Robert E. Cleland

Chapter 6 Control of Plant Cell Enlargement By Hydrogen Ions

Publisher Summary This chapter discusses the evidence that in Avena coleoptiles and pea stem tissues the hormone auxin induces cell enlargement, by activating proton extrusion, and that the resulting acidification of the wall leads to enzymic cell wall loosening and thus cell enlargement. It emphasizes the point that auxin itself does not act directly on the wall but acts at the cell surface or within the cytoplasm. When a plant cell enlarges, most of the increase in volume is due to the uptake of water into an expanding–centrally located vacuole. The direction of the cell expansion depends on the molecular architecture of the wall and can be primarily in one direction or equally in all directions. Cell enlargement can be initiated in one of the two ways: by an increase in the osmotic concentration of the cell or by an increase in cell wall extensibility (cell wall loosening). In most systems, cell enlargement is initiated by wall loosening. If a section is removed from the elongation zone of a pea stem and placed in water, the cells enlarge, but only at a slow rate. Rapid enlargement can be induced by only four agents: the group of hormones called auxins, hydrogen ions, CO, and the phytotoxin fusicoccin. If hydrogen ions are excreted from auxin-treated Avena coleoptile cells, and if the pH of the wall region falls below 5.8, the walls would undergo cell wall loosening and growth would occur.

Auxin and Cell Elongation

One of the most dramatic and rapid hormone responses in plants is the induction by auxin of rapid cell elongation in isolated stem and coleoptile sections. The response begins within 10 minutes after the addition of auxin, results in a 5-10 fold increase in the growth rate, and persists for hours or even days (22). It is hardly surprising that this may be the most studied hormonal response in plants.

Extensibility of isolated cell walls: Measurement and changes during cell elongation.

Summary1.The technique of measuring the extensibility of isolated cell walls with an extensometer (Instron technique) has been modified so that compliance values which characterize the plastic and elastic extensibility are obtained (DP and DE, respectively).2.DP and DE values are influenced by the conditions under which the measurements are made. DP is affected by the rate of extension, the applied force and the presence of the protoplast. DE is inversely proportional to the applied force but is independent of the presence of the protoplast. Because of retarded elastic behaviour and of hysteresis in the elastic extension-stress curves, measurement of DE during extension and relaxation gives different values.3.Irreversible extension as determined in this procedure appears to be due primarily to strain-hardened plastic deformation with a minor component due to some form of viscoelastic flow. Auxin increases the extensibility by acting on the strain-hardening function.4.Changes in DP and DE which occur during the course of auxin-induced cell elongation of Avena coleoptile sections have been determined. DP increases following addition of auxin, reaches a maximum after 90–120 min, and then remains constant for up to 24 hours. Sucrose has no effect on the change in DP. DE shows smaller but similar changes.5.An increase in IAA concentration up to 5×10-5 M produces similar increases in growth rate and DP. When IAA is raised to higher levels, DP remains constant while the growth rate drops.6.These results are in agreement with the concept that auxin exerts its effect on cell elongation by regulating wall extensibility but indicate that the growth rate is also influenced by other factors such as the osmotic potential of the cells.

The in-vitro acid-growth response: Relation to in-vivo growth responses and auxin action

SummaryWe have examined in detail the characteristics of the hydrogen-ion extension response in frozen-thawed Avena coleoptile sections (in-vitro acid-growth response). These data allow us to compare the in vitro response with the in-vivo extension responses initiated by auxin and hydrogen ions. The in-vitro response has three characteristics in common with the in-vivo responses: a similar Q10 (3–4 between 15 and 25°C, but almost 1 between 25 and 35°); a minimum yield stress; and a lack of stored growth (i.e., an inability to induce a potential for growth during periods of reduced wall tension). Both the in-vivo and in-vitro acid-growth responses have a threshold pH of about 4.5 and give an optimum response at pH values of 3 and below. These similarities suggest that the in-vitro and in-vivo acid-growth responses have a common wall-loosening and wall-extension mechanism. We have also examined the effects of Pronase, sodium lauryl sulfate (SLS), elevated temperatures, calcium, and potassium ions on the in-vitro acid-growth response. We suggest that hydrogen ions do not activate wall-associated enzymes, but act to hydrolyze non-enzymatically some acid-labile linkages in the cell wall. Furthermore, we suggest that auxin induces cell elongation either by causing the release of hydrogen ions from the protoplast or by causing the appearance in the wall of an enzyme which can hydrolyse the acid-labile linkages.

Two callose synthases, GSL1 and GSL5, play an essential and redundant role in plant and pollen development and in fertility.

Callose, a β-1,3-glucan that is widespread in plants, is synthesized by callose synthase. Arabidopsis thaliana contains a family of 12 putative callose synthase genes (GSL1–12). The role of callose and of the individual genes in plant development is still largely uncertain. We have now used TILLING and T-DNA insertion mutants (gsl1-1, gsl5-2 and gsl5-3) to study the role of two closely related and linked genes, GSL1 and GSL5, in sporophytic development and in reproduction. Both genes are expressed in all parts of the plant. Sporophytic development was nearly normal in gsl1-1 homozygotes and only moderately defective in homozygotes for either of the two gsl5 alleles. On the other hand, plants that were gsl1-1/+ gsl5/gsl5 were severely defective, with smaller leaves, shorter roots and bolts and smaller flowers. Plants were fertile when the sporophytes had either two wild-type GSL1 alleles, or one GSL5 allele in a gsl1-1 background, but gsl1-1/+ gsl5/gsl5 plants produced an extremely reduced number of viable seeds. A chromosome with mutations in both GSL1 and GSL5 rendered pollen infertile, although such a chromosome could be transmitted via the egg. As a result, it was not possible to obtain plants that were homozygous for mutations in both the GSL genes. Pollen grain development was severely affected in double mutant plants. Many pollen grains were collapsed and inviable in the gsl1-1/gsl1-1 gsl5/+ and gsl1-1/+ gsl5/gsl5 plants. In addition, gsl1-1/+ gsl5/gsl5 plants produced abnormally large pollen with unusual pore structures, and had problems with tetrad dissociation. In this particular genotype, while the callose wall formed around the pollen mother cells, no callose wall separated the resulting tetrads. We conclude that GSL1 and GSL5 play important, but at least partially redundant roles in both sporophytic development and in the development of pollen. They are responsible for the formation of the callose wall that separates the microspores of the tetrad, and also play a gametophytic role later in pollen grain maturation. Other GSL genes may control callose formation at different steps during pollen development.

Plasmodesmal-mediated cell-to-cell transport in wheat roots is modulated by anaerobic stress

SummaryCell-to-cell transport of small molecules and ions occurs in plants through plasmodesmata. Plant roots are frequently subjected to localized anaerobic stress, with a resultant decrease in ATP. In order to determine the effect of this stress on plasmodesmal transport, fluorescent dyes of increasing molecular weight (0.46 to 10 kDa) were injected into epidermal and cortical cells of 3-day-old wheat roots, and their movement into neighboring cells was determined by fluorescence microscopy. Anaerobiosis was generated by N2 gas or simulated by the presence of sodium azide, both of which reduced the ATP levels in the tissue by over 80%. In the absence of such stress, the upper limit for movement, or size exclusion limit (SEL), of cortical plasmodesmata was <1 kDa. The ATP analogue TNP-ADP (mw 681) moved across the plasmodesmata of unstressed roots, indicating that plasmodesmata may be conduits for nucleotide (ATP and ADP) exchange between cells. Upon imposition of stress, the SEL rose to between 5 and 10 kDa. This response of plasmodesmata to a decrease in the level of ATP suggests that they are constricted by an ATP-dependent process so as to maintain a restricted SEL. When roots are subjected to anaerobic stress, an increase in SEL may permit enhanced delivery of sugars to the affected cells of the root where anaerobic respiration could regenerate the needed ATP.

Proton excretion and cell expansion in bean leaves.

Light-induced expansion of Phaseolus vulgaris L. leaf cells is accompanied by increased cell-wall plasticity. The possibility that leaf-cell walls are loosened by excreted protons has been investigated. First, light causes acidification, detected at the leaf surface, within 5–15 min. Growth starts 10–20 min after exposure to light. Second, exogenous acid induces loosening of isolated leaf cell walls. Third, infiltration of the tissue with a neutral buffer inhibits light-induced growth. Fourth, fusicoccin stimulates growth of as well as H+ excretion by bean leaf cells, without light. These findings show that the acid-growth theory is applicable to light-induced growth of leaf cells, and indicate that light-induced proton excretion initiates cell enlargement in leaves.

Auxin-induced hydrogen ion excretion: correlation with growth, and control by external pH and water stress

Summary1.The acid-growth theory predicts that the rates of auxin-induced cell elongation and H+-excretion should be closely correlated as long as the experimental conditions remain fairly constant. To test this, Avena coleoptiles have been induced to elongate at different rates by varying the concentration of auxin, the age of the tissue, or by addition of metabolic inhibitors. As predicted, in each case there was a close correlation between the rates of H+-excretion and growth.2.The rate and direction of movement of H+ between the coleoptile and the external medium is regulated by the external pH. Coleoptiles take up H+ passively from acidic solutions and excrete H+ into basic solutions. In the absence of auxin, uptake and excretion are at equilibrium when the solution pH is near 5.7, a pH too high to allow rapid cell wall loosening. Auxin stimulates the excretion, but as the external pH drops the excretion is inhibited and a new equilibrium is established near 5.0. This allows amlows maximum wall loosening without causing toxic side-effects.3.H+-excretion is also affected by water stress. Increasing water stress induced by mannitol decreases H+-excretion in auxin-treated tissues but stimulates H+-excretion in the absence of auxin. At incipient plasmolysis H+-excretion is insensitive to auxin, suggesting that even if H+-excretion is mediated by an auxin-activated ATPase it may be impossible to demonstrate an effect of auxin on this enzyme in vitro.4.Three types of H+-excretion have been recognized in coleoptiles; auxin-induced, stress-induced, and basal level. All appear to require ATP, but only basal level H+-excretion does not also require continual protein synthesis.

Fusicoccin-induced growth and hydrogen ion excretion of Avena coleoptiles: Relation to auxin responses

SummaryThe fungal toxin fusicoccin (FC) induces both rapid cell elongation and H+-excretion in Avena coleoptiles. The rates for both responses are greater with FC than with optimal auxin, and in both cases the lag after addition of the hormone is less with FC. This provides additional support for the acid-growth theory. The FC responses resemble the auxin responses in that they are inhibited by a range of metabolic inhibitors, but the responses differ in three ways. First auxin, but not FC, requires continual protein synthesis for its action. The auxin-induced H+-excretion is inhibited by water stress or by low external pH, while the FC-induced H+-excretion is much less sensitive to either. It is concluded that auxin-induced and FC-induced H+-excretion may occur via different mechanisms.

The dosage-response curve for auxin-induced cell elongation: A reevaluation.

SummaryThe dosage-response curve for auxin-induced growth of coleoptile sections has been reevaluated, using initial growth rates than rates obtained hours after application of auxin. The curve is sigmoid rather than the “classical” bell-shape; i.e., the growth rate increases as the IAA concentration is raised from 10-8 M, but then the same optimal rate prevails over the range of 3×10-7 to 10-3 M. In the absence of sucrose this sigmoid shape persists, but with sucrose present the shape slowly changes into a modified bell-shaped curve. It is suggested that the classical bell-shaped curve is due, at least in part, to an auxin-sucrose interaction, and that it is not a true reflection of the kinetics of auxin-induced elongation.