A vegetative storage protein improves drought tolerance in maize

Summary 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. We identified and characterized a VSP in maize and demonstrated that its overexpression improved drought tolerance. Nitrogen supplementation selectively induced a mesophyll lipoxygenase (ZmLOX6), which was targeted to chloroplasts by a novel N‐terminal transit peptide of 62 amino acids. 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. Constitutive expression or targeted expression specifically to the bundle sheath cells increased its accumulation by less than twofold. The overexpressed ZmLOX6 was remobilized from the leaves like other major proteins during grain development. 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. Additional storage of nitrogen as a VSP in maize leaves ameliorated the effect of drought on grain yield.

. Nitrogen-induced proteins identified by mass spectroscopy from the leaves of maize seedlings. The induced bands were excised, treated with trypsin as described in M&M, and eluted peptides subjected to LC-MS-MS. Five different polypeptides were identified from the 100 kD band and only one from the 50 kD band.  Figure S4 Tryptic peptides revealed by mass spectroscopy corresponding to ZmLOX6 protein (a) are highlighted along with their amino acid positions in the protein in grey boxes in the bar graph below (b). The green arrow (position 1-62) represents the transit peptide (corresponding bold font in the sequence) and blue box represents the fourteen amino acids (blue italics) identified by N-terminal sequencing of the ZmLOX6 protein immunopurified from maize leaves. Potential trypsin target sites in the N-terminal portion of the predicted protein are highlighted in red (Arg) and green (Lys) fonts. Lysyl residue at position 81, where the first trypic peptide was identified, is highlighted. Peptides 2, 4, and 11 contained 1, 2, and 4 additional sites for trypsin digestion, which each resulted in independent peptides as identified by mass spectroscopy but are subsumed in the numbered peptides. A total of 19 peptides were identified. ZmLOX6 amino acid composition in terms of percent frequency and mol percentage of each amino acid in the protein is shown in panel c. The ZmLOX6 protein (GenBank accession DQ335764) has a molecular mass of 97.395 kDa and an isoelectric point of 5.48.  Regression of grain yield on the ear leaf proteins at flowering under well-watered conditions (a, c, e) and managed water stress (b, d, f). Average concentrations of the proteins were: ZmLOX6, 0.7%; ZmPEPC, 4.7%; and ZmPPDK, 4.9%. Under well-watered conditions, PEPC explained most variation, a result that agrees with the other unpublished data from our group. Under stress, however, the LOX6 protein alone explained 56% of the variation. A positive correlation suggests that the hybrids that accumulated more LOX6 protein in their leaves at flowering also had higher grain yield. Although the correlations are relatively weak, an opposite trend was observed with the PEPC and PPDK proteins. Horizontal and vertical bars within the plots are least significant difference (LSD) estimates at 5% level of significance for the traits represented on the respective axes.

Figure S8
Path coefficients representing direct and indirect contributions of leaf proteins to grain yield under well-watered conditions (a) and managed water stress (b). Above, path diagrams; below, total variation explained in grain yield and variation explained by each protein. Path coefficients are represented by single-headed arrows. Double-headed arrows are for the correlation coefficients between the two variables they connect. Each path coefficient represents the direct and indirect contribution through the other variables it is connected to. Under well-watered conditions, the total variation in grain yield explained (R 2 ) by the three proteins was 42%, with ZmPEPC explaining the most variation, 30%, a result that is supported by the other, unpublished data from our group on larger germplasm screening. Under water stress, the ZmLOX6 protein alone explained more than half of the variation in grain yield whereas all three proteins together explained 2/3 rd of the variation. ZmLOX6 was the best predictor of grain yield under water stress. Further, ZmPEPC and ZmPPDK are the C4 enzymes so altering their expression had the potential to interfere with the seminal process of photosynthesis. ZmLOX6 protein was thus a natural choice for further, in-depth exploration. Immunopurification of mature ZmLOX6 protein from maize leaf chloroplasts expressing the ZmLOX6 gene under the control of the pZmPEPC promoter. Lanes A, flow-through fraction; B to G, wash fractions; H, eluent from 100 mM glycine, pH 2.8; I, eluent from 6M guanidine-HCl wash following glycine elution; and J, eluent from untreated beads from affinity column with 100 mM glycine, pH 2.8. Arrow points to the ZmLOX6 protein.

Figure S13
Immunocytochemical localization of ZmLOX6 in a wild type maize leaf (a) and transgenic plants generated with ZmLOX6 driven by the pZmPEPC promoter (b-d). Panel B is the same as panel c in Fig. 5 and is included as a reference. Stomatal guard cell pairs are pointed out with asterisks. Color was artificially enhanced in panel d. The promoter pZmPEPC was specific to the mesophyll cells and did not appear to express in the epidermis or guard cells. It is possible, but appears unlikely, that some ZmLOX6 protein was expressed in the guard cells as this protein is expressed at low level in the mesophyll cells (a) but was below the threshold of detection with microscopy. Bar scale 20 µm.

Figure S14
Micrographs obtained from transgenic maize plants overexpressing ZmLOX6 under the control of the pZmPEPC (a) and pZmrbcS (b) promoters. Micrograph 'a' is a truncated, enlarged version of Supplementary Figure 13C. The color in the micrograph is enhanced to ensure detection of any signal in the guard cell pair. pZmPEPC promoter did not appear to express in the guard cells whereas pZmrbcS promoter did. Asterisks point to a guard cell pair in each micrograph. The AcGFP protein in Fig. 3 was expressed in the guard cells because those micrographs were obtained from transient expression of AcGFP under the control of pZmUbiintron promoter after biolistic transformation, which directly transformed the guard cells. The objective of that experiment was to demonstrate the targeting of the AcGFP protein to the chloroplasts by the novel transit peptide of 62 N-terminal amino acids of the ZmLOX6 protein we identified by N-terminal sequencing of the immunopurified protein from leaves. The micrographs in this figure and in Supplementary Figure 13, in contrast, show the expression of the respective promoters in different cell types in stable transgenic events. Bar scale: 10 µm.

Figure S15
Seedling growth assay of transgenic events expressing ZmLOX6 gene under the control of a vascular bundlespecific promoter, pZmrbcS, or mesophyll-specific promoter, pZmPEPC. Data from Table 2 were averaged across the transgenic events and controls, respectively, and the ratios of the respective traits in transgenic events against controls presented. The red dotted line represents a ratio of 1 or 100%. Total nitrogen in the transgenic events expressing the ZmLOX6 gene under the control of the pZmPEPC promoter was increased whereas it was unchanged in the events expressing it under the control of the pZmrbcS promoter. An increase in total biomass explained the increase in total nitrogen in the pZmPEPC-driven events. Expression of the ZmLOX6 gene under the control of the pZmrbcS promoter increased nitrogen concentration but it was compensated by a reduction in biomass, leaving the total seedling nitrogen unchanged. 16