Soybean plants [G. max (L.) Merrill] (Williams 82) were grown under natural conditions in the field at the Agricultural Experiment Station of Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Changchun, China. The Arabidopsis zou mutant zou-4 (Col-0 background) allele was obtained from the Gabi-Kat collection of T-DNA inserts (Rosso et al., 2003) and corresponded to GABI_584D09. Arabidopsis seeds were surface-sterilized, vernalized at 4°C for 3 days, and then allowed to germinate and grow on plant growth medium in the growth chamber maintained under a 16-h light/8-h dark (22–18°C) photoperiod. Soil-grown plants were also maintained under the same conditions.

Total RNA was isolated from soybean developing seeds under normal growth conditions using a Plant Total RNA Extraction Kit (Bioteke Corporation) and first strand cDNA was prepared from 0.5 μg of total RNA primed with an oligo-dT primer using an AMV reverse transcription system (Takara) according to the manufacturer’s instructions. The genes were cloned using PrimeSTAR HS DNA Polymerase (Takara). Two PCR primers, OL0085 and OL0086, were designed to amplify the GmZOU-1 gene. A 937-bp PCR fragment was amplified, and the PCR reactions were performed as follows: 98°C for 3 min and then 30 cycles of 98°C for 10 s, 55°C for 15 s, and 72°C for 1 min. Primers OL0087 and OL0088 were designed to amplify the GmZOU-2 gene. A 830-bp PCR fragment was amplified and the PCR reactions were performed as follows: 98°C for 3 min and then 30 cycles of 98°C for 10 s, 55°C for 15 s, and 72°C for 50 s. 3′ A overhangs were attached to the PCR fragments by using EasyTaq (TransGenBiotech) before their cloning into a pMD18-T vector (Takara).

The ZOU protein sequences from different species with similarities to AtZOU were mined from Phytozome V11¹ and GenBank (National Institutes of Health genetic sequence database). The amino acid sequences were aligned using CLUSTALW (Thompson et al., 1994). We limited the boundaries of the sequences. In total, 21 sequences (Supplementary Dataset 1) were analyzed. Phylogenetic reconstruction of the ZOU genes was performed using the neighbor-joining (NJ) method (Saitou and Nei, 1987). The bootstrap consensus tree inferred from 1,000 replicates was used to represent the evolutionary history of the analyzed taxa (Felsenstein, 1985). Branches corresponding to partitions reproduced in less than 50% bootstrap replicates were collapsed. The percentage of replicate trees in which the associated taxa were clustered together in the bootstrap test (1,000 replicates) was shown next to the branches (Felsenstein, 1985). The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances that were used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. All positions containing alignment gaps and missing data were eliminated only in pairwise sequence comparisons (pairwise deletion option). A total of 451 positions were included in the final dataset. Phylogenetic analyses were conducted using MEGA4 (Tamura et al., 2007).

In soybean, total RNA was extracted from different tissues, including root, stem, leaf, inflorescence, unopened flower, opened flower, and seeds at 5 DAP (globular stage), 9 DAP (early heart stage), 12 DAP (late heart stage), and 16 DAP (cotyledon stage; Jones and Vodkin, 2013). To obtain the endosperm and embryo separately from soybean seeds, the seeds were dissected transversely (slightly out of the middle toward the micropylar end) using forceps and a microtome blade (Leica). The embryo was picked out by forceps and the endosperm was separated with integument using a scalpel knife under the stereo microscope (Olympus SZX7), and material was flash frozen in liquid nitrogen. In Arabidopsis, total RNA was extracted from siliques with seeds developing to the heart stage (about 5 DAP). The total RNA was treated with DNase I (Takara) according to the manufacturer’s instructions. RNA concentrations were determined by using a Nanodrop Spectrophotometer (NanoDrop Technologies). First-strand cDNA was prepared from 0.5 μg of total RNA primed with oligo-dT primer using an AMV reverse transcription system (Takara). PCR reactions were prepared using SYBR Premix EX Taq (Takara) and each 25 μl reaction was triplicated (technical replicates) and for each experiment three biological replicates (i.e., independent plant samples or different plants in the same transgenic line) were made. According to the manufacturer’s protocol, the following program was used: 10 min at 95°C, followed by 40 cycles of 95°C for 10 s and 60°C for 1 min. The products were quantified using a Bio-Rad DNA Engine Opticon 2 real-time PCR machine and an associated software to assay SYBR green fluorescence. Expression levels were calculated using ACTIN11 (Soybean), which is a commonly used reference gene with stable expression in different tissues (Hu et al., 2009), and EIF4A1 (Arabidopsis) in developing seed as described (Yang et al., 2008; Xing et al., 2013; Grimault et al., 2015). The primers used are listed in Supplementary Table S1.

To construct the pAtZOU::GmZOU-1 and pAtZOU::GmZOU-2 expression vectors, cloned GmZOU-1 and GmZOU-2 CDS in pMD18-T vectors in the correct orientation were digested with SalI and SmaI and cloned into the vector pRT101 that was digested by XhoI and HincII, thereby constructing the vectors ZYH051 and ZYH052. ZYH051 and ZYH052 were then digested with PstI, and the gene fragments were separately cloned into a binary vector pCAMBIA1300 to construct the vectors, ZYH053 and ZYH054. A 1.6-kb region upstream from the AtZOU start codon was amplified by PCR (as described earlier) using primers ZOUSALIF and ZOUSALIR and subcloned into vector pSC-B (Stratagene). The vector containing AtZOU promoter was digested with SalI, and the promoter fragment was then cloned into the binary vectors ZYH053 and ZYH054, thereby constructing the pAtZOU::GmZOU-1 and pAtZOU::GmZOU-2 expression vectors ZYH055 and ZYH056. All recombinant plasmids identified from individual Escherichia coli colonies were verified by sequencing, the expression vectors described above were introduced into Agrobacterium tumefaciens GV3101 independently for plant transformation. Plant transformations were conducted according to Clough and Bent (1998). Plants were dipped twice in a solution of 10% sucrose, 0.5 × MS salts, and 0.05% silwet L-77 solution containing GV3101 Agrobacterium cells that were grown for 48 h.

Seeds from the transformed plants were screened on MS agar (Sigma) containing 2% sucrose, 20 mg L^-1 hygromycin (Roche) and 100 mg L^-1 cefotaxime (Sangong). Resistant plants were transferred to pots with soil and perlite (3:1 v/v) and grown to maturity in a growth chamber. Hygromycin-resistant plants were screened for the presence of the transgene by PCR using soybean GmZOUs-specific primers (above). Seeds were harvested and sown on MS agar containing 2% sucrose, 20 mg L^-1 hygromycin to obtain homozygous plants. Homozygous plants were transferred to soil and were grown for seed production. Seeds from these homozygous plants were used in the subsequent experiments.

The Toluidine Blue (TB) test was carried out following published procedures (Tanaka et al., 2004; Xing et al., 2013). Seeds were spread uniformly on 15-cm plates containing 1 × MS Basal Salts (Sigma), 0.3% sucrose, and 0.4% Phytagel (Sigma; pH 5.8). Stratification was conducted at 4°C for 3 days before transferring plates to a growth chamber for 7 days. Lids were removed and plates were immediately flooded with the staining solution [0.05% (w/v) TB + 0.4% (v/v) Tween-20] for 2 min. The staining solution was poured off, and the plates were immediately rinsed gently by flooding under a running tap until the water cleared (1–2 min). Seedlings were photographed, or harvested for TB quantification. To harvest, seedlings were removed individually from plates and both roots and any adhering seed coats (both of which stain darkly with TB) were completely removed before plunging the hypocotyl and cotyledons into 1 ml of 80% ethanol. Seedlings were incubated with continuous shaking for 2 h, until all blue color and chlorophyll had been removed from cotyledons. The resulting liquid was analyzed using a spectrophotometer.

To visualize and stage the developing seeds, siliques were opened with needles. The seeds were then cleared overnight in modified Hoyer’s solution (chloral hydrate: water: glycerol in proportions 8 g : 2 mL : 1 mL) and visualized under DIC optics using a Nikon Eclipse E600 microscope.

To identify the ZOU homologs in soybean, the TBLASTN program was used to query the deduced amino acid sequences of gene models from the soybean genomic sequence database (Phytozome) with that of AtZOU. The two highest scores were obtained for the genes models Glyma.02G103200 and Glyma.02G103100, and the corresponding genes were designated as GmZOU-1 and GmZOU-2, respectively. Alignment of the amino acid sequences of the GmZOU-1 and GmZOU-2 with that of AtZOU revealed the presence of a basic helix-loop-helix (bHLH) DNA-binding domain (pfam00010) and a conserved C-terminal domain (Figure 1A). Phylogenetic analysis of the all 480 bHLH family proteins in soybean and the AtZOU protein showed that both GmZOUs were in the same phylogenetic clade with AtZOU (Supplementary Figure S1).

The two copies of GmZOUs identified in soybean were located on Gm02: 9,795,987–9,799,196 (Accession Number Glyma.02G103200) and Gm02: 9,784,634–9,786,935 (Accession Number Glyma.02G103100), which is adjacent to the chromosome with the same transcription orientation. The chromosome fragment in soybean chromosome 2 containing 13 genes around the GmZOUs was aligned with the chromosome fragment containing AtZOU from the Arabidopsis genome. There were five genes in this selected Arabidopsis chromosome region that corresponded to seven homologs in this soybean chromosomal segment that contained two genes that were tandemly duplicated. In addition, these homologs showed synteny, as indicated by the same order and orientation (Figure 1B). The two chromosomal regions showed similar evolutionary origin, and the two copies of GmZOUs were the result of tandem gene duplication.

To identify the phylogenetic relationships and functional conservation of different species during evolution, the homolog of the ZOU gene in other species, including A. thaliana, salt cress (Thellungiella halophila), soybean (Glycine max), lucerne (M. truncatula), poplar (Populus trichocarpa), cassava (Manihot esculenta), rice (Oryza sativa), corn (Zea mays), sorghum (Sorghum bicolor), pine (Pinus taeda), spruce (Picea sitchensis), selaginella (Selaginella moellendorffii), and moss (Physcomitrella patens), were downloaded and filtered. Only full-length sequences and those with at least 30% similarity with the AtZOU gene were included in the analysis. A total of 21 sequences were selected and used in the subsequent NJ phylogenetic analysis. A strict consensus tree was also generated (Figure 2).

The phylogenetic tree showed that ZOU is widely conserved in plants. Within angiosperms, ZOU was found in both plants that develop persistent endosperm and plants that do not as well as in seeded plants lacking endosperm (e.g., P. sitchensis, a gymnosperm). It was also found in more basal vascular plant groups, such as the moss (e.g., P. patens) and ferns (e.g., S. moellendorffii), which lack seeds altogether. Besides that the ZOU genes in gymnosperms such as P. sitchensis and P. taeda had a closer relationship with dicotyledonous species compared to monocotyledonous species. This may be related to the specific function of the ZOU gene, which regulated the degeneration of maternal material during gametogenesis. In monocotyledonous species, the gametogenesis evolved in a specific manner wherein the maternally derived nutrients are stored in the endosperm until seed germination. Although ZOU in monocotyledonous species, like AtZOU is involved in the local lysis of endosperm to allow embryo growth, the bulk of persistent endosperm implies some functional alterations. Recently the potential functional specificity of ZmZOU unique to monocotyledon had also been shown in maize (Grimault et al., 2015).

The expression profiles of the GmZOUs in both vegetative and reproductive soybean organs, including roots, stems, leaves, inflorescences, unopened flowers, opened flowers and developing seeds with embryos in different stages were examined by qPCR analysis. The results showed that both GmZOUs were highly expressed in developing seeds and preferentially at the heart stage. GmZOU-2 was also detected in the inflorescences, unopened flowers, and opened flowers (Figure 3A).

In order to study the spatial-specific expression pattern of GmZOUs in developing seeds, gene expression analysis had been carried out on dissected seed compartments. The embryo and endosperm were separately isolated from developing seeds at late heart stage or cotyledon stage. In order to prevent contamination, the expression of embryo specific genes (Glyma03g03500 and Glyma14g35560) and endosperm specific genes (Glyma07g02220 and Glyma08g21890; Danzer et al., 2015) was also checked in these dissected seed compartments (Supplementary Figure S2). Upon verification, GmZOUs expression was detected by qPCR analysis. The results showed that both of GmZOUs were highly expressed in endosperm whereas no expression was detected in embryo (Figure 3B). The similar expression pattern with AtZOU implied functional conservation of GmZOUs in seed development.

To confirm that GmZOUs are involved in endosperm breakdown, expression vectors with GmZOU-1 and GmZOU-2 under the control of the AtZOU promoter (AtZOUpro-GmZOUs) were separately transformed into Atzou-4 mutants. After resistance screening, presence of exogenous AtZOU promoter-driven GmZOU-1 or GmZOU-2 constructs was checked in the transgenic lines containing the T-DNA insertions. The Atzou-4 mutant background of the GmZOU-1 and GmZOU-2 transgenic lines was then confirmed by PCR analysis based on the T-DNA insertion site. Furthermore, the expression of exogenous GmZOUs was also detected by qPCR (Figure 4E). Background identification and exogenous GmZOUs expression analysis identified a total of four GmZOU-1 transgenic lines and four GmZOU-2 transgenic lines, which were then used in the subsequent analysis. Among these transgenic lines, the mature seeds in the GmZOU-1 transgenic lines rescued the Atzou-4 shriveled seed phenotype into wild-type-like or malformed seeds (Figures 4A,C), whereas the mature seeds in the GmZOU-2 transgenic lines showed no distinct phenotypic differences from that of Atzou-4 mutant seeds (Figures 4B,D).

To study the embryo and endosperm growth in these transgenic lines, the developing seeds at about 10–12 days after pollination (DAP) were observed. The Col-0 wild-type embryos were in the mature stage showing a completely bent embryo, and the endosperm had already undergone complete breakdown with only one cell layer surrounding the embryo (Figures 5A,F); the embryo growth in Atzou-4 mutant was restricted at the heart/torpedo shaped embryo, and the endosperm failed to undergo a breakdown, with cellularized endosperm persisting (Figures 5B,G). The seeds of the GmZOU-2 transgenic lines showed similar features as that of the Atzou-4 mutant, with an embryo arrested in the heart/torpedo stage andx showing a sustained cellularized endosperm (Figures 5E,J). On the contrary, the embryo of the GmZOU-1 transgenic lines showed elongated and bending, with the semi-bent embryo almost occupying the entire embryonic sac (Figures 5C,D); and the endosperm showed breakdown in GmZOU-1 transgenic lines resulting in a reduction in the amount of persistent endosperm compared with Atzou-4 mutant (Figures 5H,I). The findings of seed development observation confirmed that the GmZOU-1 partially recovered embryo expansion and endosperm breakdown in Atzou-4 mutants, whereas GmZOU-2 did not influence Atzou-4 mutant seed development.

Furthermore, we also studied the embryonic cuticle in the transgenic lines as the cuticle integrity in zou was not intact with breaks in epidermal. TB, a hydrophilic dye, was used to detect the hydrophobic cuticle and epidermal defection (Tanaka et al., 2004). The seedlings of Col-0, Atzou-4, and the GmZOUs transgenic lines at about 7 days after germination were stained with TB and visualized under a dissecting microscope. Cotyledons of the Atzou-4 mutants showed strong staining, while the cotyledons of Col-0 wild-type had no visible permeability to TB. The transgenic GmZOU-2 cotyledons also showed strong staining similar to that in the Atzou-4 mutants, whereas the GmZOU-1 transgenic lines were weakly stained (Figure 5K). To further investigate this phenotype, TB uptake by seedlings was quantified by using a spectrophotometer. The results were similar to that of microscopy analysis (Figure 5L). These findings indicate that the GmZOU-1 transgenic plants also partially rescued cuticle formation, whereas GmZOU-2 did not.

The expression of presumed ZOU target genes, including ALE1. At3g06890. At5g03820, and At4g33600 (Kondou et al., 2008; Yang et al., 2008; Xing et al., 2013) were detected in the GmZOUs transgenic lines (Figure 6). The ZOU target genes At3g06890 and At4g33600 showed almost full recovery in the GmZOU-1 transgenic lines. The mRNA levels of the putative direct target ALE1 (Yang et al., 2008; Xing et al., 2013) increased sevenfold and threefold in the GmZOU-1 transgenic lines compared with levels observed in untransformed Atzou-4 mutants but reached only 30 and 13%, respectively, of the level observed in Col-0. Similarly, the expression of target gene At5g03820 in the GmZOU-1 transgenic lines reached only 17 and 9.3%, respectively, relative to that in Col-0. On the other hand, the expression of these target genes did not differ in the GmZOU-2 transgenic lines compared to that in the Atzou-4 mutant.

These results showed that the downregulated genes in the Atzou-4 mutant underwent partial recovery in the GmZOU-1 transgenic lines, whereas these did not change in the GmZOU-2 transgenic lines. In the GmZOU-1 transgenic lines, the partially recovered expression of these target genes explained the observed partial recovery of the Atzou-4 mutant phenotype. The less efficient recovery of the ZOU target genes such as ALE1 and At5g03820 implied that part of the ZOU target genes requires higher expression levels or complete ZOU function. Nonetheless, our data confirmed that GmZOU-1 is a functional ortholog of AtZOU because both soybean and Arabidopsis ZOU induced the upregulation of ZOU presumed target genes, which have been associated with endosperm breakdown and embryo cuticle development.

Searching the soybean genomic sequence database identified two AtZOU homologs in soybean, which were then further confirmed by sequence alignment and chromosome synteny analysis. The results also confirmed that the AtZOU homologs resulted from tandem duplication. To confirm functional conservation, the GmZOUs were expressed in Arabidopsis Atzou-4 mutants and only GmZOU-1 could complement the Atzou-4 mutant phenotype with endosperm breakdown, embryo expansion, and cuticle formation. Additionally, the homologs showed expression patterns that were similar to that of AtZOU, which indicated that GmZOU-1 is the ZOU ortholog in soybean. GmZOU-1 only partially complemented the Atzou-4 mutant, which was indicative of species specificity or functional diversity during evolution.

GmZOU-1 (Glyma.02G103200) and GmZOU-2 (Glyma.02G103100) resulted from tandem duplication with the same transcription orientation. Sequence alignment indicated that GmZOU-1 consisted of a classical bHLH DNA binding domain at the N terminus and a conserved functional domain at the C terminus compared to AtZOU. By contrast GmZOU-2 only had the conserved function domain, whereas the bHLH domain had lost the conserved amino acids which may influence its DNA binding capability. Transgenic analysis indicated that GmZOU-2 did not influence the Atzou-4 mutant in terms of embryonic arrest at the heart stage, sustaining the endosperm, and cuticle formation deficiency. Additionally, considering its less specific expression pattern, we concluded that GmZOU-2 is the product of gene duplication and may have lost its original function during the course of evolution.

The cuticularization of juxtaposed surfaces has been shown to be extremely important in defining organ boundaries. Mutants with compromised cuticles often show extensive organ fusions (Kurdyukov et al., 2006; Xing et al., 2013). In the GmZOU-1 transgenic lines, ALE1 expression was partially recovered, and TB staining showed that the cuticle layer of the embryonic cotyledons did not show complete recovery. The embryo with the defective cuticle readily fused with the endosperm, similar to that observed in other embryo cuticle-deficient mutants such as ale1. gso1/gso2, and zou (Tanaka et al., 2001; Tsuwamoto et al., 2008; Yang et al., 2008). We inferred that the observed organ fusion suppressed embryo expansion. The combination of organ fusion in the embryo and the arrest of endosperm degradation restricted embryonic development, which resulted in the reverse bending of the embryo and a severe malformation phenotype (Figure 5). These mechanisms may explain the development of malformed embryos in the GmZOU-1 transgenic seeds.