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AmolecularinsightintoDarwin’s“plantbrainhypothesis”throughexpressionpatternstudyoftheMKRNgeneinplantembryocomparedwithmouseembryo

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A molecular insight into Darwin’s‘plant brain hypothesis’ through expression

pattern study of the MKRN gene in plant embryocompared with mouse embryo

Vaidurya Pratap Sahi,1,2,* Hanumant Baburao Wadekar,1,2 Nagganatha Suganthan Ravi,1,2

Thangavelu Umashankar Arumugam,1,3 Eugene Hayato Morita1,2 and Shunnosuke Abe1,2

1Ehime University; Matsuyama, Japan; 2Laboratory of Molecular Cell Physiology; Faculty of Agriculture; 3Cell Free Science and Technology Research Centre; Faculty of Science

Keywords: gene-expression, root-brain hypothesis, makorin, Darwin, analogy

MKRN gene family encodes zinc ring finger proteins characterized by a unique array of motifs (C3H, RING and acharacteristic cys-his motif) in eukaryotes. To elucidate the function of the MKRN gene and to draw an analogy betweenplant root apical meristem and animal brain, we compared the gene expression pattern of MKRN in plant seeds with thatof mouse embryo. The spatio-temporal expression of MKRN in seeds of pea and rice was performed using non radioactivemRNA in situ hybridization (NRISH) with DIG and BIOTIN labeled probes for pea and rice embryos respectively. Images ofMKRN1 expression in e10.5 whole mount mouse embryo, hybridized with DIG labeled probes, were obtained from theMouse Genome Database (MGD). MKRN transcripts were expressed in the vascular bundle, root apical meristem (RAM)and shoot apical meristem (SAM) in pea and rice embryos. The spatial annotation of the MKRN1 NRISH of whole mountmouse embryo shows prominent localization of MKRN1 in the brain, and its possible expression in spinal cord and thegenital ridge. Localization of MKRN in the anterior and posterior ends of pea and rice embryo suggests to the probablerole it may have in sculpting the pea and rice plants. The expression of MKRN in RAM may give a molecular insight intothe hypothesis that plants have their brains seated in the root. The expression of MKRN is similar in functionally andanatomically analogous regions of plant and animal embryos, including the vascular bundle (spinal cord), the RAM(brain), and SAM (genital ridge) thus paving way for further inter-kingdom comparison studies.

Introduction

Development and evolutionary biologists seek to understand thelogic behind evolutionary development through inter kingdomcomparative studies of various molecular, cellular and physio-logical processes. Studies pertaining to plant and animal analogiesdate back to the time of Aristotle, Father of biology.1 Aristotle inhis work “De Partibus Animalium” and “De Incessu Animalium” isreported to have said that the anterior and posterior poles of plantsare like animals upside down.2 In modern times, it was CharlesDarwin who for the first time suggested that the root apices maybe the regions similar to brains in animal.3 A comparative study ofvarious physiological and molecular processes across kingdomswill give us a better understanding of evolution and development.Spatial and temporal gene expression pattern studies have made iteasy to characterize the developmental process during embryo-genesis and germination in plants.4,5 Such Advances in the field ofmolecular techniques has given impetus to understand theconcepts pertaining plant and animal development.

MKRN, a zinc ring finger protein characterized by a uniquearray of C3H, RING and a characteristic cys-his motif, is encodedby makorin gene family in eukaryotes. Nine MKRN family locihave been found to be scattered throughout the human genomeand MKRN1 was characterized in number of organisms, whichinclude human, mouse, wallaby, chicken, fruit fly and nematode.6

MKRN2, another gene of makorin family, resulted from geneduplication of MKRN1 some 450 million years ago, and it wascharacterized by Gray et al. (2001)7 in human, mouse and fish.In plant systems, there has been substantial work since initialcharacterization of aMKRN in Arabidopsis by Gray et al. (2000).6

Abe et al. (2007)8 reported the expression of MKRN in pea whichprovides the first experimental evidence that hints at thefunctional role of MKRN in plants followed by the charac-terization of MKRN in rice.9 They also investigated the genomicorganization of rice, pea, Arabidopsis and other metazoans, andshowed phylogenetic relationships between them.

MKRN 1 is one of those genes which act downstream ofOCT 4, a transcriptional factor which has a role in establishment

*Correspondence to: Vaidurya Pratap Sahi; Email: vpsahi@hotmail.comSubmitted: 10/31/11; Revised: 12/16/11; Accepted: 12/16/11http://dx.doi.org/10.4161/psb.7.3.19094

RESEARCH PAPER

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and maintenance of totipotency or pluripotency of embryonicand undifferentiated stem cells.10MKRN 1 has also been reportedto act as an E3 ubiquitin ligase for p21 and p23 cycles.11

Expression pattern of MKRN1 in murine embryonic nervoussystem and testis suggests that MKRN 1 may have an importantrole in embryonic development and neurogenesis. Yang et al.(2008)12 for the first time characterized the functional role ofMKRN2 in Xenopus laevis and reported that MKRN2 plays a rolein PI3 K /Akt mediated neurogenesis during embryonic develop-ment of Xenopus. Expression studies of makorin genes (MKRN 1and MKRN 2) in yellowtail fish,13 mouse and humans6,7 andXenopus12,14 have paved the way for understanding of thisgene family in animal systems. In plants, expression of MKRNtranscripts was reported in differentiating tissues as well asdifferentiated tissues during germination and early vegetativestages of rice9 and pea.15

In the present study we studied the MKRN expression patternin rice and pea seeds, using non radioactive in situ hybridizationtechnique, and compared it with the MKRN expression patterndata of a developing mouse embryo, obtained from the MouseGenome Database (MGD).

Results

Embryos from mature rice and pea seeds were compared and theirposition shown in Figure 1.

In silico analysis. On aligning the amino acid sequences ofrice MKRN (accession number Q5ZA07), pea MKRN (accessionnumber BAC81564.1) and MKRN1 of mouse (accession numberNP_061280.2), we found all the six hallmark motifs found inmakorin (Fig. 2A) conserved and sequentially arranged. Based onthe genomic sequence we deduced the structure of makorinproteins in rice, pea and mouse which shows the exon/intronpositions and the motifs (Fig. 2B).

Non radioactive in situ hybridization of rice seed. BIOTINlabeled antisense probes hybridized with the MKRN transcriptson sections of rice embryo, to give a purple color and thosehybridized with sense probes gave a light purple color (Fig. 3).Figure 3A shows the rice seed section, without probe, Figure 3B

shows the section hybridized with sense probe and Figure 3Cand D show sections hybridized with antisense probes. Sectionshybridized with antisense probes (Fig. 3D) clearly show theexpression of MKRN transcripts, in shoot apical meristem (SAM),root apical meristem (RAM) and the vascular region, whencompared with sections hybridized with sense probe (Fig. 3B).There was no expression of MKRN in the endosperm of rice seed(Fig. 3C).

Non radioactive in situ hybridization of pea seed. The DIGlabeled antisense probes hybridized with the MKRN transcriptson the section to give a purple color and the sections hybridizedwith sense probes turned out to be off white with a faint tingeof purple. In Figure 4C, F and I show sections hybridizedwith antisense probes and Figure 4B, E and H show sectionshybridized with sense probes. On comparing the sections hybri-dized with sense probes (Fig. 4B, E and H) with those hybridizedwith antisense probes (Fig. 4C, F and I) we found that theexpression of MKRN was obvious in the SAM, region of vascularbundle and the RAM. Figure 4A, D and G are sections withoutany hybridization.

Non radioactive in situ hybridization of mouse embryo. Theimage of mouse embryo (MGI:3501182) retrieved from theMouse Genome Database (MGD), Mouse Genome Informatics,The Jackson Laboratory, Bar Harbor, Maine, World Wide Web(www.informatics.jax.org), shows expression of MKRN1 in thebrain and possible expression in the regions of spinal cord andthe genital ridge.30,31

Discussion

With a number of plant and animal genome sequences available,it has become easier to understand their similarities and differ-ences. One of the ways by which plant and animal comparisonscan be made is through pattern formation.16 In Plants, the basicbody plan and stem cell populations for generation of organs,in later stages, is established during embryogenesis.17,18 In thepresent study we compared the expression pattern of MKRN inmature seeds of pea and rice with the expression pattern ofMKRN1 gene in mouse embryo.

Makorin in animals was characterized by Gray et al. (2000)6 forthe first time and MKRN in plants was first characterized in pea8

followed by rice.9 It is known that plant and animal makorinsshare a common ancestor and that plant MKRN is more similar toMKRN1 of animals.9 On aligning the rice MKRN, pea MKRNand mouse MKRN1 amino acid sequences, we found the sixhallmark motifs of makorin to be conserved.

We found the expression of MKRN in mature pea embryoand rice embryo to be similar. In both, MKRN transcripts wereexpressed prominently in the radicle, though not in the root cap.The expression was prominent in shoot apical meristem and alsoin the region of vascular bundle. Our spatial expression results areconsistent with the previous reports on the temporal expressionof MKRN in dry rice9 and pea8 seeds. Studies have shown thespatial expression of MKRN in the meristematic tissues, lateralroot primordia and the vascular regions of young germinatingrice.9 Also, expression of MKRN has been reported in the shoot

Figure 1. Position of embryo on pea and rice seeds. The arrows showthe position of embryo on rice and pea seeds.

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apical meristem, vascular region and the root tissues during earlygermination stages of pea.15 Our plant MKRN results whencompared with the expression of MKRN1 in the brain of mouseembryo (Fig. 5), shows a similar pattern of expression based onthe polar axes. The spatial annotation of MKRN1 expression inmouse embryo shows expression in brain, nervous tissues andgenital ridge (Fig. 5C). This is consistent with previous studies

on expression of murine MKRN1 in the embryonic brain,nervous tissues and the testis.6,19 Expression of MKRN1 has alsobeen reported in brain and gonads of humans, chicken, fishesand amphibians.6,19-21

Analogous expression pattern of MKRN and MKRN1 in plantsand mouse, suggests to the importance of this gene in interkingdom studies. Drawing an analogy between RAM and brain,

Figure 2. (A) Alignment of amino acid sequences of rice MKRN (O. sativa), pea MKRN (P. sativum) and mouse MKRN1 (M. musculus). The characteristicsix motifs of makorin family are shaded. The asterisk marks below the sequences denotes complete conservation, two dots shows highly similar aminoacids and one dot shows moderately similar amino acids. (B) A comparison of genomic structure of mouse MKRN1 with rice and pea MKRN.The characteristic motifs of Makorin gene family are shown in red (C3H), green (cys-his) and blue (RING). The exons are numbered and the intron sizesare given in base pairs (kb) below the line.

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Do not distribute.one might wonder as to why the plants have their brain like tissuesunderground. To this, Baluska et al. (2004)1 have suggested threereasons: first they suggest that the soil environment is more stablethan the air environment. Second, it is protected from animalsand other sources of physical destruction. Lastly, they suggestthat since the posterior end of plant (stem) bears the organs ofreproduction (flowers), the anterior end (root), logically becomesthe brain.

The roots and shoots of a plant are interconnected by thevascular system, which is composed of continuous strands ofelongated cells.22,23 Auxin, which is in abundance in the polarregions of the plant maybe referred as plant neurotrans-mitter.1,24,25 The fact that RAM is rich in auxin related activitiesfurther supports the root brain theory. They have furthersuggested that the RAM represents sink of oxygen, just like thebrain. The supporting parenchyma cells of the vascular tissues(xylem and phloem) are anatomically similar to the gilial cellswhich support the neurons.1 Plant homologoes of ionotropicglutamate receptors have been reported to be expressed in thevascular tissues.26,27 Recent advances in plant signaling andphysiological studies have revealed similarities between plant cellsand neurons.28 Evidences that neurotransmitters like acetycholineand serotonin activate ion pumps in xylem parenchyma29 furthersuggests to the functional similarity between vascular tissues andthe neurons.

Similar expression pattern of MKRN in RAM of plants andof MKRN1 in mouse brain, gives an impetus to Darwin’s “rootbrain theory.” Expression patterns of MKRN in the roots tissuesand lateral root primordia of rice9 and pea8,15 and MKRN1 inbrain of humans, mouse, yellowtail fish, zebrafish, medaka,clawed frog6,13,19,21 are analogous. Similarly based on the expres-sion of MKRN in vascular regions9,15 and the nervous tissues ofmouse,6,19 an analogy can be drawn between the vascular tissuesand the nervous tissues. Lastly, the expression of MKRN in SAMof pea15 and rice9 is similar to the expression ofMKRN1 in gonadsof animals.6,19-21

Based on the comparative study of the expression pattern ofMKRN in plants and MKRN1 in animals, it is quite possible thatthe analogy between the plant and animal, as suggested byAristotle, Darwin and many recent scientists, may find strongsupport.

Materials and Methods

In silico analysis. Protein sequences of pea and rice MKRN andmouse MKRN1 from NCBI database were taken for analysis.Using T-COFFEE, Version_8.99 multiple sequence alignment wasperformed (http://tcoffee.crg.cat/apps/tcoffee/play?name=regular).

mRNA in situ hybridization of pea seed. Pea seeds (Pisumsativum L. var Alaska) were fixed in freshly prepared 4%

Figure 3. In situ hybridization of MKRN in embryo of dry rice seed. Panel A shows section without any hybridization, panel B shows section hybridizedwith sense probe while panels C and D show sections hybridized with antisense probes. Panel C is a section of rice endosperm. The bar represents 60mm.Notifications are: SAM, Shoot Apical Meristem; RAM, Root Apical Meristem; VB, Vascular Bundle; es, endosperm.

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paraformaldehyde solution overnight and Sections of 30 mmwere obtained using Yamato cryomicrotome.15 Pre-hybridizationwashes9 were followed by hybridization of the tissue samples withDIG labeled sense and antisense probe. After the probes (2 ml)were mixed with Dako mRNA in situ hybridization solution in aplastic container, tissue sections were soaked in plastic containerand incubated at 37°C, for 15 hrs. Post-hybridization was carriedby subjecting the hybridized sections with buffers according to theprotocol provided by the company (www.genedetect.com). Afterthe post-hybridization washes the sections were washed threetimes in Buffer1 (100mM TRIS-HCl pH 7.5, 150mM NaCl)for 5min each. The washed tissues were then immersed inBuffer1 containing 0.5% Blocking reagent (Roche), followed byincubation for two hours with the anti-DIG antibody, diluted to1: 500 with buffer1 containing 0.1% triton X smf 1% Blocking

Reagent. The antibody was removed by washing the sections fourtimes with buffer1 containing 0.1% triton for 20min. Thesections were then equilibrated with Buffer 2 for 5min, followedby staining with NBT/BCIP solution (1 tablet NBT/BCIP in10 ml of distilled water) in a plastic box overnight. In order toconfirm the presence of mRNA before hybridization of sectionswith sense and antisense probes, we used polydT. We alsoprepared a section from without any probes, as a control. Thesections stained were washed in tap water and observed undermicroscope and photographs were taken using digital camerasNikon Coolpix for stereo microscope and pixera for invertedmicroscope.

mRNA in situ hybridization of rice seed. Rice seeds (Oryzasativa L. var Nipponbare) were fixed in freshly prepared 4%paraformaldehyde solution overnight and Sections of 30 mm

Figure 4. In Situ hybridization of MKRN in embryo of dry pea seed. Panels A, D and G show sections without hybridization. Panels B, E and H showsections hybridized with sense probes while sections hybridized with antisense probes are shown in panels C, F and I. Panels A, B and C are showingthe Shoot Apical Meristem, panels D, E and F the middle part of embryo which has vascular bundles and panels G, H and I are showing the root tipshowing the root cap and the Root Apical Meristem. The bar represents 30mm. Notifications are: SAM, Shoot Apical Meristem; RAM, Root Apical Meristem;VB, Vascular Bundle; RC, Root Cap

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were obtained using Yamato cryomicrotome. Pre-hybridizationwashes of the tissue samples followed by hybridization withBIOTIN labeled sense or antisense probes was done accordingto the method described by Arumugam et al. (2007).9 Post-hybridization was carried by subjecting the hybridized sectionswith buffers according to the protocol provided by the company(www.genedetect.com). After the post-hybridization washes thesections were washed three times in Buffer1 (100mM TRIS-HClpH 7.5, 150mM NaCl) for 5min. The washed tissues were thenimmersed in Buffer1 containing 0.5% Blocking reagent (Roche),followed by incubation for two hours with the streptavidin,diluted to 1: 500 with buffer1 containing 0.1% triton X smf 1%Blocking Reagent. The antibody was removed by washing thesections four times with buffer1 containing 0.1% triton for20 min. The sections were then equilibrated with Buffer 2 for

5min, followed by staining with NBT/BCIP solution (1 tabletNBT/BCIP in 10 ml of distilled water) in a plastic box over-night. In order to confirm the presence of mRNA before westarted hybridization of sections with sense and antisense probes,we used polydT. The sections stained were washed in tap waterand observed under microscope and photographs were takenusing digital cameras Nikon Coolpix for stereo microscope andpixera for inverted microscope.

Non radioactive in situ hybridization of mouse embryo. Weobtained the image (EMAGE: 1588) of mouse embryo (10.5dpc)hybridized with DIG labeled MKRN1 probes from EMAGE geneexpression database (www.emouseatlas.org/emage/).

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Figure 5. Comparative expression pattern of Makorin in (A) rice, (B) pea and (C) mouse embryo. In panel C red shows region of high expression,yellow shows moderate expression, green shows region of probable expression and blue shows regions of no or very less expression. Panel C has beentaken from the mouse image database emage.

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