Transcriptomes of Eight Arabidopsis thaliana Accessions Reveal Core Conserved, Genotype- and Organ-Specific Responses to Flooding Stress

Climate change has increased the frequency and severity of flooding events, with significant negative impact on agricultural productivity. These events often submerge plant aerial organs and roots, limiting growth and survival due to a severe reduction in light reactions and gas exchange necessary for photosynthesis and respiration, respectively. To distinguish molecular responses to the compound stress imposed by submergence, we investigated transcriptomic adjustments to darkness in air and under submerged conditions using eight Arabidopsis (Arabidopsis thaliana) accessions differing significantly in sensitivity to submergence. Evaluation of root and rosette transcriptomes revealed an early transcriptional and posttranscriptional response signature that was conserved primarily across genotypes, although flooding susceptibility-associated and genotype-specific responses also were uncovered. Posttranscriptional regulation encompassed darkness- and submergence-induced alternative splicing of transcripts from pathways involved in the alternative mobilization of energy reserves. The organ-specific transcriptome adjustments reflected the distinct physiological status of roots and shoots. Root-specific transcriptome changes included marked up-regulation of chloroplast-encoded photosynthesis and redox-related genes, whereas those of the rosette were related to the regulation of development and growth processes. We identified a novel set of tolerance genes, recognized mainly by quantitative differences. These included a transcriptome signature of more pronounced gluconeogenesis in tolerant accessions, a response that included stress-induced alternative splicing. This study provides organ-specific molecular resolution of genetic variation in submergence responses involving interactions between darkness and low-oxygen constraints of flooding stress and demonstrates that early transcriptome plasticity, including alternative splicing, is associated with the ability to cope with a compound environmental stress.

The environment that surrounds a plant changes constantly, often imposing constraints on metabolism that modify vegetative and reproductive development. Flooding can have a dramatic impact on plant performance; while it occurs regularly in some natural ecosystems, it is usually disastrous in controlled agricultural environments. Flooding restricts gas diffusion between submerged organs and the surrounding aquatic environment. The limited exchange of oxygen and CO₂ slows down aerobic respiration and photosynthesis (Mommer and Visser, 2005; Zabalza et al., 2009). Turbid and muddy floodwaters restrict light penetration, further compromising the photoautotrophic generation of critical carbohydrates (Vervuren et al., 2003). Finally, oxygen-deficient flooded soils often have a severely reduced redox potential and accumulate toxic compounds, which limit root growth (Armstrong and Armstrong, 2001).

Therefore, flooding is a compound stress, imposing multiple constraints on submerged plants. Despite this, marshes and river floodplains support a rich diversity of plant life that display a gradient of flood tolerance traits and responses (Van Eck et al., 2004; Voesenek et al., 2004). Studies on rice (Oryza sativa) and several wild species have identified two antithetical survival strategies, dependent on the selection pressure of their natural flooding regime. An escape response involving rapid shoot elongation allows plants to regain air contact by forming a snorkel during shallow and prolonged floods (Voesenek and Bailey-Serres, 2015). Deep or very short floods require a quiescent strategy, where a restriction of growth combined with conservation of energy expenditure and reserve utilization promotes survival until the floods recede (van Veen et al., 2014b). Fundamental knowledge of the genetic, physiological, and molecular regulation of these traits is not only of general interest but essential to improve the tolerance of many economically relevant crops, most of which are very sensitive to floods (Voesenek et al., 2014). The genetic and molecular regulation of flood-adaptive strategies has been studied most extensively in semiaquatic flood-tolerant species of the genera Oryza, Rorippa, and Rumex (Fukao et al., 2006; Hattori et al., 2009; Lee et al., 2009; Sasidharan et al., 2013; van Veen et al., 2013, 2014a; Narsai et al., 2015).

Our understanding of the flooding-induced low-oxygen and low-energy signaling networks also has benefited greatly from studies on flood-sensitive Arabidopsis (Arabidopsis thaliana). These investigations have identified the main players in energy and carbon signaling (Smeekens et al., 2010; Ljung et al., 2015) and revealed whole-plant and cell type-specific transcriptional and translational adjustments induced by low-oxygen stress (Mustroph et al., 2009; Juntawong et al., 2014). Importantly, oxygen-dependent degradation of the group VII family of ethylene response factors via the N-end rule pathway of protein degradation has been identified as a molecular mechanism that translates oxygen availability into transcriptional reprogramming (Gibbs et al., 2011; Licausi et al., 2011; Weits et al., 2014). Recent studies also have revealed how this molecular hypoxic response is highly regulated and fine-tuned to maintain cellular homeostasis during low-oxygen conditions (Gibbs et al., 2014; Giuntoli et al., 2014; Gonzali et al., 2015).

Despite the progress in our understanding of flooding-induced signaling pathways, much remains to be discovered regarding the molecular mechanisms that cause variation in flooding tolerance across and within species (Voesenek and Bailey-Serres, 2015). Variation in flooding responses among natural plant populations is an important tool to identify the underlying causal genes and processes (Xu et al., 2006; Magneschi et al., 2009; Chen et al., 2010; Campbell et al., 2015). Despite their relative intolerance to flooding stress, Arabidopsis accessions show considerable variation in their tolerance to complete submergence (Vashisht et al., 2011). Remarkably, this variation is not linked to differences in internal oxygen content or initial carbohydrate reserves, the two parameters generally considered to be essential for surviving flooding events.

The majority of studies investigating the molecular regulation of transcriptional reprograming in response to changes in oxygen availability in Arabidopsis have relied on hypoxia and/or used agar-based seedling assays (Baena-González et al., 2007; Branco-Price et al., 2008; Bond et al., 2009; Christianson et al., 2009; Mustroph et al., 2009; Banti et al., 2010). However, in natural conditions, flooding results in a gradual decline in oxygen levels and often is accompanied by other physiological changes, such as a rapid buildup of the gaseous hormone ethylene (Voesenek and Sasidharan, 2013). Furthermore, flooding imposes distinct environmental constraints on the root and the shoot and, thereby, also elicits different physiological responses. Accordingly, an exploration of the shoot and root responses of flooded, soil-grown plants is more relevant to understanding flooding stress as experienced in the field.

Here, we characterized the early molecular response to darkness and flooding acclimation in eight different Arabidopsis genetic backgrounds (Supplemental Table S1), varying in their tolerance to complete submergence, using poly(A)⁺ mRNA sequencing (mRNAseq). The use of soil-grown plants subjected to submergence (in the dark) mimicked naturally flooded conditions in a highly controlled way, and the inclusion of a darkness-only (without submergence) treatment allowed us to simultaneously disentangle dark effects from submergence effects (Lee et al., 2011; Vashisht et al., 2011). Given the distinct carbohydrate and oxygen status of the root and shoot (rosette) tissues under these two stress conditions, these organs were analyzed separately, and then each organ response was compared with the other. This was performed for all eight accessions. Our data suggest an important role for gluconeogenesis in short-term stress acclimation, which includes alternative splicing (AS) of transcripts encoding key regulatory enzymes and quantitative transcriptional differences between tolerant and intolerant accessions. A conservative mode of energy and resource utilization via metabolic reprogramming and constrained growth contributes toward prolonged survival underwater. Shoot-specific flooding-induced transcriptional reprogramming was primarily growth related, whereas in the root, mainly plastidial and developmental processes changed. Our results provide insight into the interactive and additive effects of the different elements of flooding stress, present a detailed picture of early molecular events mediating stress acclimation, and identify putative novel aspects of flooding tolerance.