Abstract
The Mediterranean basin is particularly sensitive to adverse outcomes of climate change and especially the incidence of high temperatures. Adverse environmental conditions will affect crop performance and productivity and will be particularly severe on woody crops, including citrus, which is the proof of concept chosen in this project to study the impact of temperature stress on reproduction and plant physiology. In particular, reproduction is affected by climate change due to morphological, physiological and molecular alterations in male and female organs. Temperature changes also affect pollen-pistil interaction and particularly the self-incompatibility reaction. In citrus (especially in mandarins) the absence of fertilization usually leads to seedless parthenocarpic fruits, which is a much-prized character. However, temperature stress is known to cause the breakdown of self-incompatibility in citrus, resulting in seedy fruit. This project aims to a better understanding of reproduction in citrus (especially in mandarins and mandarin-like varieties such as clementine) so as to evaluate the damaging effects of non-optimal temperature conditions that can occur during the reproduction cycle, from floral induction to fruit set. The project will be focussed on the effect of temperature stress in incompatibility reaction, which will be assessed in selected mandarin varieties [self-compatible (SC) and self-incompatible (SI) genotypes] exposed to different temperature regimes in crucial steps of the fruitification cycle. An integrated cytological, molecular and genetic approach will be applied to decipher the effects of temperature stress. In particular, histo-molecular analysis in planta will be performed to assess the effect of temperature stress on reproductive organs development and on fertilization process. Pollen viability and behavior will be analyzed in in vitro tests while the analysis of female gametophyte will provide information on the damage caused to male-female crosstalk. Transcriptomic and epigenomic studies during male and female gametophyte development under temperature stress will provide specific insight into the pathways involved in the response of plants to temperature stress during reproduction. Results will provide insight into the pollen-pistil interaction in mandarin varieties of selected genotypes (SC and SI) under critical environmental conditions. Comprehensive understanding of how temperature variation alters the reproductive process will unravel the physiological, molecular, and genetic basis of sterility in citrus and help define the temperature conditions causing seedy fruit production and high depreciation of their commercial value. Furthermore, the availability of reference genomes of clementine (C. clementina Hort. ex Tan.) and mandarin (C. reticulata Blanco) will allow the development of biotechnological tools useful in the selection process of new varieties with better performances under temperature stress.
Results achieved
The project clarified how sub-optimal and supra-optimal temperatures affect reproductive biology and self-incompatibility in Mediterranean mandarins and clementines. Controlled pollination experiments were performed under defined thermal regimes (15 C, 25 C and 30 C), comparing compatible (NxT) and incompatible (NxN) crosses. These analyses generated quantitative evidence on pollen tube progression, fertilization potential and reproductive success, allowing the definition of operational temperature thresholds associated with the maintenance or disruption of seedlessness and reproductive compatibility. A major result concerned the temperature-dependent composition of the stigma and style, which emerged as a key determinant of pollen-pistil interaction. Histochemical staining with periodic acid-Schiff (PAS), Toluidine Blue O and Ruthenium Red, combined with aniline blue staining for pollen tube growth, showed that 15 C provided the most favourable biochemical environment for pollen tube elongation. Under these conditions, the transmitting tissue of the style was enriched in neutral and acidic polysaccharides and pectins, supporting sustained pollen tube growth in compatible crosses and allowing prolonged progression even in incompatible crosses before arrest. At 25 C, both compatible and incompatible combinations showed a reduction in polysaccharide secretion in the style, indicating a partial weakening of the biochemical support required for efficient pollen tube growth. At 30 C, the reproductive tissues showed the strongest alterations: neutral polysaccharides accumulated mainly in the papillae, acidic polysaccharides were largely confined to the apical stigma, and the stylar canal was comparatively depleted of key compounds. Pectin levels were also reduced, especially in incompatible crosses. These changes were associated with early pollen tube arrest and reduced fertilization potential. The project also showed that temperature stress affects the female gametophyte and ovule physiology. Variations in starch deposition within ovules were observed together with changes in stigma and style polysaccharides, indicating that thermal regimes influence both pollen tube guidance and the metabolic status of the ovule. The integration of these observations provides a mechanistic explanation of the different fertility outcomes observed under temperature stress. At the molecular level, transcriptomic (RNA-seq) and DNA methylation analyses of anthers and pistils identified differentially expressed genes and epigenetic patterns associated with thermal sensitivity or resilience. These datasets were integrated with phenotypic, cytological and biochemical evidence to identify candidate genes and potential molecular and epigenetic markers for breeding. Overall, the project produced reference datasets, operational protocols and practical knowledge that can support marker-assisted selection, the development of climate-resilient citrus genotypes, and agronomic strategies aimed at maintaining fruit quality and controlling seed formation under future warming scenarios. The analysis of the stigmatic/style exudate through a proteomic approach, addressed the stigmatic exudate as a metabolically active extracellular secretion containing proteins involved in pollen adhesion, pollen-tube guidance and processes associated with compatibility or incompatibility. According to the project protocol, pre-flowering branches were isolated to avoid pollen contamination, collected shortly before anthesis and maintained under controlled conditions. The exudate was then obtained from washed pistils in phosphate buffer, collected as independent samples and stored at -20 C until analysis. The proteomic profiling is aimed at identifying the protein component associated with pollen-tube support and guidance, comparing control and thermal-stress conditions to detect candidate proteins involved in altered progamy and fertilization, and integrating these data with the parallel characterization of sugars and phenolic compounds. These results will help clarify the molecular mechanisms by which global warming and thermal stress modify pollen-pistil communication in Mediterranean mandarins, while providing candidate protein markers useful for interpreting changes in fertility and productivity under critical climatic conditions.Dettagli del progetto
Responsabile scientifico: Stefano Del Duca
Strutture Unibo coinvolte:
Dipartimento di Scienze Biologiche, Geologiche e Ambientali
Coordinatore:
Università di Siena(Italy)
Contributo totale Unibo: Euro (EUR) 66.891,00
Durata del progetto in mesi: 24
Data di inizio
12/10/2023
Data di fine:
28/02/2026
