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A differential distribution of auxin and flavonols in radiata pine stem seedlings exposed to inclination

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Abstract

Stem reorientation in response to inclination in trees has commanded the attention of the research community widely, but the molecular mechanisms of the response are still unknown. The most accepted theory is auxin redistribution in affected tissues. Flavonols have been reported as potent inhibitors of polar auxin transport; however, no reports have shown differential modulation by flavonols within the trunk. Here, transcriptional, microscopic, and chemical approaches were used to analyze the contents of flavonoids in 45° inclined radiata pine seedlings. A time course was established and samples obtained by cutting stems into apical, medial, and basal segments or by longitudinal dissection into upper and lower halves to determine polar distribution of flavonoids. The expression analysis of PrCHS, PrF3H, and PrFLS indicated induction in response to inclination; higher transcript levels were recorded at the basal zone and in the upper half of the stem. A reduction in abundance of PrARP transcripts in addition to immunospecific auxin detection at the lower half of inclined stem was found. Concomitantly, quercetin and kaempferol accumulated in the upper stem half. Our results suggest that stem reorientation is a response to a concerted and differential mechanism that modulates imbalance of auxin distribution in one side of the stem.

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Acknowledgments

This work was supported by FONDECYT [11121170, 1120635, 1150964] and post-doctoral CONICYT [PBCT-PSD61] projects and “Núcleo Científico Multidisciplinario” from Universidad de Talca, Chile. Authors would like to thank the reviewers for their highly valuable comments.

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Authors and Affiliations

  1. Laboratorio de Fisiología Vegetal y Genética Molecular, Instituto de Ciencias Biológicas, Universidad de Talca, 2 Norte 685, Talca, Chile

    Patricio Ramos, Joselin Guajardo, María Alejandra Moya-León & Raúl Herrera

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  1. Patricio Ramos
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  2. Joselin Guajardo
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  3. María Alejandra Moya-León
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  4. Raúl Herrera
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Correspondence to Patricio Ramos.

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The authors declare that they have no conflict of interest.

Additional information

Communicated by G. G. Vendramin

Data archiving statement

The sequences obtained in this study have been deposited at GenBank. The sequences of CHS, F3H, FLS, and ARP from Pinus radiata D. Don have GenBank accession numbers KF704817, KF704818, KF704819, and KF704820, respectively.

Electronic supplementary material

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Supplementary Figure 1

Multiple alignment of the amino acid sequence of PrCHS with several plant CHS proteins. Gaps are indicated by dashes, letters with black background are identical amino acids, and letters with grey background are similar amino acids. The protein sequence shows four CHS-specific conserved motifs labeled by lines. The active site (♦), catalytic residues (*) and CoA-binding sites are indicated. Arabidopsis thaliana (AED91961), Vitis vinifera (NP_001267879), Populus trichocarpa (EEE99396), Ginkgo biloba (AAT68477), Petunia x hybrida (AAB36038), Solanum tuberosum (AGU70032), Zea mays (NP_001149022), Brassica oleracea (ADD91690), Solanum lycopersicum (AEK99072), Citrus maxima (ACX37403), Trifolium subterraneum (AAA67701), Abies alba (ABD38616), Nicotiana tabacum (AAK49457), Fragaria x ananassa (AAX99413), Hordeum vulgare (CAA41250), Oryza sativa (BAB39764), Pinus pinaster (CAP09644). (GIF 2890 kb)

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(TIF 2681 kb)

Supplementary Figure 2

Multiple alignment of the amino acid sequence of PrF3H with several plant F3H proteins. Gaps are indicated by dashes, letters with black background are identical amino acids, and letters with grey background are similar amino acids. Five conserved motifs are labeled (bold lines) and the residues for ferrous iron ligation and 2-oxoglutarate binding are indicated (*). A. thaliana (Q9S818), Litchi chinensis (ADO95201), Narcissus tazetta (AFI08246), Gossypium barbadense (ABL86673), Z. mays (NP_001105695), Scutellaria viscidula (ACR56689), S. lycopersicum (AEK99074), Glycine max (ACA81459), Antirrhinum andraeanum (ABI50233), Prunus avium (AEO79981), Pyrus communis (AGL50918), Malus hybrid cultivar (ACP30361), G. biloba (AAU93347), Daucus carota (AAD56577). (GIF 1335 kb)

High resolution image

(TIF 1517 kb)

Supplementary Figure 3

Multiple alignment of the amino acid sequence of PrFLS with several plant FLS proteins. Gaps are indicated by dashes, letters with black background are identical amino acids, and letters with grey background are similar amino acids. Two highly conserved regions in flavonoid-specific dioxygenase enzymes are labeled with lines. Residues coordinating iron atoms (*) or interacting with 2-oxoglutarate (♦) are indicated. G. max (NP_001237419), Allium cepa (AAT68476), V. vinifera (BAE75810), G. biloba (ACY00393), A. thaliana (AAB41504), Malus domestica (AAD26261), F. x ananassa (AAZ78661), N. tabacum (ABE28017), Antirrhinum majus (ABB53382), P. x hybrida (CAA80264), S. tuberosum (CAA63092), P. avium (AFO67943), M. truncatula (AES71332), Scutellaria baicalensis (AHA14501) (GIF 1265 kb)

High resolution image

(TIF 1471 kb)

Supplementary Figure 4

Multiple alignment of the amino acid sequence of partial PrARP with several plant ARP proteins. Gaps are indicated by dashes, letters with black background are identical amino acids, and letters with grey background are similar amino acids. Pyrus x bretschneideri (AGI02595), N. tabacum (AAS76635), F. ananassa (AAA73872), Phaseolus vulgaris (AGV54274), M. truncatula (AES63872), Ricinus communis (EEF50833), A. thaliana (AAC26202), Pisum sativum (AAB84193). (GIF 662 kb)

High resolution image

(TIF 614 kb)

Supplementary Figure 5

Transcript abundance of flavonol genes by qPCR at different heights of inclined stems. Transcripts were quantified in stem samples of inclined seedlings at different times post inclination. Non-inclined seedlings correspond to control (C). Stem samples were obtained at three different heights: AZ, apical zone; MZ, medial zone; BZ, basal zone. CHS, Chalcone synthase; F3H, Flavanone 3-hydroxylase; FLS, Flavonol synthase; ARP, Auxin-repressed protein. For relative transcript levels, normalization was performed against housekeeping genes (Ge066D02, UBC2 and UBC7) in all samples. Values represent the mean ± S.E. of three biological replicates of each tissue. For a particular gene, different letters indicate statistical significance between tissues (P < 0.05; two-way ANOVA). (GIF 165 kb)

High resolution image

(TIF 242 kb)

Supplementary Figure 6

Effect of auxin treatment on the transcript level of ARP. Total RNA was extracted from non-inclined stem seedlings previously treated with increasing NAA or IAA concentrations for 24 h. qPCR analyses for the ARP gene were performed on untreated and auxin treated stem samples. Values represent the mean ± S.E. of three biological replicates of each tissue. Different letters indicate statistical significance between tissues (P < 0.05; one-way ANOVA). (GIF 56 kb)

High resolution image

(TIF 109 kb)

Supplementary Figure 7

Transcript analysis of monolignol biosynthetic genes in longitudinal-dissected stems by qPCR. Transcripts were quantified in stem samples of inclined seedlings at different times post inclination. Non-inclined seedlings correspond to controls (C). Stems from inclined seedlings were divided into upper and lower halves. CCR, Cinnamoyl-CoA reductase; COMT, Caffeic acid O-methyl transferase. For relative transcript level, normalization was performed against housekeeping genes (Ge066D02, UBC2 and UBC7) in all samples. Values represent the mean ± S.E. of three biological replicates of each tissue. For a particular gene, different letters indicate statistical significance between tissues (P < 0.05; two-way ANOVA). (GIF 62 kb)

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(TIF 110 kb)

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Ramos, P., Guajardo, J., Moya-León, M.A. et al. A differential distribution of auxin and flavonols in radiata pine stem seedlings exposed to inclination. Tree Genetics & Genomes 12, 42 (2016). https://doi.org/10.1007/s11295-016-1003-1

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