The phosphorylation of carboxyl-terminal eIF2α by SPA kinases contributes to enhanced translation efficiency during photomorphogenesis.

Light triggers an enhancement of global translation during photomorphogenesis in Arabidopsis, but little is known about the underlying mechanisms. The phosphorylation of the α-subunit of eukaryotic initiation factor 2 (eIF2α) at a conserved serine residue in the N-terminus has been shown as an important mechanism for the regulation of protein synthesis in mammalian and yeast cells. However, whether the phosphorylation of this residue in plant eIF2α plays a role in regulation of translation remains elusive. Here, we show that the quadruple mutant of SUPPRESSOR OF PHYA-105 family members (SPA1-SPA4) display repressed translation efficiency after light illumination. Moreover, SPA1 directly phosphorylates the eIF2α C-terminus under light conditions. The C-term-phosphorylated eIF2α promotes translation efficiency and photomorphogenesis, whereas the C-term-unphosphorylated eIF2α results in a decreased translation efficiency. We also demonstrate that the phosphorylated eIF2α enhances ternary complex assembly by promoting its affinity to eIF2ß and eIF2γ. This study reveals a unique mechanism by which light promotes translation via SPA1-mediated phosphorylation of the C-terminus of eIF2α in plants.

COP1-ERF1-SCE1 regulatory module fine-tunes stress response under light-dark cycle in Arabidopsis.

ETHYLENE RESPONSE FACTOR 1 (ERF1) plays an important role in integrating hormone crosstalk and stress responses. Previous studies have shown that ERF1 is unstable in the dark and its degradation is mediated by UBIQUITIN-CONJUGATING ENZYME 18. However, whether there are other enzymes regulating ERF1's stability remains unclear. Here, we use various in vitro and in vivo biochemical, genetic and stress-tolerance tests to demonstrate that both CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) and SUMO-CONJUGATING ENZYME 1 (SCE1) regulate the stability of ERF1. We also performed transcriptomic analyses to understand their common regulatory pathways. We show that COP1 mediates ERF1 ubiquitination in the dark while SCE1 mediates ERF1 sumoylation in the light. ERF1 stability is positively regulated by SCE1 and negatively regulated by COP1. Upon abiotic stress, SCE1 plays a positive role in stress defence by regulating the expression of ERF1's downstream stress-responsive genes, whereas COP1 plays a negative role in stress response. Moreover, ERF1 also promotes photomorphogenesis and the expression of light-responsive genes. Our study reveals the molecular mechanism of how COP1 and SCE1 counteract to regulate ERF1's stability and light-stress signalling crosstalk.

Phytochrome Signaling Networks.

The perception of light signals by the phytochrome family of photoreceptors has a crucial influence on almost all aspects of growth and development throughout a plant's life cycle. The holistic regulatory networks orchestrated by phytochromes, including conformational switching, subcellular localization, direct protein-protein interactions, transcriptional and posttranscriptional regulations, and translational and posttranslational controls to promote photomorphogenesis, are highly coordinated and regulated at multiple levels. During the past decade, advances using innovative approaches have substantially broadened our understanding of the sophisticated mechanisms underlying the phytochrome-mediated light signaling pathways. This review discusses and summarizes these discoveries of the role of the modular structure of phytochromes, phytochrome-interacting proteins, and their functions; the reciprocal modulation of both positive and negative regulators in phytochrome signaling; the regulatory roles of phytochromes in transcriptional activities, alternative splicing, and translational regulation; and the kinases and E3 ligases that modulate PHYTOCHROME INTERACTING FACTORs to optimize photomorphogenesis.

PCH1 and PCHL Directly Interact with PIF1, Promote Its Degradation, and Inhibit Its Transcriptional Function during Photomorphogenesis.

PHOTOPERIODIC CONTROL OF HYPOCOTYL 1 (PCH1) and PCH1-LIKE (PCHL) were shown to directly bind to phytochrome B (phyB) and suppress phyB thermal reversion, resulting in plants with dramatically enhanced light sensitivity. Here, we show that PCH1 and PCHL also positively regulate various light responses, including seed germination, hypocotyl gravitropism, and chlorophyll biosynthesis, by physically interacting with PHYTOCHROME INTERACTING FACTOR 1 (PIF1) and CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1). PCH1 and PCHL interact with PIF1 both in the dark and light, and regulate PIF1 abundance. Moreover, PCH1 and PCHL facilitate the physical interaction between phyB and PIF1 in vivo to promote the light-induced degradation of PIF1. PCH1 and PCHL also inhibit the DNA-binding ability of PIF1 to negatively regulate the expressions of PIF1 target genes. In addition, PCH1 and PCHL interact with COP1 and undergo degradation through the 26S proteasome pathway in the dark. Consistently, pch1 suppresses cop1 phenotype in darkness. Collectively, our study reveals a novel mechanism by which PCH1 and PCHL regulate diverse light responses not only by stabilizing phyB Pfr form but also by directly interacting with PIF1 and COP1, providing a molecular understanding of the control of hypocotyl growth by these proteins.

UBC18 mediates ERF1 degradation under light-dark cycles.

Ethylene Response Factor 1 (ERF1) plays a crucial role in biotic and abiotic stress responses. Previous studies have shown that ERF1 regulates stress-responsive gene expression by binding to different cis-acting elements in response to various stress signals. ERF1 was also reported to be unstable in the dark, and it regulates hypocotyl elongation. Here, we elucidated the mechanism underlying degradation of ERF1. Yeast two-hybrid screening showed that UBIQUITIN-CONJUGATING ENZYME 18 (UBC18) interacted with ERF1. The interaction between ERF1 and UBC18 was verified using pull-down assays and coimmunoprecipitation analyses. We then compared the ERF1 protein abundance in the UBC18 mutant and overexpression plants. Based on the results of protein degradation and in vivo ubiquitination assays, we proposed that UBC18 mediates ERF1 ubiquitination and degradation. ERF1 was more stable in UBC18 mutants and less stable in UBC18 overexpression lines compared with that in wild-type plants. ERF1 was degraded by the 26S proteasome system via regulation of UBC18 and promotes dark-repression of downstream genes and proline accumulation. UBC18 negatively regulated drought and salt stress responses by altering the abundance of ERF1 and the expression of genes downstream of ERF1.

ORA47 (octadecanoid-responsive AP2/ERF-domain transcription factor 47) regulates jasmonic acid and abscisic acid biosynthesis and signaling through binding to a novel cis-element.

ORA47 (octadecanoid-responsive AP2/ERF-domain transcription factor 47) of Arabidopsis thaliana is an AP2/ERF domain transcription factor that regulates jasmonate (JA) biosynthesis and is induced by methyl JA treatment. The regulatory mechanism of ORA47 remains unclear. ORA47 is shown to bind to the cis-element (NC/GT)CGNCCA, which is referred to as the O-box, in the promoter of ABI2. We proposed that ORA47 acts as a connection between ABA INSENSITIVE1 (ABI1) and ABI2 and mediates an ABI1-ORA47-ABI2 positive feedback loop. PORA47:ORA47-GFP transgenic plants were used in a chromatin immunoprecipitation (ChIP) assay to show that ORA47 participates in the biosynthesis and/or signaling pathways of nine phytohormones. Specifically, many abscisic acid (ABA) and JA biosynthesis and signaling genes were direct targets of ORA47 under stress conditions. The JA content of the P35S:ORA47-GR lines was highly induced under wounding and moderately induced under water stress relative to that of the wild-type plants. The wounding treatment moderately increased ABA accumulation in the transgenic lines, whereas the water stress treatment repressed the ABA content. ORA47 is proposed to play a role in the biosynthesis of JA and ABA and in regulating the biosynthesis and/or signaling of a suite of phytohormone genes when plants are subjected to wounding and water stress.

Increased glutathione contributes to stress tolerance and global translational changes in Arabidopsis.

Although glutathione is well known for its reactive oxygen species (ROS) scavenging function and plays a protective role in biotic stress, its regulatory function in abiotic stress still remains to be elucidated. Our previous study showed that exogenously applied reduced glutathione (GSH) could improve abiotic stress tolerance in Arabidopsis. Here, we report that endogenously increased GSH also conferred tolerance to drought and salt stress in Arabidopsis. Moreover, both exogenous and endogenous GSH delayed senescence and flowering time. Polysomal profiling results showed that global translation was enhanced after GSH treatment and by the induced increase of GSH level by salt stress. By performing transcriptomic analyses of steady-state and polysome-bound mRNAs in GSH-treated plants, we reveal that GSH has a substantial impact on translation. Translational changes induced by GSH treatment target numerous hormones and stress signaling molecules, which might contribute to the enhanced stress tolerance in GSH-treated plants. Our translatome analysis also revealed that abscisic acid (ABA), auxin and jasmonic acid (JA) biosynthesis, as well as signaling genes, were activated during GSH treatment, which has not been reported in previously published transcriptomic data. Together, our data suggest that the increased glutathione level results in stress tolerance and global translational changes.

Optimal beam design on intensity-modulated radiation therapy with simultaneous integrated boost in nasopharyngeal cancer.

This study aims to determine the optimal beam design among various combinations of field numbers and beam trajectories for intensity-modulated radiation therapy (IMRT) with simultaneous integrated boost (SIB) technique for the treatment of nasopharyngeal cancer (NPC). We used 10 fields with gantry angles of 155°, 130°, 75°, 25°, 0° L, 0° R, 335°, 285°, 230°, and 205° denoted as F10. To decrease doses in the spinal cord, the F10 technique was designed by featuring 2 pairs of split-opposed beam fields at 155° to 335° and 205° to 25°, as well as one pair of manually split beam fields at 0°. The F10 technique was compared with 4 other common field arrangements: F7E, 7 fields with 50° equally spaced gantry angles; F7, the basis of F10 with 155°, 130°, 75°, 0°, 285°, 230°, and 205°; F9E, 9 fields with 40° equally spaced gantry angles; and FP, 7 posterior fields with 180°, 150°, 120°, 90°, 270°, 240°, and 210°. For each individual case of 10 patients, the customized constraints derived after optimization with the standard F10 technique were applied to 4 other field arrangements. The 4 new optimized plans of each individual case were normalized to achieve the same coverage of planning target volume (PTV)63Gy as that of the standard F10 technique. The F10 field arrangement exhibited the best coverage in PTV70Gy and the least mean dose in the trachea-esophagus region. Furthermore, the F10 field arrangement demonstrated the highest level of conformity in the low-dose region and the least monitor unit. The F10 field arrangement performed more outstandingly than the other field arrangements in PTV70Gy coverage and spared the central organ. This arrangement also exhibited the highest conformity and delivery efficiency. The F10 technique is recommended as the standard beam geometry for the SIB-IMRT of NPC.

The Arabidopsis ETHYLENE RESPONSE FACTOR1 regulates abiotic stress-responsive gene expression by binding to different cis-acting elements in response to different stress signals.

ETHYLENE RESPONSE FACTOR1 (ERF1) is an upstream component in both jasmonate (JA) and ethylene (ET) signaling and is involved in pathogen resistance. Accumulating evidence suggests that ERF1 might be related to the salt stress response through ethylene signaling. However, the specific role of ERF1 in abiotic stress and the molecular mechanism underlying the signaling cross talk still need to be elucidated. Here, we report that ERF1 was highly induced by high salinity and drought stress in Arabidopsis (Arabidopsis thaliana). The salt stress induction required both JA and ET signaling but was inhibited by abscisic acid. ERF1-overexpressing lines (35S:ERF1) were more tolerant to drought and salt stress. They also displayed constitutively smaller stomatal aperture and less transpirational water loss. Surprisingly, 35S:ERF1 also showed enhanced heat tolerance and up-regulation of heat tolerance genes compared with the wild type. Several suites of genes activated by JA, drought, salt, and heat were found in microarray analysis of 35S:ERF1. Chromatin immunoprecipitation assays found that ERF1 up-regulates specific suites of genes in response to different abiotic stresses by stress-specific binding to GCC or DRE/CRT. In response to biotic stress, ERF1 bound to GCC boxes but not DRE elements; conversely, under abiotic stress, we observed specific binding of ERF1 to DRE elements. Furthermore, ERF1 bound preferentially to only one among several GCC box or DRE/CRT elements in the promoter region of its target genes. ERF1 plays a positive role in salt, drought, and heat stress tolerance by stress-specific gene regulation, which integrates JA, ET, and abscisic acid signals.

Functional characterization of an abiotic stress-inducible transcription factor AtERF53 in Arabidopsis thaliana.

AP2/ERF proteins play crucial roles in plant growth and development and in responses to biotic and abiotic stresses. ETHYLENE RESPONSE FACTOR 53 (AtERF53) belongs to group 1 in the ERF family and is induced in the early hours of dehydration and salt treatment. The functional study of AtERF53 is hampered because its protein expression in Arabidopsis is vulnerable to degradation in overexpressed transgenic lines. Taking advantage of the RING domain ligase1/RING domain ligase2 (rglg1rglg2) double mutant in which the AtERF53 can express stably, we investigate the physiological function of AtERF53. In this study, we demonstrate that expression of AtERF53 in wild-type Arabidopsis was responsive to heat and abscisic acid (ABA) treatment. From results of the cotransfection experiment, we concluded that AtERF53 has positive transactivation activity. Overexpression of AtERF53 in the rglg1rglg2 double mutant conferred better heat-stress tolerance and had resulted in higher endogenous ABA and proline levels compared to rglg1rglg2 double mutants. AtERF53 also has a function to regulate guard-cell movement because the stomatal aperture of AtERF53 overexpressed in rglg1rglg2 double mutant was smaller than that in the rglg1rglg2 double mutant under ABA treatment. In a global gene expression study, we found higher expressions of many stress-related genes, such as DREB1A, COR15A, COR15B, PLC, P5CS1, cpHSC70 s and proline and ABA metabolic-related genes. Furthermore, we identified several downstream target genes of AtERF53 by chromatin immunoprecipitation assay. In conclusion, the genetic, molecular and biochemical result might explain how AtERF53 serving as a transcription factor contributes to abiotic stress tolerance in Arabidopsis.

Arabidopsis RGLG2, functioning as a RING E3 ligase, interacts with AtERF53 and negatively regulates the plant drought stress response.

Transcriptional activities of plants play important roles in responses to environmental stresses. ETHYLENE RESPONSE FACTOR53 (AtERF53) is a drought-induced transcription factor that belongs to the AP2/ERF superfamily and has a highly conserved AP2 domain. It can regulate drought-responsive gene expression by binding to the GCC box and/or the dehydration-responsive element in the promoter of downstream genes. Overexpression of AtERF53 driven by the cauliflower mosaic virus 35S promoter resulted in an unstable drought-tolerant phenotype in T2 transgenic Arabidopsis (Arabidopsis thaliana) plants. Using a yeast two-hybrid screen, we identified a RING domain ubiquitin E3 ligase, RGLG2, which interacts with AtERF53 in the nucleus. The copine domain of RGLG2 exhibited the strongest interacting activity. We also demonstrated that RGLG2 could move from the plasma membrane to the nucleus under stress treatment. Using an in vitro ubiquitination assay, RGLG2 and its closest sequelog, RGLG1, were shown to have E3 ligase activity and mediated AtERF53 ubiquitination for proteasome degradation. The rglg1rglg2 double mutant but not the rglg2 or rglg1 single mutant exhibited a drought-tolerant phenotype when compared with wild-type plants. AtERF53-green fluorescent proteins expressed in the rglg1rglg2 double mutants were stable. The 35S:AtERF53-green fluorescent protein/rglg1rglg2 showed enhanced AtERF53-regulated gene expression and had greater tolerance to drought stress than the rglg1rglg2 double mutant. In conclusion, RGLG2 negatively regulates the drought stress response by mediating AtERF53 transcriptional activity in Arabidopsis.