Keun Pyo Lee

EXECUTER1- and EXECUTER2-dependent transfer of stress-related signals from the plastid to the nucleus of Arabidopsis thaliana

Shortly after the release of singlet oxygen (1O2), drastic changes in nuclear gene expression occur in the conditional flu mutant of Arabidopsis that reveal a rapid transfer of signals from the plastid to the nucleus. In contrast to retrograde control of nuclear gene expression by plastid signals described earlier, the primary effect of 1O2 generation in the flu mutant is not the control of chloroplast biogenesis but the activation of a broad range of signaling pathways known to be involved in biotic and abiotic stress responses. This activity of a plastid-derived signal suggests a new function of the chloroplast, namely that of a sensor of environmental changes that activates a broad range of stress responses. Inactivation of the plastid protein EXECUTER1 attenuates the extent of 1O2-induced up-regulation of nuclear gene expression, but it does not fully eliminate these changes. A second related nuclear-encoded protein, dubbed EXECUTER2, has been identified that is also implicated with the signaling of 1O2-dependent nuclear gene expression changes. Like EXECUTER1, EXECUTER2 is confined to the plastid. Inactivation of both EXECUTER proteins in the ex1/ex2/flu triple mutant is sufficient to suppress the up-regulation of almost all 1O2-responsive genes. Retrograde control of 1O2-responsive genes requires the concerted action of both EXECUTER proteins within the plastid compartment.

Chloroplasts of Arabidopsis Are the Source and a Primary Target of a Plant-Specific Programmed Cell Death Signaling Pathway

Under mild light stress, plants enhance the production of singlet oxygen that acts as a signal. Singlet oxygen–mediated signaling forms an integral part of photosynthesis that translates environmental variability affecting photosynthetic electron transport into signals that regulate the readjustment of the plant to environmental changes. Enhanced levels of singlet oxygen (1O2) in chloroplasts trigger programmed cell death. The impact of 1O2 production in chloroplasts was monitored first in the conditional fluorescent (flu) mutant of Arabidopsis thaliana that accumulates 1O2 upon a dark/light shift. The onset of 1O2 production is rapidly followed by a loss of chloroplast integrity that precedes the rupture of the central vacuole and the final collapse of the cell. Inactivation of the two plastid proteins EXECUTER (EX1) and EX2 in the flu mutant abrogates these responses, indicating that disintegration of chloroplasts is due to EX-dependent signaling rather than 1O2 directly. In flu seedlings, 1O2-mediated cell death signaling operates as a default pathway that results in seedlings committing suicide. By contrast, EX-dependent signaling in the wild type induces the formation of microlesions without decreasing the viability of seedlings. 1O2-mediated and EX-dependent loss of plastid integrity and cell death in these plants occurs only in cells containing fully developed chloroplasts. Our findings support an as yet unreported signaling role of 1O2 in the wild type exposed to mild light stress that invokes photoinhibition of photosystem II without causing photooxidative damage of the plant.

A seed coat bedding assay shows that RGL2-dependent release of abscisic acid by the endosperm controls embryo growth in Arabidopsis dormant seeds

Seed dormancy is an ecologically important adaptive trait in plants whereby germination is repressed even under favorable germination conditions such as imbibition with water. In Arabidopsis and most plant species, dormancy absolutely requires an unidentified seed coat germination-repressive activity and constitutively higher abscisic acid (ABA) levels upon seed imbibition. The mechanisms underlying these processes and their possible relationship are incompletely understood. We developed a “seed coat bedding” assay monitoring the growth of dissected embryos cultured on a layer of seed coats, allowing combinatorial experiments using dormant, nondormant, and various genetically modified seed coat and embryonic materials. This assay, combined with direct ABA measurements, revealed that, upon imbibition, dormant coats, unlike nondormant coats, actively produce and release ABA to repress embryo germination, whatever the embryo origin, i.e., from dormant, nondormant, or never dormant aba seeds, unable to synthesize ABA. The persistent high ABA levels in imbibed dormant seeds requires the permanent expression of the DELLA gene RGL2, where it remains insensitive to gibberellins (GA) unlike in nondormant seeds. These findings present the seed coat as an organ actively controlling germination upon seed imbibition and provide a framework to investigate how environmental factors break seed dormancy.

Spatially and genetically distinct control of seed germination by phytochromes A and B

Phytochromes phyB and phyA mediate a remarkable developmental switch whereby, early upon seed imbibition, canopy light prevents phyB-dependent germination, whereas later on, it stimulates phyA-dependent germination. Using a seed coat bedding assay where the growth of dissected embryos is monitored under the influence of dissected endosperm, allowing combinatorial use of mutant embryos and endosperm, we show that canopy light specifically inactivates phyB activity in the endosperm to override phyA-dependent signaling in the embryo. This interference involves abscisic acid (ABA) release from the endosperm and distinct spatial activities of phytochrome signaling components. Under the canopy, endospermic ABA opposes phyA signaling through the transcription factor (TF) ABI5, which shares with the TF PIF1 several target genes that negatively regulate germination in the embryo. ABI5 enhances the expression of phytochrome signaling genes PIF1, SOMNUS, GAI, and RGA, but also of ABA and gibberellic acid (GA) metabolic genes. Over time, weaker ABA-dependent responses eventually enable phyA-dependent germination, a distinct type of germination driven solely by embryonic growth.

1O2-mediated retrograde signaling during late embryogenesis predetermines plastid differentiation in seedlings by recruiting abscisic acid

Plastid development in seedlings of Arabidopsis thaliana is affected by the transfer of 1O2-mediated retrograde signals from the plastid to the nucleus and changes in nuclear gene expression during late embryogenesis. The potential impact of these mechanisms on plastid differentiation is maintained throughout seed dormancy and becomes effective only after seed germination. Inactivation of the 2 nuclear-encoded plastid proteins EXECUTER1 and EXECUTER2 blocks 1O2-mediated retrograde signaling before the onset of dormancy and impairs normal plastid formation in germinating seeds. This long-term effect of 1O2 retrograde signaling depends on the recruitment of abscisic acid (ABA) during seedling development. Unexpectedly, ABA acts as a positive regulator of plastid formation in etiolated and light-grown seedlings.

TIGRINA d, required for regulating the biosynthesis of tetrapyrroles in barley, is an ortholog of the FLU gene of Arabidopsis thaliana.

Regulation of tetrapyrrole biosynthesis in higher plants has been attributed to negative feedback control of steps prior to δ‐aminolevulinic acid (ALA) formation. One of the first mutants with a defect in this control had been identified in barley. The tigrina (tig) d mutant accumulates 10–15‐fold higher amounts of protochlorophyllide than wild type, when grown in the dark. The identity of the TIGRINA d protein and its mode of action are not known yet. Initially this protein had been proposed to act as a repressor of genes that encode enzymes involved in early steps of ALA formation, but subsequent attempts to confirm this experimentally failed. Here we demonstrate that the TIGRINA d gene of barley is an ortholog of the FLU gene of Arabidopsis thaliana. The FLU protein is a nuclear‐encoded plastid protein that plays a key role in negative feedback control of chlorophyll biosynthesis in higher plants. Sequencing of the FLU gene of barley revealed a frame shift mutation in the FLU gene of the tig d mutant that results in the loss of two tetratricopeptide repeats that in the FLU protein of Arabidopsis are essential for its biological activity. This mutation cosegregates strictly with the tigrina phenotype within the F1 population of a heterozygous tig d mutant, thus providing additional support for the flu gene being responsible for the tigrina phenotype of barley.

Abscisic acid transporters cooperate to control seed germination

Seed germination is a key developmental process that has to be tightly controlled to avoid germination under unfavourable conditions. Abscisic acid (ABA) is an essential repressor of seed germination. In Arabidopsis, it has been shown that the endosperm, a single cell layer surrounding the embryo, synthesizes and continuously releases ABA towards the embryo. The mechanism of ABA transport from the endosperm to the embryo was hitherto unknown. Here we show that four AtABCG transporters act in concert to deliver ABA from the endosperm to the embryo: AtABCG25 and AtABCG31 export ABA from the endosperm, whereas AtABCG30 and AtABCG40 import ABA into the embryo. Thus, this work establishes that radicle extension and subsequent embryonic growth are suppressed by the coordinated activity of multiple ABA transporters expressed in different tissues.

The Identification of in Vitro Production of Lectin from Callus Cultures of Korean Mistletoe (Viscum album L. var. coloratum)

Despite potential medical, economical, and agronomical importance, the bioprocessing of mistletoe cell cultures, from callus cultures to mass production of high-value products (e.g., lectins and viscotoxins), has been unsuccessful to date. In this study, we confirmed the potential of in vitro lectin production from callus cultures of Korean mistletoe (Viscum album L. var. coloratum).