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Quantitative phosphoproteomics reveals SnRK1-dependent phosphosites in rice sheaths during grain filling

ABSTRACT: Insufficient leaf photosynthesis promotes sheath NSC transport to the panicle during grain filling. SnRK1 regulates assimilate distribution between plant tissues and organs. However, the mechanism by which SnRK1 regulates sheath NSC transport to the panicle and its response to limited leaf assimilation remain unclear. Here, we performed leaf cutting (LC) at anthesis to simulate insufficient leaf assimilation and set no treatment as a CK. In response to LC, SnRK1 activity increased rapidly, and NSC transport was advanced. Sheath NSCs were not transported in a snrk1a mutant with deficient SnRK1 activity. These results indicated that SnRK1 activity is important for sheath NSC transport. The high correlation of T6P and sucrose and the inhibition of SnRK1 by T6P in sheaths in vitro implied that T6P slightly inhibits SnRK1 activity in rice sheaths in response to sucrose availability. The increase in SnRK1 activity was accompanied by an increase in OsSnRK1a expression and a low sucrose content. Low sucrose signaling increases SnRK1 transcription. Therefore, we speculated that OsSnRK1a expression positively regulates SnRK1 activity in response to low sucrose availability in rice sheaths. Through phosphoproteomics and further PRM verification, 20 phosphosites that depend on SnRK1 and are correlated with NSC transport were obtained. Based on the function of these sites and proteins, we found that SnRK1 regulates starch degradation, sucrose metabolism, phloem transport, sugar transport across tonoplast, and glycolysis via phosphorylation to promote sheath NSC transport. Overall, our results revealed the importance, function and regulatory mechanism of SnRK1 in sheath NSC transport.

INSTRUMENT(S): timsTOF Pro

ORGANISM(S): Oryza Sativa (rice)

TISSUE(S): Leaf Sheath

SUBMITTER: Hu Yuxiang

LAB HEAD: Yuxiang Hu

PROVIDER: PXD024675 | Pride | 2022-03-18

REPOSITORIES: Pride

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Project description:The bundle sheath cells (BSCs) layer – a presumed control point for radial transport of water and solutes between the vasculature and the leaf mesophyll cells (MCs) – is still largely understudied. Using isolated protoplasts, we found that 45% of the 90 genes differentially expressed in BSCs vs. MCs are membrane related and 20% are transport related, suggesting unique functionality of membrane transport in the BSCs, supported also by functional assays (electrophysiology and fluorescence imaging). A tight control of long distance transport is required for the optimization of the hydro-mineral homeostasis of organs in plants under fluctuating ambient conditions. The mechanism of ion selectivity and absorption from the xylem to the leaf is still poorly understood. The bundle sheath (BS), tightly enwrapping the leaf vasculature, has been suggested as a part of this mechanism, acting as a selective barrier which regulates the radial transport of water and solutes from the xylem to leaf cells. This suggestion relies on the anatomy, as well as on the recent physiological transport assays of the BS and its cells (BSCs). We hypothesized that the unique transport functionality of the BSCs is manifested in its transcriptome. To test this, we compared the transcriptomes of individually hand-picked protoplasts of GFP-labeled BSCs and non-labeled mesophyll cells (MCs) of Arabidopsis thaliana leaves. Indeed, in conformation of our hypothesis, 45% of the 90 genes differentially expressed in BSCs vs. MCs are membrane related and 20% are transport related, with the proton-pump AHA2 as a prominent example. Moreover, fluorescence imaging using the potentiometric dye (di-8 ANEPPS) revealed a more negative membrane potential of the BSCs protoplasts compared to those of MCs, and electrophysiological assays (patch-clamp) showed that the major, AKT2-like, membrane K+ conductances of BSCs and MCs had different voltage dependency ranges. Combined, these differences are compatible with an expected possibility of simultaneous but oppositely directed transmembrane K+ fluxes in BSCs and MCs in otherwise similar conditions.

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Quantitative phosphoproteomics reveals SnRK1-dependent phosphosites in rice sheaths during grain filling

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