Introduction

Water is the most limiting resource for crop production (Boyer, 1982); consequently, there is considerable interest in understanding drought tolerance and creating drought‐tolerant germplasm. Drought is a complex trait in that the timing and degree of water limitation produce a multitude of stress‐induced responses. In maize, a drought stress that occurs during the vegetative phase of growth and development can reduce plant height, decrease leaf elongation and trigger leaf wilting. If the stress continues unabated, leaf senescence ensues followed by leaf death. Drought stress occurring during the reproductive phase can cause pollen sterility, tassel necrosis, delayed silk exsertion and kernel abortion. All of these reactions can contribute to a reduction in grain yield and likely evolved as conservative, adaptive mechanisms by the plant. However, these mechanisms are less than ideal for obtaining maximum grain yield in modern production agriculture. The basic premise is that maize is too conservative in its overall response to drought for the objective of maintaining high yield in agricultural environments and that modulating this conservatism could lead to an increase in grain yield.

Phytohormones are potent molecules that control diverse plant phenotypes. One of these, ethylene, has been demonstrated to regulate many different aspects of growth and development, particularly under abiotic stresses. Ethylene is a gas that is synthesized in almost all plant tissues in the presence of oxygen (Lin et al., 2010). Methionine is the starting point in the ethylene biosynthetic pathway, and it is converted into S‐adenosylmethionine (SAM) by methionine adenosyltransferases (Yang and Hoffman, 1984). ACC synthase (ACS) catalyses the conversion of SAM into 1‐aminocyclopropane‐1‐carboxylic acid (ACC), the first committed step of ethylene biosynthesis. ACC is then converted by ACC oxidase into ethylene with ACC oxidase activity largely considered as constitutive in plants (Yang and Hoffman, 1984). Ethylene becomes the effector molecule that triggers subsequent reactions. Signalling is initiated via the interaction between the ethylene ligand and its receptors localized in the endoplasmic reticulum (Lin et al., 2010). This binding shuts down receptor signalling, releasing the pathway from inhibition and setting forth a cascade of downstream cellular actions (Alonso and Ecker, 2001; Bleecker and Kende, 2000; Klee, 2002).

The ethylene pathway has been linked to many diverse physiological processes in vegetative organs of maize. One of the best characterized is the role of ethylene in aerenchyma formation in roots under water‐logging conditions. Seedling roots exposed to hypoxia exhibit an increased rate of ethylene evolution, greater activities of ACS and ACC oxidase relative to controls and consequently greater programmed cell death that manifests itself as aerenchyma (He et al., 1996). A drought study with maize seedlings demonstrated there was an inverse relationship between endogenous ethylene levels and root elongation (Alarcón et al., 2009) and that elongation was inhibited when roots were exposed to ACC. In another example, Young et al. (2004) generated knockouts of ACSs in maize and documented that mutants had a reduction in ethylene emission as well as an inhibition in drought‐induced senescence in older leaves.

In maize, as well as other cereals, ethylene has also been linked to numerous facets of growth and development in reproductive organs. Similar to the response shown in flooded roots (He et al., 1996), elevated ethylene levels were coupled to the triggering of programmed cell death in endosperms of developing kernels (Young et al., 1997). Under water‐limiting conditions, kernel abortion in maize typically occurs at the ear tip. In a study by Feng et al. (2011), ethylene production in kernels that ultimately aborted at the ear tip declined more slowly and was maintained at a higher level than that of kernels on the rest of the ear. An in vitro study with maize kernels demonstrated that adding ACC to cultured kernels caused them to abort and those that remained viable had reduced mass (Hanft et al., 1990). Application of ACC to developing kernels of field‐grown plants reduced the mass of apical kernels that led the authors to conclude that ethylene was involved in kernel abortion (Cheng and Lur, 1996). Cox and Andrade (1988) determined that application of ethephon (a chemical that increases ethylene evolution) to field‐grown maize hybrids caused a reduction in kernel number/ear. In small grain cereals, a related set of studies has shown similar ethylene functionality. In wheat (Triticum aestivum L.), a heat stress was applied during reproductive development to heat‐tolerant and heat‐susceptible cultivars (Hays et al., 2007). This stress caused a 6‐, 7‐ and 12‐fold change in ethylene emission in developing kernels, embryos and the flag leaf, respectively, in the heat‐susceptible cultivar, but no change in ethylene levels in the heat‐tolerant one. Yang et al. (2006) exposed developing rice (Oryza sativa L.) kernels to an ethylene inhibitor and measured an increase in cell division rate, maximum cell number, grain‐filling rate and grain mass of inferior spikelets. In rice endosperms, cell division rate and starch concentration were negatively correlated with ethylene levels (Panda et al., 2009). From these studies, as well as others (Beltrano et al., 1994, 1999; Mohapatra et al., 2009; Yang et al., 2007; Zhang et al., 2009), it is apparent that ethylene physiology plays an important role in the decreased yield of cereals grown under abiotic stress conditions (Wilkinson et al., 2012).

We are interested in ethylene as it relates to improving drought tolerance in maize because of the numerous pharmacological and morphometric studies that have associated this hormone with grain yield stability. Additionally, we are interested in this phytohormone because its biosynthesis, catabolism and signalling have been well characterized at the molecular level, and consequently, the pathway is readily amenable to transgenic modification in plants. As the conversion of SAM to ACC is the rate‐limiting step in production of ethylene (Woeste et al., 1999), it follows that ACSs would be a focal point for control of ethylene biosynthesis. The objective of this research was to use a transgenic approach to reduce the synthesis of ethylene in maize via expression of an ACS RNA interference construct and to determine its subsequent effect on plant performance in abiotic stress environments.