Kris Vissenberg

In Vivo Colocalization of Xyloglucan Endotransglycosylase Activity and Its Donor Substrate in the Elongation Zone of Arabidopsis Roots

We have developed a method for the colocalization of xyloglucan endotransglycosylase (XET) activity and the donor substrates to which it has access in situ and in vivo. Sulforhodamine conjugates of xyloglucan oligosaccharides (XGO–SRs), infiltrated into the tissue, act as acceptor substrate for the enzyme; endogenous xyloglucan acts as donor substrate. Incorporation of the XGO–SRs into polymeric products in the cell wall yields an orange fluorescence indicative of the simultaneous colocalization, in the same compartment, of active XET and donor xyloglucan chains. The method is specific for XET, as shown by competition experiments with nonfluorescent acceptor oligosaccharides, by negligible reaction with cello-oligosaccharide–SR conjugates that are not XET acceptor substrates, by heat lability, and by pH optimum. Thin-layer chromatographic analysis of remaining unincorporated XGO–SRs showed that these substrates are not extensively hydrolyzed during the assays. A characteristic distribution pattern was found in Arabidopsis and tobacco roots: in both species, fluorescence was most prominent in the cell elongation zone of the root. Proposed roles of XET that include cell wall loosening and integration of newly synthesized xyloglucans could thus be supported.

Root gravitropism is regulated by a transient lateral auxin gradient controlled by a tipping-point mechanism

Gravity profoundly influences plant growth and development. Plants respond to changes in orientation by using gravitropic responses to modify their growth. Cholodny and Went hypothesized over 80 years ago that plants bend in response to a gravity stimulus by generating a lateral gradient of a growth regulator at an organs apex, later found to be auxin. Auxin regulates root growth by targeting Aux/IAA repressor proteins for degradation. We used an Aux/IAA-based reporter, domain II (DII)-VENUS, in conjunction with a mathematical model to quantify auxin redistribution following a gravity stimulus. Our multidisciplinary approach revealed that auxin is rapidly redistributed to the lower side of the root within minutes of a 90° gravity stimulus. Unexpectedly, auxin asymmetry was rapidly lost as bending root tips reached an angle of 40° to the horizontal. We hypothesize roots use a “tipping point” mechanism that operates to reverse the asymmetric auxin flow at the midpoint of root bending. These mechanistic insights illustrate the scientific value of developing quantitative reporters such as DII-VENUS in conjunction with parameterized mathematical models to provide high-resolution kinetics of hormone redistribution.

The Root Apex of Arabidopsis thaliana Consists of Four Distinct Zones of Growth Activities: Meristematic Zone, Transition Zone, Fast Elongation Zone and Growth Terminating Zone.

In the growing apex of Arabidopsis thaliana primary roots, cells proceed through four distinct phases of cellular activities. These zones and their boundaries can be well defined based on their characteristic cellular activities. The meristematic zone comprises, and is limited to, all cells that undergo mitotic divisions. Detailed in vivo analysis of transgenic lines reveals that, in the Columbia-0 ecotype, the meristem stretches up to 200 µm away from the junction between root and root cap (RCJ). In the transition zone, 200 to about 520 µm away from the RCJ, cells undergo physiological changes as they prepare for their fast elongation. Upon entering the transition zone, they progressively develop a central vacuole, polarize the cytoskeleton and remodel their cell walls. Cells grow slowly during this transition: it takes ten hours to triplicate cell length from 8.5 to about 35 µm in the trichoblast cell files. In the fast elongation zone, which covers the zone from 520 to about 850 µm from the RCJ, cell length quadruplicates to about 140 µm in only two hours. This is accompanied by drastic and specific cell wall alterations. Finally, root hairs fully develop in the growth terminating zone, where root cells undergo a minor elongation to reach their mature lengths.

A role for pectin de-methylesterification in a developmentally regulated growth acceleration in dark-grown Arabidopsis hypocotyls.

• We focused on a developmentally regulated growth acceleration in the dark-grown Arabidopsis hypocotyl to study the role of changes in cell wall metabolism in the control of cell elongation. • To this end, precise transcriptome analysis on dissected dark-grown hypocotyls, Fourier transform infrared (FT-IR) microspectroscopy and kinematic analysis were used. • Using a cellulose synthesis inhibitor, we showed that the growth acceleration marks a developmental transition during which growth becomes uncoupled from cellulose synthesis. We next investigated the cellular changes that take place during this transition. FT-IR microspectroscopy revealed significant changes in cell wall composition during, but not after, the growth acceleration. Transcriptome analysis suggested a role for cell wall remodeling, in particular pectin modification, in this growth acceleration. This was confirmed by the overexpression of a pectin methylesterase inhibitor, which caused a delay in the growth acceleration. • This study shows that the acceleration of cell elongation marks a developmental transition in dark-grown hypocotyl cells and supports a role for pectin de-methylesterification in the timing of this event.

Enzymic characterization of two recombinant xyloglucan endotransglucosylase/hydrolase (XTH) proteins of Arabidopsis and their effect on root growth and cell wall extension

Xyloglucan endotransglucosylase/hydrolases (XTHs) are enzymes involved in the modification of load-bearing cell wall components. They cleave xyloglucan chains and, often, re-form bonds to the non-reducing ends of available xyloglucan molecules in plant primary cell walls. The enzymic properties and effects on root growth of two Arabidopsis thaliana XTHs belonging to subgroup I/II, that are predominantly expressed in root hairs and in non-elongating zones of the root, were analysed here. AtXTH14 and AtXTH26 were recombinantly produced in Pichia and subsequently purified. Both proteins were found to exhibit xyloglucan endotransglucosylase (XET; EC 2.4.1.207) but not xyloglucan endohydrolase (XEH; EC 3.2.1.151) activity. Their endotransglucosylase activity was at least 70x greater on xyloglucan rather than on mixed-linkage beta-glucan. Differences were found in pH- and temperature-dependence as well as in acceptor-substrate preferences. Furthermore, the specific activity of XET was approximately equal for the two enzymes. Removal of N-linked sugar residues by Endo H treatment reduced XET activity to 60%. Constant-load extensiometry experiments revealed that the enzymes reduce the extension in a model system of heat-inactivated isolated cell walls. When given to growing roots, either of these XTH proteins reduced cell elongation in a concentration-dependent manner and caused abnormal root hair morphology. This is the first time that recombinant and purified XTHs added to growing roots have exhibited a clear effect on cell elongation. It is proposed that these specific XTH isoenzymes play a role in strengthening the side-walls of root-hairs and cell walls in the root differentiation zone after the completion of cell expansion.

Ethylene in vegetative development: a tale with a riddle

The vegetative development of plants is strongly dependent on the action of phytohormones. For over a century, the effects of ethylene on plants have been studied, illustrating the profound impact of this gaseous hormone on plant growth, development and stress responses. Ethylene signaling is under tight self-control at various levels. Feedback regulation occurs on both biosynthesis and signaling. For its role in developmental processes, ethylene has a close and reciprocal relation with auxin, another major determinant of plant architecture. Here, we discuss, in view of novel findings mainly in the reference plant Arabidopsis, how ethylene is distributed and perceived throughout the plant at the organ, tissue and cellular levels, and reflect on how plants benefit from the complex interaction of ethylene and auxin, determining their shape. Furthermore, we elaborate on the implications of recent discoveries on the control of ethylene signaling.

Apoplastic Alkalinization Is Instrumental for the Inhibition of Cell Elongation in the Arabidopsis Root by the Ethylene Precursor 1-Aminocyclopropane-1-Carboxylic Acid

In Arabidopsis (Arabidopsis thaliana; Columbia-0) roots, the so-called zone of cell elongation comprises two clearly different domains: the transition zone, a postmeristematic region (approximately 200–450 μm proximal of the root tip) with a low rate of elongation, and a fast elongation zone, the adjacent proximal region (450 μm away from the root tip up to the first root hair) with a high rate of elongation. In this study, the surface pH was measured in both zones using the microelectrode ion flux estimation technique. The surface pH is highest in the apical part of the transition zone and is lowest at the basal part of the fast elongation zone. Fast cell elongation is inhibited within minutes by the ethylene precursor 1-aminocyclopropane-1-carboxylic acid; concomitantly, apoplastic alkalinization occurs in the affected root zone. Fusicoccin, an activator of the plasma membrane H+-ATPase, can partially rescue this inhibition of cell elongation, whereas the inhibitor N,N′-dicyclohexylcarbodiimide does not further reduce the maximal cell length. Microelectrode ion flux estimation experiments with auxin mutants lead to the final conclusion that control of the activity state of plasma membrane H+-ATPases is one of the mechanisms by which ethylene, via auxin, affects the final cell length in the root.

New insights into root gravitropic signalling

An important feature of plants is the ability to adapt their growth towards or away from external stimuli such as light, water, temperature, and gravity. These responsive plant growth movements are called tropisms and they contribute to the plants survival and reproduction. Roots modulate their growth towards gravity to exploit the soil for water and nutrient uptake, and to provide anchorage. The physiological process of root gravitropism comprises gravity perception, signal transmission, growth response, and the re-establishment of normal growth. Gravity perception is best explained by the starch-statolith hypothesis that states that dense starch-filled amyloplasts or statoliths within columella cells sediment in the direction of gravity, resulting in the generation of a signal that causes asymmetric growth. Though little is known about the gravity receptor(s), the role of auxin linking gravity sensing to the response is well established. Auxin influx and efflux carriers facilitate creation of a differential auxin gradient between the upper and lower side of gravistimulated roots. This asymmetric auxin gradient causes differential growth responses in the graviresponding tissue of the elongation zone, leading to root curvature. Cell biological and mathematical modelling approaches suggest that the root gravitropic response begins within minutes of a gravity stimulus, triggering genomic and non-genomic responses. This review discusses recent advances in our understanding of root gravitropism in Arabidopsis thaliana and identifies current challenges and future perspectives.

Roles of the XTH Protein Family in the Expanding Cell

Since xyloglucan is believed to be an important structural polysaccharide in the cell wall, possibly interconnecting adjacent cellulose microfibrils, enzymes that modify xyloglucan during the cell expansion process receive much attention. One of the enzymes is xyloglucan endotransglucosylase/hydrolase (XTH), a subgroup of glycoside hydrolase family 16. XTH proteins examined so far display endotransglycosylase (XET), hydrolase (XEH), or both activities towards xyloglucans. Genome sequencing of several model plant species has revealed that XTH proteins are encoded by large multigene families. Comprehensive analyses of XTH gene expression, together with functional analyses based on loss-of-function mutants, have provided evidence in support of the hypothesis that each member of these multigene families has its own role. This is reflected by different substrate specificities and pH dependencies of several individual members. Expression of each of these genes is precisely regulated by various hormones as well as by environmental signals. Some members appear to be critical in promoting cell wall expansion and are therefore essential for cell expansion, whereas others are required for construction of cell walls in cells that have completed the expansion process.

Differences in enzymic properties of five recombinant xyloglucan endotransglucosylase/hydrolase (XTH) proteins of Arabidopsis thaliana

Xyloglucan endotransglucosylase/hydrolases (XTHs) are cell wall enzymes that are able to graft xyloglucan chains to oligosaccharides or to other available xyloglucan chains and/or to hydrolyse xyloglucan chains. As they are involved in the modification of the load-bearing cell-wall components, they are believed to be very important in the regulation of growth and development. Given the large number (33) of XTH genes in Arabidopsis and the overlapping expression patterns, specific enzymic properties may be expected. Five predominantly root-expressed Arabidopsis thaliana XTHs belonging to subgroup I/II were analysed here. These represent two sets of closely related genes: AtXTH12 and 13 on the one hand (trichoblast-enriched) and AtXTH17, 18, and 19 on the other (expressed in nearly all cell types in the root). They were all recombinantly produced in the yeast Pichia pastoris and partially purified by ammonium sulphate precipitation before they were subsequently all subjected to a series of identical in vitro tests. The kinetic properties of purified AtXTH13 were investigated in greater detail to rule out interference with the assays by contaminating yeast proteins. All five proteins were found to exhibit only the endotransglucosylase (XET; EC 2.4.1.207) activity towards xyloglucan and non-detectable endohydrolytic (XEH; EC 3.2.1.151) activity. Their endotransglucosylase activity was preferentially directed towards xyloglucan and, in some cases, water-soluble cellulose acetate, rather than to mixed-linkage β-glucan. Isoforms differed in optimum pH (5.0-7.5), in temperature dependence and in acceptor substrate preferences.