E3 SUMO ligase SIZ1 splicing variants localize and function according to external conditions.

SIZ1 (SAP and MIZ1) is a member of the Siz/PIAS-type RING family of E3 SUMO (small ubiquitin-related modifier) ligases that play key roles in growth, development, and stress responses in plant and animal systems. Nevertheless, splicing variants of SIZ1 have not yet been characterized. Here, we identified four splicing variants of Arabidopsis (Arabidopsis thaliana) SIZ1, which encode three different protein isoforms. The SIZ1 gene encodes an 873-amino acid (aa) protein. Among the four SIZ1 splicing variants (SSVs), SSV1 and SSV4 encode identical 885 aa proteins; SSV2 encodes an 832 aa protein; and SSV3 encodes an 884 aa protein. SSV2 mainly localized to the plasma membrane, whereas SIZ1, SSV1/SSV4, and SSV3 localized to the nucleus. Interestingly, SIZ1 and all SSVs exhibited similar E3 SUMO ligase activities and preferred SUMO1 and SUMO2 for their E3 ligase activity. Transcript levels of SSV2 were substantially increased by heat treatment, while those of SSV1, SSV3, and SSV4 transcripts were unaffected by various abiotic stresses. SSV2 directly interacted with and sumoylated cyclic nucleotide-gated ion channel 6 (CNGC6), a positive thermotolerance regulator, enhancing the stability of CNGC6. Notably, transgenic siz1-2 mutants expressing SSV2 exhibited greater heat stress tolerance than wild-type plants, whereas those expressing SIZ1 were sensitive to heat stress. Furthermore, transgenic cngc6 plants overaccumulating a mutated mCNGC6 protein (K347R, a mutation at the sumoylation site) were sensitive to heat stress, similar to the cngc6 mutants, while transgenic cngc6 plants overaccumulating CNGC6 exhibited restored heat tolerance. Together, we propose that alternative splicing is an important mechanism that regulates the function of SSVs during development or under adverse conditions, including heat stress.

Intronic long noncoding RNA, RICE FLOWERING ASSOCIATED (RIFLA), regulates OsMADS56-mediated flowering in rice.

Long noncoding RNAs (lncRNAs) are known to play important roles in several plant processes such as flowering, organ development and stress response. However, studies exploring the diversity and complexity of lncRNAs and their mechanism of action in plants are far fewer that those in animals. Here, we show that an intronic lncRNA in rice (Oryza sativa L.), RICE FLOWERING ASSOCIATED (RIFLA), is required for the inhibition of OsMADS56 expression. RIFLA is produced from the first intron of the OsMADS56 gene. Overexpression of RIFLA in rice repressed OsMADS56 expression but activated the expression of flowering inducers Hd3a and RFT1. Additionally, RIFLA-overexpressing transgenic rice plants flowered earlier than the wild type. Under normal conditions, the transcript level of the rice enhancer of zeste gene OsiEZ1, a homolog of Arabidopsis histone H3K27-specific methyltransferase genes SWINGER (SWN) and CURLY LEAF (CLF), was as low as that of RIFLA, whereas the transcript level of OsMADS56 was relatively high. In the osiez1 mutant, OsMADS56 expression was upregulated, whereas RIFLA expression was downregulated. Additionally, RIFLA formed a complex with OsiEZ1. Together, these results suggest that the floral repressor activity of OsMADS56 is epigenetically regulated by RIFLA and OsiEZ1.

A mutation in the pPLA-IIα gene encoding PATATIN-RELATED PHOSPHOLIPASE a causes late flowering in Arabidopsis.

Arabidopsis PATATIN-RELATED PHOSPHOLIPASE 2A (pPLA-IIα) participates in the responses to various growth conditions. The factors affecting pPLA-IIα gene expression and pPLA-IIα protein activity for gycerolipids have been studied thoroughly, but the role of pPLA-IIα during the reproductive phase remains unclear. The effect of pPLA-IIα on flowering time was therefore investigated. ppla-iiα mutants flowered later than wild-type plants under long day conditions. Expression of the floral stimulators FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) was downregulated in ppla-iiα mutants compared with their expression in wild-type plants, but expression of the floral repressor FLOWERING LOCUS C (FLC) was upregulated. In addition, expression levels of COLDAIR, a long intronic noncoding RNA, decreased in ppla-iiα mutants. Taken together, these data indicate that pPLA-IIα acts as a positive regulator of flowering time through repression of FLC expression.

Overexpression of Rice OsS1Fa1 Gene Confers Drought Tolerance in Arabidopsis.

Small peptides and proteins play critical regulatory roles in plant development and environmental stress responses; however, only a few of these molecules have been identified and characterized to date because of their poor annotation and other experimental challenges. Here, we present that rice (Oryza sativa L.) OsS1Fa1, a small 76-amino acid protein, confers drought stress tolerance in Arabidopsis thaliana. OsS1Fa1 was highly expressed in leaf, culm, and root tissues of rice seedlings during vegetative growth and was significantly induced under drought stress. OsS1Fa1 overexpression in Arabidopsis induced the expression of selected drought-responsive genes and enhanced the survival rate of transgenic lines under drought. The proteasome inhibitor MG132 protected the OsS1Fa1 protein from degradation. Together, our data indicate that the small protein OsS1Fa1 is induced by drought and is post-translationally regulated, and the ectopic expression of OsS1Fa1 protects plants from drought stress.

Mutation of the OsGlyRS3 gene affects heading date in rice.

Aminoacyl-tRNA synthetases play a critical role in protein synthesis by catalyzing the covalent attachment of amino acids to their cognate tRNAs. However, the role of aminoacyl-tRNA synthetases in the transition from vegetative to reproductive growth in plants remains poorly understood. In this study, a rice (Oryza sativa) glycyl-tRNA synthetase 3, OsGlyRS3, was found to impact heading date in rice. Flowering in osglyrs3, a mutant line containing a T-DNA insertion in OsGlyRS3, was advanced by approximately 2 weeks compared to wild type. Expression analysis of flowering regulator genes showed that transcript levels of Heading date 1 (Hd1), Heading date 3a (Hd3a), and OsMADS51 were elevated in osglyrs3. These data indicate that the loss of OsGlyRS3 activity induces the expression of flowering-activating genes, resulting in early flowering.

E3 SUMO ligase AtSIZ1 regulates the cruciferin content of Arabidopsis seeds.

Arabidopsis thaliana E3 SUMO ligase SIZ1 (AtSIZ1) controls vegetative growth and development, including responses to nutrient deficiency and environmental stresses. Here, we analyzed the effect of AtSIZ1 and its E3 SUMO ligase activity on the amount of seed proteins. Proteomic analysis showed that the level of three major nutrient reservoir proteins, CRUCIFERIN1 (CRU1), CRU2, and CRU3, was reduced in the siz1-2 mutant compared with the wild type. However, quantitative real-time PCR (qRT-PCR) analysis showed that transcript levels of CRU1, CRU2, and CRU3 genes were significantly higher in the siz1-2 mutant than in the wild type. Yeast two-hybrid analysis revealed direct interaction of AtSIZ1 with CRU1, CRU2, and CRU3. The sumoylation assay revealed that CRU2, and CRU3 proteins were modified with a small ubiquitin-related modifier (SUMO) by the E3 SUMO ligase activity of AtSIZ1. Additionally, high-performance liquid chromatography (HPLC) analysis showed that the amino acid content was slightly higher in siz1-2 mutant seeds than in wild type seeds. Taken together, our data indicate that AtSIZ1 plays an important role in the accumulation and stability of seed storage proteins through its E3 ligase activity.

Post-translational modifications of FLOWERING LOCUS C modulate its activity.

Flowering Locus C (FLC) is a key floral repressor that precisely controls flowering time. The role of FLC has been extensively studied at the transcriptional level using molecular biological and epigenetic approaches. However, how FLC functions and how its stability is controlled at the post-translational level are only beginning to be understood. Recent studies show that various post-translational modifications (PTMs) control the stability and activity of FLC. In this review, we focus on three types of PTMs that regulate FLC function: phosphorylation, ubiquitination, and sumoylation. This report should serve as a model to guide post-translational studies of other important floral regulators.

The E3 SUMO ligase AtSIZ1 functions in seed germination in Arabidopsis.

Seed germination is an important stage in the lifecycle of a plant because it determines subsequent vegetative growth and reproduction. Here, we show that the E3 SUMO ligase AtSIZ1 regulates seed dormancy and germination. The germination rates of the siz1 mutants were less than 50%, even after a short period of ripening. However, their germination rates increased to wild-type levels after cold stratification or long periods of ripening. In addition, exogenous gibberellin (GA) application improved the germination rates of the siz1 mutants to the wild-type level. In transgenic plants, suppression of AtSIZ1 caused rapid post-translational decay of SLEEPY1 (SLY1), a positive regulator of GA signaling, during germination, and inducible AtSIZ1 overexpression led to increased SLY1 levels. In addition, overexpressing wild-type SLY1 in transgenic sly1 mutants increased their germination ratios to wild-type levels, whereas the germination ratio of transgenic sly1 mutants overexpressing mSLY1 was similar to that of sly1. The germination ratios of siz1 mutant seeds in immature developing siliques were much lower than those of the wild-type. Moreover, SLY1 and DELAY OF GERMINATION 1 (DOG1) transcript levels were reduced in the siz1 mutants, whereas the transcript levels of DELLA and ABSCISIC ACID INSENSITIVE 3 (ABI3) were higher than those of the wild-type. Taken together, these results indicate that the reduced germination of the siz1 mutants results from impaired GA signaling due to low SLY1 levels and activity, as well as hyperdormancy due to high levels of expression of dormancy-related genes including DOG1.