Nguyen Tung C T, Obermeier Christian, Friedt Wolfgang, Abrams Suzanne R, Snowdon Rod J
Department of Plant Breeding, IFZ Research Centre for BioSystems, Land Use and Nutrition, Justus Liebig University Giessen, Germany.
Department of Chemistry, University of Saskatchewan Saskatoon, SK, Canada.
Front Plant Sci. 2016 Mar 15;7:322. doi: 10.3389/fpls.2016.00322. eCollection 2016.
The Brassica napus (oilseed rape) accession 1012-98 shows a disturbed germination phenotype that was thought to be associated with its lack of testa pigmentation and thin seed coat. Here, we demonstrate that the disturbed germination and seedling development are actually due to independent mutations that disrupt the balance of hormone metabolites and their regulators in the seeds. High-throughput UPLC-MS/MS hormone profiling of seeds and seedlings before and after germination revealed that 1012-98 has a severely disturbed hormone balance with extremely atypical, excessive quantities of auxin and ABA metabolites. The resulting hypersensitivity to abscisic acid (ABA) and a corresponding increase in dormancy often results in death of the embryo after imbibition or high frequencies of disturbed, often lethal developmental phenotypes, resembling Arabidopsis mutants for the auxin regulatory factor gene ARF10 or the auxin-overproducing transgenic line iaaM-OX. Molecular cloning of Brassica ARF10 orthologs revealed four loci in normal B. napus, two derived from the Brassica A genome and two from the C genome. On the other hand, the phenotypic mutant 1012-98 exhibited amplification of C-genome BnaC.ARF10 copy number along with a chimeric allele originating from recombination between homeologous A and C genome loci which lead to minor increase of Bna.ARF10 transcription on the critical timepoint for seed germination, the indirect regulator of ABI3, the germinative inhibitor. Bna.GH3.5 expression was upregulated to conjugate free auxin to IAA-asp between 2 and 6 DAS. Functional amino acid changes were also found in important DNA binding domains of one BnaC.ARF10 locus, suggesting that regulatory changes in Bna.ARF10 are collectively responsible for the observed phenotpyes in 1012-98. To our knowledge, this study is the first to report disruption of germination and seedling development in Brassica napus caused by the crosstalk of auxin-ABA and the corresponding regulators Bna.ARF10 and Bna.GH3.5.
甘蓝型油菜（油菜籽）种质1012 - 98表现出萌发异常的表型，人们认为这与其种皮缺乏色素沉着和种皮薄有关。在此，我们证明萌发和幼苗发育异常实际上是由于独立突变破坏了种子中激素代谢物及其调节因子的平衡所致。对萌发前后的种子和幼苗进行高通量超高效液相色谱 - 串联质谱（UPLC - MS/MS）激素分析表明，1012 - 98的激素平衡严重紊乱，生长素和脱落酸（ABA）代谢物的量极不典型且过量。由此导致的对脱落酸（ABA）超敏反应以及相应的休眠增加，常常导致吸胀后胚胎死亡或出现高频的发育异常、通常是致死性的发育表型，类似于拟南芥生长素调节因子基因ARF10的突变体或生长素过量产生的转基因系iaaM - OX。甘蓝型油菜ARF10直系同源基因的分子克隆揭示了正常甘蓝型油菜中的四个位点，两个来自甘蓝型油菜A基因组，两个来自C基因组。另一方面，表型突变体1012 - 98表现出C基因组BnaC.ARF10拷贝数扩增以及一个嵌合等位基因，该等位基因源于同源A和C基因组位点之间的重组，这导致在种子萌发的关键时间点（萌发抑制因子ABI3的间接调节因子）Bna.ARF10转录略有增加。在2至6天龄期，Bna.GH3.5的表达上调，将游离生长素缀合为IAA - 天冬氨酸。在一个BnaC.ARF10位点的重要DNA结合域中也发现了功能性氨基酸变化，这表明Bna.ARF10的调节变化共同导致了1012 - 98中观察到的表型。据我们所知，本研究首次报道了甘蓝型油菜中生长素 - ABA及其相应调节因子Bna.ARF10和Bna.GH3.5的相互作用导致萌发和幼苗发育受到破坏。
