SPINDLY, a negative regulator of gibberellic acid signaling, is involved in the plant abiotic stress response

Abstract

The SPINDLY (SPY) gene was first identified as a negative regulator of plant gibberellic acid (GA) signaling because mutation of this gene phenocopies plants treated with an overdose of bioactive GA and results in insensitivity to a GA inhibitor during seed germination. The SPY gene encodes an O-linked N-acetylglucosamine transferase that can modify the target protein and modulate the protein activity in cells. In this study, we describe the strong salt and drought tolerance phenotypes of Arabidopsis (Arabidopsis thaliana) spy-1 and spy-3 mutants in addition to their GA-related phenotypes. SPY gene expression was found to be drought stress inducible and slightly responsive to salt stress. Transcriptome analysis of spy-3 revealed that many GA-responsive genes were up-regulated, which could explain the GA-overdosed phenotype of spy-3. Some stress-inducible genes were found to be up-regulated in spy-3, such as genes encoding late embryogenesis abundant proteins, Responsive to Dehydration20, and AREB1-like transcription factor, which may confer stress tolerance on spy-3. CKX3, a cytokinin (CK) catabolism gene, was up-regulated in spy-3; this up-regulation indicates that the mutant possesses reduced CK signaling, which is consistent with a positive role for SPY in CK signaling. Moreover, overexpression of SPY in transgenics (SPY overexpressing [SPY-OX]) impaired plant drought stress tolerance, opposite to the phenotype of spy. The expression levels of several genes, such as DREB1E/DDF1 and SNH1/WIN1, were decreased in SPY-OX but increased in spy-3. Taken together, these data indicate that SPY plays a negative role in plant abiotic stress tolerance, probably by integrating environmental stress signals via GA and CK cross talk.

Figure 1.

spy mutants were more tolerant of high-salinity stress compared with wild-type plants. A, Morphological phenotypes of spy mutants. Three-week-old Col, spy-1, and spy-3 plants growing on GM agar plates were photographed. Ler, Ecotype Landsberg erecta. B, Schematic structure of the SPY protein and the locations of the mutations in the mutants. C, spy-1, spy-3, and wild-type (WT) plants grown on GM plates for 7 d and subsequently transferred onto 0.5× MS medium plates with or without 200 mm NaCl. D, The survival rate was calculated from independent experiments (n ≥ 3; ** P < 0.01, * P < 0.05, Student’s t test). E, Salt stress tolerance was compared between 8-d-old Col and 7-d-old spy-3 plants, as described in A.

Figure 2.

spy-1 and spy-3 mutants were more tolerant of drought stress. A, The plant survival rate under drought stress was compared in individual soil pots. B, Four-week-old Col and spy-3 plants were subjected to dehydration in pots containing the same amounts of soil. After 12 d of water withholding, the weight of each pot was recorded daily. Watering of the plants was reinitiated once they were equally dehydrated, which occurred on days 16 and 19 for wild-type and spy-3 plants, respectively. C, Representative photographs for the data in B are shown. D, Plant drought tolerance was compared in a large tray containing soil in which the plants were cultivated side by side. Water was withheld from the plants for approximately 14 d, after which the significant difference was observed between spy-3 and wild-type plants. E, Water loss rates of detached spy-1, spy-3, and wild-type shoots. Wild-type Col is shown as the control. The mean and sd were obtained from four plants in each assay. F, Leaf electrolyte leakage was compared among spy-1, spy-3, and Col plants after 2.5 h of dehydration stress. G, Three-week-old Col plants treated with 50 μm GA₃ or water after germination were subjected to drought stress, and their survival rates were compared. For all drought tolerance experiments, photographs were taken after 1 week of reinitiated watering. Survival rates were calculated from at least three independent experiments. In all panels, * indicates P < 0.05 and ** indicates P < 0.01 (by Student's t test).

Figure 3.

A, Expression of the SPY gene under various stress treatments. Twenty micrograms of total RNA from 3-week-old plants that had been treated as indicated was used for RNA-blot hybridization with a gene-specific probe. B, SPY was preferentially expressed in guard cells in leaf tissue based on data from the Arabidopsis eFP Browser.

Figure 4.

Venn diagrams of gene expression alterations in spy-3 plants under normal or dehydration stress conditions based on microarray analysis. A, Overlap between Col plants dehydrated for 3 d and nondehydrated spy-3 plants. B, Overlap between genes up- or down-regulated by GA with those of untreated spy-3 plants. C, Overlap between Col plants treated for 6 h with ABA and untreated spy-3 plants. D, Overlap between Col plants dehydrated for 3 d and spy-3 plants dehydrated for 2 h.

Figure 5.

qRT-PCR analysis of the expression of genes that were identified as being up-regulated in the spy-3 mutant under normal growing and 2-h-dehydrated conditions. Primer sequences are listed in Supplemental Table S11.

Figure 6.

Overexpression of SPY reduced plant drought stress tolerance. A, RNA gel-blot analysis of transgene expression in vector and 35S:SPY-transformed plants; 10 μg of total RNA was loaded. B, Morphological phenotype of 3-week-old SPY-OX plants as compared with the wild type. C, Survival rate of vector-transformed and SPY-OX lines. The averaged data were obtained from three independent experiments (** P < 0.01, * P < 0.05, Student’s t test). D, qRT-PCR analysis of the expression of genes that were up-regulated in spy-3 mutants but down-regulated in SPY-OX plants. White bars indicate the relative expression level in the untreated plants, and black bars indicate the relative expression level in the 2-h-dehydrated plants. Primer sequences are listed in Supplemental Table S11.