Abstract
The TTG2 transcription factor of Arabidopsis regulates a set of epidermal traits, including the differentiation of leaf trichomes, flavonoid pigment production in cells of the inner testa (or seed coat) layer and mucilage production in specialized cells of the outer testa layer. Despite the fact that TTG2 has been known for over twenty years as an important regulator of multiple developmental pathways, little has been discovered about the downstream mechanisms by which TTG2 co-regulates these epidermal features. In this study, we present evidence of phosphoinositide lipid signaling as a mechanism for the regulation of TTG2-dependent epidermal pathways. Overexpression of the AtPLC1 gene rescues the trichome and seed coat phenotypes of the ttg2-1 mutant plant. Moreover, in the case of seed coat color rescue, AtPLC1 overexpression restored expression of the TTG2 flavonoid pathway target genes, TT12 and TT13/AHA10. Consistent with these observations, a dominant AtPLC1 T-DNA insertion allele (plc1-1D) promotes trichome development in both wild-type and ttg2-3 plants. Also, AtPLC1 promoter:GUS analysis shows expression in trichomes and this expression appears dependent on TTG2. Taken together, the discovery of a genetic interaction between TTG2 and AtPLC1 suggests a role for phosphoinositide signaling in the regulation of trichome development, flavonoid pigment biosynthesis and the differentiation of mucilage-producing cells of the seed coat. This finding provides new avenues for future research at the intersection of the TTG2-dependent developmental pathways and the numerous molecular and cellular phenomena influenced by phospholipid signaling.
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Introduction
A suite of co-regulated epidermal traits in Arabidopsis has served as one of the preeminent systems for studying the genetics of cell differentiation, patterning and organ formation1,2,3. These epidermal developmental pathways lead to trichome (hair) initiation and patterning, the differentiation of the mucilage-producing outer testa (seed coat) layer, the production of flavonoid-based proanthocyanidin (PA) pigments (also known as condensed tannins) in the inner seed coat layer, and root hair patterning. Extensive genetic studies of these plant epidermal features identified the combinatorial MYB-bHLH-WDR (MBW) transcriptional complex regulatory model. The transparent testa glabra 1 (ttg1) mutant of Arabidopsis defined a key member of the MBW complex and highlighted the co-regulation of seemingly disparate epidermal traits4,5. TTG1 encodes a small WD-repeat containing protein, which serves as a platform for protein–protein interactions. TTG1 physically interacts with combinations of R2R3 MYB and bHLH transcription factors to specify specific epidermal cell fates1,2,3
An additional layer of transcriptional control of the TTG1-dependent epidermal pathways exists just downstream of the MBW complex6,7,8,9,10,11. Two transcription factors, Glabra2 (GL2), a homeodomain Leu-zipper (HD-ZIP) protein, and Transparent Testa Glabra2 (TTG2), a WRKY protein, are themselves major direct transcriptional targets of the MBW complex. GL2 and TTG2 regulate events in all of the MBW-dependent developmental programs, representing key control nodes in these programs. Both GL2 and TTG2 have positive roles in trichome initiation and development, and in seed coat mucilage production. GL2 also regulates root hair patterning while TTG2 functions in PA biosynthesis in the seed coat.
TTG2 was originally identified as a trichome and seed coat mutant from a transposon tagging screen8. The ttg2-1 loss of function mutant shows fewer and underdeveloped, mostly unbranched trichomes compared to wild-type, suggesting a primary role in trichome outgrowth and branch initiation. The seed coats of ttg2-1 mutants fail to differentiate properly; the inner testa layer lacks PA pigments while the outer layer cells do not differentiate to produce mucilage. TTG2 also contributes to maternal control of seed size by regulating integument cell elongation12. Consistent with these pleiotropic phenotypes, TTG2 is expressed in trichomes and developing seed coats8. TTG2 is also expressed in non-hair root epidermal cell files, but no mutant root hair patterning defect is obvious in ttg2-1 mutants. Additionally, TTG2 is expressed in the endosperm throughout seed development where it appears to have a role in preventing endosperm cellularization13.
TTG2 encodes a 429 amino acid protein containing two WRKY plant transcriptional regulation domains. The WRKY domain is a DNA binding domain of about 60 amino acids containing the conserved WRKY motif along with a novel zinc finger motif14,15,16. WRKY proteins show high affinity for the W box defined as (T)(T)TGAC(T/C). As a family, WRKY genes tend to mediate biotic and abiotic stress responses, so TTG2 is notable in that it functions in development as well.
Some of the molecular and biochemical mechanisms of TTG2 regulation and its relationship to the MBW complex have been uncovered. In the trichome pathway, TTG2 directly regulates the Triptychon (TRY) gene, encoding a R3 MYB negative regulator of trichome initiation17,18. In the flavonoid pigment pathway, TTG2 regulates PA precursor vacuolar transport steps encoded by Transparent Testa 12 (TT12) and AHA10/Transparent Testa 13 (TT13)19. Also, TTG2 physically interacts with TTG1, and TTG2 requires TTG1 for target gene activation in the trichome and PA pathways17,19.
Several lines of evidence, by way of gene expression analysis, suggest a possible role for PLC signaling in trichome development. Microarray experiments showed that AtPLC1 (At5G58670) is expressed about 24-fold higher in trichome cells compared to the non-trichome cells of the leaves from which they were harvested20. Promoter:GUS reporter analysis showed that AtPLC2 is expressed in trichomes21, although no trichome phenotypes in atplc2 mutant plants were reported. Also AtPLC7 promoter:GUS analysis shows expression in trichomes22. Promoter:GUS analysis has also revealed that AtPLC3 is expressed in support cells at the base of trichomes. Moreover, phospholipid signaling was originally implicated years ago in at least one of the MBW-regulated pathways by the discovery that root hair patterning requires the AtPLDζ1 gene which is a direct target of GL223.
To further study potential roles of phospholipid signaling in the TTG2-dependant epidermal traits, we focused our analysis on the AtPLC1 gene (At5G58670). We found that AtPLC1 is expressed in trichomes and this expression is reduced in trichomes of ttg2-1 mutants. Moreover, overexpression of AtPLC1 rescues the phenotypes of the ttg2-1 mutant. In the case of the seed coat PA pigment pathway, overexpression of AtPLC1 in ttg2-1 plants restored the expression of TT12 and AHA10/TT13. In addition, an AtPLC1 T-DNA insertion mutant line, plc1-1D (S025769C), shows increased trichome initiation and branching, and this phenotype is inherited dominantly. Also, the plc1-1D allele rescues the trichome phenotype of ttg2-3 mutants. Curiously, overexpression of AtPLC1 in Ler wild-type seedlings inconsistently results in underdeveloped trichomes. Taken together, these observations point to a novel TTG2 regulatory mechanism involving phospholipid signaling for the coordinate control of trichome and seed coat development.
Results
AtPLC1 is expressed in trichomes and this expression is dependent on TTG2
To confirm trichome specific expression of the AtPLC1 gene, we created promoter:GUS reporter constructs for AtPLC1. We found that Ler ecotype reporter lines harboring the AtPLC1pro:GUS construct showed trichome specific expression on the leaves of young seedlings (Fig. 1a). This expression is reduced in the trichomes of ttg2-1 reporter liners (Fig. 1b).
AtPLC1pro:GUS expression pattern in seedlings and developing seed. (a) Ler seedling with expanded first true leaves and emerging third and fourth leaves. (b) ttg2-1 seedling with expanded first true leaves and emerging third and fourth leaves. (c) Dissected Ler silique showing developing seed at approximately the late heart stage. Scale bars: 500 µm.
Because trichome and seed coat development are co-regulated by TTG2, we checked the expression of AtPLC1 in developing seed. AtPLC1pro:GUS reporter Ler lines showed no obvious expression in developing seed coats (Fig. 1c).
Overexpression of AtPLC1 in Ler suppresses trichome development
To investigate possible roles of AtPLC1 in Arabidopsis, we made constructs expressing the AtPLC1 gene under the control of the constitutive cauliflower mosaic virus 35S promoter (35S:AtPLC1). The 35S:AtPLC1 construct in Ler resulted in underdeveloped trichomes in some of the primary transgenic lines (Fig. 2). Instead of the three-branched trichomes typically found on Ler seedlings, the trichomes of 35S:AtPLC1 Ler seedlings were primarily two-branched (Fig. 2c,d). No obvious seed coat phenotypes were observed in seeds of 35S:AtPLC1 plants.
AtPLC1 overexpression in Ler seedlings. Light micrographs of expanding third and fourth leaves on (a) Ler seedling and (b) 35S:AtPLC1 Ler transgenic seedling. (c) and (d) SEM of expanding first true leaves on Ler seedling and 35S:AtPLC1 Ler transgenic seedling, respectively. Scale bars: a and b = 500 µm, c and d = 100 µm.
Overexpression of AtPLC1 in ttg2-1 plants rescues mutant trichome and seed coat phenotypes
Surprisingly, ttg2-1 plants overexpressing AtPLC1 showed rescued trichome development. The trichomes on first true leaves of ttg2-1 seedlings are typically unbranched (Fig. 3a,b), but trichomes on ttg2-1 seedlings overexpressing AtPLC1 were two and three-branched (Fig. 3c,d). On the third and fourth leaves (Fig. 4), trichome initiation, as well as branching, was increased in the overexpressor compared to untransformed ttg2-1 plants. In addition, some trichome clustering (in pairs) was observed on leaves of 35S:AtPLC1 ttg2-1 plants (Fig. 3d), indicating a disruption in trichome patterning (clustering of trichomes is not observed in wild-type plants). Lastly, trichomes on the overexpressor lines appeared to be terminally differentiated, as evidenced by a loss of the glassy appearance under light microscopy and increased or enhanced papillae on the cell surface as imaged under SEM (Fig. 4a,b,e and f).
SEMs of AtPLC1 overexpression in ttg2-1 seedlings. (a) ttg2-1 seedling with expanding first true leaves and (b) An expanded ttg2-1 first true leaf. (c) Two 35S:AtPLC1 ttg2-1 transgenic seedlings with expanding first true leaves and (d) Two expanded first true leaves from 35S:AtPLC1 ttg2-1 transgenic seedlings. Images are from a single transgenic line representative of other lines. Scale bars: 200 µm.
AtPLC1 overexpression in third and fourth leaves ttg2-1 seedlings. Light micrographs of expanding third and fourth leaves on (a) ttg2-1 seedling and (b) 35S:AtPLC1 ttg2-1 transgenic seedling. (c) ttg2-1 seedling with expanding third and fourth leaves. (d) A 35S:AtPLC1 ttg2-1 seedling with expanding third and fourth leaves. (e) Trichome on ttg2-1 mutant seedlings and (f) trichomes on 35S:AtPLC1 ttg2-1 seedling. Scale bars: a-d = 500 µm, e = 20 µm, f = 50 µm.
Seed development was also rescued in ttg2-1 plants overexpressing AtPLC1. Under SEM, the cells of the outer testa layer of wild-type dry seeds showed the characteristic appearance of raised columellae surrounded by reinforced radial walls11,24 (Fig. 5a). These cells on ttg2-1 seed showed no columella formation and indistinct radial walls (Fig. 5b). However, seeds from ttg2-1 plants overexpressing AtPLC1 showed a range of partially to mostly rescued cell morphologies (Fig. 5c).
AtPLC1 overexpression in ttg2-1 seeds. SEM of (a) Ler and (b) ttg2-1 dry seed surface. (c) three representative images of dry seed surface showing the mucilage-producing outer testa cells of 35S:AtPLC1 ttg2-1 seed. (d) Dry seed color phenotypes from left to right: Ler, ttg2-1 and seed from three lines of 35S:AtPLC1 ttg2-1 plants. (e) DMACA stained dry seed from Ler, ttg2-1, and 35S:AtPLC1 ttg2-1 plants. Scale bars: a-c = 20 µm, e = 1 mm.
In addition, seed color appeared to be mostly restored in ttg2-1 plants overexpressing AtPLC1 (Fig. 5d). DMACA reagent was also used to stain PAs and epicatechin pigment precursors in Ler, ttg2-1 and 35S:AtPLC1 ttg2-1 dry seed25,26. DMACA staining further showed restoration of PA and pigment precursor production in 35S:AtPLC1 ttg2-1 lines (Fig. 5e). ttg2-1 mutant seeds showed very low DMACA staining but seed from Ler and 35S:AtPLC1 ttg2-1 lines showed positive, dark DMACA staining.
Overexpression of AtPLC1 in ttg2-1 mutant restores the expression of TT12 and AHA10/TT13 PA pathway genes
Because AtPLC1 overexpression rescued PA pigment and pigment precursor production in ttg2-1 seeds, we employed semi-quantitative RT-PCR to check for TT12 and AHA10/TT13 expression in developing siliques. As previously reported19, TT12 and AHA10/TT13 expression was detected in Ler developing silique, but not detected in ttg2-1 developing silique (Fig. 6a and b). However, TT12 and AHA10/TT13 expression was detected in RT-PCR reactions containing first strand cDNA prepared from silique of ttg2-1 plants overexpressing AtPLC1 (Fig. 6a and b). Expression of the actin control was detected in all RT-PCR reactions (6c).
Semi-quantitative RT-PCR gene expression of TT12 and AHA10/TT13 in Ler, ttg2-1 and 35S:AtPLC1 ttg2-1. Amplification of (a) TT12, (b) AHA10/TT13 and (c) ACT2 amplicons from two biological replicates of Ler, ttg2-1, and 35S:AtPLC1 ttg2-1 first-strand cDNAs. Images (a, b and c) are from three independently run gels.
An AtPLC1 T-DNA insertion line, plc1-1D, shows increased trichome initiation and branching phenotypes that are inherited dominantly, and rescues the ttg2-3 trichome phenotype
We observed a discrepancy regarding the phenotypic results of overexpressing AtPLC1 in Ler plants (Fig. 2) vs. ttg2-1 mutants (Figs. 2 and 3). To help resolve the phenotypic discrepancy between Ler and ttg2-1 lines overexpressing AtPLC1, we acquired an AtPLC1 T-DNA homozygous insertion line (plc1-1D) in the Col-0 accession (Salk 025769C) from the Arabidopsis Biological Resource Center27 (Alonso et al.; 2003). In this line, the insertion is immediately after the 126th base pair (or 42nd codon) in the first exon22 (Kanehara et al., 2015). The plc1-1D insertion line showed an increase in trichome branching, with the four-branch class increasing to 61% from 13% observed in Col (Table 1). Moreover, five and six-branched trichomes, which are not observed in Col first true leaves, were observed on first true leaves of the T-DNA insertion line: 20% of trichomes in the plc1-1D line were five-branched while 2% were six-branched. Also, the plc1-1D line showed increased trichome initiation, with first true leaves containing about 41% more trichomes than Col first true leaves (Table 1). When Col plants were crossed to the plc1-1D line, the F1 progeny showed increased trichome branching and an increase in initiation, indicating that the plc1-1D phenotype is inherited dominantly, and that the insertion allele is likely a gain-of-function mutation (Table 1).
To investigate possible mechanisms by which the plc1-1D allele might remain functional, we conducted semi-quantitative RT-PCR to check for any expression from the AtPLC1 locus downstream of the insertion. We used a primer set 354 bp downstream of the T-DNA insertion to amplify a 727 bp fragment of AtPLC1 from first-strand seedling cDNA. Also, the primers spanned four introns of the AtPLC1 genomic locus such that amplification from genomic DNA would result in a 1042 bp amplicon (Fig. 7a). As expected, AtPLC1 expression was detected in Col-0 seedling cDNA (Fig. 7b). AtPLC1 expression was also detected in cDNA prepared form the plc1-1D mutant line, as evidenced by the amplification of the 727 bp RT-PCR fragment (Fig. 7b). This indicates that the AtPLC1 locus in the T-DNA mutant is not transcriptionally silent, leaving the possibility that the remaining 519 codons after the insertion may yield some truncated protein with potential functionality. Expression of the actin control was detected in all RT-PCR reactions.
Characterization of the AtPLC1 T-DNA mutant, plc1-1D. (a) Schematic model of the AtPLC1 gene. Shown are the general locations of T-DNA insertion site in exon 1 and the primers downstream of then insertion for detecting AtPLC1 expression in RT-PCR. Gray boxes depict exons, lines depict introns, the triangle depicts the insertion and arrows depict the RT-PCR primers. A scale bar for the exons of approximately 300 bp is shown. (b) Semi-quantitative RT-PCR gene expression of AtPLC1 in Col and plc1-1D mutant: 1. Amplification of AtPLC1 (larger band) and ACT2 (smaller band) from four biological replicates of Col first-strand seedling cDNA, 2. Amplification of AtPLC1 (larger band) and ACT2 (smaller band) from four biological replicates of plc1-1D mutant first-strand seedling cDNA. (c) Genotyping results for ttg2-3; plc1-1D double mutant. Lane 1 shows the DNA ladder. Lanes 2–4 shows TTG2 locus genotyping in Col-0 and in two double mutant candidates; lanes 5–7 show AtPLC1 locus genotyping in Col-0 and in two double mutant candidates. For both genes tested, larger band signifies wild-type alleles and smaller bands signify TDNA insertion alleles. (d) Trichome phenotypes on emerging third and fourth true leaves. From left to right: ttg2-3; plc1-1D mutant; F1 individual from a cross between ttg2-3 and plc1-1D mutants; ttg2-3; plc1-1D double mutant. Scale bars: d = 500 µm.
To further characterize the genetic nature of the plc1-1D line, we crossed the ttg2-3 T-DNA insertion mutant in the Col-0 accession (Salk 148838C) to the plc1-1D line. The ttg2-3 loss-of-function mutant has been previously described13. The trichome phenotype of ttg2-3 mutant plants was noted as showing few and aberrant trichomes. Seeds of ttg2-3 plants also lack PA pigment like ttg2-1 seed13. We note that ttg2-3 trichomes showed a range of mutant phenotypes including swollen, glassy, abnormally branched, and generally distorted trichomes (Fig. 7d).
The F1 of a cross between ttg2-3 and the plc1-1D lines showed normal looking, branched, terminally differentiated trichomes (Fig. 7d). The F1 was allowed to self and the F2 seedlings showed two qualitatively distinct trichome phenotypes: (1) the aberrant ttg2-3 parental trichome phenotype and (2) normal looking, differentiated, branched trichomes. Out of 263 F2 seedlings scored, 17 showed the ttg2-3 trichome phenotype and 246 seedlings showed the developed, terminally differentiated trichome phenotype. This phenotypic ratio is 1:14.5, close to the 1:15 F2 phenotypic ratio that would be expected if the plc1-1D allele is dominant and rescues the ttg2-3 trichome phenotype. A chi-square test of the F2 data results in a critical value of 0.02, indicating that the small deviation from the predicted 1:15 phenotypic ratio is about 90% probable on the basis of chance. Because we expect 4/16 of the F2 population to be of the genotype ttg2-3/ttg2-3 but only observe the ttg2-3 trichome phenotype at a frequency of about 1/15, this suggests that one copy of the plc1-1D allele is sufficient to rescue the trichome phenotype of ttg2-3 homozygous mutants.
To further demonstrate the rescue of ttg2-3 phenotypes by the plc1-1D allele, we isolated plants with the genotype ttg2-3/ttg2-3; plc1-1D/plc1-1D (Fig. 7c and d). These plants show wild-type looking trichomes despite being homozygous for the ttg2-3 mutant allele. Overall, these results are consistent with a gain-of-function mutation in the plc1-1D line that rescues the tt2-3 mutant. This result is also consistent with the observation that AtPLC1 overexpression rescues the ttg2-1 mutant.
Discussion
In Arabidopsis, multiple epidermal traits including trichomes, flavonoid pigment production in the shoot and inner seed coat layer, mucilage production in the outer seed coat layer, and root epidermal cell patterning, are co-regulated by MBW transcription factor complexes1,2,3. This regulatory circuit includes two key downstream transcription factor targets, TTG2 and GL2. The regulation of this suite of epidermal traits radiates from these two control nodes, with TTG2 and GL2 controlling an overlapping set of the TTG1-dependent epidermal pathway.
The discovery of AtPLDζ1 as a GL2 target over 20 years ago represents a fascinating but lone example of phospholipid signaling as part of the MBW-GL2 regulatory circuit controlling the development of the root epidermis in Arabidopsis23. AtPLDζ1 presumably cleaves phosphatidylcholine into choline and a phosphatidic acid. Phosphatidic acid has emerged as a key signaling lipid in plants, regulating an array of cellular processes including vesicle trafficking and actin cytoskeleton organization28. In the context of root epidermal cell fate, it might be that vesicle trafficking and/or translocation of proteins to the growing root hair tip necessary for cell morphogenesis is regulated by AtPLDζ123.
In this study, we introduce a link between the epidermal traits co-regulated by the MBW-TTG2 regulatory circuit and phosphoinositide lipid signaling, specifically involving a phospholipase C gene, AtPLC1. Implicating PLC signaling in the TTG2-dependent suit of epidermal pathways should provide fertile ground for future research into plant phosphoinositide regulation of molecular, cellular and developmental processes.
AtPLC1 overexpression effects MBW-TTG2 epidermal traits but with some discrepant results
Transgenic plants overexpressing AtPLC1 show changes in trichome phenotype in both the Ler wild-type and ttg2-1 mutant lines. However, Ler transgenic lines show reduced trichome development while ttg2-1 transgenic lines show substantial degrees of suppression of the mutant trichome phenotype. In addition, AtPLC1 overexpression rescues ttg2-1 mutant seed coat mucilage production and seed coat color, including the restoration of PA pathway structural gene expression in ttg2-1 developing siliques. However, no obvious reduction in seed coat development was detected in Ler plants overexpressing AtPLC1 (data not shown).
These initial results beg the questions: does AtPLC1 promote or inhibit TTG2-dependent epidermal pathways? Why the contradictory trichome phenotypes in wild-type plants and ttg2-1 mutants overexpressing AtPLC1? To begin with the latter question, it is possible the opposing trichome phenotype results are due to a difference in the genetic backgrounds of the wild-type and the mutant lines, and their interplay with regulatory circuitry having both positive and negative control mechanisms. The ttg2-1 mutant might represent a sensitized genetic background in which the promoting effects of AtPLC1 are revealed, while the wild-type genetic background is primed to reveal inhibition of the trichome pathway. In fact, such a phenomenon has been observed, in which the R2R3 MYB GL1 activates both trichome initiation and inhibition so that trichomes normally differentiate in a pattern as isolated cells rather than in clusters. Loss-of-function gl1 mutants are bald due to a lack of initiation of the trichome cell fate29. However, when GL1 is overexpressed in wild-type plants, trichome initiation is largely suppressed, due to over-activation of R3 MYB repressors that interfere with functional MBW complexes30,31. When GL1 is overexpressed in mutant plants lacking the TRY R3 MYB repressor of trichome initiation, only then is an overproduction of trichome cells observed32. Indeed, similar to GL1, TTG2 promotes trichome initiation and development, while also directly regulating the trichome inhibitor gene, TRY17. Thus, the downregulation of TRY is a potentially consequential genetic difference between Ler and ttg2-1 plants overexpressing AtPLC1. It will be interesting to see in future studies the effects of overexpressing AtPLC1 in other trichome genetic backgrounds such as R3 MYB repressor mutants as well as initiation mutants.
Regardless, this study shows that AtPLC1 can influence multiple MBW-TTG2 dependent epidermal traits; AtPLC1 overexpression not only affects trichomes, but also rescues seed coat development in ttg2-1 mutants. Moreover, expression of the TTG2 PA pigment pathway gene targets, TT12 and AHA10/TT13, is restored in ttg2-1 plants overexpressing AtPLC1. Taken together, these data suggest that AtPLC1 positively mediates TTG2 control of this co-regulated group of epidermal traits.
A T-DNA insertion line sheds more light on the biological role of AtPLC1 in TTG2-dependent epidermal pathways
A search for an AtPLC1 knockout line among the Salk T-DNA insertion collection ironically yielded a dominant mutant allele that resulted in enhanced trichome phenotypes. Nonetheless, this insertion mutant, plc1-1D (Salk 025769C), provided more insights to the role of this lipid signaling gene in epidermal development. When crossed to the Col wild-type accession, all the F1 progeny showed a phenotype intermediate between both parents, representing an enhancement of trichome development compared to the Col parent (Table 1). In addition, the plc1-1D allele appears to rescue the trichome phenotype of the ttg2-3 mutant, given the 15:1 wild-type to mutant phenotypic ratio among the F2 progeny of a cross between ttg2-3 and plc1-1D lines. Also, plants homozygous for both ttg2-3 and AtPLC1 insertion alleles show normal-looking trichomes (Fig. 7). This result is reminiscent of AtPLC1 ectopic expression rescuing the ttg2-1 mutant and again suggests that plc1-1D allele is a gain-of-function.
Lastly, these crossing data help resolve the observation that AtPLC1 overexpression in wild-type plants reduces trichome development but rescues in ttg2 mutant plants. The dominant plc1-1D allele acts as a positive regulator of trichome development in both wild-type and ttg2 plants. Overall, the varied genetic data using the dominant plc1-1D line in wild-type backcrosses and dihybrid crosses with ttg2-3, the 35S:AtPLC1 data in ttg2-1, and the AtPLC1pro:GUS data in Ler and ttg2-1 are consistent with the hypothesis that AtPLC1 genetically interacts with TTG2 to positively regulate multiple epidermal developmental pathways. As previously discussed, the MBW-TTG2 regulatory circuit both promotes and inhibits epidermal development17,19. Thus, the trichome pathway is sensitive to perturbations that disrupt the stochastic balance of co-regulated activating and inhibiting elements of the pathway. It is therefore possible that overexpression of AtPLC1 via the 35S promoter represents a strong perturbation of the system towards inhibition, while the T-DNA insertion allele represents a more subtle, localized boost in function, and thus a smaller perturbation towards promoting the trichome pathway, leading to different outcomes. Similar to previous studies of GL1, these various observations using the 35S:AtPLC1 construct, and the plc1-1D allele in wild-type plants and sensitized ttg2 mutant lines, might again be revealing a dual capability for genes that regulate MBW-controlled epidermal pathways. Future work will focus on examining more closely the possible positive and negative regulatory roles of AtPLC1 in TTG2-dependent epidermal pathways.
The phosphoinositide signaling pathway regulates various cellular and biological functions in plants consistent with putative roles in the MBW-TTG2 controlled epidermal traits
Phosphoinositides (PIs) are a class of negatively charged glycerophospholipids found in eukaryotic membranes. PIs are derived from the phosphorylation of phosphatidylinositol (PtdIns), a family of lipids characterized by the attached myo-inositol head group. The myo-inositol head group can be phosphorylated at the 3, 4 and 5 positions of the inositol ring to yield phospotidylinositol-monophosphates (PIP) and bisphosphates (PIP2). Relative to other membrane lipids, PIs are present in very small quantities that are in a dynamic state of flux. This constant turnover is due to the action of kinases, phosphatases and lipases comprising the PI pathway (for excellent reviews on the PI pathway and its roles in plant biology, see references33,34,35,36,37,38).
PIs and their signaling derivatives exert profound effects. They regulate multiple facets of cell biology such as actin cytoskeleton organization, cell wall synthesis, vacuole morphology and function, vesicle trafficking, and nuclear functions33,36,37,38. Accordingly, perturbations to the levels of PIs and their signaling products via manipulation of PI pathway genes can result in severe phenotypes. This phenotypic approach has yielded much insight to the biological roles of this important lipid-signaling pathway38.
Indeed, previous genetic studies of the PI pathway held clues indicating possible roles for lipid metabolism genes in at least some of the epidermal traits regulated by TTG2. For example, the fragile fiber7 (fra7) mutant gene was found to be allelic to AtSAC1, encoding a PI phosphatase exhibiting in vitro activity towards PI(3,5)P239. Fiber and pith cells of the fra7 mutant show aberrant cell morphogenesis, cell wall synthesis and actin organization. Interestingly, trichomes of the fra7 mutant when imaged by scanning electron microscopy appeared stunted and lacked surface papillae, indicating a lack of secondary cell wall thickening, a characteristic feature of terminal trichome differentiation.
Additionally, AtIPK1 encodes an inositol pentakisphosphate (IP5) kinase that produces inositol hexakisphosphate (IP6). Both IP5 and IP6 are important signaling molecules ultimately derived by phosphorylation of the inositol head group cleaved from the phosphoinositides PI4P and PI(4,5)P2 by PLCs. Plants harboring the atipk1-1 mutant allele show reduced growth and a long root hair phenotype associated with defects in phosphate sensing and signaling40. AtIPK1 promoter:GUS analysis showed expression in several cell types and tissues, including trichomes and developing seeds, possibly suggesting a function for this gene in some of the same cells and tissues co-regulated by TTG2.
Interestingly, IP5 is a cofactor for the F-box protein Coronatine Insensitive 1 (COI1), which mediates the jasmonic acid (JA) wounding response. The atipk1 mutant also shows increased sensitivity to JA (presumably due to the increase in IP5), and thus an enhanced wounding response and a more robust defense against insect herbivory36,41. Part of the herbivory-induced wound response mediated by JA signaling is activation of the trichome and anthocyanin MBW transcriptional complexes, subsequently resulting in increased trichome initiation and anthocyanin production42,43. This represents another possible connection between PI signaling and the epidermal pathways co-regulated by the MBW-TTG2 transcriptional network via a positive feedback loop reinforcing the wound response: JA mediated activation of the MBW complex would in turn activate TTG2 gene expression, possibly resulting in increased PLC signaling, yielding the IP5 cofactor of COI1.
The phosphoinositide bisphosphate PI(4,5)P2 and the enzymes such as PLCs that metabolize this PI are known to have roles in several biological processes. These range from pollen tube and root hair growth, hormone signaling, stomatal opening/closure, developmental phase shifts, and biotic and abiotic stress response34,35. Also, PLC signaling has been shown to regulate female gametogenesis and embryo development, and plant immunity in Arabidopsis44,45. Lastly, it’s been shown that plc5 plc7 double mutants are deficient for seed coat mucilage production22, a trait regulated by TTG2. Here, we show that genetic perturbations targeting the activity of the AtPLC1 gene affect the TTG2 co-regulated suite of epidermal traits, revealing new biological roles for PLCs and the PI pathway in plant development. However, the exact mechanisms by which PLC signaling influences these epidermal pathways remain unclear, thus providing opportunities for future work.
Possible modes of PLC-mediated PI(4,5)P2 signaling in the regulation of plant cellular functions
The canonical model for PLC signaling to emerge from animal studies involves extracellular receptor-activated PLCs cleaving PI(4,5)P2 into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG)46 (Fig. 8). IP3 is free to diffuse through the cytosol where it activates ligand-gated channels, releasing stored Ca2+ into the cytosol. This Ca2+ concentration increase in the cytosol results in signal transduction effecting many downstream components of the signaling cascade. Similarly, DAG, the lipid cleavage product, diffuses through the membrane to activate protein kinase C (PKC), with subsequent phosphorylation of downstream signal transducers prompting a variety of cellular responses.
Model for the integration of PLC-mediated phosphoinositide lipid signaling in the regulation of the TTG2-dependent epidermal cell fate pathways. Green boxes indicate the TTG2 transcription factor and the co-regulated epidermal traits. In between is the PLC lipid signaling pathway with blue boxes indicating the cellular functions influenced by the various branches and signaling metabolites of the pathway. The regulation of such cellular functions are likely important for the development of trichomes and cells of the seed coat. PLC lipid signaling is therefore a possible mechanism for the regulation of cellular functions by which TTG2 controls epidermal development. The genetic interactions between TTG2 and AtPLC1 observed in this study suggests such a mechanism. This model provides a framework for further exploring the possible roles phosphoinositide lipid signaling in the TTG2-dependent epidermal cell fate pathways.
Although early studies in plants showed that IP3 could release intracellular stores of Ca2+ into the cytosol, resulting in stomatal closure, no obvious homolog for the IP3-gated Ca2+ channel has been identified in plant genomes to date. Likewise, identification of plant genes homologous to animal PKC genes have not been forthcoming33,35,47. Instead, phosphorylated versions of both IP3 and DAG second messengers appear to be the key downstream elicitors of the cellular responses to plant PLC activity (Fig. 8). For example, IP6 can trigger Ca2+ release from intracellular stores, and IP5 and IP6 bind to auxin and jasmonate hormone receptors, respectively, ultimately resulting in the regulation of gene expression. Likewise, the PLC lipid cleavage product DAG can be phosphorylated to form phosphatidic acid, which has emerged as an important second messenger in plants28.
Alternatively, PLCs may be operating to effect signaling by a different mechanism. PI(4,5)P2 may not merely be a precursor of second messengers, but can itself as an intact phospholipid be a mediator of cellular functions. The negatively charged head group of PI(4,5)P2 can serve as a ligand for a diverse array of proteins containing one of a number of lipid binding domains36. For example, actin-binding proteins can bind to PI(4,5)P2, thus regulating actin cytoskeleton organization.
Also, organization of PI(4,5)P2 pools to specific locations on the plasma membrane, or microdomains, is a key mechanism by which PI(4,5)P2 exerts its cellular functions33,38. For example, in growing root hairs and pollen tubes, PI(4,5)P2 is localized to the apical end of the cell, establishing the necessary polarity that directs the secretory pathway to the growing end48,49,50,51. Accordingly, the enzymes that synthesize and/or degrade PI(4,5)P2 may be localized to the sub-apical region as required to maintain the appropriate lipid microdomain for normal root hair or pollen tube growth. Disruption of this apical phospholipid microdomain by an increase or decrease of the enzymes that metabolize PI(4,5)P2 results in growth defects and abnormal morphologies of root hairs and pollen tubes.
According to the observations presented here, the differentiation of trichomes and seed coat cells also seemingly depend on PI(4,5)P2. Either PI(4,5)P2 is serving as a precursor for downstream second messengers produced by PLC activity, or as an intact phospholipid whose turnover rate or membrane localization influences the cellular processes necessary for trichome and seed coat cell development, or both (Fig. 8). These observations contribute to previous studies establishing the importance of PI(4,5)P2 metabolism for normal plant cell growth. The model systems co-regulated by the MBW-TTG2 regulatory circuit provide a new venue to genetically dissect the PI signaling pathway. Identifying other PI pathway genes expressed in these epidermal cell types could expand the list of candidates with roles in these cell fate pathways. In addition, regulating PI(4,5)P2 levels via targeted expression of PI pathway genes in these model systems could yield instructive phenotypes, providing insights to the modes of PI(4,5)P2 action and expanding upon the biological roles of the PI signaling pathway33,38.
PLC-mediated PI pathway lipid signaling can regulate gene expression in the proanthocyanidin pathway
It was interesting to discover that overexpression of AtPLC1 restored TT12 and AHA10/TT13 gene expression in the ttg2-1 mutant (Fig. 5d and e). To our knowledge, there has not been a direct link between phospholipid signaling and proanthocyanidin pigment production in plants. The control of AtPLDζ1 by GL2 may suggest an indirect link between anthocyanin biosynthesis and phospholipid signaling. GL2 has been shown to negatively regulate anthocyanin biosynthesis in Arabidopsis seedlings and stems52. Given that GL2-mediated root hair patterning requires the AtPLDζ1 gene23, it is possible that phospholipid signaling also has a role in GL2 regulation of the anthocyanin pathway (although this has not been empirically demonstrated).
Thus, the observation that AtPLC1 restored pigment gene expression in the ttg2-1 mutant highlights that the regulation of gene expression is at least part of the molecular mechanisms by which AtPLC1 might be influencing TTG2-dependent epidermal traits. Phosphoinositide and inositol polyphosphate messengers in plants and animals are known to influence nuclear functions including transcription and mRNA export40,46,53,54. In the context of this nuclear pathway, these lipid-derived messengers and the enzymes catalyzing their synthesis and degradation are active in the nucleus, where they influence chromatin modification.
For example, the Arabidopsis trithorax factor (ATX1), a histone H3 trimethylase, modifies the nucleosomes of the WRKY70 gene, positively influencing its transcription. However, upon dehydration stress, PI5P levels increase due to the activity of the PI(3,5)P2 phosphatase AtMTM1. PI5P then binds to ATX1, altering its localization from the nucleus to the cytoplasm. This results in decreased methylation of the nucleosomes of WRKY70 and downregulation of its expression53.
In another example, mutations in the AtIPK1 gene leads to a decrease of IP6 levels, which correlate to altered histone composition in chromatin and ultimately increases the expression of phosphate starvation response genes40. In yeast and animal systems, PI(4,5)P2 is present within the nucleus, along with a number of enzymes involved in PI(4,5)P2 signaling, including PLCs54,55. From within the nucleus, PI(4,5)P2 signaling can influence a broad range of phenomena including chromatin structure, transcription, telomere function and mRNA processing and export. In the context of AtPLC1 and PI lipid regulation of the PA pigment pathway, it will be interesting to further investigate the nuclear functions, such as chromatin remodeling/histone modifications, that might be influencing the transcription of TT12 and AHA10/TT13.
Future work: a genetics approach targeting the misexpression of other PI enzymes could clarify modes of phosphoinositide signaling action in the regulation of the MBW-TTG2 system of epidermal pathways
A phenotypic approach upon perturbation of the PI signaling pathway has proven fruitful for identifying the relevance of phospholipids as regulators of a wide range of plant biology38. In this study, such an approach has revealed that phospholipid signaling, particularly mediated by PLC activity, can influence multiple TTG2-dependent epidermal developmental pathways. Future genetic studies will focus on identifying the possible roles for other PI pathway genes in this system of co-regulated epidermal traits, particularly (but not limited to) those genes involved in PI(4,5)P2 metabolism (Fig. 8). These studies should also have the potential to discriminate between signaling modes of action. For example, if increased AtPLC1 signaling in 35S:AtPLC1-ttg2-1 transgenic lines is occurring via the generation of inositol polyphosphates, then co-expressing phosphatases targeting these second messengers might counteract PLC signaling, resulting in a reversal of mutant phenotype rescue. Such an approach overexpressing a type I inositol polyphosphate 5-phosphatase (5PTase) encoded by the AtIP5PII gene, and targeting IP3, reversed AtPLC1-mediated seed dormancy and plant growth inhibition in the abscisic acid signaling pathway56,57.
Alternatively, if ttg2-1 mutant phenotype rescue due to AtPLC1 overexpression is a function of decreased PI(4,5)P2 levels (and not the generation of second messengers), then expressing phosphatases thought to target PI(4,5)P2, such as Suppressor of Actin 9 (SAC9) or SAC750, might similarly result in ttg2-1 mutant phenotype rescue. Similarly, a type II 5PTase encoded by Fragile Fiber 3 influences actin organization and cell wall thickening in fiber and xylem cells by targeting PI(4,5)P258, providing another means to genetically manipulate PLC signaling by reducing PI(4,5)P2 levels in the context of TTG2-dependent epidermal pathways.
Also, several PI4P 5-kinases that synthesize PI(4,5)P2 from PI4P are known to effect cellular functions such as vacuole morphology, directional vesicle trafficking, membrane recycling, localized pectin deposition, and ultimately regulate cell growth polarity and morphology of pollen tubes and root hairs48,51,59,60,61,62. If expressing these kinases in 35S:AtPLC1-ttg2-1 plants reverses the phenotype rescue, then this similarly would validate the hypothesis that levels of intact PI(4,5)P2 are important for regulating cell fate determination in the TTG2-dependent epidermal pathways. Beyond PI(4,5)P2 and PLC activity, it will be interesting to investigate other PI pathway enzymes and phospholipids known to regulate cellular functions important for the differentiation of trichomes and testa cells (such as actin organization, vesicle trafficking, cell wall synthesis and vacuole biology regulated by a variety of phosphoinositides and their metabolic enzymes).
Materials and methods
Arabidopsis accessions and transgenic lines
The mutant line ttg2-1 is in the Landsberg erecta (Ler) ecotype and has been previously described4,5,8. The plc1-1D (Salk 025769C) and ttg2-3 (Salk 148,838) insertion mutant lines in Col-0 background were obtained from the Arabidopsis Biological Resource Center27. Four 35S:AtPLC1 lines were generated in the ttg2-1 mutant background and two in the Ler background, and these transgenics were phenotypically characterized. Data documented in this study are representative of common trends observed in the transgenic lines. All plants were grown in soil at approximately 21 °C in continuous white light.
Microscopy
Scanning electron microscopy was performed as previously described24,63.
Plasmid construction
pAtPLC1pro:GUS- an approximately 2 kb fragment upstream of the AtPLC1 start codon was amplified from Col-0 genomic DNA using the primers below and recombined into pDONR222 (Invitrogen) to produce pEAtPLC1pro. pEAtPLC1pro was then used to recombine the AtPLC1 regulatory fragment into pBGWFS7 GUS vector64. Gateway recombination sequences were included on all appropriate primers but are not shown.
AtPLC1profwd: 5′attB1- CAGGAGCGATTCCTTTACTAG-3′.
AtPLC1prorev: 5′attB2- CTTGTGAAAGTTAAGCGAG-3′.
p35S:AtPLC1- the AtPLC1 Col-0 genomic locus from start to stop codons was amplified using the primers below and recombined into pDONR222 (Invitrogen) to produce pEAtPLC1. AtPLC1 was then recombined from pEAtPLC1 into pB7WG264.
AtPLC1fwd: 5′attB1- ATGAAAGAATCATTCAAAGTG-3′.
AtPLC1rev: 5′attB2- CTAACGAGGCTCCAAGACAAA-3′.
TT12 and AHA10 gene expression analysis by semi-quantitative RT-PCR
Total RNA was prepared from young developing siliques, about 2–5 daf or during the globular stage, using a Qiagen RNeasy plant mini kit. 2 μg of total RNA was used to produce first-strand cDNA in 20 μl reverse transcription reactions using a Super-Script III RT kit (Invitrogen). 50 μl PCR reactions were prepared using 1 μl cDNA reaction as template and run for 25 cycles. 5ul of completed PCR reactions were checked on 1% agarose gels. Target primers were used at 200 nM final concentration and 200 nM actin primers were used in separate control reactions. Two biological replicates were performed for each experiment. The following primers were used for target and control gene amplification:
AHA10 fwd: 5′-TGCCAACAACAGTGAACAAGTG-3′.
AHA10 rev: 5′-TTAGACAGTATGAGCTGCACGG-3′.
TT12 fwd: 5′-GGGATATGCAGTTCATGCTTGG-3′.
TT12 rev: 5′-TTAAACACCTGCGTTAGCCATC-3′.
ACT2 fwd: 5′-AGAAGTCTTGTTCCAGCCCTC-3′.
ACT2 rev: 5′-TTAGAAACATTTTCTGTGAACG-3′.
AtPLC1 Gene expression analysis by semi-quantitative RT-PCR
Total RNA was prepared from young seedlings, with expanded 1st leaves and emerging 3rd and 4th leaves, using a Qiagen RNeasy plant mini kit. 3 μg of total RNA was used to produce first-strand cDNA in 20 μl reverse transcription reactions using a Super-Script III RT kit (Invitrogen). For AtPLC1 fragment amplification, 50 μl PCR reactions were prepared using 5 μl cDNA reaction as template and run for 30 cycles. For the actin control, 50 μl PCR reactions were prepared using 0.5 μl cDNA reaction as template and run for 30 cycles. 5ul of completed PCR reactions were checked on 1% agarose gels. Target primers were used at 200 nM final concentration and 200 nM actin primers were used in separate control reactions. Four biological replicates were performed for each experiment. The following primers were used for target and control gene amplification:
PLC1 det-fwd2: 5′-GAAGCTGAAGTTCGTCATGG-3′.
PLC1 det-rev2: 5′-CATAGCCACATCCACCATTG-3′.
ACT2 fwd: 5′-AGAAGTCTTGTTCCAGCCCTC-3′.
ACT2 rev: 5′-TTAGAAACATTTTCTGTGAACG-3′.
T-DNA allele verification
Genomic DNA was prepared from 100 mg of plant tissue using the Qiagen DNeasy plant kit. We used PCR primer sequences provided by SIGnAL primer design tool for genotyping TTG2 and AtPLC1 loci towards the identification of a double T-DNA mutant line. Primer sequences are listed below:
LP-TTG2: 5′-TAAAACCAAACGACACCGTTC-3′
RP-TTG2: 5′-TCCAAGTTTGTTGACGATTCC-3′
LP-PLC1: 5′-AAACGCGTTCTCCTTAACCAG-3′
RP-PLC1: 5′-TACTTTGGGTCAACGGTTCTG-3′
LBb1.3: 5′-AAACGCGTTCTCCTTAACCAC-3′.
Research involving plants
Commonly used and publicly available strains of the laboratory model plant, Arabidopsis thaliana, were used in this study. No wild plants or endangered species of plants were used in this study. No field experiments were conducted. Therefore, this study is compliant with relevant institutional, national, and international guidelines and legislation on plant research.
Data availability
Most of the Arabidopsis strains used in this study, including the ecotypes Col and Ler and the insertion mutants ttg2-3 and plc1-1D, are available upon request at the Arabidopsis Biological Resource Center. The transgenic reporter lines and overexpression lines described in this study are avaible free upon request from the Plant Pathways Freshman Research Initiative lab by contacting Dr. Tony Gonzalez. E. coli strains carrying promoter:reporter and overexpression constructs are also available upon request from the Plant Pathways lab.
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Acknowledgements
This work was supported by the US National Science Foundation grant 0716049 and the Howard Hughes Medical Institute grant 52006985. 52008124. We would also like to acknowledge the Freshman Research Initiative for providing broad support to our undergraduate research group.
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A. G. and A. L. contributed to the conception and design of this work. A. G. drafted the manuscript and A. L. provided critical feedback and edits towards the completion of the final draft. A. G. and N. R. performed the semi-quantitative RT-PCR experiments and A. G. drafted the corresponding figures. P. O. performed the scanning electron microscopy. C. G. contributed to the creation of plasmid constructs and transgenic plant lines. M. O. contributed to data acquisition presented in Table 1. L. R. contributed to the creation of plasmid constructs and to transgenic plant data acquisition. M. N. and F. P. H. contributed to the isolation of the ttg2-3; plc1-1D double mutant, conducted the genotyping experiments and drafted the corresponding figure.
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Goldberg, A., O’Connor, P., Gonzalez, C. et al. Genetic interaction between TTG2 and AtPLC1 reveals a role for phosphoinositide signaling in a co-regulated suite of Arabidopsis epidermal pathways. Sci Rep 14, 9752 (2024). https://doi.org/10.1038/s41598-024-60530-8
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DOI: https://doi.org/10.1038/s41598-024-60530-8
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