A sorghum gigantea mutant attenuates florigen gene expression and delays flowering time

Abstract GIGANTEA (GI) is a conserved plant‐specific gene that modulates a range of environmental responses in multiple plant species, including playing a key role in photoperiodic regulation of flowering time. The C4 grass Sorghum bicolor is an important grain and subsistence crop, animal forage, and cellulosic biofuel feedstock that is tolerant of abiotic stresses and marginal soils. To understand sorghum flowering time regulatory networks, we characterized the sbgi‐ems1 nonsense mutant allele of the sorghum GIGANTEA (SbGI) gene from a sequenced M4 EMS‐mutagenized BTx623 population. sbgi‐ems1 plants flowered later than wild type siblings under both long‐day or short‐day photoperiods. Delayed flowering in sbgi‐ems1 plants accompanied an increase in node number, indicating an extended vegetative growth phase prior to flowering. sbgi‐ems1 plants had reduced expression of floral activator genes SbCO and SbEHD1 and downstream FT‐like florigen genes SbFT, SbCN8, and SbCN12. Therefore, SbGI plays a role in regulating SbCO and SbEHD1 expression that serves to accelerate flowering. SbGI protein physically interacts with the sorghum FLAVIN‐BINDING, KELCH REPEAT, F‐BOX1‐like (SbFFL) protein, a conserved flowering‐associated blue light photoreceptor, and the SbGI‐SbFFL interaction is stimulated by blue light. This work demonstrates that SbGI is an activator of sorghum flowering time upstream of florigen genes under short‐ and long‐day photoperiods, likely in association with the activity of the blue light photoreceptor SbFFL. Significance Statement This study elucidates molecular details of flowering time networks for the adaptable C4 cereal crop Sorghum bicolor, including demonstration of a role for blue light sensing in sorghum GIGANTEA activity. This work validates the utility of a large publicly available sequenced EMS‐mutagenized sorghum population to determine gene function.


| INTRODUC TI ON
GIGANTEA (GI) is a gene identified in early genetic screens for delayed flowering mutants in Arabidopsis thaliana (Koornneef et al., 1991;Redei, 1962). GI participates in flowering time control, the circadian clock, and a wide range of other physiological activities (Mishra & Panigrahi, 2015). Arabidopsis GI stimulates flowering by promoting FLOWERING LOCUS T (FT) expression under long day (LD) photoperiods through post-transcriptional inactivation of CONSTANS (CO) transcriptional repressors (Park et al., 1999;Sawa & Kay, 2011;Sawa et al., 2007;Suarez-Lopez et al., 2001). In this capacity, GI interacts with the blue light photoreceptor FLAVIN-BINDING, KELCH REPEAT, F-BOX1 (FKF1) as part of the E3 ubiquitin ligase that targets a family of CO transcriptional repressors for degradation by the 26S proteasome system (Sawa et al., 2007). GI is also implicated in direct transcriptional regulation of FT (Sawa & Kay, 2011). The CO-FT regulatory module is a highly conserved point of integration for photoperiodic flowering signals (Song et al., 2015). CO is a B-box CCT domain transcription factor and its primary role is control of FT expression (Griffiths et al., 2003;Putterill et al., 1995). FT is a PEBP-family protein (Danilevskaya et al., 2008;Turck et al., 2008), which acts as florigen, a mobile signal transmitting the flowering signal from leaves to the shoot apical meristem (SAM; Pennazio, 2004). Leaf expressed FT-like proteins in Arabidopsis, rice, tomato, and cucurbits trigger the SAM to transition from vegetative to floral developmental programs (Jaeger & Wigge, 2007;Lifschitz et al., 2006;Lin et al., 2007;Notaguchi et al., 2008;Tamaki et al., 2007).  (Izawa et al., 2011). OsGI is important for blue light-promoted induction of rice Early heading date 1 (OsEHD1) expression as part of the mechanism for critical SD day-length recognition (Itoh et al., 2010). OsEHD1 encodes a B-type response regulator that promotes expression of the FT homolog OsHd3a under SD separate from the CO homolog OsHd1 (Doi et al., 2004;Itoh et al., 2010;Zhao et al., 2015). Maize has two paralogous GI genes, GIGANTEA1 (GI1) and GIGANTEA2 (Mendoza et al., 2012;Miller et al., 2008). gi1 mutants flower earlier in LD, but not SD, and have elevated expression of FT homolog Zea mays CENTRORADIALIS 8 (ZCN8) and CO homolog CONSTANS OF Zea mays1 (CONZ1), indicating that GI1 is an upstream repressor in LD (Bendix et al., 2013). The function of maize GI1 is sufficiently conserved that it complements late flowering of an Arabidopsis gi knockout mutant (Bendix, 2015).
Sorghum is a C4 grass native to Africa that is a key grain and subsistence crop, an animal forage, and a promising cellulosic biofuel feedstock. Sorghum is highly stress tolerant, maintaining productivity in marginal soils and under arid conditions. Sorghum is originally a short day (SD) flowering plant in which long dark periods, and correspondingly short days, above a critical threshold promote flowering (Craufurd et al., 1999;Quinby, 1974). Selection of so called Maturity (Ma) loci, which reduce the SD requirement for promotion of flowering, has allowed expansion of sorghum cultivation to more northern latitudes (Quinby, 1974). Of these, the Ma1 locus has the largest impact on sorghum flowering time. Inactive ma1 alleles confer early flowering in LD conditions and played an important role in early domestication of sorghum (Quinby, 1967 (Murphy et al., 2011). SbPRR37 encodes a member of a family of transcriptional repressors originally discovered as core circadian clock genes in Arabidopsis (Farre & Liu, 2013), but SbPRR37 has no contribution to circadian clock function (Murphy et al., 2011).
Like its rice and Arabidopsis counterparts, sorghum CONSTANS (SbCO) acts upstream to promote expression of SbEhd1 and several florigen-related genes in both LD and SD photoperiods . Of the thirteen PEBP-family genes in sorghum, sorghum CENTRORADIALIS 8 (SbCN8) is the co-linear ortholog of maize ZCN8 and SbFT is the co-linear ortholog of rice Hd3a (Murphy et al., 2011). An additional PEBP-family gene orthologous between maize and sorghum is SbCN12 (Murphy et al., 2011;Yang et al., 2014). Both SbCN8 and SbCN12 possess florigen activity when overexpressed in Arabidopsis (Wolabu et al., 2016). Collectively, SbFT, SbCN8, SbCN12 are regulated by SbCO and SbEhd1 (Murphy et al., 2014;Yang et al., 2014), consistent with this set of genes acting as the CO-FT module in sorghum.
The contribution of the sorghum GI (SbGI) gene to regulation of sorghum flowering is not well-characterized. A comparative genomic study of 219 African sorghum accessions identified single nucleotide polymorphisms (SNPs) at SbGI significantly associated with photoperiod sensitivity (Bhosale et al., 2012). Two associated SNPs caused non-synonymous amino acid changes and a third represented a frameshift mutation. Like all known GI genes, SbGI expression has a diel rhythm where peak expression occurs 9-12 hr after dawn (Lai et al., 2020;Murphy et al., 2011). Diurnal expression of SbGI is close to that of maize GI1 and expression of both SbGI and GI1 is substantially higher than GI2 (Lai et al., 2020). SbPRR37 does not contribute substantially to regulation of SbGI (Murphy et al., 2011).
Here we characterize a mutant allele in the SbGI gene, sbgi-ems1, from a sequenced M4 EMS-mutagenized population (Jiao et al., 2016). Plants carrying this nonsense mutation, which truncates GI protein by two thirds, exhibit delays in flowering under LD and SD photoperiod conditions. Delayed flowering in sbgi-ems1 under LD accompanies an increase in node number, indicating an extended vegetative growth phase prior to flowering. Mutant plants had low expression of SbCN8 and SbCN12 under LD and SD photoperiods.
Also, our observations indicate that SbGI promotes expression of the SbCO under both LD and SD photoperiods, but contributes to peak SbEHD1 expression mainly under SD. Testing of the molecular activity of SbGI showed that it physically interacts with the sorghum FKF1-like (SbFFL) protein, a potential flowering-associated blue light photoreceptor, and the SbGI-SbFFL interaction is stimulated by blue light.

| Plant stocks and environmental conditions
All sorghum lines are the BTx623/ATx623 genetic background.
The ARS223 line is from a collection of 256 whole genome sequenced M4 EMS-mutagenized sorghums lines described previously (Jiao et al., 2016). Plants were screened for the sbgi-ems1 mutation in SbGI by Derived Cleaved Amplified Polymorphic Sequences PCR with the primers in Table S1. The PCR fragment amplified from the sbgi-ems1 locus is resistant to the XcmI restriction enzyme (New England Biolabs, www.neb.com) and the product from WT SbGI locus is cleaved by this enzyme. One plant carrying the sbgi-ems1 allele from the M4 ARS223 population was used as pollen donor for a cross to a male sterile ATx623 panicle. Progeny of this cross were used for subsequent experiments and backcrossing.
LD conditions in the greenhouse were 16-hr days and 8-hr nights. Natural sunlight was supplemented with LumiGrow Pro325 LEDs (www.lumig row.com) set at maximum intensity for all channels.

| Assessment of flowering time
Plants were individually scored for the number of days from sowing to reach boot stage and flowering, while field grown plants were scored for boot stage only, due poor pollen shed and stigma exertion at the UC Berkeley Oxford tract. Boot stage was scored as the first day when the entire flag leaf collar was visible in the leaf whorl.
Flowering stage was scored as the first day of anthesis for fertile plants or stigma exertion for male sterile plants.

| Analysis of gene expression by qPCR
Plants at the fifth to sixth leaf stage grown under LD greenhouse conditions were transferred to a growth chamber set to either LD (16 hr light; 8 hr darkness) or SD (10 hr light; 14 hr darkness) with daytime temperature of 28°C and nighttime temperature of 23°C until all plants reached the fifth to seventh leaf stage. These plants were sampled at 0, 6, 12, and 18 hr after lights came on. Leaf samples were taken by cutting directly across the 6th leaf ligule with scissors.
Three or two biological replicates were collected for each genotype at each time point. A biological replicate consisted of pooled tissue from three individuals of the same genotype. Leaf samples were flash frozen in liquid nitrogen.
After tissue was ground under liquid nitrogen, total RNA was extracted with TRIzol Reagent (ThermoFisher Scientific, www. therm ofish er.com) according to the manufacturer's recommendations. Total RNA (3.5 µg) for each sample was treated with dsD-Nase (ThermoFisher Scientific, www.therm ofish er.com) to remove contaminating genomic DNA and was used as a template for cDNA synthesis with the Maxima H Minus First Strand cDNA synthesis Kit (ThermoFisher Scientific, www.therm ofish er.com) according to the manufacturer's recommendations. cDNA diluted in half with water served as template for two technical replicate real-time quantitative PCR (qPCR) reactions composed and performed as previously described (Bendix et al., 2013). qPCR reactions for normalization employed PCR primers for 18S RNAs (Table S1)

| Protein interaction analysis by yeast twohybrid
The coding sequences of SbGI and SbFFL were amplified by PCR with Q5 High Fidelity Polymerase (New England Biolabs, www.
neb.com) from cDNA with the primers in Table S1. PCR products were cloned into pENTR/D-TOPO vector (ThermoFisher Scientific, www.therm ofish er.com) and sequences confirmed by Sanger sequencing. SbFFL and SbGI cDNA sequences were subcloned into bait vector pGBKT7-Rec and the prey plasmid pGADT7-Rec, respectively, with LR Clonase II (ThermoFisher Scientific, www. therm ofish er.com). Bait and prey plasmids were transformed into Y2H Gold yeast cells according to the manufacturer's recommendations (Takara Bio, www.takar abio.com). For interaction tests, two individual transformants for each plasmid combination were grown at 30°C in liquid Synthetic Dropout (SD) media lacking amino acids Leu (L) and Trp (W) supplemented with 50 μg/mL Kanamycin (SD-T-W) to an absorbance of 600 nm (A 600 ) = 1.0, then samples were prepared corresponding to cell densities with A 600 = 4, 2, 1, 0.5, 0.1, and 0.01. 10 μL of each sample was spotted on SD-L-W plates with or without 200 ng/ml of the antibiotic Aureobasidin A (Takara Bio USA, www.takar abio.com). Interaction between bait and prey proteins confers resistance to Aureobasidin A. After drying, plates were sealed with Micropore Paper Tape (3M, www.3m.com) and placed under continuous blue light provided by blue LEDs at 25-30 μmol/m 2 s or continuous darkness at 30°C in a Percival LED-30 growth chamber. Digital images of plates were taken after 3, 5, and 7 days to monitor yeast growth.

| Confocal microscopy
Small sections (0.5 cm 2 ) of infiltrated N. benthamiana leaves were infiltrated with water and were mounted on microscope slides.
Samples were imaged using a Leica SP8 confocal laser-scanning microscope equipped with a 20x water-immersion objective. The 514 nm argon laser line was used to excite YFP, and florescence was observed using the specific emission window of 520-600 nm. The laser power (Argon intensity 25%), gain (1,050), zoom (zoom factor 1), and average settings (Format 1024x1024; Speed 200; line average 2; line accuracy 1; frame average 2; frame accuracy 1) were kept consistent over the same image series to allow fluorescence intensity comparison across samples. Images were processed using the Leica Application Suite X software package.

| gi-ems1 is a nonsense EMS mutation in SbGI
A single GI gene is present in the sorghum genome on the short arm of chromosome 3 (position 3:3,821,973-3,830,666; Sobic.003G040900; SORBI_3003G040900; Lai et al., 2020). Publicly available RNAseq analysis shows that SbGI is widely expressed in juvenile and adult tissues, with expression higher in leaf, shoot, and root-related tissues compared to flower-and seed-associated tissues ( Figure S1a; Davidson et al., 2012;Olson et al., 2014;Makita et al., 2015).
The SbGI protein is 68% identical to the Arabidopsis GI protein (Data S1). SbGI shares 96.47% and 96.21% amino acid identity with maize orthologs GI1 and GI2, respectively (Data S1). Interestingly, 80.5% of the variant residues are shared between SbGI and only one of the maize orthologs, instead of residues identical between GI1 and GI2 (Data S1). Indeed, 43% of the total variants are only shared between SbGI and GI1, while 37.5% are only shared between SbGI and GI2.
To evaluate the function of SbGI, we took advantage of an uncharacterized mutant allele in a collection of M4 EMS-mutagenized BTx623 lines described previously (Jiao et al., 2016). The ARS223 line carries an EMS-induced G to A mutation in SbGI at nucleotide

| sbgi-ems1 reduces expression of key flowering time genes under LD and SD photoperiods
To understand molecular changes associated with delayed flow-  The expression of the floral repressor SbPRR37 was also tested to determine whether SbGI contributes to its regulation. In WT plants, SbPRR37 transcript had a sharp peak of expression at 12 hr after dawn under LD and SD photoperiods, while levels in sbgi-ems1 plants were diminished by 2-to 5-fold at these time points (Figure S3a,b).
These observations show that SbGI activity contributes to the upregulation of SbPRR37.

| SbGI interaction with SbFFL is blue lightstimulated
Because CO regulation by Arabidopsis GI involves physical interaction between GI and FKF1, we investigated whether SbGI physically interacts with the sorghum FKF1-like protein (Lai et al., 2020). SbFFL is over 93%-95% identical to maize orthologs FFL1 and FFL2, and 73% identical to the Arabidopsis FKF1 protein (Data S2). Evaluation of SbFFL transcript levels in leaves showed diurnal expression with a peak at 12 hr after dawn in WT plants, like Arabidopsis FKF1 (Nelson et al., 2000), and no significant change in expression in sbgi-ems1 ( Figure 5a).  et al., 2004). nY-SbGI and cY-SbGI were employed as a positive control for BiFC, since Arabidopsis GI assembles into homotetramers (Black et al., 2011) and the maize GI proteins interact with themselves and with one another (Bendix et al., 2015). As expected, transient co-expression of nY-SbGI and cY-SbGI in N. benthamiana leaves produced strong fluorescent signal in pavement cells and a subcellular compartment likely to be the nucleus ( Figure S4a), while the same pattern was not apparent when cY-SbGI was co-expressed with nY alone (Figure 5d). Co-expression of nY-SbFFL and cY-SbGI produced fluorescent signal in the cytoplasm of pavement cells (Figure 5c). Blue light treatment also resulted in fluorescence signal from subcellular compartments consistent with nuclei, similar to that seen with the nY-SbGI and cY-SbGI combination (Figure 5e, Figure S4a).

Tests of SbGI interaction with
Thus, blue light may promote SbGI-SbFFL interaction in the nucleus.
Fluorescence of comparable intensity did not appear in blue light exposed leaves where cY-SbGI and nY or nY-SbFFL and cY were expressed together (Figure 5f, Figure S4c). These results indicate that SbGI physically interacts with SbFFL and this interaction is stimulated by blue light. In addition, the SbGI-SbFFL complex is potentially more likely to be located in the nucleus under blue light than under dark conditions.

photoperiods.
A well-established role of florigen in multiple plant species is to promote the vegetative to floral transition at the SAM (Jaeger & Wigge, 2007;Lifschitz et al., 2006;Lin et al., 2007;Notaguchi et al., 2008;Tamaki et al., 2007). An additional role of florigen observed in wheat is triggering accumulation of growth-promoting gibberellins (GA) in the SAM to drive spike development and shoot growth (Pearce et al., 2013). Taking into account the phenotypes associated with delayed flowering in sbgi-ems1 -increased DTB together with greater node production and limited additional main   (Murphy et al., 2011). We also conclude that the contribution of SbGI to flowering time is independent of SbPRR37 activity.
Comparing the observations for sbgi-ems1 to previous work repression. The apparent functional dichotomy between SbGI and GI1 likely reflects a dependence on underlying regulatory network architecture instead of fundamental differences in protein activity. Indeed, GI1 expressed in Arabidopsis thaliana complements the extreme late flowering phenotype of a gi knockout mutant to the same degree as Arabidopsis GI (Bendix, 2015), indicating that GI1 shares an inherent activity similar to Arabidopsis GI.
SbGI and SbFFL proteins physically interact and this interaction is stimulated by blue light ( Figure 5). The SbGI-SbFFL complex is localized in the cytosol under dark conditions, whereas in continuous blue light the protein complex appears in both the cytoplasm and a compartment consistent with the nucleus. This blue light responsive protein-protein interaction must be intrinsic to these proteins, with SbFFL likely serving as the photoreceptor, since this activity is apparent in yeast in the absence of other plant proteins. The Arabidopsis GI interactions with FKF1 and ZTL are blue light-dependent (Kim et al., 2007;Krahmer et al., 2018;Pudasaini et al., 2017;Sawa et al., 2007). The FKF1-GI complex occurs in the cytosol and the nucleus, while the ZTL-GI interaction is proposed to be exclusively in the cytosol (Kim et al., 2007;Park et al., 2013).
Like the activity of its Arabidopsis counterparts (Sawa et al., 2007), we predict that the SbGI interaction with SbFFL facilitates SbFKF1promoted relief of SbCO transcriptional repression and influences FKF1-promoted stabilization of SbCO protein (Song et al., 2012(Song et al., , 2014.
Overall, we demonstrate that SbGI contributes to the regulatory networks controlling sorghum flowering time in a role conserved with orthologs like those from Arabidopsis, maize, and rice.
In this capacity, SbGI serves as a key upstream activator of genes promoting flowering under both LD and SD photoperiods. SbGI activity is independent of the major floral repressor PRR37 but may contribute to its expression. At least one important interacting partner of SbGI is SbFFL, which perceives blue light and responds by binding to SbGI.

CO N FLI C T S O F I NTE R E S T
The authors declare no conflicts of interest.