- Email: [email protected]
Sieve elements and companion cells — extreme division of labour
research
news
Sieve elements and companion cells extreme division of labour It is over 70 years since Ernst Miinch presented his pressure flow theory describing a mechanism for long-distance transport of photoassimilates in the phloem 1. In many plants, this pressure flow starts at the sieve element-companion cell complexes (SE-CCCs), which are symplastically isolated from the adjacent cells of the leaf. The driving force for the pressure flow is generated by pairs of primary pumps, the plasma membrane-localized H+-ATPases, and secondary active transporters, the H*-solute symporters. Together, these proteins allow solute (mostly sucrose) movement into the SE-CCCs, and so generate the necessary osmotic pressure. The gene for the first plant sucrose transporter was cloned five years ago, using a yeast expression system s, and subsequent RNA in situ hybridization studies 3 and reporter gene analyses 4 allowed the expression of sucrose carrier genes to be localized to the phloem. The essential role of the sucrose transport protein SUT1 in phloem loading was finally confirmed by antisense repression 5'6. The sieve element-companion cell complex
shown for the PP2 lectin of pumpkin, which was found in both types of cells, although its mRNA is confined to companion cells 9. The recent paper from Wolf Frommer's group ~° presents another elegant set of data confirming that plasmodesmata are more like intercellular organelles than passive tubes allowing solute flux between cells. The results are particularly illuminating with regard to the nature and physiological role of the transported molecules. The analysis included the immunohistochemical localization of SUT1 suerose-H÷-symporters in sieve elements at both the light
membrane of sieve elements of three solanaceous species. No SUT1 protein is detected inside the companion cells. This feature, and in particular the asymmetric localization of SUT1 mRNA at the openings of the plasmodesmata (with higher levels on the sieve element side), suggests that mRNA is indeed transported from the companion cells to the sieve elements, where it is subsequently translated. An asymmetric distribution of RNA has previously been demonstrated in animal cells 11, where it results in asymmetric protein synthesis and distinct polarity. The asymmetric distribution of S U T I mRNA and its accumulation at the plasmodesreal orifices could indicate a similar role for SUT1, such as in the differentiation of the SE-CCC. To substantiate this model of SUT1 mRNA translation in sieve elements, it will be important to identify the ribosomal components. Nevertheless, the possibility that SUT1 proteins in mature sieve elements originate from protein synthesis when the cells are immature, prior to nuclear degradation, was excluded by Ktihn et al.~°; they were able to demonstrate that both SUT1 protein and SUT1 mRNA are degraded during the dark and synthesized in the light. This turnover demonstrates that SUT1 is synthesized de novo in mature sieve elements, and once again underlines the importance of the protein in the regulation of assimilate partitioning and in determining the source activity of leaves.
The SE-CCCs are composed of highly specialized cells - the companion c e l l s can be regarded as power packs that energize the sieve elements, which are living tubes solely for assimilate translocation. Many intracellular structures and organelles, including nuclei, vacuoles, microtubules, rough endoplasmic reticulum, ribosomes and Golgi bodies, appear to be degraded during the course of sieve element development 7. Both cell Phloem loading types are connected by many branched plasmodesmata. As a conse- A sieve element-companioncell complex (sieve element on the right). Why are SUT1 proteins of solanaquence of the long lifetime of the enu- Courtesy of Dr Alexander Schultz and Dr H.-Dietrnar Behnke. ceous plants targeted to the plasma membrane of sieve elcleated sieve elements, which can be active for years, it has frequently been sug- and electron microscope level, as well as in ements, not companion cells? In other gested that these plasmodesmata are not situ localizations of the respective mRNAs species, both H+-ATPases and sucrose only important for the flow of assimilates, in companion cells and sieve elements. The transporters have been localized to combut also for the supply of macromolecular data demonstrate the potential for traffick- panion cells 1~1s. The reason may be that compounds, including proteins. ing of either SUT1 protein or SUT1 mRNAs the species examined load their SE-CCCs The first direct evidence for macromol- through plasmodesmata. The translation of in different ways, or that two different ecular trafficking through plasmodesmata the mRNAs only appears to occur after sucrose transporters are active in both cell came from the group of William Lucas 8, their translocation into sieve elements. The types. An argument can be made for this who revealed that recombinant transcripdata suggest that in addition to protein latter possibility, because most plants have tion factor KN1 (the product of the maize sorting between intracellular compart- two different types of SE-CCCs. In the ments, the companion cells (and maybe loading phloem of source leaf minor veins, knotted1 gene) can migrate through the other cells) can selectively traffick proteins wide companion cells accompany narrow plasmodesmata of tobacco mesophyll cells. and/or mRNAs to the plasmodesmata. sieve elements, and in many plants numerMoreover, it has been demonstrated that ous cell wall ingrowths increase the cell knotted1 mRNA can also pass through surface of the companion cells even more. plasmodesmata in the presence of KN1. Asymmetric distribution of SUT1 mRNA In the transport phloem of the major veins Macromolecular trafficking between com- Kfihn et al. ~° have specifically identified of petioles and stems, however, wide sieve panion cells and sieve elements was first SUT1 sucrose carriers in the plasma
© 1997 Elsevier Science Ltd
August 1997, Vot. 2, No. 8
285
research
news
elements are accompanied by narrow companion cells. A single sucrose transporter (either in the companion cells or in the sieve elements) would minimize the capacity for sucrose transport in one of the two SE-CCCs. Thus, maximal uptake might be achieved by employing two sucrose transporters, one in the companion cells and one in the sieve elements.
molecular analysis has forced a reappraisal of existing models and shown how little is known about this highly specialized pair of cells.
Future prospects
Friedrich-Alexander-Universit&t ErlangenNQrnberg, Botanik II - Molekulare Pflanzenphysiologie, Staudtstrasse 5, D-91058 Erlangen, Germany (tel +49 9131 85 8212; fax +49 9131 85 8751; e-mail nsauer @biologie.uni-erlangen.de)
Acknowledgements I would like to thank M. Gahrtz, R. Stadler and E. Truernit for helpful discussions.
Norbert Sauer The paper of K~hn et al. 1° raises many other questions. It will be important to clarify the physiological impact of SUT1 turnover in mature SE-CCCs, and to assess whether S U T 1 mRNA is indeed translated inside the sieve elements or if the protein migrates into sieve elements after synthesis in the companion cells. Factors responsible for the distribution of asymmetrically distributed mRNAs (including c i s - e l e m e n t s in the 3'-ends of the mRNA and the interacting t r a n s factors) have been identified in animals, and it will be interesting to see whether similar mechanisms are responsible for the asymmetric distribution in SE-CCCs. Irrespective of whether S U T 1 mRNA or the SUT1 protein is translocated from companion cells to sieve elements, it will also be important to identify the signal that is recognized by the companion cell sorting machinery, to find the mode of interaction with the plasmodesmata and to understand the mechanism of translocation. For decades, SE-CCCs have primarily been studied using the methods of light and electron microscopy. Once again,
References 1 Mt~nch,E. (1930) Die Stoffbewegungenin der Pflanze, Gustav Fischer 2 Riesmeier,J.W. et al. (1992) Isolation and characterization of a sucrose carrier cDNA from spinach by functional expressionin yeast, EMBO J. 11, 4705-4713 3 Riesmeier,J.W. et al. (1993) Potato sucrose transporter expressionin minor veins indicates a role in phloem loading,Plant Cell 5, 1591-1598 4 Truemit, E. and Sauer, N. (1995) The promoter of the Arabidopsis thaliana SUC2 sucrose-H+symporter gene directs expression of ~-glucuzonidaseto the phloem: evidencefor phloem loading and unloading by SUC2, Planta 196, 564-570 5 Riesmeier,J.W. et al. (1994) Evidencefor an essential role of the sucrose transporter in phloem loading and assimilate partitioning, EMBO J. 13, 1-7
6 Ktthn, C. et al. (1996) Companioncell-specific inhibition of the potato sucrose transporter SUT1, Plant Cell Environ. 19, 1115-1123 7 Behnke, H-D. (1989) Structure of the phloem, in Transport of Photoassimilates (Baker, D.A. and Mflburn,J.A., eds), pp. 79-137, Longman 8 Lucas, W.J. et al. (1995) Selectivetrafficking of KNOTTED1 homeodomainprotein and its mRNA through plasmodesmata, Science 270, 1980-1983 9 Bostwick,D.E. et al. (1992) Pumpkin phloem lectin genes are specificallyexpressed in companion cells,Plant Cell 4, 1539-1548 10 Ktthn, C. et al. (1997) Macromolecular traffickingindicated by localizationand turnover of sucrose transporters in enucleate sieve elements, Science 275, 1298-1300 11 St Johnston, D. (1995) The intracellular localizationof messenger RNAs, Cell 81, 161-170 12 DeWitt, N.D. and Sussman, M. (1995) Immunocytologicallocalizationof an epitope-taggedplasma membrane proton pump (H+-ATPase)in phloem companioncells, Plant Cell 7, 2053-2067 13 Bouch~-Pillon,S. et al. (1994) Immunolocalizationof the plasma membrane H+-ATPasein minor veins of Vicia faba in relation to phloem loading, Plant Physiol. 105, 691-697 14 Stadler, R. et al. (1995) Phloem loadingby the PmSUC2 sucrose carrier fromPlantago major occurs into companioncells,Plant Cell 7, 1545-1554 15 Stadler, R. and Saner, N. (1996) The Arabidopsis thaliana AtSUC2 gene is specificallyexpressed in companJoncells,Bot. Acta 109, 299-306
Engineering plant metabolism In keeping with the inclusion of skiing in the programme, the recent Keystone meeting on plant metabolic engineering* included both the highs and the lows of the subject. The presentations revealed the power and limitations of genetic technology to study and engineer plant metabolism. Resistance genes have now been cloned from several species, and it is clear that they are initiators or components of signal transduction chains leading to a range of responses, including altered gene expression and host cell death. The challenge is to identify the signal transduction events that lead to these pathogen-induced responses. Dan Klessig (Rutgers University, Piscataway, USA) has characterized a tobacco 48 KDa MAP kinase that is activated by salicylic acid or infection by tobacco mosaic virus (TMV). The MAP kinase catalyses the phosphorylation of a
DNA-binding protein, which then binds to the P R 2 d promoter. Similarly, another tobacco myb gene has been identified that specifically binds to the P R l a promoter. The transcription factor-phosphorylation theme was further elaborated on by Chris Lamb (Salk Institute, San Diego, USA), who described the reversible phosphorylation of the G/HBF1 basic leucine zipper transcription factor, which binds to an important cis element in the C H S 1 5 promotet of Phaseolus vulgaris. Using antibodies against different domains of the protein, it has been possible to show that phosphorylation induces a conformational change in G/HBF1. A Ca2+-dependent protein kinase is responsible for this phosphorylation. Using the yeast twohybrid system with G/HBF1 as 'bait', Ca 2÷dependent protein kinase and type I protein phosphatase clones have been identified. This is a very active field with lots of possible applications in crop protection.
*Metabolic Engineering in Transgenic Plants (Keystone Symposium), Copper Mountain, CO, USA, 6-11 April 1997.
In a fascinating session on gene suppression and virus defence, David Banlcombe
Defence responses
286
August1997,Vol.2, No. 8
Gene silencing
(John Innes Centre, Norwich, UK) reported on the induction of gene silencing by the potato virus X transgene expression systern. Innoculation with virus transformed to include sequences identical to endogenous genes can lead to the inactivation of these genes. This opens up new ways in which to use viruses as tools for creating gene knock-outs based on the gene silencing system. Moreover, the results suggest that a 'gene silencing signal' may move out of infected cells and induce gene repression in uninfected cells. Jos Mol (Free University, Amsterdam, The Netherlands) discussed results on petunia chalcone synthase gene silencing, and presented a model of post-transcriptional silencing of genes. The presence of inverted repeat sequences integrated into the transgenic plant genome correlates with the induction of silencing: the challenge is to explain this in molecular terms. In an exciting talk, Ramesh Kumar (Kimeragen, Newtown, USA) presented data on an efficient way to introduce sitedirected mutations into animal genomes 1. The finding that RNA-DNA hybrids are
© 1997 Elsevier Science Ltd
news
Sieve elements and companion cells extreme division of labour It is over 70 years since Ernst Miinch presented his pressure flow theory describing a mechanism for long-distance transport of photoassimilates in the phloem 1. In many plants, this pressure flow starts at the sieve element-companion cell complexes (SE-CCCs), which are symplastically isolated from the adjacent cells of the leaf. The driving force for the pressure flow is generated by pairs of primary pumps, the plasma membrane-localized H+-ATPases, and secondary active transporters, the H*-solute symporters. Together, these proteins allow solute (mostly sucrose) movement into the SE-CCCs, and so generate the necessary osmotic pressure. The gene for the first plant sucrose transporter was cloned five years ago, using a yeast expression system s, and subsequent RNA in situ hybridization studies 3 and reporter gene analyses 4 allowed the expression of sucrose carrier genes to be localized to the phloem. The essential role of the sucrose transport protein SUT1 in phloem loading was finally confirmed by antisense repression 5'6. The sieve element-companion cell complex
shown for the PP2 lectin of pumpkin, which was found in both types of cells, although its mRNA is confined to companion cells 9. The recent paper from Wolf Frommer's group ~° presents another elegant set of data confirming that plasmodesmata are more like intercellular organelles than passive tubes allowing solute flux between cells. The results are particularly illuminating with regard to the nature and physiological role of the transported molecules. The analysis included the immunohistochemical localization of SUT1 suerose-H÷-symporters in sieve elements at both the light
membrane of sieve elements of three solanaceous species. No SUT1 protein is detected inside the companion cells. This feature, and in particular the asymmetric localization of SUT1 mRNA at the openings of the plasmodesmata (with higher levels on the sieve element side), suggests that mRNA is indeed transported from the companion cells to the sieve elements, where it is subsequently translated. An asymmetric distribution of RNA has previously been demonstrated in animal cells 11, where it results in asymmetric protein synthesis and distinct polarity. The asymmetric distribution of S U T I mRNA and its accumulation at the plasmodesreal orifices could indicate a similar role for SUT1, such as in the differentiation of the SE-CCC. To substantiate this model of SUT1 mRNA translation in sieve elements, it will be important to identify the ribosomal components. Nevertheless, the possibility that SUT1 proteins in mature sieve elements originate from protein synthesis when the cells are immature, prior to nuclear degradation, was excluded by Ktihn et al.~°; they were able to demonstrate that both SUT1 protein and SUT1 mRNA are degraded during the dark and synthesized in the light. This turnover demonstrates that SUT1 is synthesized de novo in mature sieve elements, and once again underlines the importance of the protein in the regulation of assimilate partitioning and in determining the source activity of leaves.
The SE-CCCs are composed of highly specialized cells - the companion c e l l s can be regarded as power packs that energize the sieve elements, which are living tubes solely for assimilate translocation. Many intracellular structures and organelles, including nuclei, vacuoles, microtubules, rough endoplasmic reticulum, ribosomes and Golgi bodies, appear to be degraded during the course of sieve element development 7. Both cell Phloem loading types are connected by many branched plasmodesmata. As a conse- A sieve element-companioncell complex (sieve element on the right). Why are SUT1 proteins of solanaquence of the long lifetime of the enu- Courtesy of Dr Alexander Schultz and Dr H.-Dietrnar Behnke. ceous plants targeted to the plasma membrane of sieve elcleated sieve elements, which can be active for years, it has frequently been sug- and electron microscope level, as well as in ements, not companion cells? In other gested that these plasmodesmata are not situ localizations of the respective mRNAs species, both H+-ATPases and sucrose only important for the flow of assimilates, in companion cells and sieve elements. The transporters have been localized to combut also for the supply of macromolecular data demonstrate the potential for traffick- panion cells 1~1s. The reason may be that compounds, including proteins. ing of either SUT1 protein or SUT1 mRNAs the species examined load their SE-CCCs The first direct evidence for macromol- through plasmodesmata. The translation of in different ways, or that two different ecular trafficking through plasmodesmata the mRNAs only appears to occur after sucrose transporters are active in both cell came from the group of William Lucas 8, their translocation into sieve elements. The types. An argument can be made for this who revealed that recombinant transcripdata suggest that in addition to protein latter possibility, because most plants have tion factor KN1 (the product of the maize sorting between intracellular compart- two different types of SE-CCCs. In the ments, the companion cells (and maybe loading phloem of source leaf minor veins, knotted1 gene) can migrate through the other cells) can selectively traffick proteins wide companion cells accompany narrow plasmodesmata of tobacco mesophyll cells. and/or mRNAs to the plasmodesmata. sieve elements, and in many plants numerMoreover, it has been demonstrated that ous cell wall ingrowths increase the cell knotted1 mRNA can also pass through surface of the companion cells even more. plasmodesmata in the presence of KN1. Asymmetric distribution of SUT1 mRNA In the transport phloem of the major veins Macromolecular trafficking between com- Kfihn et al. ~° have specifically identified of petioles and stems, however, wide sieve panion cells and sieve elements was first SUT1 sucrose carriers in the plasma
© 1997 Elsevier Science Ltd
August 1997, Vot. 2, No. 8
285
research
news
elements are accompanied by narrow companion cells. A single sucrose transporter (either in the companion cells or in the sieve elements) would minimize the capacity for sucrose transport in one of the two SE-CCCs. Thus, maximal uptake might be achieved by employing two sucrose transporters, one in the companion cells and one in the sieve elements.
molecular analysis has forced a reappraisal of existing models and shown how little is known about this highly specialized pair of cells.
Future prospects
Friedrich-Alexander-Universit&t ErlangenNQrnberg, Botanik II - Molekulare Pflanzenphysiologie, Staudtstrasse 5, D-91058 Erlangen, Germany (tel +49 9131 85 8212; fax +49 9131 85 8751; e-mail nsauer @biologie.uni-erlangen.de)
Acknowledgements I would like to thank M. Gahrtz, R. Stadler and E. Truernit for helpful discussions.
Norbert Sauer The paper of K~hn et al. 1° raises many other questions. It will be important to clarify the physiological impact of SUT1 turnover in mature SE-CCCs, and to assess whether S U T 1 mRNA is indeed translated inside the sieve elements or if the protein migrates into sieve elements after synthesis in the companion cells. Factors responsible for the distribution of asymmetrically distributed mRNAs (including c i s - e l e m e n t s in the 3'-ends of the mRNA and the interacting t r a n s factors) have been identified in animals, and it will be interesting to see whether similar mechanisms are responsible for the asymmetric distribution in SE-CCCs. Irrespective of whether S U T 1 mRNA or the SUT1 protein is translocated from companion cells to sieve elements, it will also be important to identify the signal that is recognized by the companion cell sorting machinery, to find the mode of interaction with the plasmodesmata and to understand the mechanism of translocation. For decades, SE-CCCs have primarily been studied using the methods of light and electron microscopy. Once again,
References 1 Mt~nch,E. (1930) Die Stoffbewegungenin der Pflanze, Gustav Fischer 2 Riesmeier,J.W. et al. (1992) Isolation and characterization of a sucrose carrier cDNA from spinach by functional expressionin yeast, EMBO J. 11, 4705-4713 3 Riesmeier,J.W. et al. (1993) Potato sucrose transporter expressionin minor veins indicates a role in phloem loading,Plant Cell 5, 1591-1598 4 Truemit, E. and Sauer, N. (1995) The promoter of the Arabidopsis thaliana SUC2 sucrose-H+symporter gene directs expression of ~-glucuzonidaseto the phloem: evidencefor phloem loading and unloading by SUC2, Planta 196, 564-570 5 Riesmeier,J.W. et al. (1994) Evidencefor an essential role of the sucrose transporter in phloem loading and assimilate partitioning, EMBO J. 13, 1-7
6 Ktthn, C. et al. (1996) Companioncell-specific inhibition of the potato sucrose transporter SUT1, Plant Cell Environ. 19, 1115-1123 7 Behnke, H-D. (1989) Structure of the phloem, in Transport of Photoassimilates (Baker, D.A. and Mflburn,J.A., eds), pp. 79-137, Longman 8 Lucas, W.J. et al. (1995) Selectivetrafficking of KNOTTED1 homeodomainprotein and its mRNA through plasmodesmata, Science 270, 1980-1983 9 Bostwick,D.E. et al. (1992) Pumpkin phloem lectin genes are specificallyexpressed in companion cells,Plant Cell 4, 1539-1548 10 Ktthn, C. et al. (1997) Macromolecular traffickingindicated by localizationand turnover of sucrose transporters in enucleate sieve elements, Science 275, 1298-1300 11 St Johnston, D. (1995) The intracellular localizationof messenger RNAs, Cell 81, 161-170 12 DeWitt, N.D. and Sussman, M. (1995) Immunocytologicallocalizationof an epitope-taggedplasma membrane proton pump (H+-ATPase)in phloem companioncells, Plant Cell 7, 2053-2067 13 Bouch~-Pillon,S. et al. (1994) Immunolocalizationof the plasma membrane H+-ATPasein minor veins of Vicia faba in relation to phloem loading, Plant Physiol. 105, 691-697 14 Stadler, R. et al. (1995) Phloem loadingby the PmSUC2 sucrose carrier fromPlantago major occurs into companioncells,Plant Cell 7, 1545-1554 15 Stadler, R. and Saner, N. (1996) The Arabidopsis thaliana AtSUC2 gene is specificallyexpressed in companJoncells,Bot. Acta 109, 299-306
Engineering plant metabolism In keeping with the inclusion of skiing in the programme, the recent Keystone meeting on plant metabolic engineering* included both the highs and the lows of the subject. The presentations revealed the power and limitations of genetic technology to study and engineer plant metabolism. Resistance genes have now been cloned from several species, and it is clear that they are initiators or components of signal transduction chains leading to a range of responses, including altered gene expression and host cell death. The challenge is to identify the signal transduction events that lead to these pathogen-induced responses. Dan Klessig (Rutgers University, Piscataway, USA) has characterized a tobacco 48 KDa MAP kinase that is activated by salicylic acid or infection by tobacco mosaic virus (TMV). The MAP kinase catalyses the phosphorylation of a
DNA-binding protein, which then binds to the P R 2 d promoter. Similarly, another tobacco myb gene has been identified that specifically binds to the P R l a promoter. The transcription factor-phosphorylation theme was further elaborated on by Chris Lamb (Salk Institute, San Diego, USA), who described the reversible phosphorylation of the G/HBF1 basic leucine zipper transcription factor, which binds to an important cis element in the C H S 1 5 promotet of Phaseolus vulgaris. Using antibodies against different domains of the protein, it has been possible to show that phosphorylation induces a conformational change in G/HBF1. A Ca2+-dependent protein kinase is responsible for this phosphorylation. Using the yeast twohybrid system with G/HBF1 as 'bait', Ca 2÷dependent protein kinase and type I protein phosphatase clones have been identified. This is a very active field with lots of possible applications in crop protection.
*Metabolic Engineering in Transgenic Plants (Keystone Symposium), Copper Mountain, CO, USA, 6-11 April 1997.
In a fascinating session on gene suppression and virus defence, David Banlcombe
Defence responses
286
August1997,Vol.2, No. 8
Gene silencing
(John Innes Centre, Norwich, UK) reported on the induction of gene silencing by the potato virus X transgene expression systern. Innoculation with virus transformed to include sequences identical to endogenous genes can lead to the inactivation of these genes. This opens up new ways in which to use viruses as tools for creating gene knock-outs based on the gene silencing system. Moreover, the results suggest that a 'gene silencing signal' may move out of infected cells and induce gene repression in uninfected cells. Jos Mol (Free University, Amsterdam, The Netherlands) discussed results on petunia chalcone synthase gene silencing, and presented a model of post-transcriptional silencing of genes. The presence of inverted repeat sequences integrated into the transgenic plant genome correlates with the induction of silencing: the challenge is to explain this in molecular terms. In an exciting talk, Ramesh Kumar (Kimeragen, Newtown, USA) presented data on an efficient way to introduce sitedirected mutations into animal genomes 1. The finding that RNA-DNA hybrids are
© 1997 Elsevier Science Ltd