Mingxun Chen

The Effect of TRANSPARENT TESTA2 on Seed Fatty Acid Biosynthesis and Tolerance to Environmental Stresses during Young Seedling Establishment in Arabidopsis

In plants, fatty acids (FAs) and FA-derived complex lipids are major carbon and energy reserves in seeds. They are essential components of cellular membranes and cellular signal or hormone molecules. Although TRANSPARENT TESTA2 (TT2) is well studied for its function in regulating proanthocyanidin biosynthesis in the seed coat, little attention has been given to its role in affecting seed FA accumulation and tolerance to environmental stresses. We demonstrate that the tt2 mutation remarkably increased the seed FA content, decreased seed weight, and altered the FA composition. The increase in FA content in the tt2 seeds was due to the relative decrease of seed coat proportion as well as the more efficient FA synthesis in the tt2 embryo. Microarray analysis revealed that tt2 mutation up-regulated a group of genes critical to FA biosynthesis and embryonic development. The mutation also altered the gene expressions that respond to stress. The microarray analysis discovered that the increase in FA accumulation of the tt2 seeds were accompanied by the significant up-regulation of FUSCA3, a transcriptional factor for embryonic development and FATTY ACID ELONGASE1, which catalyzes the elongation of FA chains. Moreover, lower seed protein accumulation during seed maturation also contributed to the increased seed FA accumulation in tt2 mutants. This study advances the understanding of the TT2 gene in seed FA accumulation and abiotic stresses during seed germination and seedling establishment.

TRANSPARENT TESTA2 regulates embryonic fatty acid biosynthesis by targeting FUSCA3 during the early developmental stage of Arabidopsis seeds

TRANSPARENT TESTA2 (TT2) regulates the biosynthesis of proanthocyanidins in the seed coat of Arabidopsis. We recently found that TT2 also participates in inhibition of fatty acid (FA) biosynthesis in the seed embryo. However, the mechanism by which TT2 suppresses the accumulation of seed FA remains unclear. In this study, we show that TT2 is expressed in embryos at an early developmental stage. TT2 is directly bound to the regulatory region of FUSCA3 (FUS3), and mediates the expression of numerous genes in the FA biosynthesis pathway. These genes include BCCP2, CAC2, MOD1 and KASII, which encode proteins involved in the initial steps of FA chain formation, FAD2 and FAD3, which are responsible for FA desaturation, and FAE1, which catalyzes very-long-chain FA elongation. Loss of function of TT2 results in reduced expression of GLABRA2 but does not cause a significant reduction in the mucilage attached to the seed coats, which competes with FA for photosynthates. TT2 is expressed in both maternal seed coats and embryonic tissues, but proanthocyanidins are only found in wild-type seed coats and not in embryonic tissues. The amount of proanthocyanidins in the seed coat is negatively correlated with the amount of FAs in the embryo.

TRANSPARENT TESTA8 Inhibits Seed Fatty Acid Accumulation by Targeting Several Seed Development Regulators in Arabidopsis

A maternal factor affects seed fatty acid biosynthesis and inhibits seed lipid accumulation by targeting seed development and down-regulating a group of genes critical to embryonic development. Fatty acids (FAs) and FA-derived complex lipids play important roles in plant growth and vegetative development and are a class of prominent metabolites stored in mature seeds. The factors and regulatory networks that control FA accumulation in plant seeds remain largely unknown. The role of TRANSPARENT TESTA8 (TT8) in the regulation of flavonoid biosynthesis and the formation of seed coat color is extensively studied; however, its function in affecting seed FA biosynthesis is poorly understood. In this article, we show that Arabidopsis (Arabidopsis thaliana) TT8 acts maternally to affect seed FA biosynthesis and inhibits seed FA accumulation by down-regulating a group of genes either critical to embryonic development or important in the FA biosynthesis pathway. Moreover, the tt8 mutation resulted in reduced deposition of protein in seeds during maturation. Posttranslational activation of a TT8-GLUCOCORTICOID RECEPTOR fusion protein and chromatin immunoprecipitation assays demonstrated that TT8 represses the activities of LEAFY COTYLEDON1, LEAFY COTYLEDON2, and FUSCA3, the critical transcriptional factors important for seed development, as well as CYTIDINEDIPHOSPHATE DIACYLGLYCEROL SYNTHASE2, which mediates glycerolipid biosynthesis. These results help us to understand the entire function of TT8 and increase our knowledge of the complicated networks regulating the formation of FA-derived complex lipids in plant seeds.

TRANSPARENT TESTA GLABRA1 Regulates the Accumulation of Seed Storage Reserves in Arabidopsis

An Arabidopsis transcription factor regulates the accumulation of seed storage reserves, including storage proteins and fatty acids. Seed storage reserves mainly consist of starch, triacylglycerols, and storage proteins. They not only provide energy for seed germination and seedling establishment, but also supply essential dietary nutrients for human beings and animals. So far, the regulatory networks that govern the accumulation of seed storage reserves in plants are still largely unknown. Here, we show that TRANSPARENT TESTA GLABRA1 (TTG1), which encodes a WD40 repeat transcription factor involved in many aspects of plant development, plays an important role in mediating the accumulation of seed storage reserves in Arabidopsis (Arabidopsis thaliana). The dry weight of ttg1-1 embryos significantly increases compared with that of wild-type embryos, which is accompanied by an increase in the contents of starch, total protein, and fatty acids in ttg1-1 seeds. FUSCA3 (FUS3), a master regulator of seed maturation, binds directly to the TTG1 genomic region and suppresses TTG1 expression in developing seeds. TTG1 negatively regulates the accumulation of seed storage proteins partially through transcriptional repression of 2S3, a gene encoding a 2S albumin precursor. TTG1 also indirectly suppresses the expression of genes involved in either seed development or synthesis/modification of fatty acids in developing seeds. In addition, we demonstrate that the maternal allele of the TTG1 gene suppresses the accumulation of storage proteins and fatty acids in seeds. Our results suggest that TTG1 is a direct target of FUS3 in the framework of the regulatory hierarchy controlling seed filling and regulates the accumulation of seed storage proteins and fatty acids during the seed maturation process.

Removal of DELLA repression promotes leaf senescence in Arabidopsis

Leaf senescence is an integrated response of leaf cells to developmental age and various internal and environmental signals. However, the role of gibberellins (GA) in leaf senescence is not clear. In the current study, we investigated the effect of DELLA on leaf senescence. Compared with the wild type (WT), leaf senescence occurred earlier in the mutant ga1-3 gai-t6 rga-t2 rgl1-1 rgl2-1 (abbreviated as Q-DELLA/ga1-3) whose DELLA repression was removed, whereas leaf senescence was retarded in the mutant ga1-3 whose GA biosynthesis was blocked and whose DELLA proteins accumulated abnormally. During leaf senescence, SAG12 and SAG29 were upregulated in Q-DELLA/ga1-3 and downregulated in ga1-3 plants. The Q-DELLA/ga1-3 senescent leaves contained more sugar but less chlorophyll and fatty acids (FAs) than those of ga1-3 and WT. Both absolute and relative contents of C18:3 in Q-DELLA/ga1-3 senescent leaves were lower compared with those of the WT and ga1-3 leaves. The genes regulating FA β-oxidation in Q-DELLA/ga1-3, such as KAT2, LACS6, LACS7, ACX1, ACX2 and MAP2, were significantly upregulated. The removal of DELLA repression highly upregulated certain genes on various hormone pathways, suggesting that GA signaling acts upstream of the jasmonic acid, salicylic acid, and ethylene pathways in regulating leaf senescence.

Sequence, expression divergence, and complementation of homologous ALCATRAZ loci in Brassica napus

AbstractsThe genomic era provides new perspectives in understanding polyploidy evolution, mostly on the genome-wide scale. In this paper, we show the sequence and expression divergence between the homologous ALCATRAZ (ALC) loci in Brassica napus, responsible for silique dehiscence. We cloned two homologous ALC loci, namely BnaC.ALC.a and BnaA.ALC.a in B. napus. Driven by the 35S promoter, both the loci complemented to the alc mutation of Arabidopsis thaliana, yet only the expression of BnaC.ALC.a was detectable in the siliques of B. napus. Sequence alignment indicated that BnaC.ALC.a and BolC.ALC.a, or BnaA.ALC.a and BraA.ALC.a, possess a high level of similarity. The understanding of the sequence and expression divergence among homologous loci of a gene is of due importance for an effective gene manipulation and TILLING (or ECOTILLING) analysis for the allelic DNA variation at a given locus.

MYB89 Transcription Factor Represses Seed Oil Accumulation

An Arabidopsis transcription factor negatively regulates the accumulation of seed oil by targeting WRI1 and several other genes involved in the seed oil biosynthetic pathway. In many higher plants, seed oil accumulation is precisely controlled by intricate multilevel regulatory networks, among which transcriptional regulation mainly influences oil biosynthesis. In Arabidopsis (Arabidopsis thaliana), the master positive transcription factors, WRINKLED1 (WRI1) and LEAFY COTYLEDON1-LIKE (L1L), are important for seed oil accumulation. We found that an R2R3-MYB transcription factor, MYB89, was expressed predominantly in developing seeds during maturation. Oil and major fatty acid biosynthesis in seeds was significantly promoted by myb89-1 mutation and MYB89 knockdown; thus, MYB89 was an important repressor during seed oil accumulation. RNA sequencing revealed remarkable up-regulation of numerous genes involved in seed oil accumulation in myb89 seeds at 12 d after pollination. Posttranslational activation of a MYB89-glucocorticoid receptor fusion protein and chromatin immunoprecipitation assays demonstrated that MYB89 inhibited seed oil accumulation by directly repressing WRI1 and five key genes and by indirectly suppressing L1L and 11 key genes involved in oil biosynthesis during seed maturation. These results help us to understand the novel function of MYB89 and provide new insights into the regulatory network of transcriptional factors controlling seed oil accumulation in Arabidopsis.

Detection of Tocopherol in Oilseed Rape （Brassica napus L.） Using Gas Chromatography with Flame Ionization Detector

Abstract The variation among Chinese genotypes of Brassica napus L. for seed tocopherols content and their analysis using gas chromatography has not been comprehensively reported till to date. In the present study, the tocopherol contents of four Chinese genotypes of Brassica napus L., namely, Gaoyou 605, Zhejiang 619, Zheshuang 758, and Zheshuang 72, were evaluated using three modified sample preparation protocols (P1, P2, and P3) for tocopherol extraction. These methods were distinguished as follows. Protocol one (P1) included the evaporation of solvent after extraction without silylation. Protocol two (P2) followed the direct supernatant collection after overnight extraction without drying and silylation. Protocol three (P3) included trimethylsilylation with N, O-bis(trimethylsilyl) trifluoroacetamide. Genotypic comparison of tocopherol and its isoforms revealed that Gaoyou 605 was dominant over the other genotypes with (140.5±10.5), (316.2± 9.2), and (559.1± 24.3) μg g−1 of seed meal α-, γ-, and total (T-) tocopherol, respectively, and a 0.44±0.04 α- to γ-tocopherol ratio. The comparison of the sample preparation protocols, on the other hand, suggests that P3 is the most suitable method for the tocopherol extraction from Brassica oilseeds and for the analysis of tocopherols using gas chromatography flame ionization detector (GC-FID). Trimethylsilylation is the key step differentiating P3 from P1 and P2. Variations detected in tocopherol contents among the Chinese rapeseed (B. napus) genotypes signify the need to quantify a wide range of rapeseed germplasm for seed tocopherol dynamics in short and crop improvement in long.

TRANSPARENT TESTA GLABRA 1 ubiquitously regulates plant growth and development from Arabidopsis to foxtail millet (Setaria italica)

TRANSPARENT TESTA GLABRA 1 of Arabidopsis thaliana (AtTTG1) is a WD40 repeat transcription factor that plays multiple roles in plant growth and development, particularly in seed metabolite production. In the present study, to determine whether SiTTG1 of the phylogenetically distant monocot foxtail millet (Setaria italica) has similar functions, we used transgenic Arabidopsis and Nicotiana systems to explore its activities. We found that SiTTG1 functions as a transcription factor. Overexpression of the SiTTG1 gene rescued many of the mutant phenotypes in Arabidopsis ttg1-13 plants. Additionally, SiTTG1 overexpression fully corrected the reduced expression of mucilage biosynthetic genes, and the induced expression of genes involved in accumulation of seed fatty acids and storage proteins in developing seeds of ttg1-13 plants. Ectopic expression of SiTTG1 restored the sensitivity of the ttg1-13 mutant to salinity and high glucose stresses during germination and seedling establishment, and restored altered expression levels of some stress-responsive genes in ttg1-13 seedlings to the wild type level under salinity and glucose stresses. Our results provide information that will be valuable for understanding the function of TTG1 from monocot to dicot species and identifying a promising target for genetic manipulation of foxtail millet to improve the amount of seed metabolites.

MYB76 Inhibits Seed Fatty Acid Accumulation in Arabidopsis

The MYB family of transcription factors is important in regulatory networks controlling development, metabolism and responses to biotic and abiotic stresses in Arabidopsis. However, their role in regulating fatty acid accumulation in seeds is still largely unclear. Here, we found that MYB76, localized in the nucleus, was predominantly expressed in developing seeds during maturation. The myb76 mutation caused a significant increase in the amounts of total fatty acids and several major fatty acid compositions in mature seeds, suggesting that MYB76 functioned as an important repressor during seed oil biosynthesis. RNA sequencing and quantitative real-time PCR analysis revealed remarkable alteration of numerous genes involved in photosynthesis, fatty acid biosynthesis, modification, and degradation, and oil body formation in myb76 seeds at 12 days after pollination. These results help us to understand the novel function of MYB76 and provide new insights into the regulatory network of MYB transcriptional factors controlling seed oil accumulation in Arabidopsis.