1 | INTRODUCTION
As the first cereal crop in the world, maize production is important to ensure food security (FAO, 2020). Because of the increasing population and feed demand due to diet shift in many areas, the continuous improvement of maize yield is a great challenge in the new era. Maize is sensitive to abiotic stresses such as drought and heat, which are major environmental factors that limit crop growth and productivity (Bonfante et al., 2015; Lizaso et al., 2018; Rossini, Maddonni, & Otegui 2016; Shen et al., 2018; Waha, Müller, & Rolinski, 2013). Drought and heat stress has been becoming more sever in time and over space around the world since 1980 (Dai, Trenberth, & Qian, 2004; Sheffield, Wood, & Roderick, 2012; Wang, Marija, Dong, Susanne, & Bernd, 2014; Harrison, Tardieu, Dong, Messina, & Hammer, 2014). Heat stress is frequently associated with reduced availability of water (Lesk, Rowhani, & Ramankutty, 2016; Wang et al., 2018), and the occurrence of drought accompanied by heat stress is predicted to increase in future (IPCC, 2014). Therefore, the understanding of independent and combined drought and heat stress impacts on maize during the whole growth duration and the underlying mechanism is pivotal to promote breeding and targeted management measures.
Across the global maize production area, the combined drought and heat stress mostly occurred around flowering (Lesk, Rowhani, & Ramankutty, 2016; Wang et al., 2018). During this stage, the adverse impacts of the stresses on the male and female maize organs for the seed-set and seed formation have been frequently reported (Bassetti and Westgate, 1993; Edreira, Carpici, Sammarro, & Otegui, 2011; Marceau, Saint-Jean, Loubet, Foueillassar, & Huber, 2012; Sánchez, Rasmussen, & Porter, 2014; Wang et al., 2018). The previous studies indicated that maize kernel number was significantly reduced in the stresses because of low pollen number and pollen viability, slow silk-growth and ovary-growth rate, and weak silk receptivity (Alam et al., 2017; Dresselhaus, & Franklin-Tong, 2013; Wilhelm, Mullen, Keeling, & Singletary, 1999; Oury, Tardieu, & Turc, 2016; Oury et al., 2016). For example, the drought stress from tassel emergence to 6 days after silking decreased the kernel numbers by 42-77% (Oury et al., 2016). The heat stress in 15 days from start of silking onwards reduced the kernel numbers by 75% (Edreira, Carpici, Sammarro, & Otegui, 2011). Most of current studies has analyzed the impacts on maize kernel number by the heat and drought stresses, but the persistent impact of the stresses around flowering on the subsequent maize kernel development has received limited attention. Furthermore, limited information is available on the individual and combined impacts of heat and drought stress during the early flowering stage on the qualitative aspects in maize grain weight.
For wheat, some studies indicated that the early (around flowering) drought and heat stress shortened leaf area duration (Cristina, Daniel, Calderini, & Gustavo, 2007), decreased chlorophyll index (Hlaváováet al., 2018) and reduced net photosynthetic rate (Wang, Marija, Dong, Susanne, & Bernd, 2014). The subsequent impact of early heat stress on grain weight in field was also observed (Abeledo, Savin, & Slafer, 1999; Calderini, Savin, Abeledo, Reynolds, & Slafer, 2001; Cristina, Daniel, Calderini, & Gustavo, 2007). For barley, the heat exposed to booting-anthesis stage decreased grain weight by 13.4% (Cristina, Daniel, Calderini, & Gustavo, 2007). For rice, grain yield was reduced by 24% because of the combined drought and heat stress at anthesis (Amjikarai, Chenniappan, & Dhashnamurthi, 2018). These observations showed grain development was limited by the source (canopy structure and function), which was affected by the early stress. Whether the sink (grain) development was also limited by the early stress around flowering if source supply was not limited since grain filling remained unclear. Meanwhile, the underlying physiological mechanism for the response of subsequent grain development with both limited and non-limited source supplies to the early heat stress should be further addressed.
It has been generally accepted that the grain weight was determined during the period since grains were actually set (Egli, 2004). Therefore, most studies for grain weight focused on the post-anthesis stage for grain filling (Royo, Abaza, Blanco, García, & Luis F, 2000). In this study, we hypothesized that the independent and combined drought and heat stress around flowering stage still has significant effect on the subsequent grain filling process even with adequate source supply although the stress was relieved since the beginning of grain filling. The purpose of this study was to (i) detect and compared the impacts of the drought stress, heat stress, and combined drought and heat stress around flowering on the subsequent kernel weight, (ii) compare the response of subsequent grain development to the early stresses with limited source supply in field and adequate source supply in laboratory incubation, (iii) investigate the underlying mechanism of the above responses. This study included three experiments (Figure 1). In Experiment 1 (Exp. 1), maize plants were subjected to drought stress, heat stress and combined drought and heat stress during the short period from tassel emergence to ovaries fertilization in ponds covered with a rain shelter, and the fertilized ovaries continued to grow in field. In Experiment 2 (Exp. 2), the fertilized ovaries in Exp. 1 were vitro cultured in the chamber with favorable growth conditions in laboratory incubation. Sufficient source supply was provided for kernel growth in vitro cultured, which was proved to be an useful method for investigating the grain development (Burle, Gengenbach, Robert, 1994; Zhang et al., 2017). In Experiment 3 (Exp. 3), maize plants were subjected to similar stress with Exp. 1 around flowering in pots, the fertilized ovaries were also vitro cultured for the measurement of carbohydrate metabolism and starch synthesis in grain. Our study reports for the first time about the subsequent impacts of independent and combined drought and heat stress around flowering on maize grain filling and thus necessities the need for development of maize genotypes resilient to these stresses, especially to combined stress environments.