The pea stem

Abstract

The Casparian strip is commonly observed in the endodermis of roots of vascular plants and, in some cases, also in the stems. Pea stems develop the Casparian strip, and its development has been reported to be regulated by blue light. In addition, for the purpose of photobiological studies, pea stems provide a unique experimental system for other physiological studies of the development of the Casparian strip. In this article, I have briefly summarized (1) the effects of environmental factors on the development of the Casparian strip, (2) the advantage of using pea stems for physiological studies of the development of the Casparian strip, and (3) cellular events indicated to be involved in the development of the Casparian strip, focusing on the studies using pea stems as well as other recent studies.

Effects of Environmental Factors on the Development of the Casparian Strip

Modulations of tissue differentiation in plant organs in response to environmental factors are important for plants to respond properly to environmental changes, which is part of the so-called plant phenotypic plasticity. Among the tissues, attention has been paid to the endodermis because it plays an important role in the regulation of water and solute transport by forming an apoplastic barrier, the Casparian strip (CS).^1-6 Whether CS development is regulated in response to environmental factors has been a matter of debate.⁴ The distance from a root (or shoot) apex to where the CS becomes visible is a convenient measure to assess the effect of an environmental factor on CS development. However, this distance depends on the rate of cell differentiation (CS formation by endodermal cells) as well as on cell production rate and cell elongation rate.⁷ In the case of maize roots, the CS development was shown to be unaffected under salt stress⁴ or in the presence of ethylene by monitoring these parameters.⁷ However, the cell production rate must be measured even for the purpose of assessment of the rate of cell differentiation in this approach, which requires a laborious procedure. To overcome this difficulty, we have most recently developed a unique method called the “sandwich” technique, in which the roots are sandwiched between two different agar media and are, thereby, unilaterally exposed to different environmental conditions.⁸ Alassimone et al. (2010) also used a different but strict approach and assessed effects of inhibitors on the basis of the cell number from the onset of endodermal cell elongation.⁹

Advantage of the Experimental System Using Pea Stems for Physiological Studies of the Development of the Casparian Strip

Another approach of assessing effects of environmental factors on CS development is to find a reference point or to make a mark on a developing organ before changing the environment. This is possible when dealing with stems grown in air. The CS is a common structure in roots but has also been known to develop in the endodermis of stems in etiolated pea seedlings; however, the function of the CS in stems is not yet known.^10,11 By assessing the CS development on the basis of marks made before light treatment, we have demonstrated that its development is regulated by light¹² and introduced a new experimental system for the study of photoregulation in tissue development. Another practical advantage of using pea stems for the study of CS development is that they are suitable for surgical manipulations. Utilizing this advantage, Yokoyama and Karahara (2001) performed a surgical manipulation to induce expansion of endodermal cells before CS formation. They suggested that some positional information exists in the radial wall of endodermal cells that defines the future site of formation of the CS and its radial width, which is already present even at 4 mm below the bending point of the hook.¹³ Alassimone et al. (2010) recently found that the central-peripheral polarity of endodermal cells is established early during embryogenesis. It is possible that the site of CS formation in the radial wall of endodermal cells is also determined earlier.

Cellular Events Involved in the Development of the Casparian Strip

Two important structural characteristics of the CS, which assure its function as an apoplastic transport barrier of water and solutes, are cell wall modification by the hydrophobic substance suberin^14-16 and tight adhesion of the plasma membrane to the cell wall at the CS.^17,18 Modification of the cell wall is assessed by fluorescence microscopy.^11,19 The uppermost position, where the cell wall is modified by lignin and suberin in 6-d-old garden pea (Pisum sativum L. cv Alaska) stems grown in the dark, that had a third internode in which the distance between the base of the third internode and the point of hook curvature ranged from 40 to 60 mm, was 30.8 ± 0.8 mm (mean ± SE, n = 5) below the bending point of the hook at the start of irradiation. Tight adhesion of the plasma membrane to the cell wall was assessed by examining whether so-called band plasmolysis occurred in the presence of an osmoticum, urea, at a concentration of 8 M.¹¹ In the same 6-d-old pea stems as mentioned above, the uppermost position, where tight adhesion of the plasma membrane to the cell wall at the CS was observed, was 31.0 ± 0.8 mm (mean ± SE, n = 5) below the bending point of the hook at the start of irradiation. There was a significant difference between these positions (paired t-test, p < 0.05). This result indicates that modification of the cell wall preceded tight adhesion of the plasma membrane to the cell wall in endodermal cells. This appears reasonable because once tight adhesion of the plasma membrane is achieved, transport of materials necessary for cell wall modification outside the plasma membrane at the CS might not be possible. However, a recent study in arabidopsis roots showed that cell wall modification occurred at exactly the same time when tight adhesion of the plasma membrane occurred.⁹ At present, we do not know the cause of the difference between these reports, but it may be due to a difference in the techniques employed, organs examined, and/or plant species tested in these studies.

By utilizing the advantage of pea stems suitable for a surgical manipulation, Karahara and Shibaoka (1998) has administrated brefeldin A at a concentration of 200 µM to a position near the site of CS development in 6-d-old dark-grown pea stems to test an involvement of endocytic and/or exocytic membrane transport in the CS development.²⁰ As a result, the position at which the upward progression of the modification of the cell wall stopped was 25.2 ± 0.7 (mean ± SE, n = 5) and the position at which the upward progression of tight adhesion of the plasma membrane stopped was 26.2 ± 0.9 (mean ± SE, n = 5) below the bending point of the hook at the start of the inhibitor treatment.²⁰ Again, there was a significant difference between these positions (paired t-test, p < 0.05). This result indicates that an endocytic and/or exocytic process is involved in both modification of the cell wall and tight adhesion of the plasma membrane, also indicating a possibility that cell wall modification is necessary for tight adhesion. It is possible that the site of modification of the cell wall plays a role with regard to the target site of formation of tight adhesion of the plasma membrane. Further experiments should be performed, in which cell-wall modification is inhibited specifically, to examine whether cell wall modification is necessary for tight adhesion. Interestingly, incomplete half CS, i.e., CS formed for only one side, was observed in the stem treated with this inhibitor.²⁰ Such a CS may result when its formation is inhibited in only one of two adjacent cells, indicating that the complementary halves of an integral whole CS are formed by two adjacent endodermal cells. Alassimone et al. (2010) used the same inhibitor and observed no effect at a concentration of 50 µM on the development of the CS in arabidopsis roots. Again, we do not know the cause of the difference, but hypothesize that it may be due to a difference in the concentrations of the inhibitor used in these studies and/or the reasons mentioned above. They observed an inhibitory effect of the endocytic inhibitor wortmannin, but no effect on CS development was observed for a drug for actin depolymerization (latrunculin B) in arabidopsis roots.⁹

On the other hand, the position at which the upward progression of the tight adhesion of the plasma membrane stopped following irradiation of white light for 42 h was 2.8 ± 0.1 mm (mean ± SE, n = 5) below the bending point of the hook at the start of irradiation in pea stems. In addition, the position at which the upward progression of the modification of the cell wall stopped was exactly at the same position at which the upward progression of the tight adhesion of the plasma membrane stopped in every seedling examined. This result indicates that the regulation of CS formation by light took place at a certain developmental stage of an endodermal cell before these two processes, i.e., modification of the cell wall and tight adhesion of the plasma membrane, start. In other words, light exerts its effect at a stage at which a cell determines to (or not to) form the CS. Both, modification of the cell wall and tight adhesion of the plasma membrane to the cell wall, may start after an endodermal cell has determined to form the strip in darkness. Locations, where the cellular events involved in CS development are supposed to occur in a 6-d-old dark-grown pea stem, are summarized in Figure 1. Recently, it was shown that endodermal cell-cell contact is required for the spatial control of Casparian band development in Arabidopsis.²¹ Further, a novel protein family has been demonstrated to mediate CS formation in the endodermis of arabidopsis root,²² which will shed a new light on and facilitate the study of CS development.

Figure 1. Schematic illustration depicting locations where the cellular events involved in the development of the Casparian strip in a 6-d-old dark-grown pea stem. The locations of the endodermal cells undergoing these cellular events, as summarized in this review, are shown by the distance from the bending point of the hook of the stem (mm, mean ± SE ).

Karahara I, Takaya E, Fujibayashi S, Inoue H, Weller JL, Reid JB, et al. Development of the Casparian strip is delayed by blue light in pea stems. Planta. 2011;234:1019–30. doi: 10.1007/s00425-011-1451-7.