Comparative transcripts profiling reveals new insight into molecular processes regulating lycopene accumulation in a sweet orange (Citrus sinensis) red-flesh mutant
© Xu et al; licensee BioMed Central Ltd. 2009
Received: 21 April 2009
Accepted: 18 November 2009
Published: 18 November 2009
Interest in lycopene metabolism and regulation is growing rapidly because accumulative studies have suggested an important role for lycopene in human health promotion. However, little is known about the molecular processes regulating lycopene accumulation in fruits other than tomato so far.
On a spontaneous sweet orange bud mutant with abnormal lycopene accumulation in fruits and its wild type, comparative transcripts profiling was performed using Massively Parallel Signature Sequencing (MPSS). A total of 6,877,027 and 6,275,309 reliable signatures were obtained for the wild type (WT) and the mutant (MT), respectively. Interpretation of the MPSS signatures revealed that the total number of transcribed gene in MT is 18,106, larger than that in WT 17,670, suggesting that newly initiated transcription occurs in the MT. Further comparison of the transcripts abundance between MT and WT revealed that 3,738 genes show more than two fold expression difference, and 582 genes are up- or down-regulated at 0.05% significance level by more than three fold difference. Functional assignments of the differentially expressed genes indicated that 26 reliable metabolic pathways are altered in the mutant; the most noticeable ones are carotenoid biosynthesis, photosynthesis, and citrate cycle. These data suggest that enhanced photosynthesis and partial impairment of lycopene downstream flux are critical for the formation of lycopene accumulation trait in the mutant.
This study provided a global picture of the gene expression changes in a sweet orange red-flesh mutant as compared to the wild type. Interpretation of the differentially expressed genes revealed new insight into the molecular processes regulating lycopene accumulation in the sweet orange red-flesh mutant.
Carotenoids are widely found in plant species and are responsible for the coloration of flowers and fruits to attract pollinators and seed-dispersing animals . Carotenoids are essential components of human diets, and they have important roles in human health as antioxidants, vitamin A precursors and cancer-preventing effectors [2, 3].
More than 700 naturally occurring carotenoids have been identified . Of them, some carotenoids and their biosynthesis are well characterized; for example, α-carotene and β-carotene serve as important source of vitamin A [5, 6]; and high levels of β-carotene accumulation in Cauliflower was mediated by an Or gene . However, the majority of carotenoids, including lycopene, their biosynthesis and regulation are poorly understood. Lycopene provides the familiar red color to tomato fruits, and is the most potent antioxidant among carotenoids [8, 9]. Interest in lycopene metabolism and regulation is growing rapidly because of the overwhelming reports on the role of lycopene in human health promotion, such as the prevention of a range of chronic diseases particularly the prostate cancer [10–12]. It has been shown in tomato fruit, the accumulation of lycopene is highly correlated with up-regulation of carotenogenic genes in the upstream of lycopene and down-regulation of lycopene cyclases in the downstream [13, 14]. Biochemical and molecular studies in tomato mutants with different carotenoids accumulation have provided more in-depth insight into lycopene biosynthesis. One type is yellow-fruited tomato mutants with reduced lycopene content where two possibilities have been reported: one possibility is due to reduced expression of carotenogenic genes in the upstream of lycopene including yellow-flesh mutant due to dysfunction in phytoene synthase (PSY) , and tangerine mutant with a deletion mutation in carotenoid isomerase gene (CRTISO) ; the other possibility is due to up-regulation of lycopene downstream genes such as Delta mutant with increased expression of lycopene epsilon-cyclase gene (LYCe) . For other types of mutants characterized with increased lycopene, the underlying mechanisms are more complicated. Ronen et al. (2000) analyzed an old-gold (og) mutant and found that lycopene accumulation is due to null mutation in the gene lycopene β-cyclase (LYCb) . Three other mutants, named as high pigment (hp), are well-known for the accumulation of higher concentrations of lycopene in the fruit. Cloning of HP-1 gene showed that it encodes UV-damaged DNA binding protein (DDB1) ; HP-2 gene also encoded a light signaling regulator deetiolated1 (DET1) ; Whereas hp-3 mutation occurred in zeaxanthin epoxidase (Zep) gene, and caused abscisic acid deficiency . Collectively, physiological, genetic and molecular studies of the mutants indicated that lycopene metabolism and regulation is complicated in tomato; however, in species other than tomato, there is limited information available on the mechanism of lycopene accumulation in fruits.
Massively parallel signature sequencing technology (MPSS) is a sequence-based method recently developed that can be used for measuring gene expression [33, 34]. The MPSS method is "global" in that it can identify a nearly complete inventory of transcripts in a given sample. For plant species that lack whole genome sequence information such as citrus, MPSS can provide a broader view of the transcriptome than microarray analyses which includes known sequences . The MPSS method has been used for various transcriptional studies in plants [36–38]. Two interesting cases in Arabidopsis mutant-wildtype pairs have suggested that MPSS method is robust in identifying mutation responsive genes [39, 40].
In this study, a genome-wide gene expression study was carried out between the red-flesh mutant 'Hong Anliu' and its wild type 'Anliu' sweet orange using MPSS method, A total of 20,178 genes were analyzed, of which 2,936 genes in sense and 802 genes in antisense showed a two-fold or greater expression difference between the mutant and wild type, and 452 genes in sense and 130 genes in antisense are differentially expressed at 0.05% significance level. The results demonstrated that some genes are newly transcribed in the mutant. Our results also provided a large number of genes previously not known to be involved in the mutation trait formation. Interpretation of the data built up links between new information herein and our previous fragmentary knowledge, and provided new insight into the molecular processes regulating lycopene accumulation in the mutant fruits.
MPSS signature abundance and distribution
Summary statistics of MPSS signatures in the red-flesh mutant 'Hong Anliu' sweet orange (MT) and its wild type 'Anliu' sweet orange (WT)
Total Sequence Collected
Low Quality Signatures
The distribution of signature abundance was quite similar between MT and WT (see additional file 2). Three signatures in MT and four signatures in WT were expressed at high abundance more than 1% (>10,000 TPM). While with the decrease of abundance, the number of signatures increased dramatically. 60% of the total signatures in MT and 56% of the total signatures in WT were at abundance less than 0.0001%. Moreover, about 99% of the total signatures in both libraries was below 0.001% abundance (<10 TPM), suggesting that genes with low transcripts are abundant in both libraries.
Differential expression of MPSS signatures between mutant and wild type
The frequency of signature was regarded as relative expression level of each transcript in MT and WT libraries. Comparative analyses of the frequency of signature between MT and WT revealed that the expression ratio (MT/WT) varied greatly from 0.008 to 828. Of the common signatures in both libraries, 29,602 signatures showed a two-fold or greater (ratio>2 or <0.5) expression difference between MT and WT (see additional file 3), and are regarded as differentially expressed transcripts according to the criteria defined by Meyers et al. (2004b) .
Identification of mutation-responsive genes
To link the signatures to known genes from sweet orange, a unigene dataset comprised 26,826 contigs and 73,607 singletons was used for MPSS signature mapping analyses. All the sequences were used to produce in silico signatures in both sense and antisense directions. A total of 176,549 and 171,355 in silico signatures were identified in sense and antisense respectively. The produced in silico signatures stored in the reference signature database, through which the expressed MPSS signatures were mapped on the corresponding EST contigs and singletons based on matches between MPSS signatures and in silico signatures. The results showed that a total of 18,106 genes (16,005 in sense and 2,101 in antisense) are expressed in MT (see additional file 4); and 17,670 genes (15,697 in sense and 1,973 in antisense) are expressed in WT (see additional file 5).
List of the important KEGG pathways more than 3 differentially expressed genes affiliated
CN183092, TC22936, TC25357, TC3430, TC81
Biosynthesis of steroids
EY708186, EY737600, TC13179, TC14003, TC8568
Calcium signaling pathway
TC10446, TC2776, TC6871, TC41
TC14843, TC17929, TC5, TC5834
TC410, TC15757, TC25101, TC5900
Citrate cycle (TCA cycle)
EY670548, TC22243, TC623
CF835920, DN620599, TC12093, TC5490
TC14843, TC17929, TC22276, TC23691
Fatty acid biosynthesis
CX074436, EY700768, CV713900, EY748567, TC10790, TC13490, TC9950
BQ622999, TC18809, TC23691, TC24546
TC13190, TC25006, TC9636
Fructose and mannose metabolism
TC3179, TC599, TC8180
CX676461, TC13437, TC1366
TC10790, TC13490, TC5567, TC599, TC4947
Limonene and pinene degradation
TC12405, TC14251, TC15293, TC20473
MAPK signaling pathway
TC10975, TC11537, TC14581, TC23673
Metabolism by cytochrome P450
TC16950, TC25526, TC25649
Nicotinate and nicotinamide metabolism
DN620930, TC12023, TC19292, TC23834
CX047553, DN621543, EY666061, TC10047, TC11705, TC16046, TC165, TC2473, TC2776, TC367, TC9593, TC9609
DY305711, EY675075, TC15280, TC17840, TC225
TC5683, TC15808, TC4307
EY655396, EY667540, TC3601
928, CV713900, EY670548, TC13490, TC13985, TC2886, TC4947, TC623
EY678532, EY755319, EY756332, TC13063, TC17838, TC18427, TC20162, TC21064, TC25973, TC7029, TC8912
Starch and sucrose metabolism
TC12327, TC6279, TC7121, TC7710
Ubiquitin mediated proteolysis
EY692161, EY720797, TC11061, TC11537, TC11678, TC11708, TC11720, TC15530, TC18081, TC24614, TC458, TC8922
Functional classification of mutation-responsive genes
The biological interpretation of the significant mutation responsive genes was further completed using KEGG pathway analyses. A total of 26 different metabolic pathways were found with more than 3 genes affiliated, of which some were consistent with biological processes already revealed by GO analyses. The most represented pathways were listed in Table 2. Of these, some were related with mutation trait formation based on previous knowledge, including carotenoid biosynthesis (4 enzymes represented), citrate cycle (3), diterpenoid biosysnthesis (4), fatty acid metabolism (7), glycolysis (5), oxidative phosphorylation (12), photosynthesis (5), pyruvate metabolism (8), starch and sucrose metabolism (4), steroids biosynthesis (5), and ubiquitin mediated proteolysis (12).
Verification of the mutant trait formation related genes
Measurement of chlorophyll content and photosynthesis activity
MPSS, like expressed sequence tags (ESTs) and serial analyses of gene expression (SAGE), is a tag-based method recently developed that can be used for quantitative measurements of gene expression when combined with genomic sequence or unigene dataset [33, 43]. Comparing with ESTs and SAGE technologies, MPSS has its advantages that it can provide a more thorough and scientific representation of the absolute transcript population, and is more sensitive to genes expressed at low levels due to deep sequencing with the resultant dataset containing more than 6 million tags for each sample in this study (also discussed in ). When compare with cDNA microarray technology that requires previous knowledge of genes, the limitations to detect unknown genes was not encountered in MPSS [32, 39]. Another difference between MPSS and microarray is that statistical analyses in microarray is based on biological replicates; while statistical analyses of MPSS is usually based on enough sequences with more than 1 million tags in a given sample [35, 38, 43]. The MPSS technology has become popular in transcriptional expression studies . From the results of this study, it is easy to notice that MPSS analyses not only highlight some genes and biological processes already revealed by our microarray and proteomic data, but also reveal large amount of genes which are possibly involved in the formation of mutation trait. The data consistency from multiple approaches assures that the MPSS data produced in this study is reliable.
Identification of key genes and metabolism pathways involved in the formation of lycopene accumulation trait in the sweet orange mutant
The first noticeable pathway is the isoprenoid and carotenoid biosynthesis pathways which exist in plastid. Four differential genes are involved in the carotenoid metabolism pathway, including TC5 gene (encoding lycopene β-cyclase, LYCb), TC5834 gene (encoding capsanthin synthase, CCS), and two different abscisic acid hydroxylases (hydroxylase 2 and 4), encoded by TC14843 and TC17929 genes (Figrue 8). The transcription level of LYCb gene detected by MPSS in mutant was 6 times lower than that in wild type, consistent with our previous data by qRT-PCR . The LYCb was believed to be a rate-limiting enzyme in the conversion of lycopene to cyclic carotenes . The CCS gene is also significantly down-regulated in the mutant at 170 DAF (Figure 6). The CCS is an enzyme with a similar action mechanism to LYCb; and low transcript level of CCS is possibly responsible for the accumulation of lycopene in red grapefruits (Citrus paradisi) . Based on these data, it can be speculated that the down-regulation of lycopene downstream genes LYCb and CCS, in combination with up-regulation of lycopene upstream genes (i.g. PSY, our previous data ), will result in the accumulation of lycopene in the sweet orange mutant. It is interesting that the biosynthesis of abcisic acid (ABA) was affected in the mutant. Two genes involved in ABA biosynthesis were down-regulated, fitting well with our observation that ABA content in MT is lower than that in WT (data unpublished). The decreased biosynthesis of ABA in MT is plausible due to the decreased supply of ABA precursor, xanthoxin, which is downstream of lycopene and could be significantly affected by down regulation of LYCb gene. This could also be evidenced from another sweet orange mutant 'Cara Cara' with high lycopene accumulation that the pulp of the mutant at color-breaker stage only contained 23% of the ABA content of its normal counterpart . A recent study in tomato hp3 mutant also showed that ABA is deficient in the mutant with higher lycopene content .
The second group of significant mutation-responsive pathways includes photosynthesis, glycolysis, citrate cycle (TCA cycle) and fatty acid metabolism. Pathways in this group showed a complicated pattern. For photosynthesis, 10 genes were involved as revealed from GO and KEGG analyses; all these genes exhibited up-regulation pattern in MT fruits (Figure 8). The induction of genes associated with photosynthesis is consistent with the GO results that large number of genes are located in chloroplast; and is in line with our gas chromatographic analyses that the sugars in juice sacs is higher in MT than in WT . Moreover, our in situ analysis of the photosynthetic rate confirmed that photosynthesis in MT is stronger than that in WT (Figure 7). Another case in tomato hp-2 mutant which manifested with increased carotenoids, primarily lycopene, showed that genes involved in photosynthesis are consistently up-regulated throughout fruit ripening ; and by contrast sugar deficiency delayed lycopene accumulation in tomato . All these findings suggest the importance role of photosynthesis and sugar in the accumulation of lycopene. Similar to photosynthesis, genes involved in glycolysis showed an up-regulated pattern. The glycolysis provides substrates pyruvate for the methylerythritol phosphate (MEP) pathway. Meanwhile, our data showed three important genes participated in MEP pathway (DXS, HDS and HDR) were also up-regulated (see additional file 8). Therefore, the increased MEP biosynthesis led to the enhanced biosynthesis of carotenoids, and meanwhile resulted in the dramatical increment of lycopene due to the partial impairment of downstream flux caused by down-regulation of LYCb and CCS gene as discussed in the previous paragraph (Figure 8). This provides a molecular scenario for the formation of lycopene accumulation trait in the mutant red-flesh fruits. In the other metabolic flux of pyruvate which happens in cytosol, acetyl-COA is produced for the mevalonic acid (MVA) pathway. And the two branch flux of acetyl-COA were inhibited. One is the TCA cycle; three genes, isocitrate dehydrogenase (TC22243), malate dehydrogenase (TC623), and importin alpha-4 gene (EY670548), were down-regulated. These MPSS data correlate well with our previous data that the citric acid in the mutant is significantly lower (nearly one quarter) than that in wild type . The other branch downstream of acetyl-COA is fatty acid metabolism; all the genes involved in this pathway were down-regulated.
The third group of metabolite pathways is downstream of MVA pathway. This group is relatively simple. Two genes involved in the ubiquinone biosynthesis were up-regulated. Six genes related to sterol biosynthesis and two to brassionsteroids biosynthesis also showed an up-regulated pattern in the mutant.
When our results on red-flesh orange mutant is compare with that on tomato mutants with abnormal accumulation of lycopene, the molecular mechanism for lycopene accumulation is different. For example, lycopene accumulation in tomato old-gold mutant is due to null mutation in LYCb gene ; and high-pigment 3 mutant is due to mutation on Zep gene ; While tomato high-pigment 1 and high-pigment 2 mutation happened on light responsive genes [18, 19]. In this study, by using a pleiotropic mutant with high sugar, low acid and abnormal accumulation of lycopene, extensive molecular pathways including isoprenoid biosynthesis, carotenoid biosynthesis and photosynthesis were affected in the mutant, and enhanced photosynthesis and the partial impairment of lycopene downstream flux caused by down-regulation of LYCb and CCS genes are critical for the formation of lycopene accumulation trait in the sweet orange mutant.
The transcriptional regulation of carotenoid metabolism in sweet orange mutant
The alternation of carotenoid composition and concentration can be achieved through biosynthesis and post-biosynthesis activities including transport, storage and degradation . These activities could be regulated on transcriptional and posttranscriptional levels. In our study, high percentage of antisense transcripts, i.e. 2,101 of 18,106 genes in MT and 1,973 of 17,670 genes in WT, were observed, possibly indicating post-transcriptional regulations existed in sweet orange fruit development. In contrast, the MPSS data provided much more information for the regulation of these activities on transcription level.
A noticeable result is that considerable amount of genes, 8.6% of the total annotated genes, were involved in regulation of biological process or transcription (Figure 4). The induction of regulatory genes of transcription correlates well with the increased overall transcription in MT. The total number of transcribed genes in the mutant was 18,106, more than that in wild type 17,670, suggesting that newly initiated transcription occurs in the mutant. Moreover, analyses of the genes with transcription changes > 2 fold showed that up-regulated genes constitute 62.7% of the total changed genes. It is noticeable that MYB gene, a transcription factor (TF), showed a significant transcription changes in the mutant as revealed by MPSS data. And qRT-PCR analysis confirmed that the differential expression of MYB gene was significant at 150, 170 and 220 DAF stages, suggesting that MYB gene functions in extensive stages during fruit development. MYB genes are a superfamily of transcription factors that control many biological processes; and have also been highlighted for their regulation in pigment accumulation, primarily in anthocyanin biosynthesis [48–51]. While, to the author's knowledge, it is still unknown whether MYB genes are regulating carotenoid biosynthesis; so this study provided an important gene that possibly regulate lycopene accumulation in the orange fruits.
Genes possibly involved in the post-biosynthesis activities were also highlighted with significant transcriptional changes. An impressive category is transporter gene, which constitutes 12.1% of the total annotated genes. One gene EY748567 shows high homology with sugar transporter gene (67% similarity), suggesting that sugar translocation is active in 170 DAF stage. This is consistent with the above mentioned result that photosynthesis are active, and thus the produced sugar has to be transported to juice sac through phloem in citrus . Our physiological data also showed that the stage 170 DAF is also the most important stage for sugar accumulation (data unpublished). Another remarkable group possibly involved in post-biosynthesis activities is ubiquitination or proteolysis related genes. Twelve genes were categorized into ubiquitin mediated proteolysis (Figure 4), indicating that protein degradation may play important role in maintaining certain important gene at a constant steady-state level, as proposed by Welsch et al. (2007) who found a ring finger protein involved in degradation processes and is a stable element in the carotenogenesis of Arabidopsis leaves . In this study, the ring-finger gene (TC10250) was mainly expressed in mature stages of the fruits, possibly indicating that ring finger gene has a similar function as a stable element in carotenogenesis during orange fruit development.
Our study provides a global picture of the gene expression changes in a sweet orange red-flesh mutant comparing with wild type. The interpretation of the MPSS data uncovered a large number of genes which were previously not known to be involved in the mutation trait formation. Functional categorization of the differentially expressed genes showed that a number of important pathways, including isoprenoid biosynthesis, carotenoid biosynthesis, photosynthesis, citrate cycle (TCA) and some post-biosynthesis activities such as transporter and degradation, cross communicated and worked collaboratively in shaping the red-flesh trait of the mutant. This study provided new insight that enhanced photosynthesis and the partial impairment of lycopene downstream flux caused by down-regulation of LYCb and CCS genes are critical for the formation of lycopene accumulation trait in sweet orange fruits.
Plant material and RNA preparation
The red-flesh mutant 'Hong Anliu' and the wild type 'Anliu' sweet orange (C. sinensis L. Osbeck), cultivated at the Institute of Citrus Research located in Guilin, Guangxi Province, China, were used as materials. The mutant 'Hong Anliu' sweet orange was a bud mutation from wild type 'Anliu' sweet orange; and they are with isogenic background as revealed from our previous molecular marker evaluations . Sampling strategy is the same as previously published . Fruit samples were harvested at 150, 170, 190, and 220 days after flowering (DAF) from three different trees, at each time point 10 representative fruits from each tree were collected. All samples were separated into peel and pulp, and immediately frozen in liquid nitrogen and kept at -80°C until use. Total RNA was extracted according to Liu et al. (2006) .
Massively Parallel Signature Sequencing (MPSS)
The materials used for MPSS analyses were fruit pulps from mutant and wild type at stage 170 DAF (our previous results indicated that 170 DAF is the critical stage for transcriptional regulation; Figure 1). 20 μg of total RNA were sent to Beijing Genomics Institute (Shenzhen) where the libraries were produced and sequenced using Illumina's Genome Analyzer (Solexa). The MPSS were carried out essentially as previous studies [33, 35], with some ideas lending from LongSAGE . Briefly, the cDNA was digested with Nla III, and then ligated with the first adapter containing the recognition site of Mme I, a Type IIs endonuclease which cleave at sites 21 bp from the recognition site. After digestion by Mme I, the transcripts were ligated with the second adapter. With the sequencing primers designed based on the two adaptors, the sequence of the 21 bp representing each transcript can be determined via a series of enzymatic reactions on the microbeads. The derived reliable sequence was termed signature herein. The abundance of each signature is normalized to one million (transcripts per million, TPM) for the purpose of comparison between samples.
Analyses of MPSS data
To remove signatures that may arise from errors in the MPSS procedure, two filters were applied to the derived signatures [34, 35]. The first filter, the "reliability filter", was to remove low quality signatures containing ambiguous nucleotides or adaptor sequences. The second "significance" filter, with the intent to remove signatures that are consistently present at background levels, excluded signatures lower than 3 TPM in both libraries according to the criteria described by .
To link the expressed signatures to known genes from orange, the unigene dataset from TIGR gene index database http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=orange, 597 sweet orange transcription factors http://planttfdb.cbi.pku.edu.cn/web/index.php?sp=cs, and 964 sweet orange cDNA sequences from our lab were combined together as reference gene dataset. All the sequences were used to produce in silico signatures in both sense and antisense strands. The produced in silico signatures were stored in the reference signature database, through which the expressed MPSS signatures can be mapped on the corresponding EST contigs and singletons based on matches between MPSS signatures and in silico signatures, as described previously .
Significance level of the difference of signature frequency and transcript abundance between the two libraries was analyzed using Z-score method according to Kal et al. (1999) .
Functional assignments of differentially expressed genes
To assign putative functions to differentially expressed genes between mutant and wild type, annot8r program was run locally to BLAST against a reference database that stores UniProt entries, their associated Gene Ontology (GO), Enzyme Commission (EC) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) annotation . The GO categorization results were expressed as three independent hierarchies for biological process, cellular component, and molecular function . The biological interpretation of the differentially expressed genes was further completed by assigning to metabolic pathways using KEGG . And for the identification of pathways significantly affected by the mutation, we focused on the metabolite pathways at least 3 genes affiliated.
Real-time Quantitative RT-PCR (qRT-PCR) verification
Twenty five genes were chosen for confirmation by real-time quantitative RT-PCR. Primer pairs were designed with the Primer Express software (Applied Biosystems, Foster City, CA, USA). Primer sequences were presented in additional file 7. Quantitative Real-time PCR for gene expression analysis was performed on the ABI 7500 Real Time System (PE Applied Biosystems, Foster City, CA, USA) using actin gene as endogenous control according to Liu et al. (2007) . Briefly, the primers for the target gene and actin were diluted in the SYBER GREEN PCR Master Mix (PE Applied Biosystems) and 20 μl of the reaction mix were added to each well. Reactions were performed by an initial incubation at 50°C for 2 min and at 95°C for 1 min, and then cycled at 95°C for 15 s and 60°C for 1 min for 40 cycles. Output data was generated by the instrument on-board software Sequence Detector Version 1.3.1 (PE Applied Biosystems).
Chlorophyll content and Photosynthesis activity measurement
Chlorophyll extraction was performed according to van Schie et al. (2007) . Briefly, 0.5 gram of ground leaf tissue was incubated with 5 ml of 95% ethanol at room temperature for 10 min. Extracts were cleared by centrifugation at 12 000 g for 1 min, then diluted 10 times with 95% ethanol. Three milliliters of the diluted extracts were measured using UV-1601 spectrophotometer (Shimadzu). Total chlorophyll content was caculated using the method of Lichtenthaler (1987) .
In situ rates of photosynthesis and stomatal conductance were measured with the TPS-1 portable photosynthesis system (PP Systems, Haverhill, MA). Three different trees in the field were used for each genotype; and four leaves on a plant were measured according to Hu et al. (2007) .
Statistical analyses (descriptive and t-test) were conducted using SPSS 10.0 software. Difference of compared sets were considered significant at p < 0.05.
We thank Dr. Ayalew Ligaba Osena from Cornell University for his critical reviews and Mr. Yuan Huang from Huazhong Agricultural University for helpful assistance on the use of TPS-1 photosynthesis system. This work was supported by the Natural Science Foundation of China (NSFC Nos. 30921002, 30830078, 30800745), Specialized Research Fund for the Doctoral Program by Ministry of Education of China (No.200805041024), and Hubei Provincial Natural Science Foundation (2008CDB083).
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