Transgenic insertion of the cyanobacterial membrane protein ictB increases grain yield in Zea mays through increased photosynthesis and carbohydrate production

Abstract

The C4 crop maize (Zea mays) is the most widely grown cereal crop worldwide and is an essential feedstock for food and bioenergy. Improving maize yield is important to achieve food security and agricultural sustainability in the 21st century. One potential means to improve crop productivity is to enhance photosynthesis. ictB, a membrane protein that is highly conserved across cyanobacteria, has been shown to improve photosynthesis, and often biomass, when introduced into diverse C3 plant species. Here, ictB from Synechococcus sp. strain PCC 7942 was inserted into maize using Agrobacterium-mediated transformation. In three controlled-environment experiments, ictB insertion increased leaf starch and sucrose content by up to 25% relative to controls. Experimental field trials in four growing seasons, spanning the Midwestern United States (Summers 2018 & 2019) and Argentina (Winter 2018 & 2019), showed an average of 3.49% grain yield improvement, by as much as 5.4% in a given season and up to 9.4% at certain trial locations. A subset of field trial locations was used to test for modification of ear traits and ФPSII, a proxy for photosynthesis. Results suggested that yield gain in transgenics could be associated with increased ФPSII, and the production of longer, thinner ears with more kernels. ictB localized primarily to the microsome fraction of leaf bundle-sheath cells, but not to chloroplasts. Extramembrane domains of ictB interacted in vitro with proteins involved in photosynthesis and carbohydrate metabolism. To our knowledge, this is the first published evidence of ictB insertion into a species using C4 photosynthesis and the largest-scale demonstration of grain yield enhancement from ictB insertion in planta. Results show that ictB is a valuable yield gene in the economically important crop maize, and are an important proof of concept that transgenic manipulation of photosynthesis can be used to create economically viable crop improvement traits.

Fig 1. Leaf carbohydrate content measured at different timepoints in controlled-environment experiments.

a) starch, b) sucrose, c) hexose, d) starch:total carbohydrate ratio. Bars show the LSmean, error bars give the LSD05/2. * indicates significant difference (P<0.05) from a one-tailed two-sample t-test testing BHB1356>control.

Fig 2. % difference between BHB1356 and control for yield, grouped by location, in the four growing seasons: 2018S, 2018W, 2019S, 2019W.

Bars are mean ± standard error. * indicates significant difference (P<0.05) of the Δ (i.e. BHB1356 minus control) from 0 based on a two-tailed t-test.

Fig 3. % Change in yield for BHB1356 relative to control in multiple field trials, grouped by location, and plotted against mean location yield.

Fig 4. RNA and protein expression of ictB in different plant tissues.

a) relative RNA expression of ictB gene, normalized to expression of actin, and b-d) Western-blots showing immunoprecipitation of ictB protein in transgenics and negative control WT plants. Samples of the purified ictB protein served as a reference. Pollen and kernels were sampled at R1 and R6, respectively. RT: reverse transcriptase; ND: not detected.

Fig 5. Subcellular localization of ictB.

a) confocal images of leaf cross-sections of Z. mays transgenic event 1356, showing GFP signal from ictB protein. White arrows show bundle-sheath cells. 63X magnification. b) as in panel a), but merged with bright-field and chlorophyll autofluorescence. c) Western-blot of ictB-GFP and GFP protein in mesophyll (MES) and bundle-sheath (BS) cells. d) Western-blot of ictB protein in the microsome and cytosol protein fraction. Samples of His-ictB protein of various concentrations are included as a reference, and a WT plant sample is included as a negative control. e) Western-blot of ictB protein in chloroplast, microsome, and total protein fraction.