Plant- and stimulus-specific variations in remote-controlled sieve-tube occlusion

Abstract

Phloem injury triggers local sieve-plate occlusion including callose-mediated constriction and protein plugging of sieve pores. In intact plants, reversible sieve-plate occlusion is induced by electric potential waves (EPWs)—accompanied by Ca²⁺-influx—as result of distant burning. Here, we present additional results which pertain to (a) the variability of EPW-profiles in relation to forisome conformation in intact Vicia faba plants and (b) the differential occlusion reactions to burning and cutting in various plant species. A correlation between stimulus perception and mode of phloem loading is discussed.

Addendum to: Furch ACU, Hafke JB, Schulz A, van Bel AJE. Ca²⁺-mediated remote control of reversible sieve-tube occlusion in Vicia faba. J Exp Bot 2007; 58:2827-38.

Modes of Sieve-Plate Occlusion

Upon phloem injury, sieve elements (SEs) are occluded by combined callose-collar formation around sieve pores1,2 and protein plugging3–6 to prevent leakage of nutrients and invasion of phytopathogens.7

Given the fact that sieve pores are modified plasmodesmata (PDs),8 callose synthesis around PDs and sieve pores may strongly resemble. PD constriction is reached by Ca²⁺-dependent9 deposition of β-1,3-glucane polymers (callose) into the apoplast, caused by activation of membrane-associated callose synthase (β-1,3-glucan synthase); PD re-opening is achieved by a callose-degrading membrane-associated β-1,3-glucanase.10 In complement sieve-tubes are occluded by structural7,4 or, possibly, water-soluble proteins11 (Fig. 1). Among the structural proteins, forisomes (dispersive protein bodies, exclusively present in legume sieve tubes) were of particular importance for our studies. Forisomes disperse upon Ca²⁺-influx into SE-lumina and recondense spontaneously, probably after Ca²⁺-removal by Ca²⁺-pumps.12,13

Remote Control of Sieve-Tube Occlusion

In intact Vicia faba plants, heat stimuli induced distant SE-occlusion13 following the passage of an electrical potential wave (EPWs)14 during which Ca²⁺ flows into the SEs via activated Ca²⁺-channels.15 EPWs are generated by mechanical and physical stimuli16–18 and depend on voltage-activated and possibly mechanosensitive channels along sieve tubes.14,19

Our recent study13 showed, that forisomes at 3–4 cm from the stimulus site dispersed 15–45 sec after burning the leaf tip. The dispersion is reversible; the forisome recondensed after 7–15 min.

In the mean time, callose1 was deposited around sieve pores and pore plasmodesma units (PPUs; one-sided branched PDs between SE and CC). Callose deposition reached a maximum ca 20 min after the heat shock and was reversible too.13 Callose breakdown at sieve pores lasted for 1.5–2.0 h, whereas degradation appeared to be more rapid at PPUs (30–40 min) (Table 1). Burning experiments with Solanum lycopersicum and Ocimum basilicum showed similar, but species-specific time lapses (Table 1).

Different EPW-Profiles Correlated with the Forisome Reaction in Response to Leaf Tip Burning

The time lapse between forisome dispersion and recondensation appreciably varies (2–30 min) in Vicia faba, which suggests a variable engagement of Ca²⁺-channels/pumps. Optical examination in combination with electrophysiology disclosed a relationship between EPW-profile and forisome reaction (Figs. 2 and 3). In a typical example of a long depolarisation wave (Fig. 2), an abrupt SE membrane depolarisation from −150 mV down to −40 mV nearly coincides with forisome dispersion, 5–8 seconds after burning. The quick repolarisation changes smoothly into a plateau phase that lasts for several minutes. The forisome remained permanently dispersed during the depolarisation plateau phase (Fig. 2). This may result from massive initial Ca²⁺-influx before or permanent Ca²⁺-influx during the plateau. Hence, activity of Ca²⁺-ATPases20 may present a physiological bottleneck for removal of Ca²⁺ from SEs. With a short depolarisation (Fig. 3), the forisome only occasionally dispersed; only a marginal swelling was observed, which suggests a minor Ca²⁺-influx into the SE.

Wounding Reactions and Passage of EPWs After Cutting the Main Vein of Different Plant Species

Surprisingly, distant cutting of leaf tips of intact Vicia faba and Solanum lycopersicum plants induced neither an EPW nor distant callose deposition, but yet forisome dispersion (Vicia faba). Callose deposition was restricted to a distance of about 0.5 cm from the site of cutting and results from local wound effects. In the vicinity of cutting loss of turgor pressure may cause activation of mechano-sensitive Ca²⁺-channels21, but reduced Ca²⁺-influx into the SE lumen is not sufficient to induce callose production further away from the cut. In conclusion, forisome dispersion may have a lower Ca²⁺ threshold than callose synthesis.13

EPWs (as in Fig. 2) were recorded only at distance of 0.1–0.5 cm from the site of cutting; the electrical signal steeply decreased with the distance (Fig. 4). While a depolarisation of 54 mV was measured close to the cut, a spike with amplitude of 6 mV was observed at 2 cm from the cut. From a distance of 3 cm from the site of cutting on, no electrical signal was recorded (Fig. 4).

In contrast to lacking callose deposition in Vicia faba and Solanum lycopersicum, heavy callose deposition was induced by cutting in Ocimum basilicum and Ficus pumila (Table 2).

Concluding Remarks

1. EPW-profiles possibly correspond with the amount of free Ca²⁺-released into the SE lumen. The variability may depend on the strength of the stimulus and/or the spatial coupling between the SE measured and the site of burning. Strong innate differences between SEs are less likely.

2. EPWs triggered by cutting are more local and less persistent than those induced by burning. In the latter case, EPWs are presumably based on the collective activities of voltage- and mechano-sensitive Ca²⁺-channels; only mechano-sensitive channels would be activated by the turgor release following cutting.

3. The higher sensitivity (callose production) to cutting of symplasmic loaders may be purely coincidental in view of the few species probed. However, it may belong to a conglomerate of structural/functional properties related to the mode of phloem loading. The initial speculation22 that the differential anatomy of SE/CCs in collection phloem of symplasmic and apoplasmic loaders extends into the transport phloem zone, was substantiated by electron microscopic studies.23 Inherent consequences for photoassimilate distribution are inferred from membrane-potential measurements in transport phloem, where the capability of photoassimilate retrieval by SEs is higher in apoplasmic loaders.24 Furthermore, differences between symplasmic and apoplasmic loaders have been reported for stomatal regulation,25 invertase activity in the leaf apoplast,26 and adaptability to high-light regimes.27

Abbreviations

CC	=	companion cell
EPW	=	electrical potential wave
PD	=	plasmodesma
PPU	=	pore plasmodesma unit
SE	=	sieve element
SE/CC	=	sieve element/companion cell complex

Figures and Tables

Figure 1 Survey of the diversity of phloem-specific proteins in SEs. (1) Parietal protein clots, (1a) Fibrillar protein nets, (2) Dispersive protein bodies (=forisomes), (3) Non-dispersive protein bodies, (4) Proteins in SE plastids, (5) Soluble proteins.

Figure 2 Long-lasting electrical response of a Vicia faba SE following burning the leaf tip (at a distance of 3 cm) and photographs taken simultaneously as an optical control of forisome reaction (arrows 1–4). The forisome dispersed during the depolarisation and recondensed after repolarisation (3↑–4↑, 13.5 minutes). The forisome is marked with an asterisk. m, microcapillary for membrane potential recording.

Figure 3 Short-lasting electrical response of a Vicia faba SE following burning the leaf tip (at a distance of 3 cm) and photographs taken simultaneously as an optical control of forisome reaction (arrows 1–4). The forisome remained in the condensed state during the short plateau phase (2↑-3↑, 3 min). The forisome is marked with an asterisk. m, microcapillary for membrane potential recording.

Figure 4 Signal conduction in the form of EPW in Vicia faba SEs in dependence of the distance from the cutting site after subsequent cutting (2) with an earthed razor blade. The schematic drawing on the right side shows the experimental design. 1↑-2 cm from the cutting site; 2↑-1 cm from the cutting site; m, microcapillary for membrane potential recording; rb, razor blade.

Table 1 Time-course of sieve-tube occlusion

Plant species	Forisome dispersion/condensation	Callose production (SEs/PPUs)	Callose degradation (SEs)	Callose degradation (PPUs)
Vicia faba	15–45 sec/7–15 min	15–20 min	1.5–2 h	30–40 min
Solanum lycopersicum		20–25 min	2–3 h	40–60 min
Ocimum basilicum		15–20 min	1h	30–40 min

Time-course of sieve-tube occlusion reactions in 3 plant species after burning the leaf tip at a distance of 3 cm from the observation window in the phloem of the main vein of intact plants. PPU, pore plasmodesma unit; SE, sieve element.

Table 2 Callose deposition and mode of phloem loading

Plant species	Loading type	Burning	Cutting
Vicia faba	apoplasmatic	+	−
Solanum lycopersicum	apoplasmatic	+	−
Capsicum frutescens	apoplasmatic	+	−
Ocimum basilicum	symplasmatic	+	+
Ficus pumila	symplasmatic	+	+

Remote callose deposition in response to burning and cutting at a distance of 3 cm from the observation window in the phloem of the main vein of intact plants, in 3 apoplasmic and 2 symplasmic phloem-loaders (+ callose deposition; − no callose deposition).

Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft (BE 1925/8-2) in the frame of Schwerpunktprogramm 1108.

Addendum to: