Shinyoung Lee

Quantitative phosphoproteomics identifies SnRK2 protein kinase substrates and reveals the effectors of abscisic acid action

Sucrose nonfermenting 1 (SNF1)-related protein kinase 2s (SnRK2s) are central components of abscisic acid (ABA) signaling pathways. The snrk2.2/2.3/2.6 triple-mutant plants are nearly completely insensitive to ABA, suggesting that most of the molecular actions of ABA are triggered by the SnRK2s-mediated phosphorylation of substrate proteins. Only a few substrate proteins of the SnRK2s are known. To identify additional substrate proteins of the SnRK2s and provide insight into the molecular actions of ABA, we used quantitative phosphoproteomics to compare the global changes in phosphopeptides in WT and snrk2.2/2.3/2.6 triple mutant seedlings in response to ABA treatment. Among the 5,386 unique phosphorylated peptides identified in this study, we found that ABA can increase the phosphorylation of 166 peptides and decrease the phosphorylation of 117 peptides in WT seedlings. In the snrk2.2/2.3/2.6 triple mutant, 84 of the 166 peptides, representing 58 proteins, could not be phosphorylated, or phosphorylation was not increased under ABA treatment. In vitro kinase assays suggest that most of the 58 proteins can serve as substrates of the SnRK2s. The SnRK2 substrates include proteins involved in flowering time regulation, RNA and DNA binding, miRNA and epigenetic regulation, signal transduction, chloroplast function, and many other cellular processes. Consistent with the SnRK2 phosphorylation of flowering time regulators, the snrk2.2/2.3/2.6 triple mutant flowered significantly earlier than WT. These results shed new light on the role of the SnRK2 protein kinases and on the downstream effectors of ABA action, and improve our understanding of plant responses to adverse environments.

Type I MADS-box genes have experienced faster birth-and-death evolution than type II MADS-box genes in angiosperms

Plant MADS-box genes form a large gene family for transcription factors and are involved in various aspects of developmental processes, including flower development. They are known to be subject to birth-and-death evolution, but the detailed features of this mode of evolution remain unclear. To have a deeper insight into the evolutionary pattern of this gene family, we enumerated all available functional and nonfunctional (pseudogene) MADS-box genes from the Arabidopsis and rice genomes. Plant MADS-box genes can be classified into types I and II genes on the basis of phylogenetic analysis. Conducting extensive homology search and phylogenetic analysis, we found 64 presumed functional and 37 nonfunctional type I genes and 43 presumed functional and 4 nonfunctional type II genes in Arabidopsis. We also found 24 presumed functional and 6 nonfunctional type I genes and 47 presumed functional and 1 nonfunctional type II genes in rice. Our phylogenetic analysis indicated there were at least about four to eight type I genes and ≈15–20 type II genes in the most recent common ancestor of Arabidopsis and rice. It has also been suggested that type I genes have experienced a higher rate of birth-and-death evolution than type II genes in angiosperms. Furthermore, the higher rate of birth-and-death evolution in type I genes appeared partly due to a higher frequency of segmental gene duplication and weaker purifying selection in type I than in type II genes.

OsMADS51 Is a Short-Day Flowering Promoter That Functions Upstream of Ehd1, OsMADS14, and Hd3a

Although flowering regulatory mechanisms have been extensively studied in Arabidopsis (Arabidopsis thaliana), those in other species have not been well elucidated. Here, we investigated the role of OsMADS51, a type I MADS-box gene in the short-day (SD) promotion pathway in rice (Oryza sativa). In SDs OsMADS51 null mutants flowered 2 weeks later than normal, whereas in long days loss of OsMADS51 had little effect on flowering. Transcript levels of three flowering regulators—Ehd1, OsMADS14, and Hd3a—were decreased in these mutants, whereas those of OsGI and Hd1 were unchanged. Ectopic expression of OsMADS51 caused flowering to occur about 7 d earlier only in SDs. In ectopic expression lines, transcript levels of Ehd1, OsMADS14, and Hd3a were increased, but those of OsGI and Hd1 remained the same. These results indicate that OsMADS51 is a flowering promoter, particularly in SDs, and that this gene functions upstream of Ehd1, OsMADS14, and Hd3a. To further investigate the relationship with other flowering promoters, we generated transgenic plants in which expression of Ehd1 or OsGI was suppressed. In Ehd1 RNA interference plants, OsMADS51 expression was not affected, supporting our conclusion that the MADS-box gene functions upstream of Ehd1. However, in OsGI antisense plants, the OsMADS51 transcript level was reduced. In addition, the circadian expression pattern for this MADS-box gene was similar to that for OsGI. These results demonstrate that OsMADS51 functions downstream of OsGI. In summary, OsMADS51 is a novel flowering promoter that transmits a SD promotion signal from OsGI to Ehd1.

Rice Indeterminate 1 (OsId1) is necessary for the expression of Ehd1 (Early heading date 1) regardless of photoperiod.

Indeterminate 1 (Id1), a classical flowering gene first reported in 1946, is one of the earliest genes whose expression in leaf tissues affects the floral transition in the shoot meristem. How Id1 is integrated into the flowering process is largely unknown. In this study, we examined the genetic action of the rice (Oryza sativa) ortholog OsId1. In rice, OsId1 is preferentially expressed in young leaves, but the overall expression pattern is broader than that in maize (Zea mays). OsId1 is able to activate transcription in yeast. RNAi mutants show a delay in flowering under both short-day (SD) and long-day (LD) conditions. OsId1 regulates the expression of Ehd1 (Early heading date 1) and its downstream genes, including Hd3a (a rice ortholog of FT) and RFT1 (Rice Flowering Locus T1), under both SD and LD conditions. In rice, the expression of Ehd1 is also controlled by the photoperiodic flowering genes OsGI (a rice ortholog of GI) and OsMADS51. However, the expression of OsId1 is independent of OsGI, OsMADS51, and OsMADS50 (a rice SOC1 ortholog). This study demonstrates that the activation of Ehd1 by OsId1 is required for the promotion of flowering.

OsMADS50 and OsMADS56 function antagonistically in regulating long day (LD)-dependent flowering in rice.

In much of the tropics and subtropics, rice (Oryza sativa L.) is grown under long days (LDs). Therefore, LD must play a major role in inducing flowering signal in rice. However, little is known on LD-dependent flowering signal in the species. We previously reported that OsMADS50, which is highly homologous to Arabidopsis SOC1, functions as a positive regulator for flowering. However, its detailed photoperiodic mechanism was not yet elucidated. Here, we report the functional analysis of OsMADS50 and its closely related gene OsMADS56. Knock-out of OsMADS50 caused a late-flowering phenotype only under LD conditions. Overexpression of OsMADS56 (56OX) also resulted in delayed flowering under LD. In the osmads50 mutants and 56OX transgenic plants, transcripts of Ehd1, Hd3a and RFT1 were reduced, although that of OsLFL1 increased. On the other hand, mRNA levels of OsGI, Hd1, OsId1, OsDof12, Ghd7, Hd6 and SE5 were unchanged. These observations imply that OsMADS50 and OsMADS56 function antagonistically through OsLFL1-Ehd1 in regulating LD-dependent flowering. Yeast two-hybrid and co-immunoprecipitation analyses indicated an interaction between those two proteins as well as their formation of homodimers. These results suggest that OsMADS50 and OsMADS56 may form a complex that regulates downstream target genes.

Rice SVP‐group MADS‐box proteins, OsMADS22 and OsMADS55, are negative regulators of brassinosteroid responses

Most short vegetative phase (SVP)-group MADS-box genes control meristem identity and flowering time. Among the three SVP-group genes in rice, OsMADS47 has been reported as a negative regulator of brassinosteroid (BR) responses. Here, we investigated the functional roles of two close homologs, OsMADS22 and OsMADS55, by generating single, double and triple RNAi lines and overexpression lines. Analyses of the plants showed that their roles in regulating meristem identity are well conserved; however, the involvement of these genes in determining flowering time has diversified. Most importantly, OsMADS55 works as a major negative regulator of BR responses, and OsMADS22 functions to support OsMADS55. Whereas single OsMADS55 RNAi plants display weak BR responses in the lamina joint (LJ), OsMADS22-OsMADS55 double and OsMADS22-OsMADS47-OsMADS55 triple RNAi plants manifest dramatic BR responses with regard to LJ inclination, coleoptile elongation and senescence. Stem elongation is also notably reduced in the double and triple RNAi plants, probably because of BR oversensitivity. Expression analyses indicate the diversified roles in age-dependent BR responses. Altogether, our study demonstrates that all three rice SVP-group genes work as negative regulators of BR responses, but that their spatial and temporal roles are diversified.

Further Characterization of a Rice AGL12 Group MADS-Box Gene, OsMADS26

Plant MADS-box genes can be divided into 11 groups. Genetic analysis has revealed that most of them function in flowering-time control, reproductive organ development, and vegetative growth. Here, we elucidated the role of OsMADS26, a member of the AGL12 group. Transcript levels of OsMADS26 were increased in an age-dependent manner in the shoots and roots. Transgenic plants of both rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana) overexpressing this gene manifested phenotypes related to stress responses, such as chlorosis, cell death, pigment accumulation, and defective root/shoot growth. In addition, apical hook development was significantly suppressed in Arabidopsis. Plants transformed with the OsMADS26-GR (glucocorticoid receptor) fusion construct displayed those stress-related phenotypes when treated with dexamethasone. Microarray analyses using this inducible system showed that biosynthesis genes for jasmonate, ethylene, and reactive oxygen species, as well as putative downstream targets involved in the stress-related process, were up-regulated in OsMADS26-overexpressing plants. These results suggest that OsMADS26 induces multiple responses that are related to various stresses.

Diversified Mechanisms for Regulating Flowering Time in a Short-Day Plant Rice

Flowering in rice is influenced by not only endogenous factors that comprise an autonomous pathway, but also environmental effects, such as photoperiod, water availability, and temperature just before floral initiation. Recent molecular genetics studies have elucidated the functional roles of genes involved in the photoperiod pathway, e.g., photoreceptors, circadian clock components, and short-day (SD) promotion factors. Although these molecular players are well conserved between rice andArabidopsis, their actual genetic functions are distinct. This is exemplified byHd1 (aCO counterpart) and phytochromes, in particular, ricePHYA. Hd1 has a dual role in regulating flowering time and the expression ofHd3a (anFT counterpart) repression under long-day (LD) conditions while promotion under SDs. Models have been proposed to explain these photoperiod-dependent antagonistic activities. Some regulatory factors are present in only one of the model systems, e.g.,FLC inArabidopsis orEhd1 in rice. Furthermore, epistatic relationships vary among such flowering regulators asHd3a (FT), OsMADS50 (SOCT), andOsMADS14 (AP1). Further experiments to probe these differences will be essential to enlarging our understanding of the diversified flowering regulation mechanisms in rice.

A possible working mechanism for rice SVP-group MADS-box proteins as negative regulators of brassinosteroid responses.

Most SVP-group MADS-box genes control meristem identity and flowering time. In rice, their roles in regulating the former are well-conserved; however, their involvement in determining the latter is not significant. Characteristically, rice SVP-group MADS-box proteins work as negative regulators in brassinosteroid (BR) responses. To elucidate the molecular mechanism, we studied the localization patterns for these proteins and found that, unexpectedly, they were not specific in the nucleus, whether expressed alone or simultaneously. Interestingly however, OsMADS22 and OsMADS47 were translocated into the nucleus along with OsMADS50 while OsMADS55 inhibited the nuclear localization of OsMADS50. An overlapping cis-element exists between the CArG motif and ARF binding site on the promoter region of OsBLE, which is upregulated by BR treatment and in SVP RNAi plants. These observations suggest that BR-mediated signals may induce target gene expression by removing the SVP-group MADS-box proteins that preoccupy the promoters of BR downstream genes. Addendum to: Lee S, Choi SC, An G. Rice SVP-group MADS-box proteins, OsMADS22 and OsMADS55, are negative regulators of brassinosteroid responses. Plant J 2008; 54:93–105.

Improvement of Ventilation Flow inside a Large Factory Building Using PIV Velocity Field Measurements

Air movement in workplaces, whether resulting from a forced ventilation system or natural airflow, has a significant impact on occupational health. In a huge building of shipbuilding factory, typical harmful factors such as fume or vaporized gas from welding and cutting of steel plates give an unpleasant feeling. From field data survey, the yearly dominant wind directions around the factory building tested were north-west, north-east and south-east. Among the three wind directions, the ventilation improvement was the worst for the north-eastern wind. This study was focused on modification of opening vents in order to utilize the natural ventilation flow effectively. Instantaneous velocity fields inside the 1/1000 scale-down factory building model were measured using a 2-frame cross-correlation PIV method. The factory model was embedded in an atmospheric boundary layer simulated in a wind tunnel. The modified vents improved the internal ventilation flow with increasing the flow speed more than two times, compared with that of present vents.