Analysis of the B. napus orthologs of A. thaliana RLKs
The RLK family has been well characterized in A. thaliana, its total member was up to 624 [18, 19, 35]. As the first step to analyze their
B. napus orthologs, the Arabidopsis RLK genes were downloaded from TAIR according to the former report . The Arabidopsis
RLK family was divided into 66 subfamilies on the basis of their difference in both
the kinase sequences and domain compositions , the number of the members varied
dramatically between different subfamilies, the largest subfamily was LRR-III consisted of 47 members, whereas, 10 subfamilies only contained 1 member, respectively (Supplementary Table S1).
Then the B. napus orthologs of A. thaliana RLKs were retrieved from Brassica database, and further identified and confirmed by BLASTP and kinase domain analysis.
The gene name and E-value of the B. napus orthologs of A. thaliana RLKs were listed in Supplementary Table S1. Their total number was 2,583, more than 4.13-fold
larger than that of AtRLKs. However, B. napus orthologs of different Arabidopsis RLKs had different expansion degrees, for example, the most expanded was AT4G21380, the
number of its B. napus orthologs was 31, whereas, the number of B. napus ortholog of 28 AtRLKs had no expansion after the A. thalian-B. napus split (Fig. 1). Besides, 56 AtRLKs had no B. napus orthologs, especially all members of Error_region1 subfamily. Totally, most of AtRLKs (452/624) had 2 to 6 B. napus orthologs (Fig. 1, Supplementary Table S1).
Differentially expressed RLKs after pollination in A. thaliana and B. napus
Matsuda et al.  had characterized genes expressed in Arabidopsis stigmatic papilla
cells pre- and post-pollination, including un-pollinated (UP), compatible pollinated (CP) and incompatible pollinated (IP) papilla cells at
60 min after pollination (termed UP, CP60 and IP60, respectively). They defined differentially
expressed genes (DEGs) as genes with an RPKM value showing a ≥ 3-fold change in expression
at a P-value ≤ 0.05, and obtained 779 DEGs in CP60 and IP60 data sets compared with UP .
Here, we obtained 20 differentially expressed AtRLK genes via screening of Arabidopsis RLK family members in these DEGs. Among them,
15 were up-regulated and 2 were down-regulated in CP60 vs. UP, 12 were up-regulated
and 1 was down-regulated in IP60 vs. UP. Totally, 9 were commonly up-regulated and
1 was down-regulated in both CP60 vs. UP and IP60 vs. UP (Fig. 2, Supplementary Table
Zhang et al.  had explored time-course gene expression during compatible and incompatible pollen-stigma interactions in B. napus stigmas, including un-pollinated (UP), compatible pollinated (CP) and incompatible
pollinated (IP) stigmas at 2, 5, 10, 20, and 30 min after pollination (termed CP2,
CP5, CP10, CP20, CP30; IP2, UP5, IP10, IP20 and IP30, respectively). The DEGs (log2
fold changes ≥ 1 and a FDR ≤ 0.01) at all stages of pollination had been analyzed
. In current study, we downloaded all the DEGs data sets. To following Matsuda’s
restrictive conditions, we had screened differentially expressed B. napus AtRLK orthologs with a ≥ 3-fold change. Finally, we obtained 129 B. napus AtRLK orthologs from the DEGs, 57, 57, 57, 63 and 65 were respectively up-regulated in CP2,
CP5, CP10, CP20 and CP 30 vs. UP, whereas only 10 and 5 were down-regulated in CP20
vs. UP and CP30 vs. UP. Besides, 56, 56, 62, 61and 74 were respectively up-regulated
in IP2, IP5, IP10, IP20 and IP30 vs. UP, 1, 2 and 48 were down-regulated in IP2, IP20
and IP30 vs. UP (Fig. 2, Supplementary Table S2). Totally, 67 B. napus AtRLK orthologs were commonly up-regulated and 8 were commonly down-regulated in IP vs.
UP and CP Vs. UP (Supplementary Table S2).
129 B. napus orthologs corresponded to 79 AtRLKs. Compared with that in A. thaliana, 106 B. napus orthologs, corresponding to 59 AtRLKs, were only detected in B. napus DEGs data sets (Supplementary Table S2). 23 B. napus orthologs and their corresponded 10 AtRLKs were respectively contained in B. napus and A. thaliana DEGs data sets, and their expression level change tendency following pollination were identical in these two species. In addition, 10 AtRLKs were only found in A. thaliana DEGs data sets (Supplementary Table S2). Totally, no matter in A. thaliana or in B. napus, the number of the up-regulated RLKs in both CP vs. UP and IP vs. UP were far more than that of the down-regulated RLKs (Fig. 2, Supplementary Table S2). Especially in CP2, CP5 and CP10 vs. UP, the number
of the up-regulated B. napus orthologs were all 57, whereas, none was down-regulated. Only at the time point of
30 min after incompatible pollination, the down-regulated B. napus orthologs were amount to 48, but still less than the number of the up-regulated, 74.
Expression patterns of the differentially expressed AtRLKs in A. thaliana
In the following sections, the differentially expressed B. napus AtRLK orthologs were represented by their corresponded AtRLKs to facilitate further bioinformatics analysis. Combined with that only found in A. thaliana DEGs data sets, the total number of differentially expressed AtRLKs was 89 (Supplementary Table S2). Klepikova et al.  had analyzed global gene expression
during development of A. thalina in 79 samples covering many stages, from embryogenesis to senescence, and diverse
organs. We had downloaded the expression data of the 89 AtRLK genes (Supplementary Table S3), and found that Klepikova et al.  had not detected
AT4G39110 gene presented in all samples. Then we analyzed its expression via eFP Browser
(Arabidopsis eFP Browse 2.0), found it was preferentially expressed in mature pollen.
Subsequently, we constructed the heat map for the reminding 88 AtRLK genes expression in those 79 samples (Fig. 3). There were only 29 AtRLKs preferentially expressed in opened anthers (F.AN) and in anthers of mature flower
before opening (F.AN.ad), all these genes grouped together in one branch. In addition, AT4G25390 enriched
in F.AN and F.AN.ad, and AT5G46080 enriched in F.AN and petiole of the senescent leaf (L.PET.sn) (Fig. 3, Supplementary Table S3). Among the rest 57 AtRLKs, the most had only a very low expression level in F.AN and F.AN.ad, especially that some of them were almost not present in these two samples, such
as AT1G21230, AT3G24790, AT4G23130 and AT5G60900 (Fig. 3, Supplementary Table S3).
Besides, AT2G39660, AT5G61560 and AT2G41820 only highly enriched in stigmatic tissue
(STI) (Fig. 3, Supplementary Table S2, S3). Excluding that only found in A. thaliana DEGs data sets, B. napus orthologs of 79 AtRLKs showed obvious fold changes of their expression level at different time points after pollination (Supplementary Table S2).
Pina et al.  had analyzed the transcriptome of Arabidopsis hydrated pollen, leaves, seedlings and siliques. Here, we had download the gene expression data, and defined gene as hydrated pollen-enriched
gene if its expression level was at least 2-fold higher than that in the reminding
three tissues. Finally, we totally got 1,234 hydrated pollen-enriched genes (Supplementary
Table S4). Among them, only 29 AtRLKs also co-existed in the differentially expressed AtRLKs after pollination(Fig. 4). Compared with the former 29 AtRLKs preferentially expressed in F.AN and F.AN.ad (Fig. 3), 28 were identical, and the differentwere AT4G39110 and AT3G26940. AT4G39110 significantly enriched in hydrated pollen in comparison to leaves, seedlings and siliques in the Pina et al.  study, was
not detected in all 79 samples in Klepikova et al.  analysis. Conversely, AT3G26940
preferentially expressed in F.AN and F.AN.ad in Klepikova et al.  analysis, was not present in the 4 tissues used in the Pina
et al.  study. Among all these 30 F.AN and F.AN.ad preferentially expressed or hydrated pollen-enriched AtRLKs, 4were only up-regulated in CP60 or IP60 vs. UP in A. thaliana (Supplementary Table S2). The expression level of the rest 26AtRLKs (or their B. napus orthologs) obviously varied at different time points after pollination, even if comparing with
that at the time point of 2 min post-pollination, 1 was down-regulated and 25 were
up-regulated (Supplementary Table S2).
Comparison of the differentially expressed AtRLKs after pollinaiton with that during in vitro pollen germination and pollen tube growth
Wang et al.  had analyzed the transcriptome of A. thaliana mature pollen (MP), in vitro hydrated grains (HP) and pollen tubes grown (PT), they designed transitions from
MP to HP and from HP to PT as PG and PTG, and finally obtained 326 DEGs (fold changes > 1.63, P-value < 0.01) during PG, 1,490 DEGs during PTG, respectively. In this study, we had
screened AtRLKs with a ≥ 3-fold change from DEGs, and obtained only 1 differentially expressed AtRLK during PG and 19 during PTG (Supplementary Table S5). Their number were far less than
that of the differentially expressed AtRLKs after pollination.
Among all the differentially expressed AtRLKs after pollination and during in vitro pollen germination and pollen tube growth, no AtRLK showedobvious expression level changes both during PG and after pollination. Only 7 AtRLKs were shared by DEGs during PTG and after pollination, accounting for about 7.87%
and 36.84% of the differentially expressed AtRLKs after pollination and during PTG, respectively (Fig. 5). Of these 7 AtRLKs, both AT4G25390 and AT3G24790 were up-regulated during PTG and after compatible pollination.
AT4G13190 was up-regulated during PTG and after incompatible pollination. AT2G33580
and its B. napus ortholog were up-regulated during PTG and after compatible and incompatible pollination
(Supplementary Table S2, S5). AT3G23750 was up-regulated during PTG, but its B. napus ortholog was down-regulated after incompatible pollination. AT1G16760 and AT3G20530
were downloaded during PTG, but their B. napus orthologs were up-regulated after compatible and incompatible pollination (Supplementary
Table S2, S5).