Atrazine chlorohydrolase (AtzA) catalyzes hydrolytic dechlorination and can be used in detoxification of atrazine, a herbicide widely employed in the control of broadleaf weeds. In this study, to investigate the potential use of transgenic tobacco plants for phytoremediation of atrazine, atzA genes from Pseudomonas sp. strain ADP and Arthrobacter strain AD1 were transferred into tobacco. Three and four transgenic lines, expressing atzA-ADP and atzA-AD1, respectively, were produced by Agrobacterium-mediated transformation. Molecular characterization including PCR, RT-PCR and Southern blot revealed that atzA was inserted into the tobacco genome and stably inherited by and expressed in the progenies. Seeds of the T(1) transgenic lines had a higher germination percentage and longer roots than the untransformed plants in the presence of 40-150 mg/l atrazine. The T(2) transgenic lines grew taller, gained more dry biomass, and had higher total chlorophyll content than the untransformed plants after growing in soil containing 1 or 2 mg/kg atrazine for 90 days. No atrazine residue remained in the soil in which the T(2) transgenic lines were grown (except 401), while, in the case of the untransformed plants, 0.91 mg (81.3%) and 1.66 mg (74.1%) of the atrazine still remained in the soil containing 1 and 2 mg/kg of atrazine, respectively, indicating that the transgenic lines could degrade atrazine effectively. The transgenic tobacco lines developed could be useful for phytoremediation of atrazine-contaminated soil and water.

Summary Ubiquitination is a fundamental mechanism regulating the stability of target proteins in eukaryotes; however, the regulatory mechanism in seed longevity remains unknown. Here, we report that an uncharacterized E3 ligase, ARABIDOPSIS TÓXICOS EN LEVADURA 5 (ATL5), positively regulates seed longevity by mediating the degradation of ACTIVATOR OF BASAL TRANSCRIPTION 1 (ABT1) in Arabidopsis. Seeds in which ATL5 was disrupted showed faster accelerated aging than the wild‐type, while expressing ATL5 in atl5‐2 basically restored the defective phenotype. ATL5 was highly expressed in the embryos of seeds, and its expression could be induced by accelerated aging. A yeast two‐hybrid screen identified ABT1 as an ATL5 interacting protein, which was further confirmed by bimolecular fluorescence complementary assay and co‐immunoprecipitation analysis. In vitro and in vivo assays showed that ATL5 functions as an E3 ligase and mediates the polyubiquitination and degradation of ABT1. Disruption of ATL5 diminished the degradation of translated ABT1, and the degradation could be induced by seed ageing and occurred in a proteasome‐dependent manner. Furthermore, disruption of ABT1 enhanced seed longevity. Taken together, our study reveals that ATL5 promotes the polyubiquitination and degradation of the ABT1 protein posttranslationally and positively regulates seed longevity in Arabidopsis.

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Seed longevity is an important trait for agriculture and the conservation of genetic resources. b-1,3-Glucanases were first recognized as pathogenesis-related proteins involved in plant defense, but their roles in seeds are largely unknown. Here, we report a glycosylphosphatidylinositol-anchored b-1,3-glucanase, BG14, that degrades callose in seed embryos and functions in seed longevity and dormancy in Arabidopsis. The loss of function of BG14 significantly decreased seed longevity, whereas functional reversion (RE) and overexpression (OE) lines reversed and increased the impaired phenotype, respectively. The loss of function of BG14 enhanced callose deposition in the embryos of mature seeds, confirmed by quantitative determination and the decreased callose degrading ability in bg14. The drop-and-see (DANS) assay revealed that the fluorescence signal in bg14 was significantly lower than that observed in the other three genotypes. BG14 is located on the periphery of the cell wall and can completely merge with callose at the plasmodesmata of epidermal cells. BG14 was highly expressed in developing seeds and was induced by aging and abscisic acid (ABA). The loss of function of BG14 led to a variety of phenotypes related to ABA, including reduced seed dormancy and reduced responses to treatment with ABA or pacolblltrazol, whereas OE lines showed the opposite phenotype. The reduced ABA response is because of the decreased level of ABA and the lowered expression of ABA synthesis genes in bg14. Taken together, this study demonstrated that BG14 is a bona fide BG that mediates callose degradation in the plasmodesmata of embryo cells, transcriptionally influences ABA synthesis genes in developing seeds, and positively affects seed longevity and dormancy in Arabidopsis.

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