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Article

Overexpression of Cassava MeSTP7 Promotes Arabidopsis Seedling Development

by
Sha Geng
1,†,
Xiaotong Wang
2,3,4,†,
Wei Yan
1,
Qian Liu
1,
Na Wang
1,
Jianyu Zhang
3,4,
Jianchun Guo
2,
Jiao Liu
2,* and
Lijuan Luo
3,4,*
1
Institute of Tropical and Subtropical Cash Crops, Yunnan Academy of Agricultural Sciences, Baoshan 678000, China
2
National Key Laboratory for Tropical Crop Breeding, Sanya Research Institute, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China
3
Key Laboratory of Sustainable Utilization of Tropical Biological Resources of Hainan Province, School of Tropical Agriculture and Forestry, Hainan University, Haikou 570228, China
4
School of Breeding and Multiplication (Sanya Institute of Breeding and Multiplication), Hainan University, Sanya 572025, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Plants 2024, 13(21), 3102; https://doi.org/10.3390/plants13213102
Submission received: 9 September 2024 / Revised: 28 October 2024 / Accepted: 2 November 2024 / Published: 4 November 2024
(This article belongs to the Section Plant Development and Morphogenesis)
Browse Figures
Versions Notes

Abstract

:
The sugar transporter (STP) gene family is a key regulator of plant development, which is crucial for the efficient transport and utilization of sugars during plant growth and development. In this study, we identified the MeSTP7 gene, which is highly expressed in cassava fibrous roots, early storage roots, and under hormonal treatment, including IAA, MeJA, ABA, and GA3, and abiotic stressors, such as mannitol and NaCl. A strong response was observed with exoqenous IAA. Transfecting MeSTP7 into Arabidopsis promoted early seedling growth, particularly in lateral root development. The content of endogenous hormones (IAA and MeJA) as well as soluble sugars (sucrose, fructose, and glucose) was elevated in transgenic Arabidopsis. Hormone treatments with IAA, MeJA, GA3, and ABA on transgenic Arabidopsis revealed that transgenic Arabidopsis responded positively to added 20 μM IAA. They also exhibited co-induced regulation of lateral root formation by GA3, MeJA, and ABA. qRT-PCR analysis showed that overexpression of MeSTP7 upregulated the expression of IAA14, ARF7, and ARF19 in Arabidopsis. Under IAA treatment, the expression of these genes was similarly upregulated but downregulated under MeJA treatment. These results suggest that MeSTP7 may promote Arabidopsis seedling development by increasing the content of sucrose, glucose, and fructose in roots, which in turn influences IAA-based hormonal signaling.

1. Introduction

Cassava is rich in carbohydrates and can be cultivated in poor soil conditions, making it a valuable source of food and income for over 800 million people in rural areas [1]. It is used to produce a variety of products, such as starch, bioethanol, and other bio-based products, including feedstuffs, pharmaceuticals, cosmetics, and biopolymers [2]. However, its growth and development can be limited by various factors, including soil fertility and planting material quality [3]. Currently, there is a significant deficiency in the exploration of genes that promote the growth and development of cassava, a shortfall that has implications for both agricultural productivity and food security in regions where this crop is a dietary staple. Therefore, it is crucial to explore the genes that promote the growth and development of cassava to unlock their potential and contribute to global food security and sustainable agricultural development.
In most plants, sugar transporters mediate the long-distance transport and allocation of photosynthates from the source-to-sink organs [4,5]. Three key types of sugar transporters exist: sucrose transporters (SUTs), sugars will eventually be exported transporters (SWEETs), and monosaccharide transporters (MSTs) [6]. MSTs are the largest family of sugar transporters and are involved in the transport of a variety of monosaccharide substrates [6]. Generally, the MSTs family, based on sequence characteristics and substrate specificities, can be classified into seven subfamilies: sugar transport protein (STP/HT), polyol transporter (PLT), early response to dehydration (EDRL), plastidic glucose transporter (pGlucT), inositol or cyclic polyol transporter (INT), tonoplastic monosaccharide transporter (TMT), and vacuolar glucose transporter (VGT) [7].
Monosaccharide transport proteins, which belong to the major facilitator superfamily (MFS), are membrane proteins that typically possess 12 transmembrane domains. They are categorized as membrane-associated proteins with an amino-terminal that spans the membrane six times on one side and a carboxy-terminal that spans the membrane six times on the other side [8]. Despite having highly homologous amino and carboxyl-terminal sequences across their transmembrane domains, different monosaccharide transport proteins can exhibit significant functional differences [8]. MSTs are widely present in all plant tissues and cells and serve multiple functions. They can transport, absorb, utilize, and accumulate monosaccharides, affecting plant growth and development by participating in the extracellular offloading of phloem hexose, the carbohydrate supply during pollen development, and by providing energy and developmental signals for plants [9,10,11,12]. It also enhances plant defense and regulates resistance to abiotic stressors, such as drought and cold [13,14,15]. In Arabidopsis, 14 MSTs capable of transporting sugars have been identified as AtSTP1 to AtSTP14 [16,17]. For instance, AtSTP2 provides glucose to pollen during pollen development [18], while AtSTP6 plays a role in the sugar supply during pollen germination and pollen tube growth [19]. AtSTP1 participates in the regulation of branching in Arabidopsis by controlling the expression of genes related to hormone synthesis and signal transduction, thus influencing the sugar signaling pathway [20,21]. In rice, knocking out the sugar transporter protein OsSTP15 increases the sugar content in the lower part of the stem, which in turn increases the number of tillers and enhances grain yield [22]. In rice, OsMST8 is involved in the response to cold stress by regulating changes in sugar content [23]. The OsMST6 gene is induced by high sugar and salt stress [24]. The heterologous expression of MdHT2.2 in apples regulates apoplastic hexose levels in tomato fruit, controlling CWINV activity, altering carbohydrate distribution, and resulting in increased fruit size [25]. Furthermore, expressing MdSUT2 in tomatoes leads to increased soluble sugar levels in transgenic lines, enhancing the plants’ tolerance to adverse environmental factors such as salinity and drought [26].
Since plants are anchored in the soil and exhibit a sessile growth form, their access to water and essential nutrients relies heavily on the underground portions of the root system. Therefore, the growth and development of roots directly influence plant growth and subsequently affect the plant’s ability to respond to environmental stressors. The root system encompasses the entirety of a plant’s roots. The primary root develops from the seed embryo, and under the combined influence of internal and external factors, the primary root grows multiple lateral branches at specific angles, known as secondary or lateral roots [27]. Many dicotyledonous plants, such as Arabidopsis thaliana, Brassica napus, Solanum lycopersicum, and Daucus carota, possess distinct primary roots, with lateral roots developing from the primary root under the influence of endogenous hormones and environmental factors. Although various plant hormones regulate lateral root development, auxin is the most critical growth regulator [28]. In Arabidopsis, INDOLE-3-ACETIC ACID INDUCIBLE14 (IAA14) serves as the key transcriptional regulator of auxin signaling during the development of lateral roots [29,30,31]. Gain-of-function mutations in the SLR1/IAA14 gene in Arabidopsis can hinder the formation of lateral roots [29]. The SLR1/IAA14 gene encodes a member of the Aux/IAA protein family, which acts as a transcriptional repressor in the auxin signaling pathway. The stabilized mutant form of IAA14 (mIAA14) in the gain-of-function slr-1 mutant is believed to inactivate the functions of ARF7 and ARF19, which are transcriptional activators in auxin signaling, thereby blocking lateral root initiation [30,32]. Recent studies have shown that jasmonic acid can inhibit the formation of lateral roots induced by auxin, and this inhibitory effect is independent of the jasmonic acid receptor CORONATINE INSENSITIVE 1 (COI1) [33,34]. This suggests that jasmonic acid may regulate the formation of lateral roots by influencing the auxin signaling pathway.
Currently, there are few reports on the involvement of STPs in plant root development. Most studies focus on the role of STPs in fruit and floral organ development, as well as the establishment of stress resistance mechanisms. In this study, we found that overexpressing the cassava MeSTP7 gene in Arabidopsis enhances early seedling growth, especially lateral root development. When treated with the hormones indoleacetic acid (IAA), methyl jasmonate (MeJA), abscisic acid (ABA), and gibberellin A3 (GA3), the transgenic Arabidopsis exhibited increased sensitivity to IAA and MeJA. IAA promoted lateral root formation in the transgenic lines, while MeJA inhibited their development. These results provide new insights into the role of the MeSTP7 gene in regulating plant growth and development.

2. Results

2.1. Expression Patterns of MeSTP7 in Cassava Tissues

In order to study the expression pattern of MeSTP7 in specific tissues of cassava, we used qRT-PCR to analyze MeSTP7 gene expression in various cassava tissues, including young leaves, mature leaves, shoot apical meristem, fibrous roots, storage roots, storage root phloem, and storage root xylem. The results showed that MeSTP7 expression was relatively low in young leaves, mature leaves, and shoot apical meristem but high in fibrous root and phloem of storage roots. Among these tissues, fibrous roots exhibited the highest expression level of MeSTP7, followed by the phloem of storage roots (Figure 1).

2.2. The Expression Pattern of MeSTP7Gene Under Hormone and Abiotic Stressor Treatments

In order to explore the role of MeSTP7 in response to abiotic stressors and hormone signals, in this study, cassava seedlings were treated with exogenous hormones (GA3, ABA, IAA, and MeJA) and abiotic stressors (mannitol-simulated drought stress, NaCl salt stress). The expression pattern of MeSTP7 was analyzed by qRT-PCR. The results showed that under treatment with GA3, IAA, ABA, MeJA, mannitol, and NaCl, the expression level of MeSTP7 was upregulated to varying degrees (Figure 2). Among them, under IAA treatment, the expression of MeSTP7 reached the most significant induction at 24 h (79.6-fold). Under GA3 treatment, the expression of MeSTP7 reached the most significant induction at 2 h (4.7-fold). Under ABA treatment, the expression of MeSTP7 reached the most significant induction (9.9-fold) at 24 h. Under MeJA treatment, the expression of MeSTP7 reached the most significant induction (11.6-fold) at 12 h. Under drought stress, the expression of MeSTP7 reached the most significant induction (37-fold) at 24 h. Under salt stress, the expression of MeSTP7 reached the most significant induction at 12 h (5.3-fold). The results suggest that MeSTP7 may respond to the stimulation by IAA, MeJA, ABA, GA3, salt, and simulated drought.

2.3. Transformation of MeSTP7 in Arabidopsis

To reveal the function of MeSTP7 in plant growth and development, we constructed a plant overexpression vector pCAMBIA1300-MeSTP7:GFP (Figure 3a). The vector was used to transform Arabidopsis. Following 100 mg/L hygromycin screening, 18 independent lines of positive transformants were obtained. These lines were designated as OE1-18, and 17 of them were confirmed by PCR analysis (Figure 3b). Nine lines with bright bands were selected for q-PCR analysis to detect the expression of the MeSTP7 gene. Three lines with different expression levels were screened out: OE7, OE9, and OE11 (Figure 3c). Pure lines were further screened for subsequent experiments. Using a Luyor-3415RG dual-wavelength fluorescent protein excitation light source, green fluorescence was observed in OE7, OE9, and OE11 (Figure 3d), indicating that GFP was successfully expressed in transgenic plants. This also confirmed that MeSTP7 overexpressing Arabidopsis had been obtained.

2.4. MeSTP7 Promotes the Early Growth of Arabidopsis

Phenotypic observations of Arabidopsis overexpressing MeSTP7 at various growth stages revealed that the growth rate of these plants was significantly higher than that of the wild type (WT) at 7 days post-germination. Additionally, root fresh weight and lateral root number were markedly greater in MeSTP7-overexpressing Arabidopsis compared to WT (Figure 4a–c). We measured the levels of endogenous IAA and MeJA hormones, as well as sucrose, glucose, and fructose in Arabidopsis. Our results indicated that these levels were significantly higher in MeSTP7-overexpressing Arabidopsis compared to the WT (Figure 4d–f). At 15 days, MeSTP7-overexpressing Arabidopsis had more leaves than WT (Supplement Figure S1a). Before the 35-day bolting period, the leaf area and number of Arabidopsis were almost the same as WT. We weighed the root biomass at that time and found no significant difference (Supplementary Figure S1b–d). We observed no significant difference in flower and fruit morphology or fruit biomass at the flowering and podding stages (Supplement Figure S1f). These results indicate that the MeSTP7 gene plays a significant enhancing role in the early growth characteristics of Arabidopsis. MeSTP7-overexpressing Arabidopsis had increased levels of IAA, MeJA, sucrose, glucose, and fructose and had greater root biomass and root morphological complexity compared to WT. However, the impact of MeSTP7 on overall plant development becomes less pronounced in later growth stages.

2.5. The Number of Lateral Roots of Transgenic Arabidopsis Increased Under IAA Treatment

The fresh weight, root weight, and lateral root number of MeSTP7 transgenic Arabidopsis (OE7, OE9, and OE11) were higher than those of WT on 1/2 MS medium without IAA. On the medium supplemented with 20 and 50 μM IAA, the transgenic plants exhibited significantly better root development, higher root fresh weight, and greater lateral root number than WT. As the IAA concentration in the medium increased, the roots of wild-type Arabidopsis initially increased but then decreased, with growth being inhibited, while the transgenic Arabidopsis was unaffected (Figure 5a,b).
Gene expression analysis showed that the auxin response factors AtARF7, AtARF19, and the auxin primary response gene AtIAA14, related to lateral root development, were significantly higher in transgenic lines than in WT on 1/2 MS medium without IAA. Under 50 μM IAA treatment, the expression levels of AtARF7, AtARF19, and AtIAA14 increased in both transgenic and wild-type Arabidopsis, but the increase was greater in the transgenic lines than in WT (Figure 5c).

2.6. The Number of Lateral Roots of Transgenic Arabidopsis Decreased under MeJA Treatment

Consistent with the results of 2.4, on 1/2 MS medium without MeJA, the total plant fresh weight, root weight, and lateral root number of MeSTP7 transgenic Arabidopsis (OE7, OE9, and OE11) were higher than those of WT, and the roots were curved. On the medium supplemented with 50 and 100 μM MeJA, the strong root development ability of transgenic plant seedlings was weakened. Specifically, at 100 μM MeJA, the root weight and lateral root number were almost the same as those of WT, and the root curving effect was reduced (Figure 6a,b).
Gene expression analysis showed that the lateral root development-related genes AtARF7/19 and AtIAA14 had significantly higher levels than WT on 1/2 MS medium without MeJA, which was consistent with the results of 2.5. Under 50 μM MeJA treatment, the expression of AtARF7/19 and AtIAA14 in transgenic Arabidopsis was inhibited (Figure 6c). The results showed that the MeSTP7 gene could induce the expression of AtARF7/19 and AtIAA14, while MeJA could inhibit them.

3. Discussion

In plants, sucrose synthesized in the leaves is transported to storage tissues for unloading via both the symplastic and apoplastic pathways to support cell division and growth in meristems [5,35]. In the apoplastic pathway, once sucrose enters the apoplast, it is hydrolyzed into glucose and fructose by cell wall invertase (CWINV). These monosaccharides are then transported into cells through monosaccharide transporters (STPs), providing energy for storing cells and synthesizing essential compounds (including protein, cellulose, and starch) [36]. Monosaccharides play a crucial role as carbon sources and sugar signals during plant growth and development [37]. Therefore, monosaccharide transport proteins play a key role in plant growth and development. The genes encoding STPs are primarily localized on the plasma membrane [5]. In cassava, most STPs function in actively dividing and extending early storage roots (after 30 and 40 days of growth) [38]. AtSTP1 is highly expressed in germinating guard cells, seeds, and seedlings [12]; AtSTP6, AtSTP8, AtSTP9, AtSTP10, and AtSTP11 are expressed in pollen tubes [39]; AtSTP7 is expressed in the root tips of seedlings and in mature pollen and styles; AtSTP8 is expressed in pollen, pollen tubes, ovules, and leaf veins; AtSTP12 is expressed in pollen [17]; and AtSTP13 is expressed in flowers, stems, young leaves, mature rosette leaves, roots, and throughout the seedling [11]. In cassava, the MeSTPs gene family is expressed in various tissues, including leaves, phloem, fibrous roots, and storage roots. MeSTP7 shows high expression levels in the phloem and fibrous roots (Figure 1); MeSTP15 has the highest expression level in fibrous roots; and MeSTP17 and MeSTP19 were highly expressed in early storage roots 80 days after planting [38]. In rice, 22 members of the STPs gene family have been identified, with the majority exhibiting peak expression during flowering, gradually decreasing as the rice heads and matures [40]. In alfalfa, most MtSTPs are predominantly expressed in root nodules, with only a few members expressed in stems and roots [41]. Our study indicates that the cassava MeSTP7 gene has high expression levels during the early development of fibrous and storage roots (Figure 1). Under various hormonal treatments and abiotic stressor conditions, the transcription levels of MeSTP7 exhibited significant changes (Figure 2), suggesting its important role in plant hormone response and abiotic stressor tolerance.
In studying the source-sink relationship, scientists have found that the transport sites and directions of sugars from source tissues to sink tissues are not fixed; they change according to different stages of growth and development [42]. Various organs of the plant have differing demands at different times, leading to changes in the functioning of transporters [43]. In the apoplastic pathway of sucrose unloading in the phloem, the activities of CWINVs and STPs dominate in storage tissues that are actively dividing and extending [44], whereas sucrose transport, primarily dominated by SUTs activity, prevails in storage tissues entering a storage mode [35,45,46]. Additionally, we found that the levels of sucrose, glucose, and fructose, as well as the endogenous hormones IAA and MeJA, were significantly elevated in the roots of transgenic Arabidopsis seedlings. This indicates that the expression of the MeSTP7 gene accelerated monosaccharide transport in Arabidopsis seedlings, promoting sucrose metabolism and consequently increasing hormone levels. This also suggests that the MeSTP7 gene functions during the rapid cell division and extension phases of transgenic Arabidopsis seedlings, while the role of the STPs correspondingly decreases as the plant enters a period of stabilized development.
Sugars are not only the primary energy source for plant life activities but also act as signaling molecules that interact with hormones to regulate the processes of plant growth and development [47,48]. The sensitivity to sugars at different developmental stages plays a crucial role in plant development, making sugar transport and signaling critical during these transitions [36]. Sugar signals work in concert with plant hormones to regulate growth; for instance, sucrose can modulate the metabolism, transport, and signaling of auxin, thereby controlling the elongation growth of meristematic tissues [49]. Transcription factors such as bZIP1, bZIP11, and bZIP44 serve as negative regulators of auxin-mediated primary root growth and their expression is downregulated by sugars. Glucose interacts with the signaling and transport mechanisms of auxin to control the growth and development of seedling root systems [50]. In this study, we found that the MeSTP7 gene was strongly induced by IAA, leading to an increase in lateral roots (Figure 5). Moreover, MeJA inhibited the formation of lateral roots in MeSTP7-overexpressing Arabidopsis plants (Figure 6), consistent with the role of MeJA as an auxin inhibitor that impedes lateral root formation [33,51]. ABA suppressed MeSTP7-induced lateral root formation by weakening the IAA response network in the root apical meristem [52]. Conversely, GA3 promoted the formation of lateral roots induced by MeSTP7 (Supplementary Figure S3). Previous studies have indicated that GA3 may enhance root development in Arabidopsis by regulating IAA transport and signaling [53]. Our findings demonstrate that MeSTP7 promotes the development of lateral roots in Arabidopsis seedlings, with its expression being induced by multiple hormones. Notably, IAA strongly induced MeSTP7 expression, while MeJA treatment inhibited it. Hormonal treatments of transgenic Arabidopsis revealed that IAA and GA3 stimulated the development of lateral roots, while MeJA and ABA suppressed it. This indicates that IAA, GA3, MeJA, and ABA collectively induce and regulate the formation of lateral roots in Arabidopsis seedlings.
It is widely accepted that the development of lateral roots in Arabidopsis relies on regulation by LR/IAA14–ARF7 (ARF19) [54,55]. Under optimal growth conditions, plants utilize most of the carbohydrates produced through photosynthesis to support their growth. To achieve a balanced growth between aerial parts and root systems, auxin synthesized in the aerial parts is transported to the root apical meristem via auxin efflux carriers (such as PIN and NRT), thereby inducing the growth of the root apical meristem and enhancing the growth rate of the roots. Our quantitative analysis revealed that the transcription levels of IAA14 were significantly elevated in transgenic Arabidopsis, along with high levels of auxin response factors ARF7 and ARF19. Additionally, the transcription factor LBD16/29, regulated by ARF7, was also found to be highly expressed, as were the IAA homeostasis regulators WOX5/7 (Supplementary Figure S4). Furthermore, the expression levels of nitrate transporters and glutamine synthetase, which provide amino acids for root development, were significantly upregulated. Based on these results, we hypothesize that the overexpression of MeSTP7 increases the concentration of hexoses in the root system of Arabidopsis seedlings. This elevated sugar concentration activates ARF7/19, leading to an upregulation of IAA synthesis, promoting the transport of auxin to the root apical meristem and consequently activating the formation of lateral roots.

4. Materials and Methods

4.1. Analysis of the Tissue-Specific Expression Pattern of MeSTP7 in Cassava

Manihot esculenta Crantz SC8 is cultivated in Lingao County, Hainan Province, China. Under normal conditions, young leaves, mature leaves, shoot apical meristems, adventitious roots, and storage roots were collected from field plants 80 days after planting. Three leaves were taken from both the young and mature leaves. For the shoot apical meristem, adventitious roots, and storage roots, 10 g was collected from each tissue. The storage roots were dissected with a sterile scalpel to separate the phloem and xylem, with 10 g taken from each tissue and then quickly frozen in liquid nitrogen. RNA was extracted from the aforementioned tissues using the RNA Plant Plus reagent (Tiangen, Beijing, China) according to the manufacturer’s instructions. cDNA was synthesized using the PrimeScript™ RT kit (TaKaRa, Dalian, China), and the tissue-specific expression level of MeSTP7 was analyzed with the TaKaRa SYBR® Premix Ex Taq™ II reagent (TaKaRa, Dalian, China). MeTubulin and MeActin were used as internal controls.

4.2. Hormonal and Abiotic Stressors Treatments for Cassava

Seedlings of the SC8 variety were cultivated in a climate-controlled growth chamber located in Haikou, China, under conditions set at a temperature of 28 °C with a 16 h daily light cycle and a humidity level of 60%. Thirty-day-old seedlings were excised from the Murashige and Skoog (Coolaber, Beijing, China) medium and subjected to treatments with 100 mM mannitol and 300 mM NaCl solutions at intervals of 0, 2, 6, 12, 24, and 48 h. The 30-day-old seedlings were also treated with 100 µM GA3, 100 µM IAA, 100 µM ABA, and 100 µM MeJA solutions at the same time intervals. After each treatment, samples of the roots, stems, and leaves were collected, rapidly frozen in liquid nitrogen, and subsequently stored at −80 °C for future RNA extraction.

4.3. Construction of MeSTP7 Plant Overexpression Vector

The pCAMBIA1300 vector was used as the backbone vector, which contains dual 35S promoters and the hygromycin resistance gene Hyg R. The primers p1300-MeSTP7-F and p1300-MeSTP7-R were designed by using SnapGene4.2.4 software for PCR amplification, and the coding region fragment of the MeSTP7 gene containing the cleavage site was obtained. Using the double-enzyme digestion method, the target gene was inserted into the plant overexpression vector pCAMBIA1300 to construct the pCAMBIA1300-MeSTP7:GFP vector. The recombinant vector was transformed into E. coli (DH5α), and the monoclonal plasmid was picked up for preliminary enzyme digestion verification. The correct plasmid was sent to Sangon Biotech (Shanghai) for sequencing, and the detection primers used were 1300-F and 1300-R. The recombinant plasmid pCAMBIA1300-MeSTP7:GFP was introduced into Agrobacterium tumefaciens LBA4404 competent cells. The primers are listed in Table S1.

4.4. Genetic Transformation and Identification of MeSTP7-Overexpression Arabidopsis Transgenic Lines

The transgenic Arabidopsis lines were generated by an Agrobacterium-mediated transformation method. The Agrobacterium solution containing pCAMBIA1300-MeSTP7:GFP plasmid was cultured, and the inflorescences of Arabidopsis were infected by the floral dip method. The T0 seeds were collected and vernalized at 4 °C for 2 days after drying. The positive transformants were screened by culturing on 1/2 MS (containing 100 mg/L hygromycin) solid medium. The Arabidopsis seedlings of various lines capable of rooting on hygromycin-containing medium were identified by leaf PCR with 2× M5 HiPer Superluminal mix with blue dye (Mei5bio, Beijing, China) and the detection primers 1300-F and 1300-R. A Luyor-3415RG (LUYOR, Joliet, IL, USA) dual-wavelength fluorescent protein excitation light source was used to observe whether the transgenic plants (T1) emitted green fluorescence, and the fluorescence intensity was used to qualitatively detect MeSTP7 expression. Three transgenic lines with strong green fluorescence were selected for subsequent experiments. In order to evaluate the expression level of MeSTP7 in transgenic Arabidopsis, total RNA was isolated from transgenic Arabidopsis lines (T3) OE7, OE9, OE11, and wild-type Arabidopsis, and RNA was reversed to cDNA. SYBR® Premix Ex TaqTM II reagent (TaKaRa, Dalian, China) was used to analyze the expression level of each gene. The Arabidopsis actin gene was employed as an internal control. The 2−∆∆Ct method was used to calculate the relative expression level. Each sample of the experiment had three biological replicates, and the calculated results were plotted with Excel. The primer pairs used were as follows: qMeSTP7-F/R and qAtActin-F/R. The primers qMeSTP7-F/R and qAtActin-F/R are listed in Table S1.

4.5. Phenotypic Observation of Transgenic Arabidopsis at Different Growth Stages

The seeds of three T3 transgenic Arabidopsis lines, OE7, OE9, OE11, and wild-type Arabidopsis, were vernalized and sown in whole vermiculite. After 10 days of growth, Arabidopsis plants with the same growth vigor were selected and transplanted into a small pot (7 cm × 7 cm). One plant was planted in each pot, and the growth was recorded every 7 days until the harvest. Five biological replicates were measured for each line.

4.6. Hormonal Treatment of Transgenic Arabidopsis

Under sterile conditions, three T3 transgenic Arabidopsis lines, OE7, OE9, OE11, and wild-type Arabidopsis, were disinfected, vernalized, and grown on 1/2 MS medium for 6 days. Then, the plants with two true leaves were transplanted to a 10 cm × 10 cm square plate, treated with different concentrations of hormones, and cultured in an environment with temperatures of 22 ± 1 °C, 60% humidity, and a 16 h light/8 h dark cycle. The hormone treatment involved sterilizing the 1/2 MS medium, adding hormones after the medium cooled to below 50 °C, and then transplanting Arabidopsis into the 1/2 MS medium containing hormones for growth. The phenotype was observed after 7 days. The IAA treatment medium was 1/2 MS supplemented with 0, 20, and 50 μM IAA. The MeJA treatment medium was 1/2 MS supplemented with 0, 50, and 100 μM MeJA. The ABA treatment medium was 1/2 MS supplemented with 0, 50, and 100 μM ABA. The medium for GA3 treatment was 1/2 MS supplemented with 0, 50, and 100 μM GA3. In this experiment, three biological replicates were carried out.

4.7. Determination of Endogenous Hormone Content in Transgenic Arabidopsis

Extraction method: The analysis for IAA and MeJA was conducted by Suzhou Keming Biotechnology Co., Ltd. (Suzhou, China). IAA in plant samples was extracted using pre-cooled 80% methanol, followed by decolorization with chloroform, pH adjustment, and ethyl acetate extraction, then concentrated and dissolved in methanol for analysis. MeJA was extracted with 80% acetonitrile, purified, and eluted through a C18 column, then concentrated and dissolved in methanol for analysis.
HPLC conditions: The Rigol L3000 high-performance liquid chromatography system equipped with a Sepax-C18 column was used. For IAA, the mobile phase consisted of water, acetic acid, and methanol (ratio 60:6:40), with detection wavelengths set at 275 nm (excitation) and 345 nm (emission). For MeJA, the mobile phase consisted of 0.1% phosphoric acid in water (A) and acetonitrile (B) in a 4:6 ratio, with a detection wavelength of 210 nm.

4.8. RNA Extraction and qRT-PCR Analysis of Related Genes

Total RNA was isolated from the treated transgenic Arabidopsis lines OE7, OE9, OE11 and wild-type Arabidopsis using the RNA Plant Plus reagent (TianGen, Beijing, China) based on the manufacturer’s instructions. TaKaRa SYBR® Premix Ex TaqTM II reagent (TaKaRa, Dalian, China) was used to analyze the expression level of root development-related genes in Arabidopsis. The Arabidopsis actin gene was employed as an internal control. The 2−∆∆Ct method was used to calculate the relative expression level. Each sample of the experiment had three biological replicates, and the calculated results were plotted with Excel. The primer pairs used are shown in Table S1.

4.9. Statistical Analysis

The data are presented here as the mean ± SD, and the data from three independent experiments were analyzed with a one-way analysis of variance. The value of p ≤ 0.05 was considered significant using GraphPad Prism 8 software.

5. Conclusions

We studied the tissue expression pattern of MeSTP7 in cassava and the expression pattern of MeSTP7 under hormone treatment and abiotic stressors. It was found that MeSTP7 was highly expressed in the phloem and fibrous roots of the early storage roots of cassava, and the expression of MeSTP7 was most strongly induced by IAA. MeSTP7 was heterologously expressed in Arabidopsis, and it was found that MeSTP7 promoted the growth of Arabidopsis seedlings at an early stage. The contents of IAA, MeJA, sucrose, glucose, and fructose in the roots of transgenic Arabidopsis seedlings increased significantly. Under IAA treatment, the number of lateral roots of transgenic seedlings increased significantly. Under MeJA treatment, the number of lateral roots of transgenic Arabidopsis seedlings decreased significantly. The results of qRT-PCR showed that the expression of IAA14, ARF7, and ARF19, which are important regulators of lateral root development, was significantly increased in transgenic plants and induced by IAA, while MeJA inhibited their expression. Based on the above results, we conclude that MeSTP7 acts in the roots of Arabidopsis, accelerating the unloading of sucrose and rapidly increasing the concentrations of sucrose, glucose, and fructose in the tissues. This, in turn, alters the endogenous hormone levels of IAA and MeJA in the roots of Arabidopsis, thereby promoting the development of Arabidopsis seedlings.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants13213102/s1, Table S1: Primers used in experiments; Figure S1: Phenotypes of Arabidopsis with Overexpression of MeSTP7 at Different Growth Stages; Figure S2: The response of MeSTP7-overexpressing Arabidopsis to ABA; Figure S3: The response of MeSTP7-overexpressing Arabidopsis to GA3; Figure S4: qRT-PCR analysis of the expression of AtLBD16, AtLBD29, AtWOX5, and AtWOX7 in transgenic Arabidopsis lines.

Author Contributions

S.G., X.W., J.L., and L.L. conceived and designed the research. S.G., X.W., J.Z., and N.W. performed the experiments. J.L., J.G., Q.L., and L.L. analyzed data. S.G. and X.W. wrote the article. J.G., W.Y., and L.L. provided commentary on the manuscript. 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 (32160398, 31660412) and Yunnan Fundamental Research Projects (202301BD070001-221).

Data Availability Statement

All analyzed data for this study are included in the contents of this article and Supplementary Materials.

Acknowledgments

We are grateful for the patience and comments of the editors and the reviewers.

Conflicts of Interest

The authors declare no conflicts of interest.

References

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Figure 1. Expression analysis of the MeSTP7 gene in cassava tissues. The storage roots were selected from cassava roots that had grown for 80 days. MeTubulin and MeActin were used as internal controls. The expression level of young leaves was set to 1. The data represent the mean ± standard deviation of three biological replicates. Different lowercase letters indicate significant differences (p < 0.05).
Figure 1. Expression analysis of the MeSTP7 gene in cassava tissues. The storage roots were selected from cassava roots that had grown for 80 days. MeTubulin and MeActin were used as internal controls. The expression level of young leaves was set to 1. The data represent the mean ± standard deviation of three biological replicates. Different lowercase letters indicate significant differences (p < 0.05).
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Figure 2. The root expression pattern of the MeSTP7 gene under various treatments of cassava seedlings, including drought, salt, IAA, MeJA, ABA, and GA3. The treatment methods were as follows: gibberellin treatment was 100 μM GA3; IAA treatment was treated with 100 μM IAA; ABA treatment was 100 μM ABA; MeJA treatment was 100 μM MeJA; salt stress was 300 mM NaCl; the drought treatment was simulated by 100 mM mannitol. The MeTubulin gene and MeActin gene were normalized as an internal control. The relative expression value of the control sample was standardized to the control value 1. The data are expressed as the average of three independent organisms. Different lowercase letters indicate significant differences (p < 0.05).
Figure 2. The root expression pattern of the MeSTP7 gene under various treatments of cassava seedlings, including drought, salt, IAA, MeJA, ABA, and GA3. The treatment methods were as follows: gibberellin treatment was 100 μM GA3; IAA treatment was treated with 100 μM IAA; ABA treatment was 100 μM ABA; MeJA treatment was 100 μM MeJA; salt stress was 300 mM NaCl; the drought treatment was simulated by 100 mM mannitol. The MeTubulin gene and MeActin gene were normalized as an internal control. The relative expression value of the control sample was standardized to the control value 1. The data are expressed as the average of three independent organisms. Different lowercase letters indicate significant differences (p < 0.05).
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Figure 3. Detection of transgenic Arabidopsis expressing heterologous MeSTP7. (a) A schematic diagram of the pCAMBIA1300-MeSTP7: GFP recombinant vector. (b) PCR detection of the leaves of resistant transgenic Arabidopsis seedlings that survived on 1/2 MS medium supplemented with 100 mg/L hygromycin, T1 generation. M: DL2000; P: Positive plasmid control; 1–18: Transgenic Arabidopsis lines; WT: Columbia-0 Arabidopsis. (c) Expression of the MeSTP7 gene in selected transgenic lines. The expression of OE8 was set to 1, and the AtActin gene was normalized as an internal control. (d) Expression of GFP in T3 transgenic lines was observed using a Luyor-3415RG dual-wavelength fluorescent protein excitation light source.
Figure 3. Detection of transgenic Arabidopsis expressing heterologous MeSTP7. (a) A schematic diagram of the pCAMBIA1300-MeSTP7: GFP recombinant vector. (b) PCR detection of the leaves of resistant transgenic Arabidopsis seedlings that survived on 1/2 MS medium supplemented with 100 mg/L hygromycin, T1 generation. M: DL2000; P: Positive plasmid control; 1–18: Transgenic Arabidopsis lines; WT: Columbia-0 Arabidopsis. (c) Expression of the MeSTP7 gene in selected transgenic lines. The expression of OE8 was set to 1, and the AtActin gene was normalized as an internal control. (d) Expression of GFP in T3 transgenic lines was observed using a Luyor-3415RG dual-wavelength fluorescent protein excitation light source.
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Figure 4. MeSTP7 increases the levels of IAA, MeJA, sucrose, glucose, and fructose in Arabidopsis seedlings, promoting seedling growth. (a) Transgenic MeSTP7 and WT plants were grown on 1/2 MS agar for 7 days. (b) Statistical analysis of plant root fresh weight. (c) Statistical analysis of the number of plant lateral roots. (d) The endogenous IAA content in the roots of the plants was measured. (e) The endogenous MeJA content in the roots of the plants was measured. (f) The content of glucose, fructose, and sucrose in WT and MeSTP7-overexpression Arabidopsis plants grown in 1/2 MS medium. The data were expressed as the mean ± SEM of three biological replicates. * represents a significant difference (p < 0.05), ** represents a highly significant difference (p < 0.01).
Figure 4. MeSTP7 increases the levels of IAA, MeJA, sucrose, glucose, and fructose in Arabidopsis seedlings, promoting seedling growth. (a) Transgenic MeSTP7 and WT plants were grown on 1/2 MS agar for 7 days. (b) Statistical analysis of plant root fresh weight. (c) Statistical analysis of the number of plant lateral roots. (d) The endogenous IAA content in the roots of the plants was measured. (e) The endogenous MeJA content in the roots of the plants was measured. (f) The content of glucose, fructose, and sucrose in WT and MeSTP7-overexpression Arabidopsis plants grown in 1/2 MS medium. The data were expressed as the mean ± SEM of three biological replicates. * represents a significant difference (p < 0.05), ** represents a highly significant difference (p < 0.01).
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Figure 5. IAA treatment enhances root development in Arabidopsis with MeSTP7 overexpression. (a) Phenotype of transgenic Arabidopsis under different IAA concentration gradients. (b) The total plant fresh weight and root fresh weight of Arabidopsis under different IAA concentration gradient treatments. (c) Expression of root development-related genes under different IAA concentrations. * represents a significant difference (p < 0.05), ** represents a highly significant difference (p < 0.01).
Figure 5. IAA treatment enhances root development in Arabidopsis with MeSTP7 overexpression. (a) Phenotype of transgenic Arabidopsis under different IAA concentration gradients. (b) The total plant fresh weight and root fresh weight of Arabidopsis under different IAA concentration gradient treatments. (c) Expression of root development-related genes under different IAA concentrations. * represents a significant difference (p < 0.05), ** represents a highly significant difference (p < 0.01).
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Figure 6. MeJA treatment inhibited the formation of lateral roots in MeSTP7-overexpressing Arabidopsis plants. (a) Phenotypes of transgenic Arabidopsis under different MeJA concentration gradients. (b) Fresh weight and lateral root number of transgenic Arabidopsis under different MeJA concentration gradients. (c) Expression of root development-related genes under different MeJA concentrations. * represents a significant difference (p < 0.05), ** represents a highly significant difference (p < 0.01).
Figure 6. MeJA treatment inhibited the formation of lateral roots in MeSTP7-overexpressing Arabidopsis plants. (a) Phenotypes of transgenic Arabidopsis under different MeJA concentration gradients. (b) Fresh weight and lateral root number of transgenic Arabidopsis under different MeJA concentration gradients. (c) Expression of root development-related genes under different MeJA concentrations. * represents a significant difference (p < 0.05), ** represents a highly significant difference (p < 0.01).
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Geng, S.; Wang, X.; Yan, W.; Liu, Q.; Wang, N.; Zhang, J.; Guo, J.; Liu, J.; Luo, L. Overexpression of Cassava MeSTP7 Promotes Arabidopsis Seedling Development. Plants 2024, 13, 3102. https://doi.org/10.3390/plants13213102

AMA Style

Geng S, Wang X, Yan W, Liu Q, Wang N, Zhang J, Guo J, Liu J, Luo L. Overexpression of Cassava MeSTP7 Promotes Arabidopsis Seedling Development. Plants. 2024; 13(21):3102. https://doi.org/10.3390/plants13213102

Chicago/Turabian Style

Geng, Sha, Xiaotong Wang, Wei Yan, Qian Liu, Na Wang, Jianyu Zhang, Jianchun Guo, Jiao Liu, and Lijuan Luo. 2024. "Overexpression of Cassava MeSTP7 Promotes Arabidopsis Seedling Development" Plants 13, no. 21: 3102. https://doi.org/10.3390/plants13213102

APA Style

Geng, S., Wang, X., Yan, W., Liu, Q., Wang, N., Zhang, J., Guo, J., Liu, J., & Luo, L. (2024). Overexpression of Cassava MeSTP7 Promotes Arabidopsis Seedling Development. Plants, 13(21), 3102. https://doi.org/10.3390/plants13213102

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