Regulation of Growth Anisotropy in Well-Watered and Water-Stressed Maize Roots. II. Role of Cortical Microtubules and Cellulose Microfibrils1

We tested the hypothesis that the degree of anisotropic expansion of plant tissues is controlled by the degree of alignment of cortical microtubules or cellulose microfibrils. Previously, for the primary root of maize (Zea mays L.), we quantified spatial profiles of expansion rate in length, radius, and circumference and the degree of growth anisotropy separately for the stele and cortex, as roots became thinner with time from germination or in response to low water potential (B.M. Liang, A.M. Dennings, R.E. Sharp, T.I. Baskin [1997] Plant Physiol 115:101–111). Here, for the same material, we quantified microtubule alignment with indirect immunofluorescence microscopy and microfibril alignment throughout the cell wall with polarized-light microscopy and from the innermost cell wall layer with electron microscopy. Throughout much of the growth zone, mean orientations of microtubules and microfibrils were transverse, consistent with their parallel alignment specifying the direction of maximal expansion rate (i.e. elongation). However, where microtubule alignment became helical, microfibrils often made helices of opposite handedness, showing that parallelism between these elements was not required for helical orientations. Finally, contrary to the hypothesis, the degree of growth anisotropy was not correlated with the degree of alignment of either microtubules or microfibrils. The mechanisms plants use to specify radial and tangential expansion rates remain uncharacterized.

Arabidopsis Roots and Shoots Have Different Mechanisms for Hypoxic Stress Tolerance

Arabidopsis has inducible responses for tolerance of O2 deficiency. Plants previously exposed to 5% O2 were more tolerant than the controls to hypoxic stress (0.1% O2 for 48 h) in both roots and shoots, but hypoxic acclimation did not improve tolerance to anoxia (0% O2). The acclimation of shoots was not dependent on the roots: increased shoot tolerance was observed when the roots of the plants were removed. An adh (alcohol dehydrogenase) null mutant did not show acclimation of the roots but retained the shoot survival response. Abscisic acid treatment also differentiated the root and shoot responses; pretreatment induced root survival in hypoxic stress conditions (0.1% O2) but did not induce any increase in the survival of shoots. Cycloheximide blocked both root and shoot acclimation, indicating that both acclimation mechanisms are dependent on protein synthesis.

Use of plant roots for phytoremediation and molecular farming

Alternative agriculture, which expands the uses of plants well beyond food and fiber, is beginning to change plant biology. Two plant-based biotechnologies were recently developed that take advantage of the ability of plant roots to absorb or secrete various substances. They are (i) phytoextraction, the use of plants to remove pollutants from the environment and (ii) rhizosecretion, a subset of molecular farming, designed to produce and secrete valuable natural products and recombinant proteins from roots. Here we discuss recent advances in these technologies and assess their potential in soil remediation, drug discovery, and molecular farming.

Rapid Up-Regulation of HKT1, a High-Affinity Potassium Transporter Gene, in Roots of Barley and Wheat following Withdrawal of Potassium1

High-affinity K+ uptake in plant roots is rapidly up-regulated when K+ is withheld and down-regulated when K+ is resupplied. These processes make important contributions to plant K+ homeostasis. A cDNA coding for a high-affinity K+ transporter, HKT1, was earlier cloned from wheat (Triticum aestivum L.) roots and functionally characterized. We demonstrate here that in both barley (Hordeum vulgare L.) and wheat roots, a rapid and large up-regulation of HKT1 mRNA levels resulted when K+ was withdrawn from growth media. This effect was specific for K+; withholding N caused a modest reduction of HKT1 mRNA levels. Up-regulation of HKT1 transcript levels in barley roots occurred within 4 h of removing K+, which corresponds to the documented increase of high-affinity K+ uptake in roots following removal of K+. Increased expression of HKT1 mRNA was evident before a decline in total root K+ concentration could be detected. Resupply of 1 mm K+ was sufficient to strongly reduce HKT1 transcript levels. In wheat root cortical cells, both membrane depolarizations in response to 100 μm K+, Cs+, and Rb+, and high-affinity K+ uptake were enhanced by K+ deprivation. Thus, in both plant systems the observed physiological changes associated with manipulating external K+ supply were correlated with levels of HKT1 mRNA expression. Implications of these findings for K+ sensing and regulation of the HKT1 mRNA levels in plant roots are discussed.

Alterations in the Cytoskeleton Accompany Aluminum-Induced Growth Inhibition and Morphological Changes in Primary Roots of Maize1

Although Al is one of the major factors limiting crop production, the mechanisms of toxicity remain unknown. The growth inhibition and swelling of roots associated with Al exposure suggest that the cytoskeleton may be a target of Al toxicity. Using indirect immunofluorescence microscopy, microtubules and microfilaments in maize (Zea mays L.) roots were visualized and changes in their organization and stability correlated with the symptoms of Al toxicity. Growth studies showed that the site of Al toxicity was associated with the elongation zone. Within this region, Al resulted in a reorganization of microtubules in the inner cortex. However, the orientation of microtubules in the outer cortex and epidermis remained unchanged even after chronic symptoms of toxicity were manifest. Auxin-induced reorientation and cold-induced depolymerization of microtubules in the outer cortex were blocked by Al pretreatment. These results suggest that Al increased the stability of microtubules in these cells. The stabilizing effect of Al in the outer cortex coincided with growth inhibition. Reoriented microfilaments were also observed in Al-treated roots, and Al pretreatment minimized cytochalasin B-induced microfilament fragmentation. These data show that reorganization and stabilization of the cytoskeleton are closely associated with Al toxicity in maize roots.

Analysis of Respiratory Chain Regulation in Roots of Soybean Seedlings1

Changes in the respiratory rate and the contribution of the cytochrome (Cyt) c oxidase and alternative oxidase (COX and AOX, respectively) were investigated in soybean (Glycine max L. cv Stevens) root seedlings using the 18O-discrimination method. In 4-d-old roots respiration proceeded almost entirely via COX, but by d 17 more than 50% of the flux occurred via AOX. During this period the capacity of COX, the theoretical yield of ATP synthesis, and the root relative growth rate all decreased substantially. In extracts from whole roots of different ages, the ubiquinone pool was maintained at 50% to 60% reduction, whereas pyruvate content fluctuated without a consistent trend. In whole-root immunoblots, AOX protein was largely in the reduced, active form at 7 and 17 d but was partially oxidized at 4 d. In isolated mitochondria, Cyt pathway and succinate dehydrogenase capacities and COX I protein abundance decreased with root age, whereas both AOX capacity and protein abundance remained unchanged. The amount of mitochondrial protein on a dry-mass basis did not vary significantly with root age. It is concluded that decreases in whole-root respiration during growth of soybean seedlings can be largely explained by decreases in maximal rates of electron transport via COX. Flux via AOX is increased so that the ubiquinone pool is maintained in a moderately reduced state.

Adaptation of Active Proton Pumping and Plasmalemma ATPase Activity of Corn Roots to Low Root Medium pH1

Corn (Zea mays L.) root adaptation to pH 3.5 in comparison with pH 6.0 (control) was investigated in long-term nutrient solution experiments. When pH was gradually reduced, comparable root growth was observed irrespective of whether the pH was 3.5 or 6.0. After low-pH adaptation, H+ release of corn roots in vivo at pH 5.6 was about 3 times higher than that of control. Plasmalemma of corn roots was isolated for investigation in vitro. At optimum assay pH, in comparison with control, the following increases of the various parameters were caused by low-pH treatment: (a) hydrolytic ATPase activity, (b) maximum initial velocity and Michaelis constant (c) activation energy of H+-ATPase, (d) H+-pumping activity, (e) H+ permeability of plasmalemma, and (f) pH gradient across the membranes of plasmalemma vesicles. In addition, vanadate sensitivity remained unchanged. It is concluded that plasmalemma H+-ATPase contributes significantly to the adaptation of corn roots to low pH. A restricted net H+ release at low pH in vivo may be attributed to the steeper pH gradient and enhanced H+ permeability of plasmalemma but not to deactivation of H+-ATPase. Possible mechanisms responsible for adaptation of plasmalemma H+-ATPase to low solution pH during plant cultivation are discussed.

Evidence for the Critical Role of Sucrose Synthase for Anoxic Tolerance of Maize Roots using a Double Mutant

The induction of the sucrose synthase (SuSy) gene (SuSy) by low O2, low temperature, and limiting carbohydrate supply suggested a role in carbohydrate metabolism under stress conditions. The isolation of a maize (Zea mays L.) line mutant for the two known SuSy genes but functionally normal showed that SuSy activity might not be required for aerobic growth and allowed the possibility of investigating its importance during anaerobic stress. As assessed by root elongation after return to air, hypoxic pretreatment improved anoxic tolerance, in correlation with the number of SuSy genes and the level of SuSy expression. Furthermore, root death in double-mutant seedlings during anoxic incubation could be attributed to the impaired utilization of sucrose (Suc). Collectively, these data provide unequivocal evidence that Suc is the principal C source and that SuSy is the main enzyme active in Suc breakdown in roots of maize seedlings deprived of O2. In this situation, SuSy plays a critical role in anoxic tolerance.

Development, Characterization, and Application of a Cadmium-Selective Microelectrode for the Measurement of Cadmium Fluxes in Roots of Thlaspi Species and Wheat1

A Cd2+-selective vibrating microelectrode was constructed using a neutral carrier-based Cd ionophore to investigate ion-transport processes along the roots of wheat (Triticum aestivum L.) and two species of Thlaspi, one a Zn/Cd hyperaccumulator and the other a related nonaccumulator. In simple Cd(NO3)2 solutions, the electrode exhibited a Nernstian response in solutions with Cd2+ activities as low as 50 nm. Addition of Ca2+ to the calibration solutions did not influence the slope of the calibration curve but reduced the detection limit to a solution activity of 1 μm Cd2+. Addition of high concentrations of K+ and Mg2+ to the calibration solution to mimic the ionic composition of the cytoplasm affected neither the slope nor the sensitivity of the electrode, demonstrating the pH-insensitive electrode's potential for intracellular investigations. The electrode was assayed for selectivity and was shown to be at least 1000 times more selective for Cd2+ than for any of those potentially interfering ions tested. Flux measurements along the roots of the two Thlaspi species showed no differences in the pattern or the magnitude of Cd2+ uptake within the time frame considered. The Cd2+-selective microelectrode will permit detailed investigations of heavy-metal ion transport in plant roots, especially in the area of phytoremediation.

Re-Aeration following Hypoxia or Anoxia Leads to Activation of the Antioxidative Defense System in Roots of Wheat Seedlings1

The response of the ascorbate-glutathione cycle was investigated in roots of young wheat (Triticum aestivum L.) seedlings that were deprived of oxygen either by subjecting them to root hypoxia or to entire plant anoxia and then re-aerated. Although higher total levels of ascorbate and glutathione were observed under hypoxia, only the total amount of ascorbate was increased under anoxia. Under both treatments a significant increase in the reduced form of ascorbate and glutathione was found, resulting in increased reduction states. Upon the onset of re-aeration the ratios started to decline rapidly, indicating oxidative stress. Hypoxia caused higher activity of ascorbate peroxidase, whereas activities of monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase were diminished or only slightly influenced. Under anoxia, activities of ascorbate peroxidase and glutathione reductase decreased significantly to 39 and 62%, respectively. However, after re-aeration of hypoxically or anoxically pretreated roots, activity of enzymes approached the control levels. This corresponds with the restoration of the high reduction state of ascorbate and glutathione within 16 to 96 h of re-aeration, depending on the previous duration of anoxia. Apparently, anoxia followed by re-aeration more severely impairs entire plant metabolism compared with hypoxia, thus leading to decreased viability.

Purification of the Plasma Membrane Ca2+-ATPase from Radish Seedlings by Calmodulin-Agarose Affinity Chromatography1

The Ca2+-ATPase of the plasma membrane (PM) of germinating radish (Raphanus sativus L.) seeds was purified by calmodulin (CaM)-affinity chromatography using a batch procedure. PM purified by aqueous two-phase partitioning was solubilized with n-dodecyl β-d-maltoside and applied to a CaM-agarose matrix. After various washings with decreasing Ca2+ concentrations, the Ca2+-ATPase was eluted with 5 mm ethylenediaminetetraacetate (EDTA). The EDTA-eluted fraction contained about 25% of the loaded Ca2+-ATPase activity, with a specific activity 70-fold higher than that of the starting PM fraction. The EDTA-eluted fraction was highly enriched in a 133-kD polypeptide, which was identified as the PM Ca2+-ATPase by 125I-CaM overlay and fluorescein-isothiocyanate labeling. The PM Ca2+-ATPase cross-reacted with an antiserum against a putative Ca2+-ATPase of the Arabidopsis thaliana chloroplast envelope.

Regulation of K+ Channels in Maize Roots by Water Stress and Abscisic Acid1

Root cortical and stelar protoplasts were isolated from maize (Zea mays L.) plants that were either well watered or water stressed, and the patch-clamp technique was used to investigate their plasma membrane K+ channel activity. In the root cortex water stress did not significantly affect inward- or outward-rectifying K+ conductances relative to those observed in well-watered plants. In contrast, water stress significantly reduced the magnitude of the outward-rectifying K+ current in the root stele but had little effect on the inward-rectifying K+ current. Pretreating well-watered plants with abscisic acid also significantly affected K+ currents in a way that was consistent with abscisic acid mediating, at least in part, the response of roots to water stress. It is proposed that the K+ channels underlying the K+ currents in the root stelar cells represent pathways that allow K+ exchange between the root symplasm and xylem apoplast. It is suggested that the regulation of K+ channel activity in the root in response to water stress could be part of an important adaptation of the plant to survive drying soils.

Mapping the Functional Roles of Cap Cells in the Response of Arabidopsis Primary Roots to Gravity1

The cap is widely accepted to be the site of gravity sensing in roots because removal of the cap abolishes root curvature. Circumstantial evidence favors the columella cells as the gravisensory cells because amyloplasts (and often other cellular components) are polarized with respect to the gravity vector. However, there has been no functional confirmation of their role. To address this problem, we used laser ablation to remove defined cells in the cap of Arabidopsis primary roots and quantified the response of the roots to gravity using three parameters: time course of curvature, presentation time, and deviation from vertical growth. Ablation of the peripheral cap cells and tip cells did not alter root curvature. Ablation of the innermost columella cells caused the strongest inhibitory effect on root curvature without affecting growth rates. Many of these roots deviated significantly from vertical growth and had a presentation time 6-fold longer than the controls. Among the two inner columella stories, the central cells of story 2 contributed the most to root gravitropism. These cells also exhibited the largest amyloplast sedimentation velocities. Therefore, these results are consistent with the starch-statolith sedimentation hypothesis for gravity sensing.

‘Quite destitute and … very desirous of going to North America’ : The Roots and Repercussions of Emigration from Sutherland and Caithness

Peer reviewed