ARGONAUTE2 Enhances Grain Length and Salt Tolerance by Activating BIG GRAIN3 to Modulate Cytokinin Distribution in Rice

Maintaining stable, high yields under fluctuating environmental conditions is a long-standing goal of crop improvement but is challenging due to internal trade-off mechanisms, which are poorly understood. Here, we identify ARGONAUTE2 (AGO2) as a candidate target for achieving this goal in rice ( Oryza sativa ). Overexpressing AGO2 led to a simultaneously increase in salt tolerance and grain length. These benefits were achieved via the activation of BIG GRAIN3 ( BG3 ), encoding a purine permease potentially involved in cytokinin transport. AGO2 can become enriched on the BG3 locus and alter its histone methylation level, thus promoting BG3 expression. Cytokinin levels decreased in shoots but increased in roots of AGO2 -overexpressing plants. While bg3 knockout mutants were hypersensitive to salt stress, plants overexpressing BG3 showed strong salt tolerance and large grains. The knockout of BG3 significantly reduced grain length and salt tolerance in AGO2 -overexpressing plants. Both genes were transcriptionally suppressed by salt treatment. Salt treatment markedly increased cytokinin levels in roots but decreased them in shoots, resulting in a hormone distribution pattern similar to that in AGO2 -overexpressing plants. These findings highlight the critical roles of the spatial distribution of cytokinins in both stress responses and grain development. Therefore, optimizing cytokinin distribution represents a promising strategy for improving both grain yield and stress tolerance in rice. the positive role of AGO2 in regulating stress resistance. To explore the underlying mechanism, we analyzed plant responses to ABA, a well-known stress hormone. A2OX plants showed significantly increased ABA sensitivity in These results demonstrate that modulating cytokinin distribution


1!
With the global human population growing and the availability of arable land 2! decreasing, grain yield per unit area must increase in order to meet food 3! demands. An alternative strategy for tackling this problem is to exploit inferior 4! soil in salinized or desert land. Both solutions demand the development of 5! superior crop cultivars with both high yields and high biotic/abiotic stress 6! resistance. Environmental deterioration and climate change compound the 7! urgent requirement for dependable crops with stable, high yields (Kissoudis et 8! al., 2016). However, this represents a tremendous challenge for crop breeding 9! because high yields and high biotic/abiotic resistance are usually incompatible 10! due to internal trade-off mechanisms in plants (Deng et al., 2017). The genetic 11! basis underlying this incongruity is largely unclear, impeding molecular 12! breeding (Qian et al., 2016). weight, but is also associated with superior grain quality (Liu et al., 2018b; 16! Zhao et al., 2018). Numerous genes associated with grain size and/or weight 17! have been identified and characterized, including many quantitative trait loci.

81!
In the current study, we found that overexpressing AGO2 simultaneously  appears that the tag fusion at the C terminus of AGO2 impaired, but did not 105! abolish, protein function, indicating that this approach could be utilized as a showed that grain yield per plant was significantly higher in A2FOX than the 108! wild type but unaltered in A2OX due to decreased grain number ( Figure 1G;  peroxidation associated with salt stress (Sharma et al., 2013;Ma et al., 2018).

352!
recently showed that the activation of BG3 enlarges grain size, suggesting that 353! cytokinin transport might be involved in regulating grain size (Xiao et al., 2019).

354!
In the current study, we showed that while bg3 loss-of-function mutants are  (Ko et al., 2014;Zhang et al., 2014;Zurcher et al., 362! 2016;Xiao et al., 2019). Optimizing cytokinin distribution in plant tissues or 363! cells could be crucial for balancing yield, quality, and resistance and thus 364! should be considered as a strategy for simultaneously enhancing grain yield, 365! grain quality, and resistance to various abiotic/biotic stresses.

368!
The japonica rice (Oryza sativa) cultivar 'Zhonghua11' was used as the wild

375!
Seeds were harvested and dried for grain size measurement using an SC-G amplification. This cDNA contained a G-A variation compared to the reference 393! genome at site 2842. As this variation also exists in some other cultivars 394! ('9311', an indica cultivar, for example), it was ignored here. AGO2-knockout 395! lines were created using the CRISPR/Cas9 system . Briefly, nucleotides 46-65 of AGO3 cDNA, as described previously (Lu et al., 2017).  grown in ½MS hydroponic medium were harvested for total RNA extraction.

437!
The purified RNAs were used for library construction using a NEBNext Ultra