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. 2019 Jun 20;180(4):2240–2253. doi: 10.1104/pp.19.00013

The Photoperiodic Flowering Time Regulator FKF1 Negatively Regulates Cellulose Biosynthesis[OPEN]

Ning Yuan 1,1, Vimal Kumar Balasubramanian 1,1, Ratan Chopra 1, Venugopal Mendu 1,2,3
PMCID: PMC6670086  PMID: 31221729

Light regulates cellulose biosynthesis through FKF1, a specific blue light receptor and photoperiodic flowering time regulator.

Abstract

Cellulose synthesis is precisely regulated by internal and external cues, and emerging evidence suggests that light regulates cellulose biosynthesis through specific light receptors. Recently, the blue light receptor CRYPTOCHROME 1 (CRY1) was shown to positively regulate secondary cell wall biosynthesis in Arabidopsis (Arabidopsis thaliana). Here, we characterize the role of FLAVIN-BINDING KELCH REPEAT, F-BOX 1 (FKF1), another blue light receptor and well-known photoperiodic flowering time regulator, in cellulose biosynthesis. A phenotype suppression screen using a cellulose deficient mutant cesa1aegeus,cesa3ixr1-2 (c1,c3), which carries nonlethal point mutations in CELLULOSE SYNTHASE A 1 (CESA1) and CESA3, resulted in identification of the phenotype-restoring large leaf (llf) mutant. Next-generation mapping using the whole genome resequencing method identified the llf locus as FKF1. FKF1 was confirmed as the causal gene through observation of the llf phenotype in an independent triple mutant c1,c3,fkf1-t carrying a FKF1 T-DNA insertion mutant. Moreover, overexpression of FKF1 in llf plants restored the c1,c3 phenotype. The fkf1 mutants showed significant increases in cellulose content and CESA gene expression compared with that in wild-type Columbia-0 plants, suggesting a negative role of FKF1 in cellulose biosynthesis. Using genetic, molecular, and phenocopy and biochemical evidence, we have firmly established the role of FKF1 in regulation of cellulose biosynthesis. In addition, CESA expression analysis showed that diurnal expression patterns of CESAs are FKF1 independent, whereas their circadian expression patterns are FKF1 dependent. Overall, our work establishes a role of FKF1 in the regulation of cell wall biosynthesis in Arabidopsis.

Cellulose is the main load-bearing structural component of plant cell walls and plays important roles in plant growth and development. Cellulose, the most abundant biopolymer on earth, has tremendous economic importance in food, fuel, fiber, forage, and timber industries. Cellulose is a linear polymer of β-1,4–linked d-Glc monomers synthesized by the plasma membrane localized CELLULOSE SYNTHASE A (CESA) protein complexes (Somerville, 2006). Cellulose biosynthesis is a highly regulated process, as the cellulose amount, crystallinity, and degree of polymerization vary among the cell types within a plant (Somerville, 2006; Li et al., 2014). The Cellulose Synthase Complexes (CSCs) are composed of hetero-trimeric proteins involving CESA1, CESA3, and CESA6 for the primary cell wall; and CESA4, CESA7, and CESA8 for secondary cell wall cellulose synthesis (Gardiner et al., 2003; Taylor et al., 2003; Persson et al., 2007). Although there is a significant progress in understanding the function of individual CESA proteins, CSC composition, and transcription factor control over cellulose biosynthesis (Li et al., 2014), the regulation of cellulose synthesis by environmental cues remains elusive.

Plants modulate their growth and development based on environmental conditions; hence, they must integrate both internal and external cues for efficient utilization of resources for developmental processes, including cellulose biosynthesis. Thus far, various internal/external cues such as brassinosteroids, and abiotic and biotic stresses have been implicated in regulation of cellulose biosynthesis (Schrick et al., 2004; Xie et al., 2011; Kesten et al., 2017). Despite a wide speculation on the role of light in cellulose biosynthesis, there is no unequivocal experimental evidence establishing a direct role of light in regulation of cellulose biosynthesis. Based on CSC movement studies in elongating hypocotyls, it has been shown that red light promotes CSC velocity and cellulose synthesis in the cesa6 mutant, presumably through phosphorylation of the partially redundant isoform of CESA5 (Bischoff et al., 2011), whereas blue light causes a concomitant reorientation of both CSC trajectories and cortical microtubules (Paredez et al., 2006). In contrast, another study using elongating hypocotyl concluded that cellulose biosynthesis is primarily regulated by carbon status and is independent of the circadian clock or light (Ivakov et al., 2017). A recent study showed that exposure of plants to blue light induced expression of secondary cell wall–related transcription factors through a blue light photoreceptor CRYPTOCHROME 1 (CRY1); cry1 mutants showed an overall reduction in stem cellulose and lignin contents (Zhang et al., 2018).

Using a phenotype suppression screen of a cellulose synthase–deficient dwarf mutant, we have identified and characterized the role of a blue light receptor FKF1 (FLAVIN-BINDING KELCH REPEAT, F-BOX 1) in cellulose biosynthesis. FKF1 is primarily known for its role in photoperiodic flowering time regulation (Song et al., 2012); however, its role in regulation of cellulose biosynthesis is a surprising revelation. We have used genetic, phenocopy, molecular, and biochemical experimental approaches to establish the role of FKF1 in cellulose biosynthesis. The FKF1 T-DNA insertion mutant fkf1-t (Cheng and Wang, 2005) showed higher cellulose contents, enhanced expression levels of cellulose synthase genes, and proteins indicating a negative regulatory role of FKF1 in cellulose biosynthesis. Further, a pronounced increase in cellulose content, CESA gene, and protein expressions was observed in fkf1-t when supplemented with blue light, indicating a negative role of FKF1 in cellulose biosynthesis. The present discovery, together with the known role of CRY1 in cell wall biosynthesis, demonstrates the role of light in regulation of cellulose biosynthesis through blue light receptors.

RESULTS

Isolation and Mapping of the llf Mutant Locus

Cell wall biosynthesis is important for proper plant growth, development, and survival. Any perturbations in cellulose deposition result in pleotropic effects in Arabidopsis (Arabidopsis thaliana; Brown et al., 2005; Hernández-Blanco et al., 2007; Persson et al., 2007; Carroll et al., 2012; Harris et al., 2012). The primary cell wall CESA double mutant (nonsynonymous amino acid substitutions) cesa1aegeus,cesa3ixr1-2 (c1,c3) shows reduced cellulose crystallinity, plant height, and leaf size compared with single mutants (Harris et al., 2012); however, the molecular basis for the dwarf phenotype is unknown. We hypothesize that the signal originating from the weak cell wall causes developmental reprograming in c1,c3, which results in the dwarf phenotype. To understand the molecular basis of c1,c3 phenotype, a phenotype suppression screen was performed by creating random mutations in the genome by treating c1,c3 seeds with ethyl methanesulfonate (EMS). Screening of 4000 M2 population seedlings resulted in identification of three independent dwarf phenotype-restoration mutants. An interesting recessive mutant, large leaf (llf), named after its characteristic large leaves, was selected for molecular mapping and functional characterization (Fig. 1A). The presence of background c1,c3 mutations in the llf mutant plant was confirmed by Sanger sequencing (Supplemental Fig. S1) indicating that a mutation at the llf locus partially rescued the c1,c3 dwarf phenotype. Phenotyping of llf showed that it had increased plant height, rosette leaf length, and leaf weight compared with c1,c3 (Fig. 1B). To identify the llf locus, the next-generation whole genome resequencing based mapping approach was used as previously described (Fig. 1C; Austin et al., 2011). Bioinformatics analysis of the whole genome sequencing data from 160 llf plants identified three single nucleotide polymorphisms (SNPs) as potential candidates of the llf locus (Supplemental Fig. S2). Among the identified SNPs was a premature stop codon mutation in the open reading frame of FKF1, a blue light receptor involved in photoperiodic flowering time regulation in Arabidopsis (Nelson et al., 2000; Song et al., 2014). The causal SNP in the FKF1 sequence was further confirmed by Sanger sequencing of five independent F2 homozygous llf mutants, which confirmed the association between the llf phenotype and FKF1 mutation (Fig. 2A; Supplemental Fig. S2B). FKF1 comprises three functional domains: blue light reception and GIGANTEA (GI) interacting LOV (Light, Oxygen, or Voltage) domain, Arabidopsis S-phase kinase-associated protein 1-LIKE protein interacting F-Box domain, and Kelch repeat domain, which is involved in protein-protein interactions (Ito et al., 2012; Song et al., 2013). The premature stop codon in FKF1 (W411*) is at the 3rd Kelch repeat domain (Fig. 2A), which is involved in ubiquitin-mediated protein degradation (Sawa et al., 2007; Ito et al., 2012).

Figure 1.

Figure 1.

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Mutation of FKF1 partially rescues the dwarf phenotype of cesa1,cesa3 (c1,c3). A, Phenotypic analysis of 12-week-old Col-0, c1,c3, and llf plants. B, Quantitation of the phenotypic analysis in (A). Rosette leaf fresh weight was calculated from 10 individual plants. Error bars represent sd (n = 10). Rosette leaf length was calculated from 15 leaves from three individual plants (5 leaves from each plant). Error bars represent sd (n = 15). Plant height was calculated using 11 individual plants. Error bars represent sd (n = 11). Statistical significance is determined by one-way ANOVA. Means not sharing the same letter indicates highly significant difference (P < 0.01). C, Representation of next-gen mapping pipeline used to map llf locus. NGM, the next-generation whole genome resequencing based mapping.

Figure 2.

Figure 2.

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Genetic and molecular confirmation of the role of FKF1 in partial restoration of c1,c3 dwarf phenotype. A, Structure of the FKF1 protein and mutation sites in llf and fkf1-t. A point mutation in the FKF1 sequence at position 1455 (G to A) resulted in a premature stop codon (W411*) in the llf mutant, whereas the T-DNA is inserted in the 6th Kelch repeat domain in the fkf1-t insertion mutant. B, Phenotypic comparison of 6-week-old and 10-week-old Arabidopsis plants. The independent triple mutant c1,c3, fkf1-t showed llf phenotype, whereas overexpression of FKF1 in llf restored c1,c3 phenotype. Scale bar = 5 cm. C, Phenotypic analysis of 6-week-old and 10-week-old Arabidopsis plants. Rosette leaf length was calculated from 28 to 36 leaves from 7 to 9 individual plants (4 leaves of each plant). Error bars represent sd (n = 28–36). Flowering time of each Arabidopsis line was calculated from 9 plants based on the rosette leaf number when the plants were beginning to bolt. Error bars represent sd (n = 9). Statistically significant difference between different groups was determined by one-way ANOVA. Means not sharing the same letter are significantly different (P < 0.05).

Genetic and Molecular Confirmation of the Role of FKF1 in Partially Restoring c1,c3 Dwarf Phenotype

Because the llf mutant was generated through EMS mutagenesis, it is possible that other independent mutations could be involved in partially rescuing the c1,c3 phenotype. To rule this out, an independent triple mutant (c1,c3,fkf1-t) was generated by crossing c1,c3 with the fkf1 T-DNA insertion mutant (Fig. 2B). The resultant triple mutant exhibited delayed flowering time (higher rosette leaf number at the time of bolting) and larger rosette leaves similar to the llf mutant (Fig. 2, B and C), confirming the role of the fkf1 mutation in partially rescuing c1,c3 phenotype. Both llf and fkf1-t mutants shared similar morphophysiological characteristics, such as increased plant height, delayed flowering time, and higher overall biomass compared with c1,c3 and wild-type Columbia-0 (Col-0), respectively (Figs. 1B and 2C; Supplemental Fig. S3). Further, ectopic expression of FKF1 in the llf mutant (Cauliflower mosaic virus [CaMV]35S:FKF1/llf) restored llf plant phenotype back to c1,c3 phenotype (Fig. 2, B and C). The phenotype-revertant transgenic lines (CaMV35S:FKF1/llf) showed higher FKF1 expression compared with Col-0, c1,c3, llf and fkf1 plants (Supplemental Fig. S4), and showed restoration of the flowering time and rosette leaf size back to the parental c1,c3 mutant phenotypes (Fig. 2C). Overall, the dwarf phenotype of c1,c3 is partially rescued in the absence of FKF1 (llf and c1,c3,fkf1-t), whereas overexpression of FKF1 in llf plants (CaMV35S:FKF1/llf) restored the c1,c3 phenotype, confirming the role of FKF1 in partially rescuing the dwarf phenotype of c1,c3 in addition to its well-established role in flowering.

FKF1 Plays a Unique Role among the ZTL/FKF1/LKP2 Family in Restoring the Dwarf Phenotype of c1,c3

FKF1 belongs to ZTL/FKF1/LKP2 gene family, which contains two additional members: ZEITLUPE (ZTL) and LOV KELCH PROTEIN 2 (LKP2). ZTL/FKF1/LKP2 family proteins are involved in blue light photoreception and modulation of downstream responses, such as circadian clock and photoperiodic flowering time regulation (Baudry et al., 2010; Suetsugu and Wada, 2013; Zoltowski and Imaizumi, 2014). Despite containing identical structural domains, phylogenetic analysis of the three proteins showed that ZTL and LKP2 are more closely related to each other than to FKF1 (Fig. 3A), which is consistent with functional divergence of the ZTL/FKF1/LKP2 gene family. ZTL and LKP2 are primarily involved in circadian clock gene regulation, whereas FKF1 is mainly involved in photoperiodic flowering time regulation in Arabidopsis (Baudry et al., 2010; Suetsugu and Wada, 2013; Zoltowski and Imaizumi, 2014). Thus, we further investigated whether the function of rescuing dwarf phenotype function is specific to FKF1, or conserved among the ZTL/FKF1/LKP2 family members. To test this hypothesis, the c1,c3 mutant was crossed independently with a ZTL T-DNA insertion mutant (ztl-105) and a LKP2 T-DNA insertion mutant (lkp2-1; Supplemental Fig. S5) to generate c1,c3, ztl-105 and c1,c3, lkp2-1 (Imaizumi et al., 2005; Martin-Tryon et al., 2007). In contrast with fkf1-t, the ztl-105 and lkp2-1 mutations did not rescue the dwarf phenotype of c1,c3 (Fig. 3B), and c1,c3,ztl-105 and c1,c3,lkp2-1 mutants showed normal flowering time and rosette leaf size similar to the c1,c3 mutant (Fig. 3C). Consistent with the phylogenetic analysis, the experimental evidence clearly demonstrates a role of FKF1 among the three family members in partially rescuing c1,c3 dwarf phenotype (Schultz et al., 2001; Más et al., 2003; Lee et al., 2017).

Figure 3.

Figure 3.

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FKF1 specific role in partial rescue of c1,c3 dwarf phenotype. A, Phylogenetic analysis of the blue light receptor proteins of Arabidopsis. Phylogenetic tree was generated by maximum likelihood method. FKF1 showed higher similarity with ZTL and LKP2 compared with the other four blue light receptors (CRY1, CRY2, PHOT1, and PHOT2). B, Phenotype analyses of 8-week-old ZTL/FKF1/LKP2 family independent triple mutants (c1,c3 × fkf1-t, c1,c3 × ztl-105, and c1,c3 × lkp2-11). Triple mutants were generated by crossing the c1,c3 mutant with respective T-DNA insertion mutants. Scale bar = 5 cm. C, Phenotypic analysis of 6-week-old and 10-week-old Arabidopsis plants. Rosette leaf length was calculated from 24 to 36 leaves from 6 to 9 individual plants (4 leaves of each plant). Error bars represent sd (n = 24–36). Flowering time of each Arabidopsis line was calculated from 8 to 9 plants based on the rosette leaf number when the plants were beginning to bolt. Error bars represent sd (n = 9). Statistically significant difference between different groups was determined by one-way ANOVA. Means not sharing the same letter are significantly different (P < 0.05).

Absence of Active FKF1 Restores the Cellulose Content and Phenotype of the c1,c3 Mutant

FKF1 has an active role in flowering time regulation under long days; however, it is inactive under short days (Sawa et al., 2007). Under long-day conditions, an abundance of FKF1 and GI proteins peak during late afternoon, resulting in formation of an active FKF1-GI complex to regulate the expression of downstream CONSTANS (CO); this, in turn, regulates flowering time (Imaizumi et al., 2003; Sawa et al., 2007). However, under short days, the abundance of FKF1 protein peaks at night, and does not form an active complex with GI (Imaizumi et al., 2003; Sawa et al., 2007). As a result, the short-day grown Col-0 phenocopies fkf1-t with a similar number of leaves and flowering time (Nelson et al., 2000; Sawa et al., 2007; Shim and Imaizumi, 2015). We postulated that FKF1 is repressing the cellulose biosynthesis only under long days, as it is inactive under short days. To test this hypothesis, we compared phenotypes of Col-0, c1,c3, llf, and fkf1-t plants that were grown under long- and short-day conditions (Supplemental Fig. S6). Under long-day conditions, c1,c3 showed significantly smaller rosette leaves compared with llf (average leaf length: 1.58 vs 4.0 cm), whereas the difference in leaf size significantly reduced under short-day conditions (average leaf length: 4.48 cm vs 5.42 cm). These results clearly indicate that FKF1 mutation caused the partial phenotype restoration in llf mutants. Further, fkf1-t showed significantly larger rosette leaves than Col-0 under long-day conditions (average leaf length: 6.49 cm vs 3.79 cm), whereas under short-day conditions, the difference in leaf size between the two genotypes was reduced (average leaf length: 6.14 vs 6.10 cm) due to lack of active FKF1 protein in Col-0 (Supplemental Fig. S6).

We then investigated if the phenotype recovery is associated with restoration of cellulose content under short-day conditions. Crystalline cellulose content of Col-0, c1,c3, and llf was estimated from the leaves of 3-week-old plants grown under long-day conditions (Fig. 4A, top) and 8-week-old plants grown under short-day conditions (Fig. 4A, bottom; before transition to flowering stage). Under long-day conditions, c1,c3 plants showed reduced cellulose content compared with llf plants (Fig. 4A, top), whereas under short-day conditions these genotypes did not show any significant difference (Fig. 4A, bottom). This indicates that under long-day conditions, active FKF1 repressed the cellulose biosynthesis in c1,c3 leaves, and thus there is a significant difference in cellulose content. Under short-day conditions, there is no active FKF1 in both c1,c3 and llf, and thus there is no significant difference in cellulose content, clearly demonstrating the role of FKF1 in cellulose biosynthesis. Further, the difference in cellulose content in c1,c3 and llf plants grown under long-day conditions correlated with the soluble sugar profiles that showed a preferential accumulation of soluble Glc and Fru in c1,c3 leaves with reduced Suc levels and cellulose content (Supplemental Fig. S7). However, in llf plants, the soluble Glc and Fru levels were reduced, with an associated increase in Suc levels and cellulose contents of leaves (Supplemental Fig. S7).

Figure 4.

Figure 4.

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Lack of functional FKF1 results in increased cellulose content. A, Crystalline cellulose content of Col-0, c1,c3, and llf leaves from plants grown under long-day (top) and short-day ( bottom) conditions. Statistically significant difference between different groups was determined by one-way ANOVA. Means not sharing the same letter are significantly different (P < 0.05). B and C, Cell wall composition analysis of leaf (B) and stem (C) samples from 12-week-old Col-0 and fkf1-t plants (Gluc. Acid: GlcA, Galac. Acid: GalUA). Data shown are an average of three independent biological replicates with three technical replicates. Error bars represent sd (n = 9). Statistically significant differences were determined by Student’s t test (*P < 0.05; **P < 0.01).

Mutation of FKF1 Results in Increased Cellulose Content

To understand the biomass composition of mature plants, cell wall composition of fully grown (12-week-old) Col-0 and fkf1-t plants was analyzed. Cell wall polymer analysis showed an increase in cellulose contents of leaf (36.2%) and stem (6.3%) of fkf1-t compared with that in Col-0, whereas no change was observed in Mol% of hemicellulose or pectic sugars in both the tissues (Fig. 4, B and C). Interestingly, the lignin content of fkf1-t leaf tissue showed a significant reduction (48.9%) compared with that of Col-0; however, there was no change in the stem lignin content (Fig. 4, B and C). Overall, genetic, molecular, and biochemical analysis established a negative role of FKF1 in cellulose biosynthesis.

Delayed Flowering Is Not the Primary Reason behind Increased Cellulose Synthesis in fkf1

FKF1 is primarily involved in regulation of photoperiodic flowering time in Arabidopsis by regulating the downstream proteins such as CO and FLOWERING LOCUS T (FT; Putterill et al., 1995; Kardailsky et al., 1999; Nelson et al., 2000; Imaizumi et al., 2003; Sawa et al., 2007; Song et al., 2012). To address the question of whether delayed flowering is the reason behind increased cellulose content of fkf1-t, a late flowering mutant (ft-10) was used for a comparative analysis. The genotype of ft-10 was confirmed with primers derived from a previous study (Kim et al., 2013). Genotype-confirmed ft-10 (Supplemental Fig. S8; Supplemental Table S1) along with Col-0 and fkf1-t were grown under white-light (WL) conditions or WL supplemented with blue light (SBL) conditions for comparative gene expression analyses. We used SBL conditions for plant growth, because short-term treatment (30 min) with complete blue light showed no difference in CESA gene expression and long-term treatment (2 weeks) of complete blue light resulted in severe growth defects (Supplemental Fig. S9). Reverse transcription–quantitative PCR (RT-qPCR) gene expression data showed that fkf1-t has significantly higher expression of 5 CESAs (CESA1, CESA2, CESA3, CESA6, and CESA8) under both light conditions and 3 CESAs under WL conditions (CESA4, CESA5, and CESA10) compared with ft-10. CESA7 and CESA9 did not show any statistically significant difference between fkf1-t and ft-10 under two light conditions (Fig. 5, A and B; Supplemental Fig. S10). Although the function of CESA10 is not known, previous studies shows that CESA2 and CESA5 are partially redundant with CESA6 for primary cell wall cellulose synthesis (Desprez et al., 2007), which are also implicated in seed coat epidermal secondary cell wall biosynthesis (Mendu et al., 2011a, 2011b). Overall, the RT-qPCR results showed that fkf1-t has significantly higher CESA gene expression compared with another independent late flowering mutant, indicating a specific role of FKF1 in cellulose biosynthesis.

Figure 5.

Figure 5.

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Blue light regulates cellulose biosynthesis in an FKF1-dependent manner. A to C, Expression analyses of cellulose synthase genes (A and B) and cell wall synthesis–related genes (C) in different Arabidopsis lines under WL and SBL conditions. Leaf samples of 4-week-old Arabidopsis plants were collected 6 h after dawn and used for RT-qPCR analysis; the data are an average of two technical replicates from three biological replicates. Error bars represent sd (n = 6). Statistically significant difference between different groups was determined by one-way ANOVA. Means not sharing the same letter are significantly different (P < 0.05). GAL, GALACTURONOSYLTRANSFERASE-LIKE; INVC, ALKALINE/NEUTRAL INVERTASE C.

In addition to CESA gene expression, we have also investigated the expression of cell wall synthesis-related genes such as ALKALINE/NEUTRAL INVERTASE C, which is involved in carbon distribution in mitochondria; NAC DOMAIN CONTAINING PROTEIN 36 (NAC036), which encodes a transcription factor regulating the leaf morphogenesis and plant development; SUC SYNTHASE 1 (SUS1), which provides UDP-Glc for cellulose synthesis; and GALACTURONOSYLTRANSFERASE-LIKE 10, which is involved in cell wall organization (Amor et al., 1995; Kato et al., 2010; Kong et al., 2011; Martín et al., 2013). RT-qPCR analysis showed up-regulation of NAC036 in fkf1-t compared with that in Col-0 and ft-10 under WL conditions, and the expression of GALACTURONOSYLTRANSFERASE-LIKE 10 was significantly higher in fkf1-t compared with Col-0 under SBL conditions (Fig. 5C). In agreement with CESA gene expression, the expression level of SUS1 is significantly higher in fkf1-t compared with that in Col-0 and ft-10 under both light conditions, indicating that FKF1 might be regulating cellulose synthesis through SUS1 by providing more UDP-Glc. Further, phenotype analysis showed that fkf1-t displayed longer rosette leaves compared with Col-0 and ft-10 under WL conditions. Under SBL conditions, fkf1-t showed longer rosette leaves compared with Col-0 (Supplemental Fig. S11); however, the difference in rosette leaf length among the three Arabidopsis lines was reduced.

Light Regulates Cellulose Synthesis in an FKF1-Dependent Manner

FKF1 is involved in ubiquitination of downstream proteins through F-Box and Kelch-repeat domains (Zoltowski and Imaizumi, 2014). Therefore, we asked if CESA proteins are regulated by FKF1 as CESA proteins are turned over at regular intervals during the day (Sampathkumar et al., 2013). We investigated the protein expression of CESA3, an indispensable component of the primary cell wall cellulose synthase complex (Burn et al., 2002). Western blot analysis of plants grown under WL and SBL conditions showed significantly higher CESA3 protein in fkf1-t compared with that in Col-0 (Fig. 6A). It is unknown whether the CESAs are not turned over quickly in fkf1-t or whether the higher protein levels result from an increased transcription (Fig. 5A). Estimation of leaf and stem cellulose content of fkf1-t showed an increase in cellulose content compared with that in Col-0 under WL conditions, and a pronounced increase in cellulose content of leaf and stem of fkf1-t under SBL conditions (Figs. 4B and 6B). These results may indicate a negative role of the blue light receptor FKF1 in cellulose synthesis. Consistent with molecular and biochemical data, histochemical staining results showed a significantly higher fluorescence of cellulose in fkf1-t in comparison with Col-0 under WL (Fig. 6C). Whereas this is not significant, there is a numerical increase under SBL conditions. Because the total cross section area of Col-0 and fkf1-t is significantly different, the calculation was performed using mean intensity from an equal area (163.6 mm2) of pith cells from both genotypes. Interestingly, mean intensity values in the primary walled pith cells of Col-0 grown under SBL conditions were higher than those of Col-0 grown under WL (Fig. 6C). There was no significant difference (visible and statistical) observed in lignin staining between Col-0 and fkf1-t grown under either WL or SBL conditions (Supplemental Fig. S12).

Figure 6.

Figure 6.

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Mutation of FKF1 results in enhanced CESA protein abundance and cellulose content. A, Leaf samples of 4-week-old Arabidopsis plants were grown under WL or SBL and collected 6 h after dawn and used for Western blot analysis; the relative protein levels of CESA3 were quantified using Ponceau S stained Rubisco as an internal reference. Col-0 sample grown under SBL conditions was arbitrarily set as 1 for relative comparison, and the data were an average of three independent biological replicates. Error bars represent sd (n = 3). The statistically significant difference was determined by one-way ANOVA. Means not sharing the same letter are significantly different (P < 0.05). B, Leaf and stem samples of 5-week-old Arabidopsis plants were used for cellulose estimation. Data were an average of three technical replicates from three independent biological replicates. Error bars represent sd (n = 9). Statistically significant difference was determined by one-way ANOVA. Means not sharing the same letter are significantly different (P < 0.05). C, Stem from 12-week-old mature Col-0 and fkf1-t plants grown in both WL and SBL conditions were sectioned and stained with Congo red stain cellulose. An equal area (163.6 mm2) of pith cells from Col-0 and fkf1-t was used to calculate the mean gray area intensity value, which corresponds to the intensity of cellulose fluorescence. The images were analyzed using ImageJ software. Data shown are an average of three independent biological replicates with at least three technical replicates. Error bars represent sd (n ≥ 9). The statistically significant difference was determined by Student’s t test (*P < 0.05).

Analysis of Diurnal/Circadian Regulation of CESA Gene Expression

Previous studies suggested that FKF1 modulates physiological and developmental processes by regulating the transcription factors in a diurnal/circadian clock-dependent manner (Ito et al., 2012; Song et al., 2012, 2013). Therefore, we investigated if FKF1 regulates CESA gene expression in a diurnal/circadian dependent manner. Gene expression analysis showed that expression of FKF1, as well as CESAs, were diurnally regulated in both Col-0 and fkf1 under long-day conditions (Supplemental Fig. S13). The FKF1 transcript peaked in the afternoon (12 h after dawn) and showed nadir at the end of the day (8 h after dusk), whereas both primary and secondary CESAs peaked in the morning (6 h after dawn) and showed nadir at the end of the day (8 h after dusk). When photoperiod-entrained Col-0 and fkf1-t seedlings were transferred to continuous light, the circadian rhythm of FKF1 was still maintained in Col-0 and fkf1-t as they peaked in the afternoon (12 h after subjective dawn); however, the CESA oscillations were completely disrupted in both Col-0 and fkf1-t (Fig. 7). On the second day (24–48 h), transcripts of most CESAs still peaked in the morning (6 h after subjective dawn) in both Col-0 and fkf1-t; however, fkf1-t showed another peak 2 h after subjective dusk (42 h), which did not appear in Col-0 (Fig. 7). On the third and fourth days (48–90 h), the oscillations of CESAs displayed a complete random pattern in Col-0 (Fig. 7), whereas, in fkf1-t, all the CESAs again showed a peak 2 h after subjective dusk (66 h) and then a morning peak (78 h; Fig. 7). The results of extended light experiments suggest that FKF1 is involved in the regulation of circadian expression pattern of CESAs, and the absence of FKF1 resulted in sharp expression peaks of CESAs (peaks appeared in the morning and 2 h after subjective dusk), an expression pattern that was not observed in Col-0 plants grown under extended light conditions. It appears that CESAs are responding to other internal cue(s), potentially carbon status, as the seedlings were under continuous light resulting in accumulation of photosynthetic carbon, and it was shown that CESAs respond to internal carbon status (Ivakov et al., 2017) and peaks disappear potentially after the utilization of excess carbon.

Figure 7.

Figure 7.

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Circadian, but not diurnal, expression patterns of CESAs are FKF1 dependent. One-week-old Arabidopsis seedlings grown under long-day conditions (16-h light/8-h dark) were transferred to continuous light conditions (24-h light) for 1 d. The seedlings were then harvested at 6-h intervals for 3 d (24–90 h) for expression analysis. To avoid variation, a total of 20 seedlings were pooled and used for gene expression analysis. This experiment was repeated three independent times. Data shown was an average of two technical replicates of three biological replicates. Error bars represent sd (n = 6). The gray rectangles indicate the subjective nighttime under continuous light experiment.

DISCUSSION

Role of Light Receptors in Photosynthetic Carbon Partition

Cellulose is one of the major sinks of photosynthetic carbon, and therefore it must be regulated based on light, photosynthetic carbon availability, developmental stage, and other external/internal cues for effective utilization of carbon resources. Light receptors are known to play critical roles in carbon resource allocation by altering the metabolic activities in plants. Analysis of a phytochrome quadruple mutant (PhyABDE) showed reduced biomass and altered resource allocation, metabolic state, and day:night growth (Yang et al., 2016), and mutation of FKF1 resulted in altered starch accumulation in Arabidopsis (Mugford et al., 2014). Further, carbon partitioning studies using 14CO2 pulse-chase labeling demonstrated the role of the circadian clock in carbon partitioning (Kölling et al., 2015). Plants possess several photoreceptors that perceive different wavelengths of light and modulate plant growth and development according to the light quality and intensity (Fankhauser and Chory, 1997). Among different spectrums of light, blue light is highly energy dense (shorter wavelength than red and green), and evidence shows that rate of photosynthesis is higher under SBL conditions (Hogewoning et al., 2010). The effect of SBL on enhanced biomass production was also observed in lettuce (Lactuca sativa) and rose (Rosa × hybrida) plants (Johkan et al., 2010; Terfa et al., 2013); however, the underlying mechanism for increased photosynthesis or higher biomass remains elusive. Increased photosynthesis and higher biomass under SBL indicate a higher photosynthetic carbon deposition in plant cell walls, as biomass is primarily composed of plant cell walls. Discovery of a role of FKF1 in cellulose biosynthesis paves the way for deciphering the molecular and biochemical mechanism behind increased photosynthesis and higher plant biomass under SBL conditions. Further, a recent study showed that blue light receptor CRY1 positively regulates secondary cell wall biosynthesis and cellulose content (Zhang et al., 2018), and our studies showed that mutation in another blue light receptor FKF1 resulted in enhanced cellulose content and cellulose synthase gene expression at various stages of plant development. This may indicate potentially different mechanisms (antagonistic) are involved in the regulation of cellulose biosynthesis by these two blue light receptors. Further studies are needed to confirm the positive role of blue light in cellulose synthesis and to determine which mechanism dominates under SBL conditions. It is possible that a combination of subdued negative regulation and augmented positive regulation operates simultaneously, resulting in increased cell wall biosynthesis and biomass production under SBL growth conditions.

Integration of Day Length and Carbon Status in Determining Photoperiodic Flowering Time

The well-established role of FKF1 is in photoperiodic flowering time regulation under long-day conditions in Arabidopsis (Song et al., 2012). The current discovery established a role of FKF1 in cellulose biosynthesis through an unknown mechanism. Further, published evidence shows that fkf1 mutants are compromised in their ability to adjust starch synthesis in response to variations in day length (Mugford et al., 2014), which suggests an active metabolic role of FKF1 in determining the flowering time. Several flowering-related genes that respond to day length variation were also found to respond to carbon/carbon nanoparticles (Ohto et al., 2001; Kumar et al., 2018), which implies that these genes are responding to internal carbon status in determining the flowering time. Based on the fact that cellulose biosynthesis is a known carbon-dependent process (Ivakov et al., 2017), we speculate that the flowering time and carbon metabolic status are coordinated for effective transition from vegetative to reproductive transition, in which FKF1 appears to be playing a key role, and that these pathways are diverging downstream of FKF1. The speculation is further supported by the data from an independent late-flowering mutant (ft-10), that is downstream of FKF1 and which did not show increased transcript level of several CESA genes (Fig. 5). Further investigations are warranted to prove the role of FKF1 role in integrating the carbon status with day length measurement in regulating flowering time in Arabidopsis.

Because FKF1 was found to be a negative regulator of cellulose biosynthesis, it is probable that active FKF1 mobilizes excess carbon reserves toward the flowering shoot, and in the absence of flowering signals in fkf1-t, the excess carbon is diverted for cellulose biosynthesis in leaves. The differential functional role of FKF1 [fkf1 mutation in c1,c3 background (llf mutant) and under short/long days] in cellulose biosynthesis (Fig. 4A) clearly shows that FKF1 in its active form promotes flowering and in its inactive form or in its complete absence (such as in the fkf1 mutant) promotes vegetative growth by diverting carbon toward cellulose biosynthesis. This assumption is supported by the fact that the c1,c3 double mutants showed early flowering (with reduced leaf size and cellulose content) compared with Col-0; however, when a fkf1 mutation was introduced into the c1,c3 background (llf), the plants showed delayed flowering time (with associated increase in leaf size and cellulose content; Fig. 2). The biochemical role of FKF1 is further supported by phenotype as well as molecular analysis of an independent late-flowering mutant ft-10. Among the two late-flowering mutants considered, fkf1-t showed larger leaf length, which correlated with increased expression of CESAs and cellulose synthase-related genes (Fig. 5; Supplemental Figs. S10 and S11). Specifically, higher SUS1 expression in fkf1-t indicates potential diversion of soluble sugars toward a cellulose biosynthesis process through the SUS1-mediated pathway, as SUSY overexpression results in increased cellulose content (Coleman et al., 2009). This hypothesis must be treated with caution and needs further investigation to develop a working model.

Potential Roles of ZTL/FKF1/LKP2 Family and GI in Cellulose Biosynthesis

Because cellulose synthesis is a carbon-demanding process, it must be regulated based on internal carbon availability. The metabolic flux-based carbon signaling has been shown to regulate cellulose biosynthesis (Ivakov et al., 2017). Our studies clearly demonstrated the role of FKF1 in regulating cellulose biosynthesis, potentially integrating photoperiod and carbon status to determine the carbon partition to flowering shoots or leaf cellulose synthesis. Consistent with the assumption, the expression patterns of CESAs showed similar diurnal oscillation under long-day conditions in Col-0 and fkf1-t. However, given there was no change in external conditions, the expression levels of most CESAs were higher in fkf1-t, indicating an altered metabolic carbon status could be inducing the increase. Further, the circadian expression patterns of CESAs in the fkf1-t mutant were different from Col-0, indicating a potential direct or indirect role of FKF1 in regulating the circadian expression pattern of CESAs (Fig. 7). The ZTL/FKF1/LKP2 family members are involved in circadian clock and flowering time regulation in Arabidopsis, and ZTL is primarily involved in regulation of the circadian clock (Somers et al., 2000; Más et al., 2003; Baudry et al., 2010; Zoltowski and Imaizumi, 2014). The diurnal oscillation of ZTL protein abundance is regulated by GI, which stabilizes the ZTL protein in the afternoon (Kim et al., 2007). Both ZTL and FKF1 interact with GI and compete for the same GI pool, and the excess GI protein in fkf1-t may stabilize ZTL protein for a longer time in fkf1-t than in Col-0 causing the alternations in circadian expression patterns of CESAs between fkf1-t and Col-0.

Overall, the present investigation using genetic, biochemical, and molecular studies firmly establishes the role of FKF1 as a blue light receptor in cellulose biosynthesis. We presume that FKF1, a key flowering time regulator, integrates the carbon status and environmental cues to determine the vegetative to reproductive phase transition. In addition, carbon status was found to regulate CESA protein abundance on the plasma membrane (Ivakov et al., 2017), and it will be interesting to study CESA protein trafficking in the fkf1 mutant and to investigate the role of FKF1 role in regulating CESA abundance at the plasma membrane in response to carbon status. Also, FKF1 interacts with client proteins through its Kelch repeats, potentially regulating CESAs/carbon or other metabolic pathway proteins, in addition to flowering pathway proteins. Further, comprehensive genetic, molecular, and biochemical evidences are required to develop working models to integrate the carbon status and flowering time regulatory mechanism mediated by FKF1.

MATERIALS AND METHODS

Plant Growth and Light Conditions

Unless otherwise noted, the Arabidopsis (Arabidopsis thaliana) plants were grown in soil or on one half Murashige and Skoog + 2% (w/v) Suc medium under long-day (16-h day/8-h night) or short-day (8-h day/16-h night) conditions with 22°C/20°C day/night temperatures in plant growth chambers (Percival) with a light density of 120 µmol/m2/s (fluorescence). For experiments whereby plants were exposed to supplemental blue light, seeds were first germinated and grown in soil under a 16-h day/8-h night photoperiod with 22°C/20°C day/night temperatures in a growth chamber for 2 weeks, and the seedlings were then transferred to WL conditions or WL supplemented with blue light (SBL) conditions (16-h day/8-h night photoperiod with 22°C/20°C day/night temperatures). LumiGrow Pro 650 light emitting diode (LED) lighting systems (LumiGrow) were used to supply white and blue LED light. Extech LED light meter LT45 (Extech) was used to measure the light intensity. To set up the complete WL growth condition, white LED light was set to scale 5 (scale range is from 0 to 10), with a light intensity of 120 µmol/m2/s. For SBL growth conditions, white LED light was set at scale 5 (120 µmol/m2/s) and blue LED light was set at scale 5 (50 µmol/m2/s). The blue and white light intensities were measured separately because the light meter LT45 can read only a single light source at a time. The Lux to Photosynthetic Photon Flux Density was calculated using the online calculator: https://www.waveformlighting.com/horticulture/convert-lux-to-ppfd-online-calculator.

EMS Mutagenesis, Isolation of the llf Mutant, and Generation of Mapping Population

Double mutant (c1,c3) seeds (Harris et al., 2012) were mutagenized with EMS. Briefly, c1,c3 seeds were soaked in 100 mm phosphate buffer at 4°C for overnight and then treated with 0.4% (w/v) EMS in fresh 100 mm phosphate buffer for 8 h. Following EMS treatment, mutant seeds were neutralized with 0.5 m NaOH, washed 20 times with water, and immediately germinated on soil to produce M1 plants. Seeds from M1 plants were collected, germinated, and screened for phenotype revenants leading to the identification of the llf mutant in M2 generation. The identified llf mutant plant was crossed with Landsberg erecta ecotype to generate a mapping population. The F2 seeds from F1 plants were collected and screened for homozygous llf plants. Two sets of 80 llf homozygous plants were identified by visual screening, and leaf tissue was collected for DNA isolation and mutant locus mapping.

Next Generation Sequencing and Mapping of the llf Locus

Genomic DNA from two independent sets of 80 llf plants (total 160) were extracted using Qiagen plant DNA extraction kit and pooled and sent for sequencing at Donnelly Sequencing center at the University of Toronto. Briefly, Illumina TruSeq Nano libraries were prepared, quality was estimated, and sequencing was performed using NextSeq500 under the criteria (150 Cycle Mid Output Kit, Read 1:75bp index read 1:8 bp, Index read 2:8bp, Read 2:75bp). Raw reads (National Center for Biotechnology Information sequence read archive database: SRP150102) obtained from sequencing were processed through trimmomatic tools for quality check. Reads were then mapped on to the reference genome of Col-0 using samtools (Li et al., 2008), and SNPs were identified using the pileup function from samtools. SNPs files were then processed through the Next-generation mutation mapping pipeline as described (Austin et al., 2011) and represented in a flow chart (Fig. 1). A back-cross population was generated by crossing an llf plant with Col-0 for cosegregation analysis of the llf phenotype with the identified SNP.

Sanger Sequencing

Sequencing of DNA for SNP differences and cloning confirmations was performed using Sanger sequencing (Genewiz). Briefly, DNA was PCR amplified using gene-specific primers (Supplemental Table S1), gel purified, and sent for sequencing using respective primers. For sequence confirmation of plasmid constructs, plasmid was isolated and sent for sequencing using respective primers. The sequencing results were analyzed for the presence of mutations/sequencing confirmations using National Center for Biotechnology Information BLAST tool.

Generation of Triple Mutants and Functional Complementation of the fkf1 Mutant

T-DNA insertion mutant lines fkf1-t (SALK_059480c), lkp2-1 (SALK_036083), and ztl-105 (SALK_069091c) were obtained from The Arabidopsis Information Resource (Alonso et al., 2003) and independently crossed with homozygous c1,c3 to generate F1 plants. F2 seeds from the F1 plants were collected and plated on one half Murashige and Skoog media plates with 10 nm Isoxaben (cesa1), 5 μm Quinozyphen (cesa3), and 50 mg/L Kanamycin (T-DNA; all three chemicals are lethal doses to wild type). The surviving plants were genotyped using PCR for T-DNA insertion confirmation and sequenced for SNPs (cesa1 and cesa3) to generate homozygous triple mutants (c1,c3,fkf1-t, c1,c3,ztl-105, and c1,c3,lkp2-1). For complementation of llf with the wild-type FKF1 gene, the 1961-bp genomic DNA of FKF1 was amplified from Arabidopsis (Col-0) with Next high-fidelity 2× PCR Mater Mix (New England Biolabs). The PCR product was then digested with AscI and PacI (New England Biolabs) and cloned into the AscI and PacI digested pMDC32 binary vector (CaMV 35Sp/AscI-attR1-CMr-ccdB-attR2-PacI/nos-CaMV 35Sp/hptII/CaMV terminator), resulting in generation of pMDC32 binary vector with FKF1 (CaMV 35Sp/AtFKF1/nos-CaMV 35Sp/hptII/CaMV terminator). The pMDC32 construct was sequenced and confirmed for the presence of FKF1.

Quantitative Analysis of Gene Expression

Total RNA was extracted using Spectrum Plant Total RNA kit (Sigma-Aldrich) from 100 mg of plant samples. The RNA was then treated with On-Column DNase I (Sigma-Aldrich) to remove genomic DNA contamination. Total DNA-free RNA (1 μg) was then used to synthesize first strand complementary DNA with iScript reverse transcription supermix RT-qPCR kit (Bio-Rad). First-strand complementary DNA was diluted to 0.05 times with water and used for RT-qPCR with FastStart Essential DNA Green Master kit (Roche). LightCycler 96 (Roche) was used to detect and analyze RT-qPCR results. RT-qPCR results were determined using ΔΔCt method (Rao et al., 2013).

Western Blot Analysis

Protein estimation was performed by Western blot analysis as previously described (Yuan et al., 2018). Briefly, membrane protein was isolated from Arabidopsis tissue with Minute Plasma membrane protein isolation kit (Invent Biotechnologies). The total protein was separated on SDS-PAGE gel and transferred onto polyvinylidene difluoride membrane using the iBlot2 Western blot transfer system (Thermo Fisher Scientific). Hybridization was performed using Anti-CESA3 (Polyclonal Antibody, Abnova) as a primary antibody at a dilution of 1:2000 in 5% (w/v) BSA TBS-Tween buffer. Goat Anti-Rabbit IgG (H+L) Poly-Horseradish Peroxidase (Invitrogen) was used as a secondary antibody at a dilution of 1:3000 in TBS-Tween buffer. Signal was developed using Clarity Western enhanced chemiluminescence Substrate (Bio-Rad), and detected using Chemidoc XRS+ imager (Bio-Rad).

Estimation of Crystalline Cellulose Content

The crystalline cellulose content of leaf and stem tissues was measured using Updegraff method (Updegraff, 1969). Alcohol insoluble residue obtained from ground tissue was treated with Updegraff reagent (Acetic acid: Nitric acid: water in ratio 8:1:2 [v/v]) at 100°C for 30 min and washed two times with water followed by acetone. The pellet was dried at 37°C overnight and used for anthrone assay (0.3% [w/v] anthrone dissolved in concentrated sulfuric acid). The absorbance was measured at 620 nm using DTX880 multiwell plate reader (Beckman Coulter), and the concentration of Glc was quantified using standard curve generated from Glc standard and equated for water loss to calculate the crystalline cellulose content.

Estimation of Lignin Content

Lignin content from leaf and stem tissue was measured using thioglycolic acid (TGA) derivatization method as described (Antonova et al., 2007). Alcohol insoluble residue was digested with cell wall hydrolases (cellulase, hemicellulase, pectinase, and xylanase) at 37°C overnight to deconstruct the cell wall and allow access for TGA to react with lignin. Following digestion, the pellet was washed and treated with proteinase K at 37°C overnight to remove protein from the pellet. The pellet was then washed three times with water and treated with 10% (v/v) TGA in 3 m hydrochloric acid and incubated at 80°C for 3 h. The pellet was then rinsed with water and dried in a speedvac at 30°C for 4 h. The dried pellet was treated with 2 m sodium hydroxide at 37°C overnight to dissolve lignin from the pellet. The supernatant was isolated and treated with concentrated hydrochloric acid at 4°C for 4 h to precipitate lignin. The precipitated lignin was dried using a speedvac at 30°C for 4 h, and the dried lignin pellet was dissolved overnight in dimethyl sulfoxide at 37°C. The absorbance of lignin was measured at 280 nm using a UV spectrophotometer (Eppendorf), and the concentration of lignin was quantified using a standard curve generated from industrial lignin.

Estimation of Hemicellulose and Pectin Sugars

The composition of hemicellulose and pectic sugars was measured using modified protocol as described (Sweeley et al., 1963). The alcohol-insoluble residue was treated with 2 m HCl/methanol and incubated at 85°C for 24 h, and the supernatant was evaporated using nitrogen gas. The pellet was treated with hexamethyldisilazane:trimethylchlorosilane:pyridine in ratio 3:1:9 at 80°C for 20 min and the reaction mixture was dried using nitrogen gas. Hexane (1 mL) was added to the dried pellet, vortexed, and centrifuged to remove the supernatant. The supernatant was concentrated using nitrogen gas for about 200 µL and analyzed using gas chromatography-mass spectrometry (GC-MS). Derivatized sample (1 µL) was injected in GC-MS (Agilent 7890AGC-5975CMS) containing HP-5MS column (30 m × 250 μm × 0.25 μm) using oven temperature program, 100°C/2 min, 220°C/2 min (4°C/min rate), and 300°C/2 min (15°C/min rate) with helium as carrier gas. Pure standards for each sugar were derivatized using the same protocol and ran on GC-MS, and retention time was selected based on repeatability and mass ion generated. The sugar composition from samples was represented as Mol% of total sugars present in each sample.

Histochemical Analysis of Stem Cross Sections

Histochemical analysis of cellulose and lignin from stem cross section was performed using published protocol (Pradhan Mitra and Loqué, 2014). Briefly, stem sections from the bottom 5 cm of 12-week-old Col-0 and fkf1-t plants were obtained by cutting approximately 1-mm thick sections using a razor blade. For Congo red staining, sections were incubated with 0.5% (w/v) Congo red (Sigma-Aldrich) for 5 min and rinsed with distilled water and imaged under blue light excitation with bandpass 560/40. For Calcofluor white staining, sections were incubated with 0.02% (w/v) Calcofluor white M2R (Sigma-Aldrich) for 5 min and rinsed with distilled water and imaged under UV light. For phloroglucinol HCl staining, sections were incubated with 3% (w/v) phloroglucinol-HCl solution (Sigma-Aldrich) and directly observed under bright field lighting. Olympus BH2 fluorescent microscopy was used for imaging, and the images were analyzed using Cell-Sens standard software. The images were quantified using ImageJ software. An equal area (163.6 mm2) of pith cells from Col-0 and fkf1-t images was used to calculate the mean gray area intensity value, which corresponds to the intensity of cellulose fluorescence.

Estimation of Soluble Sugars

The soluble sugars were estimated using published protocol (Glassop et al., 2007). Briefly, 60 mg of fresh leaf tissue was collected and ground, and 350 µl of methanol was added, vortexed, and incubated at 70°C for 15 min. The samples were centrifuged, and supernatant was transferred to a fresh tube Water (350 µL) and 300 µL of chloroform were added to the supernatant, vortexed, and centrifuged to separate the polar phase containing soluble sugars. To the soluble phase, 300 µL chloroform was again added and centrifuged to retain the polar phase, which was then vacuum dried at 45°C for 2 h. Trimethlysilyl derivatization was performed in the tube with dried soluble phase using Hexamethyldisilazane: Trimethylchlorosilane: pyridine in ratio 3:1:9 at 80°C for 12 h. After derivatization, the pellet was dried and resuspended in hexane, and analyzed in GC-MS using the conditions similar to hemicelluloses and pectin sugar composition analysis.

Statistical Analysis

Student’s t test was used to calculate the difference between the means from two groups. *P < 0.05 was considered to be statistically significant. **P < 0.01 was considered to be statistically highly significant. One-way ANOVA [F(dfbetween, dfwithin) = F ration, P = p-value], where df = degrees of freedom) with post hoc comparisons using the Tukey Honestly Significant Difference test was used to determine the statistically significant difference between the means from three or more groups. Means not sharing the same letter were considered to be statistically significantly different (P < 0.05) or highly statistically significantly different (P < 0.01).

Accession Numbers

Sequence data from this article can be found in the Arabidopsis Genome Initiative database and European Molecular Biology Laboratory under the following accession numbers: FKF1 (AT1G68050), ZTL (AT5G57360), LKP2 (AT2G18915), FT (AT1G65480), CESA1 (AT4G32410), CESA2 (AT4G39350), CESA3 (AT5G05170), CESA4 (AT5G44030), CESA5 (AT5G09870), CESA6 (AT5G64740), CESA7 (AT5G17420), CESA8 (AT4G18780), CESA9 (AT2G21770), CESA10 (AT2G25540), SUS1 (AT5G20830), GATL10 (AT3G28340), ALKALINE/NEUTRAL INVERTASE C (AT3G06500), NAC036 (AT2G17040), and Actin2 (AT3G18780).

Germplasm Accession Numbers

The germplasm information of the Arabidopsis mutants can be found in The Arabidopsis information Resource under the following accession numbers: fkf1-t (SALK_059480c), lkp2-1 (SALK_036083), ztl-105 (SALK_069091c), and ft-10 (CS9869).

Supplemental Data

The following supplemental materials are available.

Acknowledgments

We thank Dr. Seth DeBolt for kindly providing cesa1,cesa3 double mutant seeds.

Footnotes

[OPEN]

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