Transformation of an artificial microRNA to soybean (Glycine max (L.) Merr.) to reduce the parasitism of Meloidogyne incognita

Effectors are specific proteins secreted by nematodes into plant cells that facilitate their parasitism to host plants. Inactivation of these effectors could reduce the parasitic ability of nematodes on plants and the damage caused by nematodes. Gene Minc14137encodes an effector unknown function that is cloned from the Meloidogyne incognita. Artificial microRNAs capable of inactivating the gene Minc14137 were synthesised and inserted into an expression vector in soybean. This construct was transformed into the soybean cotyledon node mediated by Agrobacterium tumefaciens and regenerated transgenic plants. Copy number and the expression level of miRNA in T0 transgenic plants were determined by the qPCR technique. In T1 transgenic soybean plants, the pathogenic ability of root-knot nematode is reduced by 44.6-50.5% compared to the control plants. Results show that effector MINC14137 could play an important role in the parasitism of Meloidogyne incognita.

Induction of Phytoalexins Gliceoline and Proteins Related to Defense in Soybean Cotyledon Treated With Yeast

Yeasts can induce mechanisms of plant resistance due to compounds with eliciting characteristics, so the aim of this work was to evaluate the effect of yeast on the induction of phytoalexins gliceoline, peroxidase, polyphenoloxidase and phenylalanine ammonia-lyase from soy cotyledons. To determine the defense enzymes, soybean seeds were sown and the cotyledons treated with sterile distilled water, Cryptococcus laurentii (AH 03-1), Pichia guilliermondii (AH 16-2), Rhodotorula glutinis (AH 14-3), Sporidiobolus johnsonii (AH 16-1) and Zygoascus hellenicus (AH 14-1). Biochemical analyzes of the formation of phytoalexins and the activity of the enzymes peroxidase, polyphenoloxidase, phenylalanine ammonia-lyase and total proteins were performed. For phytoalexins glycerolins the yeasts Cryptococcus laurentii (AH 03-1) and Zygoascus hellenicus (AH 14-1) promoted an increase of 83.65% and 78.75% in the formation of this compound. Cryptococcus laurentii (AH 03-1) increased peroxidase activity by 36.84%, while for polyphenoloxidase, the Pichiaguilliermondii e yeasts (AH 16-2), Rhodotorula glutinis (AH 14-3), Sporidiobolus johnsonii (AH 16-1) and Zygoascus hellenicus (AH 14-1), increased the activity of this enzyme by 33.33%, 28.00%, 33.33% and 33.33%, respectively. For phenylalanine ammonia-lyase, Cryptococcus laurentii (AH 03-1) and Zygoascushellenicus (AH 14-1) promoted an increase of 75.57% and 78.86%, respectively, in their activity. The results demonstrate the potential of yeasts studied in the induction of phytoalexins glyceolins and in the activity of peroxidase, polyphenoloxidase and phenylalanine ammonia-lyase in soybean cotyledons.

The small GTPase Rab5a and its guanine nucleotide exchange factors are involved in post-Golgi trafficking of storage proteins in developing soybean cotyledon

Storage protein is the most abundant nutritional component in soybean seed. Morphology-based evidence has verified that storage proteins are initially synthesized on the endoplasmic reticulum, and then follow the Golgi-mediated pathway to the protein storage vacuole. However, the molecular mechanisms of storage protein trafficking in soybean remain unknown. Here, we clone the soybean homologs of Rab5 and its guanine nucleotide exchange factor (GEF) VPS9. GEF activity combined with yeast two-hybrid assays demonstrated that GmVPS9a2 might specifically act as the GEF of the canonical Rab5, while GmVPS9b functions as a common activator for all Rab5s. Subcellular localization experiments showed that GmRab5a was dually localized to the trans-Golgi network and pre-vacuolar compartments in developing soybean cotyledon cells. Expression of a dominant negative variant of Rab5a, or RNAi of either Rab5a or GmVPS9s, significantly disrupted trafficking of mRFP–CT10, a cargo marker for storage protein sorting, to protein storage vacuoles in maturing soybean cotyledons. Together, our results systematically revealed the important role of GmRab5a and its GEFs in storage protein trafficking, and verified the transient expression system as an efficient approach for elucidating storage protein trafficking mechanisms in seed.

Multistep Optimization of β-Glucosidase Extraction from Germinated Soybeans (Glycine max L. Merril) and Recovery of Isoflavone Aglycones

Epicotyls from germinated soybeans (EGS) have great potential as sources of endogenous β-glucosidase. Furthermore, this enzyme may improve the conversion of isoflavones into their corresponding aglycones. β-Glucosidase may also increase the release of aglycones from the cell wall of the plant materials. Therefore, the aim of this work was to optimize both the extraction of β-glucosidase from EGS and to further examine its application in defatted soybean cotyledon to improve the recovery of aglycones, which were evaluated by ultra-high performance liquid chromatography (UHPLC). A multistep optimization was carried out and the effects of temperature and pH were investigated by applying a central composite design. The linear effect of pH and the quadratic effect of pH and temperature were significant for the extraction of β-glucosidase and recovery aglycones, respectively. Optimum extraction of β-glucosidase from EGS occurred at 30 °C and pH 5.0. Furthermore, the maximum recovery of aglycones (98.7%), which occurred at 35 °C and pH 7.0–7.6 during 144 h of germination, increased 8.5 times with respect to the lowest concentration. The higher bioaccessibility of aglycones when compared with their conjugated counterparts is well substantiated. Therefore, the data provided in this contribution may be useful for enhancing the benefits of soybean, their products, and/or their processing by-products.

Soybean (Glycine max) Tolerance to Timing Applications of Pyroxasulfone, Flumioxazin, and Pyroxasulfone + Flumioxazin

Four field studies were conducted over a 3-yr period (2011 to 2013) to determine the tolerance of four soybean cultivars to pyroxasulfone (89 and 178 g ai ha−1), flumioxazin (71 and 142 g ai ha−1), and pyroxasulfone + flumioxazin (160 and 320 g ai ha−1) applied either preplant incorporated (PPI), PRE, or at the soybean cotyledon stage (COT). When pyroxasulfone + flumioxazin was applied at 160 and 320 g ai ha−1, at the cotyledon stage soybean yield was decreased by 9 and 14%, respectively. The only other treatment that decreased soybean yield was pyroxasulfone (178 g ai ha−1) applied PPI; yield was decreased by 6% despite minimal injury and dry biomass reductions observed during the season. Soybean tolerance to pyroxasulfone or flumioxazin applied alone was generally similar and injury was less than with pyroxasulfone + flumioxazin. Similarly, herbicides applied PPI and PRE were less injurious to soybean than the COT timing. Results suggest that soybean is tolerant to PPI and PRE applications of pyroxasulfone + flumioxazin but COT applications should be avoided.