Soybean Hairy Root Transformation for the Analysis of Gene Function.

Soybean (Glycine max) is a valuable crop in agriculture that has thousands of industrial uses. Soybean roots are the primary site of interaction with soil-borne microbes that form symbiosis to fix nitrogen and pathogens, which makes research involving soybean root genetics of prime importance to improve its agricultural production. The genetic transformation of soybean hairy roots (HRs) is mediated by the Agrobacterium rhizogenes strain NCPPB2659 (K599) and is an efficient tool for studying gene function in soybean roots, taking only 2 months from start to finish. Here, we provide a detailed protocol that outlines the method for overexpressing and silencing a gene of interest in soybean HRs. This methodology includes soybean seed sterilization, infection of cotyledons with K599, and the selection and harvesting of genetically transformed HRs for RNA isolation and, if warranted, metabolite analyses. The throughput of the approach is sufficient to simultaneously study several genes or networks and could determine the optimal engineering strategies prior to committing to long-term stable transformation approaches.

RNA-Seq Dissects Incomplete Activation of Phytoalexin Biosynthesis by the Soybean Transcription Factors GmMYB29A2 and GmNAC42-1.

Glyceollins, isoflavonoid-derived antimicrobial metabolites, are the major phytoalexins in soybean (Glycine max). They play essential roles in providing resistance to the soil-borne pathogen Phytophthora sojae and have unconventional anticancer and neuroprotective activities that render them desirable for pharmaceutical development. Our previous studies revealed that the transcription factors GmMYB29A2 and GmNAC42-1 have essential roles in activating glyceollin biosynthesis, yet each cannot activate the transcription of all biosynthesis genes in the absence of a pathogen elicitor treatment. Here, we report that co-overexpressing both transcription factors is also insufficient to activate glyceollin biosynthesis. To understand this insufficiency, we compared the transcriptome profiles of hairy roots overexpressing each transcription factor with glyceollin-synthesizing roots treated with wall glucan elicitor (WGE) from P. sojae. GmMYB29A2 upregulated most of the WGE-regulated genes that encode enzymatic steps spanning from primary metabolism to the last step of glyceollin biosynthesis. By contrast, GmNAC42-1 upregulated glyceollin biosynthesis genes only when overexpressed in the presence of WGE treatment. This is consistent with our recent discovery that, in the absence of WGE, GmNAC42-1 is bound by GmJAZ1 proteins that inhibit its transactivation activity. WGE, and not GmMYB29A2 or GmNAC42-1, upregulated the heat shock family gene GmHSF6-1, the homolog of Arabidopsis HSFB2a that directly activated the transcription of several glyceollin biosynthesis genes. Our results provide important insights into what biosynthesis genes will need to be upregulated to activate the entire glyceollin biosynthetic pathway.

Metabolomics Differences of Glycine max QTLs Resistant to Soybean Looper.

Quantitative trait loci (QTLs) E and M are major soybean alleles that confer resistance to leaf-chewing insects, and are particularly effective in combination. Flavonoids and/or isoflavonoids are classes of plant secondary metabolites that previous studies agree are the causative agents of resistance of these QTLs. However, all previous studies have compared soybean genotypes that are of dissimilar genetic backgrounds, leaving it questionable what metabolites are a result of the QTL rather than the genetic background. Here, we conducted a non-targeted mass spectrometry approach without liquid chromatography to identify differences in metabolite levels among QTLs E, M, and both (EM) that were introgressed into the background of the susceptible variety Benning. Our results found that E and M mainly confer low-level, global differences in distinct sets of metabolites. The isoflavonoid daidzein was the only metabolite that demonstrated major increases, specifically in insect-treated M and EM. Interestingly, M confers increased daidzein levels in response to insect, whereas E restores M's depleted daidzein levels in the absence of insect. Since daidzein levels do not parallel levels of resistance, our data suggest a novel mechanism that the QTLs confer resistance to insects by mediating changes in hundreds of metabolites, which would be difficult for the insect to evolve tolerance. Collective global metabolite differences conferred by E and M might explain the increased resistance of EM.

Transgene CpNAC68 from Wintersweet (Chimonanthus praecox) Improves Arabidopsis Survival of Multiple Abiotic Stresses.

The NAC (NAM, ATAFs, CUC) family of transcription factors (TFs) play a pivotal role in regulating all processes of the growth and development of plants, as well as responses to biotic and abiotic stresses. Yet, the functions of NACs from non-model plant species remains largely uncharacterized. Here, we characterized the stress-responsive effects of a NAC gene isolated from wintersweet, an ornamental woody plant that blooms in winter when temperatures are low. CpNAC68 is clustered in the NAM subfamily. Subcellular localization and transcriptional activity assays demonstrated a nuclear protein that has transcription activator activities. qRT-PCR analyses revealed that CpNAC68 was ubiquitously expressed in old flowers and leaves. Additionally, the expression of CpNAC68 is induced by disparate abiotic stresses and hormone treatments, including drought, heat, cold, salinity, GA, JA, and SA. Ectopic overexpression of CpNAC68 in Arabidopsis thaliana enhanced the tolerance of transgenic plants to cold, heat, salinity, and osmotic stress, yet had no effect on growth and development. The survival rate and chlorophyll amounts following stress treatments were significantly higher than wild type Arabidopsis, and were accompanied by lower electrolyte leakage and malondialdehyde (MDA) amounts. In conclusion, our study demonstrates that CpNAC68 can be used as a tool to enhance plant tolerance to multiple stresses, suggesting a role in abiotic stress tolerance in wintersweet.

Nanoparticle-based genetic transformation of Cannabis sativa.

Cannabis sativa (Cannabis) is a multipurpose plant species consisting of specific lineages that for centuries has either been artificially selected for the production of fiber or the psychoactive drug Δ9-tetrahydrocannabinol (THC). With the recent lifting of previous legal restrictions on consuming Cannabis, there has been a resurgence of interest in understanding and manipulating Cannabis genetics to enhance its compositions. Yet, recently developed approaches are not amenable to high-throughput gene stacking to study multi-genic traits. Here, we demonstrate an efficient nanoparticle-based transient gene transformation protocol where multiple gene plasmids can be expressed simultaneously in intact Cannabis leaf cells in a very short time (5 days). Constructs encoding two soybean transcription factors were co-grafted onto poly-ethylenimine cationic polymer-modified silicon dioxide-coated gold nanoparticles (PEI-Au@SiO2). Infiltration of the DNA-PEI-Au@SiO2 into Cannabis leaf tissues resulted in the transcription of both soybean genes and the localization of fluorescent-tagged transcription factor proteins in the nuclei of Cannabis leaf cells including the trichomes, which are the cell types that biosynthesize valuable cannabinoid and terpene metabolites. Our study exemplifies a rapid transient gene transformation approach that will be useful to study the effects of gene stacking in Cannabis.

Glyceollin Transcription Factor GmMYB29A2 Regulates Soybean Resistance to Phytophthora sojae.

Glyceollin isomers I, II, and III are the major pathogen-elicited secondary metabolites (i.e. phytoalexins) of soybean (Glycine max) that, collectively with other 5-deoxyisoflavonoids, provide race-specific resistance to Phytophthora sojae. The NAC-family transcription factor (TF) GmNAC42-1 is an essential regulator of some but not all glyceollin biosynthesis genes, indicating other essential TF(s) of the glyceollin gene regulatory network remain to be identified. Here, we conducted comparative transcriptomics on soybean hairy roots of the variety Williams 82 and imbibing seeds of Harosoy 63 upon treatment with wall glucan elicitor from P. sojae and identified two homologous R2R3-type MYB TF genes, GmMYB29A1 and GmMYB29A2, up-regulated during the times of peak glyceollin biosynthesis. Overexpression and RNA interference silencing of GmMYB29A2 increased and decreased expression of GmNAC42-1, GmMYB29A1, and glyceollin biosynthesis genes and metabolites, respectively, in response to wall glucan elicitor. By contrast, overexpressing or silencing GmMYB29A1 decreased glyceollin I accumulation with marginal or no effects on the expressions of glyceollin synthesis genes, suggesting a preferential role in promoting glyceollin turnover and/or competing biosynthetic pathways. GmMYB29A2 interacted with the promoters of two glyceollin I biosynthesis genes in vitro and in vivo. Silencing GmMYB29A2 in Williams 82, a soybean variety that encodes the resistance gene Rps1k, rendered it compatible with race 1 P. sojae, whereas overexpressing GmMYB29A2 rendered the susceptible Williams variety incompatible. Compatibility and incompatibility coincided with reduced and enhanced accumulations of glyceollin I but not other 5-deoxyisoflavonoids. Thus, GmMYB29A2 is essential for accumulation of glyceollin I and expression of Phytophthora resistance.

Characterization and Quantification of Major Flavonol Glycosides in Ramps (Allium tricoccum).

The ramp (Allium tricoccum) is a traditional plant in the eastern Appalachian Mountains. Ramps have been used in traditional medicine for their health-promoting roles in lowering blood pressure and cholesterol. Information on the chemical composition of the potentially bioactive components in ramps is limited. Therefore, the aim of this work was to characterize and quantify major flavonols in ramps. Flavonoids were extracted in 50% methanol and 3% acetic acid. Characterization was conducted using UHPLC-PDA-MS and MS/MS, and quantification was performed using UHPLC-PDA detection. The major flavonol glycosides were kaempferol sophoroside glucuronide, quercetin sophoroside glucuronide, kaempferol rutinoside glucuronide, quercetin hexoside glucuronide, quercetin sophoroside, and kaempferol sophoroside. All conjugates were detected in leaves. Quercetin and kaempferol sophoroside glucuronide conjugates were detected in the stem, but no flavonol glycosides were detected in the bulb. The total amounts of the identified quercetin and kaempferol conjugates in whole ramps were 0.5972 ± 0.235 and 0.3792 ± 0.130 mg/g dry weight, respectively. Flavonol conjugates were concentrated in the leaves. To our knowledge, this work is the first to identify and quantify the major flavonol glycosides in ramps. Our findings suggest that specifically the leaves may harbor the potentially bioactive flavonols components of the plant.

Transcriptional and Chemical Changes in Soybean Leaves in Response to Long-Term Aphid Colonization.

Soybean aphids (Aphis glycines Matsumura) are specialized insects that feed on soybean (Glycine max) phloem sap. Transcriptome analyses have shown that resistant soybean plants mount a fast response that limits aphid feeding and population growth. Conversely, defense responses in susceptible plants are slower and it is hypothesized that aphids block effective defenses in the compatible interaction. Unlike other pests, aphids can colonize plants for long periods of time; yet the effect on the plant transcriptome after long-term aphid feeding has not been analyzed for any plant-aphid interaction. We analyzed the susceptible and resistant (Rag1) transcriptome response to aphid feeding in soybean plants colonized by aphids (biotype 1) for 21 days. We found a reduced resistant response and a low level of aphid growth on Rag1 plants, while susceptible plants showed a strong response consistent with pattern-triggered immunity. GO-term analyses identified chitin regulation as one of the most overrepresented classes of genes, suggesting that chitin could be one of the hemipteran-associated molecular pattern that triggers this defense response. Transcriptome analyses also indicated the phenylpropanoid pathway, specifically isoflavonoid biosynthesis, was induced in susceptible plants in response to long-term aphid feeding. Metabolite analyses corroborated this finding. Aphid-treated susceptible plants accumulated daidzein, formononetin, and genistein, although glyceollins were present at low levels in these plants. Choice experiments indicated that daidzein may have a deterrent effect on aphid feeding. Mass spectrometry imaging showed these isoflavones accumulate likely in the mesophyll cells or epidermis and are absent from the vasculature, suggesting that isoflavones are part of a non-phloem defense response that can reduce aphid feeding. While it is likely that aphid can initially block defense responses in compatible interactions, it appears that susceptible soybean plants can eventually mount an effective defense in response to long-term soybean aphid colonization.

Acidity stress for the systemic elicitation of glyceollin phytoalexins in soybean plants.

Glyceollins are the major pathogen- and stress-inducible natural products (phytoalexins) of soybean that possess broad-spectrum anticancer and neuroprotective properties. Yet like other phytoalexins, glyceollins are difficult to obtain because they are typically biosynthesized only transiently and in low amounts in plant tissues. We recently identified acidity stress (pH 3.0 growth medium) as an elicitor that exerted prolonged (week-long) inductive effects on glyceollin biosynthesis and identified the NAC family TF gene GmNAC42-1 that activates glyceollin biosynthesis in response to acidity stress or WGE from the soybean pathogen Phytophthora sojae. GmNAC42-1 was annotated as an SAR gene and SAR genes were statistically overrepresented in the transcriptomic response to acidity stress suggesting that acidity stress triggers the systemic elicitation of glyceollin biosynthesis. Here, we demonstrate that acidity stress acts as a systemic elicitor when provided to soybean roots. Acidity stress preferentially elicited specific glyceollins in different soybean organs with exceptionally high yields of glyceollin I in root tissues.

The NAC family transcription factor GmNAC42-1 regulates biosynthesis of the anticancer and neuroprotective glyceollins in soybean.

BACKGROUND: Glyceollins are isoflavonoid-derived pathogen-inducible defense metabolites (phytoalexins) from soybean (Glycine max L. Merr) that have important roles in providing defense against pathogens. They also have impressive anticancer and neuroprotective activities in mammals. Despite their potential usefulness as therapeutics, glyceollins are not economical to synthesize and are biosynthesized only transiently and in low amounts in response to specific stresses. Engineering the regulation of glyceollin biosynthesis may be a promising approach to enhance their bioproduction, yet the transcription factors (TFs) that regulate their biosynthesis have remained elusive. To address this, we first aimed to identify novel abiotic stresses that enhance or suppress the elicitation of glyceollins and then used a comparative transcriptomics approach to search for TF gene candidates that may positively regulate glyceollin biosynthesis. RESULTS: Acidity stress (pH 3.0 medium) and dehydration exerted prolonged (week-long) inductive or suppressive effects on glyceollin biosynthesis, respectively. RNA-seq found that all known biosynthetic genes were oppositely regulated by acidity stress and dehydration, but known isoflavonoid TFs were not. Systemic acquired resistance (SAR) genes were highly enriched in the geneset. We chose to functionally characterize the NAC (NAM/ATAF1/2/CUC2)-family TF GmNAC42-1 that was annotated as an SAR gene and a homolog of the Arabidopsis thaliana (Arabidopsis) indole alkaloid phytoalexin regulator ANAC042. Overexpressing and silencing GmNAC42-1 in elicited soybean hairy roots dramatically enhanced and suppressed the amounts of glyceollin metabolites and biosynthesis gene mRNAs, respectively. Yet, overexpressing GmNAC42-1 in non-elicited hairy roots failed to stimulate the expressions of all biosynthesis genes. Thus, GmNAC42-1 was necessary but not sufficient to activate all biosynthesis genes on its own, suggesting an important role in the glyceollin gene regulatory network (GRN). The GmNAC42-1 protein directly bound the promoters of biosynthesis genes IFS2 and G4DT in the yeast one-hybrid (Y1H) system. CONCLUSIONS: Acidity stress is a novel elicitor and dehydration is a suppressor of glyceollin biosynthesis. The TF gene GmNAC42-1 is an essential positive regulator of glyceollin biosynthesis. Overexpressing GmNAC42-1 in hairy roots can be used to increase glyceollin yields > 10-fold upon elicitation. Thus, manipulating the expressions of glyceollin TFs is an effective strategy for enhancing the bioproduction of glyceollins in soybean.

Hormone deficient mutants have distinct flavonoid proportion fingerprints in response to abiotic stress.

Distinct flavonoid profiles (a.k.a. 'fingerprints') are produced in the vegetative tissues of plants in response to different abiotic stresses, yet it remained unknown whether flavonoid levels or their relative their proportions are more tightly regulated in response to stress. Here we show that the relative proportions of 19 flavonoids were more stringently controlled compared to their levels in response to variety of abiotic stresses. We screened mutants that are deficient in the biosynthesis of the stress response hormones ABA, Eth, JA, and GA by growing them in an abiotic stress condition that induces the biosynthesis of a wide variety of flavonoids and found that mutants deficient in a particular hormone generally had a distinct flavonoid proportion fingerprint. Our results suggest that flavonoid proportion fingerprints of uncharacterized mutants could be used to predict gene involvement in particular hormone pathways that signal responses to abiotic stress.

Combining semi-synthesis with plant and microbial biocatalysis: new frontiers in producing a chemical arsenal against cancer.

Natural products (NPs) that exhibit anticancer activities are frequently not potent enough to be used clinically as therapeutics. Semi-synthesis and metabolic engineering are promising approaches for producing more efficacious derivatives of anticancer NPs (ACNPs), but each technique alone can be inefficient at obtaining specific ACNP derivatives that may be suspected to have enhanced anticancer activity. Here, we demonstrate that the methods of semi-synthesis and biocatalysis can be used as modules in succession and in different combinations to produce 6,8-dibromogenkwanin, a derivative of the ACNP apigenin. Further, we demonstrated that soybean seed coats can be used as a biocatalyst to convert brominated flavonoids into multiple derivatives. A strength of the combinatorial (bio)synthesis approach was that the order of the modules could be rearranged to increase the yield of the desired product. At lower treatment concentration (5 µM), 6,8-dibromogenkwanin exhibited enhanced antiproliferative activities against HT-29 colorectal adenocarcinoma cancer cells under normoxic and hypoxic conditions compared to its ACNP precursors, but not at higher concentrations. Dose-response analyses suggested that dibromogenkwanin had a distinct mode-of-action compared to apigenin. Thus, this proof-of-concept paper demonstrates combinatorial (bio)synthesis as an approach that can be used to produce novel chemistries for anticancer research.

Arabidopsis MATE45 antagonizes local abscisic acid signaling to mediate development and abiotic stress responses.

Anthocyanins provide ideal visual markers for the identification of mutations that disrupt molecular responses to abiotic stress. We screened Arabidopsis mutants of ABC (ATP-Binding Cassette) and MATE (Multidrug And Toxic compound Extrusion) transporter genes under nutritional stress and identified four genes (ABCG25,ABCG9,ABCG5, and MATE45) required for normal anthocyanin pigmentation. ABCG25 was previously demonstrated to encode a vascular-localized cellular exporter of abscisic acid (ABA). Our results show that MATE45 encodes an aerial meristem- and a vascular-localized transporter associated with the trans-Golgi, and that it plays an important role in controlling the levels and distribution of ABA in growing aerial meristems and non-meristematic tissues. MATE45 promoter-GUS reporter fusions revealed the activity localized to the leaf and influorescence meristems and the vasculature. Loss-of-function mate45 mutants exhibited accelerated rates of aerial organ initiation suggesting at least partial functional conservation with the maize ortholog bige1. The aba2-1 mutant, which is deficient in ABA biosynthesis, exhibited a number of phenotypes that were rescued in the mate45-1 aba2-1 double mutant. mate45 exhibited enhanced the seed dormancy, and germination was hypersensitive to ABA. Enhanced frequency of leaf primordia growth in mate45 seedlings grown in nutrient imbalance stress was ABA-dependent. The ABA signaling reporter construct pRD29B::GUS revealed elevated levels of ABA signaling in the true leaf primordia of mate45 seedlings grown under nutritional stress, and gradually reduced signaling in surrounding cotyledon and hypocotyl tissues concomitant with reduced expressions of ABCG25. Our results suggest a role of MATE45 in reducing meristematic ABA and in maintaining ABA distribution in adjacent non-meristematic tissues.

Distinct Mechanisms of Biotic and Chemical Elicitors Enable Additive Elicitation of the Anticancer Phytoalexin Glyceollin I.

Phytoalexins are metabolites biosynthesized in plants in response to pathogen, environmental, and chemical stresses that often have potent bioactivities, rendering them promising for use as therapeutics or scaffolds for pharmaceutical development. Glyceollin I is an isoflavonoid phytoalexin from soybean that exhibits potent anticancer activities and is not economical to synthesize. Here, we tested a range of source tissues from soybean, in addition to chemical and biotic elicitors, to understand how to enhance the bioproduction of glyceollin I. Combining the inorganic chemical silver nitrate (AgNO3) with the wall glucan elicitor (WGE) from the soybean pathogen Phytophthora sojae had an additive effect on the elicitation of soybean seeds, resulting in a yield of up to 745.1 µg gt-1 glyceollin I. The additive elicitation suggested that the biotic and chemical elicitors acted largely by separate mechanisms. WGE caused a major accumulation of phytoalexin gene transcripts, whereas AgNO3 inhibited and enhanced the degradation of glyceollin I and 6″-O-malonyldaidzin, respectively.

Using fluorescence lifetime microscopy to study the subcellular localization of anthocyanins.

Anthocyanins are flavonoid pigments that accumulate in most seed plants. They are synthesized in the cytoplasm but accumulate inside the vacuoles. Anthocyanins are pigmented at the lower vacuolar pH, but in the cytoplasm they can be visualized based on their fluorescence properties. Thus, anthocyanins provide an ideal system for the development of new methods to investigate cytoplasmic pools and association with other molecular components. We have analyzed the fluorescence decay of anthocyanins by fluorescence lifetime imaging microscopy (FLIM), in both in vitro and in vivo conditions, using wild-type and mutant Arabidopsis thaliana seedlings. Within plant cells, the amplitude-weighted mean fluorescence lifetime (τm ) correlated with distinct subcellular localizations of anthocyanins. The vacuolar pool of anthocyanins exhibited shorter τm than the cytoplasmic pool. Consistently, lowering the pH of anthocyanins in solution shortened their fluorescence decay. We propose that FLIM is a useful tool for understanding the trafficking of anthocyanins and, potentially, for estimating vacuolar pH inside intact plant cells.

Anthocyanin Vacuolar Inclusions Form by a Microautophagy Mechanism.

Anthocyanins are flavonoid pigments synthesized in the cytoplasm and stored inside vacuoles. Many plant species accumulate densely packed, 3- to 10-µm diameter anthocyanin deposits called anthocyanin vacuolar inclusions (AVIs). Despite their conspicuousness and importance in organ coloration, the origin and nature of AVIs have remained controversial for decades. We analyzed AVI formation in cotyledons of different Arabidopsis thaliana genotypes grown under anthocyanin inductive conditions and in purple petals of lisianthus (Eustoma grandiorum). We found that cytoplasmic anthocyanin aggregates in close contact with the vacuolar surface are directly engulfed by the vacuolar membrane in a process reminiscent of microautophagy. The engulfed anthocyanin aggregates are surrounded by a single membrane derived from the tonoplast and eventually become free in the vacuolar lumen like an autophagic body. Neither endosomal/prevacuolar trafficking nor the autophagy ATG5 protein is involved in the formation of AVIs. In Arabidopsis, formation of AVIs is promoted by both an increase in cyanidin 3-O-glucoside derivatives and by depletion of the glutathione S-transferase TT19. We hypothesize that this novel microautophagy mechanism also mediates the transport of other flavonoid aggregates into the vacuole.

Abiotic stresses induce different localizations of anthocyanins in Arabidopsis.

Anthocyanins are induced in plants in response to abiotic stresses such as drought, high salinity, excess light, and cold, where they often correlate with enhanced stress tolerance. Numerous roles have been proposed for anthocyanins induced during abiotic stresses including functioning as ROS scavengers, photoprotectants, and stress signals. We have recently found different profiles of anthocyanins in Arabidopsis (Arabidopsis thaliana) plants exposed to different abiotic stresses, suggesting that not all anthocyanins have the same function. Here, we discuss these findings in the context of other studies and show that anthocyanins induced in Arabidopsis in response to various abiotic stresses have different localizations at the organ and tissue levels. These studies provide a basis to clarify the role of particular anthocyanin species during abiotic stress.

Not all anthocyanins are born equal: distinct patterns induced by stress in Arabidopsis.

MAIN CONCLUSION: Different abiotic stress conditions induce distinct sets of anthocyanins, indicating that anthocyanins have different biological functions, or that decoration patterns of each anthocyanin are used for unique purposes during stress. The induction of anthocyanin accumulation in vegetative tissues is often considered to be a response of plants to biotic or abiotic stress conditions. Arabidopsis thaliana (Arabidopsis) accumulates over 20 anthocyanins derived from the anthocyanidin cyanidin in an organ-specific manner during development, but the anthocyanin chemical diversity for their alleged stress protective functions remains unclear. We show here that, when grown in various abiotic stress conditions, Arabidopsis not only often accumulates significantly higher levels of total anthocyanins, but different stress conditions also favor the accumulation of different sets of anthocyanins. For example, the anthocyanin patterns of seedlings grown at pH 3.3 or in media lacking phosphate are very similar and characterized by relatively high levels of the anthocyanins A8 and A11. In contrast, anthocyanin inductive conditions (AIC) provided by high sucrose media are characterized by high accumulation of A9* and A5 relative to other stress conditions. The modifications present in each condition correlate reasonably well with the induction of the respective anthocyanin modification enzymes. Taken together, our results suggest that Arabidopsis anthocyanin profiles provide 'fingerprints' that reflect the stress status of the plants.

Comparability of imazapyr-resistant Arabidopsis created by transgenesis and mutagenesis.

The Arabidopsis CSR1 gene codes for the enzyme acetohydroxyacid synthase (AHAS, EC 2.2.1.6), also known as acetolactate synthase, which catalyzes the first step in branched-chain amino acid biosynthesis. It is inhibited by several classes of herbicides, including the imidazolinone herbicides, such as imazapyr; however, a substitution mutation in csr1-2 (Ser-653-Asn) confers selective resistance to the imidazolinones. The transcriptome of csr1-2 seedlings grown in the presence of imazapyr has been shown in a previous study (Manabe in Plant Cell Physiol 48:1340-1358, 2007) to be identical to that of wild-type seedlings indicating that AHAS is the sole target of imazapyr and that the mutation is not associated with pleiotropic effects detectable by transcriptome analysis. In this study, a lethal null mutant, csr1-7, created by a T-DNA insertion into the CSR1 gene was complemented with a randomly-inserted 35S/CSR1-2/NOS transgene in a subsequent genetic transformation event. A comparison of the csr1-2 substitution mutant with the transgenic lines revealed that all were resistant to imazapyr; however, the transgenic lines yielded significantly higher levels of resistance and greater biomass accumulation in the presence of imazapyr. Microarray analysis revealed few differences in their transcriptomes. The most notable was a sevenfold to tenfold elevation in the CSR1-2 transcript level. The data indicate that transgenesis did not create significant unintended pleiotropic effects on gene expression and that the mutant and transgenic lines were highly similar, except for the level of herbicide resistance.

Identification of two anthocyanidin reductase genes and three red-brown soybean accessions with reduced anthocyanidin reductase 1 mRNA, activity, and seed coat proanthocyanidin amounts.

Anthocyanidin reductase (ANR; EC 1.3.1.77) catalyzes a key step in the biosynthesis of proanthocyanidins (PAs; also known as condensed tannins), flavonoid metabolites responsible for the brown pigmentation of seeds. Here, two ANR genes (ANR1 and ANR2) from the seed coat of brown soybean (Glycine max (L.) Merr.) have been isolated and their enzymatic function confirmed for the reduction of cyanidin to (-)-epicatechin in vitro. Biochemical and genetic comparisons of soybean lines differing in seed coat color revealed three red-brown lines to exhibit major reductions in the amounts of soluble PAs in the seed coat compared to brown soybean lines. Two spontaneous mutants with red-brown grain color had reduced ANR1 gene expression in the seed coat, and an EMS-mutagenized red-brown mutant had nonsynonymous substitutions that resulted in slightly reduced ANR1 activity in vitro. These results suggest that defects in the ANR1 gene can be associated with red-brown soybean grain color. These results suggest that suppressing ANR1 gene expression or activity may be a rational approach toward engineering seed coat color to enable the visual identification of genetically modified soybean grains.