Scientific Article ARANYA; Vol I (I); January - June 2011

Today’s Tears-Tomorrow’s Friend Dr. Amal Kumar Mondal1 and Dr. Sanjukta Mondal (Parui)2

1. Associate Professor and Former Head of the Department of Botany and Forestry, Plant Taxonomy, Biosystematics and Molecular Taxonomy Laboratory, VIDYASAGAR UNIVERSITY, Midnapore-721 102, West Bengal, India Email: [email protected] 2. Associate Professor, Department of Zoology, Lady Brabourne College, Kolkata-700 017, Wst Bengal, India Email: [email protected] Abstract

The Guttation, a process of exuding water from plants is not just restricted to leaf margins and tips but to entire leaf surface, and can now serve as a medium for production of recombinant proteins

Key words Guttation, hydathodes, phyllosecretion We are very familiar with the dew drops found on the grasses and the leaves of other plants, in the early morning hours, giving the plants the fresh young look, soothing our eyes during our morning walks. But these drops of water are not always dewdrops, but are instead, water exuded by the leaves of the plants - a process called guttation. It is the secretion of water on to the leaf surface, particularly along the leaf margin, through specialized pores called hydathodes. This process occurs most frequently during conditions of high humidity, when the rate of transpiration is low. It has been observed during warm humid nights. However it occurs maximum during day and minimum during nights. The term “guttation” was proposed by Burgerstien for the exudation of water from plants in the form of liquid. The leaf blade (lamina) as we know consists of an upper and lower epidermis, which is a thin, usually transparent, colorless layer of cells, and is generally referred to as the skin of the leaf blade. Sandwiched in between these two layers of epidermis is the spongy layer of tissue, called the mesophyll, in which runs a branching system of veins. The epidermis is most often covered by a layer of cutin called cuticle, which is a waxy substance secreted by the epidermal cells. The epidermis together with the cuticle prevents excessive loss of water from the leaves and also protects the leaves from injury. The plant generally transpires through pores called stomata, which are scattered throughout the epidermis. However, the numbers of stomata are more on the undersurface of the leaf, than on the upper surface, which prevents excessive loss of water from evaporating from the upper surface of the leaf, which is exposed to the sun. The stomata however does not always remain open and the opening and closing of each stomata is regulated by a pair of bean-shaped cells called guard cells, in response to heat and light. The stomata usually close at night, which further helps in water conservation. Hydathodes in plants are of two types – Epithem hydathodes and Active hydathodes. In case of Epithem hydathode, water is forced out by the root pressure whereas in case of Active hydathode, water is secreted by the force developed within the cells themselves. However root pressure is generally the main cause of guttation and the guttation rate is reduced by conditions reducing root pressure such as cold and dry aerated soil and also mineral deficiency. The force that drives the water through the root is based on differences in the water potential of the soil i.e. the root’s surrounding and the xylem sap of the root. The ions that pass through the endodermis of the root along with the water is trapped and cannot leave the stele any more. Due to osmosis, a pressure called endosmotic root pressure develops in the xylem that presses or forces the water along with the dissolved ions upwards. This root pressure acts most efficiently at night, but its efficiency decreases during the day when it’s rate becomes much smaller than the rate of transpiration. The guttated water contains various kinds of sugars, amino acids, enzymes, organic acids, vitamins, other organic compounds and minerals such as calcium. This leaves a white crust on the leaf surface when the guttated water dries. A typical example is the lime secretion of Saxifraga sp. and the salt glands of halophytes. Scientific Article Scientific Article ARANYA; Vol I (I); January - June 2011 ARANYA; Vol I (I); January - June 2011

Guttation has however not been observed universally. This process has been reported to occur in 333 Proteins of interest targetted for the outside are synthesized by ribosomes which are attached to the rough endoplasmic genera belonging to 115 families. It is restricted to certain genera of mostly herbaceous plants. Heavy guttation has reticulum. As they are synthesized, these proteins are translocated into the lumen of the endoplasmic been observed in Colocasia antiquorum, in which the amount of guttated water ranges from a few drops to 100 ml reticulum, where they are glycosylated and molecular chaperones aid the protein folding. or even more per day. Other common guttating plants include the grasses, Ambrosia trifida, Brassica oleracea, The misfolded proteins are retrotranslocated to the cytosol where they are degraded by a proteasome by the Cleome viscosa, Commelina benghalensis, Hordeum vulgare (barley), Hydrocotyle, Lactuca, Lycopersicon endoplasmic reticulum associated degradation system. The vesicles containing the properly-folded proteins then esculentum (tomata), Physocarpus, Pilea pumila, Potentilla palustris, Spilanthes, Trifolium, Tropoelum sp, Urtica enter the Golgi apparatus. In the Golgi apparatus, the glycosylation of the proteins is modified and further posttranslational dioica, Vitex trifolia, Equisitum (Pteridophyte), etc. Further, the hydathodes are restricted to the tips and margins modificat ions takes place, incl uding cleavage and functionalizat ion. The proteins are then moved of leaves, due to which the droplets are seen deposited on the margin and tips of leaves. However recently guttation into secretory vesicles which travel along the cytoskeleton to the edge of the cell where the vesicle fuses with the has been observed in some unreported genera like Ficus hispida, Ficus cunea and Fleurya interupta more cell membrane in a process called exocytocis, releasing its contents out of the cell’s environment. During this interestingly, the guttated water has been observed on the entire leaf blade and not just restricted to leaf tips and entire sequence, strict biochemical control is maintained by usage of a pH gradient. The pH of the cytosol is 7.4, margins. This study indicates that although hydathodes represent the main points of guttation fluid production, but the endoplasmic reticulum’s pH is 7.0, and the cis-golgi has a pH of 6.5. Secretory vesicles have pHs ranging guttation fluid can also be released through the cuticle or stomata, as has been reported earlier by Lausberg (1935) between 5.0 and 6.0. and Baid (1952) respectively. In the process of phyllosecretion, the proteins destined for export must first be labelled correctly so thet the As mentioned earlier, some proteins are naturally secreted into the plant guttation fluid. Proteins like catalase trans-Golgi network can dispatch the proteins to their correct destination. The labelling may be either in the form and peroxidase enzyme have been reported in the guttation fluid of Zea mays (maize), Avena sativa (oat), several of a signal sequences at the end of the polypeptide chain or signal patches, which are dispersed throughout the peroxidases in Fragaria ananassa (strawberry), Lycopersicon esculentum (tomato) and Cucumis sativus (cucumber) polypeptide chain, but are brought together when in a 3D conformation. All cells must export proteins to the and reductase in Phleum pzatense (timothy). Very recently guttation has been successfully used as a vehicle exterior of the plasma membrane, but specialized secretory cells must also control what domain of the plasma for recombinant protein production in plants by a process termed “phyllosecretion” by a group of scientists of membrane the proteins are released on. Thus, all cells have the constitutive secretory pathway, or default pathway, Biotech Center, Cook College, Rutgers University, New Jersey and Phytomedics, Dayton, New Jersey. They engineered but specialized secretory cells also have a regulated secretory pathway. In the secretory pathway, proteins are the tobacco plant (Nicotiana tabacum L. cv Wisconsin) to secrete human placental secreted alkaline phosphatase packaged into secretory vesicles, which then aggregate next to the plasma membrane where they await an extracellular (SEAP), green fluorescent protein (GFP) from jellyfish (Aequorea victoria) and xylanase from Clostridium signal to fuse with the plasma membrane and release their contents. thermocellum through the plant cell default secretion pathway. They found that recombinant proteins directed to the leaf intercellular space i.e. apoplast are effectively released into the plant guttation fluid, which can be collected Thus by using endoplasmic reticulum signal peptides fused to the recombinant protein sequences, these scientists continuously throughout the plant’s lifetime. have generated transgenic tobacco (Nicotiana tabacum L. cv Wisconsin) plants that secrete three heterologousproteins of different genetic backgrounds (bacterial xylanase, green fluorescent protein of jellyfish [Aequorea Plants serve as suitable bioreactors for the production of many valuable recombinant proteins used as pharmaceuticals, victoria], and human placental alkaline phosphatase) through the leaf intercellular space into tobacco guttation industrial enzymes, etc. Foreign genes are now being routinely expressed in many plant species to fluid. produce recombinant proteins because of their ability to carry out numerous post-translational protein modifications required for biological activity, and they can be easily cultivated. Till date numerous heterologous proteins Thus, guttation fluid can have numerous applications in future scientific investigations, apart from its role in have been expressed in different plant organs and plant cell compartments. However, the major obstacle for the helping plants to get rid of the excess water, which will no longer go as a waste. Another future prospect would be large-scale protein production in plants is the high cost of protein extraction and purification from biochemically to assess the ability of the simultaneous use of both the phyllosecretion and rhizosecretion systems. If successful, complex plant tissues. This obstacle has been partially overcome by aseptically cultivated cell cultures or plant the combination of both these techniques can significantly increase the total yield of heterologous proteinsproduction organs that secrete recombinant proteins into the surrounding medium. These in vitro systems however can be by plants in the easily accessible form of a water solution. expensive, slow growing, unstable, and relatively low yielding. In this respect the production of recombinant proteins in guttation fluid has several advantages and is comparatively a much easier process and cost effective. Production of Recombinant Proteins Recombinant proteins are proteins whose amino acid sequence is encoded by a cloned gene. This process involves The advantage of using phyllosecretion is that, this method allows continuous and non-destructive recovery of the cloning of the gene encoding the desired protein into an expression vector, which should contain a promoter so recombinant proteins from a living plant, as guttation fluid can be collected throughout a plant’s life. Guttation that the protein can be expressed. Thus through this recombinant DNA technology, a large amount of the protein fluid also has the potential of increasing yield, abolishing the need for tissue extraction, and simplifying its downstream can be produced. processing i.e. simplifies complex protein purification procedures, thus increasing the efficiency of recombinant protein production technology. It is nondestructive. Also, recombinant biopharmaceutical proteins purified from leaf exudates are less likely to be contaminated with pathogenic viruses, which may be present in the milk or urine of transgenic animals. The plant protein phyllosecretion system is easy to scale up and is less sensitive to the cytotoxicity of the final products. The guttation fluid can be easily collected, by shaking them off the leaves into a collection vessel, or a method for large-scale collection of guttation fluid can be developed like removing them from the leaf surface with a vacuum or blotting. Another system called Rhizosecretion system for the production of recombinant proteins has been developed recently, which takes advantage of the ability of roots of hydroponically cultivated plants to secrete properly targeted recombinant proteins into the surrounding medium. Now what exactly is Phyllosecretion? Phyllosecretion is a method, which exploits leaf guttation as a An example of the process of production of recombinant proteins (a) The expression vector contains lac promoter and the lacZ gene, which encodes b-galactosidase in presence medium to continuously “wash away” recombinant proteins from a living plant. Guttation fluid is transformed of lactose, which stimulates expression of b-galactosidase. into a concentrated solution of recombinant proteins. Eukaryotic cells have a highly evolved process of secretion. (b) lacZ is replaced by another gene, which encodes the protein of interest, Lactose stimulate the expression of desired proteins. Scientific Article Scientific Article ARANYA; Vol I (I); January - June 2011 ARANYA; Vol I (I); January - June 2011

Guttation has however not been observed universally. This process has been reported to occur in 333 Proteins of interest targetted for the outside are synthesized by ribosomes which are attached to the rough endoplasmic genera belonging to 115 families. It is restricted to certain genera of mostly herbaceous plants. Heavy guttation has reticulum. As they are synthesized, these proteins are translocated into the lumen of the endoplasmic been observed in Colocasia antiquorum, in which the amount of guttated water ranges from a few drops to 100 ml reticulum, where they are glycosylated and molecular chaperones aid the protein folding. or even more per day. Other common guttating plants include the grasses, Ambrosia trifida, Brassica oleracea, The misfolded proteins are retrotranslocated to the cytosol where they are degraded by a proteasome by the Cleome viscosa, Commelina benghalensis, Hordeum vulgare (barley), Hydrocotyle, Lactuca, Lycopersicon endoplasmic reticulum associated degradation system. The vesicles containing the properly-folded proteins then esculentum (tomata), Physocarpus, Pilea pumila, Potentilla palustris, Spilanthes, Trifolium, Tropoelum sp, Urtica enter the Golgi apparatus. In the Golgi apparatus, the glycosylation of the proteins is modified and further posttranslational dioica, Vitex trifolia, Equisitum (Pteridophyte), etc. Further, the hydathodes are restricted to the tips and margins modificat ions takes place, incl uding cleavage and functionalizat ion. The proteins are then moved of leaves, due to which the droplets are seen deposited on the margin and tips of leaves. However recently guttation into secretory vesicles which travel along the cytoskeleton to the edge of the cell where the vesicle fuses with the has been observed in some unreported genera like Ficus hispida, Ficus cunea and Fleurya interupta more cell membrane in a process called exocytocis, releasing its contents out of the cell’s environment. During this interestingly, the guttated water has been observed on the entire leaf blade and not just restricted to leaf tips and entire sequence, strict biochemical control is maintained by usage of a pH gradient. The pH of the cytosol is 7.4, margins. This study indicates that although hydathodes represent the main points of guttation fluid production, but the endoplasmic reticulum’s pH is 7.0, and the cis-golgi has a pH of 6.5. Secretory vesicles have pHs ranging guttation fluid can also be released through the cuticle or stomata, as has been reported earlier by Lausberg (1935) between 5.0 and 6.0. and Baid (1952) respectively. In the process of phyllosecretion, the proteins destined for export must first be labelled correctly so thet the As mentioned earlier, some proteins are naturally secreted into the plant guttation fluid. Proteins like catalase trans-Golgi network can dispatch the proteins to their correct destination. The labelling may be either in the form and peroxidase enzyme have been reported in the guttation fluid of Zea mays (maize), Avena sativa (oat), several of a signal sequences at the end of the polypeptide chain or signal patches, which are dispersed throughout the peroxidases in Fragaria ananassa (strawberry), Lycopersicon esculentum (tomato) and Cucumis sativus (cucumber) polypeptide chain, but are brought together when in a 3D conformation. All cells must export proteins to the and reductase in Phleum pzatense (timothy). Very recently guttation has been successfully used as a vehicle exterior of the plasma membrane, but specialized secretory cells must also control what domain of the plasma for recombinant protein production in plants by a process termed “phyllosecretion” by a group of scientists of membrane the proteins are released on. Thus, all cells have the constitutive secretory pathway, or default pathway, Biotech Center, Cook College, Rutgers University, New Jersey and Phytomedics, Dayton, New Jersey. They engineered but specialized secretory cells also have a regulated secretory pathway. In the secretory pathway, proteins are the tobacco plant (Nicotiana tabacum L. cv Wisconsin) to secrete human placental secreted alkaline phosphatase packaged into secretory vesicles, which then aggregate next to the plasma membrane where they await an extracellular (SEAP), green fluorescent protein (GFP) from jellyfish (Aequorea victoria) and xylanase from Clostridium signal to fuse with the plasma membrane and release their contents. thermocellum through the plant cell default secretion pathway. They found that recombinant proteins directed to the leaf intercellular space i.e. apoplast are effectively released into the plant guttation fluid, which can be collected Thus by using endoplasmic reticulum signal peptides fused to the recombinant protein sequences, these scientists continuously throughout the plant’s lifetime. have generated transgenic tobacco (Nicotiana tabacum L. cv Wisconsin) plants that secrete three heterologousproteins of different genetic backgrounds (bacterial xylanase, green fluorescent protein of jellyfish [Aequorea Plants serve as suitable bioreactors for the production of many valuable recombinant proteins used as pharmaceuticals, victoria], and human placental alkaline phosphatase) through the leaf intercellular space into tobacco guttation industrial enzymes, etc. Foreign genes are now being routinely expressed in many plant species to fluid. produce recombinant proteins because of their ability to carry out numerous post-translational protein modifications required for biological activity, and they can be easily cultivated. Till date numerous heterologous proteins Thus, guttation fluid can have numerous applications in future scientific investigations, apart from its role in have been expressed in different plant organs and plant cell compartments. However, the major obstacle for the helping plants to get rid of the excess water, which will no longer go as a waste. Another future prospect would be large-scale protein production in plants is the high cost of protein extraction and purification from biochemically to assess the ability of the simultaneous use of both the phyllosecretion and rhizosecretion systems. If successful, complex plant tissues. This obstacle has been partially overcome by aseptically cultivated cell cultures or plant the combination of both these techniques can significantly increase the total yield of heterologous proteinsproduction organs that secrete recombinant proteins into the surrounding medium. These in vitro systems however can be by plants in the easily accessible form of a water solution. expensive, slow growing, unstable, and relatively low yielding. In this respect the production of recombinant proteins in guttation fluid has several advantages and is comparatively a much easier process and cost effective. Production of Recombinant Proteins Recombinant proteins are proteins whose amino acid sequence is encoded by a cloned gene. This process involves The advantage of using phyllosecretion is that, this method allows continuous and non-destructive recovery of the cloning of the gene encoding the desired protein into an expression vector, which should contain a promoter so recombinant proteins from a living plant, as guttation fluid can be collected throughout a plant’s life. Guttation that the protein can be expressed. Thus through this recombinant DNA technology, a large amount of the protein fluid also has the potential of increasing yield, abolishing the need for tissue extraction, and simplifying its downstream can be produced. processing i.e. simplifies complex protein purification procedures, thus increasing the efficiency of recombinant protein production technology. It is nondestructive. Also, recombinant biopharmaceutical proteins purified from leaf exudates are less likely to be contaminated with pathogenic viruses, which may be present in the milk or urine of transgenic animals. The plant protein phyllosecretion system is easy to scale up and is less sensitive to the cytotoxicity of the final products. The guttation fluid can be easily collected, by shaking them off the leaves into a collection vessel, or a method for large-scale collection of guttation fluid can be developed like removing them from the leaf surface with a vacuum or blotting. Another system called Rhizosecretion system for the production of recombinant proteins has been developed recently, which takes advantage of the ability of roots of hydroponically cultivated plants to secrete properly targeted recombinant proteins into the surrounding medium. Now what exactly is Phyllosecretion? Phyllosecretion is a method, which exploits leaf guttation as a An example of the process of production of recombinant proteins (a) The expression vector contains lac promoter and the lacZ gene, which encodes b-galactosidase in presence medium to continuously “wash away” recombinant proteins from a living plant. Guttation fluid is transformed of lactose, which stimulates expression of b-galactosidase. into a concentrated solution of recombinant proteins. Eukaryotic cells have a highly evolved process of secretion. (b) lacZ is replaced by another gene, which encodes the protein of interest, Lactose stimulate the expression of desired proteins. Scientific Article Scientific Article ARANYA; Vol I (I); January - June 2011 ARANYA; Vol I (I); January - June 2011

Plants reported to show guttation indicating the group and family to which they belong Sl. No. Plant Name Group Family Sl. No. Plant Name Group Family 46. Equisitum palustre Pteridophyte Equisitaceae 1. Achyranthes aspera Dicotyledon Amaranthaceae 47. Eupatorium odoratum Dicotyledon Asteraceae 2. Ageratum conyzoides Dicotyledon Asteraceae 48. Ficus cunia Dicotyledon Moraceae 3. Ageratum conyzoides Dicotyledon Asteraceae 49. Ficus elastica Dicotyledon Moraceae 4. Alchemilla vulgaris (Lady's Mantle) Dicotyledon Rosaceae 50. Ficus religiosa Dicotyledon Moraceae 5. Alocasia sp Monocotyledon Araceae 51. Fleurya interupta Dicotyledon Urticaceae 6. Amaranthus sp Dicotyledon Amaranthaceae 52. Fragaria ananassa (strawberry) Dicotyledon Rosaceae 7. Ambosia trifida Dicotyledon Asteraceae 53. Fragaria vesca Dicotyledon Rosaceae 8. Amorphophallus sp Monocotyledon Araceae 54. Fuchsia hybrida Dicotyledon Onagraceae 9. Aneilema spiratum Monocotyledon Commelinaceae 55. Globba bulbifera Monocotyledon Zingiberaceae 10. Anthurium sp Monocotyledon Araceae 56. Gloriosa superba Monocotyledon Liliaceae 11. Arisaema speciosum Monocotyledon Araceae 57. Glycosmis pentaphylla Dicotyledon Rutaceae 12. Artemisia caruifolia Dicotyledon Asteraceae 58. Gynandropsis pentaphylla Dicotyledon Capparidaceae 13. Avena sativa (oats) Monocotyledon Poaceae 59. Hodgsonia heteroplita Dicotyledon Cucurbitaceae 14. Basella rubra Dicotyledon Basellaceae 60. Hordeum vulgare (barley), Monocotyledon Poaceae 15. Benincasa hispida Dicotyledon Cucurbitaceae 61. Hydrocotyle acetica Dicotyledon Apiaceae 16. Blumea lacera Dicotyledon Asteraceae 62. Hydrocotyle himalayaca Dicotyledon Apiaceae 17. Boerhaavia repens Dicotyledon Nyctaginaceae 63. Jussia repens Dicotyledon Onagraceae 18. Brassica oleracea Dicotyledon Brassicaceae 64. Kalanchoe spathulata Dicotyledon Crassulaceae 19. Brassica sp Dicotyledon Brassicaceae 65. Lactuca sp. Dicotyledon Asteraceae 20. Bryophylum calycinum Dicotyledon Crassulaceae 66. Lagenaria vulgaris Dicotyledon Cucurbitaceae 21. Caladium sp Monocotyledon Araceae 67. Lasia heterophylla Monocotyledon Araceae 22. Canna indica Monocotyledon Cannaceae 68. Lindenbergia sp Dicotyledon Scrophulariaceae 23. Cephalandra indica Dicotyledon Cucurbitaceae 69. Ludwigia parviflora Dicotyledon Onagraceae 24. Chenopodium album Dicotyledon Chenopodiaceae 70. Luffa aegyptiaca Dicotyledon Cucurbitaceae 25. Citrullus vulgaris Dicotyledon Cucurbitaceae 71. Lycopersicon esculentum (tomata), Dicotyledon Solanaceae 26. Cleome viscosa Dicotyledon Capparidaceae 72. Melilotus alba Dicotyledon Fabaceae 27. Clerodendron inerme Dicotyledon Verbenaceae 73. Melothria heterophylla Dicotyledon Cucurbitaceae 28. Clerodendron infortunatum Dicotyledon Verbenaceae 74. Mikenia scandens Dicotyledon Asteraceae 29. Colocasia antiquorum Monocotyledon Aracaceae 75. Monstera sp Monocotyledon Araceae 30. Commelina appendiculata Monocotyledon Commelinaceae 76. Mukia scabrella Dicotyledon Cucurbitaceae 31. Commelina benghalensis, Monocotyledon Commelinaceae 77. Musa paradisiaca Monocotyledon Musaceae 32. Commelina obliqua Monocotyledon Commelinaceae 78. Oxalis corniculata Dicotyledon Oxalidaceae 33. Corchorus acutangulus Dicotyledon Tiliaceae 79. Peperomia relexa Dicotyledon Piperaceae 34. Costus speciosus Monocotyledon Costaceae 80. Phleum pzatense (timothy) Monocot Poaceae 35. Crinum asiaticum Monocotyledon Amaryllidaceae 81. Physocarpus sp. Dicotyledon Rosaceae 36. Crotalaria juncea Dicotyledon Fabaceae 82. Pilea pumila Dicotyledon Urticaceae 37. Cucumis melo Dicotyledon Cucurbitaceae 83. Pilularia globulifera (the semi aquatic Pteridophytes Marsileaceae 38. Cucumis sativus (cucumber) Dicotyledon Cucurbotaceae fern 'pillwort') 39. Cucurbita maxima Dicotyledon Cucurbitaceae 84. Pistia stratiotes Monocotyledon Araceae 40. Curculigo orchioides Monocotyledon Amaryllidaceae 85. Pontederia sagittata Monocotyledon Pontederiaceae 41. Curcuma longa Monocotyledon Zingiberaceae 86. Potentilla palustris, (Shoreline Plants) Dicotyledon Rosaceae 42. Cyanotis axillaries Monocotyledon Commelinaceae 87. Pothos scandens Monocotyledon Araceae 43. Dracaena terniflora Monocotyledon Liliaceae 88. Ravenala madagascariensis Monocotyledon Musaceae 44. Eclipta alba Dicotyledon Asteraceae 89. Rheo discolour Monocotyledon Commelinaceae 45. Eichhornia crassipes Monocotyledon Pontederiaceae 90. Richardia ethiopica Monocotyledon Araceae Continued Continued Scientific Article Scientific Article ARANYA; Vol I (I); January - June 2011 ARANYA; Vol I (I); January - June 2011

Plants reported to show guttation indicating the group and family to which they belong Sl. No. Plant Name Group Family Sl. No. Plant Name Group Family 46. Equisitum palustre Pteridophyte Equisitaceae 1. Achyranthes aspera Dicotyledon Amaranthaceae 47. Eupatorium odoratum Dicotyledon Asteraceae 2. Ageratum conyzoides Dicotyledon Asteraceae 48. Ficus cunia Dicotyledon Moraceae 3. Ageratum conyzoides Dicotyledon Asteraceae 49. Ficus elastica Dicotyledon Moraceae 4. Alchemilla vulgaris (Lady's Mantle) Dicotyledon Rosaceae 50. Ficus religiosa Dicotyledon Moraceae 5. Alocasia sp Monocotyledon Araceae 51. Fleurya interupta Dicotyledon Urticaceae 6. Amaranthus sp Dicotyledon Amaranthaceae 52. Fragaria ananassa (strawberry) Dicotyledon Rosaceae 7. Ambosia trifida Dicotyledon Asteraceae 53. Fragaria vesca Dicotyledon Rosaceae 8. Amorphophallus sp Monocotyledon Araceae 54. Fuchsia hybrida Dicotyledon Onagraceae 9. Aneilema spiratum Monocotyledon Commelinaceae 55. Globba bulbifera Monocotyledon Zingiberaceae 10. Anthurium sp Monocotyledon Araceae 56. Gloriosa superba Monocotyledon Liliaceae 11. Arisaema speciosum Monocotyledon Araceae 57. Glycosmis pentaphylla Dicotyledon Rutaceae 12. Artemisia caruifolia Dicotyledon Asteraceae 58. Gynandropsis pentaphylla Dicotyledon Capparidaceae 13. Avena sativa (oats) Monocotyledon Poaceae 59. Hodgsonia heteroplita Dicotyledon Cucurbitaceae 14. Basella rubra Dicotyledon Basellaceae 60. Hordeum vulgare (barley), Monocotyledon Poaceae 15. Benincasa hispida Dicotyledon Cucurbitaceae 61. Hydrocotyle acetica Dicotyledon Apiaceae 16. Blumea lacera Dicotyledon Asteraceae 62. Hydrocotyle himalayaca Dicotyledon Apiaceae 17. Boerhaavia repens Dicotyledon Nyctaginaceae 63. Jussia repens Dicotyledon Onagraceae 18. Brassica oleracea Dicotyledon Brassicaceae 64. Kalanchoe spathulata Dicotyledon Crassulaceae 19. Brassica sp Dicotyledon Brassicaceae 65. Lactuca sp. Dicotyledon Asteraceae 20. Bryophylum calycinum Dicotyledon Crassulaceae 66. Lagenaria vulgaris Dicotyledon Cucurbitaceae 21. Caladium sp Monocotyledon Araceae 67. Lasia heterophylla Monocotyledon Araceae 22. Canna indica Monocotyledon Cannaceae 68. Lindenbergia sp Dicotyledon Scrophulariaceae 23. Cephalandra indica Dicotyledon Cucurbitaceae 69. Ludwigia parviflora Dicotyledon Onagraceae 24. Chenopodium album Dicotyledon Chenopodiaceae 70. Luffa aegyptiaca Dicotyledon Cucurbitaceae 25. Citrullus vulgaris Dicotyledon Cucurbitaceae 71. Lycopersicon esculentum (tomata), Dicotyledon Solanaceae 26. Cleome viscosa Dicotyledon Capparidaceae 72. Melilotus alba Dicotyledon Fabaceae 27. Clerodendron inerme Dicotyledon Verbenaceae 73. Melothria heterophylla Dicotyledon Cucurbitaceae 28. Clerodendron infortunatum Dicotyledon Verbenaceae 74. Mikenia scandens Dicotyledon Asteraceae 29. Colocasia antiquorum Monocotyledon Aracaceae 75. Monstera sp Monocotyledon Araceae 30. Commelina appendiculata Monocotyledon Commelinaceae 76. Mukia scabrella Dicotyledon Cucurbitaceae 31. Commelina benghalensis, Monocotyledon Commelinaceae 77. Musa paradisiaca Monocotyledon Musaceae 32. Commelina obliqua Monocotyledon Commelinaceae 78. Oxalis corniculata Dicotyledon Oxalidaceae 33. Corchorus acutangulus Dicotyledon Tiliaceae 79. Peperomia relexa Dicotyledon Piperaceae 34. Costus speciosus Monocotyledon Costaceae 80. Phleum pzatense (timothy) Monocot Poaceae 35. Crinum asiaticum Monocotyledon Amaryllidaceae 81. Physocarpus sp. Dicotyledon Rosaceae 36. Crotalaria juncea Dicotyledon Fabaceae 82. Pilea pumila Dicotyledon Urticaceae 37. Cucumis melo Dicotyledon Cucurbitaceae 83. Pilularia globulifera (the semi aquatic Pteridophytes Marsileaceae 38. Cucumis sativus (cucumber) Dicotyledon Cucurbotaceae fern 'pillwort') 39. Cucurbita maxima Dicotyledon Cucurbitaceae 84. Pistia stratiotes Monocotyledon Araceae 40. Curculigo orchioides Monocotyledon Amaryllidaceae 85. Pontederia sagittata Monocotyledon Pontederiaceae 41. Curcuma longa Monocotyledon Zingiberaceae 86. Potentilla palustris, (Shoreline Plants) Dicotyledon Rosaceae 42. Cyanotis axillaries Monocotyledon Commelinaceae 87. Pothos scandens Monocotyledon Araceae 43. Dracaena terniflora Monocotyledon Liliaceae 88. Ravenala madagascariensis Monocotyledon Musaceae 44. Eclipta alba Dicotyledon Asteraceae 89. Rheo discolour Monocotyledon Commelinaceae 45. Eichhornia crassipes Monocotyledon Pontederiaceae 90. Richardia ethiopica Monocotyledon Araceae Continued Continued Scientific Article Scientific Article ARANYA; Vol I (I); January - June 2011 ARANYA; Vol I (I); January - June 2011

Sl. No. Plant Name Group Family 91. Saxifraga sp. Dicotyledon Saxifragaceae 92. Sonchus oleraceus Dicotyledon Asteraceae 93. Spilanthes acmella Dicotyledon Asteraceae 94. Strelitzia reginae Monocotyledon Musaceae 95. Tradescantia virginiana (Spiderworts) Monocotyledon Commelinaceae 96. Trichosanthes dioica Dicotyledon Cucurbitaceae 97. Tridax procumbens Dicotyledon Asteraceae 98. Trifolium repens Dicotyledon Fabaceae 99. Trigonella corniculata Dicotyledon Fabaceae 100. Tropaeolum majus (Garden Nasturtium) Dicotyledon Tropaeolaceae 101. Tropaeolum sp. Dicotyledon Tropaeolaceae 102. Typhonium trilobatum Monocotyledon Araceae 103. Urtica dioica Dicotyledon Urticaceae 104. Vitis trifolia Dicotyledon Vitaceae 105. Vitis pedata Dicotyledon Vitaceae 106. Vitis quandrangularis Dicotyledon Vitaceae 107. Vitis vinifera Dicotyledon Vitaceae 108. Zea mays (maize) Monocotyledon Poaceae 109. Zingiber officinale Monocotyledon Zingiberaceae 110. Zizyphus jujuba Dicotyledon Rhamnaceae

Guttation along the leaf margin observed in the early morning hours in Vitex trifolia (Vitaceae)

The structure of a leaf showing the position of the hydathodes (A) Entire leaf (B) Anatomical structure of a leaf

Guttation along the leaf margin observed in the early morning hours in Hibiscus safdarifa (Malvaceae) Scientific Article Scientific Article ARANYA; Vol I (I); January - June 2011 ARANYA; Vol I (I); January - June 2011

Sl. No. Plant Name Group Family 91. Saxifraga sp. Dicotyledon Saxifragaceae 92. Sonchus oleraceus Dicotyledon Asteraceae 93. Spilanthes acmella Dicotyledon Asteraceae 94. Strelitzia reginae Monocotyledon Musaceae 95. Tradescantia virginiana (Spiderworts) Monocotyledon Commelinaceae 96. Trichosanthes dioica Dicotyledon Cucurbitaceae 97. Tridax procumbens Dicotyledon Asteraceae 98. Trifolium repens Dicotyledon Fabaceae 99. Trigonella corniculata Dicotyledon Fabaceae 100. Tropaeolum majus (Garden Nasturtium) Dicotyledon Tropaeolaceae 101. Tropaeolum sp. Dicotyledon Tropaeolaceae 102. Typhonium trilobatum Monocotyledon Araceae 103. Urtica dioica Dicotyledon Urticaceae 104. Vitis trifolia Dicotyledon Vitaceae 105. Vitis pedata Dicotyledon Vitaceae 106. Vitis quandrangularis Dicotyledon Vitaceae 107. Vitis vinifera Dicotyledon Vitaceae 108. Zea mays (maize) Monocotyledon Poaceae 109. Zingiber officinale Monocotyledon Zingiberaceae 110. Zizyphus jujuba Dicotyledon Rhamnaceae

Guttation along the leaf margin observed in the early morning hours in Vitex trifolia (Vitaceae)

The structure of a leaf showing the position of the hydathodes (A) Entire leaf (B) Anatomical structure of a leaf

Guttation along the leaf margin observed in the early morning hours in Hibiscus safdarifa (Malvaceae) Scientific Article Scientific Article ARANYA; Vol I (I); January - June 2011 ARANYA; Vol I (I); January - June 2011

Guttation along the leaf margin observed in the early morning hours in Vitex pedata (Vitaceae) Guttation along the leaf margin observed in the early morning hours in Globba bulbifera (Zingiberaceae)

Guttation along the leaf margin observed in the early morning hours in Mikenia scandens (Asteraceae) Guttation in Typhenium trilobatum (Araceae) showing fewer numbers of hydathodes not on margins but on the leaf surface. Scientific Article Scientific Article ARANYA; Vol I (I); January - June 2011 ARANYA; Vol I (I); January - June 2011

Guttation along the leaf margin observed in the early morning hours in Vitex pedata (Vitaceae) Guttation along the leaf margin observed in the early morning hours in Globba bulbifera (Zingiberaceae)

Guttation along the leaf margin observed in the early morning hours in Mikenia scandens (Asteraceae) Guttation in Typhenium trilobatum (Araceae) showing fewer numbers of hydathodes not on margins but on the leaf surface. Scientific Article Scientific Article ARANYA; Vol I (I); January - June 2011 ARANYA; Vol I (I); January - June 2011

Guttation is not just restricted to leaf margins but is also seen on the entire leaf surface, as seen in Ficus hispida (Moraceae) Guttation in a free floating aquatic angiosperm [Pistia stratiotes (Araceae)] – a rare view.

Guttation is not just restricted to leaf margins but is also seen on the entire leaf surface, as seen in Fleurya interupta (Urticaceae) Scientific Article Scientific Article ARANYA; Vol I (I); January - June 2011 ARANYA; Vol I (I); January - June 2011

Guttation is not just restricted to leaf margins but is also seen on the entire leaf surface, as seen in Ficus hispida (Moraceae) Guttation in a free floating aquatic angiosperm [Pistia stratiotes (Araceae)] – a rare view.

Guttation is not just restricted to leaf margins but is also seen on the entire leaf surface, as seen in Fleurya interupta (Urticaceae)