The distribution of flavonoids, isoflavonoids, and their conjugates in developing soybean (Glycine max L.) seedling organs and in root and seed exudates has been examined. Conjugates of the isoflavones daidzein and genistein are major metabolites in all embryonic organs within the dry seed and in seedling roots, hypocotyl, and cotyledon tissues at all times after germination. Primary leaf tissues undergo a programmed shift from isoflavonoid to flavonoid metabolism 3 days after germination and become largely predominated by glycosides of the flavonols kampferol, quercetin, and isorhamnetin by 5 days. Cotyledons contain relatively constant and very high levels of conjugates of both daidzein and genistein. Hypocotyl tissues contain a third unidentified compound, P19.3, also present in multiple conjugated forms. Conjugates of daidzein, genistein, and P19.3 are at their highest levels in the hypocotyl hook and fall off progressively down the hypocotyl. These isoflavones also undergo a programmed and dramatic decrease between 2 and 4 days in the hypocotyl hook. All root sections are predominated by daidzein and its conjugates, particularly in the root tip, where they reach the highest levels in the seedling. Light has a pronounced effect on the distribution of the isoflavones; in the dark, isoflavone levels in the root tips are greatly reduced, while those in the cotyledons are higher. Finally, the conjugates of daidzein and genistein and several unidentified aromatic metabolites are selectively excreted into root and seed exudates. Analysis of seed exudates suggests that this is a continuous, but saturable event.Phenylpropanoid-derived metabolites are widely distributed in the plant kingdom and have been proposed to play many diverse roles (23). They have been implicated in the protection of plants against both insects and pathogens and in the protection of plants against UV light damage. They are determinants offlower color and are involved in the attraction of pollinating insects. Because of the remarkable structural diversity shown by these metabolites and their derivatives and the complexity of their distribution, it has long been proposed that they may play additional, undefined roles in the plant, including a possible role in the regulation of growth and development (23,28). ' Partial salary and research support were provided by state and federal funds appropriated to the Ohio Agricultural and Development Center (OARDC). This publication is OARDC manuscript 243-90.
Isoflavones are thought to play diverse roles in plant-microbe interactions and are also potentially important to human nutrition and medicine. Isoflavone synthase (IFS) is a key enzyme for the formation of the isoflavones. Here, we examined the consequences of RNAi silencing of genes for this enzyme in soybean (Glycine max). Soybean cotyledon tissues were transformed with Agrobacterium rhizogenes carrying an RNAi silencing construct designed to silence expression of both copies of IFS genes. Approximately 50% of emerging roots were transformed with the RNAi construct, and most transformed roots exhibited .95% silencing of isoflavone accumulation. Silencing of IFS was also demonstrated throughout the entire cotyledon (in tissues distal to the transformation site) both by high-performance liquid chromatography analysis of isoflavones and by real-time reverse transcription-PCR. This distal silencing led to a nearly complete suppression of mRNA accumulation for both the IFS1 and IFS2 genes and of isoflavone accumulations induced by wounding or treatment with the cell wall glucan elicitor from Phytophthora sojae. Preformed isoflavone conjugates were not reduced in distal tissues, suggesting little turnover of these stored isoflavone pools. Distal silencing was established within just 5 d of transformation and was highly efficient for a 3-to 4-d period, after which it was no longer apparent in most experiments. Silencing of IFS was effective in at least two genotypes and led to enhanced susceptibility to P. sojae, disrupting both R gene-mediated resistance in roots and nonrace-specific resistance in cotyledon tissues. The soybean cotyledon system, already a model system for defense signal-response and cell-to-cell signaling, may provide a convenient and effective system for functional analysis of plant genes through gene silencing. (Kramer et al., 1984; RiveraVargas et al., 1993) and as precursors for the defenserelated coumestan and pterocarpan phytoalexins (Ebel, 1986). They also play roles as signal molecules in the induction of microbial genes involved in infection (Dixon and Sumner, 2003) and symbiosis between soybean and Bradyrhizobium japonicum (Pueppke, 1996;Ferguson and Mathesius, 2003). Finally, the isoflavones also have many activities potentially important to human nutrition and medicine, including activities as phytoestrogens, anti-cancer, and anti-oxidant compounds (for review, see Ososki and Kennelly, 2003;Cornwell et al., 2004;Huntley and Ernst, 2004).In soybean, the major isoflavones include daidzein, genistein, and glycitein. These are the predominant aromatic secondary metabolites in soybean seed and all seedling organs (Graham, 1991a). Other than the presence of relatively high levels of free daidzein in root tissues, the isoflavones in soybean seed and seedling tissues are predominantly present as glucosyl-and malonyl-glucosyl conjugates, the levels of which vary markedly in different developmental stages in various organs (Graham, 1991a). While the isoflavones are normally present at relativel...
Isoflavonoids are thought to play an important role in soybean (Glycine max) resistance to Phytophthora sojae. This was addressed by silencing two genes for their biosynthesis and a third gene controlling their elicitation. Silencing of genes for isoflavone synthase (IFS) or chalcone reductase (CHR) was achieved in soybean roots through an Agrobacterium rhizogenes-mediated RNAi approach. Effectiveness of silencing was followed both by quantitative reverse transcriptase-polymerase chain reaction and high-performance liquid chromatography analyses. Silencing either IFS or CHR led to a breakdown of Rps-mediated resistance to race 1 of P. sojae in 'W79' (Rps 1c) or 'W82' (Rps 1k) soybean. Loss of resistance was accompanied by suppression of hypersensitive (HR) cell death in both cultivars and suppression of cell death-associated activation of hydrogen peroxide and peroxidase. The various results suggest that the 5-deoxyisoflavonoids play a critical role in the establishment of cell death and race-specific resistance. The P. sojae cell wall glucan elicitor, a potent elicitor of 5-deoxyisoflavonoids, triggered a cell death response in roots that was also suppressed by silencing either CHR or IFS. Furthermore, silencing of the elicitor-releasing endoglucanase (PR-2) led to a loss of HR cell death and race-specific resistance to P. sojae and also to a loss of isoflavone and cell death responses to cell wall glucan elicitor. Taken together, these results suggest that in situ release of active fragments from a general resistance elicitor (pathogen-associated molecular pattern) is necessary for HR cell death in soybean roots carrying resistance genes at the Rps 1 locus, and that this cell death response is mediated through accumulations of the 5-deoxyisoflavones.
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