Powerful regulatory systems and post-transcriptional gene silencing resist increases in cellulose content in cell walls of barley

Abstract

Background: The ability to increase cellulose content and improve the stem strength of cereals could have beneficial applications in stem lodging and producing crops with higher cellulose content for biofuel feedstocks. Here, such potential is explored in the commercially important crop barley through the manipulation of cellulose synthase genes (CesA).

Results: Barley plants transformed with primary cell wall (PCW) and secondary cell wall (SCW) barley cellulose synthase (HvCesA) cDNAs driven by the CaMV 35S promoter, were analysed for growth and morphology, transcript levels, cellulose content, stem strength, tissue morphology and crystalline cellulose distribution. Transcript levels of the PCW HvCesA transgenes were much lower than expected and silencing of both the endogenous CesA genes and introduced transgenes was often observed. These plants showed no aberrant phenotypes. Although attempts to over-express the SCW HvCesA genes also resulted in silencing of the transgenes and endogenous SCW HvCesA genes, aberrant phenotypes were sometimes observed. These included brittle nodes and, with the 35S:HvCesA4 construct, a more severe dwarfing phenotype, where xylem cells were irregular in shape and partially collapsed. Reductions in cellulose content were also observed in the dwarf plants and transmission electron microscopy showed a significant decrease in cell wall thickness. However, there were no increases in overall crystalline cellulose content or stem strength in the CesA over-expression transgenic plants, despite the use of a powerful constitutive promoter.

Conclusions: The results indicate that the cellulose biosynthetic pathway is tightly regulated, that individual CesA proteins may play different roles in the synthase complex, and that the sensitivity to CesA gene manipulation observed here suggests that in planta engineering of cellulose levels is likely to require more sophisticated strategies.

Figure 1

Image generated using Strudel. Gray lines show homologous relationships between CesA genes in Sorghum (Sorghum bicolor), barley (Hordeum vulgare) and rice (Oryza sativa). Positions of CesA genes on the respective chromosomes are also indicated.

Figure 2

Photos of representatives from the T ₁ and T ₂ generations showing the aberrant phenotypes observed in 35S:HvCesA4 (A, B,C) and 35S:HvCesA8 (D,E,F) plants. (A) T₁ 35S:HvCesA4 plants and wild-type (WT) Golden promise on the far left. Dwarfism (d) persisted in most plants grown from parents with an aberrant phenotype except for one or two plants within the same line (e.g. plant NP, normal phenotype). The ratio of plants displaying dwarf: normal phenotype (including nulls & revertants) in T₁ is 58%: 42%. (B) Many T₂, 35S:HvCesA4 progeny were dwarfed with “brittle nodes” (d,B). About 25% of T₂ plants from each line exhibited a severe reduction in stature, was sterile (S) and some died. The plants with a severe phenotype may be homozygotes. (C) Close up view of necrosis found at the leaf-tips of a 1 month old plant that further developed into a dwarf plant with few viable grains. (D) T₁ 35S:HvCesA8 plants. Aberrant phenotypes observed were “brittle node” (B) and severely stunted plants that died young (S) (~1 month old). Plants with a “brittle node” phenotype had no reduction in stature but when pressure was applied manually, the stems snapped at the nodes. (E) T₂ 35S:HvCesA8 plants. About 25% of T₂ plants from each line were stunted and died young (S). Many were only brittle at the node (B) with no compromise in stature. (F) Comparison of two wild-type (left) and two transgenic “brittle node” stems (right). One stem each from wild-type and transgenic plant were sliced in half to reveal the stem’s internal anatomy. The bracket indicates the nodal region of the stem and at closer inspection the break-point was often found to be at the “nodal plate” (arrow).

Figure 3

Averaged transcript levels of four genes in transgenic 35S:HvCesA4 and 35S:HvCes8 T ₁ plants. X-axis depicts the transgenic lines and control plants (where n = number analysed). The transcript values were averaged for sibling lines with similar phenotype. Where possible, null segregants were selected from three different parental lines. For clarity between very high and low transcript levels, the Y-axis for normalised mRNA copies/microlitre is divided into two different scales (black and red). Error bar is the standard error of the mean (SEM) of biological variation between sibling lines. (A) Transcripts measured for SCW 35S:HvCesA4 transgenic plants were the HvCesA4 transgene and eHvCesA4, eHvCesA7 and eHvCesA8. Plants within the same line exhibited variations in phenotype. There were three independent lines with a dwarfed phenotype (black solid circle) and three other with a normal phenotype. (B) Transcripts measured for SCW 35S:HvCesA8 transgenic plants were the HvCesA8 transgene and eHvCesA4,eHvCesA7 and eHvCesA8. There were three lines that were stunted, sterile and died young (open circle), three lines with a “brittle node” phenotype (black solid circle) and three lines with a normal phenotype.

Figure 4

Cellulose content and stem strength data for T ₁ SCW 35S: HvCesA4 and 35S: HvCesA8 plants. (A) Cellulose content was measured as percent cellulose (%). There were three independent lines with a dwarfed and leaf necrosis phenotype (black solid circle) and three lines with a normal phenotype. (B) maximum flexural load, N, was a measure of stem strength. There were three independent lines with ‘brittle node’ phenotype (black solid circle) and three normal-looking transgenic plants. Plants that were severely stunted died at a young stage so were not available for cellulose content analysis. Error bars are standard error of the mean of biological replicates (n). Significant differences were determined by one-way ANOVA followed by post hoc Dunnett’s multiple comparisons test.

Figure 5

Immunofluorescent labelling of T ₂ 35S:HvCesA4 and T ₂ 35S:HvCesA8 internode cross-sections. (A) negative (same treatment as control and transgenic was applied but CBM3a was excluded), (B) control = wild type or nulls, (C) transgenic 35S:HvCesA4 plant from Line 11, (D) transgenic 35S:HvCesA4 plant from Line 15, (E) transgenic 35S:HvCesA8 plant from Line 14 and (F) transgenic plant from Line 20. Fluorescent images were taken at the same exposure and magnification for all samples. Scale bar is 100 μM. E = epidermis, VB = vascular bundle, PC = parenchyma cell.

Figure 6

Light microscopy of cross-sections of T ₁ 35S:HvCesA4 and 35S:HvCesA8 stem internodes stained with Toluidine Blue. Equivalent internodes were sectioned using vibratome (~30-50 μM thick) from (A, B) wild-type or null, (C, D) dwarfed 35S:HvCesA4 transgenic T₁ plants and (E, F) 35S:HvCesA8 transgenic T₁ plants. Red arrows indicate xylem vessels and in D, they are collapsed and irregular in shape. Scale bars denote 100 μM. E = epidermis, VB = vascular bundle, Ph = phloem tissue, Xy = meta-xylem, BS = bundle sheath, PC = parenchyma cells.

Figure 7

TEM and measurement of SCW thickening for control and T ₂ transgenic plants. (A) Xylem cell wall of WT, (B) Xylem cell wall of T₂ dwarf 35S:HvCesA4, (C) Sclerenchyma cell wall of WT and (D) Sclerenchyma cell wall of T₂ dwarf 35S:HvCesA4. Scale bar is 1 μM for (A, B) and 10 μM for (C, D). Cy = Cytoplasm of bundle sheath cell, ML = middle lamella. (E, F) Percent AIR extracted (w/w) from 35S:HvCesA4, 35S:HvCesA8 and control from stem tissues. (G) % reduction of xylem cell wall thickness as measured using ImageJ.