Supplementary Table S3

Supplementary Table S

Supplementary Table S1

Supplementary Table S

Supplementary Table S2

Supplementary Table S

Supplementary Figures

Supplementary Figure

Supplementary Table S5

Supplementary Table S

Autophagy contributes to sulfonylurea herbicide tolerance via GCN2-independent regulation of amino acid homeostasis

<p>Sulfonylurea (SU) herbicides inhibit branched-chain amino acid (BCAA) biosynthesis by targeting acetolactate synthase. Plants have evolved target-site resistance and metabolic tolerance to SU herbicides; the GCN2 (general control non-repressible 2) pathway is also involved in SU tolerance. Here, we report a novel SU tolerance mechanism, autophagy, which we call ‘homeostatic tolerance,’ is involved in amino acid signaling in <i>Arabidopsis</i>. The activation and reversion of autophagy and GCN2 by the SU herbicide tribenuron-methyl (TM) and exogenous BCAA, respectively, confirmed that TM-induced BCAA starvation is responsible for the activation of autophagy and GCN2. Genetic and biochemical analyses revealed a lower proportion of free BCAA and more sensitive phenotypes in <i>atg5</i>, <i>atg7</i>, and <i>gcn2</i> single mutants than in wild-type seedlings after TM treatment; the lowest proportion of free BCAA and the most sensitive phenotypes were found in <i>atg5 gcn2</i> and <i>atg7 gcn2</i> double mutants. Immunoblotting and microscopy revealed that TM-induced activation of autophagy and GCN2 signaling do not depend on the presence of each other, and these 2 pathways may serve as mutually compensatory mechanisms against TM. TM inhibited the TOR (target of rapamycin), and activated autophagy in an estradiol-induced <i>TOR</i> RNAi line, suggesting that TM-induced BCAA starvation activates autophagy, probably via TOR inactivation. Autophagy and GCN2 were also activated, and independently contributed to TM tolerance in plants conferring metabolic tolerance. Together, these data suggest that autophagy is a proteolytic process for amino acid recycling and contributes to GCN2-independent SU tolerance, probably by its ability to replenish fresh BCAA.</p

Differences in DNA and mRNA expression.

<p>(A) The insertion location is shown in gray rectangles in the ORF of the <i>BrTT8</i>. The black rectangles represent the exons, and TL1, TL2, TR1 and YCR1 are the primers that were developed from the corresponding exon sequences and insert sequence. The arrows are used to indicate the directions of the primers. The insertion sequences and flanking <i>BrTT8</i> intron 2 are shown in red and black, respectively. The conserved sequences at the termini of the element are underlined. Palindromic sequences that are capable of forming a hairpin are shown in blue. (B) The amplification products of the genomic DNA of the yellow-seeded (1) and black-seeded line (2) using the primers TL1 and TR1. (C) The primers TL2, TR1 and YCR1 amplified the genomic DNA from the three genotypes: (1) the homozygous yellow-seeded, (2) heterozygous black-seeded, (3) homozygous black-seeded plants. (D) mRNA levels in the immature seeds of the yellow-seeded line (three on the left) and black-seeded line (three on the right). The numbers 10, 20, and 30 signify the number of days after pollination. (E) 18S control.</p

Supplementary Table S4

Supplementary Table S

BrTT8 shows features of a bHLH DNA-Binding domain protein.

<p>(A) Amino acid comparison of the bHLH domain of <i>BrTT8</i>, <i>B. napus</i> (ABY59772.1), <i>B. rapa</i> (AEA03281), <i>B. oleracea</i> (ADP76654.1), <i>Arabidopsis</i> (NP_192720.2) and <i>Populus</i> (XP_0023067). (B) Dendrogram of the relationships among the bHLH domains from several bHLH-related proteins. For the construction of the tree, the BrTT8 protein sequence and other selected bHLH-related proteins were used. The sequence similarity was calculated using the MEGA programme to generate a branching pattern. The numbers below the branches indicate the percentages of bootstrap support after 1000 replicates. The sequences used are <i>Brassica rapa</i> AEA03281, <i>Arabidopsis</i> NP_192720.2(AtTT8), <i>Brassica napus</i> ABY59772.1, <i>Brassica oleracea</i> ADP76654.1, <i>Populus</i> XP_002306769.1, <i>Vitis vinifera</i> CBI32369.3, <i>Lotus</i> BAH28881.1(LjTT8), <i>Raphanus</i> AEO53065.1, <i>Pisum sativum</i> ADO13282.1, <i>Perilla</i> BAC56998.1(F3G1), <i>Ricinus</i> XP_002520758.1, <i>Malus</i> AEI84807.1, <i>Petunia</i> AAG25927.1(AN1), <i>Nicotiana</i> AEE99260.1, <i>Nicotiana</i> AEE99258.1(NtAN1b), <i>Hordeum vulgare</i> BAJ92594.1, <i>Lilium</i> BAE20058.1, <i>Sorghum</i> XP_002448313.1, <i>Dahlia</i> BAJ33515.1, <i>Oryza</i> NP_001053530.2, <i>Zea mays</i> NP_001105706.1, <i>Cornus</i> AAR21675.1, <i>Cornus</i> AAS86268.1, <i>Oryza</i> EEC77782.1, <i>Mimulus</i> ACA04013.1, and <i>Gynura bicolor</i> BAJ17663(GbMYC1).</p

Mapping of the <i>BrTT8</i> gene.

<p>(A) The genetic linkage map of the <i>BrTT8</i> gene. The markers gsr23, gsr44, and gsr29 are derived from Scaffold000135. (B) BLAST analysis with the <i>Arabidopsis</i> genome showed that Scaffold000135 shared similarity with a region on chromosome 4. The rectangles containing <i>Arabidopsis</i> genes present several representative genes (E<10⁻³⁰) in this region.</p