The C-terminal domains SnRK2-box and ABA-box have a role in sugarcane SnRK2s auto-activation and activity

Abstract

26Resistance to drought stress is fundamental to plant survival and development. Abscisic 27 acid (ABA) is one of the major hormones involved in different types of abiotic and 28 biotic stress responses. ABA intracellular signaling has been extensively explored in 29 Arabidopsis thaliana and occurs via a phosphorylation cascade mediated by three 30 related protein kinases, denominated SnRK2s (SNF1-related protein kinases). However, 31 the role of ABA signaling and the biochemistry of SnRK2 in crop plants remains 32 underexplored. Considering the importance of the ABA hormone in abiotic stress 33 tolerance, here we investigated the regulatory mechanism of sugarcane SnRK2s -34 known as SAPKs (Stress/ABA-activated Protein Kinases). The crystal structure of 35 ScSAPK10 revealed the characteristic SnRK2 family architecture, in which the 36 regulatory SnRK2-box interacts with the kinase domain αC helix. To study sugarcane 37 SnRK2 regulation, we produced a series of mutants for the protein regulatory domains 38 SnRK2-box and ABA-box. Surprisingly, mutations in the SnRK2-box did not 39 drastically affect sugarcane SnRK2 activity, in contrast to previous observations for the 40 homologous proteins in Arabidopsis. Also, we found that the ABA-box might have a 41 role in SnRK2 activation in the absence of PP2C phosphatase. Taken together, our 42 2 results demonstrate that both C-terminal domains of sugarcane SnRK2 proteins play a 43 fundamental role in protein activation and activity. 44 45 Keywords 46 Abscisic acid, abiotic stress, SnRK2, crop plant, kinase regulation, sugarcane 47 48 78 dephosphorylation. In the absence of ABA, PP2C is released from the complex with 79 PYL/PYR/RCAR receptors and can interact with the SnRK2 kinases, leading to kinase 80 dephosphorylation and repression of ABA-response. The interaction between kinase 81 and phosphatase is mediated by another C-terminal motif, known as ABA-box, only 82 3 preserved in the ABA-responsive members of the SnRK2 subfamily (Soon et al., 2012; 83 Umezawa et al., 2009; Vlad et al., 2009). 84 Despite extensive characterization in Arabidopsis, the protein structure and biochemical 85 regulation of ABA-responsive SnRK2s from crop plants remain poorly explored. 86 SnRK2 subfamily members have been identified in several crop plants, such as rice, 87 maize and cotton (Huai et al., 2008; Kobayashi et al., 2004; Liu et al., 2017). Just like 88 their counterparts from Arabidopsis, these proteins have been shown to mediate plant 89 responses to abiotic stress and ABA. In Saccharum officinarum L. (So) sugarcane, a 90 recent study identified ten SnRK2 subfamily members, three of which (SoSAPK8/9/10) 91 have the characteristic ABA-box in their C-terminus and, accordingly, are responsive to 92 ABA (Li et al., 2017). Despite these studies, currently, there is no structural information 93 on SnRK2 subfamily members from crop plants. Moreover, the role of the regulatory 94 domains SnRK2-box and ABA-box in protein activity and activation remain unclear for 95 sugarcane and other...

Structural and functional characterization of sugarcane SnRK2 kinases

“…As Arabidopsis, sugarcane presents three SnRK2 kinases (named as SAPKs -Stress/ABA-activated Protein Kinases) highly conserved in terms of protein sequence (sequence identity > 76 %) and structure (Figure 2A) (Righetto et al, 2019). In contrast to the model plant, sugarcane possesses only one DELLA protein which shares high sequence identity (62%) with Arabidopsis GAI and was previously reported as ScGAI (Garcia Tavares et al, 2018).…”