Florigen governs shoot regeneration

It is widely known that during the reproductive stage (flowering), plants do not root well. Most protocols of shoot regeneration in plants utilize juvenile tissue. Adding these two realities together encouraged us to study the role of florigen in shoot regeneration. Mature tobacco tissue that expresses the endogenous tobacco florigen mRNA regenerates poorly, while juvenile tissue that does not express the florigen regenerates shoots well. Inhibition of Nitric Oxide (NO) synthesis reduced shoot regeneration as well as promoted flowering and increased tobacco florigen level. In contrast, the addition of NO (by way of NO donor) to the tissue increased regeneration, delayed flowering, reduced tobacco florigen mRNA. Ectopic expression of florigen genes in tobacco or tomato decreased regeneration capacity significantly. Overexpression pear PcFT2 gene increased regeneration capacity. During regeneration, florigen mRNA was not changed. We conclude that florigen presence in mature tobacco leaves reduces roots and shoots regeneration and is the possible reason for the age-related decrease in regeneration capacity.

www.nature.com/scientificreports/ protein family) of perennial plants regulate cellular proliferation and new tissue formation and induce flowering when expressed tobacco or Arabidopsis 21,22 . However, while FT1-like expression in perennial plants precedes flowering, suggesting it functions as florigen, FT2-like genes are associated with the juvenile and vegetative period 21,23 . Thus, FLOWERING LOCUS T-like genes coordinate the repeated cycles of vegetative and reproductive growth in perennial like poplar and pear by cycled expression year-round 21,24 .
Kumar et al. 25 demonstrated that Oncidium's flowering is mediated by NO (Nitric Oxide) levels, suggesting that NO controlled the phase transition and flowering process 25 . Application of sodium nitroprusside (NO donor) to Arabidopsis vtc1 mutant caused late flowering, and expression levels of flowering-associated genes (CO, FT, and LFY) were reduced, suggesting NO signaling is vital for flowering 25,26 . The induction to flowering or vegetation pattern relies on the balance between the expression levels of genes in the PEBP gene family (phosphatidylethanolamine-binding protein) like FT1 or FT2 21,24,27 mutations these homologous genes have different consequences on flowering or vegetative growth. However, it seems that these genes determine the fate of the meristem for vegetative or reproductive growth 28 .
In the study here, we present evidence to show that the florigen gene levels in tobacco or tomato influence regeneration capacity. Overexpression of pear PcFT2 gene increased regeneration capacity while FT1 or florigen reduces regeneration capacity. During regeneration, tobacco florigen mRNA does not change. We conclude that florigen presence in mature tobacco leaves reduces roots and shoots regeneration. It may be the possible reason for the age-related decrease in regeneration capacity.

Results
Root and shoot regeneration is affected by leaf age. We tested root regeneration from leaf petioles and found that as the leaves mature and the plant approaches flowering, the number of roots regenerated declines (Fig. 1a) as well as percent regeneration (Fig. S1). Root regeneration from mature leaf segments taken from the leaf blade was much lower than juvenile leaf segments (Fig. 1b), as was percent shoot regeneration (Fig. S2a). The number of shoots regenerated from juvenile leaves (leaf number 7-8) was significantly higher than from mature leaves (leaf number 20-21) (Fig. 1c), as was percent shoot regeneration (Fig. S2b). Juvenile leaves regenerate more roots and shoots than mature leaves. The juvenile leaves' tips are much rounded than the mature leaves (Fig. 1d); as the tobacco plant ages, the leaves turn more elongated, which can be seen as the ratio between leaf width to leaf length decreases (Fig. S3). Preventing the effect of leaf age on shoot or root regenerative ability was achieved by using leaves at the same developmental stage, 3-4 days after they reached the final size. Under our conditions in the growth room, tobacco plants flowers at about 20 to 22 leaves.

Shoot regeneration and flowering are affected by Nitric Oxide. The flowering of tobacco and
Arabidopsis plants was affected by a short preincubation of the seedlings in the presence or absence of Nitric Oxide (NO) level modifiers. Growing the seedlings of tobacco (Fig. 2a) or Arabidopsis (Fig. 2b) on media containing the NO synthesis inhibitor DiPhenyleneiodonium (DiPhenyl) prior to planting in pots advances flowering of both plant genotypes ( Fig. 2a; 2b). Incubation with NO donor Molsidomine (Molsido) had a nonsignificant but repeated small flowering delay (Fig. 2a,b). Shoot regeneration was enhanced in plants treated with Molsidomine (Fig. 2c) and inhibited by DiPhenyl in non-transformed SR1 plants. Overexpression of avocado florigen in SR1 plants reduced shoot regeneration (Fig. 2c) while promoting flowering (Fig S2c). DiPhenyl inhibited shoot regeneration in florigen expressing and untransformed plants. However, florigen overexpressing plants were insensitive to treatment with NO donor (Fig. 2c).
Treating tobacco seedlings with DiPhenyl that inhibits NO synthesis 29,30 shows a similar phenotype of lacking NO as in Arabidopsis nos1 mutant 31 (Fig. 2d) i.e., impaired growth, yellowish first true leaves, reduces root size, defective abscisic acid-induced stomatal movements, and most importantly, induces early blooming 31 . Transfer of the tobacco seedlings to the soil without DiPhenyl resulted in rapid greening and growth and early flowering (Fig. 2d). The ratio between leaf width to length did not change after treatment with NO modifiers (Fig. S3a) or overexpression of florigen (Fig. S3b).

FLOWERING LOCUS T mRNA level is influenced by leaf position and NO treatment. The level
of mRNA of several FLOWERING LOCUS T (NtFT1, NtFT2, NtFT3, and NtFT4) genes of tobacco was compared between juvenile leaf and mature leaf and juvenile leaves treated with DiPhenyl or Molsido. NtFT4 mRNA (the tobacco florigen) level was high in mature leaves, and DiPhenyl treated juvenile leaves and correlated with flowering. In leaves of plants that do not flower, NtFT4 was not expressed (Fig. 3a). NtFT2 and NtFT1 are expressed in all examined leaves (Fig. 3a), and NtFT3 was only expressed in mature leaves. The NtFT gene family level does not change during the initial regeneration period, but NtTFL1 expression increases (Fig. 3b).
The expression of genes associated with FLOWERING LOCUS T, like TEMPRANILLO or SQUAMOSA PRO-MOTER BINDING, did not show the pattern exhibited by the tobacco florigen (NtFT4) (Tables S1 and S2). mRNA expression heatmap shows extensive changes between mature and juvenile leaves (Fig. 3c). The expression of 2792 genes changes between juvenile and mature leaves, while between juvenile leaves treated with DiPhenyl, only 403 genes changed expression, and between juvenile leaves treated with Molsido, only 212 genes changed expression (Fig. S4a). The gene heatmap expression of juvenile leaf treated with DiPhenyl shows specific patterns like the mature leaf. The Molsido treated leaf is similar to juvenile leaves (Fig. 3c). Six different expression patterns, i.e., C1-C6, were distinguished according to similarity or difference to juvenile leaf (Fig. S4b). Mature leaf segments collected at the time flower buds are visible show reduced shoot regeneration in non-transformed plants (Fig. 4c). However, flowering did not affect shoot regeneration in plants transformed with avocado florigen (PaFT1) (Fig. 4c). Overexpression of the pear (PcFT2) rejuvenator gene in tobacco plants resulted in increased shoot regeneration capacity in juvenile leaf segments (Fig. 4d).

Discussion
We designed this study to analyse the basis of root and shoot regeneration differences between vegetative state (Juvenile) and flowering state (Mature) plant tissues. Past reports described that flower buds' presence on a plant reduces rooting, for example, in Rhododendron, Camellia, Coleus, Vaccinium, and Taxus 19 or inhibit cambial activity in stems of flowering plants 20 . The differences between juvenile and mature tissues in the capacity to regenerate roots or shoots depend on physiological age. Most shoot or root regeneration protocols vary vastly between plant species; almost all with a few exceptions, use a tissues (or explant) that are juvenile, such as cotyledon, hypocotyl, petiole, or dormant meristem. In both plants and animals, regeneration ability declines with age 7,8,11 . FLOWERING LOCUS T (FT) is a small mobile protein that functions as a floral and developmental regulator gene family. FT protein is a critical element in annual plants' competence to flower shortly after emergence; however, perennial plants contain at least two FT genes with different functions in flowering florigen and a rejuvenator 21 . Perennials plants have an extended juvenile period lasting up to many years of vegetative growth before achieving flowering 18,22 . After the first flowering period, perennials enter into a yearly cycle of vegetative and reproductive processes. Perennials express at least two versions of FT genes, florigen and a rejuvenator gene. Both are a phosphatidylethanolamine-binding protein family (PEBP gene family) and induced flowering when  Fig. S3. (d) Phenotype difference between mature and juvenile leaves. Statistical analysis was conducted among each color group using the JMP program using Tukey analysis. Different letters depict statistically significant differences between genotypes or treatments (p{f} < 0.001).  27,32 functions to transform the leaf into a mature organ changing its shape 33 and reducing its capacity to regenerate. While the phase change from vegetative to reproductive growth in a plant is accompanied by changing leaf shape 33 , we found that inducing early flowering and reducing regeneration ability is not related to this shape change. While the shape changes are correlated with reduced mir156 and increased SPL genes 33 , this study shows that independently from flowering and other meristematic effects, FT genes function in the leaf tissue as a determinant of juvenility or maturity depending if the rejuvenator or the florigen is expressed without changing the leaf shape that is typical of plant maturity. Examining FT's immediate suspects in meristematic flowering processes did not reveal an expression pattern that is similar to FT mRNA (Table S1 and S2). Thirteen FT-like genes were identified in tobacco 34 ; out of these 13 genes, NtFT4 and NtFT5 were shown to function as florigen 35,36 . RNA-seq of leaf tissue from mature or juvenile leaves showed that NtFT4 is expressed in mature leaves and not in juvenile leaves, while NtFT5 is not expressed in the leaves. Treating the tobacco leaves with NO modifiers that promoted flowering and inhibited regeneration increased NtFT4 but did not affect the expression of NtFT5 (remained zero). Treatment of tobacco leaves with NO modifiers that inhibited flowering and enhanced regeneration and depressed NtFT4 expression, NtFT5 expression was zero. We, therefore, conclude that there are tissue-specific florigen in tobacco and maybe other plants. Some florigen genes are expressed in the leaves, and some in the stem 35   www.nature.com/scientificreports/ cellular NO increases regeneration 30 (and in this study). We postulated a connection between NO and FT gene family members; it seems that NO level controls FT mRNA expression and thus flowering and regeneration ability. Our data show that NO level affects florigen mRNA level and, as a consequence, influences regeneration and flowering. A link between flowering and root regeneration (rooting) is known for decades and used in plants' vegetative production of crop plants like trees, vegetables, and flowers. The florigen's mRNA expression level seems to explain why plants at the reproductive stage do not regenerate as well as plants in the vegetative phase when the florigen is not expressed. NO impact on seed aging progression was demonstrated by treating seeds with NO or NO donors that alleviate cell death and aging 38 . NO also delays tissue senescence 38,39 . Here we show that the effect of NO on shoot regeneration is in juvenile tissue and not in mature tissue; thus, the effects of NO on aging are not related to the effect of NO on shoot regeneration. It is probably a different and direct effect on regeneration. The main NO target in animal tissue is the soluble enzyme guanylyl cyclase; however, NO in plants is unknown 40 . The indication that NO affects different plant processes could result from many targets and effectors to NO. They vary according to a temporal and physical location. Juvenility across kingdoms is associated with enhanced regenerative ability. For example, juvenile plants exhibit a high regenerative capacity; as the plant mature, this capacity declines 11 , as shown here, and modifying mice's adolescent state affects tissue repair, a type of regeneration 41 or juvenile axolotl can regenerate a limb faster than an adult 42 . These observations show that the juvenility state of the tissue governs plants' and animals' regenerative capacity. Zhang et al. 11 speculated that the binding of SPL9 to ARR2 changes the conformation of ARR2, thereby impairing its transcriptional activation toward downstream 11 . In the flowering cascade, FT is influenced by miR156 and SPL genes 43 . Thus, as shown here, the florigen protein FT affects regeneration capacity on its own.
Our results revealed that the decrease in shoot and root regeneration in mature plant tissue is correlated with a high florigen expression. The mechanism causing the reduced shoot or root regenerative capacity in old plants and whether FT expression is connected to altered phytohormones response awaits further investigations. Ongoing analysis is the effect of cytokinins and auxin combination on shoot regeneration in mature vs. juvenile www.nature.com/scientificreports/ leaves. Knox gene family was implicated in FT function 27 and in shoot regeneration 30 . We are currently testing the effect of Knox gene family knockout using crisper technology on shoot regeneration. Shoot and root regeneration is influenced by many factors, the explants, the culture medium, phytohormones, and gelling agent, to name the most tested. FT genes expression level or presence can be used as a marker for regeneration capacity. FT gene manipulation can increase plant species propagation, especially in recalcitrant and rare and endangered plants.

Materials and methods
Seed decontamination and plant growth. Tobacco Seeds (Nicotiana tabacum L. cv. SR1) or Arabidopsis Colombia are used and grown in our lab for many years. Seeds were cleansed with sodium hypochlorite (0.5% active material) in a 1.7 mL micro-tube (Eppendorf) for 15 min. After sodium hypochlorite treatment, the seeds were washed thrice with sterile water and spread on ½-strength MS medium (Duchefa Co. Haarlem, Netherlands, Product number M0221.0050) on a Petri dishes. After germination, seedlings were transffered to polypropylene Vitro Vent containers (Duchefa, NL; 9.6 cm × 9.6 cm and 9 cm in height) with ½-strength MS medium. Molsido (five µM Molsidomine and five μM DiPhenyleneiodonium chloride) was added to the agar medium when treated with NO effectors. Plants were grown in sterile containers in a growth room with 16 hours of light and eight hours of darkness at 26 °C for weeks until leaves were ready to be harvested for regeneration. Tobacco plants were transformed as described before 30,44 with Avocado (Persea Americana), FLOWERING LOCUS T-like plasmids obtained from Ziv et al. 45 , pear (Pyrus communis) from Frieman et al. 21 , and pepper (Capsicum annum) from Borovsky et al 46 . Transgenic tomato (Solanum lycopersicon M82) seeds were obtained from Borovsky et al 46 .  Leaves preparation and shoot and root regeneration. Leaves were detached from sterile plants, and the midrib was removed. The leaf blade was cut into segments about 25 mm 2 (5 mm × 5 mm) and placed on a shoot regeneration (Reg) medium containing standard MS salts as before 30,44 . Medium was augmented with 30 g l −1 sucrose and 8 g l −1 agar and the following growth regulators: 4.57 μM IAA; 9.29 µM Kinetin, and 4.56 µM Zeatin (all from Duchefa Co.) or on 1 mg l −1 IBA for rooting. At least 20 leaf segments were placed on each petri dish with at least four plates per treatment in all experiments. Shoot regeneration from leaf segments was scored 30-32 days after putting them on the medium; root regeneration from segments was scored 10-15 days after placing the leaf segment on medium. Leaf petiole rooting was scored between 4 to 15 days.
Analyses of variance (ANOVA) were performed with the SAS/JMP software (SAS Institute Inc., Cary, NC, USA). Differences among means were calculated based on the Tukey-Kramer honestly significant difference (HSD) test for three or more treatments and T-test for two treatments 30 .
The numbers of explants and regenerated shoots or roots were scored. The regenerative capacity is represented by the number of regenerated shoots or roots per explants. At least three independent experiments (biological triplicates) were performed, and in each, at least three samples were tested.
RNA preparation and transcript detection. RNA was isolated from leaf segment using the TRI-reagent Transcriptome analysis. Raw reads were analyzed by filtering and cleaning procedure. The Trimmomatic tool 47 was used to remove Illumina adapters from the reads. FASTX (http:// hanno nlab. cshl. edu/ fastx_ toolk it/ index. html, version 0.0.13.2) was used next to trim the read-end with quality scores <30, utilising the FASTQ Quality Trimmer, and to eliminate sequences with fewer than 70% base pairs homology with a quality record ≤30 using the FASTQ Quality Filter. The selected reads were mapped to the reference genome of Nicotiana tabacum 48 ftp:// ftp. solge nomics. net/ genom es/ Nicot iana_ tabac um/ edwar ds_ et_ al_ 2017/ assem bly/) using STAR software 49,50 . Gene abundance estimation was performed using Cufflinks 51 combined with gene annotations from the Sol Genomics Network database (https:// solge nomics. net/; ftp:// ftp. solge nomics. net/ genom es/ Nicot iana_ tabac um/ edwar ds_ et_ al_ 2017/ annot ation). PCA analysis and Heatmap visualization were performed using R Bioconductor 51 . Gene expression numbers were calculated as FPKM. Distinction expression analysis was completed using the DESeq2 R package 52 . Genes that varied from the control more than twofold, with an adjusted p-value of no more than 0.05, were considered differentially expressed 53 . Venn diagrams were produced using the online tool at bioinformatics.psb.ugent.be/webtools/Venn/. KOBAS 3.0 tool 54 http:// kobas. cbi. pku. edu. cn/ kobas 3/?t=1) was applied to find the statistical enrichment of differentially expressed genes KEGG pathway and Gene Ontology (GO).