DLK2 regulates arbuscule hyphal branching during arbuscular mycorrhizal symbiosis.

D14 and KAI2 receptors enable plants to distinguish between strigolactones (SLs) and karrikins (KARs), respectively, in order to trigger appropriate environmental and developmental responses. Both receptors are related to the regulation of arbuscular mycorrhiza (AM) formation and are members of the RsbQ-like family of α,ß-hydrolases. DLK2 proteins, whose function remains unknown, constitute a third clade from the RsbQ-like protein family. We investigated whether the tomato SlDLK2 is a new regulatory component in the AM symbiosis. Genetic approaches were conducted to analyze SlDLK2 expression and to understand SlDLK2 function in AM symbiosis. We show that SlDLK2 expression in roots is AM-dependent and is associated with cells containing arbuscules. SlDLK2 ectopic expression arrests arbuscule branching and downregulates AM-responsive genes, even in the absence of symbiosis; while the opposite effect was observed upon SlDLK2 silencing. Moreover, SlDLK2 overexpression in Medicago truncatula roots showed the same altered phenotype observed in tomato roots. Interestingly, SlDLK2 interacts with DELLA, a protein that regulates arbuscule formation/degradation in AM roots. We propose that SlDLK2 is a new component of the complex plant-mediated mechanism regulating the life cycle of arbuscules in AM symbiosis.

Identification and expression analysis of the arbuscular mycorrhiza-inducible Rieske non-heme oxygenase Ptc52 gene from tomato.

Arbuscular mycorrhizal (AM) formation enhances plant growth and fitness through improved uptake of water and mineral nutrients in exchange for carbon compounds to the AM fungus. The fungal structure for the reciprocal exchange of nutrients in the symbiosis is the arbuscule, and defence genes expressed in cells containing arbuscules could play a role in the control of hyphal spread and arbuscule formation in the root. We characterized and analyzed the Ptc52 gene from tomato (SlPtc52), a member of the gene family of non-heme oxygenases, whose function has been related to the lethal leaf spot 1 (Lls1) lesion mimic phenotype in plants which is sometimes associated with enhanced disease resistance. Sequence analysis of the SlPTC52 protein revealed conserved typical motifs from non-heme oxygenases, including a Rieske [2Fe-2S] motif, a mononuclear non-heme iron-binding motif and a C-terminal CxxC motif. The level of transcript accumulation was low in stem, flower and green fruits, and high in leaves. Although SlPtc52 expression was perceptible at low levels in roots, its expression increased concomitantly with AM fungus root colonization. Tomato non-mycorrhizal hairy roots expressing the GUS protein under the control of promoter SlPtc52 exhibited GUS activity in the endodermis, the apical meristem of the root tip and in the lateral root primordium. AM fungal colonization also resulted in intensive GUS activity that clearly corresponds to cortical cells containing arbuscules. SlPtc52 gene silencing led to a delay in root colonization and a decrease in arbuscular abundance, suggesting that SlPTC52 plays a regulatory role during AM symbiosis.

Identification and Expression Analysis of GRAS Transcription Factor Genes Involved in the Control of Arbuscular Mycorrhizal Development in Tomato.

The formation and functioning of arbuscular mycorrhizal (AM) symbiosis are complex and tightly regulated processes. Transcriptional regulation mechanisms have been reported to mediate gene expression changes closely associated with arbuscule formation, where nutrients move between the plant and fungus. Numerous genes encoding transcription factors (TFs), with those belonging to the GRAS family being of particular importance, are induced upon mycorrhization. In this study, a screening for candidate transcription factor genes differentially regulated in AM tomato roots showed that more than 30% of known GRAS tomato genes are upregulated upon mycorrhization. Some AM-upregulated GRAS genes were identified as encoding for transcription factors which are putative orthologs of previously identified regulators of mycorrhization in other plant species. The symbiotic role played by other newly identified AM-upregulated GRAS genes remains unknown. Preliminary results on the involvement of tomato SlGRAS18, SlGRAS38, and SlGRAS43 from the SCL3, SCL32, and SCR clades, respectively, in mycorrhization are presented. All three showed high transcript levels in the late stages of mycorrhization, and the analysis of promoter activity demonstrated that SlGRAS18 and SlGRAS43 are significantly induced in cells containing arbuscules. When SlGRAS18 and SlGRAS38 genes were silenced using RNA interference in hairy root composite tomato plants, a delay in mycorrhizal infection was observed, while an increase in mycorrhizal colonization was observed in SlGRAS43 RNAi roots. The possible mode of action of these TFs during mycorrhization is discussed, with a particular emphasis on the potential involvement of the SHR/SCR/SCL3 module of GRAS TFs in the regulation of gibberellin signaling during mycorrhization, which is analogous to other plant developmental processes.

Role of gibberellins during arbuscular mycorrhizal formation in tomato: new insights revealed by endogenous quantification and genetic analysis of their metabolism in mycorrhizal roots.

Gibberellins (GAs) are key regulators of plant growth and development and recent studies suggest also a role during arbuscular mycorrhizal (AM) formation. Here, complementary approaches have been used to obtain a clearer picture that correlates AM fungal development inside roots with GA metabolism. An extensive analysis of genes associated with GA metabolism as well as a quantification of GA content in roots was made. Application of GA3 and its biosynthesis inhibitor prohexadione calcium (PrCa) combined with a GA-constitutive response mutant (procera) were used to determine whether fungal colonization is altered by the level of these hormones or by changes in the GA-signaling pathway. The increased levels of specific GAs from the 13-hydroxylation pathway in mycorrhizal roots correlate closely with the increased expression of genes coding enzymes from the GA biosynthetic trail. The imbalance of GAs in tomato roots caused by exogenous applications of GA3 or PrCa affects arbuscules in both negative and positive ways, respectively. In addition, procera plants were adversely affected by the mycorrhization process. Our findings demonstrate that an imbalance in favor of an increased amount of GAs negatively affects the frequency of mycorrhization and particularly the arbuscular abundance in tomato mycorrhizal roots and the results point out that AM formation is associated with a change in the 13-hydroxylation pathway of GAs.

SINGLE FLOWER TRUSS regulates the transition and maintenance of flowering in tomato.

The characterisation of the single flower truss ( sft) mutant phenotype of tomato ( Lycopersicon esculentum Mill.), as well as its genetic interactions with other mutations affecting FALSIFLORA ( FA) and SELF PRUNING ( SP) genes, has revealed that SFT is a key gene in the control of floral transition and floral meristem identity. The single sft mutation produces a late-flowering phenotype in both long-day and short-day conditions. In combination with fa, a mutation affecting the tomato gene orthologous to LFY, sft completely blocks the transition to flowering in this species. Thus, the phenotype of the sft fa double mutants indicates that SFT and FA participate in two parallel pathways that regulate the switch from vegetative to reproductive phase in tomato, and that both genes are indispensable for flowering. On the other hand, the replacement of flowers by vegetative shoots observed in the sft inflorescence suggests that SFT regulates flower meristem identity during inflorescence development of tomato. In addition to these two main functions, SFT is involved in the development of both flowers and sympodial shoots of tomato. First, the mutation produces a partial conversion of sepals into leaves in the first floral whorl, and a reduction in the number of floral organs, particularly carpels. Secondly, the sympodial development in the mutant plants is altered, which can be related to the interaction between SFT and SP, a gene controlling the number of nodes in sympodial shoots. In fact, we have found that the sft phenotype is epistatic to that of sp, and that the level of SP mRNA in the apical buds of sft around flowering is reduced. SFT can therefore co-ordinate the regulation of two simultaneous developmental processes in the tomato apical shoot, the promotion of flowering in one sympodial segment and the vegetative development of the next segment.