Discussion
To maximize the fitness, Plant response to these stresses and growth should be appropriately coordinated, but the related players and underlying mechanisms remains poorly understood. In the present study, we provided evidence that CaNAC2c act as crucial regulator in coordinating pepper growth and response to HTS and RSI.
Our data demonstrated that upon HTS or RSI, CaNAC2c is transcriptionally upregulated, leading to enhanced basal and acquired thermotolerance or resistance to RSI, but repression of growth and development, indicating role of CaNAC2c as negative regulator in pepper growth but positive regulator in pepper thermotolerance and defense response to RSI. Noticeably, CaNAC2c silencing did not produced any effect on pepper resistance to RSI, and its simultaneous silencing with CaNAC2d significantly decreased resistance to RSI, indicating thatCaNAC2c acts redundantly with CaNAC2d in pepper immunity. Similar functional redundancy has been extensively found in immunity associated genes such as basic Helix-loop-Helix (bHLH) type transcription factor(Xu et al., 2014). It can be speculated that functional redundancy in NAC2 subfamily might enable more robust transcriptional reprogramming upon pathogen recognition. The difference that CaNAC2c acts specifically in thermotolerance but in redundancy with other genes in response to pathogen attack might be due to the different nature of selection pressure derived from HTS and pathogen attack. During arm race between plants and pathogens, plant immunity has been frequently overcome by the newly occurred effectors in the pathogens via targeting specific immune components including transcription factors(Canonne et al., 2011). Thus this redundancy might be an important mechanism for the robust of immunity, removal or repression of a part of the redundant components can be functionally compensated for by others.
In addition, our data indicated that CaNAC2c acts as a regulator in the trade-off between pepper growth and thermotolerance or immunity. Growth-defense trade-offs are thought to occur in plants to maximize their fitness upon different stresses when resources are limited(Berens et al., 2019; Huot et al., 2014). The transcriptional levels ofCaNAC2c is lower in the absence of stress in pepper plants, and is upregulated by RSI or HTS, indicating that the transcriptional levelsCaNAC2c are crucial for derepression of pepper growth in absence of stress and enhanced defense response upon stress. The transcriptional modification of CaNAC2c was found to be determined by signaling mediated by JA, ABA, IAA or GA3, and the data from 5’ series deletion assay further related the transcriptional modification of CaNAC2cby exogenous applied JA, ABA, IAA or GA3 to specific regions ofCaNAC2c promoter (Figure S3), indicating that ABA and JA are involved in pepper response to HTS and to RSI, respectively. Similar observations have been found previously that ABA is involved in plant response to heat stress(Y. C. Huang et al., 2016), and JA signaling contribute to plant immunity towards necrotrophic pathogens(Campos, Kang, & Howe, 2014), and signaling mediated by auxin or gibberellins (GA3) is involved in the regulation of growth(Denance, Sanchez-Vallet, Goffner, & Molina, 2013). So, it can be speculated that tradeoff between growth and immunity or thermotolerance mediated by CaNAC2c is mainly regulated at transcriptional level through crosstalk between signaling mediated by ABA, JA, IAA or GA3.
Furthermore, our data showed that CaNAC2c acts as a positive regulator in thermotolerance and immunity against RSI. This result is similar to that of our previous studies that CaWRKY6 (Cai et al., 2015),CaWRKY40 (F. F. Dang et al., 2013) and CabZIP63 (Shen et al., 2016) act positively in pepper response to both RSI and HTS, supporting the notion that pepper response to RSI is closely linked to its response to HTS. Although, CaNAC2c is upregulated by both HTS and RSI, its targeting and transcriptional regulation might be different. For instance, CaHSFA5 is directly targeted and regulated by CaNAC2c upon HTS, but not upon RSI (Figure 5, 6 and S7). Similarly, thermotolerance related CaHSP24 , CaHSP70 andCaHSF2A were upregulated by CaNAC2c upon HTS, but not upon RSI (Figure 6F and G). By contrast, immunity related DEF1 was upregulated by CaNAC2c upon RSI but not upon HTS (Figure 6K). Importantly, immunity positively related(Alvarez et al., 1998; Yoshioka et al., 2003) and thermotolerance negatively related H2O2 accumulation(Yu et al., 2019; Zhuang et al., 2020) (Figure 1 and S6) was decreased by CaNAC2c upon HTS but enhanced upon RSI (Figure 3E-F). All these data indicate a trade-off between thermotolerance and immunity against RSI mediated by CaNAC2c, and that the targeting of CaNAC2c and its function are modified in a context dependent manner. It can be speculated that unlike that tradeoffs between growth and disease resistance or thermotolerance mediated by CaNAC2c is determined by its transcription, tradeoff between thermotolerance and immunity against RSI mediated by CaNAC2c might be mainly determined by its post-translational regulation, probably in protein-protein interaction manner(Alves et al., 2014; Chi et al., 2013). As upregulation of CaNAC2c upon RSI and HTS is regulated by JA- and ABA-signaling, respectively, potential immune reaction during HTS response or thermotolerance during RSI response might be blocked by the antagonism between ABA- and JA-signaling(Anderson et al., 2004; S. Liu, Ziegler, Zeier, Birkenbihl, & Somssich, 2017; Nahar, Kyndt, Nzogela, & Gheysen, 2012). HTS and to RSI are most frequently occurred stresses in subtropical or tropical climates where pepper originally distributed, the post-translational regulation might benefit rapid and precise defense response to a specific stress, in particular, from one stress to another.
Collectively, our data indicate that CaNAC2c acts positively in thermotolerance and in immunity against RSI while negatively in pepper growth, CaNAC2c functions differentially in a context dependent manner, tradeoffs between growth and thermotolerance or immunity mediated by CaNAC2c is determined by its transcription, while tradeoffs between thermotolerance and immunity mediated by CaNAC2c is conferred by its post-translational regulation.