Reducing cell wall feruloylation by expression of a fungal ferulic acid esterase in Festuca arundinacea modifies plant growth, leaf morphology and the turnover of cell wall arabinoxylans

Abstract

A feature of cell wall arabinoxylan in grasses is the presence of ferulic acid which upon oxidative coupling by the action of peroxidases forms diferuloyl bridges between formerly separated arabinoxylans. Ferulate cross-linking is suspected of playing various roles in different plant processes. Here we investigate the role of cell wall feruloyaltion in two major processes, that of leaf growth and the turnover of cell wall arabinoxylans on leaf senescence in tall fescue using plants in which the level of cell wall ferulates has been reduced by targeted expression of the Aspergillus niger ferulic acid esterase A (FAEA) to the apoplast or Golgi. Analysis of FAE expressing plants showed that all the lines had shorter and narrower leaves compared to control, which may be a consequence of the overall growth rate being lower and occurring earlier in FAE expressing leaves than in controls. Furthermore, the final length of epidermal cells was shorter than controls, indicating that their expansion was curtailed earlier than in control leaves. This may be due to the observations that the deposition of both ether and ester linked monomeric hydroxycinnamic acids and ferulate dimerization stopped earlier in FAE expressing leaves but at a lower level than controls, and hydroxycinnamic acid deposition started to slow down when peroxidase levels increased. It would appear therefore that one of the possible mechanisms for controlling overall leaf morphology such as leaf length and width in grasses, where leaf morphology is highly variable between species, may be the timing of hydroxycinnamic acid deposition in the expanding cell walls as they emerge from cell division into the elongation zone, controlled partially by the onset of peroxidase activity in this region.

Fig 1

Levels of FAEA enzyme activity in whole young, mature and senescing leaves (A) and in whole mature roots (B) of control, apoplast (TR27, TR27R) and Golgi (T28 and T29) FAE expressing plants. Mean ±SEM (n = 3–5). Different letters indicate significant differences from the control (Tukey’s = 0.05). One unit of FAEA activity equals 1 μg ferulic acid released from ethyl ferulate in 24 h at 28°C.

Fig 2. Levels of FAE enzyme activity in 10 mm segments along 12–14 cm leaf blades starting at the cell elongation zone of control, apoplast (T27 and T27R) and Golgi (T28 and T29) FAE expressing plants.

Mean ± SEM (n = 2–3 replicates of 25–30 leaf sections from two independent transformed plants). L = ligula; EZ = extension zone. One unit of FAEA activity equals 1 μg ferulic acid released from ethyl ferulate in 24 h at 28°C.

Fig 3

Final leaf length (A) and leaf width (B) of control, apoplast (T27, T27R), and Golgi (T28 and T29) FAE expressing plants. Mean ± SEM (n = 10–15 leaves from individual plants), and plant phenotype (C) of twenty week old control (CONT) and apoplast (T27) and Golgi (T28) FAE expressing plants.

Fig 4

Root growth kinetics (A), final root length (B) and root morphology phenotypes (C) of tillers of control, apoplast (T27 and T27R) and Golgi (T28 and T29) expressing plants grown in hydroponic culture for three weeks. Mean ± SEM (n = 3–5). Letters indicate significant difference (Tukey’s, α = 0.05) among mean values.

Fig 5

Average epidermal cell length (A), width (B), area (C) and cell number per unit area at the base, middle and tip (D), of the abaxial epidermis of fully expanded leaves of control and apoplast (T27, T27R) or Golgi (T28 and T29) FAE expressing plants. Mean ± SEM n = 10 replicates of 10 leaves per plant, 1 cm from leaf tip in rows adjacent to the leaf midrib. Letters indicate significant difference (Tukey’s, α = 0.05) among mean values.

Fig 6. Comparison of growth of newly emerging third leaves of control and apoplast (T27 and T27R) or Golgi (T28 and T29) FAE expressing plants.

Growth as increase in leaf length (A), distribution of growth within the elongation zone of leaf blades, determined as the relative segmental elongation rate (B), maximum daily extension rate (C), and leaf length at maximum extension rate (D). Mean ± SEM (n = 20–30 from each plant). Third leaves from tillers of 2–3 plants per line were measured until leaf length was constant. Letters indicate significant difference (Tukey’s, α = 0.05) among mean values.

Fig 7. Chlorophyll content of leaves of control and an FAE expressing plant (T27R) during leaf senescence.

Fig 8

Total ester+ether linked HCAs in cell wall AIR isolated from 1cm leaf sections (A), total (soluble and ionically bound) peroxidase activity (B) in 5mm sections along 14–16 cm leaves starting from the elongation zone of control and apoplast (T27 and T27R) or Golgi (T28 and T29) FAE expressing plants. Leaf sections (120–130) were pooled by location along the leaf blade from 8–9 tillers of 2 to 3 replicates per line from 2 independent FAE expressing plants.

Fig 9

Ester + ether linked HCAs (A-C) and ester linked HCAs (D-F) in cell wall AIR isolated from 10mm segments along 14–16 cm leaves starting from the elongation zone of control and apoplast (T27 and T27R) or Golgi (T28 and T29) FAE expressing plants. p-coumaric acid (A, D), ferulate monomers (B, E), and total ferulate dimers (C, F). Leaf sections (120–130) were pooled by location along the leaf blade for each of 2 to 3 replicates per line. Ferulate monomers = trans-ferulic + cis-ferulic acid. Ferulate dimers = 8-0-4’-diferulate + 5–5’diferulate + 8–5 cyclic diferulate (benzo form) + 8–5’-diferulate + an unknown ferulate dimer. Mean ± SEM (n = 4–6 from 2 independent FAE expressing plants).

Fig 10. Ester linked HCAs in mature roots.

p-coumaric acid (A), ferulate monomers (B) and ferulate dimers (C). Ferulate monomers = trans- ferulic+ cis-ferulic acid. Ferulate dimers = 8-0-4’-diferulate + 5–5’ diferulate + 8-5cyclic diferulate (benzo form) + 8–5’-diferulate + an unknown ferulate dimer. Mean ± SEM (n = 2). Different letters indicate significant differences from the control (Tukey’s = 0.05).

Fig 11. Transverse sections through the central vascular bundles of elongating leaf blades, 10 cm distal to the ligula, stained with toluidine blue.

Control (B), apoplast (T27) (A) and Golgi (T28) (C). The metaxylem element (MX), abaxial fiber bundle (B) and phloem (PH), are indicated. Bar = 50 μm.

Fig 12

Ether+ ester (A, B, C) and ester linked (D, E, and F). p-coumaric acid (A, D), ferulate monomers (B, E) and ferulate dimers (C,F) in isolated cell wall AIR of young, mature and senescing leaves of control and apoplast (T27 and T27R) or Golgi (T28 and T29) FAE expressing plants. Ferulate monomers = trans- ferulic + cis-ferulic acid. Ferulate dimers = 8-0-4’-diferulate + 5–5’diferulate + 8-5cyclic diferulate (benzo form) + 8–5’-diferulate + an unknown ferulate dimer. Mean ± SEM (n = 3). Columns with different letters indicate statistically significant differences (P < 0.05).

Fig 13

Arabinose (A) and xylose (B) composition of isolated cell wall AIR extracted from young, mature and senescing leaves of control and FAE expressing plants. Mean ± SEM (n = 3). Columns with different letters indicate statistically significant differences (P < 0.05).