Fig. 3: PHT4;4 prevents chlorosis under the combined deficiency of iron and phosphorus.

a A Manhattan plot for genome-wide association mapping using principal component 1 that explained 89.5% of expression variation of the 32 photosynthesis-related genes across 727A. thaliana accessions¹⁷. The five chromosomes are depicted by alternating light- and dark-blue colors. Dashed lines correspond to significant SNPs determined using an accelerated mixed model with adjustment for multiple tests using a FDR 5% threshold (blue) and Bonferroni α = 0.05 (red). The light-gray rectangle highlights a significant association located in an intergenic region (SNP: 4493712). Two significant associations that were followed up in this study are highlighted in red rectangles. b Representative images of wild-type Col-0, pht4;4, vtc4, and a line expressing genomic PHT4;4 in pht4;4 (pht4;4-CL) grown for 7 days in the presence of iron and phosphorus (+Fe+P) and transferred to three different media: +Fe+P, −Fe+P, or −Fe−P for 7 additional days. Scale bars: 7 mm. The experiment was repeated with similar results three times. c Total chlorophyll content in Col-0, pht4;4, vtc4, and PHT4;4-CL grown under +Fe+P, −Fe+P, −Fe−P, or −Fe+P+AsA. FW: fresh weight. Data shown are from ten plants conducted in three independent experiments. d Relative mRNA abundance of VTC genes (VTC1 (AT2G39770), VTC2 (AT4G26850), and VTC4 (AT3G02870)) in shoots of Col-0 plants grown on +Fe+P media for 7 days and transferred to +Fe+P, −Fe+P, or −Fe−P media for 52 h. Data shown are the means from 3 experiments. Error bars represent the 95% confidence interval. e Total ascorbic acid (AsA) content in Col-0, pht4;4, vtc4, and PHT4;4-CL plants grown for 7 days on +Fe+P media and transferred to +Fe+P, −Fe+P, or −Fe−P media for 52 h. Data shown are from three experiments, each with 16 plants. In box plots (c, e), center lines indicate sample medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. For c–e, letters above bars or boxes represent statistically different means at P < 0.05 (one-way ANOVA with a Duncan post hoc test). Data points are plotted as open circles. Source data are provided as a Source Data file.