- Email: [email protected]
I Can See Clearly Now – Embolism in Leaves
Spotlight
I Can See Clearly Now – Embolism in Leaves Christine Scoffoni1 and Steven Jansen2,*
quantify the amount of drought-induced embolism in leaves, highlight the potential of this non-invasive and low-cost technique to visualize xylem failure at the entire leaf level in vivo. The method developed by Brodribb et al. allows direct live visualization of putative embolism spread across distinct venation types, which is demonstrated in their paper for three fern species and two angiosperm trees. The technique is a modern, digital approach of earlier visualization techniques (see for instance references cited in [3]) and is based on stereomicroscopy, which allows non-invasive observations of embolized conduits according to differences in light transmission of veins between functional (i.e., water-filled) and air-filled conduits.
Deciphering how air enters the plant hydraulic transport tissues represents a major challenge to understanding plant drought responses. Using a non-invasive and cheap visualization technique applied to leaves, the spread of embolism is found to initiate in the midrib, increase with vein order, Three important findings have emerged and is seemingly influenced by vein from this new technique. First, large dehytopology.
dration times are necessary (up to 70 h of shoots drying on the bench) to induce embolism in leaf veins, suggesting that leaf veins are more resistant to droughtinduced embolism than was previously thought (e.g., [4]). Second, large veins are most vulnerable to embolism, while high vein orders (i.e., 3rd, 4th, and 5th vein orders) are the most resistant. Although these results contradict earlier papers using dye techniques (e.g., [5]), they are consistent with a neutron imaging study showing that embolism starts in major veins and then spreads to the minor veins under higher dehydration [6]. This second finding implies that leaves would only need to refill their major veins to become functional again. Moreover, the hydraulic vulnerability segmentation hypothesis would not apply within leaf veins because the most-distal high-order veins were not found to be most vulnerable to embolism. Hydraulic vulnerability segmentation, however, would be achieved through high vulnerability of the outside-xylem pathways in leaves [7] and/or petiole vulnerability [8]. The third important finding of the study is the role of vein topology in embolism resistance. Their results, although limited to five Recent findings from Brodribb et al. [2], species, suggest that hierarchical/reticubased on an indirect optical technique to late systems could provide an efficient
Vascular plants include a highly complex water transport system that is driven by the transpiring surfaces of leaves. The mechanism behind this transport system results in a negative (i.e., sub-atmospheric) pressure of xylem sap. Because of the highly cohesive property of the H2O molecule, water can be transported under negative pressure from the soil through the entire plant system until it reaches the sites of evaporation in leaves. Although there is general agreement about the cohesion-tension theory to explain long-distance vascular transport of water, the occurrence of gas bubbles, which can expand to block conduits and disable the hydraulic transport system via embolism formation, represents a major challenge to understanding how plants will respond to more severe and long-term periods of drought in many locations worldwide [1]. The leaf vein system can be highly reticulate, and understanding how embolism spreads across vein orders has remained challenging. Thus, visualizing embolism events and how these vary across species and during drought is of crucial importance.
way to avoid rapid and catastrophic failure of the entire vein network. This was observed in the non-hierarchical and non-reticulate venation pattern of the fern Adiantum capillus-veneris, and could allow the dense and embolism-resistant minor veins to remain functional during prolonged drought, bypassing embolized conduits in major veins. While the method presented by Brodribb et al. [3] provides promising possibilities to answer fundamental questions about embolism in leaves, there remain some shortcomings where caution is warranted. First, this method should be tested on a wider range of leaf structures and anatomies: it is unknown, for example, if this method would work on thick or leathery leaves. Most importantly, the method assumes that the captured change in refractive index relates to embolism. However, this method has not been validated yet against an independent and more direct visualization approach such as Xray microtomography (microCT), which allows direct visualization of embolized conduits and their anatomical characteristics in different vein orders (Figure 1A,B). Although X-rays are known to potentially damage DNA in living cell types, they do not affect embolism formation in stem conduits after repeated scans [9]. Validation of the optical technique with other techniques such as microCT is especially crucial if the technique is to be used to infer the hydraulic impact of embolisms on leaf hydraulic conductance [10]. For instance, the percentage of ‘embolized pixels’ that are measured would not necessarily show a 1:1 correlation with the actual percentage of embolized conduits. Hence, high conduit redundancy, especially in midribs (Figure 1C), could make it hard to accurately relate the percent of embolized pixels to actual embolized conduits. In addition, exact quantitative data of the embolized conduits will be necessary to determine their true hydraulic impact. Thus, the percentage of ‘embolized pixels’ reported by this method could overestimate the true hydraulic impact on
Trends in Plant Science, September 2016, Vol. 21, No. 9
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(A)
lower-order veins. Whether or not embolism resistance is related to conduit size and the nature of the tracheary element (tracheid vs vessel element) remains unclear and deserves more detailed anatomical observations. Detailed anatomy will also be necessary to understand conduit connectivity in vein nodes, which may (C) (B) function as a hydraulic bottleneck. To understand why major veins are more vulnerable to embolism, special attention should be paid to the ultrastructure and chemistry of air-seeding pores between cellulose microfibrils in interconduit pit membranes, which are known to play a major role in drought-induced embolism [1,12]. In fact, almost nothing is known about interconduit pit membranes in leaves with respect to their physical and chemical properties, although it is assumed that these pit membranes are Figure 1. Leaf Cross-Sections from X-Ray Computed Microtomography (microCT) and Light similar to those in secondary xylem of Microscopy. MicroCT cross-sections provide direct visualization of embolized conduits (red arrows) in the leaf the stem. midrib of Eucalyptus sp. (A) and a needle of Pinus pinaster (B) in vivo. Note, no embolized conduits are present in the minor veins (blue arrows) in (A). Light microscopy of a cross-section of Viburnum rhytidophyllum (C) reveals the high number of xylem conduits in the leaf midrib, and the wide range of their conduit dimensions. A high percentage of embolized midrib conduits would probably be necessary to induce significant hydraulic decline given the redundancy of conduits. Scale bars, 500 mm in A and B, 100 mm in C.
In conclusion, if used in combination with other techniques, this method provides a promising and cheap means to better understand water transport mechanism in plants and hydraulic failure. This could leaf hydraulic conductance. By validating (i.e., with secondary veins terminating at lead to potential applications in the field of this technique against microCT, modeling, the leaf margin) would become discon- biomimetics such as solar-driven, nanoand/or detailed anatomical observations nected from the main water source [11]. and microfluidic devices. to support the occurrence of embolism, this technique has the potential to be a The findings from Brodribb et al. [3] also Acknowledgments useful and cheap tool to investigate the provide evidence for air-seeding as the S.J. thanks the Swiss Light Source (SLS) at the Paul impact of drought on leaves. main mechanism for the spread of embo- Scherrer Institute (Villigen, Switzerland) for synchrolism. Although not discussed, the images tron radiation beamtime at the TOMCAT beamline and One of the most promising outcomes of and movies presented appear to show the European Synchrotron Radiation Facility (ESRF, this technique concerns the production of that there is also evidence for ‘novel’ for- proposal EC-1026, Grenoble, France) for access to spatiotemporal maps of embolism forma- mation of embolism in conduits that are the ID19 beamline. Drs Sarah Irvine (SLS) and Alexantion in entire leaves, which opens up pos- not connected to the embolized ones, der Rack (ESRF) are acknowledged for providing technical assistance. sibilities to investigate hydraulic aspects of which supports similar observations on a large diversity of vein topologies. It has progressive embolism spread in wood 1University of California, Los Angeles, Department of been hypothesized, for instance, that a based on microCT [9]. These observa- Ecology and Evolutionary Biology, 621 Charles E. Young brochidodromous vein pattern (i.e., with tions indicate that these conduits become Drive South, Box 951606, Los Angeles, CA 90095, USA 2 Ulm University, Institute of Systematic Botany and the secondary veins not terminating at the embolized by a mechanism other than air- Ecology, Albert-Einstein-Allee 11, 89081 Ulm, Germany leaf margins, but joining together in a seeding. *Correspondence: [email protected] (S. Jansen). series of loops or arches) would enable higher redundancy and safety against Finally, because major veins were found to http://dx.doi.org/10.1016/j.tplants.2016.07.001 damage: if a loop is damaged, water could be more vulnerable to embolism, Brodribb References still flow to the entire leaf area, whereas et al. [3] hypothesized this could be due to 1. Schenk, H.J. et al. (2015) Nanobubbles: a new paradigm for air-seeding in xylem. Trends Plant Sci. 20, 199–205 a leaf with craspedodromous venation larger conduit sizes present in these
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Trends in Plant Science, September 2016, Vol. 21, No. 9
2. Brodribb, T.J. et al. (2016) Revealing catastrophic failure of leaf networks under stress. Proc. Natl. Acad. Sci. U. S. A. 113, 4865–4869 3. Clearwater, M.J. and Goldstein, G. (2005) Embolism repair and long distance water transport. In Vascular Transport in Plants (Holbrook, N.M. and Zwieniecki, M.A., eds), pp. 375–399, Elsevier 4. Johnson, D.M. et al. (2012) Evidence for xylem embolism as a primary factor in dehydration-induced declines in leaf hydraulic conductance. Plant Cell Environ. 35, 760–769 5. Salleo, S. et al. (2001) Vulnerability to cavitation of leaf minor veins: any impact on leaf gas exchange? Plant Cell Environ. 24, 851–859 6. Defraeye, T. et al. (2014) Quantitative neutron imaging of water distribution, venation network and sap flow in leaves. Planta 240, 423–436 7. Scoffoni, C. et al. (2014) Leaf shrinkage with dehydration: coordination with hydraulic vulnerability and drought tolerance. Plant Physiol. 164, 1772–1788 8. Hochberg, U. et al. (2016) Grapevine petioles are more sensitive to drought induced embolism than stems: evidence from in vivo MRI and microCT observations of hydraulic vulnerability segmentation. Plant Cell Environ. Published online February 12, 2016. http://dx.doi.org/10. 1111/pce.12688 9. Choat, B. et al. (2016) Non-invasive measurement of vulnerability to drought induced embolism by X-ray microtomography. Plant Physiol. 170, 273–282 10. Brodribb, T.J. et al. (2016) Visual quantification of embolism reveals leaf vulnerability to hydraulic failure. New Phytol. 209, 1403–1409 11. Roth, A. et al. (1995) Hydrodynamic modeling study of angiosperm leaf venation types. Bot. Acta 108, 121–126 12. Li, S. et al. (2016) Intervessel pit membrane thickness as a key determinant of embolism resistance in angiosperm xylem. Int. Assoc. Wood Anat. J. 37, 152–171
Spotlight
Stem Cell Fate versus Differentiation: the Missing Link Judith Nardmann,1 John W. Chandler,1 and Wolfgang Werr1,* The shoot apical meristem provides a microenvironment that ensures stem cell fate and proliferation via homeostasis between WUSCHEL (WUS) activity and CLAVATA signalling. New data from maize and arabidopsis reveal that an evolutionarily conserved signal deriving from primordium cells links WUS transcription to the morphogenetic programme.
The development and growth of higher plants critically depends on meristems, the most important of which reside at the shoot or root tips and consist of highly organised cell arrays containing so-called stem-cell niches [1], which are asymmetric microenvironments that hold stem cells in place, control their proliferation, and repress their differentiation [2]. The niche concept evolved in animal models, where the number of niches and their size is tightly regulated throughout ontogeny, and each niche is associated with robust, self-reinforcing mechanisms that both ensure stem-cell fate and proliferation and also ensure via asymmetric cell divisions that on average, half the daughter cells emerge into conditions conducive to differentiation. In plants, pioneering work on the arabidopsis (Arabidopsis thaliana) shoot apical meristem (SAM) has elucidated the CLAVATA (CLV) and WUSCHEL (WUS) feedback loop, which controls SAM stem-cell number [3]. In short, stem cells at the apical tip of the SAM secrete the CLV3 ligand, which is perceived by a leucine–rich–repeat (LRR) transmembrane receptors such as the CLV1/CLV2 heterodimer in the subtending organising centre (OC), where it limits WUS transcription. The encoded WUS homeodomain transcription factor in turn is mobile, and migrates upwards into the stem cell domain, where it promotes cell proliferation. Although the CLV/WUS feedback loop can explain the maintenance and size of a durable stem-cell niche in the central zone (CZ), it does not correlate stem-cell proliferation with ongoing cellular differentiation in the SAM peripheral zone (PZ) for the elaboration of new lateral organs, that is, growth of the plant body. Novel experimental data concerning the maize (Zea mays) FASCIATED EAR3 (FEA3) gene function [4] now identify an additional signal that originates from differentiated cells of the leaf primordium to fine-tune WUS transcription.
phenotypes such as fasciated ears, increased seed rows and thick tassels were reminiscent of mutations in genes encoding maize orthologues of CLV1 or CLV2 signalling components, that is, TD1 [5] or FEA2 [6], respectively. Map-based cloning of FEA3 revealed that it encodes a receptor-like protein (LRP) with a signal peptide followed by 12 LRRs, whereas CLV2 or its maize orthologue FEA2 [6] share 16 LRRs, a trans-membrane domain, and a short cytoplasmic tail. Within the SAM, the FEA3 transmembrane receptor is expressed centrally within the CZ, but also in subtending cells during the vegetative and reproductive phases. Loss of FEA3 function results in a basal expansion of the ZmWUS1 expression domain and increases WUS transcription; the resulting increase in stem cells relates to the enlargement of the fea3 mutant SAM and the fasciation/ seed row number phenotype. The first important finding is that a signal depending on the FEA3 transmembrane receptor restricts WUS transcription at the base of the OC. A second, genetic, conclusion is that FEA3 acts independently to the CLV2 receptor, which was demonstrated by synergistic ear and tassel meristem size phenotypes of fea2/fea3 double mutants.
Thus, a novel transmembrane receptor complex that involves FEA3 actively and selectively represses WUS transcription in cells subtending the OC, and the obvious question concerning whether the ligand is a maize orthologue of CLV3 was addressed by a combined phylogenetic–experimental approach. The application of candidate CLV3/EMBRYOSURROUNDING REGION (CLE) peptides to fea3 and wild-type embryos in culture or supplied to the root growth medium excluded two potential maize CLV3 orthologues, ZmCLE7 and ZmCLE14, as ligands. However, fea3 mutant embryos or roots were nonresponsive to the maize orthologue of the rice (Oryza Interest in FEA3 started from loss-of-func- sativa) FLORAL ORGAN NUMBER2-LIKE tion mutant plants, whose inflorescence CLE PROTEIN 1 (FCP1) peptide, whereas
Trends in Plant Science, September 2016, Vol. 21, No. 9
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I Can See Clearly Now – Embolism in Leaves Christine Scoffoni1 and Steven Jansen2,*
quantify the amount of drought-induced embolism in leaves, highlight the potential of this non-invasive and low-cost technique to visualize xylem failure at the entire leaf level in vivo. The method developed by Brodribb et al. allows direct live visualization of putative embolism spread across distinct venation types, which is demonstrated in their paper for three fern species and two angiosperm trees. The technique is a modern, digital approach of earlier visualization techniques (see for instance references cited in [3]) and is based on stereomicroscopy, which allows non-invasive observations of embolized conduits according to differences in light transmission of veins between functional (i.e., water-filled) and air-filled conduits.
Deciphering how air enters the plant hydraulic transport tissues represents a major challenge to understanding plant drought responses. Using a non-invasive and cheap visualization technique applied to leaves, the spread of embolism is found to initiate in the midrib, increase with vein order, Three important findings have emerged and is seemingly influenced by vein from this new technique. First, large dehytopology.
dration times are necessary (up to 70 h of shoots drying on the bench) to induce embolism in leaf veins, suggesting that leaf veins are more resistant to droughtinduced embolism than was previously thought (e.g., [4]). Second, large veins are most vulnerable to embolism, while high vein orders (i.e., 3rd, 4th, and 5th vein orders) are the most resistant. Although these results contradict earlier papers using dye techniques (e.g., [5]), they are consistent with a neutron imaging study showing that embolism starts in major veins and then spreads to the minor veins under higher dehydration [6]. This second finding implies that leaves would only need to refill their major veins to become functional again. Moreover, the hydraulic vulnerability segmentation hypothesis would not apply within leaf veins because the most-distal high-order veins were not found to be most vulnerable to embolism. Hydraulic vulnerability segmentation, however, would be achieved through high vulnerability of the outside-xylem pathways in leaves [7] and/or petiole vulnerability [8]. The third important finding of the study is the role of vein topology in embolism resistance. Their results, although limited to five Recent findings from Brodribb et al. [2], species, suggest that hierarchical/reticubased on an indirect optical technique to late systems could provide an efficient
Vascular plants include a highly complex water transport system that is driven by the transpiring surfaces of leaves. The mechanism behind this transport system results in a negative (i.e., sub-atmospheric) pressure of xylem sap. Because of the highly cohesive property of the H2O molecule, water can be transported under negative pressure from the soil through the entire plant system until it reaches the sites of evaporation in leaves. Although there is general agreement about the cohesion-tension theory to explain long-distance vascular transport of water, the occurrence of gas bubbles, which can expand to block conduits and disable the hydraulic transport system via embolism formation, represents a major challenge to understanding how plants will respond to more severe and long-term periods of drought in many locations worldwide [1]. The leaf vein system can be highly reticulate, and understanding how embolism spreads across vein orders has remained challenging. Thus, visualizing embolism events and how these vary across species and during drought is of crucial importance.
way to avoid rapid and catastrophic failure of the entire vein network. This was observed in the non-hierarchical and non-reticulate venation pattern of the fern Adiantum capillus-veneris, and could allow the dense and embolism-resistant minor veins to remain functional during prolonged drought, bypassing embolized conduits in major veins. While the method presented by Brodribb et al. [3] provides promising possibilities to answer fundamental questions about embolism in leaves, there remain some shortcomings where caution is warranted. First, this method should be tested on a wider range of leaf structures and anatomies: it is unknown, for example, if this method would work on thick or leathery leaves. Most importantly, the method assumes that the captured change in refractive index relates to embolism. However, this method has not been validated yet against an independent and more direct visualization approach such as Xray microtomography (microCT), which allows direct visualization of embolized conduits and their anatomical characteristics in different vein orders (Figure 1A,B). Although X-rays are known to potentially damage DNA in living cell types, they do not affect embolism formation in stem conduits after repeated scans [9]. Validation of the optical technique with other techniques such as microCT is especially crucial if the technique is to be used to infer the hydraulic impact of embolisms on leaf hydraulic conductance [10]. For instance, the percentage of ‘embolized pixels’ that are measured would not necessarily show a 1:1 correlation with the actual percentage of embolized conduits. Hence, high conduit redundancy, especially in midribs (Figure 1C), could make it hard to accurately relate the percent of embolized pixels to actual embolized conduits. In addition, exact quantitative data of the embolized conduits will be necessary to determine their true hydraulic impact. Thus, the percentage of ‘embolized pixels’ reported by this method could overestimate the true hydraulic impact on
Trends in Plant Science, September 2016, Vol. 21, No. 9
723
(A)
lower-order veins. Whether or not embolism resistance is related to conduit size and the nature of the tracheary element (tracheid vs vessel element) remains unclear and deserves more detailed anatomical observations. Detailed anatomy will also be necessary to understand conduit connectivity in vein nodes, which may (C) (B) function as a hydraulic bottleneck. To understand why major veins are more vulnerable to embolism, special attention should be paid to the ultrastructure and chemistry of air-seeding pores between cellulose microfibrils in interconduit pit membranes, which are known to play a major role in drought-induced embolism [1,12]. In fact, almost nothing is known about interconduit pit membranes in leaves with respect to their physical and chemical properties, although it is assumed that these pit membranes are Figure 1. Leaf Cross-Sections from X-Ray Computed Microtomography (microCT) and Light similar to those in secondary xylem of Microscopy. MicroCT cross-sections provide direct visualization of embolized conduits (red arrows) in the leaf the stem. midrib of Eucalyptus sp. (A) and a needle of Pinus pinaster (B) in vivo. Note, no embolized conduits are present in the minor veins (blue arrows) in (A). Light microscopy of a cross-section of Viburnum rhytidophyllum (C) reveals the high number of xylem conduits in the leaf midrib, and the wide range of their conduit dimensions. A high percentage of embolized midrib conduits would probably be necessary to induce significant hydraulic decline given the redundancy of conduits. Scale bars, 500 mm in A and B, 100 mm in C.
In conclusion, if used in combination with other techniques, this method provides a promising and cheap means to better understand water transport mechanism in plants and hydraulic failure. This could leaf hydraulic conductance. By validating (i.e., with secondary veins terminating at lead to potential applications in the field of this technique against microCT, modeling, the leaf margin) would become discon- biomimetics such as solar-driven, nanoand/or detailed anatomical observations nected from the main water source [11]. and microfluidic devices. to support the occurrence of embolism, this technique has the potential to be a The findings from Brodribb et al. [3] also Acknowledgments useful and cheap tool to investigate the provide evidence for air-seeding as the S.J. thanks the Swiss Light Source (SLS) at the Paul impact of drought on leaves. main mechanism for the spread of embo- Scherrer Institute (Villigen, Switzerland) for synchrolism. Although not discussed, the images tron radiation beamtime at the TOMCAT beamline and One of the most promising outcomes of and movies presented appear to show the European Synchrotron Radiation Facility (ESRF, this technique concerns the production of that there is also evidence for ‘novel’ for- proposal EC-1026, Grenoble, France) for access to spatiotemporal maps of embolism forma- mation of embolism in conduits that are the ID19 beamline. Drs Sarah Irvine (SLS) and Alexantion in entire leaves, which opens up pos- not connected to the embolized ones, der Rack (ESRF) are acknowledged for providing technical assistance. sibilities to investigate hydraulic aspects of which supports similar observations on a large diversity of vein topologies. It has progressive embolism spread in wood 1University of California, Los Angeles, Department of been hypothesized, for instance, that a based on microCT [9]. These observa- Ecology and Evolutionary Biology, 621 Charles E. Young brochidodromous vein pattern (i.e., with tions indicate that these conduits become Drive South, Box 951606, Los Angeles, CA 90095, USA 2 Ulm University, Institute of Systematic Botany and the secondary veins not terminating at the embolized by a mechanism other than air- Ecology, Albert-Einstein-Allee 11, 89081 Ulm, Germany leaf margins, but joining together in a seeding. *Correspondence: [email protected] (S. Jansen). series of loops or arches) would enable higher redundancy and safety against Finally, because major veins were found to http://dx.doi.org/10.1016/j.tplants.2016.07.001 damage: if a loop is damaged, water could be more vulnerable to embolism, Brodribb References still flow to the entire leaf area, whereas et al. [3] hypothesized this could be due to 1. Schenk, H.J. et al. (2015) Nanobubbles: a new paradigm for air-seeding in xylem. Trends Plant Sci. 20, 199–205 a leaf with craspedodromous venation larger conduit sizes present in these
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Trends in Plant Science, September 2016, Vol. 21, No. 9
2. Brodribb, T.J. et al. (2016) Revealing catastrophic failure of leaf networks under stress. Proc. Natl. Acad. Sci. U. S. A. 113, 4865–4869 3. Clearwater, M.J. and Goldstein, G. (2005) Embolism repair and long distance water transport. In Vascular Transport in Plants (Holbrook, N.M. and Zwieniecki, M.A., eds), pp. 375–399, Elsevier 4. Johnson, D.M. et al. (2012) Evidence for xylem embolism as a primary factor in dehydration-induced declines in leaf hydraulic conductance. Plant Cell Environ. 35, 760–769 5. Salleo, S. et al. (2001) Vulnerability to cavitation of leaf minor veins: any impact on leaf gas exchange? Plant Cell Environ. 24, 851–859 6. Defraeye, T. et al. (2014) Quantitative neutron imaging of water distribution, venation network and sap flow in leaves. Planta 240, 423–436 7. Scoffoni, C. et al. (2014) Leaf shrinkage with dehydration: coordination with hydraulic vulnerability and drought tolerance. Plant Physiol. 164, 1772–1788 8. Hochberg, U. et al. (2016) Grapevine petioles are more sensitive to drought induced embolism than stems: evidence from in vivo MRI and microCT observations of hydraulic vulnerability segmentation. Plant Cell Environ. Published online February 12, 2016. http://dx.doi.org/10. 1111/pce.12688 9. Choat, B. et al. (2016) Non-invasive measurement of vulnerability to drought induced embolism by X-ray microtomography. Plant Physiol. 170, 273–282 10. Brodribb, T.J. et al. (2016) Visual quantification of embolism reveals leaf vulnerability to hydraulic failure. New Phytol. 209, 1403–1409 11. Roth, A. et al. (1995) Hydrodynamic modeling study of angiosperm leaf venation types. Bot. Acta 108, 121–126 12. Li, S. et al. (2016) Intervessel pit membrane thickness as a key determinant of embolism resistance in angiosperm xylem. Int. Assoc. Wood Anat. J. 37, 152–171
Spotlight
Stem Cell Fate versus Differentiation: the Missing Link Judith Nardmann,1 John W. Chandler,1 and Wolfgang Werr1,* The shoot apical meristem provides a microenvironment that ensures stem cell fate and proliferation via homeostasis between WUSCHEL (WUS) activity and CLAVATA signalling. New data from maize and arabidopsis reveal that an evolutionarily conserved signal deriving from primordium cells links WUS transcription to the morphogenetic programme.
The development and growth of higher plants critically depends on meristems, the most important of which reside at the shoot or root tips and consist of highly organised cell arrays containing so-called stem-cell niches [1], which are asymmetric microenvironments that hold stem cells in place, control their proliferation, and repress their differentiation [2]. The niche concept evolved in animal models, where the number of niches and their size is tightly regulated throughout ontogeny, and each niche is associated with robust, self-reinforcing mechanisms that both ensure stem-cell fate and proliferation and also ensure via asymmetric cell divisions that on average, half the daughter cells emerge into conditions conducive to differentiation. In plants, pioneering work on the arabidopsis (Arabidopsis thaliana) shoot apical meristem (SAM) has elucidated the CLAVATA (CLV) and WUSCHEL (WUS) feedback loop, which controls SAM stem-cell number [3]. In short, stem cells at the apical tip of the SAM secrete the CLV3 ligand, which is perceived by a leucine–rich–repeat (LRR) transmembrane receptors such as the CLV1/CLV2 heterodimer in the subtending organising centre (OC), where it limits WUS transcription. The encoded WUS homeodomain transcription factor in turn is mobile, and migrates upwards into the stem cell domain, where it promotes cell proliferation. Although the CLV/WUS feedback loop can explain the maintenance and size of a durable stem-cell niche in the central zone (CZ), it does not correlate stem-cell proliferation with ongoing cellular differentiation in the SAM peripheral zone (PZ) for the elaboration of new lateral organs, that is, growth of the plant body. Novel experimental data concerning the maize (Zea mays) FASCIATED EAR3 (FEA3) gene function [4] now identify an additional signal that originates from differentiated cells of the leaf primordium to fine-tune WUS transcription.
phenotypes such as fasciated ears, increased seed rows and thick tassels were reminiscent of mutations in genes encoding maize orthologues of CLV1 or CLV2 signalling components, that is, TD1 [5] or FEA2 [6], respectively. Map-based cloning of FEA3 revealed that it encodes a receptor-like protein (LRP) with a signal peptide followed by 12 LRRs, whereas CLV2 or its maize orthologue FEA2 [6] share 16 LRRs, a trans-membrane domain, and a short cytoplasmic tail. Within the SAM, the FEA3 transmembrane receptor is expressed centrally within the CZ, but also in subtending cells during the vegetative and reproductive phases. Loss of FEA3 function results in a basal expansion of the ZmWUS1 expression domain and increases WUS transcription; the resulting increase in stem cells relates to the enlargement of the fea3 mutant SAM and the fasciation/ seed row number phenotype. The first important finding is that a signal depending on the FEA3 transmembrane receptor restricts WUS transcription at the base of the OC. A second, genetic, conclusion is that FEA3 acts independently to the CLV2 receptor, which was demonstrated by synergistic ear and tassel meristem size phenotypes of fea2/fea3 double mutants.
Thus, a novel transmembrane receptor complex that involves FEA3 actively and selectively represses WUS transcription in cells subtending the OC, and the obvious question concerning whether the ligand is a maize orthologue of CLV3 was addressed by a combined phylogenetic–experimental approach. The application of candidate CLV3/EMBRYOSURROUNDING REGION (CLE) peptides to fea3 and wild-type embryos in culture or supplied to the root growth medium excluded two potential maize CLV3 orthologues, ZmCLE7 and ZmCLE14, as ligands. However, fea3 mutant embryos or roots were nonresponsive to the maize orthologue of the rice (Oryza Interest in FEA3 started from loss-of-func- sativa) FLORAL ORGAN NUMBER2-LIKE tion mutant plants, whose inflorescence CLE PROTEIN 1 (FCP1) peptide, whereas
Trends in Plant Science, September 2016, Vol. 21, No. 9
725