Involvement of a truncated MADS-box transcription factor ZmTMM1 in root nitrate foraging

A MADS-box transcription factor transcriptionally up-regulated and modulating lateral root development in response to local nitrate supply exists as a truncated form unique to grass species.


Supplementary Data
Fig. S1: The exon-intron structure of AGL17-like genes in Arabidopsis and rice.         Table S1: Gene structure of truncated AGL17-like genes and orthologs in monocots identified from comparative genome analysis. Table S2: Primers used in this study.
Dataset S1: Comparative genome analysis of AGL17-like genes in monocots.

Supplementary Fig. S1. The exon-intron structure of AGL17-like genes in Arabidopsis and rice
Gene structure of AGL17-like genes in Arabidopsis (A) and rice (B). Gene structure annotation was conducted by yrGATE algorithm. Blue blocks stand for exons and blue lines for introns. Magentacolored lines highlight the intron inserted into the gap between I-and K-domain of MIKC-type MADS-box genes. Green and red arrow heads indicate the site of start and stop codons, respectively.

Supplementary Fig. S2. Root preferential expression of ZmTMM1 in maize
Expression level of ZmTMM1 transcript was analyzed by quantitative real-time PCR and normalized by maize GAPDH (gi22302). a Samples were collected from field-grown maize plants at seedling stage (28 days after germination), b at silking stage, and c at stage of 15 days after pollination. Data represent means ± SD (n = 3 replicates; each replicate represents a single seedling). (B) Root growth of Col-0 and dko plants under homogenous nitrate supply. Plants were grown on Nfree half-strength MS agar plates supplemented with 1 mM KNO3 for 12 days. Bars represent means ± SE (n = 16 replicates; each replicate represents a single seedling). Asterisk indicates significant differences between dko and Col-0 at *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant (Student's t-test).
(C) Root response of Col-0 and dko mutant to localized nitrate supply in vertically split segmented agar plates. Arabidopsis seedlings pruned to have only two first-order LRs were transferred to split agar plates containing 1 mM KNO3 in the +N patch and 0.5 mM K2SO4 in the -N patch, respectively.
Root phenotypes were measured 6 days after transfer. Bars represent means ± SE (n = 12 replicates; each replicate represents a single seedling). Different letters represent significant differences among means at P < 0.05 (Tukey's test). transcript levels in response to local nitrate supply in WT and ZmTMM1-RNAi lines. Gene expression was detected 12h after the transfer of seedlings to local nitrate treatment using split-root systems.

Relative transcript levels of indicated genes were determined by qPCR and normalized by maize
Tubulin 4 (AJ420856). Data represent means ± SD (n = 4 replicates; each replicate represents a single seedling). Different letters indicate significant differences between the four bars in each group at P < 0.05 (Tukey's test). (C) LR development of WT and ZmTMM1-RNAi lines in response to local nitrate supply. Root growth were measured at 5 days after the transfer to the split-root system. Data represent means ± SD (n = 4 replicates; each replicate represents two seedlings). Different letters indicate significant differences between the four bars in each group at P < 0.05 (Tukey's test). Supplementary Fig. S9. Expression of Arabidopsis AGL17-like genes in response to local nitrate supply.
Arabidopsis seedlings were cultivated in a split-root system which contained 1 mM KNO3 in the +N patch and 0.5 mM K2SO4 in the -N patch. After 12 h of local nitrate treatment in the split-root system, transcipt levels of AGL16, AGL17, AGL21 and ANR1 in roots were determined by qPCR and normalized by AtUBQ10. Data represent means ± SD (n = 3 replicates; each replicate represents a single seedling).
Asterisks indicate significant differences between the gene expression in roots on +N and -N patches at: *, p < 0.05; **, p < 0.01; ns, not significant (Student's t-test).

Supplementary Table S2. Primers used in this study
Primer name Primer sequence Purpose