Abstract
Global warming is expected to have negative effects on agricultural productivity and thus on the food supply chain in the coming decades. The constant increase in average temperatures predicted for the near future will have a strong impact in some ecologically vulnerable areas of the planet, inducing a strong stress on plant species and critically impacting on their sexual reproduction. This phenomenon determines morphological, physiological, and molecular alterations in reproductive organs which negatively affect crop performance and yield, with heavy economic consequences. In Angiosperms, which are the main source of edible vegetables, seeds, and fruits, the male component of the flower is the most affected by heat stress (HS), often resulting in male sterility. While different studies have investigated these aspects, HEATSTOP proposes a novel approach to the issue, focusing on the molecular mechanisms underlying pollen-pistil interactions and investigating how they are affected by high daily temperatures during pollen development and release. For this purpose, HS-sensitive and HS-tolerant cultivars of rapeseed (Brassica napus L.), will be exposed to high temperature regimes in crucial steps of the microsporogenesis. The HS response will be monitored measuring photosynthetic efficiency in stressed and control groups during the experiment, and the impact of HS on fertility will be assessed by comparing fruit and seed parameters for the two groups at the end of the experiment. HS effects on pollen development will instead be assessed using a combination of imaging and molecular techniques, comparing pollen productivity, dimensions, viability, and germinability between stressed and control plants. Eventually, the impact of HS on male-female crosstalk will be achieved through an integrated cytological, molecular, and genetic approach, enhancing the changes in lipids, proteins, and miRNAs in pollen during germination, with a special focus on pollen-secreted molecules and nanovesicles. The latter, called pollensomes, have been recently discovered and their role in pollen germination and plant fertilization is still unknown. The present study will not only characterize the content of the pollensomes for the selected species in terms of proteins and lipids, but it will also determine for the first time the miRNAs carried by these vesicles, also detecting possible HS-induced changes in pollensomes composition. In summary, HEATSTOP will provide specific insight into the pathways involved in the plant response to HS during reproduction, and specifically on the molecular basis of HS-induced male sterility, on economically valuable crops. A comprehensive understanding of these mechanisms will unravel the physiological, molecular, and genetic basis of the consequent yield loss, paving the way for biotechnological applications aimed at breeding HS-tolerant varieties and improving crop productivity.
Results achieved
In order to clarify the molecular alterations underlying HS-induced male sterility, lipidomic and small RNA sequencing analyses were conducted on hydrated pollen (HP), germinated pollen (GP), pollensomes (PS), and the vesicle-free medium of Brassica napus L. cv Phoenix CL grown under both control and HS conditions. HS significantly reduced pollen germinability and increased PS externalization. Although particle size remained unchanged, a significant increase in PS abundance was observed in HS-derived samples. Lipid profiling revealed significant HS-induced remodelling across all samples, including an increase in saturated fatty acids in HP and GP. Notably, triacontanoic acid, the dominant lipid in control PS, was lost under HS conditions and was replaced by oleic acid. Small RNA sequencing identified 70 miRNAs, 61 of which were differentially expressed. HP showed the strongest response to HS, while PS showed opposite trends, suggesting the selective retention or export of miRNAs. HS increased the levels of miR160, miR6030a and miR319a in PS, while the release of miR399 shifted from being vesicular to non-vesicular. Target prediction revealed that these miRNAs regulate pathways associated with development, hormones, vesicles, and lipids. Overall, this study reveals that HS remodels lipid metabolism and miRNA-mediated regulation in pollen and PS, providing molecular signatures for improving crop heat tolerance. These results have been accepted for publication in the Journal of Experimental Botany. The article, titled "Heat stress-induced remodeling of lipid and microRNA networks in Brassica napus L. germinated pollen and pollen-derived small extracellular vesicles," by D'Agostino et al. The Journal of Experimental Botany has a CiteScore rank of 2025, in the Agricultural and Biological Sciences, Plant Science category, in the top decile, specifically at the 96th percentile. Furthermore, on the same samples we performed proteomic analysis identifying proteins differentially expressed under HS regime, on hydrated pollen (HP), germinated pollen (GP), pollensomes (PS). Some of these proteins are involved in metabolic processes, signalling, plant defense, and DNA and protein modification. These data will be the subject of a further publication.Dettagli del progetto
Responsabile scientifico: Stefano Del Duca
Strutture Unibo coinvolte:
Dipartimento di Scienze Biologiche, Geologiche e Ambientali
Coordinatore:
ALMA MATER STUDIORUM - Università di Bologna(Italy)
Contributo totale di progetto: Euro (EUR) 230.127,00
Contributo totale Unibo: Euro (EUR) 114.890,00
Durata del progetto in mesi: 24
Data di inizio
30/11/2023
Data di fine:
28/02/2026
