Gene complementation analysis suggests that dodder plants (Cuscuta spp.) do not depend on the host FT protein for flowering

Background:

The search for a mysterious, leaf-derived substance that may induce flowering in the shoot meristem of plants was a hot topic in the 20^th century (Somssich, 2020). Already in 1863 did the German botanist Julius Sachs speculate that such a substance must exist, and in the 1930s, Mikhail K. Chailakhyan conducted a series of elegant experiments to show that it is indeed the leaves that perceive day-length via light, and that it is sufficient to expose the leaves to a specific light regime to induce flowering in a differently treated shoot (Sachs J., 1865; Chailakhyan, 1968). Chailakhyan suggested that it could be a phytohormone that acts as messenger, and provisionally named it ‘florigen’ (blossom-former). In Lysenkoist Russia, this hormonal, rather than Marxist theory of plant development was unacceptable. Science had to be tailored to fit the communist party line, which dictated that life was controlled through external forces, not internal factors, such as hormones. Thus, his work cost Chailakhyan his PhD, job and career – but the quest to identify florigen continued to intrigue plant scientists until the early 2000s (Somssich, 2020). It was only in 2007, that four independent studies identified the gene product of the FLOWERING LOCUS T (FT) to be the mysterious florigen (Corbesier et al., 2007; Jaeger and Wigge, 2007; Mathieu et al., 2007; Tamaki et al., 2007).

Curiously, it now appears that we once again find ourselves hunting for a mobile, flower-inducing signal – and this time it may not be FT!

Cuscuta ssp. (dodder) is a leaf- and rootless parasitic plant with little photosynthetic activity that depends entirely on a host plant to supply it with water, nutrients, and carbohydrates to complete its life cycle. Following germination, the Cuscuta seedling senses a suitable host in its periphery via excreted plant volatiles that act as chemo-attractants (Kaiser et al., 2015). Among the susceptible hosts are many crop plants, such as potato, tomato, sugar beet and melons (Mishra and Kogan, 2009). After locating the host, the Cuscuta stems wind around the host shoots and develop a specialized organ at the contact site, the haustorium, to penetrate the host tissue and establish a connection to its xylem (Lee, 2007). Importantly, this interface does not just allow the parasite to drain nutrients, solutes and carbohydrates from the host, but also the exchange of macromolecules like RNA and proteins (Kaiser et al., 2015). Furthermore, the parasite also synchronizes its lifecycle with that of the host, including its flowering time. It therefore seemed reasonable to assume that the mobile FT is among the proteins transferred to the parasite to induce flowering at the same time as the host, and indeed, this is what a recent study by Shen et al. (2020) suggested.

Shen et al., (2020) proposed that Cuscuta australis uses FT from its host plant Arabidopsis to synchronize its own flowering time with that of the host (Shen et al., 2020). For the parasite, this would enable it to make use of the host’s whole life cycle for feeding, while not jeopardizing its own propagation by ending its life cycle too late. The authors provided different lines of evidence for this: the Cuscuta genome lost many genes related to flowering time regulation in Arabidopsis. C. australis FT expression was not detected in stems before, while, or after the transition to the reproductive stage. Short day conditions suppressed the endogenous expression of AtFT, and misexpression of CaFT in Arabidopsis did not induce flowering under these conditions either, suggesting that CaFT is inactive. The authors could also confirm the presence of AtFT in C. australis, interaction between AtFT and the C. australis homolog of the essential FT co-factor FD, as well as the activation of flowering related genes. Thus, it indeed appeared that host FT was transferred to the parasite and was functional there. However, the author’s conclusion, that Cuscuta ‘eavesdrops on the host’s FT signal’, was recently queried, when Bernal-Galeano et al. (2022) established an in vitro system for the growth of Cuscuta campestris, in which the parasite grows to flowering stage despite the use of a paper spindle from a cotton swab as “host” (Bernal-Galeano et al., 2022).

In the new preprint discussed here, Mäckelmann and Känel (equal contribution) et al. (2022) investigate this phenomenon further and find that C. campestris has its own FT proteins, which are sufficient to induce flowering of the parasite – even in the absence of a host FT.

Key findings:

The authors used the tobacco (Nicothiana tabacum)-C. campestris host-parasite-system for their work since tobacco is day-length neutral and double mutants of the two floral inducers (Ntft4/Ntft5) do not flower at all. Using these plants, the authors found that C. campestris flowered after ~ 40 days – both in wild type and FT-less Ntft4/Ntft5 double mutant plants.

Analysing genomic and transcriptomic data, the authors then identified hundreds of flowering time-associated protein-coding genes, and observed that only two had Cuscuta-specific deletions, while other gene losses or divergence from the Arabidopsis coding sequences where shared with other eudicots, and therefore most likely inconsequential for flowering. C. campestris has two FT homologues, CcFT1 and CcFT2, and the amino acid sequence of CcFT1 was identical to the C. australis FT homologue. They furthermore identified two and one FD-like genes in C. campestris and C. australis, respectively. qRT-PCR and RNAseq analysis showed expression of C. campestris and C. australis FD-likes in all tissues examined, while expression of their respective FT homologs was specific to haustoria.

Using BiFluorescence Complementation assays, the authors then went on to show that CcFT1 and CcFT2 can interact with the FDs from both C. campestris and tobacco, and that CcFD-like1 can interact with NtFT5, suggesting a conserved interaction across plant species. Given that the amino acid sequence of CcFT1 is identical to CaFT, it can be assumed that CaFT shares these interactions.

Finally, the authors tested the functionality of CcFT in tobacco plants by overexpressing CcFT1 in the Ntft4/Ntft5 double mutant plants and found that CcFT1 was able to restore the flowering capacity of the mutant to wild type level.

Conclusions:

The hypothesis that Cuscuta lost its ability to flower without a flowering host, is partially based on the loss of genes in Cuscuta that are involved in flowering in Arabidopsis. However, this only holds true if we consider the flowering control of Arabidopsis a universal concept. But in fact, Mäckelmann and Känel et al. (2022) could show that gene losses and divergence also occur among other flowering plants when compared to Arabidopsis, so gene loss or divergence as such cannot be used as evidence.

In contrast to Shen et al. (2020), Mäckelmann and Känel et al. (2022) could detect expression of Cuscuta FT in both species, C. australis and C. campestris, because they included the haustorial tissue. Haustoria are the interface between the parasitic plant and the host and are therefore crucial for sensing the physiological status of the hosts. Instead of the transfer of host-derived FT protein, the endogenous production of FT could be triggered in the Cuscuta haustoria by other host-derived metabolites, such as sucrose. Sucrose, coupled to its production in photosynthesis and thereby an indirect read-out of the photoperiod and flowering induction of the host, was shown before to induce the expression of FT (King et al., 2008).

In conclusion, transfer of the host FT to the parasite does not appear to be required for the parasite to flower. Nonetheless, it is true that the parasite synchronizes its flowering time to the host timing, and therefore there must be a host-derived signal to provide a cue for flowering. Thus, we once again find ourselves searching for a mobile, flower-inducing signal. A blossom-former, another florigen. This substance could be another protein, a metabolite, and possibly even a phytohormone – as suggested by Chailakhyan in 1936.

Why we think this preprint is important:

The study underlines how small deviations in an experimental setup (such as working with the different plant species C. australis and C. campestris from the same genus), or different pre-assumptions can lead to contrasting conclusions. Furthermore, it reinforces the fact that we should always consider different plant models for universal conclusions about mechanisms and pathways. At the same time, it illustrates how each study complements previous work, and how, piece by piece and publication by publication, our understanding of a subject deepens.

Future directions and question for the authors:

It was shown before that C. australis only flowers when the host plant is able to flower, and that the flowering times are highly synchronized (Shen et al., 2020). In case of C. campestris, however, flowering is possible even when grown on the Ntft4/Ntft5 double mutant. Is this due to the experimental conditions, for example the light regime, or determined by the different Cuscuta species (i.e., did the authors try to reproduce the results of Shen et al. (2020) in their lab)?

An important experiment in this regard would be the knock-out of the haustorial expressed CaFT in C. australis, to see if this mutant would still flower. If a Caft mutant still flowers, and does so in sync with the host, this would be a very good indication that it is indeed the host FT that induces flowering in C. australis. Such an experiment could be conducted by decreasing CaFT expression by RNAi or CRISPR/Cas9 with the available Cuscuta transformation protocol, which targets the adhesive discs of the haustorium, the relevant tissue in this context (Lachner et al., 2020).

If it is indeed due to the different Cuscuta species, it would be very interesting to find out why C. australis has developed this additional mechanism of adaptation. Further to this, it could be interesting to look into the role of CcFT2, since this homolog is lost in C. australis. Does misexpression of CcFT2 in C. australis change its flowering time?

 

References:

Bernal-Galeano, V., Beard, K. and Westwood, J.H. (2022) An artificial host system enables the obligate parasite Cuscuta campestris to grow and reproduce in vitro. Plant Physiol., 189, 687–702.

Chailakhyan, M.K. (1968) Internal Factors of Plant Flowering. Annu. Rev. Plant Physiol., 19, 1–37.

Corbesier, L., Vincent, C., Jang, S., et al. (2007) FT protein movement contributes to long-distance signaling in floral induction of Arabidopsis. Science (80-. )., 316, 1030–1033.

Jaeger, K.E. and Wigge, P.A. (2007) FT Protein Acts as a Long-Range Signal in Arabidopsis. Curr. Biol., 17, 1050–1054.

Kaiser, B., Vogg, G., Fürst, U.B. and Albert, M. (2015) Parasitic plants of the genus Cuscuta and their interaction with susceptible and resistant host plants. Front. Plant Sci., 6, 1–9.

King, R.W., Hisamatsu, T., Goldschmidt, E.E. and Blundell, C. (2008) The nature of floral signals in Arabidopsis. I. Photosynthesis and a far-red photoresponse independently regulate flowering by increasing expression of FLOWERING LOCUS T (FT). J. Exp. Bot., 59, 3811–3820.

Lachner, L.A.M., Galstyan, L. and Krause, K. (2020) A highly efficient protocol for transforming Cuscuta reflexa based on artificially induced infection sites. Plant Direct, 4, 1–11.

Lee, K.B. (2007) Structure and development of the upper haustorium in the parasitic flowering plant Cuscuta japonica (Convolvulaceae). Am. J. Bot., 94, 737–745.

Mathieu, J., Warthmann, N., Küttner, F. and Schmid, M. (2007) Export of FT Protein from Phloem Companion Cells Is Sufficient for Floral Induction in Arabidopsis. Curr. Biol., 17, 1055–1060.

Mishra, J.S. and Kogan, M. (2009) Biology and Management of Cuscuta species. Indian J. Weed Sci., 41, 1–11.

Sachs J. (1865) Untersuchungen über die allgemeinsten Lebensbedingungen der Pflanzen und die Functionen ihrer Organe. In Handbuch der Experimental-Physiologie der Pflanzen. Leipzig: Wilhelm Engelmann.

Shen, G., Liu, N., Zhang, J., Xu, Y., Baldwin, I.T. and Wu, J. (2020) Cuscuta australis (dodder) parasite eavesdrops on the host plants’ FT signals to flower. Proc. Natl. Acad. Sci. U. S. A., 117, 23125–23130.

Somssich, M. (2020) A Short History of Vernalization. Zenodo.

Tamaki, S., Matsuo, S., Wong, H.L., Yokoi, S. and Shimamoto, K. (2007) Hd3a Protein Is a Mobile Flowering Signal in Rice. Science (80-. )., 316, 1033–1036.

 

doi: https://doi.org/10.1242/prelights.33586

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