摘要
硫是植物生长所需要的必须营养元素,同时在植物抵御生物和非生物胁迫里起到重要作用,但是关于硫营养利用效率的分子机制的研究还少,最根本的原因在于植物对硫本身的需求量不高,所以要进行胁迫必须达到一个极低的硫浓度。在本论文里,我们利用一个理论上无硫的低硫胁迫浓度,对一个55,000独立转化株系的拟南芥激活突变体库进行高通量的筛选,获得了2株低硫耐受的突变体株系——sue3和sue4。相比较与野生型,突变体在低硫胁迫的培养基上有着更发达的主根根系,而且这种表型是对于低硫胁迫特异响应,对于其它营养胁迫都没有相应,基于以上表型我们推测也许这和硫营养的吸收能力相关。同时这两个突变体对于氧化胁迫和重金属胁迫的耐受性也远远高于野生型。对于sue3,它是一个编码VirE2-interacting蛋白1的基因,Atlg43700单基因敲除的功能缺失突变体,对于sue4,它是一个结构功能都未知的At3g55880单基因激活的功能获得性突变体,遗传试验和功能重演实验都验证了这两个突变体的低硫耐受表型,
综上所述,我们成功的建立了一个高通量筛选耐低硫突变的筛选体系,筛选到了2个确定表型的突变体株系,这些株系不仅仅是和低硫耐受相关,还很可能和硫代谢途径中硫的利用效率有关,关于这些突变体的研究,会很好的帮助我们进一步了解清楚硫胁迫过程中植物体内所发生的各种应对机制。
上面的筛选实验得到并确定由于体内At3g55880的表达量上调造成耐低硫表型的sue4突变体,通过启动子分析,蛋白定位等进一步的功能解析得到其空间表达模式可能和IAA的运出载体PIN1相重叠。细菌双杂交和双分子荧光互补实验确定SUE4和PIN1具有相互作用,并且通过进一步的分析确定很可能是由于SUE4蛋白的在sue4突变体中的上调,通过与PIN1互作,影响其根部IAA含量和分布的变化,从而产生了突变体根系构型以及低硫耐受性的表型。
Sulfur is an essential element for plant growth and development as well as for defense against biotic and abiotic stresses. But little is known about the genetic determinants for sulfate utilization efficiency, because of the technical difficulties imposed by low sulfur demand of plants.
Here we report the isolation and characterization of two low-sulfur tolerant mutants, sue3 and sue4 using a high-throughput genetic screen from an activation-tagging library of approximately 55,000 individual lines where a "sulfur-free" solid medium was devised to give the selection pressure necessary to suppress the growth of the wild type seedlings. Both mutants showed improved tolerance to low sulfur conditions and markedly increased root systems. Potentially these mutants have enhanced sulfate utilization efficiency. The mutant phenotype of both sue3 and sue4 was specific to sulfate deficiency and the mutants displayed enhanced tolerance to heavy metal and oxidative stress. Genetic analysis revealed that sue3 was caused by a single recessive nuclear mutation while sue4 was caused by a single dominant nuclear mutation. The recessive locus in sue3 is the previously identified VirE2-interacting protein 1. The dominant locus in sue4 is a function-unknown locus activated by the four enhancers on the T-DNA. The function of SUE3 and SUE4 in low sulfur tolerance was confirmed either by multiple mutant alleles or by recapitulation analysis.
Taken together, our results demonstrate that this genetic screen is a reasonable approach to isolate Arabidopsis mutants with improved low sulfur tolerance and potentially with enhanced sulfur utilization efficiency. The two loci identified in sue3 and sue4 should assist understanding the pertinent molecular mechanisms involved in low sulfur tolerance.
To investigate further the function of SUE4, the expression patteren and protein localization was analyzed via promoter-GUS reporter and GFP tagging, respectively. It was found that SUE4 showed a similar expression pattern to PIN1. SUE4 protein localization also overlapped with PIN1. This led us to test whether SUE4 and PIN1 interact. Further analysis with bacterial two hybrid and BiFC demonstrated that SUE4 interacted with the auxin efflux carrier PIN1 in vivo. This interaction likely altered IAA distribution in roots and resulted in altered root architecture of the mutant.
引文
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