Interactions between ScNAC23 and ScGAI regulate GA-mediated flowering and senescence in sugarcane

Highlights

• ScNAC23 is putatively involved in sugarcane flowering and leaf senescence.

• Ectopic ScNAC23 accelerates GA-driven flowering and leaf senescence in Arabidopsis.

• ScNAC23 participates in the GA pathway through direct interaction with ScGAI.

Abstract

Control of gene transcription is crucial to regulate plant growth and development events, such as flowering, leaf senescence, and seed germination. Here we identified a NAC transcription factor (ScNAC23) isolated from sugarcane (cv. ROC22). Analysis by qRT-PCR indicated that ScNAC23 expression was strongly induced in mature leaves and flowering varieties and was also responsive to exogenous treatment with the hormone gibberellin (GA). Ectopic expression of ScNAC23 in Arabidopsis accelerated bolting, flowering, and leaf senescence compared to wild type plants. Furthermore, Arabidopsis overexpressed ScNAC23 were more sensitive to GA than the wild type, and exogenous GA significantly accelerated flowering and senescence in the ScNAC23-overexpressed ones. A direct interaction between ScNAC23 and ScGAI, an inhibitor of GA signaling, was confirmed by yeast-two hybrid, bimolecular fluorescence complementation, and GST-pull down assay. The putative GA-ScNAC23-LFY/SAGs regulator module might provide a new sight into the molecular action of GA to accelerating flowering and leaf senescence in sugarcane.

Introduction

Sugarcane (Saccharum spp.) is grown in over 100 countries to supply more than 80 % of sugar consumed globally and is the second-largest feedstock for bioethanol production worldwide (http://faostat.fao.org/). Hybridization is crucial for breeding new high-yield cultivars. However, sugarcane flowering is unpredictable, wherein some cultivars that heavily flower one year may not flower in other years. The difficulties in an inappropriate flowering timing of desired parents have also impeded improvements in sugarcane breeding efficiency.

Flowering in sugarcane is a complex physiological process that involves multiple stages of development, and each phase has specific environmental and physiological requirements [1]. Flower induction and subsequent events are strongly affected by photoperiod, temperature, humidity, altitude, latitude, and several other factors, such as sugarcane variety and plant maturity [2,3]. Removing mature leaves (3 and 4) from US48-34 result in earlier flowering than undefoliated controls [4]. The genetic control of photoperiod-induced flowering has been fully demonstrated in other species, such as Arabidopsis, rice, sorghum, and Brachypodium [[5], [6], [7]]. In sugarcane, genes associated with photoperiod perception such as PROTEIN PHOSPHATASE 2C (ShPP2C), CHLOROPHYLL a/b-BINDING PROTEIN 2 (ShCAB2) and PHOTYOSYSTEM 1 GENE (ShPS1), and floral induction pathway, such as LEAFY (ShLFY), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (ShSOC1), APETALA 1 (ShAP1) and FLOWERING LOCUS T-A (ShFT-A), have different changes and patterns in expression over a 24-h cycle [8]. TERMINAL FLOWER 1(TFL1) and FT are phosphatidylethanolamine-binding protein (PEBP) family members, and AtTFL1 plays a role in maintaining the inflorescence in an indeterminate state. AtFT controls the timing of flowering [9]. Ectopic overexpressed ScTFL1 leads to delayed flowering in Arabidopsis [10]. Surprisingly, overexpressed ScFT1 in Arabidopsis also resulted in delayed flowering [10].

Gibberellin (GA) is a critical hormone that plays important roles throughout many plant's life cycles and is involved in seed germination, stem elongation, floral transition, fruit development, and leaf senescence [[11], [12], [13], [14], [15], [16]]. In Arabidopsis, GA is required for flowering under short-day conditions, and treatment with exogenous GA can promote early flowering [17]. Multiple genes associated with flowering in Arabidopsis, such as AtSOC1, AtCONSTANS (CO), AtFT, and ATLFY, are implicated in GA signal transduction. AtCO functions a vital role in the photoperiod response pathway, promoting flowering by inducing the direct downstream genes AtFT and AtSOC1 [18,19]. The rice mutant of early flowering1(el1) flowers 5–6 days earlier than the wild type under normal growth conditions, and EL1 expression is rapidly suppressed by GA treatment [20]. In barley, flowering time is delayed by PAC treatment and restored by application of GA₃. HvELF3 is a circadian clock gene that inhibits flowering by blocking GA production and expression of HvFT1 under non-inductive photoperiods [21]. DELLA protein is the repressor factor of GA signaling. Arabidopsis thaliana contains five DELLA genes, named GA-INSENSITIVE (GAI), REPRESSOR OF GA (RGA), RGA-LIKE1 (RGL1), RGL2, RGL3 [22], while the sole DELLA genes in rice (SLENDER RICE1, SLR1) and barley (SLENDER 1, SLN1), respectively [23,24]. Maize contains two DELLA proteins, dwarf plant8 (d8) and d9 [25], while sugarcane has one ScGAI gene [26]. GA can promote flowering through DELLA binding to miR156-targeted AtSPL transcription factors [16,27]. In sugarcane, flowering can negatively affect sugar yield and quality [28]. Therefore, identifying genes and pathways related to flowering regulation is crucial for increasing sugarcane yield and facilitating desirable traits.

The NAC family is one of the most significant families of plant-specific transcription factors. Members of the NAC family have a conserved N-terminal region termed the NAC domain (NAM, ATAF1/2, CUC) [29] and a C-terminal region that varies in both sequence and length [30]. NACs activate the expression of many downstream genes involved in multiple events in the plant life cycle, such as seed development, cell division and expansion, floral development and morphogenesis, senescence, lateral root development, and hormone signaling, and responses to abiotic and biotic stresses [[31], [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42]]. In Arabidopsis, overexpression of ATAF1 shows some developmental defects, such as sterility, reduced or absent flower initiation, and early yellowing of leaves [43]. AtLOV1, a NAC-domain transcription factor, controls floral development, and the mutant lov1-1D delays flowering, which negatively regulating AtCO expression in a GI-independent manner [44]. Constitutively OsNAC2-expressed rice is more insensitive to GA₃ and delays flowering for approximately five days [45]. Plants with OsNTL5ΔC and OsNTL5ΔC-SRDX (OsNTL5ΔC fused with the SRDX transcriptional repressor motif) exhibit a strongly late-flowering phenotype, while the OsNTL5ΔC-VP line (OsNTL5ΔC fused with the VP16 activation domain) flowers earlier [46]. Wheat allele NAC transcription factor NAM-B1 accelerates senescence, whereas the reduced RNA levels of all NAM homologs retard senescence for more than three weeks [47]. CRISPR-CAS9-mediated atnac050/052 double mutant plants and atnac050/052-RNAi plants showed early flowering phenotypes, and the two NAC transcription factors controlling flowering were associated with the AtJMJ14 in Arabidopsis [48]. Overexpression of AtNAP causes premature senescence, while expression of AtNAP with two T-DNA inserted delays leaf senescence [49]. The promoter-GUS studies of ANAC092 reveal that ANAC092 expresses in floral organs and senescent leaves, and gene expression in the anac019 mutant indicates that ANAC019 may be an activator of senescence in activating flavonoid and anthocyanin biosynthesis [37,50].

ScNAC23 was screened in a yeast cDNA library, ScGAI-C terminal, as the bait protein. The phylogenetic tree identified the orthologs (SsNAC23) and its paralogs (ATAF1, ATAF2, SbNAC49, ZmNAC109, OsNAC011, etc). SsNAC23 plays a role in abiotic and biotic stress [42], and paralogs ATAF1 also function in regulating flowering [51]. In this study, we explored the new functions of ScNAC23. ScNAC23 localized to the nucleus and exhibited transcription activity. Ectopic expression of ScNAC23 in Arabidopsis promoted flowering and leaf senescence by upregulating the expression of AtLFY, AtSAG12, and AtSAG13. Plants that overexpressed ScNAC23 were more sensitive to exogenous GA₃. We also showed that ScNAC23 interacted with the DELLA protein ScGAI directly in vitro and in vivo. Together, these results demonstrated the essential role of ScNAC23 in GA-mediated flowering and leaf senescence in sugarcane.