Combined effect of boron and salinity on water transport: The role of aquaporins

Boron toxicity is an important disorder that can limit plant growth on soils of arid and semi arid environments throughout the world. Although there are several reports about the combined effect of salinity and boron toxicity on plant growth and yield, there is no consensus about the experimental results. A general antagonistic relationship between boron excess and salinity has been observed, however the mechanisms for this interaction is not clear and several options can be discussed. In addition, there is no information, concerning the interaction between boron toxicity and salinity with respect to water transport and aquaporins function in the plants. We recently documented in the highly boron- and salt-tolerant the ecotype of Zea mays L. amylacea from Lluta valley in Northern Chile that under salt stress, the activity of specific membrane components can be influenced directly by boron, regulating the water uptake and water transport through the functions of certain aquaporin isoforms.Key words: aquaporins, boron, salinity, water relations, Zea maysHigh concentrations of boron are often associated to saline soils in semi arid and/or arid climates and frequently crops are exposed to both stresses simultaneously.¹ As there is no a unique plant response to combination of salinity and boron toxicity, several mechanisms has been proposed to explain the experimental results. Some reports showed no additive effects of boron and salinity on shoot weight of different cultivars suggesting independent of the interaction.^2–5 However, additive effects^6–9 have been also proposed and the interaction of boron and salinity declined the rate of germination and limited growth in maize and sorghum plants.¹⁰ No explanation is currently available for these contradictory observations. Recently, the Abbot method has been applied to characterize the combined effect of boron and salinity at toxic levels in pepper plants, observing mainly an antagonistic relationship regarding growth and yield.¹¹ Antagonism between salinity and boron may be the result of decreased toxicity of boron in the presence of NaCl, reduced toxicity of NaCl in the presence of boron, or both together. Letey et al.,¹² have reported that increased soil salinity may also reduce boron movement to the broccoli plants and hence result in a reduction of boron toxicity symptoms. Reduction of boron accumulation in leaves in the presence of salinity has been also reported for melon,⁵ tomato⁸ jack pine¹³ and grapesvines¹⁴ and could be the result of the reduced rates of transpiration in plants where boron is transported via xylem as consequence of the osmotic effect of the salt. On the other hand, it has been observed that concentration of Na⁺ in leaves decreased with increasing addition of boron to the soil, probably due to the inhibition in root growth and reduction in root density caused by the boron treatment.¹⁵ Grieve and Poss⁷ found in wheat plants that the Cl⁻ content in the leaves was reduced when boron was increased. Similar results were reported in pepper plants suggesting that boron could reduce Cl⁻ toxicity.¹¹ Also, in our recent report although a nutrient imbalance resulted from the effect of salinity or boron alone, a general optimisation was observed when both treatments were applied together.¹⁶Under saline conditions, an optimal water balance is important in order to maintain the plant homeostasis and aquaporins may be one of the mechanisms involved under environmental and developmental changes.^17–19 However, there is no information concerning plant water uptake and transport in response to combined excess boron and salinity.It has been reported that, at high external B concentrations, considerable B transport occurs through the plasma membrane aquaporins, and a specific membrane intrinsic protein (MIP) has been described.²⁰ Thus boron uptake across the plasma membrane, by permeation through the lipid membrane and aquaporins, may be greatly influenced by the plant tolerance to salinity, through the associated changes in root hydraulic conductivity. Wimmer et al.,²¹ showed that salinity could interact with boron toxicity by a combined effect on boron and water uptake. In addition, we reported that the reduction of aquaporin functionality in NaCl-exposed plants could induce the reduction of plant boron concentration, producing a beneficial effect.²²Recently, we showed in a tolerant ecotype of maize a different pattern for PIP1 and PIP2 protein content under the application of excess of boron in combination with salinity, suggesting a differential aquaporin response in this cultivar and pointed out the complexity of the interaction.¹⁶ These results were in consonance with the previous observation that different aquaporin isoforms may represent a response to environmental changes.^18,19,²³ Thus, we concluded that the activity of specific membrane components can be influenced by boron under salt stress regulating the functions of certain aquaporin isoforms as possible components of the salinity tolerance mechanism. However, although a fine water transport control through the aquaporins could be necessary in order to reduce the accumulation of toxic boron levels in the tissues, the contribution of each isoform to water transport through the plasma membrane under boron-salinity combination must be elucidated.     

Silicon efflux transporters isolated from two pumpkin cultivars contrasting in Si uptake

The accumulation of silicon (Si) differs greatly with plant species and cultivars due to different ability of the roots to take up Si. In Si accumulating plants such as rice, barley and maize, Si uptake is mediated by the influx (Lsi1) and efflux (Lsi2) transporters. Here we report isolation and functional analysis of two Si efflux transporters (CmLsi2-1 and CmLsi2-2) from two pumpkin (Cucurbita moschata Duch.) cultivars contrasting in Si uptake. These cultivars are used for rootstocks of bloom and bloomless cucumber, respectively. Different from mutations in the Si influx transporter CmLsi1, there was no difference in the sequence of either CmLsi2 between two cultivars. Both CmLsi2-1 and CmLsi2-2 showed an efflux transport activity for Si and they were expressed in both the roots and shoots. These results confirm our previous finding that mutation in CmLsi1, but not in CmLsi2-1 and CmLsi2-2 are responsible for bloomless phenotype resulting from low Si uptake.Key words: silicon, efflux transporter, pumpkin, cucumber, bloomSilicon (Si) is the second most abundant elements in earth''s crust.¹ Therefore, all plants rooting in soils contain Si in their tissues. However Si accumulation in the shoot differs greatly among plant species, ranging for 0.1 to 10% of dry weight.^1–3 In higher plants, only Poaceae, Equisetaceae and Cyperaceae show a high Si accumulation.^2,3 Si accumulation also differs with cultivars within a species.^4,5 These differences in Si accumulation have been attributed to the ability of the roots to take up Si.^6,7Genotypic difference in Si accumulation has been used to produce bloomless cucumber (Cucumis sativus L.).⁸ Bloom (white and fine powders) on the surface of cucumber fruits is primarily composed of silica (SiO₂).⁹ However, nowadays, cucumber without bloom (bloomless cucumber) is more popular in Japan due to its more attractive and distinctly shiny appearance. Bloomless cucumber is produced by grafting cucumber on some specific pumpkin (Cucurbita moschata Duch.) cultivars. These pumpkin cultivars used for bloomless cucumber rootstocks have lower silicon accumulation compared with the rootstocks used for producing bloom cucumber.⁹Our study showed that the difference in Si accumulation between bloom and bloomless root stocks of pumpkin cultivars results from different Si uptake by the roots.¹⁰ Si uptake has been demonstrated to be mediated by two different types of transporters (Lsi1 and Lsi2) in rice, barley and maize.^11–15 Lsi1 is an influx transporter of Si, belonging to a NIP subfamily of aquaporin family.^10,11,13,14 This transporter is responsible for transport of Si from external solution to the root cells.¹¹ On the other hand, Lsi2 is an efflux transporter of Si, belonging to putative anion transporter.¹² Lsi2 releases Si from the root cells towards the xylem. Both Lsi1 and Lsi2 are required for Si uptake by the roots.^11,12 To understand the mechanism underlying genotypic difference in Si uptake, we have isolated and functionally characterized an influx Si transporter CmLsi1 from two pumpkin cultivars used for rootstocks of bloomless and bloom cucumber.¹⁰ Sequence analysis showed only two amino acids difference of CmLsi1 between two pumpkin cultivars. However, CmLsi1 from bloom rootstock [CmLsi1(B⁺)] showed transport activity for Si, whereas that from bloomless rootstock [CmLsi1(B⁻)] did not.¹⁰ Furthermore, we found that loss of Si transport activity was caused by one amino acid mutation at the position of 242 (from proline to leucine).¹⁰ This mutation resulted in failure to be localized at the plasma membrane, which is necessary for functioning as an influx transporter. The mutated protein was localized at the ER.¹⁰ Here, we report isolation and expression analysis of Si efflux transporters from two pumpkin cultivars contrasting in Si uptake and accumulation to examine whether Si efflux transporter is also involved in the bloom and bloomless phenotypes.     

Development of casparian strip in rice cultivars

The development of Casparian strips (CSs) on the endo- and exodermis and their chemical components in roots of three cultivars of rice (Oryza sativa) with different salt tolerance were compared using histochemistry and Fourier transform infrared (FTIR) spectroscopy. The development and deposition of suberin lamellae of CSs on the endo- and exodermis in the salt-tolerant cultivar Liaohan 109 was earlier than in the moderately tolerant cultivar Tianfeng 202 and the sensitive cultivar Nipponbare. The detection of chemical components indicated major contributions to the structure of the outer part from aliphatic suberin, lignin and cell wall proteins and carbohydrates to the rhizodermis, exodermis, sclerenchyma and one layer of cortical cells in series (OPR) and the endodermal Casparian strip. Moreover, the amounts of these major chemical components in the outer part of the Liaohan 109 root were higher than in Tianfeng 202 and Nipponbare, but there was no distinct difference in endodermal CSs among the three rice cultivars. The results suggest that the exodermis of the salt-tolerant cultivar Liaohan 109 functions as a barrier for resisting salt stress.Key words: casparian strip, chemical components, development, rice, rootPlant roots are in direct contact with the soil environment and thus particularly affected by unfavorable conditions. To withstand the surrounding environment, roots have developed anatomical and physiological adaptations. The development of Casparian strips (CSs) in the root endo- and exodermis is one such strategy.^1–3 In roots of most species, the sequence of development of the endo- and exodermis is roughly the same and involves two consecutive developmental stages: (1) formation of CSs in radial and transverse walls impregnating the primary cell wall pores with lipophilic and aromatic substances and (2) deposition of suberin lamellae to the inner surface of anticlinal and tangential cell walls.^4–6A major function of the CS is to block the non-selective apoplastic bypass flow of water and ions into the stele.³ Therefore, the structure,^7–9 chemical nature,^10–12 and physiological function^13,14 of endo- and exdodermal CSs in roots have been the focus of many investigations. Although oxygen loss, drought and salinity can influence the development and chemical nature of CSs in different rice cultivars,^15–19 few investigations have considered the development and formation of endo- and exdodermal CSs in the roots of rice cultivars with different salt tolerance under normal growing conditions.In the present paper, light microscopy and Fourier transform infrared (FTIR) spectroscopy were used to examine the cytochemistry and root anatomy of isolated CSs. The aim was to compare anatomical development and chemical characteristics of the endoand exdodermal CSs of three rice (Oryza sativa L.) cultivars having different salt tolerance in north China: the salt-tolerant Liaohan 109 and two widely grown cultivars, Tianfeng 202 and Nipponbare.     

The chemical cross talk between rice and barnyardgrass

The chemical cross talk between rice and barnyardgrass which is one of the most noxious weeds in rice cultivation was investigated. Allelopathic activity of rice was increased by the presence of barnyardgrass seedlings or barnyardgrass root exudates. Rice allelochemical, momilactone B, concentration in rice seedlings and momilactone B secretion level from rice were also increased by the presence of barnyardgrass seedlings or barnyardgrass root exudates. As momilactone B possesses strong growth inhibitory activity and acts as an allelochemical, barnyardgrass-induced rice allelopathy may be due to the increased momilactone B secretion. These results suggest that rice may respond to the presence of neighboring barnyardgrass by sensing the chemical components in barnyardgrass root exudates and increase allelopathic activity by elevated production and secretion levels of momilactone B. Thus, rice allelopathy may be one of the inducible defense mechanisms by chemical-mediated plant interaction between rice and barnyardgrass and the induced-allelopathy may provide a competitive advantage for rice through suppression of the growth of barnyardgrass.Key words: allelopathy, Echinochloa, chemical interaction, induced-allelopathy, momilactone, Oryza sativaThe chemical cross talk between host and symbiotic or parasitic plants is an essential process for the development of physical connections in symbiosis and parasitism.^1–3 Barnyardgrass is one of the most common and noxious weeds in rice paddy fields.⁴ Although barnyardgrass is adapted rice production system due to its similarity in growth habit, the reason why barnyardgrass so often invades into the rice paddy fields is unknown. There might be some special interactions between both plant species.Plants are able to accumulate phytoalexins around infection sites of pathogens soon after sensing elicitors of pathogen origin. This accumulation of phytoalexins can protect the plants from further pathogen infection.^5,6 Plants are also able to activate defense mechanisms against attacking herbivores by sensing volatile compounds, such as methacrolein and methyl jasmonate, released by herbivore-attacked plant cells. The volatile-sensed plants increase the production of phenolics, alkaloids, terpenes and defense proteins, which reduce herbivory attacks.^7,8 Therefore, plants are able to elevate the defense mechanisms against several biotic stress conditions by detection of various compounds.Allelopathy is the direct influence of organic chemicals released from plants on the growth and development of other plants.^9–11 Allelochemicals are such organic chemicals involved in the allelopathy.^12,13 Allelochemicals can provide a competitive advantage for host-plants through suppression of soil microorganism and inhibition of the growth of competing plant species because of their antibacterial, antifungal and growth inhibitory activities.^3,14,15Rice has been extensively studied with respect to its allelopathy as part of a strategy for sustainable weed management, such as breeding allelopathic rice strains. A large number of rice varieties were found to inhibit the growth of several plant species when these rice varieties were grown together with these plants under the field or/and laboratory conditions.^16–20 These findings suggest that rice may produce and release allelochemicals into the neighboring environments and may inhibit the growth of the neighboring plants by the allelochemicals.Potent allelochemical, momilactone B, was isolated from rice root exudates.²¹ Momilactone B inhibits the growth of typical rice weeds like barnyardgrass and Echinochloa colonum at concentrations greater than 1 µM and the toxicity of momilactone B to rice itself was very low.²² In addition, rice plants secrete momilactone B from the roots into the rhizosphere over their entire life cycle.²² The observations suggest rice allelopathy may be primarily dependant on the secretion levels of momilactone B from the rice seedlings.^22,23Allelopathic activity of rice exhibited 5.3- to 6.3-fold increases when rice and barnyardgrass seedlings were grown together. Root exudates of barnyardgrass seedlings also increased allelopathic activity and momilactone B concentration in rice seedlings. The increasing the exudate concentration increased the allelopathic activity and momilactone B concentration in rice.²⁴ Thus, the chemical components in barnyardgrass root exudates may affect gene expressions involved in momilactone B biosynthesis. However, effects of the barnyardgrass root exudates on the secretion level of mimilactone B from rice has not yet reported.Rice seedlings were incubated in the medium containing barnyardgrass root exudates for 10 d, and secretion level of momilactone B by rice was determined (Fig. 1). The root exudates increased the secretion level significantly at concentrations greater than 30 mg/L of barnyardgrass root exudates, and increasing the concentration increased the secretion level. At concentrations of 300 mg/L of the root exudates, the secretion level was 10-fold greater than that in control (0 mg of root exudate). There was no significant difference in the osmotic potential between the medium contained barnyardgrass root exudates and control medium (all about 10 mmol/kg), and pH value of the medium was maintained at 6.0 throughout the experiments.²⁵ These results suggest that unknown chemical components in the barnyardgrass root exudates may induce the secretion of momilactone B from rice. As momilactone B possesses strong phytotoxic and allelopathic activities,^21–23,25 the elevated production and secretion of momilactone B in rice may provide a competitive advantage for root establishment through local suppression of pathogens and inhibition of the growth of competing plant species including barnyardgrass. Thus, barnyardgrass-induced rice allelopathy may be caused by the chemical components in the barnyardgrass root exudates.Open in a separate windowFigure 1Effects of barnyardgrass root exudates on momilactone B secretion level in rice. Rice seedlings were incubated in the medium containing barnyardgrass root exudates for 10 d, and secretion level of momilactone B was determined as described by Kato-Noguchi.²⁴ The experiment was repeated six times with three assays for each determination. Different letters show significant difference (p < 0.01) according to Tukey''s HSD test.Although mechanisms of the exudation are not well understood, it is suggested that plants are able to secrete a wide variety of compounds from root cells by plasmalemma-derived exudation, endoplasmic-derived exudation and proton-pumping mechanisms.^3,15 Through the root exudation of compounds, plants are able to regulate the soil microbial community in their immediate vicinity, change the chemical and physical properties of the soil, and inhibit the growth of competing plant species.^3,14,15 The present research suggests that rice may be aware of the presence of neighboring barnyardgrass by detection of certain key in barnyardgrass root exudates, and this sensorial function may trigger a signal cascade resulting in increasing rice allelopathy through increasing production of momilactone B and secretion of momilactone B into the rhizosphere. Therefore, rice allelopathy may potentially be an inducible defense mechanism by chemical-mediated plant interactions between rice and barnyardgrass.     

The shared and separate roles of aposematic (warning) coloration and the co-evolution hypothesis in defending autumn leaves

The potential anti-herbivory functions of colorful (red and yellow) autumn leaves received considerable attention in the last decade. The most studied and discussed is the co-evolutionary hypothesis, according to which autumn coloration signals the quality of defense to insects that migrate to the trees in autumn. In addition to classic aposematism (repellency due to signaling unpalatability, non profitability of consumption, or danger for whatever reasons) that operates immediately, this hypothesis also proposes that the reduced fitness of the insects is in their next generation hatching in the spring from eggs laid on the trees in autumn. Supporters of the co-evolutionary hypothesis either posited that this hypothesis differs from visual aposematism or ignored the issue of aposematism. Interestingly, other authors that cited their papers considered the co-evolutionary hypothesis as visual aposematism. Recently, the overlap between the co-evolutionary hypothesis and visual aposematism was finally recognized, with the exception of yellow autumn leaves not signaling defense to aphids, which are known to be attracted to yellow leaves. However, the detailed relationships between these two hypotheses have not been discussed yet. Here I propose that the co-evolutionary hypothesis generally equals visual aposematism in red and yellow autumn leaves towards all herbivores except for yellow not operating with aphids. The co-evolutionary signaling extends beyond classic aposematism because it may operate later and not only immediately. The possibility that for yellow autumn leaves the co-evolutionary hypothesis may also operate via olfactory aposematism should not be dismissed.Key words: aposematic, autumn coloration, co-evolution, defense, evolution, herbivory, treesColorful (red and yellow) autumn leaves dominate large areas of America, Asia and Europe, expressed by thousands of tree, shrub and climber species.^1–5 In the last decade, this phenomenon received considerable scientific attention. For a long time it was a common belief that this coloration is the by-product of the cessation of masking by chlorophylls that degrade in autumn. However, two key theoretical and experimental developments stimulated the recent wave of study of autumn leaf coloration. The first was the recognition that anthocyanins are synthesized de novo in red autumn leaves,^1,2 and the second was the formulation of the anti-herbivory co-evolutionary hypothesis.^6–8The updated version of the co-evolutionary hypothesis⁹ posits that red autumn coloration signals to all types of insects (including aphids) that migrate to the trees in autumn about their chemical defense, lower nutritional quality or imminent leaf fall, or any other characteristic that would induce a lower fitness in the insects. In addition, yellow leaves signals the same to all herbivores except aphids. A special aspect of the co-evolutionary hypothesis is that the reduced fitness of the insects is not only immediate, reducing insect feeding in autumn, but also related to the reduced development of the next generation that hatches in the following spring from eggs laid on the trees in the autumn.⁹ Originally, the co-evolutionary hypothesis addressed both red and yellow autumn leaves.^6–8 However, with the later understanding that yellow leaves usually attract rather than repel aphids,^9–13 the co-evolutionary hypothesis was later restricted to red leaves when aphids are concerned.⁹In addition to other various potential anti-herbivory roles,^14,15 red autumn leaf coloration has several potential physiological functions, such as protection from photoinhibition and photo oxidation, and other physiological functions have been proposed but not agreed upon.^{1,2,9,16–21}     

The ever expanding role of aquaglyceroporins: Confirmation of protein-facilitated boron transport

The exact mechanism of transport of boron (B) entering the plant cell as boric acid B(OH)₃, has become hotly debated with evidence for both passive and protein facilitated transport. Here we put the controversy to rest by confirming that boron influx into plants can be partially controlled by opening and closing of channel-like transport proteins. Using treatments that were likely to inhibit membrane transporters capable of facilitating B transport, we confirmed that at least 50% of B transport could be contributed by a transporter of some type in barley roots. Based on the physiochemical similarities between B(OH)₃ and other solutes that were known to be transported via aquaglyceroporins, we hypothesised that aquaglyceroporins would be likely candidates to facilitate B(OH)₃ transport into the cytoplasm. We demonstrated using functional yeast complementation that two barley root aquaglyceroporins, HvPIP1;3 and HvPIP1;4, were both capable of facilitating B transport. This finding has demonstrated yet another function of aquaglyceroporins.Key words: aquaporin, aquaglyceroporin, boron, transport, PIPsThe major intrinsic protein (MIP) superfamily contains aquaporins and the related ‘aquaglyceroporins’ (AQGP), whose numbers and functionality are rapidly expanding.¹ These transport proteins are responsible for not only the bidirectional transport of water and glycerol, but also for the transport of other small neutral uncharged solutes. Based on their size, net charge and volume compared to the diameter of the aquaglyceroporin pore, it was predicted that a range of other molecules such as arsenite (AsIII) and silicic acid Si(OH)₄ would also permeate aquaglyceroporins, and this has been confirmed.^2–5 It has long been argued that because of the strong similarity with H₂O, it could reasonably be assumed that H₂S would cross membranes via aquaporins. However, it has very recently been demonstrated that membrane fluxes of H₂S were insensitive to treatments that inhibited influx of H₂O, leading to the conclusion that H₂S simply passed through the phospholipid bilayer and not through a protein transporter.⁶Boron (B), available to plants as boric acid, B(OH)₃, can be classed as a small neutral uncharged molecule based on physiochemical similarities to glycerol and arsenite.⁷ Like H₂S, the research surrounding B transport across biological membranes has been highly debated and the literature contains conjecture about the exact mode of transport with evidence for both passive and active transport. Several studies have demonstrated substantial passive B movement through both lipid bilayers and plant membranes, consistent with measurements indicating that B has high lipid solubility which would favor permeation through such membranes.^8–12 These data suggested that protein-mediated transport into cells would be redundant and would be short-circuited by the passive leak pathway. However, other reports have indicated that B transport may have an active transport component when plants were grown under B deficient conditions.^12,13 The presence of protein-assisted passive transport has proved hard to establish.Our recent work has focused on putting this controversy to rest by attempting to modify B uptake using treatments that should not affect B transfer through the lipid phase of the membrane.⁷ Firstly, we hypothesized that aquaglyceroporins may be involved in the transport of B and examined influx, efflux and concentration-dependence of B uptake in barley roots using inhibitors known to cause the closure of aquaporins though cytoplasmic acidification (butyric acid) or metabolic inhibition (sodium azide). Results from these experiments demonstrated that a significant component of both B influx and efflux was responsive to these treatments. Metabolic inhibition by sodium azide reduced influx and efflux by 40–50%, while cytoplasmic acidification with butyric acid reduced influx to a lesser but still significant degree.⁷Secondly, in order to elucidate which transport proteins may be involved, we hypothesised more specifically that the PIP1 subgroup may be able to facilitate the movement of B(OH)₃ based on the location of such proteins on the plasma membrane. This had previously been suggested by Dordas et al.¹⁴ who showed that a maize aquaporin ZmPIP1 when expressed in Zenopus oocytes could account for at least 25% of B uptake. We selected two aquaglyceroporins isoforms previously characterised from barley roots,^15–17 HvPIP1;3 and HvPIP1;4, and functionally expressed these in a Saccharomyces cerevisiae mutant containing a deletion of the yeast native aquaglyceroporin, FPS1. Expression of these PIP1 constructs caused the yeast to become sensitive to B toxicity. Influx measurement revealed that both HvPIP1;3 and HvPIP1;4 were capable of transporting B as indicated by increases of up to 40% in the rate of B uptake. Activation in yeast of some plant Nod 26-like intrinsic proteins (NIPs) that also function as aquaglyceroporins, requires truncation of the N-terminal sequence, presumably because this region contains a control domain. In our yeast experiments, a truncated version of HvPIP1;3 (HvPIP1;3t) was engineered to determine the effect of the removal of the first 44 amino acids from the N-terminal tail on the expression and subsequent B transport capacity. Surprisingly truncation of HvPIP1;3 had little effect on either the expression or transport capacity of HvPIP1;3.As a result of this study it has been firmly established that boron entry into plants can be partially controlled by opening and closing of channel-like transport proteins. Specifically, we have demonstrated that B can be transported via two aquaglyceroporins, HvPIP1;3 and HvPIP1;4. However, we suspect that most of the HvPIP1 subgroup, which contains another 3 members, may all have some capacity to transport B based on high sequence homology amongst the PIP1 subgroup.The confirmation of the ability of PIP1s to transport B contributes greatly to the overall understanding of B transport in the plant system. Recently other aquaglyceroporins NIP5;1 and NIP6;1 have also been shown to be involved in B influx^18–20 while a separate class of non-aquaglyceroporins, that are structurally related to anion exchangers, are involved in the active efflux of B under toxicity conditions^21,22 or xylem loading under deficiency conditions.^23,24Aquaglyceroporins may have evolved to facilitate transport of beneficial and essential nutrients such as Si(OH)₄,² B(OH)₃, urea and ammonia²⁵ but other toxic molecules with similar physiochemical characteristics such as AsIII and Sb(OH)₃ may have ‘piggy backed’ on the process allowing these toxins to also enter the plant system. An understanding of selectivity mechanism that allows both essential and toxic elements to pass through the aquaglyceroporin pore and into the cytoplasm may have important implications for research into the potential bioremediation of toxic substances. It seems highly probable that other small molecules will be shown to be transported by aquaglyceroporins. There is still much to be learnt about the roles of other classes of MIPs, in particular NIPs, small basic intrinsic proteins (SIPs)²⁶ and tonoplast intrinsic proteins (TIPs) in the movement of these molecules into and within cells. No doubt the roles and functions of aquaglyceroporins within the plant system will continue to grow.     

Influence of Leaf Tolerance Mechanisms and Rain on Boron Toxicity in Barley and Wheat

Boron (B) toxicity is common in many areas of the world. Plant tolerance to high B varies widely and has previously been attributed to reduced uptake of B, most commonly as a result of B efflux from roots. In this study, it is shown that the expression of genes encoding B efflux transporters in leaves of wheat (Triticum aestivum) and barley (Hordeum vulgare) is associated with an ability of leaf tissues to withstand higher concentrations of B. In tolerant cultivars, necrosis in leaves occurred at B concentrations more than 2-fold higher than in sensitive cultivars. It is hypothesized that this leaf tolerance is achieved via redistribution of B by efflux transporters from sensitive symplastic compartments into the leaf apoplast. Measurements of B concentrations in leaf protoplasts, and of B released following infiltration of leaves, support this hypothesis. It was also shown that under B-toxic conditions, leaching of B from leaves by rain had a strong positive effect on growth of both roots and shoots. Measurements of rates of guttation and the concentration of B in guttation droplets indicated that the impact of guttation on the alleviation of B toxicity would be small.Boron (B) toxicity affects a wide variety of plants growing on soils with naturally high levels of B or when irrigated with water containing elevated levels of B (Stangoulis and Reid, 2002). Symptoms are most commonly seen as necrosis on leaf margins or leaf tips, depending on the type of leaf venation (Oertli and Kohl, 1961). Plant tolerance to high B varies considerably but is most commonly associated with reduced accumulation of B (Nable et al., 1997). Hayes and Reid (2004) identified differences in B efflux in roots as the primary determinant of the net uptake of B in barley (Hordeum vulgare). Reid (2007) established that this was also the mechanism for differences in B uptake in wheat (Triticum aestivum) and showed that there was a strong correlation between tolerance in both wheat and barley with the expression in roots of the genes TaBOR2 and HvBOR2, which encode B efflux transporters with homology to B efflux transporters in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa; Takano et al., 2002; Nakagawa et al., 2007). Since the concentration of B in shoots was closely related to the concentration of B in roots (Hayes and Reid, 2004; Reid, 2007) a simple mechanism of tolerance could be explained. A similar mechanism of tolerance was shown to occur in Arabidopsis when roots overexpressed AtBor4 (Miwa et al., 2007).Sutton et al. (2007) made a qualitative analysis of the expression in leaves of Bot1 (which is identical to HvBOR2 and to avoid confusion will henceforth be referred to as HvBOR2) and found strong expression associated with hydathodes in the leaf tip. They proposed that in addition to root-based tolerance conferred by pumping of B from roots, that further tolerance could be achieved by excretion of B from hydathodes and its subsequent removal by rain. Oertli (1962) demonstrated that in young barley seedlings, significant amounts of B could be lost from leaves in this way.In the early work on B tolerance in cereals, it was noted that toxicity for plants grown in the field was generally observed at much lower concentrations of B in leaves than for plants grown in the glasshouse. For example, Nable et al. (1990) found that a 17% reduction in yield of field-grown barley occurred with a shoot B concentration of 62 mg kg⁻¹ dry weight (DW) whereas in the glasshouse the corresponding concentration was 120 mg kg⁻¹ DW. It was concluded that the most likely cause of the difference in shoot B between the growth conditions was leaching of B from leaves by rain in the field. However, an experiment in which a comparison was made between plants on which the leaves were regularly sprayed with water or not sprayed failed to show any difference in growth, despite significant reductions in leaf B in the sprayed plants (Nable et al., 1990).Jefferies et al. (1999) identified chromosome regions associated with tolerance in barley. They found a major locus on chromosome 4 that was related to reduced B uptake and a decrease in leaf symptoms. This locus was subsequently found to contain HvBOR2 (Sutton et al., 2007), whose expression in roots could explain both reduced B uptake and the decrease in leaf symptoms. In addition to the locus on chromosome 4, there was another locus on chromosome 2 that was associated with leaf symptom score but not associated with whole shoot B concentration (Jefferies et al., 1999).In this study we have shown that the expression of B efflux transporter genes in leaves results in enhanced tolerance to B, and contrary to previous reports, that rain can significantly reduce B toxicity.     

Strigolactones' ability to regulate root development may be executed by induction of the ethylene pathway

The newly defined phytohormones strigolactones (SLs) were recently shown to act as regulators of root development. Their positive effect on root-hair (RH) elongation enabled examination of their cross talk with auxin and ethylene. Analysis of wild-type plants and hormone-signaling mutants combined with hormonal treatments suggested that SLs and ethylene regulate RH elongation via a common regulatory pathway, in which ethylene is epistatic to SLs. The SL and auxin hormonal pathways were suggested to converge for regulation of RH elongation; this convergence was suggested to be mediated via the ethylene pathway, and to include regulation of auxin transport.Key words: strigolactone, auxin, ethylene, root, root hair, lateral rootStrigolactones (SLs) are newly identified phytohormones that act as long-distance shoot-branching inhibitors (reviewed in ref. ¹). In Arabidopsis, SLs have been shown to be regulators of root development and architecture, by modulating primary root elongation and lateral root formation.^2,3 In addition, they were shown to have a positive effect on root-hair (RH) elongation.² All of these effects are mediated via the MAX2 F-box.^2,3In addition to SLs, two other plant hormones, auxin and ethylene, have been shown to affect root development, including lateral root formation and RH elongation.^4–6 Since all three phytohormones (SLs, auxin and ethylene) were shown to have a positive effect on RH elongation, we examined the epistatic relations between them by examining RH length.⁷ Our results led to the conclusion that SLs and ethylene are in the same pathway regulating RH elongation, where ethylene may be epistatic to SLs.⁷ Moreover, auxin signaling was shown to be needed to some extent for the RH response to SLs: the auxin-insensitive mutant tir1-1,⁸ was less sensitive to SLs than the wild type under low SL concentrations.⁷On the one hand, ethylene has been shown to induce the auxin response,^9–12 auxin synthesis in the root apex,^11,12 and acropetal and basipetal auxin transport in the root.^4,13 On the other, ethylene has been shown to be epistatic to SLs in the SL-induced RH-elongation response.⁷ Therefore, it might be that at least for RH elongation, SLs are in direct cross talk with ethylene, whereas the cross talk between SL and auxin pathways may converge through that of ethylene.⁷ The reduced response to SLs in tir1-1 may be derived from its reduced ethylene sensitivity;^7,14 this is in line with the notion of the ethylene pathway being a mediator in the cross talk between the SL and auxin pathways.The suggested ethylene-mediated convergence of auxin and SLs may be extended also to lateral root formation, and may involve regulation of auxin transport. In the root, SLs have been suggested to affect auxin efflux,^3,15 whereas ethylene has been shown to have a positive effect on auxin transport.^4,13 Hence, it might be that in the root, the SLs'' effect on auxin flux is mediated, at least in part, via the ethylene pathway. Ethylene''s ability to increase auxin transport in roots was associated with its negative effect on lateral root formation: ethylene was suggested to enhance polar IAA transport, leading to alterations in the quantity of auxin that unloads into the tissues to drive lateral root formation.⁴ Under conditions of sufficient phosphate, SL''s effect was similar to that of ethylene: SLs reduced the appearance of lateral roots; this was explained by their ability to change auxin flux.³ Taken together, one possibility is that the SLs'' ability to affect auxin flux and thereby lateral root formation in the roots is mediated by induction of ethylene synthesis.To conclude, root development may be regulated by a network of auxin, SL and ethylene cross talk.⁷ The possibility that similar networks exist elsewhere in the SLs'' regulation of plant development, including shoot architecture, cannot be excluded.     

Root tips moving through soil: An intrinsic vulnerability

Root elongation occurs by the generation of new cells from meristematic tissue within the apical 1–2 mm region of root tips. Therefore penetration of the soil environment is carried out by newly synthesized plant tissue, whose cells are inherently vulnerable to invasion by pathogens. This conundrum, on its face, would seem to reflect an intolerable risk to the successful establishment of root systems needed for plant life. Yet root tip regions housing the meristematic tissues repeatedly have been found to be free of microbial infection and colonization. Even when spore germination, chemotaxis, and/or growth of pathogens are stimulated by signals from the root tip, the underlying root tissue can escape invasion. Recent insights into the functions of root border cells, and the regulation of their production by transient exposure to external signals, may shed light on long-standing observations.Key words: border cells, chemotaxis, zoospores, neutrophil extracellular traps (NETs)…The evidence suggests that there has evolved within plants, mechanisms for extremely rapid adjustment to changes in the soil environment. The logical conclusion is that plants can and do selectively manipulate the ecological balances within the rhizosphere to their own advantage.¹“Sloughed root cap cells” that detach from the root tip were long presumed to be moribund tissue serving to lubricate passage of the elongating root.² The discovery nearly a century ago that these cells from Zea mays L. and Pisum sativum L. can remain 100% viable for weeks after detachment into hydroponic culture did not alter this perception.³ In recent decades, studies have shown that the cells from root caps of most species are metabolically active and can survive even after detachment into the soil.⁴ Moreover, the cell populations express distinct patterns of gene expression reflecting tissue specialization and were therefore given the name root ‘border’ cells.⁵ Like ‘border towns’ that exist at the boundary of disparate countries and cultures, border cells are part of the plant and part of the soil, yet distinct from both.The soil is a dynamic environment whose pH, surface charge, water availability, texture and composition can range markedly on a large and small scale.^1,6,7 The concept of a ‘microniche’ emphasizes that the biological requirements for a particular soil microorganism may be met within one site but not another site only a micron away.⁸ Thus, the rhizosphere—the region adjacent to root surfaces—can support much higher levels of microorganisms than bulk soil a few millimeters distant.⁹ This phenomenon is recognized to be driven by an increased availability of nutrients released from plants into the external environment.¹⁰ Less well recognized is the dynamic variation that occurs along the root surface, and its significance in patterns of disease development. As roots emerge and the new tissue differentiates progressively through stages from root cap, root apical meristem, elongation zone, and finally mature roots with lignified cell walls, the material released into the environment also changes.^11–13 More than 90% of bulk carbon released from young roots of legumes is delivered by the root cap, a 1 mm zone at the apex.¹⁴ Some pathogens are attracted specifically to the root tip region, presumably in response to such exudates.^15,16 For example, instantaneous swarming occurs when a cotton root is placed into a suspension of Pythium dissotocum zoospores (Sup. Fig. 1). This host-specific attraction is specific to the root tip region where border cells are present (Sup. Fig. 2). Border cells remain attractive to zoospores when removed from the root (Sup. Fig. 3). The nature of the attractant is not known, but its impact is localized and transient (Sup. Fig. 4).Newly generated tissue is highly susceptible to infection by pathogens, in general, so elongating root tips would be predicted to be vulnerable to invasion. And yet, root apices repeatedly have been found to escape infection and colonization.^17–19 Recent discoveries about parallels between mammalian white blood cells and root border cells may provide new insight into this apparent conundrum.²⁰ Neutrophils, a type of white blood cell, are produced in response to infection. Neutrophil extracellular traps (NETs) then attract and kill the invader through a process that requires extracellular DNA (exDNA) and an array of extracellular proteins.^21,22 Border cell production, like that of neutrophils, also is induced in response to signals from pathogens and root tip resistance to infection requires exDNA and an array of extracellular proteins.^20,23 Root tip specific chemotaxis, like that seen with Pythium zoospores, has been presumed to involve steps in a process of pathogen invasion.^15,16 It may, instead, involve a process of extracellular trapping and killing by cells designed to protect root meristems from invasion, in a manner analogous to that which occurs in mammalian defense. If tests confirm this model, the mystery of how root tips escape infection by soilborne pathogens they attract could be resolved.     

Colors of young and old spring leaves as a potential signal for ant-tended hemipterans

A new hypothesis explaining the adaptive significance of bright autumn leaf colors argues that these colors signal tree quality to myrmecophilous specialist aphids. In turn, the aphids attract aphid-tending ants during the following spring, which defend the trees from other aphids and herbivores. In this context, other types of plant coloration, such as the color change observed in young and old spring leaves, may function as a signal of plant quality for aphids and other myrmecophilous hemipterans. If these plant colors are costly for plants, then vividly colorful plants would be required to invest more in growth than in defense; as a result, colorful plants may be more palatable for honeydew-producing hemipterans, such as aphids, scale insects and treehoppers, although the relative importance of hemipterans other than aphids may be relatively low. These hemipterans may be attracted to colorful plants, after which their attendant ants would protect the plants from herbivory. However, it is necessary to examine color vision in hemipterans to support this hypothesis.Key words: ant-Hemiptera interactions, indirect effects, myrmecophiles, plant-ant mutualism, plant coloration, tritrophic interactionsRecently, the adaptive significance of plant coloration has attracted scientific interest.¹ Various theories have been postulated to explain the adaptive value of autumn leaf colors (red and yellow).² The coevolution hypothesis, the most novel and challenging theory among those proposed, argues that bright leaf colors serve as a conspicuous defense signal against autumn-colonizing insect herbivores, particularly aphids.³ According to this hypothesis, the production of autumn color pigments is an indicator of a particularly vigorous tree. Aphids, which have color vision and have long been associated with trees, migrate to winter host trees in the autumn and cause substantial damage. Therefore, vivid leaf color in the autumn would encourage aphids to colonize other less vigorously defended trees.⁴ Hamilton and Brown³ and Holopainen and Peltonen⁵ detected a higher number of specialist aphids on tree species with more intense autumn colors.After Hamilton and Brown,³ several researchers have attempted to explain the relationship between aphids and autumn color.^2,6 However, they did not account for several possibilities.⁶ First, healthy, vigorous trees may not be well defended, because they invest more in growth than in defense. Second, some aphid species avoid colonizing trees with bright colors, whereas others are attracted to bright colors. Finally, there are numerous multispecific interactions between plants, herbivores, predators and parasitoids in tree crowns. Ants prey on various arthropods living in trees, and ant-aphid mutualism affects arboreal arthropod communities. I incorporated these factors and formed a hypothesis in which autumn leaf colors signal tree quality to myrmecophilous specialist aphids. These aphids, in turn, attract aphid-tending ants during the following spring, which then defend the trees from other aphids and herbivores. Thus, autumn colors may be adaptive, because they attract myrmecophilous specialist aphids and their attendant ants, thereby reducing herbivory and interspecific competition among aphids.⁶In this addendum, I extend my former hypothesis beyond the relationship between autumn leaf colors and aphids. First, myrmecophilous aphids are not the only arthropods that benefit trees. Styrsky and Eubanks⁷ recently reviewed the literature regarding the effects of interactions between ants and honeydew-producing hemipterans on plants, and found that plants actually benefited indirectly from these interactions in most cases. This finding supports a new hypothesis focused on plant-ant mutualism via aphids. In addition, the mutualism between ants and honeydew-producing hemipterans includes many other organisms in addition to aphids, such as scale insects and treehoppers. Scale insects, especially soft scales (Coccidae) and mealybugs (Pseudococcidae), comprise many species that are tended by honeydew-collecting ants,⁸ and ant-scale insect mutualism is often beneficial for host plants.⁷ Although the female adults of scale insects are usually immobile, first-instar nymphs (crawlers) disperse by wind and locate on host plants, usually trees.⁹ The nymphs, emerging at various times from spring to autumn,¹⁰ may use plant coloration to select a suitable host. However, because specialist coccids and mealybugs represent a minority among the speciose scale insects,10 coevolutionary relationships between plants and ants via specialist scale insects may be relatively rare. The treehoppers also comprise many myrmecophilous species,^8,11 but the diversity of this group is highest in tropical regions; only a relatively small number of membracid species are present in temperate regions.¹² Therefore, scale insects and treehoppers may be attracted to autumn colors, and their attendant ants may then defend trees against other herbivorous insects. To fully account for the adaptive value of autumn colors, one would expect the importance of these hemipterans to be less than that of aphids, based on their low host-plant specificity, restricted distribution and life cycles. However, hemipterans may be associated with plant coloration in other aspects than autumn leaf color.Second, the colors of young and old spring leaves may also signal plant quality to ant-tended honeydew-producing hemipterans. The young leaves of many plants are reddish or yellowish (Fig. 1A and B).¹³ In the spring and other seasons, the old leaves of some evergreen tree species turn red or yellow (Fig. 1B). Because changes in leaf color may occur from spring to autumn, various hemipteran species may play specific roles as the season progresses. Aphids migrate in the spring and in the autumn,¹⁴ although most host-alternating aphids migrate to trees in autumn and to herbs in the late spring in temperate regions.¹⁵ If plants pay some cost for these colors¹⁶ and vivid colors indicate high plant quality for hemipterans, then changing colors may attract myrmecophilous hemipterans including aphids, scale insects and treehoppers, which may then protect plants against herbivory by other insects.Open in a separate windowFigure 1(A) Red young leaves of the evergreen oak Quercus glauca. (B) Yellowish young and reddish old leaves of the camphor tree Cinnamomum camphora.However, color vision has not been examined in detail in most hemipteran insects.^17,18 Many insects are insensitive to red, although one species of flower-visiting thrip is specifically attracted to red flowers.¹⁹ Thus, studies on color vision in hemipteran insects are required to evaluate this new hypothesis, as well as the coevolution hypothesis.     

Strigolactones as mediators of plant growth responses to environmental conditions

Strigolactones (SLs) have been recently identified as a new group of plant hormones or their derivatives thereof, shown to play a role in plant development. Evolutionary forces have driven the development of mechanisms in plants that allow adaptive adjustments to a variety of different habitats by employing plasticity in shoot and root growth and development. The ability of SLs to regulate both shoot and root development suggests a role in the plant''s response to its growth environment. To play this role, SL pathways need to be responsive to plant growth conditions, and affect plant growth toward increased adaptive adjustment. Here, the effects of SLs on shoot and root development are presented, and possible feedback loops between SLs and two environmental cues, light and nutrient status, are discussed; these might suggest a role for SLs in plants'' adaptive adjustment to growth conditions.Key words: strigolactones, light, nutrient status, root, shoot, branching, lateral roots, root hairsStrigolactones (SLs) are carotenoid-derived terpenoid lactones suggested to stem from the carotenoid pathway¹ via the activity of various oxygenases.^2,3 SLs production has been demonstrated in both monocotyledons and eudicotyledons (reviewed in ref. ⁴), suggesting their presence in many plant species.⁵ SLs are synthesized mainly in the roots and in some parts of the stem and then move towards the shoot apex (reviewed ref. ⁷).^6,8,9SLs were first characterized more than 40 years ago as germination stimulants of the parasitic plants Striga and Orobanche and later, as stimulants of arbuscular mycorrhiza hyphal branching as well (reviewed in ref. ^{4, 10–13}). Recently, SLs or derivatives thereof, have been identified as a new group of plant hormones, shown to play a role in inhibition of shoot branching,^2,3,8,9 thereby affecting shoot architecture; more recently they have also been shown to affect root growth by affecting auxin efflux.¹⁴Plants have developed mechanisms that allow adaptive adjustments to a variety of different habitats by employing plasticity in their growth and development.¹⁵ Shoot architecture is affected by environmental cues, such as light quality and quantity and nutrient status.^16–19 Root-system architecture and development are affected by environmental conditions such as nutrient availability (reviewed in ref. ^{20, 21}). At the same time, plant hormones are known to be involved in the regulation of plant growth, development and architecture (reviewed in ref. ^22–24) and to be mediators of the effects of environmental cues on plant development; one classic example is auxin''s role in the plant''s shade-avoidance response (reviewed in ref. ²⁵).The ability of SLs to regulate shoot and root development suggests that these phytohormones also have a role in the plant''s growth response to its environment. To play this putative role, SL pathways need to be responsive to plant growth conditions, and affect plant growth toward enhancing its adaptive adjustment. The present review examines the SLs'' possible role in adaptive adjustment of the plant''s response to growth conditions, by discussing their effect on plant development and the possible associations and feedback loops between SLs and two environmental cues: light and nutrient status.     

Chemical Signal as a Rapid Long-Distance Information Messenger After Local Wounding of a Plant?

A series of works have described an important role of chemical signaling compounds in generation of the stress response of plants in both the wounded and distant undamaged plant tissues. However, pure chemical signals are often not considered in the fast (minutes) long-distance signaling (systemic response) because of their slow propagation speed. Physical signals (electrical and hydraulic) or a combination of the physical and chemical signals (hydraulic dispersal of solutes) have been proposed as possible linkers of the local wound and the rapid systemic response. We have recently demonstrated an evidence for involvement of chemical compounds (jasmonic and abscisic acids) in the rapid (within 1 hour) inhibition of photosynthetic rate and stomata conductance in distant undamaged tobacco leaves after local burning. The aim of this addendum is to discuss plausible mechanisms of a rapid long-distance chemical signaling and the putative interactions between the physical and chemical signals leading to the fast systemic response.Key Words: tobacco, local burning, systemic response, hydraulic surge, electrical signal, abscisic acid, jasmonic acidPlants have evolved an amazingly complex system of defence-related strategies to protect themselves upon local wounding.^1–7 Important characteristics of self-defence responses of plants are their velocity and ubiquity. Indeed, fast (minutes to hours) responses to injurious factors have been detected in the site of injury and in distant regions (systemic response) in various plants.^8–11 These findings suggest that a signal generated by an attack to one leaf is transmitted through a whole plant. Several kinds of chemical^3,6 and physical¹² signals induced by local wounding and even their combination¹³ have been implicated. However, a little is known about the interactions of these signals and about the mechanisms of initiation of the short-term systemic responses.We have used a model system—tobacco plants exposed to the local burning—to study the signals involved in rapid wound responses of photosynthetic apparatus.¹⁴ Local burning of an upper leaf of a tobacco plant induced rapid changes in surface electrical potential (within seconds) and a pronounced fast decline in the stomatal conductance, CO₂ assimilation and transpiration (within minutes) in the basipetal direction (Fig. 1). Moreover, we have detected a fast (within minutes) transient increase in levels of endogenous abscisic acid (ABA) followed by a huge rapid rise in endogenous jasmonic acid (JA) in the leaf below the burned one. ABA and jasmonates are known to be involved in signaling pathways leading to stomatal closure and downregulation of photosynthesis.^15,16 Increases in ABA and/or JA levels have only previously been detected in remote untreated tissues several hours after local wounding^8,9 suggesting that chemical signals are too slow to induce rapid systemic response. Previous works have reported that fast physical (electrical) signals play an essential role in short-term systemic photosynthetic responses.^11,17 However, a several-minutes delayed stomata closing response after the initiation of electrical potential changes has been reported in Mimosa¹⁸ and in our case in tobacco¹⁴ plants. Therefore, the guard cell deflation is most likely triggered not only by the electrical signal, but also by indirect factors. Based on close correlations, our results now provide a new evidence for the idea that chemical signals (ABA and mainly JA) participate in mediating the short-term systemic photosynthetic responses to local burning in tobacco plants.Open in a separate windowFigure 1The model of putative signalling pathways leading to the rapid systemic responses of tobacco plants to local burning. Hypothetical (dashed lines) local responses, generation of signals and transport processes and detected (full line) systemic responses are demonstrated. For details see the text.The question is how do the physical (electrical and/or hydraulic) and chemical signals act? They may independently induce specific elements of systemic responses. However, they are more likely to act in a coordinated, interactive fashion. In this scenario (see Fig. 1), within first minutes after the local burning, hydraulic surge transmitted basipetally and acropetally through the xylem would transport chemicals released at the wound site (hydraulic dispersal¹⁹) and evoke changes in the ion fluxes in surrounding living cells leading to the local electrical activity.^12,13 The hypothesis of hydraulic dispersal is supported by our preliminary experiments with the fluorescent dye Rhodamine B applied on cut petiole of the upper leaf of tobacco plants showing that solutes can be rapidly transported (within minutes) basipetally following wounding.The rapid kinetics and transient character of ABA accumulation¹⁴ suggest that the main transport mechanism is the hydraulic dispersal in xylem. The participation of ABA in the generation of systemic electrical activity and/or vice versa cannot be ruled out.^8,20A rapid hydraulically driven transport of chemicals in the xylem of wounded plant in a reversed (basipetal) direction^19,21 to transpiration stream is not generally accepted. Exposing of leaves of undamaged plants to radioactive labelled molecules to determine the speed of chemical signal transport could be misrepresent, because hydraulic signal is not generated in undamaged plants and then the detected transport speed is too slow. Moreover, previous work²² demonstrated that neither the mass flow itself, nor the associated pressure changes induce the systemic response (the proteinase inhibitor activity). Thus, the efficacy of chemical agents in rapid systemic signaling seems to depend on transport by the mass flows associated with hydraulic signals.¹⁹However, hydraulic dispersal acts only for minutes, until all water released at the wound site is exhausted.²¹ A requirement for hydraulic dispersal of any solute is its presence in the wounded tissue at the time of wounding.¹⁹ Detected slower kinetics of JA accumulation than in the case of ABA and the huge rise of JA levels¹⁴ indicate a systemic accumulation of JA also by some additional processes.Does additional JA accumulation result from de-novo synthesis in undamaged leaves as a response to physical signal or does it result from a JA transport from the wounded leaves? In the longer time-frame the phloem transport²³ should also be considered. Experiments with tomato plants have shown that de novo JA synthesis in distant leaves is not required for the systemic response and that biosynthesis of JA at the wound site is necessary for the generation of a systemic signal.⁷ Indeed, a short-term increase in endogenous concentrations of JA has been detected in wounded tissue in Nicotiana sylvestris⁹ and rice.¹⁰However, a rapid burst in the systemic JA accumulation found in our experiments¹⁴ would implicate an ultra-rapid and extreme JA accumulation at the wound site before its transport. The systemic JA accumulation (within 1 hour¹⁴) preceded the generation of enzymes involved in the JA biosynthesis in the wounded leaf.Thus, several processes are suggested to play a role in the ultra-rapid and huge JA accumulation:

1. initiation of JA accumulation by preexisting enzymes,²⁴
2. fast release of free JA from its storage pools in cells (e.g., JA-conjugates²⁵),
3. direct uptake of elicitors (JA) by the phloem of the wounded leaf and exchange between the xylem and phloem as a consequence of severe wounds,²⁶
4. the mass flow (containing remaining JA) driven mainly acropetally in the xylem by transpiration after damping the hydraulic surge,²¹
5. JA accumulation evoked by the fast transmitting physical (electrical or hydraulic) signal that leads to imbalances in ion fluxes,^8,12,27
6. JA accumulation (and subsequent transport) directly in the phloem, where JA biosynthetic enzymes are located (at least in tomato²⁴),
7. volatile chemical compounds (methylester of JA) spreading in the surrounding air of wounded leaf could serve as signaling molecules and sources of JA.^25,28

The relevance of the above mentioned mechanisms should be checked by further research. Complex quantitative and kinetic analysis of JA and ABA content, levels of its biosynthetic derivatives (also volatiles in the surrounding air) and simultaneous physical signal detection in wounded and distant unwounded tissues would fill the remaining void about their role and interactions in the wound signal transduction networks. In addition, a suppression of other signaling pathways with similar transport kinetics (e.g., volatile compounds transmission, systemin and oligosaccharides generation and/or transport, using mutant plants) would be useful.Substantial similarity between the rapid physical (electrical) signaling in animal nervous system compared with the physical (electrical) signaling in plants has already been reported.^29,30 Interaction of chemical and electrical signals is the process well documented for post-synaptic events in animals. Our data now strengthen the role of chemical signals next to the role of physical signals in plants in the rapid systemic wound response; such a role of chemicals in plants was often underestimated up to now.     

Plasmodesmata transport of GFP and GFP fusions requires little energy and transitions during leaf expansion

Plasmodesmata (Pd) are symplastic channels between neighboring plant cells and are key in plant cell-cell signaling. Viruses of proteins, nucleic acids, and a wide range of signaling macromolecules move across Pd. Protein transport Pd is regulated by development and biotic signals. Recent investigations utilizing the Arrhenius equation or Coefficient of conductivity showed that fundamental energetic measurements used to describe transport of proteins across membrane pores or the nuclear pore can also apply to protein movement across Pd. As leaves continue to expand, Pd transport of proteins declines which may result from changes in cell volume, Pd density or Pd structure.Key words: plasmodesmata, diffusion, GFP, viral transport, PVX, triple gene blockResearchers have argued for the last decade that movement of proteins and other macromolecules across Pd is regulated by development, stress and biotic signals. There are four current models describing different mechanisms of Pd transport. First is the non cell autonomous protein (NCAP) pathway that carries ribonucleoprotein complexes across Pd. NCAPs often carry RNA in a ribonucleoprotein complex to the Pd.^1–4 This mode of transport involved targeted movement, meaning that a set of proteins must dock within the Pd to gate it open to enable transfer between cells. Proteins which are normally too large to move across Pd can gate open Pd to enable its own transfer into neighboring cells. This is contrasted by nontargetted movement, which is passive movement of proteins that are sufficiently small enough to pass between cells.^5,6 The green fluorescent protein (GFP) has been described as a protein whose movement is non-targeted, meaning that it can diffuse across Pd. Reasons that we do not see continuous movement of small proteins between cells include protein compartmentalization or subcellular targeting signals. For example proteins may be synthesized and modified via the ER and Golgi networks and then transferred into vesicles and transported within cells to their destination. These proteins would not be free in the cytosol for diffusion across Pd. Alternatively, proteins which have dominant subcellular targeting signals which direct them to certain organelles such as the nucleus, peroxisome, or other destination would not be free to move across Pd.^5,6 A third model represents proteins in the ER that move laterally along the membrane or through the ER lumen into neighboring cells. This transport is quite rapid and investigations are ongoing to determine how this is regulated.^7–11 Finally, there is vesicle transport which deliver cargo to Pd.^12,13 The origin of these vesicles is still under investigation. Much more research has been accomplished toward defining non-targeted movement and the NCAP pathway while the ER and vesicle transport pathways are only recently described and very little is known about the regulatory mechanisms underlying these pathways.Pd permeability is governed in part by architecture, but also by key regulatory factors that determine Pd conductivity. Factors such as mysoin VIII, actin and calreticulin were identified in Pd which likely regulate expansion and contraction.^14–19 In addition calcium, ATP and plant hormones can downregulate Pd permeability during development and stress.^20,21 The tools for measuring Pd permeability has been to study the transport of fluorescently tagged proteins, fluorescent dextran beads, GFP or GFP fusions following microinjection or biolistic delivery to the cytoplasm of one cell.^22–26 Then video imaging or captured still images at select time intervals are used to characterize Pd transport. Until recently researchers quantified movement by the frequency they observed a certain type of movement. Therefore our ability to describe Pd permeability has been limited.Evidence that ATP impacts Pd conductivity has led investigations to explore the energy requirements for macromolecular transport across Pd. By understanding the energy requirements for transport of various proteins and nucleic acids we can better characterize passive or active transport processes. Toward this end two recent studies detailed quantitative approaches that can be employed to describe the developmental and energy requirements cell-to-cell transport of cytosolic proteins. Both papers used biolistic bombardment to deliver plasmids expressing GFP or GFP fusions to tobacco leaf epidermal cells and then captured still images of GFP fluorescence in neighboring cells. We employed the Arrhenius equation to characterize transport of GFP or GFP fused to the Potato virus X (PVX) TGBp1 movement protein. PVX TGBp1 was selected to compare with GFP alone since it is known to gate open Pd and has ATPase activity.⁴⁵ We predicted that the abilities of GFP alone and GFP-TGBp1 to move across Pd might be different and were surprised to learn that the energy for transport of both proteins was similar. This project established the principle that GFP and GFP-TGBp1 transport is temperature dependent showing a linear relationship between protein movement and the temperatures at which leaves were incubated.Green fluorescent sites on bombarded leaves were scored for the movement or no movement. Movement is defined as evidence of fluorescence in 2 or more cells at 24 h and no movement is when fluorescence is in single cells. These were then presented as a percentage of the total. So by digitizing the representation of movement we were able to represent a linear relationship between movement and temperature. Representing movement in this way also enabled us to represent movement values on a logarithm scale necessary for a classic Arrhenius plot. The activation energy (Ea) was calculated by fitting the data to the Arrhenius equation:% movement = A exp(-Ea/RT); and the Ea for GFP and GFP-TGBp1 was approximately 38 kJ/mol and 29 kJ/mol. These low activation energies are comparable to the reported 30 kJ/mol calculated for temperature dependence of protein transport through the cytosol. Evidence that GFP movement across Pd requires slightly more energy than through the cytoplasm suggests there may be some resistance within the pore. The lower energy for GFP-TGBp1 suggests that movement is facilitated, which likely reflects Pd gating by TGBp1, enabling greater transfer between cells.Liarzi and Epel define a new coefficient of conductivity of Pd.⁴² This study also concluded that cell-to-cell transport of GFP in nontransgenic or transgenic N. benthamiana plants expressing the Tobacco mosaic virus (TMV) movement protein (MP) is temperature dependent. The method was to measure the exponential decay, which is a measure of the impedance to diffusion driven cell-to-cell movement of fluorescence. The exponential decay factor? was determined by calculating the ratio of GFP fluorescence in bombarded cell 0 and neighboring cells. This was presented as a measure of fluorescence transfer from cell 0 to cell 1 to cell 2. A coefficient for conductivity C(Pd), 1/? for GFP was reflective of diffusion. Interestingly the (TMV) MP did not increase conductivity of GFP between neighboring leaf epidermal cells indicating that movement was already maximal. Considering prior reports that the TMV MP shows preferential spread into mesophyll rather than epidermal tissues during virus infection, it is possible that preferential spread into mesophyll cells would prevent experimental efforts to achieve improved conductivity of GFP between epidermal cells.^27,28 In which case the absence of a trans effect of TMV MP on GFP conductivity in the epidermis may not be surprising. In fact, prior investigations of TMV MP gating activities were conducted in mesophyll cells.^29,30 The best explanation for the combined studies is that cytosolic GFP can diffuse across Pd , however viral proteins which gate Pd enable their own low energy transfer into neighboring cells without allowing other proteins to flood into neighboring cells. Therefore viral movement proteins, such as PVX TGBp1 and TMV MP, which gate Pd provide themselves with an energetic advantage for transport into neighboring cells which is essential for rapid dissemination of virus into further tissues.These studies provide an interesting contrast between PVX TGBp1 and TMV MP. Both proteins gate open Pd for virus cell-to-cell transport, but there seems to be differences in how these proteins function in epidermal cells. This is likely due to their different roles in promoting virus cell-to-cell movement. PVX TGBp1 protein is also a suppressor of RNA silencing. We recently proposed a model in which TGBp1 rapidly moves from cell-to-cell ahead of virus infection, to suppress the cell''s RNA degradation machinery, as a means to promote infection.³¹ The TMV MP on the other hand is reported to bind viral RNA for transfer into neighboring cells.^32,33 Therefore, the different observations of PVX TGBp1 and TMV MP transport between epidermal cells likely reflect their functional differences. Both proteins are required for virus cell-to-cell movement, but their exact roles in virus movement are not identical.As mentioned earlier, Pd permeability is downregulated during plant development. Research tracking GFP diffusion through Pd in embryonic cells, in young emerging leaves, and in fully expanded leaves showed that fluorescence is highly mobile between cells in young tissues but is restricted during maturation. Viral movement proteins such as Cucumber mosaic virus 3a, and PVX TGBp1 remain highly mobile in mature leaves because they gate open Pd under conditions that normally restrict movement of GFP.^34,35 Schoenknecht et al., undertook a straightforward investigation of leaf maturation describing Pd transport in relationship to leaf area expansion. The outcome of this study was evidence that GFP movement between cells declines as leaves expand.It is reasonable to consider that simultaneous changes in gene expression and physiology is reflected in a downward trend in Pd conductivity and an increased requirement for Pd gating to enable selected transport of macromolecules between cells. In Arabidopsis embryos there is an obvious transition between developmental stages which are also represented by a decline in the ability for GFP to diffuse across Pd.^36,37 A detailed analysis of Pd structure in source and sink tissues revealed that Pd are simple single channeled structures in sink tissues while source tissues contain predominantly “H” shaped branching Pd structures. The change in Pd structure has been correlated with changes in conductivity and is often correlated with changes in sink to source metabolism.^38,39 The sink-to-source transition in leaf development is typically monitored using phloem loading of carboxyfluorescein diacetate. Leaves where CF dye unloads are defined as sink leaves and leaves that were restricted in dye unloading were defined as source leaves. Then biolistic bombardment of GFP expressing plasmids to sink and source leaves revealed that GFP readily diffuses across Pd in sink leaves but is more often restricted in source leaves.^{26,34,40–42}Leaf development is typically defined as a transition from juvenile to adult which is represented by homeotic transformations as well as vegetative phase changes.^43,44 Source and sink regions of a leaf have been shown to correlate with changes in Pd structure and conductivity during leaf expansion. However, in our study we found that N. tabacum leaves identified as source during week 2 or 3 would continue to expand over an 8 week period to twice or three times the leaf area which provides a real indication that the source designation may not entirely reflect final leaf maturation or completion of leaf development.⁴⁵> For example, as cells transition from sink to source physiology it is suggested that the frequency of single channeled Pd declines while the frequency of branched Pd increases.³⁹ It is possible that even after leaves transition into photosynthetic sources that Pd architecture continues to change and there is a further decline in the proportion of single channel to branched channels. Therefore either the change in cell volume or Pd architecture or both can slow-down diffusion of GFP between cells.Researchers often point to the ER continuity between cells as a driving force for Pd formation and function. During cell division the cell wall is laid down and forms around the ER creating Pd channels.⁴⁶ However, it is also worth noting that the actin cytoskeleton is also present in Pd and is central to organ and reproductive development.^19,47 Actin and actin binding proteins are necessary for a number of plant processes determining the cell division plane, cell polarity, cell elongation, cytoplasmic streaming, transporting mRNAs and proteins, and defense.^48–51 Overexpression of ACT1 in Arabidopsis leaves can lead to changes in epidermal leaf shape and cause dwarfism in plants.⁵² Actin binding proteins are also necessary for organizing and remodeling the F-actin network which drives normal development of specific cell types and organs.⁵³ Actin filament bundling and remodeling are also seen in nonhost defense responses.⁵⁴ We do not know the effects of overexpressing certain actin homologues or actin remodeling on Pd formation or conductivity. Because the F-actin network is also central to Pd trafficking of proteins and macromolecules between cells it is worth considering F-actin as an early factor contributing to Pd formation which may be necessary to ensure cell-to-cell communication when cell polarity and elongation as well as defense machinery are being established.In summary, the novel quantitative tools developed for measuring protein movement across Pd reveal the temperature dependence of protein trafficking. Both the use of Arrhenius equation and C(Pd) provide new opportunities to measure the energy requirements for protein transport. These tools will enable researchers to quantify effects of environmental and developmental conditions on Pd conductivity, as well as comparing differences in Pd conductivity between plant species or induced by genetic mutations.