The molecular mechanisms behind the rapid and selective dehydration of Viola philippica fruits during seed dispersal = 호제비꽃의 종자 산포 과정 중 열매의 선택 건조 과정에 관한 분자 메커니즘

다국어 초록 (Multilingual Abstract)

In Viola philippica, seeds are dispersed explosively by the hygroscopic contraction of fruit valves. Such hygroscopic movement is made possible by the rapid and selective dehydration of the fruit. Here, I investigate the cause of selective fruit dehydration in V. philippica and its underlying molecular mechanisms.
By conducting dye tracing experiments, I discovered that xylem flow into the fruit declines just before seed dispersal. When fruit water content was measured, I found a tight temporal correlation between fruit dehydration and decline of xylem flow. Therefore, the decline of xylem flow into the fruit (i.e. hydraulic isolation) is most likely the cause of selective fruit dehydration.
To investigate the cause of fruit hydraulic isolation, I examined the tissue of hydraulic isolation, the pedicel, by resin sectioning and immunostaining. I found that pectin accumulates in the xylem lumens of the pedicel during hydraulic isolation. Furthermore, by Lugol staining of the pedicel, I found that starch accumulates specifically in the pedicel during fruit development and degrades rapidly at the onset of fruit hydraulic isolation.
In order to screen for genes responsible for fruit hydraulic isolation, I performed RNA-sequencing. Pattern clustering analysis revealed a cluster of genes specifically upregulated in the pedicel during hydraulic isolation, many of which were predicted to be involved in hypoxia and cell wall remodeling processes. Several genes putatively involved in pectin synthesis and modification were upregulated specifically in the pedicel, especially the putative pectin methylesterases VpPME1 and VpPME2. Furthermore, the putative beta-amylases VpBAM1 and VpBAM2 were found to be pedicel-specifically upregulated as well. To validate that VpPME1, VpPME2, VpBAM1 and VpBAM2 are indeed essential for fruit hydraulic isolation, knockout mutants are being generated using the CRISPR/Cas9 system and their phenotypes will be assessed in the future.
While hydraulic isolation had been observed in numerous plant species, its molecular mechanism has not been elucidated yet. The results of my study show that the accumulation of pectin inside xylem vessel elements is spatiotemporally correlated with hydraulic isolation. Furthermore, the analysis of RNA-sequencing experiments provides a foundation for the functional studies of candidate genes necessary for the detailed characterization of hydraulic isolation.

국문 초록 (Abstract)

호제비꽃은 종자를 폭발적으로 산포시키기 위해 열매를 강하게 수축시키며, 이러한 열매의 수축은 급격하고도 선택적인 열매의 건조에 의해 이루어진다. 본 연구에서는 호제비꽃 열매의 선택적인 건조를 유발하는 원인과 그 분자적 기작을 알아보고자 했다.
색소 추적 실험을 통해 호제비꽃의 종자 산포 과정 중 열매로의 물관 수송이 감소한다는 사실을 알 수 있었다. 또한 열매의 수분 함량을 측정함으로써 열매의 건조와 물관 수송의 감소 사이 높은 상관 관계가 존재함을 발견하였다. 이와 같은 결과들을 종합하였을 때, 열매 안으로의 물관 수송이 감소하는 것이 열매의 급격한 건조를 일으킨다는 것을 유추해볼 수 있었다.
물관 수송 감소의 원인을 조사하고자 물관 수송이 감소하는 조직인 꽃자루(pedicel)를 수지 섹션(resin section)과 면역 염색(immunostaining)으로 분석하였다. 그 결과, 물관 수송이 감소할 때 물관 내부에 펙틴이 다량 축적되는 것을 발견하였다. 또한 루골 염색(Lugol staining)을 통해 열매의 발달 과정 중 꽃자루에 특이적으로 녹말이 축적되며, 이 녹말은 종자가 산포할 때 급격히 분해되는 것을 관찰하였다.
물관 수송의 감소를 야기하는 유전자들을 검출하기 위해 RNA sequencing을 실시하였다. 패턴 군집 분석(pattern clustering analysis)를 통해 물관 수송 감소 중 꽃자루에서 특이적으로 발현량이 증가하는 유전자군을 동정할 수 있었으며, 이 유전자군은 저산소증(hypoxia)와 세포벽 구성 물질의 변화에 주요하게 연관되어있다고 예측되었다. 펙틴의 합성과 대사에 역할을 할 것이라 예측되는 다수의 유전자들이 꽃자루에서 발현하는 것을 발견하였으며, 그 중에서도 펙틴 메틸에스터레이즈(pectin methylesterase)인 VpPME1과 VpPME2의 발현량이 특히나 두드러졌다. 또한 베타아밀레이즈(beta-amylase)로 기능하리라 예측되는 VpBAM1과 VpBAM2의 발현량 또한 꽃자루에서 특이적으로 높았다. VpPME1, VpPME2, VpBAM1, VpBAM2의 발현이 물관 수송 감소에 필수적임을 검증하기 위해 CRISPR/Cas9을 활용한 돌연변이체를 제작하는 중이다.
물 수송 감소 현상은 과거 여러 식물 종에서 관찰된 바가 있지만, 이를 야기하는 분자적 기작은 현재로서는 밝혀진 바가 없다. 본 연구 결과는 물관 내부 펙틴의 축적이 시기적, 위치적으로 물 수송 감소 현상과 연과이 있음을 보인다. 또한, RNA-sequencing의 분석 결과는 추후 물 수송 감소 현상의 분자적 기작을 밝히기 위해 필수적인 후보 유전자들의 기능 연구에 중요한 기반을 제공하리라 생각된다.

목차 (Table of Contents)

• 1. Introduction 1
• 2. Materials and Methods 4
• 2.1 Plant materials and growth conditions 4
• 2.2 Plasmid construction and plant transformation 4
• 2.3 Dye tracing 4
• 2.4 Water content measurement 5
• 2.5 Resin section 5
• 2.6 Immunostaining 6
• 2.7 Lugol staining 7
• 2.8 RNA-seq 7
• 2.9 RT-qPCR 8
• 3. Results 10
• 3.1 The hydraulic isolation of V. philippica fruits causes its selective dehydration during explosive seed dispersal 10
• 3.1.1 The stage-specific dehydration of V. philippica fruits causes its contraction and explosive seed dispersal 10
• 3.1.2 Hydraulic isolation at the pedicel causes the selective dehydration of the fruit 14
• 3.2 Several physiological changes occur in the pedicel during hydraulic isolation 16
• 3.2.1 Pectin accumulates inside the xylem lumen of the pedicel during hydraulic isolation 16
• 3.2.2 Starch that had been accumulated specifically at the pedicel becomes degraded just before hydraulic isolation 20
• 3.3 RNA sequencing uncovered a cluster of genes specifically upregulated in the pedicel during hydraulic isolation 22
• 3.3.1 RNA sequencing was performed at three tissues during the four stages of hydraulic isolation 22
• 3.3.2 GO analysis of pedicel-upregulated genes revealed terms related to hypoxia, cell wall remodeling, and reponse to ethylene 24
• 3.4 Several genes involved in pectin synthesis and modification might be needed for hydraulic isolation 27
• 3.4.1 Several genes putatively involved in pectin synthesis and modification were upregulated in the pedicel during hydraulic isolation 27
• 3.4.2 VpPME1 and VpPME2 expression levels are extremely high in the pedicel during hydraulic isolation, but not in other tissues or developmental stages 33
• 3.4.3 VpPME1 and VpPME2 were targeted for CRISPR for the generation of stable knockout mutants 36
• 3.5 Several genes involved in starch degradation might be needed for hydraulic isolation 37
• 3.5.1 Several genes putatively functioning as beta-amylases were upregulated in the pedicel during hydraulic isolation 37
• 3.5.2 VpBAM1 and VpBAM2 were targeted for CRISPR for the generation of stable knockout mutants 40
• 4. Discussion 41
• 5. Reference 44
• 6. 국문 초록 49