Stutts, Lauren*1, Wang, Yishi2 and Stapleton, Ann E.1§
1Department of Biology and Marine Biology, 2Department of Mathematics and Statistics email: email@example.com, University of North Carolina Wilmington, Wilmington, NC 28403
§corresponding author firstname.lastname@example.org 910-962-7267
*current address Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL email: email@example.com
running title: Synergistic hormone-environmental stress interactions in maize
date of submission: November 16, 2016
12 figures, all color online-only
word count: 5118
supplemental files: one figure, one table, two data files, six results files
Plant growth regulators can ameliorate effects of combinations of abiotic and biotic stress in maize, in certain genotypes and under specific stress conditions.
Plant growth regulators have documented roles in plant responses to single stresses. In combined-stress environments, plants display novel genetic architecture for growth traits and the response to growth regulators is unclear. We investigated the role of plant growth regulators in combined-stress responses in Zea mays. Twelve maize inbreds were exposed to all combinations of the following stressors: drought, nitrogen, and density stress. Chemical treatments were utilized to alter balances of the hormones abscisic acid, gibberellic acid, and brassinosteroids. We found a significant difference between the seed weights of plants given different chemical treatments after accounting for differences in genotype and stress environments. We conclude that plant growth regulators have targets in combined-stress response pathways in Zea mays.
Key words: density stress, drought, low nitrogen, plant growth regulators, hormones, abiotic stress combinations, genotype variation, kernel weight, seed weight, maize
Of all of the world’s grains, maize production is the largest by weight, and the United States is the top exporter of this grain (Capehart) USDA (2016). The large amounts of maize produced are used globally in various roles; maize remains a major source of food for both humans and animals around the world, and is also utilized in the production of biofuels. Maize production is continuously being improved by efforts in plant breeding and genetic modification, therefore maize genetics remains a central area of research. Geneticists aim to select for traits that will result in better protection against pests, more resistance to harsh environmental conditions, and as a more nutritious food source (Carena et al. , 2010). With the continued growth of the world’s population the agriculture industry faces a higher demand for grains and smaller land resources to meet this demand. Therefore, the overarching goal is to produce crops of a higher quality at higher quantity. Knowledge about plant response to stress at the molecular level is key to meeting higher demand, as a large proportion of crops are exposed to stress annually (Lobell and Gourdji, 2012).
Response to Environmental Stress The response of crops to abiotic and biotic stress has long been a focal point for agricultural research. A solid comprehension of the mechanisms plants use to combat environmental stresses such as drought, light-stress, fungal infections, and nutrient depletion, for example, allows researchers to develop plants that will be more resistant to these stresses (Taiz and Zeiger, 2006). Exposure of plants to stress at certain points of development can have detrimental impacts on growth and crop yield (Carena et al., 2010). Significant decreases in corn grain yield and plant biomass can result from limitations in nitrogen availability, which is especially important in low-input smallholder settings (Weber et al., 2012). Loss of plant biomass can also be seen in response to varying plant density, even in some modern maize hybrids (Tokatlidis et al., 2011).
Source-sink balance is a key determinant of the final harvest weight of maize kernels, typically with an interaction between genotypes and environmental limits across years (Borrás et al., 2004; Sala et al. , 2007; Boomsma et al., 2009). Kernel weight is less affected by late abiotic stress than kernel number, and the kernel weight environmental response varied across hybrid genotypes (Slafer and Otegui, 2000). This makes kernel weight a useful trait across both basic research and applied agronomic experimentation (Kesavan et al., 2013; Zhang et al., 2016).
Increased attention has been given recently to plant responses to combined stresses (Rejeb et al., 2014). Plant physiological responses to combined-stress are not additive; when exposed to two simultaneous stresses, portions of the two single-stress response genetic pathways are expressed, but not all (Mittler, 2006; Suzuki et al. , 2014). A synergistic response to drought and low nitrogen maize can be present and has been exploited for production via agronomic advice to reduce nitrogen fertilizer application under drought conditions (Bennett et al., 1989; Weber et al., 2012; Sadras and Richards, 2014), though this synergistic response is genetically variable and thus would not apply to all production settings. Beyond the nitrogen-drought interaction, additional non-linear combinatoric responses can be used to group maize genotypes into high and low input optimal types (Ruffo et al., 2015).
A signaling network has been proposed by Makumburage et al. (Makumburage et al. , 2013), in which loci within individual stress response pathways repress loci in different stress response pathways. Evidence was provided that maize displays a novel genetic architecture in response to combined stress, relative to the architecture of genetic response to a single stress. Makumburage et al. (2013) observed that the interaction between two stress-response pathways in corn allowed improved growth under combined-stresses compared to what would be expected. Combining abiotic and biotic stressors, specifically plant density, also results in non- additive responses (Rossini et al., 2011). Information about response to combined-stresses is relevant in the agriculture industry, because crops growing in the field encounter multiple stresses simultaneously throughout their life cycle, rather than only one stress in an otherwise controlled environment.
At the cellular level, it has been observed that once a plant has been exposed to one stress, the molecular response to a second stress can be altered (Rasmussen et al., 2013). Furthermore, novel genes not expressed under either stress individually are expressed when the plant is exposed to both stresses simultaneously (Rizhsky et al., 2004; Plessis et al., 2015). Humbert et al. (Humbert et al., 2013) presented data confirming that maize transcript-level response to drought varied depending on whether the plant was also under nitrogen stress.
Genotypes For this study, we selected a range of genotypes that were from temperate, tropical and mapping populations. The B73 and Mo17 inbreds are widely studied; B73 in particular was a key to improved germplasm in the single-cross hybrid era of maize breeding (Carena et al. , 2010). Improved tropical genotype CML103 was selected by CIMMYT breeders and is included in key diversity panels such as the NAM (McMullen et al., 2009). We also chose a few genotypes from a widely used mapping population (more than 300 citations), which was derived from B73 and Mo17 with intermating to increase the number of recombination events (Lee et al., 2002).
Plant Hormones Plant hormones have been long known to be mediators between the external environment and the internal activities of plants (Wilkinson et al., 2012). Plant hormones regulate the growth and development of plants, stimulating seed germination, placement and growth of new organs, death and abscission of organs, ripening of fruit, and regulation of stomatal closure (Taiz and Zeiger, 2006). Hormones are involved in cross-talk between other pathways within the plant (Mittler et al., 2011), and often play an integrator role between multiple pathways (Jaillais and Chory, 2010; Gómez-Cadenas et al. , 2014). Plant hormones are tightly intertwined with every aspect of the organism’s life. Due to their role as pathway integrators, we have focused on hormones as candidates for the interaction seen between stress-response pathways during multiple-stress responses.
Gibberellins are a group of plant hormones known to influence plant growth and development (Taiz and Zeiger, 2006). These compounds are synthesized in the chloroplasts, endoplasmic reticulum, and cytosol of plant cells, and transported via the xylem, and play a role in modulation of abiotic stress (Colebrook et al., 2014). Abscisic acid has long been recognized for its role in plant response to water-limiting conditions. Abscisic acid is known to be a key player in regulating the opening and closing of stomata, by controlling the surrounding guard cells (Li et al., 2006). There is evidence that abscisic acid may be active in helping plants to tolerate both short-term and long-term stress (Sreenivasulu et al., 2012). Another group of plant signaling molecules known to have significant impacts on plant growth is brassinosteroids. These molecules are synthesized in the endoplasmic reticulum and bind neighboring cell surface receptors (Symons et al., 2008), thereby initiating complex signaling pathways within the plant, allowing response to environmental conditions (Belkhadir and Chory, 2006).
Plant Growth Regulators Due to their effects on plant traits, hormones are targets of researchers attempting to influence these traits. “Plant Growth Regulator” is a term given to a large group of chemicals used to alter intrinsic levels of plant hormones. Many of these chemicals are sold commercially, and target the biosynthesis or degradation of plant hormones. In this study, we used three commercial plant growth regulators, along with direct application of gibberellic acid, to change hormone levels within individual plants. The three plant growth regulators used were paclobutrazol (PAC), uniconazole (UCN), and propiconazole (PCZ). These compounds are all triazoles, which target enzymes and result in inhibition of the synthesis of various compounds, such as GA, BR, and ABA. Some triazole compounds were originally used as fungicides (by limiting GA synthesis in fungi), and were later recognized for their effects on plant growth (Rademacher et al., 1992). Paclobutrazol is commonly used to limit stem elongation in crops. The compound inhibits synthesis of gibberellic acid by preventing formation of the precursor molecule kaurenoic acid (Hedden and Graebe, 1985). The limitation of stem elongation in crops is beneficial because it prevents lodging, or stem breakage, when top-heavy plants are exposed to adverse conditions. Uniconazole is another regulator used to limit plant height. Uniconazole has also been shown to increase drought tolerance in Arabidopsis thaliana (Saito et al., 2006). These effects are achieved by inhibiting synthesis of GA and BR, and inhibiting the breakdown of ABA. Treatment of maize with propiconazole also results in dwarf phenotypes, via inhibition of BR synthesis (Hartwig et al., 2012).
In this study we investigated the role of hormones in plant responses to combined stresses, via manipulation of intrinsic hormone levels of plants grown in single-stress and combined-stress environments. We predicted that an alteration in hormone balance would alter combined-stress response pathways and ultimately alter phenotypic response. We found that for certain genotypes, a creation of hormone imbalance within the plants altered the seed weight trait response to combined-stress environments.
MATERIALS AND METHODS