Genome-Wide Analysis of SPL Gene Family and Functional Identification of JrSPL02 Gene in the Early Flowering of Walnut

: SPL transcription factors affect plant growth and development, including blooming and photoperiod control. The investigation began with transcriptome data screening of 28 JrSPL genes in walnut ( Junglans regia L.) ‘Wen185’. These genes were discovered on all chromosomes except 6 and 15. Phylogenetic study divides the 28 JrSPL genes into five groupings. The biggest cluster, cluster IV, has 12 JrSPL genes. The expression of JrSPL genes in different tissues was investigated by qRT-PCR. JrSPL02 gene expression was greater in walnut female and male flower tissues than other genes. Subcellular localization has shown the JrSPL02 gene resides in the nucleus. Jre-miR156 may target JrSPL02 ’s 3 ′ -UTR region, according to miRNA sequencing, RACE, and BiFC studies. Arabidopsis plants expressing the JrSPL02 gene flowered 3 days faster than the wild type, according to phenotypic observation. Transgenic lines had more stem branches and siliques than the control group but fewer rosette leaves. In summary, this study functionally analyses the metamorphosis of the miR156-SPL module during the blooming stage and the underlying mechanisms that govern early fruiting in early-fruiting walnuts in Xinjiang.


Introduction
The SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factor family, which harbours a conserved SBP domain, was first identified in Antirrhinum majus [1].The participation of SPL transcription factors in phase transitions plays a significant role in plants, since they serve as a pivotal regulatory hub throughout the developmental process of flowers.The SPL family genes have significant functions in the regulation of various aspects of plant development, including embryonic development, interphase length, leaf development, transition between reproductive development phases, flower and fruit development, apical dominance, accumulation of anthocyanin, response to gibberellin, transduction of light signals, and maintenance of intracellular copper ion homeostasis [2][3][4][5].The SPL genes modulate the temporal regulation of plant blooming and the development of floral organs.An instance of early blooming and aberrant inflorescence in Arabidopsis may be seen as a result of the overexpression of AtSPL3 [6].Numerous research have provided empirical evidence supporting the involvement of the SPL gene family in the intricate processes of floral growth and development.These investigations have underscored the pivotal role played by this gene family in the meticulous regulation of the blooming cycle in many plant species.Currently, SPL transcription factors have been reported in several plant species, including Arabidopsis, apple, pear, citrus, grape, peach, jujube, tea tree, and mulberry [7][8][9][10][11][12][13]. The SPL genes exhibit distinct functions across species, while most studies focus on their regulation of blooming.
CsSPLs respond to SBP genes that have been found to regulate the expression of MADS-box genes, which are crucial in flower development and transformation [14,15].
Multiple studies have shown that SPL genes play an important role in facilitating the process of plant blooming in both leaf and stem apical meristems.The BpSPL1 gene in birch has a special affinity for the BpMADS5 promoter, hence playing a role in the control of flowering development [16].According to, the genes AtSPL3, AtSPL4, and AtSPL5 in Arabidopsis are identified as direct upstream activators that redundantly promote flowering by regulating leaf-like, fruit-like, and APETALA1 traits [17].The expression levels of BrSBP9, BrSBP10, and BrSBP19 in cabbage flowers are dramatically increased compared to other tissues [18].These findings suggest that these genes play a crucial role in the regulation of the blooming process.
The maturation process of fruit trees necessitates undergoing a singular phase transition from the juvenile to the adult stage, occurring only once throughout their lifespan.The phase transition is characterised by a continuous and progressive progression.In comparison to other agricultural crops, fruit trees often exhibit a lengthier duration from the initial planting stage to the subsequent stages of blooming and fruit production.The age pathway is governed by SPL genes, which serve as crucial regulatory factors [19,20].The traslation of SPL genes in several plant species is inhibited by miR156 [19].In the model plant Arabidopsis, it has been shown that 11 out of the 17 members of the SPL gene family are known to be targeted by miR156.SPL3, SPL4, and SPL5 have be identified as key regulators of the blooming period and phase transition in Arabidopsis [7].The MdmiR156a2/26 gene in apple serves as a precursor for miR156, a regulatory molecule that plays a role in controlling the phase transition and blooming process in apple.This regulation occurs via the cleavage of MdSPL7/26 by miR156.These findings suggest that miR156, together with its target SPL genes, significantly contribute to the process of blooming.
The walnut (Juglans regia L.) is a long-lasting woody plant belonging to the Juglandaceae family.It is cultivated extensively over the globe owing to its substantial economic, ecological, and social advantages.The typical time frame for walnut trees to undergo flowering and fruit production after seeding ranges from 2 to 8 years [21].Furthermore, the process of developing a new walnut variety via breeding methods is known to need many decades.The breeding process of walnuts is severely restricted by this limitation.Xinjiang is well recognised as a prominent region in China renowned for its abundant walnut resources, making it a major contributor to the country's overall walnut production.The Xinjiang early flowering and fruit production walnut variety, known as 'Wen185', has the remarkable ability to blossom and produce fruit within 2-3 years of being sown, and in some cases, even within the same year.This characteristic makes it a valuable resource for investigating the fast phase transition process of fruit trees.
Currently, there is a limited amount of literature available about the transcription factors of SPL in the early stages of walnut cultivar 'Wen185'.This study aims to investigate the involvement of SPL transcription factors in the differentiation of flower buds during the early stages of walnut development.The research group conducted a comprehensive analysis of 28 JrSPL genes in walnut, utilising transcriptome sequencing data from the early walnut variety 'Wen185'.The expression of JrSPL02, exhibiting the greatest expression level, was subjected to functional validation in Arabidopsis.Additionally, small RNA sequencing was conducted on both juvenile and adult tissues of walnut to identify miR156.The connection between miR156 and JrSPL02 was confirmed by the implementation of RLM-RACE and luciferase activity detection studies.This study provides insights into the miR156-SPL module's role in phase transition of early Xinjiang walnut flower bud.

Identification of Walnut SPL Genes
A selection was made from the previously identified JrSPL gene family, focusing on genes that exhibited expression during the walnut bud development stage.As a result, a total of 28 JrSPLs were identified and subsequently named as walnut SPL genes (JrSPL01~JrSPL28) based on the chromosomal location of the respective SPL proteins (Table S1).The walnut SPL proteins exhibit a range of lengths, spanning from 148 to 1073 amino acids.These proteins also display varying molecular weights, falling within the range of 16.89 to 119.07 kDa.All JrSPL genes were predicted to be localized in the nucleus.An analysis was conducted to examine the distribution of JrSPL genes across chromosomes.The results revealed that all JrSPL genes are present on 16 chromosomes, with the exception of chromosomes 06 and 15, each chromosome is responsible for the distribution of 1-3 units, as depicted in (Table S1).

Phylogenetic Tree Analysis of Walnut JrSPL Gene Family Members
The analysis of gene structure reveals that, with the exception of JrSPL02, JrSPL05, and JrSPL08, which consist of a single intron, the remaining 25 genes exhibit a range of 2 to 10 introns (Figure 1A).Furthermore, it is noteworthy that only 5 JrSPL genes possess a complete UTR region.The conserved motifs of walnut SPL proteins were analysed using the MEME online software.The analysis revealed that all JrSPL proteins possess motif1, motif2, and motif4, which are closely positioned.Furthermore, among the longer JrSPL proteins (>560 aa JrSPL protein), a total of 8 proteins exhibit motif3 and motif5 (Figure 1A).The SBP domain within the SPL transcription factors in walnut demonstrates a high degree of conservation, with all instances featuring distinct zinc binding sites (Zn-1 and Zn-2) as well as nuclear localization signals (NLS).Furthermore, it has been discovered that four SPL proteins in walnut possess an additional Ankyrin repeats (ANK_2) domain located at the 3 ′ end.The cis-acting elements within the promoter region of JrSPL genes were examined (Figure 1C, Table S2).The analysis revealed that, along with a significant presence of CAAT-box and TATA-box core elements, the most prevalent elements in JrSPL promoters are associated with light response.This indicates that the expression of JrSPL genes is related with light signal pathway, aligning with their role in flower development.Furthermore, it is worth to note that certain promoters found in family members exhibit an abundance of stress response elements, such as those related to hypoxia, low temperature, or hormone response elements like gibberellin and auxin.This suggests that the expression of JrSPL is subject to regulation through other pathways.The clustering results of the JrSPL promoter elements exhibit inconsistencies with their phylogenetic results, suggesting that the evolutionary trajectories of JrSPLs function and expression pattern may diverge.
In order to investigate the phylogenetic relationship between the 28 JrSPL gene family members in walnut and SPL genes in other species (Figure 1B), this study primarily focused on Arabidopsis thaliana and another five fruit tree species (Pyrus bretschneideri Rehd., Malus pumila Mil1., Vitis vinifera L., Prunus persica, and Ziziphus jujuba) for the purpose of conducting phylogenetic tree analysis.The findings indicate that the complete evolutionary tree is partitioned into five distinct clusters.These clusters are denoted as clusters I to V. Cluster V is comprised of the largest number of members within the walnut JrSPL gene family, consisting 12 members.Cluster I consists of 7 members of the walnut JrSPL gene family.Clusters II, III, and IV exhibit a relatively uniform distribution, comprising 4, 2, and 3 members of the walnut JrSPL gene family, respectively.The genes JrSPL10, 13, 22, 23, 25, 26, and 28 within cluster I exhibit homology with Arabidopsis AtSPL8, peach PrupeSPL8, and other similar genes that are known to be associated with anther development.The genes JrSPL01, 03, 04, and 27 are found within cluster II along with Pbr038289.1 and other genes.These genes are known to be involved in the regulation of crucial targets during the flowering process.JrSPL06 and 09 are found to be distributed in cluster III along with apple MdSPL33.MdSPL33 is known to be involved in the regulation of flower quantity.The cluster labelled as V contains the genes JrSPL07, 11,12,14,15,16,18,19,20,21,24, and 27, along with PrupeSPL1 and other genes.These genes are associated with the regulation of fruit ripening.The genes JrSPL02, 05, and 08 are found within cluster IV, alongside ATSPL3/4 and other genes that are associated with flower development.In general, the SPL genes of walnut, Arabidopsis, and five different types of fruit trees exhibit an even distribution across five distinct clusters.
regulation of flower quantity.The cluster labelled as Ⅴ contains the genes JrSPL07, 11,12,14,15,16,18,19,20,21,24, and 27, along with PrupeSPL1 and other genes.These genes are associated with the regulation of fruit ripening.The genes JrSPL02, 05, and 08 are found within cluster Ⅳ, alongside ATSPL3/4 and other genes that are associated with flower development.In general, the SPL genes of walnut, Arabidopsis, and five different types of fruit trees exhibit an even distribution across five distinct clusters.

Expression Pattern Analysis of Walnut JrSPL Genes in Different Tissues
The present study aimed to analyse the expression levels of JrSPL genes during five female flower bud development stages.It was observed that 9 JrSPL genes (JrSPL02, JrSPL05, JrSPL06, JrSPL10, JrSPL17, JrSPL19, JrSPL20, JrSPL23, JrSPL26) exhibited elevated expression levels, as depicted in Figure 2A.The expression levels of JrSPL02, JrSPL05, and JrSPL09 exhibited an upward trend, whereas the expression levels of the remaining JrSPL genes demonstrated a downward trend.JrSPL02 and JrSPL05 exhibited elevated expression levels during the developmental stages of female flower buds in comparison to the remaining JrSPL genes.In order to investigate the expression characteristics of JrSPL genes in various tissues of walnut, we employed fluorescent quantitative PCR to analyse the expression levels of 9 JrSPL genes mentioned above during the development of female flower buds.This analysis was conducted on 8 different tissues and organs of walnut, namely stems, leaves, male flowers, female flowers, male flower buds, female flower buds, kernels, and apical buds.The expression of all 9 JrSPL genes is observed in various tissues (Figure 2B).Among the identified genes, JrSPL17 and JrSPL26 exhibit the highest expression levels in leaves.In contrast, JrSPL02, JrSPL10, and JrSPL23 demonstrate the highest expression levels in male flowers.Lastly, JrSPL20 displays a specific and notably high expression level in female flower buds.The expression levels of JrSPL genes in stems and kernels exhibit relatively low levels.It has come to our attention that JrSPL02 exhibits notably elevated expression levels in male flowers.female flower bud development stages.It was observed that 9 JrSPL genes (JrSPL02, JrSPL05, JrSPL06, JrSPL10, JrSPL17, JrSPL19, JrSPL20, JrSPL23, JrSPL26) exhibited elevated expression levels, as depicted in Figure 2A.The expression levels of JrSPL02, JrSPL05, and JrSPL09 exhibited an upward trend, whereas the expression levels of the remaining JrSPL genes demonstrated a downward trend.JrSPL02 and JrSPL05 exhibited elevated expression levels during the developmental stages of female flower buds in comparison to the remaining JrSPL genes.In order to investigate the expression characteristics of JrSPL genes in various tissues of walnut, we employed fluorescent quantitative PCR to analyse the expression levels of 9 JrSPL genes mentioned above during the development of female flower buds.This analysis was conducted on 8 different tissues and organs of walnut, namely stems, leaves, male flowers, female flowers, male flower buds, female flower buds, kernels, and apical buds.The expression of all 9 JrSPL genes is observed in various tissues (Figure 2B).Among the identified genes, JrSPL17 and JrSPL26 exhibit the highest expression levels in leaves.In contrast, JrSPL02, JrSPL10, and JrSPL23 demonstrate the highest expression levels in male flowers.Lastly, JrSPL20 displays a specific and notably high expression level in female flower buds.The expression levels of JrSPL genes in stems and kernels exhibit relatively low levels.It has come to our attention that JrSPL02 exhibits notably elevated expression levels in male flowers.

Walnut miRNA Sequencing and Target Gene Prediction
MicroRNA (miRNA) sequencing was conducted on adult and juvenile leaves, resulting in an average of 14,960,063 clean reads collected (Table S3).Among these reads, those falling within the 18-30 bp range accounted for 71.63%, and 48.89% of the reads could be successfully mapped to the walnut genome.The average Q30 value of the two

Walnut miRNA Sequencing and Target Gene Prediction
MicroRNA (miRNA) sequencing was conducted on adult and juvenile leaves, resulting in an average of 14,960,063 clean reads collected (Table S3).Among these reads, those falling within the 18-30 bp range accounted for 71.63%, and 48.89% of the reads could be successfully mapped to the walnut genome.The average Q30 value of the two samples was determined to be 95.85%.The identification of miRNAs was conducted on the reads that were mapped to adult and juvenile samples, yielding a total of 140 known miRNAs and 397 newly discovered miRNAs, respectively.
The miRNA expression profiles in Adult and Juvenile samples exhibit similarities, with a shared presence of 139 known miRNAs and 396 new miRNAs.Notably, only a limited number of miRNAs demonstrate specific expression in either of the samples (Figure 3A).The expression status of miRNA in both samples exhibits a high degree of similarity.The box plot illustrates the average TPM value following normalisation, revealing minimal disparity between the two samples (Figure 3B).The length of known miRNA was determined and found to be concentrated primarily at 20 or 21 base pairs (Figure 3C).The miRNAs were categorised into 8 miRNA families based on distinct miRNA gene families.Subsequently, the average TPM value for each known miRNA gene family was computed.The findings indicate that over 100 known microRNAs (miRNAs) primarily fall into 8 miRNA families.Notably, the miR166, miR167, and miR162 families are the predominant expressed miRNAs, as depicted in Figure 3A.
The results of the differential expression analysis indicate that out of the total 553 miRNAs examined, only 51 miRNAs exhibited significant expression differences between adult and juvenile samples (Figure 3D).Among these, 35 miRNAs were found to be up-regulated in adult samples, while 16 miRNAs were down-regulated.Differential expression of miRNAs was analysed, and subsequent target gene prediction and functional enrichment analyses were conducted.In the Gene Ontology (GO) biological process terms (Figure 3E), the target genes of differentially expressed miRNAs are primarily enriched in terms related to the regulation of transcription, DNA-templated, developmental processes.The analysis of KEGG enrichment revealed that the primary enriched pathways are plant hormone signal transduction, protein processing in endoplasmic reticulum, and pentose and glucuronate interconversions (Figure 3F).The results of the differential expression analysis indicate that out of the total 553 miRNAs examined, only 51 miRNAs exhibited significant expression differences between adult and juvenile samples (Figure 3D).Among these, 35 miRNAs were found to be up-regulated in adult samples, while 16 miRNAs were down-regulated.Differential expression of miRNAs was analysed, and subsequent target gene prediction and functional enrichment analyses were conducted.In the Gene Ontology (GO) biological process terms (Figure 3E), the target genes of differentially expressed miRNAs are primarily enriched in terms related to the regulation of transcription, DNA-templated, developmental processes.The analysis of KEGG enrichment revealed that the primary enriched pathways are plant hormone signal transduction, protein processing in endoplasmic reticulum, and pentose and glucuronate interconversions (Figure 3F).

Co-Expression Network Construction
To further explore the role of SPL genes in walnut flower development, we conducted a WGCNA analysis on a set of 12,664 differentially expressed genes (DEGs) across various developmental stages (Figure 4A).A total of seven JrSPL genes were categorised into two distinct modules.We have identified genes that are co-expressed with JrSPL, with a correlation coefficient greater than 0.8, in two distinct modules.Subsequently, we have constructed a co-expression network based on these findings.Module1 consists of JrSPL02, JrSPL03, JrSPL05, JrSPL10, and an additional 24 genes.The primary node within this module is JrSPL02, which has a degree of 38.JrSPL02 is found to be co-expressed with a total of 17 genes.Furthermore, it should be noted that JrSPL10 exhibits co-expression with a total of seven genes.Module2 consists of JrSPL19, JrSPL20, JrSPL26, and 14 additional genes.The central node within this module is JrSPL26, which has a degree of 33 and exhibits co-expression with 13 other genes.Additionally, we conducted a prediction of the target relationship between miRNA expressed in adult and juvenile samples and all gene cDNA in the co-expression network.We also incorporated miRNA into the co-expression network.Based on the findings, it has been determined that out of the 45 genes present in the co-expression network, only JrSPL02, JrSPL03, and JrSPL26 are projected to be influenced by known miRNA.It is noteworthy that all of these miRNAs are classified under the miR156 and miR157 families.Furthermore, it has been observed that Jr07_24410, which exhibits co-expression with JrSPL05, is anticipated to be the target gene of seven newly discovered miRNAs.On the other hand, Jr09_06540, which does not display co-expression with any JrSPL gene, is predicted to be the target gene of two newly identified miRNAs.It is noteworthy that all of these miRNAs are situated within module1.The regulatory role of MiR156 in flower development is of significant importance and has been observed to be conserved across various terrestrial plant species.The MiR156 molecule selectively targets two co-expression networks led by JrSPL genes, potentially influencing walnut flower development through distinct mechanisms.The expression level heat map indicates that out of the three JrSPL genes targeted by miR156 and miR157, only JrSPL02 exhibits a clear expression advantage in the associated co-expression network (module 1) (refer to Figure 4B,C).The aforementioned findings indicate that miR156-JrSPL02 may hold considerable importance in the context of walnut flower development.

Gene Cloning and Interaction Verification
We selected JrSPL02 for further study, as it is the core node with expression advantage in the co-expression network.The full-length cDNA of JrSPL02 was amplified by Race experiment and sequenced (Figure 5A).The alignment results show that there are 3 bp nucleotide variations between Race-JrSPL02 and the sequence in the reference genome.In addition, at the 3 ′ end, Race-JrSPL02 is actually 50 nucleotides less than the sequence in the reference genome, which corrects the annotation error of this gene in the reference genome.The 2-20th bases of JrmiR156 are complementary to the 2987-2969 bp of JrSPL02, and there is no mismatch in the complementary region.This region is located in the 3 ′ -UTR region of JrSPL02.Jre-miR156 may regulate the translation process of JrSPL02 by targeting this position, which explains their same expression pattern during flower development.Further interaction between Jre-miR156-JrSPL02 was verified by BiFC (Figure 5B).Compared with the miR-NC/miR-156-Wt co-transfection group, the relative fluorescence value of the miR-156/JrSPL02 Wt co-transfection group decreased (p < 0.01); compared with miR-NC/JrSPL02 Mut, the relative fluorescence value of miR-156/JrSPL02 Mut decreased, but there was no significant difference (p < 0.05); compared with miR-156/JrSPL02 Wt, the relative fluorescence value of miR-156/JrSPL02 Mut significantly increased (p < 0.01).These results indicate that miR-156 effectively cut the 3 ′ -UTR region of the JrSPL02 gene, and the mutation affected the binding ability between them.

Subcellular Localization of JrSPL02 Protein in Walnut
The subcellular localization of the JrSPL02 protein was examined through the transient expression of its fusion protein with green fluorescent protein (GFP) in tobacco leaf epidermal cells.The result confirmed that the JrSPL02 protein was localised within the nucleus (Figure 5C), consisting with the prediction of NLS within JrSPL02 protein (Figure 1A).

Subcellular Localization of JrSPL02 Protein in Walnut
The subcellular localization of the JrSPL02 protein was examined through the transient expression of its fusion protein with green fluorescent protein (GFP) in tobacco leaf epidermal cells.The result confirmed that the JrSPL02 protein was localised within the nucleus (Figure 5C), consisting with the prediction of NLS within JrSPL02 protein (Figure 1A).

Phenotype Identification of JrSPL02-3301 Transgenic Arabidopsis
The gene overexpression vector, JrSPL02-3301, was successfully introduced into wildtype Arabidopsis through Agrobacterium tumefaciens GV3101 (Figure 6).The wild-type and transgenic T3 generation homozygous seeds were planted in nutrient pots filled with substrate.Three candidates (L1, L2, and L3) were chosen (Table 1).The plant height, number of rosette leaves, flowering time, and number of siliques were recorded after a growth period of 40 days.The data indicates that the transgenic Arabidopsis exhibits increased height compared to the wild-type Arabidopsis.Specifically, the transgenic lines display an average of 8 rosette leaves, whereas the control group demonstrates an average of 12 rosette leaves.The transgenic lines exhibit a reduced flowering time compared to the control group.Additionally, the transgenic lines display an increased number of stem branches and siliques in comparison to the control group.These findings indicate the JrSPL02-3301 involves in the regulation of the phenotype in plant.

Discussion
The cultivation of walnut has an extensive cultural history and is associated with significant financial benefits.Distant hybridization sometimes takes place among different species within the walnut genus, resulting in the enrichment of genetic resources.This is facilitated by the fact that each species within the genus Walnut has the same number of chromosomes, allowing for successful cross-pollination and fruit production.According to a recent study conducted by Jin et al. [22], empirical evidence indicates that walnuts exhibiting early-fruiting characteristics have a very brief infancy phase, with some individual plants capable of flowering and producing fruit within 2-3 years of being sown.In contrast, late-fruiting walnut varieties take 8-10 years until they are able to yield fruit.Regardless of the implemented interventions, plants are unable to initiate the process of blooming prior to the occurrence of the stage changeover.According to Song et al. [23], the plant's ability to produce flowers and undergo blooming is contingent upon

Discussion
The cultivation of walnut has an extensive cultural history and is associated with significant financial benefits.Distant hybridization sometimes takes place among different species within the walnut genus, resulting in the enrichment of genetic resources.This is facilitated by the fact that each species within the genus Walnut has the same number of chromosomes, allowing for successful cross-pollination and fruit production.According to a recent study conducted by Jin et al. [22], empirical evidence indicates that walnuts exhibiting early-fruiting characteristics have a very brief infancy phase, with some individual plants capable of flowering and producing fruit within 2-3 years of being sown.In contrast, late-fruiting walnut varieties take 8-10 years until they are able to yield fruit.Regardless of the implemented interventions, plants are unable to initiate the process of blooming prior to the occurrence of the stage changeover.According to Song et al. [23], the plant's ability to produce flowers and undergo blooming is contingent upon the completion of the stage transition.The Xinjiang early-fruiting walnut seeds have the ability to blossom and produce fruit within a span of 2 to 3 years after being sown, and in some cases, even within the same year [24].These seeds are very valuable resources for investigating the processes behind the growth and developmental stages of walnuts.This study used Xinjiang earlyfruiting walnuts as the experimental materials to investigate the developmental process of early-fruiting walnuts.
SPL contributes to the transition from the juvenile to adult phases of plant growth, serving as a crucial regulatory centre throughout the flower development process [2].A promoter specifically expressed in the shoot apical meristem could induce the expression of SPL in apical buds, facilitating the acceleration of blooming in plants.Additional genetic tests have provided more evidence to support the notion that the SPL gene is stimulated via the activation of MADS-box genes, including AP1, LFY, FUL, and SOC1, as shown in a study conducted by Wang et al. [25].The SPL gene is distinguished by its conserved SBP domain of 76 amino acids, which is exclusive to green plants and lacks any known counterparts in bacteria or mammals [26].
The upregulation of PvSPL6 in foxtail millet promotes flowering and leads to a decrease in internode length, internode number, and plant height [27].The modulation of 15 CclSBP genes during floral induction defence is associated with the initiation of flower formation [28].The ZmSPL genes have been identified as significant regulators of the flowering period in maize.The regulation of blooming time in foxtail millet involves the direct up-regulation of SEPAL-LATA3 (SEP3) and MADS32 by SPL7 and SPL8 [29].The upregulation of SPL7 and SPL8 has been shown to accelerate the process of flowering, while the suppression of their expression may result in a delay in flowering or even the absence of blooming altogether [30].WRKY12 and WRKY13 participate in the control of age-mediated flowering under short-day conditions though physically interacting with SPL genes [31].FHY3 and FAR1 directly interact with SPL3, SPL4, and SPL5, and inhibit their binding to the promoters of several key flowering regulatory genes, thus downregulating their transcript levels and delaying flowering [32].
The transition of plants from the juvenile to adult phases is mostly facilitated by the miR156-SPL module [19,33].Several prior research have shown the collaborative role of microRNA 156 (miR156) and its target SPL transcription factor in the regulation of the blooming transition process in plants [19,25,34].The upregulation of miR156 in Arabidopsis has been shown to considerably prolong the shift from vegetative development to reproductive growth.Conversely, the overexpression of miR156 leads to the manifestation of heightened juvenile characteristics in plants [35].According to Yue et al. [36], there is a decline in the quantity of miR156 as individuals age, although the expression of its target gene SBP/SPL shows an increase.The miR156-SPL pathway in plants is crucial for regulating plant stage transition and blooming, as well as for its involvement in other biological processes at the transcriptional level or via protein interactions [37].
This work used transcriptome sequencing data obtained from early-fruiting walnut samples to identify a total of 28 SPL genes that have conserved SBP domains.In comparison to other species, walnuts have a greater abundance of SPL genes.Out of the total of 28 JrSPL proteins, it has been shown that four of them possess ANK domains.Two of the proteins, JrSPL12 and JrSPL14, possess ANK domains as well as BLAST DEXDc domains.The conservation of the SBP domain is evident across all SPL transcription factors, as they all possess distinct zinc-binding sites (Zn-1 and Zn-2) and NLS (Figure 1A).The majority of SPL genes in plants include a CCCH-type motif known as Zn-1, with the NLS overlapping with the Zn-2 motif by four residues [38,39].The JrSPL genes have significant similarity to the other fruit tree SPL genes and exhibit characteristic SBP conserved domains and placement signals, suggesting that SPL genes are largely conserved across diverse species.The development of the SPL gene family across species may exhibit species-specific patterns, perhaps influenced by several evolutionary events.The impacts, nevertheless, the functionalities of gene family members seem to be mostly consistent.
The findings of the phylogenetic tree analysis were categorised into five groups, consistent with earlier studies on SPL genes in Arabidopsis and tartary buckwheat [33,40].The similarity in structural characteristics and motif composition within the same group provides additional evidence for their placement in the phylogenetic tree (Figure 1B), suggesting a correlation between the functional evolution of SPL genes and their varied structures and conserved motifs [38,39].The results of the cluster analysis indicate a significant degree of similarity between JrSPL02 and ATSPL3/4, a gene known to play a crucial role in the regulation of flower development.The JrSPL26 protein is the homology of ATSPL8 and PrupeSPL8, which are known to be associated with anther development Moreover, these proteins are found to be closely grouped together, indicating a potential involvement of JrSPL02 and JrSPL26 in the developmental processes of the walnut stage.The transformation process has a regulatory influence on the growth and development of walnuts.
Several SPL genes exhibit distinct roles at different phases of plant development [8].For instance, the level of MdSPL6 expression in apples is elevated in both leaves and buds [41].Similarly, the expression of VvSBP2 and VvSBP15 in grapes is greater in vegetative organs, such as leaves, stems, and tendrils, while a steady reduction of expression occurs throughout the fruit ripening process [42].The analysis of transcriptome expression data revealed that a total of 9 JrSPLs genes exhibited expression.The analysis of tissuespecific expression data revealed that both JrSPL02 and JrSPL05 exhibited high expression levels in inflorescences and flower buds.However, it was observed that the total expression level of JrSPL05 was comparatively lower.The expression pattern of JrSPL26 is seen in leaves, inflorescences, and flower buds, with a comparatively lower expression level in floral organs as compared to JrSPL02 (Figure 2).Hence, based on our first assessment, it can be inferred that JrSPL02 potentially plays a crucial role in regulating the growth and maturation of walnut blossoms.
The examination of the sequencing data findings indicated that the majority of short RNA sequences in juvenile and mature exhibited a concentration within the range of 20 to 24 (Figure 3), aligning with the typical length distribution of small RNAs [43].The sequencing findings were subjected to statistical analysis, which led to the identification of a potential targeting link between JrSPL02 and 15 anticipated miR156 (Figure 4).This prediction was based on the examination of the targeted cleavage site of the JrSPL gene [44].The targeted cleavage connection between JrSPL02 and mdm-miR156u was confirmed using RLM-5 ′ -RACE analysis.The band size in question measures around 127 base pairs, with the cleavage location occurring between the bases UC.The targeted cleavage link between miR-156 and JrSPL02 was verified by the use of a dual-luciferase reporter system (Figure 5).This experimental approach provided additional evidence that miR-156 specifically cleaves inside the 3 ′ -UTR region of the JrSPL02 gene.
According to a previous study [16], the BpSPL1 gene in white birch trees has a particular binding affinity towards the promoter region of the BpMADS5 gene, therefore playing a role in the regulation of flower development.The SPL8 gene in Arabidopsis has been shown to have an impact on anther development [45].On the other hand, SPL14 has been identified as a negative regulator that influences the transition from vegetative growth to flowering, as discussed by Wu and Poethig [2].The functional deficiency of SPL9 leads to a reduced duration between the production of leaf primordia during the vegetative development phase.Additionally, it induces alterations in the structure of inflorescence and promotes branching augmentation [46].The JrSPL02 transgenic plants exhibited a reduction in the number of rosette leaves and a decrease in leaf size.Additionally, these plants had a notable acceleration in the blooming process, suggesting that the JrSPL02 gene has the capacity to facilitate early flowering in plants (Figure 6).The investigation included the measurement of root length and plant height in transgenic plants.The results indicated that transgenic plants exhibited considerably greater length in their major roots and stems compared to wild-type Arabidopsis plants.Additionally, transgenic plants had an earlier blooming period, almost three days ahead of wild-type plants.

Walnut Materials
This research employed walnut 'Wen 185' from the Ministry of Agriculture and Rural Affairs' Fruit Tree Science Observation Station in Xinjiang (Yecheng County, Kashgar).The three-year-old walnut 'Wen 185' stems, leaves, male flowers, female flowers, kernels, apical buds, male flower buds, and female flower buds were collected for tissue expression quantification from April to June 2019.From 27 April, female flower buds were taken every 15-20 days for transcriptome sequencing (qP1, qP2, qS1, qS2, qS3).The transcriptome data was previously published [47].We gathered 20-30 female flower buds each stage.After isolation, all tissues were rapidly frozen in liquid nitrogen and stored at −80 • C.

Characterization of SPL Genes
These genes' chromosomal location and gene structure were derived from the genome annotation file using the walnut JrSPL gene ID.To explore the evolutionary relationshipof walnut JrSPL with Arabidopsis, pear, apple, grape, peach, jujube, and other species, ClustalW software matched their SPL protein sequences.MEGA 7 software was used to build an evolutionary tree using neighbor-joining with bootstrap set to 1000.The conserved motifs of walnut JrSPL proteins were identified and analysed using MEME (http://meme-suite.org/,accessed on 7 July 2021).The JrSPL genes' 2 kb promoter sequences upstream of the start codon were extracted and uploaded to PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/htmL/searchCARE.html,accessed on 7 July 2021).These potential promoter sequences' cis-acting elements were predicted and visualised using TBtools (version 1).

Fluorescent Real-Time Quantitative PCR
Walnut sample RNA was extracted using the EASYspin Plant RNA Rapid Extraction Kit from Beijing Tiangen Biotech Co., Ltd (Beijing, China).RNA integrity and concentration were determined using 1% agarose gel electrophoresis.For fluorescence quantitative expression analysis, whole RNA was reverse transcribed into cDNA and kept at −20 • C. The JrSPL gene sequences were used to create qRT-PCR primers (Table S4), using walnut 18S as the internal reference gene.All primers were diluted according to primer synthesis guidelines.Following the TAKARA SYBR Premix Ex Taq kit instructions, the reaction system was performed using a Bio-rad CFX96 Real Time Systerm (Hercules, CA, USA).Each experiment included 3 technical and 3 biological replicates, and the data was analysed using the 2 −∆∆Ct technique.

Small RNA Sequencing and Co-Expression Network Construction
Small RNA was isolated from early walnut tissue from juvenile and adult zones using CTAB + Edler RN40 (Edler, RN40)/TRIzol.After extraction, small RNA was verified for purity and prepared for adapter attachment process.To convert short RNA into cDNA, the First Strand Synthesis Reaction buffer, Murine RNase Inhibitor, and M-MµLV Reverse Transcriptase (RNase H-) reagent kit were used.The short RNA library was created and PE150 sequenced using the Illumina NovaSeq 6000 (Illumina, San Diego, CA, USA).
The data were annotated and aligned for short RNA sequences, and TargetFinder software (version 1.7) projected target genes using known and newly predicted miRNA and species gene sequence information.To get target gene annotations, BLAST software was used to match projected target gene sequences to NR, Swiss-Prot, GO, COG, KEGG, KOG, and Pfam databases.The co-expression network and miRNA-RNA targeting connection were created using Cytoscape software, and the WGCNA analysis was the same as previously published [49].

PPM-RLM-5 ′ RACE
The connecting reaction scheme for RNA Oligo was shown in Table S5.After adding RNA to a tube containing RNA Oligo (0.25 µg), it was mixed by pipetting, centrifuged briefly, heated at 65 • C for 5 min in a metal bath, then put on ice for 2 min before centrifugation.Table S6 shows the reverse transcription reaction setup.The solution was pipetted, centrifuged briefly, incubated at 50 • C for 50 min in a constant temperature metal bath, at 70 • C for 15 min, and then put on ice for 2 min and centrifuged at 12,000 rpm for 2 min.After adding 1 µL of RNase H (2U) to a constant temperature metal bath at 37 • C for 20 min, the product was amplified by PCR and stored at −80 • C. The 5-RACE PCR first-round system: 5 ′ RACE template 0.5 µL, 10 µM 5 ′ GeneRacer outer primer 1.0 µL, JrSPL02-R1 (10 µM) 1.0 µL, Invitrogen Platinum PCR Supermix High Fidelity

Dual Fluorescence Reporter Gene Experiment
A seamless cloning procedure joined the JrSPL02 gene's 3 ′ -UTR sequence and the mutation site to the pmirGLO vector.Table S6 shows the mutation location.293TT cells were grown in 10% FBS-DMEM high glucose medium.When confluence reached 90%, cells were digested and transferred to a 24-well plate until density reached 80%.The miRNA-NC or miR-156 mimics, recombinant plasmid pmirGLO-JRSPL02 Wt, and mutated plasmid were transfected into 293TT cells in a 24-well plate using lipo3000 transfection reagent.After 48 h after transfection, cells were washed twice with PBS and 250 µL of 1 × PLB lysis solution was added to each well.Shakers shook the samples at room temperature for 30 min.To measure luciferase activity, 100 µL of LAR II and 20 µL of lysis buffer were added to samples on a 96-well black plate.Exporting and analysing data using GraphPad prism 8.0.

Subcellular Localization
The upstream and downstream primers of JrSPL02 were used to amplify the complete gene length to study its subcellular distribution (Table S5).HindIII and BamHI were used to doubly digest the PCR product containing JrSPL02 and the binary vector pCAMBIA1300-35S-GFP.Using T4 DNA ligase, the digested PCR product and vector were linked and introduced into E. coli DH5α competent cells after purification.Single clone colonies were selected for PCR and sequencing after transformation.To grow the culture, the properly sequenced colonies were shaker-cultured, and JrSPL02-pCAMBIA1300-35S-GFP was removed.The recombinant plasmid and empty vector were introduced separately into Agrobacterium GV3101.After 48 h, single-clone colonies were selected for PCR verification.
After sequencing, bacteria were agitated and put into tobacco.Laser confocal microscope photos were taken after 48 h of culture.

Arabidopsis Genetic Transformation and Phenotype Identification
Based on the results of previous research, the amplified product of the JrSPL02 gene was recovered and connected to the 3301 empty vector after double digestion with HindIII and BamHI.After correct sequencing, the JrSPL02-pBI121 overexpression vector was constructed.It was injected into Agrobacterium GV3101 competent cells, screened for positive clones with kanamycin, and PCR validated the bacterial liquid.The flower dipping approach converted wild-type Arabidopsis (Columbia type) and collected T0 generation seeds in a mixture.Next, T0 transgenic plants were tested on 1/2 MS medium with 25 mg/L kanamycin.After two real leaves grew, they were put in a hole plate.We gathered positive plant seeds and labelled them T1.Genomic DNA was isolated using cetyltrimethylammonium bromide (CTAB) [50], and primers (Table S4) were used to PCR identify JrSPL02F and JrSPL02R to acquire homozygous plants for phenotypic identification.Wild-type and homozygous T3 seedlings were planted in nutritious soil.After 2 days at 4 • C to break dormancy, they were put in a light incubator (22 • C, 16 h light/8 h dark) for 15 days of vertical development.Flowering time, plant height, branches, and rosette leaves were recorded.

Conclusions
The aforementioned data suggest that the JrSPL genes play a role not only in the initiation of blooming, but also in the regulation of growth and development of vegetative organs in walnut.The presence of the regulatory influence exerted by the miR156-SPL module has been seen in early-fruiting walnuts.This finding serves as a significant point of reference and foundation for conducting further investigations into the metamorphosis of the miR156-SPL module during the blooming stage and the underlying mechanisms governing early fruiting in Xinjiang's early-fruiting walnuts.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/horticulturae10020158/s1,Table S1: Identification of JrSPL genes; Table S2: Cis-promoter elements analysis of JrSPL gene in Walnut; Table S3: Statistics of miRNA sequencing results; Table S4: List of the primers used in this work; Table S5: PPM-RLM-5 ′ RACE template synthesis; Table S6: The mutation location for JrSPL02.Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Figure 1 .
Figure 1.Characteristics, phylogenetic and cis-acting element analysis of JrSPL genes.(A) Gene structure, motif, and domain distribution of JrSPL.(B) Phylogenetic analysis of JrSPL genes among multiple species.(C) Number of cis-acting elements in the promoter region of JrSPL.The color of the heat map represents the number of corresponding components, which is shown in the box.

Figure 1 .
Figure 1.Characteristics, phylogenetic and cis-acting element analysis of JrSPL genes.(A) Gene structure, motif, and domain distribution of JrSPL.(B) Phylogenetic analysis of JrSPL genes among multiple species.(C) Number of cis-acting elements in the promoter region of JrSPL.The color of the heat map represents the number of corresponding components, which is shown in the box.

Figure 2 .
Figure 2. Expression of JrSPL in walnut flower bud development and multiple tissues.(A) Heat map of gene expression of JrSPL in walnut development process.(B) Quantitative expression of highly expressed JrSPL in multiple walnut tissues during flower bud development.T-tests were employed to assess the significance of differences in expression levels, with a significance level set at 0.01, significant differences are shown in lowercase letters a-d above the graphs.Mf: Male flower; Ff: Female flower; Mfb: Male flower bud; Ffb: Female flower bud; Sk: seed kernel; Tb: terminal bud.

Figure 2 .
Figure 2. Expression of JrSPL in walnut flower bud development and multiple tissues.(A) Heat map of gene expression of JrSPL in walnut development process.(B) Quantitative expression of highly expressed JrSPL in multiple walnut tissues during flower bud development.t-tests were employed to assess the significance of differences in expression levels, with a significance level set at 0.01, significant differences are shown in lowercase letters a-d above the graphs.Mf: Male flower; Ff: Female flower; Mfb: Male flower bud; Ffb: Female flower bud; Sk: seed kernel; Tb: terminal bud.

Figure 3 .
Figure 3. Statistics of miRNA sequencing results and functional enrichment analysis of target genes.(A) Venn diagram of known and new-miRNA in adult and juvenile groups, the numbers represent the amount of miRNA, and heat map of average expression of different miRNA families in two groups of samples, the redder color represents higher expression, the bluer color represents

Figure 3 .
Figure 3. Statistics of miRNA sequencing results and functional enrichment analysis of target genes.(A) Venn diagram of known and new-miRNA in adult and juvenile groups, the numbers represent the amount of miRNA, and heat map of average expression of different miRNA families in two groups of samples, the redder color represents higher expression, the bluer color represents lower expression.(B) Box plot of miRNA expression.(C) Length distribution of identified miRNA.(D) Volcano plot of differential miRNA.(E) Enrichment results of differential miRNA target genes in GO biological process term.(F) Enrichment results of differential miRNA target genes in KEGG.

Figure 4 .
Figure 4. Co-expression network of JrSPL and target relationship of miRNA.(A) Co-expression network of JrSPL in flower bud development process, miRNA is introduced into the network whether there is a targeting relationship.Both module1 and module2, genes are shown with circles, the color of the gene node represents the degree of differential expression, with a redder shade indicating higher expression levels during the S stages, and a bluer shade indicating lower expression levels during the S stages.The size of the gene nodes represents the degree values of network.miRNA is shown with inverted triangles, the color of the miRNA nodes indicates whether they are known or new miRNAs.The thickness of the line represents the size of the correlation.(B) Heat map of expression of module 1 nodes in the flower bud development process, walnuts SPL been marked with red.(C) Heat map of expression of module 2 nodes in the flower bud development process, walnuts SPL been marked with red.

Figure 4 .
Figure 4. Co-expression network of JrSPL and target relationship of miRNA.(A) Co-expression network of JrSPL in flower bud development process, miRNA is introduced into the network whether there is a targeting relationship.Both module1 and module2, genes are shown with circles, the color of the gene node represents the degree of differential expression, with a redder shade indicating higher expression levels during the S stages, and a bluer shade indicating lower expression levels during the S stages.The size of the gene nodes represents the degree values of network.miRNA is

Horticulturae 2024 , 17 Figure 6 .
Figure 6.Results of PCR and Transgenic plants.(A) PCR plot of transgenic positive seedlings.M: Marker Lanes.1-12: PCR of positive transgenic seedlings.(B) Flowering time of wild type plants.I, II and III represent duplicates of the transgenic line, respectively.(C) Flowering time of Jrspl02-3301 transgenic plants.

Figure 6 .
Figure 6.Results of PCR and Transgenic plants.(A) PCR plot of transgenic positive seedlings.M: Marker Lanes.1-12: PCR of positive transgenic seedlings.(B) Flowering time of wild type plants.I, II and III represent duplicates of the transgenic line, respectively.(C) Flowering time of Jrspl02-3301 transgenic plants.

Author
Contributions: Conceptualization, K.M. and P.G.; Data curation, K.M., Y.Z. and L.H.; Formal analysis, K.M.; Methodology, Y.Z. and L.H.; Project administration, K.M.; Software, Y.Z.; Supervision, P.G.; Validation, C.G. and L.H.; Visualization, Y.Z.; Writing-original draft, K.M.; Writing-review & editing, J.H. and P.G.All authors have read and agreed to the published version of the manuscript.Funding: This research was funded by the Project of National Natural Science Foundation of China (31960584), Xinjiang Academy of Agricultural Sciences Young Science and Technology Backbone Innovation Ability Training Project (xjnkq-2023007), the central government guided local scientific and technological development special funds in 2022, the Xinjiang Production and Construction Corps' Key Industry R & D Program of the Southern Xinjiang (2022DB022).

Table 1 .
Phenotypic statistics of transgenic materials.
* represents significant difference.The p value of difference is 0.01.

Table 1 .
Phenotypic statistics of transgenic materials.
* represents significant difference.The p value of difference is 0.01.