Tracheary element differentiation is correlated with inhibition of cell expansion in xylogenic mesophyll suspension cultures

Abstract

To test the hypothesis that xylogenesis is coupled to cell growth suppression, cell expansion in Zinnia elegans L. var. Envy mesophyll suspension cultures was manipulated by varying the extracellular osmolarity and the effect on xylogenesis was examined. Cell expansion and tracheary element differentiation were inversely related along a gradient of extracellular osmolarity ranging from 200 to 400 mOsm, supporting the hypothesis that tracheary element differentiation is coupled to cessation of cell expansion. Above 300 mOsm, reduction in the number of cells that differentiated into tracheary elements coincided with an increase in the number of plasmolyzed cells as extracellular osmolarity was increased, indicating that plasmolysis inhibits tracheary element differentiation, although not specifically. Using the plasmolysis method we showed that cellular osmolarity within populations of isolated Zinnia mesophyll cells ranges from 250 to 600 mOsm with a mean of 425 mOsm. The broad range in cellular osmolarity within Zinnia mesophyll cell populations, coupled with inhibition of differentiation in the low range due to cell expansion and in the high range due to plasmolysis, may help explain why tracheary element differentiation in Zinnia suspension cultures is never complete nor perfectly synchronous and enable further optimization of this culture system.

Introduction

Cell differentiation in plants occurs primarily in meristematic regions, where it is coordinated with cell division and cell expansion. Xylogenic suspension cultures provide a convenient system for studying this coordination since all three processes take place in the cultures and cells are accessible to experimental manipulation and observation [11], [13], [20], [28]. These cultures are prepared by mechanically isolating mesophyll cells from Zinnia elegans L. and incubating them in a medium containing auxin and cytokinin [12]. In our laboratory, secondary cell wall deposition is visible with fluorescence microscopy in some cells at 48–52 h and up to 75% of the cells by 60–64 h [25].

Various lines of evidence indicate that xylogenesis is coupled to suppression of cell expansion. Using xylogenic mesophyll suspension cultures, we have shown that stimulating cell expansion by buffering the culture medium [25] or supplementing the cultures with conditioned medium [24] delays and inhibits tracheary element differentiation. These observations led us to suggest that cessation of cell expansion is a prerequisite for deposition of the tracheary element secondary cell wall [24]. Recently it was reported that several mutants of Arabidopsis exhibit both reduced cell expansion and overproduction of lignified cells, including tracheary elements [3].

To test the hypothesis that xylogenesis is coupled to cell growth suppression in Zinnia mesophyll suspension cultures, we sought ways to manipulate cell expansion directly. The driving force for cell expansion is turgor pressure in excess of the cell wall yield threshold (growth-effective turgor), which is generated by a difference between cellular and extracellular water potential [19]. Thus, adding a non-cell-permeant osmoticum to the culture medium to lower the extracellular water potential and reduce turgor pressure below the yield threshold can inhibit cell expansion. Although the rate of cell expansion in planta is controlled primarily through metabolic processes that regulate stress relaxation in the cell wall [7], varying the external osmolarity can provide a relatively direct method for experimentally inhibiting cell expansion in culture.

It is known that decreasing the extracellular osmolarity inhibits tracheary element differentiation in Zinnia and other tissue cultures. Fukuda and Komamine [12] showed that addition of mannitol to the culture medium was required to achieve high percentages of differentiation in Zinnia cultures. While developing a simplified medium for these cultures, we showed that reducing the mannitol concentration by 50% substantially delayed and reduced the final percentage of differentiation [26]. After finding that polyethylene glycol could mimic the xylogenic effects of sucrose in callus cultures, Doley and Leyton [9] suggested that sugars induce differentiation by decreasing the extracellular osmotic potential. Torrey et al. [30] further suggested that the reduced osmotic potential exerted its xylogenic effect through inhibition of cell expansion. However, these results do not address the possibility that extracellular osmotic potential exerts independent effects on cell expansion and tracheary element differentiation. If differentiation is truly coupled to cessation of cell expansion, we predict a direct relationship between inhibition of cell expansion and stimulation of tracheary element differentiation with increasing extracellular osmolarity.

This study may also provide the means for improving the utility of xylogenic Zinnia suspension cultures. These cultures have become an important model for investigating the determination of cell fate in plants because the proportion of cells that differentiate is high and the onset of differentiation following induction is rapid and fairly synchronous [13]. Despite rigorous attempts to optimize the culture conditions [6], [12], [21], [26], [29], timing, synchrony and percent differentiation vary substantially within and among the various laboratories using this system. We have suspected that slight changes in the osmolarity resulting from variation in medium sterilization methods and storage conditions may explain some of this variation in our laboratory. In addition, the conditions under which the Zinnia plants are grown or the developmental stage of the leaves at the time of harvest may affect the intracellular osmotic potential of the isolated mesophyll cells and thus the rate of cell expansion and percent differentiation.

Here we report that tracheary element differentiation is negatively correlated with cell expansion and also that freshly isolated Zinnia mesophyll cells exhibit a wide range in cellular osmolarity. We also show that the differentiation-enhancing effect of the osmoticum is counteracted at high concentrations due to plasmolysis.