PagSWEET17a Mediates Sugar Transport in Root and Affects Drought Tolerance in Populus alba × P. glandulosa

: Sugars are the principal carbon and energy sources and serve as osmotic regulators and radical scavengers, thus playing an important role in plant responses to drought. Sugar transporters steering the distribution of sugar are vital players involved in tolerance to drought. Sugars Will Eventually be Exported Transporters (SWEETs) facilitate both the inﬂux and efﬂux of mono-and/or disaccharides and control both inter and intracellular distribution of sugars. PagSWEET17a in Populus alba × P. glandulosa is one of four orthologous genes of AtSWEET17 in Arabidopsis . Unlike AtSWEET17, which is a vacuolar localized transporter, PagSWEET17a is localized to the endoplasmic reticulum (ER). Here, the role of PagSWEET17a in poplar responses to drought stress was investigated. PagSWEET17a was speciﬁcally expressed in cambium cells in younger root but mainly located in phloem ﬁbers, and xylem vessels and ﬁber cells in the root undergoing secondary growth. Loss of PagSWEET17a inhibited the growth of roots in poplar seedlings and led to a decreased tolerance to drought. Analysis of sugar proﬁles revealed that accumulations of predominant sugars were signiﬁcantly suppressed in both knockout (KO) mutant lines under drought. PagSWEET17a might contribute to poplar tolerance to drought by promoting drought-induced root expansion and diminishing oxidative damages caused by the stress.


Introduction
Poplar is an important tree species in terms of ecological and economic importance worldwide. Drought is one of the major environmental stress factors that seriously affects the growth and development of poplar trees. In China, nearly half of the land has an arid or semi-arid climate, which critically decreases the survival rate of poplar trees and limits the productivity of poplar. Genetic engineering is one of the most powerful and promising techniques to improve resistance or tolerance to drought in poplar [1]. Exploring the molecular mechanisms underlying drought tolerance and finding more key genes involved in this biological process will facilitate the development of drought resistance engineering and the breeding of drought-tolerant poplar varieties.
Sugars fulfill many different functions in plants. They are not only the predominant carbon and energy source for plants but also act as osmotic regulators to participate in abiotic stresses [2]. As compatible solutes, sugars are induced by drought to promote the removal of active oxygen and reduce cell penetration to maintain water content in plants [3,4]. In plants, sugars in cells are generally hydrated, which endows them with solubility and prevents free permeation across biological membranes. Transport of sugar between subcellular organelles and among cells lacking plasmodesmata is mediated by transporters [5,6]. Sugar transport controls the compartmentation and homeostasis of sugar and thus influences plant tolerance to drought. Sugars Will Eventually be Exported Transporters (SWEETs) are one kind of major transporters associated with the apoplasmic transport of sugars [7,8]. Unlike sucrose transporters (SUTs) [9] and monosaccharide transporters (MSTs) [10], which generally have 12-helical transmembrane domains (TMs), SWEETs have only seven TMs and belong to the MtN3 family [11]. Recent studies have shown that SWEETs mediate both the influx and the efflux of hexose or sucrose and facilitate a plant's ability to tolerate drought [12,13].
In Arabidopsis, several SWEET proteins have been reported to be involved in response to drought. AtSWEET17, a vacuolar fructose facilitator, contributes to the modulation of the root system during drought [14]. By participating in carbon redistribution in the whole plant under drought stress, AtSWEET17 also plays a role in cauline branch emergence and growth [15]. AtSWEET11 and AtSWEET12, which are involved in sucrose phloem loading, were significantly upregulated in the leaves of water-deficient plants and facilitated sugar export from the leaves to the roots to maintain an efficient root system under drought [16]. Abscisic acid (ABA) is the key regulator of responses to drought and plays a vital role in the acclimatization of plants to stress [17]. Further study demonstrated that ABA could activate SnRK2 protein kinases which control the phosphorylation of SWEET11/12, enhance sucrose transport, and contributes to improved root growth and drought tolerance [18]. Arabidopsis transgenic plants overexpressing AtSWEET4 or AtSWEET16 also exhibit an altered sugar distribution and are more tolerant to drought [19,20]. In rice, OsSWEET13 and OsSWEET15 are activated by OsbZIP72, which is an ABA-responsive transcription factor maintaining sugar homeostasis in plant cells and improving the resistance to drought [13]. Heterologous expression of DsSWEET17 from Dianthus spiculifolius and MdSWEET17 from apple (Malus × domestic) in Arabidopsis also identified that both genes conferred tolerance to drought [12,21].
The above research on SWEETs involved in drought mainly focuses on herbaceous plants. Unlike annual herbaceous plants, perennial woody plants exhibit unique developmental processes such as secondary growth, indicating that the mechanisms underlying carbohydrate allocation induced by drought may be different from those of herbaceous plants. In poplar, the SWEET family has undergone significant expansion, with a total of 27 members. Four orthologous genes of AtSWEET17 were identified in poplar, namely PtSWEET17a, PtSWEET17b, PtSWEET17c and PtSWEET17d. PagSWEET17a is an endoplasmic reticulum (ER) localized protein and is highly expressed in the root [22]. In this study, the role of PagSWEET17a in drought was investigated. It was found that PagSWEET17a was specifically expressed in vascular tissues of roots, and its expression was induced by drought simulated by polyethylene glycol (PEG) treatment. Exogenous sugar application showed that PagSWEET17a might be involved in the transport of glucose and sucrose. Under drought treatment, the mutant seedlings showed a higher level of malondialdehyde (MDA) compared to Hybrid poplar (Populus alba × P. glandulosa) clone 84K and significantly decreased biomass. Sugar content analysis confirmed that drought-induced upregulation of soluble sugar content in 84K, but this increase was significantly suppressed in mutants. These results suggested that PagSWEET17a contributed to poplar tolerance to drought by controlling sugar transport in the root.

Plant Materials and Growth Conditions
Hybrid poplar (P. alba × P. glandulosa) clone 84K was used for expression analysis and genetic transformation. The P PagSWEET17a ::GUS transgenic plants originated from our previous work [22]. In order to obtain the PagSWEET17a knockout (KO) plants, the webbased software package CRISPR-GE (http://skl.scau.edu.cn, accessed on 15 April 2018) [23] was used to design the target sgRNAs, and then the resulting specific primers (Table S1) were annealed into two pairs of oligonucleotides to help specifically target PagSWEET17a and sgRNA cassettes assembled into the pYLCRISPR/Cas9P35S-H vector [24], based on Golden Gate cloning [25].
All the above materials were grown on 1/2 MS solid medium (5 g/L agar, 30 g/L sucrose, 50 mg/L indole butyric acid, 20 mg/L naphthyl acetic acid, pH 5.8-6.0) and transferred to soil in separate pots when they were older than 1 month. All seedlings were grown under 16/8 h (light/dark) photoperiod and 75% relative humidity at 24 • C.

Stress Treatments and Phenotype Measurements
84K and KO lines were grown in soil pots (0.8-L) for 6 weeks and watered with deionized water before the drought treatment. At each subsequent watering, the 20% (w/v) solution of PEG 6000 (this concentration was derived from pre-experiments) was used instead of deionized water to simulate drought, while the control group remained watered as normal; this process lasted for approximately 4 weeks. After treatment, the height and diameter of the plants were measured, and their roots were collected to determine the sugar content. In addition, their leaves were used to determine the MDA contents and peroxidase (POD) enzyme activities using customized kits (Solarbio, Beijing, China) following the manufacturer's instructions.
Furthermore, to study the effect of drought on the root growth of each material, 1-month-old seedlings were replaced in larger pots (2.5-L, compared to the simulated drought treatment). Unlike the above treatments, the group receiving the drought treatment received only minimal water for survival (1/4 of fully watered control). After a period of time, the roots of each material were observed and photographed. Apart from this, for the drought treatment of the 4-week-old tissue-culture seedlings, they were removed from the medium and their roots were subsequently submerged in the 20% PEG solution used to simulate drought. Similarly, the control group was submerged in water. The treatment experiments were repeated three times, and six plants were used for each line per experiment.

Histochemical and Immunofluorescence Analysis
β-Glucuronidase (GUS) staining was performed as our previous study with a few changes [22]. In brief, 2-week-old seedlings were incubated in 90% (v/v) cold acetone for at least 20 min on ice and washed three times with staining buffer (0.2 M NaH 2 PO 4 , 0.2 M Na 2 HPO 4 , 2 mM K 3 [Fe(CN) 6 ], 2 mM K 4 [Fe(CN) 6 ]). The washed samples were then transferred to a staining solution (staining buffer with 1 mM X-Gluc) and were vacuumed until the samples were embedded into the staining solution. After incubation at 37 • C for 12 h, the roots were destained with 75% ethanol. Then, the stained roots were embedded in 5% agar and were cross-sectioned at 50 µm with a vibratome (Leica, Wetzlar, Germany).
The methods for immunolocalization of PagSWEET were carried out exactly as previously published methods [26]. A cross-section of 70 µm was made from younger and older roots of 2-month-old 84K plants. The antibodies were diluted as follows: 1:200 for anti-PagSWEET17a and 1:400 for fluorescein-labeled goat anti-rabbit IgG (Servicebio, Wuhan, China). Finally, the samples were observed using a Zeiss LSM 880 confocal microscope (ZEISS, Oberkochen, Germany).

RNA Extraction and RT-qPCR Analysis
The tissue-culture seedlings were subjected to the PEG solution for 0, 2, 4, 8, 12, 24 and 36 h for drought treatment or to water as a control. Materials were harvested at each time point and immediately frozen in liquid nitrogen for extracting RNA.
Total RNA was extracted using the RNA Easy Fast Kit (TIANGEN, Beijing, China) according to the manufacturer's instructions. Then, 1 µg of RNA was used to synthesize cDNA using the PrimeScript™ RT reagent Kit (TaKaRa, Dalian, China) and later diluted for analyzing the relative expression levels of PagSWEET17a, PagNCED3 and PagSnRK2. PagActin was used as an internal control. The primer sequences used in RT-qPCR are listed in Table S1. RT-qPCR analysis was performed on a Light Cycler 480 (Roche, Penzberg, Germany) using the SYBR Premix Ex Taq™ Kit (TaKaRa). The following program was used: 95 • C for 3 min, 45 cycles at 95 • C for 20 s denaturation, 60 • C for 5 s annealing, and 72 • C for 20 s elongation, with a final elongation step at 72 • C for 3 min. The relative gene expression levels were calculated by the 2 −∆∆Ct method [27]. Each sample was subjected to four technical replicates and three biological replicates.

Analysis of the Sugar Contents
The monosaccharides and oligosaccharides were extracted and measured according to the previous method with some modifications [28,29]. The liquid nitrogen frozen material was made into a powder, and then 10 mg was weighed and quickly dissolved in 1 mL of extraction buffer (methanol: water, 4:1). The extraction was then sonicated at room temperature for 1 h, followed by overnight at 4 • C. After drying the samples, a derivatization process was carried out. Fifty microlitres of methoxylamine hydrochloride/pyridine at a concentration of 20 mg/mL was added to the dry samples and incubated at 40 • C for 2 h. Then 80 µL of BSTFA was added to the mixture and kept at the same temperature for 1 h. Finally, 100 µL of hexane was used to dilute the solution before analysis. All reagents used in this experiment were from Sigma Company (Sigma, Los Angeles, CA, USA). The detection was carried out on an Agilent 7890A GC system connected to an Agilent 7000B triple quadrupole MSD (Agilent, Santa Clara, CA, USA) with electron impact ionization mode. One microliter of each sample was injected into splitless mode into the DB-5ms column (30 m × 0.25 mm × 0.25 µm, Agilent) fused silica capillary column. Helium was used as the carrier gas for GC at a flow rate of 1.0 mL/min. The injector temperature was 280 • C, and the oven program was as follows: 80 • C for 5 min, linear ramp at a rate of 7 • C/min to 310 • C, held at 310 • C for 15 min. The transfer line temperature was 280 • C.

Sugar Treatment Assay
For sugar treatment tests, shoot segments about 3 cm in length with two young leaves were cut from seedlings and grown vertically for 2 weeks on 1/2 MS medium (sucrose-free) containing 3% mannitol, 3% fructose, 3% glucose, 3% sucrose, with the medium without sugar serving as a control. The roots were then observed for growth, and the experiment was repeated three times.

Graphing and Statistical Analysis
One-way ANOVA was used in this study, and either Dunnett's test or Tukey's test was applied as appropriate. Statistical analysis and graphical representation were performed using GraphPad Prism 8. The data were presented as the mean ± SD in accordance with established convention. In addition, to visualize the changes in sugar contents, a heat map was generated using TBtools [30].

PagSWEET17a Might Mediate Sugar Transport in Poplar Root
Expression analysis on different tissues showed that PagSWEET17a was more highly expressed in the root than in other tissues [22]. In the present study, the detailed expression profile of PagSWEET17a in root was analyzed using P PagSWEET17a ::GUS transgenic plants.
A cross-section of the root in 2-week-old tissue-culture seedlings showed GUS signals in the cambial zone in younger root ( Figure 1a). Immunofluorescence assays on 2-month-old poplar seedlings identified that, in the younger root, PagSWEET17a was mainly located in the cambium, while in the mature roots undergoing secondary growth, it was located in the phloem fibers, xylem vessels, and xylem fiber cells (Figure 1b,c). To investigate the role of PagSWEET17a in the sugar transport of poplar roots, function-deficient mutants (KO-4 and KO-13) were generated by the CRISPR/Cas9 system. Seedlings of mutants and 84K were grown on 1/2 MS medium and applied with different sugars. Compared with the nontransgenic 84K, seedlings of both mutants showed less adventitious roots when sucrose or glucose was applied individually (Figures 2 and S1), indicating that PagSWEET17a might mediate the transport of sugars in roots. Compared with the non-transgenic 84K, seedlings of both mutants showed less advent tious roots when sucrose or glucose was applied individually (Figures 2 and S1), indica ing that PagSWEET17a might mediate the transport of sugars in roots.

Expression of PagSWEET17a in Root Was Drought-Induced
One of the main functions of roots is to supply water for the plant, and root charac teristics play an important role in plant response to drought. To investigate whether Pag SWEET17a is involved in response to drought, tissue-culture seedlings of 84K were grow in a hydroponic system and applied drought stress by the addition of PEG 6000. After th first 2 h and 4 h of drought, the expression of PagSWEET17a decreased to 10% and 50% o the control value (0 h) at the beginning of the experiment, respectively. However, withi the next 4 h, a rapid increase of the corresponding mRNA was obvious, reaching 3 time of the value present at 0 h drought stress. The expression level reached its peak at 12 when the value was six times that of 0 h. Afterward, the mRNA level began to decreas but still remained at a relatively high level at 24 h and 36 h, about three times that of 0 h respectively ( Figure 3).  Compared with the non-transgenic 84K, seedlings of both mutants showed less adventitious roots when sucrose or glucose was applied individually (Figures 2 and S1), indicating that PagSWEET17a might mediate the transport of sugars in roots.

Expression of PagSWEET17a in Root Was Drought-Induced
One of the main functions of roots is to supply water for the plant, and root characteristics play an important role in plant response to drought. To investigate whether Pag-SWEET17a is involved in response to drought, tissue-culture seedlings of 84K were grown in a hydroponic system and applied drought stress by the addition of PEG 6000. After the first 2 h and 4 h of drought, the expression of PagSWEET17a decreased to 10% and 50% of the control value (0 h) at the beginning of the experiment, respectively. However, within the next 4 h, a rapid increase of the corresponding mRNA was obvious, reaching 3 times of the value present at 0 h drought stress. The expression level reached its peak at 12 h when the value was six times that of 0 h. Afterward, the mRNA level began to decrease but still remained at a relatively high level at 24 h and 36 h, about three times that of 0 h, respectively ( Figure 3).

Expression of PagSWEET17a in Root Was Drought-Induced
One of the main functions of roots is to supply water for the plant, and root characteristics play an important role in plant response to drought. To investigate whether PagSWEET17a is involved in response to drought, tissue-culture seedlings of 84K were grown in a hydroponic system and applied drought stress by the addition of PEG 6000. After the first 2 h and 4 h of drought, the expression of PagSWEET17a decreased to 10% and 50% of the control value (0 h) at the beginning of the experiment, respectively. However, within the next 4 h, a rapid increase of the corresponding mRNA was obvious, reaching 3 times of the value present at 0 h drought stress. The expression level reached its peak at 12 h when the value was six times that of 0 h. Afterward, the mRNA level began to decrease but still remained at a relatively high level at 24 h and 36 h, about three times that of 0 h, respectively (Figure 3).

KO Lines of PagSWEET17a Exhibit Decreased Drought Tolerance
Drought treatment on 4-week-old tissue-culture seedlings showed that the expression of PagNCED3, the first committed and key regulated step in the synthesis of ABA [31], was significantly induced by drought on 84K and reached its expression peak at 12 h (Figure 4a). However, the induction of PagNCED3 was clearly inhibited in the stressed mutants (Figure 4b), suggesting a different response from 84K. Then, the mutants and non-transgenic 84K seedlings were grown in soil and applied to drought stress for 4 weeks. The results showed drought-induced larger root biomass in seedlings of 84K, but these changes were not evident in both KO mutant lines (Figures 5a and S2). For the aerial parts, the seedlings of non-transgenic 84K showed mild growth retardation, showing reduced height and diameter after simulated drought treatment, while in the mutants, especially in KO-4, growth retardation was further increased compared to their normal watering control (Figure 5b-d). The activities of POD, which facilitate the removal of reactive oxygen species and protect plant cells from damage [32], were elevated fourfold by drought in 84K, but in treated mutants, only twofold increases were induced (Figure 6a). Accordingly, MDA, a product of lipid peroxidation indicating the level of lipid damage, showed less of an increase in the treated 84K plants than in the treated mutants ( Figure  6b).

KO Lines of PagSWEET17a Exhibit Decreased Drought Tolerance
Drought treatment on 4-week-old tissue-culture seedlings showed that the expression of PagNCED3, the first committed and key regulated step in the synthesis of ABA [31], was significantly induced by drought on 84K and reached its expression peak at 12 h ( Figure 4a). However, the induction of PagNCED3 was clearly inhibited in the stressed mutants (Figure 4b), suggesting a different response from 84K. Then, the mutants and non-transgenic 84K seedlings were grown in soil and applied to drought stress for 4 weeks. The results showed drought-induced larger root biomass in seedlings of 84K, but these changes were not evident in both KO mutant lines (Figures 5a and S2). For the aerial parts, the seedlings of non-transgenic 84K showed mild growth retardation, showing reduced height and diameter after simulated drought treatment, while in the mutants, especially in KO-4, growth retardation was further increased compared to their normal watering control (Figure 5b-d). The activities of POD, which facilitate the removal of reactive oxygen species and protect plant cells from damage [32], were elevated fourfold by drought in 84K, but in treated mutants, only twofold increases were induced (Figure 6a). Accordingly, MDA, a product of lipid peroxidation indicating the level of lipid damage, showed less of an increase in the treated 84K plants than in the treated mutants (Figure 6b).

KO Lines of PagSWEET17a Exhibit Decreased Drought Tolerance
Drought treatment on 4-week-old tissue-culture seedlings showed that the expression of PagNCED3, the first committed and key regulated step in the synthesis of ABA [31], was significantly induced by drought on 84K and reached its expression peak at 12 h (Figure 4a). However, the induction of PagNCED3 was clearly inhibited in the stressed mutants (Figure 4b), suggesting a different response from 84K. Then, the mutants and non-transgenic 84K seedlings were grown in soil and applied to drought stress for 4 weeks. The results showed drought-induced larger root biomass in seedlings of 84K, but these changes were not evident in both KO mutant lines (Figures 5a and S2). For the aerial parts, the seedlings of non-transgenic 84K showed mild growth retardation, showing reduced height and diameter after simulated drought treatment, while in the mutants, especially in KO-4, growth retardation was further increased compared to their normal watering control (Figure 5b-d). The activities of POD, which facilitate the removal of reactive oxygen species and protect plant cells from damage [32], were elevated fourfold by drought in 84K, but in treated mutants, only twofold increases were induced (Figure 6a). Accordingly, MDA, a product of lipid peroxidation indicating the level of lipid damage, showed less of an increase in the treated 84K plants than in the treated mutants ( Figure  6b).  week-old 84K seedlings subjected to drought treatment. The bars represented means from n = 3 biological replicates ± SD. Different letters above the bars denoted significant differences according to ordinary one-way ANOVA with post-hoc Tukey testing (p < 0.05). (b) Expression of PagNCED3 in 84K and KO lines after 12 h of drought treatment. The bars represented means ± SD, and the asterisks indicated significant differences from 84K according to one-way ANOVA followed by Dunnett's post-hoc test (** p < 0.01).

Mutantion in PagSWEET17a Decreased Accumulation of Soluble Sugars in Root
In order to investigate the changes in soluble sugars related to PagSWEET17a induced by drought, the sugar profiles of 84K and KO lines under simulated drought and normal watering conditions were investigated. The contents of 14 soluble sugars, which are commonly found in plant species, were quantified. These sugars included six-carbon sugars, five-carbon sugars, disaccharides, trimeric sugars, sugar alcohols, and phosphorylated sugars. The results showed that the sugar profiles of all seedlings were clearly  to ordinary one-way ANOVA with post-hoc Tukey testing (p < 0.05). (b) Expression of PagNCED3 in 84K and KO lines after 12 h of drought treatment. The bars represented means ± SD, and the asterisks indicated significant differences from 84K according to one-way ANOVA followed by Dunnett's post-hoc test (** p < 0.01).  Error bars, SD (one-way ANOVA/Dunnett, **** p < 0.0001; *** p < 0.001; ** p < 0.01) and asterisks indicated significant differences from 84K.

Mutantion in PagSWEET17a Decreased Accumulation of Soluble Sugars in Root
In order to investigate the changes in soluble sugars related to PagSWEET17a induced by drought, the sugar profiles of 84K and KO lines under simulated drought and normal watering conditions were investigated. The contents of 14 soluble sugars, which are commonly found in plant species, were quantified. These sugars included six-carbon sugars, five-carbon sugars, disaccharides, trimeric sugars, sugar alcohols, and phosphorylated sugars. The results showed that the sugar profiles of all seedlings were clearly Error bars, SD (one-way ANOVA/Dunnett, **** p < 0.0001; *** p < 0.001; ** p < 0.01) and asterisks indicated significant differences from 84K.

Mutantion in PagSWEET17a Decreased Accumulation of Soluble Sugars in Root
In order to investigate the changes in soluble sugars related to PagSWEET17a induced by drought, the sugar profiles of 84K and KO lines under simulated drought and normal watering conditions were investigated. The contents of 14 soluble sugars, which are commonly found in plant species, were quantified. These sugars included six-carbon sugars, five-carbon sugars, disaccharides, trimeric sugars, sugar alcohols, and phosphorylated sugars. The results showed that the sugar profiles of all seedlings were clearly disturbed by drought. Compared with the control group with normal watering, the contents of 12 out of 14 sugars in drought-treated samples were significantly upregulated (Figure 7). Seven sugars, including glucose, mannose, galactose, fructose, maltose, melezitose and raffinose, exhibited lower levels in drought-treated mutants compared to 84K in stress. Further analysis of the fold-changes identified that drought-induced smaller changes of glucose, maltose, galactose, fructose, sucrose, and raffinose in both mutant lines than in non-transgenic 84K (Figure 8). disturbed by drought. Compared with the control group with normal watering, the contents of 12 out of 14 sugars in drought-treated samples were significantly upregulated (Figure 7). Seven sugars, including glucose, mannose, galactose, fructose, maltose, melezitose and raffinose, exhibited lower levels in drought-treated mutants compared to 84K in stress. Further analysis of the fold-changes identified that drought-induced smaller changes of glucose, maltose, galactose, fructose, sucrose, and raffinose in both mutant lines than in non-transgenic 84K (Figure 8).   disturbed by drought. Compared with the control group with normal watering, the contents of 12 out of 14 sugars in drought-treated samples were significantly upregulated (Figure 7). Seven sugars, including glucose, mannose, galactose, fructose, maltose, melezitose and raffinose, exhibited lower levels in drought-treated mutants compared to 84K in stress. Further analysis of the fold-changes identified that drought-induced smaller changes of glucose, maltose, galactose, fructose, sucrose, and raffinose in both mutant lines than in non-transgenic 84K (Figure 8).  and raffinose (f) in mutant lines and non-transgenic 84K. Error bars, SD (one-way ANOVA/Dunnett, ** p < 0.01) and asterisks indicated significant differences from 84K.

Discussion
Under drought conditions, plants reprogram their metabolism and development to initiate an economical mode of growth and protect themselves from the adverse effects of the environment. In this process, sugars fulfill many vital functions. They are the main carbon and energy sources, serve as signaling molecules, and act as protective compounds during drought stress [33]. Sugar transporters mediate the distribution of sugar, control the subcellular and tissue-specific carbohydrate profiles, and are important players in plant tolerance to drought [34]. Here, the role of PagSWEET17a in poplar responses to drought stress was investigated. The results showed that the expression of PagSWEET17a was significantly induced by drought. Loss of function in PagSWEET17a suppressed the drought-induced upregulation of sugar, leading to a decreased tolerance to stress.
The root is an important organ for plants to absorb water and plays a crucial role in responses to drought. Well-developed root systems will enable plants to make water absorption as efficiently as possible and facilitate survival under drought [35]. GUS staining of P PagSWEET17a ::GUS transgenic plants and immunolocalization with an antibody identified that PagSWEET17a was specifically expressed in cambial cells in younger roots, while in older roots, it expressed in phloem fiber cells, xylem vessels and fiber cells. Previous studies have identified that sugar concentration in cambium or other meristematic tissues is not only an inducing signal for cell expansion and cell wall synthesis but also provides carbon and energy source for these activities influencing the development of plant organs [36]. In Arabidopsis, AtSWEET17 was mainly expressed in the root vasculature and meristematic cells of the root tip. Knockout of AtSWEET17 reduced the number of lateral roots and impaired the tolerance to drought [14]. In this study, it was found that PagSWEET17a might not influence the development of lateral roots, but affect that of adventitious roots ( Figure S1). The missing function of PagSWEET17a decreased the number of adventitious roots and root biomass of seedlings and diminished the drought stress tolerance in poplar trees. The difference in function between AtSWEET17 and PagSWEET17a might be attributed to gene expansion in perennial poplar trees. Unlike only one AtSWEET17 member in Arabidopsis, there are four PagSWEET17s in poplar [22]. Although PagSWEET17a was highly expressed in root vascular, PagSWEET17b, PagSWEET17c, and PagSWEET17d were also expressed in root, suggesting that their functions in the development of root undergo differentiation. Further research will be conducted to clarify their specific roles in root growth and drought response.
Under drought conditions, plants adjust their metabolism to cope with drought and maintain their normal growth. Carbohydrate metabolism forms the core of the reprogramming of plant metabolism in the responses to drought [34]. To maintain soluble sugar concentrations and subsequent osmotic adjustment, plants generally degrade starch into soluble sugars induced by drought stress [37]. Research on crops and woody plants have shown that drought can reduce starch concentration in leaf and increase levels of soluble sugars [13,14,38]. In addition to the reprogramming of carbon metabolism, plants also modulate the priority of carbon allocation to deal with the imbalance between carbon supply and carbon demand resulting from diminished photosynthesis led by drought. The growth of the roots will be promoted by increasing the transport of sugars from the aerial organs to the roots. The result of this strategy is an increase in the root-shoot ratio and a decrease in the above-ground growth of plants [16]. Indeed, in this study, drought-induced a larger root (Figure 5a and Figure S2) in seedlings of 84K, while this phenomenon is not evident in the mutants. Analysis of sugar profiles identified that 12 out of the 14 detected sugars were significantly upregulated by drought in roots of KO lines and 84K (Figure 7), confirming the findings of previous studies. Several sugars, including sucrose, glucose, fructose, galactose and raffinose showed a decreased accumulation in the treated mutants compared to the stressed 84K. Particularly, the level of raffinose, which acts as an important player in crop tolerance to drought [39,40], was increased 13-fold by drought in 84K but only upregulated ninefold in both KO lines. These results indicated that PagSWEET17a might play an important role in carbon allocation from shoot to root. Therefore, the loss of function of PagSWEET17a limited the accumulation of soluble sugars and, therefore, impaired poplar tolerance to drought.
Apart from stimulating root growth and osmotic adjustment, sugars also function in the protection of cells from drought-induced damage [2]. Sugar accumulation maintains the turgidity of cells and prevents dehydration of membranes and proteins [41]. Soluble sugars also can prevent the oxidation of cell membranes by scavenging excess reactive oxygen species (ROS) under stress [33,42,43]. The abundant sugars, glucose, fructose, sucrose, fructans and raffinose have been shown to be excellent scavengers of ROS [39,40,44,45]. Furthermore, the accumulation of low concentrations of sugars stimulates the activity of some antioxidant enzymes, such as peroxidase, catalase and superoxide dismutase, which help to maintain membrane integrity and prevent protein denaturation [46]. The present study found that, although several sugars were increased in both 84K and mutants induced by drought, the fold changes in mutants were significantly lower than those of 84K. The activity of POD, an antioxidant enzyme involved in reducing oxidative stress, in drought-treated seedlings of 84K was almost twofold higher than those of mutants. Correspondingly, the content of malondialdehyde, which is an indicator of oxidative stress, was significantly higher in the latter than in the former, suggesting that higher oxidative damage existed in treated mutants. It was possible that PagSWEET17a contributed to reduction of oxidative damage induced by drought by facilitating sugar accumulation in roots.
The phytohormone ABA was known to play an important role in modulating plant responses to drought. Sugar allocation and accumulation are important events involved in tolerance to drought. Previous studies have shown that interactions between ABA and sugar may be antagonistic or synergistic depending on the concentration and kind of sugar [47]. In this study, it was determined that the synthesis of ABA in pagsweet17a mutants, which contain lower levels of several sugars, was clearly inhibited compared to 84K. However, it is unclear how the loss of PagSWEET17a decreases ABA synthesis. Recently, studies on Arabidopsis found that AtSWEET11 and AtSWEET12 are phosphorylated by ABAactivated SnRK2 and facilitate sugar transport from shoots to roots promoting preferential growth of roots [17]. Interestingly, in the present study, the expression of PagSnRK2 showed no significant difference between drought-treated mutants and 84K ( Figure S3). Future work will focus on revealing the relationships among ABA-PagSnRK2-PagSWEET17a. Although the underlying mechanism is not clear, the role of PagSWEET17a in drought has been identified in this study; that is, PagSWEET17a contributes to tolerance to drought in poplar by mediating the transport and accumulation of sugar in roots. This gene could be a candidate gene for genetic engineering to develop improved drought-tolerant poplar trees.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/f14071445/s1, Figure S1: Number of adventitious roots in seedlings of 84K and KO plants, Figure S2: Fold change of root biomass (dry weight) induced by drought in seedlings of 84K and KO lines, Figure S3: Relative expression levels of PagSnRK2 in 84K and KO lines after 12 h of drought treatment, Table S1: Primer sequences used for CRISPR/Cas9 system and qPCR, Table S2: Sugar contents in the roots of normal watering and drought-treated seedlings.