Introduction

Orchids are horticultural plants with important ornamental value and economic value (Li et al. 2021). Belonging to one of the largest families of angiosperms, orchids encompass approximately 880 genera and 27,800 species (Givnish et al. 2016). Phalaenopsis equestris, known as the “Queen of Orchids,” is globally cultivated (Lin et al. 2016; Zhang et al. 2018). Irradiation mutagenesis breeding, characterized by high variation frequencies and diverse variations, enables the production of stable offspring within a short breeding period. This method can induce changes in plant morphology, physiology, and biochemistry, leading to the development of new varieties with unique traits that are not achievable through other breeding methods. Consequently, it has been widely utilized for enhancing horticultural crops (Yamaguchi 2018). Among the mutagens used for this type of breeding, γ-ray is considered to be the most effective (Kovacs and Keresztes 2002). It can affect the process of plant growth and development. However, the molecular mechanisms underlying the changes in plant phenotypes induced by gamma irradiation have been little explored.

Low-dose irradiation has been shown to stimulate various physiological metabolic processes in cells, including increased enzyme activities, photosynthetic and respiratory rates, growth promotion, and enhanced immunity (Calabrese and Baldwin 2000; Upton 2001). In tomatoes, low-dose irradiation has been found to promote flowering and early fruit set (Alarkon et al. 1987) and stimulate stem growth, root branching, and root length in tissue culture seedlings (Charbaji and Nabulsi 1999). Similarly, low-dose irradiation has been reported to increase the growth rate, height, and fresh weight of wheat (Singh and Datta 2010), as well as accelerate the growth of rice and mung beans, resulting in increases in plant height, number of inflorescences, tillering, and number and length of pods and seeds (Maity et al. 2005).

The leaf, as the primary photosynthetic organ of plants, exhibits significant variations in morphology across different species, developmental stages, and growth conditions (Tsukaya 2014). Leaves play a crucial role in contributing to plant biomass, as the energy and carbohydrates produced during photosynthesis support the plant’s growth and life cycle (Demura and Ye 2010). After the establishment of leaf polarity in most plants, the leaf undergoes cell proliferation and expansion to attain its final size and shape (Yamaguchi et al. 2010). Leaf initiation begins with a group of primitive initiating cells that emerge laterally in the shoot apical meristem (Kalve et al. 2014). These primitive cells within the leaf primordia undergo extensive cell division, leading to an increase in the number of cells that ultimately determine the size of the leaf (Gonzalez et al. 2012). The cell wall, primarily composed of cellulose and pectin, serves as the interface between protoplasts and the extracellular environment in plant cells. It plays a crucial role in perceiving external changes and influences morphological characteristics and mechanical properties during growth and development (Szymanski and Cosgrove 2009; Somerville et al. 2004). Deletion of cellulase in Arabidopsis thaliana mutants leads to reduced cellulose contents, as well as decreased leaf and cotyledon areas (Williamson et al. 2001). Pectin is essential for cell growth and tissue morphological development (Anderson 2016, 2019). Transcription factors associated with various hormone pathways, such as abscisic acid, gibberellin, auxin, and cytokinin pathways, play significant roles in plant leaf development (Saidi and Hajibarat 2021). PeEXPA10 is an expansion that contributes to cell wall expansion and affects leaf growth and development by modulating the cell wall structure. The ectopic expression of EXP10 in A. thaliana increases the size of leaves and cells and the length of petioles, whereas the down-regulated expression of EXP10 has the opposite effects (Cosgrove 2015). In addition, the TCP family of transcription factors can regulate plant leaf morphology by affecting cell proliferation and differentiation (Aguilar-Martínez and Sinha 2013). NGA genes have a more general role in controlling lateral organ growth. TCP-NGA regulatory interactions may be conserved in angiosperms, including important crop species, and the regulation of leaf development is an important mechanism (Ballester et al. 2015). The leaves of the Arabidopsis nga mutant are wider and shorter than those of the wild type, and these phenotypes are also observed in other floral organs, especially sepals and petals (Fourquin and Ferrándiz 2014).In contrast, NGA overexpression produced small, narrow leaves and petals (Alvarez et al. 2009; Kwon et al. 2009; Trigueros et al. 2009). Some mir319-regulated TCP factors are activators of NGA expression, and NGA factors mediate the role of TCP in leaf morphogenesis to some extent. In Arabidopsis, TCP2 and TCP3, which belong to the CIN branch of the TCP family, are targets of miR319. TCP2 and TCP3 are able to activate the NGA3 promoter. And this TCP-NGA regulatory module appears to be conserved in distantly related species, including crops such as tomato, rice or soybean (Ballester et al. 2015).

In this study, P. equestris plants were irradiated with 60Co-γ-rays (40 Gy), which increased the soluble sugar content, antioxidant capacity, and leaf growth rate. The genes that were DEGs involved in leaf development and growth were screened. Additionally, the expression patterns and functions of PeNGA and PeEXPA10 were examined. This study revealed the molecular mechanism underlying the rapid growth of irradiated P. equestris plants. The generated data may be useful for optimizing the γ-ray mutagenesis of P. equestris cultivars and the selection of new germplasm with ideal traits.

Materials and methods

Plant growth conditions and irradiation treatment

The irradiated materials were from the greenhouse of the Plant Germplasm Resources Center of Shanghai Normal University. Twelve P. equestris of the same growth period were selected, 6 of which were used as control (CK-1, CK-2, CK-3, CK-4, CK-5, CK-6), and 6 of which were used for radiation treatment (FZ- 40 Gy-1, FZ-40 Gy-2, FZ-40 Gy-3, Fz-40 Gy-4, FZ-40 Gy-5, FZ-40 Gy-6). Irradiation treatment: a dose of 40 Gy using 60Co-γ-rays, with a dose rate of 150 Gy/h and an irradiation time of 16 min, was employed in the beam energy Irradiation Center of Shanghai Academy of Agricultural Sciences. Three strains of P equestris of CK and three strains of P equestris treated with 40 Gy were used for RNA-seq analysis.

P.equestris was cultivated under the standard greenhouse conditions, sunshine maintained for 12–14 h per day with the light intensity in 12,000–20000 lx; the temperature was set at 23 ◦C–27 ◦C; the relative humidity in the greenhouse was maintained at 60–80%.

P. equestris were used for VIGS gene silencing experiment, in the same growth period, and the number of infected plants was n > 10, respectively. Under the same growth conditions, the new leaves were observed, and the leaf growth data were recorded every seven days.

Determination of physiological indexes

Malondialdehyde (MDA) content determination (spectrophotometric method) (Li et al. 2000); Superoxide dismutase (SOD) activity (Lolium tetrazolium colourimetric method) (Li et al. 2000); Peroxidase (POD) activity (guaiacol method) (Li et al. 2000); Catalase (CAT) activity(ultraviolet absorption (UV)) ( Li et al. 2000);Soluble sugar content (ananthous colourimetric method) (Wei et al. 2014).

RNA-seq

We collected leaves, which are characterized by rapid growth and wild-type, for RNA-seq analysis. RNA purification, reverse transcription, library construction, and sequencing were performed at Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd.

According to the manufacturer’s instructions, using an Illumina TruSeqTM RNA Sample Preparation Kit (San Diego, CA, USA), we prepared the RNA-Seq transcriptome libraries, which were then sequenced in a single lane on an Illumina Hiseq ten/NovaSeq 6000 sequencer for 2 × 150 bp paired-end reads. The RNA-seq raw read data were processed using the software fastx_toolkit_0.0.14 (http://hannonlab.cshl.edu/fastx_toolkit/), SeqPrep (https://github.com/jstjohn/SeqPrep), and Sickle (https://github.com/najoshi/sickle) to evaluate and discard sequences of low quality and those affected by adaptor contamination. Quality control is shown in Supplementary Data Table 1,4. The clean reads were mapped to the reference genome sequence of P equestris. NR, Swiss-Prot, Pfam, COG, GO, and KEGG databases were used for gene annotation.

RNA sequencing data were uploaded to the NCBI database (accession number PRJNA899319). Expression data were converted to FPKM values and gene expression differences were calculated using TBtools Biologist software, with heat maps displayed in red and blue showing high and weak expression of genes, respectively.

Quantification of gene expression levels

HISAT2 (http://ccb.jhu.edu/software/hisat2/index.shtml) was used to count the number of reads mapped to each gene. The Read Counts of each sample gene/transcript were obtained using the results of the comparison to the genome as well as the genome annotation file. They were then converted to FPKM, which led to the normalized gene/transcript expression levels.

Differential expression analysis

Based on the results of expression quantification, differential gene analysis between groups was performed to obtain genes that were differentially expressed between the two groups, with the differential analysis software: DESeq2, and the screening threshold: |log2FC|> = 1 & padjust < 0.05(Qi et al. 2020; Anders and Huber 2012).

GO and KEGG enrichment analysis of DEGs

GO enrichment analysis of genes/transcripts in the gene set was performed using the software Goatools (https: //github.com/tanghaibao/GOatools), which is usually corrected for p-values by default and considered significantly enriched when the corrected P-value (FDR) is < 0.05 (Young et al. 2010). The KEGG PATHWAY enrichment analysis was performed by scripting the genes/transcripts in the gene set using the same principle as GO functional enrichment analysis.

Functional verification of virus-induced gene silencing (VIGS)

A specific fragment (300 bp) of PeEXPA10, PeNGA gene close to the 3´ end was inserted into the pCymMv vector by homologous recombination using ClonExpress®II One Step Cloning Kit (Vazyme Biotech). The VIGS silencing vectors pCymMv -PeEXPA10 and pCymMv -PeNGA were constructed. The recombinant expression transformants were placed in 5 ml of LB medium containing 100 μM Acetosyringone and 50 μg/ml kanamycin, so as to complete the activation of Agrobacterium cells, which were then cultured at 28℃ and 200 rpm for 13–16 h. When the OD600 of Agrobacterium cells reached 0.8–1.0, we suspended the cell pellet of Agrobacterium in 300 µl MS medium, and then we added 100 µM Acetosyringone to the MS medium and resuspended the cell pellet. Finally, we injected suspension into the leaves of Phalaenopsis. The plant materials were cultured for 30–40 days, accompanied by the continuous observation of leaves. Then the infection was placed in an appropriate culture environment to observe the growth of new leaves.

Observation of growth rate

The leaf growth rate of the plants was observed using a consistent method. The data on leaf length and width of plants before irradiation were recorded in the irradiation experiment. Then, the data on the length and width of the leaves were recorded after ten days of growth. The area was estimated from literature reports (S = 0.375L2; S, Square; L, Length) (Zhang et al. 2016), and then the ratio obtained using the area after ten days/area after irradiation was used as data for growth rate.

Similarly, in the VIGS experiment, the length and width of the leaves were recorded at 7-day intervals after VIGS infestation, and then the area was estimated using the same method. The data of leaf growth rate were obtained by dividing the area of 7D, 14D, 21D, 28D, 35D by the area of 0D, and were analyzed and made through the drawing software of PRISM.

Observation of epidermal cells

The newly born leaves of pCymMv silencing lines and CK were selected. Some epidermal tissues were cut on the leaf epidermis with a cutting knife, then placed on a slide with water, and covered with the cover glass to make simple plant tissue sections. The plant tissue section was placed on the microscope table to be observed with a 40 × lens, and the picture was saved. The cell number was counted, the more complete epidermal cells that could be seen in the microscopic field were counted as the whole number, while the epidermal cells with more defective shapes were not counted, and the data were repeated 10 times and three sets of reasonable data were selected as the final data, and the data were analyzed and made through the drawing software of PRISM.

RNA extraction and qPCR

The latest leaves of Phalaenopsis at different developmental stages (S0-S4) were collected to detect gene expression. RNA was extracted by mixing CK and irradiated plant samples. Total RNA was extracted using a SteadyPure Plant RNA Extraction Kit (Code: AG21019; Accurate Biotechnology Co., Ltd., Hunan, China). cDNA synthesis was performed with the RT reagent kit (Takara) according to the manufacturer’s protocol. Real-time PCRs were done on a Chromo 4™ continuous fluorescence detector with the SYBR RT-PCR Kit (Takara) in a 20 µL reaction volume, which contained 10 µL of SYBR Green I PCR mix, 0.5 µM each of forward and reverse primer, 1 µL of cDNA template and appropriate amounts of sterile ddH2O. Amplification conditions were: 2 min at 95 ◦C; 40 cycles of 15 s at 95 ◦C, 30 s at 58 ◦C, and 30 s at 72 ◦C. Fold changes of RNA transcripts were calculated by the 2 − ∆∆Ct method. We used PeActin as the internal control. The entire experiment was repeated three times. Primers used in the qPCR were listed in Supplementary Data Table 5.

Results

Leaf growth was accelerated and physiological metabolism was enhanced after the irradiation with 60Co-γ-rays

P. equestris plants were irradiated with 60Co-γ-rays at a dose of 40 Gy and then maintained in a greenhouse to monitor their growth. At 10 days post-irradiation, the leaf growth rate of the irradiated group was significantly higher than that of the CK (Fig. 1A). Similarly, the SOD, POD, and CAT activities as well as the malondialdehyde and soluble sugar contents were significantly higher in the irradiated group than in the CK (Fig. 1B).

Fig. 1
figure 1

Effect of 60Co-γ-40 Gy irradiation on the growth of P. equestris leaves. A Comparison of growth rates between CK and 10 days after irradiation. B Analysis of physiological indexes of leaves after irradiation with 60Co-γ-40 Gy. An asterisk (*) in-dicates significant differences among treatments (*p < 0.05; **p < 0.01. ***p < 0.001. ****p < 0.0001). All data were represented as means ± standard deviations (triplicate). Error bars indicate the standard error of the mean

Full size image

Identification of DEGs in irradiated leaves

To explore how irradiation promoted the growth and development of P. equestris leaves, a transcriptome sequencing analysis was performed using the P. equestris plants irradiated with a 40 Gy dose.

The transcriptome sequencing of 6 samples (CK, FZ, respectively, three repetitions) was completed and a total of 47.5 Gb Clean Data was obtained, with each sample reaching over 7.67 Gb Clean Data and the percentage of Q30 bases above 94.42%. The statistical power of this experimental design, calculated in RNASeqPower is 0.867.

Three replicates of the control and irradiated groups were analyzed and the transcriptome sequencing error was small (Supplementary Table S1). The principal component analysis of the RNA-seq data (Fig. 2A) detected obvious differences between the irradiated group and the CK. Moreover, the repeatability was relatively high. The correlation and cluster analyses of the six samples showed that the control material and the irradiated material formed separate clusters (Fig. 2B), indicating that the transcriptome data were appropriate for the subsequent analysis.

Fig. 2
figure 2

Statistics of DEGs in RNA-seq. A Principal component analysis (PCA) chart. B Heat map of DEGs between FZ group and CK group. C Venn diagram of the number of DEGs between the FZ group and the CK group. D Volcanic map of DEGs in FZ and CK group. E Differential gene map with variable shear

Full size image

A total of 16,908 genes were identified in the irradiated group and the non-irradiated CK of which 15,521 genes were co-expressed, 570 genes were specifically expressed in CK, and 817 genes were specifically expressed in the irradiated group (Fig. 2C). The volcano plot indicated that the expression level changes were greater for the up-regulated genes in the control and irradiated groups than for the down-regulated genes (Fig. 2, D). There were 1070 DEGs, including 774 up-regulated genes and 296 down-regulated genes.

The proportions of alternative splicing events were as follows (Fig. 2E): skipped exon (SE), 37.7953%; retained intron (RI), 7.6990%; alternative 5′ splice site (A5SS), 15.6605%; alternative 3′ splice site (A3SS), 33.8583%; and mutually exclusive exon (MXE), 4.9869%. Thus, the most common alternative splicing events were SE and A3SS, which may have resulted in changes to protein structures and functions and altered plant growth.

Functional classification of DEGs according to GO and KEGG analyses

To characterize the genes induced by irradiation, the DEGs were functionally annotated on the basis of enriched GO terms and KEGG pathways. The GO enrichment analysis (Fig. 3A) classified the DEGs into the three main GO categories (molecular function, cellular component, and biological process). The 466 genes in the biological process category were annotated with various GO terms, including “detoxification,” “developmental process,” “response to stimulus,” “cellular component organization or biogenesis,” “biological regulation,” “localization,” “cellular process,” and “metabolic process.” The KEGG pathway analysis divided the DEGs in the irradiated and CK into the following five main categories (Fig. 3, B): “metabolism,” “genetic information processing,” “environmental information processing,” “cellular processes,” and “organismal systems.” A total of 151 up-regulated genes and 111 down-regulated genes were classified. The main enriched pathways related to “metabolism” were “amino acid metabolism,” “biosynthesis of other secondary metabolites,” “carbohydrate metabolism,” and “lipid metabolism.” The main enriched pathways in the “genetic information processing” category were “folding, sorting and degradation,” “replication and repair,” and “translation.” The enriched “environmental information processing” pathways were “membrane transport” and “signal transduction.” Only one enriched pathway was detected in the “cellular processes” (“transport and catabolism”) and “organismal systems” (“environmental adaptation”) categories.

Fig. 3
figure 3

Annotation analysis of DEGs in RNA-seq. A GO classification statistics. B KEGG classification statistics

Full size image

The DEGs between the CK and irradiated samples were classified by GO and KEGG enrichment analyses. The significantly enriched GO terms were “external encapsulating structure organization,” “cell wall organization,” “extracellular region,” “cell wall,” “external encapsulating structure,” “plant-type cell wall,” “carbohydrate metabolic process,” “polysaccharide metabolic process,” “cellular carbohydrate metabolic process,” and “cell wall organization or biogenesis.” More specifically, 52, 23, and 23 genes were annotated with “carbohydrate metabolic process,” “cell wall,” and “external encapsulating structure,” respectively (Fig. 4A). The significantly enriched KEGG pathways were “nitrogen metabolism,” “tropane, piperidine, and pyridine alkaloid biosynthesis,” “plant hormone signal transduction,” “pentose and glucuronate interconversions,” “cysteine and methionine metabolism,” “ether lipid metabolism,” “cutin, suberin and wax biosynthesis,” “steroid biosynthesis,” “tyrosine metabolism,” and “phenylpropanoid biosynthesis.” A total of 18, 13, 10, and 10 genes were assigned to the “plant hormone signal transduction,” “phenylpropanoid biosynthesis,” “pentose and glucuronate interconversions,” and “cysteine and methionine metabolism” pathways (Fig. 4B).

Fig. 4
figure 4

GO and KEGG enrichment analysis of DEGs in RNA-seq. A GO enrichment of DEGs. B KEGG enrichment of DEGs

Full size image

Combined analysis of RNA-seq data and physiological indices

To explore the reasons for the increased activities of the ROS-scavenging enzymes in the leaves following the 40 Gy irradiation treatment, the DEGs related to the regulated synthesis of antioxidant enzymes mediating ROS homeostasis were analyzed (Fig. 5A). Compared with CK samples, peroxidase (LOC110032151, LOC110021580, LOC110021581, and LOC110027479), superoxide dismutase (LOC110031523 and LOC110019501), ascorbate oxidase (LOC110020259, and LOC110036698), and ascorbate transporter (LOC110028342) genes were expressed at higher levels in the irradiated group. This may help to explain the increased ROS-scavenging capacity of the irradiated samples. However, the expression of a CAT gene (LOC110025729) decreased in response to the irradiation (Fig. 5A).

Fig. 5
figure 5

Analysis of DEGs related to reactive oxygen metabolism pathway and glucose metabolism pathway in RNA-seq. A Heat map analysis of DEGs encoding antioxidant enzymes in CK and FZ-40 Gy. B Heat map analysis of DEGs encoding glucose metabolism pathway between CK and FZ-40 Gy. C Heat map analysis of DEGs encoding pectin and cellulose metabolism between CK and FZ-40 Gy. The related information of genes used for heat map analysis is shown in Supplementary Data Table S2

Full size image

Starch, which is an important storage material in plant organs, is produced from soluble sugars. The transcriptome analysis of the irradiated plants revealed significant irradiation-induced increases in the expression of a starch synthase gene (LOC110034665) and genes related to starch, fructose, trehalose, galactose, and sucrose metabolism (LOC110027413, LOC110021337, LOC110027041, LOC110024845, LOC110027033, LOC110037880, and LOC110031226) (Fig. 5, B). Plant cell walls comprise cellulose and pectin. The transcriptome data indicated the irradiation of the plants significantly increased the expression of pectinase genes (LOC110029424, LOC110039337, LOC110033510, LOC110019941, LOC110017824, LOC110018751, and LOC110034709) and cellulase genes (LOC110020528, LOC110039125, LOC110027740, and LOC110023601) (Fig. 5, C).

Transcriptome sequencing analyses identified DEGs related to the rapid plant growth and development after the irradiation

The mechanism mediating the rapid growth of P. equestris leaves after the irradiation treatment was elucidated by analyzing the transcriptome data. Rapid leaf development is related to cell proliferation and expansion, the cell division cycle, and the regulation of various growth hormones (Hepworth and Lenhard 2014; Bar and Ori 2014; Vercruysse et al. 2020). Functional genes related to plant hormone signal transduction, plant cell proliferation and expansion, and the cell cycle, were screened. The UniProt website and the available literature were used to clarify gene functions and identify relevant genes (Fig. 6, A). Among the genes in the IAA pathway, the expression levels of auxin response genes (PeIAA3-1, PeIAA3-2, PeAUX6B, PeAUX22D, PeAUX10A, PeSAUR64, and PeSAUR60) increased after the irradiation, as did the expression of genes encoding auxin-binding proteins (PeABP19a-1, PeABP19a-2, and PeABP19a-3). The PeLOG1 gene encodes a cytokinin 5′-phosphoribosyl monophosphate hydrolase, which catalyzes the last step of the bioactive cytokinin synthesis pathway. The expression of this gene increased significantly in the irradiated samples. The expression of PeHK6, which encodes a histidine kinase, also increased in the irradiated group. The expression levels of GA regulatory protein-encoding genes (PeGASA12, PeGASA6, and PeGASA5) increased after the irradiation. In the ABA signaling pathway, the expression levels of PeCYP707A3 (involved in ABA degradation) and PePYL4 (ABA receptor) increased following the irradiation, which was in contrast to the decreased expression levels of PePP2C (activated by ABA) and PeSAPK10 (involved in ABA signaling). Among the jasmonic acid (JA) signaling pathway genes, the irradiation reduces the expression of PeJAR1, which mediates the synthesis of JA, and increases the expression of PeTRFY10a, which encodes an inhibitor of jasmonic acid reactions. The expression levels of cyclin genes (PeCYCA3-1, PeCYCD2-2, PeCYCD4-1, and PeCCYCP3-1) also increased after the irradiation. The expression of PeNGA (associated with inhibited cell proliferation) decreased significantly in the irradiated plants, whereas the expression of PeEXPA10 (related to cell expansion) increased. Ten of these genes (PeCYCA3-1, PeCYCD2-2, PeEXPA10, PeGAST1, PeGASA12, PeSAUR50, PePP2C, PeHK6, PeLOG1, and PeNGA) were analyzed by qPCR; the resulting data were consistent with the transcriptome data (Fig. 6, B).

Fig. 6
figure 6

Screening and expression verification of genes related to leaf development inRNA-seq. A Heat map analysis of DEGs in leaf development between CK and FZ-40 Gy (Supplementary Data Table S3). B qPCR analysis of DEGs. An asterisk (*) indicates significant differences among treatments (*p < 0.05; **p < 0.01. ***p < 0.001. ****p < 0.0001). All data were represented as means ± standard deviations (triplicate). Error bars indicate the standard error of the mean

Full size image

Silencing of PeEXPA10 resulted in delayed leaf growth

The results of the RNA-seq and qPCR analyses suggested that irradiation significantly promoted the degradation of pectin and cellulose (i.e., cell wall components). We speculated that PeEXPA10, which is related to cell wall ductility, may be one of the genes contributing to the rapid growth of leaves. The 40 Gy irradiation increased the PeEXPA10 expression level. Leaf development was divided into five stages, namely S0, S1, S2, S3, and S4. The PeEXPA10 gene was highly expressed in the leaves at the S0 stage, but its expression level decreased as the leaves developed (S0–S4) (Fig. 7A). Virus-induced gene silencing (VIGS) was used to verify the effects of PeEXPA10 on leaf growth. Specifically, PeEXPA10 was silenced and the growth of new leaves was observed every 7 days (Fig. 7B). Compared with the CK leaves, the growth rate of the pCymMv-PeEXPA10 leaves decreased significantly in the third week (Fig. 7C). In addition, there were significantly more epidermal cells in the pCymMv-PeEXPA10 plants than in the CK plants (Fig. 7D, E). The qPCR analysis confirmed the PeEXPA10 expression levels were significantly lower in the pCymMv-PeEXPA10 plants than in the CK plants (Fig. 7F). The qPCR data also indicated that the expression levels of PeAN, PeAPL, PeTOR, and PeROT3, which are related to cell expansion, were significantly down-regulated in the pCymMv-PeEXPA10 plants (Fig. 7G).

Fig. 7
figure 7

Functional verification of PeEXPA10 in Phalaenopsis leaf growth. A Relative expression of PeEXPA10 in leaves at different stages (S0–S4). B After infection, the growth status of infected plants was compared with that of no-load control plants (leaves indicated by arrows were the target, and the ratio was 1:1 cm). C The growth rate of leaves from day 0 to day 35 after infection. D Epidermal cells of leaves of silenced lines and control plants under the microscope (40X microscope). (E) The number of epidermal cells was counted. F qPCR verification was performed in silenced lines. G qPCR analysis of genes in leaves of silenced lines and control plants. An as-terisk (*) indicates significant differences among treatments (*p < 0.05; **p < 0.01. ***p < 0.001. ****p < 0.0001). All data were represented as means ± standard deviations (triplicate). Error bars indicate the standard error of the mean

Full size image

Silencing of PeNGA accelerated leaf growth

According to the RNA-seq and qPCR analyses, PeNGA expression decreased significantly after the irradiation. We hypothesized that PeNGA and PeEXPA10 may have the opposite effects on leaf growth. The expression of PeNGA in P. equestris leaves at different developmental stages (S0–S4) was analyzed by qPCR (Fig. 8A). The gene was expressed at very low levels at the S0 stage, but its expression level increased from S0 to S4. The involvement of PeNGA in leaf growth and development was examined via VIGS. The pCymMv-PeNGA vector was constructed to silence PeNGA expression in P. equestris, after which the growth of new leaves was analyzed every 7 days (Fig. 8B). The leaf growth rate was significantly higher for the pCymMv-PeNGA plants than for the CK plants in the second week (Fig. 8C). There was no significant difference in the number of epidermal cells between the pCymMv-PeNGA and CK plants (Fig. 8D, E). Accordingly, leaf growth was due to cell proliferation rather than cell expansion. The significant decrease in PeNGA expression in the pCymMv-PeNGA plants was verified by the qPCR analysis (Fig. 8F). Furthermore, the cyclin gene (PeCYCA3-1, PeCYCD1, PeCYCP3-1, and PeCYCP48) expression levels were significantly up-regulated in the pCymMv-PeNGA plants (Fig. 8G).

Fig. 8
figure 8

Functional verification of PeNGA in Phalaenopsis leaf growth. A Relative expression of PeNGA in leaves at different periods (S0–S4). B After infection, the growth status of infected plants was compared with that of no-load control plants (leaves indicated by arrows were the target, and the ratio was 1:1 cm). C The growth rate of leaves from day 0 to day 35 after infection. D Epidermal cells of leaves of silenced lines and control plants under the microscope (20X microscope). E The number of epidermal cells was counted. F qPCR verification was performed in silenced lines. G qPCR analysis of genes in leaves of silenced lines and control plants. An asterisk (*) indicates significant differences among treatments (*p < 0.05; **p < 0.01. ***p < 0.001. ****p < 0.0001). All data were represented as means ± standard deviations (triplicate). Error bars indicate the standard error of the mean

Full size image

Discussion

Radiation breeding, a method of physical mutagenesis breeding, can generate new germplasm of rare mutants that are challenging to obtain through other techniques (Yamaguchi, 2018). In this study, P. equestris was exposed to 40 Gy of 60Co-γ rays, resulting in rapid leaf growth (Fig. 1A, B). The malondialdehyde content and the activities of SOD, POD, CAT, and GPX in the irradiated materials showed significant increases. Carbohydrates, essential energy sources, play a crucial role in plant growth and development (Zhang et al. 2019). RNA-seq analysis of DEGs in irradiated mutagenized plants and the CK revealed a significant increase in genes associated with starch synthesis in leaves. The expressions of genes related to starch, sucrose, galactose, and fructose metabolism were also significantly elevated, suggesting an acceleration in plant growth and metabolic rate (Fig. 5B).

Plant growth and development are intricately linked to the regulation of hormone network factors. Reduction in abscisic acid concentration in plants can decelerate leaf senescence (Xue et al. 2010). Transcriptome analysis of irradiated plants revealed significant differential expression of ABA signaling pathway genes (PeCYP707A3, PePYL4, PePP2C, and PeSAPK10) (Fig. 6A), all of which have been reported to play a role in leaf development in previous studies (Spartz et al. 2014; Santiago et al. 2009; Han et al. 2017). In the CK, significant genes involved in leaf development (PeHK6, PeLOG1) were also identified (Fig. 6A) (Kuroha et al. 2009; Choi et al. 2012). Auxin influences cell division, extension formation, and differentiation, serving as a signal for cell proliferation to determine the final size and shape of organs (Leyser 2001). Transcriptome analysis of IAA signaling pathway genes (PeIAA3-1, PeIAA3-2, PeAUX6B, PeAUX22D, PeAUX10A, PeSAUR64, and PeSAUR60) (Fig. 6A) revealed their key role in leaf development, as reported in previous studies (Hou et al. 2013; Kant et al. 2009; Chae et al. 2012). Additionally, significant up-regulation of auxin-binding proteins (PeABP19a-1, PeABP19a-2, and PeABP19a-3) was observed after irradiation (Fig. 6A). Gibberellic acid (GA), a plant hormone belonging to diterpenes, plays a crucial role in the entire life cycle of plants, promoting cell elongation and differentiation (Davière et al. 2008). The transcriptome revealed upregulation of GA signaling pathway genes (PeGASA12, PeGASA6, and PeGASA5) (Fig. 6A), with GASA genes potentially playing a significant role in leaf development, as indicated in previous studies (Zhang and Wang 2017; Zhong et al. 2015; Sun et al. 2013). Jasmonic acid has a clear inhibitory effect on plant seedling growth, particularly in the inhibition of leaf growth (Dathe et al. 1981). Additionally, JA pathway genes (PeJAR1, TRFY10a) were significantly expressed (Fig. 6A), and they also play a crucial role in leaf development (Wasternack and Jasmonates 2013). It can be inferred that IAA, GA, and CK may promote rapid leaf growth, while ABA and JA play inhibitory roles.

Cellulose and pectin are the primary components of the plant cell wall. Increased levels of cellulase and pectinase enhance plant cell plasticity, resulting in cell wall loosening, which in turn promotes cell expansion and increases the leaf area of the plant (Anderson 2019). Transcriptome data analysis revealed a significant increase in the expression levels of genes related to pectinase and cellulase (Fig. 4 C). PeEXPA10 plays a crucial role in regulating cell wall expansion. EXP10 is a non-enzymatic, cell wall-active protein that can relax plant cell walls (Kende et al. 2004). Previous studies have demonstrated that plants ectopically expressing EXP10 exhibit larger leaves and longer petioles with more giant cells, while downregulation of EXP10 has the opposite effect (Cosgrove 2015). Following irradiation, the expression of PeEXPA10 increased, potentially in relation to the elevated levels of pectinase and cellulase, leading to cell wall relaxation and leaf enlargement. This study observed a significantly lower growth rate in PeEXPA10 gene-silenced plants compared to the CK (Fig. 7C), with the epidermal cells becoming notably smaller (Fig. 7D). These findings suggest that the absence of the PeEXPA10 gene diminished cell expansion capability, resulting in smaller leaves. It has been reported that the reduction of PP2C leads to the activation of cell wall-associated EXP through extracellular acidification induced by auxin, an ATPase that imports H + ions and can promote cell wall loosening. However, further investigation is required to elucidate these mechanisms (Spartz et al. 2014). After irradiation, the expression of the negatively regulated transcription factor PeNGA decreased. The growth rate of PeNGA gene-silenced plants was significantly higher than that of the CK (Fig. 8C). There was no significant change in cell size (Fig. 8D). In Arabidopsis thaliana, increased petals in nga1-1 mutants were also attributed to increased cell number, indicating that AtNGA1 may serve as a negative regulator of cell proliferation in lateral organs. In this study, qPCR analysis revealed a significant increase in the expression of cyclin in the silenced lines (Fig. 8G). Therefore, the deletion of the PeNGA gene may have alleviated the restriction on cell proliferation, resulting in an accelerated cell proliferation rate. The transcriptome analysis showed that the expression levels of cyclin genes PeCYCA3-1, PeCYCD2-2, PeCYCD4-1, and PeCYCP3-1 increased (Fig. 8G). Cyclin (CYC) and Cyclin-dependent kinase (CDK) are the central regulators of the plant cell cycle (Jackson 2008). These genes primarily participate in the G1 phase and the transition from G1 to S phase, playing a crucial role in cell cycle regulation (Vandepoele et al. 2002).

Based on previous research progress and literature reports, combined with our results, we have determined that PeEXPA10 may function downstream of the abscisic acid pathway and affect the structure of the cell wall. It may also play a role in hormonal pathways, glucose metabolism and reactive oxygen metabolism pathways. Moreover, cell division and proliferation occur continuously and cross various developmental pathways. We have screened the PeNGA gene and verified its association with cell division in plants. Although we have only listed the TCP-NGA and cytokinesis pathways in the model, there is still a relationship with the hormone pathway, reactive oxygen metabolism pathway, and sugar metabolism pathway. The individual developmental pathways of plants are complex and interconnected. In conclusion, the developmental process of plants is a complex and extensive network, and we have only partially predicted and verified gene functions. Further studies are needed to explore this topic in greater depth.

This study focused on the rapid growth phenotype and the physiological and metabolic changes in leaves induced by 60Co-γ-40 Gy irradiation. RNA-seq technology was employed to identify key regulatory genes associated with plant hormone signal transduction, glucose metabolism, reactive oxygen species metabolism, and leaf development in response to irradiation stress. Based on the findings, a pathway model of irradiated leaf development was simulated (Fig. 9). Further research is needed to understand how these identified genes regulate the cell cycle for proliferation and expansion. The essential role of PeNGA and PeEXPA10 in rapid leaf development was demonstrated using VIGS technology. However, further investigation is needed to understand the intricate molecular regulatory mechanisms underlying 60Co-γ-40 Gy irradiated mutagenesis, particularly in relation to the interaction between genes and transcription factors, as well as the regulatory dynamics between plant hormones during leaf development. Additionally, it would be intriguing to explore the feasibility of concurrent cell wall expansion and cell proliferation, and to elucidate the coordination and antagonism among key genes. Irradiation offers a promising avenue for plant breeding, yet the underlying mechanisms of this phenomenon warrant continued exploration in the future.

Fig. 9
figure 9

Leaf development pathway model of P equestris. Red arrows indicate genes with increased expression after irradiation, while blue arrows indicate genes with decreased expression after irradiation

Full size image

Conclusions

This study revealed the effects of 60Co-γ-40 Gy irradiation on leaf development and physiological metabolism of Phalaenopsis. In addition, we used RNA-seq to demonstrate that the differential expression of glucose metabolism genes, ROS metabolism genes, and leaf development-related genes in irradiated leaves led to these changes. The VIGS technique also confirmed that PeNGA and PeEXPA10 play an essential role in the growth and development of phalaenopsis leaves. Therefore, the interaction and regulation between genes and transcription factors after 60Co-γ-40 Gy radiation mutagenesis and the molecular regulatory mechanism of leaf growth and development need to be further studied.