Next Article in Journal
Identification of the GDP-L-Galactose Phosphorylase Gene as a Candidate for the Regulation of Ascorbic Acid Content in Fruits of Capsicum annuum L.
Previous Article in Journal
Evidence of Disruption in Neural Regeneration in Dry Eye Secondary to Rheumatoid Arthritis
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 24
Issue 8
10.3390/ijms24087533
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Genome-Wide Identification, Expression, and Response to Fusarium Infection of the SWEET Gene Family in Garlic (Allium sativum L.)

by
Mikhail A. Filyushin
*,
Olga K. Anisimova
,
Anna V. Shchennikova
and
Elena Z. Kochieva
Federal Research Center “Fundamentals of Biotechnology” of the Russian Academy of Sciences, 119071 Moscow, Russia
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2023, 24(8), 7533; https://doi.org/10.3390/ijms24087533
Submission received: 22 March 2023 / Revised: 17 April 2023 / Accepted: 18 April 2023 / Published: 19 April 2023
(This article belongs to the Section Molecular Plant Sciences)
Browse Figures
Versions Notes

Abstract

:
Proteins of the SWEET (Sugar Will Eventually be Exported Transporters) family play an important role in plant development, adaptation, and stress response by functioning as transmembrane uniporters of soluble sugars. However, the information on the SWEET family in the plants of the Allium genus, which includes many crop species, is lacking. In this study, we performed a genome-wide analysis of garlic (Allium sativum L.) and identified 27 genes putatively encoding clade I–IV SWEET proteins. The promoters of the A. sativum (As) SWEET genes contained hormone- and stress-sensitive elements associated with plant response to phytopathogens. AsSWEET genes had distinct expression patterns in garlic organs. The expression levels and dynamics of clade III AsSWEET3, AsSWEET9, and AsSWEET11 genes significantly differed between Fusarium-resistant and -susceptible garlic cultivars subjected to F. proliferatum infection, suggesting the role of these genes in the garlic defense against the pathogen. Our results provide insights into the role of SWEET sugar uniporters in A. sativum and may be useful for breeding Fusarium-resistant Allium cultivars.

1. Introduction

Carbohydrates are important biomolecules for energy generation and storage in plants and, as such, are involved in plant development and stress response [1]. Furthermore, sugars define the economic value of many crops. Thus, the content and ratio of fructose and glucose in sink organs significantly affect the taste and quality of grape [2] and tomato [3]) fruit, potato tubers [4], and carrot roots [1]).
Glucose, a basic monosaccharide produced by photosynthesis, is used for the synthesis of fructose and sucrose [5]; then, the three sugars are transported from the leaves through the phloem to accumulating organs (flowers, fruit, seeds, and roots), where they are stored (partly as starch) to support organ growth and development [6,7]. To date, three families of sugar transporters have been characterized: monosaccharide transporters (MSTs), sucrose transporters (SUTs), and Sugar Will Eventually be Exported Transporters (SWEETs) [6,8].
Despite their extensive expansion and functional diversity, the SWEET genes are considered to be evolutionarily conserved. A study of this gene family in 31 representative plant species demonstrated a dramatic increase in the number of SWEET members in higher plants, largely due to tandem and segmental duplication under purifying selection [9]. Structurally, SWEET transporters are unique due to the presence of up to seven transmembrane domains, which may have arisen during the evolutionary duplication of a prokaryotic ancestral gene. In the plant, SWEETs are involved in maintaining a variety of physiological processes such as pollen nutrition, nectar secretion, seed filling, phloem loading, and pathogen feeding [10].
The members of the SWEET family transport mono- and disaccharides through the plasma membrane in both directions, thus mediating the uptake as well as the efflux of soluble sugars in plant tissues [11,12]. According to their sugar specificity, SWEETs are subdivided into four clades: clades I and II transport hexoses, and clades III and IV—sucrose and fructose, respectively [13,14,15]. SWEETs are known to have organ-specific expression, which, together with their distinct substrate specificity, accounts for diverse physiological functions illustrated by studies in a model species, Arabidopsis thaliana L. [16,17,18,19,20,21,22,23,24]. Thus, AtSWEET1 and 2 transport glucose in the rhizosphere and contribute to pathogen resistance. AtSWEET4–8 and 13 also transfer glucose; in addition, AtSWEET5 transports galactose, for which it has dose-dependent sensitivity during pollen germination, whereas AtSWEET8 along with AtSWEET13 is involved in sucrose transport associated with pollen fertility. Four other AtSWEETs are also specific for sucrose: AtSWEET9 participates in sucrose secretion from the nectary parenchyma into the extracellular space, where it is hydrolyzed to form a mixture of sucrose, glucose, and fructose; whereas AtSWEET11, 12, and 15 provide sucrose outflow from the seed coat to the embryo. AtSWEET16 and 17 are associated with fructose transport in leaf and root tonoplasts, where their expression is sensitive to drought. In tomato (Solanum lycopersicum L.), the SlSWEET1a gene is linked to the Fgr (fructose to glucose ratio) locus modulating the partitioning of hexoses in the fruit [25,26,27].
Accumulating evidence indicates that SWEETs play a key role not only in sugar transfer, signaling, and plant growth but also in stress tolerance and plant-pathogen interactions [13,28,29]. Many of the 29 tomato SWEET genes showed sensitivity to exogenous fructose and sucrose treatment, as well as to high and low-temperature stresses [21]. In Medicago truncatula, a large number of 25 SWEET genes were found to be upregulated in response to cold, drought, and salt [30]. It has been shown that variations in SWEET gene expression can affect resistance to infection through changes in sugar efflux and distribution [29,31,32]. Plant pathogens can release effectors that divert SWEET gene expression to benefit infection. Thus, Xanthomonas oryzae, the causal agent of bacterial blight, secretes transcription activator-like (TAL) effectors which specifically bind promoters of individual SWEET genes in rice, whereas mutations in the effector-binding elements of the SWEET promoters confer resistance to blight [13,28,29]. Based on this effect, blight-resistant rice lines have been obtained through SWEET promoter editing [31] and gene silencing [32].
The most important sink organ of the plant, the root, imports sugar from the aerial parts of the plant through the phloem and uses it for metabolism and storage, and also releases it into the rhizosphere, where it is assimilated by beneficial microorganisms and pathogens. Sugar transport in all cases is mediated by membrane transporters, including SWEETs [33].
Fusarium oxysporum, a root-colonizing fungal pathogen causing root rot, has also been shown to affect SWEET gene activity. Thus, inoculation of tobacco (Nicotiana tabacum L.) with F. oxysporum leads to the downregulation of the NtSWEET1, NtSWEET3b, and NtSWEET12 genes and RNAi-mediated suppression of NtSWEET1 makes tobacco roots more susceptible to rot, indicating the role of NtSWEET1 in tobacco anti-fungal resistance [34]. It has been reported that in sweet potato (Ipomoea batatas (L.) Lam.), F. oxysporum infection significantly upregulates the IbSWEET10 gene and that its overexpression or silencing confers resistance or susceptibility to F. oxysporum, respectively [35]. In watermelon (Citrullus lanatus Thunb.), ClaSWEET genes are involved in the resistance to F. oxysporum as well as in the response to drought, salt, and cold stresses [36]. Infection of potato (Solanum tuberosum L.) with Fusarium solani, a necrotroph, or F. oxysporum f. sp. tuberose, a hemibiotrophic, induces StSWEET7a while suppressing most other StSWEET genes, and StSWEET7a overexpression in potato roots promotes their colonization by F. oxysporum f. sp. tuberosi through an increase in the sink strength [37]. It is speculated that clade III SWEET genes are involved in the biotrophic phase of host-pathogen interaction [38,39]; thus, these genes are upregulated by hemibiotrophic bacteria (Xanthomonas or Pseudomonas) or fungi (Golovinomyces cichoracearum) in rice (OsSWEET11 and OsSWEET14) [28], cassava (MeSWEET10a) [38], and Arabidopsis (AtSWEET10, AtSWEET12, and AtSWEET15) [16]. At the same time, clade II SWEET genes are suggested to play a role in promoting pathogen virulence (AtSWEET4, AtSWEET5, AtSWEET7, and AtSWEET8 in Arabidopsis [16]) or be involved in plant response to necrotrophs (SWEET4 in Arabidopsis and grape [39,40,41]).
Garlic (Allium sativum L.) is the second most popular spice and the second most important crop in the Allium genus [42,43], with production of over 30 million tons per year (http://www.fao.org/; accessed on 26 January 2023). Garlic is known to have health-promoting and anti-fungal properties, producing compounds that can regulate glucose metabolism and transport and exert therapeutic effects in cerebral ischemia and obesity [44,45] and volatile and non-volatile molecules that act as fungicides [46]. Garlic cloves are rich in carbohydrates, including high-molecular weight fructans and fructooligosaccharides [47,48], as well as soluble sugars such as sucrose, fructose, and glucose [49,50], which constitute up to 75% of the bulb dry matter. Most sugars are photoassimilate-derived, suggesting the existence of an extensive carbohydrate transportation system; however, the information on sugar transporters in garlic is lacking. In particular, nothing is known about SWEETs, not only in garlic, but also in other Allium species.
The aim of the present study was to search for and characterize the A. sativum genes belonging to the SWEET family and investigate their tissue expression patterns. As garlic is susceptible to Fusarium infection known to affect SWEET expression, we also examined SWEET gene activity in response to F. proliferatum attack in garlic cultivars resistant and susceptible to Fusarium basal rot (FBR).

2. Results

2.1. Identification of SWEET Genes in the A. sativum Genome

Twenty-seven sequences of full-length SWEET genes were found by in silico analysis of the A. sativum cv. Ershuizao genome (PRJNA606385) and transcriptome (PRJNA607255) were annotated as A. sativum (As) SWEET1–27 (Table 1). Given the low homology between the genes of garlic and the model species A. thaliana, combined with the lack of data on SWEET genes in Allium species, the gene numbering was based on the order of their location on the chromosomes, as was done, for example, for maize Zea mays SWEETs [51].
The chromosome distribution of the 27 SWEET genes was not random: most of them (16) were located on chromosomes 2 (AsSWEET2, AsSWEET3, AsSWEET10, and AsSWEET4–9 cluster) and 8 (AsSWEET26 and AsSWEET20–25 cluster). Ten genes were scattered among chromosomes 1, 3, and 5–7; two of the three genes located on chromosome 7 (AsSWEET18 and AsSWEET19) were tandemly clustered (Figure 1a). The AsSWEET27 gene was found in scaffold and did not match any chromosome (assembly Garlic.V2.fa; Table 1).
The AsSWEET genes significantly varied in size, ranging from 953 bp (AsSWEET16) to 6556 bp (AsSWEET1), and in the number of exons (3–7) and introns (2–6): most genes (18; ~66.7%) contained 6 exons/5 introns, and 2, 5, 1, and 1 genes contained 7, 5, 4, and 1 exons, respectively. No splice variants were found. The coding sequences (CDSs) varied from 654 bp (AsSWEET13) to 1002 bp (AsSWEET14) and consisted of 3 (AsSWEET13) to 7 (AsSWEET1 and AsSWEET23) exons (Table 1, Figure 1b). An additional search of the transcriptome data for AsSWEET mRNA isoforms revealed only one transcript variant for each of the 27 genes.

2.2. Characterization of Putative SWEET Proteins in Garlic

The AsSWEET proteins predicted based on the CDSs consisted of 217–333 amino acids (Table 1). Analysis of AsSWEET physicochemical properties indicated that the proteins differed in isoelectric points (pIs) (from 5.84 in AsSWEET16 to 10.01 in AsSWEET21), hydrophobicity index (GRAVY) (from 0.203 in AsSWEET14 to 0.860 in AsSWEET12), and molecular weight (MW) (from 23.991 kDa in AsSWEET13 to 34.077 kDa in AsSWEET23) (Table 1).
The secondary structures of AsSWEETs were characterized by the presence of two MtN3_slv functional domains typical for sugar efflux transporters and several transmembrane helices: seven in most proteins (77.8%), six in AsSWEET13–15, AsSWEET23, and AsSWEET24, and five in AsSWEET16 (Figure 2a), with corresponding consensuses (Figure 2b). In terms of Gene Ontology (GO), all AsSWEET proteins were predicted to have sugar transmembrane transporter activity and be involved in carbohydrate transport.
Phylogenetic analysis of AsSWEETs revealed six groups comprising 12 (group 1), 2 (AsSWEET23, AsSWEET24; group 2), 2 (AsSWEET20–22, 25; group 3), 4 (AsSWEET10, AsSWEET12–14; group 4), 2 (AsSWEET26, AsSWEET27; group 5), and 3 (AsSWEET1, AsSWEET15, AsSWEET17; group 6) proteins (Figure 3a). The group division was supported by MEME-based analysis, which revealed 10 conserved motifs (Figure 3b). Among them, motifs 2 and 4 were common for all AsSWEETs, and motifs 1, 3, and 5 were present in most of them with few exceptions: motif 1 was absent in AsSWEET13 and AsSWEET16; motif 2, in AsSWEET1; and motif 5, in AsSWEETs of groups 2 and 4. Motifs 6 and 7 were characteristic for group 1, being present in 10 (except AsSWEET2 and 16) and 8 (except AsSWEET3, AsSWEET6, AsSWEET11, and AsSWEET16) proteins, respectively. In contrast, motif 8 was absent in the AsSWEETs of group 1 but present in those of the other groups (except for AsSWEET13 and AsSWEET14). Motif 9 was found in all AsSWEETs of group 3 and in some of groups 1 and 6, and motif 10, in two members of groups 1 and 3, respectively (Figure 3b).
Thus, besides three tandemly organized AsSWEET gene clusters on chromosomes 2, 8, and 7 (Figure 1a), we could also distinguish five phylogenetic clusters (AsSWEET26, AsSWEET27; AsSWEET14, AsSWEET12, AsSWEET10, AsSWEET13; AsSWEET1, AsSWEET15, AsSWEET17; AsSWEET18, AsSWEET19, AsSWEET2; and AsSWEET6, AsSWEET3, AsSWEET11) (Figure 3), the members of which were located on different chromosomes and might have been originated as a result of segmental/transposed gene duplication events.
Next, we analyzed the relationships of the predicted AsSWEETs and the SWEETs of A. thaliana, S. lycopersicum, and Z. mays since Arabidopsis, tomato, and maize are considered models for studying dicot and monocot species, respectively (1) and there are no data on SWEET genes in monocot Allium species for comparison (2). The results indicated that AsSWEETs of groups 5/6, 4, 1, and 2/3 (Figure 3a) corresponded to A. thaliana SWEET clades I, II, III, and IV, respectively, and that the tandemly organized AsSWEET gene clusters (Figure 1a) corresponded to specific functional A. thaliana SWEET clades: AsSWEET4–9, AsSWEET18, and AsSWEET19, to clade III and AsSWEET20–25, to clade IV (Figure 4).
It was found that, within clades I, II, and IV, the garlic genes split into subclades based on similarity to genes of Arabidopsis, tomato (dicot), and maize (monocot). In clade I, AsSWEET1 was grouped with At/SlSWEET3 and ZmSWETT4/ZmSWETT16, AsSWEET26/AsSWEET27 with At/SlSWEET2 and ZmSWETT15/ZmSWETT17; and AsSWEET15/AsSWEET17 with At/SlSWEET1 and ZmSWETT13. In clade II, AsSWEET10/AsSWEET12/AsSWEET13 formed subclade with At/SlSWEET6, AtSWEET7 and ZmSWETT11; and AsSWEET14 formed subclade with At/SlSWEET5, AtSWEET4, AtSWEET8, SlSWEET16a, and ZmSWETT17/ZmSWEET18. In clade IV, AsSWEET23/AsSWEET24 clustered with AtSWEET16, At/SlSWEET17, and ZmSWETT14/ZmSWEET15; and AsSWEET20–22 and AsSWEET25 with SlSWEET16 and ZmSWETT1 (Figure 4).
Clade III had the largest number of members, but the SWEETs of garlic, as well as maize, were grouped predominantly separately from the SWEET proteins of dicotyledonous plant species. All AsSWEETs (AsSWEET4–9, AsSWEET18, and AsSWEET19) are included in the Z. mays ZmSWEET3, ZmSWEET6, ZmSWEET9, ZmSWEET19, and ZmSWEET320 subclade (Figure 4). Similar clustering of clade III proteins, preferentially separating monocot and dicot proteins, is shown, for example, in [54].
In addition, NCBI-BLAST was used to determine the closest homologs of AsSWEET proteins in two model plant species, A. thaliana and S. lycopersicum (Table S1). It was found that the proteins of garlic have only 49–67% identity with the SWEETs of tomato and Arabidopsis. Clade III AsSWEETs were the most similar to AtSWEET11 and AtSWEET12 (except for AsSWEET16, which was close to AtSWEET14) in A. thaliana and to SlSWEET10c, SlSWEET12b, SlSWEET11c in S. lycopersicum. Clade I proteins were the closest homologs of AtSWEET3/SlSWEET3 (AsSWEET1), AtSWEET1/SlSWEET1b (AsSWEET15 and AsSWEET17) and AtSWEET2/SlSWEET2a-2 (AsSWEET26 and AsSWEET27). The highest similarity of clade II and IV proteins was observed with AtSWEET7/SlSWEET7a and AtSWEET17/SlSWEET16, respectively (Supplementary Table S1).

2.3. Cis-Acting Elements in the AsSWEET Promoters

Considering the role of SWEETs in plant stress- and hormone-related responses [14,15,16,17,18,19,20,21,22], we searched the AsSWEET promoter regions (~1 kb upstream of the initiation codon) for the stress- and hormone-sensitive cis-regulatory elements. The results showed that the promoters of the AsSWEET6, AsSWEET8, and AsSWEET23 genes were the most enriched in cis-elements, containing 12, 11, and 9 of them, respectively (Figure 5).
Nine types of hormone-responsive cis-elements, including those involved in the response to abscisic acid (ABA), auxin (AuxRR), methyl JA (MeJA), salicylic acid (SA), gibberellic acid (GA), and ethylene (ET), were identified in AsSWEET genes (Figure 5). Each AsSWEET promoter had 2–5 elements, except for AsSWEET4 and 5, which had none. The largest number of one-type hormone-sensitive elements (ABA-responsive) was detected in AsSWEET6 (clade III) (Figure 5).
In total, 10 types of cis-elements related to stresses such as anaerobic conditions, drought, cold, wounding, and pathogens were identified in AsSWEET promoters, all of which contained at least one (Figure 5). Two genes, AsSWEET4 and AsSWEET5, contained only stress-related elements; the former, along with AsSWEET8 and AsSWEET9, had the largest number (4) of elements responsive to anaerobic conditions. The largest number of STREs was found in AsSWEET8 (5), followed by AsSWEET23 (3). AsSWEET16 and AsSWEET17 contained only wound-responsive elements—two Wun-motifs and one W-box, respectively; the other wounding/pathogen-responsive motifs (WRE3 and/or box S) were found only in AsSWEET26 and 27. A drought-responsive element was identified only in AsSWEET10 (Figure 5).

2.4. Tissue Expression Patterns of AsSWEET Genes

Analysis of the A. sativum cv. Ershuizao transcriptome (PRJNA607255) revealed that seven AsSWEET genes (AsSWEET1, AsSWEET2, AsSWEET12–14, AsSWEET23, and AsSWEET25) were not transcribed in garlic. The majority of the transcribed genes had the highest mRNA levels in the leaves, except for AsSWEET16 (maximum in the buds), AsSWEET15, AsSWEET17, AsSWEET27 (flowers), AsSWEET18, AsSWEET19, AsSWEET22 (sprouts), and AsSWEET26 (roots). High levels of AsSWEET expression were also detected in the pseudostem (except for AsSWEET15, AsSWEET16, and AsSWEET27). AsSWEET3, AsSWEET4, AsSWEET7, AsSWEET22, and AsSWEET27 were not expressed in the stage 1/2 bulbs, whereas AsSWEET4, AsSWEET7, AsSWEET15–17, and AsSWEET20–24 had very low or no expression in the roots (Figure 6).
Organ-specific expression was observed for the following AsSWEET genes: AsSWEET3, AsSWEET9, AsSWEET11, and AsSWEET26 (roots), AsSWEET3, AsSWEET5–7, AsSWEET9, AsSWEET16, AsSWEET18–22, and AsSWEET26 (sprouts), AsSWEET6, AsSWEET10, AsSWEET15, AsSWEET17, and AsSWEET27 (flowers), AsSWEET3, AsSWEET5, AsSWEET6, AsSWEET16, AsSWEET17, and AsSWEET26 (buds), AsSWEET17, AsSWEET20, AsSWEET21, and AsSWEET26 (bulbs of all stages), and AsSWEET4, AsSWEET7–9, and AsSWEET27 (bulbs at the final [6,7,8] stages) (Figure 6). These data indicated that genes of each SWEET clade were expressed in the analyzed organs. Given the previously shown role of SWEETs in the transport of hexoses (clades I and II), sucrose (clade III), and fructose (clade IV) [11,12,13], we may assume that AsSWEETs of clades I, II, and IV are involved in the transport of both hexoses and clade III AsSWEETs—in the sucrose transport.
Tissue expression patterns differed for the genes of two chromosomal clusters (AsSWEET4–9 and AsSWEET20–25), whereas those of the third cluster genes (AsSWEET18, AsSWEET19) were similar. Presumably segmentally duplicated genes (AsSWEET26, AsSWEET27; AsSWEET11, AsSWEET15, AsSWEET17; and AsSWEET6, AsSWEET3, AsSWEET11) also showed different tissue expression patterns. In phylogenetic group 4, only AsSWEET10 was expressed; in group 1, AsSWEET2, as a possible segmentally duplicated variant of AsSWEET18 and AsSWEET19, was not transcribed (Figure 6).
The expression of 11 genes (AsSWEET1, AsSWEET15, AsSWEET17, and AsSWEET26 of clade I, AsSWEET13 of clade II, AsSWEET3, AsSWEET5, AsSWEET9, AsSWEET11, and AsSWEET18/19 of clade III, and AsSWEET24 of clade IV) was verified by quantitative real-time (qRT)-PCR in the tissues of A. sativum cv. Sarmat (Figure 7). It was confirmed that AsSWEET1 and AsSWEET13 were not transcribed in any garlic tissue. The results also revealed that the expression of most analyzed genes in the roots, cloves, leaves, and pseudostems was similar in cv. Ershuizao and Sarmat (Figure 6 and Figure 7); however, three genes showed different expression levels: AsSWEET5 in the pseudostem (high/traces, respectively), AsSWEET15 in the roots (traces/high, respectively), and AsSWEET11 and AsSWEET24 in the leaves and pseudostems (high/low, respectively).
AsSWEET3, AsSWEET9, AsSWEET15, AsSWEET17, and AsSWEET26 were expressed in all analyzed tissues with the maximal level in the roots (AsSWEET26), leaves (AsSWEET5 and AsSWEET15), peduncles (AsSWEET3, AsSWEET9, AsSWEET11, AsSWEET17, and AsSWEET24), and receptacles (AsSWEET9 and AsSWEET18–19). AsSWEET5 was expressed only in the leaves. The expression of AsSWEET9 and AsSWEET17 was characteristic for the cloves and air bulbs, whereas that of AsSWEET5 and AsSWEET11 was absent in these organs, and that of AsSWEET5, AsSWEET18–19, and AsSWEET24 was absent in the roots (Figure 7).

2.5. AsSWEET Gene Expression in Response to F. proliferatum Infection

Considering that SWEETs are involved in plant resistance to Fusarium spp. [34], the expression of the genes which showed differential tissue expression patterns (AsSWEET3, AsSWEET5, AsSWEET9, AsSWEET11, AsSWEET18, and AsSWEET19 [clade III], AsSWEET15, AsSWEET17, and AsSWEET26 [clade I], and AsSWEET24 [clade IV]) (Figure 7) was compared in the roots of garlic cultivars resistant (cv. Sarmat) and susceptible (cv. Strelets) to FBR. The plants were infected with F. proliferatum and analyzed 24 and 96 h post-inoculation (hpi), covering the peak of pathogenesis-related (PR) gene expression in response to hemibiotrophic pathogens [55,56,57,58].
The expression of AsSWEET5, AsSWEET18, AsSWEET19, and AsSWEET24 was not detected either in non-infected or infected roots, which is consistent with the previous results (Figure 7), whereas that of the other six genes (AsSWEET3, AsSWEET9, AsSWEET11, AsSWEET15, AsSWEET17, and AsSWEET26) was affected by F. proliferatum infection (Figure 8). At 24 hpi, AsSWEET3, AsSWEET11, AsSWEET15, and AsSWEET26 were significantly upregulated in both cultivars, especially in cv. Strelets, where AsSWEET9 was induced about 8-fold, AsSWEET3—over 20-fold, and AsSWEET11, AsSWEET15, and AsSWEET26—about 30-fold compared to the uninfected control (Figure 8). Thus, the AsSWEET genes were strongly upregulated in the FBR-susceptible cultivar at the early stage of Fusarium infection. Later (96 hpi), the expression level of all AsSWEET genes in cv. Strelets decreased but still remained significantly higher than in control; at the same time, in cv. Sarmat, the expression of AsSWEET3 and AsSWEET11 was increased 140–150 times (Figure 8). These results indicated cultivar-dependent activation of AsSWEET genes in response to Fusarium infection.

2.6. Effect of F. proliferatum Infection on Sucrose, Glucose, and Fructose Contents in Garlic

Next, we compared the content of soluble sugars (glucose, fructose, and sucrose) in infected and non-infected garlic cultivars. The results indicated that at 24 hpi, the levels of all sugars were significantly decreased in the roots of FBR-resistant cv. Sarmat but not in those of FBR-susceptible cv. Strelets where the levels of glucose and sucrose were unchanged and that of fructose significantly increased (Figure 9). No significant changes in the sugar content were observed in both cultivars at 96 hpi. This may indicate that the FBR-resistant cv. Sarmat does not supply Fusarium with sugars, while the FBR-sensitive cv. Strelets supplies pathogen in an early response to infection but may stop efflux at 96 hpi.

2.7. Cloning and Characterization of CDSs and Regulatory Regions of AsSWEET Genes Differentially Expressed in FBR-Sensitive and -Resistant Cultivars after F. proliferatum Infection

Considering the differential expression of AsSWEET3, AsSWEET9, AsSWEET11, and AsSWEET26 genes in cv. Sarmat and Strelets in response to F. proliferatum infection (Figure 8), we cloned and sequenced the CDSs and regulatory regions of these genes and compared them with those of cv. Ershuizao (used as reference).
The results indicated that each cloned CDS had 2–5 single nucleotide polymorphisms (SNPs) (Table 2). Fifteen SNPs were shared by cv. Sarmat and Strelets; among them, only one (c.293A>G in AsSWEET3) was non-synonymous and led to amino acid substitution p. K98R (Table 2). In cv. Sarmat, the ASWEET3 gene contained an additional non-synonymous SNP (c. 758T>C leading to p. V253A). The other cultivar-specific polymorphisms were found in AsSWEET11, which in cv. Sarmat contained a non-synonymous SNP c.734A>T (p. E245V) and in cv. Strelets—synonymous c. 69C>T and non-synonymous c. 761T>C (p. I254T) SNPs (Table 2).
Analysis of the regulatory regions (~1 kb, including promoter; ~0.7 kb in case of AsSWEET11) indicated that those of AsSWEET3 had a 340 bp deletion and 3 SNPs in both cv. Sarmat and Strelets; in addition, this gene had a 3 bp insertion in cv. Sarmat and carried 2 SNPs in cv. Strelets. In both cv. Sarmat and Strelets, the promoter regions of AsSWEET9 had a 2 bp deletion and 12 SNPs, those of AsSWEET11–2 SNPs, and those of AsSWEET26–8 SNPs. In addition, the AsSWEET26 promoter of cv. Sarmat carried four extra SNPs and a 10 bp deletion, whereas that of cv. Strelets had 10 extra SNPs (Table 2).
The identified polymorphisms led to changes in the composition of cis-regulatory elements (Table 3). Thus, the AsSWEET3 promoter in both cultivars lacked the F-box element and that in cv. Strelets contained the WRE3 element. In both cv. Sarmat and Strelets, the promoter of the AsSWEET9 gene acquired box 4 but lost several AE-boxes, that of AsSWEET11 lost a STRE, and that of AsSWEET26 acquired TGA, CGTCA, and P-box elements and extra MYC elements but lost some ARE and MYB elements; furthermore, in cv. Sarmat, the AsSWEET9 promoter lacked the AuxRR-core element.

3. Discussion

SWEET uniporters that mediate the bidirectional transfer of soluble sugars between different tissues are important not only for plant growth and development but also for the immune response [13,28,29,31,32].
Accumulating evidence indicates that during infection, plant pathogens selectively target SWEET transporters to access host carbohydrate reserves and obtain soluble sugars [39]. Thus, Xanthomonas spp. are known to upregulate the expression of specific SWEET genes; conversely, the blockage of SWEET activation can increase plant resistance to infection and reduce pathogen growth and virulence [39]. It is suggested that the suppression of SWEET genes in plants can reduce their sensitivity to infection and may potentially be employed as a strategy to breed resistant crop cultivars [31,32,39]. However, this approach is not entirely correct since symbiotic arbuscular mycorrhizal (AM) fungi, which can significantly enhance plant growth and adaptability by colonizing their roots, also obtain carbohydrates from the root cortex apoplast, including through the SWEET activities. For example, colonization of potato roots by the AM fungus Rhizophagus irregularis causes significant changes in the transcription of 22 out of 35 genes of the SWEET family [59]. Therefore, functional studies of the specific SWEET genes can help to determine the balanced effect on the transporter activity, which would contribute to plant protection, on the one hand, and, on the other hand, would not break the symbiotic potential of plant-microbial communities.
The present study was the first attempt to identify and characterize SWEET genes in the Allium genus in general and in garlic in particular. Overall, we found 27 SWEET genes in A. sativum cv. Ershuizao through genome-wide analysis (Table 1). Multiple SWEET genes are characteristic of many species, including agricultural crops; thus, there are 17 such genes in A. thaliana, 25 in Musa acuminate Colla, 29 in S. lycopersicum, and 108 in Triticum aestivum L. [60]. However, if in tomato, for example, the size of gene families can be regulated through sexual reproduction and introgressive hybridization [60,61,62], it is not the case in garlic, which is an asexually reproducing (apomictic) species whose genotypes presumably originate through mutations in a single clone, leading to significant phenotypic diversity [63]. It can be speculated that the high number of SWEET genes in garlic is a result of accelerated random mutagenesis and somaclonal variation observed especially in the genes related to the basic processes of plant development and adaptability [60,61,62]. Alternatively, it is possible that the SWEET gene family in A. sativum had evolved before this species lost the ability for sexual reproduction.
Our structural analysis revealed AsSWEET genes of all currently known SWEET clades (I–IV) (Figure 2, Figure 3 and Figure 4), suggesting functional conservation of the bidirectional transport of soluble sugars: hexoses (clades I and II), fructose (clade IV), and sucrose (clade III) [13,14,15].
The observed clustering of AsSWEET20–25 (clade IV) and AsSWEET18/19 and AsSWEET4–9 (clade III) on chromosomes 8, 7, and 2, respectively (Figure 1a) suggests their evolutionary origin through tandem duplication and points on the functional redundancy of paralogous transporters within these groups. Differences in the expression patterns of AsSWEET genes in the three clusters, including complete silencing of some members (Figure 6 and Figure 7), may reflect the neofunctionalization effect of duplicated genes due to mutations during adaptation to environmental changes. Such mutations can occur in the CDSs as well as in the regulatory sequences, as evidenced by the obvious differences in the patterns of cis-regulatory elements in the AsSWEET promoter regions (Figure 5).
The presence of hormone- and stress-responsive elements in AsSWEET promoters (Figure 5) (which is also characteristic of garlic PR genes [54,55,56]) is consistent with the involvement of SWEETs in hormone-mediated as well as in hormone-independent signaling pathways regulating plant responses to abiotic and biotic stresses, including Fusarium infection [28,29,35,36,37,55,56,57].
FBR of the bulbs and Fusarium wilt of the leaves can lead to significant (up to 60%) losses of the garlic crop [64,65,66,67,68,69]. F. proliferatum, one of the Fusarium species infecting garlic [57], is a common hemibiotrophic pathogen that utilizes plant sugars both in the biotrophic and necrotrophic phases [70]. Therefore, we hypothesized that this fungus could activate carbohydrate metabolism in garlic through the regulation of AsSWEET genes. Analysis of AsSWEET transcription in the roots of FBR-resistant and -susceptible garlic cultivars at the biotrophic (24 hpi) and pre-necrotrophic (96 hpi) phases of F. proliferatum infection revealed the upregulation of six genes, AsSWEET3, AsSWEET9, AsSWEET11, AsSWEET15, AsSWEET17, and AsSWEET26, in response to infection in both cultivars (Figure 8). Among these genes, AsSWEET3, AsSWEET9, and AsSWEET11 belong to clade III (Figure 4), the members of which are known to be induced in plants at the biotrophic phase of pathogen infection [39]. However, there was a significant difference in the activation level of AsSWEET3, AsSWEET9, and AsSWEET11 between FBR-resistant and -susceptible cultivars both at 24 and 96 hpi (Figure 8), which is consistent with the role of clade III SWEETs in response to Fusarium infection. Thus, the expression of these genes was much more significantly upregulated in the sensitive cv. Strelets than in the resistant cv. Sarmat at 24 hpi (Figure 8), suggesting pathogen-induced activation in order to access host sugars and promote colonization at the biotrophic stage. At the same time, AsSWEET3, AsSWEET9, and AsSWEET11 were much stronger upregulated in the resistant cv. Sarmat than in the susceptible cv. Strelets at the pre-necrotrophic (96 hpi) stage of infection (Figure 8). It is possible that the late-stage induction of AsSWEET3, AsSWEET9, and AsSWEET11 in the resistant cultivar could represent an attempt of the host plant to stimulate sugar outflow from the infected roots in order to restrict pathogen growth, which is in agreement with a decrease in fructose, glucose and sucrose levels in the roots of cv. Sarmat but not in those of cv. Strelets (Figure 9), as well as the fact that sugars act as signaling molecules and interact with the hormonal network that regulates the immune system of plants [71]. Differential expression patterns of SWEET genes have also been reported in Fusarium-resistant and -susceptible cultivars of watermelon [36]. Thus, our results, together with previous data, indicate distinct roles of individual SWEETs in plant resistance to fungal infection.
The three genes of clade I (AsSWEET15, AsSWEET17, and AsSWEET26) upregulated by F. proliferatum infection showed overall similar dynamics and degree of activation in both cultivars (Figure 8), suggesting that the regulatory pathways underlying AsSWEET15, AsSWEET17, and AsSWEET26 transcriptional induction in response to infection are not involved in the garlic resistance to Fusarium.
Despite the differential transcriptional response to the infection of AsSWEET3, AsSWEET9, AsSWEET11, and AsSWEET26, sequence alignment revealed a high degree of structural similarity in their cis-regulatory regions and protein products in FBR-susceptible and -resistant garlic cultivars (Table 2 and Table 3). This fact points to intricate molecular mechanisms controlling SWEET gene expression in garlic, which are likely genotype-dependent and defines the strength of garlic resistance to Fusarium infection.
Earlier studies of FBR-resistant and -susceptible garlic cultivars have identified a set of A. sativum PR genes encoding chitinases, glucanases, and thaumatin-like proteins, which also have infection stage-dependent activation patterns and could be involved in anti-fungal immune defense [55,56,57]. Together with these findings, our present results suggest that AsSWEET genes can be a part of a complex protection system evolved in garlic to resist biotic as well as abiotic stresses, which requires further investigation.

4. Materials and Methods

4.1. Identification and Structural Analysis of the A. sativum SWEET Genes

The search for the full-length SWEET genes was performed in the A. sativum cv. Ershuizao genome (PRJNA606385, assembly Garlic.V2.fa) according to the annotated transcriptome (PRJNA607255) (genome and transcriptome were prepared by Sun et al. (2020) [52]); only sequences with start and stop codons and two MtN3_slv domains (pfam03083) in the translated version were selected. Comparative gene and protein structural analyses were conducted with MEGA 7.0.26 [53]. The phylogenetic dendrogram was constructed based on protein sequences using MEGA 7.0.26 (Neighbor-Joining method); confidence for tree topologies was estimated by bootstrap values of 1000 replicates. Used in analysis A. thaliana and S. lycopersicum SWEET numbering is given according to [27] and [21,72], respectively.
The chromosomal localization map was drawn using MG2C v. 2.1 (http://mg2c.iask.in/mg2c_v2.1/; accessed on 26 January 2023). To predict exon–intron structures, AsSWEET genes, and their CDSs were analyzed using GSDS v2.0 [73]. Putative proteins were characterized by MW, pI, and GRAVY (ExPASy ProtParam; https://web.expasy.org/protparam/; accessed on 26 January 2023), conserved domains (NCBI-CDD, https://www.ncbi.nlm.nih.gov/cdd; accessed on 26 January 2023), consensuses (MEME 5.5.1, http://meme-suite.org/tools/meme; accessed on 26 January 2023), GO biological processes (PANNZER2; http://ekhidna2.biocenter.helsinki.fi/sanspanz/; accessed on 26 January 2023), and transmembrane helices (TMHMM-2.0; https://services.healthtech.dtu.dk/service.php?TMHMM-2.0; accessed on 26 January 2023).

4.2. In Silico mRNA Expression Analysis

AsSWEET gene expression in garlic tissues was analyzed based on A. sativum cv. Ershuizao RNA-seq dataset (ID: PRJNA607255), which was normalized as Fragments Per Kilobase of transcript per million mapped reads (FPKM) [52] and included data on the gene expression profiles in the roots, bulbs (8 developmental stages), leaves, pseudostems, buds, flowers, and sprouts. According to Sun et al. (2020) [52], buds were collected at the bud stage of garlic plants planted in October 2018; samples of roots, leaves, sprouts, pseudostems (212 days after sowing), and flowers (217 days after sowing) were collected in May 2019; samples of bulbs of eight growth stages were taken every five days from April 11 to May 16, 2019. Transcripts with an average FPKM value of ≥10 in at least one tissue type were selected and used for heatmap construction with Heatmapper [74].

4.3. Plant and Fungi Material and F. proliferatum Infection

FBR-resistant cv. Sarmat and FBR-susceptible cv. Strelets used in this study are winter garlic cultivars of Russian breeding. Bulbs from the 2022 harvest were kindly provided by the Federal Scientific Vegetable Center (Moscow region, Russia).
The F. proliferatum strain was originally isolated from the bulbs of garlic cv. Strelets was kindly provided by the Group of Experimental Mycology, Winogradsky Institute of Microbiology (Research Center of Biotechnology of the RAS, Moscow, Russia). According to the pathogenicity test, the first signs of the disease appear on the clove surface 5 days after infection [55].
F. proliferatum infection was performed as previously described [56]. Cloves were surface sterilized in 70% ethanol for 3 min, rinsed with sterile water, placed in Petri dishes with wet filter paper, and incubated at +25 °C in the dark for 72 h until active root growth was observed. Then, cloves were infected by soaking in F. proliferatum conidial suspension (~106 conidia mL−1) for 5 min, transferred to fresh Petri dishes, and incubated at +25 °C in the dark for 24 and 96 h (n = 3 cloves per each time point); uninfected cloves were used as control. The roots, pseudostems, and cloves were collected at each time point, frozen in liquid nitrogen, and stored at −80 °C.

4.4. RNA Extraction and qRT-PCR Analysis

Total RNA was extracted from garlic tissues (0.1 g of each) using the RNeasy Plant Mini Kit (QIAGEN, Hilden, Germany), cleaned from genomic DNA (RNase free DNase set; QIAGEN), qualified by gel electrophoresis, and used for first-strand cDNA synthesis (GoScript Reverse Transcription System; Promega, Madison, WI, USA) with an oligo-dT primer. RNA and cDNA concentrations were quantified by fluorimetry (Qubit® Fluorometer, Thermo Fisher Scientific, Waltham, MA, USA). qRT-PCR was performed in a CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) with 3.0 ng cDNA, SYBR Green RT-PCR mixture (Syntol, Moscow, Russia), and specific primers. Primers were designed based on A. sativum cv. Ershuizao transcriptomic data (PRJNA607255) (Table S2) as a result of comparative analysis of AsSWEET mRNAs; the most variable regions of each gene were used to select primers so that there was at least one intron between them. Further, manual revision of sequence polymorphisms and additional evaluation was performed using Primer3 (http://frodo.wi.mit.edu/primer3/, accessed on 15 October 2022). Primers were experimentally tested at three concentrations of cDNA (mix from various organs)—10, 1, and 0.1 ng per well in three technical replicates. Primer pairs with only one significant peak occurring in the melt curve above the instrument detection limit were selected; the efficiency of the validated pairs of primers was 95–110%, R2 = 0.943–0.999. Given the high identity (over 98%) of the AsSWEET18 and AsSWEET19 mRNAs, common primers were designed for these genes (Table S2). The following cycling conditions were used: initial denaturation at 95 °C for 5 min and 40 cycles of denaturation at 95 °C for 15 s, and annealing/extension at 60 °C for 40 s.
mRNA expression of AsSWEET genes was normalized to that of two reference genes, GAPDH and UBQ [56], which were previously used separately as a reference in onion (Allium cepa L.) and garlic [75,76]. We used the combination of GAPDH and UBQ based on studies showing that the combination of two stably expressed reference genes, rather than one, improves the accuracy of qPCR (for example, [77]); previously, we have already successfully applied this approach to the analysis of garlic gene expression [55,56,57]. The results were statistically analyzed with GraphPad Prism version 8 (GraphPad Software Inc., San Diego, CA, USA; https://www.graphpad.com/scientific-software/prism/ [accessed on 10 January 2023]). The data were expressed as the mean ± standard error (SE) based on three technical replicates of two biological replicates for each combination of cDNA and primer pairs. The unequal variance (Welch’s) t-test was applied to assess differences in gene expression; p < 0.01 was considered to indicate statistical significance.

4.5. Gene Amplification and Sequencing

To amplify the AsSWEET CDSs, gene-specific primers were designed based on A. sativum cv. Ershuizao transcriptomic data (NCBI project accession number: PRJNA607255) (Supplementary Table S2). PCR amplification was performed with 30 ng cDNA from the roots of each cultivar at the following conditions: initial denaturation at 95 °C for 5 min, 35 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 2 min, and final extension at 72 °C for 5 min. PCR products of the expected size were purified by using the QIAEX® II Gel Extraction kit (QIAGEN, Hilden, Germany), cloned in the pGEM®-T Easy vector (Promega, Madison, WI, USA), and sequenced (3–5 clones for each accession) on ABI Prism 3730 DNA Sequencer (Applied Biosystems, Waltham, MA, USA) using the same primers.

4.6. Promoter Analysis

The search for cis-elements in the promoters (~1.0 kb regions upstream of the initiation codon) was performed using the PlantCARE database of cis-regulatory elements, enhancers, and repressors; (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/; accessed on 26 January 2023). Promoter sequences and defining of hormone- and stress-responsive cis-elements are provided in Supplementary Tables S3 and S4, respectively.

4.7. Sucrose, Glucose, and Fructose Contents

About 0.2 g of freeze-dried ground roots was extracted twice with 200 µL of 80% methanol; the obtained extracts were pooled, evaporated, re-dissolved in 30% methanol to the final concentration of 50 mg fresh weight per 100 µL extract, and subjected to mass spectral analysis using ultra-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF-MS/MS) according to [https://lcms.cz/labrulez-bucket-strapi-h3hsga3/1866243_lcms_148_how_potato_fights_its_enemies_02_2019_ebook_rev_01_9d3990d6c4/1866243-lcms-148-how-potato-fights-its-enemies-02-2019-ebook-rev-01.pdf; accessed on 1 December 2022].

5. Conclusions

We identified and characterized 27 genes encoding sugar uniporters of the SWEET family in the A. sativum cv. Ershuizao genome and cloned AsSWEET3, AsSWEET9, AsSWEET11, and AsSWEET26 CDSs and promoter regions from FBR-resistant and -susceptible garlic cultivars. Structural analysis of AsSWEET genes indicated conserved functional involvement in the bidirectional transport of soluble sugars: hexoses (clade I AsSWEET1, AsSWEET15, AsSWEET17, AsSWEET26, and AsSWEET27 and clade II AsSWEET10, and AsSWEET12–14), fructose (clade IV AsSWEET20–25), and sucrose (clade III AsSWEET2–9, AsSWEET11, AsSWEET16, AsSWEET18, and AsSWEET19). The AsSWEET promoters contained hormone- and stress-related elements associated with the response to fungal pathogens. Comparative AsSWEET transcriptional profiling in the F. proliferatum-infected roots of FBR-resistant and -susceptible cultivars suggests that clade III AsSWEET3, AsSWEET9, and AsSWEET11 genes could be involved in the garlic response to Fusarium infection. Our results provide the first insights into the functions of SWEET genes in the plants of the Allium genus and may be used in breeding programs to increase the resistance of Allium crops to Fusarium infections.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms24087533/s1.

Author Contributions

Investigation, M.A.F. and O.K.A.; formal analysis: E.Z.K., A.V.S. and M.A.F.; writing, A.V.S., M.A.F. and E.Z.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Ministry of Science and Higher Education of the Russian Federation in accordance with agreement № 075-15-2022-318 on 20 April 2022 on providing a grant in the form of subsidies from the Federal budget of the Russian Federation. The grant was provided for state support for the creation and development of a World-class Scientific Center, “Agrotechnologies for the Future”.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

AsSWEET CDSs of A. sativum cv. Sarmat/Strelets are available at NCBI (https://www.ncbi.nlm.nih.gov/ accessed on 1 March 2023) (see Table 2).

Acknowledgments

We would like to thank Marina Chuenkova for English language editing. This work was performed using the experimental climate control facility at the Institute of Bioengineering (Research Center of Biotechnology, Russian Academy of Sciences).

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

References

  1. Liu, Y.-J.; Wang, G.-L.; Ma, J.; Xu, Z.-S.; Wang, F.; Xiong, A.-S. Transcript profiling of sucrose synthase genes involved in sucrose metabolism among four carrot (Daucus carota L.) cultivars reveals distinct patterns. BMC Plant Biol. 2018, 18, 8. [Google Scholar] [CrossRef]
  2. Duarte-Delgado, D.; Ñústez-López, C.E.; Narváez-Cuenca, C.E.; Restrepo-Sánchez, L.P.; Melo, S.E.; Sarmiento, F.; Kushalappa, A.C.; Mosquera-Vásquez, T. Natural variation of sucrose, glucose and fructose contents in Colombian genotypes of Solanum tuberosum Group Phureja at harvest. J. Sci. Food Agric. 2016, 96, 4288–4294. [Google Scholar] [CrossRef] [PubMed]
  3. Zhang, Z.; Zou, L.; Ren, C.; Ren, F.; Wang, Y.; Fan, P.; Li, S.; Liang, Z. VvSWEET10 mediates sugar accumulation in grapes. Genes 2019, 10, 255. [Google Scholar] [CrossRef] [PubMed]
  4. Ji, Y.; Nuñez Ocaña, D.; Choe, D.; Larsen, D.H.; Marcelis, L.F.M.; Heuvelink, E. Far-red radiation stimulates dry mass partitioning to fruits by increasing fruit sink strength in tomato. New Phytol. 2020, 228, 1914–1925. [Google Scholar] [CrossRef] [PubMed]
  5. Ruan, Y.L. Sucrose metabolism: Gateway to diverse carbon use and sugar signaling. Annu. Rev. Plant Biol. 2014, 65, 33–67. [Google Scholar] [CrossRef]
  6. Yoon, J.; Cho, L.H.; Tun, W.; Jeon, J.S.; An, G. Sucrose signaling in higher plants. Plant Sci. 2021, 302, 110703. [Google Scholar] [CrossRef]
  7. Sami, F.; Siddiqui, H.; Hayat, S. Interaction of glucose and phytohormone signaling in plants. Plant Physiol. Biochem. 2019, 135, 119–126. [Google Scholar] [CrossRef]
  8. Chen, L.Q.; Cheung, L.S.; Feng, L.; Tanner, W.; Frommer, W.B. Transport of sugars. Annu. Rev. Biochem. 2015, 84, 865–894. [Google Scholar] [CrossRef]
  9. Li, X.; Si, W.; Qin, Q.; Wu, H.; Jiang, H. Deciphering evolutionary dynamics of SWEET genes in diverse plant lineages. Sci. Rep. 2018, 8, 13440. [Google Scholar] [CrossRef]
  10. Jeena, G.S.; Kumar, S.; Shukla, R.K. Structure, evolution and diverse physiological roles of SWEET sugar transporters in plants. Plant Mol. Biol. 2019, 100, 351–365. [Google Scholar] [CrossRef]
  11. Julius, B.T.; Leach, K.A.; Tran, T.M.; Mertz, R.A.; Braun, D.M. Sugar transporters in plants: New insights and discoveries. Plant Cell Physiol. 2017, 58, 1442–1460. [Google Scholar] [CrossRef] [PubMed]
  12. Chen, L.Q. SWEET sugar transporters for phloem transport and pathogen nutrition. New Phytol. 2014, 201, 1150–1155. [Google Scholar] [CrossRef]
  13. Chen, L.-Q.; Qu, X.-Q.; Hou, B.-H.; Sosso, D.; Osorio, S.; Fernie, A.R.; Frommer, W.B. Sucrose efflux mediated by SWEET proteins as a key step for phloem transport. Science 2012, 335, 207–211. [Google Scholar] [CrossRef]
  14. Patil, G.; Valliyodan, B.; Deshmukh, R.; Prince, S.; Nicander, B.; Zhao, M.; Sonah, H.; Song, L.; Lin, L.; Chaudhary, J.; et al. Soybean (Glycine max) SWEET gene family: Insights through comparative genomics, transcriptome profiling and whole genome re-sequence analysis. BMC Genom. 2015, 16, 520. [Google Scholar] [CrossRef] [PubMed]
  15. Klemens, P.A.; Patzke, K.; Deitmer, J.; Spinner, L.; Le Hir, R.; Bellini, C.; Bedu, M.; Chardon, F.; Krapp, A.; Neuhaus, H.E. Overexpression of the vacuolar sugar carrier AtSWEET16 modifies germination, growth, and stress tolerance in Arabidopsis. Plant Physiol. 2013, 163, 1338–1352. [Google Scholar] [CrossRef]
  16. Chen, L.Q.; Hou, B.H.; Lalonde, S.; Takanaga, H.; Hartung, M.L.; Qu, X.Q.; Guo, W.J.; Kim, J.G.; Underwood, W.; Chaudhuri, B.; et al. Sugar transporters for intercellular exchange and nutrition of pathogens. Nature 2010, 468, 527–532. [Google Scholar] [CrossRef]
  17. Eom, J.S.; Chen, L.Q.; Sosso, D.; Julius, B.T.; Lin, I.W.; Qu, X.Q.; Braun, D.M.; Frommer, W.B. SWEETs, transporters for intracellular and intercellular sugar translocation. Curr. Opin. Plant Biol. 2015, 25, 53–62. [Google Scholar] [CrossRef]
  18. Isoda, R.; Palmai, Z.; Yoshinari, A.; Chen, L.Q.; Tama, F.; Frommer, W.B.; Nakamura, M. SWEET13 transport of sucrose, but not gibberellin, restores male fertility in Arabidopsis sweet13;14. Proc. Natl. Acad. Sci. USA 2022, 119, e2207558119. [Google Scholar] [CrossRef] [PubMed]
  19. Chen, H.Y.; Huh, J.H.; Yu, Y.C.; Ho, L.H.; Chen, L.Q.; Tholl, D.; Frommer, W.B.; Guo, W.J. The Arabidopsis vacuolar sugar transporter SWEET2 limits carbon sequestration from roots and restricts Pythium infection. Plant J. 2015, 83, 1046–1058. [Google Scholar] [CrossRef]
  20. Valifard, M.; Le Hir, R.; Müller, J.; Scheuring, D.; Neuhaus, H.E.; Pommerrenig, B. Vacuolar fructose transporter SWEET17 is critical for root development and drought tolerance. Plant Physiol. 2021, 187, 2716–2730. [Google Scholar] [CrossRef]
  21. Feng, C.Y.; Han, J.X.; Han, X.X.; Jiang, J. Genome-wide identification, phylogeny, and expression analysis of the SWEET gene family in tomato. Gene 2015, 573, 261–272. [Google Scholar] [CrossRef]
  22. Wang, J.; Yu, Y.C.; Li, Y.; Chen, L.Q. Hexose transporter SWEET5 confers galactose sensitivity to Arabidopsis pollen germination via a galactokinase. Plant Physiol. 2022, 189, 388–401. [Google Scholar] [CrossRef] [PubMed]
  23. Lin, I.; Sosso, D.; Chen, L.Q.; Gase, K.; Kim, S.G.; Kessler, D.; Klinkenberg, P.M.; Gorder, M.K.; Hou, B.H.; Qu, X.Q.; et al. Nectar secretion requires sucrose phosphate synthases and the sugar transporter SWEET9. Nature 2014, 508, 546–549. [Google Scholar] [CrossRef]
  24. Chen, L.Q.; Lin, I.W.; Qu, X.Q.; Sosso, D.; McFarlane, H.E.; Londoño, A.; Samuels, A.L.; Frommer, W.B. A cascade of sequentially expressed sucrose transporters in the seed coat and endosperm provides nutrition for the Arabidopsis embryo. Plant Cell 2015, 27, 607–619. [Google Scholar] [CrossRef]
  25. Shammai, A.; Petreikov, M.; Yeselson, Y.; Faigenboim, A.; Moy-Komemi, M.; Cohen, S.; Cohen, D.; Besaulov, E.; Efrati, A.; Houminer, N.; et al. Natural genetic variation for expression of a SWEET transporter among wild species of Solanum lycopersicum (tomato) determines the hexose composition of ripening tomato fruit. Plant J. 2018, 96, 343–357. [Google Scholar] [CrossRef] [PubMed]
  26. Levin, I.; Gilboa, N.; Yeselson, E.; Shen, S.; Schaffer, A.A. Fgr, a major locus that modulates fructose to glucose ratio in mature tomato fruit. Theor. Appl. Genet. 2000, 100, 256–262. [Google Scholar] [CrossRef]
  27. Ho, L.H.; Klemens, P.A.W.; Neuhaus, H.E.; Ko, H.Y.; Hsieh, S.Y.; Guo, W.J. SlSWEET1a is involved in glucose import to young leaves in tomato plants. J. Exp. Bot. 2019, 70, 3241–3254. [Google Scholar] [CrossRef]
  28. Antony, G.; Zhou, J.; Huang, S.; Li, T.; Liu, B.; White, F.; Yang, B. Rice xa13 recessive resistance to bacterial blight is defeated by induction of the disease susceptibility gene Os-11N3. Plant Cell 2010, 22, 3864–3876. [Google Scholar] [CrossRef]
  29. Chu, Z.; Yuan, M.; Yao, J.; Ge, X.; Yuan, B.; Xu, C.; Li, X.; Fu, B.; Li, Z.; Bennetzen, J.L.; et al. Promoter mutations of an essential gene for pollen development result in disease resistance in rice. Genes Dev. 2006, 20, 1250–1255. [Google Scholar] [CrossRef] [PubMed]
  30. Hu, B.; Wu, H.; Huang, W.; Song, J.; Zhou, Y.; Lin, Y. SWEET gene family in Medicago truncatula: Genome-wide identification, expression and substrate specificity analysis. Plants 2019, 8, 338. [Google Scholar] [CrossRef]
  31. Li, T.; Liu, B.; Spalding, M.H.; Weeks, D.P.; Yang, B. High-efficiency TALEN-based gene editing produces disease-resistant rice. Nat. Biotechnol. 2012, 30, 390–392. [Google Scholar] [CrossRef] [PubMed]
  32. Li, C.; Wei, J.; Lin, Y.; Chen, H. Gene silencing using the recessive rice bacterial blight resistance gene xa13 as a new paradigm in plant breeding. Plant Cell Rep. 2012, 31, 851–862. [Google Scholar] [CrossRef]
  33. Hennion, N.; Durand, M.; Vriet, C.; Doidy, J.; Maurousset, L.; Lemoine, R.; Pourtau, N. Sugars en route to the roots. Transport, metabolism and storage within plant roots and towards microorganisms of the rhizosphere. Physiol. Plant. 2019, 165, 44–57. [Google Scholar] [CrossRef]
  34. Gai, X.T.; Jiang, N.; Ma, J.; Wang, A.; Lu, C.; Xuan, Y.H.; Xia, Z.Y. NtSWEET1 promotes tobacco resistance to Fusarium oxysporum-induced root rot disease. Plant Signal. Behav. 2021, 16, 1970940. [Google Scholar] [CrossRef] [PubMed]
  35. Li, Y.; Wang, Y.; Zhang, H.; Zhang, Q.; Zhai, H.; Liu, Q.; He, S. The Plasma Membrane-Localized Sucrose Transporter IbSWEET10 Contributes to the Resistance of Sweet Potato to Fusarium oxysporum. Front. Plant Sci. 2017, 8, 197. [Google Scholar] [CrossRef] [PubMed]
  36. Xuan, C.; Lan, G.; Si, F.; Zeng, Z.; Wang, C.; Yadav, V.; Wei, C.; Zhang, X. Systematic genome-wide study and expression analysis of SWEET gene family: Sugar transporter family contributes to biotic and abiotic stimuli in Watermelon. Int. J. Mol. Sci. 2021, 22, 8407. [Google Scholar] [CrossRef]
  37. Tamayo, E.; Figueira-Galán, D.; Manck-Götzenberger, J.; Requena, N. Overexpression of the potato monosaccharide transporter StSWEET7a promotes root colonization by symbiotic and pathogenic fungi by increasing root sink strength. Front. Plant Sci. 2022, 13, 837231. [Google Scholar] [CrossRef]
  38. Cohn, M.; Bart, R.S.; Shybut, M.; Dahlbeck, D.; Gomez, M.; Morbitzer, R.; Hou, B.H.; Frommer, W.B.; Lahaye, T.; Staskawicz, B.J. Xanthomonas axonopodis virulence is promoted by a transcription activator-like effector-mediated induction of a SWEET sugar transporter in Cassava. Mol. Plant Microbe Interact. 2014, 27, 1186–1198. [Google Scholar] [CrossRef] [PubMed]
  39. Chandran, D. Co-option of developmentally regulated plant SWEET transporters for pathogen nutrition and abiotic stress tolerance. IUBMB Life 2015, 67, 461–471. [Google Scholar] [CrossRef]
  40. Chong, J.; Piron, M.C.; Meyer, S.; Merdinoglu, D.; Bertsch, C.; Mestre, P. The SWEET family of sugar transporters in grapevine: VvSWEET4 is involved in the interaction with Botrytis cinerea. J. Exp. Bot. 2014, 65, 6589–6601. [Google Scholar] [CrossRef]
  41. Ferrari, S.; Galletti, R.; Denoux, C.; De Lorenzo, G.; Ausubel, F.M.; Dewdney, J. Resistance to Botrytis cinerea induced in Arabidopsis by elicitors is independent of salicylic acid, ethylene, or jasmonate signaling but requires PHYTOALEXIN DEFICIENT3. Plant Physiol. 2007, 144, 367–379. [Google Scholar] [CrossRef] [PubMed]
  42. Netzel, M.E. Garlic: Much more than a common spice. Foods 2020, 9, 1544. [Google Scholar] [CrossRef] [PubMed]
  43. Stavělíková, H. Morphological characteristics of garlic (Allium sativum L.) genetic resources collection–Information. Hort. Sci. (Prague) 2008, 35, 130–135. [Google Scholar] [CrossRef]
  44. Gomez, C.D.; Aguilera, P.; Ortiz-Plata, A.; López, F.N.; Chánez-Cárdenas, M.E.; Flores-Alfaro, E.; Ruiz-Tachiquín, M.E.; Espinoza-Rojo, M. Aged garlic extract and S-allylcysteine increase the GLUT3 and GCLC expression levels in cerebral ischemia. Adv. Clin. Exp. Med. 2019, 28, 1609–1614. [Google Scholar] [CrossRef]
  45. Ban, J.O.; Lee, D.H.; Kim, E.J.; Kang, J.W.; Kim, M.S.; Cho, M.C.; Jeong, H.S.; Kim, J.W.; Yang, Y.; Hong, J.T.; et al. Antiobesity effects of a sulfur compound thiacremonone mediated via down-regulation of serum triglyceride and glucose levels and lipid accumulation in the liver of db/db mice. Phytother. Res. 2012, 26, 1265–1271. [Google Scholar] [CrossRef]
  46. Mylona, K.; Garcia-Cela, E.; Sulyok, M.; Medina, A.; Magan, N. Influence of two garlic-derived compounds, propyl propane thiosulfonate (PTS) and propyl propane thiosulfinate (PTSO), on growth and mycotoxin production by Fusarium species in vitro and in stored cereals. Toxins 2019, 11, 495. [Google Scholar] [CrossRef]
  47. Rizzalli, R.H.; Villalobos, F.J.; Orgaz, F. Radiation interception, radiation-use efficiency and dry matter partitioning in garlic (Allium sativum L.). Eur. J. Agron. 2002, 18, 33–43. [Google Scholar] [CrossRef]
  48. Baumgartner, S.; Dax, T.G.; Praznik, W.; Falk, H. Characterisation of the high-molecular weight fructan isolated from garlic (Allium sativum L.). Carbohydr. Res. 2000, 328, 177–183. [Google Scholar] [CrossRef]
  49. Guevara-Mashayekhi, K.; Mohammadi Chiane, S.; Mianabadi, M.; Ghaderifar, F.; Mousavizadeh, S.J. Change in carbohydrate and enzymes from harvest to sprouting in garlic. Food Sci. Nutr. 2015, 4, 370–376. [Google Scholar] [CrossRef]
  50. Figueroa, T.; López-Hernández, L.; Lopez, M.G.; Hurtado, M.D.; Vázquez-Barrios, M.E.; Guevara-Olvera, L.; Guevara González, R.G.; Rivera-Pastrana, D.M.; Torres-Robles, H.; Mercado-Silva, E.M. Conditioning garlic “seed” cloves at low temperature modifies plant growth, sugar, fructan content, and sucrose sucrose fructosyl transferase (1-SST) expression. Sci. Hortic. 2015, 189, 150–158. [Google Scholar] [CrossRef]
  51. Vinodh Kumar, P.N.; Mallikarjuna, M.G.; Jha, S.K.; Mahato, A.; Lal, S.K.; Yathish, K.R.; Lohithaswa, H.C.; Chinnusamy, V. Unravelling structural, functional, evolutionary and genetic basis of SWEET transporters regulating abiotic stress tolerance in maize. Int. J. Biol. Macromol. 2023, 229, 539–560. [Google Scholar] [CrossRef]
  52. Sun, X.; Zhu, S.; Li, N.; Cheng, Y.; Zhao, J.; Qiao, X.; Lu, L.; Liu, S.; Wang, Y.; Liu, C.; et al. A Chromosome-level genome assembly of garlic (Allium sativum) provides insights into genome evolution and allicin biosynthesis. Mol. Plant 2020, 13, 1328–1339. [Google Scholar] [CrossRef] [PubMed]
  53. Kumar, S.; Stecher, G.; Tamura, K. MEGA7: Molecular evolutionary genetics analysis version 7.0. molecular biology and evolution. Mol. Biol. Evol. 2016, 33, 1870–1874. [Google Scholar] [CrossRef] [PubMed]
  54. Zhu, J.; Zhou, L.; Li, T.; Ruan, Y.; Zhang, A.; Dong, X.; Zhu, Y.; Li, C.; Fan, J. Genome-wide investigation and characterization of sweet gene family with focus on their evolution and expression during hormone and abiotic stress response in maize. Genes 2022, 13, 1682. [Google Scholar] [CrossRef]
  55. Anisimova, O.K.; Shchennikova, A.V.; Kochieva, E.Z.; Filyushin, M.A. Pathogenesis-related genes of PR1, PR2, PR4, and PR5 families are involved in the response to Fusarium infection in garlic (Allium sativum L.). Int. J. Mol. Sci. 2021, 22, 6688. [Google Scholar] [CrossRef]
  56. Filyushin, M.A.; Anisimova, O.K.; Kochieva, E.Z.; Shchennikova, A.V. Genome-wide identification and expression of chitinase class I genes in garlic (Allium sativum L.) cultivars resistant and susceptible to Fusarium proliferatum. Plants 2021, 10, 720. [Google Scholar] [CrossRef]
  57. Anisimova, O.K.; Kochieva, E.Z.; Shchennikova, A.V.; Filyushin, M.A. Thaumatin-like protein (TLP) genes in garlic (Allium sativum L.): Genome-wide identification, characterization, and expression in response to Fusarium proliferatum infection. Plants 2022, 11, 748. [Google Scholar] [CrossRef]
  58. Bartholomew, E.S.; Black, K.; Feng, Z.; Liu, W.; Shan, N.; Zhang, X.; Wu, L.; Bailey, L.; Zhu, N.; Qi, C.; et al. Comprehensive analysis of the chitinase gene family in cucumber (Cucumis sativus L.): From gene identification and evolution to expression in response to Fusarium oxysporum. Int. J. Mol. Sci. 2019, 20, 5309. [Google Scholar] [CrossRef]
  59. Manck-Götzenberger, J.; Requena, N. Arbuscular mycorrhiza symbiosis induces a major transcriptional reprogramming of the potato SWEET sugar transporter family. Front. Plant Sci. 2016, 7, 487. [Google Scholar] [CrossRef] [PubMed]
  60. Gautam, T.; Saripalli, G.; Gahlaut, V.; Kumar, A.; Sharma, P.K.; Balyan, H.S.; Gupta, P.K. Further studies on sugar transporter (SWEET) genes in wheat (Triticum aestivum L.). Mol. Biol. Rep. 2019, 46, 2327–2353. [Google Scholar] [CrossRef] [PubMed]
  61. Labate, J.A.; Robertson, L.D. Evidence of cryptic introgression in tomato (Solanum lycopersicum L.) based on wild tomato species alleles. BMC Plant Biol. 2012, 12, 133. [Google Scholar] [CrossRef] [PubMed]
  62. Zhou, Y.; Zhao, X.; Li, Y.; Xu, J.; Bi, A.; Kang, L.; Xu, D.; Chen, H.; Wang, Y.; Wang, Y.G.; et al. Triticum population sequencing provides insights into wheat adaptation. Nat. Genet. 2020, 52, 1412–1422. [Google Scholar] [CrossRef]
  63. Kıraç, H.; Dalda Şekerci, A.; Coşkun, Ö.F.; Gülşen, O. Morphological and molecular characterization of garlic (Allium sativum L.) genotypes sampled from Turkey. Genet. Resour. Crop Evol. 2022, 69, 1833–1841. [Google Scholar] [CrossRef]
  64. Kalman, B.; Abraham, D.; Graph, S.; Perl-Treves, R.; Meller Harel, Y.; Degani, O. Isolation and identification of Fusarium spp., the causal agents of onion (Allium cepa) basal rot in northeastern Israel. Biology 2020, 9, 69. [Google Scholar] [CrossRef]
  65. Cramer, C.S. Breeding and genetics of Fusarium basal rot resistance in onion. Euphytica 2000, 115, 159–166. [Google Scholar] [CrossRef]
  66. Gálvez, L.; Urbaniak, M.; Waśkiewicz, A.; Stępień, Ł.; Palmero, D. Fusarium proliferatum—Causal agent of garlic bulb rot in Spain: Genetic variability and mycotoxin production. Food Microbiol. 2017, 67, 41–48. [Google Scholar] [CrossRef] [PubMed]
  67. Chand, S.K.; Nanda, S.; Mishra, R.; Joshi, R.K. Multiple garlic (Allium sativum L.) microRNAs regulate the immunity against the basal rot fungus Fusarium oxysporum f. sp. cepae. Plant Sci. 2017, 257, 9–21. [Google Scholar] [CrossRef]
  68. Leyronas, C.; Chrétien, P.L.; Troulet, C.; Duffaud, M.; Villeneuve, F.; Morris, C.E.; Hunyadi, H. First report of Fusarium proliferatum causing garlic clove rot in France. Plant Dis. 2018, 102, 2658. [Google Scholar] [CrossRef]
  69. Tonti, S.; Prà, M.D.; Nipoti, P.; Prodi, A.; Alberti, I. First report of Fusarium proliferatum causing rot of stored garlic bulbs (Allium sativum L.) in Italy. J. Phytopathol. 2012, 160, 761–763. [Google Scholar] [CrossRef]
  70. Lalak-Kańczugowska, J.; Witaszak, N.; Waśkiewicz, A.; Bocianowski, J.; Stępień, Ł. Plant metabolites affect Fusarium proliferatum metabolism and in vitro fumonisin biosynthesis. Int. J. Mol. Sci. 2023, 24, 3002. [Google Scholar] [CrossRef]
  71. Wei, Y.; Xiao, D.; Zhang, C.; Hou, X. The expanded SWEET gene family following whole genome triplication in Brassica rapa. Genes 2019, 10, 722. [Google Scholar] [CrossRef]
  72. Morkunas, I.; Ratajczak, L. The role of sugar signaling in plant defense responses against fungal pathogens. Acta Physiol. Plant. 2014, 36, 1607–1619. [Google Scholar] [CrossRef]
  73. Hu, B.; Jin, J.; Guo, A.Y.; Zhang, H.; Luo, J.; Gao, G. GSDS 2.0: An Upgraded Gene Feature Visualization Server. Bioinformatics 2015, 31, 1296–1297. [Google Scholar] [CrossRef] [PubMed]
  74. Babicki, S.; Arndt, D.; Marcu, A.; Liang, Y.; Grant, J.R.; Maciejewski, A.; Wishart, D.S. Heatmapper: Web-enabled heat mapping for all. Nucl. Acids Res. 2016, 44, W147–W153. [Google Scholar] [CrossRef]
  75. Schwinn, K.E.; Ngo, H.; Kenel, F.; Brummell, D.A.; Albert, N.W.; McCallum, J.A.; Pither-Joyce, M.; Crowhurst, R.N.; Eady, C.; Davies, K.M. The onion (Allium cepa L.) R2R3-MYB gene MYB1 regulates anthocyanin biosynthesis. Front. Plant Sci. 2016, 7, 1865. [Google Scholar] [CrossRef] [PubMed]
  76. Liu, M.; Wu, Z.; Jiang, F. Selection and validation of garlic reference genes for quantitative real-time PCR normalization. Plant Cell Tissue Organ Cult. 2015, 122, 435–444. [Google Scholar] [CrossRef]
  77. Chen, M.; Wang, Q.; Li, Y.; Gao, L.; Lv, F.; Yang, R.; Wang, P. Candidate reference genes for quantitative gene expression analysis in Lagerstroemia indica. Mol. Biol. Rep. 2021, 48, 1677–1685. [Google Scholar] [CrossRef] [PubMed]
Ijms 24 07533 g001 550
Figure 1. Location and structure of the identified AsSWEET genes. (a) Chromosomal distribution of the AsSWEET1–26 genes; AsSWEET27 is absent as a scaffold-localized gene. The scale on the left indicates chromosome size according to the A. sativum cv. Ershuizao genome (PRJNA606385, assembly Garlic.V2.fa) [52]; chr, chromosome; Mb, megabase. (b) Predicted exon–intron structures of the AsSWEET1–27 genes.
Figure 1. Location and structure of the identified AsSWEET genes. (a) Chromosomal distribution of the AsSWEET1–26 genes; AsSWEET27 is absent as a scaffold-localized gene. The scale on the left indicates chromosome size according to the A. sativum cv. Ershuizao genome (PRJNA606385, assembly Garlic.V2.fa) [52]; chr, chromosome; Mb, megabase. (b) Predicted exon–intron structures of the AsSWEET1–27 genes.
Ijms 24 07533 g001
Ijms 24 07533 g002 550
Figure 2. Structure of the predicted AsSWEET proteins. (a) Sequence alignment. Gray-shaded regions are 70–100% identical; red blocks on the top indicate positions of transmembrane (TM) helices 1–7. (b) Consensus sequences of TM helices 1–7.
Figure 2. Structure of the predicted AsSWEET proteins. (a) Sequence alignment. Gray-shaded regions are 70–100% identical; red blocks on the top indicate positions of transmembrane (TM) helices 1–7. (b) Consensus sequences of TM helices 1–7.
Ijms 24 07533 g002
Ijms 24 07533 g003 550
Figure 3. Phylogenetic analysis of the AsSWEET proteins. (a) The unrooted dendrogram based on amino acid sequences was constructed using the neighbor-joining method in MEGA7.0.26 [53]. Percentages of replicate trees in which the associated sequences clustered together in the bootstrap test (1000 replicates) are shown next to the branches. (b) The distribution of conserved motifs was revealed using MEME 5.4.1. The length of each box is proportional to the size of the motif.
Figure 3. Phylogenetic analysis of the AsSWEET proteins. (a) The unrooted dendrogram based on amino acid sequences was constructed using the neighbor-joining method in MEGA7.0.26 [53]. Percentages of replicate trees in which the associated sequences clustered together in the bootstrap test (1000 replicates) are shown next to the branches. (b) The distribution of conserved motifs was revealed using MEME 5.4.1. The length of each box is proportional to the size of the motif.
Ijms 24 07533 g003
Ijms 24 07533 g004 550
Figure 4. Phylogenetic relationships of SWEET proteins from A. sativum (red) and A. thaliana (black). Analysis was performed using the neighbor-joining method in MEGA7.0.26 [53]. Percentages of replicate trees in which the associated sequences clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Clades I–IV refer to SWEET clades previously identified in A. thaliana [16]. The numbering of maize genes is given in accordance with [51].
Figure 4. Phylogenetic relationships of SWEET proteins from A. sativum (red) and A. thaliana (black). Analysis was performed using the neighbor-joining method in MEGA7.0.26 [53]. Percentages of replicate trees in which the associated sequences clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Clades I–IV refer to SWEET clades previously identified in A. thaliana [16]. The numbering of maize genes is given in accordance with [51].
Ijms 24 07533 g004
Ijms 24 07533 g005 550
Figure 5. Hormone- and stress-related cis-acting elements found in the promoter regions of AsSWEET genes. MeJA, methyl jasmonate. The color scheme (from pale to dark) corresponds to the numbers of cis˗elements (from low to high).
Figure 5. Hormone- and stress-related cis-acting elements found in the promoter regions of AsSWEET genes. MeJA, methyl jasmonate. The color scheme (from pale to dark) corresponds to the numbers of cis˗elements (from low to high).
Ijms 24 07533 g005
Ijms 24 07533 g006 550
Figure 6. Heatmap of AsSWEET expression in A. sativum cv. Ershuizao (PRJNA607255). AsSWEET mRNA levels were analyzed in the roots, bulbs (stages 1–8 corresponding to 192-, 197-, 202-, 207-, 212-, 217-, 222-, and 227-day-old bulbs, respectively), leaves, pseudostems (ps.stem), buds, flowers, and sprouts. The color scheme indicates gene expression gradient from low (red) to high (green).
Figure 6. Heatmap of AsSWEET expression in A. sativum cv. Ershuizao (PRJNA607255). AsSWEET mRNA levels were analyzed in the roots, bulbs (stages 1–8 corresponding to 192-, 197-, 202-, 207-, 212-, 217-, 222-, and 227-day-old bulbs, respectively), leaves, pseudostems (ps.stem), buds, flowers, and sprouts. The color scheme indicates gene expression gradient from low (red) to high (green).
Ijms 24 07533 g006
Ijms 24 07533 g007 550
Figure 7. Transcription of the selected AsSWEET genes in A. sativum cv. Sarmat tissues. “AsSWEET18–19” means the sum expression of AsSWEET18 and AsSWEET19 genes; the high identity (over 98%) of their mRNAs forced the design of common primers. The data were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and ubiquitin (UBQ) mRNA levels; a–h p < 0.01 indicates significant differences between tissues; ps.stems, pseudostems.
Figure 7. Transcription of the selected AsSWEET genes in A. sativum cv. Sarmat tissues. “AsSWEET18–19” means the sum expression of AsSWEET18 and AsSWEET19 genes; the high identity (over 98%) of their mRNAs forced the design of common primers. The data were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and ubiquitin (UBQ) mRNA levels; a–h p < 0.01 indicates significant differences between tissues; ps.stems, pseudostems.
Ijms 24 07533 g007
Ijms 24 07533 g008 550
Figure 8. Expression of the selected AsSWEET genes in the roots of A. sativum FBR-resistant cv. Sarmat and FBR-susceptible Strelets in response to F. proliferatum infection. The plants were incubated with F. proliferatum conidia and analyzed for the transcription of the indicated genes at 24 and 96 h post-inoculation (hpi). The data were normalized to GAPDH and UBQ mRNA levels and presented as fold change (mean ± SE) of control (24 h in cv. Sarmat taken as 1); * p < 0.01 compared to uninfected control.
Figure 8. Expression of the selected AsSWEET genes in the roots of A. sativum FBR-resistant cv. Sarmat and FBR-susceptible Strelets in response to F. proliferatum infection. The plants were incubated with F. proliferatum conidia and analyzed for the transcription of the indicated genes at 24 and 96 h post-inoculation (hpi). The data were normalized to GAPDH and UBQ mRNA levels and presented as fold change (mean ± SE) of control (24 h in cv. Sarmat taken as 1); * p < 0.01 compared to uninfected control.
Ijms 24 07533 g008
Ijms 24 07533 g009 550
Figure 9. Changes in glucose, fructose, and sucrose contents in the roots of FBR-resistant cv. Sarmat and FBR-susceptible cv. Strelets in response to F. proliferatum infection. The plants were incubated with F. proliferatum conidia and analyzed for sugar contents at 24 and 96 h post-inoculation (hpi). The data are presented as fold change (mean ± SE) of control (24 h in cv. Sarmat taken as 1); * p < 0.01 compared to uninfected control.
Figure 9. Changes in glucose, fructose, and sucrose contents in the roots of FBR-resistant cv. Sarmat and FBR-susceptible cv. Strelets in response to F. proliferatum infection. The plants were incubated with F. proliferatum conidia and analyzed for sugar contents at 24 and 96 h post-inoculation (hpi). The data are presented as fold change (mean ± SE) of control (24 h in cv. Sarmat taken as 1); * p < 0.01 compared to uninfected control.
Ijms 24 07533 g009
Table
Table 1. Characteristics of the SWEET genes identified in the genome of A. sativum cv. Ershuizao.
Table 1. Characteristics of the SWEET genes identified in the genome of A. sativum cv. Ershuizao.
GeneGene/Transcript ID [31]LocalizationLength, bpNumber
of Exons		CDS, bpProtein, aaMW, kDapIGRAVYMtN3_slv DomainTM Helix
AsSWEET1Asa1G04426.1/Asa2G00182.1ch1: 1194647983–1194654538 (−)6556769923225.9989.180.60715–99, 138–2237
AsSWEET2Asa2G01431.1/Asa3G01319.1ch2: 399216486–399218187 (+)1702574124627.6478.950.62812–89, 123–2057
AsSWEET3Asa2G02061.1/Asa3G02137.1ch2: 572338389–572339990 (−)1602683427731.0339.430.66612–99, 133–2157
AsSWEET4Asa2G05047.1/Asa3G05154.1ch2: 1381249160–1381250776 (−)1617679226329.7489.120.75912–99, 133–2157
AsSWEET5Asa2G05048.1/Asa3G05155.1ch2: 1382002246–1382003909 (+)1664677425729.0449.370.74812–99, 133–2167
AsSWEET6Asa2G05049.1/Asa3G05156.1ch2: 1382472847–1382474148 (+)1302582527430.7118.550.60412–98, 133–2157
AsSWEET7Asa2G05056.1/Asa3G05153.1ch2: 1383831994–1383833643 (+)1650679226329.7549.200.69812–99, 133–2157
AsSWEET8Asa2G05057.1/Asa3G05172.1ch2: 1383951410–1383953045 (+)1636679226329.7509.230.74912–99, 133–2157
AsSWEET9Asa2G05060.1/Asa3G05167.1ch2: 1384535061–1384536711 (+)1651678926229.5449.260.71412–99, 133–2157
AsSWEET10Asa2G07032.1/Asa3G07188.1ch2: 1914150050–1914153509 (+)3460574424727.0989.350.97211–98, 134–2187
AsSWEET11Asa3G02607.1/Asa8G00525.1ch3: 721990358–721991933 (+)1576681927230.4569.300.69612–99, 133–2157
AsSWEET12Asa3G03715.1/Asa8G02144.1ch3: 1020592700–1020594065 (−)1366576225328.0128.890.86011–94, 132–2187
AsSWEET13Asa5G00452.1/Asa6G00626.1ch5: 108642296–108644275 (−)1980365421723.9919.360.8351–65, 103–1896
AsSWEET14Asa5G01067.1/Asa6G01212.1ch5: 246733367–246737173 (−)38076100233337.1859.750.203116–183, 220–3016
AsSWEET15Asa5G05357.1/Asa5G00822.1ch5: 1446881843–1446885138 (−)3296581327029.2399.360.55240–122, 159–2416
AsSWEET16Asa6G00924.1/Asa6G07296.1ch6: 237675722–237676674 (+)953469923225.9615.840.6131–42, 76–1625
AsSWEET17Asa7G01759.1/Asa1G02255.1ch7: 480861997–480863949 (+)1953673524427.2819.280.6447–95, 129–2147
AsSWEET18Asa7G03377.1/Asa1G00572.1ch7: 914393920–914395735 (−)1816677725829.0409.070.79612–99, 133–2147
AsSWEET19Asa7G03379.1/Asa1G00574.1ch7: 914587432–914589248 (+)1817677725829.0409.070.79612–99, 133–2157
AsSWEET20Asa8G00607.1/Asa6G05670.1ch8: 193855534–193857365 (+)1832686428731.8529.540.5156–93, 129–2127
AsSWEET21Asa8G00611.1/Asa7G00384.1ch8: 194959619–194961439 (+)1821684928231.36910.010.6126–93, 129–2127
AsSWEET22Asa8G00624.1/Asa7G00398.1ch8: 197356527–197358249 (−)1722670523425.8329.680.7656–93, 129–2107
AsSWEET23Asa8G00626.1/Asa7G00406.1ch8: 197411881–197415626 (−)3746793631134.0778.870.7976–91, 127–2086
AsSWEET24Asa8G00629.1/Asa8G00420.1ch8: 198119328–198122294 (−)2967684027930.6966.820.77825–111, 157–2336
AsSWEET25Asa8G00631.1/Asa7G00403.1ch8: 198345649–198347027 (+)1379670523425.9079.580.7956–93, 129–2137
AsSWEET26Asa8G02736.1/Asa7G02662.1ch8: 756634184–756635906 (−)1723671123626.3808.790.85219–105, 139–2247
AsSWEET27Asa0G02772.1/Asa8G01762.1scaffold24049: 67334–68424 (−)1091572023926.7618.190.74121–102, 141–2257
MW, molecular weight; pI, isoelectric point; GRAVY, grand average hydropathy; CDS, coding sequence; MtN3_slv, superfamily of sugar efflux transporters for intercellular exchange; TM, transmembrane.
Table
Table 2. Polymorphisms in the AsSWEET coding sequences and regulatory regions in cv. Sarmat and Strelets compared to cv. Ershuizao.
Table 2. Polymorphisms in the AsSWEET coding sequences and regulatory regions in cv. Sarmat and Strelets compared to cv. Ershuizao.
GeneNCBI ID (Sarmat/Strelets)SNP (aa Substitution) in CDSSNPs and Indels in Regulatory Region
cv. Sarmatcv. Streletscv. Sarmatcv. Strelets
AsSWEET3OQ607029/OQ607030c. 282T>C, c. 293A>G (p. K98R), c. 351G>A, c. 387T>C, c. 711T>C-768A>T, -769A>G, -778C>T, del. ˗239.. ˗579 (340 bp), insertion 3 bp (˗704)˗188 T>A, ˗768A>T, ˗769A>G, ˗778C>T, ˗832T>C, del. ˗239.. ˗579 (340 bp)
c. 758T>C (p. V253A)
AsSWEET9OQ607031/OQ607032c. 153C>T, c. 336A>T, c. 582G>A˗307G>A, ˗411C>T, ˗412T>C, ˗419A>G, ˗429C>T, ˗450T>G, ˗454A>G, ˗456C>G, ˗459A>T, ˗475A>G, ˗910C>T, del. ˗595.. ˗596 (2 bp)˗307G>A, ˗411C>T, ˗412T>C, ˗419A>G, ˗429C>T, ˗450T>G, ˗454A>G, ˗456C>G, ˗459A>T, ˗475A>G, ˗910C>T, del. ˗595.. ˗596 (2 bp)
AsSWEET11OQ607033/OQ607034c. 72C>T, c. 75T>C, c. 324G>A, c. 813C>A, c. 734A>T˗501C>T, ˗551T>C˗501C>T, ˗551T>C
c. 734A>T (p. E245V)c. 69C>T, c. 761T>C (p. I254T)
AsSWEET26OQ607035/OQ607036c. 204C>A, c. 534T>C˗225G>A, ˗291T>A, ˗515T>G, ˗528C>T, ˗564G>A, ˗578A>G, ˗596T>G, ˗671G>T, ˗673T>C, ˗791T>A, ˗849T>C, ˗856T>C, del. ˗624.. ˗633 (10 bp)˗222T>C, ˗259G>A, ˗355A>G, ˗515T>G, ˗516T>C, ˗533T>A, ˗539T>G, ˗578A>G, ˗591G>A, ˗596T>G, ˗604A>C, ˗653A>G, ˗667T>C, ˗671G>T, ˗673T>C, ˗791T>A, ˗849T>C, ˗856T>C
Note: Non-synonymous SNPs in CDS and corresponding amino acid substitutions, as well as cultivar-specific polymorphisms in gene regulatory regions are marked in bold.
Table
Table 3. Hormone- and stress-related cis-acting elements found in the upstream region of AsSWEET3, 9, 11, and 26 genes.
Table 3. Hormone- and stress-related cis-acting elements found in the upstream region of AsSWEET3, 9, 11, and 26 genes.
ElementsAsSWEET3AsSWEET9AsSWEET11AsSWEET26
ErshuizaoSarmatStreletsErshuizaoSarmatStreletsErshuizaoSarmatStreletsErshuizaoSarmatStrelets
Hormone-relatedABRE 111
AuxRR-core 1 1
TGA-element 11
CGTCA-motif111111 11
TCA-element222111
ERE111
P-box 11
Stress-relatedARE111444 211
STRE1 211111
LTR 111
WRE3 1 111
WUN-motif 111
GC-motif 222
OtherAAGAA-motif111111111111
Box 4111 11 111
F-box1
AE-box 322111
CAT-box 111
G-box 111 222
ATC-motif 111
TCT-motif
Circadian 111
AT-rich element 111
CTAG-motif 111
O2-site 111
Box S 111
MYC333 333122
MYB 222 312
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Filyushin, M.A.; Anisimova, O.K.; Shchennikova, A.V.; Kochieva, E.Z. Genome-Wide Identification, Expression, and Response to Fusarium Infection of the SWEET Gene Family in Garlic (Allium sativum L.). Int. J. Mol. Sci. 2023, 24, 7533. https://doi.org/10.3390/ijms24087533

AMA Style

Filyushin MA, Anisimova OK, Shchennikova AV, Kochieva EZ. Genome-Wide Identification, Expression, and Response to Fusarium Infection of the SWEET Gene Family in Garlic (Allium sativum L.). International Journal of Molecular Sciences. 2023; 24(8):7533. https://doi.org/10.3390/ijms24087533

Chicago/Turabian Style

Filyushin, Mikhail A., Olga K. Anisimova, Anna V. Shchennikova, and Elena Z. Kochieva. 2023. "Genome-Wide Identification, Expression, and Response to Fusarium Infection of the SWEET Gene Family in Garlic (Allium sativum L.)" International Journal of Molecular Sciences 24, no. 8: 7533. https://doi.org/10.3390/ijms24087533

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop