RESUMEN
Leaves are derived from heterotrophic meristem tissue that, at some point, must make the transition to autotrophy via the initiation of photosynthesis. However, the timing and spatial coordination of the molecular and cellular processes underpinning this switch are poorly characterized. Here, we report on the identification of a specific stage in rice (Oryza sativa) leaf development (P3/P4 transition) when photosynthetic competence is first established. Using a combined physiological and molecular approach, we show that elements of stomatal and vascular differentiation are coordinated with the onset of measurable light absorption for photosynthesis. Moreover, by exploring the response of the system to environmental perturbation, we show that the earliest stages of rice leaf development have significant plasticity with respect to elements of cellular differentiation of relevance for mature leaf photosynthetic performance. Finally, by performing an RNA sequencing analysis targeted at the early stages of rice leaf development, we uncover a palette of genes whose expression likely underpins the acquisition of photosynthetic capability. Our results identify the P3/P4 transition as a highly dynamic stage in rice leaf development when several processes for the initiation of photosynthetic competence are coordinated. As well as identifying gene targets for future manipulation of rice leaf structure/function, our data highlight a developmental window during which such manipulations are likely to be most effective.
Asunto(s)
Clorofila/metabolismo , Perfilación de la Expresión Génica/métodos , Oryza/genética , Fotosíntesis/genética , Hojas de la Planta/genética , Clorofila/química , Fluorescencia , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Luz , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Oryza/crecimiento & desarrollo , Oryza/metabolismo , Fotosíntesis/efectos de la radiación , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/metabolismo , Estomas de Plantas/genética , Estomas de Plantas/metabolismo , Estomas de Plantas/ultraestructura , Haz Vascular de Plantas/genética , Haz Vascular de Plantas/crecimiento & desarrollo , Haz Vascular de Plantas/metabolismo , Plastidios/genética , Plastidios/metabolismo , Plastidios/ultraestructura , Factores de TiempoRESUMEN
Next generation DNA sequencing (NGS) has the potential to improve the diagnostic and prognostic utility of newborn screening programmes. This study assesses the feasibility of automating NGS on dried blood spot (DBS) DNA in a United Kingdom National Health Service (UK NHS) laboratory. An NGS panel targeting the entire coding sequence of five genes relevant to disorders currently screened for in newborns in the UK was validated on DBS DNA. An automated process for DNA extraction, NGS and bioinformatics analysis was developed. The process was tested on DBS to determine feasibility, turnaround time and cost. The analytical sensitivity of the assay was 100% and analytical specificity was 99.96%, with a mean 99.5% concordance of variant calls between DBS and venous blood samples in regions with ≥30× coverage (96.8% across all regions; all variant calls were single nucleotide variants (SNVs), with indel performance not assessed). The pipeline enabled processing of up to 1000 samples a week with a turnaround time of four days from receipt of sample to reporting. This study concluded that it is feasible to automate targeted NGS on routine DBS samples in a UK NHS laboratory setting, but it may not currently be cost effective as a first line test.