Lack of Vacuolar H+ -Pyrophosphatase and Cytosolic Pyrophosphatases Causes Fatal Developmental Defects in Arabidopsis thaliana
Lack of Vacuolar H+ -Pyrophosphatase and Cytosolic Pyrophosphatases Causes Fatal Developmental Defects in Arabidopsis thaliana
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DOI：
10.3389/fpls.2020.00655
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发表时间：
2020-05-26
影响因子：
5.6
通讯作者：
Segami, Shoji
中科院分区：
文献类型：
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作者：
Fukuda, Mayu;Mieda, Marika;Segami, Shoji
无机焦磷酸盐（PPi）的胞质水平受到精细调节，其中PPi主要由液泡H+-焦磷酸酶（H+-PPase，VHP 1/FUGU 5/AVP 1）水解，其次由五种胞质可溶性焦磷酸酶（sPPases; PPa 1-PPa 5）水解。据报道，H+-PPase功能丧失突变体（fugu 5s）在培养基中以硝酸盐为唯一氮源时，其莲座叶表现出萎缩表型。对于这种表型，两个问题仍然没有答案：为什么萎缩取决于芽和介质之间的物理接触，铵如何防止这种萎缩。为了理解驱动这种表型的机制，我们详细分析了突变体在无铵培养基上的生长和表型。fugu 5 -1在叶片中显示角质层缺陷、细胞肿胀、β-葡聚糖水平降低和脉畸形，表明细胞壁弱化和细胞致死。基于在双突变体fugu 5 -1 ppa 1和fugu 5 -1 ppa 4中观察到的与fugu 5 -1相比更严重的萎缩，氮依赖性表型可能与PPi代谢有关。为了阐明铵在这一过程中的作用，我们研究了sPPase mRNA水平的波动和替代PPi去除因子的可能性，如其他类型的焦磷酸酶。首先，我们发现sPPases的蛋白质和mRNA水平均不受氮源的影响。其次，为了评估其他PPi去除因素的影响，我们在含铵培养基上检测了H+-PPase和两种sPPase的三重敲除突变体的表型。fugu 5 ppa 1 ppa 2和fugu 5 ppa 1 ppa 4都具有几乎致死的胚胎表型，存活者表现出明显的侏儒症和异常形态。此外，fugu 5 ppa 1（+/-）ppa 4在叶缘表现出严重的萎缩。其他三重突变体，fugu 5 ppa 1 ppa 5和fugu 5 ppa 2 ppa 4，表现出根毛死亡，几乎不育，由于变形的雌蕊，分别，即使在标准培养基上生长。总之，这些结果表明H+-PPase和sPPase共同作用以维持PPi稳态，其他PPi去除剂的存在不太可能，并且铵可能会抑制氮代谢期间PPi的产生，而不是刺激PPi水解。
The cytosolic level of inorganic pyrophosphate (PPi) is finely regulated, with PPi hydrolyzed primarily by the vacuolar H+-pyrophosphatase (H+-PPase, VHP1/FUGU5/AVP1) and secondarily by five cytosolic soluble pyrophosphatases (sPPases; PPa1-PPa5) in Arabidopsis thaliana. Loss-of-function mutants of H+-PPase (fugu5s) have been reported to show atrophic phenotypes in their rosette leaves when nitrate is the sole nitrogen source in the culture medium. For this phenotype, two questions remain unanswered: why does atrophy depend on physical contact between shoots and the medium, and how does ammonium prevent such atrophy. To understand the mechanism driving this phenotype, we analyzed the growth and phenotypes of mutants on ammonium-free medium in detail. fugu5-1 showed cuticle defects, cell swelling, reduced beta-glucan levels, and vein malformation in the leaves, suggesting cell wall weakening and cell lethality. Based on the observation in the double mutants fugu5-1 ppa1 and fugu5-1 ppa4 of more severe atrophy compared to fugu5-1, the nitrogen-dependent phenotype might be linked to PPi metabolism. To elucidate the role of ammonium in this process, we examined the fluctuations of sPPase mRNA levels and the possibility of alternative PPi-removing factors, such as other types of pyrophosphatase. First, we found that both the protein and mRNA levels of sPPases were unaffected by the nitrogen source. Second, to assess the influence of other PPi-removing factors, we examined the phenotypes of triple knockout mutants of H+-PPase and two sPPases on ammonium-containing medium. Both fugu5 ppa1 ppa2 and fugu5 ppa1 ppa4 had nearly lethal embryonic phenotypes, with the survivors showing striking dwarfism and abnormal morphology. Moreover, fugu5 ppa1(+/-) ppa4 showed severe atrophy at the leaf margins. The other triple mutants, fugu5 ppa1 ppa5 and fugu5 ppa2 ppa4, exhibited death of root hairs and were nearly sterile due to deformed pistils, respectively, even when grown on standard medium. Together, these results suggest that H+-PPase and sPPases act in concert to maintain PPi homeostasis, that the existence of other PPi removers is unlikely, and that ammonium may suppress the production of PPi during nitrogen metabolism rather than stimulating PPi hydrolysis.