The phosphoproteomic and interactomic landscape of qGL3/OsPPKL1-mediated brassinosteroid signaling in rice.

Oryza sativa L. (rice) is one of the most important crops in the world, and grain size is a major component determining rice yield. Recent studies have identified a number of grain size regulators, which are involved in phytohormone signaling, G protein signaling, the mitogen-activated protein kinase signaling pathway, the ubiquitin-proteasome pathway or transcriptional regulation. In a previous study, we cloned qGL3/OsPPKL1 encoding a rice protein phosphatase that negatively modulates brassinosteroid (BR) signaling and grain length. Here, to further explore the qGL3-mediated BR signaling network, we performed phosphoproteomic screenings using two pairs of rice materials: the indica rice cultivar 9311 and its near-isogenic line NILqgl3 and the japonica rice cultivar Dongjin and its qGL3 knockout mutant m-qgl3. Together with qGL3-interacting proteins, we constructed the qGL3-mediated network, which reveals the relationships between BR signaling and other critical signaling pathways. Transgenic plants of these network components showed BR-related alterations in plant architecture. From this network, we validated a qGL3-interacting protein, O. sativa VERNALIZATION INSENSITIVE 3-LIKE 1 (OsVIL1), and demonstrated that qGL3 dephosphorylates OsVIL1 to modulate BR signaling. The qGL3-dependent network uncovered in this study increases our understanding of BR signaling and provides a profound foundation for addressing how BR modulates plant architecture in rice.

qGL3/OsPPKL1 induces phosphorylation of 14-3-3 protein OsGF14b to inhibit OsBZR1 function in brassinosteroid signaling.

Brassinosteroids (BRs) play essential roles in regulating plant growth and development, however, gaps still remain in our understanding of the BR signaling network. We previously cloned a grain length quantitative trait locus qGL3, encoding a rice (Oryza sativa L.) protein phosphatase with Kelch-like repeat domain (OsPPKL1), that negatively regulates grain length and BR signaling. To further explore the BR signaling network, we performed phosphoproteomic analysis to screen qGL3-regulated downstream components. We selected a 14-3-3 protein OsGF14b from the phosphoproteomic data for further analysis. qGL3 promoted the phosphorylation of OsGF14b and induced the interaction intensity between OsGF14b and OsBZR1. In addition, phosphorylation of OsGF14b played an important role in regulating nucleocytoplasmic shuttling of OsBZR1. The serine acids (Ser258Ser259) residues of OsGF14b play an essential role in BR-mediated responses and plant development. Genetic and molecular analyses indicated that OsGF14b functions as a negative regulator in BR signaling and represses the transcriptional activation activity of OsBZR1. Collectively, these results demonstrate that qGL3 induces the phosphorylation of OsGF14b, which modulates nucleocytoplasmic shuttling and transcriptional activation activity of OsBZR1, to eventually negatively regulate BR signaling and grain length in rice.

OsBAK2/OsSERK2 expression is repressed by OsBZR1 to modulate brassinosteroid response and grain length in rice.

Brassinosteroids (BRs) are a class of polyhydroxylated steroidal phytohormones that are essential for plant growth and development. Rice BRASSINOSTEROID-INSENSITIVE1 (BRI1)-ASSOCIATED RECEPTOR KINASES (OsBAKs) are plasma membrane-localized receptor kinases belonging to the subfamily of leucine-rich repeat receptor kinases. It has been found that in Arabidopsis, BRs induce the formation of a BRI1-BAK1 heterodimer complex and transmit the cascade signal to BRASSINAZOLE RESISTANT1/bri1-EMS-SUPPRESSOR1 (BZR1/BES1) to regulate BR signaling. Here, in rice (Oryza sativa ssp. japonica), we found that OsBZR1 binds directly to the promoter of OsBAK2, but not OsBAK1, and represses the expression of OsBAK2 to form a BR feedback inhibition loop. Additionally, the phosphorylation of OsBZR1 by OsGSK3 reduced its binding to the OsBAK2 promoter. The osbak2 mutant displays a typical BR-deficiency phenotype and negative modulates the accumulation of OsBZR1. Interestingly, the grain length of the osbak2 mutant was increased whereas in the cr-osbak2/cr-osbzr1 double mutant, the reduced grain length of the cr-osbzr1 mutant was restored, implying that the increased grain length of osbak2 may be due to the rice somatic embryogenesis receptor kinase-dependent pathway. Our study reveals a novel mechanism by which OsBAK2 and OsBZR1 engage in a negative feedback loop to maintain rice BR homeostasis, facilitating a deeper understanding of the BR signaling network and grain length regulation in rice.

A Cys2/His2 Zinc Finger Protein Acts as a Repressor of the Green Revolution Gene SD1/OsGA20ox2 in Rice (Oryza sativa L.).

Gibberellins (GAs) play important roles in the regulation of plant growth and development. The green revolution gene SD1 encoding gibberellin 20-oxidase 2 (GA20ox2) has been widely used in modern rice breeding. However, the molecular mechanism of how SD1/OsGA20ox2 expression is regulated remains unclear. Here, we report a Cys2/His2 zinc finger protein ZFP207 acting as a transcriptional repressor of OsGA20ox2. ZFP207 was mainly accumulated in young tissues and more specifically in culm nodes. ZFP207-overexpression (ZFP207OE) plants displayed semidwarfism phenotype and small grains by modulating cell length. RNA interference of ZFP207 caused increased plant height and grain length. The application of exogenous GA3 could rescue the semidwarf phenotype of ZFP207OE rice seedlings. Moreover, ZFP207 repressed the expression of OsGA20ox2 via binding to its promoter region. Taken together, ZFP207 acts as a transcriptional repressor of SD1/OsGA20ox2 and it may play a critical role in plant growth and development in rice through the fine-tuning of GA biosynthesis .

A microRNA biogenesis-like pathway for producing phased small interfering RNA from a long non-coding RNA in rice.

Phased small interfering RNAs (phasiRNAs) are a class of non-coding RNAs that perform essential functions in plants. Unlike microRNA biogenesis from a hairpin structure, the production of phasiRNAs usually requires a phase initiator and an RNA-dependent RNA polymerase (RDR) to form double-strand RNAs. By using full-length rice cDNA (KL-cDNA) to identify phasiRNA loci, we found that a putative non-coding sequence with a long hairpin structure generates the phasiRNAs, which we name Long Hairpin-structure containing non-coding RNA (LHR). The biogenesis of LHR-derived phasiRNAs was dependent on rice DCL4, but not on RDR2/6, DCL1, or DCL3. Since all of the LHR-phasiRNAs (-5p from the forward strand and -3p from the reverse strand of the dsRNAs) are mapped to the forward strand of LHR, LHR-phasiRNAs should be derived from its hairpin structure, similar to a microRNA precursor. A degradome-based validation suggested that several thylakoid-related genes were targeted by LHR-phasiRNAs. In addition, the production of LHR-phasiRNAs was completely abolished in the lhr mutant, which also exhibited decreased plant height, leaf size, and grain weight, probably through the regulation of photosynthesis. Based on our results, we propose a microRNA biogenesis-like pathway for producing phased siRNAs that expands our understanding of the current model of phased siRNA biogenesis in plants.

A Zinc Finger Transcriptional Repressor Confers Pleiotropic Effects on Rice Growth and Drought Tolerance by Down-Regulating Stress-Responsive Genes.

The C2H2-type zinc finger proteins (ZFPs) are involved in a wide range of plant development and stress responses. Many studies have shown the positive roles of ZFP genes in stress tolerance. However, overexpression of ZFP genes usually leads to the side effect of growth retardation. Here we report a new member of the ZFP family, Oryza sativa drought-responsive zinc finger protein 1 (OsDRZ1), positively regulating both stress tolerance and plant architecture in rice (Oryza sativa L.). OsDRZ1 was expressed throughout all tissues examined and could be induced by multiple abiotic stresses. OsDRZ1 protein was localized mostly in the nucleus. Unlike most reported rice ZFPs functioning as transcriptional activators, OsDRZ1 is a transcriptional repressor. Overexpression of OsDRZ1 in rice increased seedling drought tolerance and the transgenic plants appeared to accumulate more free proline and fewer reactive oxygen species (ROS), and elevate the activities of antioxidant enzymes. In contrast, RNA interference (RNAi) of OsDRZ1 led to lower activities of antioxidative response and more sensitivity to drought. RNA sequencing analysis revealed that the genes down-regulated by OsDRZ1 were mostly down-regulated by drought, implying the critical role of OsDRZ1 in modulating drought-responsive gene expression. A cupin gene OsGLP1 (germin-like protein1) was identified as one of the potential target genes of OsDRZ1, as suggested by real-time PCR and transient expression analysis in rice protoplasts. Moreover, overexpression of OsDRZ1 did not lead to growth inhibition but the promotion of rice growth, implying the potential application prospective of OsDRZ1 in engineering drought-tolerant crops.

An A20/AN1-type zinc finger protein modulates gibberellins and abscisic acid contents and increases sensitivity to abiotic stress in rice (Oryza sativa).

The plant hormones gibberellins (GA) and abscisic acid (ABA) play important roles in plant development and stress responses. Here we report a novel A20/AN1-type zinc finger protein ZFP185 involved in GA and ABA signaling in the regulation of growth and stress response. ZFP185 was constitutively expressed in various rice tissues. Overexpression of ZFP185 in rice results in a semi-dwarfism phenotype, reduced cell size, and the decrease of endogenous GA3 content. By contrast, higher GA3 content was observed in RNAi plants. The application of exogenous GA3 can fully rescue the semi-dwarfism phenotype of ZFP185 overexpressing plants, suggesting the negative role of ZFP185 in GA biosynthesis. Besides GA, overexpression of ZFP185 decreased ABA content and expression of several ABA biosynthesis-related genes. Moreover, it was found that ZFP185, unlike previously known A20/AN1-type zinc finger genes, increases sensitivity to drought, cold, and salt stresses, implying the negative role of ZFP185 in stress tolerance. ZFP185 was localized in the cytoplasm and lacked transcriptional activation potential. Our study suggests that ZFP185 regulates plant growth and stress responses by affecting GA and ABA biosynthesis in rice.

An Adenylate Kinase OsAK3 Involves Brassinosteroid Signaling and Grain Length in Rice (Oryza sativa L.).

BACKGROUND: Grain size is one of the major determinants of cereal crop yield. As a class of plant polyhydroxysteroids, brassinosteroids (BRs) play essential roles in the regulation of grain size and plant architecture in rice. In a previous research, we cloned qGL3/OsPPKL1 encoding a protein phosphatase with Kelch-like repeat domains, which negatively regulates BR signaling and grain length in rice. RESULTS: Here, we screened qGL3-interacting proteins (GIPs) via yeast two-hybrid assay and analyzed the phenotypes of the T-DNA insertion mutants of GIPs. Among these mutants, mutant osak3 presents shorter grain length and dwarfing phenotype. OsAK3 encodes an adenylate kinase, which regulates grain size by controlling cell expansion of rice spikelet glume. Overexpression of OsAK3 resulted in longer grain length. OsAK3 interacts with qGL3 in vivo and in vitro. Lamina inclination, coleoptile elongation and root inhibition experiments showed that the osak3 mutant was less sensitive to exogenous brassinolide (BL) treatment. The transcriptional level of OsAK3 was up-regulated under BL induction. In addition, RNA-Seq data indicate that OsAK3 is involved in a variety of biological processes that regulate BR signaling and grain development in rice. CONCLUSIONS: Our study reveals a novel BR signaling component OsAK3 in the regulation of grain length, and provides novel clues for uncovering the potential functions of OsAK3 in rice growth and development.