Research project - WATAHIKI Laboratory Hokkaido University

Auxin signal transduction

  Plants are commonly considered as “Still lives”, however, we are just not able to sense their movement in our temporal sense.  You may see the dynamic movement and development of plants in time-lapse movie.  Plant hormone, “Auxin”, acts central roles of these “Morphogenesis” in plant.  This laboratory focuses on “Early-Auxin Inducible Genes” and investigates the mechanisms how auxin works for making new organ (called “Organogenesis”).  Arabidopsis is one of our experimental plants for forward genetics and reverse genetics but Potato and Carrot are participated in our project as well.
     Plants are auxotroph organisms and sustain our life as well as environment. Understandings of plant development will become increasingly important in future. My research aims for contribution to agriculture and sustainable environment from the academic knowledge and application of auxin research.

生长素信号转导的研究
植物通常被认为是一种静物,这是因为受视觉感官的限制我们无法感受到植物的移动。使用时序电影拍摄技术,可以观察植物的动态移动和动态生长发育过程。生长素在控制植物形态发生形成过程中起着核心作用。本实验室以模式植物拟南芥为研究对象,利用正向遗传学和反向遗传学的实验手段,从事生长素早期诱导基因的研究和生长素调控新器官发生形成的研究。此外也以马铃薯和胡萝卜为研究对象,开展生长素信号转导的研究。
   植物是一种营养缺陷型生物体,维持着我们的生命和生存环境。深入理解植物生长发育机理将会变得越来越重要。本实验室着重于把学术知识和生长素的研究成果应用于农业生产和可持续环境发展的研究目标。

Root regeneration

  A plant’s root system is highly regenerative. It plays a critical role in absorbing water and nutrients from the soil and therefore its loss can be an immediate threat to their lives. The plasticity of the root system also helps plants adopt to adverse conditions such as drought. An agricultural technique called root pruning, or root cutting, uses this natural robustness to control plant growth. It has also been used in horticulture to control plant size and vigor as seen in Bonsai. Previous studies have suggested that root regeneration occurs through the induction of lateral root (LR) formation, and that auxin, a well-studied growth hormone involved in various processes of plant development, plays a role in the process. However, the molecular mechanism behind root regeneration has remained largely unknown.

 

Using Arabidopsis as a model,  root cutting induces both LR formation and the growth of existing roots. Experiments investigating gene expressions and using mutants identified YUCCA9 as the primary gene responsible for auxin biosynthesis during root-system regeneration after root cutting. In collaboration with Professor Masashi Asahina of Teikyo University, the team also found an evident increase in the level of auxin after cutting.Increased levels of AUX/IAA19, which indicate an activation of auxin signaling, were observed in the cut-end of root-cut plants compared to intact plants. Scale bar = 0.1 mm. (Xu D. et al., Plant and Cell Physiology, September 1, 2017)

Auxin commonly shows an uneven distribution in plant bodies as a result of polar transportation, leading to gravity- or light-induced bending of the plant. The team found that the polar transport system is required for root regeneration as well. Interestingly, the defective LRs of some auxin signaling mutants can be recovered by root cutting, suggesting the robustness of the auxin signaling induced by root cutting. They also showed a redundancy of auxin biosynthesis genes by mutant analysis. The primary gene of auxin biosynthesis which is responsible for root regeneration upon root damage. This finding could lead to the development of new methods for suppressing or enhancing root regeneration, and thus controlling plant growth in agriculture and horticulture.

生长素信号转导的研究
植物通常被认为是一种静物,这是因为受视觉感官的限制我们无法感受到植物的移动。使用时序电影拍摄技术,可以观察植物的动态移动和动态生长发育过程。生长素在控制植物形态发生形成过程中起着核心作用。本实验室以模式植物拟南芥为研究对象,利用正向遗传学和反向遗传学的实验手段,从事生长素早期诱导基因的研究和生长素调控新器官发生形成的研究。此外也以马铃薯和胡萝卜为研究对象,开展生长素信号转导的研究。
植物是一种营养缺陷型生物体,维持着我们的生命和生存环境。深入理解植物生长发育机理将会变得越来越重要。本实验室着重于把学术知识和生长素的研究成果应用于农业生产和可持续环境发展的研究目标。

Remodeling of auxin response

   Auxin-insensitive mutants, massugu1 (msg1) and massugu2 (msg2) showed defect in gravitropism in etiolated hypocotyl. These mutant tend to grow straight (massugu in Japanese) irrespective of the direction of gravity. Both mutants are isolated by auxin-lanolin curvature test and auxin sensitivity reduction in hypocotyl (Watahiki and Yamamoto, 1997). msg1 is a recessive mutant and encodes ARF7 transcriptional activator, while msg2 is a dominant mutant and encodes AUX/IAA19, which is induced by auxin and function as the suppressor of ARF7  through TOPLESS protein. Dominant phenotype of msg2 caused by point mutation of AUX/IAA19 domainII, which is responsible for the interaction with TIR1/AFBs. How dominant mutation suppresses auxin response? We hypothesize that auxin response of AUX/IAA19 is triggered by release of the suppression of ARF7 transcriptional activator (see “Binding with ARF and functional suppression” in figure below). ①Tir1/AFBs auxin receptor and AUX/IAA19 protein binds each other by small chemical, auxin (indole-3-acetic acid). AUX/IAA19 protein may be ubiquitinated by SCF complex and ubiquitinated AUX/IAA19 degraded by 26S proteasome pathway. ② the decreased cellular (nuclear) concentration of AUX/IAA19 protein will lead to release the suppression of ARF7 function. And then activated ARF7 transcriptional activator promotes target genes for expression. ③ However, ARF7 also activates AUX/IAA19 gene expression and de-novo protein of AUX/IAA19 will suppress ARF7 again.  After all, auxin concentration controls cellular (nuclear) concentration of AUX/IAA19 negatively and further regulates transcriptional activity of ARF7. So, dominant mutation of domainII in msg2 mutant may explain that protein degradation of msg2 is not occurred by increase of cellular auxin level.

生长素响应模式重塑的研究
     拟南芥生长素不敏感突变体massugu1 (msg1)和massugu2 (msg2),其黄化胚轴失去了向地性的响应,并表现出不受重力方向影响的直立生长(massugu的日文意思)的趋势。这两个突变体是通过auxin-lanolin curvature test和拟南芥胚轴对生长素响应敏感性降低实验被发现分离出来的 (Watahiki and Yamamoto, 1997)。msg1是隐性突变体,其基因编码的蛋白是ARF7转录激活因子,相比之下msg2是显性突变体,其基因AUX/IAA19受生长素的诱导,AUX/IAA19蛋白可以与 TOPLESS蛋白结合作为共阻遏物,抑制ARF转录激活因子的活性。显性突变体msg2是由于AUX/IAA19蛋白 domainII发生了点突变,从而影响AUX/IAA19蛋白和TIR1/AFBs蛋白的互作。显性突变体msg2是怎样抑制生长素响应的呢?我们推断AUX/IAA19基因对生长素的响应,是通过释放对ARF7转录激活因子的抑制而进行的 (请参照下面的Binding with ARF and functional suppression图片)。首先①生长素受体Tir1/AFBs蛋白和AUX/IAA19蛋白通过生长素相互连接在一起。AUX/IAA19蛋白可以被SCF complex泛素化,泛素化的AUX/IAA19蛋白随后被26S 蛋白酶体系统所降解;其次②因为降解AUX/IAA19 蛋白细胞浓度(核细胞)会降低,从而促使AUX/IAA19 蛋白对ARF7的抑制被释放,进而被激活的ARF7转录激活因子会诱导其目的基因的表达;再次③活化后ARF7将激活目的基因AUX/IAA19的表达,从而新合成的AUX/IAA19蛋白又会重新抑制ARF7的功能。以上是生长素响应模式途径,总而言之,生长素负调控AUX/IAA19 蛋白细胞浓度(核细胞),从而更进一步调控ARF7转录激活因子活性。因此,利用domainII 点突变显性突变体msg2可以用来解释msg2蛋白降解是不受细胞生长素水平升高的影响。

 

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    Although this model may apply if we do not consider of the time, the expression of AUX/IAA19 is transient (increase expression and then decrease) by application of auxin. Recent our study suggests that the auxin responsiveness of plant tissue can be discriminated in basal state and stationary state. The organogenesis progresses by stepwise stages and auxin may contribute to each step. We speculate the conversion of “Basal state” and “Stationary state” could explain this stepwise progression of organogenesis. We named this conversion of auxin responsiveness as “Remodeling of auxin response” and dedicate to investigate the molecular mechanisms.

     如果不考虑时间因素的影响,以上生长素响应模式途径是合理的,但是当受到生长素处理时, AUX/IAA19基因在植物细胞体内的表达是瞬时的(先升高后降低)。近来,我们的研究结果表明:植物组织对生长素的响应能力可以分成基础阶段和定态阶段两个阶段。器官发生形成是一个逐步进行的过程,并且生长素可能参与每个阶段。我们推断植物对生长素响应由基础阶段向定态阶段的转变,可以解释为什么植物器官发生形成是逐步进行的。我们将生长素响应能力的转变命名为“生长素响应模式重塑”,并致力于探索其分子机理。

Visualization of auxin response

 The activity of auxin-inducible gene promoter could be visualized by gene reporters. Aux/IAA19 promoter which shows “Remodeling of auxin response” is mainly employed for our research with the fusion of β-glucuronidase (GUS), Green fluorescence protein (GFP), Luciferase (Luc) which emit bioluminescence.

生长素响应可视化的研究
       生长素响应基因启动子活性可以通过与报告基因的连接实现可视化。将Aux/IAA19基因启动子与化学荧光报告基因β-葡萄糖醛酸酶(GUS),生物荧光报告基因绿色荧光蛋白(GFP)或者荧光素酶(Luc)相连接,可以观察Aux/IAA19基因启动子对生长素响应模式是重塑的。
 

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       Sequential photos of Arabidopsis influorescence stem which shows negative gravitropism (left) and bioluminescence of pIAA19:Luc which responds to auxin redistribution alongside with stem. Note that lower side of the stem shows high expression compared with upper side during stem bending but less difference in stood stem.

     拟南芥花序茎反向地性生长连续图和pIAA19:Luc对沿茎侧生长素重新分配响应的荧光连续图。可以发现对于弯曲的茎,pIAA19:Luc表达量在茎的下侧高于茎的上侧;对于直立的茎,pIAA19:Luc表达量在茎的下侧和上侧没有明显区别。

Grwoth of pollen tube and calcium oscillation

      Pollen tube is one of the paticular cells in plant. The tip of pollentube expands its surface area and forms cylindrical shape cell. This manner of growth called “tip growth” and trichome or root hair formation is also indicated . Active docking of vesicles occurs in the tip region of pollen tube and expands the tip but it is not well known how the diameter of tube or rate of growth are regulated. Calcium oscillation of pollen tube tip occurs with periodical changes of growth rate. However how its oscillation maintained is not known.

花粉管生长和钙震荡的研究
      植物花粉管是一种特殊的细胞。花粉管的尖端会扩张其表面区域并形成圆柱型的细胞。这种生长方式被称为“尖端生长”,在表皮毛和根毛形成过程中也存在这种生长方式。活性小泡会富集在花粉管的尖端并促使其扩张,目前对于花粉管直径,和花粉管生长速率调控机理还有待更深入的研究。花粉管尖端钙震荡会随花粉管尖端生长速率周期的变化而变化,但是对于花粉管尖端钙震荡维持的机理还需要进一步的研究。
 

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Oscillation free calcium concentration in pollen tube tip
花粉管尖端自由钙离子浓度震荡图

YOUTUBE(res-movie2)