Antagonistic MADS-box transcription factors SEEDSTICK and SEPALLATA3 form a transcriptional regulatory network that regulates seed oil accumulation.

Transcriptional regulation is essential for balancing multiple metabolic pathways that influence oil accumulation in seeds. Thus far, the transcriptional regulatory mechanisms that govern seed oil accumulation remain largely unknown. Here, we identified the transcriptional regulatory network composed of MADS-box transcription factors SEEDSTICK (STK) and SEPALLATA3 (SEP3), which bridges several key genes to regulate oil accumulation in seeds. We found that STK, highly expressed in the developing embryo, positively regulates seed oil accumulation in Arabidopsis (Arabidopsis thaliana). Furthermore, we discovered that SEP3 physically interacts with STK in vivo and in vitro. Seed oil content is increased by the SEP3 mutation, while it is decreased by SEP3 overexpression. The chromatin immunoprecipitation, electrophoretic mobility shift assay, and transient dual-luciferase reporter assays showed that STK positively regulates seed oil accumulation by directly repressing the expression of MYB5, SEP3, and SEED FATTY ACID REDUCER 4 (SFAR4). Moreover, genetic and molecular analyses demonstrated that STK and SEP3 antagonistically regulate seed oil production and that SEP3 weakens the binding ability of STK to MYB5, SEP3, and SFAR4. Additionally, we demonstrated that TRANSPARENT TESTA 8 (TT8) and ACYL-ACYL CARRIER PROTEIN DESATURASE 3 (AAD3) are direct targets of MYB5 during seed oil accumulation in Arabidopsis. Together, our findings provide the transcriptional regulatory network antagonistically orchestrated by STK and SEP3, which fine tunes oil accumulation in seeds.

BnaC06.WIP2-BnaA09.STM transcriptional regulatory module promotes leaf lobe formation in Brassica napus.

The lobed leaves of rapeseed (Brassica napus L.) offer significant advantages in dense planting, leading to increased yield. Although AtWIP2, a C2H2 zinc finger transcription factor, acts as a regulator of leaf development in Arabidopsis thaliana, the function and regulatory mechanisms of BnaWIP2 in B. napus remain unclear. Here, constitutive expression of the BnaC06.WIP2 paralog, predominantly expressed in leaf serrations, produced lobed leaves in both A. thaliana and B. napus. We demonstrated that BnaC06.WIP2 directly repressed the expression of BnaA01.TCP4, BnaA03.TCP4, and BnaC03.TCP4 and indirectly inhibited the expression of BnaA05.BOP1 and BnaC02.AS2 to promote leaf lobe formation. On the other hand, we discovered that BnaC06.WIP2 modulated the levels of endogenous gibberellin, cytokinin, and auxin, and controlled the auxin distribution in B. napus leaves, thus accelerating leaf lobe formation. Meanwhile, we revealed that BnaA09.STM physically interacted with BnaC06.WIP2, and ectopic expression of BnaA09.STM generated smaller and lobed leaves in B. napus. Furthermore, we found that BnaC06.WIP2 and BnaA09.STM synergistically promoted leaf lobe formation through forming transcriptional regulatory module. Collectively, our findings not only facilitate in-depth understanding of the regulatory mechanisms underlying lobed leaf formation, but also are helpful for guiding high-density breeding practices through improving leaf morphology in B. napus.

Linum usitatissimum FAD2A and FAD3A enhance seed polyunsaturated fatty acid accumulation and seedling cold tolerance in Arabidopsis thaliana.

Flax (Linum usitatissimum) seed oil is rich in polyunsaturated fatty acids (PUFAs), particularly linolenic acid, which is converted from linoleic acid. Studies have indicated that the biosynthesis of linoleic acid and linolenic acid is controlled by FAD2 and FAD3, respectively. However, the functional distinctions of different LuFAD2 and LuFAD3 copies from L. usitatissimum in governing the biosynthesis of linoleic acid or linolenic acid, respectively, remain unclear. In this study, five LuFAD2 and three LuFAD3 cDNAs were cloned from the L. usitatissimum cultivar 'Longya 10', and GC-MS results demonstrated that LuFAD2A and LuFAD3A play predominant roles in the accumulation of linoleic acid and linolenic acid, respectively. Their simultaneous overexpression in Arabidopsis thaliana seeds led to a significant increase in fatty acid contents, especially PUFAs. Additionally, LuFAD2A and LuFAD3A promoted the biosynthesis of jasmonic acid by increasing the levels of linolenic acid, which, in turn, enhanced plant cold tolerance. When the amount of linolenic acid is not sufficient, plants adapt to low temperature via the accumulation of anthocyanins. These findings provide insights into the higher accumulation of PUFAs in L. usitatissimum seeds, and provide potential targets for improving oil quality of other oil-producing crops through molecular manipulation.

Functional characterization of a heterologously expressed Brassica napus WRKY41-1 transcription factor in regulating anthocyanin biosynthesis in Arabidopsis thaliana.

Previous studies have shown that a plant WRKY transcription factor, WRKY41, has multiple functions, and regulates seed dormancy, hormone signaling pathways, and both biotic and abiotic stress responses. However, it is not known about the roles of AtWRKY41 from the model plant, Arabidopsis thaliana, and its ortholog, BnWRKY41, from the closely related and important oil-producing crop, Brassica napus, in the regulation of anthocyanin biosynthesis. Here, we found that the wrky41 mutation in A. thaliana resulted in a significant increase in anthocyanin levels in rosette leaves, indicating that AtWRKY41 acts as repressor of anthocyanin biosynthesis. RNA sequencing and quantitative real-time PCR analysis revealed increased expression of three regulatory genes AtMYB75, AtMYB111, and AtMYBD, and two structural genes, AT1G68440 and AtGSTF12, all of which contribute to anthocyanin biosynthesis, in the sixth rosette leaves of wrky41-2 plants at 20 days after germination. We cloned the full length complementary DNA of BnWRKY41-1 from the C2 subgenome of the B. napus genotype Westar and observed that, when overexpressed in tobacco leaves as a fusion protein with green fluorescent protein, BnWRKY41-1 is localized to the nucleus. We further showed that overexpression of BnWRKY41-1 in the A. thaliana wrky41-2 mutant rescued the higher anthocyanin content phenotype in rosette leaves of the mutant. Moreover, the elevated expression levels in wrky41-2 rosette leaves of several important regulatory and structural genes regulating anthocyanin biosynthesis were not observed in the BnWRKY41-1 overexpressing lines. These results reveal that BnWRKY41-1 has a similar role with AtWRKY41 in regulating anthocyanin biosynthesis when overexpressed in A. thaliana. This gene represents a promising target for genetically manipulating B. napus to increase the amounts of anthocyanins in rosette leaves.