doi:10.1093/plphys/kiab421 PLANT PHYSIOLOGY 2021: 187: 1221–1234 Woodland strawberry axillary bud fate is dictated by a crosstalk of environmental and endogenous factors Research Article Javier Andrés,1 Julie Caruana ,2,3 Jiahui Liang ,4 Samia Samad ,5 Amparo Monfort ,6,7 Zhongchi Liu ,2 Timo Hytönen1,8 and Elli A. Koskela1,6,*,† 1 Department of Agricultural Sciences, Faculty of Agriculture and Forestry, University of Helsinki, Finland 2 Department of Cell Biology and Molecular Genetics, University of Maryland, Maryland 20742, USA 3 American Society for Engineering Education, Washington, District of Columbia, USA 4 Department of Fruit Science, College of Horticulture, China Agricultural University, China 5 Department of Biosystems and Technology, Swedish University of Agricultural Sciences, Alnarp SE-230 53, Sweden 6 Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, 08193 Bellaterra, Barcelona, Spain 7 Institut de Recerca i Tecnologia Agroalimentàries (IRTA), 08193 Barcelona, Spain 8 NIAB East Malling Research, West Malling, ME19 6BJ, UK *Author for communication: elli.koskela@helsinki.fi † Senior author. J.A., S.S., J.L., and E.A.K.: carried out the experimental work; J.C.: produced the FvGA20ox4-RNAi plant materials under the supervision of Z.L.; A.M.: su- pervised the experiments carried out at CRAG; T.H. and E.A.K.: supervised the experiments carried out at UH; E.A.K., J.A., and T.H.: drafted the manu- script with input from all the co-authors. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plphys/pages/General-Instructions) is: Timo Hytönen (timo.hytonen@helsinki.fi). Abstract Plant architecture is defined by fates and positions of meristematic tissues and has direct consequences on yield potential and environmental adaptation of the plant. In strawberries (Fragaria vesca L. and F.  ananassa Duch.), shoot apical mer- istems can remain vegetative or differentiate into a terminal inflorescence meristem. Strawberry axillary buds (AXBs) are lo- cated in leaf axils and can either remain dormant or follow one of the two possible developmental fates. AXBs can either develop into stolons needed for clonal reproduction or into branch crowns (BCs) that can bear their own terminal inflores- cences under favorable conditions. Although AXB fate has direct consequences on yield potential and vegetative propaga- tion of strawberries, the regulation of AXB fate has so far remained obscure. We subjected a number of woodland straw- berry (F. vesca L.) natural accessions and transgenic genotypes to different environmental conditions and growth regulator treatments to demonstrate that strawberry AXB fate is regulated either by environmental or endogenous factors, depend- ing on the AXB position on the plant. We confirm that the F. vesca GIBBERELLIN20-oxidase4 (FvGA20ox4) gene is indispens- able for stolon development and under tight environmental regulation. Moreover, our data show that apical dominance inhibits the outgrowth of the youngest AXB as BCs, although the effect of apical dominance can be overrun by the activity of FvGA20ox4. Finally, we demonstrate that the FvGA20ox4 is photoperiodically regulated via FvSOC1 (F. vesca SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1) at 18 C, but at higher temperature of 22 C an unidentified FvSOC1-independent pathway promotes stolon development. Received May 09, 2021. Accepted August 26, 2021. Advance access publication September 1, 2021 C The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists. V Open Access This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 1222 | PLANT PHYSIOLOGY 2021: 187; 1221–1234 Andrés Jimenez et al. Introduction tremuloides). Specific components of the regulatory network Plant architecture is defined by meristem fates that can take controlling bud outgrowth are conserved between hybrid as- on different avenues, depending on plant species and the pen and annual plants; the putative tree orthologs of BRC1 position of the meristem. Shoot apical meristems (SAMs) inhibit bud outgrowth (Muhr et al., 2018) and are regulated can either remain vegetative and keep on producing new by strigolactones (Muhr et al., 2016). However, aspen BRC1 vegetative plant tissues, or, when induced to flower, differen- is also regulated by a negative feedback loop involving puta- tiate into an inflorescence meristem. Axillary meristems tive tree orthologs of TERMINAL FLOWER1 (TFL1) and (AXMs) are initiated in leaf axils and form axillary buds APETALA1 (LIKE-APETALA1/LAP1) (Maurya et al., 2020). (AXBs) with distinguishable anatomical features such as leaf Aspen TFL1 represses LAP1 expression, which in turn is a primordia. In literature, the terms AXM and AXB are some- negative regulator of both TFL1 and BRC1. SDs (short days) times used interchangeably, causing confusion. In this work, and low temperature upregulate TFL1, leading to downregu- we use the term AXB to describe a macroscopically visible lation of LAP1 and de-repression of BRC1. Upregulation of but visually undifferentiated organ located in the leaf axil. BRC1 by conditions mimicking oncoming winter inhibits AXBs can either grow out as vegetative or generative bud outgrowth and suppresses branching (Maurya et al., branches or remain dormant, depending on the SAM status 2020). (reviewed in Leyser, 2003). Dormancy was originally defined The role of gibberellins in the outgrowth of AXBs in hy- as “lack of visible growth in a meristematic tissue” (Lang, brid aspen was recently suggested by Katyayini et al. (2020). 1987), and in this work, we apply this definition to AXBs. In They found that different bioactive GA forms have opposing the economically important Rosaceae family, AXBs can ei- roles in regulating AXB outgrowth as a response to shoot ther remain dormant or form a short shoot with short decapitation. In growth-arrested AXBs of hybrid aspen, GA3 internodes and leaf rosette-like appearance. The third fate is and GA6 upregulate catabolic GA2ox genes, whose protein to form long shoots characterized by long internodes. products deactivate GA1 and GA4. Thus, AXB quiescence is Because different shoot types have different capabilities for maintained despite active ongoing GA biosynthesis and is forming inflorescence meristems and have different growth released upon loss of apical dominance by strong downregu- vigor, AXB fate has major consequences on plant architec- lation of GA2ox genes (Katyayini et al., 2020). However, care ture, yield potential and environmental adaptation of the should be taken when interpreting these results because plant (Costes et al., 2014). In strawberry, AXB fate directly they are based on exogenous hormonal applications. affects yield potential, as inflorescences are formed at the In both woodland strawberry (Fragaria vesca) and culti- apices of short shoots called branch crowns (BCs) (Battey et vated strawberry (F.  ananassa), AXB fate is controlled by al., 1998). Long shoots of strawberry, known as stolons or environmental and genetic factors. In general, flowering in- runners, are indispensable for clonal propagation of the ducing conditions promote the formation of BCs analogous crop. Although a handful of genes regulating AXB fate in to short spur shoots in Rosaceous fruit trees. Strawberry strawberry has been identified, environmental regulation of inflorescences arise terminally at the SAM of the main AXB fate in this important crop remains obscure. crown and the uppermost AXB(s) of the main crown form In Arabidopsis, AXB outgrowth depends on both endoge- BC(s), thus following a sympodial growth pattern (Darrow, nous and environmental factors. The most important en- 1966; Guttridge, 1985). Under nonflowering-inducing condi- dogenous factor is apical dominance exerted by the SAM tions, AXBs may remain dormant in a visually undifferenti- controlling lateral bud outgrowth (Cline, 1997). Apical domi- ated state or produce stolons, a form of asexual nance functions, at least in part, via auxin produced by the reproduction (Brown and Wareing, 1965; Darrow, 1966; SAM. Auxin travels downward through the phloem, pro- Hytönen et al., 2009). It remains unknown whether the envi- moting the synthesis of strigolactones while inhibiting the ronmental conditions favoring floral induction regulate the activity of cytokinins. Unlike auxin, these hormones can AXB fate directly, or whether the floral induction at the move into AXBs to inhibit and promote bud outgrowth, re- SAM induces BC development indirectly, by e.g. releasing spectively (Skoog and Thimann, 1934; Dun et al., 2012; AXBs from growth arrest caused by apical dominance. Rameau et al., 2015; Qiu et al., 2019). Both strigolactones Strawberry genotypes are classified based on their photo- and cytokinins have been shown to regulate the expression periodic flowering responses into either seasonal or perpet- of BRC1 (BRANCHED1; Dun et al., 2012), a TCP (TEOSINTE ual flowering types. In seasonal flowering F. vesca genotypes, BRANCHED1, CYCLOIDEA, PCF) transcription factor that is SDs and cool temperatures promote flowering and AXB de- expressed in growth-arrested AXBs both in monocots and velopment into BCs, while LDs (long days) and warm tem- dicots (Takeda et al., 2003; Aguilar-Martinez et al., 2007; peratures enhance stolon development. In contrast, Braun et al., 2012). perpetual flowering F. vesca genotypes initiate inflorescences The above-mentioned mechanisms for controlling AXB and develop BCs in LD conditions, while SDs and warm fate have been elucidated in annual species. A genetic temperature promote stolon development (Brown and framework for controlling AXB fate in perennial species has Wareing, 1965; Mouhu et al., 2009; Sønsteby and Heide, started to emerge only recently, mainly based on results 2008). In seasonal flowering F.  ananassa cultivars, the en- obtained in hybrid aspen (Populus tremula  Populus vironmental responses are similar to those of seasonal F. Woodland strawberry axillary bud fate regulation PLANT PHYSIOLOGY 2021: 187; 1221–1234 | 1223 vesca. Seasonal flowering F.  ananassa cultivars are in- expression either by overexpression or silencing obstructs duced to flower and develop BCs under SDs, and their sto- the photoperiodic regulation of vegetative development in lon development is promoted by LDs and warm strawberry. Altered FvSOC1 expression levels in FvSOC1 temperature (Heide, 1977). In perpetual flowering F.  ana- transgenic plants changed the activity of several GA path- nassa cultivars, the environmental responses are more vari- way genes in leaf tissues (Mouhu et al., 2013). able than in perpetual flowering F. vesca genotypes. For FvSOC1 is photoperiodically regulated via the FvCO–FvFT1 example, SDs and high temperature (27 C) strongly pro- (F. vesca CONSTANS1; F. vesca FLOWERING LOCUST1) moted stolon development in the perpetual flowering F1 hy- pathway that is active exclusively under LD conditions brid “Elan” (Sønsteby and Heide, 2007a, 2007b). However, (Rantanen et al., 2014; Kurokura et al., 2017). FvSOC1 relays LDs at 27 C promoted stolon development in the perpetual photoperiodic signals not only to the GA pathway, but also flowering cultivars ‘Rita’, ‘Ridder’, and ‘Flamenco’, whereas to the photoperiodic flowering pathway by upregulating the cultivar ‘Rondo’ appeared to develop stolons indepen- FvTFL1, a strong floral repressor (Koskela et al., 2012; Mouhu dently of photoperiod (Sønsteby and Heide, 2007b). et al., 2013). In seasonal flowering accessions, activity of the Several studies have shown an association between AXB FvFT1–FvSOC1–FvTFL1 pathway represses flowering in LDs. fate and GA in the two strawberry species. The early study Perpetual flowering accessions carry a mutation in the by Thompson and Guttridge (1959) demonstrated that GA FvTFL1 coding sequence that inhibits its function as a floral application to a SD-grown seasonal flowering F.  ananassa repressor. Since the flowering decision depends on the bal- cultivar can mimic the effect of LD conditions; GA-treated ance between FvFT1 and functional FvTFL1, the abundance plants developed stolons instead of BCs and did not initiate of FvFT1 under LD conditions coupled to the presence of flowers in contrast to nontreated control plants. This finding nonfunctional FvTFL1 enhances flowering in perpetual flow- was later expanded by Hytönen et al. (2009), who showed ering accessions (Koskela et al., 2012; Mouhu et al., 2013; that AXB development into stolons in F.  ananassa Rantanen et al., 2015) requires bioactive GA1 and that inhibiting GA biosynthesis A complicating factor in studying vegetative responses in by prohexadione-calcium (Pro-Ca, an inhibitor of GA biosyn- strawberries is the dependence of AXB fate on the SAM thesis) increases the proportion of AXBs developing into state. Once the SAM is committed to forming an inflores- BCs. Recently, the requirement for bioactive GA for stolon cence meristem, apical dominance is released and the up- development in F. vesca was suggested by Tenreira et al. permost AXBs of the main crown develop into BCs (2017), who provided evidence that the stolonless pheno- (Darrow, 1966; Guttridge 1985, Sugiyama et al., 2004). type observed in certain perpetual flowering F. vesca acces- However, the SAM fate cannot be the only factor regulating sions is caused by a loss-of-function mutation in FvGA20ox4. AXB fate, because AXBs do develop into BCs photoperiod- GA20-oxidases are GA biosynthetic enzymes that produce dependently also in genotypes where the SAM is forced to precursors of bioactive GA that are then converted to bioac- remain in vegetative state by overexpressing FvTFL1 (Koskela tive GA1 and GA4 by GA3-oxidases. Further down in the et al., 2012). pathway, GA2-oxidases control the pool of bioactive GA by In this article, we study the environmental regulation of catalyzing their inactivation (Hedden, 2020). The importance AXB fate independently of that of the SAM by using a num- of GA signaling downstream of the GA biosynthetic path- ber of natural F. vesca accessions and transgenic genotypes way was demonstrated by Caruana et al. (2018), who with different environmental responses and flowering char- showed that the stolonless F. vesca phenotype can be re- acteristics. Our analyses on FvGA20ox4 transgenic plants also versed by a loss of function mutation in FveRGA1, encoding confirm that FvGA20ox4 is indispensable for stolon develop- a DELLA growth repressor in GA signaling pathway (Peng et ment and show that the activity of FvGA20ox4 is tightly reg- al., 1997). Due to the mutation, the GA signaling pathway is ulated by both photoperiod and temperature. Finally, we constitutively switched on, resulting in continuous stolon show that FvGA20ox4 is photoperiodically regulated via development in the normally stolonless perpetual flowering FvSOC1 at 18 C and provide evidence for an FvSOC1-inde- F. vesca accession (Caruana et al., 2018). A recent study by pendent pathway controlling vegetative development at (Feng et al., 2021) described a mutant of loss of axillary mer- 22 C. istems (lam), defective in AXM initiation. However, the lam mutation did not affect AXB fate in the tested F. vesca gen- Results otypes, nor was the effect of the mutation tested under dif- ferent environmental conditions. FvGA20ox4 expression is required for stolon Although the importance of the GA pathway for deter- development mining AXB fate in strawberry has been demonstrated, the Strawberry AXB can develop a stolon, a BC, or remain dor- upstream regulation of the GA pathway remains a less- mant (Darrow, 1966), and a recent study showed evidence, studied topic, especially in terms of environmental regula- by using a natural stolonless F. vesca mutant grown under tion. However, it has been shown that the F. vesca MADS natural light conditions, that GA biosynthetic enzyme box transcription factor FvSOC1 relays photoperiodic signals FvGA20ox4 was needed for stolon development (Tenreira et to the GA pathway at 18 C and that altering FvSOC1 al., 2017). However, the role of FvGA20ox4 in stolon 1224 | PLANT PHYSIOLOGY 2021: 187; 1221–1234 Andrés Jimenez et al. development in F. vesca has not been confirmed by trans- perpetual flowering and stolon-forming H4 and obtained genesis, nor have the phenotypes of FvGA20ox4-deficient two transgenic lines with strong and gene-specific silencing genotypes been examined under a controlled environment. of FvGA20ox4 (Figure 2; Supplemental Figure S1). We To understand the function of FvGA20ox4 in the control of attempted to force stolon development in these plants by AXB fate, we first explored photoperiodic and temperature growing them in SDs at 17 C and found that one of the regulation of FvGA20ox4 mRNA expression and AXB fate in FvGA20ox4-RNAi lines completely lacked stolons, and an- the perpetual flowering and stolon-forming wild-type (WT) other line with weaker silencing of FvGA20ox4 rarely pro- genotype H4 (Mouhu et al., 2009; for the characteristics of duced stolons (Figure 2). In WT H4, in contrast, 80% of the F. vesca genotypes used in this work, see Supplemental the AXBs developed into stolons. The FvGA20ox4-RNAi lines Table S1). By analyzing shoot apex samples containing developed BCs at the same frequency as the WT (Figure 2), young AXBs, we observed a much higher FvGA20ox4 expres- suggesting that silencing of FvGA20ox4 had a specific effect sion level in SDs at 24 C than in other temperature/photo- on stolon development without interfering with BC develop- period combinations (Figure 1). Because a previous study in ment. However, the AXBs that were inhibited from develop- seasonal flowering F. vesca showed that FvSOC1 upregulates ing a stolon due to silencing of FvGA20ox4 remained FvGA20ox4 in leaves (Mouhu et al., 2013), we also analyzed dormant, resulting in a higher proportion of dormant AXBs FvSOC1 expression. We found that LD conditions upregu- in RNAi lines compared with WT (Figure 2). These data in- lated FvSOC1 in shoot apices at both 24 C and 11 C dicate that the absence of FvGA20ox4 expression in AXBs (Figure 1), revealing a disconnection between FvSOC1 and does not directly lead to BC development, nor does it in- FvGA20ox4 at 24 C. Concordantly with FvGA20ox4 expres- hibit it. All WT and RNAi plants remained vegetative until sion, the majority of AXBs in plants grown under SDs at the end of the experiment confirming that the silencing of 24 C developed a stolon (Figure 1), showing that the higher FvGA20ox4 affected AXB fate directly and not indirectly FvGA20ox4 mRNA expression level in SDs at 24 C is associ- through floral development at SAM. Taken together, our ated with AXB development into stolons in H4, while a results on RNAi lines demonstrated that FvGA20ox4 is higher percentage of AXBs developed into BCs in LDs needed for stolon development and that additional factor(s) (Figure 1). All plants grown under LDs at 24 C flowered af- are required for BC development in the absence of ter 7 weeks of the treatment, while all the plants grown un- FvGA20ox4 expression. der SDs or at 11 C remained vegetative, supporting previous findings that flower-inducing conditions promote BC devel- FvGA20ox4 activity releases axillary bud dormancy opment, and in vegetative stage, most AXBs develop into a Since the silencing of FvGA20ox4 increased the proportion stolon (Brown and Wareing, 1965; Kurokura et al., 2017). of dormant AXBs in perpetual flowering and stolon-forming The cool temperature of 11 C promoted AXB dormancy in- H4 (Figure 2), we carried out growth regulator treatments dependently of the photoperiod, while at 24 C AXB dor- to further test the connection between GA and AXB dor- mancy was promoted by LDs (Figure 1). Taking these data mancy. First, we sprayed young H4 seedlings with Pro-Ca, a together, FvGA20ox4 expression and thereby AXB fate are GA biosynthesis inhibitor (Evans et al., 1999), and observed tightly controlled by both temperature and photoperiod in AXB fates in SD conditions to maintain the plants in vegeta- the perpetual flowering F. vesca, and this regulation mecha- tive phase. While untreated plants profusely produced sto- nism is uncoupled from FvSOC1 expression at 24 C lons, Pro-Ca-treated plants did not develop any stolons To confirm the function of FvGA20ox4 in controlling AXB (Figure 3). BCs were rarely observed in control plants during fate, we transformed FvGA20ox4-RNAi construct into the the 2 weeks of observations in this experiment, and Pro-Ca Figure 1 Stolon development is environmentally regulated via the activation of FvGA20ox4 in perpetual flowering woodland strawberry. Relative expression of FvGA20ox4 (A) and FvSOC1 (B). Percentage of axillary buds developing stolons (C), or BCs (D), or remaining dormant (E). WT Hawaii-4 seedlings (n = 13–20) were grown in growth chambers equipped with LED lamps under 18 or 12-h photoperiod at 24 C or 11 C for 10 weeks and axillary bud fates were recorded until the end of the experiment. For relative expression, three apices per each biological replicate (n = 3) were collected after 5 weeks. In (A and B), center lines represent the median and upper and lower hinges represent the first and third quar- tiles. Upper and lower whiskers extend from the upper hinge to 150% of the interquartile range and from the lower hinge to –150% of the inter- quartile distance, respectively. Points beyond the whiskers are outliers according to Tukey. In (C–E), error bars represent the standard error of the mean and different letters indicate significant differences calculated by logistic regression and Tukey’s test (P 5 0.05). Woodland strawberry axillary bud fate regulation PLANT PHYSIOLOGY 2021: 187; 1221–1234 | 1225 Figure 2 FvGA20ox4 is required for stolon formation in perpetual flowering woodland strawberry. Relative FvGA20ox4 expression (A). Percentage of axillary buds developing stolons (B), or BCs (C), or remaining dormant (D). WT Hawaii-4 and FvGA20ox4-RNAi seedlings (n = 10) were grown in a growth chamber equipped with LED tubes under 12-h photoperiod at 17 C. Axillary bud fates were recorded and apex samples collected when the plants had developed five fully opened leaves. For relative expression, six to eight apices per each biological replicate (n = 4) were col- lected. In (A), center lines represent the median and upper and lower hinges represent the first and third quartiles. Upper and lower whiskers ex- tend from the upper hinge to 150% of the interquartile range and from the lower hinge to –150% of the interquartile distance, respectively. In (B– D), error bars represent the standard error of the mean and statistically significant differences, calculated by logistic regression and Dunnett’s test, are indicated by asterisks (***P 5 0.0001; **P 5 0.001; *P 5 0.01); H4 = Hawaii-4. treatment only slightly although significantly increased the Supplemental Table S2). The fates of the older AXBs were proportion of AXBs developing into BCs (Figure 3). The ma- not affected by decapitation or LD in either accession. These jority of AXBs remained dormant after the Pro-Ca treatment data demonstrate that the removal of apical dominance by supporting the hypothesis that GA is required to break AXB decapitation is sufficient to release dormancy of the 1–2 dormancy in F. vesca in vegetative phase (Figure 3). youngest AXBs and to induce BC development under SD To test this hypothesis from different angle, we carried conditions in perpetual flowering F. vesca accessions. On the out GA3 treatment on seedlings of stolonless perpetual other hand, decapitation does not increase the proportion flowering genotype ‘Reine des Vallées’ (RV) (Supplemental of AXBs developing stolons in H4. Taken together, Figure S2) grown in SDs. The GA3 treatment strongly pro- FvGA20ox4 promotes stolon development irrespectively of moted AXB outgrowth as stolons in vegetative seedlings, apical dominance, whereas BC development from the youn- whereas no AXB outgrowth was observed in control plants gest AXBs is inhibited by apical dominance. during the first 2 weeks in SD (Figure 3). Also, a transfer of nontreated RV plants from SDs to flower-inductive LDs rap- Direct environmental control of FvGA20ox4 idly released AXB dormancy, but in these conditions, AXBs expression and AXB fate in the seasonal flowering developed into BCs (Supplemental Figures S2 and S3). In woodland strawberry conclusion, our growth regulator experiments and studies in We have so far confirmed that the expression of FvGA20ox4 FvGA20ox4 RNAi lines revealed that both active GA biosyn- is indispensable for SD- and warm temperature-promoted thesis in the AXB and application of synthetic GA can over- stolon development in perpetual flowering F. vesca acces- come AXB dormancy in F. vesca . This effect has not been sions. As opposed to perpetual flowering accessions, stolon observed in previous studies that used flower-induced plants development in seasonal flowering F. vesca genotypes is pro- (Tenreira et al., 2017). moted by LD conditions and high temperature (Heide and AXB dormancy may be caused by apical dominance Sønsteby, 2007; Mouhu et al., 2013). Because of the oppos- exerted by the actively growing vegetative SAM or by envi- ing photoperiodic responses, we wanted to gain evidence of ronmental conditions. AXBs can be released from apical the role of FvGA20ox4 in the control of AXB fate in seasonal dominance by decapitation (Cline, 1997). To dissect the ef- flowering F. vesca . First, we assessed the expression of all fect of apical dominance from the environmental effect on four FvGA20ox genes present in the latest F. vesca genome AXB dormancy, we followed AXB fates in perpetual flower- annotation (v.4.0.2; Li et al., 2019; Supplemental Table S3). ing H4 and RV plants grown at 24 C under three different Only FvGA20ox2 and FvGA20ox4 expression was detected in treatments: SDs, LDs, and decapitation in SDs. In the stolon- the SAMs and AXBs collected from plants grown under SDs less RV, both LD conditions and decapitation of SD-grown or LDs at 18 C (Supplemental Figure S4). In AXBs collected plants promoted the development of the youngest AXBs from LD-grown plants, the mRNA level of FvGA20ox4 was into BCs, whereas intact SD-grown plants had a higher pro- more than one thousand times higher than that of portion of dormant AXBs (Figure 4; Supplemental Table S2). FvGA20ox2 (Supplemental Figure S4), suggesting that Also in the stolon-forming H4, LDs and decapitation in SDs FvGA20ox4 encodes a major GA20ox enzyme catalyzing the promoted BC development, although the effect was less biosynthesis of the precursors of bioactive GA in AXB. clear due to profuse stolon development (Figure 4; Moreover, the expression of FvGA20ox4 was more than 1226 | PLANT PHYSIOLOGY 2021: 187; 1221–1234 Andrés Jimenez et al. approximately 100 times higher in AXBs compared with shoot apex samples (Supplemental Figure S4). According to these data, gibberellin biosynthesis in the AXBs of seasonal and perpetual flowering F. vesca accessions is dependent on the activity of the same GA20ox enzyme, encoded by the FvGA20ox4 gene. Next, we wanted to study how the photoperiod affects AXB fate in the seasonal flowering F. vesca, and whether this effect is direct or dependent on the SAM fate. If the fates of the AXBs were uncoupled from the fate of the SAM, SDs should inhibit stolon development and promote BC devel- opment even if the SAM remains vegetative. To test this hy- pothesis, we compared photoperiodic responses of seasonal flowering FIN56 accession with genotypes that remain vege- tative in SDs at 18 C including FvTFL1-overexpression lines in FIN56 background (Koskela et al., 2012) and a natural vernalization-requiring accession NOR1 which shows abnor- mally high FvTFL1 expression in SDs (Koskela et al., 2017). Photoperiod had a highly significant impact on AXB fate, triggering similar responses in FIN56, FvTFL1-overexpression lines, and NOR1. In all genotypes, more stolons developed in LDs compared with SDs, while SDs enhanced BC forma- tion and increased the proportion of dormant buds (Figure 5; Supplemental Figure S5). However, the effect of Figure 3 Gibberellic acid is required to release axillary bud dormancy SDs on BC development was more pronounced in FIN56 in perpetual flowering woodland strawberry. Percentage of axillary than in the other genotypes. Flowering was observed only in buds developing stolons (A and D), BCs (B and E), or remaining dor- FIN56; all FIN56 plants flowered in SDs, and in LDs only two mant (C and F). In (A–C), WT Hawaii-4 seedlings (n = 17) were grown plants (13.3%) flowered. In summary, stolon development in a growth chamber equipped with LED lamps under 12-h photope- was inhibited by SDs independently of the SAM fate, riod at 24 C and sprayed with Pro-Ca. In (D–F), RV seedlings (n = 35) were grown under 12-h photoperiod at 24 C and sprayed with GA3. whereas BC development depended both on the photope- In both experiments, the seedlings were sprayed when they had two riod and on the SAM status. to three fully opened leaves and axillary bud fates were recorded 2 We also explored the effect of photoperiod on the mRNA weeks later when the plants had developed five to six leaves. Only the expression of FvGA20ox4 and other genes of the GA biosyn- axillary buds developed after the treatment were taken into account. thetic, catabolism, and signaling pathways in seasonal flower- Error bars represent the standard error of the mean and statistically ing FIN56 and FvTFL1 overexpression lines. In consistence significant differences, calculated by logistic regression and Dunnett’s with the role of FvGA20ox4 in the control of AXB fate in test, are indicated by asterisks (***P 5 0.0001; **P 5 0.001; *P 5 0.01); the perpetual flowering woodland strawberry (Figure 1), we H4 = Hawaii-4. Figure 4 Decapitation releases axillary buds from apical dominance and leads to BC development. Short day-grown seedlings of perpetual flower- ing Hawaii-4 and RV were either decapitated at 4–6 leaf stage, left intact and maintained under short-day conditions, or moved to long-day condi- tions. The plants were grown at 24 C in growth chambers equipped with LED tubes. Axillary bud fates were scored weekly until nearly all available axillary buds in the decapitated seedlings had differentiated. Each square depicts the fate of an individual axillary bud; H4 = Hawaii-4, D = dor- mant, S = stolon. Woodland strawberry axillary bud fate regulation PLANT PHYSIOLOGY 2021: 187; 1221–1234 | 1227 Figure 6 Cool temperature stops stolon development. Stolon-propa- Figure 5 Photoperiod controls axillary bud fate at 18 C independently gated plants of seasonal flowering wild type FIN56, FvTFL1-OX line #2 of shoot apical meristem fate. Percentage of axillary buds developing in FIN56 background, and NOR1 were subjected to photoperiod treat- stolons (A), BCs (B), or remaining dormant (C). Percentage of flower- ments (18 or 12-h daylight) at 10 C for 5 weeks. Plants were grown in ing plants (D). Relative expression of FvGA20ox4 (E), FvGA3ox1 (F), growth chambers equipped with LED lamps during the treatments FvGA2ox1 (G), and FvGAI (H) in axillary bud samples. Stolon-propa- and then transferred to a greenhouse under long days at 18 C. gated plants from FvTFL1-OX lines #4 and #5 in FIN56 background, Axillary bud observations were recorded up to Week 6. Error bars rep- and WT FIN56 were grown in a greenhouse under 18 or 12-h photo- resent standard error (n = 14–16). period at 18 C for 6 weeks. Axillary bud observations were recorded up to week 4 (n = 10–15) and flowering was scored up to Week 10. In (A–D), error bars represent the standard error of the mean and differ- downregulated in SDs in both FIN56 and FvTFL1 overexpres- ent letters indicate significant differences calculated by logistic regres- sion lines (Figure 5). However, no major differences between sion and Tukey’s test (P 5 0.05). For relative expression, axillary bud photoperiods were found in the expression of FvGAI samples including five axillary buds per each biological replicate (Figure 5; Supplemental Table S3), which encodes a major (n = 4) were collected on week 4. In (E–H), center lines represent the DELLA protein in F. vesca (Caruana et al., 2018; Li et al., median and upper and lower hinges represent the first and third quar- 2018). These data demonstrate that the GA biosynthetic tiles. Upper and lower whiskers extend from the upper hinge to 150% pathway in AXBs of seasonal flowering F. vesca is regulated of the interquartile range and from the lower hinge to –150% of the directly by photoperiod and does not depend on floral in- interquartile distance, respectively. Points beyond the whiskers are duction in the SAM. outliers according to Tukey. So far, we concentrated on the role of photoperiod in the regulation of AXB fate. To understand the role of tempera- found strong downregulation of FvGA20ox4 mRNA levels in ture in the control of AXB fate in more detail, we explored SDs compared with LDs in both FIN56 and the transgenic the responses of seasonal flowering FIN56, one FvTFL1 over- lines (Figure 5) confirming the direct photoperiodic regula- expression line, and NOR1 to 10 C temperature in SDs and tion of this gene in AXBs. Among the other GA pathway LDs. Stolon development ceased after 4 weeks in all geno- genes studied by Mouhu et al. (2013) in leaf tissues, types under both photoperiods (Figure 6), and a relatively FvGA3ox1, FvGA2ox1, and FvGAI (F. vesca GIBBERELLIC ACID high proportion of AXBs developed into BCs in FIN56 and INSENSITIVE) exhibited high expression levels in AXB sam- NOR1 (Supplemental Figure S6). In FIN56, almost all plants ples (Figure 5). Both FvGA3ox1 and FvGA2ox1 were clearly flowered in both photoperiods, but in other genotypes, only 1228 | PLANT PHYSIOLOGY 2021: 187; 1221–1234 Andrés Jimenez et al. a single NOR1 plant flowered (Supplemental Table S4), the expression of the floral marker gene Fragaria vesca showing that 10 C affected AXB fate independently of the APETALA1 (FvAP1; Koskela et al., 2012) in SAM samples. In SAM developmental status or photoperiod in NOR1 and SDs at 17 C, FvAP1 mRNA level remained low until Day 14, FvTFL1 overexpression plants. but increased abruptly thereafter indicating that flower in- Next, we carried out time-course expression analyses in duction happened between Days 14 and 21, while the lack AXB samples in parallel with stolon observations to explore of the FvAP1 activation at 23 C showed that plants how the temporal regulation of FvGA20ox4 associates with remained in vegetative phase at this temperature (Figure 7). AXB fate in seasonal flowering FIN56 transferred from LD In all the analyses above, independently of the photoperi- conditions to SDs at 17 C or 23 C. In SDs at 17 C, stolon odic response type of the plants (perpetual or seasonal flow- development ceased after 14 d (Figure 7). This was associ- ering), stolon development was associated with the ated with an almost complete shutdown of FvGA20ox4 vegetative status of the SAM, which is in line with previous mRNA expression already during the first week of the treat- studies (Mouhu et al., 2013; Kurokura et al., 2017; Tenreira ment, indicating that the strong photoperiodic regulation of et al., 2017). However, our results show that the downregu- FvGA20ox4 controls AXB fate in FIN56 at 17 C (Figure 7). lation of FvGA20ox4 in AXBs and the cessation of stolon de- However, in SDs at 23 C FvGA20ox4 expression level velopment occur before the upregulation of FvAP1 in the remained high and stolon development continued until the SAM of SD-grown FIN56 suggesting temporal separation in end of the experiment demonstrating a strong temperature the environmental control of AXB and SAM fates. effect on the expression of FvGA20ox4 and AXB fate. To explore the connection of these AXB observations The role of FvSOC1 in the regulation of AXB fate with the developmental status of the SAM, we also analyzed To investigate the genetic regulation of AXB fate upstream of the GA pathway, we focused on FvSOC1. We reanalyzed photoperiodic responses of FvSOC1 overexpression and si- lenced lines generated by Mouhu et al. (2013) and explored the expression of GA pathway genes in their AXBs. In con- sistence with previous results (Mouhu et al., 2013), we found that both FvSOC1 overexpression and silenced lines had lost the normal photoperiodic control of AXB fate and flowering at 18 C. In the overexpression lines, almost all AXBs devel- oped a stolon in both photoperiods, and none of the plants flowered (Figure 8). FIN56 developed stolons from about half of the AXBs in LDs, while the FvSOC1-RNAi silenced line developed significantly less stolons in these conditions. However, SDs reduced the proportion of AXBs developing a stolon in FIN56, but not in the silenced line. Furthermore, the silenced line produced slightly more BCs under LDs than FIN56, and more AXBs remained dormant in silenced line in both photoperiods. All FIN56 plants flowered in SDs, while the FvSOC1 silenced line flowered in both photoperiods. These data demonstrate that FvSOC1 is required for relaying information on photoperiod to the AXBs, and that artifi- cially increasing FvSOC1 abolishes both photoperiodic and temperature regulation of AXB fate. Next, we analyzed the expression of FvSOC1 and GA path- way genes in AXBs of seasonal flowering FIN56 plants grown at 18 C. FvSOC1 mRNA level was clearly higher in FvSOC1 overexpression lines and lower in the silenced line than in FIN56, and clear downregulation of this gene was found in SDs compared with LDs in both FIN56 and the silencing line (Figure 8). The expression of FvGA20ox4 and FvGA3ox1 fol- Figure 7 FvGA20ox4 is environmentally regulated in seasonal flower- lowed the patterns of FvSOC1 expression in both transgenic ing FIN56. The number of stolons developed per plant (A), relative lines and FIN56 grown in different photoperiods (Figure 8) FvGA20ox4 expression in axillary bud samples (B), and relative FvAP1 expression in apex samples (C). FIN56 plants (n = 18) were grown un- supporting the role of FvSOC1 in the activation of GA bio- der 12-h daylight at 17 C and 23 C for 4 weeks and axillary bud fates synthetic genes in AXBs. The FvGA2ox1 gene encoding a GA were recorded weekly. For relative expression, samples including three catabolic enzyme followed the same pattern of photoperi- axillary buds (B) or three apices (C) per each biological replicate odic regulation as the biosynthetic genes in the WT FIN56, (n = 3) were collected weekly. Error bars represent the standard error with LDs upregulating expression (Figure 8). However, in the of the mean. FvSOC1 overexpression and silenced lines, the photoperiodic Woodland strawberry axillary bud fate regulation PLANT PHYSIOLOGY 2021: 187; 1221–1234 | 1229 Figure 8 FvSOC1 regulates gibberellic acid biosynthesis in axillary buds at 18 C. Percentage of axillary buds developing stolons (A), BCs (B), or remaining dormant (C). Percentage of flowering plants (D). Relative expression of FvSOC1 (E), FvGA20ox4 (F), FvGA3ox1 (G), FvGA2ox1 (H), and FvGAI (I) in axillary bud samples. Stolon-propagated plants (n = 10 – 15) of seasonal flowering WT FIN56, FvSOC1–OX7, FvSOC1–OX12, and FvSOC1–RNAi2 in FIN56 background were grown in the greenhouse under 18 or 12-h photoperiod at 18 C for 6 weeks. Axillary bud observations were recorded up to Week 4 and flowering was scored up to Week 10. In (A–D), error bars represent the standard error of the mean and different letters indicate significant differences calculated by logistic regression and Tukey’s test (P 5 0.05). For relative expression, axillary bud samples in- cluding five axillary buds per each biological replicate (n = 4) were collected on week 4. In (E–I), center lines represent the median and upper and lower hinges represent the first and third quartiles. Upper and lower whiskers extend from the upper hinge to 150% of the interquartile range and from the lower hinge to –150% of the interquartile distance, respectively. Points beyond the whiskers are outliers according to Tukey. regulation of FvGA2ox1 was clearly abolished. Furthermore, developed stolons profusely, independently of the environ- lower expression level of FvGAI was detected in FvSOC1 si- mental conditions. In contrast to the results presented here lenced line compared with FIN56 (Figure 8), and the overex- at 18 C (Figure 8) and in Mouhu et al. (2013), the FvSOC1– pression lines had slightly higher levels of FvGAI mRNA than RNAi3 plants grown in LDs at 22 C developed stolons at the the wild type. same frequency as the WT FIN56 (Supplemental Figure S7). Once we revealed the key role of FvSOC1 in the photoperi- This means that in FIN56, FvSOC1 is not required for stolon odic regulation of AXB fate at 18 C, we tested its function at development at 22 C, and that an FvSOC1-independent path- different temperatures by growing WT FIN56, FvSOC1-OX way controls AXB fate at warm temperature in the seasonal and RNAi plants at 10 C and 22 C in both LDs and SDs and flowering woodland strawberry. observed AXB fates. Due to lack of plant material, the FvSOC1 silenced line (FvSOC1–RNAi3) we used in this experiment was not tested at 18 C temperature in the experiments described Discussion in this article, but the line was earlier shown to develop less Studies in F. vesca on the effect of photoperiod and tempera- stolons than the WT FIN56 when grown in LDs at 18 C ture have previously focused on flowering responses, while (Mouhu et al., 2013). The transcript levels of FvSOC1 in the data on how the environment affects vegetative growth re- FvSOC1–RNAi lines grown at 23 C have been previously char- main scarce. Moreover, previous studies (Sugiyama et al., acterized by Rantanen et al. (2015), who showed that FvSOC1 2004; Tenreira et al., 2017) have speculated that the AXB fate is silenced in these lines at 23 C. In FIN56, 10 C inhibited and depends on the flowering decision made at the SAM. In other 22 C promoted stolon development independently of the words, floral-inducing environmental conditions would have photoperiod (Supplemental Figure S7). The FvSOC1-OX plants an indirect effect on AXB fate because floral induction at 1230 | PLANT PHYSIOLOGY 2021: 187; 1221–1234 Andrés Jimenez et al. SAM forces BC development from AXBs. Here, we use func- Axillary bud fate in the seasonal flowering tional genetics, growth regulator treatments, and controlled- strawberry is predominantly environmentally climate experiments to demonstrate that environmental con- regulated ditions affect the activity of the GA biosynthetic pathway and Earlier studies in the seasonal flowering F. vesca accession AXB fate directly, and show that the fate of the SAM affects FIN56 have demonstrated that LDs promote stolon develop- only on the fates of the youngest AXBs. We also suggest a ment and activate the expression of FvGA20ox4 in leaf tis- role for FvGA20ox4 in releasing strawberry AXBs from growth sues (Mouhu et al., 2013). We show here for the first time arrest caused by apical dominance and provide evidence for that FvGA20ox4 expression is activated by LDs in AXBs of an FvSOC1-independent high-temperature activated pathway FIN56 at 18 C, while at 23 C high expression level is found promoting stolon development. even in SDs (Figures 7 and 8, F). To summarize, FvGA20ox4 activity associates with stolon development in all the tested genotypes and environmental conditions (Figures 5, 7, and FvGA2o0x4 activity overrides the effect of apical 8). This is hardly a surprising finding, given that exogenous dominance and promotes stolon development application of bioactive GA has been shown to promote Previous studies on the role of GA in strawberry vegetative stolon development in a number of studies (Thompson and responses have suggested that the presence of bioactive GA in Guttridge, 1959; Hytönen et al., 2009; Mouhu et al., 2013). the AXBs leads to stolon development, while in the absence of Therefore, we wanted to go a step further and study how GA BCs are formed (Hytönen et al., 2009; Tenreira et al., 2017; the environment affects AXB fate in general, and to dissect Feng et al., 2021). However, these studies have either used the indirect environmental effect via floral induction (in flower-induced plants (Tenreira et al., 2017; Feng et al., 2021), other words, the loss of apical dominance) from the direct or have not observed the frequency of dormant AXBs on the environmental effect on AXB fate in the seasonal flowering main crown (Hytönen et al., 2009). Our current results are F. vesca. based on careful observations of AXB fates of the main crown To achieve this, we studied the association between AXB of both flower-induced and noninduced plants and provide ev- fates and floral induction under different environmental idence for a fine-tuned regulatory mechanism that involves the conditions using genotypes with contrasting flowering char- FvGA20ox4 as an on/off switch controlling stolon development acteristics. Stolon development is inhibited at 9 C or by SDs but also suggests the presence of an active mechanism control- at 15 C, the conditions that induce flowering in seasonal ling BC development and AXB dormancy. flowering woodland strawberry genotypes (Heide and In consistence with a number of earlier studies that Sønsteby, 2007). Our data corroborate these results in FIN56 showed GA as a factor promoting stolon development (e.g. at 10 C (Figure 6) or SDs at 17–18 C (Figures 5 and 7), and Thompson and Guttridge, 1959; Hytönen et al, 2009; also demonstrates that the inhibition of stolon development Mouhu et al., 2013; Tenreira et al., 2017), our experiments occurs independently of the SAM fate in FvTFL1-OX plants revealed a clear connection between stolon development and NOR1 accession that remain vegetative under these and the activity of FvGA20ox4 in all examined woodland conditions. Moreover, the inhibition of FvGA20ox4 expres- strawberry genotypes (Figures 1–3, 5, 7, and 8). In the per- sion and stolon development in FIN56 by SDs occurs weeks petual flowering H4, stolon development was enhanced un- before floral induction takes place (Figure 7), showing that der SDs at warm temperatures. However, SD conditions these processes can be temporally separated. failed to induce stolon development on FvGA20ox4–RNAi We have confirmed that FvGA20ox4 is indispensable for lines, confirming that FvGA20ox4 is required for stolon de- stolon development and it is environmentally regulated, but velopment. BC development was not affected in the we do not know the identity of the gene(s) regulating BC FvGA20ox4–RNAi plants (Figure 2), indicating that silencing development in F. vesca. A good starting point for such FvGA20ox4 had a specific effect on stolon development. The studies would be the analysis of AXB fate-related candidate AXBs that were restrained from developing stolons due to genes identified by Samad et al. (2017). Their QTL analysis silencing of FvGA20ox4 remained dormant (Figure 2). We revealed an association between AXB fate and strawberry were able to reproduce the dormant-AXB phenotype in TCP transcription factor FvTCP7 on LG4, as well as with two WTs of two LD-flowering accessions H4 and RV grown un- DORMANCY-ASSOCIATED MADS (DAM) BOX genes in LG5 der nonflower-inductive SDs by treating the plants with (Samad et al., 2017). As FvTCP7 belongs to the same tran- growth regulators. It is noteworthy that the AXBs of vegeta- scription factor family as BRC1, a major regulator of bud tively growing plants developed stolons in the presence of outgrowth in Arabidopsis (Aguilar-Martinez et al., 2007), bioactive GA, but in the absence of GA, they remain dor- studying the function of FvTCP7 could provide important mant instead of developing into BCs (Figure 3). These results insights into the control of BC formation in Fragaria. DAM are at odds with the idea presented by Tenreira et al. genes have been associated with dormancy in a number of (2017), who suggested that AXBs develop into BCs as a de- perennial species (reviewed in Falavigna et al., 2019). fault setting whenever bioactive GA is absent. Functional studies of the LG5 DAM genes reported by Woodland strawberry axillary bud fate regulation PLANT PHYSIOLOGY 2021: 187; 1221–1234 | 1231 Samad et al. (2017) are needed to confirm their roles in con- data suggest that while environmental conditions are the trolling AXB fate. major regulators of BC development in seasonal flowering F. vesca, floral induction directly regulates the fates of the FvSOC1 is required for stolon development only at youngest and uppermost AXBs. intermediate temperature In other sympodially growing species, such as cotton LDs have been shown to activate FvSOC1, leading to upregu- (Gossypium hirsutum) or tomato (Lycopersicon esculentum), lation of FvGA20ox4 in leaf tissues at 18 (Mouhu et al., the major determinants of the sympodial growth pattern 2013). Concordantly, in our photoperiodic experiments are the CETS genes (Banfield and Brady, 2000), to which FvSOC1 expression associated with the level of FvGA20ox4 TFL1 also belongs (Kobayashi et al., 1999). In both species, si- and FvGA3ox1 expression in AXBs in the seasonal flowering lencing the homologs of TFL1 leads to AXB release from api- FIN56 plants grown at 18 C (Figure 8). Moreover, overex- cal dominance and the abolishment of the sympodial pressing or silencing FvSOC1 abolished the normal photope- growth habit (Pnueli et al., 2001; McGarry et al., 2016). In riodic responses (Figure 8), demonstrating that FvSOC1 is strawberry, the maintenance of apical dominance and the required for relaying photoperiodic information to regulate sympodial growth habit does not depend on FvTFL1 activity, GA biosynthesis by transcriptional regulation of FvGA20ox4 because the perpetual flowering accessions with nonfunc- at 18 C. However, in seasonal flowering woodland straw- tional mutated FvTFL1 follow exactly the same sympodial berry, temperature of 22 C promoted stolon development growth mode (Brown and Wareing, 1965). Possible regula- in both the WT and FvSOC1-silenced lines independently of tors of strawberry sympodial growth are the two photoperiod (Supplemental Figure S7). In perpetual flower- CENTRORADIALIS-LIKE genes whose functions remain ing F. vesca, FvGA20ox4 expression and stolon development unexplored. were activated under SDs at 24 C, although FvSOC1 was The second strongest effect on AXB fate is exerted by en- expressed only weakly under these conditions (Figure 1). To vironmental conditions promoting stolon development. The summarize, our data provide evidence for an FvSOC1-inde- environmental conditions that favor vegetative growth de- pendent and high temperature-activated pathway that pro- pend on the genotype; for perpetual flowering LD acces- motes stolon development at temperatures over 22 C. This sions, it is the combination of SDs and warm temperature pathway shares an obvious analogy with the high (Figure 1), while in seasonal flowering accessions stolon de- temperature-activated and FvSOC1-independent pathway velopment is promoted by LDs and/or warm temperature that upregulates FvTFL1 and inhibits flowering at 23 C (Figures 5, 7, and 8; Hartmann, 1947; Konsin et al., 2001; (Rantanen et al., 2015). These two pathways, one promoting Hytönen et al., 2004). Despite the contrasting environmental FvGA20ox4 expression and one upregulating FvTFL1 at tem- conditions, the mechanism for boosting stolon development peratures over 22 C, are likely to share regulatory compo- is the same; GA biosynthetic pathway is activated in the nents whose identities warrant further investigations. vast majority of nondifferentiated AXBs, resulting in prolifer- ative stolon development (Figures 1, 2, and 7). Abundant Regulation of axillary bud fate in strawberry clonal reproduction has been shown to increase compensa- In this work, we aimed at elucidating regulation of AXB fate, tory growth in case of damage to apical meristems in grasses and establishing whether AXB fate depends more on envi- (Liu et al., 2007). Therefore, the capability of strawberries of ronmental or endogenous factors. Our data suggest that developing stolons is likely to be an important ecological ad- both types of factors are involved in the regulation AXB aptation that has contributed to the wide geographical dis- fate, and their effect depends on the position of the AXB on tribution of strawberry species. the plant. BC development from older AXBs is controlled by specific The strongest effect is exerted by the SAM, but only on genotype-dependent environmental conditions (Figures 1, 2, the youngest, uppermost AXBs. The fates of these buds de- and 5; Supplemental Figure S6). It is interesting that the en- pend directly on the fate of the SAM; if the SAM is induced vironmental conditions promoting BC development also in- to form an inflorescence, the uppermost AXBs form BCs, crease the proportion of dormant axillary buds (Figures 1, 2, maintaining sympodial growth pattern. Our data on perpet- and 5), both in perpetual flowering H4 and seasonal flower- ual flowering F. vesca accessions (Figure 4) suggest that this ing FIN56 accessions as well as in FvTFL1 overexpression effect is caused by apical dominance, whose effect can be lines in the FIN56 background. This phenomenon is likely to overridden by the activity of FvGA20ox4 to promote stolon be related to the maintenance of the perennial life cycle; in development in the uppermost AXBs. Release from apical both diploid F. vesca and octoploid F.  ananassa, the dominance, i.e. floral induction, appears to promote BC de- same environmental conditions promote both floral induc- velopment also in the topmost AXBs of seasonal flowering tion and BC development (e.g. Hytönen and Kurokura, F. vesca, as the SD-grown floral-induced FIN56 plants devel- 2020). A similar phenomenon has been recently described in oped a significantly higher proportion of BCs than the non- the perennial Arabis alpina, in which floral initiation and the flowering genotypes FvTFL1-OX and NOR1 (Figure 5). These initiation of new AXBs take place during vernalization. In A. 1232 | PLANT PHYSIOLOGY 2021: 187; 1221–1234 Andrés Jimenez et al. alpina, the AXBs initiated before the cold period are des- #K240020). After cloning into pENTR and confirmation by tined to remain dormant, while the buds initiated during sequencing, the fragment was cloned into the binary vector and after the cold grow out as vegetative branches that are pH7GWIWG2-7F2.1 by LR Clonase (Invitrogen, catalog capable of flowering post-vernalization (Vayssières et al., #11791-100). The plasmid was transformed into 2020). Thus, maintaining a pool of undifferentiated dormant Agrobacterium tumefaciens GV3101 by electroporation. AXBs under floral-inducing conditions is a strategy to ensure Transformation of the RNAi construct into F. vesca H4 vegetative growth the following season in herbaceous peren- WT plants was carried out as previously described (Kang et nials including A. alpina and F. vesca . al., 2013; Caruana et al., 2018) using cotyledons as starting material. Individual transgenic lines were identified and con- Materials and Methods firmed by GFP fluorescence and by PCR of genomic DNA using primers targeting the hygromycin gene (Supplemental Plant material and growing conditions Table S3). Seasonal flowering woodland strawberry (F. vesca L.) acces- sion FIN56 (PI551792) and perpetual flowering F. vesca culti- Expression analysis var ‘Hawaii-4’ (PI551572) were obtained from the National Total RNA extraction, cDNA synthesis and real-time PCR Clonal Germplasm Repository, Corvallis, USA. Seeds of the were performed as described by Mouhu et al. (2009). The perpetual flowering cultivar ‘RV’ (PI551824) were obtained number of technical replicates per sample was three and from Dr Amparo Monfort (Centre for Research in FvMSI1 was used as a stable reference gene for standardiza- Agricultural Genomics, Barcelona, Spain). Transgenic lines in tion. Relative expression was calculated using the DDCt FIN56 background have been previously reported (Koskela method (Pfaffl, 2001) unless stated otherwise in the figure et al. 2012; Mouhu et al. 2013). FIN56 and transgenic plants legend. RT-qPCR primer sequences used were the same as in FIN56 background were clonally propagated from mother in Mouhu et al. (2009) and Koskela et al. (2012), except for plants grown in greenhouse under LDs (18 h) at 18 ± 2 C. the FvGA20ox4 expression analysis shown in Figure 2A, RV, H4, and transgenic plants in H4 background were pro- where the primers specified in Supplemental Table S3 were duced from seeds that were scarified with a 1M H2SO4 solu- used. Sampling details including time points and the num- tion for 5 min and soaked in water at 28 C overnight before ber of biological replicates are specified in the figure legends. germination. Seeds were germinated under LDs at 22 C until cotyledons were visible. Transgenic seedlings were selected Statistical methods based on GFP fluorescence. Logistic regression was used to test the main factors and ei- Growth chambers were equipped with LED lamps (AP67, ther Tukey HSD or Dunnets was used for subgroup analysis. Valoya, Finland) or LED tubes, supplying 200 mmol m–2 s–1 Statistics were done using R 4.0.3 (R Core Team, 2020), the of photosynthetic photon flux density (PPFD). In the green- stats (v3.6.2; R Core Team, 2020) and the DescTools house, natural lighting was complemented with 120 mmol (v0.99.40; Andri et mult. al. S, 2021) packages. m–2 s–1 PPFD from high-pressure sodium lamps (Airam 400W, Kerava, Finland) and 8 lmol m–2 s– 1 PPFD from in- Accession numbers candescent lights were used to extend day length in some Sequence data from this article can be found in the experiments. Experiment-specific light and temperature con- GenBank/EMBL data libraries under accession numbers avail- ditions are described in the figure legends. Plants were prop- able in Supplemental Table S4. agated and grown on Jiffy peat disks (Jiffy Products International BV) or in pots filled with peat (Kekkilä, Supplemental data Finland) and fertilized when needed. Seedlings had devel- The following materials are available in the online version of oped at least 2–3 leaves before environmental treatments. this article. For the growth regulator treatments, GA3 (Duchefa) and Supplemental Figure S1. FvGA20ox4 is the main FvGA20- Pro-Ca (BAS125; BASF) solutions were prepared as described oxidase controlling axillary bud fate in the perpetual flower- in Mouhu et al. (2013) and seedlings were sprayed at 2–3 ing F. vesca. leaves stage and 2 weeks later. Phenotypic observations Supplemental Figure S2. ’RV’ flowers exclusively under were performed as described in the figure legends. long days. Supplemental Figure S3. ‘RV’ starts forming BCs rapidly Generation of transgenic constructs and plant upon exposure to long days. transformation Supplemental Figure S4. FvGA20ox4 is the main FvGA20- To produce the FvGA20ox4–RNAi construct, a 479-bp se- oxidase controlling axillary bud fate in the seasonal flowering quence within the first exon of gene09034 (F. vesca v2.0.a2; F. vesca. Li et al. 2018) was amplified from H4 genomic DNA using Supplemental Figure S5. Photoperiod controls axillary Phusion DNA polymerase (NEB, catalog #M0530S) using the meristem fate independently of flowering in seasonal flower- primers described in Supplemental Table S3. The forward ing FIN56 and NOR1. primer included the “cacc” sequence required for cloning Supplemental Figure S6. Cool temperatures promote BC into the pENTRTM/D-TOPO Cloning Kit (Invitrogen, catalog formation in seasonal flowering FIN56 and NOR1. Woodland strawberry axillary bud fate regulation PLANT PHYSIOLOGY 2021: 187; 1221–1234 | 1233 Supplemental Figure S7. FvSOC1 is not required for sto- Cline MG (1997) Concepts and terminology of apical dominance. Am J Bot 84: 1064–1069 lon development at warm temperature in the seasonal flow- Costes E, Crespel L, Denoyes B, Morel P, Demene MN, Lauri PE, ering FIN56. Wenden B (2014) Bud structure, position and fate generate vari- Supplemental Table S1. Characteristics of F. vesca geno- ous branching patterns along shoots of closely related rosaceae types used in this study. species: a review. Front Plant Sci 5666 Supplemental Table S2. Statistical analysis of axillary bud Darrow GM (1966) The Strawberry. History, Breeding and Physiology. Holt, Reinhart and Winston, New York, NY fate in H4 and RV. Dun EA, de Saint Germain A, Rameau C, Beveridge CA (2012) Supplemental Table S3. Primers used in this study. Antagonistic action of strigolactone and cytokinin in bud out- Supplemental Table S4. Gene IDs in F. vesca genome growth control. Plant Physiol 158: 487–498 v4a2. Evans JR, Balles JA, Brinkman BA, Harris VE, Helm JD, Kirksey KB, McKemie TE, Thomas GG, Rademacher W (1999) 225 Prohexadione-Ca: Reduction in Vegetative Growth and Pruning of Funding Apple Trees. Hort Science 34: 481A J.A. was funded by Doctoral Programme of Plant Sciences Falavigna VS, Guitton B, Costes E, Andrés F (2019) I want to (bud) break free: the potential role of DAM and SVP-like genes in regulat- (University of Helsinki, Finland). J.L. was supported by a grant ing dormancy cycle in temperate fruit trees. Front Plant Sci 9: 1990 from China Scholarship council (201906350014). The work Feng J, Cheng L, Zhu Z, Yu F, Dai C, Liu Z, Guo W-W, Wu X-M, carried out by J.C. and Z.L. was supported by an USDA-NIFA Kang C (2021) GRAS transcription factor LOSS OF AXILLARY grant (11889048) and USDA_Maryland MAES Hatch Project MERISTEMS is essential for stamen and runner formation in wild (MD-CBMG-19008; to Z.L.). S.S. received a personal grant strawberry. Plant Physiol 186: 1970–1984 Guttridge CG (1985) Fragaria x ananassa. In AH Halevy ed, Handbook from the Fondazione Edmund Mach, Italy (GMPF Ph.D. of Flowering, vol. 3. CRC Press, Boca Raton, FL, pp 16–33. Fellowship) and belonged to the Doctoral Program of Plant Hartmann HT (1947) Some effects of temperature and photoperiod Sciences (University of Helsinki, Finland). A.M. acknowledges on flower formation and runner production in the strawberry. financial support from the Spanish Ministry of Science and Plant Physiol 22: 407–420 Innovation-State Research Agency (AEI), through the “Severo Hedden P (2020) The current status of research on gibberellin bio- synthesis. Plant Cell Physiol 61: 1832–1849 Ochoa Programme for Centres of Excellence in R&D” SEV- Heide OM (1977) Photoperiod and temperature interactions in the 2015-0533 and CEX2019-000902-S. The work was funded by growth and flowering of strawberries. Plant Physiol 40: 21–26 the Academy of Finland (Grant 317306 to T.H.). E.A.K. was Heide OM, Sønsteby A (2007) Interactions of temperature and pho- funded by a personal grant from Ella och Georg Ehrnrooth toperiod in the control of flowering of latitudinal and altitudinal foundation (Finland). populations of wild strawberry (Fragaria vesca). Physiologia Plantarum 130: 280–289 Conflict of interest statement. None declared. 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