Pallaoro, M. | bioRxiv Science

bioRxiv2026

AI-enabled simultaneous phenotyping of leaf vein and stomatal traits uncovers independent genetic control in maize

BackgroundLeaves maintain hydraulic homeostasis during photosynthesis through the coordinated action of stomata, which regulate gas exchange and transpiration, and veins, which supply water to the leaf lamina. While functional links between stomatal and vascular traits are known in dicots, their potential genetic coordination in C4 crops remains poorly understood. We investigated the genetic architecture of these traits in maize using a Multi-parent Advanced Generation Inter-Cross (MAGIC) population and a low-cost, high-throughput phenotyping platform integrating leaf clearing, digital microscopy, artificial intelligence, and image analysis ResultsWe phenotyped 285 recombinant inbred lines and the MAGIC founder lines, generating 8,072 images from 2,026 leaf samples taken from seedlings grown in controlled conditions. A YOLOv8-based model automatically detected stomata, while a custom and efficient image-processing pipeline quantified vein traits and stomatal spatial distribution patterns along cell bundles. This enabled simultaneous characterization of stomatal density, size, and distribution together with vein density, thickness, and bundle-associated spatial patterning. Substantial phenotypic variation was observed among genotypes, with strong correlations between abaxial and adaxial traits but no significant correlations between stomatal and vein traits. QTL mapping identified 37 genomic regions associated with stomatal and vein traits, including loci containing known developmental regulators such as stomatal density and distribution1 and stomagen1, as well as novel loci controlling stomatal spatial patterns, divergence between leaf surfaces and veins traits. ConclusionsThese results support independent genetic control of stomata and veins and decoupled contribution to water-use efficiency, providing a novel genetic framework to independently optimize leaf hydraulic capacity and gas exchange in target environments.

plant biology↗

bioRxiv2023

Age and anatomical region related differences in vascularization of the porcine meniscus using micro-computed tomography imaging

Meniscal lesions in vascularized regions are known to regenerate while lack of vascular supply leads to poor healing. Here we developed and validated novel methodology for three-dimensional structural analysis of meniscal vascular structures with high-resolution micro-computed tomography ({micro}CT). We collected porcine medial menisci from 10 neonatal (not-developed meniscus, n-) and 10 adults (fully developed meniscus, a-). The menisci were cut into anatomical regions (anterior horn (n-AH & a-AH), central body (n-CB & a-CB), and posterior horn (n-PH & a-PH). Specimens were cut in half, fixed, and one specimen underwent critical point drying and {micro}CT imaging, while other specimen underwent immunohistochemistry and vascularity biomarker CD31 staining for validation of {micro}CT. Parameters describing vascular structures were calculated from {micro}CT. The vascular network in neonatal spread throughout meniscus, while in adult was limited to a few vessels in outer region, mostly on femoral side. a-AH, a-CB, and a-PH had three, five, and seven times greater vascular volume than neonate, respectively. Moreover, thickness of blood vessels, in three regions, was six times higher in adult than in newborn. Finally, a-PH appeared to have thicker blood vessels than both a-AH and a-CB. For the first time, critical point drying-based {micro}CT imaging allowed detailed three-dimensional visualization and quantitative analysis of vascularized meniscal structures. We showed more vascularity in neonatal menisci, while adult menisci had fewer and thicker vascularity especially limited to the femoral surface which is involved in load transmission response, thus suggesting how nutritional support in this area of the outer zone is more necessary.

biophysics↗