Identification and characterization of the soybean IPK1 ortholog of a low phytic acid mutant reveals an exon-excluding splice-site mutation.

Phytic acid (myo-inositol 1, 2, 3, 4, 5, 6 hexakisphosphate) is an important constituent of soybean meal. Since phytic acid and its mineral salts (phytates) are almost indigestible for monogastrics, their abundance in grain food/feed causes nutritional and environmental problems; interest in breeding low phytic acid has therefore increased considerably. Based on gene mapping and the characteristics of inositol polyphosphates profile in the seeds of a soybean mutant line Gm-lpa-ZC-2, the soybean ortholog of inositol 1,3,4,5,6 pentakisphosphate (InsP(5)) 2-kinase (IPK1), which transforms InsP(5) into phytic acid, was first hypothesized as the candidate gene responsible for the low phytic acid alteration in Gm-lpa-ZC-2. One IPK1 ortholog (Glyma14g07880, GmIPK1) was then identified in the mapped region on chromosome 14. Sequencing revealed a G → A point mutation in the genomic DNA sequence and the exclusion of the entire fifth exon in the cDNA sequence of GmIPK1 in Gm-lpa-ZC-2 compared with its wild-type progenitor Zhechun No. 3. The excluded exon encodes 37 amino acids that spread across two conserved IPK1 motifs. Furthermore, complete co-segregation of low phytic acid phenotype with the G → A mutation was observed in the F(2) population of ZC-lpa x Zhexiandou No. 4 (a wild-type cultivar). Put together, the G → A point mutation affected the pre-mRNA splicing and resulted in the exclusion of the fifth exon of GmIPK1 which is expected to disrupt the GmIPK1 functionality, leading to low phytic acid level in Gm-lpa-ZC-2. Gm-lpa-ZC-2, would be a good germplasm source in low phytic acid soybean breeding.

The reduction of reactive oxygen species formation by mitochondrial alternative respiration in tomato basal defense against TMV infection.

The role of mitochondrial alternative oxidase (AOX) and the relationship between systemic AOX induction, ROS formation, and systemic plant basal defense to Tobacco mosaic virus (TMV) were investigated in tomato plants. The results showed that TMV inoculation significantly increased the level of AOX gene transcripts, ubiquinone reduction levels, pyruvate content, and cyanide-resistant respiration (CN-resistant R) in upper, un-inoculated leaves. Pretreatment with potassium cyanide (KCN, a cytochrome pathway inhibitor) greatly increased CN-resistant R and reduced reactive oxygen species (ROS) formation, while application of salicylhydroxamic acid (SHAM, an AOX inhibitor) blocked the AOX activity and enhanced the production of ROS in the plants. Furthermore, TMV systemic infection was enhanced by SHAM and reduced by KCN pretreatment, as compared with the un-pretreated TMV counterpart. In addition, KCN application significantly diminished TMV-induced increase in antioxidant enzyme activities and dehydroascorbate/total ascorbate pool, while an opposite change was observed with SHAM-pretreated plants. These results suggest that the systemic induction of the mitochondrial AOX pathway plays a critical role in the reduction of ROS to enhance basal defenses. Additional antioxidant systems were also coordinately regulated in the maintenance of the cellular redox homeostasis.

Systemic induction and role of mitochondrial alternative oxidase and nitric oxide in a compatible tomato-Tobacco mosaic virus interaction.

The role of mitochondrial alternative oxidase (AOX) and the relationship between AOX and nitric oxide (NO) in virus-induced systemic defense to Tobacco mosaic virus (TMV) were investigated in susceptible tomato (Solanum lycopersicum) plants. TMV inoculation to the lower leaves induced a rapid NO synthesis and AOX activation in upper uninoculated leaves as early as 0.5 day postinoculation. Application of exogenous potassium cyanide (KCN, a cytochrome pathway inhibitor) at nonlethal concentrations and NO donor diethylamine NONOate (DEA/NO) to the upper uninoculated leaves greatly induced accumulation of AOX transcript, reduced TMV viral RNA accumulation, and increased the leaf photochemical quantum yield at photosystem II. Pretreatment with NO scavenger almost completely blocked TMV-induced AOX induction and substantially increased TMV susceptibility. Salicylhydroxamic acid (SHAM, an AOX inhibitor) pretreatment reduced the DEA/NO-induced cyanide-resistant respiration and partially compromised induced resistance to TMV. Conversely, KCN and SHAM pretreatment had very little effect on generation of NO, and pretreatment with NO scavenger did not affect KCN-induced AOX induction and TMV resistance. These results suggest that TMV-induced NO generation acts upstream and mediates AOX induction which, in turn, induces mitochondrial alternative electron transport and triggers systemic basal defense against the viral pathogen.

Effects of two low phytic acid mutations on seed quality and nutritional traits in soybean (Glycine max L. Merr).

Reduction of phytic acid in soybean seeds has the potential to improve the nutritional value of soybean meal and lessen phosphorus pollution in large scale animal farming. The objective of this study was to assess the effect of two new low phytic acid (LPA) mutations on seed quality and nutritional traits. Multilocation/season comparative analyses showed that the two mutations did not affect the concentration of crude protein, any of the individual amino acids, crude oil, and individual saturated fatty acids. Among other traits, Gm-lpa-TW75-1 had consistently higher sucrose contents (+47.4-86.1%) and lower raffinose contents (-74.2 to -84.3%) than those of wild type (WT) parent Taiwan 75; Gm-lpa-ZC-2 had higher total isoflavone contents (3038.8-4305.4 microg/g) than its parent Zhechun # 3 (1583.6-2644.9 microg/g) in all environments. Further tests of homozygous F(3) progenies of the cross Gm-lpa-ZC-2 x Wuxing # 4 (WT variety) showed that LPA lines had a mean content of total isoflavone significantly higher than WT lines. This study demonstrated that two LPA mutant genes have no negative effects on seed quality and nutritional traits; they instead have the potential to improve a few other properties. Therefore, these two mutant genes are valuable genetic resources for breeding high quality soybean varieties.

Decreased energy synthesis is partially compensated by a switch to sucrose synthase pathway of sucrose degradation in restricted root of tomato plants.

Tomato (Solanum lycopersium L.) plants were grown hydroponically to investigate the changes of energy metabolism and adaptive mechanism in response to root restriction. Root restriction resulted in a significant increase in root lipid peroxidation and reduction in leaf net CO(2) assimilation rate, which was accompanied by increase of alcohol dehydrogenase (ADH; EC 1.1.1.1) and lactate dehydrogenase (LDH; EC 1.1.1.27) activities. Total, cytochrome pathway, and alternative pathway respirations were all decreased in the roots after 15 days of root restriction treatment. Accompanied with the decrease of ATP content, ratio of invertase/sucrose synthase activity was increased in the restricted roots together with a decrease in glucose content and an increase in fructose content. We concluded that the decreased energy synthesis under root restriction condition was partially compensated by the energy-conserving sucrose synthase pathway of sucrose metabolism.

Putrescine enhancement of tolerance to root-zone hypoxia in Cucumis sativus: a role for increased nitrate reduction.

Cucumber (Cucumis sativus L.) plants were subjected to hypoxic stress with or without a pretreatment of putrescine (Put) to investigate whether nitrate reduction is involved in the enhancement effects of Put on tolerance to root-zone hypoxia. Both hypoxic stress and exogenous Put application significantly increased the contents of endogenous Put, spermidine and spermine. Plants grown under hypoxic conditions exhibited reductions in plant growth rate, NAD+/NADH ratio, ATP concentration, and consequent lowered cell viability in roots. The detrimental effects, however, were significantly alleviated by the addition of Put into the nutrient solution 24 h before the administration of hypoxia. Transcript levels of NR (nitrate reductase) and its cofactor binding domain genes FAD (FAD binding) and CYP51G1 (Heme binding), the activity of nitrate reductase (NR, EC 1.6.6.1) and the nitrate reduction process were each greatly enhanced by Put application, particularly in roots exposed to hypoxia. Lactate dehydrogenase (EC 1.1.1.27) activity was independent of aeration condition and Put application, whereas alcohol dehydrogenase (EC 1.1.1.1) activity was significantly increased after exposure to hypoxia, but did not increase after Put application. Put failed to alleviate the hypoxia injury of root electrolyte leakage when NR was inhibited by tungstate in the nutrient solution. These results suggest that Put enhances tolerance to hypoxia by increasing the transcript levels of NR and its cofactor binding domain genes, thereby stimulating the activities of NR and nitrate reduction to maintain the redox and energy status.