WANG Cui ,SUN Jin-jing ,YANG Xue-yong ,WAN Li ,ZHANG Zhong-hua ,ZHANG Hui-min

1 College of Horticulture,Qingdao Agricultural University/Engineering Laboratory of Genetic Improvement of Horticultural Crops of Shandong Province/Laboratory of Quality &Safety Risk Assessment for Fruit (Qingdao),Ministry of Agriculture and Rural Affairs,Qingdao 266109,P.R.China

2 Institute of Vegetables and Flowers,Chinese Academy of Agricultural Sciences/Key Laboratory of Biology and Genetic Improvement of Horticultural Crops of the Ministry of Agriculture and Rural Affairs/Sino-Dutch Joint Laboratory of Horticultural Genomics,Beijing 100081,P.R.China

Abstract In situ mRNA hybridization (ISH) is a powerful tool for examining the spatiotemporal expression of genes in shoot apical meristems and flower buds of cucumber.The most common ISH protocol uses paraffin wax;however,embedding tissue in paraffin wax can take a long time and might result in RNA degradation and decreased signals.Here,we developed an optimized protocol to simplify the process and improve RNA sensitivity.We combined embedding tissue in low melting-point Steedman’s wax with processing tissue sections in solution,as in the whole-mount ISH method in the optimized protocol.Using the optimized protocol,we examined the expression patterns of the CLAVATA3 (CLV3) and WUSCHEL (WUS) genes in shoot apical meristems and floral meristems of Cucumis sativus (cucumber) and Arabidopsis thaliana (Arabidopsis).The optimized protocol saved 4-5 days of experimental period compared with the standard ISH protocol using paraffin wax.Moreover,the optimized protocol achieved high signal sensitivity.The optimized protocol was successful for both cucumber and Arabidopsis,which indicates it might have general applicability to most plants.

Keywords: cucumber,in situ hybridization,Steedman’s wax,paraffin wax

1.lntroduction

In situmRNA hybridization (ISH) is widely used to detect gene expression patterns in plants (Brahic and Haase 1978;Singer and Ward 1982),because it provides a precise view of when and where a gene is expressed in the tissue.With the development of single-cell sequencing and spatial transcriptome technologies (Zhang T Qet al.2021),examining gene expression patterns has become a challenge using the standard ISH protocol.

The standard ISH protocol often includes paraffin sectioning or cryosectioning of plant tissues.For most tissues and cell types,sectioning facilitates permeation of the probe into the sample (de Almeida Engleret al.2001;Breweret al.2006).However,the paraffin sectioning protocol is time consuming because paraffin wax takes a long time to infiltrate into the tissues.Moreover,cryosectioning destroys histological details (Kerket al.2003;Nakazonoet al.2003).Although whole-mount ISH was developed forArabidopsis thaliana(Arabidopsis) (Hejátkoet al.2006),it is not widely used because it is suitable only for permeable tissues,but not for complex structural tissues.

Steedman’s wax can also be used in ISH,as an alternative to paraffin wax (Schneitzet al.1998).Originally,Steedman’s wax was developed as a general-purpose histological embedding medium composed of polyethylene glycol distearate and 1-hexadecanol since it reduces the hardening and shrinkage of tissues (Steedman 1957;Sidmanet al.1961;Brownet al.1989).Steedman’s wax has a lower melting point than paraffin wax,and it easily and rapidly infiltrates into tissues (Sidmanet al.1961;Norenburg and Barrett 1987).RNA recovered from tissues embedded in Steedman’s wax is of a higher quality than that isolated from tissues embedded in paraffin wax (Hua and Hibberd 2019;Choeet al.2020).As Steedman’s wax is soluble in alcohols,the wax can infiltrate the tissue directly (Sidmanet al.1961;Norenburg and Barrett 1987).By contrast,paraffin wax is insoluble in alcohols so infiltration must be mediated by xylene.Thus,using Steedman’s wax in ISH can shorten and simplify the experimental procedure and result in a more sensitive mRNA signal.However,the adoption of Steedman’s wax has been hindered by difficulties in mounting tissue sections on microscope slides (Sidmanet al.1961).

Cucumber is a globally cultivated vegetable crop in the Cucurbitaceae family bearing unisexual flowers.The important agronomical traits including fruit and unisexual flower development are associated with the development of shoot apical meristems (SAMs) and flower meristems(FMs).Functional genes have been well studied in SAM and FM development,and theWUS-CLV3pathway plays an essential role in SAM and FM development in cucumber(Cheet al.2020).Meanwhile,CsACS2,CsACS1G,CsACS11,CsWIP1andCsACO2control unisexual flower development (Boualemet al.2008,2016;Chenet al.2016;Zhang H Met al.2021).These functional genes are commonly expressed in the specific cell types of SAM and FM.TheWUS-CLV3pathway is essential to SAM development and flower bud differentiation in most plants(Schoofet al.2000;Nardmann and Werr 2006;Wanget al.2012;Somssichet al.2016;Cheet al.2020).

Here,we developed a functional and reproducible ISH procedure using Steedman’s wax.Our procedure takes advantage of the low melting point of Steedman’s wax and uses the whole-mount ISH method.In order to test our optimized protocol using Steedman’s wax,we used it to examine the expression patterns ofCLV3andWUSin the SAMs and the FMs of cucumber and Arabidopsis.The results showed that our protocol using Steedman’s wax is faster than the previous protocol using paraffin wax and has high sensitivity.Our protocol was successful with both cucumber and Arabidopsis,and therefore it should be generally applicable to most plants.

2.Materials and methods

2.1.Materials

Cucumber seedlings from the inbred line CU2 were grown in a glasshouse at the Institute of Vegetables and Flowers,Chinese Academy of Agricultural Sciences,Beijing,China.Shoot apexes from cucumber seedlings at the six-leaf stage were used for gene expression analysis.Arabidopsis Col-0 seedlings were grown in a growth room with a 16-h-light/8-h -dark cycle at 22-24°C.Shoot apexes from bolting Arabidopsis plants were used for gene expression analysis.

2.2.General lSH procedure

The standard ISH protocol using paraffin wax includes the following four steps: (a) probe preparation;(b) tissue fixation,dehydration,infiltration,embedding,and sectioning;(c) tissue pre-treatment and hybridization;and (d) antibody incubation,signal detection,and imaging.We modified and optimized steps (b) and (c) for our new protocol using Steedman’s wax.The details of our optimized protocol using Steedman’s wax are shown in Fig.1 and Appendix A.

Fig.1 A flowchart showing the processes of in situ hybridization by paraffin wax and Steedman’s wax.PFA,paraformaldehyde;DIG-AP,anti-digoxigenin-alkaline phosphatase-conjugate;NBT/BCIP,nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate.

2.3.Probe design and preparation

The primer sequences forCLV3andWUSin the two different species of cucumber and Arabidopsis were reported previously (Mayeret al.1998;Schoofet al.2000;Chenet al.2016;Zhaoet al.2018;Cheet al.2020).Primer information is given in Appendix A.

cDNA was reverse-transcribed from the RNA of the SAMs of cucumber and Arabidopsis.The probe sequences forCsCLV3,CsWUS,AtCLV3,andAtWUSwere amplified from the cDNA using the corresponding primers and inserted into the pEASY-Blunt vector with the T7 promoter (Transgene,pEASY-Blunt cloning kit;#CB501-02).The pEASY-Blunt vectors carryingCsCLV3,CsWUS,AtCLV3,andAtWUSwere linearized by restriction endonuclease digestion(Appendix B).Digoxigenin-labeled RNA antisense probes and sense probes were produced byin vitrotranscription using DIG-RNA-Labeling Mixture (Roche,#11277073910).The detailed steps are provided in Appendix A.

2.4.Fixation,dehydration,infiltration,embedding,and sectioning

The SAM and FM tissue samples of cucumber and Arabidopsis were placed in 4% paraformaldehyde (PFA)fixative solution.The tissue samples were placed under a vacuum on ice for several 1 h periods until the samples sank in the solution.The fixed tissue samples were then dehydrated on ice using an ethanol concentration series for 1 h for each step.

The most important difference between the standard protocol and our optimized protocol was in the tissue infiltration step.In the standard protocol,dehydrated tissue is transitioned to paraffin wax by passing it through a series of ethanol/xylene (1:3,1:1,3:1 (v/v)),xylene,and paraffin/xylene (1:3,1:1,3:1 (v/v)) solutions of increasing concentration for 1 h for each step.Then,the samples are incubated in pure paraffin wax at 60°C for 3 days.In our optimized protocol,dehydrated tissue samples are placed directly in Steedman’s wax and infiltrated for 1 day.Because no transition solutions are needed and the infiltration step is more rapid,the protocol time is shortened from 3 days to 1 day.In detail,the tissue samples were placed in a tube containing two phases -Steedman’s wax on the bottom and ethanol on the top (1:1 (v/v)),and incubated at 42°C for 12 h,during which time the samples remained suspended at the interface between the two phases.During an additional 12 h of incubation,the samples slowly sank down into the pure wax phase.

Another difference between the two protocols is in the sectioning step.In the standard protocol,ribbons of 10 μm paraffin wax sections are flattened onto microscope slides before further processing.In our optimized protocol,we omitted this step and instead handled tissue samples in solution throughout the procedure,using the whole-mount ISH method.The embedded tissue samples were cut into 10-20 μm sections with a Leica RM 2015 Microtome and transferred to small Petri dishes for hybridization (see below).The detailed steps are provided in Appendix A.

2.5.Tissue pre-treatment and hybridization

In the standard protocol,tissue section pre-treatment involves de-waxing with xylene and rehydration in an ethanol concentration series.However,in our optimized protocol,tissue sections could be rehydrated without first removing the wax.

For convenience,tissue sections were placed in cell strainers in Petri dishes or multi-well cell culture plates.For pre-treatment,tissue sections were rehydrated in an ethanol concentration series,digested with proteinase K,and re-fixed with PFA.Antisense probes and sense probes were denatured and mixed with the hybridization solution.Tissue sections were incubated with probe hybridization solution in Petri dishes or multi-well cell culture plates sealed with parafilm at 50°C overnight for hybridization.The detailed steps are provided in Appendix A.

2.6.Antibody incubation and signal detection

Tissue sections were washed in 0.2× SSC and 1× NTE,and then treated with RNase A to remove excess probe.Sections were incubated with blocking reagent,bovine serum albumin(BSA) solution,and anti-DIG-AP-antibody to detect the DIGtagged probe.Finally,for the staining step,sections were stained with NBT/BCIP in detection buffer.When the signal was strong enough,the reaction was terminated using TE buffer.The sections were then transferred from cell strainers onto slides,and images were taken using a Leica DM5500B microscope equipped with a digital camera.The detailed steps are provided in Appendix A.

3.Results

3.1.Summary of the optimized protocol

The standard ISH protocol using paraffin wax generally takes 10 days,but our optimized protocol using Steedman’s wax only took 6 days (Fig.1).In the tissue infiltration and embedding step,tissue infiltration took 3 days with the standard protocol but only 1 day with our optimized protocol.For the tissue sectioning step,the standard protocol calls for sections to be mounted onto microscope slides.However,in our protocol,we handled sections in solution,which saved about 1 day.Moreover,signal detection in the standard protocol usually takes 1-3 days.However,signal detection with our optimized protocol took only several hours for strongly expressed genes and 1 day at most for weakly expressed genes.In summary,our optimized protocol was 4-5 days shorter than the standard protocol (Fig.1).

3.2.Optimized protocol effective for detecting expression patterns of CLV3 and WUS in cucumber

To determine whether we could obtain high-sensitivity RNA signals using our optimized protocol,we performedin situhybridization to monitor the expression patterns ofCLV3andWUSin cucumber.TheCsCLV3gene was specifically expressed in the central region of the SAM (Fig.2-A;Appendix C-a) and expression extended upward to the apex of the FM in stages 2 and 2-3 (Fig.2-B and C,Appendix C-b).CsWUSwas specifically expressed in the central region underneath the central zone in the SAM in early stage 1 (Fig.2-D;Appendix C-c) and the FM in stages 1 and 2 (Fig.2-E and F;Appendix C-d).These results were consistent with previous studies (Cheet al.2020),indicating that the optimized protocol had a high sensitivity to the RNA signals.

Fig.2 Expression patterns of CLV3 and WUS in early-stage flower buds in cucumber detected by the optimized protocol.A-C,in situ expression of CsCLV3 in shoot apex and flower buds in cucumber.A,a shoot apex containing flower buds in early development.SAM,shoot apical meristems.B,flower buds in early stage 2 of development.C,flower buds in early stage 2 to 3 of development.D-F,in situ expression of CsWUS in shoot apex and flower buds in cucumber.D,a shoot apex containing flower buds in early stage 1.E,flower buds in early stage 1 of development.F,flower buds in early stage 2 of development.Scale bars=100 μm.

3.3.Optimized protocol effective for detecting expression patterns of CLV3 and WUS in Arabidopsis

To verify whether the method is applicable in other species,we also detected the expression patterns of theCLV3andWUSgenes in the model species Arabidopsis.AtCLV3transcripts specifically accumulated in the stem cells in the outer three cell layers of SAM (Fig.3-A),the inflorescence meristem (IM) (Fig.3-B) and the FM in stage 1 (Fig.3-C;Appendix B-e).Our results showed that theAtWUSgene was specifically expressed in a small group of cells underneath the third layer in the SAM (Fig.3-D),the IM(Fig.3-E) and the second layer in the FMs in stages 1 and 2 (Fig.3-E and F;Appendix C-f).The expression patterns ofAtCLV3andAtWUSdetected using our optimized protocol were consistent with those of previous studies(Mayeret al.1998;Schoofet al.2000).In summary,the optimized protocol with Steedman’s wax is also applicable in Arabidopsis.

Fig.3 Expression patterns of CLV3 and WUS in shoot and flower meristems of Arabidopsis detected by the optimized protocol.A-C,in situ expression of AtCLV3 in shoot and flower meristems of Arabidopsis.A,shoot meristem.B,inflorescence meristem (IM).C,flower meristem in early stage 1 (1) of development.D-F,in situ expression of AtWUS in shoot and flower meristems of Arabidopsis.D,shoot meristem.E,IM and flower meristem in early stage 1 (1) of development.F,flower meristem in early stage 2 (2) of development.Scale bars=50 μm.

4.Discussion

In this study,we optimized thein situhybridization protocol using Steedman’s wax,and the optimized protocol simplified the experimental process,shortening it by 4-5 days compared with the standard protocol using paraffin wax.We have used the optimized protocol to examine the gene expression patterns ofCLV3andWUSin shoot apical meristems and flower meristems in cucumber.The results showed that the expression patterns ofCLV3andWUSwere consistent with those of a previous study (Cheet al.2020).However,our optimized protocol had higher RNA signals while saving 4-5 days of experimental period compared to the standard protocol.We also examined the gene expression patterns ofCLV3andWUSin shoot apical meristems and flower meristems in Arabidopsis.The results indicated the optimized high-sensitivity protocol should be generally applicable to most plants.

Steedman’s wax has significant advantages over paraffin wax.Its melting point is 37°C,which is lower than that of paraffin wax (60°C).It also has a good solubility in alcohols,ethers,esters,ketones,and hydrocarbons (Sidmanet al.1961;Norenburg and Barrett 1987).Consequently,higher RNA quality is retained in plant tissues embedded in Steedman’s wax compared with those in paraffin wax(Hua and Hibberd 2019).Moreover,unlike paraffin wax,tissue infiltration with Steedman’s wax does not require mediation by xylene.Therefore,a transition step between dehydration and tissue infiltration and a dewaxing step are not needed,which simplifies the procedure.Despite these obvious advantages,the adoption of Steedman’s wax was previously hindered by difficulties in mounting tissue sections onto slides.Several researchers have tried to adhere the sections to slides using various agents,such as gelatin,but were not successful (Sidmanet al.1961).

We overcame the problems associated with Steedman’s wax by combining it with the whole-mount ISH method.In our optimized protocol,the sections are handled in liquid,as in the whole-mount method,instead of mounting them to slides.This method avoids the adhesion problem of Steedman’s wax-embedded sections.At the same time,using sections increases the accessibility of probes and antibodies to tissue samples compared with the standard whole-mount ISH method.Our optimized protocol took 6 days,while the standard paraffin wax protocol required at least 10 days.However,our optimized protocol is not suitable for structurally loose or discontinuous tissues because the samples are not mounted on slides.

During protocol development,we made several observations that are relevant for achieving high-quality signals using our optimized protocol with Steedman’s wax.First,fixation time is critical.Insufficient fixation of tissues results in mRNA degradation and displacement.However,over-fixation leads to a weak or absent signal,especially for weakly expressed genes.In our optimized protocol,the fixation of shoot apical meristems and floral meristems,which are poorly penetrable tissues (especially for cucumber),required the use of a vacuum for 4-10 steps of 1 h each before the tissues sank to the bottom of the tubes.Second,in the tissue embedding steps,the polymerization time and temperature surrounding the tissue being embedded are important to ensure that tissue samples are not too soft or too hard to cut.For our samples,polymerization took 12 h,although it can be prolonged to 1-2 days if necessary.Third,because the samples were handled in liquid in tissue culture dishes,the use of cell strainers throughout the procedure is recommended.Fourth,before the hybridization step,there is an optional step in which appropriate sections can be selected with a microscope after ribbons of Steedman’s wax have been dewaxed in pure ethanol.Fifth,the probe concentration should be adjusted based on the relative abundance of the target RNA.When detecting genes with low expression levels,we recommend a final probe concentration of approximately 5 μg mL-1and a hybridization temperature of 50°C to enhance the signal.

5.Conclusion

We present an optimized protocol for studying the expression patterns of genes in plant tissues using Steedman’s wax.The optimized protocol simplified the experimental process,shortening it by 4-5 days and improving the RNA signals compared with the standard protocol using paraffin wax.It provides a useful tool for studying the expression patterns of genes in shoot apical meristems and flower buds of cucumber.We further demonstrated that this protocol can be applied to Arabidopsis,and is thus likely to be suitable for applications in most plants.

Acknowledgements

We thank Dr.Hu Bowen (Hunan Agricultural University,China) for valuable suggestions on this program.This work was supported by the National Natural Science Foundation of China (32002036).

Declaration of competing interest

The authors declare that they have no conflict of interest.

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Appendicesassociated with this paper are available on http://www.ChinaAgriSci.com/V2/En/appendix.htm

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