Production of recombinant proteins in tobacco guttation fluid

Guttation, the loss of water and dissolved materials from uninjured plant organs, is a common phenomenon in higher plants. By using endoplasmic reticulum signal peptides fused to the recombinant protein sequences, we have generated transgenic tobacco (Nicotiana tabacum L. cv Wisconsin) plants that secrete three heterologous proteins of different genetic backgrounds (bacterial xylanase, green fluorescent protein of jellyfish [Aequorea victoria], and human placental alkaline phosphatase) through the leaf intercellular space into tobacco guttation fluid. Production rates of 1.1 microg/g of leaf dry weight per day were achieved for alkaline phosphatase with this protein comprising almost 3% of total soluble protein in the guttation fluid. Guttation fluid can be collected throughout a plant's life, thus providing a continuous and nondestructive system for recombinant protein production. Guttation fluid has the potential of increasing the efficiency of recombinant protein production technology by increasing yield, abolishing extraction, and simplifying its downstream processing.     

Salt stress aggravates boron toxicity symptoms in banana leaves by impairing guttation

Boron (B) is known to accumulate in the leaf margins of different plant species, arguably a passive consequence of enhanced transpiration at the ends of the vascular system. However, transpiration rate is not the only factor affecting ion distribution. We examine an alternative hypothesis, suggesting the participation of the leaf bundle sheath in controlling radial water and solute transport from the xylem to the mesophyll in analogy to the root endodermis. In banana, excess B that remains confined to the vascular system is effectively disposed of via dissolution in the guttation fluid; therefore, impairing guttation should aggravate B damage to the leaf margins. Banana plants were subjected to increasing B concentrations. Guttation rates were manipulated by imposing a moderate osmotic stress. Guttation fluid was collected and analysed continuously. The distribution of ions across the lamina was determined. Impairing guttation indeed led to increased B damage to the leaf margins. The kinetics of ion concentration in guttation samples revealed major differences between ion species, corresponding to their distribution in the lamina dry matter. We provide evidence that the distribution pattern of B and other ions across banana leaves depends on active filtration of the transpiration stream and on guttation.     

A hydathode inoculation technique for the simulation of natural black rot infection of cabbage by Xanthomonas campestris pv. campestris

A technique was developed for the reliable inoculation of Xanthomonas campestris pv. campestris into the cabbage host, in a manner which simulates a natural process of penetration. By introducing the inoculum into guttation droplets on the leaf margin, the bacteria are taken into the plant via the hydathodes, thereby avoiding mechanical injury to the plant and the possibility of producing artefacts. This technique, while being highly efficient, possesses several advantages over commonly used methods of inoculation.     

Release of Guttation Fluid from Passive Hydathodes of Intact Barley Plants. I. Structural and Cytological Aspects

The structural details of the guttating tips of 7-day-old barleyleaves were studied as a basis for a subsequent report on thephysiology of guttation. The walls of the vessels at the tipsof leaves bear many pits and are rather thin, appearing neithercutinized nor lignified. This could facilitate a direct passageof solutes out of the xylary system through the leaf apoplastand out to the leaf surface via hydathode openings. The latterare formed by modified stomatal guard cells, and there are nospecially differentiated epithem, epithelium or gland hair likestructures that could serve an active elimination of guttation.Xylem parenchyma cells and the peculiar mesophyll cells withdense cytoplasm, numerous mitochondria, an extended ER systemand a considerable formation of small vesicles in the leaf tipcould modify the content of the guttated fluid along the routeof transport. Hordeum vulgare L., barley, hydathodes, guttation     

Phylloplane proteins: emerging defenses at the aerial frontline?

The phylloplane, or leaf surface, is an interkingdom crossroads between plants and microorganisms, and secretion of antimicrobial biochemicals to aerial surfaces is thought to be one defensive strategy by which plants deter potential pathogens. Secondary metabolites on leaf surfaces are well documented but antimicrobial phylloplane proteins have only recently been identified. In this review, we describe the physical structures and biochemicals of the phylloplane and briefly discuss protein-based surface defenses of animals. We also review the emerging evidence pertaining to antimicrobial phylloplane proteins and mechanisms by which proteins can be released to the phylloplane, including biosynthesis (e.g. phylloplanins) by specific trichomes and delivery in guttation fluid from hydathodes. Future research should lead to exciting advances in our understanding of the phylloplane and to useful biotechnological interventions.     

Guttation 1: chemistry, crop husbandry and molecular farming

Guttation is one of the most conspicuous visible phenomena in plants occurring in a wide range of plants. The guttation fluids, though look clear and translucent, carry a number of organic and inorganic constituents. The organic component may include sugars, amino acids, general proteins, antimicrobial phylloplane proteins, transport proteins for transporting sucrose, purine and cytokinins, toxic elements etc. and enzymes such as peroxidases, dehydrogenases, ATPases, in addition to mRNA, ATP, reductants and other important ingredients of plant life. Guttation fluids also contain a number of natural plant hormones such as auxins, gibberellins, cytokinins, abscisic acid etc., apart from several vitamins. Recent discoveries have revealed the presence of a number of salts, ions, nutrients and macromolecules in guttation fluid playing significant role in enhancing disease resistance, tolerance to toxic elements, photosynthetic efficiency, biomass production and economic yield of agricultural crops. In the light of aforementioned discoveries in guttation transgenic plants have been created to serve as bio-factories for producing various kinds of phytochemicals of immense agricultural, pharmaceutical, nutriceutical, therapeutic, cosmeceutic and commercial significance impacting food productivity and human health adding happiness to life.     

Hydathode trichomes actively secreting water from leaves play a key role in the physiology and evolution of root-parasitic rhinanthoid Orobanchaceae

Background and Aims Root hemiparasites from the rhinanthoid clade of Orobanchaceae possess metabolically active glandular trichomes that have been suggested to function as hydathode trichomes actively secreting water, a process that may facilitate resource acquisition from the host plant’s root xylem. However, no direct evidence relating the trichomes to water secretion exists, and carbon budgets associated with this energy-demanding process have not been determined.Methods Macro- and microscopic observations of the leaves of hemiparasitic Rhinanthus alectorolophus were conducted and night-time gas exchange was measured. Correlations were examined among the intensity of guttation, respiration and transpiration, and analysis of these correlations allowed the carbon budget of the trichome activity to be quantified. We examined the intensity of guttation, respiration and transpiration, correlations among which indicate active water secretion.Key Results Guttation was observed on the leaves of 50 % of the young, non-flowering plants that were examined, and microscopic observations revealed water secretion from the glandular trichomes present on the abaxial leaf side. Night-time rates of respiration and transpiration and the presence of guttation drops were positively correlated, which is a clear indicator of hydathode trichome activity. Subsequent physiological measurements on older, flowering plants indicated neither intense guttation nor the presence of correlations, which suggests that the peak activity of hydathodes is in the juvenile stage.Conclusions This study provides the first unequivocal evidence for the physiological role of the hydathode trichomes in active water secretion in the rhinanthoid Orobanchaceae. Depending on the concentration of organic elements calculated to be in the host xylem sap, the direct effect of water secretion on carbon balance ranges from close to neutral to positive. However, it is likely to be positive in the xylem-only feeding holoparasites of the genus Lathraea, which is closely related to Rhinanthus. Thus, water secretion by the hydathodes might be viewed as a physiological pre-adaptation in the evolution of holoparasitism in the rhinanthoid lineage of Orobanchaceae.     

Suppression of bacterial blight by a bacterial community isolated from the guttation fluids of anthuriums

Growth and survival of Xanthomonas campestris pv. dieffenbachiae in guttation fluids (xylem sap exuded from leaf margins) of anthuriums were suppressed by several bacterial strains indigenous to leaves of various anthurium cultivars. Inhibition of growth was not observed in filter-sterilized guttation fluids and was restored to original levels only by reintroducing specific mixtures of bacteria into filter-sterilized guttation fluids. The inhibitory effect was related to the species in the bacterial community rather than to the total numbers of bacteria in the guttation fluids. One very effective bacterial community consisted of five species isolated from inhibitory guttation fluids of two susceptible anthurium cultivars. The individual strains in this community had no effect on the pathogen, but the mixture was inhibitory to X. campestris pv. dieffenbachiae in guttation fluids. The populations of the individual strains remained near the initial inoculum levels for at least 14 days. The effect of the five inhibitory strains on reducing disease in susceptible anthurium plants was tested by using a bioluminescent strain of X. campestris pv. dieffenbachiae to monitor the progression of disease in leaves nondestructively. Invasion of the pathogen through hydathodes at leaf margins was reduced by applying the strain mixture to the leaves. When the strain mixture was applied directly to wounds created on the leaf margins, the pathogen failed to invade through the wounds. This bacterial community has potential for biological control of anthurium blight.     

Overexpression of GLUTAMINE DUMPER1 leads to hypersecretion of glutamine from Hydathodes of Arabidopsis leaves

Secretion is a fundamental process providing plants with the means for disposal of solutes, improvement of nutrient acquisition, and attraction of other organisms. Specific secretory organs, such as nectaries, hydathodes, and trichomes, use a combination of secretory and retrieval mechanisms, which are poorly understood at present. To study the mechanisms involved, an Arabidopsis thaliana activation tagged mutant, glutamine dumper1 (gdu1), was identified that accumulates salt crystals at the hydathodes. Chemical analysis demonstrated that, in contrast with the amino acid mixture normally present in guttation droplets, the crystals mainly contain Gln. GDU1 was cloned and found to encode a novel 17-kD protein containing a single putative transmembrane span. GDU1 is expressed in the vascular tissues and in hydathodes. Gln content is specifically increased in xylem sap and leaf apoplasm, whereas the content of several amino acids is increased in leaves and phloem sap. Selective secretion of Gln by the leaves may be explained by an enhanced release of this amino acid from cells. GDU1 study may help to shed light on the secretory mechanisms for amino acids in plants.     

Temporal generation of multiple antifungal proteins in primed seeds

A drastic increase of antifungal activity was demonstrated during plant seed germination and in seed protein extract in vitro. Multiple antifungal proteins with a wide spectrum of activity were generated and identified. Chromatographic and electrophoretic analysis demonstrated that during seed germination, more fractions with potent antifungal activity were generated, and the antifungal activity shifted from small molecules to high molecular proteins. This germination-related increase of antifungal activity were observed in all three plants tested, i.e., cheeseweed, cigar tree and wheat. This rapid increase of antifungal activity was also observed with incubation of seed proteins in vitro, suggesting that at least part of the antifungal protein generation is independent of gene expression. Seven antifungal proteins with activities against five different plant pathogens were isolated from the active fractions. However, random digestion of purified seed protein with multiple proteinases failed to generate any antifungal proteins. It is suggested that during plant seed germination, a regulated biochemical process takes place that results in the generation of multiple peptides or proteins with antifungal activities. This onset of antifungal proteins is transitional in nature, but could play an important role in the protection of plants in early stage of development when the more sophisticated defense system has yet to develop.     

Smart pipes: The bundle sheath role as xylem-mesophyll barrier

Signs of abiotic toxicity often appear first at the margins of leaves and gradually spread toward the midrib. It has been suggested that the bundle sheath tissue surrounding the shoot vascular system acts as a solute transport-regulating barrier that prevents excessive quantities of toxic ions from entering the leaf and pushes them toward the hydathodes. We examined this hypothesis by examining the distribution of toxic boron (B) in mutant Arabidopsis leaves with flooded mesophyll and comparing it with that observed in control leaves that exuded guttation drops. As opposed to the control plants, which showed classical symptoms of B toxicity (necrosis starting at the leaf margins), in the mutants, necrosis was first observed inside the leaf. We will discuss this result and how it supports the hypothesis that the bundle sheath serves as a selective barrier filtering the xylem-to-leaf radial transport flow and pushing toxic solutes toward the hydathodes.     

F-box proteins everywhere

The ubiquitin proteasome system is a key regulator of many biological processes in all eukaryotes. This mechanism employs several types of enzymes, the most important of which are the ubiquitin E3 ligases that catalyse the attachment of polyubiquitin chains to target proteins for their subsequent degradation by the 26S proteasome. Among the E3 families, the SCF is the best understood; it consists of a multi-protein complex in which the F-box protein plays a crucial role by recruiting the target substrate. Strikingly, nearly 700 F-box proteins have been predicted in Arabidopsis, suggesting that plants have the capacity to assemble a multitude of SCF complexes, possibly controlling the stability of hundreds of substrates involved in a plethora of biological processes. Interestingly, viruses and even pathogenic bacteria have also found ways to hijack the plant SCF and to reprogram it for their own purposes.     

Heat‐shock proteins and cross‐tolerance in plants

Environmental stresses dramatically affect plant survival and productivity. Because plants are immobile, presumably different strategies are required for protection against transient stresses. Under stress, plants synthesize specific proteins, and their accumulation has a role in protecting the tissue from possible damage. An increasing number of studies show the existence of cross‐tolerance in plants: Exposure of tissue to moderate stress conditions often induces resistance to other stresses. Many varied mechanisms explaining the phenomenon of cross‐tolerance have been proposed, and they often, but not always, suggest that specific proteins are induced by one kind of stress and are involved in the protection against other kinds. Although various cross‐protections have been demonstrated in a number of plants, a common mechanism has not been found. This review discusses heat‐shock proteins and their possible roles in protecting the plant under heat and other stresses.     

Transport of cytokinins mediated by purine transporters of the PUP family expressed in phloem,hydathodes, and pollen of Arabidopsis

Nucleobases and derivatives like cytokinins and caffeine are translocated in the plant vascular system. Transport studies in cultured Arabidopsis cells indicate that adenine and cytokinin are transported by a common H+-coupled high-affinity purine transport system. Transport properties are similar to that of Arabidopsis purine transporters AtPUP1 and 2. When expressed in yeast, AtPUP1 and 2 mediate energy-dependent high-affinity adenine uptake, whereas AtPUP3 activity was not detectable. Similar to the results from cell cultures, purine permeases (PUP) mediated uptake of adenine can be inhibited by cytokinins, indicating that cytokinins are transport substrates. Direct measurements demonstrate that AtPUP1 is capable of mediating uptake of radiolabeled trans-zeatin. Cytokinin uptake is strongly inhibited by adenine and isopentenyladenine but is poorly inhibited by 6-chloropurine. A number of physiological cytokinins including trans- and cis-zeatin are also efficient competitors for AtPUP2-mediated adenine uptake, suggesting that AtPUP2 is also able to mediate cytokinin transport. Furthermore, AtPUP1 mediates transport of caffeine and ribosylated purine derivatives in yeast. Promoter-reporter gene studies point towards AtPUP1 expression in the epithem of hydathodes and the stigma surface of siliques, suggesting a role in retrieval of cytokinins from xylem sap to prevent loss during guttation. The AtPUP2 promoter drives GUS reporter gene activity in the phloem of Arabidopsis leaves, indicating a role in long-distance transport of adenine and cytokinins. Promoter activity of AtPUP3 was only found in pollen. In summary, three closely related PUPs are differentially expressed in Arabidopsis and at least two PUPs have properties similar to the adenine and cytokinin transport system identified in Arabidopsis cell cultures.     

Group 1 LEA proteins, an ancestral plant protein group, are also present in other eukaryotes, and in the archeae and bacteria domains

Water is an essential element for living organisms, such that various responses have evolved to withstand water deficit in all living species. The study of these responses in plants has had particular relevance given the negative impact of water scarcity on agriculture. Among the molecules highly associated with plant responses to water limitation are the so-called late embryogenesis abundant (LEA) proteins. These proteins are ubiquitous in the plant kingdom and accumulate during the late phase of embryogenesis and in vegetative tissues in response to water deficit. To know about the evolution of these proteins, we have studied the distribution of group 1 LEA proteins, a set that has also been found beyond the plant kingdom, in Bacillus subtilis and Artemia franciscana. Here, we report the presence of group 1 LEA proteins in green algae (Chlorophyita and Streptophyta), suggesting that these group of proteins emerged before plant land colonization. By sequence analysis of public genomic databases, we also show that 34 prokaryote genomes encode group 1 LEA-like proteins; two of them belong to Archaea domain and 32 to bacterial phyla. Most of these microbes live in soil-associated habitats suggesting horizontal transfer from plants to bacteria; however, our phylogenetic analysis points to convergent evolution. Furthermore, we present data showing that bacterial group 1 LEA proteins are able to prevent enzyme inactivation upon freeze–thaw treatments in vitro, suggesting that they have analogous functions to plant LEA proteins. Overall, data in this work indicate that LEA1 proteins’ properties might be relevant to cope with water deficit in different organisms.     

Long-Distance Water Transport in Aquatic Plants

Acropetal mass flow of water is demonstrated in two submerged angiosperms, Lobelia dortmanna L. and Sparganium emersum Rehman by means of guttation measurements. Transpiration is absent in truly submerged plants, but the presence of guttation verifies that long-distance water transport takes place. Use of tritiated water showed that the water current arises from the roots, and the main flow of water is channeled to the youngest leaves. This was confirmed by measurement of guttation, which showed the highest rates in young leaves. Guttation rates were 10-fold larger in the youngest leaf of S. emersum (2.1 [mu]L leaf-1 h-1) compared with the youngest leaf of L. dortmanna (0.2 [mu]L leaf-1 h-1). This is probably due to profound species differences in the hydraulic conductance (2.7 x 10-17 m4 Pa-1 s-1 for S. emersum and 1.4 x 10-19 m4 Pa-1 s-1 for L. dortmanna). Estimates derived from the modified Hagen-Poiseuille equation showed that the maximum flow velocity in xylem vessels was 23 to 84 cm h-1, and the required root pressure to drive the flow was small compared to that commonly found in terrestrial plants. In S. emersum long-distance transport of water was shown to be dependent on energy conversion in the roots. The leaves ceased to guttate when the roots were cooled to 4[deg]C from the acclimatization level at 15[deg]C, whereas the guttation was stimulated when the temperature was increased to 25[deg]C. Also, the guttation rate decreased significantly when vanadate was added to the root medium. The observed water transport is probably a general phenomenon in submerged plants, where it can act as a translocation system for nutrients taken up from the rich root medium and thereby assure maximum growth.     

Ingress of Salmonella enterica Typhimurium into Tomato Leaves through Hydathodes

Internal contamination of Salmonella in plants is attracting increasing attention for food safety reasons. In this study, three different tomato cultivars “Florida Lanai”, “Crown Jewel”, “Ailsa Craig” and the transgenic line Sp5 of “Ailsa Craig” were inoculated with 1 µl GFP-labeled Salmonella Typhimurium through guttation droplets at concentrations of 10⁹ or 10⁷ CFU/ml. Survival of Salmonella on/in tomato leaves was detected by both direct plating and enrichment methods. Salmonella cells survived best on/in the inoculated leaves of cultivar “Ailsa Craig” and decreased fastest on/in “Florida Lanai” leaves. Increased guttation in the abscisic acid over-expressing Sp5 plants may have facilitated the entrance of Salmonella into leaves and the colonization on the surface of tomato leaves. Internalization of Salmonella Typhimurium in tomato leaves through guttation drop inoculation was confirmed by confocal laser microscopy. For the first time, convincing evidence is presented that S. enterica can enter tomato leaves through hydathodes and move into the vascular system, which may result in the internal translocation of the bacteria inside plants.     

Removal of salt from xylem sap by leaves and stems of guttating plants

Although root pressure and guttation presumably result from a high concentration of salt in the root xylem, the guttation fluid is very dilute. Measurements of the osmotic potential of the guttation liquid and of exudates at various levels in guttating plants indicate that salt is removed from the xylem in the upper part of plants, particularly in the leaves. The concentration of salt solutions forced through individual leaves by an artificial root pressure has no influence on the osmotic potential of the guttation fluid. This suggests that leaves play an important role in removing salt from the xylem of guttating plants.     

Enhanced quantitative resistance against fungal disease by combinatorial expression of different barley antifungal proteins in transgenic tobacco

cDNAs encoding three proteins from barley ( Hordeum vulgare ), a class-II chitinase (CHI), a class-II β-1,3-glucanase (GLU) and a Type-I ribosome-inactivating protein (RIP) were expressed in tobacco plants under the control of the CaMV 35S-promoter. High-level expression of the transferred genes was detected in the transgenic plants by Northern and Western blot analysis. The leader peptides in CHI and GLU led to accumulation of these proteins in the intercellular space of tobacco leaves. RIP, which is naturally deposited in the cytosol of barley endosperm cells, was expressed either in its original cytosolic form or fused to a plant secretion peptide (spRIP). Fungal infection assays revealed that expression of the individual genes in each case resulted in an increased protection against the soilborne fungal pathogen Rhizoctonia solani , which infects a range of plant species including tobacco. To create a situation similar to 'multi-gene' tolerance, which traditional breeding experience has shown to provide crops with a longer-lasting protection, several of these antifungal genes were combined and protection against fungal attack resulting from their co-expression in planta was evaluated. Transgenic tobacco lines were generated with tandemly arranged genes coding for RIP and CHI as well as GLU and CHI. The performance of tobacco plants co-expressing the barley transgenes GLU/ CHI or CHI/RIP in a Rhizoctonia solani infection assay revealed significantly enhanced protection against fungal attack when compared with the protection levels obtained with corresponding isogenic lines expressing a single barley transgene to a similar level. The data indicate synergistic protective interaction of the co-expressed anti-fungal proteins in vivo .