CO2 concentration affects seed shattering in cultivated and weedy rice
Here we showed that weedy rice biotypes demonstrated lower BTS at e[CO2] than a[CO2], and rice cultivars, presented lower shattering with higher BTS at e[CO2]. Nunes et al., (2015) reported low shattering capacity for rice cultivars, while for weedy rice biotypes, a high level of shattering was reported. The low shattering observed for rice cultivars is related to the crop's domestication, which is linked to reducing the natural shattering of seeds (Li et al., 2011).
The mechanistic basis for the increase in shattering at the higher CO2 concentration is not clear. The increase in [CO2] has been reported to influence several biochemical and physiological plant processes, affecting secondary metabolic processes, acting in different pathways, and interfering with cellular metabolism (Kimball, 2016; Noctor and Mhamdi, 2017; Xu et al., 2015). The shattering process involves the degradation of a specific zone between the pedicel and the grain, coordinated by methods involving the action on hydrolytic enzymes, where the middle lamella and the cell wall are degraded (Roberts et al., 2002). Also, a hormonal balance between ethylene and auxin influences the beginning of the shattering process, and environmental factors can affect this balance. Another critical point is the wide genetic and phenotypic diversity present in weedy rice biotypes (Singh, 2013). All these factors indicate that many processes can influence shattering characteristics and that environmental by [CO2] interactions will require additional study.
The positive effect of high CO2 in increasing cultivated rice yield while decreased in weedy rice
Different profiles at e[CO2] was observed between cultivated rice yield and weedy rice. Variability in panicle number and grain yield was observed for most of the weedy rice biotypes tested. Weedy rice biotypes are widely described to present high variability in several traits. For rice cultivars, the results were different, most responded with a greater panicles number and grain yield in e[CO2], except for the IRGA 424 RI genotype, in which there was no difference. Similar results were observed by Xu et al., (2018), indicating no difference in the production of panicles number and grain yield. Observing the rice cultivars, except for IRGA 424 RI, they were responsive to e[CO2], which corroborates with studies developed with rice culture (Ainsworth et al., 2006; Cai et al., 2016; Hasegawa et al., 2013).
The increase in grain yield under e[CO2] is related to increased photosynthetic rate, production of organic compounds by plant, and source-sink ratio (Cai et al., 2016). With enhance of [CO2], there is an increase in carboxylation, and suppression of the oxygenation activity of ribulose-1,5-bisphosphate carboxylase/oxygenase decreases photorespiration (Busch et al., 2018); thus, there is an increase in photosynthetic rate. Li et al., (2017) obtained an increase in the panicle number and grain yield in rice plants grown in e[CO2]. In the present study, cultivars were more responsive to [CO2] than weedy rice biotypes. Weedy rice results were also found in evaluating rice genotypes where the seed mass similar and the above-ground mass was increased in the CO2 (Wang et al., 2020). In the present study, weedy rice biotypes tended to produce more biomass but were inefficient in the source-sink for grain production.
Increased CO2 concentration affects seed shattering by altering gene expression pattern
The molecular control of seed shattering has been unveiled and genes involved in abscission layer cell differentiation, lignin deposition and abscission layer cell separation were detected in rice. qSH1 is the major QTL for seed shattering in japonica rice and encodes for a BELL1-type homeobox protein and is involved with abscission layer cell differentiation (Konishi et al., 2006). qSH1 and OSH15, a KNOX protein, form a dimer that promote abscission layer cell differentiation (Yoon et al., 2017). The dimer qSH1 and OSH15 transcriptionally regulates SHAT1 (Zhou et al., 2012). SHAT1 encodes a transcription factor with an APETALA2 domain and regulate downstream genes involved in cell abscission layer differentiation. SHAT1 can also be activated by SH4 to activates qSH1 to maintain SH4 expression (Zhou et al., 2012). This fine-tuned network promotes abscission layer cell differentiation, the first phase of seed shattering. Here, we show that the transcriptional regulation of these genes is affected by increasing in CO2 concentration. We also show that in e[CO2] condition all analyzed genotypes, weedy and cultivated rice, increase shattering level.
Specifically related to qSH1 transcriptional regulation, no expression pattern was detected between weedy and cultivated rice (Fig. 4A). A previous study also evaluated the Batatais cultivar, AV60, and other weedy rice genotypes and found that the qSH1 gene was not differentially expressed among the genotypes (Nunes et al., 2014) and that the SNP associated with seed shattering was not present in the weedy rice (Nunes et al., 2015). Here, the qSH1 was expressed mainly in weedy rice biotypes in a[CO2] condition and all evaluated genotypes in e[CO2] condition, including those with a low shattering level. Thus, it seems that qSH1 is not a suitable marker gene for seed shattering in e[CO2] condition. The high qSH1 expression in genotypes showing low shattering does not agree with the phenotype and may be an effect of other genes acting repressing the shattering (Htun et al., 2014).
As expected, OSH15 show a similar profile as observed for qSH1 in a[CO2] and e[CO2] conditions (Fig. 4B). However, it is interesting to note the higher transcript amount of OSH15 related to qSH1 in e[CO2] condition where all genotypes also showed increased shattering level. It can be explained by the fact that OSH15 displays two different roles in regulating seed shattering, in forming a dimer with qSH1 to promote abscission layer cell differentiation and also in forming a dimer with SH5, a BELL homeobox protein acting to inhibit lignin biosynthesis in abscission layer (Yoon et al., 2017). The interaction between OSH15 and SH5 may explain the upregulation of OSH15 in those genotypes showing low shattering degree (Batatais) (Fig. 1). SHAT1 is affected by e[CO2] rice and weedy rice genotypes and its expression pattern at a[CO2] condition in most of weedy rice genotypes highlight its role in seed shattering (Fig. 4D). The upregulation of SHAT1 in cultivated rice genotypes, mainly in Batatais, may be due the fact that SHAT1 can display other roles independent of SH4 as previously reported by Zhou et al., (2012). SH4 has a different transcriptional profile from the other players involved in AZ cell differentiation. The alternated profile identified in weedy rice and Batatais where SH4 was downregulated in a[CO2] and upregulated in e[CO2] corroborates with the increased seed shattering. The low seed shattering profile of cultivated rice at a[CO2], despite SH4 upregulation, may be due the high expression of OsCPL1.
If in one hand, qSH1, OSH15, SH4 and SHAT1 work together to promote abscission layer formation; the OsCPL1 works oppositely. OsCPL1 is a recessive shattering gene that codifies for a carboxy-terminal domain (CTD) phosphatase-like 1 protein that represses the differentiation of the abscission layer reducing seed shattering (Ji et al., 2010). OsCPL1 show increase transcript accumulation in almost all genotypes and [CO2] conditions (Fig. 4E). Higher OsCPL1 transcript amount in e[CO2] and the high shattering level in all genotypes in e[CO2], maybe a case of post-transcriptional or post-translational modifications affecting OsCPL1 product in play its role in repressing abscission layer cell differentiation (Nunes et al., 2014).
Homologous to qSH1, the SH5 gene encodes a BEL1-type homeobox protein that inhibit lignin biosynthesis at abscission layer and therefore promotes seed shattering (Yoon et al., 2014). Interestingly, SH5 is downregulated in almost all genotypes analyzed here at a[CO2] condition, while SH5 is upregulated in all analyzed genotypes under e[CO2] condition (Fig. 4F). Therefore, the transcriptional regulation of SH5 is sensitive to [CO2] changes, with a vital effect on seed shattering in a future environment with increased [CO2]. That effect was highlighted in Batatais genotype that have a high shattering level in e[CO2] (Fig. 1). In addition, the SH5 sensitivity to increased [CO2] is also harmful since SH5 can also induce the expression of SHAT1 and Sh4, two important players for the proper abscission layer formation (Yoon et al., 2014). Also, considering that the abscission layer is a specialized cell layer located in the rachilla and is composed of small cells with thin nonlignified walls surrounded by larger lignified cells (Yu et al., 2020), the expression of SH5 and OSH15 can be acting directly in inhibiting the deposition of lignin, increasing shattering. Moreover, these genes effects can stand out from the others, resulting in suffer repression due to the expression of others genes, since shattering has a complex and polygenic character (Yoon et al., 2017).
Finally, abscission layer cell separation is the last step for seed abscission. Cell wall degrading enzymes can be involved in this process. OsXTH8 is a cell wall remodeling enzyme that catalyzes cleavage of xyloglucan polymers (Jan et al., 2004). OsXTH8 was reported to be highly expressed in abscission layer, and it is proposed to facilitates the separation of the grain (Nunes et al., 2014). As observed for SH5, OsXTH8 expression was also highly sensitive to [CO2] changes (Fig. 4G). The high expression of OsXTH8 in Batatais cultivar, which has low shattering, suggests that OsXTH8 maybe not the main factor acting in seed shattering and/or some post-transcriptional or post-translational mechanism may be acting avoiding OsXTH8 effect.
Seed shattering in a future atmosphere CO2 concentration
The exact and detailed mechanism of how abscission layer formation and degradation occur is not fully understood. The genes identified in these processes may not act alone since other processes, such as synthesizing new cells and enzymes, can have a fundamental role. Also, a hormonal gradient between ethylene and auxin interconnects the process, acting precisely in the synthesis and secretion of enzymes that act on cell wall degradation and proteins that remodel the cell wall (Taiz et al., 2017). However, studies that underlie phytohormones' involvement in the specification of the abscission layer suggest that a precise balance between their biosynthesis and responses is of fundamental importance (Dong and Wang, 2015). Still, there is no complete elucidation of the molecular mechanisms and interactions of plant hormones underlying the abscission layer differentiation.
The plant's machinery works powered by the photosynthetic process, from where it obtains energy toward the formation of fundamental organic compounds toward the plant. When the plant is exposed to e[CO2], gene expression is altered (Leakey et al., 2009; Tallis et al., 2010) indicating that changes in gene regulation can be a mechanism linked to adaptation to increased [CO2] (Watson-Lazowski et al., 2016). Some transcriptomic studies have identified processes that respond to increased [CO2] with changes in gene expression such as photosynthesis (De Souza et al., 2008), respiration (Leakey et al., 2009) and leaves development (Ainsworth et al., 2006). In the latter, transcripts for ribosomal proteins, cell cycle, and cell wall loosening, necessary in cytoplasmic growth and cell proliferation, were highly expressed in growing soybean leaves grown in e[CO2]. In rice plants, gene expression obtained from plants grown in e[CO2] revealed to many gene expression, including senescence-associated protein 5, a gene associated with leaf senescence that triggers cell growth and structure (Fukayama et al., 2009). Thus, the expression of genes from the senescence-associated family can influence the shattering process in rice. Still, in a study developed with tomato leaves (Pan et al., 2019) the transcription of multiple genes related to ethylene synthesis and heat shock proteins was induced by elevated CO2 (800 µmol mol-1), acting in abscission in leaves. Therefore, there could be an effect of CO2 on the hormonal balance in the abscission layer, affecting rice shattering.
Here, we identified that seed shattering-related genes are sensitive to changes in [CO2]. We detected substantial increases in transcript accumulation of qSH1, OSH15, SHAT1, OsCPL1, SH5 and OsXTH8 which are related to abscission layer formation, lignin biosynthesis inhibition, and abscission layer cell separation in e[CO2] condition. Besides, the abscission layer inhibitor showed a smaller increase in its expression in the e[CO2] condition. Moreover, the weedy rice biotypes showed high seed shattering-related gene expression in e[CO2]. This expression profile corroborates with the BTS values detected in the e[CO2] condition, whereas all analyzed genotypes showed increased shattering. Altogether, we demonstrate the harmful shattering profile, mainly related to weedy rice, in a future increased [CO2] environment (Fig. 5).
Atmospheric carbon dioxide directly determines the rate of photosynthesis in plants, affecting plant productivity and fitness, and can act as a selective pressure, driving evolution. Changes in gene expression, linked to critical adaptive characteristics, represent phenotypic plasticity and offer clues to the selection targets during long-term (multigenerational) adaptation. Also, it is possible that, in addition to gene regulation, the mutation acts to give rise to new locally adapted alleles (Watson-Lazowski et al., 2016). The availability of new molecular tools, particularly high‐throughput and inexpensive RNA and DNA sequencing, suggests that further studies can be developed, using previously impossible approaches that combine phenotyping, functional genomics, and population genetic analysis in non-model systems.