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.

Comparative transcriptome analysis reveals candidate genes for cold stress response and early flowering in pineapple.

Pineapple originates from tropical regions in South America and is therefore significantly impacted by cold stress. Periodic cold events in the equatorial regions where pineapple is grown may induce early flowering, also known as precocious flowering, resulting in monetary losses due to small fruit size and the need to make multiple passes for harvesting a single field. Currently, pineapple is one of the most important tropical fruits in the world in terms of consumption, and production losses caused by weather can have major impacts on worldwide exportation potential and economics. To further our understanding of and identify mechanisms for low-temperature tolerance in pineapple, and to identify the relationship between low-temperature stress and flowering time, we report here a transcriptomic analysis of two pineapple genotypes in response to low-temperature stress. Using meristem tissue collected from precocious flowering-susceptible MD2 and precocious flowering-tolerant Dole-17, we performed pairwise comparisons and weighted gene co-expression network analysis (WGCNA) to identify cold stress, genotype, and floral organ development-specific modules. Dole-17 had a greater increase in expression of genes that confer cold tolerance. The results suggested that low temperature stress in Dole-17 plants induces transcriptional changes to adapt and maintain homeostasis. Comparative transcriptomic analysis revealed differences in cuticular wax biosynthesis, carbohydrate accumulation, and vernalization-related gene expression between genotypes. Cold stress induced changes in ethylene and abscisic acid-mediated pathways differentially between genotypes, suggesting that MD2 may be more susceptible to hormone-mediated early flowering. The differentially expressed genes and module hub genes identified in this study are potential candidates for engineering cold tolerance in pineapple to develop new varieties capable of maintaining normal reproduction cycles under cold stress. In addition, a total of 461 core genes involved in the development of reproductive tissues in pineapple were also identified in this study. This research provides an important genomic resource for understanding molecular networks underlying cold stress response and how cold stress affects flowering time in pineapple.

Weed-induced changes in the maize root transcriptome reveal transcription factors and physiological processes impacted early in crop-weed interactions.

A new paradigm suggests weeds primarily reduce crop yield by altering crop developmental and physiological processes long before the weeds reduce resources through competition. Multiple studies have implicated stress response pathways are activated when crops such as maize are grown in close proximity with weeds during the first 4-8 weeks of growth-the point at which weeds have their greatest impact on subsequent crop yields. To date, these studies have mostly focused on the response of above-ground plant parts and have not examined the early signal transduction processes associated with maize root response to weeds. To investigate the impact of signals from a below-ground competitor on the maize root transcriptome when most vulnerable to weed pressure, a system was designed to expose maize to only below-ground signals. Gene set enrichment analyses identified over-represented ontologies associated with oxidative stress signalling throughout the time of weed exposure, with additional ontologies associated with nitrogen use and transport and abscisic acid (ABA) signalling, and defence responses being enriched at later time points. Enrichment of promoter motifs indicated over-representation of sequences known to bind FAR-RED IMPAIRED RESPONSE 1 (FAR1), several AP2/ERF transcription factors and others. Likewise, co-expression networks were identified using Weighted-Gene Correlation Network Analysis (WGCNA) and Spatiotemporal Clustering and Inference of Omics Networks (SC-ION) algorithms. WGCNA highlighted the potential roles of several transcription factors including a MYB 3r-4, TB1, WRKY65, CONSTANS-like5, ABF3, HOMEOBOX 12, among others. These studies also highlighted the role of several specific proteins involved in ABA signalling as being important for the initiation of the early response of maize to weeds. SC-ION highlighted potential roles for NAC28, LOB37, NAC58 and GATA2 transcription factors, among many others.

Hybrid spatial-temporal Mueller matrix imaging spectropolarimeter for high throughput plant phenotyping.

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)×10-3 and (7.1±3.1)×10-3, respectively. Finally, we provide preliminary field data (depolarization, retardance, and diattenuation) to establish baselines of barren and non-barren Zea maize hybrids (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.

The Game of Timing: Circadian Rhythms Intersect with Changing Environments.

Recurring patterns are an integral part of life on Earth. Through evolution or breeding, plants have acquired systems that coordinate with the cyclic patterns driven by Earth's movement through space. The biosystem responses to these physical rhythms result in biological cycles of daily and seasonal activity that feed back into the physical cycles. Signaling networks to coordinate growth and molecular activities with these persistent cycles have been integrated into plant biochemistry. The plant circadian clock is the coordinator of this complex, multiscale, temporal schedule. However, we have detailed knowledge of the circadian clock components and functions in only a few species under controlled conditions. We are just beginning to understand how the clock functions in real-world conditions. This review examines what we know about the circadian clock in diverse plant species, the challenges with extrapolating data from controlled environments, and the need to anticipate how plants will respond to climate change.

Auxin-cytokinin interplay shapes root functionality under low-temperature stress.

Low-temperature stress alters root system architecture. In particular, changes in the levels and response to auxin and cytokinin determine the fate of root architecture and function under stress because of their vital roles in regulating root cell division, differentiation, and elongation. An intricate nexus of genes encoding components of auxin and cytokinin biosynthesis, signaling, and transport components operate to counteract stress and facilitate optimum development. We review the role of auxin transport and signaling and its regulation by cytokinin during root development and stem cell maintenance under low-temperature stress. We highlight intricate mechanisms operating in root stem cells to minimize DNA damage by altering phytohormone levels, and discuss a working model for cytokinin in low-temperatures stress response.

Conserved plant transcriptional responses to microgravity from two consecutive spaceflight experiments.

Introduction: Understanding how plants adapt to the space environment is essential, as plants will be a valuable component of long duration space missions. Several spaceflight experiments have focused on transcriptional profiling as a means of understanding plant adaptation to microgravity. However, there is limited overlap between results from different experiments. Differences in experimental conditions and hardware make it difficult to find a consistent response across experiments and to distinguish the primary effects of microgravity from other spaceflight effects. Methods: Plant Signaling (PS) and Plant RNA Regulation (PRR) were two separate spaceflight experiments conducted on the International Space Station utilizing the European Modular Cultivation System (EMCS). The EMCS provided a lighted environment for plant growth with centrifugal capabilities providing an onboard 1 g control. Results and discussion: An RNA-Seq analysis of shoot samples from PS and PRR revealed a significant overlap of genes differentially expressed in microgravity between the two experiments. Relative to onboard 1 g controls, genes involved in transcriptional regulation, shoot development, and response to auxin and light were upregulated in microgravity in both experiments. Conversely, genes involved in defense response, abiotic stress, Ca++ signaling, and cell wall modification were commonly downregulated in both datasets. The downregulation of stress responses in microgravity in these two experiments is interesting as these pathways have been previously observed as upregulated in spaceflight compared to ground controls. Similarly, we have observed many stress response genes to be upregulated in the 1 g onboard control compared to ground reference controls; however these genes were specifically downregulated in microgravity. In addition, we analyzed the sRNA landscape of the 1 g and microgravity (µ g) shoot samples from PRR. We identified three miRNAs (miR319c, miR398b, and miR8683) which were upregulated in microgravity, while several of their corresponding target genes were found to be downregulated in microgravity. Interestingly, the downregulated target genes are enriched in those encoding chloroplast-localized enzymes and proteins. These results uncover microgravity unique transcriptional changes and highlight the validity and importance of an onboard 1 g control.

The intersection between circadian and heat-responsive regulatory networks controls plant responses to increasing temperatures.

Increasing temperatures impact plant biochemistry, but the effects can be highly variable. Both external and internal factors modulate how plants respond to rising temperatures. One such factor is the time of day or season the temperature increase occurs. This timing significantly affects plant responses to higher temperatures altering the signaling networks and affecting tolerance levels. Increasing overlaps between circadian signaling and high temperature responses have been identified that could explain this sensitivity to the timing of heat stress. ELF3, a circadian clock component, functions as a thermosensor. ELF3 regulates thermoresponsive hypocotyl elongation in part through its cellular localization. The temperature sensitivity of ELF3 depends on the length of a polyglutamine region, explaining how plant temperature responses vary between species. However, the intersection between the circadian system and increased temperature stress responses is pervasive and extends beyond this overlap in thermosensing. Here, we review the network responses to increased temperatures, heat stress, and the impacts on the mechanisms of gene expression from transcription to translation, highlighting the intersections between the elevated temperature and heat stress response pathways and circadian signaling, focusing on the role of ELF3 as a thermosensor.

Multistatic fiber-based system for measuring the Mueller matrix bidirectional reflectance distribution function.

Bidirectionality effects can be a significant confounding factor when measuring hyperspectral reflectance data. The bidirectional reflectance distribution function (BRDF) can effectively characterize the reflectivity of surfaces to correct remote sensing measurements. However, measuring BRDFs can be time-consuming, especially when collecting Mueller matrix BRDF (mmBRDF) measurements of a surface via conventional goniometric techniques. In this paper, we present a system for collecting mmBRDF measurements using static optical fiber detectors that sample the hemisphere surrounding an object. The entrance to each fiber contains a polarization state analyzer configuration, allowing for the simultaneous acquisition of the Stokes vector intensity components at many altitudinal and azimuthal viewing positions. We describe the setup, calibration, and data processing used for this system and present its performance as applied to mmBRDF measurements of a ground glass diffuser.

Evaluating the Effects of the Circadian Clock and Time of Day on Plant Gravitropic Responses.

Circadian rhythms are regular oscillations of an organism's physiology with a period of approximately 24 h. In the model plant Arabidopsis thaliana, circadian rhythms regulate a suite of physiological processes, including transcription, photosynthesis, growth, and flowering. The circadian clock and external rhythmic factors have extensive control of the underlying biochemistry and physiology. Therefore, it is critical to consider the time of day when performing gravitropism experiments, even if the circadian clock is not a focus of study. We describe the critical factors and methods to be considered and methods to investigate the possible circadian regulation of gravitropic responses.

Uncovering Transcriptional Responses to Fractional Gravity in Arabidopsis Roots.

Although many reports characterize the transcriptional response of Arabidopsis seedlings to microgravity, few investigate the effect of partial or fractional gravity on gene expression. Understanding plant responses to fractional gravity is relevant for plant growth on lunar and Martian surfaces. The plant signaling flight experiment utilized the European Modular Cultivation System (EMCS) onboard the International Space Station (ISS). The EMCS consisted of two rotors within a controlled chamber allowing for two experimental conditions, microgravity (stationary rotor) and simulated gravity in space. Seedlings were grown for 5 days under continuous light in seed cassettes. The arrangement of the seed cassettes within each experimental container results in a gradient of fractional g (in the spinning rotor). To investigate whether gene expression patterns are sensitive to fractional g, we carried out transcriptional profiling of root samples exposed to microgravity or partial g (ranging from 0.53 to 0.88 g). Data were analyzed using DESeq2 with fractional g as a continuous variable in the design model in order to query gene expression across the gravity continuum. We identified a subset of genes whose expression correlates with changes in fractional g. Interestingly, the most responsive genes include those encoding transcription factors, defense, and cell wall-related proteins and heat shock proteins.

Warm nights disrupt transcriptome rhythms in field-grown rice panicles.

In rice, a small increase in nighttime temperature reduces grain yield and quality. How warm nighttime temperatures (WNT) produce these detrimental effects is not well understood, especially in field conditions where the typical day-to-night temperature fluctuation exceeds the mild increase in nighttime temperature. We observed genome-wide disruption of gene expression timing during the reproductive phase in field-grown rice panicles acclimated to 2 to 3 °C WNT. Transcripts previously identified as rhythmically expressed with a 24-h period and circadian-regulated transcripts were more sensitive to WNT than were nonrhythmic transcripts. The system-wide perturbations in transcript levels suggest that WNT disrupt the tight temporal coordination between internal molecular events and the environment, resulting in reduced productivity. We identified transcriptional regulators whose predicted targets are enriched for sensitivity to WNT. The affected transcripts and candidate regulators identified through our network analysis explain molecular mechanisms driving sensitivity to WNT and identify candidates that can be targeted to enhance tolerance to WNT.

Quantification of gray mold infection in lettuce using a bispectral imaging system under laboratory conditions.

Gray mold disease caused by the fungus Botrytis cinerea damages many crop hosts worldwide and is responsible for heavy economic losses. Early diagnosis and detection of the disease would allow for more effective crop management practices to prevent outbreaks in field or greenhouse settings. Furthermore, having a simple, non-invasive way to quantify the extent of gray mold disease is important for plant pathologists interested in measuring infection rates. In this paper, we design and build a bispectral imaging system for discriminating between leaf regions infected with gray mold and those that remain unharmed on a lettuce (Lactuca spp.) host. First, we describe a method to select two optimal (high contrast) spectral bands from continuous hyperspectral imagery (450-800 nm). We then explain the process of building a system based on these two spectral bands, located at 540 and 670 nm. The resultant system uses two cameras, with a narrow band-pass spectral filter mounted on each, to measure the bispectral reflectance of a lettuce leaf. The two resulting images are combined using a normalized difference calculation that produces a single image with high contrast between the leaves' infected and healthy regions. A classifier was then created based on the thresholding of single pixel values. We demonstrate that this simple classification produces a true-positive rate of 95.25% with a false-positive rate of 9.316% in laboratory conditions.

Genome-wide association study and gene network analyses reveal potential candidate genes for high night temperature tolerance in rice.

High night temperatures (HNT) are shown to significantly reduce rice (Oryza sativa L.) yield and quality. A better understanding of the genetic architecture of HNT tolerance will help rice breeders to develop varieties adapted to future warmer climates. In this study, a diverse indica rice panel displayed a wide range of phenotypic variability in yield and quality traits under control night (24 °C) and higher night (29 °C) temperatures. Genome-wide association analysis revealed 38 genetic loci associated across treatments (18 for control and 20 for HNT). Nineteen loci were detected with the relative changes in the traits between control and HNT. Positive phenotypic correlations and co-located genetic loci with previously cloned grain size genes revealed common genetic regulation between control and HNT, particularly grain size. Network-based predictive models prioritized 20 causal genes at the genetic loci based on known gene/s expression under HNT in rice. Our study provides important insights for future candidate gene validation and molecular marker development to enhance HNT tolerance in rice. Integrated physiological, genomic, and gene network-informed approaches indicate that the candidate genes for stay-green trait may be relevant to minimizing HNT-induced yield and quality losses during grain filling in rice by optimizing source-sink relationships.

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.

Directions for research and training in plant omics: Big Questions and Big Data.

A key remit of the NSF-funded "Arabidopsis Research and Training for the 21st Century" (ART-21) Research Coordination Network has been to convene a series of workshops with community members to explore issues concerning research and training in plant biology, including the role that research using Arabidopsis thaliana can play in addressing those issues. A first workshop focused on training needs for bioinformatic and computational approaches in plant biology was held in 2016, and recommendations from that workshop have been published (Friesner et al., Plant Physiology, 175, 2017, 1499). In this white paper, we provide a summary of the discussions and insights arising from the second ART-21 workshop. The second workshop focused on experimental aspects of omics data acquisition and analysis and involved a broad spectrum of participants from academics and industry, ranging from graduate students through post-doctorates, early career and established investigators. Our hope is that this article will inspire beginning and established scientists, corporations, and funding agencies to pursue directions in research and training identified by this workshop, capitalizing on the reference species Arabidopsis thaliana and other valuable plant systems.

Novel transcriptional responses to heat revealed by turning up the heat at night.

KEY MESSAGE: The circadian clock controls many molecular activities, impacting experimental interpretation. We quantify the genome-wide effects of time-of-day on the heat-shock response and the effects of "diurnal bias" in stress experiments. Heat stress has significant adverse effects on plant productivity worldwide. Most experiments examining heat stress are performed during daytime hours, generating a 'diurnal bias' in the pathways and regulatory mechanisms identified. Such bias may confound downstream interpretations and limit our understanding of the full response to heat stress. Here we show that the transcriptional and physiological responses to a sudden heat shock in Arabidopsis are profoundly sensitive to the time of day. We observe that plant tolerance and acclimation to heat shock vary throughout the day and are maximal at dusk. Consistently, over 75% of heat-responsive transcripts show a time of day-dependent response, including many previously characterized heat-response genes. This temporal sensitivity implies a complex interaction between time and temperature where daily variations in basal transcription influence thermotolerance. When we examined these transcriptional responses, we uncovered novel night-response genes and cis-regulatory elements, underpinning new aspects of heat stress responses not previously appreciated. Exploiting this temporal variation can be applied to most environmental responses to understand the underlying network wiring. Therefore, we propose that using time as a perturbagen is an approach that will enhance our understanding of plant regulatory networks and responses to environmental stresses.

New candidate loci and marker genes on chromosome 7 for improved chilling tolerance in sorghum.

Sorghum is often exposed to suboptimal low temperature stress under field conditions, particularly at the seedling establishment stage. Enhancing chilling tolerance will facilitate earlier planting and so minimize the negative impacts of other stresses experienced at later growth stages. Genome-wide association mapping was performed on a sorghum association panel grown under control (30/20 °C; day/night) and chilling (20/10 °C) conditions. Genomic regions on chromosome 7, controlling the emergence index and seedling (root and shoot) vigor, were associated with increased chilling tolerance but they did not co-localize with undesirable tannin content quantitative trait loci (QTLs). Shoot and root samples from highly contrasting haplotype pairs expressing differential responses to chilling stress were used to identify candidate genes. Three candidate genes (an alpha/beta hydrolase domain protein, a DnaJ/Hsp40 motif-containing protein, and a YTH domain-containing RNA-binding protein) were expressed at significantly higher levels under chilling stress in the tolerant haplotype compared with the sensitive haplotype and BTx623. Moreover, two CBF/DREB1A transcription factors on chromosome 2 showed a divergent response to chilling in the contrasting haplotypes. These studies identify haplotype differences on chromosome 7 that modulate chilling tolerance by either regulating CBF or feeding back into this signaling pathway. We have identified new candidate genes that will be useful markers in ongoing efforts to develop tannin-free chilling-tolerant sorghum hybrids.

Analysis of differential gene expression and alternative splicing is significantly influenced by choice of reference genome.

RNA-seq analysis has enabled the evaluation of transcriptional changes in many species including nonmodel organisms. However, in most species only a single reference genome is available and RNA-seq reads from highly divergent varieties are typically aligned to this reference. Here, we quantify the impacts of the choice of mapping genome in rice where three high-quality reference genomes are available. We aligned RNA-seq data from a popular productive rice variety to three different reference genomes and found that the identification of differentially expressed genes differed depending on which reference genome was used for mapping. Furthermore, the ability to detect differentially used transcript isoforms was profoundly affected by the choice of reference genome: Only 30% of the differentially used splicing features were detected when reads were mapped to the more commonly used, but more distantly related reference genome. This demonstrated that gene expression and splicing analysis varies considerably depending on the mapping reference genome, and that analysis of individuals that are distantly related to an available reference genome may be improved by acquisition of new genomic reference material. We observed that these differences in transcriptome analysis are, in part, due to the presence of single nucleotide polymorphisms between the sequenced individual and each respective reference genome, as well as annotation differences between the reference genomes that exist even between syntenic orthologs. We conclude that even between two closely related genomes of similar quality, using the reference genome that is most closely related to the species being sampled significantly improves transcriptome analysis.

Neural Net Classification Combined With Movement Analysis to Evaluate Setaria viridis as a Model System for Time of Day of Anther Appearance.

In many plant species, the time of day at which flowers open to permit pollination is tightly regulated. Proper time of flower opening, or Time of Day of Anther Appearance (TAA), may coordinate flowering opening with pollinator activity or may shift temperature sensitive developmental processes to cooler times of the day. The genetic mechanisms that regulate the timing of this process in cereal crops are unknown. To address this knowledge gap, it is necessary to establish a monocot model system that exhibits variation in TAA. Here, we examine the suitability of Setaria viridis, the model for C4 photosynthesis, for such a role. We developed an imaging system to monitor the temporal regulation of growth, flower opening time, and other physiological characteristics in Setaria. This system enabled us to compare Setaria varieties Ames 32254, Ames 32276, and PI 669942 variation in growth and daily flower opening time. We observed that TAA occurs primarily at night in these three Setaria accessions. However, significant variation between the accessions was observed for both the ratio of flowers that open in the day vs. night and the specific time of day where the rate is maximal. Characterizing this physiological variation is a requisite step toward uncovering the molecular mechanisms regulating TAA. Leveraging the regulation of TAA could provide researchers with a genetic tool to improve crop productivity in new environments.