Automated Phenotypic Trait Extraction for Rice Plant Using Terrestrial Laser Scanning Data.

To quickly obtain rice plant phenotypic traits, this study put forward the computational process of six rice phenotype features (e.g., crown diameter, perimeter of stem, plant height, surface area, volume, and projected leaf area) using terrestrial laser scanning (TLS) data, and proposed the extraction method for the tiller number of rice plants. Specifically, for the first time, we designed and developed an automated phenotype extraction tool for rice plants with a three-layer architecture based on the PyQt5 framework and Open3D library. The results show that the linear coefficients of determination (R2) between the measured values and the extracted values marked a better reliability among the selected four verification features. The root mean square error (RMSE) of crown diameter, perimeter of stem, and plant height is stable at the centimeter level, and that of the tiller number is as low as 1.63. The relative root mean squared error (RRMSE) of crown diameter, plant height, and tiller number stays within 10%, and that of perimeter of stem is 18.29%. In addition, the user-friendly automatic extraction tool can efficiently extract the phenotypic features of rice plant, and provide a convenient tool for quickly gaining phenotypic trait features of rice plant point clouds. However, the comparison and verification of phenotype feature extraction results supported by more rice plant sample data, as well as the improvement of accuracy algorithms, remain as the focus of our future research. The study can offer a reference for crop phenotype extraction using 3D point clouds.

Combined Metabolite and Transcriptomic Profiling Unveil a Potential Gene Network Involved in the Triterpenoid Metabolism of Rose roxburghii.

Rose roxburghii, a horticulturally significant species within the Rosa genus of the Rosaceae family, is renowned for its abundance of secondary metabolites and ascorbate, earning it the title 'king of vitamin C'. Despite this recognition, the mechanisms underlying the biosynthesis and regulation of triterpenoid compounds in R. roxburghii remain largely unresolved. In this study, we conducted high-performance liquid chromatography profiling across various organs of R. roxburghii, including fruit, root, stem, and leaves, revealing distinct distributions of triterpenoid compounds among different plant parts. Notably, the fruit exhibited the highest total triterpenoid content, followed by root and stem, with leaf containing the lowest levels, with leaf containing the lowest levels. Transcriptomic analysis unveiled preferential expression of members from the cytochrome P450 (CYP) and glycosyltransferase (UGT) families, likely contributing to the higher accumulation of both ascorbate and triterpenoid compounds in the fruits of R. roxburghii compared to other tissues of R. roxburghii. Transcriptomic analysis unveiled a potential gene network implicated in the biosynthesis of both ascorbate and triterpenoid compounds in R. roxburghii. These findings not only deepen our understanding of the metabolic pathways in this species but also have implications for the design of functional foods enriched with ascorbate and triterpenoids in R. roxburghii.

Dark secrets of phytomelatonin.

Phytomelatonin is a newly identified plant hormone, and its primary functions in plant growth and development remain relatively poorly appraised. Phytomelatonin is a master regulator of reactive oxygen species (ROS) signaling and acts as a darkness signal in circadian stomatal closure. Plants exhibit at least three interrelated patterns of interaction between phytomelatonin and ROS production. Exogenous melatonin can induce flavonoid biosynthesis, which might be required for maintenance of antioxidant capacity under stress, after harvest, and in leaf senescence conditions. However, several genetic studies have provided direct evidence that phytomelatonin plays a negative role in the biosynthesis of flavonoids under non-stress conditions. Phytomelatonin delays flowering time in both dicot and monocot plants, probably via its receptor PMTR1 and interactions with the gibberellin, strigolactone, and ROS signaling pathways. Furthermore, phytomelatonin signaling also functions in hypocotyl and shoot growth in skotomorphogenesis and ultraviolet B (UV-B) exposure; the G protein α-subunit (Arabidopsis GPA1 and rice RGA1) and constitutive photomorphogenic1 (COP1) are important signal components during this process. Taken together, these findings indicate that phytomelatonin acts as a darkness signal with important regulatory roles in circadian stomatal closure, flavonoid biosynthesis, flowering, and hypocotyl and shoot growth.

An update on the function and regulation of methylerythritol phosphate and mevalonate pathways and their evolutionary dynamics.

Isoprenoids are among the largest and most chemically diverse classes of organic compounds in nature and are involved in the processes of photosynthesis, respiration, growth, development, and plant responses to stress. The basic building block units for isoprenoid synthesis-isopentenyl diphosphate and its isomer dimethylallyl diphosphate-are generated by the mevalonate (MVA) and methylerythritol phosphate (MEP) pathways. Here, we summarize recent advances on the roles of the MEP and MVA pathways in plant growth, development and stress responses, and attempt to define the underlying gene networks that orchestrate the MEP and MVA pathways in response to developmental or environmental cues. Through phylogenomic analysis, we also provide a new perspective on the evolution of the plant isoprenoid pathway. We conclude that the presence of the MVA pathway in plants may be associated with the transition from aquatic to subaerial and terrestrial environments, as lineages for its core components are absent in green algae. The emergence of the MVA pathway has acted as a key evolutionary event in plants that facilitated land colonization and subsequent embryo development, as well as adaptation to new and varied environments.

A CRISPR/LbCas12a-based method for highly efficient multiplex gene editing in Physcomitrella patens.

Due to their high efficiency, specificity, and flexibility, programmable nucleases, such as those of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a (Cpf1) system, have greatly expanded the applicability of editing the genomes of various organisms. Genes from different gene families or genes with redundant functions in the same gene family can be examined by assembling multiple CRISPR RNAs (crRNAs) in a single vector. However, the activity and efficiency of CRISPR/Cas12a in the non-vascular plant Physcomitrella patens are largely unknown. Here, we demonstrate that LbCas12a together with its mature crRNA can target multiple loci simultaneously in P. patens with high efficiency via co-delivery of LbCas12a and a crRNA expression cassette in vivo. The mutation frequencies induced by CRISPR/LbCas12a at a single locus ranged from 26.5 to 100%, with diverse deletions being the most common type of mutation. Our method expands the repertoire of genome editing tools available for P. patens and facilitates the creation of loss-of-function mutants of multiple genes from different gene families.

Comprehensive analysis of the Ppatg3 mutant reveals that autophagy plays important roles in gametophore senescence in Physcomitrella patens.

BACKGROUND: Autophagy is an evolutionarily conserved system for the degradation of intracellular components in eukaryotic organisms. Autophagy plays essential roles in preventing premature senescence and extending the longevity of vascular plants. However, the mechanisms and physiological roles of autophagy in preventing senescence in basal land plants are still obscure. RESULTS: Here, we investigated the functional roles of the autophagy-related gene PpATG3 from Physcomitrella patens and demonstrated that its deletion prevents autophagy. In addition, Ppatg3 mutant showed premature gametophore senescence and reduced protonema formation compared to wild-type (WT) plants under normal growth conditions. The abundance of nitrogen (N) but not carbon (C) differed significantly between Ppatg3 mutant and WT plants, as did relative fatty acid levels. In vivo protein localization indicated that PpATG3 localizes to the cytoplasm, and in vitro Y2H assays confirmed that PpATG3 interacts with PpATG7 and PpATG12. Plastoglobuli (PGs) accumulated in Ppatg3, indicating that the process that degrades damaged chloroplasts in senescent gametophore cells was impaired in this mutant. RNA-Seq uncovered a detailed, comprehensive set of regulatory pathways that were affected by the autophagy mutation. CONCLUSIONS: The autophagy-related gene PpATG3 is essential for autophagosome formation in P. patens. Our findings provide evidence that autophagy functions in N utilization, fatty acid metabolism and damaged chloroplast degradation under non-stress conditions. We identified differentially expressed genes in Ppatg3 involved in numerous biosynthetic and metabolic pathways, such as chlorophyll biosynthesis, lipid metabolism, reactive oxygen species removal and the recycling of unnecessary proteins that might have led to the premature senescence of this mutant due to defective autophagy. Our study provides new insights into the role of autophagy in preventing senescence to increase longevity in basal land plants.

Functional redundancy and divergence of ß-carbonic anhydrases in Physcomitrella patens.

MAIN CONCLUSION: ß-carbonic anhydrases, which function in regulating plant growth, C/N status, and stomata number, showed functional redundancy and divergence in Physcomitrella patens. Carbonic anhydrases (CAs) catalyze the interconversion of CO2 and HCO3-. Plants have three evolutionarily unrelated CA families: α-, ß-, and γ-CAs. ßCAs are abundant in plants and are involved in CO2 assimilation, stress responses, and stomata formation. Recent studies of ßCAs have mainly examined C3 or C4 plants, whereas their functions in non-vascular plants are mostly unknown. In this study, phylogenetic analysis revealed that the evolution of ßCAs were conserved between subaerial green algae and bryophytes after terrestrialization event, and ßCAs from some cyanobacteria might begin evolving for the adaptation of terrestrial environment/habitat. In addition, we investigated the physiological roles of ßCAs in the basal land plant Physcomitrella patens. High PpßCA expression levels in different tissues suggest that PpßCAs play important roles in development in P. patens. Plants treated with 1-10 mM NaHCO3 had higher fresh and dry weight, PpßCA expression, total CA activity, and photosynthetic yield (Fv/Fm) compared with water-treated plants. However, treatment with 10 mM NaHCO3 influenced the C/N status. Further study of six Ppßca single-gene mutants revealed that PpßCAs have functional redundancy and divergence in regulating the C/N ratio of plants and stomatal formation. This study provides new insight into the physiological roles of ßCAs in basal land plants.

Genome-Wide Analysis of the MYB Transcription Factor Superfamily in Physcomitrella patens.

MYB transcription factors (TFs) are one of the largest TF families in plants to regulate numerous biological processes. However, our knowledge of the MYB family in Physcomitrella patens is limited. We identified 116 MYB genes in the P. patens genome, which were classified into the R2R3-MYB, R1R2R3-MYB, 4R-MYB, and MYB-related subfamilies. Most R2R3 genes contain 3 exons and 2 introns, whereas R1R2R3 MYB genes contain 10 exons and 9 introns. N3R-MYB (novel 3RMYB) and NR-MYBs (novel RMYBs) with complicated gene structures appear to be novel MYB proteins. In addition, we found that the diversity of the MYB domain was mainly contributed by domain shuffling and gene duplication. RNA-seq analysis suggested that MYBs exhibited differential expression to heat and might play important roles in heat stress responses, whereas CCA1-like MYB genes might confer greater flexibility to the circadian clock. Some R2R3-MYB and CCA1-like MYB genes are preferentially expressed in the archegonium and during the transition from the chloronema to caulonema stage, suggesting their roles in development. Compared with that of algae, the numbers of MYBs have significantly increased, thus our study lays the foundation for further exploring the potential roles of MYBs in the transition from aquatic to terrestrial environments.

A new fluorescence-based method to monitor the pH in the thylakoid lumen using GFP variants.

The ΔpH-dependent/Tat pathway is unique for using only the proton motive force for driving proteins transport across the thylakoid membrane in chloroplasts. 9-aminoacridine fluorescence quenching is widely used to monitor the ΔpH developed across the thylakoid membrane in the light. However, this method suffers from limited sensitivity to low ΔpH values and to spurious fluorescence signals due to membrane binding. In order to develop a more sensitive method for monitoring the real pH of the thylakoid lumen without these problems we transformed Arabidopsis thaliana with a ratiometric pH-sensitive GFP variant (termed pHluorin) targeted to the lumen by the prOE17 transit peptide. Positive transgenic plants displayed localization of pHluorin in the chloroplast by confocal microscopy, and fractionation experiments revealed that it is in the lumen. The pHluorin signal was the strongest in very young plants and diminished as the plants matured. The pHluorin released from the lumen displayed the expected fluorescence intensity changes in response to pH titration. The fluorescence signal in isolated chloroplasts responded to illumination in a manner consistent with light-dependent lumen acidification. Future experiments will exploit the use of this new pH-indicating probe of the thylakoid lumen to examine the influence of the thylakoid ΔpH on ATP synthesis and protein transport.

Mitochondrial energy-dissipation pathway and cellular redox disruption compromises Arabidopsis resistance to turnip crinkle virus infection.

Members of the plant mitochondrial energy-dissipation pathway (MEDP) coordinate cellular energy metabolism, redox homeostasis and the balance of ROS production. However, the roles of MEDP members, particularly uncoupling protein (UCP), in resistance to turnip crinkle virus infection (TCV) are poorly understood. Here, we showed that disrupting some MEDP genes compromises plant resistance to TCV viral infection and this is partly associated with damaged photosynthetic characteristics, altered cellular redox and increased ROS production. Experiments using mutant plants with impaired cellular compartment redox poising further demonstrated that impaired chloroplast/mitochondria and cystosol redox increases the susceptibility of plants to viral infection. Our results illustrate a mechanism by which MEDP and cellular compartment redox act in concert to regulate plant resistance to viral infections.

Unraveling the evolution and regulation of the alternative oxidase gene family in plants.

Alternative oxidase (AOX) is a diiron carboxylate protein present in all plants examined to date that couples the oxidation of ubiquinol with the reduction of oxygen to water. The predominant structure of AOX genes is four exons interrupted by three introns. In this study, by analyzing the genomic sequences of genes from different plant species, we deduced that intron/exon loss/gain and deletion of fragments are the major mechanisms responsible for the generation and evolution of AOX paralogous genes. Integrating gene duplication and structural information with expression profiles for various AOXs revealed that tandem duplication/block duplication contributed greatly to the generation and maintenance of the AOX gene family. Notably, the expression profiles based on public microarray database showed highly diverse expression patterns among AOX members in different developmental stages and tissues and that both orthologous and paralogous genes did not have the same expression profiles due to their divergence in regulatory regions. Comparative analysis of genes in six plant species under various perturbations indicated a large number of protein kinases, transcription factors and antioxidant enzymes are co-expressed with AOX. Of these, four sets of transcription factors--WRKY, NAC, bZIP and MYB--are likely involved in the regulating the differential responses of AOX1 genes to specific stresses. Furthermore, divergence of AOX1 and AOX2 subfamilies in regulation might be the main reason for their differential stress responses.

Roles of mitochondrial energy dissipation systems in plant development and acclimation to stress.

BACKGROUND: Plants are sessile organisms that have the ability to integrate external cues into metabolic and developmental signals. The cues initiate specific signal cascades that can enhance the tolerance of plants to stress, and these mechanisms are crucial to the survival and fitness of plants. The adaption of plants to stresses is a complex process that involves decoding stress inputs as energy-deficiency signals. The process functions through vast metabolic and/or transcriptional reprogramming to re-establish the cellular energy balance. Members of the mitochondrial energy dissipation pathway (MEDP), alternative oxidases (AOXs) and uncoupling proteins (UCPs), act as energy mediators and might play crucial roles in the adaption of plants to stresses. However, their roles in plant growth and development have been relatively less explored. SCOPE: This review summarizes current knowledge about the role of members of the MEDP in plant development as well as recent advances in identifying molecular components that regulate the expression of AOXs and UCPs. Highlighted in particular is a comparative analysis of the expression, regulation and stress responses between AOXs and UCPs when plants are exposed to stresses, and a possible signal cross-talk that orchestrates the MEDP, reactive oxygen species (ROS), calcium signalling and hormone signalling. CONCLUSIONS: The MEDP might act as a cellular energy/metabolic mediator that integrates ROS signalling, energy signalling and hormone signalling with plant development and stress accumulation. However, the regulation of MEDP members is complex and occurs at transcriptional, translational, post-translational and metabolic levels. How this regulation is linked to actual fluxes through the AOX/UCP in vivo remains elusive.

The roles of autophagy in development and stress responses in Arabidopsis thaliana.

Autophagy is a dynamic process that involves the recycling process of the degradation of intracellular materials. Over the past decade, our molecular and physiological understanding of plant autophagy has greatly been increased. Most essential autophagic machineries are conserved from yeast to plants. The roles that autophagy-related genes (ATGs) family play in the lifecycle of the Arabidopsis are proved to be similar to that in mammal. Autophagy is activated during certain stages of development, senescence or in response to starvation, or environmental stress in Arabidopsis. In the progression of autophagy, ATGs act as central signaling regulators and could develop sophisticated mechanisms to survive when plants are suffering unfavorable environments. It will facilitate further understanding of the molecular mechanisms of autophagy in plant. In this review, we will discuss recent advances in our understanding of autophagy in Arabidopsis, areas of controversy, and highlight potential future directions in autophagy research.

The role of phytomelatonin receptor 1-mediated signaling in plant growth and stress response.

Phytomelatonin is a pleiotropic signaling molecule that regulates plant growth, development, and stress response. In plant cells, phytomelatonin is synthesized from tryptophan via several consecutive steps that are catalyzed by tryptophan decarboxylase (TDC), tryptamine 5-hydroxylase (T5H), serotonin N-acyltransferase (SNAT), and N-acetylserotonin methyltransferase (ASMT) and/or caffeic acid-3-O-methyltransferase (COMT). Recently, the identification of the phytomelatonin receptor PMTR1 in Arabidopsis has been considered a turning point in plant research, with the function and signal of phytomelatonin emerging as a receptor-based regulatory strategy. In addition, PMTR1 homologs have been identified in several plant species and have been found to regulate seed germination and seedling growth, stomatal closure, leaf senescence, and several stress responses. In this article, we review the recent evidence in our understanding of the PMTR1-mediated regulatory pathways in phytomelatonin signaling under environmental stimuli. Based on structural comparison of the melatonin receptor 1 (MT1) in human and PMTR1 homologs, we propose that the similarity in the three-dimensional structure of the melatonin receptors probably represents a convergent evolution of melatonin recognition in different species.

Phytomelatonin interferes with flavonols biosynthesis to regulate ROS production and stomatal closure in tobacco.

Flavonols are well-known antioxidants that prevent stomatal closure via interfering with ROS signaling. Phytomelatonin regulates stomatal closure, but the signaling pathways are still largely unknown. Here, we investigated the role of flavonols in phytomelatonin-mediated stomatal closure in tobacco plants. The application of melatonin induced stomatal closure through NADPH oxidase-mediated ROS production. Transgenic tobacco plants overexpressing soybean GmSNAT1 (coding for serotonin N-acetyltransferase that catalyzes the penultimate step in phytomelatonin biosynthesis) had higher phytomelatonin concentration, accumulated more ROS in guard cells and were more sensitive to melatonin-induced stomatal closure than the wild-type plants, which was associated with the higher expression of PMTR1-homologous genes. Exogenous melatonin decreased flavonol concentrations in guard cells and the expression of flavonoid-related genes in wild-type and transgenic tobacco plants, and these inhibitory effects were more obvious in GmSNAT1-overexpressing plants than the wild type. However, the melatonin-mediated stomatal closure and ROS production were diminished by the application of kaempferol (a type of flavonol). Additionally, transgenic tobacco plants with increased expression of NtFLS (encoding flavonol synthase) were less sensitive to melatonin-induced stomatal closure. In conclusion, phytomelatonin hampers the biosynthesis of flavonols in guard cells, which results in high concentration of ROS and induces stomatal closure in tobacco plants.

Current research and future directions of melatonin's role in seed germination.

Seed germination is a complex process regulated by internal and external factors. Melatonin (N-acetyl-5-methoxytryptamine) is a ubiquitous signaling molecule, playing an important role in regulating seed germination under normal and stressful conditions. In this review, we aim to provide a comprehensive overview on melatonin's effects on seed germination on the basis of existing literature. Under normal conditions, exogenous high levels of melatonin can suppress or delay seed germination, suggesting that melatonin may play a role in maintaining seed dormancy and preventing premature germination. Conversely, under stressful conditions (e.g., high salinity, drought, and extreme temperatures), melatonin has been found to accelerate seed germination. Melatonin can modulate the expression of genes involved in ABA and GA metabolism, thereby influencing the balance of these hormones and affecting the ABA/GA ratio. Melatonin has been shown to modulate ROS accumulation and nutrient mobilization, which can impact the germination process. In conclusion, melatonin can inhibit germination under normal conditions while promoting germination under stressful conditions via regulating the ABA/GA ratios, ROS levels, and metabolic enzyme activity. Further research in this area will deepen our understanding of melatonin's intricate role in seed germination and may contribute to the development of improved seed treatments and agricultural practices.

Nitric oxide acts downstream of reactive oxygen species in phytomelatonin receptor 1 (PMTR1)-mediated stomatal closure in Arabidopsis.

Reactive oxygen species (ROS) and nitric oxide (NO) are important signaling molecules regulating stomatal movements in plants. Melatonin (N-acetyl-5-methoxytryptamine) was found to induce stomatal closure via phytomelatonin receptor 1 (PMTR1)-mediated activation of ROS production. Here, we evaluated the interaction between ROS and NO in the melatonin-induced stomatal closure in Arabidopsis. The results showed that the exogenous melatonin-induced stomatal closure and NO production were abolished by carboxy-PTIO (cPTIO, a NO scavenger). Additionally, the mutant lines nitrate reductase 1 and 2 (nia1nia2) and NO-associated 1 (noa1) did not show melatonin-induced stomatal closure, indicating that the melatonin-mediated stomatal closure is dependent on NO. The application of H2O2 induced the NO production and stomatal closure in the presence or absence of melatonin. However, the melatonin-induced NO production was impaired in the rhohC and rbohD/F (NADPH oxidase respiratory burst oxidase homologs) mutant plants. Furthermore, the ROS levels in nia1nia2 and noa1 did not differ significantly from the wild type plants, indicating that NO is a downstream component in the melatonin-induced ROS production. Exogenous melatonin did not induce NO and ROS production in the guard cells of pmtr1 mutant lines, suggesting NO occurs downstream of ROS in the PMTR1-mediated stomatal closure in Arabidopsis. Taken together, the results presented here suggest that melatonin-induced stomatal closure via PMTR1-mediated signaling in the regulation of ROS and NO production in Arabidopsis.

Physiological and Molecular Changes in Cherry Red Tobacco in Response to Iron Deficiency Stress.

The genotype CR60 is a spontaneous Cherry Red variant (containing granular red dapples on flue-cured leaves) of the Yunyan 87 (Y87) tobacco; it accumulates higher concentration of iron (Fe) in leaves than Y87, but the physiological differences between them remain largely unknown. We investigated the physiological and molecular mechanisms of CR60 in response to Fe deficiency under hydroponic conditions. Our results showed no significant phenotypic difference between Y87 and CR60 at optimal (40 µM) and high Fe (160 and 320 µM) concentrations. By contrast, CR60 exhibited higher tolerance to Fe deficiency (0 µM) than Y87, as shown by higher concentrations of chlorophyll in CR60 leaves after 21-day Fe-deficiency stress. Transcriptome profiling coupled with RT-PCR analyses found that the expression of IRT1 and several genes associated with chlorophyll biosynthesis and photosynthesis (e.g., PRO, GSA, FD1, PsbO, and PC) was higher in CR60 than Y87. These results indicated that CR60 maintains sufficient Fe uptake, chlorophyll biosynthesis and photosynthetic rate when subjected to Fe starvation.

Orphan gene PpARDT positively involved in drought tolerance potentially by enhancing ABA response in Physcomitrium (Physcomitrella) patens.

Almost all genomes have orphan genes, the majority of which are not functionally annotated. There is growing evidence showed that orphan genes may play important roles in the environmental stress response of Physcomitrium patens. We identified PpARDT (ABA-responsive drought tolerance) as a moss-specific and ABA-responsive orphan gene in P. patens. PpARDT is mainly expressed during the gametophytic stage of the life cycle, and the expression was induced by different abiotic stresses. A PpARDT knockout (Ppardt) mutant showed reduced dehydration-rehydration tolerance, and the phenotype could be rescued by exogenous ABA. Meanwhile, transgenic Arabidopsis lines exhibiting heterologous expression of PpARDT were more sensitive to exogenous ABA than wild-type (Col-0) plants and showed enhanced drought tolerance. These indicate that PpARDT confers drought tolerance among land plants potentially by enhancing ABA response. Further, we identified genes encoding abscisic acid receptor PYR/PYL family proteins, and ADP-ribosylation factors (Arf) as hub genes associated with the Ppardt phenotype. Given the lineage-specific characteristics of PpARDT, our results provide insights into the roles of orphan gene in shaping lineage-specific adaptation possibly by recruiting common pre-existed pathway components.

Combined Proteomic and Physiological Analysis of Chloroplasts Reveals Drought and Recovery Response Mechanisms in Nicotiana benthamiana.

Chloroplasts play essential roles in plant metabolic processes and stress responses by functioning as environmental sensors. Understanding chloroplast responses to drought stress and subsequent recovery will help the ability to improve stress tolerance in plants. Here, a combined proteomic and physiological approach was used to investigate the response mechanisms of Nicotiana benthamiana chloroplasts to drought stress and subsequent recovery. Early in the stress response, changes in stomatal movement were accompanied by immediate changes in protein synthesis to sustain the photosynthetic process. Thereafter, increasing drought stress seriously affected photosynthetic efficiency and led to altered expression of photosynthesis- and carbon-fixation-related proteins to protect the plants against photo-oxidative damage. Additional repair mechanisms were activated at the early stage of recovery to restore physiological functions and repair drought-induced damages, even while the negative effects of drought stress were still ongoing. Prolonging the re-watering period led to the gradual recovery of photosynthesis at both physiological and protein levels, indicating that a long repair process is required to restore plant function. Our findings provide a precise view of drought and recovery response mechanisms in N. benthamiana and serve as a reference for further investigation into the physiological and molecular mechanisms underlying plant drought tolerance.