1. Introduction

The accelerating pace of rising atmospheric carbon dioxide (CO2) concentration has been a serious global climatic issue for a couple of decades. Despite the Kyoto Protocol being adopted in 1997 and entered into force in 2005, followed by Paris Agreement in November 2016, the atmospheric CO2 concentration ([CO2]) shows no sign of slowing down in its increasing rate. The current historical record of atmospheric [CO2] reached a climax of 417.07 ppm in May 2020 (ESRL, Mauna Loa, Hawaii, USA). In terms of crop production, the elevated atmospheric CO2 concentration (eCO2) and the consequential temperature hike will bring in more extreme weathers (such as drought and flood, extreme heatwave and freezing temperature, hurricane and hails), frequent crop diseases and pests occurrences, and recurrent adverse crop growth conditions. All these will reduce crop productivity, quality, and stability, especially for wheat and maize (Easterling et al, 2005; Rosenzweig et al, 2014; Li et al, 2015; Wang et al, 2019a). It seems that rice and soy­bean are the mere major crops that may marginally benefit from the eCO2, especially when considering the collateral temperature and ozone hikes (Long et al, 2006; Ainsworth, 2008; Kimball, 2016; Usui et al, 2016; Zhao et al, 2017). To mitigate the threat and secure the crop production, it is critically important to breed crop varieties adaptable for the future [CO2] and temperature conditions, and to integrate a novel cultivation management system to take the advantage of the fertilization effect of this irreversible eCO2 change (Long et al, 2004). However, genes responsive for eCO2 adaptation are largely unknown (Morita et al, 2015; Nakano et al, 2017; Hasegawa et al, 2019), thus, we need to clarify targets for breeding purpose. Top priority should be given to those yield-limiting agronomic characters that showing beneficially responsive to eCO2.
Rice (Oryza sativa L.) is a staple cereal crop for more than half of the world population, especially in the densely populated Asian regions. Despite the divergent responses of the productivity to eCO2 by different crops, it is generally concluded that rice may marginally benefit from the CO2 fertilization in temperate and tropical regimes (Ruiz-Vera et al, 2013; Rosenzweig et al, 2014; Kimball et al, 2016). Most studies conclude that the eCO2 increases grain yield of rice (Hasegawa et al, 2017). For example, multiple-year free-air CO2enrichment (FACE) field experiments conducted at different locations with various indica , japonica and hybrid rice varieties concluded that, eCO2 at 550 ppm can enhance rice grain yield up to 5~35% compared to the ambient [CO2] (Kim et al, 2003; Kimball et al, 2016). Among the four major rice yield components, namely, panicles per area, spikelets per panicle, seed setting rate (%), and grain weight, eCO2 consistently increases panicles per area, while the other three yield components show both negative and positive responses (Kim et al, 2003a; Huang et al, 2004; Lai et al, 2014; Hasegawa et al, 2019). The panicle per area is a fundamental factor that being determined at the earlier stage of rice growth. It impacts the other three yield components at later stage of growth and arbitrates final grain yield. Therefore, endeavor to achieve a stable panicle number is always a top priority in rice production management.
In addition to the main stem, rice plant produces tillers (branches) that may eventually develop into panicle florescence to generate grain yield (Wang and Li, 2011). The tiller number of a rice plant is not only a basis for panicle number, but also an indicator of plant growth status. Rice plants with more tillers at the early stages usually indicate they are in a healthy developmental path toward higher yield. Tiller number also shows a significant accrual response to eCO2 (Kim et al, 2003b; Huang et al. 2004). Since the discovery of MOC1 in tillering regulation (Li et al, 2003), recent advances in molecular genetics have clarified that more than 60 genes involving in tiller regulation in rice plants (Wang et al, 2011; Wang et al, 2018a). However, most of the knowledge is derived from mutant or gene manipulation experiments, limited info is available on how they coordinate in a regular variety (Zhang et al, 2019).
Moreover, nitrogen (N) is a major macronutrient that constrains tiller growth. Reports have clarified that eCO2 alters the element stoichiometry in plants, especially a reduction of N content was consistently observed from grasses to crops and trees (Luo et al, 2006; Norby et al, 2010; Deng et al, 2015). Insufficient N availability is a constraint on the growth of perennial grass species in response to eCO2 (Reich et al, 2006; Mueller et al, 2013). Multiple reviews summarize that quite a range of crops and model plants displaying certain pattern of interaction between N requirement and eCO2, though interpretation differs but N constraint is a consistency (Stitt and Krapp, 1999; Wang et al, 2010; Bloom et al, 2015; Rubio-Asensio and Bloom 2017; Andrew et al, 2019). eCO2 reduces the N content in rice plant as well (Makino et al, 1997; Kim et al, 2001; Lieffering et al, 2004; Zhang et al, 2013; Wang et al, 2019b). However, low N content in rice plants is supposed to inhibit tiller occurrence (Jiang et al, 1997). Despite the expected inhibition effect on tillering by reduced N content in rice plants under the eCO2, multiple reports have confirmed that tillers are promoted by eCO2 (Jitla et al, 1997; Huang et al, 2004; Yang et al, 2007; Jiang et al, 2020). However, the underlying physiological and molecular mechanisms remain unclear.
We hypothesized that N distribution changes in favor of tillering under eCO2 condition. To test this, we investigated the interactive effects of CO2 and N rate on rice tillering in growth chambers. Our objectives were 1) to analyze the interaction effect of eCO2 and N application rate on rice tillering at early stage; 2) to decipher the change in the N distribution among different organs; and 3) to investigate the molecular change in response to the eCO2. Clarifying the molecular adaptation mechanism of rice to the eCO2 would directly enable breeders to target certain genes in order to breed varieties that can better benefit from the CO2 fertilization effect. The mechanism may also help interpreting the adaptation of other terrestrial plants to the eCO2 and develop new approaches to mitigate the eCO2.