Developmental changes occur in maize ( <i> Zea mays </i> L.) as it transitions from juvenile stages to the mature plant. Changes also occur as newly formed cells mature into adult cells. Maize leaf blades including the midribs and sheaths undergo cell wall changes as cells transition to fully mature cell types. As is common in grasses during cell wall maturation , the lignin in the plant tissue is acylated with <i> p </i> -coumarates ( <i> p </i> CA). This work characterizes cell walls in maize that make up leaf blade, leaf midrib, and sheath tissues corresponding to tissue development. Maize plants grown in the greenhouse were harvested; leaf, leaf midrib, and sheath tissues from nodes 9 through 14 tissues were analyzed for cell wall composition. Cell wall carbohydrates varied with the type of maize tissue, but there was little change within a tissue type among the different nodes. Lignin concentrations were lowest in the leaf blade (70 - 88 g·kg - 1 CW) followed by the sheath (123 - 140 g·kg - 1 CW) and highest in the midrib (140 - 168 g·kg - 1 CW). Incorporation of <i> p </i> CA into cell walls paralleled the lignification. Ferulates (FA) remained relatively constant as a proportion of the cell wall (3.1 - 6.4 g·kg - 1 CW) across nodes and across all tissue types. The range of FA was 3.8 vs 5.7 g·kg - 1 CW averaged over all nodes with leaf blades being the lowest. Lignin composition did not change significantly with cell wall maturation within a given tissue type. The aerial portions of maize plants excluding the stem showed little difference in cell wall composition along the different nodes. Higher levels of ferulates were found in the sheath and leaf midrib compared to the leaf blade tissues. Leaf midribs from the upper nodes of the plant contained the highest levels of lignin. Perhaps a reflection of the function to keep leaves extended and in an upward angle to help maximize photosynthetic capacity.
This Maize (Zea mays L.) is a prominent grain crop both in the United States and other countries throughout the world. In the dairy industry, it is an important nutritional biomass crop for milk production being preserved primarily as corn silage. With interest in biomass for energy production, there is potential for increased demand and competition in crop production between biomass for animal use and as a bioenergy source. No matter its final use it is important to maximize energy conversion efficiency from the crop to insure sufficient production is available to meet all food, fiber, and bioenergy needs. The fiber portion of plants is made of cell walls providing a structural framework to keep the plant upright and leaves extended to maximize photosynthetic capacity for continued growth and development and reproductive fitness. In grasses, like corn, accumulation of biomass occurs from the combined developmental processes of new cell formation, cell elongation, and cell wall thickening.
In maize development, leaves can be divided into groups based upon where they develop along the growing stem. Juvenile leaves are those that develop from seedling stage through internode 10 and have slightly different compositional characteristics compared to the adult leaves (above internode 10). Previous studies of cell wall composition in sequential leaves along the maize stem although limited, indicate variation exists. Bergvinson et al. [
Composition of cell walls can impact resistance to insect herbivory [
Corn inbred B73 (obtained from Mycogen) was grown in greenhouses at the U.S. Dairy Forage Research Center, Madison, WI. Plants were established (two per pot) in late winter of 2009 in three gallon pots, watered as needed to maintain adequate soil moisture and fertilized once weekly (Pete’s Soluble 10-10-10). Pots were labeled as one of three replicates and randomized within the greenhouse space. Four individual plants were harvested for each experimental replicate. Plants were cut through the seventh node (just above the soil line) with leaves and sheaths carefully removed from each stem. Nodes were labeled from 8 to the upper most internode that had undergone elongation (internode 14). Growing conditions can impact the leaf or internode number before plant development switches from a vegetative to a reproductive stage. Since all plants for this study were grown under controlled conditions in the greenhouse (14/10 day/night light regime, 26˚C - 28˚C night 30˚C - 34 ˚C day temperature), all plants developed relatively uniformly and leaves and sheaths were collected through node 14 and the tassel was removed for analysis. As leaves were removed from the stem the midrib of each was carefully excised from the leaf blade. Individual, leaf blades, sheaths, and midribs for each node were pooled across the four plants within an experimental replicate and processed, immediately frozen in liquid nitrogen and stored separately at −80˚C.
The Frozen samples (sheaths, leaf blades, midribs) were further processed by grinding in a Freezer Mill (6750, Spex Sample Prep) using a pre-cool interval of 10 minutes followed by three cycles of two-minute grind (oscillation rate setting of 10)-one minute rest interval. After milling samples were immediately transferred to 50 mL centrifuge tubes in liquid nitrogen before storing at -80˚C until further processing.
A sub-sample of frozen ground tissue material (sheath, leaf blade or midrib) was accurately weighed into 50 mL Oakridge tubes (5 g). Thawed samples were extracted three times with 50 mM NaCl buffer (2.5 mL∙g−1 sample). Samples were suspended in buffer, shaken (175 rpm) at 40˚C for 30 min before centrifuging at 2000 × g for 15 min (25˚C) and the buffer extract carefully removed from the insoluble residue/pellet. The three buffer extracts were combined, frozen in liquid nitrogen and stored at −80˚C until processed for soluble phenolics. Tris-Acetate buffer (50 mM, pH 6.7) was then added to each insoluble residue, and samples were placed in a 90˚C water bath for 2 h. Samples were transferred to a 55˚C water bath and incubated for 2 h after adding amylase (Sigma A3403 10 U/tube) and amyloglucosidase (Fluka 10115 10 U/tube) for starch removal. Ethyl alcohol (EtOH, 95%) was added to each tube to make the final EtOH concentration 80%. Samples were stirred with a spatula before centrifuging at 25000 × g for 15 min. Insoluble residues/pellets recovered from the starch extraction procedure were washed extensively (1 mL solvent/g fresh tissue). The solvent series included: 80% EtOH (2X), chloroform: methanol 2:1 (CHCl3: MeOH; 1X), and acetone (3X) to remove cytoplasmic contaminants [
Carbohydrate analyses. Cell wall residues were dried overnight in a 55˚C oven prior to weighing for wall analysis. Samples (~50 mg) were accurately weighed into Pyrex culture tubes (20 × 125mm) with Teflon lined caps tubes. Four to five glass beads were added to each tube to aid in mixing during hydrolysis. Samples were hydrolyzed using the Saeman method [
Lignin determination and analysis. Isolated cell walls were accurately weighed (~25 mg/sample) into glass culture tubes (16 mm × 150 mm, with Teflon lined caps) for lignin determination. The acetyl bromide method was use as modified by Hatfield et al [
Cell wall phenolics. Cell walls were analyzed for ester- and ether-linked phenolics using the procedure of Grabber et al [
To release ether linked phenolics the residual cell wall sample in the vials was allowed to stand in a hood overnight to evaporate small amounts of residual ether. To the cell wall suspensions 1.5 mL of 12M NaOH was added along with 100 mg of 2-hydroxycinnamic acid (1 mg∙mL−1 in 2N NaOH) and 100 mg of synthetic 5 - 5 diferulate (1 mg∙mL−1 in 2N NaOH). Vials were sealed and placed inside a second larger reaction vessel (5 Telfon vials per large reaction vessel) along with a few mL of water. The reaction vessels were heated in a forced air oven (170˚C) for 2 h. At the completion of the heating cycle, reaction vessels were cooled under cold water. Individual Teflon vials were opened and contents quantitatively transferred to Pyrex culture tubes (20 × 250 mm, with Teflon lined caps) with dH2O (2 × 1 mL). Released phenolics were extracted and analyzed as with the ester-linked materials using GLC-FID under similar conditions.
Data from individual experiments were subjected to analysis of variance (ANOVA) at the a = 0.05 significance level.
There are distinct changes in cell wall components as the corn plant matures. [
One would expect cell wall components (i.e., total neutral sugars, total uronosyls, total lignin and phenolics) to account for greater than 90% of the total dry matter isolated as corn cell wall. In this study, total neutral sugars and lignin account for nearly 90% of the total cell wall (
Using fresh (frozen at −80˚C) starting material helps in the cell wall clean up process to eliminate more of the cytosolic contaminates. Based on the cell wall analyses this would mean that of the original dry matter samples for leaf blade material was approximately 35% - 40% cell wall material and the remaining would be cell solubles made up of cytoplasmic materials. There was no consistent pattern of cell wall component changes within the leaf blade materials from the lowest node (most mature part of the plant) to the least mature the upper
Tissue | Corn internode | Neutral Sugars | Lignin g∙kg−1 | Uronosyls | Phenolics | Total Cell Wall |
---|---|---|---|---|---|---|
Leaf | Node 9 | 441.8 ± 39.3 | 73.6 ± 12.6 | 27.4 ± 0.5 | 4.8 ± 0.3 | 547.6 |
Node 10 | 546.9 ± 164.4 | 87.3 ± 6.5 | 24.1 ± 1.3 | 5.6 ± 0.8 | 663.8 | |
Node 11 | 487.3 ± 23.0 | 88.6 ± 6.0 | 21.6 ± 3.8 | 5.8 ± 1.2 | 603.2 | |
Node 12 | 522.4 ± 12.0 | 91.5 ± 9.2 | 23.7 ± 0.7 | 7.2 ± 0.8 | 644.9 | |
Node 13 | 521.6 ± 58.3 | 82.7 ± 5.0 | 23.9 ± 1.0 | 7.9 ± 1.6 | 636.0 | |
Node 14 | 534.0 ± 65.4 | 87.5 ± 13.1 | 20.8 ± 1.5 | 8.0 ± 0.7 | 650.3 | |
Midrib | Node 9 | 824.6 ± 60.3 | 140.4 ± 14.2 | 32.5 ± 2.4 | 12.3 ± 1.0 | 1009.8 |
Node 10 | 597.4 ± 49.1 | 150.8 ± 2.9 | 29.5 ± 2.8 | 13.9 ± 0.4 | 791.6 | |
Node 11 | 724.3 ± 170.2 | 146.9 ± 30.0 | 29.9 ± 1.9 | 15.1 ± 0.5 | 916.3 | |
Node 12 | 774.1 ± 62.2 | 152.6 ± 20.1 | 26.6 ± 3.4 | 17.2 ± 3.8 | 970.4 | |
Node 13 | 673.8 ± 87.0 | 170.1 ± 10.3 | 28.0 ± 2.8 | 15.3 ± 0.2 | 887.3 | |
Node 14 | 740.1 ± 104.7 | 158.4 ± 12.4 | 27.9 ± 1.9 | 16.9 ± 0.7 | 943.3 | |
Sheath | Node 9 | 767.4 ± 27.3 | 140.3 ± 8.7 | 38.9 ± 6.4 | 12.4 ± 1.5 | 958.1 |
Node 10 | 734.2 ± 20.7 | 134.3 ± 8.9 | 35.0 ± 6.8 | 13.3 ± 1.6 | 916.8 | |
Node 11 | 716.0 ± 87.3 | 127.2 ± 21.8 | 40.8 ± 2.1 | 14.8 ± 2.3 | 898.8 | |
Node 12 | 708.2 ± 44.1 | 139.6 ± 2.7 | 39.4 ± 2.6 | 14.2 ± 1.1 | 895.4 | |
Node 13 | 805.2 ± 118.1 | 136.4 ± 30.5 | 36.0 ± 5.8 | 14.6 ± 1.9 | 992.2 | |
Node 14 | 835.0 ± 47.5 | 123.1 ± 1.6 | 36.3 ± 2.1 | 14.9 ± 1.1 | 1009.3 |
most node (number 14). This may be expected because the function of the leaf blade tissue (largely mesophyll cells) is to carry out photosynthesis to supply metabolites for the rest of the growing plant. Since leaf development occurs from an intercalary meristem at the base of the leaf and the majority of the leaf used in this study examined fully expanded leaves one may not expect much of a change in cell wall composition. MacAdams and Grabber found grass leaf maturation occurred fairly rapidly once new cells were formed at the base of the leaf [
A detailed comparison of the individual chemical entities making up a given plant part (e.g., individual sugars within the total cell wall) does show minor shifts. A comparison of the three major sugars present in grass cell walls arabinose (Ara), xylose (Xyl), and glucose (Glc), which account for over 90% of the total neutral sugar composition, do show changes especially in the leaf blade tissues (
Lignin accumulation within the leaf blade, leaf midrib, and sheath showed no consistent overall trends among the tissue types. The leaf blades tended to have levels that held steady at around 8% to 9% irrespective of the node the leaf came from (
Although lignin subunits (syringyl vs guaiacyl) can change the amount of branching, it remains unclear as to the full impact of changing the proportion of S to G will have upon the overall functionality of the resulting lignin polymer. The leaf and midrib also show greater S:G in the older tissue at the base of the stem which has been shown to occur in older tissue with more secondary wall formation.
Phenolic components in grass cell walls are primarily hydroxycinnamates ferulates (FA) and p-coumarates (pCA). Both are incororated into the wall matrix as ester linked conjugates. FA is primarily attached to Ara side chains of arabinoxylan (AX) and glucuronoarabinoxylans (GAX). In the case of FA they can undergo dehydrogenative radical coupling reactions to form a cross-linked network of ferulate dimers binding xylan polymers together. Due to the electro-
Internode | Leaf S:G | Midrib S:G | Sheath S:G |
---|---|---|---|
9 | 2.21 ± 0.02 | 1.65 ± 0.81 | 5 0.9 ± 0.08 |
10 | 1 1.7 ± 0.08 | 5 1.1 ± 0.04 | 8 0.7 ± 0.02 |
11 | 7 1.2 ± 0.02 | 5 0.7 ± 0.01 | 6 0.8 ± 0.05 |
12 | 6 1.7 ± 0.02 | 6 1.0 ± 0.08 | 6 0.5 ± 0.01 |
13 | 9 0.7 ± 0.40 | 4 0.7 ± 0.02 | 8 0.7 ± 0.04 |
14 | 9 0.9 ± 0.13 | 0 0.8 ± 0.09 | 1 0.9 ± 0.06 |
chemical nature of FA radicals they can freely enter into radical mediated coupling reactions with radical species of guaiacyl and syringyl units on developing lignin polymers. This in turn produces a complex cross-linked wall matrix involving polysaccharides and lignin. In the case of pCA it can be incorporated into the cell wall matrix either as ester linked to Ara on AX or GAX polysaccharides or ester linked monolignols, primarily sinapyl alcohol. The pCA remains only attached by an ester linkage to the monolignol or Ara of the initial conjugate used to shuttle it out into the cell wall. Electrochemical properties of the pCA-monolignol conjugate (primarily pCA-SA) result in only the monolignol portion entering into radical mediated coupling reaction with other monolignols or FA. This results in pCA remaining attached only by the original ester bond to lignin polymers. Typically pCA does not become involved in cross-coupling types of reactions to link different components together within the wall matrix. [
Levels of pCA tend to track with concentration of lignin within the different plant tissue types (
Ester Linked | Ether linked | |||||||
---|---|---|---|---|---|---|---|---|
pCA | FA | pCA | FA | |||||
Tissue | Node | g∙kg−1 CW | g∙kg−1 CW | g∙kg−1 CW | g∙kg−1 CW | |||
Leaf | 9 | 1.70 ± 0.11 | 2.93 ± 0.18 | 0.03 ± 0.01 | 0.13 ± 0.01 | |||
10 | 2.06 ± 0.34 | 3.39 ± 0.46 | 0.03 ± 0.01 | 0.14 ± 0.03 | ||||
11 | 2.19 ± 0.34 | 3.41 ± 0.84 | 0.03 ± 0.01 | 0.15 ± 0.01 | ||||
12 | 2.75 ± 0.22 | 4.28 ± 0.49 | 0.04 ± 0.01 | 0.17 ± 0.03 | ||||
13 | 3.18 ± 0.70 | 4.50 ± 0.86 | 0.04 ± 0.01 | 0.15 ± 0.01 | ||||
14 | 3.28 ± 0.32 | 4.50 ± 0.32 | 0.04 ± 0.01 | 0.16 ± 0.01 | ||||
Midrib | 9 | 7.48 ± 0.71 | 4.56 ± 0.22 | 0.06 ± 0.01 | 0.24 ± 0.04 | |||
10 | 8.38 ± 0.19 | 5.17 ± 0.17 | 0.07 ± 0.00 | 0.27 ± 0.02 | ||||
11 | 9.26 ± 0.17 | 5.43 ± 0.24 | 0.08 ± 0.03 | 0.31 ± 0.08 | ||||
12 | 10.78 ± 2.10 | 5.86 ± 1.55 | 0.10 ± 0.01 | 0.42 ± 0.06 | ||||
13 | 9.69 ± 0.07 | 5.25 ± 0.11 | 0.09 ± 0.02 | 0.31 ± 0.04 | ||||
14 | 10.67 ± 0.22 | 5.86 ± 0.40 | 0.07 ± 0.01 | 0.31 ± 0.04 | ||||
Sheath | 9 | 6.74 ± 1.23 | 5.40 ± 0.23 | 0.05 ± 0.01 | 0.17 ± 0.03 | |||
10 | 7.23 ± 0.87 | 5.76 ± 0.64 | 0.06 ± 0.01 | 0.23 ± 0.05 | ||||
11 | 8.31 ± 0.70 | 6.12 ± 1.53 | 0.06 ± 0.02 | 0.25 ± 0.08 | ||||
12 | 8.20 ± 0.32 | 5.68 ± 0.76 | 0.06 ± 0.00 | 0.25 ± 0.01 | ||||
13 | 8.30 ± 1.32 | 5.93 ± 0.48 | 0.09 ± 0.01 | 0.30 ± 0.06 | ||||
14 | 9.13 ± 0.17 | 5.37 ± 0.89 | 0.09 ± 0.02 | 0.31 ± 0.05 | ||||
Ferulates on the other hand have less variation among the different tissue types. Leaf blade tissue ranged from 3 to 4.5 g∙kg−1 CW, midrib 4.6 to 5.9 g∙kg−1 CW, and sheath 5.4 to 6.1 g∙kg−1 CW (
A comparison of leaf blade, leaf midrib, and sheath tissues of the corn plant revealed different chemical compositions within the tissue types. However, within a given tissue type there were no obvious large changes in this chemical make-up from the bottom older nodes to the top younger nodes. The leaf blade, leaf midrib and sheath tissues along the stem do not reflect the changes seen in the “actively growing” (elongation and expansion) of the stem internodes [
Hatfield, R.D. and Marita, J.M. (2017) Maize Development: Cell Wall Changes in Leaves and Sheaths. American Journal of Plant Sciences, 8, 1248-1263. https://doi.org/10.4236/ajps.2017.86083