A normalization method that controls for total RNA abundance affects the identification of differentially expressed genes, revealing bias toward morning-expressed responses.

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.

Time of day and genotype sensitivity adjust molecular responses to temperature stress in sorghum.

Sorghum is one of the four major C4 crops that are considered to be tolerant to environmental extremes. Sorghum shows distinct growth responses to temperature stress depending on the sensitivity of the genetic background. About half of the transcripts in sorghum exhibit diurnal rhythmic expressions emphasizing significant coordination with the environment. However, an understanding of how molecular dynamics contribute to genotype-specific stress responses in the context of the time of day is not known. We examined whether temperature stress and the time of day impact the gene expression dynamics in thermo-sensitive and thermo-tolerant sorghum genotypes. We found that time of day is highly influencing the temperature stress responses, which can be explained by the rhythmic expression of most thermo-responsive genes. This effect is more pronounced in thermo-tolerant genotypes, suggesting a stronger regulation of gene expression by the time of day and/or by the circadian clock. Genotypic differences were mostly observed on average gene expression levels, which may be responsible for contrasting sensitivities to temperature stress in tolerant versus susceptible sorghum varieties. We also identified groups of genes altered by temperature stress in a time-of-day and genotype-specific manner. These include transcriptional regulators and several members of the Ca2+ -binding EF-hand protein family. We hypothesize that expression variation of these genes between genotypes along with time-of-day independent regulation may contribute to genotype-specific fine-tuning of thermo-responsive pathways. These findings offer a new opportunity to selectively target specific genes in efforts to develop climate-resilient crops based on their time-of-day and genotype variation responses to temperature stress.

Redox-engineering enhances maize thermotolerance and grain yield in the field.

Increasing populations and temperatures are expected to escalate food demands beyond production capacities, and the development of maize lines with better performance under heat stress is desirable. Here, we report that constitutive ectopic expression of a heterologous glutaredoxin S17 from Arabidopsis thaliana (AtGRXS17) can provide thermotolerance in maize through enhanced chaperone activity and modulation of heat stress-associated gene expression. The thermotolerant maize lines had increased protection against protein damage and yielded a sixfold increase in grain production in comparison to the non-transgenic counterparts under heat stress field conditions. The maize lines also displayed thermotolerance in the reproductive stages, resulting in improved pollen germination and the higher fidelity of fertilized ovules under heat stress conditions. Our results present a robust and simple strategy for meeting rising yield demands in maize and, possibly, other crop species in a warming global environment.

Deep learning based high-throughput phenotyping of chalkiness in rice exposed to high night temperature.

BACKGROUND: Rice is a major staple food crop for more than half the world's population. As the global population is expected to reach 9.7 billion by 2050, increasing the production of high-quality rice is needed to meet the anticipated increased demand. However, global environmental changes, especially increasing temperatures, can affect grain yield and quality. Heat stress is one of the major causes of an increased proportion of chalkiness in rice, which compromises quality and reduces the market value. Researchers have identified 140 quantitative trait loci linked to chalkiness mapped across 12 chromosomes of the rice genome. However, the available genetic information acquired by employing advances in genetics has not been adequately exploited due to a lack of a reliable, rapid and high-throughput phenotyping tool to capture chalkiness. To derive extensive benefit from the genetic progress achieved, tools that facilitate high-throughput phenotyping of rice chalkiness are needed. RESULTS: We use a fully automated approach based on convolutional neural networks (CNNs) and Gradient-weighted Class Activation Mapping (Grad-CAM) to detect chalkiness in rice grain images. Specifically, we train a CNN model to distinguish between chalky and non-chalky grains and subsequently use Grad-CAM to identify the area of a grain that is indicative of the chalky class. The area identified by the Grad-CAM approach takes the form of a smooth heatmap that can be used to quantify the degree of chalkiness. Experimental results on both polished and unpolished rice grains using standard instance classification and segmentation metrics have shown that Grad-CAM can accurately identify chalky grains and detect the chalkiness area. CONCLUSIONS: We have successfully demonstrated the application of a Grad-CAM based tool to accurately capture high night temperature induced chalkiness in rice. The models trained will be made publicly available. They are easy-to-use, scalable and can be readily incorporated into ongoing rice breeding programs, without rice researchers requiring computer science or machine learning expertise.

A universal method for high-quality RNA extraction from plant tissues rich in starch, proteins and fiber.

Using existing protocols, RNA extracted from seeds rich in starch often results in poor quality RNA, making it inappropriate for downstream applications. Though some methods are proposed for extracting RNA from plant tissue rich in starch and other polysaccharides, they invariably yield less and poor quality RNA. In order to obtain high yield and quality RNA from seeds and other plant tissues including roots a modified SDS-LiCl method was compared with existing methods, including TRIZOL kit (Invitrogen), Plant RNeasy mini kit (Qiagen), Furtado (2014) method, and CTAB-LiCl method. Modifications in the extraction buffer and solutions used for RNA precipitation resulted in a robust method for extracting RNA in seeds and roots, where extracting quality RNA is challenging. The modified SDS-LiCl method revealed intense RNA bands through gel electrophoresis and a nanodrop spectrophotometer detected ratios of ≥ 2 and 1.8 for A260/A230 and A260/A280, respectively. The absence of starch co-precipitation during RNA extraction resulted in enhanced yield and quality of RNA with RIN values of 7-9, quantified using a bioanalyzer. The high-quality RNA obtained was demonstrated to be suitable for downstream applications, such as cDNA synthesis, gene amplification, and RT-qPCR. The method was also effective in extracting RNA from seeds of other cereals including field-grown sorghum and corn. The modified SDS-LiCl method is a robust and highly reproducible RNA extraction method for plant tissues rich in starch and other secondary metabolites. The modified SDS-LiCl method successfully extracted high yield and quality RNA from mature, developing, and germinated seeds, leaves, and roots exposed to different abiotic stresses.

Does susceptibility to heat stress confound screening for drought tolerance in rice?

Drought affected rice areas are predicted to double by the end of this century, demanding greater tolerance in widely adapted mega-varieties. Progress on incorporating better drought tolerance has been slow due to lack of appropriate phenotyping protocols. Furthermore, existing protocols do not consider the effect of drought and heat interactions, especially during the critical flowering stage, which could lead to false conclusion about drought tolerance. Screening germplasm and mapping-populations to identify quantitative trait loci (QTL)/candidate genes for drought tolerance is usually conducted in hot dry seasons where water supply can be controlled. Hence, results from dry season drought screening in the field could be confounded by heat stress, either directly on heat sensitive processes such as pollination or indirectly by raising tissue temperature through reducing transpirational cooling under water deficit conditions. Drought-tolerant entries or drought-responsive candidate genes/QTL identified from germplasm highly susceptible to heat stress during anthesis/flowering have to be interpreted with caution. During drought screening, germplasm tolerant to water stress but highly susceptible to heat stress has to be excluded during dry and hot season screening. Responses to drought and heat stress in rice are compared and results from field and controlled environment experiments studying drought and heat tolerance and their interaction are discussed.