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Alteration of flavonoid accumulation patterns in transparent testa mutants disturbs auxin transport, gravity responses, and imparts long-term effects on root and shoot architecture

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Abstract

Flavonoids have broad cross-kingdom biological activity. In Arabidopsis, flavonoid accumulation in specific tissues, notably the root elongation zone and root/shoot junction modulate auxin transport, affect root gravitropism, and influence overall plant architecture. The relative contribution made by aglycones and their glycosides remains undetermined, and the longer-term phenotypic effects of altered flavonoid accumulation are not fully assessed. We tested Arabidopsis thaliana mutants that accumulate different flavonoids to determine which flavonoids were causing these affects. Tandem mass spectrometry and in situ fluorescence localisation were used to determine the in vivo levels of aglycones in specific tissues of 11 transparent testa mutants. We measured rootward and shootward auxin transport, gravitropic responses, and identified the long-term changes to root and shoot architecture. Unexpected aglycone species accumulated in vivo in several flavonoid-pathway mutants, and lower aglycone levels occurred in transcription factor mutants. Mutants accumulating more quercetin and quercetin-glycosides changed the greatest in auxin transport, gravitropism, and aerial tissue growth. Early flavonoid-pathway mutants showed aberrant lateral root initiation patterns including clustered lateral root initiations at a single site. Transcription factor mutants had multiple phenotypes including shallow root systems. These results confirm that aglycones are present at very low levels, show that lateral root initiation is perturbed in early flavonoid-pathway mutants, and indicate that altered flavonoid accumulation affects multiple aspects of plant architecture.

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Abbreviations

DPBA:

Diphenylboric acid 2-aminoethyl ester

IAA:

Indole-3-acetic acid

RDEZ:

Root distal elongation zone

RSJ:

Root/shoot junction

N:

Naringenin

DHK:

Dihydrokaempferol

DHQ:

Dihydroquercetin

K:

Kaempferol

Q:

Quercetin

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Acknowledgments

An Australian Research Council Discovery Project DP1096299 supported this work. The Australian Research Council Centre of Excellence for Integrated Legume Research (project no. CEO348212) is acknowledged for supporting the summer scholar Natalia Sampio. The authors declare that they have no conflict of interest. Discussions with colleagues in the Centre of Excellence are appreciated. Teresa Neeman of ANU Math Dept. is thanked for statistical analyses of aerial phenotyping data from the CSIRO plant phenomics facility. Gabriel James is thanked for assistance regarding mass spectrometry data analysis.

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  1. Plant Sciences Division, Research School of Biology, College of Medicine, Biology, and Environment, The Australian National University, Linneaus Bldg #134, Linneaus Way, Canberra, ACT, 0200, Australia

    Charles S. Buer, Farzanah Kordbacheh & Michael A. Djordjevic

  2. RSB Mass Spectrometry Facility, Research School of Biology, College of Medicine, Biology, and Environment, The Australian National University, Canberra, ACT, 0200, Australia

    Thy T. Truong & Charles H. Hocart

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  1. Charles S. Buer
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  2. Farzanah Kordbacheh
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Correspondence to Michael A. Djordjevic.

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425_2013_1883_MOESM1_ESM.jpg

Suppl Fig. S1 a Aglycones in crude extracts are not reliably detected or quantified by HPLC. HPLC traces of crude extracts from roots for selected tt mutants. The wild type trace overlays the mutant trace. Elution points for the aglycone standards are indicated. The retention times were: N, 38.7 min.; DHK, 29.2 min.; DHQ, 24.1 min.; K, 40.8 min.; and Q, 36.3 min. Identification was verified by the UV spectra associated with the aglycone. b Flavonoid aglycone levels determined by HPLC following hydrolysis. The question mark indicates a co-eluting compound making peak area analysis impossible (JPEG 958 kb)

425_2013_1883_MOESM2_ESM.jpg

Suppl Fig. S2 MS/MS product ion spectra of selected flavonoid standards. The insets show possible fragmentation patterns of the compound. The yellow highlighted masses are those used to differentiate the compounds during targeted MS/MS analyses (JPEG 1144 kb)

425_2013_1883_MOESM3_ESM.jpg

Suppl Fig. S3 The DPBA-flavonoid fluorescence in RSJs and the RDEZ of the tt mutants. DPBA-flavonoid fluorescence varies according to the flavonoid compound that accumulates. The RDEZ is where gravitropic bending occurs and the RSJ provides the transition from shoot to root. The mutants in row (a) are the flavonoid-pathway mutants, and those in (b) are in the transcription factor mutants, except Ler, which is the wild type. The mutants tt4 and tt6 have very dim fluorescence from background sinapate esters and N-glycosides, respectively. The tt5 mutant has fluorescence from the spontaneous reaction forming N, from N-chalcone, and subsequent downstream products from N (Cisak and Mielczarek 1992; Mol et al. 1985). Seedlings were analysed for fluorescence 5-day following germination. The scale bar = 100 μm (JPEG 1268 kb)

425_2013_1883_MOESM4_ESM.jpg

Suppl Fig. S4 Representative root phenotype montages of tt4 and Ler grown in jars containing medium solidified with Phytagel. Seedlings were grown for 4 weeks post germination. Scale bar = 100 μm. Seedlings were grown for 3 weeks. Scale bar = 5 mm (JPEG 963 kb)

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Buer, C.S., Kordbacheh, F., Truong, T.T. et al. Alteration of flavonoid accumulation patterns in transparent testa mutants disturbs auxin transport, gravity responses, and imparts long-term effects on root and shoot architecture. Planta 238, 171–189 (2013). https://doi.org/10.1007/s00425-013-1883-3

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