Abstract
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 Δ1-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 tRNApro AGG accumulation and triggers the phosphorylation of eukaryotic initiation factor 2α (eIF2α) 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.
Highlights
In maize (Zea mays), a series of seed mutants with a starchy endosperm have been identified, and the cause of the opaque/floury kernel phenotype has been investigated through analyzing the functions of cloned genes
To identify the effect caused by the pro1 mutation on proline biosynthesis, we examined the proline content in protein-bound (PBAAs) and free amino acids (FAAs) in endosperms of mature kernels
Zein gene expression was examined during kernel development at the transcriptional level by quantitative real-time RT-PCR, and the results indicated that the transcription of a- (19 and 22 kD), b- (15 kD), d- (10 kD), and g-zein (27 and 50 kD) classes is downregulated in pro1-ref, especially at 18 d after pollination (DAP) (Figure 10B)
Summary
In maize (Zea mays), a series of seed mutants with a starchy endosperm have been identified, and the cause of the opaque/floury kernel phenotype has been investigated through analyzing the functions of cloned genes. Downregulation of zein gene (a-, b-, d-, and g-zein genes) expression by RNA interference reproduced an opaque phenotype (Segal et al, 2003; Wu and Messing, 2010). These results indicate that the opaque/floury phenotypes of opaque (o2), floury (fl2), Defective endosperm B30, and Mucronate mutants are caused by either quantitative or qualitative alterations in zein proteins. Proline responding (pro1), allelic to o6 (Manzocchi et al, 1986), is a classical recessive opaque mutant. Recovery of the normal phenotype in mutant seedlings occurs after they are supplemented with external L-proline (Racchi et al, 1978; Tonelli et al, 1984). Tonelli et al (1986) predicted that pro might be associated with a defect in proline biosynthesis, but that has not been validated
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