Functional characterization of galactinol synthase and raffinose synthase in desiccation tolerance acquisition in developing Arabidopsis seeds.

Raffinose family oligosaccharides (RFOs) accumulate during seed development, and have been thought to be associated with the acquisition of desiccation tolerance (DT) by seeds. Here, comprehensive approaches were adopted to evaluate the changes of DT in developing Arabidopsis seeds of wild type, overexpression (OX-AtGS1/GS2/RS5), and mutant lines by manipulating the expression levels of the GALACTINOL SYNTHASE (GS) and RAFFINOSE SYNTHASE (RS) genes. Our results indicate that seeds of the double mutant (gs1, gs2) and rs5 delayed the timing of DT acquisition as compared to wild type. Subsequent detection confirmed that seeds from OX-AtGS1/GS2 plants with high levels of galactinol, raffinose, and stachyose, and OX-AtRS5 plants possess more raffinose and stachyose but less galactinol compared to wild type. These lines all showed greater germination percentage and shorter time to 50% germination after desiccation treatment at 11 and 15 days after flower (DAF). Further analysis revealed that the role of RFOs is time limited and mainly affects the middle stage (9-16 DAF) of seed development by enhancing seed viability and the ratio of GSH to GSSH in cells, but there is no significant difference in DT of mature seeds. In addition, RFOs could reduce damage to seeds caused by oxidative stress. We conclude that GALACTINOL SYNTHASE and RAFFINOSE SYNTHASE play important roles in DT acquisition during Arabidopsis seed development, and that galactinol and RFOs are crucial protective compounds in the response of seeds to desiccation stress.

Modulating role of ROS in re-establishing desiccation tolerance in germinating seeds of Caragana korshinskii Kom.

In close agreement with visible germination, orthodox seeds lose desiccation tolerance (DT). This trait can be regained under osmotic stress, but the mechanisms are poorly understood. In this study, germinating seeds of Caragana korshinskii Kom. were investigated, focusing on the potential modulating roles of reactive oxygen species (ROS) in the re-establishment of DT. Germinating seeds with 2 mm long radicles can be rendered tolerant to desiccation by incubation in a polyethylene glycol (PEG) solution (-1.7 MPa). Upon PEG incubation, ROS accumulation was detected in the radicles tip by nitroblue tetrazolium chloride staining and further confirmed by confocal microscopy. The PEG-induced re-establishment of DT was repressed when ROS scavengers were added to the PEG solution. Moreover, ROS act downstream of abscisic acid (ABA) to modulate PEG-mediated re-establishment of DT and serve as a new inducer to re-establish DT. Transcriptomic analysis revealed that re-establishment of DT by ROS involves the up-regulation of key genes in the phenylpropanoid-flavonoid pathway, and total flavonoid content and key enzyme activity increased after ROS treatment. Furthermore, DT was repressed by an inhibitor of phenylalanine ammonia lyase. Our data suggest that ROS play a key role in the re-establishment of DT by regulating stress-related genes and the phenylpropanoid-flavonoid pathway.

Functional characterization of BnHSFA4a as a heat shock transcription factor in controlling the re-establishment of desiccation tolerance in seeds.

Desiccation tolerance (DT) is the crucial ability of seeds to resist desiccation. However, the regulatory mechanisms of seed DT are not fully understood. In this study, two heat shock cis-elements (HSEs) were identified in the Brassica napus galactinol synthase (BnGolS1) promoter and shown to bind the heat shock transcription factor A4a (BnHSFA4a). Transcriptional expression of BnHSFA4a was induced at the early stage of DT acquisition, prior to increased BnGolS1 activity and galactinol production. Ectopic overexpression of BnHSFA4a (oxBnHSFA4a) in Arabidopsis enhanced DT, particularly during DT re-establishment. OxBnHSFA4a up-regulated the expression of GolS1, GolS2, and raffinose synthase 2 (BnRS2) in Arabidopsis and increased the enzymatic activity of GolS and RS and the concentration of raffinose family oligosaccharides (RFOs). Additionally, the overexpression lines exhibited increased antioxidant abilities. In contrast, the Arabidopsis mutant athsfa4a was more sensitive to dehydration, showing decreases in the efficiency of DT re-establishment, RFO contents, and oxidation resistance. Complementation analysis indicated that DT was rescued in athsfa4a/BnHSFA4a seeds to similar levels compared with those of Col-0. Taken together, these results indicated that BnHSFA4a probably functions in the regulation of GolS expression and activity, and activation of the antioxidative system and other stress response factors to improve DT.

Identification of desiccation tolerance transcripts potentially involved in rape (Brassica napus L.) seeds development and germination.

To investigate regulatory processes and protective mechanisms leading to desiccation tolerance (DT) in seeds, cDNA amplified fragment length polymorphism (cDNA-AFLP) in conjunction with 128 primer combinations was used to detect differential gene expression in rape seeds in response to DT during seed development and germination. We obtained approximately 8000 transcript-derived fragments (TDFs), of which 394 TDFs with differential expression patterns ("sustained expression", "up-regulated", "couple with seed DT", and "down-regulated") were excised from gels and re-amplified by polymerase chain reaction (PCR). After sequencing and comparison with the National Center for Biotechnology Information database, 176 TDFs presented significant similarity with known genes that could be classified into the following categories: metabolism and energy, stress resistance and defense, storage, signal transduction, and other functional categories. Using semiquantitative reverse-transcription PCR and real-time PCR approaches, the significance of the differences was further confirmed in fresh seeds and dehydrated seeds. The genes that encode superoxide dismutase, peroxiredoxin, caleosin, oleosin S3, steroleosin, late embryogenesis abundant protein, glutathione reductase, ß-glucosidase, S23 transcriptional repressor, and some heat-shock proteins could be associated with DT. The results of this study will aid in the identification of candidate genes for future experiments that seek to understand seed DT.