Phylogenetic analysis showed that TaCKX1 shares high sequence similarity with orthologues from other monocot species, including HvCKX1 in barley, ZmCKX1 in maize and OsCKX1 in rice (Figure 2), suggesting that comparative phylogenetic analysis based on sequence similarity is a powerful method to identify homologous genes and to predict their physiological functions in target species. Identification of orthologues across species is essential to predict gene function in newly sequenced genomes such as wheat for which a draft genome has recently been released (http://www.cerealsdb.uk.net/search_reads.htm). Indeed, when Gu et al. combined a phylogenetic analysis with comparative expression of Arabidopsis, poplar, maize and rice CKXs in various tissues, they observed a general association of sequence similarity with expression patterns. Phylogenetic analysis has been widely used to classify gene families and predict their functional orthologues, and has been successfully used to generate functional categories within large gene families such as the WRKY and bHLH transcription factor families [60, 61]. However, phylogenetic analysis does not allow the differential expression of homoeologous genes to be determined.
In this study, we isolated most members of the major multi-gene families, TaIPTs, TaCKXs, TaZOGs, and TaGLUs involved in cytokinin synthesis and metabolism. Potential functional homoeologues from the three genomes, A, B and D, were pre-screened based on their expression in selected tissue types and in developmental stages closely related to grain yield determination. By using a novel strategy with specific PCR primers shared among the three homoeologues of each family member, we have been able to reveal detailed expression profiles of family members of the four multigene families during leaf, spike and seed development using qRT-PCR. Such information is required before determining which are the key genes and homoeologues contributing to cytokinin homeostasis within specific organs and developmental stages. These key genes then become the target for further investigation and genetic manipulation toward yield improvement in a breeding programme.
In plants, the rate limiting enzyme in cytokinin biosynthesis is considered to be IPT, which functions by attaching the isopentenyl side chain to the N6 moiety of ADP or ATP. At least three of the six IPT genes tested in this study, TaIPT2
TaIPT5 and TaIPT8 showed specific expressed patterns during spike, carpel, seed and leaf development. All three genes were expressed at the highest levels at early seed development immediately after anthesis (Figure 7A), a developmental window coincident with the cell division period, during which the cell number of the endosperm is determined, and during which endogenous cytokinin levels peak [e.g. [19–21]. Expression of IPT family members was also detected in young spikes and developing carpels, suggesting that these genes should be further investigated as players in determining the number as well as the size of the reproductive organs. Interestingly, these TaIPT genes expressed at relatively low levels during the expansion and the fully expanded phase of the leaf. Together with similar results recently reported in maize [32, 33], this suggests that it would be possible to manipulate IPT activity during reproductive development without significantly affecting normal leaf functions. An initial target for enhanced expression could be TaIPT8, which does not express highly in leaves, thereby avoiding creating competition between sink and source.
The CKX gene family members have been shown to express in different plant tissues and to play essential roles in controlling cytokinin levels during plant growth and development . Information about the expression and physiological function of TaCKXs was only recently available and only for TaCKX1, 2 and 3[47–49]. While investigating the expression of ten sets of TaCKX genes, we showed that at least three genes are specifically or preferentially expressed during seed development (Figure 7B). The strong and specific expression of the TaCKX1 gene during early seed development provides a useful candidate target. By perturbing cytokinin homeostasis during this key yield determining stage, i.e. by down regulating the expression of any one of CKX1, 2 or 6 during seed development and disturbing the normal mechanism of cytokinin homeostasis, the resulting elevated cytokinin level may lead to enhanced seed number and size . Indeed, Zalewski et al. were able to increase grain yield of barley by down regulating the TaCKX1 orthologue, HvCKX1, using RNAi.
O-glucosylation is a major step in the metabolism of cytokinins. Since O-glucosylation is reversible and O-glucosides are resistant to cleavage of the N
6-side chain by CKX, this conversion forms part of the homeostatic control of active cytokinin levels through temporary storage of cytokinins . Interestingly, TacisZOG1 showed elevated expression pre-anthesis (Figure 3), at a time when endogenous O-glucosides start to accumulate in the developing ovule . Extremely high level of expression was detected for two cis-ZOG genes, TacisZOG1 and TacisZOG2-1. The dramatic increase of TacisZOG expression during leaf senescence in the present study agrees with early observations of high levels of O-glucosides in senescing leaves  and cytokinin inactivation by O-glucosylation promoted by senescence-related processes in petunia . In contrast, Vyroubalová et al. only detected low expression of ZmcisZOG in old leaves of maize. This difference might be due to differences in cytokinin metabolism during senescence in these species .
d-glucosidases are members of the GH1 family, which is implicated in the hydrolysis of a number of plant secondary metabolites, including the regulation of the biological activity of cytokinins . Until now, Zm-p60/ZmGLU1 in maize, TaGLU1 in wheat and Bgl4 in Brassica napus are the only enzymes for which the ability to release cytokinins from O-glucoside conjugation has been demonstrated [50, 65–67]. In wheat, only one homoeologous set of GLU genes, TaGLU1a, b and c, has been investigated , but with no information shown on in planta expression during reproductive and leaf development. Our qRT-PCR data showed high expression of TaGLU1-1, the equivalent of TaGLU1a in Sue et al. , in 0.5 cm spike, 10 cm spike and 2 cm leaf. Using a ZmGLU1P-GUS construct, Gu et al. observed a significant up-regulation of the maize β-glucosidase gene, ZmGLU1, in immature transgenic tobacco seeds. Vyroubalová et al. also detected high expression of ZmGLU in immature ears and young leaves of maize. Therefore, we speculated that this gene may play an important role in reproductive organ initiation and seed development.
Interestingly, and in agreement with the pattern shown in our preliminary study , at anthesis, when expression of IPT
CKX and ZOG genes was uniformly low in the flag leaf, the TaGLU1 gene showed a marked peak of expression, with over a 300-fold up-regulation. From anthesis, the flag leaf provides significant resources to the developing seed. We postulate that the high β-glucosidase gene activity at this stage could lead to the release of cytokinin from conjugation in the leaf and provide a source of cytokinin for the developing carpels/seeds. Clearly, experiments to support this supposition still need to be performed.
The modest correlation of response regulators with TaIPT gene expression during the early stage of seed development when endogenous cytokinin levels are substantially elevated [19–21] indicates that response regulators may not be good surrogates of endogenous cytokinin content. However, the elevated expression of members of all four gene families at 2 daa is indicative of dynamic turnover of cytokinins at this key stage of seed development.
An interesting trend shown by our data, and that of our preliminary study , is that several of the highly expressed genes in the seeds have low expression in the leaves and vice versa, further suggesting that expression of individual family members of the cytokinin synthesis and metabolism genes are tissue specific. As a monocarpic plant, wheat development is characterized by a rapid translocation of metabolites from leaves to developing grains after anthesis. Consequently, delayed leaf senescence at a late stage of seed development may not be beneficial to the final grain yield due to the decreased nutrient translocation from leaves to seeds as suggested by Sýkorová et al.. The independent and variable expression profiles of different members within or among cytokinin regulatory gene families provides useful flexibility for independently manipulating the endogenous bioactive cytokinin levels in reproductive and vegetative tissues to achieve maximal seed yield.
The expression levels shown in this study represent at least one of the three homoeologues for each locus in all cases apart from the TaZOG2-1
TaGLU1-1 and TaGLU1-2 genes, in which the three homoeologous genes showed different expression profiles. The third homoeologue in these cases displayed little activity in the initial screen for highly expressed genes. The expression among homoeologous genes of other cytokinin regulatory genes may also vary, or individual homoeologous genes could even be silenced in some or all of the tissue samples analysed. Homoeologous gene silencing and/or differential expression is a phenomenon widely documented in bread wheat [43–46] and other polyploid species including tetraploid cotton , and tetraploid Tragopogon mirus.
Given that homoeologous expression can be differentially regulated by genetic and epigenetic mechanisms in a tissue and developmentally-specific manner [43–45], detection and pyramiding of interesting homoeologues for improvement of target traits has recently been used in wheat breeding programs . For instance, in an attempt to improve the bread-making quality of hexaploid wheat, homoeologous recombination has been used to increase the copy number of the stronger flour quality contributing homoeologue, Glu-D1, of the HMW-GS gene. A partially isohomoeoallelic line, in which the chromosome 1A homoeologue was replaced by the chromosome 1D homoeologue, showed significantly improved bread making quality. The improved quality can be explained by the duplication of the Glu-D1 homoeologue [73, 74]. Using a different approach, TILLING in combination with homoeologue-specific primers, Slade et al. obtained a triple homozygote loss-of-function of the Wx gene and obtained a line with waxy phenotype and improved grain quality,
Therefore, from a breeding perspective, the above strategy, of accumulating targeted homoeologues, could also be applied to the cytokinin synthesis and metabolic gene families. To improve seed yield, higher endogenous cytokinin levels could be achieved through tissue-specific increased expression of multiple homoeologues of the IPT genes TaIPT2, TaIPT5 and TaIPT8, or through decreased homoeologue expression of CKX genes TaCKX1 and TaCKX2. This could be achieved using a number of genetic tools, such as over-expression, down-regulation, TILLING and MAS strategies. However, while the differential expression profile of each homoeologue elucidated in this study could be directly used for genetic manipulation towards the improvement of the grain yield, precise genome allocation of these positive homoeologues is important for further understanding the genetic control, and for facilitating the efficient accumulation, of these homoeologues within the same locus and across different loci.