Increased temperature (eT) and water stress (wS), alone and in combination, are among the factors that have the greatest influence on plant development and reproduction in the tropics. Using the T-FACE (Temperature Free-Air Controlled Enhancement) facility to increase canopy temperature integrated with a drip irrigation setup, under field conditions, we investigated the effects of water deficit and warming (+2 °C) on the pollen grains of Stylosanthes capitata, a leguminous species native to Brazil with significant economic and ecological importance. Plants were subjected to four treatments: aToW (ambient temperature with optimal water conditions), aTwS (ambient temperature with water stress), eToW (elevated temperature with optimal water conditions), and eTwS (elevated temperature combined with water stress). Flowers collected in the experiment were dehydrated in increasing ethanol series, pre-included in absolute ethanol and resin, and embedded in synthetic resin for pollen structure analysis within the flower's keel. Sections were cut with a Leica rotary microtome (2-4 microns) and stained with Toluidine Blue (TB) and Periodic Acid-Schiff (PAS). Histochemical staining included Alcian Blue, Sudan Black, Sudan III, Coomassie Blue, and Basic Fuchsin. PAS and PAS+TB reactions identified positive starch with Lugol's stain added. Analyses were conducted using a Nikon Microphot-FXA Fluorescence Light Microscope (LM). The results revealed a significant interaction between elevated temperature and water deficit on starch density within the pollen grains. As an immediate response to the imposed stresses, the starch within the pollen grain acts as an energy source to sustain pollen tube growth and facilitate successful fertilization. Moreover, the combination of these stresses led to the compaction of the intine in the pollen grain aperture region, which may hinder pollen tube germination and disrupt the reproductive processes of the species. These findings suggest that the physiological changes induced by water deficit, in combination with elevated temperature (+2 °C), can significantly impact the reproductive success of S. capitata.

This study was supported by FAPESP (Grant 08/58075-8), CNPq/ANA/MCTI (Grant 446357/2015-4), CNPq fellowship (Grants 141921/2019-6 and 304686/2022-0), and CAPES - Finance Code 001.