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

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.

Localization of Arabidopsis FORKED1 to a RABA-positive compartment suggests a role in secretion.

When FORKED1 (FKD1) is mutated, asymmetric localization of PINFORMED1 (PIN1), particularly to the apical side of cells, fails to occur properly in developing veins, resulting in an open vein pattern. FKD1 encodes a protein with a Pleckstrin homology-like (PL) domain, suggesting interaction with phosphoinositides. FKD1 has been previously found to interact with an ADP ribosylation factor GTPase-activating protein (ARF-GAP) important for vein patterning, SCARFACE/VAN3 (SFC). We find that FKD1-green fluorescent protein (GFP) localizes to the plasma membrane and to punctae labeled by SFC-yellow fluorescent protein (YFP). Supporting the idea that the FKD1 PL domain recognizes phosphatidylinositol 4-phosphate [PtdIns(4)P], FKD1-GFP co-localizes with PtdIns(4)P markers, and is more cytosolic when in a background mutant for the PtdIns(4,5)P2 hydrolases CVP2 and CVL1. Both FKD1 and SFC partially co-localize with markers for the trans-Golgi network (TGN), at which endocytic and secretory pathways merge. FKD1-labeled punctae rarely co-localize with the endocytic marker FM4-64, suggesting that FKD1 is not involved primarily in the endocytic pathway. FKD1 and SFC co-localize with members of the RABA group of RAB-GTPases, which are proposed to act in the post-Golgi secretory pathway. The compartments labeled by FKD1 and SFC do not localize to membrane compartments induced by the fungal toxin brefeldin A (BFA). Collectively, our data suggest that FKD1 and SFC act in a BFA-insensitive secretory pathway.

Cotyledon vascular pattern2-mediated inositol (1,4,5) triphosphate signal transduction is essential for closed venation patterns of Arabidopsis foliar organs.

Vein patterns in leaves and cotyledons form in a spatially regulated manner through the progressive recruitment of ground cells into vascular cell fate. To gain insight into venation patterning mechanisms, we have characterized the cotyledon vascular pattern2 (cvp2) mutants, which exhibit an increase in free vein endings and a resulting open vein network. We cloned CVP2 by a map-based cloning strategy and found that it encodes an inositol polyphosphate 5' phosphatase (5PTase). 5PTases regulate inositol (1,4,5) triphosphate (IP(3)) signal transduction by hydrolyzing IP(3) and thus terminate IP(3) signaling. CVP2 gene expression is initially broad and then gradually restricted to incipient vascular cells in several developing organs. Consistent with the inferred enzymatic activity of CVP2, IP(3) levels are elevated in cvp2 mutants. In addition, cvp2 mutants exhibit hypersensitivity to the plant hormone abscisic acid. We propose that elevated IP(3) levels in cvp2 mutants reduce ground cell recruitment into vascular cell fate, resulting in premature vein termination and, thus, in an open reticulum.

The identification of CVP1 reveals a role for sterols in vascular patterning.

Vascular cell axialization refers to the uniform alignment of vascular strands. In the Arabidopsis cotyledon vascular pattern1 (cvp1) mutant, vascular cells are not arranged in parallel files and are misshapen, suggesting that CVP1 has a role in promoting vascular cell polarity and alignment. Characterization of an allelic series of cvp1 mutations revealed additional functions of CVP1 in organ expansion and elongation. We identified CVP1 and found that it encodes STEROL METHYLTRANSFERASE2 (SMT2), an enzyme in the sterol biosynthetic pathway. SMT2 and the functionally redundant SMT3 act at a branch point in the pathway that mediates sterol and brassinosteroid levels. The SMT2 gene is expressed in a number of developing organs and is regulated by various hormones. As predicted from SMT2 enzymatic activity, the precursors to brassinosteroid are increased at the expense of sterols in cvp1 mutants, identifying a role for sterols in vascular cell polarization and axialization.