grasviq: an image analysis framework for automatically quantifying vein number and morphology in grass leaves

Abstract

Summary Beyond facilitating transport and providing mechanical support to the leaf, veins have important roles in the performance and productivity of plants and the ecosystem. In recent decades, computational image analysis has accelerated the extraction and quantification of vein traits, benefiting fields of research from agriculture to climatology. However, most of the existing leaf vein image analysis programs have been developed for the reticulate venation found in dicots. Despite the agroeconomic importance of cereal grass crops, like Oryza sativa (rice) and Zea mays (maize), a dedicated image analysis program for the parallel venation found in monocots has yet to be developed. To address the need for an image‐based vein phenotyping tool for model and agronomic grass species, we developed the grass vein image quantification (grasviq) framework. Designed specifically for parallel venation, this framework automatically segments and quantifies vein patterns from images of cleared leaf pieces using classical computer vision techniques. Using image data sets from maize inbred lines and auxin biosynthesis and transport mutants in maize, we demonstrate the utility of grasviq for quantifying important vein traits, including vein density, vein width and interveinal distance. Furthermore, we show that the framework can resolve quantitative differences and identify vein patterning defects, which is advantageous for genetic experiments and mutant screens. We report that grasviq can perform high‐throughput vein quantification, with precision on a par with that of manual quantification. Therefore, we envision that grasviq will be adopted for vein phenomics in maize and other grass species.

Hormonal control of medial–lateral growth and vein formation in the maize leaf

“…The vt2 mutant has wider leaves and more numerous 3° veins (see juvenile leaf phenotypes in Fig. 5d–f and Robil et al ., 2021; adult leaf phenotypes in Fig. S10d–f).…”

“…S10d–f). Despite the increased vein number, the mutant displays normal leaf and vascular anatomy, including normal vein size and spacing (Figs 5c, S10c; quantification in Robil et al ., 2021). These phenotypes suggest that extra cell files form in the median cell layer between 2° veins in vt2 .…”

“…Transgenic and mutant lines were backcrossed with B73 at least three times. Plants were grown in a glasshouse as described previously (Robil et al ., 2021) and were collected at the V2 stage with emerging third leaf (L3) for confocal analyses and hormone quantification and at V5–V6 stage with fully emerged L5 for genetic analyses. Adult phenotypes were analyzed from plants grown at the Genetics Farm in Columbia, MO, USA, during the summer.…”

“…The abnormal leaf width in maize auxin, CK, and GA biosynthesis and signaling mutants indicates that these hormones regulate ML leaf growth. The auxin biosynthesis mutant, vanishing tassel2 ( vt2 ), which encodes the maize co‐ortholog of TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1 (TAA1), has wider leaves than normal (Phillips et al ., 2011; Robil et al ., 2021). The GA biosynthesis mutants, anther ear1 ( an1 ), dwarf plant1 ( d1 ), d3 , and d 5, and the GA transcriptional repressor DELLA mutants, Dwarf plant8 ( D8 ) and D9 (Phinney et al ., 1986; Lawit et al ., 2010), also have wider leaves.…”

A rapid method to quantify vein density in C4plants using starch staining

“…Although, the powerful programme LeafVeinCNN (Xu et al ., 2021) enables high-throughput analysis of traits including vein order, areole number, vein width and vein density it currently requires manual real-time supervision of individual images and can be computationally slow due to the number of networks in use and the number of measurements being attained per image (Xu et al ., 2021). Further, to our knowledge, only one tool has been developed to assess vein structure in monocotyledons (Robil et al ., 2021). As C 4 photosynthesis is found in both monocotyledons and dicotyledons it would be useful to have a tool that permits analysis of vein density in both clades.…”