Mesophyll conductance limits photosynthesis in fluctuating light under combined drought and heat stresses.

Drought and heat stresses usually occur concomitantly in nature, with increasing frequency and intensity of both stresses expected due to climate change. The synergistic agricultural impacts of these compound climate extremes are much greater than those of the individual stresses. However, the mechanisms by which drought and heat stresses separately and concomitantly affect dynamic photosynthesis have not been thoroughly assessed. To elucidate this, we used tomato (Solanum lycopersicum) seedlings to measure dynamic photosynthesis under individual and compound stresses of drought and heat. Individual drought and heat stresses limited dynamic photosynthesis at the stages of diffusional conductance to CO2 and biochemistry, respectively. However, the primary limiting factor for photosynthesis shifted to mesophyll conductance under the compound stresses. Compared with the control, photosynthetic carbon gain in fluctuating light decreased by 38%, 73%, and 114% under the individual drought, heat, and compound stresses, respectively. Therefore, compound stresses caused a greater reduction in photosynthetic carbon gain in fluctuating light conditions than individual stress. These findings highlight the importance of mitigating the effects of compound climate extremes on crop productivity by targeting mesophyll conductance and improving dynamic photosynthesis.

Metabolic and transcriptomic analyses elucidate a novel insight into the network for biosynthesis of carbohydrate and secondary metabolites in the stems of a medicinal orchid Dendrobium nobile.

Dendrobium nobile is an important medicinal and nutraceutical herb. Although the ingredients of D. nobile have been identified as polysaccharides, alkaloids, amino acids, flavonoids and bibenzyls, our understanding of the metabolic pathways that regulate the synthesis of these compounds is limited. Here, we used transcriptomic and metabolic analyses to elucidate the genes and metabolites involved in the biosynthesis of carbohydrate and several secondary metabolites in the stems of D. nobile. A total of 1005 metabolites and 31,745 genes were detected in the stems of D. nobile. The majority of these metabolites and genes were involved in the metabolism of carbohydrates (fructose, mannose, glucose, xylulose and starch), while some were involved in the metabolism of secondary metabolites (alkaloids, ß-tyrosine, ferulic acid, 4-hydroxybenzoate and chrysin). Our predicted regulatory network indicated that five genes (AROG, PYK, DXS, ACEE and HMGCR) might play vital roles in the transition from carbohydrate to alkaloid synthesis. Correlation analysis identified that six genes (ALDO, PMM, BGLX, EGLC, XYLB and GLGA) were involved in carbohydrate metabolism, and two genes (ADT and CYP73A) were involved in secondary metabolite biosynthesis. Our analyses also indicated that phosphoenol-pyruvate (PEP) was a crucial bridge that connected carbohydrate to alkaloid biosynthesis. The regulatory network between carbohydrate and secondary metabolite biosynthesis established will provide important insights into the regulation of metabolites and biological systems in Dendrobium species.

Simulation and optimization of scrap wagon dismantling system based on Plant Simulation.

Based on the existing plant layout and process flow, a simulation analysis was conducted using the Plant Simulation platform with the utilization efficiency of each station and production capacity of the dismantling system as indicators. A problem with long-term suspension in the disassembly process was determined. Based on the two optimization directions of increasing material transportation equipment and expanding the buffer capacity, a cost-oriented optimization model is established. A genetic algorithm and model simulation were used to solve the model. An optimization scheme that satisfies the production needs and has the lowest cost is proposed. The results show that the optimized dismantling system solves the suspended work problem at the dismantling station and a significant improvement in productivity and station utilization efficiency compared with the previous system.

Age-related differences in physiological and metabolic responses of Pleione aurita (Orchidaceae) pseudobulbs to drought stress and recovery.

The pseudobulb is a storage organ for water and nutrients that plays a crucial role in the growth and survival of epiphytic orchids. However, the role of water and metabolites in pseudobulb during adaptation to environmental stress are rarely detected through control experiments. In the present study, water-related physiological traits and metabolite changes in the pseudobulbs at the flowering stage and full leaf expansion stage for Pleione aurita were investigated after drought stress and recovery treatments. We found that the composition of non-structural carbohydrates (starch vs. soluble sugar) varied over the lifetime of pseudobulbs, and older pseudobulbs stored more water, whereas younger pseudobulbs stored more dry matter. When plants were subjected to drought stress and subsequent recovery, multiple metabolites in the pseudobulbs including non-structural carbohydrates, flavonoids, phenolic acids, as well as amino acids and their derivatives responded positively to these water level fluctuations. For those metabolites that differently accumulated in both stress and recovery processes, old pseudobulbs contained a higher number of these key metabolites than did the connected younger pseudobulbs. In addition, young and old pseudobulbs use different metabolic pathways to both respond and recover to drought. These results indicate that orchid pseudobulbs cope with water level fluctuations by mobilizing metabolite reserves and that pseudobulbs of different ages exhibit different physiological and metabolic responses to drought stress. These findings broadens our understanding of the role pseudobulbs play in the survival of orchids growing in epiphytic habitats.

High-quality Cymbidium mannii genome and multifaceted regulation of crassulacean acid metabolism in epiphytes.

Epiphytes with crassulacean acid metabolism (CAM) photosynthesis are widespread among vascular plants, and repeated evolution of CAM photosynthesis is a key innovation for micro-ecosystem adaptation. However, we lack a complete understanding of the molecular regulation of CAM photosynthesis in epiphytes. Here, we report a high-quality chromosome-level genome assembly of a CAM epiphyte, Cymbidium mannii (Orchidaceae). The 2.88-Gb orchid genome with a contig N50 of 22.7 Mb and 27 192 annotated genes was organized into 20 pseudochromosomes, 82.8% of which consisted of repetitive elements. Recent expansions of long terminal repeat retrotransposon families have made a major contribution to the evolution of genome size in Cymbidium orchids. We reveal a holistic scenario of molecular regulation of metabolic physiology using high-resolution transcriptomics, proteomics, and metabolomics data collected across a CAM diel cycle. Patterns of rhythmically oscillating metabolites, especially CAM-related products, reveal circadian rhythmicity in metabolite accumulation in epiphytes. Genome-wide analysis of transcript and protein level regulation revealed phase shifts during the multifaceted regulation of circadian metabolism. Notably, we observed diurnal expression of several core CAM genes (especially ßCA and PPC) that may be involved in temporal fixation of carbon sources. Our study provides a valuable resource for investigating post-transcription and translation scenarios in C. mannii, an Orchidaceae model for understanding the evolution of innovative traits in epiphytes.

Physiological response and photosynthetic recovery to an extreme drought: Evidence from plants in a dry-hot valley savanna of Southwest China.

The frequency of extreme drought events has been rising worldwide, but due to its unpredictability, how plants will respond remains poorly understood. Here, we aimed to characterize how the hydraulics and photosynthesis of savanna plants respond to extreme drought, and tested whether they can subsequently recover photosynthesis after drought. There was an extreme drought in 2019 in Southwest (SW) China. We investigated photosynthetic gas exchange, leaf-, stem-, and whole-shoot hydraulic conductance of 18 plant species with diverse leaf habits (deciduous, semi-deciduous and evergreen) and growth forms (tree and shrub) from a dry-hot valley savanna in SW China for three rainy seasons from 2019 to 2021. We also compared photosynthetic gas exchange to those of a regular year (2014). We found that leaf stomatal and hydraulic conductance and maximum photosynthetic rate were significantly lower during the drought in 2019 than in the wetter years. In 2019, all studied plants maintained stomatal conductance at their minimum level observed, which could be related to high vapor pressure deficits (VPD, >2 kPa). However, no significant difference in stem and shoot hydraulic conductance was detected across years. The reductions in leaf hydraulic conductance and stomatal regulation under extreme drought might help keep the stem hydraulic function. Stomatal conductance and photosynthesis after drought (2020 and 2021) showed comparable or even higher values compared to that of 2014, suggesting high recovery of photosynthetic gas exchange. In addition, the response of hydraulic and photosynthetic traits to extreme drought was convergent across leaf habits and growth forms. Our results will help better understand the physiological mechanism underlying the response of savanna ecosystems to climate change.

Drought stress delays photosynthetic induction and accelerates photoinhibition under short-term fluctuating light in tomato.

Fluctuating light (FL) and drought stress usually occur concomitantly. However, whether drought stress affects photosynthetic performance under FL remains unknown. Here, we measured gas exchange, chlorophyll fluorescence, and P700 redox state under FL in drought-stressed tomato (Solanum lycopersicum) seedlings. Drought stress significantly delayed the induction kinetics of stomatal and mesophyll conductances after transition from low to high light and thus delayed photosynthetic induction under FL. Therefore, drought stress exacerbated the loss of carbon gain under FL. Furthermore, restriction of CO2 fixation under drought stress aggravated the over-reduction of photosystem I (PSI) upon transition from low to high light. The resulting stronger FL-induced PSI photoinhibition significantly suppressed linear electron flow and PSI photoprotection. These results indicated that drought stress not only caused a larger loss of carbon gain under FL but also accelerated FL-induced photoinhibition of PSI. Furthermore, drought stress enhanced relative cyclic electron flow in FL, which partially compensated for restricted CO2 fixation and thus favored PSI photoprotection under FL. To our knowledge, we here show new insight into how drought stress affects photosynthetic performance under FL.

DNA barcoding of Cymbidium by genome skimming: Call for next-generation nuclear barcodes.

Cymbidium is an orchid genus that has undergone rapid radiation and has high ornamental, economic, ecological and cultural importance, but its classification based on morphology is controversial. The plastid genome (plastome), as an extension of plant standard DNA barcodes, has been widely used as a potential molecular marker for identifying recently diverged species or complicated plant groups. In this study, we newly generated 237 plastomes of 50 species (at least two individuals per species) by genome skimming, covering 71.4% of members of the genus Cymbidium. Sequence-based analyses (barcoding gaps and automatic barcode gap discovery) and tree-based analyses (maximum likelihood, Bayesian inference and multirate Poisson tree processes model) were conducted for species identification of Cymbidium. Our work provides a comprehensive DNA barcode reference library for Cymbidium species identification. The results show that compared with standard DNA barcodes (rbcL + matK) as well as the plastid trnH-psbA, the species identification rate of the plastome increased moderately from 58% to 68%. At the same time, we propose an optimized identification strategy for Cymbidium species. The plastome cannot completely resolve the species identification of Cymbidium, the main reasons being incomplete lineage sorting, artificial cultivation, natural hybridization and chloroplast capture. To further explore the potential use of nuclear data in identifying species, the Skmer method was adopted and the identification rate increased to 72%. It appears that nuclear genome data have a vital role in species identification and are expected to be used as next-generation nuclear barcodes.

Complementary water and nutrient utilization of perianth structural units help maintain long floral lifespan in Dendrobium.

Most orchids have high ornamental value with long-lived flowers. However, the mechanisms by which orchids maintain floral longevity are poorly understood. Here, we hypothesized that floral longevity in Dendrobium is maintained by high resource investment and complementary water and nutrient utilization in different structural units of the perianth. To test this hypothesis, we determined which water- and nutrient-related traits are correlated with flower longevity in 23 Dendrobium species or cultivars, and examined variations of the related traits during flower development of one long-lived cultivar. We found that floral longevity was correlated with dry mass per unit area of perianths and total flower biomass, which indicates that maintaining floral longevity requires increased resource investment. During development of long-lived flowers, labella showed a high capacity for water storage and nutrient reutilization, which could partly remedy high water demand and biomass investment. Sepals and petals, in contrast, had stronger desiccation avoidance and higher metabolic activity with lower biomass investment. These findings indicate that Dendrobium flowers maintain longevity by complementary water and nutrient utilization strategies in the sepals, petals and labella, with labella consuming more water and nutrients to extend flower display, and sepals and petals using a more conservative strategy.

The Dynamic Changes of Alternative Electron Flows upon Transition from Low to High Light in the Fern Cyrtomium fortune and the Gymnosperm Nageia nagi.

In photosynthetic organisms except angiosperms, an alternative electron sink that is mediated by flavodiiron proteins (FLVs) plays the major role in preventing PSI photoinhibition while cyclic electron flow (CEF) is also essential for normal growth under fluctuating light. However, the dynamic changes of FLVs and CEF has not yet been well clarified. In this study, we measured the P700 signal, chlorophyll fluorescence, and electrochromic shift spectra in the fern Cyrtomium fortune and the gymnosperm Nageia nagi. We found that both species could not build up a sufficient proton gradient (∆pH) within the first 30 s after light abruptly increased. During this period, FLVs-dependent alternative electron flow was functional to avoid PSI over-reduction. This functional time of FLVs was much longer than previously thought. By comparison, CEF was highly activated within the first 10 s after transition from low to high light, which favored energy balancing rather than the regulation of a PSI redox state. When FLVs were inactivated during steady-state photosynthesis, CEF was re-activated to favor photoprotection and to sustain photosynthesis. These results provide new insight into how FLVs and CEF interact to regulate photosynthesis in non-angiosperms.

Biomass and Active Compounds Accumulation of the Medicinal Orchid Pleione bulbocodioides in Response to Light Intensity and Irrigation Frequency.

Pseudobulbs of Pleione species are widely used as traditional medicine in Asian countries, but the mechanism of active compound accumulation remains unclear. In the present study, we investigated the accumulation of biomass and three active compounds (dactylorhin A, militarine and batatasin III) of Pleione bulbocodioides in response to different light intensities and irrigation frequencies. We found that single high light (65 % of full sunlight) or drought stress (14-day irrigation interval) increased active compounds accumulation but the combined effect of these two treatments decreased the total content of these three active compounds. This decrease was due to the plants under combined stress having a significantly lower photosynthetic rate, leaf area and longevity, leading to a dramatic decrease in pseudobulb biomass. Among all treatments, the highest total content of active compounds was observed in plants subjected to the high light level with a high water level (3-day irrigation interval), and plants under medium light intensity (30 % of full sunlight) also had considerable content of active compounds accumulation. To balance the quality and quantity of Pleione pseudobulbs during artificial cultivation, 30∼65 % of full sunlight with the avoidance of drought stress is recommended. Our results suggest the accumulation of the three active compounds is significantly influenced by light intensity and irrigation frequency, which may contribute to the artificial cultivation and quality control of medicinal Pleione.

Photosynthetic Induction Under Fluctuating Light Is Affected by Leaf Nitrogen Content in Tomato.

The response of photosynthetic CO2 assimilation to changes of illumination affects plant growth and crop productivity under natural fluctuating light conditions. However, the effects of nitrogen (N) supply on photosynthetic physiology after transition from low to high light are seldom studied. To elucidate this, we measured gas exchange and chlorophyll fluorescence under fluctuating light in tomato (Solanum lycopersicum) seedlings grown with different N conditions. After transition from low to high light, the induction speeds of net CO2 assimilation (A N ), stomatal conductance (g s ), and mesophyll conductance (g m ) delayed with the decline in leaf N content. The time to reach 90% of maximum A N , g s and g m was negatively correlated with leaf N content. This delayed photosynthetic induction in plants grown under low N concentration was mainly caused by the slow induction response of g m rather than that of g s . Furthermore, the photosynthetic induction upon transfer from low to high light was hardly limited by photosynthetic electron flow. These results indicate that decreased leaf N content declines carbon gain under fluctuating light in tomato. Increasing the induction kinetics of g m has the potential to enhance the carbon gain of field crops grown in infertile soil.

Leaf physiological and anatomical responses of two sympatric Paphiopedilum species to temperature.

Paphiopedilum dianthum and P. micranthum are two endangered orchid species, with high ornamental and conservation values. They are sympatric species, but their leaf anatomical traits and flowering period have significant differences. However, it is unclear whether the differences in leaf structure of the two species will affect their adaptabilities to temperature. Here, we investigated the leaf photosynthetic, anatomical, and flowering traits of these two species at three sites with different temperatures (Kunming, 16.7 ± 0.2 °C; Puer, 17.7 ± 0.2 °C; Menglun, 23.3 ± 0.2 °C) in southwest China. Compared with those at Puer and Kunming, the values of light-saturated photosynthetic rate (Pmax), stomatal conductance (gs), leaf thickness (LT), and stomatal density (SD) in both species were lower at Menglun. The values of Pmax, gs, LT, adaxial cuticle thickness (CTad) and SD in P. dianthum were higher than those of P. micranthum at the three sites. Compared with P. dianthum, there were no flowering plants of P. micranthum at Menglun. These results indicated that both species were less resistance to high temperature, and P. dianthum had a stronger adaptability to high-temperature than P. micranthum. Our findings can provide valuable information for the conservation and cultivation of Paphiopedilum species.

Regulation of Leaf Angle Protects Photosystem I under Fluctuating Light in Tobacco Young Leaves.

Fluctuating light is a typical light condition in nature and can cause selective photodamage to photosystem I (PSI). The sensitivity of PSI to fluctuating light is influenced by the amplitude of low/high light intensity. Tobacco mature leaves are tended to be horizontal to maximize the light absorption and photosynthesis, but young leaves are usually vertical to diminish the light absorption. Therefore, we tested the hypothesis that such regulation of the leaf angle in young leaves might protect PSI against photoinhibition under fluctuating light. We found that, upon a sudden increase in illumination, PSI was over-reduced in extreme young leaves but was oxidized in mature leaves. After fluctuating light treatment, such PSI over-reduction aggravated PSI photoinhibition in young leaves. Furthermore, the leaf angle was tightly correlated to the extent of PSI photoinhibition induced by fluctuating light. Therefore, vertical young leaves are more susceptible to PSI photoinhibition than horizontal mature leaves when exposed to the same fluctuating light. In young leaves, the vertical leaf angle decreased the light absorption and thus lowered the amplitude of low/high light intensity. Therefore, the regulation of the leaf angle was found for the first time as an important strategy used by young leaves to protect PSI against photoinhibition under fluctuating light. To our knowledge, we show here new insight into the photoprotection for PSI under fluctuating light in nature.

Selection and Validation of Reference Genes for Quantitative Real-Time PCR Analysis of Development and Tissue-Dependent Flower Color Formation in Cymbidium lowianum.

The development and tissue-dependent color formation of the horticultural plant results in various color pattern flowers. Anthocyanins and carotenoids contribute to the red and yellow colors, respectively. In this study, quantitative real-time polymerase chain reaction (qRT-PCR) is used to analyze the expression profiles of anthocyanin and carotenoids biosynthesis genes in Cymbidium lowianum (Rchb.f.) Rchb.f. Appropriate reference gene selection and validation are required before normalization of gene expression in qRT-PCR analysis. Thus, we firstly selected 12 candidate reference genes from transcriptome data, and used geNorm and Normfinder to evaluate their expression stability in lip (divided into abaxial and adaxial), petal, and sepal of the bud and flower of C. lowianum. Our results show that the two most stable reference genes in different tissues of C. lowianum bud and flower are EF1δ and 60S, the most unstable reference gene is 26S. The expression profiles of the CHS and BCH genes were similar to FPKM value profiles after normalization to the two most stable reference genes, EF1δ and 60S, with the upregulated CHS and BCH expression in flower stage, indicating that the ABP and CBP were activated across the stages of flower development. However, when the most unstable reference gene, 26S, was used to normalize the qRT-PCR data, the expression profiles of CHS and BCH differed from FPKM value profiles, indicating the necessity of selecting stable reference genes. Moreover, CHS and BCH expression was highest in the abaxial lip and adaxial lip, respectively, indicating that the ABP and CBP were activated in abaxial and adaxial lip, respectively, resulting in a presence of red or yellow segments in abaxial and adaxial lip. This study is the first to provide reference genes in C. lowianum, and also provide useful information for studies that aim to understand the molecular mechanisms of flower color formation in C. lowianum.

Diurnal Response of Photosystem I to Fluctuating Light Is Affected by Stomatal Conductance.

Upon a sudden transition from low to high light, electrons transported from photosystem II (PSII) to PSI should be rapidly consumed by downstream sinks to avoid the over-reduction of PSI. However, the over-reduction of PSI under fluctuating light might be accelerated if primary metabolism is restricted by low stomatal conductance. To test this hypothesis, we measured the effect of diurnal changes in stomatal conductance on photosynthetic regulation under fluctuating light in tomato (Solanum lycopersicum) and common mulberry (Morus alba). Under conditions of high stomatal conductance, we observed PSI over-reduction within the first 10 s after transition from low to high light. Lower stomatal conductance limited the activity of the Calvin-Benson-Bassham cycle and aggravated PSI over-reduction within 10 s after the light transition. We also observed PSI over-reduction after transition from low to high light for 30 s at the low stomatal conductance typical of the late afternoon, indicating that low stomatal conductance extends the period of PSI over-reduction under fluctuating light. Therefore, diurnal changes in stomatal conductance significantly affect the PSI redox state under fluctuating light. Moreover, our analysis revealed an unexpected inhibition of cyclic electron flow by the severe over-reduction of PSI seen at low stomatal conductance. In conclusion, stomatal conductance can have a large effect on thylakoid reactions under fluctuating light.

Mycorrhizal Fungal Partners Remain Constant during a Root Lifecycle of Pleione bulbocodioides (Orchidaceae).

Mycorrhizal mutualisms are vital for orchids through germination to adulthood. Fungal species diversity and community composition vary across seasons and plant development stages and affect plant survival, adaptation, and community maintenance. Knowledge of the temporal turnover of mycorrhizal fungi (OMF) remains poorly understood in the eco-physiologically diverse orchids (especially in epiphytic orchids), although it is important to understand the function and adaptation of mycorrhizae. Some species of Pleione are epiphytic plants with annual roots and may recruit different fungal partners during their root lifecycle. Based on continuous samplings of Pleione bulbocodioides during a whole root lifecycle, we characterized the fungal temporal dynamics using Illumina sequencing of the ITS2 region. Our data showed that the plants of P. bulbocodioides were quickly colonized by OMF at root emergence and had a constant OMF composition throughout one root lifecycle, although the OMF richness declined with root aging after a peak occurrence during root elongation. In contrast, the richness of root-inhabiting fungal endophytes kept increasing with root aging and more drastic turnovers were found in their species compositions. Our findings of OMF temporal turnover contribute to further understanding of mycorrhizal associations and adaptation of Orchidaceae and will benefit orchid resource conservation and utilization.

Allometry Between Vegetative and Reproductive Traits in Orchids.

In flowering plants, inflorescence characteristics influence both seed set and pollen contribution, while inflorescence and peduncle size can be correlated with biomass allocation to reproductive organs. Peduncles also play a role in water and nutrient supply of flowers, and mechanical support. However, it is currently unclear whether inflorescence size is correlated with peduncle size. Here, we tested whether orchids with large diameter peduncles bear more and larger flowers than those with smaller peduncles by analyzing 10 traits of inflorescence, flower, and leaf in 26 species. Peduncle diameters were positively correlated with inflorescence length and total floral area, indicating that species with larger peduncles tended to have larger inflorescences and larger flowers. We also found strongly positive correlation between inflorescence length and leaf area, and between total floral area and total leaf area, which suggested that reproductive organs may be allometrically coordinated with vegetative organs. However, neither flower number nor floral dry mass per unit area were correlated with leaf number or leaf dry mass per unit area, implying that the function between leaf and flower was uncoupled. Our findings provided a new insight for understanding the evolution of orchids, and for horticulturalists interested in improving floral and inflorescence traits in orchids.

Roles of alternative electron flows in response to excess light in Ginkgo biloba.

Ginkgo biloba L., the only surviving species of Ginkgoopsida, is a famous relict gymnosperm, it may provide new insight into the evolution of photosynthetic mechanisms. Flavodiiron proteins (FDPs) are conserved in nonflowering plants, but the role of FDPs in gymnosperms has not yet been clarified. In particular, how gymnosperms integrate FDPs and cyclic electron transport (CET) to better adapt to excess light is poorly understood. To elucidate these questions, we measured the P700 signal, chlorophyll fluorescence and electrochromic shift signal under fluctuating and constant light in G. biloba. Within the first seconds after light increased, G. biloba could not build up a sufficient proton gradient (ΔpH). Concomitantly, photo-reduction of O2 mediated by FDPs contributed to the rapid oxidation of P700 and protected PSI under fluctuating light. Therefore, in G. biloba, FDPs mainly protect PSI under fluctuating light at acceptor side. Under constant high light, the oxidation of PSI and the induction of non-photochemical quenching were attributed to the increase in ΔpH formation, which was mainly caused by the increase in CET rather than linear electron transport. Therefore, under constant light, CET finely regulates the PSI redox state and non-photochemical quenching through ΔpH formation, protecting PSI and PSII against excess light. We conclude that, in G. biloba, FDPs are particularly important under fluctuating light while CET is essential under constant high light. The coordination of FDPs and CET fine-tune photosynthetic apparatus under excess light.

Pre-illumination at high light significantly alleviates the over-reduction of photosystem I under fluctuating light.

Photosystem I (PSI) is the primary target of photoinhibition under fluctuating light (FL). In angiosperms, cyclic electron flow (CEF) around PSI is thought to be the main player protecting PSI under FL. The activation of CEF is linked to the thylakoid stacking, which is in turn affected by light intensity. However, it is unknown how pre-illumination affects the CEF activation and PSI redox state under FL. To address this question, we conducted a spectroscopic analysis under FL in Bletilla striata and Morus alba after pre-illumination at moderate light (ML, 611 µmol photons m-2 s-1) or high light (HL, 1455 µmol photons m-2 s-1). Our results indicated that both species displayed a transient over-reduction of PSI after a transition from low to high light, but the extent of PSI over-reduction under FL was largely alleviated by pre-illumination at HL when compared with pre-illumination under ML. Furthermore, pre-illumination at HL accelerated the activation rate of CEF but did not affect the activation of non-photochemical quenching and linear electron flow from photosystem II under FL. Therefore, such increased CEF activity by pre-illumination under HL alleviated PSI over-reduction under FL by facilitating the electron sink downstream of PSI. Taking together, pre-illumination at HL protects PSI under FL through acceptor-side regulation.