Pro1基因編碼一個△1-吡咯啉-5-羧酸合成酶(P5CS)
對玉米經典突變pro1基因進行圖位克隆發現，玉米Pro1基因編碼一個△1-吡咯啉-5-羧酸合成酶(P5CS) (Figure 2A)，它是催化以谷氨酸為前體合成脯氨酸過程中的限速酶(Figure 2B, C, D, E)。Pro1基因功能的喪失，造成突變體中脯氨酸合成與積累的下降。
Figure 2 Phylogenetic Analysis of Pro1 and Biochemical Assay of Recombinant P5CS Protein.
(A) Phylogenetic relationships of Pro1 (Zm P5CS) and its homologs.
Maize Pro1 and identified P5CS in rice (monocot) and Arabidopsis (dicot) plants were aligned by MUSCLE method in MEGA 5.2 software package. The phylogenetic tree was constructed using MEGA 5.2, and P5CS of Caenorhabditis elegans, Homo sapiens, and Mus musculus were used as outgroups (see Methods). The numbers at the nodes (100) represent the percentage of 1,000 bootstraps. Zm: Zea mays; Os: Orya sativa; At: Arabidopsis thaliana; Ce: Caenorhabditis elegans; Hs: Homo sapiens; Mm: Mus musculus.
(B) SDS-PAGE gel of purified GST-P5CS protein stained with Coomassie blue. Left lane, GST- P5CS; right lane, molecular weight markers.
(C) and (D) Standard curve and its control of Pi determination based on a malachite green colorimetric assay
(E) Kinetic analysis of P5CS was performed using glutamate concentrations from 0 to 350 mM. Km and Vmax were determined from nonlinear regression to the Michaelis-Menten equation for concentrations of glutamate up to 350 mM from five replicate experiments. For each sample, five independent biological replicates were performed. Error bars represent SD.
Proline1基因功能的減弱/喪失抑制了玉米籽粒和幼苗中L-脯氨酸的合成
Pro1基因(Zm P5CS2)功能的減弱/喪失可能抑制了玉米中L-脯氨酸的合成。通過對玉米成熟籽粒進行總的氨基酸和游離氨基酸分析表明，與野生型相比，pro1-ref突變體成熟籽粒中總的L-脯氨酸的相對含量下降了35.9%(Figure 3A)，總的游離L-脯氨酸的含量下降了56.0%(Figure 3B)。苗期(14DAG)氨基酸分析表明，與野生型相比，pro1-ref幼苗中總的L-脯氨酸的相對含量下降了45.2%(Figure 3C)，總的游離L-脯氨酸的含量下降了100.0%(Figure 3D)。由此表明，Proline1基因功能的減弱/喪失抑制了玉米籽粒和幼苗中L-脯氨酸的合成。
Figure 3 Analysis of Total and Free Proline from Mature Endosperms and 14 DAG Seedling of Wild Type and pro1-ref.
(A) and (B) Comparison of total and free proline from mature endosperm of wild type and pro1-ref . wt, wild type; pro1, pro1-ref.
(C) and (D) Comparison of total and free proline from 14 DAG seedlings of wild type and pro1-ref. wt, wild type; pro1, pro1-ref.
For each sample, three independent biological replicates were performed. Bars represent average values ± SD, n=3 replicates (** refers to P < 0.01, Student’s test).
pro1突變體中脯氨酸缺乏引起的空載tRNApro AGG累積從而造成了eIF2α)的磷酸化
脯氨酸的缺乏造成pro1突變體細胞中空載tRNApro AGG累積，由此引發真核生物翻譯起始因子2的α亞基(eIF2α)磷酸化(Figure 4A, B, C, D, and E)，造成突變體中蛋白合成的普遍下降。
Figure 4 Uncharged tRNApro Accumulation Caused by Proline Deficiency Triggers the Phosphorylation of eIF2α
(A) Free proline content analysis of 12 DAP (I), 18 DAP (II), and 24 DAP (III) endosperm from wild type and pro1-ref. For each sample, three independent biological replicates were performed. Bars represent average values ± SD, n=3 replicates (** refers to P < 0.01, Student’s test). wt, wild type; pro, pro1-ref.
(B) Northern Blot analysis of charged and uncharged tRNApro accumulation in developing endosperm. wt, wild type; pro, pro1-ref.
(C) Northern Blot analysis of charged and uncharged tRNApro accumulation in 14 DAG seedlings of wild type and pro1-ref cultivated with 0 mM, 1 mM and 3 mM L-proline. wt, wild type; pro, pro1-ref.
(D) Immunoblot analysis of eIF2α phosphorylation during kernel development. wt, wild type; pro, pro1-ref.
(E) Immunoblot analysis of eIF2α phosphorylation in 14 DAG seedlings of wild type and pro1-ref cultivated with 0 mM, 1 mM and 3 mM L-proline. wt, wild type; pro, pro1-ref.