Proline, an important amino acid, accumulates in many plant species. Besides its role in plant cell responses to environmental stresses, the potential biological functions of proline in growth and development are unclear. Here, we report cloning and functional characterization of the maize (Zea mays) classic mutant proline responding1 (pro1) gene. This gene encodes a D 1-pyrroline-5-carboxylate synthetase that catalyzes the biosynthesis of proline from glutamic acid. Loss of function of Pro1 significantly inhibits proline biosynthesis and decreases its accumulation in the pro1 mutant. Proline deficiency results in an increased level of uncharged tRNA pro AGG accumulation and triggers the phosphorylation of eukaryotic initiation factor 2a (eIF2a) in the pro1 mutant, leading to a general reduction in protein synthesis in this mutant. Proline deficiency also downregulates major cyclin genes at the transcriptional level, causing cell cycle arrest and suppression of cell proliferation. These processes are reversible when external proline is supplied to the mutant, suggesting that proline plays a regulatory role in the cell cycle transition. Together, the results demonstrate that proline plays an important role in the regulation of general protein synthesis and the cell cycle transition in plants. INTRODUCTION In maize (Zea mays), a series of seed mutants with a starchy endosperm have been identified, and the cause of the opa-que/floury kernel phenotype has been investigated through analyzing the functions of cloned genes. Previous studies suggested that most of the opaque and floury mutants (opa-que2, floury2, Mucronate, and Defective endosperm B30) involve genes that regulate zein synthesis, such as regulatory genes or structural genes for these storage proteins (Schmidt et al., 1987; Coleman et al., 1995; Gillikin et al., 1997; Kim et al., 2004, 2006). Downregulation of zein gene (a-, b-, d-, and g-zein genes) expression by RNA interference also reproduced an opaque phenotype (Segal et al., 2003; Wu and Messing, 2010). These results indicate that the opaque/floury phenotypes of opaque2 (o2), floury2 (fl2), Defective endo-sperm B30, and Mucronate mutants are caused by either quantitative or qualitative alterations in zein proteins. However , other opaque/floury mutants, such as o1, o5, and fl1, show no notable alterations in zein proteins (Holding et al.

Zeins are the major seed storage proteins in maize (Zea mays). They are synthesized on the endoplasmic reticulum (ER) and deposited into protein bodies. Failure of signal peptide cleavage from zeins can cause an opaque endosperm in the mature kernel; however, the cellular and molecular mechanisms responsible for this phenotype are not fully understood. In this study, we report the cloning and characterization of a novel, semidominant opaque mutant, floury4 ( fl4). fl4 is caused by a mutated z1A 19-kD a-zein with defective signal peptide cleavage. Zein protein bodies in fl4 endosperm are misshapen and aggregated. Immunolabeling analysis indicated that fl4 participates in the assembly of zeins into protein bodies, disrupting their proper spatial distribution. ER stress is stimulated in fl4 endosperm, as illustrated by dilated rough ER and markedly up-regulated binding protein content. Further analysis confirmed that several ER stress pathways are induced in fl4 endosperm, including ERassociated degradation, the unfolded protein response, and translational suppression by the phosphorylation of eukaryotic translational initiation factor2 a-subunit. Programmed cell death is also elevated, corroborating the intensity of ER stress in fl4. These results provide new insights into cellular responses caused by storage proteins with defective signal peptides.

The actin-based myosin system is essential for the organization and dynamics of the endomembrane system and transport network in plant cells. Plants harbour two unique myosin groups, class VIII and class XI, and the latter is structurally and functionally analogous to the animal and fungal class V myosin. Little is known about myosins in grass, even though grass includes several agronomically important cereal crops. Here, we identified 14 myosin genes from the genome of maize (Zea mays). The relatively larger sizes of maize myosin genes are due to their much longer introns, which are abundant in transposable elements. Phylogenetic analysis indicated that maize myosin genes could be classified into class VIII and class XI, with three and 11 members, respectively. Apart from subgroup XI-F, the remaining subgroups were duplicated at least in one analysed lineage, and the duplication events occurred more extensively in Arabidopsis than in maize. Only two pairs of maize myosins were generated from segmental duplication. Expression analysis revealed that most maize myosin genes were expressed universally, whereas a few members (XI-1, -6, and -11) showed an anther-specific pattern, and many underwent extensive alternative splicing. We also found a short transcript at the O1 locus, which conceptually encoded a headless myosin that most likely functions at the transcriptional level rather than via a dominant-negative mechanism at the translational level. Together, these data provide significant insights into the evolutionary and functional characterization of maize myosin genes that could transfer to the identification and application of homologous myosins of other grasses.