2. Development and Findings

Tumors of higher plants are insufficiently studied, but represent a very interesting phenomenon, since they arise due to the release of a population of cells out of a very reliably arranged systemic control of the proliferation of plant cells. Spontaneous tumors in higher plants are especially rare, and the exact mechanisms of development of most of them remain unclear. In recent years, technologies of next-generation sequencing (NGS) have proven to be instrumental for studying various biological phenomena in different plants ^[8][9]. RNA sequencing (RNA-seq) is used to assess the functional state of biological tissue based on the variety and abundance of RNA molecules in the sample, so RNA-seq makes it possible to identify candidates for the role of regulators of plant tumor formation. To date, RNA-seq approaches were used for studying the gene expression during the development of only one type of plant tumor, the crown gall caused by 

A. tumefaciens ^[10][11]. In our work, we used RNA-seq to analyze the changes in gene expression in the spontaneous tumor of the radish inbred line compared with the lateral root of the same line of radish.

Arabidopsis, such as genes of CDKBs and its interacting partners, B-class cyclins, which provide the G2-M transition ^[12], as well as such important regulators of cytokinesis as centromere-associated histone H3 variant CEN3, which represents the assembly site for the kinetochore complex of active centromeres ^[13] or syntaxin KNOLLE, which is essential for phragmoplast formation ^[14]. The upregulation of genes involved in the control of the cell cycle and cytokinesis reflects the high frequency of cell divisions in spontaneous tumors (

Figure 1). In addition, among the upregulated “cell division genes”, there were also inhibitors of CDKs, an increase in the expression of which is usually associated with an increase in the frequency of endomitoses, e.g., during the formation of one of the types of plant tumors—root galls induced by nematodes ^[15][16]. Indeed, in the spontaneous radish tumors, a high level of polyploid cells was noted compared to normal root tissues (

Figure 1). Finally, in our early studies using qPCR, we revealed an increase in the expression levels of genes associated with cell division, including cell cycle genes and some of meristem regulators, in the spontaneous tumors of radish ^[5][17]. Thus, data on the upregulation of genes involved in the control of cell division in spontaneous tumors of radish are not unexpected for us.

At the same time, the pathways associated with the response to biotic and abiotic stresses were mostly downregulated in the spontaneous tumors. Among genes involved in these pathways, the most widely represented were genes controlling the biosynthesis of JA and the response to them, as well as genes that regulate the synthesis of specific secondary metabolites that are synthesized when plants are wounded or eaten by insects, for example, glucosinolates and lignin ^[18][19]. Thus, among downregulated genes, there were radish homologs of the 

Arabidopsis genes that regulate all stages of the JA biosynthesis from the beginning until the end ^[20], as well as genes that regulate the biosynthesis of glucosinolates ^[21] and common precursors of glucosinolates and IAA ^[22][23], and biosynthesis of lignin ^[24].

These data were also not very unexpected, since the intensification of cell division can lead to tissue juvenilization. The work of age-dependent resistance mechanisms leads to a gain or reinforcement of disease resistance in mature plant organs, whereas juvenile organs with a high level of cell divisions are more susceptible to pathogens ^[25]. In the adult plant, the activation of age-dependent resistance takes place under the control of genes involved in the regulation of flowering, such as LEAFY ^[25]. Therefore, the downregulation of genes involved in the flowering control which was revealed in the tumor tissue, may also contribute to the suppression of the age-dependent resistance in the tumor.

The results on changes in the expression of genes that regulate the level of CK and GA and the response to them were quite unexpected for us: based on the data obtained, the balance of these hormones in tumors should sharply shift towards GA. CK and GA are known to antagonistically interact in the control of meristem development, where CK promotes cell division, and GA is responsible for cell growth and differentiation in the periphery ^[26][27]. Therefore, one should rather expect the activation of CK biosynthesis and suppression of GA biosynthesis in the tumor. Moreover, our previous data indicate the sharp increase of free CK content in the spontaneous tumor of radish ^[6], and CK were shown to induce tumor formation in radish in vitro ^[4][28]. It is possible that the downregulation of “CK genes” and an upregulation of “GA genes” revealed by RNA-seq was the result of the work of hypothetical negative feedbacks that reduce the hyperactivation of CK biosynthesis in the radish tumor.

Finally, in the spontaneous tumors, the downregulation of genes that are involved in the control of photomorphogenesis and flowering was revealed. These data were rather unexpected and we can explain the expression of flowering-associated genes in the radish root only by suggesting the broader functions of these genes, e.g., in the radish storage root development. On the other hand, it was reported that individual circadian clock regulators can also participate in providing the plant stress response and affecting SA and JA signaling ^[29][30]. Therefore, these photomorphogenesis- and flowering-associated genes may be downregulated in the tumor together with other stress-related genes.