NAC-type transcription factors regulate accumulation of starch and protein in maize seeds

First author: Zhiyong Zhang; Affiliations: Rutgers University (罗格斯大学): Piscataway, USA

Corresponding author: Joachim Messing

Grain starch and protein are synthesized during endosperm development, prompting the question of what regulatory mechanism underlies the synchronization of the accumulation of secondary and primary gene products. We found that two endosperm-specific NAC transcription factors, ZmNAC128 and ZmNAC130, have such a regulatory function. Knockdown of expression of ZmNAC128 and ZmNAC130 with RNA interference (RNAi) caused a shrunken kernel phenotype with significant reduction of starch and protein. We could show that ZmNAC128 and ZmNAC130 regulate the transcription of Bt2 and then reduce its protein level, a rate-limiting step in starch synthesis of maize endosperm. Lack of ZmNAC128 and ZmNAC130 also reduced accumulation of zeins and nonzeins by 18% and 24% compared with nontransgenic siblings, respectively. Although ZmNAC128 and ZmNAC130 affected expression of zein genes in general, they specifically activated transcription of the 16-kDa γ-zein gene. The two transcription factors did not dimerize with each other but exemplified redundancy, whereas individual discovery of their function was not amenable to conventional genetics but illustrated the power of RNAi. Given that both the Bt2 and the 16-kDa γ-zein genes were activated by ZmNAC128 or ZmNAC130, we could identify a core binding site ACGCAA contained within their target promoter regions by combining Dual-Luciferase Reporter and Electrophoretic Mobility Shift assays. Consistent with these properties, transcriptomic profiling uncovered that lack of ZmNAC128 and ZmNAC130 had a pleiotropic effect on the utilization of carbohydrates and amino acids.

籽粒淀粉和蛋白在种子胚乳发育过程中被合成，那么是哪些调控机制在控制这些次生和初生基因产物的积累？作者在本文中发现两个胚乳特异性表达的NAC转录因子：ZmNAC128和ZmNAC130具有这样的调控功能。通过RNA干扰技术敲低ZmNAC128和ZmNAC130基因的表达会导致玉米籽粒皱缩，并且籽粒的淀粉和蛋白含量都显著降低。ZmNAC128和ZmNAC130能够调控Bt2基因的转录，并且降低Bt2蛋白水平，而Bt2是玉米胚乳中淀粉合成的一个限速酶。相比于非转基因植株，ZmNAC128和ZmNAC130的缺失同样能够降低18%的玉米醇溶蛋白和24%的非玉米醇溶蛋白。尽管，ZmNAC128和ZmNAC130一般来说能够影响玉米醇溶蛋白相关基因的表达，但其能够特异性激活16-kDa γ-zein基因的转录。这两个NAC转录因子并不会二聚化，但二者之间存在功能冗余，通过传统遗传学的方法并不能很好地区分开他们各自的功能，而需要借助于RNA干扰技术。由于Bt2和16-kDa γ-zein基因均能够被ZmNAC128和ZmNAC130所激活，作者在这两个基因的上游启动子区鉴定到了一个保守的结合位点ACGCAA，并通过双荧光素酶报告和电泳迁移率试验进行了验证。转录组的分析显示ZmNAC128和ZmNAC130的缺失对于转基因植株的碳水化合物和氨基酸利用存在多效性影响。

通讯：Joachim Messing（https://www.waksman.rutgers.edu/messing）

研究方向：玉米种子贮藏蛋白在种子发育和分子育种中的作用。

doi: https://doi.org/10.1073/pnas.1904995116

Journal: PNAS

First Published: May 20, 2019