SnRK2 Protein Kinases and mRNA Decapping Machinery Control Root Development and Response to Salt

Abstract

SNF1-RELATED PROTEIN KINASES 2 (SnRK2) are important components of early osmotic and salt stress signaling pathways in plants. The Arabidopsis (Arabidopsis thaliana) SnRK2 family comprises the abscisic acid (ABA)-activated protein kinases SnRK2.2, SnRK2.3, SnRK2.6, SnRK2.7, and SnRK2.8, and the ABA-independent subclass 1 protein kinases SnRK2.1, SnRK2.4, SnRK2.5, SnRK2.9, and SnRK2.10. ABA-independent SnRK2s act at the posttranscriptional level via phosphorylation of VARICOSE (VCS), a member of the mRNA decapping complex, that catalyzes the first step of 5'mRNA decay. Here, we identified VCS and VARICOSE RELATED (VCR) as interactors and phosphorylation targets of SnRK2.5, SnRK2.6, and SnRK2.10. All three protein kinases phosphorylated Ser-645 and Ser-1156 of VCS, whereas SnRK2.6 and SnRK2.10 also phosphorylated VCS Ser-692 and Ser-680 of VCR. We showed that subclass 1 SnRK2s, VCS, and 5' EXORIBONUCLEASE 4 (XRN4) are involved in regulating root growth under control conditions as well as modulating root system architecture in response to salt stress. Our results suggest interesting patterns of redundancy within subclass 1 SnRK2 protein kinases, with SnRK2.1, SnRK2.5, and SnRK2.9 controlling root growth under nonstress conditions and SnRK2.4 and SnRK2.10 acting mostly in response to salinity. We propose that subclass 1 SnRK2s function in root development under salt stress by affecting the transcript levels of aquaporins, as well as CYP79B2, an enzyme involved in auxin biosynthesis.

Figure 1.

Components of SnRK2 subclass 1-regulated 5′ mRNA decay pathways contribute to root development and root system architecture responses to salt stress. Root system architecture of quintuple snrk2.1/2.4/2.5/2.7/2.9/2.10, amiRNA lines VCS#2 and VCS#4, xrn4-5, and xrn4-6. Seedlings (10 d old) were transferred to 0 and 125 mm NaCl at the 4-d-old stage. Main root length, average lateral root length per main root length, and lateral root density of Col-0, snrk2.1/2.4/2.5/2.7/2.9/2.10 (A), amiRNA lines VCS2 and VCS4 (B), xrn4-5 and xrn4-6 (C) on media supplemented with 0 or 125 mm NaCl are shown. Boxplots denotes span from 25th to the 75th percentile and are centered to the data median. Asterisk denotes p-value of pairwise comparison by least square method: ***<0.001, **<0.01, *<0.05, n > 30.

Figure 2.

SnRK2 subclass 1 protein kinases control various biological processes. A, Salt stress regulated expression profiles in Col-0 and snrk2.4, snrk2.4/2.10, and snrk2.1/2.4/2.5/2.7/2.9/2.10. Heatmap presents log2 fold changes (log2FC) in expression of the genes significantly affected by 1-h 150 mm NaCl treatment in Col-0 and corresponding log2FC value in mutants tested. Gray color indicates genes for which expression was not significantly changed (absolute value of log2FC > 1). B, GO categories enriched among the salt stress–regulated genes with altered expression in tested mutants. Heatmap presents corrected p-value (q-value) of the enrichment. Categories that were not enriched in individual genotypes are represented by gray squares (NA). C, Number of salt stress–regulated genes with expression altered in the tested mutants (left). Number of genes acting downstream of subclass 1 SnRK2 protein kinases that are substrates of VCS and have been reported to participate in root development (right) are shown. A detailed list of the genes is presented in Supplemental Table S16.

Figure 3.

Salt-induced expression of PLASMA MEMBRANE INTRINSIC PROTEINS (PIP2;5, PIP2;3), BETA GLUCOSIDASE 6 (BGLU6), CYTOCHROME P450, FAMILY 79, SUBFAMILY B, POLYPEPTIDE 2 (CYP79B2) is dependent on SnRK2 subclass 1 protein kinases signaling. Expression of PIP2;5 (A), PIP2;3 (B), BGLU6 (C), CYP79B2 (D), as a ratio of the expression under salt stress salt and control (left), under control conditions (middle), and upon salt treatment (right) in Col-0, snrk2.4, snrk2.4/2.10, snrk2.1/2.4/2.5/2.9/2.10, and xrn4-5 lines is shown. Values present are averages of normalized expression levels of three biological replicates, and error bars denote se. Statistical comparison was done by one-way ANOVA followed by lsd post hoc test (P < 0.05). Different letters indicate significant differences.

Figure 4.

Mode of action of salt stress–induced modulations of root system architecture (RSA) governed by SnRK2 protein kinases. SnRK2 protein kinases are autophosphorylated upon salt stress. Both ABA-independent (in yellow) and ABA-dependent (in pink) SnRK2 protein kinases can phosphorylate VCS. Additional phosphorylation targets were already identified for SnRK2.6, and for ABA-independent SnRK2s, other phosphorylation substrates remain unknown. Phosphorylation of VCS may affect proper functioning of the decapping complex and lead to either inhibition or enhancement of 5′ mRNA decay. The abundance of PIP2;3, PIP2;5, and CYP79B2 transcripts, among others, depends on the subclass 1 SnRK2s, yet it remains unknown whether it is a consequence of the phosphorylation any of the kinase targets and whether it is direct or indirect regulation. PIP2;3 and PIP2;5 regulate formation and elongation of lateral roots (LR) via control of water fluxes in lateral root primordia (LRP) and CYP79B2 via local auxin biosynthesis. XRN4 activity controls abundance of PIP2;3, PIP2;5, and CYP79B2 via an unknown mechanism. Transcripts for which abundance is affected by salt stress and that modulate main root (MR) elongation remain unknown. Drawings of proteins and processes were reproduced from the model published in Kawa and Testerink (2017). Dashed lines indicate proposed processes that have not been experimentally validated.