PNAS爱荷华州立大学PPDK基因与玉米品质性状的精准调控

普通玉米籽粒中是氨基酸组成不均衡，尤其是必需氨基酸——赖氨酸含量低，蛋白质品质较差。玉米胚乳中重要的转录调控因子Opaque 2 基因发生突变会使玉米中的赖氨酸含量大幅度上升，可极大地改善玉米籽粒的蛋白质品质，利用 opaque2 ( o2) 突变体是目前获得高赖氨酸优质蛋白玉米的主要手段。但 o2玉米常会产生一些不良的农艺性状，尤其会形成粉质胚乳。作为胚乳中重要的转录调控因子， o2 蛋白通过调控醇溶蛋白(Zeins)、淀粉合成（SSIII）以及丙酮酸磷酸双激酶( PPDK)的表达，进而影响胚乳品质特性。明确o2蛋白的调控网络对玉米品质改良具有重要意义。目前， Zeins和SSIII突变体的作用机制业已明确，但PPDK的调控机理还不完全清晰。

近日，爱荷华州立大学的科研人员首次报道了PPDK参与玉米胚乳品质性状的调控模式。玉米中有两个PPDK基因，分别为pdk1 和pdk2。研究人员通过构建pdk1 和 pdk2基因的单突变和双突变，系统解析了PPDK基因在胚乳发育中的作用，结果显示，PPDK 基因通过调控糖酵解通路，间接影响胚乳品质，而不影响籽粒生物量的积累。该研究表明，与之前通过调控转录因子进行玉米品质遗传改良相比，通过精准调控具体的某个代谢路径更为有效。

PNAS; published ahead of print December 18, 2017

Functions of maize genes encoding pyruvate phosphate dikinase in developing endosperm

Authors

Ryan R. Lappea, John W. Baierb, Susan K. Boehleinb, Ryan Huffmanc, Qiaohui Lina, Fabrice Wattebledd, A. Mark Settlesb, L. Curtis Hannahb, Ljudmilla Borisjuke, Hardy Rolletscheke, Jon D. Stewartf, M. Paul Scottc,g, Tracie A. Hennen-Bierwagena, and Alan M. Myersa,

Significance

Mutations affecting the transcription factor encoded by the gene o2 are important in maize agriculture because they result in improved grain nutritional quality. The mutations also cause detrimental effects by reducing kernel hardness and diminishing agronomic quality and food applications. The undesirable characteristics are not fully understood because the o2 product regulates multiple targets that could contribute to the phenotype. This study investigated one target that had not been previously mutated, pyruvate phosphate dikinase (PPDK), and showed that PPDK deficiency in isolation causes the negative phenotype associated with reduced kernel hardness. Thus, maize improvement may be better accomplished by targeting individual metabolic pathways determining protein and amino acid balance rather than pleiotropic regulators such as the o2 transcription factor.

Abstract

Maize opaque2 (o2) mutations are beneficial for endosperm nutritional quality but cause negative pleiotropic effects for reasons that are not fully understood. Direct targets of the bZIP transcriptional regulator encoded by o2 include pdk1 and pdk2 that specify pyruvate phosphate dikinase (PPDK). This enzyme reversibly converts AMP, pyrophosphate, and phosphoenolpyruvate to ATP, orthophosphate, and pyruvate and provides diverse functions in plants. This study addressed PPDK function in maize starchy endosperm where it is highly abundant during grain fill. pdk1 and pdk2 were inactivated individually by transposon insertions, and both genes were simultaneously targeted by endosperm-specific RNAi. pdk2 accounts for the large majority of endosperm PPDK, whereas pdk1 specifies the abundant mesophyll form. The pdk1– mutation is seedling-lethal, indicating that C4 photosynthesis is essential in maize. RNAi expression in transgenic endosperm eliminated detectable PPDK protein and enzyme activity. Transgenic kernels weighed the same on average as
nontransgenic siblings, with normal endosperm starch and total N contents, indicating that PPDK is not required for net storage compound synthesis. An opaque phenotype resulted from complete PPDK knockout, including loss of vitreous endosperm character similar to the phenotype conditioned by o2-. Concentrations of multiple glycolytic intermediates were elevated in transgenic endosperm, energy charge was altered, and starch granules were more numerous but smaller on average than normal. The data indicate that PPDK modulates endosperm metabolism, potentially through reversible adjustments to energy charge, and reveal that o2- mutations can affect the opaque phenotype through regulation of PPDK in addition to their previously demonstrated effects on storage protein gene expression.

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