Volume 8, Issue 12, Pages 1710-1724 (December 2015) Tribenuron-Methyl Induces Male Sterility through Anther-Specific Inhibition of Acetolactate Synthase Leading to Autophagic Cell Death  Lun Zhao, Xue Jing, Li Chen, Yingjun Liu, Yanan Su, Tingting Liu, Changbin Gao, Bin Yi, Jing Wen, Chaozhi Ma, Jinxing Tu, Jitao Zou, Tingdong Fu, Jinxiong Shen  Molecular Plant  Volume 8, Issue 12, Pages 1710-1724 (December 2015) DOI: 10.1016/j.molp.2015.08.009 Copyright © 2015 The Author Terms and Conditions

Figure 1 Comparison of TM-Treated and Control Rapeseed Plants. (A and B) Inflorescences of control (A) and TM-treated (B) rapeseed plants. (C–H) Flowers and pollen grains from control and TM-treated plants. Compared with the control (C–E), TM-treated plants presented unelongated filaments (F) and abnormally stained and extremely crinkled pollen grains (G and H). Scale bars, 20 μm. (I–P) Cross sections of anthers from control (I–L) and TM-treated (M–P) plants at the young microspore stage (I and M), vacuolated microspore stage (J and N), pollen mitosis I stage (K and O), and pollen mitosis II stage (L and P). Insets show magnifications of the sections surrounded by dashed lines. Arrowheads indicate the abnormally vacuolated microspores or tapetum cells. E, epidermis; En, endothecium; T, tapetum; Msp, microspore; DMsp, degraded microspore; PG, pollen grain; DPG, degraded pollen grain. Scale bars, 20 μm. Magnification area of mesophyll cells is indicated by a dashed box. Molecular Plant 2015 8, 1710-1724DOI: (10.1016/j.molp.2015.08.009) Copyright © 2015 The Author Terms and Conditions

Figure 2 In Vivo ALS Activity in Various Tissues and the Proportion of BCAAs in the Anthers of Control and TM-Treated Rapeseed. (A) Comparison of the in vivo ALS activity in the leaves, stems, and anthers of control and TM-treated rapeseed. All data are relative to that in control stems. (B) Decreased proportions of BCAAs in the total free amino acids extracted from the anthers of TM-treated rapeseed. Values are the mean ± SE from at least two independent experiments. Statistical significance was assessed by Student's t-test (*P < 0.05; **P < 0.01). Molecular Plant 2015 8, 1710-1724DOI: (10.1016/j.molp.2015.08.009) Copyright © 2015 The Author Terms and Conditions

Figure 3 Constitutive or Anther-Specific Expression of csr1-1D Rescued the TM-Induced Male Sterile Phenotype. (A) Schematic map of vector constructs. Arrows indicate the promoters: EF, constitutive expression; AMS, microspore- and tapetum cell-specific expression at the late stages of anther development; AP3, expression in the early stages of stamen and petal formation. csr1-1D and polyA are shown as open boxes. (B) Expression analyses of csr1-1D in the indicated tissues using RT–PCR. Five independent transformed lines were analyzed for each construct. Wild-type (WT) was used as a negative control. (C) Expression and in vitro ALS activity analyses of csr1-1D in distinct tissues from a pAMS:csr1-1D transgenic rapeseed line. In vitro ALS activity data are relative to the corresponding controls. (D–F) Inflorescences and pollen activity of one representative transgenic rapeseed line harboring the indicated constructs. Plants harboring the pEF::csr1-1D (D) or pAMS::csr1-1D (E) construct were male fertile (elongated filaments and red-stained mature pollen grains) after TM application, whereas plants harboring the pAP3::csr1-1D construct (F) were male sterile (unelongated filaments and abnormally stained pollen grains). Insets show magnifications of the flowers indicated by the arrows. Scale bars, 20 μm. Molecular Plant 2015 8, 1710-1724DOI: (10.1016/j.molp.2015.08.009) Copyright © 2015 The Author Terms and Conditions

Figure 4 Selective Induction of Male Sterile Branches by Daubing Stems with TM. Sterile anthers (red) only occurred on TM-daubed branches (A and B) or on the main stem and on the branches above the TM-daubed places on the main stem (C), whereas the rest of the branches were fertile (yellow). TM might be transported in a polar manner, as indicated by the red arrows on the branches. MS, male sterile anther; F, fertile anther. Molecular Plant 2015 8, 1710-1724DOI: (10.1016/j.molp.2015.08.009) Copyright © 2015 The Author Terms and Conditions

Figure 5 Bel Expression in the Mesophyll or Phloem Rescued the TM-Induced Male Sterile Phenotype. (A) Schematic map of vector constructs and Bel expression analyses in the indicated tissues. Arrows show the tissue-specific promoters: pLhCB2.1 for mesophyll-specific expression, pSUC2 for expression in phloem, and pAP3 and pAMS (described in Figure 3); Bel and NOS terminator are shown as open boxes. Five independent transformed lines were analyzed for each construct, except the pAMS::Bel construct (one line). (B–E) The inflorescences and pollen activity of one representative transgenic rapeseed line harboring the indicated constructs. Plants harboring the pLhCB2.1::Bel (B), pSUC2::Bel (C), or pAP3::Bel (D) construct showed 70%–86% fertile pollen grains after TM spraying. In contrast, plants harboring the pAMS::Bel construct (E) presented sterile anthers. Insets are magnifications of the flowers indicated by the arrows. Scale bars, 20 μm. Molecular Plant 2015 8, 1710-1724DOI: (10.1016/j.molp.2015.08.009) Copyright © 2015 The Author Terms and Conditions

Figure 6 Increased Autophagic Activity in TM-Treated Microspores and Tapetum Cells of Arabidopsis. (A) Transcript analysis of ATG8 in TM-treated Arabidopsis flower buds. Expression data relative to control were normalized to the expression of Actin. (B) Immunoblot analysis of free GFP accumulation in control and TM-treated Arabidopsis flower buds. β-Actin was included as a loading control. Relative autophagic activity is shown as the ratio of free GFP normalized to that of the control. (C) Representative TEM images of microspores and tapetum from control and TM-treated Arabidopsis at the indicated stages. Vacuolar or autophagic cell death was observed in the microspores and tapetum cells of TM-treated plants. Massive cup-shaped phagophores (pp), classic double-membrane autophagosomes (ap), and autophagic bodies (ab) appeared in the TM-treated anthers. Arrowheads show double-membrane structures. m, mitochondrion; V, vesicle; N, nucleus; T, tapetum; Msp, microspore; DMsp, degraded microspore; PW, pollen wall; P, plastid; PG, pollen grain; DPG, degraded pollen grain. Scale bars correspond to 2 μm (microspores and tapetum cells) and 0.5 μm (autophagic structures). (D) Relative autophagic activity in TM-treated microspores and tapetum cells normalized to the corresponding activities in the control. Molecular Plant 2015 8, 1710-1724DOI: (10.1016/j.molp.2015.08.009) Copyright © 2015 The Author Terms and Conditions

Figure 7 3-Methyladenine Partially Reduced TM-Induced Pollen Abortion. (A) Immunoblot analysis of free GFP accumulation and quantification of relative autophagic activity in Arabidopsis flower buds after the indicated treatment. Treatment with 1 mM 3-MA partly inhibited and 5 mM 3-MA partially and almost entirely inhibited the accumulation of free GFP induced by TM. (B) Pollen viability after the indicted treatment. Treatment with 1 mM 3-MA significantly increased the pollen viability of TM-treated Arabidopsis, whereas 5 mM 3-MA did not. The asterisks indicate a significant difference (P < 0.05; Student's t test). Molecular Plant 2015 8, 1710-1724DOI: (10.1016/j.molp.2015.08.009) Copyright © 2015 The Author Terms and Conditions

Figure 8 A Proposed Model for TM-Induced Plant Male Sterility. Most foliar-sprayed TM was polar-transported to the anthers mainly through the mesophyll and phloem. ALS is firstly expressed in the tapetum and tetrads at the tetrad stage, and maintained at a high level at the microspore and pollen mitosis stages. Anther-specific inhibition of ALS by the accumulated TM caused BCAA starvation in anther, and triggered excessive autophagy. During autophagy, bulk cytoplasmic contents were degraded, which ultimately led to sterile anthers. Molecular Plant 2015 8, 1710-1724DOI: (10.1016/j.molp.2015.08.009) Copyright © 2015 The Author Terms and Conditions