rolA 12

rolA encodes a small protein of approximate 11 kDa molecular mass.

Oligonucleotide sequences of rolA gene in all type of Ri plasmids share high homology (Nilsson and Olsson 1997). rolA was initially demonstrated as a gene related to rhizogenesis; however, later research discovered that rolA is a minor factor in rooting. In addition to rhizogenesis, rolA has been proposed to stimulate secondary metabolites in many types of plant tissues. The detailed descriptions are presented below.

2.1.1. rolA affects plant morphogenesis

A. tumefaciens harboring rolA with its promoter was able to induce rooting on tobacco leaf discs (Spena et al. 1987; Vilaine et al. 1987), but not on Kalanchoë leaves (Spena et al. 1987). rolA-deficient A. rhizogenes induced thicker and more curled hairy roots (White et al. 1985). The phenotypic changes in rolA-transgenic tobacco include highly wrinkled leaves, shorter internodes, and more condensed inflorescences with larger flowers (Schmülling et al. 1988; Sinkar et al. 1988); by

contrast, it was also reported to have smaller flowers with lower male fertility (Sun et al. 1991; Martin-Tanguy et al. 1993; Michael and Spena 1995). rolA-transgenic tomato had longer internodes, a smaller root system, smaller wrinkled leaves, smaller flowers, and lower pollen germination rate (van Altvorst et al. 1992).

2.1.2. rolA and plant hormone

rolA could increase the sensitivity to auxin in transgenic plant (Maurel et al.

1991; Vansuyt et al. 1992). Besides, rolA-expressing tobacco showed a similar phenotype with wild-type plant treated with gibberellic acid (GA) biosynthesis inhibitor; however, treating GA with rolA-transgenic tobacco only partially restored the phenotypic change (Dehio et al. 1993). Mortiz and Schmülling discovered that two active GAs, GA1 and GA20, were reduced in rolA-transgenic tobacco plant, and the precursors GA53 and GA19 were accumulated, indicating blocking GA synthetic pathway partially explains the phenotypic change caused by rolA (1998). Other hormone levels were measured in rolA-transgenic tobacco (Dehio et al. 1993), but no conclusive result could be proposed from the above reports.

2.1.3. rolA promoter

Transformation of rolA along with its 473-nucleotide upstream sequence, which is similar to some upstream sequences of auxin-responsible genes, was sufficient to cause the phenotypic change in tobacco (Carneiro and Vilaine 1993). In the same report, they demonstrated that stem had the most abundant rolA mRNA level, which was 5-fold and 50-fold higher than those in leaf and in root, respectively. rolA transcripts containing a 5’-untranslated region (5’-UTR), which would be spliced in Arabidopsis, was proposed to be an indispensable fragment to rolA expression, and it might act as a cis-acting regulatory factor (Magrelli et al. 1994). Pandolfini and

coworkers found that rolA mRNA could be transcribed in bacteria. However, the rolA transcripts were abolished while the 5‘-UTR was deleted (2000). This 5’-UTR has been proposed as a bacterial promoter. In 1996, Guivarc’h and coworkers expressed rolA driven by its 477 bp or 366 bp long upstream fragments, and they discovered the longer promoter would induce wrinkled leaves and short internodes in transgenic tobacco, whereas the shorter promoter only cause a dwarf phenotype with normal leaves (1996). In summary, rolA driven by its own promoter expresses in both prokaryotic and eukaryotic cells under the regulation of 5’-UTR, and the tissue-specific activation pattern is regulated by its 477 bp long promoter sequence.

2.1.4. RolA biological functions

Through sequence analysis and structure modeling, RolA was proposed to be a DNA- binding protein owing to the fact that it is a alkaline protein structurally homologous with papillmavirus E2 DNA-binding protein (Levesque et al. 1988;

Rigden and Carneiro 1999). However, the RolA-GUS transgenic tobacco cells showed the lowest GUS activity in nucleus and the highest in plasma membrane system (Vilaine et al. 1998). There is no transmembrane signals in RolA protein, which indicated RolA is a non-integrated membrane protein. Moreover, RolA might expresses in not only plant but also bacterial cell (Guivarc’h et al., 1996). These studies propose that RolA might possess multiple functions. From these evidence, we could hypothesize that RolA is a membrane-associated protein in plant cell, and it might be a transcriptional factor in bacterial cell. Combining these evidence, we proposed a possible role of RolA in plant. RolA is associated with plasma membrane in ground state, and it could be translocated into nucleus to regulate transcription process via unknown signaling stimulation. Nevertheless, RolA is still an functionally

known protein with the positive effects on rhizogenesis, development, and hormone homeostasis.

2.2. rolB

rolB encodes a protein of 259 amino acids in A. rhizogenes strain A4. It is discovered in all types of Ri plasmids. rolB is the most well-studied gene among the rol genes; however, rather conflicting results have been obtained. Recently, studies of rolB have been focused on its ability to induce secondary metabolites in plants. The way how rolB affects plant is illustrated in Figure 1-2, and the details are presented as follows.

Figure 1-2 The overview of rolB-mediated plant responses. rolB can be activated by auxin-dependent signals or auxin-independent transcript factors BBF1 and RBF1.

The RolB expression would inhibit the growth of R. cordifolia calli and induce tobacco leaf necrosis. The RolB can interact with tobacco 14-3-3 protein which resulting in nucleus translocation; however, abolishing the interaction by several point mutations only make little damage on the rooting. Besides, RolB can enhance the secondary metabolites and increase the auxin perceptions in transformants.

Increasing the auxin perceptions promotes plant de novo pluripotent meristem formation, resulting in rhizogenesis and other organogenesis.

2.2.1. rolB affects plant morphogenesis

rolB is the earliest research of interest among the rol genes. By deleting rolB from A. rhizogenes A4, the ability to induce hairy root on K. daigremontiana was abolished (White et al. 1985). Besides, rolB alone was capable of inducing tobacco rhizogenesis with almost the same efficiency as wild-type A. rhizogenes (Cardarelli et al. 1987; Spena et al. 1987). These reports indicated that rolB was the most important gene in rooting among the rol genes. Altamura and coworkers reported that rolB could strongly promote meristem formation by bypassing the regulatory factors of all types of organs (1994; 1998), and the results suggested that rolB regulates plant re-differentiation toward rooting.

rolB-transgenic tobacco presented smaller leaves with lower length-to-width ratio and highly branched plentiful roots (Cardarelli et al. 1987). Tobacco expressing rolB driven by Cauliflower mosaic virus (CaMV) 35S promoter showed bigger flower and early necrotic leaf (Schmülling et al. 1988). rolB-transgenic tomato showed reduced internode length and apical dominance with smaller flowers, lower pollen viability, and smaller fruits (van Altvirst et al. 1992; Arshad et al. 2014). On the other hand, rolB induces apical dominance in rose (van der Salm et al. 1997).

These indicate rolB functions in a species-dependent and tissue-dependent manner.

2.2.2. rolB and auxin

The phenotypic change in rolB-transgenic plants suggest that rolB is an-auxin responsive protein which mediates auxin signaling. Membrane potential measurement showed that rolB-transformed cells could increase auxin sensitivity up to 10000-fold (Maurel et al., 1991), and the polarization of auxin in rolB-expressing protoplast

could be blocked by a larger number of anti-auxin binding protein antibodies (Venis et al. 1992).

RolB was at first characterized as a glucosidase that hydrolyses indole glucosides in vitro, and rolB-transformed plant would increase auxin sensitivity by increasing IAA directly (Estruch et al. 1991c). However, two independent research groups invalidated the hypothesis later. Nilsson and coworkers demonstrated that wild-type and rolB-transformed tobacco showed the same contents of free IAA, and they had the same capacity of hydrolyzing IAA conjugates (1993a). Increasing auxin sensitivity in rolB-transformed tobacco was independent to intracellular auxin concentration because neither the accumulation nor the metabolism of endogenous auxin was affected; instead, rolB might increase the auxin perception (Delbarre et al.

1994; Maurel et al. 1994). This hypothesis was consistent with the experiment that the plasma membranes of rolB-transformed tobacco cells had additional auxin binding ability (Filippini et al. 1994).

There is other evidence supporting that rolB has a close connection with auxin.

Expressing rolB in tomato ovary by the tissue-specific promoter results in fruit parthenocarpy (Carmi et al. 2003), which has the similar effect with accumulating auxin in ovary by expressing bacterial auxin synthetic gene iaaH (indole acetamide hydrolase) driven by the same ovary-specific promoter along with treating its substrate (Szechtman et al. 1997). Expression of rolB in tobacco anther cells reduces stamen elongation and delays dehiscence (Cecchetti et al. 2004), which is considered as the result of lacking auxin polar transport system (Okada et al. 1991). In 1994, Altamura and coworkers demonstrated rolB promotes de novo primordia formation from tobacco thin cell layer in not only root but also flower (1994), which is

consistent with the reports that auxin controls the development of tobacco cells toward roots and flowers (Smulders et al. 1988; Smulders et al. 1990). In addition, auxin plays a crucial role in floral meristem formation and subsequent flower primordia formation (reviewed by Cheng and Zhao 2007). All in all, rolB has the auxin-like effects on plant fruit, ovary, and flower development, which supports the concept that rolB enhances the auxin perception in transformants.

Besides auxin, rolB was reported to have the correlation with cytokinin in promoting shoot formation from thin cell layers (Altamura et al. 1998). However, little connection between rolB and cytokinin has been proposed.

2.2.3. rolB promoter

rolB and rolC share a bidirectional promoter. Respective rolB and rolC native promoters drive uidA (β-D-glucuronidase) showed a similar expression pattern in shoot phloem but distinguishably in roots. rolB promoter activity shows mainly in the root primordia, including both primary and lateral primordia, and root cap, whereas rolC promoter activity does in phloem and in the apical meristems (Schmülling et al.

1989). The expression pattern of rolB indicates it has a close relation to cell differentiation and proliferation in the root. Overall, these two genes share a bidirectional promoter but they are regulated distinguishably.

rolB seems to be an auxin-regulated gene. In tobacco mesophyll protoplast, the expression level of rolB could be stimulated 20- to 100-fold by auxin treatment, whereas rolC expression increases only 5-fold (Maurel et al. 1990). In the same report, they discovered that treating exogenous auxin makes rolB express not only in root primordia but also in root vascular tissue and pericycle cells. Maurel and coworkers proposed the full activation of rolB by auxin is 12 to 18 hours after

treating, indicating that rolB belongs to an auxin late responsive gene. On the other hand, rolB could increase auxin perception within 8 hours after auxin treatment (1994). We can hypothesize the following two points. First, a low level of rolB expression is enough to increase auxin sensitivity in plant, and second, the activation of rolB might not only participate in amplifying auxin signals but also regulate other physiological behaviors independent to auxin signals.

There is much other evidence supporting that rolB is responsive to auxin but regulates not only auxin-related physiology. An auxin antagonist oligogalacturonide polymer is capable of inhibiting the rhizogenesis of rolB, and this effect disappears while rolB is driven by tetracycline-inducible promoter (Bellincampi et al. 1996).

Expressing rolB under control of its native promoter resulted in root or flower primordia formation, which is similar to treating exogenous auxin, whereas expression under CaMV 35S promoter in Hieracium piloselloides resulted in multi-potency (Koltunow et al. 2001). These phenomena showed the activation and the function of rolB have a close relationship with auxin.

Chimeric fusion of uidA with different lengths of upstream non-coding sequence of rolB shows that a 1185 bp length promoter region triggers the highest GUS activity (Capone et al. 1991; Capone et al. 1994). However, the 623 bp length promoter sequence drives a comparable activity. In addition, they identified five cis-elements, including regions -623 to -341, -341 to -306, -216 to -158, and the other two within regions about 70 and 80 bp around the CAAT and the TATA box, and they are so named as domain A-E, respectively. De Paolis and coworkers isolated a protein, which binds to the ACTTTA motif within domain B of rolB promoter via a single zinc finger structure. This protein was designated NtBBF1, representing N. tabacum rolB

domain B factor 1 (1996). NtBBF1 is essential for tissue-specific expression of rolB (Baumann et al. 1999). However, NtBBF1 is not an auxin-regulated gene, which indicates rolB is regulated at least by an unknown factor related to auxin and NtBBF1. In addition, another trans-acting element NtRBF1 (N. tabacum rol binding factor 1) can bind to -533 to -530 region of rolB promoter in non-meristem cells, and there is no differences between the concentrations of NtRBF1 in rolB-transformed and non-transformed tobacco plants (Filetici et al. 1997). Collectively, rolB is an auxin-inducible gene which increases auxin perception, but rolB can also be activated by an auxin-independent pathway and regulates auxin-independent responses in plants.

2.2.4. RolB biological function

Protein crude extract from RolB-expressing Escherichia coli has higher phosphatase activity than the extract from empty plasmid transformed E. coli, and the phosphatase is inhibited by tyrosine phosphatase inhibitor (Filippini et al. 1996).

Moriuchi and colleagues reported that RolB was a nucleus-localized protein that could interact with tobacco 14-3-3 κI, κII, ωI, ωII, ωIII, and ε (2004). In the same report, they generated a series of point mutations in RolB, and some of them could abolish the interaction; however, these point mutation reduce, but not abolish, root induction ability. The findings indicated that the physical interaction of RolB and 14-3-3 proteins are not essential for rhizogenesis.