植物磷元素的测定方法

测定植物样品活性无机磷酸盐

1. 取样：称量样品鲜重，干重或冷冻后的重量（每个EP 管取10-20mg 根或茎）

2. 提取：向含有新鲜或冰冻样品EP 管加入500纯水，85℃温育45min (干燥或粉末状样品需离心)

3.Pi 含量测定：

储备液配置：

1. 仲钼酸铵溶液配置：

-将0.44g 仲钼酸铵溶于97.3mL 纯水

-接入2.66ML （36N ）浓硫酸定容至100Ml. 所配溶液避光可存放几个月。

2.10%维生素C 溶液配置

-将1个维生素C 钠盐溶于10mL 纯水

-该溶液需现配现用

步骤

在EP 管中混合以下溶液（新制备的样品解离液可加入600μL仲钼酸铵溶液

，100μL10%维生素C 溶液及纯水）

和纯水）

-600μL仲钼酸铵溶液

-100μL10%维生素C 溶液

-300μL基本溶液

1样品为根：200μL纯水加+100μL提取后溶液

2样品为茎：250μL纯水+50μL提取后溶液

3 样品溶液已制备好：直接加50和100μL体积即可

42温育1h 后测820nm 吸光度

pho2, a Phosphate Overaccumulator, Is Caused by aNonsense Mutation in a MicroRNA399 Target Gene1[W]

We recently demonstrated that microRNA399 (miR399) controls inorganic phosphate (Pi) homeostasis by regulating theexpression of UBC24 encoding a ubiquitin-conjugating E2 enzyme in Arabidopsis (Arabidopsis thaliana). Transgenic plantsoverexpressing miR399 accumulated excessive Pi in the shoots and displayed Pi toxic symptoms. In this study, we revealedthat a previously identified Pi overaccumulator, pho2, is caused by a single nucleotide mutation resulting in early terminationwithin the UBC24 gene. The level of full-length UBC24 mRNA was reduced and no UBC24 protein was detected in the pho2mutant, whereas up-regulation of miR399 by Pi deficiency was not affected. Several characteristics of Pi toxicity in the pho2mutant were similar to those in the miR399-overexpressing and UBC24 T-DNA knockout plants: both Pi

uptake andtranslocation of Pi from roots to shoots increased and Pi remobilization within leaves was impaired. These phenotypes of thepho2 mutation could be rescued by introduction of a wild-type copy of UBC24. Kinetic analyses revealed that greater Pi uptakein the pho2 and miR399-overexpressing plants is due to increased Vmax. The transcript level of most PHT1 Pi transporter genesas not significantly altered, except PHT1;8 whose expression was enhanced in Pi-sufficient roots of pho2 and miR399-overexpressing compared with wild-type plants. In addition, changes in the expression of several organelle-specific Pitransporters were noticed, which may be associated with the redistribution of intracellular Pi under excess Pi. Furthermore,miR399 and UBC24 were colocalized in the vascular cylinder. This observation not only provides important insight into theinteraction between miR399 and UBC24 mRNA, but also supports their systemic function in Pi translocation andremobilization.

PHO2, MicroRNA399, and PHR1 Define aPhosphate-Signaling Pathway in Plants1[W][OA]

Inorganic phosphate (Pi)-signaling pathways in plants are still largely unknown. The Arabidopsis (Arabidopsis thaliana) pho2mutant overaccumulates Pi in leaves in Pi-replete conditions. Micrografting revealed that a pho2 root genotype is sufficient toyield leaf Pi accumulation. In pho2 mutants, Pi does not repress a set of Pi starvation-induced genes, including AtIPS1, AT4, andPi transporters Pht1;8 and Pht1;9. Map-based cloning identified PHO2 as At2g33770, an unusual E2 conjugase gene. It wasrecently shown that Pi deprivation induces mature microRNA (miRNA [miR399]) and that overexpression of miR399 inPi-replete conditions represses E2 conjugase expression and leads to high leaf Pi concentrations, thus phenocopying pho2. Weshow here that miR399 primary transcripts are also strongly induced by low Pi and rapidly repressed after addition of Pi.PHO2 transcripts change reciprocally to miR399 transcripts in Pi-deprived plants and in miR399 overexpressers. However,responses after Pi readdition and in b-glucuronidase reporter lines suggest that PHO2 expression is also regulated by Pi in amanner unrelated to miR399-mediated transcript cleavage. Expression of miR399 was strongly reduced in Pi-deprivedArabidopsis phr1 mutants, and a subset of Pi-responsive genes repressed in Pi-deprived phr1 mutants was up-regulated inPi-replete pho2 mutants. This places miR399 and PHO2 in a branch of the Pi-signaling network downstream of PHR1. Finally,putative PHO2 orthologs containing five miR399-binding sites in their 5#-untranslated regions were identified in other higherplants, and Pi-dependent miR399 expression was demonstrated in rice (Oryza sativa), suggesting a conserved regulatorymechanism.