Transcription factors MhyFIL1 and MhyFIL3 (Monotropa hypopitys) determine the asymmetric development of above-ground lateral organs in plants

It is believed that the complete mycoheterotroph pinesap Monotropa hypopitys adaptively evolved from a photosynthetic mycorrhizal ancestor, which had lost its photosynthetic apparatus and vegetative organs (stem and leaves). The aerial part of the plant is a reproductive axis with sterile bracts and inflorescence with a flower type canonical for higher plants. The origin of leaves and leaf-like lateral organs is associated, among other factors, with the evolution of the YABBY genes, which are divided into “vegetative” and evolutionarily recent “reproductive” genes, with regard to their expression profiles. The study of the vegetative YABBY genes in pinesap will determine whether their functions (identification of cell identity on the abaxial surface of the lateral organs) are preserved in the leafless plant. In this study, the structural and phylogenetic analysis of the pinesap vegetative genes MhyFIL1 and MhyFIL3 is performed, the main conserved domains and motifs of the encoded proteins are characterized, and it is confirmed that the genes belong to the vegetative clade YABBY3/FIL. The effect of heterologous ectopic expression of the MhyFIL1 and MhyFIL3 genes on the phenotype of transgenic tobacco Nicotiana tabacum is evaluated. The leaves formed by both types of plants, 35S::MhyFIL1 and 35S::MhyFIL3, were narrower than in control plants and were twisted due to the changed identity of adaxial surface cells. Also, changes in the architecture of the aerial part and the root system of transgenic plants, including aberrant phyllotaxis and arrest of the shoot and root apical meristem development, were noted. Some of the 35S::MhyFIL1 and 35S::MhyFIL3 plants died as early as the stage of the formation of the first leaves, others did not bloom, and still others had a greatly prolonged vegetation period and formed fewer flowers than normal ones. The flowers had no visible differences from the control except for fragile pedicles. Thus, the absence of structural changes from the M. hypopitys flower in comparison to autotrophic species and the effect of MhyFIL1/3 heterologous expression on the development of tobacco plants indicate the preservation of the functions of the vegetative YABBY genes by the MhyFIL1/3 genes in pinesap. Moreover, the activity of YABBY transcription factors of the FIL clade in M. hypopitys is not directly related to the loss of the ability of pinesap to form leaves during the evolutionary transition from autotrophic nutrition to heterotrophy.


Introduction
The most significant event in plant evolution is considered to be the emergence of photosynthesis, due to which most modern plants are autotrophs and only about 1 % of flowering plants are heterotrophic. Among the latter, a special place is occupied by complete mycoheterotrophs, which, in the course of adaptation to adverse environmental conditions, have acquired the ability to obtain nutrients through mycorrhiza (a symbiotic association of roots with fungi). The range of adaptation consequences due to photosynthesis incapability includes the degradation and rearrangement of the genome, large-scale loss of functional genes, etc. (Wicke et al., 2016;Graham et al., 2017).
The monotropoid type of mycorrhiza is characteristic only for members of the subfamily Monotropoideae of the Ericaceae family (Leake, 1994), including Monotropa hypopitys (syn. Hypopitys monotropa). Compared with the related photosynthetic species Pyrola rotundifolia, achlorophyllous M. hypopitys is characterized by considerable structural rearrangements in the genome, an increased rate of accumulation of nucleotide substitutions in the genes, a significant reduction in the plastome, and a loss of the photosynthesis apparatus from both the plastome and the nuclear genome Graham et al., 2017). Such changes often lead to degradation and/or modification of vegetative structures (Graham et al., 2017). Thus, pinesap is not only deprived of chlorophyll but it does not form aboveground vegetative organs. The reproductive axis bearing sterile bracts and inflorescence develops bypassing the vegetative stage, from adventitious buds in the pinesap mycorrhiza root system (Wallace, 1975;Merckx et al., 2013).
The development of photosynthesis is closely related to the evolution of the leaf, which changed from radially symmetric to asymmetric, thus increasing the insolation of its surface (Stewart, Rothwell, 1993;Cronk, 2001;Bowman et al., 2002;Beerling, Fleming, 2007). It is believed that the asymmetric leaf of seed plants originated in part due to the duplication and diversification of YABBY genes (Eckardt, 2010). The evolution of the ancestral YABBY gene produced a family of genes with different specializations, which could be associated with further transformations of the leaf and the emergence of other asymmetric organs that formed the flower (Mathews, Kramer, 2012).
The abaxial-adaxial asymmetry of all lateral organs is characteristic of most extant plants. One of the main factors determining the identity of the abaxial surface of organs is the family of YABBY transcription factors (Bowman et al., 2002;Bartholmes et al., 2012). In angiosperms, this family is divided into five subfamilies: three "vegetative" -YABBY1/ YABBY3 (FILAMENTOUS FLOWER (FIL)), YABBY2/ FASCIATED (FAS) and YABBY5, and two "reproductive" -CRABS CLAW (CRC) and INNER NO OUTER (INO) (Yamada et al., 2011;Bartholmes et al., 2012;Finet et al., 2016). "Reproductive" YABBYs have a narrow specialization, while "vegetative" YABBYs are involved in determining the polar development of vegetative and reproductive organs and are also important for proper organization and phyllotaxis of the shoot apical meristem (McConnell, Barton, 1998;Bartholmes et al., 2012). Thus, the "vegetative" YABBY genes preserve the expression profile of the ancestral gene, although they cannot completely replace the "reproductive" YABBYs (Yamada et al., 2011;Bartholmes et al., 2012).
The study of the YABBY genes of the complete mycoheterotroph M. hypopitys could clarify the possibility of preserving the ancestral functions by the "vegetative" YABBYs upon loss of the vegetative organs. The YABBY5 (MhyYAB5) and YABBY3/FIL (MhyFIL1, MhyFIL2, and MhyFIL3) genes with opposite expression patterns have been identified in pinesap . In bracts, which are evolutionarily closer to leaves than to floral organs, only trace amounts of MhyFIL2 transcripts are observed, and the expression levels of MhyFIL1 and MhyFIL3 are 5-10 times lower than that of MhyYAB5 . In the absence of leaves in pinesap, the reduced MhyFIL1 and MhyFIL3 expression in bracts suggests a loss of part of the "vegetative" YABBY function.
In this study, we perform a functional analysis of the vegetative YABBY genes, MhyFIL1 and MhyFIL3, in leafless pinesap 407 генетика растений / plant genetics M. hypopitys. The study of homologs of genes determining leaf asymmetry in higher plants in a complete mycoterotroph can expand the understanding of the evolution of the YABBY transcription factor family in the course of dramatic adaptive rearrangement of the plant.

Materials and methods
We invoked data from the transcriptome analysis of M. hypopitys roots, sterile bracts, and flowers (at the stage of anthesis) . To amplify and clone the coding sequence of the pinesap YABBY genes MhyFIL1 and MhyFIL3, primers were designed on the basis of previously identified gene transcripts : forward -5'-catcatgtcctcctcaaattctt-3' (for both genes), and reverse -5'-cttcttgattagtagggggacaca-3' (MhyFIL1) and 5'-cttcttgattagtagggggagacc-3' (MhyFIL3). Total RNA was isolated from pinesap flowers, where the expression of the MhyFIL genes was highest (the RNeasy Plant Mini Kit, QIA-GEN, USA), and used for cDNA synthesis (Reverse Transcription System, Promega, USA). The complete coding sequences of the MhyFIL1 and MhyFIL3 genes were amplified at the following PCR conditions schedule: denaturation 95 °С 5 min; 30 cycles of denaturation (94 °С, 30 s), annealing (55 °С, 30 s) and elongation (72 °С, 1 min); extension (72 °С, 7 min). Amplificates of the expected size were purified (MinElute Gel Extraction Kit; QIAGEN, USA), cloned into the pGEM ® -T Easy (Promega, Madison, WI, USA), and sequenced (Core Facility "Bioengineering", FRC "Fundamentals of Biotechnology" RAS). Sequence analysis of the fragments confirmed the cloning of the MhyFIL1 and MhyFIL3 cDNAs. The nucleotide and amino acid sequences were analyzed with the following software: Clone Manager 7.11 (http://clonemanager-professional.software.informer.com/), NCBI-CDD (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) and MEME 5.0.1 (Bailey, Elkan, 1994). Alignment of sequences of genes and proteins encoded by them was performed using ClustalX (Larkin et al., 2007) and MEGA 6.0. (Tamura et al., 2013). For phylogenetic analysis, NCBI BLAST (http://blast.ncbi.nlm.nih.gov/) and MEGA 6.0. (Tamura et al., 2013) were applied with tree generation by the maximum likelihood method based on the JTT model (Zuckerkandl, Pauling, 1965). To analyze the function of the MhyFIL1 and MhyFIL3 transcription factors, two types of transgenic Nicotiana tabacum plants with constitutive expression of the MhyFIL1 and MhyFIL3 genes were obtained. Two binary vectors were constructed based on the pBin19 plasmid, containing the cDNA (MhyFIL1/MhyFIL3) expression cassette in sense orientation under the control of the 35S CaMV promoter and NOS terminator. Agrobacterium tumefaciens AGL0 strains carrying the constructed plasmids were used for tobacco leaf disc transformation with further regeneration according to the previously described protocol (Goloveshkina et al., 2012). Regenerants were analyzed for the transgene presence in the genome by PCR with primers specific for the 3'-sequence of each gene (see above) and the 35S promoter (5'-caatcccactatccttcgcaagaccc-3'). The phenotypes of plants that gave a positive PCR signal were compared with the control (nontransgenic tobacco). The following parameters were assessed: the vegetation period (from planting in the greenhouse to budding), the structure of the aboveground vegetative part of the plant, and the phenotypes of the roots, leaves, and floral organs.

Results
Structural and phylogenetic analyses of MhyFIL proteins  by NCBI-BLAST, NCBI-CDD and MEGA 6.0 confirm that MhyFILs belong to the YABBY3/ FIL clade (Fig. 1). MhyFIL3 is closer to the ancestor than two other proteins, MhyFIL1 and MhyFIL2. As expected, the closest relatives of MhyFIL are representatives of the YABBY3/FIL clade in species of the Ericales order (basal Asterids), which include pinesap (see Fig. 1). Members of the YABBY3/FIL clade of other asterids form a sister subcluster (see Fig. 1). Inside the clade YABBY3/FIL, proteins of rosids (Arabidopsis thaliana) form a basal subcluster to analyzed asterid proteins (see Fig. 1). Analysis of putative conserved motifs (MEME 5.0.1) in the analyzed proteins reveals two sequences characteristic of all YABBY transcription factors and corresponding to the zinc finger and YABBY domains (Bartholmes et al., 2012). YABBY3/FIL proteins differ from members of other clades by the presence of six clade-specific (interdomain and C-terminal) motifs, and proteins of asterids, including Ericales, have an N-terminal motif, which is absent from A. thaliana YABBY3/FIL proteins (Rosids) (see Fig. 1). According to the conserved motif scheme obtained, all three MhyFILs are structurally closer to FIL than to YABBY3 (A. thaliana) (see Fig. 1).
For functional analysis of transcription factors MhyFIL1 and MhyFIL3, transgenic N. tabacum plants with individual constitutive expression of the cDNA of each of the MhyFIL1 and MhyFIL3 genes were obtained. Independent transgenic regenerants T 0 35S::MhyFIL1 (3 plants) and 35S::MhyFIL3 (12 plants), which rooted and formed true green leaves, were adapted to greenhouse conditions and then compared with the control (nontransgenic tobacco).
In contrast to the control, the obtained tobacco plants, 35S::MhyFIL1 and 35S::MhyFIL3, developed the bushy structure (instead of a single stem), had a significantly longer vegetation (on average, 282 days vs. the control 48 days), and formed abaxially twisted leaves (with an altered identity of the adaxial surface), and a strongly thickened and shortened root with abnormal leaf-like outgrowths (instead of an extensive root system) (Fig. 2).
Reproductive axes that developed on one of the shoots of the bushy 35S::MhyFIL1 and 35S::MhyFIL3 plants produced flowers outwardly similar to wild flowers, but often with rotting/brittle pedicles. Seeds were obtained from only six 35S::MhyFIL3 and two 35S::MhyFIL1 plants. In the T 1 generation, changes in plant morphology increased. Only one bushy plant 35S::MhyFIL3 formed a wild-type shoot that blossomed and gave seeds. The obtained seeds germinated, but the seedlings were characterized by abnormal development of roots (severe shortening and arrest in development) and shoot meristems (maximum shoot height 1.5-3.0 cm, bushiness, early development stop), which led to the death of the seedlings. In this regard, further analysis of the transgenic phenotype was impossible.
A microscopic analysis of the leaf surface of transgenic plants in comparison with the control confirmed that the cell identity on the adaxial side was partially changed as a MhyFIL1 and MhyFIL3 determine the asymmetry of the above-ground lateral organs in Monotropa hypopitys The Pinus taeda YAB sequence was used as an outgroup. The lengths of the branches estimated as the genetic distance (the number of substitutions per site), and the essential bootstrap values for 1000 replicates are shown at the base of the branches. The NCBI accession numbers are given against the names of proteins. To the right of the dendrogram -a scheme of conserved motifs of the analyzed proteins obtained as a result of the MEME 5.0.1 (http://meme-suite.org/tools/meme) analysis is represented. Below are the sequences of two motifs corresponding to the zinc finger (ZF) and YABBY domains.
result of heterologous transgene expression. There appeared stomata-like structures, which normally should not be on the upper surface of the leaf. Probably, they were the cause of leaf twisting.

Discussion
It is believed that mycoheterotrophic plants adaptively evolved from photosynthetic mycorrhiza lines, and the growth of such plants at poor insolation led to the inactivation and loss of the photosynthesis apparatus (Bidartondo, 2005;Buchanan-Wollaston et al., 2005;Zhang, Zhou, 2013;Ravin et al., 2016). In pinesap M. hypopitys, this was probably the cause of the subsequent disappearance of the unnecessary aboveground vegetative structures, including leaves (Wallace et al., 1975;Merckx et al., 2013). The achlorophyllous pinesap reproductive axis is often mistaken for a stem with leaves. However, the presence of MADS-box gene transcripts homologous to APETALA3, TM6 and SEPALLATA3 in sterile bracts ("leaves"), whereas in higher plants these genes are expressed only in flowers, is one of the signatures of the reproductive nature of the M. hypopitys aerial part .
The origin of asymmetric leaves and their further transformations, including the emergence of asymmetric flowering organs, as mentioned above, are associated, in part, with the evolutionary duplication and diversification of plant-specific YABBY genes (Eckardt, 2010;Mathews, Kramer, 2012). The structure and function of these genes are described in detail in a photosynthetic plants, model and other species (Bowman, 2000;Bowman et al., 2002;Finet et al., 2016;Strable et al., 2017). In complete mycoheterotrophs, YABBY genes are also present and transcribed . It is not clear, however, whether the functions of the vegetative YABBY genes are preserved in these leafless plants.

ZF YABBY генетика растений / plant genetics
It is known that the simultaneous knockout of all "vegetative" YABBY genes in an A. thaliana plant leads to the formation of narrow twisted or radially symmetric leaves, since all leaf cells become adaxial (Stahle et al., 2009). Theoretically, in case of overexpression of such genes, the formation of radially symmetric leaves should also be expected, the only difference being their abaxial identity. The observed narrowing and curling of leaves in 35S::MhyFIL1/3 plants confirm this assumption. Interestingly, the effects described above also occurred with the heterologous overexpression of the FIL genes BraYAB1-702 (Brassica rapa) and TaYAB1 (Triticum aestivum) in transgenic A. thaliana plants (Zhao et al., 2006;. Both species (B. rapa and T. aestivum) are photosynthetic autotrophs; therefore, the similarity of the effect of constitutive expression of the BraYAB1-702 and TaYAB1 genes in A. thaliana with the effect of the overexpression of MhyFIL1/3 in transgenic tobacco plants indicates the preservation of the ancestral role of the FIL genes in determining the identity of cells of the abaxial leaf surface.
It is also known that the correct morphogenesis of the meristem depends on the correct activity of the FIL genes (Bartholmes et al., 2012). For instance, A. thaliana with a double mutation, fil yab3, among other defects, demonstrates aberrant phyllotaxis (Goldshmidt et al., 2008). It is shown that transcription factor FIL nonautonomously and consistently affects the phyllotaxis and growth of lateral organs, coordinating the expression of markers (WUSCHEL, CLAVATA3 (CLV3)) of the central zone of the shoot apical meristem (Goldshmidt et al., 2008). The ectopic expression of SrGRAM (FIL-like gene in Streptocarpus rexii) completely suppressed the development of the A. thaliana shoot meristem (Tononi et al., 2010). The disturbance of the aboveground architecture of the 35S::MhyFIL1/3 transgenic plants and the resulting protracted vegetation may thus be caused by aberrant phyllotaxis up to the arrest of the shoot apical meristem development caused by ectopic MhyFIL1/3 overexpression.
It is worth highlighting the dramatic changes in the root structure of 35S::MhyFIL1/3 plants. In previously published papers, there was no information about what happens to the roots of such plants. The researchers may have omitted this aspect, since normally YABBY genes are expressed only in leaves and flowers, and therefore their functions are associated exclusively with these organs (Siegfried et al., 1999;Sarojam et al., 2010). Indeed, various combinations of yabby-mutations in A. thaliana do not affect root development (Boter et al., 2015). It is known that the apical meristems of the root and shoot are supported in a similar way, and CLV3 and WUS-CHEL-RELATED HOMEOBOX 5 (WOX5) genes are markers of the quiescent center of the root meristem (Fiers et al., 2005;Stahl et al., 2009;Chu et al., 2013). Hence, it is reasonable to assume that the root phenotype in 35S::MhyFIL1/3 plants is a result of suppression of the apical root meristem development due to the interference of the MhyFIL1/3 transcription factor in the regulation of the expression of N. tabacum genes homologous to CLV3 and WOX5.

Conclusion
The obtained results may indicate that, despite the absence of aboveground vegetative organs from pinesap, the function of the MhyFIL1/3 genes as "vegetative" YABBY genes is preserved. In M. hypopitys, transcription factors FIL1 and FIL3 still determine the asymmetric development of the lateral organs of the plant aerial part, which follows from the normal structure of pinesap floral organs, as well as the characteristics of the influence of heterologous MhyFIL1/3 gene expression on the development of tobacco, in particular, its leaves. Thus, the activity of the MhyFIL1/3 genes is not directly related to the loss of the pinesap ability to produce leaves during the evolutionary transition from autotrophic nutrition to heterotrophy.