A maize ADP-ribosylation factor ZmArf2 increases organ and seed size by promoting cell expansion in Arabidopsis.

ADP-ribosylation factors (ARFs) are small GTP-binding proteins that regulate a wide variety of cell functions. Previously, we isolated a new ARF, ZmArf2, from maize (Zea mays). Sequence and expression characteristics indicated that ZmArf2 might play a critical role in the early stages of endosperm development. In this study, we investigated ZmArf2 function by analysis of its GTP-binding activity and subcellular localization. We also over-expressed ZmArf2 in Arabidopsis and measured organ and cell size and counted cell numbers. The expression levels of five organ size-associated genes were also determined in 35S::ZmArf2 transgenic and wild-type plants. Results showed that the recombinant ZmArf2 protein purified from Escherichia coli exhibited GTP-binding activity. Subcellular localization revealed that ZmArf2 was localized in the cytoplasm and plasma membrane. ZmArf2 over-expression in Arabidopsis showed that 35S::ZmArf2 transgenic plants were taller and had larger leaves and seeds compared to wild-type plants, which resulted from cell expansions, not an increase in cell numbers. In addition, three cell expansion-related genes, AtEXP3, AtEXP5 and AtEXP10, were upregulated in 35S::ZmArf2 transgenic lines, while the expression levels of AtGIF1 and AtGRF5, were unchanged. Collectively, our studies suggest that ZmArf2 has an active GTP-binding function, and plays a crucial role in growth and development in Arabidopsis through cell expansion mediated by cell expansion genes.

Dynamic Proteomic Characteristics and Network Integration Revealing Key Proteins for Two Kernel Tissue Developments in Popcorn.

The formation and development of maize kernel is a complex dynamic physiological and biochemical process that involves the temporal and spatial expression of many proteins and the regulation of metabolic pathways. In this study, the protein profiles of the endosperm and pericarp at three important developmental stages were analyzed by isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with LC-MS/MS in popcorn inbred N04. Comparative quantitative proteomic analyses among developmental stages and between tissues were performed, and the protein networks were integrated. A total of 6,876 proteins were identified, of which 1,396 were nonredundant. Specific proteins and different expression patterns were observed across developmental stages and tissues. The functional annotation of the identified proteins revealed the importance of metabolic and cellular processes, and binding and catalytic activities for the development of the tissues. The whole, endosperm-specific and pericarp-specific protein networks integrated 125, 9 and 77 proteins, respectively, which were involved in 54 KEGG pathways and reflected their complex metabolic interactions. Confirmation for the iTRAQ endosperm proteins by two-dimensional gel electrophoresis showed that 44.44% proteins were commonly found. However, the concordance between mRNA level and the protein abundance varied across different proteins, stages, tissues and inbred lines, according to the gene cloning and expression analyses of four relevant proteins with important functions and different expression levels. But the result by western blot showed their same expression tendency for the four proteins as by iTRAQ. These results could provide new insights into the developmental mechanisms of endosperm and pericarp, and grain formation in maize.