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Spectral analysis combined with advanced linear unmixing allows for histolocalization of phenolics in leaves of coffee trees.

Conéjéro G, Noirot M, Talamond P, Verdeil JL - Front Plant Sci (2014)

Bottom Line: Lastly, young leaves of Coffea pseudozanguebariae (PSE), C. eugenioides (EUG), C. arabica (ARA) and C. canephora (CAN) were compared.This confirmed the presence of xanthone in PSE and EUG, but especially its precise tissue localization.This non-invasive optical technique does not require pretreatment and is an effective experimental tool to differentiate multiple naturally-occuring fluorochores in living tissues.

View Article: PubMed Central - PubMed

Affiliation: Plant Cell Imaging platform PHIV UMR AGAP (Cirad, SupAgro, INRA), UMR B&PMP (INRA, CNRS, UM2, SupAgro) Montpellier, France.

ABSTRACT
An imaging method using spectral analysis combined with advanced linear unmixing was used to allow histolocalization of natural autofluorescent compounds such as hydroxycinnamic acid (chlorogenic acid) and xanthone (mangiferin) in living cells and tissues (mature coffee leaves). The tested method included three complementary steps: 1/ visualization of natural autofluorescence and spectrum acquisition with a multiphoton microscope; 2/ identification of some compounds using previous information on the chemical composition of the tissue, obtained from litterature; and 3/ localization of candidate compounds by spectral imaging. The second part of the study consisted of describing the histochemical structure of leaves during their development. This revealed very fast histochemical differentiation of leaves during the first week after their emergence. Lastly, young leaves of Coffea pseudozanguebariae (PSE), C. eugenioides (EUG), C. arabica (ARA) and C. canephora (CAN) were compared. This confirmed the presence of xanthone in PSE and EUG, but especially its precise tissue localization. This also highlighted the paternal CAN origin of the leaf structure in the allotetraploid species ARA. The limits and advantages of the method without staining are discussed relative to classical epifluorescence microscopy under UV light. This non-invasive optical technique does not require pretreatment and is an effective experimental tool to differentiate multiple naturally-occuring fluorochores in living tissues.

No MeSH data available.


Cross-sections of young leaves of PSE (C. pseudozanguebariae) (I), EUG (C. eugenioides) (II), ARA (C. Arabica “Bourbon”) (III) and CAN (C. canephora) (IV). The advanced linear unmixing process was carried out using chlorophyll, 5-CQA and mangiferin reference spectra. AdE, adaxial epidermis; AbE, abaxial epidermis. Scale bar = 50μm.
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Figure 6: Cross-sections of young leaves of PSE (C. pseudozanguebariae) (I), EUG (C. eugenioides) (II), ARA (C. Arabica “Bourbon”) (III) and CAN (C. canephora) (IV). The advanced linear unmixing process was carried out using chlorophyll, 5-CQA and mangiferin reference spectra. AdE, adaxial epidermis; AbE, abaxial epidermis. Scale bar = 50μm.

Mentions: Histochemical comparison using the advanced linear unmixing process concerned leaves of PSE, EUG, ARA “Bourbon” (ARA) and CAN. In mature leaves, the histochemical structure was similar in the four species (data not shown). This was not the case in young leaves (phase φ3). In this case, two groups of species could be defined, i.e., PSE and EUG vs ARA and CAN. In PSE (Figure 6I) and EUG (Figure 6II), there was strong histochemical differentiation between adaxial and abaxial epidermal tissues at the vacuole level, based on 5-CQA and mangiferin, respectively (Figure 6I). Nevertheless, mangiferin was also present in the spongy parenchyma and, at a lower level, in palisade parenchyma. See also splitted image of PSE leaf in Figure BII (supplementary material). In contrast, ARA (Figure 6III) and CAN (Figure 6IV) did not show mangiferin in adaxial and abaxial epidermal tissues and 5-CQA was strongly present in vacuoles of both of these tissues. In fact, mangiferin was only observable in parenchyma, but as small vesicles.


Spectral analysis combined with advanced linear unmixing allows for histolocalization of phenolics in leaves of coffee trees.

Conéjéro G, Noirot M, Talamond P, Verdeil JL - Front Plant Sci (2014)

Cross-sections of young leaves of PSE (C. pseudozanguebariae) (I), EUG (C. eugenioides) (II), ARA (C. Arabica “Bourbon”) (III) and CAN (C. canephora) (IV). The advanced linear unmixing process was carried out using chlorophyll, 5-CQA and mangiferin reference spectra. AdE, adaxial epidermis; AbE, abaxial epidermis. Scale bar = 50μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3927124&req=5

Figure 6: Cross-sections of young leaves of PSE (C. pseudozanguebariae) (I), EUG (C. eugenioides) (II), ARA (C. Arabica “Bourbon”) (III) and CAN (C. canephora) (IV). The advanced linear unmixing process was carried out using chlorophyll, 5-CQA and mangiferin reference spectra. AdE, adaxial epidermis; AbE, abaxial epidermis. Scale bar = 50μm.
Mentions: Histochemical comparison using the advanced linear unmixing process concerned leaves of PSE, EUG, ARA “Bourbon” (ARA) and CAN. In mature leaves, the histochemical structure was similar in the four species (data not shown). This was not the case in young leaves (phase φ3). In this case, two groups of species could be defined, i.e., PSE and EUG vs ARA and CAN. In PSE (Figure 6I) and EUG (Figure 6II), there was strong histochemical differentiation between adaxial and abaxial epidermal tissues at the vacuole level, based on 5-CQA and mangiferin, respectively (Figure 6I). Nevertheless, mangiferin was also present in the spongy parenchyma and, at a lower level, in palisade parenchyma. See also splitted image of PSE leaf in Figure BII (supplementary material). In contrast, ARA (Figure 6III) and CAN (Figure 6IV) did not show mangiferin in adaxial and abaxial epidermal tissues and 5-CQA was strongly present in vacuoles of both of these tissues. In fact, mangiferin was only observable in parenchyma, but as small vesicles.

Bottom Line: Lastly, young leaves of Coffea pseudozanguebariae (PSE), C. eugenioides (EUG), C. arabica (ARA) and C. canephora (CAN) were compared.This confirmed the presence of xanthone in PSE and EUG, but especially its precise tissue localization.This non-invasive optical technique does not require pretreatment and is an effective experimental tool to differentiate multiple naturally-occuring fluorochores in living tissues.

View Article: PubMed Central - PubMed

Affiliation: Plant Cell Imaging platform PHIV UMR AGAP (Cirad, SupAgro, INRA), UMR B&PMP (INRA, CNRS, UM2, SupAgro) Montpellier, France.

ABSTRACT
An imaging method using spectral analysis combined with advanced linear unmixing was used to allow histolocalization of natural autofluorescent compounds such as hydroxycinnamic acid (chlorogenic acid) and xanthone (mangiferin) in living cells and tissues (mature coffee leaves). The tested method included three complementary steps: 1/ visualization of natural autofluorescence and spectrum acquisition with a multiphoton microscope; 2/ identification of some compounds using previous information on the chemical composition of the tissue, obtained from litterature; and 3/ localization of candidate compounds by spectral imaging. The second part of the study consisted of describing the histochemical structure of leaves during their development. This revealed very fast histochemical differentiation of leaves during the first week after their emergence. Lastly, young leaves of Coffea pseudozanguebariae (PSE), C. eugenioides (EUG), C. arabica (ARA) and C. canephora (CAN) were compared. This confirmed the presence of xanthone in PSE and EUG, but especially its precise tissue localization. This also highlighted the paternal CAN origin of the leaf structure in the allotetraploid species ARA. The limits and advantages of the method without staining are discussed relative to classical epifluorescence microscopy under UV light. This non-invasive optical technique does not require pretreatment and is an effective experimental tool to differentiate multiple naturally-occuring fluorochores in living tissues.

No MeSH data available.