A cucumber protein, Phloem Phosphate Stress Repressed 1, rapidly degrades in response to a phosphate stress condition

Under depleted external phosphate (Pi), many plant species adapt to this stress by initiating downstream signalling cascades. In plants, the vascular system delivers nutrients and signalling agents to control physiological and developmental processes. Currently, limited information is available regarding the direct role of phloem-borne long-distance signals in plant growth and development under Pi-stress conditions. Here, we report on the identification and characterization of a cucumber protein, Cucumis sativus Phloem Phosphate-Stress-Repressed 1 (CsPPSR1), whose level in the phloem translocation stream rapidly responds to imposed Pi-limiting conditions. CsPPSR1 degradation is mediated by the 26S proteasome; under Pi-sufficient conditions, CsPPSR1 is stabilized by its phosphorylation, within the sieve tube system, through the action of CsPPSR1 Kinase. Further, we discovered that CsPPSR1 Kinase was susceptible to Pi-starvation-induced degradation, in the sieve tube system. Our findings ..., In-gel kinase assays were performed as follows: Briefly, E. coli-purified GFP and CsPPSR1-4M8H were used as substrates. Fast protein liquid chromatography (FPLC) fractionated cucumber phloem sap proteins were separated on a substrate-containing SDS-PAGE (200 µg/ml of each substrate). Conditioned gels were incubated with 50 µM ATP and [γ32P]-ATP (20 Ci/mL) for in-gel phosphorylation. The reaction was terminated by incubating the gels with gel wash solution (5% TCA [V/V] and 1% [W/V] sodium pyrophosphate) and phosphorimaging using a Storm 860 system (GE Healthcare Lifesciences). To identify the kinase that phosphorylated CsPPSR1, named CsPPSRK, fractions with CsPPSR1-specific phosphorylation activity were separated on a 13% SDS-PAGE gel. Then the approximate 40 kDa protein band with phosphorylation activity was excised for mass spectrometry analysis. These mass spectral data were interrogated to identify sequences associated with a protein kinase of the appropriate mass., , # A cucumber protein, Phloem Phosphate Stress Repressed 1, rapidly degrades in response to a phosphate stress condition

Specific kinase to phosphorylate Phloem Phosphate Stress Repressed 1 (CsPPSR1) was identified through in-gel kinase assay. The SDS-PAGE resolved FPLC fraction that contains the specific signal to phosphorylate CsPPSR1 as substrate was analyzed by LC-MS/MS and the raw data sets were deposited into DRYAD.

## Description of the data and file structure

The MASCOT file is PPAN1314.mzid.gz_GPM00500046882.MGF.
The Spectra raw information is in PPAN1314.mzid.

PPAN1314.mzid.gz_GPM00500046882.MGF file was used to encode mass spectra results; multiple spectra were encoded in one MGF. Every spectrum begins with the BEGINS IONS assessment and finishes with END IONS. PEPMASS is the precursor mass of the representative spectra. This MGF file describes Ions information within two columns: the first column is the ion mass (Unit: Dalton, Da), the second column is the ion charge (U...

在外部无机磷酸盐（Pi）匮乏的条件下，多数植物物种会启动下游信号转导级联反应以适应该胁迫环境。植物维管系统负责输送养分与信号分子，从而调控生理与发育进程。目前，关于韧皮部介导的长距离信号在磷酸盐胁迫条件下对植物生长发育的直接作用，相关研究报道仍较为有限。
本研究鉴定并表征了一种黄瓜蛋白——黄瓜韧皮部磷酸盐胁迫抑制蛋白1（Cucumis sativus Phloem Phosphate-Stress-Repressed 1，CsPPSR1），其在韧皮部转运流中的丰度会快速响应施加的限磷条件。CsPPSR1的降解由26S蛋白酶体介导；在磷充足条件下，筛管系统中的CsPPSR1可通过CsPPSR1激酶的催化作用发生磷酸化，从而维持蛋白稳定性。进一步研究发现，CsPPSR1激酶在筛管系统中易受磷饥饿诱导的降解。我们的研究发现……
凝胶内激酶实验操作如下：简要而言，以大肠杆菌纯化的GFP与CsPPSR1-4M8H作为底物。经快速蛋白液相色谱（Fast Protein Liquid Chromatography，FPLC）分级分离的黄瓜韧皮部汁液蛋白，在含底物的SDS-PAGE凝胶（每升凝胶中底物浓度为200 μg/ml）上进行分离。将凝胶置于含50 μM ATP与[γ-³²P]-ATP（20 Ci/mL）的体系中孵育，以进行凝胶内磷酸化反应。通过凝胶洗涤液（体积分数5%三氯乙酸[TCA，V/V]、质量体积分数1%焦磷酸钠）终止反应后，使用Storm 860系统（GE Healthcare Lifesciences）进行磷成像分析。为鉴定磷酸化CsPPSR1的激酶（命名为CsPPSRK），将具有CsPPSR1特异性磷酸化活性的组分通过13% SDS-PAGE凝胶分离，切下分子量约为40 kDa且具有磷酸化活性的蛋白条带进行质谱分析。对所得质谱数据进行检索，以鉴定匹配该分子量的蛋白激酶序列。

# 黄瓜蛋白韧皮部磷酸盐胁迫抑制蛋白1（Phloem Phosphate Stress Repressed 1，CsPPSR1）可响应磷酸盐胁迫快速降解
通过凝胶内激酶实验，研究人员鉴定得到了可磷酸化CsPPSR1的特异性激酶。以CsPPSR1为底物的SDS-PAGE分离后的FPLC分级组分，经液相色谱-串联质谱（LC-MS/MS）分析，原始数据集已上传至DRYAD数据库。

## 数据与文件结构说明
该MASCOT文件为PPAN1314.mzid.gz_GPM00500046882.MGF。
原始光谱信息存储于PPAN1314.mzid文件中。
PPAN1314.mzid.gz_GPM00500046882.MGF文件用于编码质谱结果，单个MGF文件可编码多条光谱。每条光谱以 BEGIN IONS 标识起始，以 END IONS 标识结束。PEPMASS代表代表性光谱的前体离子质量。该MGF文件以两列形式描述离子信息：第一列为离子质量（单位：道尔顿，Da），第二列为离子电荷（U……）