Expression characterization and cross-species complementation uncover the functional conservation of YABBY genes for leaf abaxial polarity and carpel polarity establishment in Saccharum spontaneum
BMC Plant Biology volume 22, Article number: 124 (2022)
Cell polarity establishment and maintenance is indispensable for plant growth and development. In plants, the YABBY transcription factor family has a distinct role in leaf asymmetric polarity establishment and lateral organ initiation. However, for the important sugar crop Saccharum, little information on YABBY genes is available.
In this study, a total of 20 sequences for 7 SsYABBY genes were identified in the sugarcane genome, designated as SsYABBY1-7 based on their chromosome locations, and characterized by phylogenetic analysis. We provided a high-resolution map of SsYABBYs’ global expression dynamics during vegetative and reproductive organ morphogenesis and revealed that SsYABBY3/4/5 are predominately expressed at the seedling stage of stem and leaf basal zone; SsYABBY2/5/7 are highly expressed in ovules. Besides, cross-species overexpression and/or complementation verified the conserved function of SsYABBY2 in establishing leaf adaxial-abaxial polarity and ovules development. We found that the SsYABBY2 could successfully rescue the leaves curling, carpel dehiscence, and ovule abortion defects in Arabidopsis crc mutant.
Collectively, our study demonstrates that SsYABBY genes retained a conserved function in establishing and preserving leaf adaxial-abaxial polarity and lateral organ development during evolution.
Establishing cell polarity, asymmetric division, and determining cell fates are essential phases in organ formation and development [1,2,3]. Polarity establishment and maintenance is a result of polarity formation initiated by a polarizing signal . For example, the HD-ZIP III REVOLUTA (REV) and KANADI (KAN1) regulate leaf abaxial-adaxial polarity in Arabidopsis . Auxin Response Factors (ARF3 and ARF4) and miR166, together with KANADI and YABBY genes, control the abaxial cell fate identity in Arabidopsis [5,6,7]. Previous studies demonstrated that YABBY transcription factors are essential for polarity establishment and maintenance [8, 9]. YABBY proteins contain a C2C2 domain and a YABBY domain [10,11,12], and are classified into five different groups (FIL/YAB3, YAB2, YAB5, INO, and CRC subgroups) in several plant species .
In Arabidopsis, FIL/YAB3, YAB2, and YAB5 redundantly regulate lateral organs development . The triple mutant yab135 (fil-8 yab3-2 yab5-1) and quadruple mutant yab1235 (fil-8 yab2-1 yab3-2 yab5-1) lacked apical dominance, and loss of lamina expansion and polarity [13, 14]. In rice, the FIL ortholog TONGARI-BOUSHI (TOB1, TOB2, and TOB3) also showed conserved functions in flower meristems and lateral organ primordia . INO controls the outer ovule integument development in Arabidopsis. The ino-1 mutant exhibits the absence of the outer integument and the typical hoodlike structure characteristic of wild-type ovules, suggesting that INO participates in the polar determination of abaxial identity in the ovule [11, 12, 16]. CRC is involved in establishing carpels polarity and nectary specification in Arabidopsis . The CRC ortholog in rice, DL (DROOPING LEAF) mutation causes carpels completely transformed into stamens . A similar phenotype of carpel morphogenesis was also observed in the ortholog of CRC in peas [18, 19]. While in maize, the CRC homolog gene DRL1 (Drooping Leaf1) expressed in incipient and emergent leaf primordia functions modulating plant architecture [20,21,22]. In tomatoes, SlYABBY2b regulates fruit size by controlling carpel number during flowering and fruit development [23, 24]. Additionally, AaYABBY5 promotes artemisinin biosynthesis by increasing the expression of artemisinin biosynthesis genes (ADS, CYP71, AV1, DBR2 and ALDH1) in Artemisia annua .
Sugarcane is an economically important Poaceae family crop that produces around 80 % of the world's sucrose and has a market worth of approximately $150 billion/year . Sugarcane cultivars are mainly hybrids derived from its progenitor species, S. officinarum and S. spontanuem [27, 28]. Sexual propagation is based on the normal growth of male and female gametophytes, which could considerably improve sugarcane quality and heterogeneity of generations. Due to reproductive organ degeneration, little progress has been achieved in sugarcane germplasm improvement by sexual propagation. YABBY genes have a wide range of roles in shoot apical and floral meristems; however, it is unclear how YABBY proteins operate in reproductive organs and leaf development in sugarcane.
This study performed the genomic analysis of gene phylogeny, gene structure, and expression patterns of YABBY genes during sugarcane leaf and ovule development. We have provided comprehensive information on the sugarcane YABBY genes and determined the critical role of SsYABBY2 in leaf and ovule development. Our findings imply that sugarcane YABBYs control leaf polarity development and may also participate in ovule development.
Identification and characterization of YABBY genes in S. spontaneum
A total of 27 candidate YABBY gene sequences were identified using HMM search (PF04690) in the sugarcane genome. The SMART and Pfam programs were further used to check the accuracy of SsYABBY member sequences, and 7 sequences that lack a complete YABBY domain were removed. Finally, 20 SsYABBY genes, including their alleles, were selected for detailed analysis. According to their chromosomal positions, we designated these genes as SsYABBY1-SsYABBY7. The detailed information of SsYABBY proteins is listed in Table 1, including gene accession number, chromosomal position, protein length, MW, pI, and numbers of exons. The length of putative SsYABBYs ranged from 333 (SsYABBY3-2) to 1467 (SsYABBY7-2) amino acids with the MW ranging from 12476.3 Da to 51860.6 Da, whereas the pI of SsYABBYs ranged from 7.15 (SsYABBY7-5) to 11.08 (SsYABBY6) (Table 1).
The characteristics of SsYABBY proteins were investigated using 20 SsYABBY protein sequences and aligning them to deduce their domains. Like other YABBY proteins found in plants, all SsYABBYs contain two conserved DNA-binding domains: a C2C2 zinc finger domain and a YABBY domain (Fig. 1A). SsYABBY3-2 and SsYABBY3-3 have incomplete C2C2 domains. SsYABBY6 shows more variability in C2C2 and YABBY domains, indicating its functional diversity (Fig. 1A). A phylogenetic tree of SsYABBYs further demonstrates the conservation of SsYABBYs amino acids and divided them into four subfamilies (FIL/YAB3, YAB2, CRC, and INO subfamilies). These subfamilies share a similar exon-intron gene structure. For example, the YAB2 subfamily possesses six exons with five introns, while FIL/YAB3 subfamily has seven exons with six introns (Fig. 1B).
Phylogenetic analysis, gene duplication, and synteny analysis
A comprehensive phylogenetic tree was constructed using the maximum-likelihood (ML) method with 20 alleles of SsYABBYs and 29 YABBYs from monocots and dicots to investigate the evolutionary relationship between sugarcane and other plants. The phylogenetic tree displayed that the SsYABBY genes could be classified into four clades: FIL/YAB3 clade, YAB2 clade, YAB5 clade, CRC clade, and INO clade (Fig. 2). The SsYABBY genes, as expectedly, were clustered with the genes of S. bicolor and O. sativa, indicating a closer relationship to monocotyledon. The FIL/YAB3 clade contained the most YABBY members (19), followed by the YAB2 clade (18), CRC clade (7), INO clade (5), and YAB5 clade (2). The INO clade had only one gene for each species, and a similar event was also observed in CRC clade except for SsYABBY2 alleles. This result indicated that these YABBY members might play similar biological functions. Surprisingly, no SsYABBY genes or monocotyledon YABBY genes belonged to the YAB5 clade. In contrast, the FIL/YAB3 clade and YAB2 clade contained more YABBY genes, showing an obvious gene expansion (Fig. 2). These results indicated that YABBY genes gained functional diversity during their species evolution. Also, 20 syntenic gene pairs were identified by MCScanX software, with 13 allele pairs and 7 nonallelic pairs (Fig. 3A; Table S1). We found only one tandem duplication (SsYABBY3-1/SsYABBY3-2), indicating that segmental duplication is the most common method for the SsYABBY gene expansion. The Ka/Ks ratios were calculated to estimate the selection pressure of these homologous gene pairs to better comprehend the evolutionary force of SsYABBYs. The results showed that Ka/Ks ratios of all SsYABBY homologous genes were less than 1 (Table S1), indicating that SsYABBY genes might experience strong purifying selective pressure during their evolution.
To better understand the evolutionary mechanism of SsYABBY genes, the comparative syntenic blocks were constructed between S. spontaneum and two monocotyledons S. bicolor and O. sativa. A total of 19 syntenic orthologous gene pairs were identified between S. spontaneum and O. sativa (Fig. 3B; Table S2), showing multiple SsYABBY genes matched one OsYABBY gene. For S. spontaneum and its most relative S. bicolor, 12 syntenic orthologous gene pairs were found offering two or three SsYABBYs syntenic with one SbYABBY (Fig. 3B; Table S3).
Subcellular localization analysis of SsYABBY proteins
To investigate the molecular characteristics of SsYABBYs, four representative SsYABBY genes (SsYABBY1, SsYABBY2, SsYABBY5, and SsYABBY6) from each subfamily were selected for further subcellular localization analysis based on their phylogenetic relationship. As expected, the GFP signals of SsYABBY1/2/5/6-GFP showed that these SsYABBY proteins were nucleus-localized, which is consistent with the previous reports (Fig. 4). Interestingly, the GFP signal of SsYABBY1/2/5 was also detected in the cell membrane, and the fluorescence signal could be well co-localized with that of the membrane marker PM-mCherry (Fig. 4). These results indicated the functional diversity of SsYABBYs in the membranes and nucleus.
Expression profiles of SsYABBYs in different tissues and development stages
To investigate the putative function of SsYABBY genes, the spatiotemporal expression patterns of all 7 SsYABBY genes were analyzed in different development stages and different tissues. For the vegetative growth from juvenile to adult stages, three different stem development stages of stems, including seedling stem, premature stem, and mature stem, were used to analyze the expression levels of all SsYABBY genes (Fig. 5A; Table S4). YAB2 clade members SsYABBY3, SsYABBY4, and SsYABBY5 expressed highly in the seedling stem. Among the SsYABBYs, the expression level of SsYABBY4 was highest during the stem development progress, while SsYABBY1, SsYABBY2, SsYABBY6, and SsYABBY7 expressed lower, suggesting their limited roles in stem development stages.
To better reveal the function of SsYABBY genes during the photosynthesis, the expression level of SsYABBYs was checked in leaf segments with a continuous leaf developmental gradient (basal zone, transitional zone, maturing zone, and mature zone) (Fig. 5B; Table S5). The results showed that SsYABBY3, SsYABBY4, and SsYABBY5 were mainly expressed from the basal zone to the mature zone. SsYABBY3 and SsYABBY4 showed higher expression levels in the basal zone, and the expression levels decreased gradually as the leaf matured. SsYABBY5 showed an increased expression from the basal zone to the transitional zone and decreased expression from the transitional zone to the mature zone. The expression levels of SsYABBY1, SsYABBY2, SsYABBY6, and SsYABBY7 were low or undetectable in these leaf segments, except for SsYABBY7, which was expressed only in the basal zone, suggesting their functional limitation during the photosynthesis.
For the meristematic and reproductive tissues, the functional divergence of SsYABBY genes was analyzed in sugarcane ovaries at 5 different ovule development stages (AC, MMC, Meiosis, Mitosis, and Mature). SsYABBY2 and SsYABBY5 were mainly expressed in these different ovule development stages. The expression level of SsYABBY1 was the highest in the MMC stage but lowest in the Mature stage. Notably, SsYABBY3, SsYABBY4, SsYABBY6, and SsYABBY7 were enriched in the mitosis stage but were lower or undetectable in AC and MMC stages of ovule development (Fig. 5C, E; Table S6).
For the root, stem, and leaf tissues, the expression levels of SsYABBY genes were investigated by RT-qPCR analysis with primers in Table S7. As shown in Fig. 5D, all 7 SsYABBYs except SsYABBY7 were predominately expressed in leaf tissues. SsYABBY1, SsYABBY2, SsYABBY6, and SsYABBY7 were mainly expressed in root, and SsYABBY2, SsYABBY4, SsYABBY6, and SsYABBY7 were expressed in stems. All together, SsYABBY genes were lowly expressed in roots, and SsYABBY3, SsYABBY4, and SsYABBY5 were mainly responsible for the stem and leaf development. SsYABBY2, SsYABBY5, and SsYABBY7 were mainly associated with ovule development. (Fig. 5D).
SsYABBY2 regulates asymmetric leaf division and ovule polarity establishment
The expression patterns of SsYABBY genes suggested that SsYABBY are responsible for the development of the vegetative and reproductive tissues. To further study the functional roles of SsYABBYs in the vegetative and reproductive tissues, SsYABBY2, which belongs to the CRC clade, was selected to explore its role in leaf development and ovule development using cross-species expression and/or complementation methods. The full-length SsYABBY2 cDNAs were introduced into Arabidopsis wild-type (WT) and crc mutant plants under the control of the constitutive 35S promoter using the floral dip method. A total of 10 WT-overexpression and 12 crc mutant complementary T3 transgenic lines were obtained, and three corresponding independent lines were used for further phenotype investigation. Compared with WT plants, the SsYABBY2-OE transgenic plants (3-week-old) showed prominent inward curled rosette leaves (Fig. 6A, B), and the leaves were curled from the abaxial side to adaxial side and became slender configuration (Fig. 6B, C). Moreover, the leaf abaxial-adaxial polarity deficiency phenotype became more severe with the leaf development from juvenile into mature (Fig. 6C). Compared with WT, the leaf length and leaf width of overexpression lines was significantly decreased (Fig. 6E-F), however, the leaf length/width ratio was slightly increased (Fig. 6G). Additionally, the overexpression plants (6-week-old) exhibited growth retardation, delayed flowering time, and slightly reduced fertility (Fig. 6D).
In Arabidopsis, CRC plays an essential role in carpel morphogenesis and nectary specification . Loss of CRC function resulted in a series of aberrant phenotypes, including cotyledons curled, nectaries loss, reduced ovule number, medially split, and reduced style tissue. We introduced the full-length of SsYABBY2 cDNAs driven by the constitutive 35S promoter into Arabidopsis crc mutant plants and obtained 12 crc mutant complemental lines in the T3 generation. As shown in Fig. 7, the defective phenotypes of crc mutant were completely recovered by expression of 35S::SsYABBY2. For example, the compact inflorescence, petal number, and style cracking were recovered in 35S::SsYABBY2 complemental lines (Fig. 7). These results show functional conservation of SsYABBY2 in the establishment of leaf asymmetric division and carpels polarity.
SsYABBYs interaction protein prediction
To further test the functional conservation of SsYABBYs, candidate interaction proteins of SsYABBYs were predicted by protein-protein interaction (PPI) analysis (Fig. 8A). As shown in Fig. 8A, SsYABBY2 and SsYABBY5 were highly expressed genes in the ovule and associated with several MADS-box proteins (MADS2, MADS4, MADS7, MADS16, MONOCULM3 (MOC3), STAMENLESS1 (SL1), ARGONAUTE14 (AGO14), and WUSCHEL (WUS)) to form a highly interactive cluster. These genes were connected with predominant expression in the floral organs and reproductive organ tissues, suggesting the conserved functions of SsYABBY2 and SsYABBY5 in the development of reproductive tissues (Fig. 8A, Table S8-S10). In leaf for the adaxial-abaxial polarity development, 4 proteins (AH2, HOX32, GRF1, and APO1) were identified as the candidate interactors of SsYABBY3/4/5, with different levels of connectivity among each other (Fig. 8A, Table S8-S10). For stem development, hormone metabolism-associated proteins, such as GA2ox6, GA3ox2, HOX4, WOX12, and RS2, were among the candidate interactions of SsYABBY3/4 proteins (Fig. 8A, Table S8-S10). Interestingly, the expression levels of all the interacting proteins were also enriched in these corresponding tissues (Fig. 8B, Table S8-S10). Moreover, Yeast-2-hybrid (Y2H), bimolecular fluorescence complementation (BiFC), and Dual-luciferase reporter assays (LUC) assays were adopted to confirm these protein interactions. As expected, SsYABBY2 directly interacted with SsMADS4, SsYABBY5 physically interacted with SsMADS4 and SsHOX32, and SsYABBY7 can interact with SsGAox6 (Fig 8C).
During the development, cells acquiring distinct fates greatly depends on the cell polarity establishment and maintenance [1, 3]. In plants, cell polarity is a fundamental feature in almost all aspects of cellular function, including cell expansion, division, differentiation, and morphogenesis [2, 3, 29]. In many species, the YABBY transcription factors were reported to play fundamental roles in the adaxial-abaxial polarity establishment and lateral organs development [2, 10,11,12, 15,16,17]. In this study, a total of 20 YABBY genes, including alleles, were genome-widely identified in S. spontaneum. More SsYABBYs existence in the sugarcane genome compared with the number of YABBY genes in S. bicolor (8) and O. sativa (8) indicated that the SsYABBY genes underwent the gene duplication events along with the sugarcane genomic autopolyploidization.
According to the phylogenetic and gene structure analysis, SsYABBY genes could be classified into four subgroups. Members clustered together into a subgroup shared similar gene structure and functions, indicating the functional conservation of these SsYABBY genes (Figs. 1 and 2). Notably, no SsYABBY gene could be clustered into the YAB5 subgroup; besides, no monocotyledon YABBY members were found in this subgroup (Fig. 2). This result is in line with the previous reports, suggesting that the YAB5 clade genes may generate along with the evolutionary divergence of monocotyledon and dicotyledons [8, 10, 30, 31]. The diversity of gene structures also plays an important role in expanding gene family members and the generation of novel genes. In our study, the gene structure of the SsYABBY1-2, SsYABBY3-2, SsYABBY3-3, and SsYABBY3-4 was variable (Fig. 1), implying the distinct function of SsYABBY genes. This phenomenon may be illustrated by the gene rearrangement and/or different chromosome fragments fusion during the sugarcane genomic autopolyploidization [32, 33].
Gene duplication events are the contributors to evolutionary momentum, and duplicate genes are mainly derived from whole-genome duplication (WGD), tandem, and segmental duplication [34,35,36]. In this study, just one pair of tandem repeat genes was identified, while twenty-three pairs of segmental duplications were found. Furthermore, the synteny analysis between sugarcane and its relative species (rice and sorghum) displayed the close evolutional and functional relevance in three species, suggesting those YABBY genes shared a similar function. For example, OsYABBY1 regulated the differentiation of reproductive cells . OsDL controls the stamen and carpel specification as a novel gene in rice . Interestingly, a couple of SsYABBYs showed collinear regions with OsYABBYs, suggesting the roles of SsYABBY genes in cell differentiation and carpel polarity.
To further understand the functional divergence of SsYABBY genes, transcriptome profiles of sugarcane stems from juvenile to mature, leaves from basal zone to apex zone, and different ovule development stages were investigated to clarify their roles of SsYABBY genes in sugar transport, photosynthesis, and establishment of leaf and ovule polarity. The expression levels of SsYABBY genes showed that YAB2 clade members (SsYABBY3, SsYABBY4, and SsYABBY5) were highly expressed in the seedling-stem stage (Fig. 5A). Similarly, genes belonging to this clade showed high expression in four zones of leaf segments, and enriched in the basal zone and transition zone (Fig. 5B). These results implied the potential function of these genes in sugar transport and photosynthesis. Previous functional study of YABBY genes in Incarvillea arguta showed that overexpression of IaYAABY2 altered the leaf and sepal polarity and increased the anthocyanin content level and photosynthesis capability of plants . Additionally, YAB2, YAB3, and FIL expressed in the abaxial domain of lateral organs, including cotyledons, leaves, and floral organs in Arabidopsis; thus, they worked as “vegetative YABBY genes” . However, for reproductive tissues, SsYABBY2 and SsYABBY5 (belonging to CRC and YAB2 clade) were predominately expressed during ovule development. SsYABBY3 and SsYABBY7 expressed weakly from AC to mature stages (Fig. 5C, E). The differential expression levels of SsYABBY genes suggest that SsYABBY genes are potentially involved in sugar transport, leaf morphogenesis, and ovule polarity.
To better understand the functional roles of SsYABBY genes in the adaxial-abaxial polarity establishment and lateral organs development, the CRC clade gene, SsYABBY2 was selected for further exploration of its function. Ectopic expression of VpYABBY1 altered leaf adaxial-abaxial polarity in Arabidopsis . Overexpression of soybean (Glycine max) GmFILa in Arabidopsis resulted in the abaxial polarity change of leaf epidermal, prolonged flowering, and inhibited apical meristem development . Consistent with the previous reports, the SsYABBY2-OE lines showed the prominent curled rosette leaves from the abaxial side to the adaxial side, and finally, leaves grow into a slender configuration (Fig. 6A-C). SsYABBY2-OE lines also showed meristem inhibition and delayed flowering (Fig. 6D).
In addition, CRC clade is specifically expressed in reproductive organs, such as carpels and ovules, so-called “flower specific YABBY genes” in Arabidopsis [41, 42]. In pea, the ortholog of CRC is also involved in carpel morphogenesis [18, 19]. For monocots, such as, Drooping Leaf (DL) in rice is orthologous with CRC. The loss-of-function dl mutation caused a complete homeotic transformation of carpels into stamens . In maize, the CRC homolog gene DRL1 (Drooping Leaf1), expressed in incipient and emergent leaf primordia, modulating leaf development and plant architecture [20,21,22]. In our study, SsYABBY2 was also preferentially transcribed in ovaries during ovules development (Fig. 5C), and ectopic expression of SsYABBY2 in Arabidopsis crc mutant could rescue the defective phenotype of carpel dehiscent in crc mutant (Fig. 7B). The adaxial to abaxial curled leaves and shortened silique defects of crc mutant were also completely recovered by SsYABBY2 expression (Fig. 7). Altogether these results further confirmed the functional specificity of CRC clade genes in the establishment and maintenance of the ovule polarity.
PPI of SsYABBYs also demonstrated the conserved function of SsYABBY genes for vegetative and reproductive development. For example, the MADS-box transcription factor genes MADS6, MADS16, and MADS3 function redundantly in the identity of the carpel/ovule development and floral meristem determinacy with the YABBY homologous gene DL . OsSL1 also regulated SPW1/OsMADS16 expression, specifying lodicule and stamen identities . We also found the strong expression levels of these homologous genes in the reproductive tissues of S. spontaneum (Table S8-S10), validating the conserved functions of these genes in floral organs development. Previous studies reported that AH2 deficiency leads to abaxial mesophyll cell programmed death for leaf polarity development and suppresses the abaxial development [45, 46]. OsAPO1 controls spikelet number and overexpression of APO1 causes an increase in inflorescence branches and spikelet . For stems development, some phytohormone-related genes (HOX4, GA2ox6, and GA3ox2) were predicted as the interacting partners of SsYABBY3/4/5 (Fig. 8A, Table S8-S10). In rice, these genes regulated gibberellin (GA) signaling and fine-tune GA responses [48,49,50,51], causing different degrees of dwarfing and increasing the number of tillers, which suggested that the GA signal pathway may play a crucial role during the stem development of S. spontaneum.
In conclusion, the present study identified and analyzed 20 SsYABBY genes in the S. spontaneum genome, which were classified into 5 subgroups. Phylogenetic and syntenic analysis verified that gene duplication contributed to expanding the SsYABBY gene family. Expression pattern analysis suggested that SsYABBY3/4/5 plays an important role in photosynthesis. SsYABBY2/5/7 may be responsible for leaf adaxial–abaxial polarity and carpel polarity establishment. Functional characterization indicated that SsYABBY2 is involved in the leaf morphogenesis and carpel polarity establishment (Fig. 9). Taken together, this systematic study provides a fine-scale map of transcriptional changes of SsYABBY genes in global tissues in sugarcane and uncovers a large number of candidate developmental regulators orchestrating the development of the different tissues in Saccharum spp.
Material and methods
The sugarcane (S. spontaneum L.) cultivar Yuetang 91-976 was grown and collected by State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources (Guangxi, China), and samples from this cultivar were used for all experiments. As previously reported , samples were collected from 5 different development stages of the ovule (including Archesporial Cell (AC), Megaspore Mother Cell (MMC), Meiosis, Mitosis, and Mature and 4 different leaf developmental stages (basal zone, transitional zone, maturing zone, mature zone), as detailed described by Mao et al., (2021) and Zhang et al., (2016). In this present study, Arabidopsis thaliana plants (Col-0) and crc-1 mutant ordered from AraShare, were grown under 16 h light/8 h dark photoperiod conditions at 22°C.
Sequence identification of YABBY genes in S. spontaneum
The Saccharum Genome database (http://sugarcane.zhangjisenlab.cn/sgd/html/index.html) was used for retrieving the genomic sequences . The sequence data of Sorghum bicolor and other species were downloaded from Phytozome v13 . The HMM model of the YABBY domain (PF04690) was used as the query sequence to search the sugarcane genome database. All candidate YABBY genes were further analyzed by the CDD program to confirm the C2C2 domain and YABBY domain. The SsYABBY proteins information such as isoelectric point (pI), molecular weight (MW), and protein length were predicted using ExPASy-Compute pI/MW.
Sequence alignment, gene structure, and phylogenetic analysis of SsYABBY genes
Multiple sequences alignment of YABBY protein from A. thaliana, O. sativa, V. vinifera, S. bicolor, and S. spontaneum were calculated by MUSCLE and visualized by Jalview . The default setting parameters was the maximum number (20), minimum width (6), and maximum width (50). The structure of SsYABBYs was displayed using the TBtools software . The cis-acting elements of YABBY gene promoters were predicted by PlantCARE, and transcription factors were predicted by PlantRegMap . The selection and substitution rates, the non-synonymous (Ka), synonymous (Ks), and Ka/Ks substitution ratios of the homologous gene pairs of sugarcane and sorghum were calculated by Ka/Ks calculation program. A phylogenetic tree was constructed by the MEGA 7.0 program using the ML method based on the JTT substitution model .
Collinearity analysis of SsYABBY genes
The loci of SsYABBY genes were retrieved from the sugarcane annotation GFF3 files. TBtools were used to visualize gene locations on the sugarcane chromosomes . For collinearity analysis, the gene pairs with a cut-off e-value of 1×10−5, used for MCScanX analysis, generating collinearity blocks. CIRCOS software was used for collinearity mapping within the sugarcane, rice, and sorghum genome .
Transcriptome profiles analysis of YABBYs and RT-qPCR
The RNA-seq data of leaf development (including basal zone, a transitional zone, a maturing zone, a mature zone) were downloaded from the Saccharum Genome database (http://sugarcane.zhangjisenlab.cn/sgd/html/index.html). The RNA-seq data of female reproductive development (including AC, MMC, Meiosis, Mitosis, and Mature) were downloaded from the European Nucleotide Archive (ENA, accession number PRJEB44944). The RNA-seq clean reads were obtained by Trimmomatic software and mapped to the reference genome by Hisat2 . DESeq2 and fragments per kilobase million values (FPKM) were used to analyze gene expression levels . The FPKM value of YABBY genes was transformed using the log2-transformed method, and the expression patterns were generated using the heatmap package in R software.
RT-qPCR assays were performed in three different tissues (root, stem, leaf) and five female reproductive stages (AC, MMC, Meiosis, Mitosis, Mature). The total RNA was isolated using RNA Extraction Kit (R6827-01, OMEGA, China), and further analyzed by gel electrophoresis and NanoDrop2000 (Thermo Fisher, China). First-strand cDNA was synthesized with the TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (TransGen Biotech). RT-qPCR was carried out using SYBR-green fluorescence (TaKaRa Biotechnology) on a Multicolor Real-Time PCR Detection System (Bio-Rad) with a 40 cycle of 95°C for 30 s; 95°C for 5 s and 60°C for 40 s. Each sample was replicated three times, and the 2-ΔΔCT method was used for calculating the gene expression levels .
Vector construction, subcellular localization, and transgenic analysis
The full-length coding region of SsYABBY genes without terminator code was amplified using primers listed in Supplementary Table S1. The PCR fragments were cloned into the pENTR/D-TOPO vector and sequenced, and then recombined into the destination vector pGWB605 by LR reaction. The resulting plasmid pGWB605-SsYABBY-GFP and empty vector pGWB605-GFP were transformed into Agrobacterium tumefaciens strain GV3101 and injected into leaves of Nicotiana benthamiana (4-week-old). After 36-48 h treatment, GFP signals were checked under a Leica confocal microscope with excited at 514 nm. The Agrobacterium tumefaciens strain GV3101 with SsYABBY2 was used to transform the crc mutant and wild-type plants using a floral dip procedure (Clough and Bent, 1998).
PPI network construction of SsYABBYs
A precomputed global resource, the Search Tool of the Retrieval of Interacting Genes (STRING) (http://string-db.org/) database is used for evaluating protein-protein interaction (PPI) information . We used the STRING online tool to predict the PPI pairs of SsYABBY proteins with a combined score of > 0.4. Cytoscape v3.8.2 was used for PPI network construction (https://cytoscape.org/) .
Yeast two-hybrid assay
The full-length CDS of SsYABBY2, SsYABBY5, and SsYABBY7 was cloned into the pGADT7 vector at the NdeI site for fusion with the GAL4 activation domain. The full-length CDS of SsMADS4, SsHOX32, and SsGAox6 were cloned into the pGBKT7 vector at the NdeI site for fusion with the GAL4 DNA-binding domain. Approximately 0.1 μg plasmids of bait and prey were co-transformed into the yeast strain AH109 using the Matchmaker™GAL4 Two-Hybrid System according to the manufacturer’s instructions (Clontech, USA). After growth at 28 °C for 3 days, yeast transformants were diluted and transferred to medium supplemented with SD/-Leu-Trp for growth. The yeast transformants on medium supplemented with SD/-Leu-Trp-His-Ade and 3-amino-1,2,4-triazole (3-AT) for protein interaction selection. The primers used to generate the constructs are listed in Table S7.
Dual-luciferase reporter assay
The full-length CDS of SsYABBY2/5/7 without the stop codon was cloned into the pCAMBIA 1300-nLUC vector to generate the pCAMBIA 1300-SsYABBY2/5/7-nLUC construct. The full-length CDS of SsMADS4, SsHOX32, and SsGAox6 without the stop codon was cloned into the pCAMBIA 1300-cLUC vector to generate the related construct. Transient expression assays were performed as described previously . Briefly, the recombinant constructs were transformed into Agrobacterium strain GV3101 and infiltrated into tobacco leaves. After 2 days of incubation, LUC and REN activities were measured using a SpectraMax® i3xMulti-Mode detection platform (Molecular Devices, USA) with a Dual-Luciferase Reporter Assay Kit (Pro-mega, USA). The LUC to REN ratio was calculated as a measure of the transcriptional activity. The primers are listed in Table S7.
Bimolecular fluorescence complementation assay
The open reading frames of full-length SsYABBY2/5/7, SsMADS4, SsHOX32, and SsGAox6 were amplified using sugarcane genomic DNA as a template. The primers are listed in Table S7. The BiFC assay was performed as previously described .
Availability of data and materials
All data generated or analyzed during this study are included in this published article and its supplementary information files. The RNA-seq data of female reproductive development have been deposited in the EMBL Nucleotide Sequence Database (ENA) with accession no. PRJEB44944 (https://www.ebi.ac.uk/ena/browser/view/PRJE44944), which will be available publicly upon acceptance of the article. The RNA-seq data of leaf development were downloaded from the Saccharum Genome database (http://sugarcane.zhangjisenlab.cn/sgd/html/index.html).
Rose L, Gonczy P. Polarity establishment, asymmetric division and segregation of fate determinants in early C. elegans embryos. WormBook, ed. The C. elegans Research Community, WormBook; 2014. p. 1–43.
Yang Z. Cell polarity signaling in Arabidopsis. Annu Rev Cell Dev Biol. 2008;24:551–75.
Yang K, Wang L, Le J, Dong J. Cell polarity: Regulators and mechanisms in plants. J Integr Plant Biol. 2020;62(1):132–47.
Huang T, Harrar Y, Lin C, Reinhart B, Newell NR, Talavera-Rauh F, et al. Arabidopsis KANADI1 acts as a transcriptional repressor by interacting with a specific cis-element and regulates auxin biosynthesis, transport, and signaling in opposition to HD-ZIP III factors. Plant Cell. 2014;26(1):246–62.
Bowman JL, Eshed Y, Baum SF. Establishment of polarity in angiosperm lateral organs. Trends Genet. 2002;18(3):134–41.
Husbands AY, Chitwood DH, Plavskin Y, Timmermans MC. Signals and prepatterns: new insights into organ polarity in plants. Genes Dev. 2009;23(17):1986–97.
Du Y, Lunde C, Li Y, Jackson D, Hake S, Zhang Z. Gene duplication at the Fascicled ear1 locus controls the fate of inflorescence meristem cells in maize. Proc.Natl Acad Sci U S A. 2021;118(7):e2019218118.
Zhang S, Wang L, Sun X, Li Y, Yao J, van Nocker S, et al. Genome-Wide Analysis of the YABBY Gene Family in Grapevine and Functional Characterization of VvYABBY4. Front Plant Sci 2019;10:1207.
Kumaran MK, Bowman JL, Sundaresan V. YABBY polarity genes mediate the repression of KNOX homeobox genes in Arabidopsis. Plant Cell. 2002;14(11):2761–70.
Bowman JL. CRABS CLAW, a gene that regulates carpel and nectary development in Arabidopsis, encodes a novel protein with zinc finger and helix-loop-helix domains. Development. 1999;126(11):2387–96.
Siegfried KR, Eshed Y, Baum SF, Otsuga D, Drews GN, Bowman JL. Members of the YABBY gene family specify abaxial cell fate in Arabidopsis. Development. 1999;126(18):4117–28.
Bowman JL. The YABBY gene family and abaxial cell fate. Curr Opin Plant Biol 2000;3(1):17–22.
Stahle MI, Kuehlich J, Staron L, von Arnim AG, Golz JF. YABBYs and the transcriptional corepressors LEUNIG and LEUNIG_HOMOLOG maintain leaf polarity and meristem activity in Arabidopsis. Plant Cell. 2009;21(10):3105–18.
Sarojam R, Sappl PG, Goldshmidt A, Efroni I, Floyd SK, Eshed Y, et al. Differentiating Arabidopsis shoots from leaves by combined YABBY activities. Plant Cell. 2010;22(7):2113–30.
Tanaka W, Toriba T, Hirano HY. Three TOB1-related YABBY genes are required to maintain proper function of the spikelet and branch meristems in rice. New Phytol. 2017;215(2):825–39.
Villanueva JM, Broadhvest J, Hauser BA, Meister RJ, Schneitz K, Gasser CS. INNER NO OUTER regulates abaxial– adaxial patterning in Arabidopsis ovules. Genes Dev. 1999;13(23):3160–9.
Yamaguchi T, Nagasawa N, Kawasaki S, Matsuoka M, Nagato Y, Hirano HY. The YABBY gene DROOPING LEAF regulates carpel specification and midrib development in Oryza sativa. Plant Cell. 2004;16(2):500–9.
Yang Z, Gong Q, Wang L, Jin Y, Xi J, Li Z, et al. Genome-Wide Study of YABBY Genes in Upland Cotton and Their Expression Patterns under Different Stresses. Front. Genet. 2018;9:33.
Fourquin C, Primo A, Martinez-Fernandez I, Huet-Trujillo E, Ferrandiz C. The CRC orthologue from Pisum sativum shows conserved functions in carpel morphogenesis and vascular development. Ann Bot-London 2014;114(7):1535–44.
Nagasawa N, Miyoshi M, Sano Y, Satoh H, Hirano H, Sakai H, et al. SUPERWOMAN1 and DROOPING LEAF genes control floral organ identity in rice. Development. 2003;130(4):705–18.
Ohmori Y, Toriba T, Nakamura H, Ichikawa H, Hirano H. Temporal and spatial regulation of DROOPING LEAF gene expression that promotes midrib formation in rice. Plant J 2011;65(1):77–86.
Strable J, Wallace JG, Unger-Wallace E, Briggs S, Bradbury PJ, Buckler ES, et al. Maize YABBY Genes drooping leaf1 and drooping leaf2 Regulate Plant Architecture. Plant Cell. 2017;29(7):1622–41.
Bartley G, Ishida B. Developmental gene regulation during tomato fruit ripening and in-vitro sepal morphogenesis. BMC Plant Biol. 2003;3:4.
Cong B, Barrero LS, Tanksley SD. Regulatory change in YABBY-like transcription factor led to evolution of extreme fruit size during tomato domestication. Nat Genet 2008;40(6):800–4.
Kayani SI, Shen Q, Ma Y, Fu X, Xie L, Zhong Y, et al. The YABBY Family Transcription Factor AaYABBY5 Directly Targets Cytochrome P450 Monooxygenase (CYP71AV1) and Double-Bond Reductase 2 (DBR2) Involved in Artemisinin Biosynthesis in Artemisia Annua. Front Plant Sci 2019;10:1084.
Ali A, Khan M, Sharif R, Mujtaba M, Gao SJ. Sugarcane Omics: An Update on the Current Status of Research and Crop Improvement. Plants. 2019;8(9):344.
Zhang J, Arro J, Chen Y, Ming R. Haplotype analysis of sucrose synthase gene family in three Saccharum species. BMC Genomics. 2013;14:314.
Su Y, Xu L, Fu Z, Yang Y, Guo J, Wang S, et al. ScChi, encoding an acidic class III chitinase of sugarcane, confers positive responses to biotic and abiotic stresses in sugarcane. Int J Mol Sci. 2014;15(2):2738–60.
Muroyama A, Bergmann D. Plant Cell Polarity: Creating Diversity from Inside the Box. Annu. Rev. Cell Dev. Biol. 2019;35:309–36.
Kim KH, Hwang JH, Han DY, Park M, Kim S, Choi D, et al. Major Quantitative Trait Loci and Putative Candidate Genes for Powdery Mildew Resistance and Fruit-Related Traits Revealed by an Intraspecific Genetic Map for Watermelon (Citrullus lanatus var. lanatus). PLoS One. 2015;10(12):e0145665.
Lian Q, Fu Q, Xu Y, Hu Z, Zheng J, Zhang A, et al. QTLs and candidate genes analyses for fruit size under domestication and differentiation in melon (Cucumis melo L.) based on high resolution maps. BMC Plant Biol. 2021;21(1):126.
Zhang J, Zhang X, Tang H, Zhang Q, Hua X, Ma X, et al. Allele-defined genome of the autopolyploid sugarcane Saccharum spontaneum L. Nat. Genet. 2018;50(11):1565–73.
Li Z, Hua X, Zhong W, Yuan Y, Wang Y, Wang Z, et al. Genome-Wide Identification and Expression Profile Analysis of WRKY Family Genes in the Autopolyploid Saccharum spontaneum. Plant Cell Physiol. 2020;61(3):616–30.
Guo B, Wei Y, Xu R, Lin S, Luan H, Lv C, et al. Genome-wide analysis of APETALA2/ethylene-responsive factor (AP2/ERF) gene family in barley (Hordeum vulgare L.). PLoS One. 2016;11(9):e0161322.
Zhang Z, Li X. Genome-wide identification of AP2/ERF superfamily genes and their expression during fruit ripening of Chinese jujube. Sci Rep. 2018;8(1):1–16.
Qiao X, Yin H, Li L, Wang R, Wu J, Wu J, Zhang S. Different Modes of Gene Duplication Show Divergent Evolutionary Patterns and Contribute Differently to the Expansion of Gene Families Involved in Important Fruit Traits in Pear (Pyrus bretschneideri). Front Plant Sci. 2018;9:161.
Toriba T, Harada K, Takamura A, Nakamura H, Ichikawa H, Suzaki T, et al. Molecular characterization of the YABBY gene family in Oryza sativa and expression analysis of OsYABBY1. Mol. Genet Genom. 2007;277(5):457–68.
Sun X, Guan Y, Hu X. Isolation and Characterization of IaYABBY2 Gene from Incarvillea arguta. Plant Mole Biol Rep. 2014;32(6):1219–27.
Xiang J, Liu RQ, Li TM, Han LJ, Zou Y, Xu TF, et al. Isolation and characterization of two VpYABBY genes from wild Chinese Vitis pseudoreticulata. Protoplasma. 2013;250(6):1315–25.
Yang H, Shi G, Li X, Hu D, Cui Y, Hou J, et al. Overexpression of a soybean YABBY gene, GmFILa, causes leaf curling in Arabidopsis thaliana. BMC Plant Biol. 2019;19(1):234.
Zhang Y, Wang P, Xia H, Zhao C, Hou L, Li C, et al. Comparative transcriptome analysis of basal and zygote-located tip regions of peanut ovaries provides insight into the mechanism of light regulation in peanut embryo and pod development. BMC Genom. 2016;17(1):1–13.
Scorza LC, Hernandes-Lopes J, Melo-de-Pinna GF, Dornelas MC. Expression patterns of Passiflora edulis APETALA1/FRUITFULL homologues shed light onto tendril and corona identities. EvoDevo. 2017;8(1):1–15.
Li H, Liang W, Hu Y, Zhu L, Yin C, Xu J, et al. Rice MADS6 interacts with the floral homeotic genes SUPERWOMAN1, MADS3, MADS58, MADS13, and DROOPING LEAF in specifying floral organ identities and meristem fate. The Plant Cell. 2011;23(7):2536–52.
Xiao H, Tang J, Li Y, Wang W, Li X, Jin L, et al. STAMENLESS 1, encoding a single C2H2 zinc finger protein, regulates floral organ identity in rice. Plant J. 2009;59(5):789–801.
Zhang G, Xu Q, Zhu X, Qian Q, Xue H. SHALLOT-LIKE1 is a KANADI transcription factor that modulates rice leaf rolling by regulating leaf abaxial cell development. Plant Cell. 2009;21(3):719–35.
Ren DY, Cui YJ, Hu HT, Xu QK, Rao YC, Yu XQ, et al. AH2 encodes a MYB domain protein that determines hull fate and affects grain yield and quality in rice. Plant J. 2019;100(4):813–24.
Ikeda K, Ito M, Nagasawa N, Kyozuka J, Nagato Y. Rice ABERRANT PANICLE ORGANIZATION 1, encoding an F-box protein, regulates meristem fate. Plant J. 2007;51(6):1030–40.
Dai M, Hu Y, Ma Q, Zhao Y, Zhou DX. Functional analysis of rice HOMEOBOX4 (Oshox4) gene reveals a negative function in gibberellin responses. Plant Mol Biol 2008;66(3):289–301.
Huang J, Tang D, Shen Y, Qin B, Hong L, You A, et al. Activation of gibberellin 2-oxidase 6 decreases active gibberellin levels and creates a dominant semi-dwarf phenotype in rice (Oryza sativa L.). J. Genet Genomics 2010;37(1):23–36.
Tong H, Xiao Y, Liu D, Gao S, Liu L, Yin Y, et al. Brassinosteroid regulates cell elongation by modulating gibberellin metabolism in rice. Plant Cell. 2014;26(11):4376–93.
Zhou W, Malabanan PB, Abrigo E. OsHox4 regulates GA signaling by interacting with DELLA-like genes and GA oxidase genes in rice. Euphytica. 2014;201(1):97–107.
Maokai Yan XJ, Liu Y, Chen H, Ye T, Hou Z, Su Z, et al. Identification and evaluation of the novel genes for transcript normalization during female gametophyte development in sugarcane, vol. 9; 2021. p. e12298.
Goodstein D, Shu S, Howson R, Neupane R, Hayes R, Fazo J, et al. Phytozome: a comparative platform for green plant genomics. Nucleic Acids Res. 2012;40:D1178–86.
Sudhir K, Glen S, Koichiro T. MEGA7. Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mole Biol Evol. 2016;(7):1870–4.
Chen C, Chen H, Zhang Y, Thomas HR, Frank MH, He Y, et al. TBtools: an integrative toolkit developed for interactive analyses of big biological data. Mole Plant. 2020;13(8):1194–202.
Tian F, Yang DC, Meng YQ, Jin J, Gao G. PlantRegMap: charting functional regulatory maps in plants. Nucleic Acids Res. 2019;48:D1104–13.
Krzywinski M, Schein J, Birol I, Connors J, Gascoyne R, Horsman D, et al. Circos: an information aesthetic for comparative genomics. Genome Res. 2009;19(9):1639–45.
Kim D, Paggi JM, Park C, Bennett C, Salzberg SL. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat Biotechnol. 2019;37(8):907–15.
Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15(12):1–21.
Ling H, Wu Q, Guo J, Xu L, Que Y. Comprehensive selection of reference genes for gene expression normalization in sugarcane by real time quantitative RT-PCR. PLoS One. 2014;9(5):e97469.
Szklarczyk D, Franceschini A, Wyder S, Forslund K, Heller D, Huerta-Cepas J, et al. STRING v10: protein-protein interaction networks, integrated over the tree of life. Nucleic Acids Res. 2015;43:D447–52.
Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003;13(11):2498–504.
Hellens RP, et al. Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants. Plant Methods. 2005;18(1):13.
Scacchi E. Dynamic, auxin-responsive plasma membrane-to-nucleus movement of Arabidopsis BRX. Development. 2009;136:2059–67.
We thank all of the colleagues in our laboratory for providing useful discussions and technical assistance. We are very grateful to the editor and reviewers for critically evaluating the manuscript and providing constructive comments for its improvement.
This work was supported by Science and Technology Major Project of Guangxi (Gui Ke 2018-266-Z01), National Natural Science Foundation of China (31800262; U1605212; 31761130074; 31600249; 31700279), China Postdoctoral Science Foundation (2018M632564), Weng Hongwu Academic Innovation Research Fund of Peking University, and a Guangxi Distinguished Experts Fellowship.
Ethics approval and consent to participate
The sugarcane (S. spontaneum L.) cultivar Yuetang 91-976 was grown and collected by State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources (Guangxi, China). The plant materials do not include any wild species at risk of extinction. No specific permits are required for sample collection in this study. We comply with relevant institutional, national, and international guidelines and legislation for plant study. The authors declare that they have no conflict of interest.
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She, Z., Huang, X., Aslam, M. et al. Expression characterization and cross-species complementation uncover the functional conservation of YABBY genes for leaf abaxial polarity and carpel polarity establishment in Saccharum spontaneum. BMC Plant Biol 22, 124 (2022). https://doi.org/10.1186/s12870-022-03501-3
- YABBY genes
- Expression analysis
- Polarity establishment