Cell wall modifications in maize pulvini in response to gravitational stress.

Changes in cell wall polysaccharides, transcript abundance, metabolite profiles, and hormone concentrations were monitored in the upper and lower regions of maize (Zea mays) pulvini in response to gravistimulation, during which maize plants placed in a horizontal position returned to the vertical orientation. Heteroxylan levels increased in the lower regions of the pulvini, together with lignin, but xyloglucans and heteromannan contents decreased. The degree of substitution of heteroxylan with arabinofuranosyl residues decreased in the lower pulvini, which exhibited increased mechanical strength as the plants returned to the vertical position. Few or no changes in noncellulosic wall polysaccharides could be detected on the upper side of the pulvinus, and crystalline cellulose content remained essentially constant in both the upper and lower pulvinus. Microarray analyses showed that spatial and temporal changes in transcript profiles were consistent with the changes in wall composition that were observed in the lower regions of the pulvinus. In addition, the microarray analyses indicated that metabolic pathways leading to the biosynthesis of phytohormones were differentially activated in the upper and lower regions of the pulvinus in response to gravistimulation. Metabolite profiles and measured hormone concentrations were consistent with the microarray data, insofar as auxin, physiologically active gibberellic acid, and metabolites potentially involved in lignin biosynthesis increased in the elongating cells of the lower pulvinus.


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Mid-season root lodging in cereals can cause considerable reductions in grain yield.
Depending upon the stage of development, the lodged plants may or may not attain a vertical orientation, which is important in mitigating yield loss. Even partial recovery to the upright orientation, referred to as goose-necking in plant breeding, can help minimize yield losses (Dhugga, 2007). In the work reported here, we examined changes that occur when maize plants are subjected to gravitational stress through placing them in a horizontal orientation.
Alterations in cell wall polysaccharide composition and architecture in response to such gravitational stresses have been studied in woody plants, where responses vary between species. In gymnosperms, gravitational forces increase cell growth in the lower region of a tree branch and result in the formation of compression wood in that region. In angiosperms gravitational responses include the formation of tension wood in the upper regions of branches. Compression wood typically has thickened secondary walls in its tracheids, and higher cellulose microfibril angles and lignin contents (Wilson and Archer, 1977). In contrast, tension wood in angiosperms is characterized by a gelatinous layer in the secondary wall, in which cellulose content increases, but cellulose microfibril angle and lignin content decrease (Wilson and Archer, 1977;Andersson-Gunneras et al., 2006).
Information on changes that occur in cell wall polysaccharides in the grasses in response to gravistimulation is more limited. Gravitropic responses are initiated in leaf sheath pulvini in festucoid grasses such as barley, wheat and oats, and in both leaf sheath pulvini and culm pulvini in panicoid grasses such as in maize (Brown et al., 1959). Collings et al. (1998) examined the growth dynamics and cytoskeleton organization when maize shoots were subjected to gravitational stimulation by placing them in a horizontal position. These authors reported the induction of a gradient of cell elongation in different regions of stem nodes, where maximal elongation was observed in the cells of the lowermost pulvini but where negligible cell elongation was detected in the cells of the uppermost pulvini. Differential cell 6 elongation in the lower and upper zones of the pulvinus causes the shoots to return to the upright orientation (Kaufman et al., 1987;Collings et al., 1998).
At the polysaccharide level, Montague (1995) reported that [ 14 C]-glucose was incorporated into cell wall fractions in whole oat pulvini in response either to gravistimulation or upon treatment with the phytohormone, auxin. Biosynthesis of both cellulose and wall matrix polysaccharides increased in the pulvini. Furthermore, Gibeaut et al. (1990) noted that (1,3;1,4)-β-D-glucan content was higher in the lower pulvini than in the upper regions of oat pulvini, which was consistent with their observations that (1,3;1,4)-β-D-glucan synthase activities increased in the lower regions of the pulvini. In an apparently contradictory report, Lu et al. (1992) reported that during the biosynthesis of (1,3;1,4)-β-D-glucan in oat plants in response to gravistimulation, an equal amount of [ 14 C] glucose was incorporated into the upper and lower regions of oat pulvini, and that the activities of β-D-glucan synthases I and II were similar in the two regions (Lu et al., 1992).
If it is accepted that cells in the lower region of the pulvinus elongate at a higher rate than those in the upper pulvinus during the re-orientation of plant growth from the horizontal to the vertical position (Kaufman et al., 1987;Collings et al., 1998), then one might anticipate that differential stimulation of the upper and lower regions by auxin would be involved in this process. Long et al. (2002) suggested that there is a transient gradient of IAA across maize pulvinal tissue shortly after gravitational stimulation, which would support this suggestion.
Here, changes that occur in the pulvinus during the re-orientation of maize plants from the horizontal to the vertical position have been defined. Detailed temporal and spatial changes in wall composition have been monitored using linkage analysis of wall polysaccharides and chemical analyses of lignin contents. Transcriptional activities of approximately 45,000 maize genes in the upper and lower regions of the pulvinus have been monitored through microarray analyses and changes in transcript levels were consistent with changes observed in the amounts and fine structures of major wall components. The transcriptional patterns of genes involved in the interconversion of sugar nucleotides, together with metabolite profiles, could also be reconciled with changes in the relative proportions of heteroxylans and glucosecontaining wall polysaccharides, and lignin. The microarray data further suggested that the transcription of genes encoding enzymes involved in phytohormone biosynthesis differentially changed in the upper and lower regions of the pulvinus, and in the cases of auxin, gibberellins and abscisic acid, the transcript profiles of the 'biosynthetic genes' were shown to correspond with the actual levels of these hormones measured in tissue extracts and with the expected biological functions of the hormones.

Cells in the Lower Pulvinus Elongate During Gravitational Stimulation
The bending capacity of maize pulvinus during the gravitropic responses observed here varied depended on the position of the pulvinus in the plant and on the age of the plant. Thus, pulvini from node 9 to node 13, where node 1 was the lowest on the plant, were responsive to gravitational stimulation in 60-65 day-old maize plants ( Fig. 1A and Supplemental Fig. S1), while pulvini from node 8 and below did not show signs of curvature. The thickness of the pulvinus from node 10 was 9-11 mm on the lowermost surface in the gravitationally stimulated maize plants, but only 3-4 mm on the uppermost surface; the latter was the same thickness as that measured in control plants (Fig. 1B).
The thicknesses of the upper and lower sides of stem pulvini from node 10 were correlated with the lengths of cells in these regions. Although the lengths of cells in the pulvini varied considerably, in the lower regions of pulvini in gravistimulated maize plants most sclerenchyma cells were 400 µm or longer, while in control plants most of the sclerenchyma cells in pulvini were in the range of 150-250 µm in length (Supplemental Table SI). Overall, the average lengths of sclerenchyma cells from node 10 were 200 µm, 220 µm and 498 µm in pulvini of controls, upper regions and lower regions, respectively. Parenchyma cells were about 3.5 fold longer in the lower pulvini than in control pulvini (Supplemental Table SI).
Thus, there was a 2.5-3.5 fold increase in the length of these cell types, which was consistent with the differences in length of the pulvini overall, as described above. Cell elongation in leaf sheath pulvini followed the same pattern as in stem pulvini (data not shown).

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In both control and gravistimulated plants, pulvini from the lower nodes on the plant had a higher resistance to puncture penetration than pulvini from the top nodes (Fig. 2). In addition, the lower pulvini in the gravistimulated plants were mechanically stronger than upper pulvini, as measured by resistance to the puncture probe (Fig. 2). The upper pulvini from nodes 9 and 10 of the gravistimulated plants showed similar resistance to the puncture probe as control pulvini. The increase in puncture strength in the lower pulvini was up to approximately 45%, compared with controls ( Fig. 2). In nodes 11 and 12 puncture strength is higher in both the lower and upper pulvini and it may be that these nodes, being younger and weaker than nodes 9 and 10, would need to increase strength on both sides to sustain a vertical orientation in a bent plant.

Lignin Contents of Walls Increased in the Lower Pulvinus
There was a substantial, statistically significant increase in lignin content (approximately 40%) in the lower pulvinus of nodes 9 and 11 in gravitationally stimulated maize stalks (Fig.   3A), compared with the upper pulvinus. The increase in lignin on the upper side relative to the control was not statistically significant. There was also an increase in ester-linked ferulic acid and p-coumaric acid on the lower side of the pulvinus (data not shown).

Heteroxylan Levels and Fine Structures Change in Walls from Lower Pulvini
Monosaccharide linkage analyses showed no significant difference in cellulose content in control, upper and lower stem pulvini, as determined by levels of (1,4)-glucosyl residues in the cell wall preparations (Tables I and II), and this was confirmed using acetic acid/nitric acid procedures (Updegraff, 1969;Fig. 3B). Similarly, no significant differences were detected in the levels of either pectic arabinan or type II arabinogalactan (Table II).
However, the linkage analyses indicated that the total xylose content of walls increased in the lower pulvini of both nodes, but more particularly in node 11 (Table I), where increases of 10 about 50% were observed in total xylosyl residues in the lower pulvinus compared with values obtained for controls and upper pulvini from node 11 (Table I). This was mainly attributable to an increase in (1,4)-xylosyl residues, which represent un-substituted backbone xylosyl residues in the polysaccharide. In pulvini from node 11, levels of both total xylosyl residues and (1,4)-linked xylosyl residues were significantly higher in the lower pulvini, compared with the upper pulvini; increases were less pronounced in node 9 where increases in both the upper and lower pulvinus were observed (Table I). Consistent with the increases in (1,4)-linked xylosyl residues, total arabinosyl residues decreased in lower pulvini from node 11 and this was at least partly attributable to a reduction in terminal arabinosyl residues (Table I). This resulted in an increase in ratio of xylosyl to arabinosyl residues from about 2.0:1 to about 3.9:1 in the lower pulvini from node 11 ( Table I). As noted above, the changes in pentose sugar contents in pulvini from node 9 were not as pronounced as in pulvini from node 11, but they nevertheless moved in the same direction. It is important to note that the amounts of terminal arabinofuranosyl derivatives were generally close to the total amounts of substituted xylopyranosyl derivatives, namely the levels of (2,4)-, (3,4)-and (2,3,4)xylopyranosyl derivatives (Table 1). The latter correspond to 2-, 3-and (2,3)-substituted xylosyl residues of the (1,4)-xylan backbone of the polysaccharide. This indicated that most of the substituents on the xylan chain were arabinosyl residues and for this reason we have referred to the polysaccharide in this paper as an arabinoxylan. However, we acknowledge that some glucuronosyl substituents might be present.
Mannosyl residues decreased in the lower pulvini from both nodes, and the analyses indicated that (1,4)-linked mannosyl residues were largely responsible for these decreases (Table I).
When the linkage analysis data were used to estimate the relative abundance of different classes of polysaccharides in the walls of the upper and lower pulvini, the results indicated that levels of heteroxylans increased by about 35% in the lower regions of pulvini in node 11, and that galactoglucomannans and xyloglucans decreased by about 50% and 30%, respectively, in lower pulvini of node 11; it should be noted that the levels of galactoglucomannans and xyloglucans in the walls were relatively low (Table II).
In the interpretations suggested above for the data presented in Tables I and II, it is important to emphasize that only values that are statistically significant to the p < 0.05 level, which are indicated in bold font in the tables, were considered.

Consistent with Increases in Pentose-Containing Polysaccharides
As noted above and shown in Table II (Zhang et al., 2005;Sharples and Fry, 2007;Reiter, 2008).
Microarray data analysis showed that UGPP, UGE, UGAE and UXS genes were all upregulated by more than 2-fold in the lower pulvini 24 h after the initiation of gravistimulation, based on the relative abundance of specific mRNAs (Table III). Eight probes on the microarray corresponded to UGPP genes. One of the UGPP genes (probe 4201585) showed a significant increase in mRNA levels in lower pulvini from both stems and leaves at 24 h (Table III; data for leaves not shown), consistent with increased channelling of carbon into sugar nucleotides. There were no significant changes in transcription of this gene in upper pulvini. Six probes were identified for the UGE genes. The mRNA levels of one of the UGE genes (probe 4303210) increased by about 2.5-fold in both the upper and lower regions of the pulvinus by 24 h (Table III).
The microarray contained seven probes for UGAE genes. The transcript levels of one of the genes (probe 4249531) increased by 6.4-fold in lower pulvini from stems at 1 h and remained high at 24 h ( Table III). One of the seven microarray probes for UXS genes (probe 4241027) detected increases of 2.2-fold and 4.1-fold in mRNA levels for the gene in lower stem pulvini at 6 and 24 h, respectively (Table III). Although there was an increase in transcript level of this gene in upper pulvini at 1 h, this had returned to control levels by 24 h (Table III). The increases in abundance of UXS gene transcripts implied increased production of UDP-Xyl and UDP-Ara, and were consistent with the increased levels of pentose-containing polysaccharides in the walls of the lower pulvini.

Microarray Analyses of Transcription of Glycosyltransferase Genes
The microarray contained approximately 40 probes for cellulose synthase (CesA) genes, including ZmCesA1, ZmCesA2,ZmCesA4,ZmCesA5,ZmCesA6,ZmCesA8,ZmCesA9,ZmCesA11 and ZmCesA12. No (Table IV). A family GT8 glycosyltransferase gene was transcribed at significantly 13 higher levels than controls in both the upper and lower pulvini, and at different times after the imposition of the gravitational stress. In the upper pulvinus, levels of the GT8 gene transcripts decreased after 6 h (Table IV) (Table IV). Transcripts for the third family GT61 gene (probe 4285578) increased two-fold in the lower pulvinus, but remained unchanged in the upper pulvinus (Table IV). Finally, a gene encoding a GT34 enzyme was transcribed at high levels after 1 h only, but in both the upper and lower pulvinus (Table IV).

Altered Transcription of Genes Involved in Heteroxylan Modification
Arabinofuranosidases from several families of glycosyl hydrolases (GH) catalyse the removal of α-arabinofuranosyl groups from the (1,4)-β-xylan backbone of arabinoxylans (Cantarel et al., 2009). Three probes on the microarray were annotated as arabinofuranosidase genes.
Transcriptional activities of two of these genes (probes 4295907 and 4292768), both of which belong to family GH51 and are likely to be arabinoxylan arabinofuranohydrolases (AXAH; Lee et al., 2001), did not change significantly in either the upper or lower pulvini (Table V).
However, mRNA levels of the other gene (probe 4211175), which encodes a GH3 family enzyme, increased substantially in the lower pulvinus at 24 h (Table V). Thus, the changes in this transcript are consistent with the decreased arabinosyl content of the arabinoxylan in the lower pulvinus (Tables I and II). In contrast, mRNA levels of a gene detected with probe 4280469, which also encodes a family GH3 enzyme but is annotated as a β-xylosidase, increased by more than three-fold in both lower and upper pulvini at 6 h ( Table V). The mRNA level in the lower pulvinus returned to the level in control plants, but the mRNA level 14 in the upper pulvinus remained 3.7 fold higher than the level in the control pulvinus at 24 h (Table V).
The mRNA level of one of these genes (probe 4291762) increased substantially in both lower and upper stem pulvini (Table V). Transcript levels of another xylanase inhibitor gene (probe 4217504) were significantly higher in the lower pulvinus at 6 h ( Table V). Both genes encode xylanase inhibitors of the XIP class and belong to the GH18 family (Cantarel et al., 2009).

Increased Transcriptional Activities of Other Genes Involved in Wall-loosening in the Lower Pulvinus
Enzymes and non-enzymic proteins such as polysaccharide endo-and exohydrolases, XETs and expansins have been implicated in cell wall loosening during cell growth in plants (Catala et al., 1997;Cosgrove, 2000;Hrmova et al., 2009). Thirteen probes were annotated as either endo-or exo-β-D-glucanase genes on the microarray. The mRNA level of a family GH9 gene (probe 4314428) increased significantly in lower stem pulvini, but not in the upper pulvinus (Supplemental Table SII). A family GH3, putative β-glucosidase gene (probe 4250320) also showed increased transcriptional activity in the lower pulvinus, but not in the upper pulvinus (Supplemental Table SIII). Eleven probes were identified as (1,3;1,4)-βglucanase genes on the microarray. One of these (probe 4297356) showed a significant increase in mRNA levels in the lower pulvinus (Supplemental Table SII). Thirty four probes were annotated as encoding (1,3)-β-D-glucanase genes. Two of these (probes 4240483 and 4218229) showed a significant increase in transcript levels in the lower pulvinus (Supplemental Table SII). Another two (probes 4279302 and 4302273) showed increased transcription in both lower and upper pulvini (Supplemental Table SII).
Forty one microarray probes were annotated as β-expansin genes. Four (probes 4224980, 4258316, 4259490 and 4271529) showed more than four-fold increases in transcript levels in the lower pulvinus (Supplemental Table SIII). An additional two probes with the same accession number as probe 4259490 also indicated increases in lower pulvini (data not shown). Another two of the putative β-expansin genes (probes 4222177 and 4280874) showed increased transcriptional activities in both lower and upper pulvini. One putative βexpansin gene (probe 4307195) showed decreased transcription activity in both the upper and lower pulvini (Supplemental Table SIII).
Nineteen of the microarray features encoded family GH16 XET genes. One (probe 4278710) showed a significant increase in transcript levels in the lower pulvinus (Supplemental Table   SIV), while another (probe 4265553) showed increased transcriptional activity in both the lower and upper pulvini. Two probes with the same Genebank accession number were annotated as a family GH16 xyloglucan (1,4)-β-glucan endohydrolases (probe 4238076).
Transcripts of this gene increased in both the lower (6 h) and upper (1 h) pulvinus (Supplemental Table SIV).

Transcript Levels of Genes Involved in Lignin Metabolism
Microarray analyses showed that of more than 50 probes representing lignin biosynthetic genes (Supplemental Table SV), levels of mRNA for a cinnamoyl-CoA reductase (CCR) gene (probe 4245740) increased significantly (p < 0.05) 1 h after the commencement of gravistimulation, in both the lower and upper pulvini (Supplemental Table SV). Transcripts for this gene remained 2.4-fold higher in the lower pulvinus at 24 h, compared with control plants, but returned to control levels in the upper pulvini during this time. Transcripts of a cinnamate-4-hydroxylase (C4H) gene (probe 4268958) increased substantially after 1 h in upper pulvini. Similarly, putative hydroxycinnamoyl transferase (HCT) gene transcripts appeared to increase early after gravistimulation (probe 4229300). In each of these three cases, increases in transcript abundance was higher in the upper pulvinus (Supplemental Table SV), which can be contrasted to the actual measurements of lignin content, which showed higher levels in the lower pulvinus (Fig. 3A). Transcript levels for three peroxidase genes (probes 4267626, 4213200 and 4225259) increased in the lower pulvinus, usually 6-24 h after the initiation of gravistimulation (Supplemental Table SV).

Transcript Levels of Genes Involved in Hormone Metabolism Changed in the Pulvinus
Among the genes involved in auxin biosynthesis, mRNA levels for anthranilate 5phosphoribosyltransferase (probe 4250382) increased about 2.7-fold in lower stem pulvini at 6 h and remained high at 24 h, but did not change in the upper pulvinus (Table VI). No changes in transcription levels were found for other genes involved in auxin biosynthesis.
The transcript levels of an auxin conjugate hydrolase gene (probe 4282474) increased 12-to 40-fold in both lower and upper pulvini at 1 h, but returned to control levels at 6 h ( Table VI).
Transcripts of an ent-kaurene synthase gene (probe no 4308055), which encodes an enzyme that catalyses the formation of ent-kaurene from geranylgeranyl diphosphate during GA biosynthesis (Hedden and Kamiya, 1997), increased 3-to 4-fold in both upper and lower stem pulvini 6 h after gravistimulation (Table VI). Transcript levels of a GA 20-oxidase gene (probe 4218360) increased more than 3-fold at 1 h and remained high at 24 h in the lower pulvinus (Table VI). Although the mRNA level of the GA 20-oxidase gene also increased at 1 and 6 h, it had decreased to control levels by 24 h. The increase in transcription of a GA 2oxidase gene (probe 4235513) was more than 13-fold at 1 h and remained high at 6 h in both upper and lower stem pulvini (Table VI). However, the mRNA level decreased to control levels in the upper pulvinus but remained high in the lower pulvinus at 24 h.

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The microarray included probes of putative violaxanthin de-epoxidase, molybdenum cofactor sulfurase and 9-cis-epoxycarotenoid dioxygenase, which are enzymes involved in the ABA biosynthetic pathway. In addition, genes encoding abscisic acid 8-hydroxylase and cytochrome 450 monooxygenase, which are involved in ABA catabolism, were represented on the microarray. Of these genes, mRNA of an ABA hydroxylase gene (probe 4316583) increased substantially in both upper and lower pulvini (Table VI).
The transcripts of a cytokinin oxidase gene (probe 4296711) increased by more than fourfold in both lower and upper pulvini at 6 h (Table VI), remained high in the lower pulvinus, but returned to control levels in the upper pulvinus at 24 h. Another cytokinin oxidase gene (probe 4291827) showed increased transcription, but only in the lower pulvinus. There were no changes in transcripts of cytokinin biosynthetic genes.
As an indication of ethylene metabolism in the gravitationally stimulated pulvini, transcript levels of two ACC synthase genes were monitored with the microarray. The mRNA levels of one of these genes (probe 4286393) was about seven-fold higher in upper than lower pulvini at 1 h (Table VI), while the mRNA level of the other ACC synthase gene (probe 4205461) was higher in lower pulvini (Table VI). When mRNA levels of two ACC oxidase genes (probes 4291681 and 4266528) were measured, both were 10-fold higher in the upper pulvinus than in the lower pulvinus (Table VI). If the increases in transcripts of ACC synthase and ACC oxidases were translated to increased enzyme activities, these changes would indicate that there might be a higher concentration of ethylene in upper pulvini in the early stage of the gravitropic response.

Microarray Data Confirmed by Quantitative-PCR
To confirm the microarray analyses, changes in transcript levels of selected genes were monitored in upper, lower and control pulvini by quantitative-PCR (Q-PCR), using experimental and data normalization procedures described by Burton et al. (2004). The direction and approximate quanta of changes in transcript levels were confirmed for all genes analysed, and some examples are presented in Supplemental Fig. S2.

Hormones were Measured in Extracts of the Pulvinus
The changes in mRNA levels described above for genes that have been implicated in the synthesis and degradation of various phytohormones suggested that temporal and spatial changes were occurring in hormone concentrations. To check this, the actual concentrations of a range of auxins, cytokinins, ABA and GA were measured in tissue extracts, together with related metabolic intermediates and conjugated forms of these hormones. Ethylene was not measured in the experiments described here.
Auxin Pulvinus from control maize plants contained 100-130 ng/g dry weight IAA, but this was almost halved after 1 h gravistimulation. However, IAA levels increased to 300 and 460 ng/g dry weight at 6 h after gravistimulation in upper and lower pulvini, respectively (Table   VII). Levels of IAA conjugates including IAA-Ala, IAA-Asp, IAA-Leu and IAA-Glu were very low, with less than 10 ng/g dry weight in the maize pulvinus (Table VII; data not shown except IAA-Glu). Although there are two types of IAA conjugates in plants, namely ester and amide conjugates, ester conjugates were not measured here due to the technical difficulties with the assay. However, the IAA ester conjugates are about five times more abundant than amide conjugates in maize vegetative tissues (Bandurski and Schulze, 1977).
Abscisic Acid (ABA) Maize pulvini contained 150-200 ng/g dry weight ABA in control plants, while in response to gravistimulation, ABA levels decreased slightly at 1 h in the upper pulvinus and increased by two-and six-fold at 6 h in upper and lower pulvini, respectively (Table VII). However, high levels of ABA degradation products were also detected at 6 h in the lower pulvinus. Dihydrophaseic acid (DPA) was the major ABA metabolite in the pulvinus (Table VII). Its level was seven-to nine-fold higher than other 19 ABA metabolites, which implied that ABA degradation was occurring via 8'-hydroxylation and conversion of 8' hydroxyl ABA to DPA via phaseic acid (PA) (Nambara and Marion-Poll, 2005)( Table VII). The levels of 7'-hydroxy ABA and 9'-hydroxy ABA were extremely low.

Gibberellin (GA)
GA4 and GA7 are active hormones derived via the non-13hydroxylation pathway from metabolites of GA12, GA15, GA24 and GA9, while GA1 and GA3 are also active hormones, but are derived from the early-13 hydroxylation pathway in which GA53, GA44, GA19 and GA20 are intermediate metabolites (Hedden and Kamiya, 1997;Hedden and Phillips, 2000). Only GA9 and GA4 from the non-13-hydroxylation pathway were quantifiable by the hormone profiling method. Gravistimulation increased GA4 and GA9 from undetected levels to about 17 and 50 ng/g dry weight in the lower pulvinus at 6 h, respectively. All four metabolites from the early-13 hydroxylation pathway were quantified. The GA53 metabolite, which initiates the early 13-hydroxylation pathway (Hedden and Kamiya, 1997), increased in upper pulvini 6 h after gravistimulation. However, it decreased sharply in the lower pulvinus at 6 h (Table VII). Further, GA20 was undetectable in control and upper pulvini but increased to about 40 ng/g dry weight in the lower pulvinus. The levels of GA44 and GA19 did not change in the lower pulvinus after gravistimulation.

Cytokinin
The maize extracts contained substantial amounts of cis-zeatin (cZ) (Veach et al., 2003), which is converted to cis-zeatin-O-glucoside (cZOG) as a storage conjugate by ciszeatin-O-glycosyltransferase (cZOGT). The reverse reaction from cZOG to active cZ is catalysed by a family GH1 β-glucosidase. The cZ can also be irreversibly removed by the action of cytokine oxidase (CKX) (Veach et al., 2003). The cZOG was most abundant in maize pulvini, while the level of trans-zeatin-O-glucoside (tZOG) was about eight-fold lower than cZOG in maize pulvini (Table VII). There were no differences in levels of tZOG and 20 cZOG in control, upper and the lower pulvinus. Isopentenyladenosine (iPA) and trans-zeatin riboside (tZR) levels were very low in control, upper and lower pulvini. Active cZ was not detected by the hormone profiling method used here.

Metabolite Profiles were Consistent with Wall Changes in Lower Pulvini
Metabolite profiles were compared in the upper and lower regions of pulvini from node 10 at 12 h and 24 h after the plants were placed in the horizontal position and a summary of changes observed at 24 h is presented in Fig. 4. A relatively small number of metabolites changed significantly after 12 h, including phosphorylated sugars of central metabolic pathways, phenyalanine and ethanolamine (Supplemental Table SVI S2). These changes are consistent with, but not necessarily indicative of, increased sugar metabolism through the TCA cycle, increased protein biosynthesis and increased synthesis of monolignols. It is possible that increased TCA cycle activity and protein synthesis would be required for the increased cell elongation observed in the lower pulvini, and that the increased phenylalanine and tyrosine levels were related to the increased lignin content in lower pulvini ( Fig. 3A). The increase in inositol at 12 h but not 24 h is consistent with reports by Perera et al. (1999), who showed that an initial increase in inositol 1,4,5-trisphosphate returned to base levels by 30 h after gravistimulation. Heteroxylans play key structural roles in the matrix phase of cell walls from both dicots and monocots, but are especially abundant in walls of the Poaceae (Carpita, 1996;Fincher, 2009;Scheller and Ulvshov, 2010). Levels of heteroxylans increased about 35% in the lower pulvinus of gravistimulated maize plants (Table II). Cellulose, which together with arabinoxylan accounts for 70-80% by weight of the walls (Table II), did not change 22 significantly in either the upper or lower pulvini of the gravistimulated maize stalks and this is consistent with the observations of Lu et al. (1992) in gravistimulated pulvini of oats. It is noteworthy that the absolute levels of cellulose assayed via the acetic acid/nitric acid method are in the range 35-40% by weight (Fig. 3B), while those deduced from the linkage analyses are in the range 50-55% by weight (Table II). The discrepancy between these values is probably attributable, in part at least, to differences in the units of expression of cellulose content. In the acetic acid/nitric acid method, cellulose is expressed as a percentage of total cell wall weight (the ethanol insoluble residue), which contains lignin, while in the linkage analysis, it is expressed as a percentage of measured monosaccharides, not including lignin.

DISCUSSION
The discrepancy could also be partly attributable to different forms of cellulose. Crystalline cellulose is measured by the acetic acid/nitric acid method, while both crystalline and noncrystalline cellulose will be measured in the linkage analyses (Burton et al., 2010b). Levels of the minor components of the walls, namely the heteromannans, xyloglucans and (1,3;1,4)β-glucans, appeared to be similar or slightly lower in the walls of the lower pulvini of the plants (Table II). Although the genes that mediate arabinoxylan biosynthesis have not been  (Table IV).
Perhaps the most important observations with respect to the composition of the cell walls in the pulvini were that the fine structure of the arabinoxylan changed and that the changes could be reconciled with the increased strength of the lower pulvini. More specifically, the degree of substitution of the arabinoxylan with arabinofuranosyl residues decreases markedly in the lower pulvinus, particularly in node 11 (Table I), which appears to have the highest angle of curvature (Fig. 1A). This is reflected in higher levels of (1,4)-xylosyl residues, fewer terminal arabinofuranosyl residues, and a higher Xyl:Ara ratio (Table I)  The transcript analyses revealed changes in the activity of a number of other genes that are involved in cell wall synthesis, wall loosening and re-modeling, and wall degradation. Firstly, the microarray data suggest that transcript levels of genes encoding sugar nucleotide interconverting enzymes altered in a manner consistent with the channelling of more carbon through the sugar nucleotide pathway overall via increased transcription of the UGPP gene, and the non-reversible commitment of carbon to UDP-Xyl and UDP-Ara formation through the action of UXS. In each case these changes can be rationalized with the increases observed in the increased arabinoxylan contents in the cell walls of the lower pulvinus (Table   II).
Secondly, levels of mRNA for several β-expansin genes increased from 4-to 27-fold in the lower pulvinus (Supplemental Table SIII), while mRNA for two XET genes increased 5-to 11-fold in the lower pulvinus (Supplemental Table SIV). Both β-expansins and XETs have been implicated in cell wall loosening, which would be required for differential elongation of  Table SII (1,3)-β-glucanase genes appeared to be transcribed in a highly co-ordinate manner in response to gravitational stress (Fig. 5).
The increased abundance of mRNA for (1,3;1,4)-β-glucan endohydrolases in the lower pulvinus (Supplemental Table SII) might be responsible for the decrease in (1,3;1,4)-β-glucan in the walls of the lower pulvinus, although the decrease is small in absolute terms (Table II).
These enzymes are active in vegetative tissues of barley and appear to be under hormonal control (Slakeski and Fincher, 1992). The small but significant decreases in (1,3;1,4)-βglucan content in the walls of the lower maize stalk pulvinus can be contrasted to increases in (1,3;1,4)-β-glucan content of walls in the lower leaf sheath pulvini of detached segments of oat stems (Gibeaut et al., 1990). These differences are probably attributable to significant differences in the experimental procedures used to impose the gravitational stress.
The microarray results for genes involved in lignin metabolism (Supplemental Table SV) (Table VI). Maize vegetative tissues contain up to 13-times higher concentrations of esterified IAA conjugates than free IAA (Bandurski and Schulze, 1977) and the increase in IAA concentration in the lower pulvinus at 6 h presumably resulted from the action of the auxin conjugate hydrolases (Table VII). It appears there might have been a pause in auxin synthesis or redistribution in the first hour, perhaps to adapt to stress, but auxin subsequently increased as expected, more so on the lower side than the upper side of the pulvinus. One might speculate that this reflects the redistribution of auxin efflux carriers (PIN proteins) after the plants were placed in a horizontal position (Noh et al., 2003;Petrasek et al., 2006). No changes were observed in mRNA levels for the 10 PIN genes measured by the microarray.

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Transcript levels of the anthranilate phosphoribosyltransferase gene, which is involved in auxin biosynthesis, increased about 2.7-fold in the lower pulvinus but remained essentially unchanged in the upper pulvinus (Table VI). Similarly, mRNA for two auxin response factors (ARF) increased more than 2-fold in the lower pulvinus, while mRNA for the Aux/IAA protein, which suppresses the auxin response, decreased at 6 h in the lower pulvinus. Based on these data, auxin levels would be expected to be higher in the lower pulvinus at 6 h, and this was indeed observed (Table VI).
In the case of ABA, which normally functions as a general growth inhibitor (Nambara and  (Table VII). Given the inhibitory functions of ABA in cell elongation, together with the fact that ABA levels are determined by a precise balance between biosynthesis and catabolism of the hormone (Nambara and Marion-Poll, 2005), it is difficult to interpret the increased levels of ABA in the lower pulvinus (Table VII), where cell elongation is occurring at a rapid rate. However, the observation might reflect the net effect of balances in hormones, if a 4-fold increase in auxin was too much for sustainable cell expansion and a 1.5-fold increase in ABA was necessary to partly offset increased auxin.

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It is possible that the ABA concentrations observed in tissue extracts here are related to GA levels, given that ABA and GA often act as antagonists in plants. The functions of GA are extensive, but include the promotion of stem elongation (Hedden and Kamiya, 1997). On this basis one might expect active GA levels to be higher in the elongating cells of the lower pulvinus. Transcript levels of ent-kaurene synthase, which is involved in GA 12 -aldehyde biosynthesis, increased in both the upper and lower pulvini, as did the levels of mRNA for a number of GA oxidases (Table VI). The GA 20-oxidase enzyme catalyses successive oxidations of C-20 methyl groups of GAs in non-13-hydroxylation and early 13hydroxylation pathways (Hedden and Kamiya, 1997). The non-13 hydroxylation pathway generates active GA1 and GA3, while the early 13-hydroxylation pathway produces active GA4 and GA7. However, there were also substantial increases in GA intermediates such as GA53 at 6 h (Table VII) in the upper pulvinus, but GA53 decreased significantly in the lower pulvinus. This indicated a clear difference in GA metabolism in the two regions of the straightening maize pulvini. Overall, the GA forms that increased in the lower pulvinus were GA4, GA9 and GA20 and, although these were not the most abundant forms of GA, they might be the active forms or the immediate precursors of active forms in the elongating cells of the lower pulvinus (Table VII).
Cytokinins (CK), which generally promote cell division rather than cell elongation, were also measured in the pulvini of the maize plants. Active CKs include N 6 -(delta 2-isopentenyl)adenine (iPA), trans-zeatin (tZOG), cis-zeatin (cZOG), and dihydrozeatin (DZ) (Sakakibara, 2006). These are synthesized by a chain of reactions catalysed by phosphatases, adenosine kinases and adenine phosphoribosyltransferases. CKs are inactivated by cleavage of side chains with CK oxidases and CK dehydrogenases. Given that we did not detect high levels of cell division in either the upper or lower pulvini, one might expect either decreased synthesis of the hormone or its more rapid degradation. At the transcript level, the 30 microarray analyses showed that mRNAs for cytokinin oxidases 2 and 3, which remove CKs, were significantly elevated in the lower pulvini at 6 h (Table VI). This would suggest a reduction on active CK levels in the lower pulvini, consistent with cell elongation without associated cell division in these regions of the gravistimulated maize stalks. Transcription of genes encoding enzymes involved in CK biosynthesis, such as adenine phosphoribosyl transferase, did not appear to change significantly in the upper or lower pulvini. Despite the apparent increases in transcriptional activity of genes involved in CK degradation in the lower pulvinus (Table VI), we could not detect any significant changes in active CK levels measured in tissue extracts (Table VII).
Finally, changes were detected in transcripts of genes normally associated with ethylene biosynthesis, although we were not able to measure ethylene concentrations in extracts of the maize pulvini. In particular, transcripts of one ACC synthase gene (probe 428693) increased dramatically in upper stem pulvini at 1h, and to a lesser greater extent in lower pulvini, while increases in transcripts of a second ACC synthase (probe 4205461) were much higher in the lower pulvinus, particularly in the first 1 h (Table VI). Levels of ACC oxidase transcripts also increased substantially after 1 h, and increases were generally greater in the upper pulvinus (Table VI). Since both ACC synthase and ACC oxidase are involved in ethylene synthesis, these data would indicate that ethylene levels could be elevated in both regions of the pulvinus, but particularly in the upper pulvinus (Peck et al., 1998). It is clear that the changes in transcript levels of these two genes occur much earlier after gravistimulation than do those associated with ABA, GA and auxin metabolism. Ethylene has been implicated in many biological functions, including gravitropic responses and growth inhibition (Peck et al., 1998;Visser and Pierik, 2007), but its precise mode of action in the pulvinus of gravitationally stressed maize stalks cannot yet be defined in detail, although it appears it 31 might initiate a stress response in the affected zone of the stalk. The 4-to 5-fold increase in mannitol and sorbitol is consistent with this stress on the lower side of the pulvinus.
In summary, we have quantitated a significant number of changes that occur in the pulvinus of maize plants subjected to gravitational stress, and have summarized the major changes diagrammatically in Fig. 6. and lower pulvini, respectively (Fig. 1B). The central part of the pulvinus was discarded. For control plants, which were not subjected to gravistimulation, one-third of pulvinus was collected from either side of pulvinus and pooled. The central part of the disk was also discarded.

Cell Wall Preparation and Analysis
Pulvini were ground on liquid nitrogen and extracted with 80% ethanol 5-7 times. The ethanol insoluble residues were extracted with acetone and methanol once each and dried in a dessicator with dried silica gel. The ethanol-insoluble residues were used for cellulose, lignin content and polysaccharide linkage analysis. Cellulose was determined according to Updegraff (1969) with modifications as described in Burton et al. (2010).
Klason lignin was determined as described by Theander and Westerlund (1986) with the following modifications. Approximately 20 mg of the ethanol-insoluble residues of cell wall preparations (in duplicate) was hydrolysed with 0.2 mL 12M sulphuric acid for 1h at 35ºC, which was then diluted to 2 M and the sample heated at 121ºC for 1 h. The hot acid-insoluble 33 residue was collected on pre-weighed 25 mm 542 Whatman filter paper discs using a Whatman 1225 sampling manifold (Whatman Asia Pacific, Australia). The filters were washed in place with water and dried in a vacuum oven (40ºC) overnight before weighing.
Monosaccharide linkage analysis was performed on five biological replicates by methylation with methyl iodide in sodium hydroxide and DMSO as described by Ciucanu and Kerek (1984) followed by acid hydrolysis, reduction and acetylation as described in Sims and Bacic (1995). Monosaccharide linkages and relative polysaccharide proportions were deduced from the partially methylated alditol acetates that were separated and analysed by GC-MS as described by Zhu et al. (2005).

Measurement of Cell Length by Light Microscopy
Pulvini from the uppermost and lowermost surfaces of maize stems were sectioned longitudinally and examined under a light microscope. Cell length was measured using Image-Pro Plus software (MediaCybernetics, Inc USA) after calibrating with a cytometer.

Metabolite Profiling
Maize pulvini from node 10 were ground in liquid nitrogen. Metabolites from five biological The value of each compound was calculated on a weight basis relative to internal standards.
The fold differences between control and upper or between control and lower pulvini were calculated relative to the appropriate time point control. Only those metabolites which were statistically significant (P < 0.05) are listed in Supplemental Table SVI.

Measurement of Puncture Strength
Maize plants were harvested two weeks after the imposition of the gravitropic stress and the leaf sheath was pealed to reveal the pulvinus. A small punctual probe (2 mm 2 at tip) was mounted on the Instron 5543 single column testing system (Instron, 825 University Ave., Norwood, MA, USA) and flexure stress at maximum load was determined with a flat-faced, tungsten puncture probe of 1 mm 2 surface area and an anvil movement rate of 100 mm/min.
The penetration was set to stop when the peak penetration load dropped by 30%.

Plant Hormone Profiling
Maize pulvini were freeze-dried and homogenized.  3,4,7,8,9,19,20,24,29,34,44,51 and 53. They were either synthesized according to Abrams et al. (2003) and Zaharia et al. (2005) Table II Deduced cell wall polysaccharide contents in maize pulvini Monosaccharide linkages were summarized according to their representation of individual polysaccharides as described by Shea et al (1989) and Zhu et al (2005). Values in bold are statistically significant (p<0.05) using a student t-test.  The values are fold of changes compared with control. Significant changes (> 2 fold and p <0.05) are indicated in bold. Functions of genes were analysed according to Hayes et al. (2010) and are shown in the 'Annotation' column with GeneBank numbers in parentheses.

Probe
Lower Upper Annotation

Figure 1 Gravitropic response of maize stems
A maize plant (65 days old) was placed on the floor at an angle of about 20 degrees for 14 days. Pulvini from node 9 to node 12 were responsive to gravistimulation, but pulvini below node 9 did not show signs of curvature. Leaves have been removed to show stem curvature more clearly. The outline of a single pulvinus from node 10 is indicated by dashed lines (B). When excised, the pulvinus is a wedge-shaped disk with thin tissue on the upper region and thick tissue on the lower region. Approximately one-third of the upper region and one third of the lower region were collected, designated as upper and lower pulvinus, and used for cell wall polysaccharide and gene expression analysis (C). The middle part of the pulvinus was discarded.

Figure 2 Puncture strength of maize stem pulvini
Maize plants (65 days old) were placed at an angle of 20 o to the horizontal for 2 weeks. Puncture strength was tested on fresh pulvini from nodes 9 to 12, using a puncture probe.
Pulvini from control plants that had not been subjected to gravistimulation were also tested.  (Table II) produced compatible results with this acid digestion method. Hormones mediate changes in cell wall polysaccharide biosynthesis and increases in stem strength in the lower maize pulvinus. Maize stems contained abundant IAA ester conjugates. An increase of IAA conjugate hydrolase (IAA con hyd) mRNA at 1 h indicated that storage IAA was mobilized upon gravistimulation, which subsequently mediated changes in several classes of cell wall metabolic gene expression. These included nucleotide sugar inter-conversion genes, cell wall loosening genes, glycosyltransferases (GT) and arabinofuranosidases (AF). A consequence was an increase in unsubstituted heteroxylan, lignin and eventually stem strength. However, ethylene or the balance of ethylene with other phytohormones might play a role in an inhibition of cell wall changes and cell elongation in upper pulvinus, as indicated by the substantial increases in expression of ACC synthase (ACC syn) and ACC oxidase (ACC oxi) genes in both upper and lower pulvinus at early gravistimulation.

Supplemental Table SI. Cell elongation in control, upper and lower pulvini
Cell length was measured under a light microscope with the aid of Image-Pro Plus software. The cells were grouped according their length and the percentage of a particular group of cells was expressed.

Organs
Cell Types Cell  Expression of ZmCesA4, ZmCesA8, ZmCesA11 and ZmCesA12 at 24 h and anthranilate phosphoribosyltransferase (ZmPRT) and auxin conjugate hydrolase (ZmACH) genes at 1 h and 6 h was quantified by Q-PCR (B and D, respectively). The data were expressed as ratios for the gravistimulated to control plants for a comparison between microarray (A and C) and Q-PCR data.