Determination of quality constituents in the young leaves of albino tea cultivars

Determination of quality constituents in the young leaves of albino tea
cultivars
Lin Feng a ,Ming-Jun Gao b ,⇑,Ru-Yan Hou a ,Xiao-Yi Hu c ,Liang Zhang a ,Xiao-Chun Wan a ,Shu Wei a ,⇑
a
Key Laboratory of Tea Biochemistry and Biotechnology,Anhui Agricultural University,130Changjiang Blvd West,Hefei,Anhui 230036,China b
Agriculture and Agri-Food Canada,Saskatoon Research Centre,107Science Place,Saskatoon,SK S7N 0X2,Canada c
College of Forestry and Landscape Architecture,Anhui Agricultural University,130Changjiang Blvd West,Hefei,Anhui 230036,China
a r t i c l e i n f o Article history:
Received 23August 2013
Received in revised form 15December 2013Accepted 15January 2014
Available online 23January 2014Keywords:
Camellia sinensis Albino tea
Metabolic profiling Multivariate analysis Tea quality
a b s t r a c t
Albino tea has received increased attention due to its brisk flavour.To identify changes in the key chem-ical constituents conveying important qualities to albino tea,the metabolite profiles of four albino cultivars and one green tea cultivar were pared to the green tea control,significantly decreased contents of chlorophyll (Chl)(p <0.01),total carotenoids (p <0.05),caffeine (p <0.01),and total catechins (p <0.05)were found in albino tea leaves with a few exceptions,whereas increases were noted in the Chl a/b ratio and the contents of both zeaxanthin and free amino acids,including theanine.Multivariate analysis identified catechins and carotenoids as the most important contributors to the met-abolic profile variance between the albino and green tea cultivars.High levels of amino acids,along with low levels of chlorophylls,catechins and caffeine,contribute to the qualities of albino tea,which include reduced astringency and bitterness,along with a strong umami taste.
Ó2014Elsevier Ltd.All rights reserved.
1.Introduction
Tea is one of the most widely consumed non-alcoholic bever-ages in the world and imparts marked benefits to human health,such as providing antioxidant activity and reducing the risk of car-diovascular disease and some forms of cancer (Cabrera,Artacho,&Gimenez,2006).Under certain environmental conditions,such as low temperature or abnormal light intensity,the albino tea culti-vars generate yellowish to off-white leaves which are deficient in chlorophyll (Du et al.,2006).Compared to normal green cultivars,albino tea germplasm is precious,due to the special flavour,dis-tinct leaf colour and scarcity.Studies have been published for only a select few of the albino tea cultivars which are commercially grown.Chemical analyses and expression profiling of the genes related to chlorophyll biosynthesis in these albino cultivars have begun to reveal the basis of tea leaf albinism at the levels of bio-chemistry and molecular biology (Du et al.,2006,2008;Wei,Wang,&Zhou,2012).However,changes in comprehensive meta-bolic profiles of albino tea cultivars and their impact on tea quality need to be determined to further improve tea quality.
The quality of processed tea is basically determined by the chemical constituents in tea leaves (Cabrera et al.,2006;Chaturve-dula &Prakash,2011).The compounds determining tea flavour and astringency include catechins (flavan-3-ols),including (À)-epigal-locatechin gallate (EGCG),(+)-catechin (C),(+)-gallocatechin (GC)and their oxidation products (Chaturvedula &Prakash,2011;Liang,Ma,Lu,&Wu,2006;Narukawa,Kimata,Noga,&Watanabe,2010).In addition,amino acids are the main contributors towards the umami taste of tea infusions (Alcázar et al.,2007).Theanine (c -glut-amyl-L -ethylamide)is the most abundant free amino acid in tea and can dramatically increase the brothy taste of tea infusions by effec-tively counteracting the astringency and bitterness (Chen,Duan,&Jiang,2011;Chu,Kobayashi,Juneja,&Yamamoto,1997).The chlo-rophylls (Chl)and carotenoids,which function as light-harvesting pigments in plants,are the determinants of tea leaf colour.
In order to determine the changes in the key chemical constit-uents affecting albino tea quality,we generated metabolic profiles of four albino cultivars and one green tea cultivar,using high performance liquid chromatography (HPLC).The data were further analysed by using multivariate analysis approaches to reveal the key determinants leading to albino tea qualities and metabolic var-iance.Our results suggest that decreased abundances of carote-noids and chlorophylls are accompanied with a corresponding increase in the abundances of free amino acids (including thea-nine)in albino leaves.These results provide insight into the forma-tion of tea quality-influencing metabolites in albino tea leaves.
/10.1016/j.foodchem.2014.01.0440308-8146/Ó2014Elsevier Ltd.All rights reserved.
⇑Corresponding authors.Tel.:+13063859369;fax:+13063859482(M.-J.Gao).Tel./fax:+8655165783941(S.Wei).
E-mail addresses:ming-jun.gao@agr.gc.ca (M.-J.Gao),weishu@ (S.Wei).
2.Materials and
2.1.Plant materials
Three-year old
(Ab)(as called White
‘Tiantai huangcha’(Th),
of‘Fuding dabaicha’
management at the
University,Hefei,China
The fully expanded
in the spring when
‘Yu’were yellowish,
green(Fig.1).A leaf
plant)collected from
three biological
ples were immediately
the lab and stored atÀ
2.2.Chemicals and
The following
(St.Louis,MO,USA):
chin(C),(À
epigallocatechin(EGC),
gallocatechin(GC),
(Arg),asparagine(Asn),
acid(Glu),glycine
(Leu),lysine(Lys),
(Pro),serine(Ser),
tyrosine(Tyr)and
CaroteNature
thin(b-Cry),lutein,
Leaf carotenoids
previously published
Hannoufa,2007)with
extracts were analysed
reverse-phase C30(4.6
ada)with a column
by comparing their
of known standards.
constructed from
tracted and analysed
with three independent
Chemical assays of
were conducted as
&Hu,2012).Leaf
measured by a
Crozier,2010),using
Phenomenex,China).
paring their retention
authentic standards.
constructed from the
2.3.Statistical and
Data were
biological replicates
was performed by
the software SPSS17.0
matrix was subjected
software(Umetrics AB,
components analysis(PCA)was run to obtain a general overview
of the variance of metabolites in a data table by forming a low dimensional model plane;a score plot was then generated to visualise the relationships among samples in the model plane. Supervised orthogonal partial least-squares discriminant analysis
1.Leaf phenotypes of the3-year-old albino and6-year-old normal cultivars.(A and B)Tiantai huangcha’(Th);(C and D),‘Huangjinya’(Hu)(E and
‘Yujinxiang’(Yu);(G and H),‘Anji baicha’(Ab);(I and J),Fuding dabaicha’(Fd).
100L.Feng et al./Food Chemistry155(2014)98–104
(OPLS-DA),a more advanced multivariate method for understand-opened leaves in late spring,the leaf phenotypes of the four albino
of chlorophylls and predominant carotenoids in albino tea leaves.(A)Chlorophyll contents;(B)Chl a/b
0.01.
3.2.Changes in the abundances of chlorophylls and carotenoids in albino cultivars
The levels of Chl a,Chl b and Chl a+b in the leaves of all the four albino cultivars were significantly lower than those in the leaves of the normal green cultivar‘Fd’(p<0.01)(Fig.2A).Obviously,such reductions in the abundance of chlorophylls led to the change in leaf colour of the albino cultivars.However,the Chl a/b ratios in the albino cultivars were significantly higher than that in‘Fd’(p<0.05);‘Ab’was the exception,whose ratio remained un-changed(Fig.2B).Reduced abundance of Chl leads to lower levels of LHC proteins and poor photosynthetic efficiency and capacity (Kim et al.,2009)because both Chl a and Chl b function as light-harvesting pigments in the photosynthetic apparatus(Tanaka& Tanaka,2007).Moreover,plants grown in high light have increased Chl a/b ratios,PSII/PSI ratios,as well as increased ATP synthase and Rubisco activities;however,they have reduced chlorophyll and carotenoids,violaxanthin and b-cryptoxanthin,were also found, though the latter was nearly pared to‘Fd’, reduction in the total carotenoid content in albino cultivars‘Ab’,‘Hu’,‘Th’and‘Yu’was28.8%,38.2%,41.7%,and54.8%,respectively (Fig.2C).The contents of violaxanthin,lutein,b-carotene and b-cryptoxanthin were markedly decreased in albino tea leaves (p<0.05).The identified tea carotenoids,lutein,violaxanthin and b-carotene,are components of the light harvest antenna com-plexes,which contain well balanced chlorophyll molecules and other components(Demmig-Adams,Gilmore,&Adams,1996;Me-ier,Tzfadia,Vallabhaneni,Gehring,&Wurtzel,2011;Polívka& Frank,2010).The coordinated biosynthesis of antenna complex components,such as proteins,chlorophylls and carotenoids,is crit-ical for the biogenesis of the photosynthetic apparatus.Therefore, it is reasonable that,in albino tea leaves examined in this study, reduction in levels of chlorophylls were accompanied with a corre-sponding reduction in levels of the carotenoids associated with
abundances of catechins(A–C)and caffeine(D)in albino and normal tea cultivars.Significance level:⁄p<0.05;
L.Feng et al./Food Chemistry155(2014)98–104101
Zeaxanthin accumulation can be triggered by adverse environmen-tal conditions,such as excess light,desiccation,and low tempera-ture(Fernández-Marín,Míguez,Becerril,&García-Plazaola,2011). We speculated that increased levels of zeaxanthin in albino leaves would be one of the adaptive responses to photooxidative stress for maintaining photosynthetic efficiency and capacity.(Ekborg-Ott,Taylor,&Armstrong,1997;Le Gall et al.,2004).We suggest that theanine,Glu,and Ser are key components for provid-ing the strong umamiflavour of green tea made from albino cultivars.
Theanine is synthesised from Glu and ethylamine by theanine synthetase(Deng et al.,2012).The Glu is derived from the gluta-mine synthase(GS)/glutamate synthase(GOGAT)cycle,and it is
plot(A)and corresponding loading plot(B)derived from the integrated HPLC data sets of albino and normal tea cultivars.Data
replicates.Abbreviations of compounds are outlined in the Materials and methods.
102L.Feng et al./Food Chemistry155(2014)98–104
varied from103to150mg gÀ1DW among the different cultivars, with albino cultivars having significantly lower content than the normal one(p<0.05;Fig.3A);our results are in accordance with previous reports(Du et al.,2006;Wei et al.,2012).The composition of catechins was also determined.EGCG was the most abundant while C was the least abundant in the leaves of all cultivars exam-ined(Fig.3B and C).Compared to the contents in the normal culti-var,EGCG content in leaves of the four albino cultivars,and EGC in 3.5.Differentiation of cultivars and identification of the metabolites contributing to cluster formation
Orthogonal partial least-squares discriminant analysis(OPLS-DA)was performed on the metabolite profiles obtained from the five cultivars to uncover the subtle changes in the occurrence and abundance of specific compounds.The OPLS-DA score plot showed a clear separation between the normal cultivar‘Fd’and
metabolic profiles(p<0.05or p<0.01)of albino tea leaves and the network of related metabolic pathways.2-OG,2-oxogluatarate;
5-aminolevulinic acid;ch,chloroplast;Chl a,chlorophyll a;Chl b,chlorophyll b;dKae,dihydrokaempferol;GAP,glyceraldehyde
geranylgeranyl diphosphate;GOGAT,glutamate synthase;GS,glutamine synthetase;hSer,homoserine;lCya,leucocyanidin;lDel, oxaloacetate;PRPP,phosphoribosyl pyrophosphate;Pchlide,protochlorophyllide;PEP,phosphoenolpyruvic acid;Phy-PP,phytyl
L.Feng et al./Food Chemistry155(2014)98–104103
compounds exhibited the greatest contribution to the cluster for-mation within the metabolite profiles.Similar results were ob-tained with respect to the identification of metabolites with the most influence on class separation and the greatest importance for the differentiation of analysed tea cultivars(Supplementary Fig.1),based on the principal components analysis(PCA)(Roessner et al.,2001;Trygg et al.,2007).
Taken together,our results,for significant changes in metabolic profiles of albino tea leaves and the network of related metabolic pathways,are summarised in Fig.5.The increase in the abundance of total amino acids(especially theanine and Glu)and decrease in the levels of catechins and caffeine in the albino leaves contributed to their reduced astringency and bitterness and imparted a strong umami taste.As a high Chl a/b ratio is usually associated with in-creased photosynthetic capacity and increased zeaxanthin is important for photoprotection by dissipating excess light energy, our results suggest that albino tea plants can modulate their pho-tosynthetic apparatus to compensate for the changes in photosyn-thetic efficiency and capacity in albino leaves.OPLS-DA analysis revealed significant differences between the metabolic profiles of albino and normal tea cultivars and identified the metabolites pre-dominantly responsible for the observed variance.We speculate that the biosynthetic inhibition of chlorophylls,carotenoids and caffeine in albino leaves might lead to a shift in the metabolic net-work towards amino acid biosynthesis.
4.Conclusion
Compared to the normal cultivar,albino tea leaves had a higher chlorophyll a/b ratio and increased abundance of zeaxanthin and free amino acids,along with lower levels of total carotenoids,cat-echins and caffeine.We suggest that changes in some key metab-olites,such as EGCG,theanine,Glu and caffeine,are responsible for the decreased astringency and bitterness and enhanced umami taste of albino tea.Moreover,OPLS-DA identified key determinants between albino and normal control tea cultivars. Acknowledgements
We thank Dr.Hua Yang and Lin-Long Ma at the Anhui Agricul-tural University for genetics analysis and data processing.This work was funded by the National Science Foundation in China (#31070614),the Research Fund for the Doctoral Program of High-er Education of The ministry of Education(#20123418110002),the Program for Changjiang Scholars and Innovative Research Team in Universities(IRT1101),and the‘‘Twelfth Five-Year’’National Key Basic Research and Development Project(973)in China (2012CB722903).
Appendix A.Supplementary data
Supplementary data associated with this article can be found,in the online version,at /10.1016/j.foodchem.2014.
01.044.
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