A customized gene expression microarray reveals that the brittle stem phenotype fs2 of barley is attributable to a retroelement in the HvCesA4 cellulose synthase gene

Abstract

The barley (Hordeum vulgare) brittle stem mutants, fs2, designated X054 and M245, have reduced levels of crystalline cellulose compared with their parental lines Ohichi and Shiroseto. A custom-designed microarray, based on long oligonucleotide technology and including genes involved in cell wall metabolism, revealed that transcript levels of very few genes were altered in the elongation zone of stem internodes, but these included a marked decrease in mRNA for the HvCesA4 cellulose synthase gene of both mutants. In contrast, the abundance of several hundred transcripts changed in the upper, maturation zones of stem internodes, which presumably reflected pleiotropic responses to a weakened cell wall that resulted from the primary genetic lesion. Sequencing of the HvCesA4 genes revealed the presence of a 964-bp solo long terminal repeat of a Copia-like retroelement in the first intron of the HvCesA4 genes of both mutant lines. The retroelement appears to interfere with transcription of the HvCesA4 gene or with processing of the mRNA, and this is likely to account for the lower crystalline cellulose content and lower stem strength of the mutants. The HvCesA4 gene maps to a position on chromosome 1H of barley that coincides with the previously reported position of fs2.

Figure 1.

Comparisons of strength and cellulose content of barley brittle culm mutants and their parental lines. A, Maximum “load-to-bend” of the second and fourth internodes of the barley lines Ohichi (J755) and Shiroseto (J156) compared with the mutant lines X054 and M245, respectively. In both mutant lines and in both internodes, the strength of the stems of the mutant lines is significantly lower than that of the parental lines. B, Maximum load-to-break of dry leaves from the barley lines Ohichi (J755) and Shiroseto (J156) compared with the mutant lines X054 and M245, respectively. Again, the strength of the tissues from the mutant lines is lower than that of the parental lines. C, Crystalline cellulose determination (Updegraff, 1969) of stem cell walls from brittle stem mutants, where E = lower, elongation zone, M = upper, maturation zone, and T = transition zone. Both mutant lines showed reduced cellulose levels relative to their corresponding parental line. For each line, the lower, elongation zone showed the lowest cellulose content of the three zones tested. D, Fourth internode from a barley stem, showing the approximate positions of tissue sampling.

Figure 2.

Transcript abundance of cellulose synthase genes. A, Normalized abundance of mRNA levels for cellulose synthase genes in the two barley brittle culm mutants and their parental lines, showing reduced abundance of HvCesA4 transcripts in the mutant lines. B, Normalized abundance of mRNA levels for cellulose synthase genes in the two barley brittle culm mutants and their parental lines, showing a slight up-regulation in HvCesA7 and HvCesA8 transcripts in the mutant lines.

Figure 3.

Structure of the HvCesA4 gene in the barley brittle stem mutants. The HvCesA4 genes of both mutant lines have a 964-bp insertion in their first intron. The sequence of the insertion corresponds to a solo-LTR of the Sasanda class of type I Copia-like retrotransposons. Orange boxes represent exons, and black arrowheads indicate the positions of the primers used to amplify products from the cDNA (see Fig. 4).

Figure 4.

Products amplified from barley cDNA preparations using PCR primers spanning the first intron of the HvCesA4 gene. A, The bottom arrow indicates the 379-bp product amplified from cDNA, which was sequenced and confirmed to be the product of a normally spliced mRNA. The other two arrows (approximately 1,400 and 800 bp) indicate products assumed to arise from cDNAs synthesized from incorrectly spliced RNAs that still carry all or part of the LTR insertion in the first intron. The O and S lanes indicate the products from the Ohichi and Shiroseto parent lines, respectively, and M245 and X054 are the PCR products from the corresponding brittle culm mutant lines. B, A schematic diagram (not to scale) of the potential origin of the 503-bp cDNA product that was consistently isolated from PCR of the cDNA corresponding to transcripts of the M245 and X054 HvCesA4 mutants. Note that the 503-bp fragment is not clearly visible in A. Exons 1 and 2 are shown in black, and the normal splicing positions are indicated by N. The aberrant 503-bp product (spliced at position A) retains the first intron (green), but the retroelement (blue) is absent. The positions of the normal (N) and retroelement (A) consensus lariat sequences (CTRAY, where R = A or G and Y = C or T) are shown. In the mutant lines, a lariat could form either between the 5′ end of the intron and the lariat motif sequence in intron 1 during normal splicing (N) or between the 5′ end of the retroelement LTR and the lariat within the retroelement during aberrant splicing (A), leaving intron 1 in the mature mRNA.

Figure 5.

Genetic map location of the HvCesA4 gene on chromosome 1H of barley. The genetic map of chromosome 1H of barley shows the colocation of the HvCesA4 cellulose synthase gene and the locus for the fragile stem phenotype (fs2), which are flanked by the markers awrm1 and psr158. Selected double haploid lines from the Clipper × Sahara (CxS) mapping population (Karakousis et al., 2003) were used to map the HvCesA4 gene.