As regards plant chimeras, some researchers have studied their phenotypes, fruit qualities and genome compositions [2-4], but they rarely studied the genetic regularity and variant expressions of biochemical substances. In this work, we firstly systematically compared the primary metabolites, volatiles and carotenoids between a novel citrus chimera OCC and its donor plants, identifying that the genetic contributions and inheritance patterns from two donors are not equal in plant chimeras. Some substances content herein detected in OCC were inclined to specific layer source donors; some were intermediate between two donors; but some were deviated from both of two donors, presenting the significantly higher or lower level than that of any of the donor plants. Besides, there were some characteristic substances were exclusively presented in OCC or in two donor plants.
Metabolic Interrelationships between OCC and its layer source donor
In citrus chimeras, the juice sacs were developed from L1 cell layer and the peels were from L2 cell layers, the expression of genetic substances in different tissues of OCC showed transgressive (i.e., not intermediate) with its layer source donors. According to the significant difference analysis, some compounds, such as myo-Inositol (Table 3) and d-limonene (Table 5), in OCJ and OJ had no significant difference but were commonly significantly higher than that in CJ; by contrast, some compounds, such as quininic acid (Table 2) and Germacrene D (Table 4), had no significant difference in OCP and CP but significantly higher than that in OP. The results indicated that there were a number of specific chemical-related genes in cell layer L1 (mainly controlled juice sacs) of OCJ were inherited from O, and others in L2 (mainly controlled peels) were inherited from C. This observation was consistent with the study that the juice sacs of a citrus periclinal chimera Ekuliku, obtained by grafting, were developed from L1 donor Nankan (C. unshiu), while the epicarp and mesocarp were both developed from L2&L3 donor Hamlin (C. sinensis) [19]. Moreover, our results also attested to the genetic regularity previously described in the leaf morphology variation of Brassica chimeras, that the variation was only controlled by the cells of the donor red cabbage and was reproducible and directional in progenies [20]. Furthermore, in the present study, we also found that the contents of the same chemical substances in different tissues of OCC were seperately apt to donor O or donor C. For example, in peels (Table 2), 4-aminobutanoic acid and palmitic acid were only occurred in OCP and CP, but in juice sacs (Table 3), 4-aminobutanoic acid and palmitic acid were only occurred in OCJ and OJ. That is to say, the appearance of 4-aminobutanoic acid and palmitic acid in the peels and juice sacs of the chimera OCC were largely depended on each layer-determined donor C and O, respectively. Similar study also noted that among the 8 high reproducibility primers, STS primers and OPH20 primers amplified 600 bp bands only appeared in chimera Ekuliku and donor Nankan, but not in another donor Hamlin, that is, these two primers were specific to Ekuliku and Nankan [19]. This specific inheritance pattern in chimera was proved at the metabolic level in this research. Therefore, the part of substances herein investigated were genotype-specific and largely controlled by independent layer source donor.
Significant deviations in metabolites between OCC and both donors
However, the expression quantities of a number of chemicals (including primary metabolites and volatiles) in OCC were not “loyalty” to layer source donor, but deviated far from both two donors (i.e., significantly higher or lower than donors). Among them, 13 compounds and 7 compounds in OCC showed significantly higher and significantly lower level than those in any of the donors, respectively. This observation was similar to two citrus hybrids, that 56 of the 113 volatile profiles in the hybrids were significantly higher or lower than in parents [21]. Looking back to this study, for instance, Germacrene D (Table 5) in OCJ was 6-17 times higher than that in CJ and OJ, respectively. The quantities of arabinose was over 3-9 times higher than in CJ and OJ (Table 3), and this profile have been reported as a good source of dietary fiber and could be available for juice production [22]. And as reported, scyllo-Inositol (Table 2, 3) could be responsible for the tolerance of citrus destructive disease HLB [23], even though its concentration was not the highest in OCC, it still had a relatively higher level. For these substances with a significantly higher or lower content than two donors, we predicted it was possible related to gene expression that regulates the metabolic status of those substances. In citrus, CsMYBF1 is an important transcription factor of R2R3-MYB, the expression pattern of CsMYBF1 in the citrus fruit may be related to the content of metabolites. In recent study through isolation of R2R3-MYB transcription factor CsMYBF1 from citrus, and analysed CsMYBF1 overexpression lines in transgenic tomato and the RNAi (RNA interference) lines in citrus, it was found that CsMYBF1 induced an up-regulation of the primary metabolites and phenylpropanoic acid pathway in the tomato, on the contrary, the RNAi of CsMYBF1 in the citrus callus resulted in down-regulation of many of the phenylpropanoic acid pathway genes and reduced the content of hydroxycinnamic acid and flavonol [24]. Accordingly, we speculated that because of the interaction between genetic materials from different genetic backgrounds in chimeras, the expression of these deviated chemical-related genes may be overexpressed, and some may be surpressed. Therefore, these genes could have been expressed normally in a single chimeric parent, O or C, but when these genes are simultaneously went into the chimera OCC, they would co-expressed and interacted in the same cell layer of OCC, at which time the role of relevant genes will be strengthened, and of course, the role of them may also be diminished, so that the substance content would show significantly higher or lower in OCC than that of two O and C. In terms of this kind of peculiar expression of genes in chimera, Fernandez and his colleagues [25] investigated the weight reduction in the berry of a grape chimera,which was caused by unusual VvpI gene expression in L1, L2 or in both cell layers, which lead to phenotypic variations (fleshless) in progeny. This study can also prove that the overexpression or surpression of the gene expressions may exist in cell layers of OCC, resulting in the significantly different content of the relevant metabolic substance from O and C.
Characteristic metabolite in OCC
The interaction between tissues with different backgrounds in chimeras may be a new source of genetic variation has been previously recognized [26-27]. In this study, a novel substances α-ylangene was only exclusively detected in the chimera OCC. α-ylangene was a unique compound, which was hardly ever been reported in any citrus volatiles, but was a main sesquiterpenoid at the post-maturation stage in grapes [28]. Zhou and her colleagues investigated two citrus chimeras (L1-L2-L3=N-F-F and F-N-N; N represents ‘Natsudaidai’, represents ‘Fukuhara’), they did found some variations that the chimeras NFF and FNN not only had the specific bands of two donor plants, but also had their own unique new bands by RAPD analysis [2]. It is suggested that the chimeras interacted at DNA level. Therefore, at metabolic level, α-ylangene was never detected in two donors but presented in the chimera OCC could be another evidence for the genetic variation during the chimeras’ development, and intercellular movements may be the reason for it.
Furthermore, the finding of this variation raised the question of what circumstances happened during so-called interactions. By using artificial plant chimeras, it has been found that transcription factors can move from one cell to another in a plant, and maintain biological activity [29, 30]. For example, the floral transcription factors LFY and AP1 have been demonstrated can participate in cell-to-cell signal transmission between and within different layers of the meristem. These two transcription factors could activate homeotic genes and then led to phenotypic variation in flower structure [30]. In recent years, the heritable variations caused by gene mutations and intercellular trafficking were extensively studied in chimeras. A grape periclinal chimera ‘Malian’, whose flesh is bronze, a spontaneous mutation appeared in L2 cell layer, and thereafter this mutant invaded L1 cell layer to give rise to a new phenotype, white flesh [31]. And there were many studies have been reported that berry color variants in grape Pinot can be mapped back to the mutation on a single locus named “berry color locus”, which is consist of four tandem MYB transcription factors on chromosome 2 [32–34]. Meanwhile, a peach mutant, where the mutation carried a PRUPE.6G281100 allele entered L2 cell layer, then led to a phenotype change of peach from flat to round [35]. In this study, according to previous studies on the metabolic pathway of α-ylangene[36], it is possible that due to the interaction between genetic materials from different backgrounds, which led to an up-regulation of a series of genes involved in sesquiterpene metabolism and the α-ylangene pathway, and induced a strong accumulation of α-ylangene, or it may be due to the lack of a series of genes consuming α-ylangene accumulation.
The presence and absence of some typical metabolites
In the context of this study, notably, the presence and absence of some typical chemical substances were also observed in OCC and two donor plants. In carotenoids, for example, α-carotene was commonly undetectable in OCP and CP, but specific to OJ and OCJ. And phytoene was only exclusively undetectable in CJ. A related study also in agreement with that α-carotene was only detected in the juice sac of ‘Rio Red’ among six grapefruits [37]. 2,4-di-t-butylphenol, an phenol which uncommonly appeared in many citrus species, was herein detected in OCP and CP, and this profile was only reported in harvested ‘Huanong’ red pomelo [38]. Observations here also consistent with the study that two DNA bands of 930bp and 1,500 bp were specifically presented in chimeric donor N but not in donor F, and were hereditarily stable in two citrus chimeras NFF and FNN [2]. In this work, it is possible that the gene for the synthesis of α-carotene in the chimera OCC peel was completely dependent on the L2 source donor C. Even if the other donor O has this gene for synthesizing the α-carotene, the OCC cannot inherited from it or the relevant gene had successfully passed to OCC, but it just expressed in other tissues instead of peels, so that the substance cannot be synthesized in OCP. On the contrary, in juice sacs, α-carotene appeared in OCJ, this may because OCJ independently inherited the relevant gene from L1 source donor O. This independent inheritance was agreement with a citrus chimera Ekuliku, by comparing the certain quantitative traits with its donor plants, the color of the skin and flesh of the chimera was more independent because of its “loyalty” to the traits of their layer source donors [19]. These observations were consistent with the conclusion that juice sacs originate from L1 and peels originate from L2 [1–4, 19]. Additionally, in response to the absence and presence of these typical chemicals, such as α-carotene, it is suggested that which can be used as a characteristic pigment and a biochemical marker for genotype differentiation and assessment of citrus breeding programs.
Speculation of genetic laws in chimeras’ metabolites
Up to now, there is limited knowledge available with regards to the inheritance regularity of chemical compounds in plant chimeras. While small RNAs expressions and DNA methylation have recently been considered to be involved in stock-scion to describe genetic variations in graft chimeras. For instance, researchers have found some conserved miRNAs were differentially expressed in graft chimera (Brassica juncea + B. oleracea) progeny rTTT (sexual self-crossing of the chimera) and donor plant TTT (B. juncea), which may contributed to the changes in expression of their target genes [20]. In this work, the content of some chemicals in OCC were between two donors but some were deviated far from both of donor parents. These phenomenons might be related to the gene expressions of these chemicals. Then, we are wondering whether the genes from O and C are co-expressed in OCC, or only expressed one gene from one donor. Furthermore, in the graft chimeras Brassica juncea and B.oleracea, sequence analysis revealed that DNA methylation will affect the flowering time–and gibberellin response-related genes expressions, and may lead to the phenotypic variations in progenies [39]. According to the studies discussed above, chemical substances variations herein observed in OCC also suggested that whether the gene sequences of same gene in two donors were consistent, if not, will DNA methylation exist to influence chemical-related gene expression in OCC, and how these genes coordinate to regulate substances quantities are valuable to study more.