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 Taxus19 or inhibit cambial activity in stems of flowering plants20. 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 age7,8,11.
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 rejuvenator21. Perennials plants have an extended juvenile period lasting up to many years of vegetative growth before achieving flowering18,22. 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 expressed in tobacco or Arabidopsis21,22. However, while FT1, the florigen expression in perennial plant's expression, precedes flowering, the FT2 or the rejuvenator expression is during the vegetative period 21,23. Thus, florigen and a rejuvenator are both homologs to the A. thaliana gene FLOWERING LOCUS T (AtFT1) gene, coordinating the repeated cycles of vegetative and reproductive growth in woody perennial like poplar24 (Populus spp.) or pear21 by cycling expression year-round. Florigen action in the meristem to induce flowering27,32 functions to transform the leaf into a mature organ changing its shape33 and reducing its capacity to regenerate. While the phase change from vegetative to reproductive growth in a plant is accompanied by changing leaf shape33, 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 genes33, 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 tobacco34; out of these 13 genes, NtFT4 and NtFT5 were shown to function as florigen35,36. 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 stem35.
NO (nitric Oxide) levels are involved in flowering, as demonstrated in Oncidium's25 and Arabidopsis37. Application of NO donor on Arabidopsis vtc1 mutant caused late flowering, and the expression levels of flowering-associated genes (AtCO, AtFT, and AtLFY) were reduced, suggesting NO signaling is part of flowering25,26. Reducing the amount of NO in Arabidopsis plants promoted flowering, increasing NO inhibited flowering37, as was shown here. In parallel, reducing the amount of NO decreases regeneration, and increasing the amount of cellular NO increases regeneration30 (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.
Juvenility across kingdoms is associated with enhanced regenerative ability. For example, juvenile plants exhibit a high regenerative capacity; as the plant mature, this capacity declines11, as shown here, and modifying mice's adolescent state affects tissue repair, a type of regeneration38 or juvenile axolotl can regenerate a limb faster than an adult39. 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 downstream11. In the flowering cascade, FT is influenced by miR156 and SPL genes40. 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. 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.