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.

Enolase2 regulates seed fatty acid accumulation via mediating carbon partitioning in Arabidopsis thaliana.

In many higher plants, fatty acid (FA) biosynthesis is coordinately regulated at multiple levels by intricate regulatory networks. However, the factors and their regulatory mechanisms underlying seed oil accumulation are still limited. Here, we identified that loss of glycolytic metalloenzyme enolase2 (AtENO2) activity increased the contents of total FAs and salicylic acid (SA) but reduced the accumulation of flavonoids and mucilage by regulating the expression of key genes involved in their biosynthesis pathway in Arabidopsis thaliana seeds. AtENO2 physically interacts with the transcription factor AtTGA5, which may participate in the regulation of SA levels. Non-targeted metabolomics analysis of eno2- and WT also showed that the levels of three flavonoids, quercetin-3-galactoside, quercitrin, and epicatechin, were significantly decreased in eno2- , and the flavonoid biosynthesis pathway was also enriched in the KEGG analysis. Meanwhile, the mutation of AtENO2 delayed silique ripening, thereby prolonging silique photosynthesis time, allowing siliques to generate more photosynthesis products for FA biosynthesis. These results reveal a molecular mechanism by AtENO2 to regulate seed oil accumulation in A. thaliana, providing potential targets for improving crop seed oil quality.

AtENO2 functions in the development of male gametophytes in Arabidopsis thaliana.

Pollen fertility is an important factor affecting the seed setting rate and seed yield of plants. The Arabidopsis thaliana enolase gene ENO2 (AtENO2) can affect the pollen morphology, germination, and pollen tube growth. AtENO2 encodes two proteins AtENO2 and AtMBP-1. To examine the effect of AtENO2 protein on pollen development, the 2nd ATG of the AtENO2 coding sequence for AtMBP-1 was mutated by site-directed mutagenesis, and transgenic plants expressing only AtENO2 but not AtMBP-1 were obtained. Phenotypic analysis indicated that AtENO2 was essential in the pollen development. The mechanisms of AtENO2 on pollen development were analyzed. AtENO2 can affect development of the pollen intine, and the mechanism may be that AtENO2 regulated the methyl esterification of pectin in pollen intine through ARF3 and AtPMEI-pi. The -734 ∼ -573 sequence of AtENO2 promoter is the main transcriptional regulatory region of AtENO2 affecting pollen development. The functional cis-acting element may be GTGANTG10(GTGA), and the trans-acting factors may be KAN, AS2 and ARF3/ETT. Moreover, the deletion of AtENO2 can cause significant difference in the expression of multiple genes related to pollen exine development. These results are useful for further studying the function of AtENO2 and exploring the mechanism of plant pollen development.

The pip1s Quintuple Mutants Demonstrate the Essential Roles of PIP1s in the Plant Growth and Development of Arabidopsis.

Plasma membrane intrinsic proteins (PIPs) transport water, CO2 and small neutral solutes across the plasma membranes. In this study, we used the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 system (CRISPR/Cas9) to mutate PIP1;4 and PIP1;5 in a pip1;1,2,3 triple mutant to generate a pip1;1,2,3,4,5 (pip1s-) quintuple mutant. Compared to the wild-type (WT) plant, the pip1s- mutants had smaller sized rosette leaves and flowers, less rosette leaf number, more undeveloped siliques, shorter silique and less seeds. The pollen germination rate of the pip1s- mutant was significantly lower than that of the WT and the outer wall of the pip1s- mutant's pollen was deformed. The transcriptomic analysis showed significant alterations in the expression of many key genes and transcription factors (TFs) in the pip1s- mutant which involved in the development of leaf, flower and pollen, suggesting that the mutant of PIP1s not only directly affects hydraulics and carbon fixation, but also regulates the expression of related genes to affect plant growth and development.

Cordycepin induces apoptosis in human bladder cancer cells via activation of A3 adenosine receptors.

Bladder cancer is a neoplasm originated from bladder epithelial cells. The therapy for bladder cancer is so far not satisfactory. In this study, we examined the effects of Cordyceps militaris hot water extracts containing cordycepin on human bladder cells. Cordyceps militaris hot water extracts containing cordycepin was used to treat human T24 bladder carcinoma cells, and we found that Cordyceps militaris hot water extracts containing cordycepin decreased T24 cell survival in a dose-dependent manner, which was seemingly mediated by activation of A3 adenosine receptor and the subsequent inactivation of Akt pathways, resulting in increases in cleaved Caspase-3 and apoptosis. Overexpression of A3 adenosine receptor in T24 cells mimicked the effects of Cordyceps militaris hot water extracts, while A3 adenosine receptor depletion abolished the effects of Cordyceps militaris hot water extracts containing cordycepin. Together, these data suggest that Cordyceps militaris hot water extracts containing cordycepin may be a promising treatment for bladder cancer via A3 adenosine receptor activation.