Comparing regional transcript profiles from maize primary roots under well-watered and low water potential conditions.

Regionally distinct elongation responses to water stress in the maize primary root tip have been observed in the past. A genetic basis for such differential responses has been demonstrated. Normalized bar-coded cDNA libraries were generated for four regions of the root tip, 0-3 mm (R1), 3-7 mm (R2), 7-12 mm (R3), and 12-20 mm (R4) from the root apex, and transcript profiles for these regions were sampled. This permitted a correlation between transcript nature and regional location for 15 726 expressed sequence tags (ESTs) that, in approximately equal numbers, derived from three conditions of the root: water stress (water potential: -1.6 MPa) for 5 h and for 48 h, respectively, and well watered (5 h and 48 h combined). These normalized cDNA libraries provided 6553 unigenes. An analysis of the regional representation of transcripts showed that populations were largely unaffected by water stress in R1, correlating with the maintenance of elongation rates under water stress known for R1. In contrast, transcript profiles in regions 2 and 3 diverged in well-watered and water-stressed roots. In R1, transcripts for translation and cell cycle control were prevalent. R2 was characterized by transcripts for cell wall biogenesis and cytoskeleton formation. R3 and R4 shared prevalent groups of transcripts responsible for defence mechanisms, ion transport, and biogenesis of secondary metabolites. Transcripts which were followed for 1, 6, and 48 h of water stress showed distinct region-specific changes in absolute expression and changes in regulated functions.

The maize root transcriptome by serial analysis of gene expression.

Serial Analysis of Gene Expression was used to define number and relative abundance of transcripts in the root tip of well-watered maize seedlings (Zea mays cv FR697). In total, 161,320 tags represented a minimum of 14,850 genes, based on at least two tags detected per transcript. The root transcriptome has been sampled to an estimated copy number of approximately five transcripts per cell. An extrapolation from the data and testing of single-tag identifiers by reverse transcription-PCR indicated that the maize root transcriptome should amount to at least 22,000 expressed genes. Frequency ranged from low copy number (2-5, 68.8%) to highly abundant transcripts (100-->1,200; 1%). Quantitative reverse transcription-PCR for selected transcripts indicated high correlation with tag frequency. Computational analysis compared this set with known maize transcripts and other root transcriptome models. Among the 14,850 tags, 7,010 (47%) were found for which no maize cDNA or gene model existed. Comparing the maize root transcriptome with that in other plants indicated that highly expressed transcripts differed substantially; less than 5% of the most abundant transcripts were shared between maize and Arabidopsis (Arabidopsis thaliana). Transcript categories highlight functions of the maize root tip. Significant variation in abundance characterizes transcripts derived from isoforms of individual enzymes in biochemical pathways.

Growth of Arabidopsis thaliana seedlings under water deficit studied by control of water potential in nutrient-agar media.

We have characterized the growth responses of Arabidopsis thaliana seedlings to water deficit. To manipulate the water potential, we developed a method whereby the nutrient-agar medium could be supplemented with polyethylene glycol (PEG 8000); PEG was introduced into gelled media by diffusion, which produced media with water potential as low as -1.6 MPa. For dark-grown plants, hypocotyl growth had a hyperbolic dependence on water potential, and was virtually stopped by -1 MPa. In contrast, primary root elongation was stimulated by moderate deficit and even at -1.6 MPa was not significantly less than the control. That these results did not depend on a direct effect of PEG was attested by obtaining indistinguishable results when a dialysis membrane impermeable to PEG was placed between the medium and the seedlings. For light-grown seedlings, moderate deficit also stimulated primary root elongation and severe deficit reduced elongation only partially. These changes in elongation were paralleled by changes in root system dry weight. At moderate deficit, lateral root elongation and initiation were unaffected and at higher stress levels both were inhibited. Primary root diameter increased steadily with time in well-watered controls and under water deficit increased transiently before stabilizing at a diameter that was inversely proportional to the deficit. Along with stimulated primary root elongation, moderate water deficit also stimulated the rate of cell production. Thus, A. thaliana responds to water deficit vigorously, which enhances its use as a model to uncover mechanisms underlying plant responses to water deficit.

Abscisic acid accumulation maintains maize primary root elongation at low water potentials by restricting ethylene production.

Previous work showed that primary root elongation in maize (Zea mays L.) seedlings at low water potentials (psi(w)) requires the accumulation of abscisic acid (ABA) (R.E. Sharp, Y. Wu, G.S. Voetberg, I.N. Saab, M.E. LeNoble [1994] J Exp Bot 45: 1743-1751). The objective of the present study was to determine whether the inhibition of elongation in ABA-deficient roots is attributable to ethylene. At a psi(w) of -1.6 MPa, inhibition of root elongation in dark-grown seedlings treated with fluridone to impose ABA deficiency was largely prevented with two inhibitors of ethylene synthesis (aminooxyacetic acid and aminoethoxyvinylglycine) and one inhibitor of ethylene action (silver thiosulfate). The fluridone treatment caused an increase in the rate of ethylene evolution from intact seedlings. This effect was completely prevented with aminooxyacetic acid and also when ABA was supplied at a concentration that restored the ABA content of the root elongation zone and the root elongation rate. Consistent results were obtained when ABA deficiency was imposed using the vp5 mutant. Both fluridone-treated and vp5 roots exhibited additional morphological symptoms of excess ethylene. The results demonstrate that an important role of ABA accumulation in the maintenance of root elongation at low psi(w) is to restrict ethylene production.

Root Growth Maintenance at Low Water Potentials (Increased Activity of Xyloglucan Endotransglycosylase and Its Possible Regulation by Abscisic Acid).

Previous work suggested that an increase in cell wall-loosening contributes to the maintenance of maize (Zea mays L.) primary root elongation at low water potentials ([psi]w). It was also shown that root elongation at low [psi]w requires increased levels of abscisic acid (ABA). In this study we investigated the effects of low [psi]w and ABA status on xyloglucan endotransglycosylase (XET) activity in the root elongation zone. XET is believed to contribute to wall-loosening by reversibly cleaving xyloglucan molecules that tether cellulose microfibrils. The activity of XET per unit fresh weight in the apical 10 mm (encompassing the elongation zone) was constant at high [psi]w but increased by more than 2-fold at a [psi]w of -1.6 MPa. Treatment with fluridone to decrease ABA accumulation greatly delayed the increase in activity at low [psi]w. This effect was largely overcome when internal ABA levels were restored by exogenous application. Spatial distribution studies showed that XET activity was increased in the apical 6 mm at low [psi]w whether expressed per unit fresh weight, total soluble protein, or cell wall dry weight, corresponding to the region of continued elongation. Treatment with fluridone progressively inhibited the increase in activity with distance from the apex, correlating with the pattern of inhibition of elongation. Added ABA partly restored activity at all positions. The increase in XET activity at low [psi]w was due to maintenance of the rate of deposition of activity despite decreased deposition of wall material. The loss of activity associated with decreased ABA was due to inhibition of the deposition of activity. The results demonstrate that increased XET activity is associated with maintenance of root elongation at low [psi]w and that this response requires increased ABA.

Response of Fructan to Water Deficit in Growing Leaves of Tall Fescue.

Changes in dry matter and water-soluble carbohydrate components, especially fructan, were examined in the basal 25 mm of expanding leaf blades of tall fescue (Festuca arundinacea Schreb.) to assess their roles in plant response to water deficit. Water was withheld from vegetative plants grown in soil in controlled-environment chambers. As stress progressed, leaf elongation rate decreased sooner in the light period than it did in the dark period. The decrease in growth rate in the dark period was associated with a decrease in local relative elongation rates and a shortening of the elongation zone from about 25 mm (control) to 15 mm. Dry matter content of the leaf base increased 23% during stress, due mainly to increased water-soluble carbohydrate near the ligule and to increased water-soluble, carbohydrate-free dry matter at distal positions. Sucrose content increased 258% in the leaf base, but especially (over 4-fold) within 10 mm of the ligule. Hexose content increased 187% in the leaf base. Content of total fructan decreased to 69% of control, mostly in regions farther from the ligule. Fructan hydrolysis could account for the hexose accumulated. Stress caused the osmotic potential to decrease throughout the leaf base, but more toward the ligule. With stress there was 70% less direct contribution of low-degree-of-polymerization fructan to osmotic potential in the leaf base, but that for sucrose and hexose increased 96 and 67%, respectively. Thus, fructan metabolism is involved but fructan itself contributes only indirectly to osmotic adjustment.

Spatial distribution of turgor and root growth at low water potentials.

Spatial distributions of turgor and longitudinal growth were compared in primary roots of maize (Zea mays L. cv FR27 x FRMo 17) growing in vermiculite at high (-0.02 megapascals) or low (-1.6 megapascals) water potential. Turgor was measured directly using a pressure probe in cells of the cortex and stele. At low water potential, turgor was greatly decreased in both tissues throughout the elongation zone. Despite this, longitudinal growth in the apical 2 millimeters was the same in the two treatments, as reported previously. These results indicate that the low water potential treatment caused large changes in cell wall yielding properties that contributed to the maintenance of root elongation. Further from the apex, longitudinal growth was inhibited at low water potential despite only slightly lower turgor than in the apical region. Therefore, the ability to adjust cell wall properties in response to low water potential may decrease with cell development.

Characterization of fructan from mature leaf blades and elongation zones of developing leaf blades of wheat, tall fescue, and timothy.

Water-soluble carbohydrate composition of mature (ceased expanding) leaf blades and the elongation zone of developing leaf blades was characterized in wheat (Triticum aestivum L.), tall fescue (Festuca arundinacea Schreb.), and timothy (Phleum pratense L.). These species were chosen because they differ in mean degree of polymerization (DP) of fructan in the mature leaf blade. Our objective was to compare the nature and DP of the fructan. Vegetative plants were grown with a 14-hour photoperiod and constant 21 degrees C at the leaf base. Gel permeation chromatography of leaf blade extracts showed that the apparent mean fructan DP increased in the order wheat < tall fescue < timothy. Apparent mean DP of elongation zone fructan was higher than that of leaf blade fructan in wheat and timothy, but the reverse occurred for tall fescue. Low DP (10) pools were found in both tissues of tall fescue and wheat, but concentration of low DP fructan was very low in either tissue of timothy. All three species have high DP fructan. Comigration with standards on thin-layer chromotography showed that wheat contained 1-kestose and a noninulin fructan oligomer series. Tall fescue contained neokestose, 1-kestose and higher oligosaccharides that comigrated with neokestose-based compounds and inulins. Thin-layer chromatography showed that small amounts of fructose-containing oligosaccharides were present in timothy.

Diurnal Growth of Tall Fescue Leaf Blades : II. Dry Matter Partitioning and Carbohydrate Metabolism in the Elongation Zone and Adjacent Expanded Tissue.

The spatial distributions of net deposition rates of water soluble carbohydrate-free dry matter (WSC-free DM) and WSC were evaluated within and above the elongation zone of tall fescue (Festuca arundinacea Schreb.) leaf blades during light and darkness. Imported DM used for WSC-free DM synthesis during darkness (67% of the total in experiment I and 59% in experiment II) was greater than during light (47% in both experiments), suggesting that the 65% higher leaf elongation rate during darkness was accompanied by higher rates of synthesis of cellular structural components. Deposition rates of WSC in the basal and central part of the elongation zone (0-20 mm from the ligule) were similar during light and darkness, but above 20 millimeters WSC deposition occurred during light and WSC loss occurred during darkness. WSC deposition and loss throughout the elongation zone and the recently expanded tissue were mostly due to net synthesis and degradation of fructan. Fructan was predominantly low molecular weight and contributed about 50% of the total osmotic partial pressure of WSC. In the most actively growing region, where fructan synthesis was most rapid, no diurnal change occurred in molecular weight distribution of fructan. WSC solute concentrations were diluted in the most actively growing tissue during darkness because net monosaccharide and fructan deposition were unaltered and sucrose deposition was decreased, but growth-associated water deposition was increased by 77%. Net rates of fructan synthesis and degradation were not related to tissue sucrose concentration, but appeared to respond to the balance between assimilate import and assimilate use in synthesis of cellular structural components (i.e. WSC-free DM) and deposition of monosaccharides. Fructan synthesized in tissue during most active elongation was degraded when the respective tissue reached the distal limit of the elongation zone where assimilate import in darkness was insufficient to maintain synthetic processes associated with further differentiation of cells.