拟南芥SPL1基因参与调节低磷条件下的根际酸化反应

doi:10.11983/CBB15080

摘要/Abstract

摘要： 根际酸化是植物适应低磷胁迫的重要策略, 但植物是如何感知和转导低磷信号, 进而促进根际酸化的分子机制至今还不十分清楚。利用pH指示剂(溴甲酚紫)显色法从拟南芥(Arabidopsis thaliana) T-DNA插入突变体库中分离得到了1株低磷诱导根际酸化缺失突变体spl1。在含溴甲酚紫的低磷培养基上培养8小时, 野生型拟南芥根际培养基的颜色变为黄色, 而突变体spl1根际培养基的颜色没有明显变化, 表明spl1的低磷根际酸化反应能力降低。当低磷胁迫处理延长20天, spl1叶片的花青素积累明显高于野生型。同时也出现, 即使在磷营养正常条件下, spl1突变体也表现出根毛数量与长度增加的特征。进一步的研究表明, 在低磷条件下, spl1突变体根部的磷含量略高于野生型, 与磷转运相关基因的表达量明显高于野生型。分子遗传学分析结果表明, SPL1基因受低磷胁迫诱导, 主要在拟南芥的叶片和花等组织中表达, 其编码的蛋白广泛分布在细胞的各个部位。以上结果表明, SPL1参与介导低磷诱导的拟南芥根际酸化反应, 调节多种低磷胁迫反应及低磷条件下磷饥饿诱导基因的表达。

关键词: 拟南芥, 根际酸化, spl1, 低磷

Abstract: Under low phosphate (Pi), the rhizosphere acidification of plants is a vital strategy to cope with low Pi stress. However, how plants perceive and transduce the low Pi signal and acidify the rhizosphere is not clear. A mutant, spl1, with decreased rhizosphere acidification induced by low Pi, was isolated from a T-DNA library by using the pH indicator (bromocresol purple). After 8 h incubation in low Pi medium containing bromocresol purple, the color of the rhizosphere of wild-type (WT) seedlings on the low Pi medium turned yellow, whereas that of the rhizosphere of spl1 seedlings did not change, which suggested decreased rhizosphere acidification capacity of spl1. The anthocyanin content was higher for spl1 than the WT with 20 days of low Pi treatment. The number and the length of root hairs of spl1 increased under normal conditions. Further research suggested that the Pi content of the spl1 mutant was slightly decreased in shoot but increased in root under Pi deficiency. Moreover, the expression of Pi transport-related genes in the spl1 mutant increased under Pi deficiency. Molecular genetics analysis revealed that the expression of SPL1 was induced by low Pi stress. SPL1 was mainly expressed in Arabidopsis leaves and flower tissues and the SPL1 protein was widely distributed in each part of the cell. These results indicate that SPL1 is involved in low Pi-induced rhizosphere acidification, regulation of multiple low Pi responses and Pi starvation-induced gene expression.

Key words: Arabidopsis, rhizosphere acidification, spl1, low Pi

雷凯健, 任晶, 朱园园, 安国勇. 拟南芥SPL1基因参与调节低磷条件下的根际酸化反应. 植物学报, 2016, 51(2): 184-193.

Kaijian Lei, Jing Ren, Yuanyuan Zhu, Guoyong An. SPL1 is Involved in the Regulation of Rhizosphere Acidification Reaction Under Low Phosphate Condition in Arabidopsis. Chinese Bulletin of Botany, 2016, 51(2): 184-193.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB15080

https://www.chinbullbotany.com/CN/Y2016/V51/I2/184

图/表 7

图1 拟南芥spl1突变体的鉴定及根际酸化缺失表型分析 (A) spl1-1和spl1-2的T-DNA插入位点示意图; (B) RT-PCR分析SPL1的表达; (C) 溴甲酚紫显色法分析WT和spl1在MS及低磷(LP)培养基上(12.5 μmol∙L−1 H2PO4−)的根际酸化反应(Bar=1 cm); (D) WT和spl1的酸化定量。*表示与野生型相比差异显著(P<0.05)。

Figure 1 Arabidopsis thaliana spl1 mutants show the phenotype of rhizosphere acidification deficiency and decreased acidi- fication capacity (A) T-DNA insertion sites in spl1-1 and spl1-2; (B) Reverse transcriptase-PCR analysis of SPL1 expression; (C) The rhizosphere acidification reaction of WT and spl1 mutants under MS and low phosphate (LP) conditions (12.5 μmo∙L−1 H2PO4−) (Bar=1 cm); (D) The acidification capacity of WT and spl1 mutants. Asterisk represent statistically differences compared with the wild type (P<0.05).

图2 低磷条件下拟南芥突变体spl1-1和spl1-2花青素的积累 (A) 在正常和低磷条件下, WT、spl1-1和spl1-2的叶片表型特征; (B) 低磷处理20天, 野生型(WT)、spl1-1和spl1-2的花青素含量变化。*表示与野生型相比差异显著(P<0.05)。

Figure 2 The anthocyanin accumulation in Arabidopsis thaliana spl1-1 and spl1-2 under low Pi condition (A) The anthocyanin accumulation in wild type (WT), spl1-1, and spl1-2 plants during Pi sufficient and Pi deprivation; (B) Anthocyanin content was determined in WT and spl1 plants grown with normal and low Pi (LP) on the twentieth day of Pi starvation. Asterisk represents statistically differences compared with the wild type (P<0.05).

图3 不同条件下拟南芥突变体spl1的无机磷含量变化生长7天的拟南芥幼苗移植到MS或低磷(LP)培养基上处理14天, 然后测定无机磷含量。*表示与野生型相比差异显著(P<0.05)。

Figure 3 The Pi content analysis of Arabidopsis thaliana spl1 mutants Seven-day-old seedlings were transferred to MS or low Pi (LP) medium for 14 d and then harvested for Pi content analy- sis. Asterisk represents statistically differences compared with the wild type (P<0.05).

图4 SPL1突变对根毛形态特征的影响 (A) 不同磷营养条件下, WT、spl1-1和spl1-2的根毛表型特征(Bar=1 mm); (B), (C) 在MS及低磷(LP)条件下的根毛数目(B)及根毛长度(C)统计结果(P<0.05)。*表示与对照相比差异显著。

Figure 4 SPL1 alters root hair architecture (A) Images of root tips with intact root hair from WT, spl1-1 and spl1-2 plants (Bar=1 mm); (B), (C) Number of root hair (B) and root hair length (C) under MS and low Pi (LP) condition. *Data significantly different from the corresponding controls are indicated (P<0.05).

图5 低磷对SPL1基因表达的影响(A)及磷饥饿诱导基因在拟南芥spl1突变体中的表达(B)

Figure 5 Expression analyses of the SPL1 gene (A) in Arabidopsis seedlings in response to low phosphate (LP) stress and expression pattern of Pi starvation induced genes in spl1 mutants (B)

图6 SPL1基因的组织表达特性 (A) SPL1::GUS转基因植株的GUS染色; (B) 用qRT-PCR方法分析SPL1基因在根、茎、叶和花中的组织表达

Figure 6 Tissue-specific expression of SPL1 (A) Histochemical localization of GUS activity directed by SPL1::GUS fusions in transgenic Arabidopsis; (B) qRT-PCR showed the relative abundant of SPL1 gene in different tissues, including root, stem, leaf and flower

图7 pHBT (A−D)及SPL1 (E−H)蛋白的细胞定位特性 (A), (E) 绿色荧光图; (B), (F) 叶绿体自发荧光图; (C), (G) 透射光图; (D), (H) 叠加图。Bar=5 µm

Figure 7 Localization of GFP signals from pHBT empty vector (A−D) and SPL1 (E−H) fused with GFP (A), (E) Fluorescence images under confocal microscopy; (B), (F) Chloroplast fluorescence; (C), (G) Bright-field images of the cells; (D), (H) Merged fluorescence. Bar=5 µm

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