Skip to main content

Advertisement

Overexpression of a soybean YABBY gene, GmFILa, causes leaf curling in Arabidopsis thaliana

Abstract

Background

YABBY genes play important roles in the growth and polar establishment of lateral organs such as leaves and floral organs in angiosperms. However, the functions of YABBY homologous genes are largely unknown in soybean.

Results

In this study, we identified GmFILa encoding a YABBY transcription factor belonging to FIL subfamily. In situ mRNA hybridization analysis indicated that GmFILa had specific expression patterns in leaf as well as in flower bud primordia. Ectopic expression of GmFILa in Arabidopsis thaliana altered the partial abaxialization of the adaxial epidermises of leaves. Besides, GmFILa transgenic plants also exhibited longer flowering period and inhibition of shoot apical meristem (SAM) development compared to the wild type plants. Digital expression data and quantitative real-time polymerase chain reaction (qRT-PCR) analysis demonstrated that the expression of GmFILa was induced by biotic and abiotic stresses and hormone treatments. Transcriptome analysis suggested that overexpressing GmFILa yielded 82 significant differentially expressed genes (DEGs) in Arabidopsis leaves, which can be classified into transcription factors, transporters, and genes involved in growth and development, metabolism, signal transduction, redox reaction and stress response.

Conclusions

These results not only demonstrate the roles of GmFILa involved in leaf adaxial-abaxial polarity in Arabidopsis, but also help to reveal the molecular regulatory mechanism of GmFILa based on the transcriptomic data.

Background

Several regulators controlling leaf abaxial-adaxial polarity and leaf growth have been identified in Arabidopsis, such as AS2 (ASYMMETRIC LEAVES2), class III HD-Zip, KANADI, ARF3/4 (AUXIN RESPONSE FACTOR), YABBY and small non-coding RNAs [1,2,3,4,5,6]. Among these different types of regulators, YABBY family is specific to seed plants [7], and contains zinc finger-like and YABBY domains [8, 9]. The analysis of the zinc finger domain showed that it could work in protein-protein interactions for the formations of homo- and heterodimers as well as protein self-association [10]. Evolutionary analysis indicated that YABBY gene family consists of five members, including FILAMENTOUS FLOWER/YABBY3 (FIL/YAB3), YABBY2 (YAB2), YABBY5 (YAB5), CRABS CLAW (CRC), and INNER NO OUTER (INO). In Arabidopsis, FIL/YAB3, YAB2 and YAB5 are expressed in the abaxial domain of lateral organs including cotyledons, leaves and floral organs, thus they were served as “vegetative YABBY genes”; whereas the other two (CRC and INO) are restrictedly expressed in the abaxial domains of carpels and the outer integument of ovules, respectively [11, 12].

Based on the discoveries over these years, many YABBY genes from different plant species have been shown to be involved in plant growth and development, particularly in lamina growth, establishment of leaf adaxial-abaxial polarity, SAM development and floral organ identity [11,12,13,14,15,16]. In Arabidopsis, fil yab3 double mutant exhibited obvious phenotypes in the vegetative organs including linear cotyledons and leaves, abnormal vasculature and abaxial leaf surface, and ectopic SAM structures [12]; triple (fil yab3 yab5) and quadruple (fil yab2 yab3 yab5) mutants showed more severe phenotypes than the double mutant: diminutive and bushy plants lacking apical dominance and displaying a dramatic loss of lamina expansion and polarity defects in lateral organs [7]; while in fil single mutant, the flowers and floral organs were strongly affected, for example, increased sepals and carpels, missing petals, and radially symmetric stamens [11, 12, 17, 18]. Three FIL/TOB clade YABBY genes, TONGARI-BOUSHI (TOB1, TOB2 and TOB3), were indicated to regulate spikelet and branch meristems in rice [19,20,21]. Rice OsYABBY4 gene belonging to FIL/YAB3 subfamily, exhibits possible functions in vasculature development [22], and regulates plant height, internode and floral organs development through modulating the gibberellin pathway [23]. CtYABBY1, a FIL homology, is sensitive to temperature variation and plays an important role in male sterility and fertility restoration in Chinese cabbage [15]. A YABBY-like gene fasciated (fas) from tomato (Solanum lycopersicum) regulates carpel number, fruit development and fruit size [24, 25]. Overexpressing an Incarvillea arguta YAB2 subfamily gene IaYABBY2 in Arabidopsis altered the adaxial-abaxial polarity of leaves and sepals, affected the development of florescence, and increased the anthocyanin content level and photosynthesis capability of plants after differential environment stress [26]. Two wild Chinese Vitis pseudoreticulata genes, VpYABBY1 and VpYABBY2, belonging to FIL and YAB2 subfamily, were shown to have divergent functions in the control of lateral organ development: VpYABBY1 regulates leaf adaxial-abaxial polarity, while VpYABBY2 may play an important role in carpel growth and grape berry morphogenesis [27]. ZmYAB2.1/ZmSh1–1, belonging to YAB5 subfamily, was identified as a candidate gene controlling nonshattering ears in maize [28], and was also reported to interact epistatically with teosinte-branched1 (tb1) to regulate the length of internodes within the ear [16]. In spearmint (Mentha spicata), a novel gene MsYABBY5 (belonging to YAB5 subfamily), was proved to be a repressor of secondary metabolism (terpene level) [29]. Arabidopsis CRC was reported to participate in the nectary development and carpel identity [8, 30]. In maize, drooping leaf (drl) gene, the homology of Arabidopsis CRC, was shown to regulate plant architecture through affecting leaf length and width, leaf angle, and internode length and diameter [31]. In rice, the drooping leaf gene (DL) not only regulates the leaf midrib formation, but also controls the specification of carpel in the flower [32,33,34]. The Arabidopsis INO was demonstrated to be necessary for polarity determination in the ovule [35].

Although many plant YABBY genes have been functionally studied, their roles in soybean are rarely reported. Zhao et al. [36] found soybean gene GmYABBY10 might be a negative regulator of plant tolerance to drought and salt stress. In this study, a YABBY gene, designated as GmFILa, was isolated and functionally studied in soybean. Moreover, microarray analysis was performed to uncover the regulation mechanism of GmFILa in the transgenic Arabidopsis. Our results suggest the roles of soybean GmFILa in regulating leaf polarity development and potential functions in stress tolerance.

Results

Duplication pattern, phylogeny, gene structure, and expression analyses of soybean YABBYs

Until now, a total of 17 YABBY genes were identified in soybean [36]. Compared with Arabidopsis (5 members) [12], rice (Oryza stative L.) (8 members) [37], maize (Zea mays L.) (13 members) [38] and tomato (9 members) [39], soybean contains the most numerous members in YABBY gene family, which may be due to the two large-scale genome replications in soybean [40]. Therefore, we analyzed the duplication patterns of soybean YABBY genes and found that all GmYABBY genes were derived from segmental duplications without tandem duplications (Additional file 1: Figure S1 and Table S1). This suggests that segmental duplication might be the main cause of expansion in soybean YABBY family.

A neighbor-joining (NJ) phylogenetic tree was constructed based on soybean and 29 known YABBY proteins from monocotyledonous and dicotyledonous plants (Fig. 1a, Additional file 2: Table S4). As with the previous report that YABBY family consists of five subclasses including FIL/YAB3, YAB2, YAB5, INO and CRC [12], soybean YABBY gene family was also divided into five subgroups. As shown in the tree, YABBY genes from monocots and dicots clustered independently in each subgroup (Fig. 1a), indicating that they have functional differentiation. In addition, the functions of soybean YABBY genes could be inferred from the known plant YABBYs through phylogenetic relationships.

Fig. 1
figure1

Phylogeny and subcellular localization of GmFILa. a Phylogenetic relationships of GmYABBYs with 29 YABBYs from other plants. Soybean YABBYs were named based on the previous report [36]. The phylogenetic tree was constructed with neighbor-joining method in MEGA 6.0 software and was divided into five subgroups marked with different colors. b GmFILa-GFP fusion protein and GFP alone were transiently expressed in onion epidermal cells, respectively. UV, images of GFP fluorescence; Light, bright field images of cell morphology; Merge, merged images. Scale bar was indicated in each panel

Exon-intron structure divergences usually represent the evolutionary relationships within gene families. In soybean YABBY gene family, exon number ranges from six to seven, and intron number is either five or six (Additional file 1: Figure S2). From the phylogenetic tree, most members within the same subgroup contain conserved exon/intron structures and similar gene lengths (Additional file 1: Figure S2), while genes in different subgroups show some differences.

Tissue expression profiles of 16 soybean YABBYs were gained based on the RNA sequencing (RNA-seq) data from SoyBase. Obviously, they were divided into two categories: the expression of CRC and INO is restricted to young leaf and/or flower; whereas “vegetative YABBY genes” have higher expression levels than CRC and INO, and express in most of the tissues, including leaf, flower, pod and seed, but not in root and nodule (Additional file 1: Figure S3). Expression profiles of eight GmYABBYs were analyzed in Plant Expression Database (Additional file 1: Figure S4). FIL/YAB3 (containing GmYABBY16/GmFILa, GmYABBY3 and GmYABBY5) and YAB5 (GmYABBY13) members were shown to have abundant expression levels in SAM and axillary meristem, but low in non-apical meristem; by the contrast, YAB2 (GmYABBY9 and GmYABBY12) and INO (GmYABBY1 and GmYABBY8) genes were found to be highly expressed in non-apical meristem (Additional file 1: Figure S4b). All eight genes have higher expression in sporophytic tissue compared with mature pollen (Additional file 1: Figure S4c). The expression in embryonic development showed that several GmYABBYs are highly expressed in young trifoliate leaf compared with other tissues during the globular and heart stages, and exhibit high expression in embryo proper at the cotyledon stage (Additional file 1: Figure S4d).

Identification of GmFILa

A soybean curled-cotyledon mutant (cco), induced by sodium azide (NaN3) and 60Coγ ray from soybean cultivar Nannong 94–16, was previously identified in our group. Shi et al. [41] revealed that the transcript level of GmYABBY16 is significantly increased in cco mutant compared to its wild type through the RNA-seq data analysis and semi-quantitative RT-PCR (sqPCR) examination. Thus, GmYABBY16 was selected for functional characterization, especially on the regulation of cotyledon and leaf development. As GmYABBY16 belongs to FIL subfamily, it was further named as GmFILa.

The coding sequence (CDS) of GmFILa (Glyma.17G138200) was cloned via reverse transcription PCR (RT-PCR) from the leaf of soybean Nannong 94–16 cultivar (Additional file 1: Figure S5), which is 648 bp in length and encodes 215 amino acids along with a protein mass of 24.02 kDa and an isoelectric point (PI) of 7.16. Physicochemical properties analysis revealed that GmFILa is a hydrophilic (with GRAVY value of − 0.341) and unstable protein (with instability index of 52.46). The GmFILa protein was predicted to have conserved YABBY domain in the N-termini and a zinc finger-like motif in the C-termini by alignments with several other plant YABBY proteins (Fig. 2). Phylogenetic analysis showed that GmFILa was grouped together with PapsFIL (identity of 67.53%) from Papaver somniferum, VpYABBY1 (identity of 72.56%) from wild Chinese Vitis pseudoreticulata and GRAM (identity of 75%) from Antirrhinum majus (Fig. 1a). Among these orthologs, PapsFIL was shown to regulate highly lobed leaf patterning [42]; overexpression of VpYABBY1 in Arabidopsis caused the partial abaxialization of the adaxial epidermises of leaves [27]; and GRAM promotes lateral growth and abaxial cell fate in the growing leaf primordia [13].

Fig. 2
figure2

Alignment of plant YABBY protein sequences. GeneDoc software was used to show the Clustal alignment figure of soybean GmFILa and other YABBY orthologs from different plants. Conserved zinc-finger and YABBY domains are underlined with different lines. YABBY protein sequences are listed in Additional file 2: Table S4

GmFILa is a nuclear-localized protein

To obtain 35S:GmFILa-GFP, the coding region of GmFILa was fused to the green fluorescent protein (GFP) reporter gene which is under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Further, the recombinant construct and empty vector (35S:GFP) were transformed into onion epidermal cells, respectively. Confocal images revealed that the GmFILa-GFP fusion protein was localized exclusively to the nucleus, by contrast, the empty vector was uniformly distributed throughout the whole cell (Fig. 1b). This observation indicated that GmFILa is a nuclear-localized protein, implying that GmFILa, like other YABBYs [22, 27], functions as a transcription factor.

Tissue expression pattern analysis of GmFILa

From RNA-seq data, GmFILa is highly expressed in leaf, followed by seed, flower, and pod tissues, but not in root and nodule (Additional file 1: Figure S3). The qRT-PCR examination indicated that GmFILa has the highest expression level in seed at 20 days after flowering (DAF), followed by leaf, flower, stem, pod shell (20DAF) and root (Fig. 3d). This discrepancy might be due to the differences in the RNA samples (G. max A81–356022 for RNA-Seq and Nannong 94–16 for qRT-PCR) or methods. Based on microarray data, we analyzed the detailed expression of GmFILa in the tissues of seed, flower, and meristem (Fig. 3a-c). During seed embryo development, GmFILa has relatively high expression in whole seed, young trifoliate leaf and embryo proper compared with other tissues. At globular stage, GmFILa has the highest expression in young trifoliate leaf; at heart stage, the expression decreases in whole seed and increases in embryo proper; at cotyledon stage, the expression of GmFILa is increased in whole seed, but absent in trifoliate leaf (Fig. 3a).

Fig. 3
figure3

Tissue-specific expression analysis of GmFILa. a-c Digital expression of GmFILa in different tissues from microarray data [70]. The Y-axis represents the log2 ratios for the MAS-normalized values. d Tissue-specific expression profiles of GmFILa with qRT-PCR amplification in seedling root, stem, leaf, flower, seed and pod shell. The expression of GmFILa in root was used as a control (expression value = 1). Paired-samples t-test (two-tail) was selected for statistical analysis. * 0.01 < P < 0.05; ** P < 0.01

The mRNA in situ hybridization was employed to precisely examine the expression of GmFILa in soybean leaf and flower tissues (Fig. 4). Before the complete formation of the leaf primordia, two incipient leaf primordia are formed on both sides of the apical meristem (AM). First, the transcripts of GmFILa were distributed throughout the incipient leaf primordia (Fig. 4a); further, GmFILa was gradually expressed in abaxial cells with the development of leaf primordia (Fig. 4a-c). During flower bud differentiation, GmFILa was mainly expressed at the top of the flower bud primordia, carpel primordia, abaxial domains of bract and sepal (Fig. 4d, e). These expression patterns suggested that GmFILa plays an important role in the stimulation of lateral organs and subsequent growth of abaxial region.

Fig. 4
figure4

In situ hybridization of GmFILa in soybean leaves and flowers. a-c The longitudinal sections of leaf primordia. d-e The longitudinal sections of flower bud primordia. AN, leaf anlagen; Ab, abaxial; Ad, adaxial; P, leaf primordia; AM, apical meristem; Le, developing leaf; Br, bract; Fb, flower bud primordia; Se, sepal; Ca, carpel primordia. Bars:100 μm

Promoter cis-elements prediction and inducible expression analysis of GmFILa

The sequence analysis in GmFILa promoter region showed some cis-acting elements related to drought, light, and hormone (such as auxin and gibberellic acid) responses (Table 1). Besides, Dof transcription factor binding site was also found in the promoter region. Collectively, GmFILa might be induced by a variety of regulatory factors associated with stresses or hormones.

Table 1 Sequence analysis of GmFILa promoter

By investigating the microarray data, we found that the expression of GmFILa was up-regulated in leaf tissue after P.pachyrhizi inoculation (Fig. 5a) for 72 h. And its expression was down-regulated when treated with abiotic stresses like drought, salt and heat shock (Fig. 5b, c, e). Other stresses including metal ions (Fig. 5d, g) and alkaline (Fig. 5f) treatments could also induce the expression of GmFILa. Further, qRT-PCR was used to investigate the expression of GmFILa exposed to several treatments. As shown in Fig. 5h-k, GmFILa could be induced by polyethylene glycol (PEG), indole acetic acid (IAA), abscisic acid (ABA) and salicylic acid (SA), implying that GmFILa might be involved in responses to both stress and hormone treatments.

Fig. 5
figure5

Induction expression analysis of GmFILa. a-g Digital expression of GmFILa in different treatments from microarray data [70]. Y-axis represents the log2 ratios for the expression values. h-k qRT-PCR examination of GmFILa in response to drought (PEG) and three hormone treatments (IAA, ABA and SA). The expression of GmFILa at 0 h was used as a control (expression value = 1). Paired-samples t-test (two-tail) was selected for statistical analysis. * 0.01 < P < 0.05; ** P < 0.01

Phenotype investigation of Arabidopsis transformed with soybean GmFILa

The CDS of GmFILa was cloned into pBI121 vector to generate the construct 35S:GmFILa, and then the recombinant construct was transferred into the Arabidopsis using Agrobacterium-mediated transformation method. A total of 10 transgenic lines were obtained and three of which with relatively higher expression levels (Additional file 1: Figure S6) were thus further used for phenotype investigation. Compared with wild-type (WT) plants, all homozygous transgenic lines (10-day-old) from T4 generation exhibited outward curled cotyledons (Fig. 6a); then the growing leaves were curled from the adaxial side to abaxial side and became long-narrow (Fig. 6b-d), this phenotype became more obvious with the increase of the leaf ages (Fig. 6b-d). Further, measurement of leaf traits with 35-day-old seedlings indicated that the leaf number and length of transgenic Arabidopsis plants were significantly increased, while the leaf width was significantly reduced (Fig. 6h-j) compared with WT. In addition, the SAM of transgenic plants was slightly inhibited (Fig. 6e, f), and the flowering stage was clearly delayed (Fig. 6e, f); however, the plants can eventually bear fruits (Fig. 6g).

Fig. 6
figure6

Phenotypic analysis of GmFILa transgenic Arabidopsis plants. a The cotyledons of 10-day-old seedlings. b The leaves of 15-day-old seedlings. c The leaves of 25-day-old seedlings. d The phenotype of adaxial (left) and abaxial (right) axis of leaves. e-f Phenotype of 41-day-old wild and GmFILa transgenic plants. g Phenotype of GmFILa transgenic and WT plants during fruiting stage. h Leaf number statistics of the rosette leaves of 35-day-old seedlings. i Leaf length statistics of the rosette leaves of 35-day-old seedlings. j Leaf width statistics of the rosette leaves of 35-day-old seedlings. WT: Wild type; 35S:GmFILa: transgenic Arabidopsis with GmFILa gene. Paired-samples t-test (two-tail) was selected for statistical analysis. * 0.01 < P < 0.05; ** P < 0.01

As the morphological development of multicellular organisms depends on the cell morphology in the tissue layer, such as pavement cells of leaf epidermises, we thus initiated to compare the epidermal cell morphology of transgenic and WT plants. In the WT plants, the adaxial epidermal cells of rosette leaves were regular and uniform, while the abaxial cells were quite irregular (Fig. 7a). However, unlike WT, the adaxial surfaces of 35S:GmFILa transgenic plants are similar to the abaxial surfaces with irregular cell shapes, greatly varied cell sizes and disordered arrangement (Fig. 7a). Therefore, it may be concluded that the changes in epidermises led to the leaf curling phenotype in GmFILa transgenic Arabidopsis. Further, the paraffin section examination showed that 35S:GmFILa plants contained normal cell layers in mesophylls as with WT, indicating that overexpression of GmFILa in Arabidopsis does not affect the internal structure of leaves (Fig. 7b). Conclusively, we demonstrate that overexpression of soybean GmFILa causes partial abaxialization of adaxial leaf epidermises in Arabidopsis.

Fig. 7
figure7

Histological analysis of rosette leaves of WT and 35S:GmFILa transgenic Arabidopsis. a Observation of leaf epidermal cells in WT and GmFILa transgenic Arabidopsis plants. ab, abaxial epidermal cells; ad, adaxial epidermal cells. Bars: 100 μm. b Observation of the transverse section of rosette leaves in WT and 35S:GmFILa plants. ad, adaxial; ab, abaxial; pa, palisade mesophyll; sp., spongy mesophyll. Bars: 50 μm

Microarray analysis of GmFILa transgenic plants

Leaf transcriptomes of WT and GmFILa-overexpressing Arabidopsis plants were compared through microarray analysis. To validate the reliability of microarray data, nine probe sets (genes) were selected for qRT-PCR examination (Fig. 8). Our results showed that At4g39950/CYP79B2 (auxin biosynthesis), At5g13360 (auxin-responsive protein), At5g61600/ERF104 (ethylene response factor), At2g17500/PILS5 (auxin efflux carrier family protein), At1g02220/ANAC003 (NAC domain containing protein) and At5g67450/AZF1 (zinc-finger protein), were all up-regulated in 35S:GmFILa plants compared with WT, whereas At4g22620 (SAUR-like auxin-responsive protein) and At1g43160/RAP2.6 (ethylene response factor) were down-regulated, suggesting that these hormone biosynthesis and signal transduction related genes were directly or indirectly regulated by GmFILa. The relative expression levels of most examined genes generally agreed with the microarray data except for At1g06160, which had different expression changes in transgenic line 1 (Fig. 8).

Fig. 8
figure8

Verification of microarray data results using qRT-PCR. Nine DEGs were selected for confirming their relative expression levels in leaf tissue of GmFILa transgenic and WT Arabidopsis plants. Paired-samples t-test (two-tail) was used. * 0.01 < P < 0.05; ** P < 0.01

With P-value < 0.05 and |FC| ≥ 2, a total of 82 probe sets exhibited significant changes in transgenic lines, with 62 being up-regulated and 20 down-regulated, suggesting that transforming GmFILa into Arabidopsis affected expression of a number of endogenous genes (Additional file 3: Table S5). Based on the gene annotation, these DEGs could be grouped into different functional categories: transcription factors, transporters, and genes involved in growth and development, metabolism, signal transduction, redox reaction and stress response (Fig. 9a). Functional analysis showed that 13 (10%) of the DEGs were related to growth and development, including eight up-regulated genes and five down-regulated genes. At4g30410/IBL1 and At1g58340/AtZF14, which belongs to bHLH transcription factor and MATE transporter gene family, respectively, were all reported to negatively regulate plant cell elongation in Arabidopsis [43, 44]. At2g06850/AtXTH4 was demonstrated to have possible role in cell wall rigidification [45]. At2g37430/ZAT11, At2g22850/bZIP6 and At1g53700/WAG1 are several genes related to plant root development [46,47,48]. A NAC transcription factor gene, At3g15510/NAC2, not only regulates lateral root, flower and embryogenesis developments, but also responds to salt stress [49,50,51,52]. Several DEGs involved in auxin (At4g39950/CYP79B2; At2g17500/PILS5) [53, 54], jasmonic acid (At3g55970/ATJRG21) [55] and ethylene (At1g06160/ORA59; At5g61600/ERF104) [56, 57] signal pathways were up-regulated in GmFILa overexpressing Arabidopsis. Some metabolic pathway-related DEGs were up-regulated in transgenic Arabidopsis including At2g29470/GSTU3 (glutathione transferase) [58], At5g22300/NIT4 (nitrilase) [59] and At5g27420/CNI (ubiquitin ligase) [60]. Many genes related to biotic/abiotic stresses were also found to be up-regulated in transgenic Arabidopsis, such as At4g11650/AtOSM34 [61], At2g35980/AtNDR1 [62], At3g15510/AtNAC2 [51], and At5g67450/AtZF1 [63].

Fig. 9
figure9

Cluster analysis of DEGs in wild type and transgenic Arabidopsis plants based on microarray data. a The pie chart represents differentially regulated (P-value < 0.05 and |FC| ≥ 2) genes in different functional categories. Gene number was indicated in parenthesis. When some genes obtain more than one functional category, they would be counted in every corresponding category. Details were listed in Additional file 3: Table S5. b Gene Ontology classification of the DEGs between transgenic and WT plants. The X-axis is the definition of GO terms, and Y-axis is the percentage of genes mapped by the GO term. The blue represents “input list” and refers to DEGs; the green represents “background or reference,” which means background genes. The percentage for the input list is calculated by the number of DEGs mapped to the GO term divided by the number of all DEGs in the input list. The same calculation was applied to the reference list to generate its percentage. Details provided in Additional file 4: Table S6. c The KEGG enrichment pathways of DEGs in GmFILa transgenic Arabidopsis plants. The X-axis represents the enrichment degree, which is determined by the correct P-values; the Y-axis represents the pathway terms; the color of the spots indicates the enrichment factor, which represents the ratio of number of DEGs compared with genome background in a pathway, and the spots size represents the number of significant DEGs. The picture was drawn by local program. Details provided in Additional file 5: Table S7

Further, gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) analyses were used to identify the key processes for transgenic plants. 82 DEGs had corresponding GO annotations (Fig. 9b) and were significantly enriched in 18 GO terms (corrected P-value < 0.05; FDR) (Additional file 4: Table S6). Of the 18 GO terms, most of the DEGs’ encoded products were associated with “cell part” and “cell”, followed by “catalytic activity” and “stress response”, indicating that many of the DEGs might be involved in plant growth and development. Furthermore, 41 DEGs were classified as associated with 25 relevant KEGG pathways (Additional file 5: Table S7), which are directly or indirectly essential for plant growth and development. A total of three functional KEGG pathways were significantly enriched (P-value < 0.05) (Fig. 9c) including biosynthesis of secondary metabolites, sesquiterpenoid and triterpenoid biosynthesis, and glutathione metabolism. There were eight genes involved in secondary metabolism pathway. The nitrilase (nitrile aminohydrolase) hydrolyze indole-3-acetonitrile to the phytohormone indole-3-acetic acid in vitro, and the sites of nitrilase expression may represent the sites of auxin biosynthesis in Arabidopsis [64]. In our microarray data, one nitrilase gene (NIT4) was up-regulated, suggesting that auxin synthesis might be also influenced in transgenic plants. At4g39950/CYP79B2 (cytochrome P450), a critical enzyme in auxin biosynthesis in vivo, was up-regulated in GmFILa transgenic plants. Plants overexpressing CYP79B2 contain increased free auxin levels and thus exhibit a series of auxin overproduction phenotypes including long hypocotyls and epinastic cotyledons [53].

Discussion

Tissue expression patterns of soybean YABBY genes

Many plant YABBY genes have been studied to reveal their functions in regulating plant leaf [42], meristem [19, 65], flower organ [30], and fruit [25] development. As the tissue-specific gene expression pattern, to some extent, can reflect their potential functions, we thus investigated the expression patterns of all soybean YABBY genes in various tissues. Digital expression data showed that there existed two types of tissue expression characteristics among soybean YABBY genes. INO and CRC members were only expressed in several specific tissues and exhibited relatively lower expression level compared with the vegetative YABBY genes. This result suggests that the differential expression characteristics of YABBY genes may determine their different functions as shown in the phylogenetic tree. Duplicated genes tend to share common or similar functions, thus the tissue-specific expression of segmental duplicated genes was compared. Three pairs of duplicated genes (GmYABBY2 and GmYABBY4, GmYABBY13 and GmYABBY15, and GmYABBY9 and GmYABBY11) were all shown to have similar tissue expression patterns, which may indicate their functional redundancy.

Soybean GmFILa is involved in the establishment of abaxial-adaxial polarity in Arabidopsis leaf

YABBY gene family is responsible for the development of abaxial cell fate in lateral organs of Arabidopsis. Many YABBY genes in other plant species have been suggested with their functions in different various processes of growth and development [12, 14, 23, 26]. However, much less is reported about the functions of soybean YABBY genes during developmental processes. GmFILa, belonging to FIL/YAB3 subgroup, is closely clustered with three known YABBY genes (PapsFIL, VpYABBY1 and GRAM), which are all associated with leaf polarity and morphology development [13, 27, 42], suggesting that GmFILa may possess similar function to these homologs in leaf growth regulation. In situ hybridization analysis suggested that GmFILa was expressed in abaxial cell layers with the development of leaf primordia. Overexpressing GmFILa in Arabidopsis produced narrow and curled leaf morphology via altering the adaxial-abaxial polarity. This phenotype was also observed in Arabidopsis plants transformed with OsYABBY4 from rice [22], VpYABBY1 from Vitis pseudoreticulata [27], and BraYAB1–702 from Chinese cabbage [65]. These results may suggest that FIL homologs in angiosperm gained the conserved functions in the regulation of leaf development and the establishment of abaxial-adaxial polarity.

GmFILa might be involved in the development of soybean cco mutant

Soybean cco mutant shows a series of aberrant phenotypes compared with its wild type, including curled cotyledons, longer growth periods, reduced root systems, and small plants [41, 66]. RNA-seq and sqPCR analysis suggested that the transcript level of GmFILa was significantly increased in cco mutant than wild type in pod tissues at 7 DAF [41]. Also, our experiments showed that 35S:GmFILa transgenic Arabidopsis altered the cotyledons and leafs morphology, and delayed the flowering. These results, indirectly, indicate that GmFILa might be involved in the soybean cco mutant development, particularly in the regulation of cotyledon development and growth period.

Overexpression of GmFILa altered the expression of genes involved in growth and development

We used microarray data to explain the mechanism by which GmFILa affects the morphological phenotypes in transgenic Arabidopsis (Additional file 6: Table S8). The results showed that overexpression of GmFILa significantly altered the expression of a series of endogenous genes in Arabidopsis. There were 82 genes in total found to be significantly differentially expressed in transgenic lines. These differentially expressed genes were involved in growth and development, metabolism, signal transduction, redox reaction and stress response. Some of important growth and development related genes are worth mentioning, include transcription factors (ERF/AP2 transcription factor, NAC domain containing protein, C2H2 and C2HC zinc fingers superfamily protein) [46, 51], signal transduction components (protein-serine/threonine kinase, thioredoxin superfamily protein) [48, 67], hormone regulator (auxin synthesis protein, jasmonate-regulated protein) [53, 55], transporter (auxin efflux carrier family protein, MATE efflux family protein) [44, 54] and metabolism participants (nitrilase, RING type ubiquitin ligase) [59, 60]. At5g16440/IPP1, the isopentenyl/dimethylallyl diphosphate isomerase (IPI), was found to be up-regulated in transgenic Arabidopsis. IPI, catalyzing the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), was reported to regulate plant growth by using IPI-defective mutants: loss of two IPI genes confer dwarfism and male sterility in Arabidopsis plants under long-day conditions [68]. Taken together, GmFILa likely affects transgenic Arabidopsis phenotypes via affecting the expression of genes in different biological processes.

Interestingly, microarray data indicated that many DEGs (30%) in GmFILa transgenic plants were involved in stress response, suggesting that GmFILa might regulate the stress tolerance. Moreover, promoter cis-acting elements analysis, digital expression data and qRT-PCR all revealed that GmFILa could be induced by drought stress, we thus conducted drought experiment using 25-day-old WT and GmFILa transgenic plants. However, after 2 weeks of drought treatment, no significant differences in phenotypes were detected between WT and transgenic plants, all of them began to turn yellow and purple (Additional file 1: Figure S7). Therefore, more experiments involving drought and other abiotic stresses need to be conducted for verification.

Conclusion

In summary, our work provides the first insight into the functional role of soybean GmFILa gene. Ectopic expression of GmFILa causes alteration in leaf polarity, SAM development and flowering time; and influences the expression of 82 genes of different biological processes in Arabidopsis leaf. GmFILa might also be involved in plant stress tolerance. Further study with soybean transgenic plants will help us to gain a better understanding of the function of GmFILa.

Methods

Plant materials

Soybean seeds from cultivars Nannong 94–16 and Williams82 were provided by Soybean Research Institute, Nanjing Agricultural University, China. The ecotype Columbia-0 (Col-0) of Arabidopsis thaliana, kept in our laboratory, was used as wild type (WT).

Plant growth conditions

The soybean seeds (Nannong 94–16) were grown under field conditions at Jiangpu experimental station, Nanjing Agricultural University, Nanjing, China. Different tissues from various developmental stages were used to examine the expression pattern of GmFILa. Roots, stems and leaves were collected at the third euphylis expanding stage. Mature flowers were sampled at flowering stage. Seeds and pod shells were harvested at 20 days after flowering (DAF). All the samples were frozen in liquid nitrogen and then stored at − 80 °C for later RNA extraction.

Arabidopsis seeds were first incubated for 48–72 h at 4 °C and then grown in a growth room under conditions of 16/8 h light/dark, 23/22 °C, with 70% relative humidity. The leaf number, leaf length and leaf width of transgenic and wild Arabidopsis plants were measured using 35-day-old seedlings; 15 plants of each genotype were analyzed.

Isolation of the GmFILa gene

The coding sequence (CDS) of GmFILa was isolated from leaf tissue of soybean cultivar Nannong 94–16 via reverse transcription PCR (RT-PCR) with specific primers (Additional file 1: Table S2). Then the resulting fragment was cloned into pMD19-T vector (Vazyme, Nanjing, China) and sequenced for confirmation (Invitrogen, Shanghai, China).

Analyses of GmFILa protein characteristics and duplication pattern of YABBYs

Isoelectric points, protein molecular weights and other protein physicochemical properties were estimated using the ProtParam tool (http://web.expasy.org/protparam/) on the ExPASy proteomics server (http://expasy.org/). Tandem duplication was analyzed based on the method that the distance of two adjacent genes on the same chromosome is less than 200 kb [69]; and segmental duplication was predicted through Plant Genome Duplication Database (http://chibba.agtec.uga.edu/duplication/).

Phylogenetic and structural analyses

The protein sequences (Additional file 2: Table S4) of 17 soybean YABBYs and 29 published plant YABBY genes were aligned by ClustalW in MEGA Version 6.0 with the default parameters. A neighbor-joining (NJ) phylogenetic tree was constructed with MEGA 6.0 with the bootstrap of 1000 replications. Soybean YABBY genes were named based on the previous report [36]. Gene structures were drawn with the help of GSDS (http://gsds.cbi.pku.edu.cn/).

Digital expression data analysis

RNA sequencing (RNA-Seq) data, downloaded from SoyBase (http://www.soybase.org/soyseq/), was mainly used to identify the tissue expression of GmYABBYs. Soybean microarray expression data, downloaded from Plant Expression Database (http://www.plexdb.org) [70], was specially utilized to analyze the stress response expression patterns of GmFILa and other GmYABBYs.

RNA extraction and gene expression analysis

Total RNA was extracted using Plant RNA Extract Kit (TianGen, Beijing, China) according to the manufacturer’s instructions and cDNA was synthetized with M-MLV reverse transcriptase (TaKaRa, Dalian, China). Quantitative real-time polymerase chain reaction (qRT-PCR) was carried out with ABI 7500 system (Applied Biosystems, Foster City, CA, USA) using ChamQ™ SYBR qPCR Master Mix (Vazyme, Nanjing, China). The PCR was performed with the following parameters: 94 °C for 1 min and 40 cycles of 95 °C for 15 s, 60 °C for 15 s, 72 °C for 45 s followed by a final extension at 72 °C for 10 min. The relative expression levels of GmFILa were normalized using soybean endogenous gene tubulin (GenBank accession no. AY907703) and were estimated utilizing the 2-ΔΔCt method [71]. Semi-quantitative RT-PCR (sqPCR) was conducted with 2 × Hieff™ PCR Master Mix (Yeasen, Shanghai, China), and Arabidopsis tubulin gene (AT5G62690) was chosen as an internal control. The PCR protocol was 95 °C for 5 min and 30 cycles of 94 °C for 30 s, 56 °C for 40 s, 72 °C for 1 min followed by a final extension at 72 °C for 10 min. All the gene-specific primer pairs were listed in Additional file 1: Tables S2 and S3.

Subcellular localization assay of GmFILa protein

The full length of GmFILa coding region without a stop codon was inserted into the pBI121-GFP vector to produce the construct 35S:GmFILa-GFP. Both recombinant construct and empty vector 35S:GFP (control) were transferred to onion epidermal cells via particle bombardment method. Laser confocal microscopy (Leica TCS SP2, Mannheim, Germany) was used for image observation.

mRNA in situ hybridization

Sample (leaf and flower tissues from soybean Nannong 94–16) preparations and mRNA in situ hybridization were performed as previously described [72]. RNA antisense and sense probes were obtained from a 138 bp fragment of the 3′ region of the GmFILa cDNA labeled with digoxigenin.

Cis-acting elements in the GmFILa promoter region

A 2000 bp fragment upstream the ATG start codon of GmFILa was used to evaluate the cis-acting elements based on PLACE database (http://www.dna.affrc.go.jp/PLACE/) and Plant CARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).

Drought and hormone treatments

Soybean Williams82 cultivar was used for drought and hormone treatment experiments. Seeds growth condition and different treatments methods were conducted according to our previous study [73].

Ectopic expression in Arabidopsis

The GmFILa CDS was PCR-amplified and introduced into the pBI121 vector. This recombinant construct was transformed into Arabidopsis using Agrobacterium-mediated transformation following the floral dip method [74]. Transgenic plants were then screened on solid Murashige and Skoog (MS) medium containing 50 μg/ml kanamycin (Kana). Resistant seedlings were transferred to soil and further verified by PCR and sqPCR.

Arabidopsis leaf epidermal cells observation

The 25-day-old leaves of GmFILa transgenic and wild type Arabidopsis plants were stained with FM4–64 with concentration of 25 μg/ml. After 3 h of dyeing, the plant leaves were observed under confocal microscope (Leica TCS SP2, Mannheim, Germany).

Leaf paraffin section

The leaves of 25-day-old seedlings were collected from WT and transgenic plants, fixed in FAA (5% formalin, 5% glacial acetic acid and 90% ethanol) at room temperature for more than 24 h, and dehydrated via a graded ethanol series. Further, the samples were embedded in paraffin, sectioned at 6 μm (Leica, RM2135), and stained with safranin. Finally, the stained sections were observed and photographed with light microscope (Leica DMLB).

Microarray analysis

Leaves of wild Arabidopsis and GmFILa transgenic plants were sampled for RNA extraction using the tri-reagent (Invitrogen, Gaithersburg, MD, USA). RNA was cleaned using the NucleoSpin® RNA clean-up kit (MACHEREY-NAGEL, Germany), and RNA quality and quantity were assessed with ultraviolet spectrophotometer (NanoDrop Technologies, ND-1000) and formaldehyde agarose gel electrophoresis. Affymetrix Arabidopsis Gene Expression Microarray Chip was used for total mRNA hybridization, which was performed by CapitalBio Technology. Fragmentation, hybridization, and washing were carried out using the Hybridization, Wash, and Stain Kit (Affymetrix Technologies) according to the manufacturer’s protocol. Subsequently, the arrays were scanned using GeneChip® Scanner 3000, and images signals (.JPG format) were converted to the digital signals with AGCC software (Affymetrix®GeneChip® Command Console® Software). The data normalization was achieved by using RMA algorithm. Significantly differentially expressed genes (DEGs) between WT and transgenic plants were selected according to these criteria: (a) |Fold Change| (FC) ≥ 2, FC represents expression fold change between wild and transgenic plants; (b) the P-value < 0.05; (c) three biological replicates were performed.

Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis

All the significant DEGs were mapped to GO database using agriGO tool (http://bioinfo.cau.edu.cn/agriGO/analysis.php). The significantly enriched GO terms were evaluated based on P-value (< 0.05). KOBAS 2.0 program (http://kobas.cbi.pku.edu.cn) was used to identify the significantly enriched pathways (P-value < 0.05) in differently expressed genes compared with genome background.

Abbreviations

ABA:

Abscisic acid

AM:

Apical meristem

CaMV:

Cauliflower mosaic virus

CDS:

Coding sequence

Col-0:

Columbia-0

DAF:

Day after flowering

DEG:

Differentially expressed genes

GFP:

Green fluorescent protein

GO:

Gene ontology

h:

Hour

IAA:

Indole acetic acid

Kana:

Kanamycin

KEGG:

Kyoto encyclopedia of genes and genomes

MS:

Murashige and Skoog

NJ:

Neighbor-joining

PEG:

Polyethylene glycol

PI:

Isoelectric point

qRT-PCR:

Quantitative real-time polymerase chain reaction

RNA-seq:

RNA sequencing

RT-PCR:

Reverse transcription PCR

SA:

Salicylic acid

SAM:

Shoot apical meristem

sqPCR:

Semi-quantitative RT-PCR

WT:

Wild type

References

  1. 1.

    Juarez MT, Kui JS, Thomas J, Heller BA, Timmermans MC. microRNA-mediated repression of rolled leaf1 specifies maize leaf polarity. Nature. 2004;428(6978):84–8.

  2. 2.

    Chitwood DH, Guo M, Nogueira FT, Timmermans MC. Establishing leaf polarity: the role of small RNAs and positional signals in the shoot apex. Development. 2007;134(5):813–23.

  3. 3.

    Emery JF, Floyd SK, Alvarez J, Eshed Y, Hawker NP, Izhaki A, et al. Radial patterning of Arabidopsis shoots by class III HD-ZIP and KANADI genes. Curr Biol. 2003;13(20):1768–74.

  4. 4.

    Iwakawa H, Iwasaki M, Kojima S, Ueno Y, Soma T, Tanaka H, et al. Expression of the ASYMMETRIC LEAVES2 gene in the adaxial domain of Arabidopsis LEAVES represses cell proliferation in this domain and is critical for the development of properly expanded leaves. Plant J. 2007;51(2):173–84.

  5. 5.

    Eckardt NA. YABBY genes and the development and origin of seed plant leaves. Plant Cell. 2010;22(7):2103.

  6. 6.

    Pekker I, Alvarez JP, Eshed Y. Auxin response factors mediate Arabidopsis organ asymmetry via modulation of KANADI activity. Plant Cell. 2005;17(11):2899–910.

  7. 7.

    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.

  8. 8.

    Bowman JL, Smyth DR. 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.

  9. 9.

    Bowman JL. The YABBY gene family and abaxial cell fate. Curr Opin Plant Biol. 2000;3(1):17–22.

  10. 10.

    Kanaya E, Watanabe K, Nakajima N, Okada K, Shimura Y. Zinc release from the CH2C6 zinc finger domain of FILAMENTOUS FLOWER protein from Arabidopsis thaliana induces self-assembly. J Biol Chem. 2001;276(10):7383–90.

  11. 11.

    Sawa S, Okada K. FILAMENTOUS FLOWER controls the formation and development of Arabidopsis inflorescences and floral meristems. Plant Cell. 1999;11(1):69–86.

  12. 12.

    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.

  13. 13.

    Golz JF, Roccaro M, Kuzoff R, Hudson A. GRAMINFOLIA promotes growth and polarity in Antirrhinum leaves. Development. 2004;131(15):3661–70.

  14. 14.

    Han HQ, Liu Y, Jiang MM, Ge HY, Chen HY. Identification and expression analysis of YABBY family genes associated with fruit shape in tomato (Solanum lycopersicum L.). Genet Mol Res. 2015;14(2):7079–91.

  15. 15.

    Zhang XL, Zhang LG. Molecular cloning and expression of the male sterility-related CtYABBY1 gene in flowering Chinese cabbage (Brassica campestris L. ssp chinensis var. parachinensis). Genet Mol Res. 2014;13(2):4336–47.

  16. 16.

    Yang CJ, Kursel LE, Studer AJ, Bartlett ME, Whipple CJ, Doebley JF. A gene for genetic background in Zea mays: fine-mapping enhancer of teosinte branched1.2 to a YABBY class transcription factor. Genetics. 2016;204(4):1573–85.

  17. 17.

    Chen Q, Atkinson A, Otsuga D, Christensen T, Reynolds L, Drews GN. The Arabidopsis FILAMENTOUS FLOWER gene is required for FLOWER formation. Development. 1999;126(12):2715–26.

  18. 18.

    Lugassi N, Nakayama N, Bochnik R, Zik M. A novel allele of FILAMENTOUS FLOWER reveals new insights on the link between inflorescence and floral meristem organization and FLOWER morphogenesis. BMC Plant Biol. 2010;10(1):1–13.

  19. 19.

    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.

  20. 20.

    Tanaka W, Toriba T, Ohomori Y, Hirano HY. Formation of two florets within a single spikelet in the rice tongari-boushi1 mutant. Plant Signal Behav. 2012;7(7):793–5.

  21. 21.

    Tanaka W, Hirano HY. The YABBY gene TONGARI-BOUSHI1 is involved in lateral organ development and maintenance of meristem organization in the rice spikelet. Plant Cell. 2012;24(1):80–95.

  22. 22.

    Liu HL, Xu YY, Xu ZH, Chong K. A rice YABBY gene, OsYABBY4, preferentially expresses in developing vascular tissue. Dev Genes Evol. 2007;217(9):629–37.

  23. 23.

    Yang C, Ma Y, Li J. The rice YABBY4 gene regulates plant growth and development through modulating the gibberellin pathway. J Exp Bot. 2016;67(18):5545–56.

  24. 24.

    Huang Z, Knaap EVD. Tomato fruit weight 11.3 maps close to fasciated on the bottom of chromosome 11. Theor Appl Genet. 2011;123(3):465–74.

  25. 25.

    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.

  26. 26.

    Sun X, Guan Y, Hu X. Isolation and characterization of IaYABBY2 gene from Incarvillea arguta. Plant Mol Biol Rep. 2014;32(6):54144–8.

  27. 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.

  28. 28.

    Lin Z, Li X, Shannon LM, Yeh CT, Wang ML, Bai G, et al. Parallel domestication of the shattering1 genes in cereals. Nat Genet. 2012;44(6):720–4.

  29. 29.

    Wang Q, Reddy VA, Panicker D, Mao HZ, Kumar N, Rajan C, et al. Metabolic engineering of terpene biosynthesis in plants using a trichome-specific transcription factor MsYABBY5 from spearmint (Mentha spicata). Plant Biotechnol J. 2016;14(7):1619–32.

  30. 30.

    Alvarez J, Smyth DR. CRABS CLAW and SPATULA, two Arabidopsis genes that control carpel development in parallel with AGAMOUS. Development. 1999;126(11):2377–86.

  31. 31.

    Strable J, Wallace JG, Unger-Wallace E, Briggs S, Bradbury P, Buckler ES, et al. Maize YABBY genes drooping leaf1 and drooping leaf2 regulate plant architecture. Plant Cell. 2017;29(7):1622–41.

  32. 32.

    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.

  33. 33.

    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.

  34. 34.

    Ohmori Y, Abiko M, Horibata A, Hirano HY. A transposon, Ping, is integrated into intron 4 of the DROOPING LEAF gene of rice, weakly reducing its expression and causing a mild drooping LEAF phenotype. Plant Cell Physiol. 2008;49(8):1176–84.

  35. 35.

    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.

  36. 36.

    Zhao SP, Lu D, Yu TF, Ji YJ, Zheng WJ, Zhang SX, et al. Genome-wide analysis of the YABBY family in soybean and functional identification of GmYABBY10 involvement in high salt and drought stresses. Plant Physiol Bioch. 2017;119:132–46.

  37. 37.

    Toriba T, Harada K, Takamura A, Nakamura H, Ichikawa H, Suzaki T, et al. Molecular characterization the YABBY gene family in Oryza sativa and expression analysis of OsYABBY1. Mol Gen Genomics. 2007;277(5):457–68.

  38. 38.

    Min GE, Yuan-Da L, Zhang TF, Tan LI, Zhang XL, Zhao H. Genome-wide identification and analysis of YABBY gene family in maize. (in Chinese with English abstract). Jiangsu J of Agr Sci. 2014;30(6):1267–72.

  39. 39.

    Huang Z, Van Houten J, Gonzalez G, Xiao H, van der Knaap E. Genome-wide identification, phylogeny and expression analysis of SUN, OFP and YABBY gene family in tomato. Mol Gen Genomics. 2013;288(3–4):111–29.

  40. 40.

    Schmutz J, Cannon SB, Schlueter J, Ma J, Mitros T, Nelson W, et al. Genome sequence of the palaeopolyploid soybean. Nature. 2010;463(7278):178–83.

  41. 41.

    Shi G, Huang F, Yu G, Xu G, Yu J, Hu Z, et al. RNA-Seq analysis reveals that multiple phytohormone biosynthesis and signal transduction pathways are reprogrammed in curled-cotyledons mutant of soybean [Glycine max (L.) Merr.]. BMC Genomics. 2014;15(1):510.

  42. 42.

    Vosnakis N, Maiden A, Kourmpetli S, Hands P, Sharples D, Drea S. A FILAMENTOUS FLOWER orthologue plays a key role in leaf patterning in opium poppy. Plant J. 2012;72(4):662–73.

  43. 43.

    Zhiponova MK, Morohashi K, Vanhoutte I, Machemernoonan K, Revalska M, Van MM, et al. Helix-loop-helix/basic helix-loop-helix transcription factor network represses cell elongation in Arabidopsis through an apparent incoherent feed-forward loop. Proc Natl Acad Sci U S A. 2014;111(7):2824–9.

  44. 44.

    Wang R, Liu X, Liang S, Ge Q, Li Y, Shao J, et al. A subgroup of MATE transporter genes regulates hypocotyl cell elongation in Arabidopsis. J Exp Bot. 2015;66(20):6327–43.

  45. 45.

    Xu Z, Wang M, Shi D, Zhou G, Niu T, Hahn MG, et al. DGE-seq analysis of MUR3-related Arabidopsis mutants provides insight into how dysfunctional xyloglucan affects cell elongation. Plant Sci. 2017;258:156–69.

  46. 46.

    Liu XM, An J, Han HJ, Kim SH, Lim CO, Yun DJ, et al. ZAT11, a zinc finger transcription factor, is a negative regulator of nickel ion tolerance in Arabidopsis. Plant Cell Rep. 2014;33(12):2015–21.

  47. 47.

    Lee JY, Colinas J, Wang JY, Mace D, Ohler U, Benfey PN. Transcriptional and posttranscriptional regulation of transcription factor expression in Arabidopsis roots. Proc Natl Acad Sci U S A. 2006;103(15):6055–60.

  48. 48.

    Santner AA, Watson JC. The WAG1 and WAG2 protein kinases negatively regulate root waving in Arabidopsis. Plant J. 2006;45(5):752–64.

  49. 49.

    Wellmer F, Riechmann JL, Alves-Ferreira M, Meyerowitz EM. Genome-wide analysis of spatial gene expression in Arabidopsis flowers. Plant Cell. 2004;16(5):1314–26.

  50. 50.

    Mandaokar A, Thines B, Shin B, Markus Lange B, Choi G, Koo YJ, et al. Transcriptional regulators of stamen development in Arabidopsis identified by transcriptional profiling. Plant J. 2006;46(6):984–1008.

  51. 51.

    He XJ, Mu RL, Cao WH, Zhang ZG, Zhang JS, Chen SY. AtNAC2, a transcription factor downstream of ethylene and auxin signaling pathways, is involved in salt stress response and lateral root development. Plant J. 2005;44(6):903–16.

  52. 52.

    Kunieda T, Hara-Nishimura I. NAC family proteins NARS1/NAC2 and NARS2/NAM in the outer integument regulate embryogenesis in Arabidopsis. Plant Cell. 2008;20(10):2631–42.

  53. 53.

    Zhao Y, Hull AK, Gupta NR, Goss KA, Alonso J, Ecker JR, et al. Trp-dependent auxin biosynthesis in Arabidopsis: involvement of cytochrome P450s CYP79B2 and CYP79B3. Genes Dev. 2002;16(23):3100–12.

  54. 54.

    Barbez E, Kubeš M, Rolčík J, Béziat C, Pěnčík A, Wang B, et al. A novel putative auxin carrier family regulates intracellular auxin homeostasis in plants. Nature. 2012;485(7396):119–22.

  55. 55.

    Nickstadt A, Thomma BP, Feussner I, Kangasjärvi J, Zeier J, Loeffler C, et al. The jasmonate-insensitive mutant jin1 shows increased resistance to biotrophic as well as necrotrophic pathogens. Mol Plant Pathol. 2004;5(5):425–34.

  56. 56.

    Pré M, Atallah M, Champion A, Vos MD, Pieterse CMJ, Memelink J. The AP2/ERF domain transcription factor ORA59 integrates jasmonic acid and ethylene signals in plant defense. Plant Physiol. 2008;147(3):1347–57.

  57. 57.

    Bethke G, Unthan T, Uhrig JF, Pöschl Y, Gust AA, Scheel D, et al. Flg22 regulates the release of an ethylene response factor substrate from MAP kinase 6 in Arabidopsis thaliana via ethylene signaling. Proc Natl Acad Sci U S A. 2009;106(19):8067–72.

  58. 58.

    Wagner U, Edwards R, Dixon DP, Mauch F. Probing the diversity of the Arabidopsis glutathione S-transferase gene family. Plant Mol Biol. 2002;49(5):515–32.

  59. 59.

    Piotrowski M, Schönfelder S, Weiler EW. The Arabidopsis thaliana isogene NIT4 and its orthologs in tobacco encode β-cyano-L-alanine hydratase/nitrilase. J Biol Chem. 2001;276(4):2616–21.

  60. 60.

    Sato T, Maekawa S, Yasuda S, Sonoda Y, Katoh E, Ichikawa T, et al. CNI1/ATL31, a RING-type ubiquitin ligase that functions in the carbon/nitrogen response for growth phase transition in Arabidopsis seedlings. Plant J. 2009;60(5):852–64.

  61. 61.

    Hao J, Wu W, Wang Y, Yang Z, Liu Y, Lv Y, et al. Arabidopsis thaliana defense response to the ochratoxin A-producing strain (Aspergillus ochraceus 3.4412). Plant Cell Rep. 2015;34(5):705–19.

  62. 62.

    Zheng MS, Takahashi H, Miyazaki A, Hamamoto H, Shah J, Yamaguchi I, et al. Up-regulation of Arabidopsis thaliana NHL10 in the hypersensitive response to Cucumber mosaic virus infection and in senescing leaves is controlled by signalling pathways that differ in salicylate involvement. Planta. 2004;218(5):740–50.

  63. 63.

    Kodaira KS, Yamaguchi-Shinozaki K. Arabidopsis Cys2/His2 zinc-finger proteins AZF1 and AZF2 negatively regulate abscisic acid-repressive and auxin-inducible genes under abiotic stress conditions. Plant Physiol. 2011;157(2):742–56.

  64. 64.

    Bartel B, Fink GR. Differential regulation of an auxin-producing nitrilase gene family in Arabidopsis thaliana. Proc Natl Acad Sci U S A. 1994;91(14):6649–53.

  65. 65.

    Zhang XL, Yang ZP, Zhang J, Zhang LG. Ectopic expression of BraYAB1-702, a member of YABBY gene family in Chinese cabbage, causes leaf curling, inhibition of development of shoot apical meristem and flowering stage delaying in Arabidopsis thaliana. Int J Mol Sci. 2013;14(7):14872–91.

  66. 66.

    Yu J, Han S, Shi G, Yu D. Characterization of a novel curled-cotyledons mutant in soybean [Glycine max (L.) Merr.]. Afr J Biotechnol. 2012;11(83):14889–98.

  67. 67.

    Huang LJ, Ning L, Thurow C, Wirtz M, Hell R, Gatz C. Ectopically expressed glutaredoxin ROXY19 negatively regulates the detoxification pathway in Arabidopsis thaliana. BMC Plant Biol. 2016;16(1):200.

  68. 68.

    Okada K, Kasahara H, Yamaguchi S, Kawaide H, Kamiya Y, Nojiri H, et al. Genetic evidence for the role of isopentenyl diphosphate isomerases in the mevalonate pathway and plant development in Arabidopsis. Plant Cell Physiol. 2008;49(4):604–16.

  69. 69.

    Yang S, Feng Z, Zhang X, Jiang K, Jin X, Hang Y, et al. Genome-wide investigation on the genetic variations of rice disease resistance genes. Plant Mol Biol. 2006;62(1–2):181–93.

  70. 70.

    Sudhansu D, John VH, Lu H, Wise RP, Dickerson JA. PLEXdb: gene expression resources for plants and plant pathogens. Nucleic Acids Res. 2012;40(D1):D1194–201.

  71. 71.

    Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods. 2001;25(4):402–8.

  72. 72.

    Coen ES, Romero JM, Doyle S, Elliott R, Murphy G, Carpenter R. floricaula: a homeotic gene required for flower development in antirrhinum majus. Cell. 1990;63(6):1311–22.

  73. 73.

    Yang H, Shi G, Du H, Wang H, Zhang Z, Hu D, et al. Genome-wide analysis of soybean LATERAL ORGAN BOUNDARIES domain-containing genes: a functional investigation of GmLBD12. Plant Genome. 2017;10(1). https://doi.org/10.3835/plantgenome2016.07.0058.

  74. 74.

    Mara C, Grigorova B, Liu Z. Floral-dip transformation of Arabidopsis thaliana to examine pTSO2::β-glucuronidase reporter gene expression. J Vis Exp. 2010;40:1952.

Download references

Acknowledgements

Not applicable.

Funding

This work was financially supported in part by National Natural Science Foundation of China (31571688, 31871649), Ministry of Science and Technology (2016YFD0101005) and Key Transgenic Breeding Program of China (2016ZX08004–003, 2016ZX08009003–004). The funders had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

Availability of data and materials

The microarray data relating to this article has been added to the additional material.

Author information

This study was designed by FH and DY. HY and GS conducted the experiments and analyzed the expression profile data. HY wrote this manuscript. FH and GS revised the manuscript. XL, DH, YC and JH assisted with doing the experiments. All authors read and approved the final manuscript.

Correspondence to Fang Huang.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Additional files

Additional file 1:

Figure S1. Chromosomal distribution and duplication of soybean YABBY genes. Figure S2. GmYABBYs gene structure analysis. Figure S3. Digital tissue expression profiles for soybean YABBY genes. Figure S4. Expression of eight soybean YABBY genes in different tissues/organs based on Plant Expression Database. Figure S5. PCR amplification of GmFILa CDS from soybean leaf. Figure S6. Identification of GmFILa transgenic Arabidopsis plants. Figure S7. Drought tolerance examination of GmFILa transgenic and wild type Arabidopsis plants. Table S1. Duplication analysis of the 17 soybean YABBY genes. Table S2. Primer pairs of GmFILa used for experiments. Table S3. Primer pairs of Arabidopsis genes used for experiments. (PDF 3710 kb)

Additional file 2:

Table S4. Protein sequences of YABBY genes in various plants. (XLSX 14 kb)

Additional file 3:

Table S5. Annotation of 82 DEGs between WT and GmFILa transgenic Arabidopsis plants. (XLSX 21 kb)

Additional file 4:

Table S6. The 93 GO terms associated with 82 genes. (XLSX 40 kb)

Additional file 5:

Table S7. The 25 KEGG pathways associated with 41 genes. (XLSX 39 kb)

Additional file 6:

Table S8. The microarray data (RMA normalized). (XLSX 1398 kb)

Rights and permissions

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Keywords

  • Soybean
  • YABBY
  • GmFILa
  • Leaf
  • Adaxial-abaxial polarity
  • Arabidopsis