Development-Related miRNA Expression and Target Regulation during Staggered In Vitro Plant Regeneration of Tuxpeño VS-535 Maize Cultivar

Abstract

In vitro plant regeneration addresses basic questions of molecular reprogramming in the absence of embryonic positional cues. The process is highly dependent on the genotype and explant characteristics. However, the regulatory mechanisms operating during organ differentiation from in vitro cultures remain largely unknown. Recently, miRNAs have emerged as key regulators during embryogenic callus induction, plant differentiation, auxin responses and totipotency. Here, we explored how development-related miRNA switches the impact on their target regulation depending on physiological and molecular events taking place during maize Tuxpeño VS-535 in vitro plant regeneration. Three callus types with distinctive regeneration potential were characterized by microscopy and histological preparations. The embryogenic calli (EC) showed higher miRNA levels than non-embryogenic tissues (NEC). An inverse correlation for miR160 and miR166 targets was found during EC callus induction, whereas miR156, miR164 and miR394 displayed similar to their targets RNA accumulation levels. Most miRNA accumulation switches took place early at regenerative spots coincident with shoot apical meristem (SAM) establishment, whereas miR156, miR160 and miR166 increased at further differentiation stages. Our data uncover particular miRNA-mediated regulation operating for maize embryogenic tissues, supporting their regulatory role in early SAM establishment and basipetala growth during the in vitro regeneration process.

Epigenetic Regulation of Auxin-Induced Somatic Embryogenesis in Plants

“…Transcriptomic datasets on Zea mays embryogenic cultures have shown that the expression pattern of individual auxin-related miRNAs is dependent on the genotype and the age of a culture [66][67][68]78]. The diversity of the miRNA expression profiles in embryogenic cultures, for example, the up-vs. downregulation of the same miRNA, could reflect the complexity of the miRNA-mediated regulatory pathways that fine-tune the somatic cell transcriptome towards embryogenic development in response to specific endo-and exogenous culture factors.…”

“…Numerous MIRNA genes, including members of the same gene family of SE-modulated expression, have been reported in transcriptomic analyses of different plant cultures [55,58,66,68,73,79]. The expression of different MIRNA gene family members is strictly regulated during plant development in an organ-and tissue-specific manner [80,81], and thus reporter line analyses could be important in identifying the specific MIRNA genes that contribute to SE induction.…”

“…The auxin-related miRNAs frequently target the fundamental components of the auxin signaling pathway, including the ARFs, which are involved in SE induction [27,93]. Accordingly, insights into embryogenic cultures of Arabidopsis and other plants indicated that miR160 could directly regulate the expression of ARF10, ARF16, and ARF17; miR167 seems to the control expression of ARF6 and ARF8, while miR390 possibly downregulates the ARF2, ARF3, ARF4 transcripts [55,63,64,68,81,85,86].…”

tasiR-ARFs Production and Target Regulation during In Vitro Maize Plant Regeneration

“…In such a way, the tissues may elicit developmental responses when endogenous auxin redistributes. The current panorama on specific EC features depicts greater accumulation of tasiR-ARF biogenesis components and ZmARF3 transcripts, expression of PIN auxin polar transporters and enrichment in auxin response-related miRNAs and targets [32]. This information provides insights into the genetic program underlying de novo plant formation, where leaf initiation and morphogenesis appear as primary and essential processes.…”

“…All analyses were performed with maize calli induced from Tuxpeño VS-535 immature embryos. After subcultures for 8-12 months on N6 proliferation medium (see methods) calli were separated in embryogenic (EC) and non-embryogenic (NEC) types according to their morphology (Supplementary Materials Figure S1; [32]). For plant regeneration, phytohormones were gradually depleted under a photoperiod.…”