CSN5A Subunit of COP9 Signalosome Temporally Buffers Response to Heat in Arabidopsis

Abstract

The COP9 (constitutive photomorphogenesis 9) signalosome (CSN) is an evolutionarily conserved protein complex which regulates various growth and developmental processes. However, the role of CSN during environmental stress is largely unknown. Using Arabidopsis as model organism, we used CSN hypomorphic mutants to study the role of the CSN in plant responses to environmental stress and found that heat stress specifically enhanced the growth of csn5a-1 but not the growth of other hypomorphic photomorphogenesis mutants tested. Following heat stress, csn5a-1 exhibits an increase in cell size, ploidy, photosynthetic activity, and number of lateral roots and an upregulation of genes connected to the auxin response. Immunoblot analysis revealed an increase in deneddylation of CUL1 but not CUL3 following heat stress in csn5a-1, implicating improved CUL1 activity as a basis for the improved growth of csn5a-1 following heat stress. Studies using DR5::N7-VENUS and DII-VENUS reporter constructs confirm that the heat-induced growth is due to an increase in auxin signaling. Our results indicate that CSN5A has a specific role in deneddylation of CUL1 and that CSN5A is required for the recovery of AUX/IAA repressor levels following recurrent heat stress to regulate auxin homeostasis in Arabidopsis.

Keywords: COP9 signalosome; VENUS reporter construct; auxin signaling; cullin deneddylation; hypomorphic mutants.

Figure 1

Phenotypic characterization of COP9 (constitutive photomorphogenesis 9) signalosome (CSN) hypomorphic strains (csn5a-1 and csn1-10) under various environmental stress: (a–c) Phenotype of plants 70 days after sowing (DAS): Col-0, csn1-10, and csn5a-1 plants respectively under salt (200 mM NaCl, 21 d), drought (21 d), and heat (2 h, 44 °C, 7 d) stress. White arrows indicate enhanced growth of csn5a-1 under heat stress (2 h, 44 °C, 7 d) in comparison to the control condition. (d–f) Phenotype of plants 60 DAS: Col-0, csn1-10, and csn5a-1 respectively under UV-C (5000 erg, 1 min), flood (7 d), and cold (4 °C overnight, 7 d) stress. (g) Phenotype of plants 30 DAS: csn1-10, Col-0, and csn5a-1 seedlings underwent temperature stresses of 28 °C (2 h, 14–21 DAS) and 44 °C (2 h, 14–21 DAS). Scale bars, 5 cm.

Figure 2

Phenomics study showing time series of relative fluorescence decline ratio in steady-state (Rfd_Lss) for the photo-morphogenesis repressor mutants following heat stress (2 h, 44 °C, 14–21 DAS). (a) Rfd_Lss increases significantly in csn5a-1 after 7 d heat stress and continues to be higher for 11D before getting equal to csn5a-1 control. (b) Rfd_Lss increases in Col-0 after 7 d heat stress but becomes equal to Col-0 control after 4D. (c) Rfd_Lss increases in csn1-10 after 7 d heat stress but becomes equal to csn1-10 control after 4D. (d) Rfd_Lss increases in csn5b-1 after 7 d heat stress but becomes equal to csn5b-1 control after 4D. (e) Rfd_Lss does not change in cop1-4 after 7 d heat stress. (f) Rfd_Lss does not change in cop1-4 after 7 d heat stress. Error bars represent SEM of biological replicates (n = 4). Student’s t test * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 3

Image and graph of abaxial epidermal cell size in 6th true leaf of plants 30 DAS having undergone 7 d heat treatment: Cell size increases in csn5a-1 after 7 d heat treatment. (a) Differential interference contrast (DIC) image of abaxial epidermal cell after 7 d heat treatment of 30 DAS csn1-10 (left), Col-0 (middle), and csn5a-1 (right). Cells colored in different color are most likely representing, 2n (green), 4n (orange), 8n (red), and stomata (yellow). Scale bar, 100 µm. (b) Graph displaying average abaxial cell size in µm². (c) Graph displaying the number of abaxial cells per 6th true leaf. (d) Graph displaying area of 6th true leaf. Error bars represent SEM of biological replicates (n = 2–4). Student’s t test ** p < 0.01; *** p < 0.001.

Figure 4

Difference in the percentage of nuclear ploidy after 7 d heat treatment in csn5a-1 30 DAS. (a) Percentage of diploid, tetraploid, and octaploid nuclei in 30 DAS Col-0, csn5a-1, and csn1-10 seedlings following 7 d heat treatment showing increase in the ploidy of csn5a-1 following heat treatment. (b) Graph showing increase in the level of higher ploidy (4n and 8n) of csn5a-1 (middle) following heat treatment whereas Col-0 (left) and csn1-10 (right) does not show increase in higher ploidy (4n and 8n). Error bars represent SEM of biological replicates (n = 3). Student’s t test * p < 0.05; ** p < 0.01.

Figure 5

Increased growth of csn5a-1 following 7 d heat treatment is due to an increase in deneddylation activity (against CUL1) and auxin response. (a) Immunoblot using equal protein concentrations with CSN5 antibodies did not show any change in the protein band of csn5a-1 after heat treatment. (b) Immunoblot using equal protein concentrations with CUL3 antibodies did not show any change in cullin neddylation/deneddylation (93/85 kDa) ratio in either in csn5a-1 or Col-0 after heat treatment. (c) Immunoblot using equal protein concentrations with CUL1 showed increase in deneddylation activity of csn5a-1 and Col-0. Rubisco large subunit stained with ponceau is used as loading control. NT, non-treated. (d) Expression of auxin responsive genes (SAUR19 (Small auxin-up RNA 19) and EXPA4 (Expansin A4) of which the expression was downregulated in csn5a-1 control plants compared to WT (Col-0) control plants increases after heat treatment. Expression of WT (Col-0) control plant is taken as the baseline (0) in the log2 fold change. Error bars represent SEM of biological replicates (n = 3–5). Student’s t test * p < 0.05; ** p < 0.01.

Figure 6

Auxin activity increases in csn5a-1 following 7 d heat treatment: (a) Relative integrated density in the roots of DR5::N7-VENUS and csn5a-1 × DR5::N7-VENUS, 3 h after 7 d heat treatment. (b) Relative integrated density of DR5::N7-VENUS and csn5a-1 × DR5::N7-VENUS, 3 d after 7 d heat treatment. Cell wall was stained with propidium iodide. (c) White dots represent the area used to quantify DR5 expression levels. Scale bars, 20 µm. Error bars represent SEM of biological replicates (n = 4–7). Student’s t test * p < 0.05.

Figure 7

Auxin repressor activity remains low even after 3 d of 7 d heat treatment. (a) Relative integrated density in the roots of DII-VENUS and csn5a-1 × DII-VENUS, 3 h after 7 d heat treatment. (b) Relative integrated density of DII-VENUS and csn5a-1 × DII-VENUS, 3 days after 7 d heat treatment. Cell wall was stained with propidium iodide. (c) White dots represent the area used to quantify VENUS expression levels. Scale bars, 50 µm. Error bars represent SEM of biological replicates (4–7). Student’s t test * p < 0.05; *** p < 0.001.