KED gene expression in early response to wounding stress in tomato plants.

The wounding-responsive KED gene, named for its coding for a lysine (K), glutamic acid (E), and aspartic acid (D)-rich protein, is widely present among land plants. However, little is known about its regulation or function. In this study, we found that transcription of the tomato (Solanum lycopersicum) KED gene, SlKED, was rapidly and transiently elevated by wounding or ethephon treatment. Compared to the wild-type plants, the CRISPR/Cas9-mediated SlKED knockout plants did not exhibit altered expression patterns for genes involved in hormone biosynthesis or stress signaling, suggesting a lack of pleiotropic effect on other stress-responsive genes. Conversely, jasmonic acid did not appear to directly regulate SlKED expression. Wounded leaves of the KED-lacking plants exhibited higher binding of Evans blue dye than the wild-type, indicating a possible role for KED in healing damaged tissues. The SlKED knockout plants showed a similar dietary effect as the wild-type on the larval growth of tobacco hornworm. But a higher frequency of larval mandible (mouth) movement was recorded during the first 2 minutes of feeding on the wounded KED-lacking SlKED knockout plants than on the wounded KED-producing wild-type plants, probably reflecting an initial differential response by the feeding larvae to the SlKED knockout plants. Our findings suggest that SlKED may be an ethylene-mediated early responder to mechanical stress in tomato, acting downstream of the wound stress response pathways. Although its possible involvement in response to other biotic and abiotic stresses is still unclear, we propose that SlKED may play a role in plant's rapid, short-term, early wounding responses, such as in cellular damage healing.

Evolutionary analysis of KED-rich proteins in plants.

During the course of evolution, organisms have developed genetic mechanisms in response to various environmental stresses including wounding from mechanical damage or herbivory-caused injury. A previous study of wounding response in the plant tobacco identified a unique wound-induced gene, aptly named KED due to its coding for a protein that has an unusually high content of amino acids lysine (K), glutamic acid (E) and aspartic acid (D). However, by far little is known about this intriguing gene. In this study, we investigated the evolutionary aspects of the KED-rich coding genes. We found that a consistent pattern of wound-induced KED gene expression is maintained across representative species of angiosperm and gymnosperm. KED genes can be identified in species from all groups of land plants (Embryophyta). All the KED proteins from vascular plants (Tracheophyta) including angiosperm, gymnosperm, fern and lycophyte share a conserved 19-amino acid domain near the C-terminus, whereas bryophytes (moss, liverwort and hornwort) possess KED-rich, multi-direct-repeat sequences that are distinct from the vascular plant KEDs. We detected KED-rich sequences in Charophyta species but not in Chlorophyta wherever genome sequences are available. Our studies suggest diverse and complex evolution pathways for land plant KED genes. Vascular plant KEDs exhibit high evolutionary conservation, implicating their shared function in response to wounding stress. The extraordinary enrichment of amino acids K, E and D in these groups of distinct and widely distributed proteins may reflect the structural and functional requirement for these three residues during some 600 million years of land plant evolution.

Tissue-Preferential Activity and Induction of the Pepper Capsaicin Synthase PUN1 Promoter by Wounding, Heat and Metabolic Pathway Precursor in Tobacco and Tomato Plants.

A promoter is an essential structural component of a gene that controls its transcription activity in different development stages and in response to various environmental stimuli. Knowledge of promoter functionality in heterologous systems is important in the study of gene regulation and biotechnological application. In order to explore the activity of the pepper capsaicin synthase gene (PUN1) promoter, gene constructs of pPUN1::GUS (for ß-glucuronidase) and pPUN1::NtKED (for a tobacco wound-responsive protein) were introduced into tobacco and tomato, respectively, and their activities were examined. Higher levels of GUS staining intensity and transcription were detected in ovary, anther and pollen than other tissues or organs in tobacco plants. Likewise, transgenic tomato fruits had a higher level of pPUN1::NtKED gene expression than the leaf and flower. The PUN1-driven gene expression can be transiently induced by wounding, heat (40 °C) and the capsaicinoid biosynthetic pathway precursor phenylalanine. When compared to the reported pPUN1::GUS-expressing Arabidopsis, the PUN1 promoter exhibited a more similar pattern of activities among pepper, tobacco and tomato, all Solanaceae plants. Our results suggest the potential utility of this tissue-preferential and inducible promoter in other non-pungent Solanaceae plants for research of gene function and regulation as well as in the biotechnological applications.

Tobacco plants expressing the maize nitrate transporter ZmNrt2.1 exhibit altered responses of growth and gene expression to nitrate and calcium.

BACKGROUND: Nitrate uptake is a highly regulated process. Understanding the intricate interactions between nitrate availability and genetically-controlled nitrate acquisition and metabolism is essential for improving nitrogen use efficiency and increasing nitrate uptake capacity for plants grown in both nitrate-poor and nitrate-enriched environments. In this report, we introduced into tobacco (Nicotiana tabacum) the constitutively expressed maize high-affinity transporter ZmNrt2.1 gene that would bypass the tight control for the endogenous nitrate-responsive genes. By using calcium inhibitors and varying levels of NO3-, Ca2+ and K+, we probed how the host plants were affected in their nitrate response. RESULTS: We found that the ZmNrt2.1-expressing plants had better root growth than the wild type plants when Ca2+ was deficient regardless of the nitrate levels. The growth restriction associated with Ca2+-deficiency can be alleviated with a high level of K+. Furthermore, the transgenic plants exhibited altered expression patterns of several endogenous, nitrate-responsive genes, including the high- and low-affinity nitrate transporters, the Bric-a-Brac/Tramtrack/Broad protein BT2 and the transcription factor TGA-binding protein TGA1, in responding to treatments of NO3-, K+ or inhibitors for the calcium channel and the cytosolic Ca2+-regulating phospholipase C, as compared to the wild type plants under the same treatments. Their expression was not only responsive to nitrate, but also affected by Ca2+. There were also different patterns of gene expression between roots and shoots. CONCLUSION: Our results demonstrate the ectopic effect of the maize nitrate transporter on the host plant's overall gene expression of nitrate sensing system, and further highlight the involvement of calcium in nitrate sensing in tobacco plants.