Uncoupling FT Protein Transport from its Function

Flowering in vascular plants is determined by intricate mechanisms controlled by external environmental conditions, such as light and temperature, as well as by internal autonomous cues. One of the main convergence points in the floral regulatory network is FLOWERING LOCUS T (FT) in Arabidopsis and its orthologs in other plants [e.g. Heading date 3a (Hd3a) for rice] (Putterill and Varkonyi-Gasic 2016).

The concept of a long-distance signaling component referred to as florigen arose in the 1930s (Zeevaart 2006). It took almost 70 years to identify the molecular nature of florigens, which we now know are FT protein and its orthologs (Corbesier et al. 2007, Tamaki et al. 2007). FT protein moves from the leaf phloem where it is synthesized to the shoot apical meristem (SAM) to initiate the floral transition. Although its mobile nature is an important characteristic of the florigen, little is known regarding the transportation mechanisms of FT. Currently, only two proteins, both of which directly interact with FT, have been characterized as regulators of FT movement. One is FT-INTERACTING PROTEIN 1 (FTIP1), the endoplasmic reticulum- and plasmodesmata-localized protein important for uploading FT from the phloem companion cells to the sieve elements (Liu et al. 2012). The other is SODIUM POTASSIUM ROOT DEFECTIVE 1 (NaKR1) protein, which regulates FT movement in the phloem stream (Zhu et al. 2016). We are in the early stages of understanding FT transportation, with some important questions remaining unanswered, such as how quickly does FT move from companion cells to the SAM? Is the FT movement passive or active? How is it regulated?

In this issue, Endo et al. (2018) provide more precise kinetic information regarding FT movement as well as insights into the amino acids that are important for FT protein transport in Arabidopsis. They systematically assessed how quickly FT protein is transported from the leaf to the SAM by separating the transport process into two steps: (i) uploading of FT from the companion cells and (ii) unloading of FT from the phloem to the SAM. In addition, they identified FT protein mutations that give rise to defective protein transport without affecting floral induction. To strictly control the expression of FT in the leaf, the authors used a heat shock-induced transient expression system in which FT expression was controlled by the promoter of the heat shock-inducible gene HEAT SHOCK PROTEIN 18.2 (HSP 18.2). By utilizing this FT-inducible system in an ft-1 background, they assessed how long plants require to transfer a sufficient amount of FT protein from the leaf to the SAM for floral induction. Once they induced FT protein expression by transient heat shock in single leaves, they removed the FT-induced leaves at 0, 4, 8, 12 or 24 h after the heat shock treatment to measure the minimum amount of time required for FT to reach the SAM and induce flowering. This experiment revealed that induction of flowering as well as FT protein accumulation at the SAM occurred at least 8 h after the induction of FT. This implies that it takes 8 h for sufficient FT protein to be transported out from the leaves through the phloem (Fig. 1). The results also suggest that unloading of FT protein from the phloem at the SAM is mediated in an active manner rather than by simple passive diffusion, which corroborates previous findings.

Fig. 1.

FT movement from the companion cells (CCs) in leaves to the shoot apical meristem (SAM). Both wild-type (magenta ovals) and movement-defective (pale pink ovals) FT proteins expressed in the CCs interact with FTIP1 (orange ovals), and are uploaded to the sieve elements (SEs) of the phloem. Uploading of FT to the SEs and movement of FT through the SEs to the SAM takes at least 8 h. Wild-type FT, but not movement-defective FT, is unloaded into the SAM. Unloading of FT from the phloem to the SAM at the shoot apex takes an additional 4 h. The active transport mechanism of FT (depicted by ‘?’) around the SAM is currently unknown.

It has been estimated that FT moves at a velocity of 30–50 cm h^–1, similar to the speed of the phloem flux (Takeba and Takimoto 1966, Savage et al. 2013). Once FT reaches the end of the phloem near the SAM, it needs to be unloaded. The authors also examined how long plants require in order to accumulate a detectable amount of FT at the SAM after FT is induced in the leaves. Heat shock-induced FT protein in a leaf was detectable in the SAM 12 h (but not 8 h) after heat shock treatment. In summary, using a combination of classical physiology and a modern genetic inducible FT expression system, the authors neatly showed that 8 h is sufficient time for enough FT protein to be uploaded to the phloem and migrate towards the SAM, and a further 4 h (in total 12 h) to be actively unloaded from the phloem and reach cells in the SAM (Fig. 1).

In addition to estimating the amount of time required for FT transport from the leaves to the SAM, the authors further analyzed the mechanisms of FT protein movement. If FT is not transferred by simple diffusion, then how is its movement directed in an active manner? FT homologs do not always move long distances. TERMINAL FLOWER 1 (TFL1), which shares high similarity in amino acid sequence with FT but functions as a floral repressor, is expressed around the SAM and moves only a short distance (Conti and Bradley 2007). Thus the authors hypothesized that amino acids unique to FT (compared with TFL1) may confer FT long-distance mobility. There are 47 amino acids that differ between FT and TFL1. The authors independently substituted most of these amino acids with alanine and tested whether any of those alanine-substituted mutants altered the mobility of FT.

To identify specifically which FT amino acids are important for transport, the authors made the best use of their heat shock FT-inducible system. They reasoned that FT mutants defective only in transport cannot induce flowering when FT is expressed in leaves, but can induce flowering when it is expressed in the SAM. Based on this idea, they induced these FT mutated proteins in leaves and also in entire plants (including the SAM), and then analyzed flowering time. Through this screening, they found at least five amino acids which are likely to be important for FT movement. Although the flowering time results of this experiment were published in the current paper, this screening was performed a while ago and was initially reported in their collaborative manuscript published in 2013. In this prior publication, evidence for the existence of a selective FT unloading mechanism at the SAM was shown in Cucurbita moschata (Yoo et al. 2013). It was reported that movement-defective FT proteins failed to accumulate beyond the phloem-unloading zone at the apex. Although the authors tested the impact of expressing defective C. moschata FT and Arabidopsis FT proteins, both carrying the same amino acid mutations, in C. moschata, it was uncertain whether the functions of these amino acids were conserved in the different plants. In the present work, using the heat shock-inducible FT method in Arabidopsis, Endo et al. (2018) demonstrated that at least three FT mutant proteins defective in movement were not detectable in the Arabidopsis SAM. Considering that the same mutant FT proteins can be found in the phloem sap when expressed in C. moschata, it may be concluded that these amino acids are important for the unloading of FT from the phloem to the SAM at the plant shoot apex (Fig. 1).

Endo et al. (2018) also started to explore the potential mechanisms of FT unloading. Even though FTIP1 and NaKR1 were identified as regulators of FT movement, it does not mean that they are the only factors regulating FT movement, as the flowering phenotypes of both ftip1 and nakr1 mutant plants are less severe than that of ft mutants (Liu et al. 2012, Zhu et al. 2016). The authors tested whether any of the FT protein mutations generated affected the direct binding with FTIP1. All unloading-defective mutant FT proteins were still able to interact with FTIP1 in yeast, indicating that these mutations may affect the interaction with other unknown factors involved in unloading FT around the SAM.

It is also noted that all three amino acids important for movement locate closely to the external loop and the putative ligand-binding pocket of FT, both of which are important for its floral induction function (Ahn et al. 2006, Ho and Weigel 2014). When all three amino acids were substituted simultaneously with alanines, the mutant was not able to initiate floral induction in C. moschata (Yoo et al. 2013). These results imply that collectively these amino acids may also affect FT activity. Further structure-based studies will help reveal the relationship between protein conformation and movement or activity.

In order to induce flowering at the proper time, FT moves to the SAM from the leaf phloem where it is synthesized. Since FT transport processes (i.e. uploading to the phloem and unloading from the phloem) are probably actively regulated, flowering time could be controlled not only by FT expression levels but also by FT transport. Therefore, the long-distance delivery of FT protein as well as environmental regulation of mRNA expression can also be considered important processes for flowering. Although FT transport is much less characterized compared with FT transcriptional regulation, understanding this entire process will be of great interest. As a first step, Endo et al. (2018) mapped amino acids important for FT transport but not for flowering initiation activity. This information will be useful for future studies regarding FT movement mechanisms, especially how FT is actively unloaded at the SAM, and for potentially finding the specific interacting proteins for these amino acids.

Funding

This work was supported by the National Science Foundation (NSF) [grant IOS-1656076]; the National Institute of Health (NIH) [grant GM079712]; and the Rural Development Administration, Republic of Korea [Next-Generation BioGreen 21 Program grant SSAC, PJ013386 to T.I.].

Disclosures

The authors have no conflicts of interest to declare.