Ryoung Shin

Overexpression of the Tobacco Tsi1 Gene Encoding an EREBP/AP2-Type Transcription Factor Enhances Resistance against Pathogen Attack and Osmotic Stress in Tobacco

Using mRNA differential display analysis, we isolated a salt-induced transcript that showed a significant sequence homology with an EREBP/AP2 DNA binding motif from oilseed rape plants. With this cDNA fragment as a probe, cDNA clone Tsi1 (for Tobacco stress-induced gene1) was isolated from a tobacco cDNA library. RNA gel blot analysis indicated that transcripts homologous with Tsi1 were induced not only in NaCl-treated leaves but also in leaves treated with ethephon or salicylic acid. Transient expression analysis using a Tsi1::smGFP fusion gene in BY-2 cells indicated that the Tsi1 protein was targeted to the nucleus. Fusion protein of Tsi1 with GAL4 DNA binding domain strongly activated transcription in yeast, and the transactivating activity was localized to the 13 C-terminal amino acids of Tsi1. Electrophoretic mobility shift assays revealed that Tsi1 could bind specifically to the GCC and the DRE/CRT sequences, although the binding activity to the former was stronger than that to the latter. Furthermore, Agrobacterium-mediated transient expression and transgenic plants expressing Tsi1 demonstrated that overexpression of the Tsi1 gene induced expression of several pathogenesis-related genes under normal conditions, resulting in improved tolerance to salt and pathogens. These results suggest that Tsi1 might be involved as a positive trans-acting factor in two separate signal transduction pathways under abiotic and biotic stress.

Expression of KT/KUP Genes in Arabidopsis and the Role of Root Hairs in K+ Uptake

Potassium (K+) is the most abundant cation in plants and is required for plant growth. To ensure an adequate supply of K+, plants have multiple mechanisms for uptake and translocation. However, relatively little is known about the physiological role of proteins encoded by a family of 13 genes, named AtKT/KUP, that are involved in K+ transport and translocation. To begin to understand where and under what conditions these transporters function, we used reverse transcription-PCR to determine the spatial and temporal expression patterns of each AtKT/KUP gene across a range of organs and tested whether selected AtKT/KUP cDNAs function as K+ transporters in Escherichia coli. Many AtKT/KUPs were expressed in roots, leaves, siliques, and flowers of plants grown under K+-sufficient conditions (1.75 mm KCl) in hydroponic culture. AtHAK5 was the only gene in this family that was up-regulated upon K+ deprivation and rapidly down-regulated with resupply of K+. Ten AtKT/KUPs were expressed in root hairs, but only five were expressed in root tip cells. This suggests an important role for root hairs in K+ uptake. The growth and rubidium (Rb+) uptake of two root hair mutants, trh1-1 (tiny root hairs) and rhd6 (root hair defective), were studied to determine the contribution of root hairs to whole-plant K+ status. Whole-plant biomass decreased in the root hair mutants only when K+ concentrations were low; Rb+ (used as a tracer for K+) uptake rates were lower in the mutants at all Rb+ concentrations. Seven genes encoding AtKUP transporters were expressed in E. coli (AtKT3/KUP4, AtKT/KUP5, AtKT/KUP6, AtKT/KUP7, AtKT/KUP10, AtKT/KUP11, and AtHAK5), and their K+ transport function was demonstrated.

Ethylene Mediates Response and Tolerance to Potassium Deprivation in Arabidopsis

Potassium deprivation leads to large reductions in plant growth and yields. How plants sense and transduce the stress signals initiated by potassium deprivation is poorly understood. Both ethylene production and the transcription of genes involved in ethylene biosynthesis increase when plants are deprived of potassium. To elucidate the role of ethylene in low potassium signaling pathways, we used both genetic and chemical approaches. Our results showed that ethylene is important in tolerance to low potassium and for changes in both root hair and primary root growth in Arabidopsis thaliana. We show that ethylene acts upstream of reactive oxygen species in response to potassium deprivation. The expression of High-Affinity K+ Transporter5 was used as a marker of potassium deprivation and was found to be dependent on ethylene signaling. In the ethylene insensitive2-1 (ein2-1) mutant, the ethylene-mediated low potassium responses were not completely eliminated, suggesting that some potassium deprivation–induced responses are either ethylene independent or EIN2 independent. Ethylene signaling is a component of the plants response to low potassium that stimulates the production of reactive oxygen species and is important for changes in root morphology and whole plant tolerance to low potassium conditions.

Differential Metal Selectivity and Gene Expression of Two Zinc Transporters from Rice

Zinc is an essential mineral for a wide variety of physiological and biochemical processes. To understand zinc transport in cereals, we identified putative zinc transporters in gene databases. Three full-length cDNAs were identified and characterized from rice (Oryza sativa). Two of the cDNAs partially complemented a yeast (Saccharomyces cerevisiae) mutant deficient in zinc uptake at low concentrations. The two transporters showed many similarities in function but differed in ionic selectivity and pH optimum of activity. Expression patterns also differed between the two genes. One gene was broadly expressed under all conditions, and the other gene was mainly induced by zinc deficiency to higher levels in roots than in leaves. Although the timing of expression differed between the two genes, localization of expression overlapped in roots. Comparisons of the protein sequences, ionic selectivity, and gene expression patterns of the two transporters suggest that they may play different roles in the physiology of the whole plant.

The high affinity K+ transporter AtHAK5 plays a physiological role in planta at very low K+ concentrations and provides a caesium uptake pathway in Arabidopsis.

Caesium (Cs(+)) is a potentially toxic mineral element that is released into the environment and taken up by plants. Although Cs(+) is chemically similar to potassium (K(+)), and much is known about K(+) transport mechanisms, it is not clear through which K(+) transport mechanisms Cs(+) is taken up by plant roots. In this study, the role of AtHAK5 in high affinity K(+) and Cs(+) uptake was characterized. It is demonstrated that AtHAK5 is localized to the plasma membrane under conditions of K(+) deprivation, when it is expressed. Growth analysis showed that AtHAK5 plays a role during severe K(+) deprivation. Under K(+)-deficient conditions in the presence of Cs(+), Arabidopsis seedlings lacking AtHAK5 had increased inhibition of root growth and lower Cs(+) accumulation, and significantly higher leaf chlorophyll concentrations than wild type. These data indicate that, in addition to transporting K(+) in planta, AtHAK5 also transports Cs(+). Further experiments showed that AtHAK5 mediated Cs(+) uptake into yeast cells and that, although the K(+) deficiency-induced expression of AtHAK5 was inhibited by low concentrations of NH(4)(+) in planta, Cs(+) uptake by yeast was stimulated by low concentrations of NH(4)(+). Interestingly, the growth of the Arabidopsis atakt1-1 mutant was more sensitive to Cs(+) than the wild type. This may be explained, in part, by increased expression of AtHAK5 in the atakt1-1 mutant. It is concluded that AtHAK5 is a root plasma membrane uptake mechanism for K(+) and Cs(+) under conditions of low K(+) availability.

Plant extracellular ATP signalling by plasma membrane NADPH oxidase and Ca2+ channels

Extracellular ATP regulates higher plant growth and adaptation. The signalling events may be unique to higher plants, as they lack animal purinoceptor homologues. Although it is known that plant cytosolic free Ca2+ can be elevated by extracellular ATP, the mechanism is unknown. Here, we have studied roots of Arabidopsis thaliana to determine the events that lead to the transcriptional stress response evoked by extracellular ATP. Root cell protoplasts were used to demonstrate that signalling to elevate cytosolic free Ca2+ is determined by ATP perception at the plasma membrane, and not at the cell wall. Imaging revealed that extracellular ATP causes the production of reactive oxygen species in intact roots, with the plasma membrane NADPH oxidase AtRBOHC being the major contributor. This resulted in the stimulation of plasma membrane Ca2+-permeable channels (determined using patch-clamp electrophysiology), which contribute to the elevation of cytosolic free Ca2+. Disruption of this pathway in the AtrbohC mutant impaired the extracellular ATP-induced increase in reactive oxygen species (ROS), the activation of Ca2+ channels, and the transcription of the MAP kinase3 gene that is known to be involved in stress responses. This study shows that higher plants, although bereft of purinoceptor homologues, could have evolved a distinct mechanism to transduce the ATP signal at the plasma membrane.

Phosphoproteomic identification of targets of the Arabidopsis sucrose nonfermenting-like kinase SnRK2.8 reveals a connection to metabolic processes

SnRK2.8 is a member of the sucrose nonfermenting-related kinase family that is down-regulated when plants are deprived of nutrients and growth is reduced. When this kinase is over expressed in Arabidopsis, the plants grow larger. To understand how this kinase modulates growth, we identified some of the proteins that are phosphorylated by this kinase. A new phosphoproteomic method was used in which total protein from plants overexpressing the kinase was compared with total protein from plants in which the kinase was inactivated. Protein profiles were compared on two-dimensional gels following staining by a dye that recognizes phosphorylated amino acids. Candidate target proteins were confirmed with in vitro phosphorylation assays, using the kinase and target proteins that were purified from Escherichia coli. Seven target proteins were confirmed as being phosphorylated by SnRK2.8. Certain targets, such as 14-3-3 proteins, regulate as yet unidentified proteins, whereas other targets, such as glyoxalase I and ribose 5-phosphate isomerase, detoxify byproducts from glycolysis and catalyze one of the final steps in carbon fixation, respectively. Also, adenosine kinase and 60S ribosomal protein were confirmed as targets of SnRK2.8. Using mass spectrometry, we identified phosphorylated residues in the SnRK2.8, the 14-3-3κ, and the 14-3-3χ. These data show that the expression of SnRK2.8 is correlated with plant growth, which may in part be due to the phosphorylation of enzymes involved in metabolic processes.

Induction of pepper cDNA encoding a lipid transfer protein during the resistance response to tobacco mosaic virus

Pepper (Capsicum annuum) plants exhibit hypersensitive response (HR) against infection by many tobamoviruses. A clone encoding a putative nonspecific lipid transfer protein (CaLTP1) was isolated by differential screening of a cDNA library from resistant pepper leaves when inoculated with tobacco mosaic virus (TMV) pathotype P0. The predicted amino acid sequence of CaLTP1 is highly similar to that of the other plant LTPs. Southern blot analysis showed that a small gene family of LTP-related sequences was present in the pepper genome. Transcripts homologous to CaLTP1 accumulated abundantly in old leaves and flowers. CaLTP1 expression was induced in the incompatible interaction with TMV-P0 but was not induced in the compatible interaction with TMV-P1.2. In correlation with the temporal progression of HR in the inoculated leaves, CaLTP1 transcripts started to accumulate at 24 h after TMV-P0 inoculation, reaching a maximal level at 48 h. A strain of Xanthomonas campestris pv. vesicatoria (Xcv) that carries the bacterial avirulence gene, avrBs2, was infiltrated into leaves of a pepper cultivar containing the Bs2 resistance gene. A marked induction of CaLTP1 expression was observed in Xcv-infiltrated leaves. Effects of exogenously applied abiotic elicitors on CaLTP1 expression were also examined. Salicylic acid caused a rapid accumulation of CaLTP1 transcripts in pepper leaves and ethephon treatment also induced the expression of the CaLTP1 gene. Transient expression in the detached pepper leaves by biolistic gene bombardment indicated that CaLTP1 is localized mostly at the plant cell surface, possibly in the cell wall. These results suggest possible role(s) for LTPs in plant defense against pathogens including viruses.

Transport, signaling, and homeostasis of potassium and sodium in plants

Potassium (K⁺) is an essential macronutrient in plants and a lack of K⁺ significantly reduces the potential for plant growth and development. By contrast, sodium (Na⁺), while beneficial to some extent, at high concentrations it disturbs and inhibits various physiological processes and plant growth. Due to their chemical similarities, some functions of K⁺ can be undertaken by Na⁺ but K⁺ homeostasis is severely affected by salt stress, on the other hand. Recent advances have highlighted the fascinating regulatory mechanisms of K⁺ and Na⁺ transport and signaling in plants. This review summarizes three major topics: (i) the transport mechanisms of K⁺ and Na⁺ from the soil to the shoot and to the cellular compartments; (ii) the mechanisms through which plants sense and respond to K⁺ and Na⁺ availability; and (iii) the components involved in maintenance of K⁺/Na⁺ homeostasis in plants under salt stress.

Ectopic expression of Tsi1 in transgenic hot pepper plants enhances host resistance to viral, bacterial, and oomycete pathogens.

In many plants, including hot pepper plants, productivity is greatly affected by pathogen attack. We reported previously that tobacco stress-induced gene 1 (Tsi1) may play an important role in regulating stress responsive genes and pathogenesis-related (PR) genes. In this study, we demonstrated that overexpression of Tsi1 gene in transgenic hot pepper plants induced constitutive expression of several PR genes in the absence of stress or pathogen treatment. The transgenic hot pepper plants expressing Tsi1 exhibited resistance to Pepper mild mottle virus (PMMV) and Cucumber mosaic virus (CMV). Furthermore, these transgenic plants showed increased resistance to a bacterial pathogen, Xanthomonas campestris pv. vesicatoria and also an oomycete pathogen, Phytophthora capsici. These results suggested that ectopic expression of Tsi1 in transgenic hot pepper plants enhanced the resistance of the plants to various pathogens, including viruses, bacteria, and oomycete. These results suggest that using transcriptional regulatory protein genes may contribute to developing broad-spectrum resistance in crop plants.