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Communication

A Type A Response Regulator Is Involved in Growth in Salix Matsudana Koidz

by
Peng Yin
†,
Lei Wang
†,
Junkang Zhang
,
Xue Wang
,
Di Wu
and
Jichen Xu
*
State Key Laboratory of Tree Genetics and Breeding, College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Forests 2024, 15(1), 4; https://doi.org/10.3390/f15010004
Submission received: 14 November 2023 / Revised: 8 December 2023 / Accepted: 18 December 2023 / Published: 19 December 2023
(This article belongs to the Special Issue Latest Progress in Research on Forest Tree Genomics)
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Abstract

:
The cytokinin signaling pathway is important for plant growth and development. To understand the regulatory process, a type A response regulator, SmRR5, in Salix matsudana Koidz., was characterized and functionally analyzed. Gene expression tests showed that SmRR5 was distinctly higher in the leaves and roots of the fast-growing S. matsudana variety 9901 than in those of the slow-growing variety Yanjing (YJ). The transcript abundance was highest in the meristem zone (MEZ), followed by the elongation zone (EZ) and maturation zone (MAZ) in 9901 roots, but it was identically low in YJ roots. Overexpression of SmRR5 in tobacco plants significantly improved plant height, maximum root length (MRL), lateral root number (LRN), fresh weight (FW), dry weight (DW), and flowering time compared with wild-type plants. Transcript profiling revealed that multiple genes associated with flowering (SWEET1, FPF1, and COL12), plant growth (YUCCA8, PIN5, and ARF9a), and adventitious root (AR) formation (Hox3, MYC2, and AGL46) were highly expressed in the overexpression of leaves and roots. Thus, SmRR5 effectively facilitated plant growth and development.

1. Introduction

As an important phytohormone, cytokinin plays an indispensable role in regulating plant growth and development, such as root elongation, shoot initiation, and development, through signal transduction series [1]. The cytokinin response regulator (RR) is a key factor in the process and is mainly classified into type A RRs (RRAs) and type B RRs (RRBs) based on the protein domain architecture and amino acid composition. RRAs are relatively small fragments with a conserved aspartate-aspartate-lysine (D-D-K) motif and a truncated C-terminal extension [2]. RRBs are transcription factors with a DNA-binding domain involved in RRA expression regulation [3].
A previous study showed that a sextuple T-DNA insertion mutant of RRAs, arr3, 4, 5, 6, 8, and 9, exhibited a superior ability to initiate callus shooting but had inhibited roots. With 10 nm BA supplementing the mutant plants, arr7 expression significantly increased, indicating cytokinin sensitivity [4]. As cytokinin has been implicated in shoot regeneration in Arabidopsis, ARR7 was detected abundantly at the transition stage. The in situ hybridization assay revealed that 69% of the root stem cells in Arabidopsis mutants arr7 and arr15 exhibited strong patterning defects. However, several key transcription factors for root stem cell specification and function were expression-abolished or reduced, such as SCR, PLT1, and WOX5, suggesting that arr7 and arr15 are involved in regulating root stem cell specification and early embryogenesis [5]. Under normal growth conditions, the overexpression of ARR3, ARR16, and ARR17 in Arabidopsis showed relatively stronger phenotypes in primary root growth, with root length over 30%, 20%, and 20% longer than that of wild-type plants. Most transgenic Arabidopsis plants overexpressing type A RR genes flowered earlier than the wild-type when grown under continuous white light, with the ARR4-transgenic line showing the strongest early flowering phenotype and the ARR3-transgenic line showing a moderate phenotype [6]. Four copies of ARR17 show high levels of expression in female S. purpurea. This suggested that ARR17 is a master regulator, possibly through gene dosage, to activate a switch from male-to-female development [7]. Three type A genes (PtRR2, PtRR3, and PtRR10) were significantly more abundant in the phloem than in the xylem of populus, while only one, the type A PtRR5, was significantly higher in the xylem than in the phloem. Five (PtRR1, PtRR2, PtRR5, PtRR6, and PtRR10) were preferentially expressed in nodes over young or mature leaves. PtRR4 was significantly more abundant in roots than in other tissues [8]. Additionally, RRAs are extensively involved in abiotic stress responses. For instance, AtRR5, AtRR6, AtRR7, and OsRR7 are induced by cold, drought, salt, and dehydration stress [2,9,10]. Overexpression of RR5, RR7, and RR15 in Arabidopsis significantly improved cold tolerance [9]. ZmRR1 positively regulates the expression of the DREB1 and CesA genes and enhances plant cold resistance [11]. RR genes are recognized as indispensable in plants.
Salix matsudana Koidz. is an important afforestation and industrial timber tree species with abundant germplasm resources. The derived varieties, 9901 and Yanjiang (YJ), were widely planted in China, especially 9901, which is fast-growing and has earlier flowering than YJ [12]. A previous transcriptome analysis demonstrated a differently expressed RR gene, SmRR5, between the two varieties (2.15-fold in 9901 than in YJ) [13]. This study aimed to investigate the role of SmRR5 and advance our understanding of type A response regulators in willow growth regulation. It would fascinate the molecular breeding program by expanding the planting range of willow varieties, increasing the biomass of the wooden plants, and speeding the breeding steps efficiently.

2. Materials and Methods

2.1. Plant Materials

Salix matsudana Koidz. cuttings were planted in plastic pots with sandy soil in a growth chamber with a 16/8 h light/dark cycle (2000 lux) (Shangsheng Instrument Company, Zhejiang, China) and a relative humidity of 75–80% at 25 °C. The plants were irrigated with water every three days and fertilized with Hoagland’s solution weekly.

2.2. Gene Expression Analysis

Fresh leaves and roots from one-month-old plants were sampled for RNA extraction using a polysaccharide polyphenol kit (TIANGEN Company, Beijing, China) and reverse transcribed to cDNA. Based on the genome database of S. matsudana [14], specific primers of the SmRR5 gene were designed (SmRR5F, 5′-ATGGGTTCGAACAGTATCGT-3′; SmRR5R, 5′-TCAAATCAGAATATGATCGA-3′). The PCR assay was conducted for the expression test with the following procedure: 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min, 35 cycles. Ubiquitin (UBQ) was used as a reference gene (UBQF, 5′-AGAAGGAGTCAGCAACGATG-3′; UBQR, 5′-CATTAGGTTCTGAACAGCAGG-3′).

2.3. Gene Cloning and Construction of Expression Vector

The gene primers with additional restriction enzyme digestion sites on both sides of SmRR5 were designed (SmRR5-HindF, 5′CCCAAGCTTATGGGTTCGAACAGTATCGT-3′; SmRR5-XbaR,5′-TGCTCTAGATCAAATCAGAATATGATCGA-3′). The full-length gene fragment was amplified with the cDNA sample of 9901 willow and ligated to the pGEM-T easy vector (Promega, Madison, WI, USA) following the manufacturer’s instructions. The cloned plasmid and gene expression vector pEZR(K)-LC were both digested with the restriction enzymes HindIII and XbaI, and ligated to form the recombinant expression vector. The recombinant vector was introduced into Agrobacterium tumefaciens GV3101 by electroporation. The positive clone was identified by PCR.

2.4. Gene Sequence and Homologous Analysis

Sequence analysis of SmRR5 was conducted with DNAMAN v7.0 software. The physical and chemical properties of the SmRR5 protein were analyzed by the online software ProtParam (Protein Identification and Analysis Tools on the ExPASy Server) (https://web.expasy.org/protparam/, accessed on 1 May 2022) (Swiss Institute of Bioinformatics, Lausanne, Switzerland). Ten type A RR gene sequences of Arabidopsis (AtRR) were obtained from the genome database TAIR10 (https://www.arabidopsis.org, accessed on 1 May 2022). Multiple sequence alignments and neighbor-joining (NJ) analyses were conducted among SmRR5 and AtRR genes by using MEGA7 [15], with the bootstrap value set at 1000 repetitions.

2.5. SmRR5 Gene Transformation to Tobacco Plants

The recombinant GV3101 bacterial strain was cultivated in YEB medium containing 50 mg/L kanamycin and 10 mg/L rifampicin at 28 °C overnight and then transferred to antibiotic-free YEB liquid medium until the OD600 reached 0.6–0.8. Wild-type young tobacco leaves (Nicotiana tabacum L.) were cut into small pieces (2 × 2 cm) and immersed in agrobacterium solution for 10 min, then transferred to antibiotic-free differentiation medium (MS + 3% (w/v) sucrose + 0.2 mg/L NAA + 2 mg/L 6-BA + 0.3% (w/v) phytagel) in the dark for 3 days and further cultured in MS selection medium (MS + 3% (w/v) sucrose + 0.2 mg/L NAA + 2 mg/L 6-BA + 50 mg/L kanamycin + 200 mg/L timentin + 0.3% (w/v) phytagel) for approximately 30 days. The regenerated seedlings were transferred to root-inducing medium (MS + 3% (w/v) sucrose + 0.2 mg/L NAA + 50 mg/L kanamycin + 200 mg/L timentin + 0.3% (w/v) phytagel) for 2 weeks [16]. The DNA and total RNA were extracted from the rooted plants. PCR tests with the specific primers of SmRR5 were conducted for the identification of the positive transgenic lines. The clonal plants for each transgenic line were obtained through stem segment cutting and cultured in vermiculite matrix for growth analysis. The tobacco plants were irrigated daily and fertilized with 1/2 Hoagland’s nutrient solution weekly.

2.6. Transcriptome Analysis

The total RNA from tobacco leaves was used for RNA library construction using the NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA) [12] and was sequenced with an Illumina platform (Biomaker Company). The clean reads were stored in FASTQ format and mapped to the Nicotiana tabacum L. reference genome [17] using HISAT2. The expression level of the genes was calculated by Stringtie. The diferentially expressed genes (DEGs) were determined using edgeR with a false discovery rate (FDR) ≤ 0.05 and |Log2 Fold Change| ≥ 1.
Gene Ontology (GO) enrichment analysis of the DEGs was based on the Wallenius noncentral hypergeometric distribution method in GOseq [18]. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment [19] was conducted using Blast2GO software (BioBam Bioinformatics SL, Valencia, Spain). The heatmap was drawn by TBtools according to the gene expression in different samples [20].

3. Results

3.1. Expression Pattern of SmRR5 in S. matsudana

The expression patterns of SmRR5 in two S. matsudana varieties were examined with the primers SmRR5-F/SmRR5-R (Figure 1). Under normal growth conditions, the abundance of SmRR5 transcripts was significantly high in the fast-growing 9901 leaves, 2.29 times that of YJ. Furthermore, SmRR5 in the meristem zone (MEZ) of 9901 roots had high expression, 2.19 and 5.86 times that of the elongation zone (EZ) and maturation zones (MAZ), respectively. However, SmRR5 had a lower expression in YJ roots and was identical in all three root regions. SmRR5 may be involved in willow plant growth and development.

3.2. Cloning and Characterization of SmRR5 in 9901 Willow Plants

The full-length fragment of SmRR5 was amplified and cloned from a 9901-cDNA sample with primers SmRR5-HindF/SmRR5-XbaR. The sequencing results confirmed a length of 726 bp, encoding 241 amino acids (Figure 2). The SmRR5 protein contains several rich amino acids, such as Ser (S) (17.40%) and Leu (L) (9.10%). The physical and chemical property analysis showed a molecular weight of 26.67 kD, an isoelectric point of 5.00, an aliphatic index of 82.86, a grand average of hydropathicity of −0.369, and an instability index of 75.05, indicating that the SmRR5 protein is an acidic, hydrophilic, thermophilic, and structurally unstable protein.
The structure analysis showed SmRR5 as a typical A-type RR gene with an REC domain that is essentially responsible for receiving the upstream signal in a two-component system but is deficient in the DNA-binding domain. To assess the homolog of SmRR5 with Arabidopsis A-type RR genes, a phylogenetic tree was constructed by the neighbor-joining method, showing that SmRR5 is phylogenetically close to ARR3 and ARR4, with an amino acid identity of 56.79% and 52.91%, respectively (Figure 3).

3.3. Performance of SmRR5 Transgenic Tobacco Plants

The SmRR5 gene was assayed for transformation into tobacco plants, with three positive lines (L1, L2, and L3) (Figure 4b). After one month of growth, the transgenic plant height (PH) was 28.9–30.3 cm, which was significantly greater than that of the wild-type plants (22.4 cm) (Table 1) (Figure 4c). The maximum root length (MRL) and lateral root number (LRN) were 1.32–1.47 times and 1.43–1.51 times that of wild-type plants, respectively (Figure 4d). The fresh weight (FW) and dry weight (DW) were 1.96–2.09 times and 1.77–1.96 times that of wild-type plants, respectively. The flowering times (FT) of the transgenic lines were 10 days earlier than those of wild-type plants (Figure 5a). Thus, SmRR5 zealously promoted plant growth and development.

3.4. Gene Network of SmRR5 for Plant Growth Regulation

To explore the network of SmRR5 modulating plant growth, transcriptome sequencing was conducted with the leaf and root samples of the transgenic (L2) and wild-type plants. A total of 1040 and 1031 genes were identified as upregulated and downregulated, respectively, in transgenic leaf tissues compared with WT. Conversely, 2717 and 1459 genes were upregulated and downregulated, respectively, in transgenic root tissues. Of these, 1525 and 2309 differentially expressed genes (DEGs) between transgenic and wild-type plant leaf and roots were annotated in the GO database. They were classified into three categories: biological process, cellular component, and molecular function (Figure 5a,c). The major terms were similar in leaf and root samples, including cellular, metabolic, single-organism processes, cells, cell parts, membranes, binding, and catalytic activities.
The KEGG pathway analysis of DEGs displayed differences in leaf and root samples (Figure 5b,d). Starch and sucrose metabolism, glycolipid metabolism, amino sugar and nucleotide sugar metabolism, glycerophospholipid metabolism, and ribosome biogenesis were specifically enriched in leaf tissues. However, the plant−pathogen interaction, MAPK signaling pathway, plant hormone signal transduction, phenylpropanoid biosynthesis, and starch and sucrose metabolism were enriched in root tissues.
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Figure 5. GO annotation (a,c) and KEGG pathway enrichment (b,d) analysis of the DEGs in leaf (a,b) and root (c,d) tissues of the transgenic plants.
Figure 5. GO annotation (a,c) and KEGG pathway enrichment (b,d) analysis of the DEGs in leaf (a,b) and root (c,d) tissues of the transgenic plants.
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Several DEGs related to plant growth and development were identified (Figure 6), such as ABCB9, YUCCA8, and PIN5, which had abundant expression levels 5.74-, 4.05-, and 3.70-fold higher in the SmRR5 overexpressing leaves, respectively, compared to WT plants. DEGs related to flowering, such as FPF1, SWEET10, and COL16, had an expression abundance of 15.35-, 11.38-, and 7.06-fold higher in SmRR5-overexpressing leaves, respectively, compared to WT plants. Additionally, DEGs associated with AR formation and root primordium identity/initiation, including LBD27, MYC2, ABCB, SAUR, AGL62, HOX3, AFB2, and JAZ7, had expression levels 5.42-, 5.21-, 3.84-, and 2.10-fold higher in SmRR5-overexpressing leaves, respectively, than in WT plants.

4. Discussion

4.1. RR Genes Are Involved in Plant Growth Regulation

As key factors of the cytokinin signaling pathway, response regulator family members play an important role in plant growth and development. For instance, the RR1 gene from Arabidopsis activates a repressor of the auxin signaling gene SHY2/IAA and negatively regulates the auxin transporter PIN. This promotes the cell differentiation of root meristems [21]. RR1, RR2, RR10, and RR12 in Arabidopsis spatially activate the WUSCHEL (WUS) expression by binding to HD-ZIP III transcription factors, promoting plant shoot regeneration [22]. The overexpression of AtRR4 markedly promoted shoot formation, while AtRR8 functioned reversely, suggesting an antagonistic effect on cytokinin signaling [23]. Overexpression of RR3 and RR5 improved rice plant growth with longer lateral roots when treated with exogenous cytokinin [24].

4.2. SmRR5 Is an Efficient Gene Resource to Advance Plant Growth

In this study, the overexpression of SmRR5 in tobacco plants effectively improved plant growth and was also involved in reproduction. Several traits were significantly advanced, including plant height, root length, and flowering date. The contributions matched its performance well in willow plants. 9901 is a well-known, fast-growing willow variety with early flowering and an advanced rooting system compared to YJ. Correspondingly, SmRR5 had a high expression in leaves and roots, especially in the root apical meristem of 9901, while it was weak in YJ. This finding also matches the transcriptomic data showing that SmRR5 expression in 9901 willow is 2.15-fold higher than that in YJ. Additionally, our previous assays demonstrated that the 9901 cutting was fast-rooting compared to YJ in hydroponic culturing, and 9901/YJ grafting significantly promoted rooting compared to YJ/YJ [12]. SmRR5 might participate in these processes and is worth applying in wooden plant breeding programs.

4.3. SmRR5 Functions via Advancing Plant Growth-Related Genes

The response regulators are categorized into two groups. RRBs have a phosphorylatable receiver domain and a DNA-binding domain, while RRAs only have a receiver domain [25]. Thus, RRBs work as transcription factors, while RRAs act as function proteins and directly regulate downstream genes in cytokinin pathways. For instance, maize ZmRR1 positively regulates the expression of ZmDREB1 and CesA to enhance chilling tolerance [9]. RR4 in Arabidopsis thaliana specifically interacts with the extreme amino-terminus of the phytochrome B photoreceptor and modulates red light signaling [26]. Overexpression of RhRR1 from Rosa hybrida promotes early flowering of Arabidopsis by increasing the expression of flowering regulatory genes, including Flowering locus D (FD), GA requiring 1 (GA1), and Lumini-dependens (LD) [27]. OsRR9 and OSRR10 from rice inhibited the expression of ion transporter genes and decreased the stress tolerance of plants [28]. In this study, we conducted a transcriptome analysis of SmRR5-overexpressing plants and identified some differentially expressed genes, such as FPF1, COL12, COL16, and CIA2, that promoted earlier flowering [29,30]. LBD, ABCB8, AGL, and SAUR were associated with AR initiation [31,32,33]. Further efforts should be made to build a bridge between SmRR5 and downstream genes to better understand its regulatory pathway in cytokinin signaling.

5. Conclusions

SmRR5, a type A cytokinin response regulator, could prompt plant growth and flowering and advance the plant root system by activating the expression of related genes. It is potentially a gene resource in plant molecular breeding programs, especially for wooden plant production.

Author Contributions

Conceptualization, J.X.; methodology, P.Y.; software, L.W.; investigation, J.Z., X.W. and D.W.; writing—original draft preparation, P.Y. and L.W.; writing—review and editing, J.X.; supervision, J.X.; funding acquisition, J.X. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (31870648).

Data Availability Statement

The SmARR5 sequence presented in this study are openly available in GeneBank (accession#: OR678209). The other A RR gene sequences of Arabidopsis (AtRR) were obtained from the genome database TAIR10 (https://www.arabidopsis.org).

Acknowledgments

The authors would like to thank Aylen B. for the language editing service.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Expression profile of SmRR5 in the willow variety 9901 and YJ. (a) Schematic drawing of the 9901 leaves (L), root maturation zone (MAZ), root elongation zone (EZ), and root meristem zone (MEZ); (b,c), PCR results and quantification data of SmRR5 in different tissues.
Figure 1. Expression profile of SmRR5 in the willow variety 9901 and YJ. (a) Schematic drawing of the 9901 leaves (L), root maturation zone (MAZ), root elongation zone (EZ), and root meristem zone (MEZ); (b,c), PCR results and quantification data of SmRR5 in different tissues.
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Figure 2. Nucleotides and amino acids of SmRR5. The underlined residues represent the conserved receiver domain. ★ indicates three invariant residues ‘DDK’ among all type A RRs.
Figure 2. Nucleotides and amino acids of SmRR5. The underlined residues represent the conserved receiver domain. ★ indicates three invariant residues ‘DDK’ among all type A RRs.
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Figure 3. Phylogenetic relationship and gene structure analysis of SmRR5 and Arabidopsis RRA genes.
Figure 3. Phylogenetic relationship and gene structure analysis of SmRR5 and Arabidopsis RRA genes.
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Figure 4. Performance of SmRR5 transgenic tobacco lines (L1–L3) and wild-type plants (WT). (a,c,d): flowering, plant height, and root phenotype of transgenic and WT lines. (b) PCR results of SmRR5 with the cDNA samples of tobacco seedlings with actin as the reference gene.
Figure 4. Performance of SmRR5 transgenic tobacco lines (L1–L3) and wild-type plants (WT). (a,c,d): flowering, plant height, and root phenotype of transgenic and WT lines. (b) PCR results of SmRR5 with the cDNA samples of tobacco seedlings with actin as the reference gene.
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Figure 6. Expression pattern and regulation of DEGs related to plant growth and development in SmRR5-overexpressing tobacco plants.
Figure 6. Expression pattern and regulation of DEGs related to plant growth and development in SmRR5-overexpressing tobacco plants.
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Table 1. Growth traits of WT and SmRR5 overexpression plants.
Table 1. Growth traits of WT and SmRR5 overexpression plants.
PH (cm)MRL (cm)LRNFW (g)DW (mg)FT (Day)
WT22.4 ± 0.99.7 ± 0.913.8 ± 1.90.099 ± 0.0234.7 ± 0.7102.3 ± 4.9
L128.9 ± 1.0 **14.3 ± 1.2 **20.8 ± 3.0 **0.194 ± 0.043 **8.6 ± 1.8 **91.6 ± 2.9 **
L230.3 ± 1.6 **13.9 ± 0.9 **19.8 ± 2.2 **0.207 ± 0.014 **9.2 ± 0.8 **90.6 ± 2.9 **
L329.5 ± 1.3 **12.8 ± 1.0 **20.6 ± 1.9 **0.194 ± 0.020 **8.3 ± 1.6 **92.1 ± 3.0 **
Data indicate means ± STDEV (n = 5). Values labeled with “**” indicate significant differences between WT and SmRR5-overexpressing plants (L1–L3) (p < 0.01) according to the LSD test (SPSS 19.0). PH, plant height; FT, flowering time; MRL, maximum root length; LRN, lateral root number; FW, fresh weight; DW, dry weight.
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Yin, P.; Wang, L.; Zhang, J.; Wang, X.; Wu, D.; Xu, J. A Type A Response Regulator Is Involved in Growth in Salix Matsudana Koidz. Forests 2024, 15, 4. https://doi.org/10.3390/f15010004

AMA Style

Yin P, Wang L, Zhang J, Wang X, Wu D, Xu J. A Type A Response Regulator Is Involved in Growth in Salix Matsudana Koidz. Forests. 2024; 15(1):4. https://doi.org/10.3390/f15010004

Chicago/Turabian Style

Yin, Peng, Lei Wang, Junkang Zhang, Xue Wang, Di Wu, and Jichen Xu. 2024. "A Type A Response Regulator Is Involved in Growth in Salix Matsudana Koidz" Forests 15, no. 1: 4. https://doi.org/10.3390/f15010004

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