Daminozide Enhances Vigor and Steviol Glycoside Yield of Stevia (Stevia rebaudiana Bert.) Propagated in the Temporary Immersion Bioreactor

Stevia (Stevia rebaudiana Bertoni) contains sweet compound widely used as natural sweetener, steviol glycoside (SG). SG is a diterpenoid secondary metabolite synthesized from ent-kaurenoic acid, the same precursor of Gibberellin (GA). Therefore, in this study, a GA inhibitor, Daminozide (0, 10, 20 ppm) was used to block ent-kaurenoic acid conversion towards GA synthesis in attempt to increase SG content of stevia propagated in Temporary Immersion Bioreactor (TIB). Daminozide in 10 mg/L was observed to be the optimum concentration which increased biomass weight and SG content (stevioside and rebaudioside A) up to 40%. The treatment also increased transcripts accumulation of genes enrolled in SG biosynthesis, such as SrKA13H, SrUGT85C2, and SrUGT76G1, indicating SG pathway become more active due to the inhibition of GA pathway. Furthermore, the inhibition of GA was also indicated by the upregulated expression of GA biosynthesis gene (GA3ox) as the result of feedback regulation, and the downregulated expression of GA catabolism gene (GA2ox2) as the result of feed-forward regulation caused by inhibitor treatment.

Tomato SlBES1.8 influences leaf morphogenesis by mediating gibberellin metabolism and signaling

Leaf morphogenetic activity determines its shape diversity. However, our knowledge to the regulatory mechanism in maintaining leaf morphogenetic capacity is still limited. In tomato, gibberellin (GA) negatively regulates leaf complexity by shortening the morphogenetic window. We here reported a tomato BRI1-EMS-SUPPRESSOR 1 (BES1) transcription factor, SlBES1.8, that promoted the simplification of leaf pattern in a similar manner as GA functions. Enhanced level of SlBES1.8 dramatically decreased the sensibility of tomato to GA whereas increased the sensibility to the GA biosynthesis inhibitor, PAC. In line with the phenotypic observation, the endogenous bioactive GA contents were increased in OE-SlBES1.8 lines, which certainly promoted the degradation of the GA signaling negative regulator, SlDELLA. Moreover, transcriptomic analysis uncovered a set of overlapping genomic targets of SlBES1.8 and GA, and most of them were regulated in the same way. Expression studies showed the repression of SlBES1.8 to the transcriptions of two GA deactivated genes, SlGA2ox2 and SlGA2ox6, and one GA receptor, SlGID1b-1. Further experiments confirmed the direct regulation of SlBES1.8 to their promoters. On the other hand, SlDELLA physically interacted with SlBES1.8 and further inhibited its transcriptional regulation activity by abolishing SlBES1.8-DNA binding. Conclusively, by mediating GA deactivation and signaling, SlBES1.8 greatly influenced tomato leaf morphogenesis.

COP1 promotes seed germination by destabilizing RGA-LIKE2 (RGL2) in Arabidopsis

Under favorable moisture, temperature and light conditions, gibberellin (GA) biosynthesis is induced and triggers seed germination. A major mechanism by which GA promotes seed germination is by promoting the degradation of the DELLA protein RGL2, a major repressor of germination in Arabidopsis seeds. Analysis of seed germination phenotypes of constitutively photomorphogenic 1 (cop1) mutants and complemented COP1-OX/cop1-4 lines in response to GA and paclobutrazol (PAC) suggested a positive role for COP1 in seed germination and a relation with GA signaling. cop1-4 mutant seeds showed PAC hypersensitivity, but transformation with a COP1 overexpression construct rendered them PAC insensitive, with a phenotype similar to that of rgl2 mutant (rgl2-SK54) seeds. Furthermore, cop1-4 rgl2-SK54 double mutants showed a PAC-insensitive germination phenotype like that of rgl2-SK54, identifying COP1 as an upstream negative regulator of RGL2. COP1 interacts directly with RGL2 and in vivo this interaction is strongly enhanced by SPA1. COP1 directly ubiquitinates RGL2 to promote its degradation. Moreover, GA stabilizes COP1 with consequent RGL2 destabilization. By uncovering this COP1-RGL2 regulatory module, we reveal a novel mechanism whereby COP1 positively regulates seed germination and controls the expression of germination-promoting genes.

OsABF1 Represses Gibberellin Biosynthesis to Regulate Plant Height and Seed Germination in Rice (Oryza sativa L.)

Gibberellins (GAs) are diterpenoid phytohormones regulating various aspects of plant growth and development, such as internode elongation and seed germination. Although the GA biosynthesis pathways have been identified, the transcriptional regulatory network of GA homeostasis still remains elusive. Here, we report the functional characterization of a GA-inducible OsABF1 in GA biosynthesis underpinning plant height and seed germination. Overexpression of OsABF1 produced a typical GA-deficient phenotype with semi-dwarf and retarded seed germination. Meanwhile, the phenotypes could be rescued by exogenous GA3, suggesting that OsABF1 is a key regulator of GA homeostasis. OsABF1 could directly suppress the transcription of green revolution gene SD1, thus reducing the endogenous GA level in rice. Moreover, OsABF1 interacts with and transcriptionally antagonizes to the polycomb repression complex component OsEMF2b, whose mutant showed as similar but more severe phenotype to OsABF1 overexpression lines. It is suggested that OsABF1 recruits RRC2-mediated H3K27me3 deposition on the SD1 promoter, thus epigenetically silencing SD1 to maintain the GA homeostasis for growth and seed germination. These findings shed new insight into the functions of OsABF1 and regulatory mechanism underlying GA homeostasis in rice.

Multi-Omics Analyses Reveal Systemic Insights into Maize Vivipary

Maize vivipary, precocious seed germination on the ear, affects yield and seed quality. The application of multi-omics approaches, such as transcriptomics or metabolomics, to classic vivipary mutants can potentially reveal the underlying mechanism. Seven maize vivipary mutants were selected for transcriptomic and metabolomic analyses. A suite of transporters and transcription factors were found to be upregulated in all mutants, indicating that their functions are required during seed germination. Moreover, vivipary mutants exhibited a uniform expression pattern of genes related to abscisic acid (ABA) biosynthesis, gibberellin (GA) biosynthesis, and ABA core signaling. NCED4 (Zm00001d007876), which is involved in ABA biosynthesis, was markedly downregulated and GA3ox (Zm00001d039634) was upregulated in all vivipary mutants, indicating antagonism between these two phytohormones. The ABA core signaling components (PYL-ABI1-SnRK2-ABI3) were affected in most of the mutants, but the expression of these genes was not significantly different between the vp8 mutant and wild-type seeds. Metabolomics analysis integrated with co-expression network analysis identified unique metabolites, their corresponding pathways, and the gene networks affected by each individual mutation. Collectively, our multi-omics analyses characterized the transcriptional and metabolic landscape during vivipary, providing a valuable resource for improving seed quality.

Rice LEAFY COTYLEDON1 hinders photosynthesis in the embryo development to promote seed dormancy

LEAFY COTYLEDON1 (LEC1) is the central regulator of seed development. During seed development, rice embryo photosynthesis is completely blocked, which is different from Arabidopsis green embryo. However, effects of LEC1 on photosynthesis in developing seeds is largely elusive. We generated OsLEC1 mutants using the CRISPR/Cas9 technique. Oslec1 mutant seeds lost the ability of dormancy and triggered photosynthesis in embryos at the early developing stage. Transcriptome analysis demonstrated that Oslec1 mutation promoted photosynthesis and altered diverse hormonal pathways and stress response contributing to seed dormancy. Further, genome-wide identification of OsLEC1 binding sites demonstrated that OsLEC1 directly bound to genes involved in photosynthesis, photomorphogenesis, as well as abscisic acid (ABA) and gibberellin (GA) pathways, in seed maturation. We illustrated an OsLEC1-controlling gene network during seed development, including the interconnection between photosynthesis and ABA/GA biosynthesis/signalling. Our findings suggested that OsLEC1 is an inhibitor of photosynthesis during embryo development to promote rice seed maturation. This study would provide new understanding for the OsLEC1 regulatory mechanisms on photosynthesis in the monocot seed development.

Modulation of Rice Leaf Angle and Grain Size by Expressing OsBCL1 and OsBCL2 under the Control of OsBUL1 Promoter

Leaf angle and grain size are important agronomic traits affecting rice productivity directly and/or indirectly through modulating crop architecture. OsBC1, as a typical bHLH transcription factor, is one of the components comprising a complex formed with LO9-177 and OsBUL1 contributing to modulation of rice leaf inclination and grain size. In the current study, two homologues of OsBC1, OsBCL1 and OsBCL2 were functionally characterized by expressing them under the control of OsBUL1 promoter, which is preferentially expressed in the lamina joint and the spikelet of rice. Increased leaf angle and grain length with elongated cells in the lamina joint and the grain hull were observed in transgenic rice containing much greater gibberellin A3 (GA3) levels than WT, demonstrating that both OsBCL1 and OsBCL2 are positive regulators of cell elongation at least partially through increased GA biosynthesis. Moreover, the cell elongation was likely due to cell expansion rather than cell division based on the related gene expression and, the cell elongation-promoting activities of OsBCL1 and OsBCL2 were functional in a dicot species, Arabidopsis.

A Cytokinin Analog Thidiazuron Suppresses Shoot Growth in Potted Rose Plants via the Gibberellic Acid Pathway

Application of thidiazuron (N-phenyl-N′-1,2,3-thiadiazol-5-ylurea, TDZ), a cytokinin analog, to inhibit the leaf yellowing that occurs after pinching potted rose plants, resulted in compact plants with shorter shoots and thicker internodes. Two weeks after treatment with 100 μM of TDZ, new shoots were half as long as those in control plants, and stem diameters were about 40% greater. This effect of TDZ is associated with changes in cell architecture. Although TDZ treatment stimulated ethylene production by the plants, inhibitors of ethylene biosynthesis (2-aminoethoxyvinyl glycine) or action (silver thiosulfate) did not affect the response of plants to TDZ. We found that TDZ treatment significantly suppressed the expression of bioactive gibberellic acid (GA) biosynthesis genes encoding GA3 and GA20 oxidases and slightly increased the expression of GA catabolism genes encoding GA2 oxidase. Application of GA3 and TDZ together resulted in normal elongation growth, although stem diameters were still somewhat thicker. Our results suggest that TDZ regulates shoot elongation and stem enlargement in potted rose plants through the modulation of bioactive GA biosynthesis.

Melatonin Pretreatment Confers Heat Tolerance and Repression of Heat-Induced Senescence in Tomato Through the Modulation of ABA- and GA-Mediated Pathways

Heat stress and abscisic acid (ABA) induce leaf senescence, whereas melatonin (MT) and gibberellins (GA) play critical roles in inhibiting leaf senescence. Recent research findings confirm that plant tolerance to diverse stresses is closely associated with foliage lifespan. However, the molecular mechanism underlying the signaling interaction of MT with GA and ABA regarding heat-induced leaf senescence largely remains undetermined. Herein, we investigated putative functions of melatonin in suppressing heat-induced leaf senescence in tomato and how ABA and GA coordinate with each other in the presence of MT. Tomato seedlings were pretreated with 100 μM MT or water and exposed to high temperature (38/28°C) for 5 days (d). Heat stress significantly accelerated senescence, damage to the photosystem and upregulation of reactive oxygen species (ROS), generating RBOH gene expression. Melatonin treatment markedly attenuated heat-induced leaf senescence, as reflected by reduced leaf yellowing, an increased Fv/Fm ratio, and reduced ROS production. The Rbohs gene, chlorophyll catabolic genes, and senescence-associated gene expression levels were significantly suppressed by MT addition. Exogenous application of MT elevated the endogenous MT and GA contents but reduced the ABA content in high-temperature-exposed plants. However, the GA and ABA contents were inhibited by paclobutrazol (PCB, a GA biosynthesis inhibitor) and sodium tungstate (ST, an ABA biosynthesis inhibitor) treatment. MT-induced heat tolerance was compromised in both inhibitor-treated plants. The transcript abundance of ABA biosynthesis and signaling genes was repressed; however, the biosynthesis genes MT and GA were upregulated in MT-treated plants. Moreover, GA signaling suppressor and catabolic gene expression was inhibited, while ABA catabolic gene expression was upregulated by MT application. Taken together, MT-mediated suppression of heat-induced leaf senescence has collaborated with the activation of MT and GA biosynthesis and inhibition of ABA biosynthesis pathways in tomato.