- Email: [email protected]
GM crops for pathogen resistance without gene flow
56
News & Comment
TRENDS in Plant Science Vol.7 No.2 February 2002
Journal Club
Renewed debate over transpiration and long-distance transport of minerals in plants Although little research is available on the subject, it is commonly accepted that transpiration, the evaporative loss of water from plant leaves, is required for the longdistance transport of inorganic nutrients in the xylem of higher plants. However, over the history of plant nutrition, there has been no lack of researchers who have questioned this precept. For example, Gottlieb Haberlandt, investigating the transpiration rate of the leaves of tropical plants in 1892, was the first to state clearly a view opposed to the commonly accepted one: ‘The low rate of transpiration, which is typical for the abundantly growing plants of the moist lowlands of Java certainly is a strong argument against the still very popular assumption that the transpiration stream as vehicle for nutrient salts is of major importance for plant nutrition’. Today it is recognized that the metabolic uptake of ions and the passive uptake of water are independent processes. Recently, Widmar Tanner and Harry Beevers [1] conducted new experiments to examine the relationship between transpiration and long-distance mineral transport in plants. As they did previously in 1990, the authors assumed that the movement
of water in the xylem as a result of transpiration inevitably results in a corresponding movement of dissolved ions and that longdistance transport is normally accelerated by this process. However, 11 years on, they question whether transpiration per se is essential for the movement of ions towards the shoot and therefore whether non-transpiring plants would be at a disadvantage. The major difficulty in studying the contribution of transpiration to long-distance mineral transport is that it is not possible to maintain 100% relative humidity around plant leaves in the light. To solve this problem, sunflowers were grown in hydroculture with relative humidity kept at 98.5% during the night and at 52.0% during the day. Experimental and control plants were exposed to mineral solution only at night (MN-plants) and only during the day (MD-plants), respectively. While transpiration in the MD-plants during the day was almost 12 times the transpiration of MN-plants during the night, the production of biomass and the concentration of cations in leaves and stems were similar in all plants. The MN-plants showed a reduction of only 17% in the amount of K+ taken up under non-transpiring conditions.
In short, the authors concluded that transpiration is not essential for long-distance transport of mineral elements in plants. ‘…transpiration is not essential for longdistance transport of mineral elements in plants.’ Tanner and Beevers suggest that 1 l of water, corresponding to non-transpirational components of water flow in the xylem (growth water + ‘Münch’s counterflow’), together with 1 l of water corresponding to the residual transpiration can transport the same amount of mineral in MN-plants. Although this work is not free of criticisms, as was the case in 1990, the researchers should be applauded for their courage in taking up this matter. 1 Tanner, W. and Beevers, H. (2001) Transpiration, a prerequisite for long-distance transport of minerals in plants? Proc. Natl. Acad. Sci. U. S. A. 98, 9443–9447
Juan M. Ruiz [email protected] Luis Romero [email protected]
GM crops for pathogen resistance without gene flow Containment of genes is a serious concern when growing gene-modified (GM) crops because gene flow of foreign genes from GM crops can create pathogen-resistant weeds or genetic pollution among other crops. ‘…transformed tobacco plants were protected against the bacterial pathogen…and the fungal pathogen…by a high-dose release of the toxin at the site of infection by chloroplast lysis.’ However, having crops with increased resistance to pathogens or reducing mycotoxin contamination caused by fungi remains a desirable objective. Earlier, Henry Daniell showed that transformation of the chloroplast genome could be engineered with useful genes that would not drift to weeds and other crops because plastid DNA, maternally http://plants.trends.com
inherited, is lost during pollen maturation. Whereas chloroplast transformation has been used before to generate biopharmaceuticals, and herbicide- and insecticide-resistant crops, transforming the plant chloroplast to produce toxins that would protect plants from attack by bacterial and fungal parasites had not been reported until now. Gerald DeGray et al. [1], using Daniell’s technique, have expressed a gene for a 22mer analog (MSI-99) of magainin-2, inserted into the chloroplast genome of tobacco (Nicotiana tabacum var. Petit Havana) plants. Magainin-2 is a 23mer defense peptide secreted from the skin of the African clawed frog (Xenopus laevis). The chloroplast genome of tobacco was transformed biolistically using a chloroplastspecific transformation vector that contained the MSI-99 gene with a chloroplast-preferred
ribosome-binding site (GGAGG), and the aadA gene that conferred resistance to spectinomycin. After selection on spectinomycin-containing media, all transformants were grown up and shown to mature normally. Nuclear transgenic plants did not confer resistance to spectinomycin and were eliminated. Southern blots confirmed integration of MSI-99 into the chloroplast genome and achievement of homoplasmy, whereas northern blots confirmed transcription. Accumulation of MSI-99 in transgenic chloroplasts did not affect normal growth and development of the transgenic plants, showing that this toxic magainin can be expressed at high levels without disrupting the chloroplast membrane. The authors speculate that membrane stability of the magainin analog is due to the different lipid content of plastid membranes
1360-1385/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved.
News & Comment
TRENDS in Plant Science Vol.7 No.2 February 2002
57
In Brief
compared with membranes of susceptible target microbes. In planta assays showed that transformed tobacco plants were protected against the bacterial pathogen, Pseudomonas syringae pv. tabaci, and the fungal pathogen, Colletotrichum destructivum, by a high-dose release of the toxin at the site of infection by chloroplast lysis. In addition, leaf extracts from transgenic plants inhibited the growth of pre-germinated spores of the fungi Aspergillus flavus, Fusarium moniliforme and Verticillium dahliae. The research reported by DeGray et al. extends the strategy of transforming the
chloroplast genome to include peptides useful for protection of plants against bacterial and fungal pathogens, while preventing the escape of pollen-mediated transgenes. However, using this strategy to engineer food crops, will raise the issue of safety of products intended for use as food or feed. 1 DeGray, G. et al. (2001) Expression of an antimicrobial peptide via the chloroplast genome to control phytopathogenic bacteria and fungi. Plant Physiol. 127, 852–862
Richard C. Staples [email protected]
Circadian regulation of gene families encoding light sensor proteins Phytochrome and cryptochrome are chromoproteins responsible for sensing the environmental light signal and initiating a series of reactions responsible for the subsequent developmental responses. Cryptochrome homologues have been identified in animal and human cells. The proteins encoded by two gene families are involved, to diverse extents, in the circadian regulation of gene expression. In a recent article, Réka Tóth and colleagues [1] have used gene fusions to the luciferase reporter to investigate the regulation of PHY and CRY genes in Arabidopsis thaliana. ‘…PHYA rhythmicity is maintained in excised leaves better than in control attached…’ All five CRY and two PHY genes show diurnal variation, with peaks of expression at different time points during the light period. Upon transfer to constant conditions (light or dark), circadian regulation is maintained for all genes, with the exception of PHYC ; nevertheless, mRNA accumulation follows a circadian rhythm for all seven genes under constant light. The system allows the identification of spatial patterns of expression showing, for example, high promoter activity for PHYA and CRY2 in shoot meristems and in root tips, and lower activity in cotyledons, hypocotyls and roots. CRY1 is expressed in aerial tissues; PHYB in all tissues, in particular in shoot meristems and root tips and PHYC, PHYD and PHYE in cotyledons and root tips. Peaks of transcriptional activity for each gene during http://plants.trends.com
the day reflect differences in regulation and might correlate with diversity in protein stability and biological function. In an accompanying article, Anthony Hall et al. [2] give further details about the regulation of expression of PHYA. In addition to the reported expression in hypocotyls, leaves and root tips, by a modified LUC+ reporter gene, expression is detected in a precise group of cells in the columella root cap. The authors investigate other characteristics of PHYA circadian regulation in intact plants or in isolated organs. All three gene transcripts for PHYA show rhythmicity upon transfer to constant conditions (light or dark). Interestingly, PHYA rhythmicity is maintained in excised leaves better than in control attached leaves (damping is reduced). Also, higher expression levels are maintained in leaves in the dark, in agreement with previous results showing light-mediated repression of phytochrome gene expression. In excised roots and hypocotyls, PHYA rhythmicity was different compared with that in leaves, indicating the existence of multiple factors responsible for organ-specific regulation and systemic regulation of the circadian clock. 1 Tóth, R. et al. (2001) Circadian clock-regulated expression of phytochrome and cryptochrome genes in Arabidopsis. Plant Physiol. 127, 1607–1616 2 Hall, A. et al. (2001) Conditional circadian regulation of PHYTOCHROME A gene expression. Plant Physiol. 127, 1808–1818
Emilio Cervantes [email protected]
AIDS herbal therapy Sutherlandia frutescens microphylla is gaining international attention as a cheap, readily grown herbal medicine that can improve the health of AIDS patients through weight gain and energy boosts. The plant is native to South Africa where indigenous people refer to the plant as insisa: the one that dispels darkness. South African scientists and medical practitioners worry that insisa will be patented and become available only to those who can afford it. Insisa contains canavanine, pinitol and GABA, chemicals already patented by drug companies. A team of traditional healers, scientists and general practitioners have joined together to try to ensure that insisa remains in the public domain. The medicine will enter drug trials later this year. [Duval Smith, A. (2001) The Independent (London) 30 November, p. 18] TS
Pretty? Deadly! It has long been known that humans and insects see flower colors differently. For example, flowers of Hypericum calycinum appear uniformly yellow to humans, but distinctly different to the ultraviolet (UV) vision of insects. Investigating the chemicals that are responsible for the UV-patterning of flowers, Matthew Gronquist and colleagues of Cornell University (Ithaca, NY, USA) have discovered that some of them might also have a hitherto unappreciated defensive role [Proc. Natl. Acad. Sci. U. S. A. (2001) 98, 13745–13750]. Of the two classes of UV pigments in Hypericum – flavonoids and dearomatized isoprenylated phloroglucinols – dearomatized isoprenylated phloroglucinols are toxic to rattlebox moth caterpillars. Say it with flowers! NC
Needles and scales An international team has discovered a new conifer tree, which, when mature, carries both short fir-like needles and scaly cypress-like leaves. The scientists named the tree Golden Vietnamese cypress because its wood has a golden color. They also established that the tree forms a new genus, Xanthocyparis. However, the tree is endangered, with only a few semi-mature species found in Vietnam close to the Chinese–Vietnam border. It is only the second novel conifer found in the past 50 years. [Cookson, C. (2001) Financial Times (London) 23 November, p. 8] TS
1360-1385/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved.
News & Comment
TRENDS in Plant Science Vol.7 No.2 February 2002
Journal Club
Renewed debate over transpiration and long-distance transport of minerals in plants Although little research is available on the subject, it is commonly accepted that transpiration, the evaporative loss of water from plant leaves, is required for the longdistance transport of inorganic nutrients in the xylem of higher plants. However, over the history of plant nutrition, there has been no lack of researchers who have questioned this precept. For example, Gottlieb Haberlandt, investigating the transpiration rate of the leaves of tropical plants in 1892, was the first to state clearly a view opposed to the commonly accepted one: ‘The low rate of transpiration, which is typical for the abundantly growing plants of the moist lowlands of Java certainly is a strong argument against the still very popular assumption that the transpiration stream as vehicle for nutrient salts is of major importance for plant nutrition’. Today it is recognized that the metabolic uptake of ions and the passive uptake of water are independent processes. Recently, Widmar Tanner and Harry Beevers [1] conducted new experiments to examine the relationship between transpiration and long-distance mineral transport in plants. As they did previously in 1990, the authors assumed that the movement
of water in the xylem as a result of transpiration inevitably results in a corresponding movement of dissolved ions and that longdistance transport is normally accelerated by this process. However, 11 years on, they question whether transpiration per se is essential for the movement of ions towards the shoot and therefore whether non-transpiring plants would be at a disadvantage. The major difficulty in studying the contribution of transpiration to long-distance mineral transport is that it is not possible to maintain 100% relative humidity around plant leaves in the light. To solve this problem, sunflowers were grown in hydroculture with relative humidity kept at 98.5% during the night and at 52.0% during the day. Experimental and control plants were exposed to mineral solution only at night (MN-plants) and only during the day (MD-plants), respectively. While transpiration in the MD-plants during the day was almost 12 times the transpiration of MN-plants during the night, the production of biomass and the concentration of cations in leaves and stems were similar in all plants. The MN-plants showed a reduction of only 17% in the amount of K+ taken up under non-transpiring conditions.
In short, the authors concluded that transpiration is not essential for long-distance transport of mineral elements in plants. ‘…transpiration is not essential for longdistance transport of mineral elements in plants.’ Tanner and Beevers suggest that 1 l of water, corresponding to non-transpirational components of water flow in the xylem (growth water + ‘Münch’s counterflow’), together with 1 l of water corresponding to the residual transpiration can transport the same amount of mineral in MN-plants. Although this work is not free of criticisms, as was the case in 1990, the researchers should be applauded for their courage in taking up this matter. 1 Tanner, W. and Beevers, H. (2001) Transpiration, a prerequisite for long-distance transport of minerals in plants? Proc. Natl. Acad. Sci. U. S. A. 98, 9443–9447
Juan M. Ruiz [email protected] Luis Romero [email protected]
GM crops for pathogen resistance without gene flow Containment of genes is a serious concern when growing gene-modified (GM) crops because gene flow of foreign genes from GM crops can create pathogen-resistant weeds or genetic pollution among other crops. ‘…transformed tobacco plants were protected against the bacterial pathogen…and the fungal pathogen…by a high-dose release of the toxin at the site of infection by chloroplast lysis.’ However, having crops with increased resistance to pathogens or reducing mycotoxin contamination caused by fungi remains a desirable objective. Earlier, Henry Daniell showed that transformation of the chloroplast genome could be engineered with useful genes that would not drift to weeds and other crops because plastid DNA, maternally http://plants.trends.com
inherited, is lost during pollen maturation. Whereas chloroplast transformation has been used before to generate biopharmaceuticals, and herbicide- and insecticide-resistant crops, transforming the plant chloroplast to produce toxins that would protect plants from attack by bacterial and fungal parasites had not been reported until now. Gerald DeGray et al. [1], using Daniell’s technique, have expressed a gene for a 22mer analog (MSI-99) of magainin-2, inserted into the chloroplast genome of tobacco (Nicotiana tabacum var. Petit Havana) plants. Magainin-2 is a 23mer defense peptide secreted from the skin of the African clawed frog (Xenopus laevis). The chloroplast genome of tobacco was transformed biolistically using a chloroplastspecific transformation vector that contained the MSI-99 gene with a chloroplast-preferred
ribosome-binding site (GGAGG), and the aadA gene that conferred resistance to spectinomycin. After selection on spectinomycin-containing media, all transformants were grown up and shown to mature normally. Nuclear transgenic plants did not confer resistance to spectinomycin and were eliminated. Southern blots confirmed integration of MSI-99 into the chloroplast genome and achievement of homoplasmy, whereas northern blots confirmed transcription. Accumulation of MSI-99 in transgenic chloroplasts did not affect normal growth and development of the transgenic plants, showing that this toxic magainin can be expressed at high levels without disrupting the chloroplast membrane. The authors speculate that membrane stability of the magainin analog is due to the different lipid content of plastid membranes
1360-1385/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved.
News & Comment
TRENDS in Plant Science Vol.7 No.2 February 2002
57
In Brief
compared with membranes of susceptible target microbes. In planta assays showed that transformed tobacco plants were protected against the bacterial pathogen, Pseudomonas syringae pv. tabaci, and the fungal pathogen, Colletotrichum destructivum, by a high-dose release of the toxin at the site of infection by chloroplast lysis. In addition, leaf extracts from transgenic plants inhibited the growth of pre-germinated spores of the fungi Aspergillus flavus, Fusarium moniliforme and Verticillium dahliae. The research reported by DeGray et al. extends the strategy of transforming the
chloroplast genome to include peptides useful for protection of plants against bacterial and fungal pathogens, while preventing the escape of pollen-mediated transgenes. However, using this strategy to engineer food crops, will raise the issue of safety of products intended for use as food or feed. 1 DeGray, G. et al. (2001) Expression of an antimicrobial peptide via the chloroplast genome to control phytopathogenic bacteria and fungi. Plant Physiol. 127, 852–862
Richard C. Staples [email protected]
Circadian regulation of gene families encoding light sensor proteins Phytochrome and cryptochrome are chromoproteins responsible for sensing the environmental light signal and initiating a series of reactions responsible for the subsequent developmental responses. Cryptochrome homologues have been identified in animal and human cells. The proteins encoded by two gene families are involved, to diverse extents, in the circadian regulation of gene expression. In a recent article, Réka Tóth and colleagues [1] have used gene fusions to the luciferase reporter to investigate the regulation of PHY and CRY genes in Arabidopsis thaliana. ‘…PHYA rhythmicity is maintained in excised leaves better than in control attached…’ All five CRY and two PHY genes show diurnal variation, with peaks of expression at different time points during the light period. Upon transfer to constant conditions (light or dark), circadian regulation is maintained for all genes, with the exception of PHYC ; nevertheless, mRNA accumulation follows a circadian rhythm for all seven genes under constant light. The system allows the identification of spatial patterns of expression showing, for example, high promoter activity for PHYA and CRY2 in shoot meristems and in root tips, and lower activity in cotyledons, hypocotyls and roots. CRY1 is expressed in aerial tissues; PHYB in all tissues, in particular in shoot meristems and root tips and PHYC, PHYD and PHYE in cotyledons and root tips. Peaks of transcriptional activity for each gene during http://plants.trends.com
the day reflect differences in regulation and might correlate with diversity in protein stability and biological function. In an accompanying article, Anthony Hall et al. [2] give further details about the regulation of expression of PHYA. In addition to the reported expression in hypocotyls, leaves and root tips, by a modified LUC+ reporter gene, expression is detected in a precise group of cells in the columella root cap. The authors investigate other characteristics of PHYA circadian regulation in intact plants or in isolated organs. All three gene transcripts for PHYA show rhythmicity upon transfer to constant conditions (light or dark). Interestingly, PHYA rhythmicity is maintained in excised leaves better than in control attached leaves (damping is reduced). Also, higher expression levels are maintained in leaves in the dark, in agreement with previous results showing light-mediated repression of phytochrome gene expression. In excised roots and hypocotyls, PHYA rhythmicity was different compared with that in leaves, indicating the existence of multiple factors responsible for organ-specific regulation and systemic regulation of the circadian clock. 1 Tóth, R. et al. (2001) Circadian clock-regulated expression of phytochrome and cryptochrome genes in Arabidopsis. Plant Physiol. 127, 1607–1616 2 Hall, A. et al. (2001) Conditional circadian regulation of PHYTOCHROME A gene expression. Plant Physiol. 127, 1808–1818
Emilio Cervantes [email protected]
AIDS herbal therapy Sutherlandia frutescens microphylla is gaining international attention as a cheap, readily grown herbal medicine that can improve the health of AIDS patients through weight gain and energy boosts. The plant is native to South Africa where indigenous people refer to the plant as insisa: the one that dispels darkness. South African scientists and medical practitioners worry that insisa will be patented and become available only to those who can afford it. Insisa contains canavanine, pinitol and GABA, chemicals already patented by drug companies. A team of traditional healers, scientists and general practitioners have joined together to try to ensure that insisa remains in the public domain. The medicine will enter drug trials later this year. [Duval Smith, A. (2001) The Independent (London) 30 November, p. 18] TS
Pretty? Deadly! It has long been known that humans and insects see flower colors differently. For example, flowers of Hypericum calycinum appear uniformly yellow to humans, but distinctly different to the ultraviolet (UV) vision of insects. Investigating the chemicals that are responsible for the UV-patterning of flowers, Matthew Gronquist and colleagues of Cornell University (Ithaca, NY, USA) have discovered that some of them might also have a hitherto unappreciated defensive role [Proc. Natl. Acad. Sci. U. S. A. (2001) 98, 13745–13750]. Of the two classes of UV pigments in Hypericum – flavonoids and dearomatized isoprenylated phloroglucinols – dearomatized isoprenylated phloroglucinols are toxic to rattlebox moth caterpillars. Say it with flowers! NC
Needles and scales An international team has discovered a new conifer tree, which, when mature, carries both short fir-like needles and scaly cypress-like leaves. The scientists named the tree Golden Vietnamese cypress because its wood has a golden color. They also established that the tree forms a new genus, Xanthocyparis. However, the tree is endangered, with only a few semi-mature species found in Vietnam close to the Chinese–Vietnam border. It is only the second novel conifer found in the past 50 years. [Cookson, C. (2001) Financial Times (London) 23 November, p. 8] TS
1360-1385/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved.