JASMONATE ZIM-domain protein 3 regulates photomorphogenesis and thermomorphogenesis through inhibiting PIF4 in Arabidopsis.

Light and temperature are 2 major environmental factors that affect the growth and development of plants during their life cycle. Plants have evolved complex mechanisms to adapt to varying external environments. Here, we show that JASMONATE ZIM-domain protein 3 (JAZ3), a jasmonic acid signaling component, acts as a factor to integrate light and temperature in regulating seedling morphogenesis. JAZ3 overexpression transgenic lines display short hypocotyls under red, far-red, and blue light and warm temperature (28 °C) conditions compared to the wild type in Arabidopsis (Arabidopsis thaliana). We show that JAZ3 interacts with the transcription factor PHYTOCHROME-INTERACTING FACTOR4 (PIF4). Interestingly, JAZ3 spontaneously undergoes liquid-liquid phase separation (LLPS) in vitro and in vivo and promotes LLPS formation of PIF4. Moreover, transcriptomic analyses indicate that JAZ3 regulates the expression of genes involved in many biological processes, such as response to auxin, auxin-activated signaling pathway, regulation of growth, and response to red light. Finally, JAZ3 inhibits the transcriptional activation activity and binding ability of PIF4. Collectively, our study reveals a function and molecular mechanism of JAZ3 in regulating plant growth in response to environmental factors such as light and temperature.

The RNA helicase UAP56 and the E3 ubiquitin ligase COP1 coordinately regulate alternative splicing to repress photomorphogenesis in Arabidopsis.

Light is a key environmental signal that regulates plant growth and development. While posttranscriptional regulatory mechanisms of gene expression include alternative splicing (AS) of pre-messenger RNA (mRNA) in both plants and animals, how light signaling affects AS in plants is largely unknown. Here, we identify DExD/H RNA helicase U2AF65-associated protein (UAP56) as a negative regulator of photomorphogenesis in Arabidopsis thaliana. UAP56 is encoded by the homologs UAP56a and UAP56b. Knockdown of UAP56 led to enhanced photomorphogenic responses and diverse developmental defects during vegetative and reproductive growth. UAP56 physically interacts with the central light signaling repressor constitutive photomorphogenic 1 (COP1) and U2AF65. Global transcriptome analysis revealed that UAP56 and COP1 co-regulate the transcription of a subset of genes. Furthermore, deep RNA-sequencing analysis showed that UAP56 and COP1 control pre-mRNA AS in both overlapping and distinct manners. Ribonucleic acid immunoprecipitation assays showed that UAP56 and COP1 bind to common small nuclear RNAs and mRNAs of downstream targets. Our study reveals that both UAP56 and COP1 function as splicing factors that coordinately regulate AS during light-regulated plant growth and development.

Condensation of SEUSS promotes hyperosmotic stress tolerance in Arabidopsis.

Osmotic stress imposed by drought and high salinity inhibits plant growth and crop yield. However, our current knowledge on the mechanism by which plants sense osmotic stress is still limited. Here, we identify the transcriptional regulator SEUSS (SEU) as a key player in hyperosmotic stress response in Arabidopsis. SEU rapidly coalesces into liquid-like nuclear condensates when extracellular osmolarity increases. The intrinsically disordered region 1 (IDR1) of SEU is responsible for its condensation. IDR1 undergoes conformational changes to adopt more compact states after an increase in molecular crowding both in vitro and in cells, and two predicted α-helical peptides are required. SEU condensation is indispensable for osmotic stress tolerance, and loss of SEU dramatically compromises the expression of stress tolerance genes. Our work uncovers a critical role of biomolecular condensates in cellular stress perception and response and expands our understanding of the osmotic stress pathway.

Functional analysis of ZmCOP1 and ZmHY5 reveals conserved light signaling mechanism in maize and Arabidopsis.

Plants have evolved light signaling mechanisms to optimally adapt developmental patterns to the ambient light environments. CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) and LONG HYPOCOTYL5 (HY5) are two critical components in the light signaling pathway in Arabidopsis thaliana. COP1 acts as an E3 ubiquitin ligase that targets positive regulators, such as HY5, leading to their degradation in darkness. However, functional analysis of the COP1-HY5 module in maize (Zea mays) has not been reported. Here, we investigated the expression patterns and roles of the COP1 and HY5 orthologs, ZmCOP1 and ZmHY5, in regulating photomorphogenesis. These two genes have high amino acid identities with their Arabidopsis homolog and were both regulated by light. Subcellular localization assay showed that ZmCOP1 was distributed in the cytosol and ZmHY5 localized in the nucleus. Exogenous expression of ZmCOP1 rescued the physiological defects of the cop1-4 mutant, and expression of ZmHY5 complemented the long hypocotyl phenotype of the hy5-215 mutant in Arabidopsis. Yeast two-hybrid and fluorescence resonance energy transfer assays showed that ZmCOP1 interacted with ZmHY5. Our study gains insight into the conserved function and regulatory mechanism of the COP1-HY5 signaling pathway in maize and Arabidopsis.

A PIF1/PIF3-HY5-BBX23 Transcription Factor Cascade Affects Photomorphogenesis.

Light signaling plays an essential role in controlling higher plants' early developmental process termed as photomorphogenesis. Transcriptional regulation is a vital mechanism that is orchestrated by transcription factors and other regulatory proteins working in concert to finely tune gene expression. Although many transcription factors/regulators have been characterized in the light-signaling pathway, their interregulation remains largely unknown. Here, we show that PHYTOCHROME-INTERACTING FACTOR3 (PIF3) and PIF1 transcription factors directly bind to the regulatory regions of ELONGATED HYPOCOTYL5 (HY5) and a B-box gene BBX23 and activate their expression in Arabidopsis (Arabidopsis thaliana). We found that BBX23 and its close homolog gene BBX22 play a redundant role in regulating hypocotyl growth, and that plants overexpressing BBX23 display reduced hypocotyl elongation under red, far-red, and blue light conditions. Intriguingly, BBX23 transcription is inhibited by light, whereas its protein is degraded in darkness. Furthermore, we demonstrate that HY5 physically interacts with BBX23, and these two proteins coordinately regulate the expression of both light-induced and light-repressed genes. BBX23 is also recruited to the promoter sequences of the light-responsive genes in a partial HY5-dependent manner. Taken together, our study reveals that the transcriptional cascade consisting of PIF1/PIF3, HY5, and BBX23 controls photomorphogenesis, providing a transcriptional regulatory layer by which plants fine-tune their growth in response to changing light environment.

Arabidopsis chromatin remodeling factor PICKLE interacts with transcription factor HY5 to regulate hypocotyl cell elongation.

Photomorphogenesis is a critical plant developmental process that involves light-mediated transcriptome changes, histone modifications, and inhibition of hypocotyl growth. However, the chromatin-based regulatory mechanism underlying this process remains largely unknown. Here, we identify ENHANCED PHOTOMORPHOGENIC1 (EPP1), previously known as PICKLE (PKL), an ATP-dependent chromatin remodeling factor of the chromodomain/helicase/DNA binding family, as a repressor of photomorphogenesis in Arabidopsis thaliana. We show that PKL/EPP1 expression is repressed by light in the hypocotyls in a photoreceptor-dependent manner. Furthermore, we reveal that the transcription factor ELONGATED HYPOCOTYL5 (HY5) binds to the promoters of cell elongation-related genes and recruits PKL/EPP1 through their physical interaction. PKL/EPP1 in turn negatively regulates HY5 by repressing trimethylation of histone H3 Lys 27 at the target loci, thereby regulating the expression of these genes and, thus, hypocotyl elongation. We also show that HY5 possesses transcriptional repression activity. Our study reveals a crucial role for a chromatin remodeling factor in repressing photomorphogenesis and demonstrates that transcription factor-mediated recruitment of chromatin-remodeling machinery is important for plant development in response to changing light environments.

The maize d2003, a novel allele of VP8, is required for maize internode elongation.

The d2003 is a natural dwarf mutant from maize inbred line K36 and has less than one-third of K36 plant height with severely shortened internodes. In this study, we reported the cloning of d2003 gene using positional cloning. The results showed that there was a single-base insertion in the coding region of Viviparous8 (VP8) in d2003 mutant, which resulted in a premature stop codon. Further genetic allelism tests confirmed that d2003 mutation is a novel allele of VP8. VP8 is mainly expressed in the stem apex, young leaves, and developing vascular tissues, and its expression levels in nodes are significantly higher than that in internodes at 12-leaf stage. Subcellular localization demonstrated that the VP8 protein is localized to the endoplasmic reticulum and the N-terminal 26 amino acids (aa) of VP8 protein are essential to its localization in ER. Further transgenic experiments showed that lack of the 26 aa leads to loss of VP8 function in Arabidopsis amp1 phenotype rescue. These results strongly suggested that the N-terminal 26 aa is critical for VP8 protein localization, and the correct protein localization of VP8 in ER is necessary for its function.

The CDC48 complex mediates ubiquitin-dependent degradation of intra-chloroplast proteins in plants.

Chloroplasts are the site of numerous biochemical reactions including photosynthesis, but they also produce reactive oxygen species (ROS) that negatively affect chloroplast integrity. The chaperone-like CDC48 complex plays critical roles in ubiquitin-dependent protein degradation in yeast and mammals, but its function in plants is largely unknown. Here, we show that defects in CDC48A and its cofactors UFD1 and NPL4 lead to the accumulation of ubiquitinated chloroplast proteins in Arabidopsis thaliana. We reveal that two plastid genome-encoded proteins, RbcL and AtpB, associate with the CDC48 complex. Strikingly, RbcL and AtpB are ubiquitinated and degraded by the 26S proteasome pathway upon ROS stress, and these processes are impaired by defects of the CDC48 complex. Functional analysis demonstrates that the CDC48 complex is required for plant tolerance to ROS. This study reveals a role for the plant CDC48 complex in modulating ubiquitin-dependent degradation of intra-chloroplast proteins in response to oxidative stress.

An F-box gene, CPR30, functions as a negative regulator of the defense response in Arabidopsis.

Arabidopsis gain-of-resistance mutants, which show HR-like lesion formation and SAR-like constitutive defense responses, were used well as tools to unravel the plant defense mechanisms. We have identified a novel mutant, designated constitutive expresser of PR genes 30 (cpr30), that exhibited dwarf morphology, constitutive resistance to the bacterial pathogen Pseudomonas syringae and the dramatic induction of defense-response gene expression. The cpr30-conferred growth defect morphology and defense responses are dependent on ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), PHYTOALEXIN DEFICIENT 4 (PAD4), and NONRACE-SPECIFIC DISEASE RESISTANCE 1 (NDR1). Further studies demonstrated that salicylic acid (SA) could partially account for the cpr30-conferred constitutive PR1 gene expression, but not for the growth defect, and that the cpr30-conferred defense responses were NPR1 independent. We observed a widespread expression of CPR30 throughout the plant, and a localization of CPR30-GFP fusion protein in the cytoplasm and nucleus. As an F-box protein, CPR30 could interact with multiple Arabidopsis-SKP1-like (ASK) proteins in vivo. Co-localization of CPR30 and ASK1 or ASK2 was observed in Arabidopsis protoplasts. Based on these results, we conclude that CPR30, a novel negative regulator, regulates both SA-dependent and SA-independent defense signaling, most likely through the ubiquitin-proteasome pathway in Arabidopsis.

Genome-wide transcriptome analysis of two maize inbred lines under drought stress.

Drought stress greatly affects plant growth and crop yield. To understand the transcriptome dynamics during drought stress in maize seedlings, genome-wide gene expression profiling was compared between the drought-tolerant line Han21 and drought-sensitive line Ye478 using Affymetrix Maize Genome Array containing 17,555 probe sets. The results showed that in response to drought, the Han21 line had fewer probe sets with significant expression change than the Ye478 line and both lines had a common set of ~2,600 regulated probe sets under drought stress. The potential components of the abscisic acid signaling pathway were significantly identified from the common probe sets. A total of 827 probe sets with significantly differential expression between the two lines under drought stress were identified. The differential expression levels of cell wall-related and transporter genes may contribute to the different tolerances of the two lines. Additionally, we found that, compared to the sensitive line Ye478, the transcriptional levels of drought-responsive probe sets in the tolerant line Han21 recovered more quickly after re-watering, and more probe sets in the tolerant line Han21 were exclusively up-regulated at the re-watering stage. Our study provides a global gene expression dynamics of two maize inbred lines during drought stress and re-watering and will be valuable for further study of the molecular mechanisms of drought tolerance in maize.

SIZ1-Mediated SUMO Modification of SEUSS Regulates Photomorphogenesis in Arabidopsis.

Small ubiquitin-like modifier (SUMO) post-translational modification (SUMOylation) plays essential roles in regulating various biological processes; however, its function and regulation in the plant light signaling pathway are largely unknown. SEUSS (SEU) is a transcriptional co-regulator that integrates light and temperature signaling pathways, thereby regulating plant growth and development in Arabidopsis thaliana. Here, we show that SEU is a substrate of SUMO1, and that substitution of four conserved lysine residues disrupts the SUMOylation of SEU, impairs its function in photo- and thermomorphogenesis, and enhances its interaction with PHYTOCHROME-INTERACTING FACTOR 4 transcription factors. Furthermore, the SUMO E3 ligase SIZ1 interacts with SEU and regulates its SUMOylation. Moreover, SEU directly interacts with phytochrome B photoreceptors, and the SUMOylation and stability of SEU are activated by light. Our study reveals a novel post-translational modification mechanism of SEU in which light regulates plant growth and development through SUMOylation-mediated protein stability.

SEUSS and PIF4 Coordinately Regulate Light and Temperature Signaling Pathways to Control Plant Growth.

Plants continuously monitor environmental conditions (such as light and temperature) and adjust their growth and development accordingly. The transcription factor PHYTOCHROME-INTERACTING FACTOR4 (PIF4) regulates both light and temperature signaling pathways. Here, we identified ENHANCED PHOTOMORPHOGENIC2 (EPP2) as a new repressor of photomorphogenesis in red, far-red, and blue light. Map-based cloning revealed that EPP2 encodes the SEUSS (SEU) transcription regulator. The C terminus of SEU has transcriptional activation activity, and SEU physically interacts with PIF4. Moreover, SEU promotes the expression of many genes, including auxin biosynthetic and responsive genes, and regulates IAA levels in plants. SEU associates with the regulatory regions of INDOLE-3-ACETIC ACID INDUCIBLE6 (IAA6) and IAA19 in a PIF4-independent manner, whereas the binding of PIF4 to these genes requires SEU. Furthermore, mutations in SEU affect H3K4me3 methylation at IAA6 and IAA19, and SEU positively regulates warm temperature-mediated hypocotyl growth together with PIF4. Collectively, our results reveal that SEU acts as a central regulator integrating light and temperature signals to control plant growth by coordinating with PIF4.

Cloning and characterization of a novel CBL-interacting protein kinase from maize.

A novel CBL-interacting protein kinase (CIPK) gene, ZmCIPK16, was isolated from maize (Zea mays), which has been certified to have two copies in the genome. The ZmCIPK16 is strongly induced in maize seedlings by PEG, NaCl, ABA, dehydration, heat and drought, but not by cold. A yeast two-hybrid assay demonstrated that ZmCIPK16 interacted with ZmCBL3, ZmCBL4, ZmCBL5, and ZmCBL8. Bimolecular fluorescence complementation (BiFC) assays prove that ZmCIPK16 can interact with ZmCBL3, ZmCBL4, ZmCBL5, and ZmCBL8 in vivo. Subcellular localization showed that ZmCIPK16 is distributed in the nucleus, plasma membrane and cytoplasm; this is different from the specific localization of ZmCBL3, ZmCBL4, and ZmCBL5, which are found in the plasma membrane. The results also showed that overexpression of ZmCIPK16 in the Arabidopsis sos2 mutant induced the expression of the SOS1 gene and enhanced salt tolerance. These findings indicate that ZmCIPK16 may be involved in the CBL-CIPK signaling network in maize responses to salt stress.

Cloning and characterization of the SnRK2 gene family from Zea mays.

The SnRK2 gene family is a group of plant-specific protein kinases that has been implicated in ABA and abiotic stress signaling. We found 11 SnRK2s in maize, assigned names from ZmSnRK2.1 to ZmSnRK2.11 and cloned ten of them. By analyzing the gene structure of all the SnRK2s from Arabidopsis, rice, and maize, we found seven exons that were conserved in length among most of the SnRK2s. Although the C-terminus was divergent, we found seven conserved motifs. Of these, motif 1 was common to all of the SnRK2 genes. Based on phylogenetic analysis using the kinase domain and motif 1, the SnRK2s were divided into three groups. Motifs 4 and 5 were found specifically in group I, and many genes of this group have been confirmed to be induced by ABA. This result suggests that these two motifs mediate the ABA response. The expression patterns of ZmSnRK2 genes were characterized by using quantitative real-time RCR, which revealed that ZmSnRK2 genes were induced by one or more abiotic stress treatments and therefore may play important roles in maize stress responses.

Annotation and expression profile analysis of 2073 full-length cDNAs from stress-induced maize (Zea mays L.) seedlings.

Full-length cDNAs are very important for genome annotation and functional analysis of genes. The number of full-length cDNAs from maize (Zea mays L.) remains limited. Here we report the construction of a full-length enriched cDNA library from osmotically stressed maize seedlings by using the modified CAP trapper method. From this library, 2073 full-length cDNAs were collected and further analyzed by sequencing from both the 5'- and 3'-ends. A total of 1728 (83.4%) sequences did not match known maize mRNA and full-length cDNA sequences in the GenBank database and represent new full-length genes. After alignment of the 2073 full-length cDNAs with 448 maize BAC sequences, it was found that 84 full-length cDNAs could be mapped to the BACs. Of these, 43 genes (51.2%) have been correctly annotated from the BAC clones, 37 genes (44.0%) have been annotated with a different exon-intron structure from our cDNA, and four genes (4.76%) had no annotations in the TIGR database. Expression analysis of 2073 full-length maize cDNAs using a cDNA macroarray led to the identification of 79 genes upregulated by stress treatments and 329 downregulated genes. Of the 79 stress-inducible genes, 30 genes contain ABRE, DRE, MYB, MYC core sequences or other abiotic-responsive cis-acting elements in their promoters. These results suggest that these cis-acting elements and the corresponding transcription factors take part in plant responses to osmotic stress either cooperatively or independently. Additionally, the data suggest that an ethylene signaling pathway may be involved in the maize response to drought stress.