The TOR-EIN2 axis mediates nuclear signalling to modulate plant growth.

The evolutionarily conserved target of rapamycin (TOR) kinase acts as a master regulator that coordinates cell proliferation and growth by integrating nutrient, energy, hormone and stress signals in all eukaryotes1,2. Research has focused mainly on TOR-regulated translation, but how TOR orchestrates the global transcriptional network remains unclear. Here we identify ethylene-insensitive protein 2 (EIN2), a central integrator3-5 that shuttles between the cytoplasm and the nucleus, as a direct substrate of TOR in Arabidopsis thaliana. Glucose-activated TOR kinase directly phosphorylates EIN2 to prevent its nuclear localization. Notably, the rapid global transcriptional reprogramming that is directed by glucose-TOR signalling is largely compromised in the ein2-5 mutant, and EIN2 negatively regulates the expression of a wide range of target genes of glucose-activated TOR that are involved in DNA replication, cell wall and lipid synthesis and various secondary metabolic pathways. Chemical, cellular and genetic analyses reveal that cell elongation and proliferation processes that are controlled by the glucose-TOR-EIN2 axis are decoupled from canonical ethylene-CTR1-EIN2 signalling, and mediated by different phosphorylation sites. Our findings reveal a molecular mechanism by which a central signalling hub is shared but differentially modulated by diverse signalling pathways using distinct phosphorylation codes that can be specified by upstream protein kinases.

The protein phosphatase PC1 dephosphorylates and deactivates CatC to negatively regulate H2O2 homeostasis and salt tolerance in rice.

Catalase (CAT) is often phosphorylated and activated by protein kinases to maintain hydrogen peroxide (H2O2) homeostasis and protect cells against stresses, but whether and how CAT is switched off by protein phosphatases remains inconclusive. Here, we identified a manganese (Mn2+)-dependent protein phosphatase, which we named PHOSPHATASE OF CATALASE 1 (PC1), from rice (Oryza sativa L.) that negatively regulates salt and oxidative stress tolerance. PC1 specifically dephosphorylates CatC at Ser-9 to inhibit its tetramerization and thus activity in the peroxisome. PC1 overexpressing lines exhibited hypersensitivity to salt and oxidative stresses with a lower phospho-serine level of CATs. Phosphatase activity and seminal root growth assays indicated that PC1 promotes growth and plays a vital role during the transition from salt stress to normal growth conditions. Our findings demonstrate that PC1 acts as a molecular switch to dephosphorylate and deactivate CatC and negatively regulate H2O2 homeostasis and salt tolerance in rice. Moreover, knockout of PC1 not only improved H2O2-scavenging capacity and salt tolerance but also limited rice grain yield loss under salt stress conditions. Together, these results shed light on the mechanisms that switch off CAT and provide a strategy for breeding highly salt-tolerant rice.

Data-Independent Acquisition Proteomics Reveals the Effects of Red and Blue Light on the Growth and Development of Moso Bamboo (Phyllostachys edulis) Seedlings.

Moso bamboo is a rapidly growing species with significant economic, social, and cultural value. Transplanting moso bamboo container seedlings for afforestation has become a cost-effective method. The growth and development of the seedlings is greatly affected by the quality of light, including light morphogenesis, photosynthesis, and secondary metabolite production. Therefore, studies on the effects of specific light wavelengths on the physiology and proteome of moso bamboo seedlings are crucial. In this study, moso bamboo seedlings were germinated in darkness and then exposed to blue and red light conditions for 14 days. The effects of these light treatments on seedling growth and development were observed and compared through proteomics analysis. Results showed that moso bamboo has higher chlorophyll content and photosynthetic efficiency under blue light, while it displays longer internode and root length, more dry weight, and higher cellulose content under red light. Proteomics analysis reveals that these changes under red light are likely caused by the increased content of cellulase CSEA, specifically expressed cell wall synthetic proteins, and up-regulated auxin transporter ABCB19 in red light. Additionally, blue light is found to promote the expression of proteins constituting photosystem II, such as PsbP and PsbQ, more than red light. These findings provide new insights into the growth and development of moso bamboo seedlings regulated by different light qualities.

The transcriptional dynamics during de novo shoot organogenesis of Ma bamboo (Dendrocalamus latiflorus Munro): implication of the contributions of the abiotic stress response in this process.

De novo shoot organogenesis is an important biotechnological tool for fundamental studies in plant. However, it is difficult in most bamboo species, and the genetic control of this highly dynamic and complicated regeneration process remains unclear. In this study, based on an in-depth analysis at the cellular level, the shoot organogenesis from calli of Ma bamboo (Dendrocalamus latiflorus Munro) was divided into five stages. Subsequently, single-molecule long-read isoform sequencing of tissue samples pooled from all five stages was performed to generate a full-length transcript landscape. A total of 83 971 transcripts, including 73 209 high-quality full-length transcripts, were captured, which served as an annotation reference for the subsequent RNA sequencing analysis. Time-course transcriptome analysis of samples at the abovementioned five stages was conducted to investigate the global gene expression atlas showing genome-wide expression of transcripts during the course of bamboo shoot organogenesis. K-means clustering analysis and stage-specific transcript identification revealed important dynamically expressed transcription regulators that function in bamboo shoot organogenesis. The majority of abiotic stress-responsive genes altered their expression levels during this process, and further experiments demonstrated that exogenous application of moderate but not severe abiotic stress increased the shoot regeneration efficiency. In summary, our study provides an overview of the genetic flow dynamics during bamboo shoot organogenesis. Full-length cDNA sequences generated in this study can serve as a valuable resource for fundamental and applied research in bamboo in the future.

Using phage-assisted continuous evolution (PACE) to evolve human PD1.

PD1/PDL1 pathway plays a critical role in cancer immune responses. The immune checkpoint inhibitors of PD1/PDL1 have been well explored and developed for immunotherapies of solid tumors. Recently, various monoclonal antibodies targeting the PD1/PDL1 pathway have emerged and achieved remarkable success in clinical trials. However, challenges with these monoclonal antibodies have appeared during cancer therapies, including predictors of response, patient selection, and innate resistance. Thus, a competitive antagonist of native PD1/PDL1, with smaller size and lower side-effect, is required for future cancer therapies. In this study, we utilized a protein evolution system of phage-assisted continuous evolution (PACE) to evolve PD1 continuously. Our results indicated that the newly evolved PD1 bound to PDL1 with higher affinity. The interactome analysis further suggested that these evolved PD1s exhibited higher specificity with PDL1. Therefore, these evolved PD1s may be applied as a new tool for tumor immunotherapy.

Arabidopsis LSH8 Positively Regulates ABA Signaling by Changing the Expression Pattern of ABA-Responsive Proteins.

Phytohormone ABA regulates the expression of numerous genes to significantly affect seed dormancy, seed germination and early seedling responses to biotic and abiotic stresses. However, the function of many ABA-responsive genes remains largely unknown. In order to improve the ABA-related signaling network, we conducted a large-scale ABA phenotype screening. LSH, an important transcription factor family, extensively participates in seedling development and floral organogenesis in plants, but whether its family genes are involved in the ABA signaling pathway has not been reported. Here we describe a new function of the transcription factor LSH8 in an ABA signaling pathway. In this study, we found that LSH8 was localized in the nucleus, and the expression level of LSH8 was significantly induced by exogenous ABA at the transcription level and protein level. Meanwhile, seed germination and root length measurements revealed that lsh8 mutant lines were ABA insensitive, whereas LSH8 overexpression lines showed an ABA-hypersensitive phenotype. With further TMT labeling quantitative proteomic analysis, we found that under ABA treatment, ABA-responsive proteins (ARPs) in the lsh8 mutant presented different changing patterns with those in wild-type Col4. Additionally, the number of ARPs contained in the lsh8 mutant was 397, six times the number in wild-type Col4. In addition, qPCR analysis found that under ABA treatment, LSH8 positively mediated the expression of downstream ABA-related genes of ABI3, ABI5, RD29B and RAB18. These results indicate that in Arabidopsis, LSH8 is a novel ABA regulator that could specifically change the expression pattern of APRs to positively mediate ABA responses.

The Arabidopsis HY2 Gene Acts as a Positive Regulator of NaCl Signaling during Seed Germination.

Phytochromobilin (PΦB) participates in the regulation of plant growth and development as an important synthetase of photoreceptor phytochromes (phy). In addition, Arabidopsis long hypocotyl 2 (HY2) appropriately works as a key PΦB synthetase. However, whether HY2 takes part in the plant stress response signal network remains unknown. Here, we described the function of HY2 in NaCl signaling. The hy2 mutant was NaCl-insensitive, whereas HY2-overexpressing lines showed NaCl-hypersensitive phenotypes during seed germination. The exogenous NaCl induced the transcription and the protein level of HY2, which positively mediated the expression of downstream stress-related genes of RD29A, RD29B, and DREB2A. Further quantitative proteomics showed the patterns of 7391 proteins under salt stress. HY2 was then found to specifically mediate 215 differentially regulated proteins (DRPs), which, according to GO enrichment analysis, were mainly involved in ion homeostasis, flavonoid biosynthetic and metabolic pathways, hormone response (SA, JA, ABA, ethylene), the reactive oxygen species (ROS) metabolic pathway, photosynthesis, and detoxification pathways to respond to salt stress. More importantly, ANNAT1-ANNAT2-ANNAT3-ANNAT4 and GSTU19-GSTF10-RPL5A-RPL5B-AT2G32060, two protein interaction networks specifically regulated by HY2, jointly participated in the salt stress response. These results direct the pathway of HY2 participating in salt stress, and provide new insights for the plant to resist salt stress.

Functions of COP1/SPA E3 Ubiquitin Ligase Mediated by MpCRY in the Liverwort Marchantia polymorpha under Blue Light.

COP1/SPA1 complex in Arabidopsis inhibits photomorphogenesis through the ubiquitination of multiple photo-responsive transcription factors in darkness, but such inhibiting function of COP1/SPA1 complex would be suppressed by cryptochromes in blue light. Extensive studies have been conducted on these mechanisms in Arabidopsis whereas little attention has been focused on whether another branch of land plants bryophyte utilizes this blue-light regulatory pathway. To study this problem, we conducted a study in the liverwort Marchantia polymorpha and obtained a MpSPA knock-out mutant, in which Mpspa exhibits the phenotype of an increased percentage of individuals with asymmetrical thallus growth, similar to MpCRY knock-out mutant. We also verified interactions of MpSPA with MpCRY (in a blue light-independent way) and with MpCOP1. Concomitantly, both MpSPA and MpCOP1 could interact with MpHY5, and MpSPA can promote MpCOP1 to ubiquitinate MpHY5 but MpCRY does not regulate the ubiquitination of MpHY5 by MpCOP1/MpSPA complex. These data suggest that COP1/SPA ubiquitinating HY5 is conserved in Marchantia polymorpha, but dissimilar to CRY in Arabidopsis, MpCRY is not an inhibitor of this process under blue light.

Arabidopsis cryptochrome 1 interacts with SPA1 to suppress COP1 activity in response to blue light.

Plant photoreceptors mediate light suppression of the E3 ubiquitin ligase COP1 (CONSTITUTIVE PHOTOMORPHOGENIC 1) to affect gene expression and photomorphogenesis. However, how photoreceptors mediate light regulation of COP1 activity remains unknown. We report here that Arabidopsis blue-light receptor cryptochrome 1 (CRY1) undergoes blue-light-dependent interaction with the COP1-interacting protein SPA1 (SUPPRESSOR OF PHYTOCHROME A). We further show that the CRY1-SPA1 interaction suppresses the SPA1-COP1 interaction and COP1-dependent degradation of the transcription factor HY5. These results are consistent with a hypothesis that photoexcited CRY1 interacts with SPA1 to modulate COP1 activity and plant development.

A CRY-BIC negative-feedback circuitry regulating blue light sensitivity of Arabidopsis.

Cryptochromes are blue light receptors that regulate various light responses in plants. Arabidopsis cryptochrome 1 (CRY1) and cryptochrome 2 (CRY2) mediate blue light inhibition of hypocotyl elongation and long-day (LD) promotion of floral initiation. It has been reported recently that two negative regulators of Arabidopsis cryptochromes, Blue light Inhibitors of Cryptochromes 1 and 2 (BIC1 and BIC2), inhibit cryptochrome function by blocking blue light-dependent cryptochrome dimerization. However, it remained unclear how cryptochromes regulate the BIC gene activity. Here we show that cryptochromes mediate light activation of transcription of the BIC genes, by suppressing the activity of CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), resulting in activation of the transcription activator ELONGATED HYPOCOTYL 5 (HY5) that is associated with chromatins of the BIC promoters. These results demonstrate a CRY-BIC negative-feedback circuitry that regulates the activity of each other. Surprisingly, phytochromes also mediate light activation of BIC transcription, suggesting a novel photoreceptor co-action mechanism to sustain blue light sensitivity of plants under the broad spectra of solar radiation in nature.

New insights into the mechanisms of phytochrome-cryptochrome coaction.

Contents Summary 547 I. Introduction 547 II. Phytochromes mediate light-induced transcription of BICs to inactivate cryptochromes 548 III. PPKs phosphorylate light-signaling proteins and histones to affect plant development 548 IV. Prospect 550 Acknowledgements 550 References 550 SUMMARY: Plants perceive and respond to light signals by multiple sensory photoreceptors, including phytochromes and cryptochromes, which absorb different wavelengths of light to regulate genome expression and plant development. Photophysiological analyses have long revealed the coordinated actions of different photoreceptors, a phenomenon referred to as the photoreceptor coaction. The mechanistic explanations of photoreceptor coactions are not fully understood. The function of direct protein-protein interaction of phytochromes and cryptochromes and common signaling molecules of these photoreceptors, such as SPA1/COP1 E3 ubiquitin ligase complex and bHLH transcription factors PIFs, would partially explain phytochrome-cryptochrome coactions. In addition, newly discovered proteins that block cryptochrome photodimerization or catalyze cryptochrome phosphorylation may also participate in the phytochrome and cryptochrome coaction. This Tansley insight, which is not intended to make a comprehensive review of the studies of photoreceptor coactions, attempts to highlight those recent findings and their possible roles in the photoreceptor coaction.

Trp triad-dependent rapid photoreduction is not required for the function of Arabidopsis CRY1.

Cryptochromes in different evolutionary lineages act as either photoreceptors or light-independent transcription repressors. The flavin cofactor of both types of cryptochromes can be photoreduced in vitro by electron transportation via three evolutionarily conserved tryptophan residues known as the "Trp triad." It was hypothesized that Trp triad-dependent photoreduction leads directly to photoexcitation of cryptochrome photoreceptors. We tested this hypothesis by analyzing mutations of Arabidopsis cryptochrome 1 (CRY1) altered in each of the three Trp-triad tryptophan residues (W324, W377, and W400). Surprisingly, in contrast to a previous report all photoreduction-deficient Trp-triad mutations of CRY1 remained physiologically and biochemically active in Arabidopsis plants. ATP did not enhance rapid photoreduction of the wild-type CRY1, nor did it rescue the defective photoreduction of the CRY1(W324A) and CRY1(W400F) mutants that are photophysiologically active in vivo. The lack of correlation between rapid flavin photoreduction or the effect of ATP on the rapid flavin photoreduction and the in vivo photophysiological activities of plant cryptochromes argues that the Trp triad-dependent photoreduction is not required for the function of cryptochromes and that further efforts are needed to elucidate the photoexcitation mechanism of cryptochrome photoreceptors.

TCP2 positively regulates HY5/HYH and photomorphogenesis in Arabidopsis.

Light regulates plant growth and development via multiple photoreceptors including phytochromes and cryptochromes. Although the functions of photoreceptors have been studied extensively, questions remain regarding the involvement of cryptochromes in photomorphogenesis. In this study, we identified a protein, TEOSINTE-LIKE1, CYCLOIDEA, and PROLIFERATING CELL FACTOR 2 (TCP2), which interacts with the cryptochrome 1 (CRY1) protein in yeast and plant cells via the N-terminal domains of both proteins. Transgenic plants overexpressing TCP2 displayed a light-dependent short hypocotyl phenotype, especially in response to blue light. Moreover, light affected TCP2 expression in a wavelength-dependent manner and TCP2 positively regulates mRNA expression of HYH and HY5. These results support the hypothesis that TCP2 is a transcription activator which acts downstream of multiple photoreceptors, including CRY1.

Optogenetic induction of caspase-8 mediated apoptosis by employing Arabidopsis cryptochrome 2.

Apoptosis, a programmed cell death mechanism, is a regulatory process controlling cell proliferation as cells undergo demise. Caspase-8 serves as a pivotal apoptosis-inducing factor that initiates the death receptor-mediated apoptosis pathway. In this investigation, we have devised an optogenetic method to swiftly modulate caspase-8 activation in response to blue light. The cornerstone of our optogenetic tool relies on the PHR domain of Arabidopsis thaliana cryptochrome 2, which self-oligomerizes upon exposure to blue light. In this study, we have developed two optogenetic approaches for rapidly controlling caspase-8 activation in response to blue light in cellular systems. The first strategy, denoted as Opto-Casp8-V1, entails the fusion expression of the Arabidopsis blue light receptor CRY2 N-terminal PHR domain with caspase-8. The second strategy, referred to as Opto-Casp8-V2, involves the independent fusion expression of caspase-8 with the PHR domain and the CRY2 blue light-interacting protein CIB1 N-terminal CIB1N. Upon induction with blue light, PHR undergoes aggregation, leading to caspase-8 aggregation. Additionally, the blue light-dependent interaction between PHR and CIB1N also results in caspase-8 aggregation. We have validated these strategies in both HEK293T and HeLa cells. The findings reveal that both strategies are capable of inducing apoptosis, with Opto-Casp8-V2 demonstrating significantly superior efficiency compared to Opto-Casp8-V1.

Comparative Proteomics Combined with Morphophysiological Analysis Revealed Chilling Response Patterns in Two Contrasting Maize Genotypes.

Maize yield is significantly influenced by low temperature, particularly chilling stress at the maize seedling stage. Various physiological approaches have been established to resist chilling stress; however, the detailed proteins change patterns underlying the maize chilling stress response at the seedling stage remain unknown, preventing the development of breeding-based methods to resist chilling stress in maize. Thus, we performed comprehensive physiological, comparative proteomics and specific phytohormone abscisic acid (ABA) assay on different maize inbred lines (tolerant-line KR701 and sensitive-line hei8834) at different seedling stages (the first leaf stage and third leaf stage) under chilling stress. The results revealed several signalling proteins and pathways in response to chilling stress at the maize seedling stage. Meanwhile, we found ABA pathway was important for chilling resistance of tolerant-line KR701 at the first leaf stage. Related chilling-responsive proteins were further catalogued and analysed, providing a resource for further investigation and maize breeding.

Arabidopsis cryptochrome 2 forms photobodies with TCP22 under blue light and regulates the circadian clock.

Cryptochromes are blue light receptors that regulate plant growth and development. They also act as the core components of the central clock oscillator in animals. Although plant cryptochromes have been reported to regulate the circadian clock in blue light, how they do so is unclear. Here we show that Arabidopsis cryptochrome 2 (CRY2) forms photobodies with the TCP22 transcription factor in response to blue light in plant cells. We provide evidence that PPK kinases influence the characteristics of these photobodies and that together these components, along with LWD transcriptional regulators, can positively regulate the expression of CCA1 encoding a central component of the circadian oscillator.

Roles of a Cryptochrome in Carbon Fixation and Sucrose Metabolism in the Liverwort Marchantia polymorpha.

In vascular plants, cryptochromes acting as blue-light photoreceptors have various functions to adapt plants to the fluctuating light conditions on land, while the roles of cryptochromes in bryophytes have been rarely reported. In this study, we investigated functions of a single-copy ortholog of cryptochrome (MpCRY) in the liverwort Marchantia polymorpha. Knock-out of MpCRY showed that a large number of the mutant plants exhibited asymmetric growth of thalli under blue light. Transcriptome analyses indicated that MpCRY is mainly involved in photosynthesis and sugar metabolism. Further physiological analysis showed that Mpcry mutant exhibited a reduction in CO2 uptake and sucrose metabolism. In addition, exogenous application of sucrose or glucose partially restored the symmetrical growth of the Mpcry mutant thalli. Together, these results suggest that MpCRY is involved in the symmetrical growth of thallus and the regulation of carbon fixation and sucrose metabolism in M. polymorpha.

Functional analyses unveil the involvement of moso bamboo (Phyllostachys edulis) group I and II NIN-LIKE PROTEINS in nitrate signaling regulation.

For rapid growth, moso bamboo (Phyllostachys edulis) requires large amounts of nutrients. Nitrate is an indispensable molecular signal to regulate nitrogen absorption and assimilation, which are regulated by group III NIN-LIKE PROTEINs (NLPs). However, no Phyllostachys edulis NLP (PeNLP) has been characterized. Here, eight PeNLPs were identified, which showed dynamic expression patterns in bamboo tissues. Nitrate did not affect PeNLP mRNA levels, and PeNLP1, -2, -5, -6, -7, and -8 successfully restored nitrate signaling in Arabidopsis atnlp7-1 protoplasts through recovering AtNiR and AtNRT2.1 expression. Four group I and II PeNLPs (PeNLP1, -2, -5, and -8) interacted with the nitrate-responsive cis-element of PeNiR. Moreover, nitrate triggered the nuclear retention of PeNLP8. PeNLP8 overexpression in Arabidopsis significantly increased the primary root length, lateral root number, leaf area, and dry and wet weight of the transgenic plants, and PeNLP8 expression rescued the root architectural defect phenotype of atnlp7-1 mutants. Interestingly, PeNLP8 overexpression dramatically reduced nitrate content but elevated total amino acid content in Arabidopsis. Overall, the present study unveiled the potential involvement of group I and II NLPs in nitrate signaling regulation and provided genetic resources for engineering plants with high nitrogen use efficiency.

Phenotype and TMT-based quantitative proteomics analysis of Brassica napus reveals new insight into chlorophyll synthesis and chloroplast structure.

The conversion of light energy into chemical energy in leaves is very important for plant growth and development. During this process, chlorophylls and their derivatives are indispensable as their fundamental role in the energy absorption and transduction activities. Chlorophyll variation mutants are important materials for studying chlorophyll metabolism, chloroplast biogenesis, photosynthesis and related physiological processes. Here, a chlorophyll-reduced mutant (crm1) was isolated from ethyl methanesulfonate (EMS) mutagenized Brassica napus. Compared to wild type, crm1 showed yellow leaves, reduced chlorophyll content, fewer thylakoid stacks and retarded growth. Quantitative mass spectrometry analysis with Tandem Mass Tag (TMT) isobaric labeling showed that totally 4575 proteins were identified from the chloroplast of Brassica napus leaves, and 466 of which displayed differential accumulations between wild type and crm1. The differential abundance proteins were found to be involved in chlorophyll metabolism, photosynthesis, phagosome and proteasome. Our results suggest that the decreased abundance of chlorophyll biosynthetic enzymes, proteins involved in photosynthesis might account for the reduced chlorophyll content, impaired thylakoid structure, and reduction of plant productivity. The increased abundance of proteins involved in phagosome and proteasome pathways might allow plants to adapt the proteome to environmental conditions to ensure growth and survival due to chlorophyll reduction. BIOLOGICAL SIGNIFICANCE: Photosynthesis, which consists of light and dark reactions, is fundamental to biomass production. Chloroplast is regarded as the main site for photosynthesis. During photosynthesis, the pigment chlorophyll is essential for light harvesting and energy transfer. This work provides new insights into protein expression patterns, and enables the identification of many attractive candidates for investigation of chlorophyll biosynthesis, chloroplast structure and photosynthesis in Brassica napus. These findings may be applied to improve the photosynthetic efficiency by genetic engineering in crops.

Large Scale Profiling of Protein Isoforms Using Label-Free Quantitative Proteomics Revealed the Regulation of Nonsense-Mediated Decay in Moso Bamboo (Phyllostachys edulis).

Moso bamboo is an important forest species with a variety of ecological, economic, and cultural values. However, the gene annotation information of moso bamboo is only based on the transcriptome sequencing, lacking the evidence of proteome. The lignification and fiber in moso bamboo leads to a difficulty in the extraction of protein using conventional methods, which seriously hinders research on the proteomics of moso bamboo. The purpose of this study is to establish efficient methods for extracting the total proteins from moso bamboo for following mass spectrometry-based quantitative proteome identification. Here, we have successfully established a set of efficient methods for extracting total proteins of moso bamboo followed by mass spectrometry-based label-free quantitative proteome identification, which further improved the protein annotation of moso bamboo genes. In this study, 10,376 predicted coding genes were confirmed by quantitative proteomics, accounting for 35.8% of all annotated protein-coding genes. Proteome analysis also revealed the protein-coding potential of 1015 predicted long noncoding RNA (lncRNA), accounting for 51.03% of annotated lncRNAs. Thus, mass spectrometry-based proteomics provides a reliable method for gene annotation. Especially, quantitative proteomics revealed the translation patterns of proteins in moso bamboo. In addition, the 3284 transcript isoforms from 2663 genes identified by Pacific BioSciences (PacBio) single-molecule real-time long-read isoform sequencing (Iso-Seq) was confirmed on the protein level by mass spectrometry. Furthermore, domain analysis of mass spectrometry-identified proteins encoded in the same genomic locus revealed variations in domain composition pointing towards a functional diversification of protein isoform. Finally, we found that part transcripts targeted by nonsense-mediated mRNA decay (NMD) could also be translated into proteins. In summary, proteomic analysis in this study improves the proteomics-assisted genome annotation of moso bamboo and is valuable to the large-scale research of functional genomics in moso bamboo. In summary, this study provided a theoretical basis and technical support for directional gene function analysis at the proteomics level in moso bamboo.