Adult A. capillus-veneris plants were cultivated in greenhouses at Peking University (Beijing, China). Spores were collected and cultivated into cordate prothalli in sugar-free Knop’s agar medium as described (Li et al., 2013). For the sugar treatment, cordate prothalli were picked up with sterile hypodermic needles under anatomical lens and placed on Knop’s agar medium containing various sugar concentrations. When both antheridia and archegonia appeared, prothalli were sprinkled with sterile water to create appropriate conditions for fertilization. All prothallus cultivation experiments were conducted in a clean bench.

Total RNA from all samples was extracted with the PureLink^TM Plant RNA Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The total RNA samples were then treated with RQ1 RNase-Free DNase (Promega, Madison, WI, USA) to remove DNA contaminants. Each sample was reverse-transcribed into cDNA using the SuperScript First-Strand System for RT-PCR (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. The quality of the RNA and cDNA was assessed by agarose gel electrophoresis. The RNA concentration was determined using a NanoDrop ND 1000 Spectrophotometer (Nano-Drop, Wilmington, DE, USA). qRT-PCR was performed with a 7500 Real-Time PCR System (Applied Biosystems, USA). The amplification reaction was carried out in a total volume of 20 μL, with 0.5 μL of each primer (10 μM), 1 μL of cDNA, 10 μL of Power SYBR Green I Master Mix kit (Bio-Rad, Hercules, CA, USA), and 8 μL RNase-free water. The PCR program was as follows: denaturing at 95°C for 10 min, followed by 40 two-step cycles (95°C for 15 s and 60°C for 64 s) and a final extension at 72°C for 5 min. Relative quantification of each gene was performed using the comparative threshold cycle method as described by Livak and Schmittgen (2001). It is reported that AcCRYPTOCHROME GENE 2 (AcCRY2) stayed at the same level through the haploid and diploid phases (Imaizumi et al., 2000), thus we used AcCRY2 as the internal control. Each sample was quantified at least in triplicate. The primer sets for each gene are listed in Supplementary Table 1.

Prothalli and pinnae at various stages were fixed in 4% paraformaldehyde (PFA) overnight at 4°C. After fixation, tissue samples were washed, dehydrated, and embedded in wax for sectioning and in situ hybridization as described by Zhang et al. (2013). AcLEC1-specific regions were amplified with primer sets 5′-GAAGATAGCAGATGATGCCAAGG-3′ and 5′-ATGAATCCCCCCGATACTACTAA-3′ and transcribed in vitro as probes using the Digoxigenin RNA labeling kit (Roche, Mannhein, Germany).

Based on a report of sugar-dependent LEC1 expression in A. thaliana (Tsukagoshi et al., 2007), A. capillus-veneris prothalli were treated transiently with sucrose or glucose. Cordate prothalli were immersed in buffer containing 30 g/L glucose for 30 min. Sugar-free buffer served as control. After rinsing in water, the treated cordate prothalli were transferred to a sugar-free medium. Prothalli were harvested 30 min, 1, 2 h, 1, 2, and 6 days after treatment followed by qRT-PCR to determine AcLEC1 expression levels.

Under sterile conditions, cordate prothalli growing on sugar-free medium were picked and transferred to medium containing 30 g/L glucose or 30 g/L sucrose. Sugar-free medium served as the control treatment. Prothalli were harvested 5, 10, 15, 20, and 25 days after transfer (DAT) for examination of AcLEC1 gene expression by qRT-PCR.

The dissected samples were dehydrated, followed by submersion in a series of gradient alcohol-isoamyl acetate solutions as described previously (Li et al., 2013). Subsequently, the samples were dried by critical-point drying in CO₂ (Hitachi HCP-2) for 6 h, mounted, and sputter-coated with gold/palladium (Hitachi E-1010). All samples were viewed under a Hitachi S-4800 SEM at 10.0 kV.

Fertilized prothalli with apparently swollen archegonia were selected as samples. This method ensured that every prothallus sample was under similar developmental stage. For each treatment group, five fertilized prothalli were selected as samples and were observed under SEM. For each sample, three 25-mm² area were randomly picked, in which the number of antheridia and archegonia were counted and the density was calculated. The diameter of mature archegonia or antheridia on the prothallus was measured by taking plotting scale as reference.

The control and sugar-treated prothalli were harvested 10, 20, and 30 DAT, dehydrated in a series of ethanol solutions, followed by exposure to a series of alcohol-acetone solutions as describe (Li et al., 2013). Next, the tissue samples were exposed to a series of acetone-Spurr’s resin solutions (acetone:resin ratio: 2:1, 1:1, 1:2, 0:1, 0:1, and 0:1), which were replaced every 8 h. Finally, the samples were embedded in Spurr’s resin. Sections (3 μm thick) were cut using a microtome (Leitz 1512, Germany) as described previously (Hu and Xu, 1990; Li et al., 2013). The periodic acid-Schiff (PAS) staining, Sudan black B staining, and Coomassie brilliant blue staining were chosen to label polysaccharides, lipids, and protein bodies, respectively, as described by Hu and Xu (1990). Images were captured with a light microscope (Zeiss Axioskop 2 Plus, Germany) using Axioplan software.

In Arabidopsis, 184 seed-specific genes, excluding 30 genes that encode transcription factors, are known to be involved in storage products accumulation (Le et al., 2010). We screened the datasets of seed-specific genes (Mu et al., 2008; Le et al., 2010) and selected the genes involved in storage products accumulation by gene annotation. After gaining the gene candidates, we screened the A. capillus-veneris expressed sequence tags (ESTs) database by BLAST to find out the high identities ESTs of A. capillus-veneris. The genes with identities higher than 50% were considered to be storage products accumulation related genes in A. capillus-veneris. The three high identities genes were listed in Supplementary Table 2.

Previously, Li et al. (2013) have described the entire life cycle of A. capillus-veneris under cultivate conditions on the duration and morphogenetic characteristics. To clarify the expression pattern of AcLEC1 during the life cycle of A. capillus-veneris, it is necessary to further divide the life cycle into development stages with distinguishable morphological characteristics for unambiguous sample collection. Based on the morphogenetic features, we divided the life cycle of A. capillus-veneris into 11 stages (Figure 1).

It needs to be mentioned that the above dividing system for developmental stages was mainly designed for the convenience of clarifying the expression pattern of AcLEC1. In general, it is more reasonable to set the zygote as a start point to describe a life cycle (Bai, 1999, 2015a,b, 2016, 2017). However, for the convenience of experimental operation, here we used spores as a start point. It should be better to adjust the start point into zygote if it becomes more accessible along with technology development. Another pragmatic consideration is that the above dividing system did not further divide prothallus into more stages although several morphologically distinguishable stages can be easily identified, such as club-shaped, early heart-shaped and so on (Li et al., 2013). Such a simplification mainly because of the growth condition for these stages are similar in terms of their effects on AcLEC1 expression.

To test the hypothesis that the specific spatiotemporal pattern of LEC1-type HAP3 gene expression is critical for proper execution of the seed maturation process, we examined the expression pattern of AcLEC1 in A. capillus-veneris. Samples of 10 stages during the entire life cycle and stem were collected according to Figure 1. Quantitative reverse transcription-PCR (qRT-PCR) was employed to determine AcLEC1 expression levels. Figure 2A shows that AcLEC1 mRNA was rarely detectable in the prothallus samples and 1-leaf-sporophyte samples. In contrast, AcLEC1 mRNA was detected at various levels in the stem and pinna at various stages. The highest expression level was detected in the sample of phase 3 pinna where sporangia forms (Figures 1, 2A). Such expression pattern is consistent to previous finding that LEC1 genes are expressed in aerial tissues and at high level in sporangia (Kirkbride et al., 2013).

To further examine the preference of AcLEC1 expression in difference tissues, in situ hybridization was carried out. Signal of AcLEC1 mRNA was detected from juvenile to phase 3 pinna (Figures 2B–F). Consistent to the highest expression level detected with qRT-PCR, the in situ hybridization confirmed the highest signals in phase 3 pinna (Figures 2E,F). It is interesting that no AcLEC1 mRNA is detected after spore release in phase 4 pinna (Figures 2A,G).

The above data demonstrated that under normal growth conditions, there is no AcLEC1 expression in prothallus development (Figure 2A). Therefore, we can use prothalli to examine whether sugar can induce AcLEC1 expression.

In our pilot experiments, we found 20 and 30 g/L sugar can induce AcLEC1 expression but 50 g/L can inhibit prothalli growth. So we used concentration of 30 g/L as a treatment. Firstly, we examined whether the AcLEC1 expression can be transiently induced. Figure 3A shows that AcLEC1 expression can be induced upon 30 min treatment, higher than the control. Such expression is dependent on existence of the sugar (Glucose here), as the expression decrease upon withdraw of the sugar.

To observe the effects of sugar treatments on prothallus development, we further examined the AcLEC1 expression in prothallus development under continuous sugar-medium culture. Considering the developmental process from spore germination to cordate prothallus taking 20 days and from cordate prothallus to fertilization taking about 25 days (Li et al., 2013), here we examined AcLEC1 expression every 5 days starting from the cordate prothallus culture. Figure 3B shows that AcLEC1 gene expression was significantly induced after 10-days culture on the sugar media, both sucrose and glucose. Interestingly the expression level on sucrose medium showed one peak at 15-days, but that on glucose medium showed two peaks, one at 10-days culture and another at 20-days culture.

To verify the AcLEC1 expression during prothallus development, in situ hybridization was carried out. Strong signals of AcLEC1 probe were detected in prothalli, especially in archegonia and antheridia (Figures 3C–F).

To examine the effects of sugar treatments on prothallus development, we analyzed the differentiation status of prothallus development. As described in Li et al. (2013), prothallus development goes through filament, clavate, broadened and cordate stages after spore germination. Following cordate stage, differentiation of prothallus mainly exhibits as initiation and differentiation of antheridia and archegonia (Li et al., 2013). As the prothalli were cultured from young cordate stage, we monitored the rate of initiation of antheridia and archegonia under sugar treatments. Figures 4A–D show typical differentiation status of prothalli, in which A shows the cordate prothallus without antheridium or archegonium initiation; B shows antheridium initiation (red arrowhead pointed); C shows both antheridium (pointed by red arrowhead) and archegonium initiation (pointed by black arrow); and D shows opened archegonia with embryogenesis (magnified in the inset). For convenience, each status from A to D framed with different colors.

Using the above morphological criteria, percentage of correspondent differentiation status in all the examined prothalli were counted (Figure 4E). On control medium, majority of cultured prothalli entered the stage B (antheridium initiation) in 5 DAT. In 15 DAT, all prothalli entered the stage C. In comparison, the differentiation process slowed down on 30 g/L sucrose medium, indicated by larger proportions of 5 DAT prothalli retaining at stage A and 10 DAT prothalli retaining at stage B. The differentiation of prothalli was more severely postponed on the 30 g/L glucose medium.

While the differentiation rate was decreased upon the sugar treatment, the densities of antheridia and archegonia were increased. Based on SEM observation, we can count the number of antheridia and archegonia for density calculation. Figures 5A–C (five panels in each lane represent the differentiation status of antheridia and archegonia at 10, 20, 30, 40, and 50 DAT) shows that comparing to the sugar-free Knop’s culture, density of both antheridia and archegonia increases. The quantitation of the densities at the fertilization stage (40 DAT) were shown in Figure 5D.

In addition, although no distinguishable alteration in architecture of antheridium nor archegonium were found, the size of archegonia and antheridia were increased, significantly for antheridia (Figures 5E,F respectively).

Among the three physiological and biochemical processes consisting the seed maturation process, the accumulation of storage products is the most prominent characteristic. To examine whether the sugar treatments can trigger the accumulation of storage products, we analyzed the accumulation of representative storage macromolecules, e.g., polysaccharides, proteins, and lipids during prothalli development by histochemical stain.

Figures 6A–C show the accumulation of polysaccharides with PAS stain during prothalli development at three different stages (10, 20, and 30 DAT), and on three different mediums, i.e., the sugar-free Knop’s (as control), 30 g/L sucrose and 30 g/L glucose. It is obvious that no PAS signals were detected at 10 DAT prothalli on all three mediums. However, after 20 DAT, obvious PAS signals were detected on the sugar-treated prothalli, not on the control prothalli. The strongest staining was detected on the prothalli at 30 DAT on the sucrose medium (Figure 6B, 30 DAT). This suggests the unambiguous accumulation of polysaccharides in the prothalli under sugar treatment.

Figures 6D–F show the accumulation of protein with Coomassie brilliant blue stain during prothalli cultured under sugar treatments. While not that obvious signals detected for storage protein as did for starch in prothalli under sugar treatments, the signals (stained to blue) were more intensive in sugar-treated prothalli than control.

Figures 6G–I show the accumulation of lipid with Sudan black B stain. The typical lipid signal by Sudan black B stain should be gray-blue and the background should be sky blue or blue (Supplementary Figure 1A). The signals in sugar-treated prothalli is much intensive than control (Supplementary Figures 1B,C).

To further explore whether the accumulation of storage products in prothalli grown on sugar mediums is similar to that in seed maturation process, we carried out a molecular analysis.

Firstly, we used three Arabidopsis seed-specific genes, SUCROSE-PHOSPHATE SYNTHASE (SPS), CRUCIFERIN2 (CRU2), and FATTY ACID ELONGASE1 (FAE1), as queries to screen A. capillus-veneris homologs from NCBI EST database. These three genes are respectively involved in accumulation of starch (SPS), storage protein (CRU2) and lipid (FAE1). The ESTs homologous in A. capillus-veneris to these three genes were therefore designated as AcSPS, AcCRU2, and AcFAE1, respectively. Afterward, we analyzed the expression pattern during prothallus development.

Figure 7 shows that consistent to the accumulation of storage macromolecules in the sugar-treated prothalli, the AcSPS expression was significantly induced in the prothalli on the sucrose medium at 20 DAT (Figure 7A). The induction of AcCRU2 is highest in the prothalli on the glucose medium at 20 DAT (Figure 7B). High induction of AcFAE1 was also observed in the prothalli on the sucrose medium, but delayed to 25 DAT (Figure 7C).