Abstract
Maize somatic embryogenesis (SE) is induced from the immature zygotic embryo in darkness and under the appropriate hormones' levels. Small RNA expression is reprogrammed and certain miRNAs become particularly enriched during induction while others, characteristic to the zygotic embryo, decrease. To explore the impact of different environmental cues on miRNA regulation in maize SE, we tested specific miRNA abundance and their target gene expression in response to photoperiod and hormone depletion for two different maize cultivars (VS-535 and H-565). The expression levels of miR156, miR159, miR164, miR168, miR397, miR398, miR408, miR528, and some predicted targets (SBP23, GA-MYB, CUC2, AGO1c, LAC2, SOD9, GR1, SOD1A, PLC) were examined upon staged hormone depletion in the presence of light photoperiod or darkness. Almost all examined miRNA, except miR159, increased upon hormone depletion, regardless photoperiod absence/presence. miR528, miR408, and miR398 changed the most. On the other hand, expression of miRNA target genes was strongly regulated by the photoperiod exposure. Stress-related miRNA targets showed greater differences between cultivars than development-related targets. miRNA/target inverse relationship was more frequently observed in darkness than light. Interestingly, miR528, but not miR159, miR168 or miR398, was located on polyribosome fractions suggesting a role for this miRNA at the level of translation. Overall our results demonstrate that hormone depletion exerts a great influence on specific miRNA expression during plant regeneration independently of light. However, their targets are additionally influenced by the presence of photoperiod. The reproducibility or differences observed for particular miRNA-target regulation between two different highly embryogenic genotypes provide clues for conserved miRNA roles within the SE process.
Highlights
Maize (Zea mays L.) is one of the most important widely cultivated cereal crops, used as valuable source for human food, livestock feed, and raw material for the industry (Huang et al, 2002)
In a previous study performed on VS-535-derived embryogenic callus (EC), we found that development-related miRNAs such as miR156, miR159, miR164 and miR168 decreased as the length of subculture increased, while stress-related miRNAs such as miR397, miR398, miR408, and miR528 remained highly expressed (Dinkova and Alejandri-Ramirez, 2014)
Embryogenesis Staged hormones depletion combined with light photoperiod promotes plant regeneration through somatic embryogenesis embryogenesis (SE) in maize (Jakubeková et al, 2012; Garrocho-Villegas et al, 2012)
Summary
Maize (Zea mays L.) is one of the most important widely cultivated cereal crops, used as valuable source for human food, livestock feed, and raw material for the industry (Huang et al, 2002). Besides its agricultural and economic relevance, it has been a major model system in plant genetics and improvement. Common methods for maize transformation involve a process known as somatic miRNA regulation in maize somatic embryogenesis embryogenesis (SE). SE begins with callus induction, a process characterized by re-organization and re-structuring of somatic cells in the presence of the synthetic auxin 2,4-D (Zimmerman, 1993). The second step of SE is plant regeneration. Plants can be regenerated when somatic embryos are depleted of external hormones and are exposed to photoperiod (Garrocho-Villegas et al, 2012). SE has been advantageously used in many plant species for clonal propagation, plant transformation and genetic improvement (Stasolla and Yeung, 2003)
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