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Evaluated in the field over locations and years, transgenic hybrids overexpressing ZmLOX6 in the mesophyll cells significantly outyielded nontransgenic sibs under managed drought stress imposed at flowering. The overexpressed ZmLOX6 was remobilized from the leaves like other major proteins during grain development. Constitutive expression or targeted expression specifically to the bundle sheath cells increased its accumulation by less than twofold. When ectopically expressed under the control of various tissue‐specific promoters, it accumulated to a fivefold higher level upon expression in the mesophyll cells than the wild‐type plants. Nitrogen supplementation selectively induced a mesophyll lipoxygenase (ZmLOX6), which was targeted to chloroplasts by a novel N‐terminal transit peptide of 62 amino acids. We identified and characterized a VSP in maize and demonstrated that its overexpression improved drought tolerance. Vegetative storage proteins (VSPs) are known to serve as nitrogen reserves in many dicot plants but remain undiscovered in grasses, most widely grown group of crops globally. Table S2 Seedling growth assay of transgenic maize events expressing ZmLOX6 gene under the control of the p ZmPEPC and p ZmrbcS promoters. Table S1 Agronomic traits for the field‐grown transgenic maize hybrids overexpressing ZmLOX6. Lack of expression in stomata is clear.įigure S14 Micrographs from transgenic maize leaves overexpressing ZmLOX6 under the control of the p ZmPEPC and p ZmrbcS promoters.įigure S15 Seedling growth of transgenic events expressing ZmLOX6 under the control of p ZmPEPC and p ZmrbcS promoters. Figure S1 Maize seedlings grown in vermiculite with various amounts of nitrogen.įigure S2 Separated leaves from 16‐day‐old plants, and a corresponding Coomassie bluestained gel of proteins extracted from these leaves.įigure S3 Nitrogen‐induced leaf proteins from maize identified by mass spectroscopy.įigure S4 Tryptic peptides of ZmLOX6 protein revealed by mass spectroscopy, and signal peptide identified by N‐terminal sequencing of the purified mature protein.įigure S5 Protein expression and antibody specificity of anti‐ZmLOX6 antibody.įigure S6 ELISA assay development for the ZmLOX6, ZmPEPC, and ZmPPDK proteins.įigure S7 Regression of grain yield on the ear leaf proteins at flowering under well‐watered conditions and managed drought stress.įigure S8 Path coefficient analysis of the influence of leaf proteins on grain yield under wellwatered conditions and managed drought stress.įigure S9 Quantification of the ZmLOX6 protein by ELISA in different zones of a maize leaf from plants grown at different levels of nitrogen.įigure S10 Tissue localization of ZmLOX6 after dissection of vascular bundles and mesophyll cells.įigure S11 Immunocytochemical localization of ZmLOX6 in a maize leaf and root with light and electron microscopy, respectively.įigure S12 Immunopurification of mature ZmLOX6 protein from maize leaf chloroplasts.įigure S13 Immunocytochemical localization of ZmLOX6 in a maize leaf expressing the ZmLOX6 gene driven by the pZ mPEPC promoter.