Clonal Transgenerational Effects of Parental Grazing Environment on Offspring Shade Avoidance

: Grassland plants that endure livestock grazing exhibit a dwarf phenotype, which can be transmitted to clonal offspring. Yet to date, it remains poorly understood whether such transgenerational dwarf effects alter the plants’ response to shade. Here, we conducted a common garden experiment under sunlight and shade conditions with clonal Leymus chinensis offspring, the parents of which had endured livestock overgrazing (OG) and non-grazing (NG) in the ﬁeld, respectively. Plant morphological, physiological, and transcriptomic analyses were carried out. The results indicated that NG offspring showed greater shade avoidance than OG offspring. That is, NG offspring exhibited greater plasticity of vegetative height and leaf width, which may be contributed to their greater photosynthetic capacity and gibberellin (GA3) content compared with OG offspring when treated with shade. In addition, RNA-Seq proﬁling showed that differentially expressed genes in NG offspring were mainly enriched in RNA modiﬁcation and metabolic processes, which facilitated rapid response to shade. Phytochrome interacting factors (PIFs) promoted downstream shade marker genes in NG offspring by signiﬁcantly downregulating the expression of PHYC , SPY , and DELLA . Our ﬁndings suggest that light conditions should be taken into account to better understand transgenerational dwarf effects induced by livestock grazing on grassland ecosystems. These results provide new insights into the inducible factors of phenotypic variations in grassland plants that experience grazing.


Introduction
Grasslands, which cover almost 40% of the Earth's surface [1][2][3], provide important ecosystem services, such as cultural inheritance, food production, water regulation, carbon storage, and climate mitigation.However, global grasslands have suffered degradation in recent decades, with up to 49% of these areas being affected to varying degrees [4][5][6].The degradation of grasslands presents a significant threat to ecological security, and seriously affects the livelihoods of millions of individuals who rely on grasslands [7].Grassland degradation is mainly due to climate change and improper management practices (e.g., overgrazing) [8].In grassland ecosystems, grazing is the most common and important land use practiced [6,9].However, livestock overgrazing can result in deteriorated soil conditions [10,11] and decreased grassland productivity and coverage [12], thereby damaging the grassland ecosystem's structure and functions.Not surprisingly, this remarkable aspect of grassland degradation has attracted extensive attention and a great deal of interest.
Grassland plants tend to exhibit phenotypic plasticity when faced with different kinds of stressful conditions [13].For example, livestock overgrazing reduces individual plant height, shortens and narrows leaves, and decreases stem length [14][15][16].Except for the immediate responses to trampling and herbivory in the field, some studies have demonstrated that these dwarf characteristics contribute to the regulation of photosynthetic and hormonal pathways [15,17].The dwarf characteristics may help plants to escape herbivores as much as possible, which is supposed to be a grazing avoidance strategy.Moreover, plants can "remember" the grazing stress history and the dwarf phenotype across clonal generations even when the grazing pressure is removed [17,18], a phenomenon that is called stress memory or clonal transgenerational effects [19][20][21].Among the published studies that show such transgenerational dwarfism effects are induced by livestock herbivory (see references above), very few have directly tested the effects on key ecological interactions such as the light environment.
Plant growth and development are strongly influenced by light; they need to capture adequate light to fix inorganic carbon through photosynthetic processes [22,23].The light conditions in natural habitats exhibit variability [23] due to the modulation of incident solar radiation in terms of both quantity and spectral quality by plant canopy and neighboring shade [24].In grassland ecosystems, overgrazing usually reduces vegetation coverage [25][26][27], resulting in a relaxation in the competition for light [28].Grazingexcluded plants are more likely to be exposed to gaps in dense vegetation during their lifetime compared with overgrazed plants.
The close proximity of competitors reduces the red/far-red (R: FR) light ratio, and can trigger shade-avoidance syndrome (SAS) due to the reduction of red light and a relative increase in far-red light [29,30].Plants can sense neighbor competitors through light-quality signals.Phytochromes, such as PHYB and PHYC, are photoreceptors, and they are used to monitor light intensity changes and regulate the stability or activity of phytochrome interacting factors (PIFs) when plants are under shade conditions [31,32].As negative regulators of photomorphogenesis, PIFs can maintain plants' dark-grown morphology.PIF-governed modulation of the gene expression of gibberellins (GAs) and the auxin-related signaling pathway is the major driver for shade avoidance [33].More evidence showed that GAs and auxin are required to regulate hypocotyl elongation in shade avoidance [34,35].DELLA proteins are GA-signaling repressors that can block plant growth and development [36][37][38], and light can promote DELLA protein accumulation by reducing GA levels [39].In the presence of GA, GA receptors, GID1 proteins, elevate their direct interaction with DELLA; in the absence of GA, nuclear-localized DELLA proteins accumulate to higher levels and abrogate light control of hypocotyl elongation.DELLA proteins can restrain plant growth largely through a diverse group of transcriptional regulators and gene expression [38,40].
Overgrazing enables grassland plants to decrease light competition, which slows plant growth, resulting in dwarf traits.Some studies have suggested that the effects of the light conditions experienced by parent plants may persist across offspring generations [20,41].Thus, this study focused on two questions: (i) Do grazing-excluded offspring exhibit stronger shade avoidance compared with overgrazed offspring?(ii) If so, how?We attempted to address these questions by comparing the responses of grazing-excluded and overgrazed offspring to shade under a common garden experiment with a rhizome species, Leymus chinensis, which is the dominant species in the Mongolia grassland.

Field Site and Parental Generation Materials
The parental generation plants grew in the semi-arid steppe ecosystem of the Inner Mongolia Grassland Ecosystem Research Station (43 • 38 30 N, 116 • 42 20 E) in northern China.This research area has a semi-arid continental climate, with an average annual precipitation (2012-2018) of about 340 mm [42] and an average annual temperature of 0.3 • C. In addition, the monthly average temperature ranges from −21.6 • C in January to 19.0 • C in July [43].Plant communities are dominated by the rhizome grass L. chinensis and bunch grasses Agropyron cristatum and Stipa grandis.The soil type is classified as dark Agronomy 2024, 14, 1085 3 of 16 chestnut, with 48.6% sand, 25.3% clay, and 26.1% silt in the top 10 cm of soil [44].The study sites consisted of two adjacent areas, non-grazing (NG) (600 × 400 m) and overgrazing (OG) (600 × 100 m), respectively.They were separated by a pasture fence.NG plots had been fenced since 1983 to avoid livestock grazing; OG plots had been grazed at a stocking rate of ~3 sheep units per hectare [45].Livestock grazing begins in early June and then ends in early October every year.The recommended stocking rate is 1.5 sheep units per hectare, and the OG area has become severely degraded.In September 2020, L. chinensis clonal offspring were randomly collected from the research plots, respectively (2 parental treatments × 16; N = 32) (Figure 1).China.This research area has a semi-arid continental climate, with an average annual precipitation (2012-2018) of about 340 mm [42] and an average annual temperature of 0.3 °C.In addition, the monthly average temperature ranges from −21.6 °C in January to 19.0 °C in July [43].Plant communities are dominated by the rhizome grass L. chinensis and bunch grasses Agropyron cristatum and Stipa grandis.The soil type is classified as dark chestnut, with 48.6% sand, 25.3% clay, and 26.1% silt in the top 10 cm of soil [44].The study sites consisted of two adjacent areas, non-grazing (NG) (600 × 400 m) and overgrazing (OG) (600 × 100 m), respectively.They were separated by a pasture fence.NG plots had been fenced since 1983 to avoid livestock grazing; OG plots had been grazed at a stocking rate of ~3 sheep units per hectare [45].Livestock grazing begins in early June and then ends in early October every year.The recommended stocking rate is 1.5 sheep units per hectare, and the OG area has become severely degraded.In September 2020, L. chinensis clonal offspring were randomly collected from the research plots, respectively (2 parental treatments × 16; N = 32) (Figure 1).

Common Garden Experiment of Offspring Generation
These collected offspring were then transplanted into individual plastic pots (25 cm in diameter and 16.5 cm deep) for cultivation under natural light conditions for nearly one year in the common garden, to avoid the resource-based maternal effect.The soil in the pots was taken from the garden, and then sieved and mixed to ensure uniformity.These pots were maintained in a well-watered condition by regular watering during the cultivation period.In the following spring, we performed a pairing shade experiment on L. chinensis.In detail, two uniformly sized young offspring in the same plot were selected and transplanted into individual pots (18 cm in diameter and 11 cm deep) and grown under the same conditions (2 parental treatments × 14 parent plants × 2 offspring treatments; N = 56) (Figure 1).(Several parent plants were excluded because they were not viable.)After 15 days of cultivation, one of these offspring pairs was placed on a big metal frame covered with a black shading net (shading rate 60% of natural light) (S), and the other one was still placed under natural light conditions (CK).After 2 months of sunlight and shade treatments, morphological traits, such as vegetative height, stem diameter, leaf length, and leaf width of the tallest clonal individual, as well as the number of ramets in each pot (with 14 replicates per treatment), were assessed as indicators of phenotypic variation.The second leaf from the top of each plant was chosen for the measurement of leaf

Common Garden Experiment of Offspring Generation
These collected offspring were then transplanted into individual plastic pots (25 cm in diameter and 16.5 cm deep) for cultivation under natural light conditions for nearly one year in the common garden, to avoid the resource-based maternal effect.The soil in the pots was taken from the garden, and then sieved and mixed to ensure uniformity.These pots were maintained in a well-watered condition by regular watering during the cultivation period.In the following spring, we performed a pairing shade experiment on L. chinensis.In detail, two uniformly sized young offspring in the same plot were selected and transplanted into individual pots (18 cm in diameter and 11 cm deep) and grown under the same conditions (2 parental treatments × 14 parent plants × 2 offspring treatments; N = 56) (Figure 1).(Several parent plants were excluded because they were not viable.)After 15 days of cultivation, one of these offspring pairs was placed on a big metal frame covered with a black shading net (shading rate 60% of natural light) (S), and the other one was still placed under natural light conditions (CK).After 2 months of sunlight and shade treatments, morphological traits, such as vegetative height, stem diameter, leaf length, and leaf width of the tallest clonal individual, as well as the number of ramets in each pot (with 14 replicates per treatment), were assessed as indicators of phenotypic variation.The second leaf from the top of each plant was chosen for the measurement of leaf traits.Then, fresh plant leaves were collected, promptly frozen in liquid nitrogen, and stored at −80 • C, for analysis of hormone levels, transcriptome sequencing, and qPCR assays.

Gas Exchange Measurements
After 2 months of shading treatment, the net photosynthetic rate (Pn), transpiration rate (E), stomatal conductance (gs), and inter-cellular CO 2 concentration (Ci) were determined.On a clear morning, the second plant leaf from the top was chosen.The photosynthetic characteristics were studied using the LI-6800 (LI-COR, Lincoln, NE, USA) photosynthetic measurement system from 9:00 to 11:00.There were 14 replicates per treatment.Water use efficiency (WUE) was then calculated by the formula WUE = Pn/E.The photosynthetically active radiation and CO 2 concentration were set according to the literature [46].

Determination of Hormone Contents
The contents of gibberellin (GA3), auxin (IAA), and abscisic acid (ABA) in L. chinensis leaves were determined.There were three samples of each group under different conditions.For each sample, frozen leaves (0.1 g) were placed in a 2 mL centrifuge tube, respectively.Methanol-acetonitrile-aqueous solution (40:40:20, v/v) was then added, mixed for 2 min, and the samples were extracted for 12 h at 4 • C under light protection conditions.At 4 • C, the mixed solution was subsequently centrifuged at 14,000 r/min for 10 min.Finally, its supernatant was used for the determination of three endogenous hormone (GA3, IAA, and ABA) contents using HPLC-MS/MS.The chromatographic conditions were set according to Qu et al. [17].

RNA Extraction, Library Construction, and Sequencing
To confirm the effects of transcriptional memory induced by the parental grazing environment (e.g., light availability) on offspring shade avoidance in L. chinensis, RNA-seq on leaves collected from plants under shade and sunlight conditions was carried out.There were three replicates per group, resulting in 12 datasets.First, a TRIzol reagent kit (Tiangen, Beijing, China) was used to extract the total RNA according to the manufacturer's protocol.The amount and quality of total RNA were assessed using a NanoDrop spectrophotometer (Thermo Scientific, Waltham, MA, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), respectively.The RIN (RNA integrity number) value of the samples directly reflects the quality of RNA [47,48].We observed that the RIN values were higher than 6.0 for all samples, indicating that these samples were of good quality (Supplementary Table S1).Then, mRNA was enriched by Oligo (dT) beads and fragmented using a fragmentation buffer.Next, random hexamer-primed reverse transcription was performed to obtain first-strand cDNA, and second-strand cDNA was synthesized by RNase H and DNA polymerase I. Finally, the cDNA fragments were purified using a QiaQuick PCR extraction kit (Qiagen, Venlo, The Netherlands), end-repaired, and ligated to Illumina sequencing adapters.After agarose gel electrophoresis, suitable products were collected to construct the final cDNA libraries.The libraries were then sequenced on an Illumina HiSeq2500 by Gene Denovo Co. (Guangzhou, China).All raw transcriptome data were deposited in the NCBI SRA database under accession number PRJNA988834.

RNA-Seq Data Processing and Functional Annotation
To obtain high-quality clean reads, raw reads were further filtered by fastp [49] (V 0.18.0).Next, they were de novo assembled into contiguous sequences (contigs) using the Trinity program [50], once the rRNA-mapped reads were removed.After that, contigs were assembled into Unigene, the abundances of which were estimated by expectation maximization (RSEM) [51].Differentially expressed genes (DEGs) with a false discovery rate (FDR) ≤ 0.05 and |log 2 fold change| ≥ 1 were identified by DESeq2 [52], and they were then analyzed for pathway enrichment by gene ontology (GO) [53].GO annotation and functional classification were analyzed by Blast2GO [54,55] and WEGO [56], respectively.

Quantitative Real-Time PCR Validation
Three L. chinensis leaf samples of each treatment under sunlight and shade conditions were analyzed.Based on the transcriptome data, 8 genes related to GA synthesis, metabolism, and signaling were selected, and they were detected by quantitative real-time PCR (qRT-PCR) analysis.The Actin gene in L. chinensis was used as the internal control for normalization.Amplification primers of these genes were designed by the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov,accessed on 3 September 2022).The gene sequences used were obtained from RNA-seq results, and the primers are provided in Supplementary Table S1.RNA was extracted as previously described, and then the cDNA Synthesis Kit (Takara, Bio Inc., Shiga, Japan) was used to synthesize cDNA.qPCR was performed on a CFX96 Connect TM Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) using the TB Green premix ExTaq (Tli RNaseH Plus) Kit (Takara, Bio Inc., Shiga, Japan) according to the manufacturer's instructions.The 2 − Ct method was used to calculate relative gene expression [57].The Pearson correlation analysis exhibited high similarity (r = 0.872) between the RNA-seq and qRT-PCR results, which indicated that the RNA-Seq data were effective (Supplementary Figure S1).

Statistical Analysis
The morphological responses to shade were analyzed by phenotypic plasticity index (PI), PI = |(S − CK)|, which was calculated according to the literature [58].Morphological responses to parental overgrazing were analyzed using an independent samples t-test (p < 0.05), while the responses to shade were analyzed using a paired samples t-test.Physiological responses to overgrazing and shade treatments were analyzed by independent samples t-test (p < 0.05).IBM SPSS Statistics 19.0 was used to complete all of the analyses in the study.The figures for morphology, photosynthesis, hormone content, and gene relative expression levels were generated in Origin 9.1.

Morphological Changes Induced by Shade in Offspring
Parental overgrazing evidently decreased the size of individual offspring, including plant vegetative height and leaf width (p < 0.05, Figure 2A,E), and increased the leaf inclination angle (epinastic) (p < 0.001, Figure 2C).Shade significantly increased both NG and OG vegetative height, leaf length, and leaf width, while it decreased the ramet number, leaf inclination angle, and stem diameter (p < 0.05, Figure 2A-F), except that the NG leaf inclination angle and OG leaf width did not reach a significant level (p > 0.05, Figure 2C,E).NG offspring grew taller and their leaf width was larger compared with OG offspring under shade treatments, and parental overgrazing decreased the plasticity and absolute variation of L. chinensis offspring height and leaf width (Figure 2A,E).

Shade alters Leaf Photosynthesis and GA3 Content in Offspring
To understand the underlying mechanism for these morphological alterations, we analyzed leaf photosynthesis and the GA3 content in NG and OG offspring.Leaf photosynthesis parameters did not differ significantly between NG and OG offspring under the indicates the absolute variation to shade."ns" indicates no significant difference; "*" near the blue and purple lines indicates significant differences between full light and shade treatments; "*" between the blue and purple error lines indicates significant differences between the two offspring groups under full light or shade treatments.Each plot displays the mean ± s.e.

Shade Alters Leaf Photosynthesis and GA3 Content in Offspring
To understand the underlying mechanism for these morphological alterations, we analyzed leaf photosynthesis and the GA3 content in NG and OG offspring.Leaf photosynthesis parameters did not differ significantly between NG and OG offspring under the control (full light) treatment (p > 0.05), except that WUE was evidently reduced by parental overgrazing (p < 0.05, Figure 3).Pn of NG offspring was not strongly affected, and WUE was significantly reduced by shade treatment, while gs, Ci, and E were significantly higher in shade than in full light treatment.In contrast, OG offspring photosynthesis was obviously inhibited, with strongly decreased Pn and E under shade conditions (p < 0.01, Figure 3A,E).
Under full light conditions, the GA3 content in OG offspring was reduced dramatically by 24.5% compared to that in NG offspring, although this difference failed to reach statistical significance (p > 0.05, Figure 4).Shade increased the content of GA3 by 34.4% in NG offspring, while no obvious change was observed in OG offspring (Figure 4).The content of GA3 in NG offspring was significantly higher than that in OG offspring (p < 0.05, Figure 4), but not the IAA and ABA contents (p > 0.05, Supplementary Figure S2) when plants were exposed to shade conditions.△ indicates the absolute variation to shade."ns" indicates no significant difference; "*" near the blue and purple lines indicates significant differences between full light and shade treatments; "*" between the blue and purple error lines indicates significant differences between the two offspring groups under full light or shade treatments.Each plot displays the mean ± s.e.

Shade alters Leaf Photosynthesis and GA3 Content in Offspring
To understand the underlying mechanism for these morphological alterations, we analyzed leaf photosynthesis and the GA3 content in NG and OG offspring.Leaf photosynthesis parameters did not differ significantly between NG and OG offspring under the control (full light) treatment (p > 0.05), except that WUE was evidently reduced by parental overgrazing (p < 0.05, Figure 3).Pn of NG offspring was not strongly affected, and WUE was significantly reduced by shade treatment, while gs, Ci, and E were significantly higher in shade than in full light treatment.In contrast, OG offspring photosynthesis was obviously inhibited, with strongly decreased Pn and E under shade conditions (p < 0.01, Figure 3A,E).Under full light conditions, the GA3 content in OG offspring was reduced dramatically by 24.5% compared to that in NG offspring, although this difference failed to reach statistical significance (p > 0.05, Figure 4).Shade increased the content of GA3 by 34.4% in NG offspring, while no obvious change was observed in OG offspring (Figure 4).The content of GA3 in NG offspring was significantly higher than that in OG offspring (p < 0.05, Figure 4), but not the IAA and ABA contents (p > 0.05, Supplementary Figure S2) when plants were exposed to shade conditions.

Transcriptional Responses in Offspring Leaves
Responsive to parental and offspring treatments, 12 RNA-seq libraries were constructed and sequenced in total to identify the DEGs in plant leaves (Supplementary Fig-

Transcriptional Responses in Offspring Leaves
Responsive to parental and offspring treatments, 12 RNA-seq libraries were constructed and sequenced in total to identify the DEGs in plant leaves (Supplementary Figure S3).A total of 381 DEGs (156 upregulated and 225 downregulated) were identified in the group CK-NG vs. CK-OG, but 1301 DEGs (591 up regulated and 710 downregulated) were identified in the group CK-NG vs. S-NG, and 909 DEGs (500 up regulated and 409 downregulated) were identified in the group CK-OG vs. S-OG (FDR < 0.05 and |log 2 FC| > 1) (Supplementary Figure S4).
GO enrichment analysis of the DEGs of the two offspring groups under full light conditions showed that the highly enriched terms of the biological process (BP) category were antibiotic metabolic and catabolic processes, defense response, and the glycogen biosynthetic process (Figure 5A).In NG offspring, GO enrichment analysis of DEGs under shade conditions showed that the highly enriched terms of the BP category were RNA modification and metabolic processes, nucleic acid phosphodiester bond hydrolysis, response to abiotic stimulus, the photosynthesis process, and chloroplast organization (Figure 5B).In OG offspring, the DEGs were mainly enriched in protein-chromophore linkage, protein folding, the photosynthesis process, and abiotic stimulus (Figure 5C).The DEGs in NG and OG offspring under shade conditions were mainly enriched in phosphorylation, protein phosphorylation, and modification processes, and the photosynthesis process (Figure 5D).

The Expression Levels of GA Signaling Pathway Genes
To further explore the molecular basis of shade avoidance conferred by shade events, the expression levels of GA signaling pathway genes in L. chinensis offspring leaves were analyzed by qPCR.Expression levels of GA negative regulatory gene SPY, phytochrome gene PHYC, and GA signaling repressor gene DELLA showed significant decreases (p < 0.05) in NG offspring when exposed to shade events (Figure 6A-C).Expression levels of SPY and PIF4 were dramatically reduced (p < 0.05) (Figure 6A,D), while PHYC and DELLA were not significantly regulated (p > 0.05) in OG offspring under shade conditions (Figure 6B,C).
analyzed by qPCR.Expression levels of GA negative regulatory gene SPY, phytochrome gene PHYC, and GA signaling repressor gene DELLA showed significant decreases (p < 0.05) in NG offspring when exposed to shade events (Figure 6A-C).Expression levels o SPY and PIF4 were dramatically reduced (p < 0.05) (Figure 6A,D), while PHYC and DELLA were not significantly regulated (p > 0.05) in OG offspring under shade conditions (Figure 6B,C).

Discussion
Our study provides clear evidence that the parental overgrazing environment can trigger clonal transgenerational plasticity with dwarf vegetative height, smaller leaves and increased leaf angle in the perennial grassland species L. chinensis (Figure 2), which i in line with previous studies [19,59,60].Similar results were found in the clonal specie Alternanthera philoxeroides, where parental herbivory could affect the growth and defense of offspring plants [61].Emerging research has shown that such transgenerational effect are mediated by epigenetic modifications such as DNA methylation, histone modification and chromatin remodeling, without alteration of the nucleotide sequence that can persis across multiple generations [62][63][64][65].Our previous research further demonstrated tha clonal transgenerational dwarfism effects induced by grazing history in L. chinensis were mediated by DNA methylation [46].DNA methylation, catalyzed by DNA methyltrans ferases, is currently the most extensively investigated epigenetic mechanism [66][67][68].

Discussion
Our study provides clear evidence that the parental overgrazing environment can trigger clonal transgenerational plasticity with dwarf vegetative height, smaller leaves, and increased leaf angle in the perennial grassland species L. chinensis (Figure 2), which is in line with previous studies [19,59,60].Similar results were found in the clonal species Alternanthera philoxeroides, where parental herbivory could affect the growth and defense of offspring plants [61].Emerging research has shown that such transgenerational effects are mediated by epigenetic modifications such as DNA methylation, histone modification, and chromatin remodeling, without alteration of the nucleotide sequence that can persist across multiple generations [62][63][64][65].Our previous research further demonstrated that clonal transgenerational dwarfism effects induced by grazing history in L. chinensis were mediated by DNA methylation [46].DNA methylation, catalyzed by DNA methyltransferases, is currently the most extensively investigated epigenetic mechanism [66][67][68].
Individual dwarf variation in L. chinensis is supposed to be a grazing avoidance strategy and generally assumed to result from livestock herbivory.Here, the results of our study suggested that light conditions could be a crucial factor affecting plant morphological characteristics in grasslands.In addition, in the annual plant Polygonum persicaria, the content-dependent effects of the parental light environment on seedling development were also mediated by DNA methylation [69].It was suggested that light conditions in the field might be one of the important factors inducing DNA methylation in grassland plants, but empirical evidence remains to be further studied.
In detail, the results in this study showed that shade triggers morphological shade avoidance syndrome (SAS) both in NG and OG offspring, with higher plant vegetative height, leaf length, leaf width, and lower ramet number, stem diameter, and leaf inclination angle (Figure 2), which can be interpreted as active plastic responses [24,70].To cope with spatial and temporal variations in light, plants often display a broad spectrum of phenotypic plasticity [71].Earlier studies have also indicated that plants tend to produce larger leaves and longer internodes to intercept light efficiently [72,73].Shade with a reduction in the R/FR ratio negatively affected vegetative ramet number [70].Reallocation of energy resources from storage organs (e.g., root) to stem and leaf enabled the shaded plants to outgrow their competitors and enhance light capture [74].
Notably, NG offspring had amplified morphological SAS compared to OG offspring, with greater plasticity in vegetative height and leaf width when faced with shade, that is, NG offspring were more adaptable to low-light conditions.Such a difference may be a function of the parental environment, particularly light in the field.Transgenerational effects are thought to play an important role in the environmental adaption of clonal plants [75][76][77].The offspring phenotype can be modified by the environment experienced by their parental generation, and offspring tend to perform better when they grow under conditions similar to those of parent plants [21,78].In grasslands, livestock overgrazing creates better light conditions for plants due to the decrease in vegetation coverage and the reduction of neighbor shade; hence, OG plants could obtain more available light than NG plants.Thus, the parental low-light environment equipped NG offspring with greater adaptability to shade than OG offspring.Previous similar research showed that plant offspring of shade-grown parents produced greater leaf area and larger biomass compared with the offspring of sun-grown parents, and these shade-adaptive effects could be pronounced only in shade [69].Heger [79] also demonstrated in an annual grassland plant that light availability experienced in the field affected the ability of the following generations to respond to shade.
Variations in light variability have been shown to differentially induce gene transcription in plant leaves [80,81].Comparative RNA-seq analyses in conifers under full light and shade conditions showed that transcriptional regulations were primarily involved in pigment biosynthesis and hormone signaling [82].In full light, photoactivated PHYs directly interact with PIFs, causing their rapid ubiquitination, phosphorylation, and subsequent degradation, which could induce transcriptional reprogramming [83].A previous genomewide analysis in Arabidopsis revealed that there are more than 300 direct target genes transcriptionally controlled by PIFs, responding to different light environment [80].In this study, different transcriptional changes were induced by the parental grazing environment between NG and OG L. chinensis offspring when treated with shade (Figures 5 and 6).GO enrichment analysis of the DEGs of OG offspring showed that protein-chromophore linkage and protein folding were mainly enriched under shade conditions (Figure 5).However, RNA modification and metabolic processes and nucleic acid phosphodiester bond hydrolysis were highly enriched in NG offspring.Nucleic acid phosphodiester bonds are formed by catalyzing 3 -hydroxyls and 5 -phosphates in nucleic acid residues by DNA and RNA ligases [84,85].RNA modification and metabolic processes, as critical post-transcriptional regulators of gene expression programs, are required for plant normal development and stress response [86][87][88].RNA modification provides a direct and fast way to manipulate the existing transcriptome, thus increasing translation efficiency response to rapidly changing conditions [89,90].It was obvious that transcriptional memory is a very important factor to better cope with shade in NG offspring.
DEGs both in NG and OG offspring were highly enriched in photosynthesis, light harvesting, and response to light stimulus under shade situations (Figure 5).When plants were faced with shade, the transcript gene abundance related to photosynthesis, light harvesting, and reaction was generally increased to acclimate to the low-light environment for optimizing the capture of the limited light resource [91].Light deficiency limited photosynthetic efficiency in OG offspring, as shown in Magnolia sinostellata [92] and Arachis hypogaea [93].By contrast, NG offspring likely showed a stronger shade avoidance response to shade, and photosynthetic capacity was not adversely affected.It was suggested that NG offspring could use light energy more efficiently than OG offspring.
The shade avoidance response is tightly coordinated by interactions between light signals and hormones.As an important endogenous hormone, GA can greatly increase cell division number through activating intermediate meristems [94] and enhance plant tolerance to various biotic or abiotic stresses [95].The GA3 content in NG offspring leaves was significantly higher than that in OG offspring leaves under shade conditions.Shade increased the content of GA3 by 34.4% in NG offspring leaves (Figure 4), and similar results were obtained in a previous study of soybean leaves [96].The GA response to shade is an active stress response that allows plants to have better adaptability.Moreover, the exogenous application of GA could enhance the adaptability of plants to low-light conditions by improving photosynthesis [97,98].Therefore, NG offspring showing greater shade avoidance might be through GA-related signaling pathways and photosynthesis regulation.
In NG offspring, there was higher GA3 content than in OG offspring under shade treatments, and GA signal transduction was activated as the expression of negative regulatory gene SPY was significantly reduced (Figure 7).GA functions through the degradation of DELLA protein, which can also function to interact with PIF4 transcription factors and repress their activity.PIF4 represents a direct link between phytochromes/DELLA and the regulation of shade marker genes [99], such as photosynthetic genes [100], and promotes shade avoidance [101,102].Our study suggested that DELLA protein may be degraded in NG offspring, but not in OG offspring.In addition, the reduced expression of phytochrome gene PHYC in NG offspring not only reflected a decreased shade sensing by plants but also a relieved repression of PIF4 (Figure 7).The perception of light signals by the phytochrome family of photoreceptors has a crucial influence on almost all aspects of growth and development throughout a plant's life cycle [83].Moreover, the expression of PIF4 in OG offspring was reduced, but not in NG offspring.Combined, NG offspring with a higher GA3 content showed stronger shade avoidance than OG offspring under shade conditions by significantly downregulating the expression of PHYC, SPY, and DELLA (Figures 6 and 7).
cell division number through activating intermediate meristems [94] and enhanc tolerance to various biotic or abiotic stresses [95].The GA3 content in NG offspring was significantly higher than that in OG offspring leaves under shade conditions increased the content of GA3 by 34.4% in NG offspring leaves (Figure 4), and sim sults were obtained in a previous study of soybean leaves [96].The GA response t is an active stress response that allows plants to have better adaptability.Moreo exogenous application of GA could enhance the adaptability of plants to low-ligh tions by improving photosynthesis [97,98].Therefore, NG offspring showing shade avoidance might be through GA-related signaling pathways and photosy regulation.
In NG offspring, there was higher GA3 content than in OG offspring unde treatments, and GA signal transduction was activated as the expression of negativ latory gene SPY was significantly reduced (Figure 7).GA functions through the d tion of DELLA protein, which can also function to interact with PIF4 transcription and repress their activity.PIF4 represents a direct link between phytochromes/ and the regulation of shade marker genes [99], such as photosynthetic genes [10 promotes shade avoidance [101,102].Our study suggested that DELLA protein degraded in NG offspring, but not in OG offspring.In addition, the reduced exp of phytochrome gene PHYC in NG offspring not only reflected a decreased shade by plants but also a relieved repression of PIF4 (Figure 7).The perception of light by the phytochrome family of photoreceptors has a crucial influence on almost all of growth and development throughout a plant's life cycle [83].Moreover, the exp of PIF4 in OG offspring was reduced, but not in NG offspring.Combined, NG off with a higher GA3 content showed stronger shade avoidance than OG offspring shade conditions by significantly downregulating the expression of PHYC, SP DELLA (Figures 6 and 7).

Conclusions
Our findings demonstrated that the parental grazing environment changed clonal offspring shade avoidance in L. chinensis.NG offspring showed greater shade avoidance than OG offspring via transcriptional memory and photosynthetic and gibberellin pathways.These results strongly indicated that clonal transgenerational dwarf effects could be partly caused by low-light competition, except for livestock herbivory in grasslands.That is, parental overgrazing enabled L. chinensis to better adapt to the light environment (lower light competition), which reduced the plants' competition for light, thus contributing to

Figure 1 .
Figure 1.Schematic diagram of the experimental design to test clonal transgenerational effects of parental grazing environment on offspring shade avoidance in L. chinensis.

Figure 1 .
Figure 1.Schematic diagram of the experimental design to test clonal transgenerational effects of parental grazing environment on offspring shade avoidance in L. chinensis.

Figure 2 .
Figure 2. Parental overgrazing effects on plant morphology of offspring in shade (S) and full light (CK) treatments.(A), Height; (B), Ramet number; (C), Leaf angle; (D), Leaf length; (E), Leaf width; (F), Stem diameter.The blue and purple lines indicate the responses of NG and OG offspring to shade, respectively.△indicates the absolute variation to shade."ns" indicates no significant difference; "*" near the blue and purple lines indicates significant differences between full light and shade treatments; "*" between the blue and purple error lines indicates significant differences between the two offspring groups under full light or shade treatments.Each plot displays the mean ± s.e.

Figure 2 .
Figure 2. Parental overgrazing effects on plant morphology of offspring in shade (S) and full light (CK) treatments.(A), Height; (B), Ramet number; (C), Leaf angle; (D), Leaf length; (E), Leaf width; (F), Stem diameter.The blue and purple lines indicate the responses of NG and OG offspring to shade, respectively.indicatesthe absolute variation to shade."ns" indicates no significant difference; "*" near the blue and purple lines indicates significant differences between full light and shade treatments; "*" between the blue and purple error lines indicates significant differences between the two offspring groups under full light or shade treatments.Each plot displays the mean ± s.e.

Figure 2 .
Figure 2. Parental overgrazing effects on plant morphology of offspring in shade (S) and full light (CK) treatments.(A), Height; (B), Ramet number; (C), Leaf angle; (D), Leaf length; (E), Leaf width; (F), Stem diameter.The blue and purple lines indicate the responses of NG and OG offspring to shade, respectively.△indicates the absolute variation to shade."ns" indicates no significant difference; "*" near the blue and purple lines indicates significant differences between full light and shade treatments; "*" between the blue and purple error lines indicates significant differences between the two offspring groups under full light or shade treatments.Each plot displays the mean ± s.e.

Figure 3 . 16 Figure 3 .
Figure 3. Effects of parental overgrazing on offspring photosynthesis in shade (S) and full light (CK) treatments.(A), Pn, net photosynthetic rate; (B), gs, stomatal conductance; (C), Ci, intercellular carbon dioxide concentration; (D), E, transpiration rate; (E), WUE, water use efficiency.The blue and purple lines indicate the responses of NG and OG offspring to shade, respectively."ns" indicates no significant difference; "*" near the blue and purple lines indicates significant differences between full light and shade treatments; "*" between the blue and purple error lines indicates significant differences between the two offspring groups under full light or shade treatments.

Figure 4 .
Figure 4. Effects of parental grazing environment on offspring GA3 content in shade (S) and full light (CK) treatments.The blue and purple lines indicate the responses of NG and OG offspring to shade, respectively."ns" indicates no significant difference; "*" between the blue and purple error lines indicates a significant difference between the two offspring groups under full light or shade treatments.
ure S3).A total of 381 DEGs (156 upregulated and 225 downregulated) were identified in the group CK-NG vs. CK-OG, but 1301 DEGs (591 up regulated and 710 downregulated) were identified in the group CK-NG vs. S-NG, and 909 DEGs (500 up regulated and 409

Figure 4 .
Figure 4. Effects of parental grazing environment on offspring GA3 content in shade (S) and full light (CK) treatments.The blue and purple lines indicate the responses of NG and OG offspring to shade, respectively."ns" indicates no significant difference; "*" between the blue and purple error lines indicates a significant difference between the two offspring groups under full light or shade treatments.

Figure 6 .
Figure 6.Real-time quantitative PCR analysis for offspring leaf genes involved in the shade-related signaling pathway under shade (S) and full light (CK) treatments.(A), SPY; (B), PHYC; (C), DELLA (D), PIF4.The values are the mean of the three samples ± standard error.Different letters indicat significant differences (p ≤ 0.05), while the same letter indicates that there is no significant differenc (p > 0.05).

Figure 6 .
Figure 6.Real-time quantitative PCR analysis for offspring leaf genes involved in the shade-related signaling pathway under shade (S) and full light (CK) treatments.(A), SPY; (B), PHYC; (C), DELLA; (D), PIF4.The values are the mean of the three samples ± standard error.Different letters indicate significant differences (p ≤ 0.05), while the same letter indicates that there is no significant difference (p > 0.05).

Figure 7 .
Figure 7.A model depicting the shade-avoidance response in NG offspring.Significantly downregulated genes induced by shade are surrounded by blue lines, and not significantly regulated genes are surrounded by black lines.Red arrow heads and black end lines indicate positive and negative regulation, respectively.