Regulation of Seed Vigor by Manipulation of Raffinose Family Oligosaccharides in Maize and Arabidopsis thaliana

Raffinose family oligosaccharides （RFOs） accumulate in seeds during maturation desiccation in many plant species. However, it remains unclear whether RFOs have a role in establishing seed vigor. GALACTINOL SYNTHASE （GOLS）, RAFFINOSE SYNTHASE （RS）, and STACHYOSE SYNTHASE （STS） are the enzymes responsible for RFO biosynthesis in plants. Interestingly, only raffinose is detected in maize seeds, and a unique maize RS gene （ZmRS） was identified. In this study, we found that two independent mutator （Mu）-interrupted zmrs lines, containing no raffinose but hyperaccumulating galactinol, have significantly reduced seed vigor, compared with null segregant controls. Unlike maize, Arabidopsis thaliana seeds contain several RFOs （raffinose, stachyose, and verbascose）. Manipulation of A. thaliana RFO content by overexpressing ZmGOLS2, ZmRS, or AtSTS demonstrated that co-overexpression of ZmGOLS2 and ZmRS, or overexpression of ZmGOLS2 alone, significantly increased the total content of RFOs and enhanced Arabidopsis seed vigor. Surprisingly, while overexpression of ZmRS increased seed raffinose content, its overexpression dramatically decreased seed vigor and reduced the seed amounts of galactinol, stachyose, and verbascose. In contrast, the atrs5 mutant seeds are similar to those of the wild type with regard to seed vigor and RFO content, except for stachyose, which accumulated in atrs5 seeds. Total RFOs, RFO/sucrose ratio, but not absolute individual RFO amounts, positively correlated with A. thaliana seed vigor, to which stachyose and verbascose contribute more than raffinose. Taken together, these re- sults provide new insights into regulatory mechanisms of seed vigor and reveal distinct requirement for RFOs in modulating seed vigor in a monocot and a dicot.

Effects of soybean raffinose on growth performance,digestibility,humoral immunity and intestinal morphology of growing pigs

There are appreciable does of raffinose in soybean,but the impacts of raffinose on pigs are poorly investigated.We used 2 experiments to investigate the influence of soybean raffinose on growth per-formance,digestibility,humoral immunity and intestinal morphology of growing pigs.In Exp.1,a total of 30 crossbred(Duroc×Landrace×Yorkshire)barrows(21.93±0.43 kg)were randomly divided into 3 groups,and were fed with the control diet,the control diets supplemented with 0.2%and 0.5%raffinose,respectively,for 21 d.Results showed that the addition of 0.2%or 0.5%raffinose reduced(P<0.05)average daily feed intake(ADFI),average daily gain(ADG)and nutrient digestibility,and dietary 0.5%raffinose increased the ratio of feed to gain(P<0.05).For serum indexes,dietary 0.5%raffinose decreased growth hormone and increased glucagon-like peptide-2,immunoglobulin G,tumor necrosis factor-α(TNF-α)and interleukin-6 concentration(P<0.05).In Exp.2,a total of 24 crossbred barrows(38.41±0.45 kg)were randomly divided into 3 groups,and were fed with the control diet(ad libitum),the raffinose diet(0.5%raffinose,ad libitum),and the control diet in the same amount as the raffinose group(feed-pair group)for 14 d,respectively.Compared with the control diet,dietary 0.5%raffinose decreased ADFI(P<0.05).Intriguingly,the raffinose group had lower ADG than the feed-pair group,lower nutrient digestibility,lower amylase activity in duodenum,lower amylase,lipase and trypsin ac-tivities in jejunum and higher TNF-αconcentration in serum compared with the other 2 groups,and a higher ratio of villus height to crypt depth compared with the control group(P<0.05).These results showed that soybean raffinose could reduce feed voluntary intake and body gain while improving in-testinal morphology without a significant negative influence on immunity.Taken together,dietary raffinose could decrease growth performance by reducing both feed intake and nutrient digestibility while inducing humoral immune response of growing pigs.

Multiplex genome editing targeting soybean with ultra-low anti-nutritive oligosaccharides

Soybean is the primary source of plant protein for humans.Owing to the indigestibility of the raffinose family of oligosaccharides(RFO),raffinose and stachyose are considered anti-nutritive factors in soybean seeds.Low-RFO soybean cultivars are generated by mutagenesis of RFO biosynthesis genes,but the carbohydrate profiles invite further modification to lower RFOs.This study employed a pooled multiplex genome editing approach to target four seed-specifically expressed genes mediating RFO biosynthesis,encoding three raffinose synthases(RS2,RS3,and RS4)and one stachyose synthase.In T1progeny,rs2/rs3 and rs4/sts homozygous double mutants and a rs2/rs3/rs4/sts quadruple mutant(rfo-4m)were characterized.The rs2/rs3 mutant showed reduced raffinose and stachyose contents,but the rs4/sts mutant showed only reduced stachyose in seeds.The RFO contents in the rfo-4m mutant were almost eliminated.Metabolomic analysis showed that the mutation of four RFO biosynthesis genes led to a shift of metabolic profile in the seeds,including the accumulation of several oligosaccharides-related metabolites.These mutants could contribute to precision breeding of soybean cultivars for soy food production.

Influence of Planting Date on Seed Protein, Oil, Sugars, Minerals, and Nitrogen Metabolism in Soybean under Irrigated and Non-Irrigated Environments

Information on the effect of planting date and irrigation on soybean [Glycine max (L.) Merr.] seed composition in the Early Soybean Production System (ESPS) is deficient, and what is available is inconclusive. The objective of this research was to investigate the effects of planting date on seed protein, oil, fatty acids, sugars, and minerals in soybean grown under irrigated (I) and non-irrigated (NI) conditions. A 2-yr field experiment was conducted in Stoneville, MS in 2007 and 2008. Soybean was planted during second week of April (early planting) and second week of May (late planting) each year. Results showed that under irrigated condition, early planting increased seed oil (up to 16% increase) and oleic acid (up to 22.8% increase), but decreased protein (up to 6.6% decrease), linoleic (up to 10.9% decrease) and linolenic acids (up to 27.7% decrease) compared to late planting. Under I conditions, late planting resulted in higher sucrose and raffinose and lower stachyose compared with early planting. Under NI conditions, seed of early planting had higher protein (up to 4% increase) and oleic acid (up to 25% increase) and lower oil (up to10.8% decrease) and linolenic acids (up to 13% decrease) than those of late planting. Under NI, stachyose concentration was higher than sucrose or raffinose, especially in early planting. Under I, early planting resulted in lower leaf and seed B, Fe, and P concentrations compared with those of late planting. Under NI, however, early planting resulted in higher accumulation of leaf B and P, but lower seed B and P compared with those of late planting. This research demonstrated that both irrigation and planting date have a significant influence on seed protein, oil, unsaturated fatty acids, and sugars. Our results suggest that seed of late planting accumulate more B, P, and Fe than those of early planting, and this could be a beneficial gain. Limited translocation of nutrients from leaves to seed under NI is undesirable. Soybean producers may use this information to maintain yield and seed quality, and soybean breeders to select for seed quality traits and mineral translocation efficiency in stress environments.

Quantitative Trait Loci Underlying Seed Sugars Content in “MD96-5722” by “Spencer” Recombinant Inbred Line Population of Soybean

Sucrose, raffinose, and stachyose are important soluble sugars in soybean [Glycine max (L.) Merr.] seeds. Seed sucrose is a desirable trait for taste and flavor. Raffinose and stachyose are undesirable in diets of monogastric animals, acting as anti-nutritional factors that cause flatulence and abdominal discomfort. Therefore, reducing raffinose and stachyose biosynthesis is considered as a key quality trait goal in soy food and feed industries. The objective of this study was to identify genomic regions containing quantitative trait loci (QTL) controlling sucrose, raffinose, and stachyose in a set of 92 F5:7 recombinant inbred lines (RILs) derived from a cross between the lines “MD96-5722” and “Spencer” by using 5376 Single Nucleotide Polymorphism (SNP) markers from the Illumina Infinium SoySNP6K BeadChip array. Fourteen significant QTL were identified and mapped on eight different linkage groups (LGs) and chromosomes (Chr). Three QTL for seed sucrose content were identified on LGs N (Chr3), K (Chr9), and E (Chr15). Seven QTL were identified for raffinose content on LGs D1a (Chr1), N (Chr3), C2 (Chr6), K (Chr9), B2 (Chr14), and J (Chr16). Four QTL for stachyose content were identified on LG D1a (Chr1), C2 (Chr6), H (Chr12), and B2 (Chr14). Selection for beneficial alleles of these QTLs could facilitate breeding strategies to develop soybean lines with higher concentrations of sucrose and lower levels of raffinose and stachyose.