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The distribution of bioactive gibberellins along peach annual shoots is closely associated with PpGA20ox and PpGA2ox expression profiles
BMC Genomics volume 23, Article number: 730 (2022)
The rapid growth of annual shoots is detrimental to peach production. While gibberellin (GA) promotes the rapid growth of peach shoots, there is limited information on the identity and expression profiles of GA-metabolism genes for this species.
All six GA biosynthetic gene families were identified in the peach genome, and the expression profiles of these family members were determined in peach shoots. The upstream biosynthetic gene families have only one or two members (1 CPS, 2 KSs, and 1 KO), while the downstream gene families have multiple members (7 KAOs, 6 GA20oxs, and 5 GA3oxs). Between the two KS genes, PpKS1 showed a relatively high transcript level in shoots, while PpKS2 was undetectable. Among the seven KAO genes, PpKAO2 was highly expressed in shoots, while PpKAO1 and − 6 were weakly expressed. For the six GA20ox genes, both PpGA20ox1 and − 2 were expressed in shoots, but PpGA20ox1 levels were higher than PpGA20ox2. For the five GA3ox genes, only PpGA3ox1 was highly expressed in shoots. Among these biosynthesis genes, PpGA20ox1 and PpGA3ox1 showed a gradual decrease in transcript level along shoots from top to bottom, and a similar trend was observed in bioactive GA1 and GA4 distribution. Among the GA-deactivation genes, PpGA2ox6 was highly expressed in peach shoots. PpGA2ox1 and − 5 transcripts were relatively lower and showed a similar pattern to PpGA20ox1 and PpGA3ox1 in peach shoots. Overexpression of PpGA20ox1, − 2, or PpGA2ox6 in Arabidopsis or tobacco promoted or depressed the plant growth, respectively, while PpGA3ox1 did not affect plant height. Transient expression of PpGA20ox1 in peach leaves significantly increased bioactive GA1 content.
Our results suggest that PpGA20ox and PpGA2ox expression are closely associated with the distribution of active GA1 and GA4 in peach annual shoots. Our research lays a foundation for future studies into ways to effectively repress the rapid growth of peach shoot.
Peach [Prunus persica (L.) Batsch.] is one of the most economically important fruit tree species. Its annual shoots grow rapidly in spring and summer, which causes many problems in peach production. This annual growth can close the tree canopy, which restricts the ventilation and light of the orchard, and requires increased labor for pruning trees. Paclobutrazol (PBZ), a biosynthetic inhibitor of GA, is usually used to slow peach stem growth. The primary function of GA in higher plants can be generalized as stimulating growth through the enhancement of cell elongation and cell division . Therefore, it is likely that GA is an important hormone regulating the rapid growth of peach annual shoot.
Bioactive GAs are diterpene plant hormones. Their biosynthesis starts from geranylgeranyl diphosphate (GGDP), which is converted to ent-kaurene by two terpene synthases, ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS), and then transformed into GA12 by two cytochrome P450 monooxygenases, ent-kaurene oxidase (KO) and ent-kaurenoic acid oxidase (KAO). Finally, GA12 is transformed into active GA4 by two 2-oxoglutarate-dependent dioxygenases, GA 20-oxidase (GA20ox) and GA 3-oxidase (GA3ox). GA12 also could be transformed into GA53 firstly by GA 13-oxidase (GA13ox) and then transformed into GA1 and GA3 by GA20ox and GA3ox [1, 2]. In addition, GA deactivation is an important mechanism for controlling endogenous GA content. The 2β-hydroxylation of active GA, a process catalyzed by GA 2-oxidase (GA2ox), is a primary way to inactivate GAs . The deactivating function of CYP714 on active GAs has been demonstrated in rice and Arabidopsis [4,5,6]. An in vitro enzyme activity assay showed that EUI (CYP714D1) significantly reduced the biological activity of GA4 . Mutation in GA biosynthetic/deactivation genes affects the endogenous GA content and therefore plant stature. A single nucleotide substitution in exon 5 of OsKO2 leads to replacement of a highly conserved arginine with a serine, resulting in a dwarf phenotype . The Arabidopsis ga20ox1 mutant contains lower bioactive GA and shows a semi-dwarf phenotype [8, 9]. Four independent mutations in ZmGA3ox2 give rise to a dwarf phenotype of maize . OsGA3ox2 is located at the D18 locus, which is associated with a dwarf phenotype in rice due to a frameshift mutation . Rht18 semi-dwarfism in wheat is caused by increased GA2oxA9 transcript levels and decreased endogenous GA contents .
GA transport is another way that plants modulate local bioactive GA levels. GA transport, in both acro- and basipetal directions, has been demonstrated to be essential for several developmental processes . GAs are mobile signals from shoot to hypocotyl and can trigger local xylem expansion . The acropetal translocation of GAs from wild-type rootstock to scions lacking a GA biosynthetic enzyme rescues the phenotypes .
Apart from the content of bioactive GAs, the activity and stability of each active GA form are important factors affecting GA signaling. More than 130 GA structures have been identified, with the most common active forms being GA1, GA3, and GA4 . GA1 and GA4 are deactivated by GA2ox, while GA3, which is synthesized by GA3ox from GA20 via the intermediate GA5, is not deactivated by GA2ox . Therefore, GA3 is more stable than GA1 and GA4. However, the activity of these three GA active forms differs. The universal occurrence of GA1 and GA4 in plants suggests that these are the functionally active forms. Additionally, GA4 appears to be more active than GA1 in rice .
GA is the primary growth-promoting hormone of peach annual shoots, but the genes involved in biosynthesis and degradation are poorly understood. To properly regulate the growth of annual shoots, it is necessary to identify the genes that encode the enzymes that synthesize and deactivate GA. Our previous study identified seven PpGA2ox genes in peach . The present study identifies the GA biosynthetic genes and analyzes their transcription levels as well as those of the deactivating PpGA2oxs in six internodes from the top to the bottom of peach shoots. In addition, the contents of the GAs in different internodes were analyzed. Additionally, PpGA20ox1, PpGA20ox2, PpGA3ox1, and PpGA2ox6 were overexpressed in Arabidopsis or tobacco. Finally, PpGA20ox1 was transiently expressed in peach leaves, and the bioactive GAs were measured.
Identification of GA biosynthetic genes in the peach genome
Six GA biosynthesis-related gene families (CPS, KS, KO, KAO, GA20ox, and GA3ox) were analyzed in peach. We identified one CPS, two KSs, one KO, seven KAOs, six GA20oxs, and five GA3oxs in the peach genome (Table 1; Fig. 1A). The number and length of the exons in each gene were predicted. There were 12 exons in PpCPS, which is three fewer than in Arabidopsis. PpKS1 had the same number of exons as AtKS, while PpKS2 had four fewer than AtKS. Among the seven KAO genes, PpKAO1 to − 6 contained eight exons and had the same numbers as AtKAO1 and − 2. All eleven GA20ox genes in peach and Arabidopsis had three exons. Five PpGA3ox had two exons, the same as AtGA3ox1, − 2, and − 4. The GA biosynthetic genes showed high conservation of exon length, with any changes in exon length occurring mainly in the first and last exons.
Interestingly, PpKO had 8 exons in peach (Fig. S1) and 7 exons in Arabidopsis. RT-PCR was used to amplify the coding sequence of PpKO and its gene structure was confirmed. The sixth exon in AtKO was 357 bp in length, which is equal to the sum of the sixth and seventh exons of PpKO. In the GA3ox family, only AtGA3ox3 contains three exons, one more exon than the other GA3ox members. Alignment of the PpGA20ox with the AtGA20ox protein sequences identified two conserved motifs (NYYPXCQKP and LPWKET) postulated to be involved in binding the 2-oxoglutarate and the GA substrate . The protein encoded by PpGA20ox4 lacks the LPWKET motif (Fig. S2), implying that the function of PpGA20ox4 may be changed.
Phylogenetic trees were constructed to analyze the evolutionary relationship of the six GA biosynthesis gene families. A phylogenetic tree was constructed using the amino acid sequence of just the GA20oxs from peach, grapevine, tomato, Arabidopsis, rice, maize, and the moss Brachypodium distachyon (Fig. 1B). All of the GA20ox protein sequences fell into one of two groups (group I and group II). Both groups contained GA20oxs from both Eudicots and Monocots, and seven analyzed species contained members from the two groups (Table 2). These results suggest that the GA20ox family evolved from two different ancestral GA20ox genes. PpGA20ox1 and PpGA20ox2 were clustered into group I, which also contained OsGA20ox1 and AtGA20ox1. PpGA20ox3, − 4, − 5, and − 6 were clustered into group II, which contained OsGA20ox2, the SEMI-DWARF1 (SD1) gene in rice. Another phylogenetic tree of the CPS gene family showed that PpCPS clustered with CPS from apple and strawberry (Fig. S3A). Peach KS and KO genes clustered together with KS and KO of apple and strawberry (Fig. S3B and C). Five PpKAOs (PpKAO1, − 2, − 3, − 4, and − 5) clustered with the KAOs from different plants, and the remaining 2 PpKAOs (PpKAO6 and − 7) were separated out (Fig. S3D). The GA3ox phylogenetic tree divided all GA3oxs into two groups, which contained the GA3oxs from Eudicots and Monocots, respectively (Fig. S3E).
Transcription profile of GA biosynthetic genes in different tissues
Because KS, KAO, GA20ox, and GA3ox gene families are comprised of multiple members, the transcription profiles of these genes were analyzed using the transcriptomes derived from stem, flowers, fruit, and fruit pits (Fig. 2). All genes showed at least some expression. Among the seven KAOs, PpKAO2 was highly expressed in all analyzed samples, while PpKAO3 was mainly expressed in flower and fruit pits. Among the five GA3oxs, PpGA3ox1 was mainly expressed in flowers and stems, and PpGA3ox3 was expressed in flowers. Between the two PpKSs, PpKS1 was expressed in all analyzed tissues. Among the six PpGA20oxs, PpGA20ox2 was expressed in all analyzed samples, while PpGA20ox1 was mainly expressed in stems. PpCPS was expressed in all analyzed tissues, while PpKO was highly expressed in stem and fruit pit.
Determination of active GA levels in annual peach shoot
Annual shoots of peach grow quickly in the spring and summer, implying active synthesis of GAs in the shoots. Sylleptic shoots undergoing vigorous growth were used to determine the content of active GAs. Segments (0.2 cm in length) were collected from the center of six internodes from the tip to the lower internodes of the branch and were successively named S1, S2, S3, S4, S5, and S6 (Fig. 3A). Due to their low concentrations, every two internodes were combined to determine the GA content. The GA1 content gradually decreased from apex to bottom, with a level 6 times higher in tip internodes (1.5 ng/g FW) than in the lowest internodes (0.25 ng/g FW) (Fig. 3B). GA4 is highest in the middle (1.01 ng/g FW) and lowest in the bottom internodes (0.21 ng/g FW) (Fig. 3D). These results demonstrated that GA1 and GA4 are at high levels in the final internodes of annual shoots.
Interestingly, the GA3 content gradually increased from the tip to the branch bottom (Fig. 3C). The GA3 level in the top internodes was significantly lower than were the levels of GA1 and GA4. The GA3 level in the bottom internodes (2.84 ng/g FW) was 11.36 times higher than in the top internodes (0.25 ng/g FW). These results demonstrated that the lower internodes accumulated a large amount of active GA3.
Transcription level of GA biosynthetic and deactivation genes in six internodes along the elongating shoot from top to bottom
The transcript levels of the GA biosynthetic genes were analyzed in six internodes along the elongating shoot from top to bottom (Fig. 4). PpCPS showed a gradually increasing trend from S2 to S6, and was at its highest level in S6. PpKS1 showed a clearly higher level in all six internodes than PpKS2 and had the highest level in S2. PpKO was expressed in all six internodes. PpKAO1 and − 2 were expressed in all six internodes, with PpKAO2 higher in all analyzed internodes. Expression of PpKAO3, − 4, − 5, − 6, and − 7 was relatively low in all six internodes.
Among the six GA20ox genes, PpGA20ox1 was highly expressed in the tip internodes (S1 and S2) and weakly in lowest internodes. PpGA20ox2 was expressed in all analyzed internodes. PpGA20ox5 and PpGA20ox6 are tandem repeat genes, with a coding sequence similarity of 99% and similar transcription profiles in the stem. The level of PpGA20ox3 was very low in all six internodes. Among the five GA3ox genes, PpGA3ox1 shows a gradually decreasing trend in transcript level from top to bottom, and PpGA3ox5 is present at a very low level in all six internodes.
Our previous study identified seven PpGA2ox genes in peach . Among the seven GA2ox genes, PpGA2ox1 and PpGA2ox5 showed a gradually decreasing trend in transcript level from top to bottom. PpGA2ox6 had the highest expression level in all analyzed internodes compared with the other six GA2ox genes. Some PpGA2oxs genes showed very little transcription.
Overexpression of PpGA20ox1, − 2, − 5, PpGA3ox1, and PpGA2ox6 in Arabidopsis or tobacco
To determine if the putative PpGA20oxs encode functional enzymes, 35S-PpGA20ox1 and − 2 were transformed into wild-type Arabidopsis (WT) (Fig. 5). Empty vector (EV) served as control. Three transgenic lines each of PpGA20ox1 and − 2 were obtained (Fig. S4C and D). The T3 seedlings were grown for 50 d, then the length of the main stem was measured. The stems of all transgenic lines ranged from 36.6–46 cm in length, which is significantly longer than that of WT and control (Fig. 5A, D). PpGA20ox1 and − 2 were also transformed into an Arabidopsis ga20ox mutant (CS92956) (Fig. S4A and B; Fig. 5B, E) and the phenotype and plant height of 42-day-old seedlings were analyzed. Overexpression of GA20ox1 and − 2 recovered the length of the main stems to that of wild type. Based on the phylogenetic tree of GA20ox, PpGA20ox1 and − 2 belong to group I, and PpGA20ox3, − 4, − 5, and − 6 are clustered into groups II. 35S-PpGA20ox5 was transformed into wild type, and the phenotype and plant height of 28-day-old seedlings were analyzed. The transgenic lines had the same phenotype as wild type overexpressing PpGA20ox1 and − 2 (Fig. S5).
PpGA3ox1 was transformed into Arabidopsis (Fig. 5C, F). Three PpGA3ox1 transgenic lines were obtained (Fig. S4E). The lengths of the main stem at 40 d were not significantly different between any of the transgenic lines and WT or EV. PpGA2ox-6 was transformed into tobacco (Nicotiana tabacum) (Fig. S4; Fig. 5G, H). Two transgenic lines were selected and showed a dwarf phenotype compared with the control.
Transient expression of PpGA20ox1 in peach leaves
The effect of PpGA20ox1 on bioactive GA contents was confirmed in peach by a transient expression assay. 35S-PpGA20ox1 and EV were transiently expressed in peach leaves. The contents of three active GAs were analyzed (Fig. 6). The result showed that there was a significant difference (P < 0.05) in the content of GA1, which was 3.7 times higher in leaves overexpressing PpGA20ox1 than in the control. GA3 was slightly lower in leaves overexpressing PpGA20ox1 compared to the control, but didn’t differ significantly between 35S-PpGA20ox1 and EV. The content of GA4 was too low to detect. Our results demonstrated that overexpressing PpGA20ox1 increases the GA1 content in peach leaves.
The rapid growth of annual shoots in peach is detrimental to agricultural production. Finding ways to repress this rapid growth is necessary. As GAs play important roles in the rapid growth process, it is important to study the genes involved in GA biosynthesis and degradation. In this study, GA biosynthetic gene families were firstly determined. To find out the candidate members involved in GA metabolism in peach shoots, the expression patterns of the GA metabolic genes were determined along a growing stem. Then, these found out candidate members were transformed into Arabidopsis or tobacco to determine gene function.
The PpGA20ox1 transcript pattern reflects active GA1 and GA4 distribution in annual peach shoots
Six gene families (CPS, KS, KO, KAO, GA20ox and GA3ox) are involved in GA biosynthesis. Among these genes, only PpGA20ox1 and PpGA3ox1 showed a gradual decrease in transcription level from shoot tip to base, a gradient that is similar to the distribution of bioactive GA1 and GA4 in the annual shoots. The results suggested that PpGA20ox1 and PpGA3ox1 may be the key genes controlling GA contents.
The function of GA20ox has been extensively studied in many plants, and overexpression of GA20ox can increase endogenous GAs in Arabidopsis, potato, tomato, rice, Artemisia annua, tobacco, carrizo citrange, cotton, poplar, kenaf, and switchgrass [18,19,20,21,22,23,24,25,26,27,28,29]. However, overexpression of AtGA3ox, the last enzyme in the GA biosynthesis pathway, showed no significant change in active GAs or plant stature in hybrid aspen . Our results also demonstrated that PpGA20ox1 and − 2, but not PpGA3ox1, promoted the growth of Arabidopsis. To demonstrate the function of PpGA20ox in peach, PpGA20ox1 was transiently expressed in peach leaves. Overexpression of PpGA20ox1 significantly increased GA1 content. Altogether, our results suggest that PpGA20ox1 encodes a key enzyme for GA biosynthesis in annual peach shoot.
Intriguingly, PpGA3ox1 had a similar expression pattern as PpGA20ox1 in the annual peach shoots. These similar GA20ox and GA3ox expression patterns are reported in several studies. In Arabidopsis, DELLA-dependent regulation also promotes the simultaneous transcription of GA20ox and GA3ox [31,32,33,34]. Treatment with low ratio of red: far red light upregulated the transcript levels of GA20ox and GA3ox simultaneously in Rumex palustris . Inversely, the transcript levels of GA20ox and GA3ox are downregulated due to mutation of the DELLA proteins LA and CRY in pea . Our previous study showed that the transcript levels of PpGA20ox1 and PpGA3ox1 are significantly upregulated in the dwarf peach ‘FenHuaShouXingTao’ that carries a mutation in the GA receptor PpGID1c . All these results suggest that GA3ox is also important for GA homeostasis.
GA3 content is relatively higher in the sixth internode of peach shoots
GA3 was reported to highly accumulate in the lower internodes of poplar branches . GA transport is well-demonstrated, and many forms of GA are deemed as mobile entities. In general, precursors are suggested to be the mobile forms . GA20 acts as the major mobile form in pea . GA12 is the major form transported over a long distance from roots to shoots in Arabidopsis ; in cucumber, GA9 is produced in the ovaries and moves to the sepals and the petals, whereas GA9 is converted into the bioactive GA4 to regulate organ growth . GA3, an active GAs, is synthesized by GA3ox from GA20 via GA5 . It is well known that GA1 and GA4, but not GA3, can be converted by GA2ox into the inactive GAs, namely GA8 and GA34 . Our previous study demonstrated that three peach GA2oxs could inactivate GA1, but not GA3 . In this study, GA3 content gradually increased from the tip to the branch bottom. PpGA2ox6 was highly expressed in all analyzed internodes of peach shoot. In addition, GA5, an intermediate product from GA20 to GA3, has been shown to resist deactivation by GA2ox and could be transported into the meristem . Together, the high accumulation of GA3 in the lower internodes may be partly attributed to GA3 transportation in the annual shoots of peach.
In this study, peach GA biosynthetic genes were analyzed in detail. The upstream biosynthetic genes have only one or two copies (1 CPS, 2 KSs and 1 KO), while the downstream genes have multiple copies (7 KAOs, 6 GA20oxs and 5 GA3oxs). Among the six biosynthetic genes, only PpGA20ox1 and PpGA3ox1 show a gradual decrease in the transcript level from apex to bottom of annual shoots of peach, which was synchronized with the GA1 and GA4 distribution. Among the GA metabolic genes, PpGA2ox1 and PpGA2ox5 showed similar transcript patterns to PpGA20ox1 and PpGA3ox1. Overexpression of PpGA20ox1, − 2 or PpGA2ox6 in Arabidopsis or tobacco promoted or repressed plant growth, respectively, while PpGA3ox1 had no effect on the plant growth. Transient expression of PpGA20ox1 in peach leaves significantly increased the content of GA1. Our study demonstrates that PpGA20ox and PpGA2ox encode key enzymes that are associated with active GA distribution in annual shoots. This information may prove helpful for devising ways to regulate the endogenous GA levels, and thus annual growth, in peach shoots.
Peach trees of the cultivars ‘QiuMiHong’ (QMH) and ‘YuJinMi3’ (YJM3) are maintained in the Fruit Tree Germplasm Repository of Henan Agricultural University (Henan Province, China). The stem and fruit samples were collected from ‘QMH’ and fruit pits were collected from ‘YuJinMi3’. Fruit developmental stages were divided according to Gabotti et al. . From the S3 to S4 stage, fruit mesocarp tissues were collected at 118 d (S3-I), 120 d (S4-I), 122 d (S4-II), and 124 d (S4-III) after full bloom. Five fruit pit samples at 15, 25, 35, 45, and 55 d after the full bloom period were collected. From the top to bottom of stems showing rapid growth, six internodes were collected and divided into three parts [top (S1-S2), middle (S3-S4), and bottom (S5-S6)]. The samples of stems and fruit pits were prepared between April and June 2019, while fruit samples were prepared on April 28, 2020. All samples were used for transcriptome analysis and the stem samples were also used for GA content analysis.
Tobacco [Nicotiana tabacum] ‘K326’ and peach seedlings were cultured in a growth chamber at 24 °C under a photoperiod of 16 h/8 h. Arabidopsis seedlings were cultured in greenhouse at 23 °C, 16 h of light/8 h of dark, relative humidity of 55%.
The peach genome sequence was downloaded from the GDR database (https://www.rosaceae.org). A standalone BLAST software was used to conduct local blast searches using the coding sequence of CPS (At4g02780), KS (At1g79460), KO (At5g25900), KAO (At1g05160), GA20ox (AT4G25420) and GA3ox (At1g15550) as the query sequences against the peach genome (Table S1). For CPS, KS, and KO, genes with the highest sequence similarity to the homologous genes in Arabidopsis were assigned as significant. PpKS2, a tandem duplicated gene, was also mentioned in this study. For KAO, scores higher than 545 with an “E” value of 0 were assigned as significant. For GA20ox and GA3ox, scores higher than 281 with an “E” value of e-93 were assigned as significant. The gene structure was confirmed using the sequence alignment/map (SAM) files of the transcriptomes and using RT-PCR to amplify the coding sequence. Finally, the Pfam (http://pfam.sanger.ac.uk/search) and SMART (http://smart.emblheidelberg.de/) databases were used to confirm the conserved domains.
Construction of phylogenetic tree
Phylogenetic trees were constructed using the amino acid sequences of CPS, KS, KO, KAO, GA20ox and GA3ox from different plants. The sequences were downloaded from the NCBI database (https://www.ncbi.nlm.nih.gov/) and the Phytozome database (https://phytozome-next.jgi.doe.gov/). Sequence alignment was performed using Clustal X. Neighbor-joining trees were constructed using the heuristic search strategies of MEGA version 5. Bootstrap values were calculated from 1000 replicate analyses. For the phylogenetic tree of GA20ox, a 2OG-Fe (II) oxygenase gene of apple (GenBank accession no. KF562006) served as an outgroup.
RNA extraction and RNA seq
Total RNA was extracted using the RNA extraction kit (Zoman, Beijing, China). The integrity of each RNA was assessed by electrophoresis on a 1.2% agarose gel, and RNA quality was evaluated in a NanoDrop 2000c spectrophotometer (Thermo Scientific, Waltham, MA). RNA samples, with A260/280 and A260/230 ratios between 1.8–2.2, were selected for the construction of cDNA libraries and RNA-sequencing. mRNA was purified and then assessed using an Agilent Technologies 2100 Bioanalyzer (Agilent, United States) to generate a transcriptome. The constructed RNA library was sequenced using a BGI-SEQ-500 platform at Shenzhen Genomics Institute (BGI, Shenzhen, China) after a library quality test. Low-quality reads and adapters of the RNA-Seq raw data were removed first for all samples to obtain high-quality clean data when RNA-seq was completed. Clean data were compared to the reference genome by HISAT software, and then the R package HTSeq was used to calculate the fragments per kilobase of exon model per million mapped fragments (FPKM) for each transcript.
Expression profiles of GA biosynthetic gene families
The transcriptional data of GA biosynthetic gene families, CPS, KS, KO, KAO, GA20ox, GA3ox, and GA2ox, were extracted from the transcriptome data of stems, fruits, flowers and fruit pits. Among these transcriptome dataset, flowers transcriptome data were sequenced by Lian et al. . Flowers were collected from two peach cultivars, ‘CN14’ and ‘HuangShuiMi’ (HSM). ‘CN14’ is a semi-dwarf cultivar and ‘HSM’ is a standard cultivar. Dwarfing alleles in ‘CN14’ can cause major changes in GA biosynthetic gene expression levels, so the transcriptional data of GA biosynthetic gene families from ‘HSM’ are used in this study. Heat maps was drawn based on the log2(FPKM) using pheatmap (v 1.0.12) in the R statistical language.
Analysis of bioactive GA content
Approximately 1.5 g of the tissues were ground in liquid nitrogen. The ground powder was transferred into a 50-mL centrifuge tube and mixed with 10 mL of extraction solution (isopropanol/ hydrochloric acid mixture). The mixture was shaken at 4 °C for 30 min. Dichloromethane (20 mL) was added to the mixture, which was further shaken at 4 °C for 30 min. The mixture was centrifuged at 10000×g for 5 min at 4 °C and separated into two layers. The lower layer was dried using an N2 gas stream and redissolved in 400 μL methanol containing 0.1% formic acid. The extracts were filtered through 0.22 μm Millipore membranes and analyzed using an HPLC ESI-MS/MS system (Agilent, America) equipped with a Poroshell 120 SB-C18 column (2.1 × 150 mm, with a particle size of 2.7 μm; Agilent, America). The column was sequentially eluted using mobile phase A (methanol containing 0.1% formic acid) and mobile phase B (water containing 0.1% formic acid). The linear gradient for phase B was as follows: 0–2 min, 80%; 2–14 min, 80–20%; 14–15 min, 20%; 15.1 min, 80%; and 15.1–20 min, 80%. Mass spectra were acquired in positive ion mode. The mass spectrometer ion spray temperature was 400 °C, the ion spray voltage was 4500 V, and the curtain gas was 15 psi. Standard curves were constructed and used to quantify the content of GA1, GA3 and GA4. Deuterated GA4 (10 ng) was added into 1 mL of the extracts, and the parent/ daughter ions of deuterated GA4 were 335.1/245.2 and 335.1/213.1. Parent/Daughter ions for GA1 were 347.4/259.2 and 347.4/273.1, GA3 were 345.2/143.0 and 345.2/239.2, and GA4 were 331.4/243.2 and 331.4/213.1.
Analysis of gene transcription level
Quantitative real-time RT-PCR (qRT-PCR) was carried out with the StepOnePlus Real-Time PCR System (Applied Biosystems, Foster, CA) as described previously  using an ABI PRISM 7500 FAST Sequence Detection System (Applied Biosystems, USA). The primer sequences are shown in Table S2. The peach actin gene PpGAPDH (Prupe.8G132000) was used to normalize the RNA-expression levels. Melt curve analysis was performed at the end of 40 cycles to ensure the proper amplification of target fragments. The melt curve had a single, narrow, sharp peak, indicating that the primers used for qRT-PCR were of good quality and met the requirements of qRT-PCR assay. Each experiment was repeated three times with three biological replicates.
Expression vector construction and plant transformation
The whole coding sequences of PpGA20ox1, − 2, − 5, PpGA3ox1 and PpGA2ox6 were amplified using cDNA synthesized from shoot tips of ‘QMH’ as templates. The PCR product and pSAK277 vector were digested with a restriction enzyme, and then joined together using ligases (NEB, Beijing). Arabidopsis transformation was performed according to the floral dip method . For transgenic plant selection, T0 seeds were sterilized and germinated on Murashige and Skoog (MS) medium containing 12 μg mL− 1 kanamycin. Kanamycin-resistant plants were transplanted to soil and placed in a growth chamber at 25 °C and 65–80% relative humidity. Phenotype analysis was conducted in the T3 generation.
Tobacco (Nicotiana tabacum) was transformed using the leaf-disc method . More than five transgenic plants were selected using Murashige and Skoog (MS) medium containing 50 μg mL− 1 kanamycin and were confirmed by PCR using specific primers for the PpGA2ox6 gene.
Transient expression of PpGA20ox1 in peach leaves
The vector 35S:PpGA20ox1 was introduced into the GV3101 Agrobacterium strain by heat shock transformation. Peach seedlings were cultivated for 18 d and then immersed into the bacterial suspension carrying either a control plasmid or recombinant plasmid at room temperature. A vacuum chamber attached to a vacuum pump (Model#SHZ-D, China) was used to create a vacuum (0.07 MPa) for 30 min to aid plant uptake of the suspension. Finally, the peach seedlings were cultured in a growth chamber at a temperature of 22 °C under a 16-h light/8-h dark cycle for 2 d. Leaves were collected for analysis of gene transcription levels and GA content.
Availability of data and materials
The RNA-seq data used in this study are available through BioProject accession number PRJNA723104 on NCBI SRA (Sequence Read Archive) and included in the following published article:
Lian, X., Zhang, H., Jiang, C., Gao, F., Yan, L., Zheng, X., Cheng, J., Wang, W., Wang, X., Ye, X., Li, J., Zhang, L., Li, Z., Tan, B., Feng, J. (2021). De novo chromosome-level genome of a semi-dwarf cultivar of Prunus persica identifies the aquaporin PpTIP2 as responsible for temperature-sensitive semi-dwarf trait and PpB3–1 for flower type and size. Plant biotechnology journal, https://doi.org/10.1111/pbi.13767. Advance online publication. https://doi.org/10.1111/pbi.13767.
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We thank Anita K Snyder for critical reading of the manuscript.
The work was conducted at the Henan Provincial Key Laboratory of Fruit and Cucurbit Biology and supported by the Joint Funds of the National Natural Science Foundation of China (U1804114), the National Key Research and Development Program of China (2019YFD1000104), the Henan Province Outstanding Foreign Scholar Program (GZS2020007), and the Special Fund for Henan Agriculture Research System (S2014–11-G02).
Ethics approval and consent to participate
All methods and protocols were carried out in accordance with the relevant guidelines and regulations. The plant tissue used in this study are collected from peach trees (preserved in Fruit Tree Germplasm Repository of Henan Agricultural University) and Arabidopsis thaliana (Ecotype Columbia) (kept by Henan Provincial Key Laboratory of Fruit and Cucurbit Biology).
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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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Verification of PpKO gene structure using the alignment results of reads obtained from the transcriptome of peach flowers, stems, fruits and pits. Figure S2. Sequence alignment of GA20ox from Arabidopsis and peach. The LPWKET motif are underlined. Figure S3. The phylogenetic trees of CPS (A), KS (B), KO (C), KAO (D) and GA3ox (E). The amino acid sequences were downloaded from Phytozome database and the Gene ID are listed after the gene name. Figure S4. qPCR analysis of transgenic lines. Two transgenic lines of PpGA20ox1 (A) and -2 (B) in ga20ox mutant (CS92956) of Arabidopsis. Three transgenic lines of PpGA20ox1 (C) and -2 (D) in Arabidopsis (Ecotype Columbia). Two lines of PpGA3ox1 (E) in Arabidopsis (Ecotype Columbia). Two lines of PpGA2ox6 in Nicotiana tabacum. Figure S5. Overexpression of PpGA20ox5 in Arabidopsis. Table S1. Sequence of genes involved in GA biosynthesis of peach. Table S2. List of primers used in this study.
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Zhang, M., Ma, Y., Zheng, X. et al. The distribution of bioactive gibberellins along peach annual shoots is closely associated with PpGA20ox and PpGA2ox expression profiles. BMC Genomics 23, 730 (2022). https://doi.org/10.1186/s12864-022-08943-5
- Prunus persica
- Annual shoot