Co-ordinate regulation of cytokinin gene family members during flag leaf and reproductive development in wheat

Abstract

Background: As the global population continues to expand, increasing yield in bread wheat is of critical importance as 20% of the world's food supply is sourced from this cereal. Several recent studies of the molecular basis of grain yield indicate that the cytokinins are a key factor in determining grain yield. In this study, cytokinin gene family members in bread wheat were isolated from four multigene families which regulate cytokinin synthesis and metabolism, the isopentenyl transferases (IPT), cytokinin oxidases (CKX), zeatin O-glucosyltransferases (ZOG), and β-glucosidases (GLU). As bread wheat is hexaploid, each gene family is also likely to be represented on the A, B and D genomes. By using a novel strategy of qRT-PCR with locus-specific primers shared among the three homoeologues of each family member, detailed expression profiles are provided of family members of these multigene families expressed during leaf, spike and seed development.

Results: The expression patterns of individual members of the IPT, CKX, ZOG, and GLU multigene families in wheat are shown to be tissue- and developmentally-specific. For instance, TaIPT2 and TaCKX1 were the most highly expressed family members during early seed development, with relative expression levels of up to 90- and 900-fold higher, respectively, than those in the lowest expressed samples. The expression of two cis-ZOG genes was sharply increased in older leaves, while an extremely high mRNA level of TaGLU1-1 was detected in young leaves.

Conclusions: Key genes with tissue- and developmentally-specific expression have been identified which would be prime targets for genetic manipulation towards yield improvement in bread wheat breeding programmes, utilising TILLING and MAS strategies.

Figure 1

The maximum likelihood (ML) phylogenetic tree for IPT proteins inArabidopsis thaliana(AtIPT),Oryza sativa(OsIPT), Triticum aestivum(TaIPT), andZea mays(ZmIPT). The tree was rooted using IPT protein from Rhodococcus fascians (RfIPT). Node values are percentages of bootstraps generated with 1000 bootstrap replicates.

Figure 2

The maximum likelihood (ML) phylogenetic tree for CKX proteins inArabidopsis thaliana(AtCKX),Oryza sativa(OsCKX), Triticum aestivum(TaCKX), andZea mays(ZmCKX). The tree was rooted using CKX protein from Rhodococcus fascians (RfCKX). Node values are percentages of bootstraps generated with 1000 bootstrap replicates.

Figure 3

The maximum likelihood (ML) phylogenetic tree for ZOG proteins inArabidopsis thaliana(AtZOG, UGT),Triticum aestivum(TaZOG, TacZOG),Zea mays(ZmZOG, ZmcZOG) andPhaseolus lunatus(ZOG1). The tree was rooted using cytokinin-N-glucosyltransferase 1 protein from Zea mays (ZmZNG1). Node values are percentages of bootstraps generated with 1000 bootstrap replicates.

Figure 4

The maximum likelihood (ML) phylogenetic tree for representative GLU proteins. At, Arabidopsis thaliana; Os, Oryza sativa; Ta, Triticum aestivum; Zm, Zea mays. The tree was rooted using β-galactosidase 3 protein from Oryza sativa (OsBGal3). Node values are percentages of bootstraps generated with 1000 bootstrap replicates.

Figure 5

Total chlorophyll content in flag leaf samples used for mRNA quantification of cytokinin regulatory genes in wheat. Flag leaf sample order: 2 and 15 cm in length, expanded, days before and after anthesis.

Figure 6

Developmental stages of spike, carpel and seed samples used for mRNA quantification of cytokinin regulatory genes in wheat observedusing SEM (A) and light microscopy (B-L).A, spike 0.5 cm in length; B, spike 2 cm in length; C, spike 10 cm in length; D, carpel 14 days before anthesis (dba); E, carpel 7 dba; F, carpel 1 dba; G, 1 day after anthesis (daa); H, 2 daa; I, 4 daa; J, 7 daa; K, 14 daa; L, 21 daa. a, anther; c, carpel; e, endosperm; f1-f5, florets 1–5 in a spikelet; fm, floral meristem; slm, spikelet meristem. Scale bars: A = 50 μm, B-E = 500 μm, F-K = 1 mm.

Figure 7

Quantitative expression profiles of selected putative cytokinin regulatory genes during carpel and seed development in bread wheatA, TaIPT genes; B, TaCKX genes; C, TaZOG genes; D, TaGLU genes; E, TaRR genes. Data values are means of relative mRNA levels in fold changes detected using qRT-PCR, using at least two technical replicates for each of the two biological replicates. Error bars represent the SD calculated for the combined technical and biological replicates. GAPDH, β-actin, 18 S rRNA, and protein phosphatase gene (PP2A), were used as internal controls. Before quantification of the expression level of each of the target genes, the Ct numbers for each target gene were corrected by using the average correction factor determined for each of the four reference genes.

Figure 8

Quantitative expression profiles of selected putative cytokinin regulatory genes during flag leaf development in bread wheat. See Figure 7 for legend.