AcFT promotes kiwifruit in vitro flowering when overexpressed and Arabidopsis flowering when expressed in the vasculature under its own promoter

Abstract Kiwifruit (Actinidia chinensis) has three FLOWERING LOCUS T (FT) genes, AcFT, AcFT1, and AcFT2, with differential expression and potentially divergent roles. AcFT was previously shown to be expressed in source leaves and induced in dormant buds by winter chilling. Here, we show that AcFT promotes flowering in A. chinensis, despite a short sequence insertion not present in other FT‐like genes. A 3.5‐kb AcFT promoter region contained all the regulatory elements required to mediate vascular expression in transgenic Arabidopsis thaliana (Arabidopsis). The promoter activation was initially confined to the veins in the distal end of the leaf, before extending to the veins in the base of the leaf, and was detected in inductive and noninductive photoperiods. The 3‐kb and 2.7‐kb promoter regions of AcFT1 and AcFT2, respectively, demonstrated different activation patterns in Arabidopsis, corresponding to differential expression in kiwifruit. Expression of AcFT cDNA from the AcFT promoter was capable to induce early flowering in transgenic Arabidopsis in noninductive photoperiods. Further, expression of AcFT cDNA fused to the green fluorescent protein was detected in the vasculature and was also capable to advance flowering in noninductive photoperiods. Taken together, these studies implicate AcFT in regulation of kiwifruit flowering time and as a candidate for kiwifruit florigen.

I hope the revised manuscript meets with your approval.

Kind regards, Erika Varkonyi-Gasic
Reviewer #1: This manuscript showed FT-mediated regulation of growth and flowering in kiwifruit using the own and/or Arabidopsis promoters combined with several FT genes of kiwifruit/Arabidopsis under flowering inductive and non-inductive conditions. The subject falls within the general scope of the journal, and the article is a new and original contribution. The interpretations and conclusions are sound enough and are justified by the data, and are consistent with the objectives. The title clearly reflects the contents of the paper and the abstract is sufficiently informative and can be read in isolation. Therefore, the manuscript contains new interesting insights to warrant publication. Before acceptance, try to address the below minor questions/suggestions. 1) In natural field condition, when does floral induction (or visible floral initiation) take place in kiwifruit? The authors focused on day length (SD/LD) in this study; how about the involvement of temperature in flower induction? Please briefly describes the basic phenology in kiwifruit flowering.
 We have included a paragraph that briefly describes kiwifruit phenology.
Temperature (cold, i.e. winter chilling) is indeed essential for kiwifruit flowering, whilst photoperiod (the shortening of the day-length) drives growth cessation and budset (reference describing this included). The reason for focusing on photoperiod was the work we performed in Arabidopsis, where we wanted to be able to maintain the 'non-flowering' state of our model. The activity of AcFT promoter in conditions when endogenous FT is not expressed in Arabidopsis provided the opportunity to test if "native" expression levels and domains have the ability to drive flowering. We could not perform the chilling work in Arabidopsis; as a herbaceous plant, it would not survive the conditions that drive budbreak and flowering in kiwifruit.
 The first exon (exon 1) and the first intron (intron 1), now described in figure legend.
3) The authors stated in lines 221-223, "A translational fusion including the AcFT first exon, first intron and the first eight codons of the second exon was also evaluated to examine whether regulatory regions exist in the first intron". Which data (Fig. S2?) corresponds to this experiment? If Fig. S2, please clearly indicate the experimental details in figure legend of Figure S2.  The figure presents the range, if more than one plant flowered at the same time, they are presented as a single dot (this is now described in the legend). We wanted to show that our promoter was capable to deliver sufficient Arabidopsis FT to drive flowering in Arabidopsis (in conditions when the Arabidopsis FT promoter is inactive and all control plants flower late). But an unintentional mistake has been made, it is three rather than four out of seven lines (now corrected). We have increased the size of the graph in Figure 4b for easier interpretation.
5) The authors stated in lines 290-292, "All Col-0 proAcFT:AcFT plants flowered in LD after the plants produced between 7 and 10 leaves, whilst ten Col-0 controls flowered after producing 8-10 leaves". Figure showed the results in SD condition; which data are comparable to this experiment in LD? Data not shown?
 Data not shown. As non-essential, now removed from the text.

Reviewer #2:
This is an interesting and well-done piece of work and manuscript. I have no substantial suggestions. In my copy of the manuscript, Figure 3 is not in English. I have no idea what language it is in.