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SUMMARY RNA‐Sequencing is widely used to investigate changes in gene expression at the transcription level in plants. Most plant RNA‐Seq analysis pipelines base the normalization approaches on the assumption that total transcript levels do not vary between samples. However, this assumption has not been demonstrated. In fact, many common experimental treatments and genetic alterations affect transcription efficiency or RNA stability, resulting in unequal transcript abundance. The addition of synthetic RNA controls is a simple correction that controls for variation in total mRNA levels. However, adding spike‐ins appropriately is challenging with complex plant tissue, and carefully considering how they are added is essential to their successful use. We demonstrate that adding external RNA spike‐ins as a normalization control produces differences in RNA‐Seq analysis compared to traditional normalization methods, even between two times of day in untreated plants. We illustrate the use of RNA spike‐ins with 3' RNA‐Seq and present a normalization pipeline that accounts for differences in total transcriptional levels. We evaluate the effect of normalization methods on identifying differentially expressed genes in the context of identifying the effect of the time of day on gene expression and response to chilling stress in sorghum. 

Many correlations exist between spectral reflectance or transmission with various phenotypic responses from plants. Of interest to us are metabolic characteristics, namely, how the various polarimetric components of plants may correlate to underlying environmental, metabolic, and genotypic differences among different varieties within a given species, as conducted during large field experimental trials. In this paper, we overview a portable Mueller matrix imaging spectropolarimeter, optimized for field use, by combining a temporal and spatial modulation scheme. Key aspects of the design include minimizing the measurement time while maximizing the signal-to-noise ratio by mitigating systematic error. This was achieved while maintaining an imaging capability across multiple measurement wavelengths, spanning the blue to near-infrared spectral region (405–730 nm). To this end, we present our optimization procedure, simulations, and calibration methods. Validation results, which were taken in redundant and non-redundant measurement configurations, indicated that the polarimeter provides average absolute errors of (5.3±2.2)×10⁻³and (7.1±3.1)×10⁻³, respectively. Finally, we provide preliminary field data (depolarization, retardance, and diattenuation) to establish baselines of barren and non-barrenZea maizehybrids (G90 variety), as captured from various leaf and canopy positions during our summer 2022 field experiments. Results indicate that subtle variations in retardance and diattenuation versus leaf canopy position may be present before they are clearly visible in the spectral transmission.