Comprehensive analysis of the differentially expressed receptor-like kinase genes in responding to pollination in A. thaliana and B. napus

Successful sexual reproduction in flowering plant requires extensive communications between male and female organs and tissues. Although RLKs have been proved to play critical roles in these communications, so far the identified RLKs are still limited. Here we performed a comprehensive analysis on differentially expressed RLKs in responding to pollination in Arabidopsis thaliana and Brassica napus to contibute to further analysis on RLKs’ function in male-female communication. Result In this study, a total of 2,583 B. napus AtRLK orthologs were obtained. 89 AtRLKs showed obvious expression level changes after pollination in A. thaliana or in B. napus. Although 30 differentially expressed AtRLKs were opened anthers and anthers of mature flower before opening preferentially expressed or hydrated pollen-enriched, up to 79 AtRLKs corresponding to 129 B. napus orthologs showed obvious expression level changes at different time points after pollination. Among 89 differentially expressed AtRLKs after pollination, only 7 AtRLKs were shared by differently expressed genes during in vitro pollen tube growth, 3 of 7 AtRLKs’ expression level change tendency after pollination and during PTG were different.


Backgroud
The stigma of flowing plants is the receptive portion of the pistil for pollination [1,2]. Brassicaceae species have a typical dry stigma, which lacks of free-flowing secretions on its surface [3][4][5]. This dry type stigma has evolved sophisticated mechanisms to discriminate between compatible and incompatible pollen for successful fertilization. Once pollen lands on the dry stigma, the incompatible pollen cannot get water from the stigma, so its hydration and germination is inhibited, only compatible pollen can induce the dry stigma to release water and other factors, and can fully hydrate and germinate [5][6][7][8][9]. After compatible pollen germination, it must experience the following sequential processes, including pollen tube formation, penetration the intercellular space of the stigma, growth down through the transmitting tissue of the style, entry into the ovule and discharge of the sperm cells to execute double fertilization [10,11]. Double fertilization is a unique characteristic of angiosperms, which requires a series of complicated communications between male and female reproductive organs and tissues [12][13][14][15][16][17].
Arabidopsis thaliana genome contains more than 620 RLK members that represents nearly 2.5% of A.
thaliana protein coding genes [18,19], Oryza sativa has nearly twice as many RLK members as A.
thaliana does [35]. Amount to 76 AtRLK genes are preferentially expressed in semi-in vivo growth pollen tubes in A. thaliana [28]. Thus, whether other RLKs also function in the communications between male and female tissues are still need to be further investigated. Now, the availability of genome sequences for some crucifer species are very helpful for genome-wide characterization of RLK family members: A. thaliana [36], Brassica napus [37]. Furthermore, genetic basis for A. thaliana transition from outcrossing to selfing had been revealed,it can be reverted to full SI by transformation with SRK-SCR from its relative A. lyrata and A. halleri [38][39][40][41][42]. Using transgenic SI plants, Matsuda et al. [43] had reported the characterization of gene expression profiles of un-pollinated (UP), compatible pollinated (CP) and incompatible pollinated (IP) papilla cells in Arabidopsis. Besides, independent mutation in SCR caused the self-compatibility in B. napus line "Westar", which transgenic line by complementing the function of SCR showed strong SI [44,45]. Using this ideal material, Zhang et al. [46] had explored the gene expression in B. napus stigmas during compatible and incompatible pollen-stigma interactions. Releases of these transcriptional data provided an excellent opportunity for a comprehensive analysis of differentially expressed genes in response to pollination in these two species. In this study, the differentially expressed AtRLKs and B. napus AtRLK orthologs after pollination were screened at the transcriptional level in A. thaliana and B. napus, the expression pattern of the differentially expressed AtRLKs in A. thaliana were analyzed, the differentially expressed AtRLKs during in vitro pollen germination and pollen tube growth were obtained, and then were compared with thatafter pollination. Finally, all our results showed that 89 AtRLKs were differentially expressed in responding to pollination in A. thaliana and B. napus, which should be mainly caused by pollen-stigma interaction.

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 [35]. The Arabidopsis RLK family was divided into 66 subfamilies on the basis of their difference in both the kinase sequences and domain compositions [35], 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  Zhang et al. [46] 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 [46]. 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 [49] 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 Table S2).

, Supplementary
Pina et al. [47] 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 pollenenriched 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  Table S2).
Comparison of the differentially expressed AtRLKs after pollinaiton with that during in vitro pollen germination and pollen tube growth 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  Table S2, S5).

Discussion
The dry stigma of Brassicaceae species could accept compatible pollen while reject self-incompatible pollen from the same species or pollen from unrelated species, the polarized secretion in the dry stigmatic papillae cells after pollination is a highly regulated process [5,[7][8][9]. Unlike sperm cells in animals, the sperms in flowering plants are immobile, they must be exactly delivered to the ovuleenclosed female gametophyte in the ovary by guided pollen tube growth for successful double fertilization [16,17]. All these mentioned processes require extensive communications between male and female organs and tissues. Although several RLKs had been proved to play key roles in these communications [16,17,22,23], a family-wide analysis on RLK genes expression level changes after pollination reminded to be performed. To improve this situation, here we finished a comprehensive analysis on differentially expressed RLKs in responding to pollination in A. thaliana and B. napus, aiming to provide useful informtaion for further analysis on RLKs' function in male-female communication.
Differentially expansion of B. napus AtRLK orthologs The B. napus genome contained about 101,040 genes, 3.96 times larger than that of A. thaliana [36,37]. Here, we found the number of B. napus AtRLK orthologs was over 4.13-fold larger than that of Arabidopsis RLKs. This should be not a simple consequence of the predicted gene number in B. napus genome lager than that in A. thaliana, for the reason that the number of B. napus orthologs varied dramatically between different Arabidopsis RLKs (Fig. 1, Supplementary Table S1). The divergence of Arabidopsis and Brassica had occurred about 14.5 to 20.4 million years ago [50], B. napus was formed about 7,500 years ago by hybridization between B. rapa and B. oleracea, followed by chromosome doubling [37]. It had been proved that both tandem and large-scale duplications mainly contributed to the expansion of the RLKfamily within Arabidopsis, while tandem duplications played a major role in the RLK expansion in rice [19,35]. Thus, although we had not analyzed the mechanism for RLKfamily expansion in B. napus, we thought RLK family should undergo their independent evolution and expansion after A. thaliana-B. napus split, which might result in the dramatic variation of B. napus orthologs numbers of different Arabidopsis RLKs.

Differentially expressed RLKs after pollination in A. thaliana and B. napus
Matsuda et al. [43] had analyzed the differentially expressed genes in Arabidopsis papillae cells at 60 min post pollination, but some processes had finished at this time point, including polarized secretion in stigmatic papillae, pollen adhesion, hydration, germination and penetration [51][52][53]. Fortunately, Zhang et al. [46] had explored gene expression in B. napus stigmas at multiple time points after pollination, which was very helpful for the analysis of the consecutive changes of gene expression during the early stages of pollen-stigma interaction. Here, we made a comprehensive analysis on the differentially expressed AtRLKs and B. napus AtRLK orthologs after pollination. We found that some differentially expressed AtRLKs were only detected in A. thaliana, some only in B. napus, which maybe be caused by the different time points used in these two studies. However, when AtRLKs and its B. napus orthologs were detected in A. thaliana and B. napus DEGs data sets, their change tendencies were identical (Fig. 2, Supplementary Table S2). We also found that the number of up-regulated RLKs after pollination was far larger than that of the down-regulated, no matte after compatible or incompatible pollination in both A. thaliana and B. napus, which meant some biological processes should be activated by pollination, as we known, the SI signaling cascade, polarized secretion and pollen hydration, and so on (Fig 2, Supplementary Table S2).
After compatible pollination, a violent change of RLKs occurred at 2 min, and then the number of the varied RLKs had no change at 2, 5 and 10 min, subsequently, a moderate increase was detected at 20 and 30 min, a drastic reduction was finally detected at 60 min. This change tendency of the number of varied RLKs was in accordance with the morphologic observations. Secretory activity in stigmatic papillae of A. thaliana and B. napus was rapidly induced by compatible pollen [53]. In addition, both A. thaliana and B. napus compatible pollen had hydrated at 4 min post pollination [51,52]. Therefore, multiple processes including pollen adhesion, foot formation, polarized secretion and hydration should have occurred within 4 min, the violent changes of RLKs at 2 min should mainly be involved in those processes. Hydration would continue to 10 min following pollination [51], it might be the main reason for no change of the varied RLKs number at 2, 5 and 10 min. Following, the pollen tube had emerged at 20 min post pollination, and the pollen tube penetrated the papillae cell wall within 30 min after pollination [53][54][55], the interaction between stigma and the emerged pollen tube might result in the moderate increase of the number of the varied RLKs during corresponding stages. During 30 min to 60 min, the former processes had completed and pollen tube continued to grow, which may be responsible for the drastic reduction of the number of the varied RLKs at 60 min. The change tendency of the number of varied RLKs after incompatible pollination was very similar with that after compatible pollination. A minor difference was that a moderate increase in the varied RLKs number emerged from 10 min after incompatible pollination. Although we had known a signaling cascade within stigmatic papillae was induced by incompatible pollen [7,26], the polarized secretion in the dry stigmatic papillae cannot be induced by incompatible pollen, and the pollen hydration and germination were inhibited [51,53]. In our knowledge, morphologic observations need to be further performed to illustrate the changes the number of the varied RLKs at different time points after incompatible pollination.

Differentially expressed RLKs after pollination should be mainly induced by pollen-stigma interaction
The major differentially expressed RLKs after pollination were up-regulated, to explore the reasons, their expression pattern and the changes of their expression level during in vitro PG and PTG were analyzed. Based on Klepikova et al. [49] and Pina et al. [47] studies, 29 of 89 differentially expressed AtRLKs preferentially expressed in F.AN and F.AN.ad (Fig. 3, Supplementary Table S3), also only 29 of the 89were hydrated pollen-enriched (Fig. 4, Supplementary Table S4 Table S2). Furthermore, excluding these F.AN and F.AN.ad preferentially expressed or hydrated pollen-enriched RLKs, the most of the reminding differentially expressed RLKs after pollination had a very low expression level in F.AN and F.AN.ad, some of them were even not present in these two samples (Fig. 3, Supplementary Table S3).
Thus, all these results indicated that the up-regulated RLKs after pollination should not be primarily caused by addition of pollen-enriched RLKs.
Based on Wang et al. [48] study, only 7 AtRLKs were shared by differentially expressed AtRLKs after pollination and during pollen tube growth, accounting for about 7.87% of that after pollination. In addition, among these 7 AtRLKs AT4G13190 and AT2G33580 were up-regulated during PTG, but their expression levels were up-regulated after incompatible pollination. As we all known, the germination of the incompatible pollen was inhibited on the dry stigma surface. Furthermore, both AT1G16760 and AT3G20530 were down-regulated during PTG, but their B. napus ortholog were up-regulated during the stages of compatible and incompatible pollination (Supplementary Table S2, S5). Therefore, the low repetition of differentially expressed AtRLKs after pollination with that during in vitro pollen germination and pollen tube growth, and the difference on the expression level change tendency of the shared AtRLKs meant that the up-regulated AtRLKs after pollination should not be mainly contributed by pollen germination and pollen tube growth. Taken together, all the above results suggested that the differentially expressed RLKs after pollination should be mainly induced by pollenstigma interaction.

Functions of the differentially expressed RLKs inmale-female communication
Among the differently expressed AtRLKs after pollination, AT5G28680 was ANX2, and AT4G39110 was BUPS1. Former report had proved that ANX2 and ANX1 were male factors controlling pollen tube behavior by directing rupture at proper timing [32,56]. Further analysis showed that ANX1 and ANX2 interacted with BUPS1 and BUPS2 to form the ANX-BUPS receptor complex, which bound to the pollen tube-expressed RALF4/19 to maintain pollen tube integrity, once RALK4/19 was replaced by the ovuleproduced RALF34, pollen tube bursting would be triggered [33,34]. AT5G16500 and AT3G02810 were LIP1 and LIP2, respectively. They were essential components of the pollen tube receptor complex to perceive the female signal AtLURE1 for micropylar pollen tube guidance [28]. AT5g45840 and AT4G18640 were MDIS1 and MDIS2. These two proteins along with MDIS1-interacting receptor like kinase MIK1 and MIK2 also participated in micropylar pollen tube guidance, especially, MDIS1 and MIK directly bound to AtLURE1 [29]. AT5G35390, AT3G20190 and AT1G50610 were PRK1, PRK4 and PRK5. PRK1, PRK2 and PRK5 were involved in the control of polarized pollen tube growth [57], PRK1, PRK3, PRK6 and PRK8 functioned synergistically in sensing of LURE1 in Arabidopsis [30]. Of the above studied AtRLKs, several were excluded from the differently expressed AtRLKs after pollination, such as BUPS2, PRK6. We thought the main reasons were that their expression level showed no obvious changes after pollination or their change fold was < 3. Another well characterized RLK was SRK, which also was excluded from the current study. Firstly, A. thaliana Columbia strain contained a nonfunctional SRK [58]. Secondly, BnSRK was highly expressed in un-pollinated stigma, but it expression level showed no obvious changes after pollination [46]. In our opinion, the following work efforts should focus on the functional analysis of the rest differentially expressed AtRLKs in male-female communications, aiming to strengthen our understanding of the molecular and cellular events behind the pollination in the Brassicaceae species.

Conclusion
In present study, we reported that 89 AtRLKs showed obvious expression level changes after pollination in A. thaliana or in B. napus, which should be mainly induced by pollen-stigma interaction, not by addition of pollen-enriched RLKs or pollen germination and pollen tube growth.  [47] report. Here we defined gene as hydrated pollenenriched gene if its expression level was at least 2-fold higher than that in leaves, seedlings and siliques. For the details pertaining to hydrated pollen-enriched genes, see Supplementary Table S4. The co-existed AtRLKs in hydrated pollen-enriched genes and the differentially expressed AtRLKs in response to pollination were analyzed using online Venny 2.1.0 (http://bioinfogp.cnb.csic.es/tools/venny/index.html).

Differentially expressed AtRLKs during in vitro pollen germination and pollen tube growth
We downloaded all the differentially expressedgenes withfold changes > 1.63 (P-value < 0.01) during in vitro Arabidopsis pollen germination (PG) and pollen tube growth (PTG) from the Supplemental  The differentially expressed AtRLKs or B. napus orthologs identified in CP and IP samples Comparison of hydrated pollen-enriched genes with the differentially expressed AtRLKs after pollination Figure 5 Comparison of differentially expressed AtRLKs during PTG with that after pollination

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