g Bo Jin

SIZ1-Mediated Sumoylation of ICE1 Controls CBF3/DREB1A Expression and Freezing Tolerance in Arabidopsis

SIZ1 is a SUMO E3 ligase that facilitates conjugation of SUMO to protein substrates. siz1-2 and siz1-3 T-DNA insertion alleles that caused freezing and chilling sensitivities were complemented genetically by expressing SIZ1, indicating that the SIZ1 is a controller of low temperature adaptation in plants. Cold-induced expression of CBF/DREB1, particularly of CBF3/DREB1A, and of the regulon genes was repressed by siz1. siz1 did not affect expression of ICE1, which encodes a MYC transcription factor that is a controller of CBF3/DREB1A. A K393R substitution in ICE1 [ICE1(K393R)] blocked SIZ1-mediated sumoylation in vitro and in protoplasts identifying the K393 residue as the principal site of SUMO conjugation. SIZ1-dependent sumoylation of ICE1 in protoplasts was moderately induced by cold. Sumoylation of recombinant ICE1 reduced polyubiquitination of the protein in vitro. ICE1(K393R) expression in wild-type plants repressed cold-induced CBF3/DREB1A expression and increased freezing sensitivity. Furthermore, expression of ICE1(K393R) induced transcript accumulation of MYB15, which encodes a MYB transcription factor that is a negative regulator of CBF/DREB1. SIZ1-dependent sumoylation of ICE1 may activate and/or stabilize the protein, facilitating expression of CBF3/DREB1A and repression of MYB15, leading to low temperature tolerance.

A New Dynamin-Like Protein, ADL6, Is Involved in Trafficking from the trans-Golgi Network to the Central Vacuole in Arabidopsis

Dynamin, a high-molecular-weight GTPase, plays a critical role in vesicle formation at the plasma membrane during endocytosis in animal cells. Here we report the identification of a new dynamin homolog in Arabidopsis named Arabidopsis dynamin-like 6 (ADL6). ADL6 is quite similar to dynamin I in its structural organization: a conserved GTPase domain at the N terminus, a pleckstrin homology domain at the center, and a Pro-rich motif at the C terminus. In the cell, a majority of ADL6 is associated with membranes. Immunohistochemistry and in vivo targeting experiments revealed that ADL6 is localized to the Golgi apparatus. Expression of the dominant negative mutant ADL6[K51E] in Arabidopsis protoplasts inhibited trafficking of cargo proteins destined for the lytic vacuole and caused them to accumulate at the trans-Golgi network. In contrast, expression of ADL6[K51E] did not affect trafficking of a cargo protein, H+-ATPase:green fluorescent protein, destined for the plasma membrane. These results suggest that ADL6 is involved in vesicle formation for vacuolar trafficking at the trans-Golgi network but not for trafficking to the plasma membrane in plant cells.

Trafficking of Phosphatidylinositol 3-Phosphate from the trans-Golgi Network to the Lumen of the Central Vacuole in Plant Cells

Very limited information is available on the role of phosphatidylinositol 3-phosphate (PI[3]P) in vesicle trafficking in plant cells. To investigate the role of PI(3)P during the vesicle trafficking in plant cells, we exploited the PI(3)P-specific binding property of the endosome binding domain (EBD) (amino acids 1257 to 1411) of human early endosome antigen 1, which is involved in endosome fusion. When expressed transiently in Arabidopsis protoplasts, a green fluorescent protein (GFP):EBD fusion protein exhibited PI(3)P-dependent localization to various compartments—such as the trans-Golgi network, the prevacuolar compartment, the tonoplasts, and the vesicles in the vacuolar lumen—that varied with time. The internalized GFP:EBD eventually disappeared from the lumen. Deletion experiments revealed that the PI(3)P-dependent localization required the Rab5 binding motif in addition to the zinc finger motif. Overexpression of GFP:EBD inhibited vacuolar trafficking of sporamin but not trafficking of H+-ATPase to the plasma membrane. On the basis of these results, we propose that the trafficking of GFP:EBD reflects that of PI(3)P and that PI(3)P synthesized at the trans-Golgi network is transported to the vacuole through the prevacuolar compartment for degradation in plant cells.

Sumoylation of ABI5 by the Arabidopsis SUMO E3 ligase SIZ1 negatively regulates abscisic acid signaling

SUMO (small ubiquitin-related modifier) conjugation (i.e., sumoylation) to protein substrates is a reversible posttranslational modification that regulates signaling by modulating transcription factor activity. This paper presents evidence that the SUMO E3 ligase SIZ1 negatively regulates abscisic acid (ABA) signaling, which is dependent on the bZIP transcripton factor ABI5. Loss-of-function T-DNA insertion siz1–2 and siz1–3 mutations caused ABA hypersensitivity for seed germination arrest and seedling primary root growth inhibition. Furthermore, expression of genes that are ABA-responsive through ABI5-dependent signaling (e.g., RD29A, Rd29B, AtEm6, RAB18, ADH1) was hyperinduced by the hormone in siz1 seedlings. abi5–4 suppressed ABA hypersensitivity caused by siz1 (siz1–2 abi5–4), demonstrating an epistatic genetic interaction between SIZ1 and ABI5. A K391R substitution in ABI5 [ABI5(K391R)] blocked SIZ1-mediated sumoylation of the transcription factor in vitro and in Arabidopsis protoplasts, indicating that ABI5 is sumoylated through SIZ1 and that K391 is the principal site for SUMO conjugation. In abi5–4 plants, ABI5(K391R) expression caused greater ABA hypersensitivity (gene expression, seed germination arrest and primary root growth inhibition) compared with ABI5 expression. Together, these results establish that SIZ1-dependent sumoylation of ABI5 attenuates ABA signaling. The double mutant siz1–2 afp-1 exhibited even greater ABA sensitivity than the single mutant siz1, suggesting that SIZ1 represses ABI5 signaling function independent of AFP1.

The SUMO E3 ligase, AtSIZ1, regulates flowering by controlling a salicylic acid-mediated floral promotion pathway and through affects on FLC chromatin structure

Loss-of-function siz1 mutations caused early flowering under short days. siz1 plants have elevated salicylic acid (SA) levels, which are restored to wild-type levels by expressing nahG, bacterial salicylate hydroxylase. The early flowering of siz1 was suppressed by expressing nahG, indicating that SIZ1 represses the transition to flowering mainly through suppressing SA-dependent floral promotion signaling under short days. Previous results have shown that exogenous SA treatment does not suppress late flowering of autonomous pathway mutants. However, the siz1 mutation accelerated flowering time of an autonomous pathway mutant, luminidependens, by reducing the expression of FLOWERING LOCUS C (FLC), a floral repressor. This result suggests that SIZ1 promotes FLC expression, possibly through an SA-independent pathway. Evidence indicates that SIZ1 is required for the full activation of FLC expression in the late-flowering FRIGIDA background. Interestingly, increased FLC expression and late flowering of an autonomous pathway mutant, flowering locus d (fld), was not suppressed by siz1, suggesting that SIZ1 promotes FLC expression by repressing FLD. Consistent with this, SIZ1 facilitates sumoylation of FLD that can be suppressed by mutations in three predicted sumoylation motifs in FLD (i.e. FLDK3R). Furthermore, expression of FLDK3R in fld protoplasts strongly reduced FLC transcription compared with expression of FLD, and this affect was linked to reduced acetylation of histone 4 in FLC chromatin. Taken together, the results suggest that SIZ1 is a floral repressor that not only represses the SA-dependent pathway, but also promotes FLC expression by repressing FLD activity through sumoylation, which is required for full FLC expression in a FRIGIDA background.

The Arabidopsis GTL1 Transcription Factor Regulates Water Use Efficiency and Drought Tolerance by Modulating Stomatal Density via Transrepression of SDD1

This work provides evidence that Arabidopsis GTL1 functions as a focal regulator of water use efficiency and water stress tolerance. The results establish a potential paradigm for how the environment influences stomatal development to reduce transpiration under low water availability conditions. A goal of modern agriculture is to improve plant drought tolerance and production per amount of water used, referred to as water use efficiency (WUE). Although stomatal density has been linked to WUE, the causal molecular mechanisms have yet to be determined. Arabidopsis thaliana GT-2 LIKE 1 (GTL1) loss-of-function mutations result in increased water deficit tolerance and higher integrated WUE by reducing daytime transpiration without a demonstrable reduction in biomass accumulation. gtl1 plants had higher instantaneous WUE that was attributable to ~25% lower transpiration and stomatal conductance but equivalent CO2 assimilation. Lower transpiration was associated with higher STOMATAL DENSITY AND DISTRIBUTION1 (SDD1) expression and an ~25% reduction in abaxial stomatal density. GTL1 expression occurred in abaxial epidermal cells where the protein was localized to the nucleus, and its expression was downregulated by water stress. Chromatin immunoprecipitation analysis indicated that GTL1 interacts with a region of the SDD1 promoter that contains a GT3 box. An electrophoretic mobility shift assay was used to determine that the GT3 box is necessary for the interaction between GTL1 and the SDD1 promoter. These results establish that GTL1 negatively regulates WUE by modulating stomatal density via transrepression of SDD1.

SIZ1 Small Ubiquitin-Like Modifier E3 Ligase Facilitates Basal Thermotolerance in Arabidopsis Independent of Salicylic Acid

Small ubiquitin-like modifier (SUMO) conjugation/deconjugation to heat shock transcription factors regulates DNA binding of the peptides and activation of heat shock protein gene expression that modulates thermal adaptation in metazoans. SIZ1 is a SUMO E3 ligase that facilitates SUMO conjugation to substrate target proteins (sumoylation) in Arabidopsis (Arabidopsis thaliana). siz1 T-DNA insertional mutations (siz1-2 and siz1-3; Miura et al., 2005) cause basal, but not acquired, thermosensitivity that occurs in conjunction with hyperaccumulation of salicylic acid (SA). NahG encodes a salicylate hydroxylase, and expression in siz1-2 seedlings reduces endogenous SA accumulation to that of wild-type levels and further increases thermosensitivity. High temperature induces SUMO1/2 conjugation to peptides in wild type but to a substantially lesser degree in siz1 mutants. However, heat shock-induced expression of genes, including heat shock proteins, ascorbate peroxidase 1 and 2, is similar in siz1 and wild-type seedlings. Together, these results indicate that SIZ1 and, by inference, sumoylation facilitate basal thermotolerance through processes that are SA independent.

A Vacuolar β-Glucosidase Homolog That Possesses Glucose-Conjugated Abscisic Acid Hydrolyzing Activity Plays an Important Role in Osmotic Stress Responses in Arabidopsis

This work presents evidence for a novel abscisic acid production pathway involving At-BG2, a β-glucosidase, in the vacuole. This result suggests that abscisic acid is produced in multiple places by multiple pathways in response to abiotic stress. The phytohormone abscisic acid (ABA) plays a critical role in various physiological processes, including adaptation to abiotic stresses. In Arabidopsis thaliana, ABA levels are increased both through de novo biosynthesis and via β-glucosidase homolog1 (BG1)-mediated hydrolysis of Glc-conjugated ABA (ABA-GE). However, it is not known how many different β-glucosidase proteins produce ABA from ABA-GE and how the multiple ABA production pathways are coordinated to increase ABA levels. Here, we report that a previously undiscovered β-glucosidase homolog, BG2, produced ABA by hydrolyzing ABA-GE and plays a role in osmotic stress response. BG2 localized to the vacuole as a high molecular weight complex and accumulated to high levels under dehydration stress. BG2 hydrolyzed ABA-GE to ABA in vitro. In addition, BG2 increased ABA levels in protoplasts upon application of exogenous ABA-GE. Overexpression of BG2 rescued the bg1 mutant phenotype, as observed for the overexpression of NCED3 in bg1 mutants. Multiple Arabidopsis bg2 alleles with a T-DNA insertion in BG2 were more sensitive to dehydration and NaCl stress, whereas BG2 overexpression resulted in enhanced resistance to dehydration and NaCl stress. Based on these observations, we propose that, in addition to the de novo biosynthesis, ABA is produced in multiple organelles by organelle-specific β-glucosidases in response to abiotic stresses.

SIZ1 regulation of phosphate starvation-induced root architecture remodeling involves the control of auxin accumulation

Phosphate (Pi) limitation causes plants to modulate the architecture of their root systems to facilitate the acquisition of Pi. Previously, we reported that the Arabidopsis (Arabidopsis thaliana) SUMO E3 ligase SIZ1 regulates root architecture remodeling in response to Pi limitation; namely, the siz1 mutations cause the inhibition of primary root (PR) elongation and the promotion of lateral root (LR) formation. Here, we present evidence that SIZ1 is involved in the negative regulation of auxin patterning to modulate root system architecture in response to Pi starvation. The siz1 mutations caused greater PR growth inhibition and LR development of seedlings in response to Pi limitation. Similar root phenotypes occurred if Pi-deficient wild-type seedlings were supplemented with auxin. N-1-Naphthylphthalamic acid, an inhibitor of auxin efflux activity, reduced the Pi starvation-induced LR root formation of siz1 seedlings to a level equivalent to that seen in the wild type. Monitoring of the auxin-responsive reporter DR5::uidA indicated that auxin accumulates in PR tips at early stages of the Pi starvation response. Subsequently, DR5::uidA expression was observed in the LR primordia, which was associated with LR elongation. The time-sequential patterning of DR5::uidA expression occurred earlier in the roots of siz1 as compared with the wild type. In addition, microarray analysis revealed that several other auxin-responsive genes, including genes involved in cell wall loosening and biosynthesis, were up-regulated in siz1 relative to wild-type seedlings in response to Pi starvation. Together, these results suggest that SIZ1 negatively regulates Pi starvation-induced root architecture remodeling through the control of auxin patterning.

yucca6, a Dominant Mutation in Arabidopsis, Affects Auxin Accumulation and Auxin-Related Phenotypes

Auxin plays critical roles in many aspects of plant growth and development. Although a number of auxin biosynthetic pathways have been identified, their overlapping nature has prevented a clear elucidation of auxin biosynthesis. Recently, Arabidopsis (Arabidopsis thaliana) mutants with supernormal auxin phenotypes have been reported. These mutants exhibit hyperactivation of genes belonging to the YUCCA family, encoding putative flavin monooxygenase enzymes that result in increased endogenous auxin levels. Here, we report the discovery of fertile dominant Arabidopsis hypertall1-1D and hypertall1-2D (yucca6-1D, -2D) mutants that exhibit typical auxin overproduction phenotypic alterations, such as epinastic cotyledons, increased apical dominance, and curled leaves. However, unlike other auxin overproduction mutants, yucca6 plants do not display short or hairy root phenotypes and lack morphological changes under dark conditions. In addition, yucca6-1D and yucca6-2D have extremely tall (>1 m) inflorescences with extreme apical dominance and twisted cauline leaves. Microarray analyses revealed that expression of several indole-3-acetic acid-inducible genes, including Aux/IAA, SMALL AUXIN-UP RNA, and GH3, is severalfold higher in yucca6 mutants than in the wild type. Tryptophan (Trp) analog feeding experiments and catalytic activity assays with recombinant YUCCA6 indicate that YUCCA6 is involved in a Trp-dependent auxin biosynthesis pathway. YUCCA6:GREEN FLUORESCENT PROTEIN fusion protein indicates YUCCA6 protein exhibits a nonplastidial subcellular localization in an unidentified intracellular compartment. Taken together, our results identify YUCCA6 as a functional member of the YUCCA family with unique roles in growth and development.