An Integrative Approach to Assess the Impact of Heat Stress on C3 and C4 Pseudocereals of Amaranthaceae

Climate change, growing population, and the depletion of natural resources present urgent challenges for global agriculture and food security. Rising temperatures impose severe limitations on crop productivity, highlighting the need for thermotolerant crops that can sustain yield stability under heat stress. Conventional cereals, although essential to human diets, often have limited adaptability and narrow nutritional diversity. In this context, pseudocereals like Chenopodium quinoa (C3) and Amaranthus hypochondriacus (C4) are gaining significance due to their exceptional nutritional profiles, resilience in marginal environments, and potential as contrasting dicot models for studying C3–C4 photosynthetic adaptations in crops—a field traditionally dominated by monocot research. This doctoral research examined the heat stress responses of quinoa and amaranth at physiological, biochemical, anatomical, and molecular levels to understand their different tolerance mechanisms and identify potential genes for thermotolerance. At the seedling stage, quinoa showed strong sensitivity to heat, with decreases in net CO₂ uptake, chlorophyll fluorescence (Fv/Fm), and biomass growth, along with damage to grana thylakoids, stomatal closure, and excessive buildup of reactive oxygen species (ROS). These responses revealed a limited antioxidative defense system, leading to unstable redox balance and reduced photosynthesis. In contrast, amaranth maintained its photosynthetic efficiency and employed various adaptive strategies, including chloroplast enlargement with a maintained thylakoid structure, increased stomatal density for improved gas exchange, and active transcription of heat shock transcription factors (HSFs) and heat shock proteins (HSPs). These changes collectively improved carboxylation efficiency and biomass, demonstrating the innate heat tolerance of C4 metabolism. During the reproductive stage, quinoa’s vulnerability was further increased under higher nighttime temperatures, which caused disproportionately greater setbacks in productivity compared to daytime heat. Nighttime heat stress disrupted the plant's carbon balance by decreasing CO₂ assimilation and increasing respiratory and photorespiratory carbon loss, thereby restricting the availability of photoassimilate for reproductive development. Furthermore, panicles exhibited reduced starch biosynthesis, decreased pollen viability, and impaired grain filling, all of which contributed to yield loss. These adverse effects were exacerbated by ROS accumulation, lipid peroxidation, and insufficient activation of antioxidative enzymes. Combined transcriptomic and metabolomic analyses confirmed these findings, showing disruptions in central carbon metabolism, photoassimilate distribution, and stress-response signaling pathways. In amaranth, genome-wide characterization identified thirteen Hsfs, among which AhHsf-7A emerged as a central regulatory hub. Its expression was strongly induced. AhHsf-7A is the closest ortholog of AtHsfA1d, a well-established master regulator of the heat stress response in Arabidopsis. Therefore, to assess their functional relevance, overexpression lines of AtHsfA1d and AhHsf-7A were used for comparative evaluation. Transgenic lines overexpressing AhHsf-7A exhibited enhanced thermotolerance, as reflected in improved photosynthetic efficiency, reduced ROS accumulation, and greater survival compared to wild-type and hsfa1d mutants. Notably, AhHsf-7A conferred stronger protection than AtHsfA1d, underscoring the superior regulatory capacity of the C4-derived gene and highlighting its translational potential for engineering heat resilience in C3 crops. Overall, this research demonstrates that heat resilience in plants requires the combined regulation of molecular signals, chloroplast structure, and whole-plant function. While quinoa highlights the vulnerability of C3 pseudocereals to heat stress, Amaranth serves as a resilient C4 model system and a valuable genetic resource for enhancing crop heat tolerance. These results improve our understanding of how C3 and C4 plants adapt to stress in dicots and identify potential targets for molecular breeding and genetic engineering to create climate-resilient crops, thereby contributing to the assurance of sustainable food and nutrition security amid global warming.