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JXB：華中農(nóng)大丨南瓜和黃瓜耐鹽性差異的分子機(jī)制（附NMT實(shí)驗(yàn)體系）
期刊：Journal of Experimental Botany
主題：南瓜和黃瓜耐鹽性差異的分子機(jī)制
標(biāo)題：Tissue-specific respiratory burst oxidase homologue -dependent H2O2 signaling to the plasma membrane H+-ATPase confers potassium uptake and salinity tolerance in Cucurbitaceae
影響因子：5.360
檢測(cè)指標(biāo)： K+流速
檢測(cè)部位：距離根尖1mm
K+流實(shí)驗(yàn)處理方法：
5日齡黃瓜幼苗，75mM NaCl處理24小時(shí)
K+流實(shí)驗(yàn)測(cè)試液成分：
0.5mM KCl，0.1mM CaCl2，pH 5.7
作者：華中農(nóng)業(yè)大學(xué)別之龍、黃遠(yuǎn)
英文摘要
Potassium (K+) is a critical determinant ofsalinity tolerance, and H2O2 has been recognised as an important signalingmolecule that mediates many physiological responses. However, the details onhow H2O2 signaling regulates potassium uptake in the root under salt stressremain elusive. In this study, the salt sensitive cucumber and salt tolerantpumpkin which belong to the same family cucurbitaceae were used to answer theabove question.
Weshow that higher salt tolerance in pumpkin was related to its superior abilityfor K+ uptake and higher H2O2 accumulation in the root apex. Transcriptomeanalysis showed that salinity induced 5886 (3005 up and 2811 down) and 4679(3965 up and 714 down) differentially expressed genes (DEGs) in cucumber andpumpkin, respectively. DEGs encoding NADPH oxidase (RBOHD), 14-3-3 protein(GRF12), plasma membrane H+- ATPase (AHA1) and potassium transporter (HAK5)showed higher expression in pumpkin than cucumber under salinity stress.Treatment with a NADPH oxidase inhibitor diphenylene iodonium resulted in alower RBOHD, GRF12, AHA1 and HAK5 expression, reduced plasma membrane H+-ATPase activity, and smaller K+ uptake, resulting in a loss of salinitytolerance trait in pumpkin. The opposite results were obtained when the plantswere pre-treated with exogenous H2O2. Knocking out of RBOHD in pumpkin byCRISPR-Cas9 editing of coding sequences resulted in lower root apex H2O2 and K+content and GRF12, AHA1 and HAK5 expression, ultimately resulting in asalt-sensitive phenotype. However, ectopic expression of pumpkin RBOHD inArabidopsis led to the opposite effect.
Taken together, this study shows that RBOHD-dependent H2O2 signaling in the root apex is important for the pumpkin salttolerance and suggests a novel mechanism that confers this trait, namelyRBOHD-mediated transcriptional and post-translational activation of plasmamembrane H+-ATPase operating upstream of HAK5 K+ uptake transporters.
中文摘要（谷歌機(jī)翻）
鉀（K+）是耐鹽性的關(guān)鍵決定因素，H2O2已被認(rèn)為是介導(dǎo)許多生理反應(yīng)的重要信號(hào)分子。然而，關(guān)于H2O2信號(hào)如何在鹽脅迫下調(diào)節(jié)根中鉀吸收的細(xì)節(jié)仍然難以捉摸。在這項(xiàng)研究中，使用屬于同一科葫蘆科的鹽敏感黃瓜和耐鹽南瓜來回答上述問題。
我們表明，南瓜中較高的耐鹽性與其對(duì)根尖的K +吸收能力和較高的H2O2積累有關(guān)。轉(zhuǎn)錄組分析顯示鹽度分別誘導(dǎo)黃瓜和南瓜中5886（3005上和下2811下）和4679（3965上和下714）差異表達(dá)基因（DEG）。在鹽脅迫下，編碼NADPH氧化酶（RBOHD），14-3-3蛋白（GRF12），質(zhì)膜H+- ATP酶（AHA1）和鉀轉(zhuǎn)運(yùn)蛋白（HAK5）的DEGs在南瓜中的表達(dá)高于黃瓜。用NADPH氧化酶抑制劑二亞苯基碘處理導(dǎo)致較低的RBOHD，GRF12，AHA1和HAK5表達(dá)，降低的質(zhì)膜H+- ATP酶活性和較小的K+攝取，導(dǎo)致南瓜中鹽度耐受性狀的喪失。當(dāng)用外源H2O2預(yù)處理植物時(shí)獲得相反的結(jié)果。通過編碼序列的CRISPR-Cas9編輯敲除南瓜中的RBOHD導(dǎo)致較低的根尖H2O2和K+含量以及GRF12，AHA1和HAK5表達(dá)，終導(dǎo)致鹽敏感表型。然而，擬南芥中南瓜RBOHD的異位表達(dá)導(dǎo)致相反的效果。
總之，本研究表明，根尖中依賴RBOHD的H2O2信號(hào)傳導(dǎo)對(duì)南瓜耐鹽性具有重要意義，并提示了一種賦予這種特性的新機(jī)制，即RBOHD介導(dǎo)的質(zhì)膜H+-ATPase的轉(zhuǎn)錄和翻譯后激活。 HAK5 K+攝取轉(zhuǎn)運(yùn)蛋白的上游。
Figure 7. Relative expression of GRF (14-3-3 protein), AHA (plasma membrane H+-ATPase) and HAK5 (high affinity K+ transporter) in the root apex of cucumber (A, C, E) and pumpkin (B, D, F) exposed to 75 mM NaCl for 24 h. (G) to (J): plasma membrane H+-ATPase activity and net K+ flux measured after 24 h of exposure to 75 mM NaCl stress from the root apex pre-treated for 1 h in solutions containing specific chemicals (DPI, an NADPH oxidase inhibitor, H2O2) in cucumber (G, I) and pumpkin (H, J). Values are the mean ± SE (n=4). Different letters indicate significant difference (P < 0.05) according to Duncan's multiple range tests. The gene ID for cucumber GRF, AHA and HAK5 is Csa3G890040 (GRF8), Csa1G045600 (AHA11) and Csa3G835810 (HAK5), and it is CmoCh01G016540 (GRF12), CmoCh11G003690 (AHA1) and CmoCh08G004000 (HAK5) for pumpkin.