping of Arabidopsis thaliana seedlings in liquid culture

Background: Phenotyping for plant stress tolerance is an essential component of many research projects. Because screening of high numbers of plants and multiple conditions remains technically challenging and costly, there is a need for simple methods to carry out large-scale phenotyping in the laboratory.Methods: We developed a method for phenotyping the germination and seedling growth of Arabidopsis (Arabidopsis thaliana) Col-0 in liquid culture. Culture was performed under rotary shaking in multiwell plates, using Evian natural mineral water as a medium. Nondestructive and accurate quantification of green pixels by digital image analysis allowed monitoring of growth. Results: The composition of the water prevented excessive root elongation growth that would otherwise lead to clumping of seedlings observed when classic nutrient-rich medium or deionized water is used. There was no need to maintain the cultures under aseptic conditions, and seedlings, which are photosynthetic, remained healthy for several weeks. Several proof-of-concept experiments demonstrated the usefulness of the approach for environmental stress phenotyping. Conclusion: The system described here is easy to set up, cost-effective, and enables a single researcher to screen large numbers of lines under various conditions. The simplicity of the method clearly makes it amenable to high-throughput phenotyping using robotics

Arabidopsis seedlings display a remarkable resilience under severe mineral starvation using their metabolic plasticity to remain self-sufficient for weeks

During the life cycle of plants, seedlings are considered vulnerable because they are at the interface between the highly stress tolerant seed embryos and the established plant, and must develop rapidly, often in a challenging environment, with limited access to nutrients and light. Using a simple experimental system, whereby the seedling stage of Arabidopsis is considerably prolonged by nutrient starvation, we analysed the physiology and metabolism of seedlings maintained in such conditions up to 4 weeks. Although development was arrested at the cotyledon stage, there was no sign of senescence and seedlings remained viable for weeks, yielding normal plants after transplantation. Photosynthetic activity compensated for respiratory carbon losses, and energy dissipation by photorespiration and alternative oxidase appeared important. Photosynthates were essentially stored as organic acids, while the pool of free amino acids remained stable. Seedlings lost the capacity to store lipids in cytosolic lipid droplets, but developed large plastoglobuli. Arabidopsis seedlings arrested in their development because of mineral starvation displayed therefore a remarkable resilience, using their metabolic and physiological plasticity to maintain a steady state for weeks, allowing resumption of development when favourable conditions ensue

The Ubiquitous Distribution of Late Embryogenesis Abundant Proteins across Cell Compartments in Arabidopsis Offers Tailored Protection against Abiotic Stress

Late embryogenesis abundant (LEA) proteins are hydrophilic, mostly intrinsically disordered proteins, which play major roles in desiccation tolerance. In Arabidopsis thaliana, 51 genes encoding LEA proteins clustered into nine families have been inventoried. To increase our understanding of the yet enigmatic functions of these gene families, we report the subcellular location of each protein. Experimental data highlight the limits of in silico predictions for analysis of subcellular localization. Thirty-six LEA proteins localized to the cytosol, with most being able to diffuse into the nucleus. Three proteins were exclusively localized in plastids or mitochondria, while two others were found dually targeted to these organelles. Targeting cleavage sites could be determined for five of these proteins. Three proteins were found to be endoplasmic reticulum (ER) residents, two were vacuolar, and two were secreted. A single protein was identified in pexophagosomes. While most LEA protein families have a unique subcellular localization, members of the LEA_4 family are widely distributed (cytosol, mitochondria, plastid, ER, and pexophagosome) but share the presence of the class A α-helix motif. They are thus expected to establish interactions with various cellular membranes under stress conditions. The broad subcellular distribution of LEA proteins highlights the requirement for each cellular compartment to be provided with protective mechanisms to cope with desiccation or cold stress

Variability within a pea core collection of LEAM and HSP22, two mitochondrial seed proteins involved in stress tolerance

LEAM, a late embryogenesis abundant protein, and HSP22, a small heat shock protein, were shown to accumulate in the mitochondria during pea (Pisum sativum L.) seed development, where they are expected to contribute to desiccation tolerance. Here, their expression was examined in seeds of 89 pea genotypes by Western blot analysis. All genotypes expressed LEAM and HSP22 in similar amounts. In contrast with HSP22, LEAM displayed different isoforms according to apparent molecular mass. Each of the 89 genotypes harboured a single LEAM isoform. Genomic and RT-PCR analysis revealed four LEAM genes differing by a small variable indel in the coding region. These variations were consistent with the apparent molecular mass of each isoform. Indels, which occurred in repeated domains, did not alter the main properties of LEAM. Structural modelling indicated that the class A α-helix structure, which allows interactions with the mitochondrial inner membrane in the dry state, was preserved in all isoforms, suggesting functionality is maintained. The overall results point out the essential character of LEAM and HSP22 in pea seeds. LEAM variability is discussed in terms of pea breeding history as well as LEA gene evolution mechanisms