Overexpression of the Stress-Inducible SsMAX2 Promotes Drought and Salt Resistance via the Regulation of Redox Homeostasis in Arabidopsis.

Recent studies have demonstrated that strigolactones (SLs) also participate in the regulation of stress adaptation; however, the regulatory mechanism remains elusive. In this study, the homolog of More Axillary Branches 2, which encodes a key component in SL signaling, in the perennial oil plant Sapium sebiferum was identified and functionally characterized in Arabidopsis. The results showed that the expression of SsMAX2 in S. sebiferum seedlings was stress-responsive, and SsMAX2 overexpression (OE) in Arabidopsis significantly promoted resistance to drought, osmotic, and salt stresses. Moreover, SsMAX2 OE lines exhibited decreased chlorophyll degradation, increased soluble sugar and proline accumulation, and lower water loss ratio in response to the stresses. Importantly, anthocyanin biosynthesis and the activities of several antioxidant enzymes, such as superoxide dismutase (SOD), peroxidase (POD), and ascorbate peroxidase (APX), were enhanced in the SsMAX2 OE lines, which further led to a significant reduction in hydrogen peroxide levels. Additionally, the SsMAX2 OE lines exhibited higher expression level of several abscisic acid (ABA) biosynthesis genes, suggesting potential interactions between SL and ABA in the regulation of stress adaptation. Overall, we provide physiological and biochemical evidence demonstrating the pivotal role of SsMAX2 in the regulation of osmotic, drought, and salt stress resistance and show that MAX2 can be a genetic target to improve stress tolerance.

Comparative transcriptome analysis reveals the regulatory networks of cytokinin in promoting the floral feminization in the oil plant Sapium sebiferum.

BACKGROUND: Sapium sebiferum, whose seeds contain high level of fatty acids, has been considered as one of the most important oil plants. However, the high male to female flower ratio limited the seed yield improvement and its industrial potentials. Thus, the study of the sex determination in S. sebiferum is of significant importance in increasing the seed yield. RESULTS: In this study, we demonstrated that in S. sebiferum, cytokinin (CK) had strong feminization effects on the floral development. Exogenous application with 6-benzylaminopurine (6-BA) or thidiazuron (TDZ) significantly induced the development of female flowers and increased the fruit number. Interestingly, the feminization effects of cytokinin were also detected on the androecious genotype of S. sebiferum which only produce male flowers. To further investigate the mechanism underlying the role of cytokinin in the flower development and sex differentiation, we performed the comparative transcriptome analysis of the floral buds of the androecious plants subjected to 6-BA. The results showed that there were separately 129, 352 and 642 genes differentially expressed at 6 h, 12 h and 24 h after 6-BA treatment. Functional analysis of the differentially expressed genes (DEGs) showed that many genes are related to the hormonal biosynthesis and signaling, nutrients translocation and cell cycle. Moreover, there were twenty one flowering-related genes identified to be differentially regulated by 6-BA treatment. Specifically, the gynoecium development-related genes SPATULA (SPT), KANADI 2 (KAN2), JAGGED (JAG) and Cytochrome P450 78A9 (CYP79A9) were significantly up-regulated, whereas the expression of PISTILLATA (PI), TATA Box Associated Factor II 59 (TAFII59) and MYB Domain Protein 108 (MYB108) that were important for male organ development was down-regulated in response to 6-BA treatment, demonstrating that cytokinin could directly target the floral organ identity genes to regulate the flower sex. CONCLUSIONS: Our work demonstrated that cytokinin is a potential regulator in female flower development in S. sebiferum. The transcriptome analysis of the floral sex transition from androecious to monoecious in response to cytokinin treatment on the androecious S. sebiferum provided valuable information related to the mechanism of sex determination in the perennial woody plants.

BRIEF-REPORT New set of microsatellites for Chinese tallow tree, Triadica sebifera.

Chinese tallow (Triadica sebifera) is an important crop and ornamental tree. After it was introduced into the USA, it gradually became a noxious invasive tree in south-eastern America since the middle of the 1900s. Because only six microsatellites were reported previously in T. sebifera, to better understand the genetic diversity and population dynamics of such species, we reported here 28 new microsatellite markers. For these 28 microsatellites, the number of alleles per locus ranged from 2-16. The expected heterozygosity and the expected heterozygosity corrected for sample size varied from 0.0796 to 0.9081 and from 0.0805 to 0.9176, respectively. These microsatellites will provide additional choice to investigate the genetic diversity and structure in T. sebifera.

Geographic distribution of genetic variation among native and introduced populations of Chinese tallow tree, Triadica sebifera (Euphorbiaceae).

PREMISE OF THE STUDY: Invasive plants often display genetically determined variation in patterns of growth and resource allocation between native and introduced genotypes, as well as among genotypes within different regions of the introduced range. We examined patterns of genetic variation within and among native and introduced populations of the tetraploid Chinese tallow tree (Triadica sebifera, Euphorbiaceae) to determine whether nonselective evolutionary processes or the introduction history could contribute to previously observed phenotypic differences between native and introduced populations as well as among introduced populations. METHODS: We used six microsatellite markers to study 12 native populations in China, 51 introduced populations in the southeastern USA, and one introduced population in Australia. KEY RESULTS: Genetic diversity was greater within and among native populations than introduced populations. Within the southeastern USA, populations in Georgia and South Carolina differed substantially in their genetic composition and had greater genetic diversity than the rest of the southeastern USA. Greater genetic similarity between some populations in the native range and introduced range indicate a common provenance for Georgia and South Carolina populations that could have come from any of several western or southern Chinese populations and a different provenance for other southeastern USA populations and the Australian population, which were most similar to more northeastern Chinese populations. CONCLUSIONS: Differences among introduced populations in potentially adaptive traits (e.g., herbivore tolerance, herbivore resistance, growth rates) may result in part from the introduction history, in particular from differences present among source populations in the native range.

Alteration of the fatty acid composition of Brassica napus L. via overexpression of phospholipid: Diacylglycerol acyltransferase 1 from Sapium sebiferum (L.) Roxb.

Sapium sebiferum (L.) Roxb. plays an important role in traditional Chinese medicine and is one of major woody oil tree in China. Phospholipid: diacylglycerol acyltransferase 1 (PDAT1), as an important catalytic enzyme for the formation of triacylglycerol (TAG), is mainly responsible for the transfer of an acyl group from the sn-2 position of phospholipids to the sn-3 position of sn-1, 2-diacylglycerol (DAG) to produce TAG and sn-1 lysophospholipids. The importance of PDAT1 in triacylglycerol biosynthesis has been illustrated in previous research, and at least 67 PDAT1 sequences have been identified from 31 organisms. However, little is known about the gene encoding PDAT1 in S. sebiferum (SsPDAT1), which is involved in seed oil biosynthesis. To explore the functional characteristics of SsPDAT1, we cloned and analyzed the full-length cDNA in the coding region of SsPDAT1, which consists of 2040 bp and encodes a putative protein of 680 amino acid (aa) residues. Thin-layer chromatography (TLC) analysis showed that recombinant SsPDAT1 could restore TAG accumulation in TAG-deficient mutant yeast (Saccharomyces cerevisiae) H1246, which revealed the enzyme activity of SsPDAT1. Moreover, transgenic Brassica napus L. W10 plants overexpressing SsPDAT1 showed significant increases of 19.6-28.9 % in linoleic acid levels but decreases of 27.3-37.1 % in linolenic acid. Furthermore, the total oil content increased by 8.1 %-10.8 % in SsPDAT1 transgenic seeds. These results confirmed the role of SsPDAT1 in stabilizing oil biosynthesis and suggested that SsPDAT1 could be exploitable to specifically regulate the oil composition of plants. These experimental results provide a new concept that may enable the industrial development of plants with high-linoleic-acid oil through overexpression of SsPDAT1 in S. sebiferum L.

Enhancing freezing tolerance of Brassica napus L. by overexpression of a stearoyl-acyl carrier protein desaturase gene (SAD) from Sapium sebiferum (L.) Roxb.

Sapium sebiferum (L.) Roxb. is an important woody oil tree and traditional herbal medicine in China. Stearoyl-acyl carrier protein desaturase (SAD) is a dehydrogenase enzyme that plays a key role in the transformation of saturated fatty acids into unsaturated fatty acids in oil; these fatty acids greatly influence the freezing tolerance of plants. However, it remains unclear whether freezing tolerance can be regulated by the expression level of SsSAD in S. sebiferum L. Our research indicated that SsSAD expression in S. sebiferum L. increased under freezing stress. To further confirm this result, we constructed a pEGAD-SsSAD vector and transformed it into B. napus L. W10 by Agrobacterium tumefaciens-mediated transformation. Transgenic plants that overexpressed the SsSAD gene exhibited significantly higher linoleic (18:2) and linolenic acid (18:3) content and advanced freezing tolerance. These results suggest that SsSAD overexpression in B. napus L. can increase the content of polyunsaturated fatty acids (PUFAs) such as linoleic (18:2) and linolenic acid (18:3), which are likely pivotal in improving freezing tolerance in B. napus L. plants. Thus, SsSAD overexpression could be useful in the production of freeze-tolerant varieties of B. napus L.

Molecular characterization of a CONSTANS gene from Sapium sebiferum (L.) Rxob.

Sapium sebiferum (L.) Roxb [S. sebiferum L.] is not only one of the most important economic woody oil trees, but is also a significant traditional herbal medicine in China. The CONSTANS (CO) gene is a key regulator of the long day-dependent flowering pathway in Arabidopsis and other plants. To gain insight into the role of CO in woody oil trees, SsCO from S. sebiferum L. was isolated and characterized in this study. The corresponding SsCO protein, with 340 amino acid residues, included two putative zinc finger motifs B-Box1 and B-Box2 in the N-terminal region and a conserved CCT domain in the C-terminal region. SsCO expression was high in flowers and exhibited distinct circadian regulation. In addition, SsCO had a transcriptional activation effect in yeast strains. Moreover, heterologous expression of SsCO complemented the late-flowering phenotype of the Arabidopsis CO mutant co-1. These results indicate that SsCO is a transcription factor and may regulate the photoperiodic flowering time and SsCO is regulated by circadian rhythms in Sapium sebiferum L.

Flower bud transcriptome analysis of Sapium sebiferum (Linn.) Roxb. and primary investigation of drought induced flowering: pathway construction and G-quadruplex prediction based on transcriptome.

Sapium sebiferum (Linn.) Roxb. (Chinese Tallow Tree) is a perennial woody tree and its seeds are rich in oil which hold great potential for biodiesel production. Despite a traditional woody oil plant, our understanding on S. sebiferum genetics and molecular biology remains scant. In this study, the first comprehensive transcriptome of S. sebiferum flower has been generated by sequencing and de novo assembly. A total of 149,342 unigenes were generated from raw reads, of which 24,289 unigenes were successfully matched to public database. A total of 61 MADS box genes and putative pathways involved in S. sebiferum flower development have been identified. Abiotic stress response network was also constructed in this work, where 2,686 unigenes are involved in the pathway. As for lipid biosynthesis, 161 unigenes have been identified in fatty acid (FA) and triacylglycerol (TAG) biosynthesis. Besides, the G-Quadruplexes in RNA of S. sebiferum also have been predicted. An interesting finding is that the stress-induced flowering was observed in S. sebiferum for the first time. According to the results of semi-quantitative PCR, expression tendencies of flowering-related genes, GA1, AP2 and CRY2, accorded with stress-related genes, such as GRX50435 and PRXⅡ39562. This transcriptome provides functional genomic information for further research of S. sebiferum, especially for the genetic engineering to shorten the juvenile period and improve yield by regulating flower development. It also offers a useful database for the research of other Euphorbiaceae family plants.

Cloning and characterization of a novel Delta12-fatty acid desaturase gene from the tree Sapium sebiferum.

A new full-length cDNA (Ssd12) encoding a Delta12-fatty acid desaturase (Delta12-FAD) was cloned from Sapium sebiferum using RT-PCR and RACE methods. Ssd12 contained a 1146 bp open reading frame encoding a protein of 381 amino acids. The amino acid sequence showed a much higher match with microsomal Delta12-FAD amino acid sequences than chloroplast Delta12-FAD amino acid sequences. Genomic Southern blot analysis suggested that Ssd12 had at least two copies. Ssd12 transcripts were detected in roots, leaves, stems, and seeds by real time PCR.