14-3-3 proteins are evolutionarily conserved regulators that orchestrate diverse physiological processes by binding to phosphorylated client proteins. These interactions modulate client activity, localization, stability, or interactome composition, thereby coordinating plant growth, development, metabolism, and stress responses. Mounting evidence underscores their direct involvement in phytohormone signaling and stress-response pathways, in which 14-3-3 binding often constitutes a key regulatory event. This review highlights recent advances elucidating the pivotal role of 14-3-3 proteins in regulating phytohormone networks, biotic and abiotic stress tolerance, and evolutionary adaptations. We position 14-3-3 proteins as crucial integrators of internal signals and external cues, serving as central hubs that rewire cellular networks to enable adaptation to complex, changing environments.

The phytochrome B (phyB)-transcription factor signaling module plays a critical role in regulating photomorphogenesis. In this study, TCP21, a member of the Class I TCP transcription factor family, was identified as a direct interacting partner of phyB. TCP21 negatively regulates photomorphogenesis through downregulating HY5 expression and repressing the transcriptional activity of HY5. Moreover, phyB physically interacts with TCP21 and inhibits its transcriptional activity. Notably, phyB outcompetes HY5 for binding to TCP21, thereby attenuating the interaction between TCP21 and HY5 and alleviating TCP21-mediated repression of HY5 DNA-binding ability. Collectively, these findings establish TCP21 as a key component within the phyB-HY5 signaling module, contributing to plant adaptation to dynamic light environments.

NNLO QCD corrections for the pion electromagnetic form factor at large momentum transfer have been recently performed in [Phys. Rev. Lett. 132, 201901 (2024); Phys. Rev. Lett. 134, 221901 (2025)], revealing that the NLO and NNLO contributions are positive and sizeable. Unfortunately, these predictions have been obtained using the conventional scale-setting method and thus they are plagued by large renormalization scale ambiguities. In this paper, we analyze the pion electromagnetic form factor at NNLO by applying the Principle of Maximum Conformality (PMC), which is introduced with the aim of resolving renormalization scheme and scale ambiguities. By applying the PMC, a more precise perturbative QCD (pQCD) prediction for the pion EMFF Q^2F_π (Q^2) without conventional renormalization scale ambiguity can be achieved. This improved pQCD prediction is highly beneficial for the precise determination of the pion light-cone distribution amplitude. We then conduct a comprehensive comparison between theoretical predictions and experimental measurements of the pion EMFF Q^2F_π (Q^2) .

CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1), a repressor of seedling photomorphogenesis, is tightly controlled by light. In Arabidopsis, COP1 primarily acts as a part of large E3 ligase complexes and targets key light-signaling factors for ubiquitination and degradation. Upon light perception, the action of COP1 is precisely modulated by active photoreceptors. During seedling development, light plays a predominant role in modulating seedling morphogenesis, including inhibition of hypocotyl elongation, cotyledon opening and expansion, and chloroplast development. These visible morphological changes evidently result from networks of molecular action. In this review, we summarize current knowledge about the molecular role of COP1 in mediating light-controlled seedling development.

The ubiquitin-proteasome system (UPS) represents a highly conserved protein degradation pathway that plays an essential role in maintaining the homeostasis of cellular proteins. This system ensures precise regulation of key regulators within the light signaling pathway, thereby enabling plants to dynamically switch between skotomorphogenesis (growth in the dark) and photomorphogenesis (growth in the light). In darkness, the negative E3 ligases (e.g. CRL4COP1-SPA) target photomorphogenesis-promoting regulators (e.g. ELONGATED HYPOCOTYL5) for ubiquitination and degradation, consequently repressing photomorphogenesis. Conversely, under light conditions, the positive E3 ligases (e.g. CRL1EBF1/2) promote the ubiquitination and degradation of photomorphogenesis-inhibitory regulators (e.g. phytochrome-interacting factors), ensuring proper seedling photomorphogenic development. This mini-review provides a concise overview of the ubiquitin-proteasome system in plants, focusing on recent advances in understanding the role of the UPS in regulating photomorphogenesis. Additionally, we highlight current challenges in further exploring the role of the UPS in photomorphogenesis.

Phytochrome B (phyB), the predominant red-light photoreceptor, interacts with various proteins, initiating a set of molecular events in plant cells that promote light responses. In this study, we identified AHL21, a member of the AT-hook motif nuclear-localized (AHL) family, as a phyB-interacting protein in Arabidopsis. AHL21 positively regulates photomorphogenesis by directly binding to PHYTOCHROME INTERACTING FACTOR 4 (PIF4) promoter and downregulating its expression. Moreover, AHL21 physically interacts with PIF4 and represses its transcriptional activation activity. Hence, AHL21 acts genetically upstream of PIF4. In addition, phyB directly interacts with both AHL21 and PIF4 and functions as a molecular glue that enhances the AHL21-PIF4 interaction. Collectively, AHL21 functions as a key component of the phyB-PIF4 signaling module to enable plants to adapt to fluctuating light conditions.

Intron retention (IR), a well-conserved form of alternative splicing, is widespread among eukaryotic organisms. It serves as an orchestrated program for regulating gene expression. A previously reported role of IR is to induce intron-retained transcript (IRT) degradation via the nonsense-mediated mRNA decay (NMD) pathway, resulting in the downregulation of gene expression. However, accumulating evidence indicates that most IRTs are detained in the nucleus, and thus, IR can downregulate gene expression through the storage of IRTs in the nucleus. Although the importance of IRTs in gene expression regulation is well established, the detailed mechanisms remain unclear. Here, we propose a potential model to explain how IRTs are retained in the nucleus and respond to environmental changes or developmental transitions. Plenty of future studies are still ahead of us to fully dissect the biological function of IR and the underlying mechanisms.

Light and brassinosteroids (BR) are indispensable for plant growth and control cell division in the apical meristem. However, how external light signals cooperate with internal brassinosteroids to program root meristem development remains elusive. We reveal that the photoreceptor phytochrome B (phyB) guides the scaffold protein RACK1 to coordinate BR signaling for maintaining root meristematic activity. phyB and RACK1 promote early root meristem development. Mechanistically, RACK1 could reinforce the phyB-SPA1 association by interacting with both phyB and SPA1, which indirectly affects COP1-dependent RACK1 degradation, resulting in the accumulation of RACK1 in roots. Subsequently, RACK1 interacts with BES1 to repress its DNA-binding activity toward the target gene CYCD3;1, leading to the release of BES1-mediated inhibition of CYCD3;1 transcription, and hence the promotion of root meristem development. Our study provides mechanistic insights into the regulation of root meristem development by combination of light and phytohormones signals through the photoreceptors and scaffold proteins.

Intron retention (IR) is the most common alternative splicing event in Arabidopsis. An increasing number of studies have demonstrated the major role of IR in gene expression regulation. The impacts of IR on plant growth and development and response to environments remain underexplored. Here, we found that IR functions directly in gene expression regulation on a genome-wide scale through the detainment of intron-retained transcripts (IRTs) in the nucleus. Nuclear-retained IRTs can be kept away from translation through this mechanism. COP1-dependent light modulation of the IRTs of light signaling genes, such as PIF4, RVE1, and ABA3, contribute to seedling morphological development in response to changing light conditions. Furthermore, light-induced IR changes are under the control of the spliceosome, and in part through COP1-dependent ubiquitination and degradation of DCS1, a plant-specific spliceosomal component. Our data suggest that light regulates the activity of the spliceosome and the consequent IRT nucleus detainment to modulate photomorphogenesis through COP1.

B-box containing proteins (BBXs) integrate light and various hormonal signals to regulate plant growth and development. Here, we demonstrate that the photomorphogenic repressors BBX28 and BBX29 positively regulate brassinosteroid (BR) signaling in Arabidopsis thaliana seedlings. Treatment with the BR brassinolide stabilized BBX28 and BBX29, which partially depended on BR INSENSITIVE1 (BRI1) and BIN2. bbx28 bbx29 seedlings exhibited larger cotyledon aperture than the wild-type when treated with brassinazole in the dark, which partially suppressed the closed cotyledons of brassinazole resistant 1-1D (bzr1-1D). Consistently, overexpressing BBX28 and BBX29 partially rescued the short hypocotyls of bri1-5 and bin2-1 in both the dark and light, while the loss-of-function of BBX28 and BBX29 partially suppressed the long hypocotyls of bzr1-1D in the light. BBX28 and BBX29 physically interacted with BR-ENHANCED EXPRESSION1 (BEE1), BEE2, and BEE3 and enhanced their binding to and activation of their target genes. Moreover, BBX28 and BBX29 as well as BEE1, BEE2, and BEE3 increased BZR1 accumulation to promote the BR signaling pathway. Therefore, both BBX28 and BBX29 interact with BEE1, BEE2, and BEE3 to orchestrate light and BR signaling by facilitating the transcriptional activity of BEE target genes. Our study provides insights into the pivotal roles of BBX28 and BBX29 as signal integrators in ensuring normal seedling development.

CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) mediates various cellular and physiological processes in plants by targeting a large number of substrates for ubiquitination and degradation. In this study, we reveal that a substitution of Pro for Leu at amino acid position 409 in WRKY32 largely suppresses the short hypocotyls and expanded cotyledon phenotypes of cop1-6. WRKY32(P409L) promotes hypocotyl growth and inhibits the opening of cotyledons in Arabidopsis. Loss of WRKY32 function mutant seedlings display elongated hypocotyls, whereas overexpression of WRKY32 leads to shortened hypocotyls. WRKY32 directly associates with the promoter regions of HY5 to activate its transcription. COP1 interacts with and targets WRKY32 for ubiquitination and degradation in darkness. WRKY32(P409L) exhibits enhanced DNA binding ability and affects the expression of more genes compared with WRKY32 in Arabidopsis. Our results not only reveal the basic role for WRKY32 in promoting photomorphogenesis, but also provide insights into manipulating plant growth by engineering key components of light signaling.

Light and the circadian clock are two essential external and internal cues affecting seedling development. COLD-REGULATED GENE27 (COR27), which is regulated by cold temperatures and light signals, functions as a key regulator of the circadian clock. Here, we report that COR27 acts as a negative regulator of light signaling. COR27 physically interacts with the CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1)-SUPPRESSOR OF PHYTOCHROME A1 (SPA1) E3 ubiquitin ligase complex and undergoes COP1-mediated degradation via the 26S proteasome system in the dark. cor27 mutant seedlings exhibit shorter hypocotyls, while transgenic lines overexpressing COR27 show elongated hypocotyls in the light. In addition, light induces the accumulation of COR27. On one hand, accumulated COR27 interacts with ELONGATED HYPOCOTYL5 (HY5) to repress HY5 DNA binding activity. On the other hand, COR27 associates with the chromatin at the PHYTOCHROME INTERACTING FACTOR4 (PIF4) promoter region and upregulates PIF4 expression in a circadian clock-dependent manner. Together, our findings reveal a mechanistic framework whereby COR27 represses photomorphogenesis in the light and provide insights toward how light and the circadian clock synergistically control hypocotyl growth.

Light-mediated seedling development is coordinately controlled by a variety of key regulators. Here, we identified two B-box (BBX)-containing proteins, BBX30 and BBX31, as repressors of photomorphogenesis. ELONGATED HYPOCOTYL5, a central regulator of light signaling, directly binds to the G-box cis-element present in the promoters of BBX30 and BBX31 and negatively controls their transcription levels in the light. Seedlings with mutations in BBX30 or BBX31 are hypersensitive to light, whereas the overexpression of BBX30 or BBX31 leads to hypo-photomorphogenic growth in the light. Furthermore, transgenic and phenotypic analysis revealed that the B-box domain of BBX30 or BBX31 is essential for their respective functioning in the regulation of photomorphogenic development in plants. In conclusion, BBX30 and BBX31 act as key negative regulators of light signaling, and their transcription is repressed by ELONGATED HYPOCOTYL5 through directly associating with their promoters.

Phytochrome B (phyB) absorbs red light signals and subsequently initiates a set of molecular events in plant cells to promote photomorphogenesis. Here we show that phyB directly interacts with B-BOX CONTAINING PROTEIN 4 (BBX4), a positive regulator of red light signaling, and positively controls its abundance in red light. BBX4 associates with PHYTOCHROME INTERACTING FACTOR 3 (PIF3) and represses PIF3 transcriptional activation activity and PIF3-controlled gene expression. The degradation of BBX4 in darkness is dependent on CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) and the 26S proteasome system. Collectively, BBX4 acts as a key component of the phyB-PIF3-mediated signaling module and fine tunes the red light action. phyB promotes the accumulation of BBX4, which in turn serves to repress PIF3 action through direct physical interaction to promote photomorphogenic development in red light.

Traditional metrology solutions are facing a range of challenges at the 1X node such as three dimensional (3D) measurement capabilities, shrinking overlay and critical dimension (CD) error budgets driven by multi-patterning and via in trench CD measurements. Hybrid metrology offers promising new capabilities to address some of these challenges but it will take some time before fully realized. This paper explores new capabilities currently offered on the in-line Critical Dimension Scanning Electron Microscope (CD-SEM) to address these challenges and enable the CD-SEM to move beyond measuring bottom CD using top down imaging.Device performance is strongly correlated with Fin geometry causing an urgent need for 3D measurements. New beam tilting capabilities enhance the ability to make 3D measurements in the front-end-of-line (FEOL) of the metal gate FinFET process in manufacturing. We explore these new capabilities for measuring Fin height and build upon the work communicated last year at SPIE1. Furthermore, we extend the application of the tilt beam to the back-endof- line (BEOL) trench depth measurement and demonstrate its capability in production targeting replacement of the existing Atomic Force Microscope (AFM) measurements by including the height measurement in the existing CDSEM recipe to reduce fab cycle time.In the BEOL, another increasingly challenging measurement for the traditional CD-SEM is the bottom CD of the self-aligned via (SAV) in a trench first via last (TFVL) process. Due to the extremely high aspect ratio of the structure secondary electron (SE) collection from the via bottom is significantly reduced requiring the use of backscatter electrons (BSE) to increase the relevant image quality. Even with this solution, the resulting images are difficult to measure with advanced technology nodes. We explore new methods to increase measurement robustness and combine this with novel segmentation-based measurement algorithm generated specifically for BSE images. The results will be contrasted with data from previously used methods to quantify the improvement. We also compare the results to electrical test data to evaluate and quantify the measurement performance improvements.Lastly, according to International Technology Roadmap for Semiconductors (ITRS) from 2013, the overlay 3 sigma requirement will be 3.3 nm in 2015 and 2.9 nm in 2016. Advanced lithography requires overlay measurement in die on features resembling the device geometry. However, current optical overlay measurement is performed in the scribe line on large targets due to optical diffraction limit. In some cases, this limits the usefulness of the measurement since it does not represent the true behavior of the device. We explore using high voltage imaging to help address this urgent need. Novel CD-SEM based overlay targets that optimize the restrictions of process geometry and SEM technique were designed and spread out across the die. Measurements are done on these new targets both after photolithography and etch. Correlation is drawn between the two measurements. These results will also be compared to conventional optical overlay measurement approaches and we will discuss the possibility of using this capability in high volume manufacturing.

At 1X node, 3D FinFETS raise a number of new metrology challenges. Gate height and fin height are two of the most important parameters for process control. At present there is a metrology gap in inline in-die measurement of these parameters. In order to fill this metrology gap, in-column beam tilt has been developed and implemented on Applied Materials V4i+ top-down CD-SEM for height measurement. A low tilt (5°) beam and a high tilt (14°) beam have been calibrated to obtain two sets of images providing measurement of sidewall edge width to calculate height in the host. Evaluations are done with applications in both gate height and fin height. TEM correlation with R2 being 0.89 and precision of 0.81nm have been achieved on various in-die features in gate height application. Fin height measurement shows less accuracy (R2 being 0.77) and precision (1.49 nm) due to challenges brought by fin geometry, yet still promising as first attempt. Sensitivity to DOE offset, die-to-die and in-die variation is demonstrated in both gate height and fin height. Process defect is successfully captured from inline wafers with gate height measurement implemented in production. This is the first successful demonstration of inline in-die gate height measurement for 14nm FinFET process control.

At 1x node, a three-dimensional (3-D) FinFET process raises a number of new metrology challenges for process control, including gate height and fin height. At present, there is a metrology gap in inline in-die measurement of these parameters. To fill this metrology gap, in-column beam tilt has been implemented on Applied Materials V4i+ critical dimension scanning electron microscope for height measurement. Low-tilt (5 deg) and high-tilt (14 deg) beam angles have been calibrated to obtain the height and the sidewall angle information. Evaluation of its feasibility and production worthiness is done with applications in both gate height and fin height measurements. Transmission electron microscope correlation with an R-2 equal to 0.89 and a precision of 0.81 nm have been achieved on various in-die features in a gate height application. The initial fin height measurement shows less accuracy (R-2 being 0.77) and precision (1.49 nm) due to greater challenges brought by the fin profile, yet it is promising for the first attempt. Sensitivity to design of experiment offset die-to-die and in-die variations is demonstrated in both gate height and fin height. The process defect is successfully captured with inline gate height measurement. This is the first successful demonstration of inline in-die gate height measurement for a 14-nm FinFET process control. (C) 2014 Society of Photo-Optical Instrumentation Engineers (SPIE)