472 research outputs found
Molecular Genetic Analysis of Trichome Development in Arabidopsis thaliana
No full textSeveral mutants with aberrant trichome patterning and differentiation are known in Arabidopsis. The genes involved in these processes code for proteins with diverse functions such as transcription, protein degradation, microtubule arrangement, cell wall remodeling etc. Even though the role of these genes in trichome development have been studied by genetic and biochemical methods, the factors that in turn regulate them at the transcriptional, translational and post-translational levels have been less studied. Here, using mutational analysis, we have studied two such factors that control the trichome development genes and dissected the molecular mechanisms adopted by them.
In the first part of the work, we have addressed the role of class II TCP genes in general, and TCP4 in specific, in trichome differentiation, and in the second part, we have addressed the role of TNI/UBP14 in controlling the proteins involved in trichome development.
TCPs negatively regulate trichome development
The TCP genes encode DNA-binding transcription factors and regulate multiple aspects of plant development including leaf shape and size. The Arabidopsis genome encodes 24 TCP genes, classified into two major groups; class I and class II, based on their sequence similarity. Among
the eleven class II members, five (TCP2, 3, 4, 10, 24) are post-transcriptionally regulated by miR319. Role of miR319-targeted TCP genes are well established in leaf morphogenesis at the cellular level as a negative regulators of cell proliferation and as a positive regulators of cellular
differentiation. It has been shown that in TCP loss-of-function, there is prolonged cell division and in their gain-of-function, there is a precocious differentiation at both organ and cellular levels. However, these studies focused mainly on the role of the TCP genes in the pavement cell
differentiation without much attention to other epidermal cells with specialized structure and function, namely trichomes and stomata.
Detailed phenotypic analysis of the class II TCP loss-of-function as well as and gain-of-function lines suggested that these TCP genes negatively regulate both trichome initiation and trichome differentiation. To learn more on the molecular mechanism of TCP4-mediated trichome
development, we analyzed the results of the DNA microarray analysis where the transcriptome of the 9-day old seedlings of jaw-D;pTCP4:mTCP4:GR genotype was compared before and after TCP4 induction and identified four genes involved in trichome development, that was up regulated by TCP4. These genes are GLABROUS INFLORESCENCE STEMS (GIS), TRICHOMELESS1 & 2 (TCL1, 2) and ZINC FINGER PROTEIN8 (ZFP8) that have been previously shown to regulate both trichome initiation and differentiation. Of the four genes, GIS is reported to regulate the trichome branching. Analysis of GIS transcript levels in the TCP loss of- function mutants like jaw-D and tcp2;4;10 showed a down-regulation while it was upregulated in the gain-of-function lines TCP4:VP16, pBLS:rTCP4:GFP and upon TCP4 induction in the pTCP4:mTCP4:GR line. GIS transcript was also up-regulated when TCP4 was induced in the absence of additional protein synthesis, suggesting that the TCP4 protein is directly responsible for GIS activation. Further, TCP4 is capable of binding to its cognate sites present on the GIS locus. While GIS is massively activated in the TCP4:VP16 line leading to reduced trichome branching, The TCP4:VP16 protein failed to suppress trichome branching in the
absence of GIS as seen in the gis;TCP4:VP16 line. Taken together, these results provide evidence that GIS is required for the TCP4-mediated inhibition of trichome differentiation. Previous analysis of several trichome mutants showed that there is a strong correlation between
the endoreduplication status of a trichome and its extent of branching. However, there are several other genes including STI, BLT, NOK and GIS that regulate trichome branching independent of endoreduplication. However TCPs, regulate GIS transcription, independent of endoreduplication
pathway.
In addition to branching, analysis of trichome development in the TCP loss and gain-of-function mutants has shown that TCPs negatively regulate trichome initiation as well. From the analysis of the microarray results mentioned above, all the four identified genes - TCL1, 2, ZFP8, GIS -
are reported to be involved in regulating trichome density. Among these, TCL1, and 2 suppress trichome initiation whereas ZFP8 & GIS promote it. It is possible that TCPs are involved in maintaining the homeostatic regulation of trichome density by activating both positive and
negative regulators of trichome initiation. Gene expression analysis has shown that TCP4 directly up-regulate the expression of all these genes (Results in Chapter IV; Krishna Reddy Challa, PhD thesis, 2014). While TCP4:VP16 suppressed trichome density in Col-0, tcl1 mutation did not show increased trichome density, possibly because of a functional redundancy between TCL1 and TCL2. However, the tcl1;TCP4:VP16 plants failed to reduce trichome density, suggesting that TCP4 required TCL1 for inhibition of trichome initiation and TCL2 is not capable of compensating for the loss of TCL1. Analysis of the tcp2;4;10;zfp8 mutant showed that ZFP8 acts downstream to the TCP genes. Taken together, we conclude that TCPs act upstream to GIS, ZFP8, TCL1/2 to maintain a balanced distribution and differentiation of trichome cells.
TARANI (TNI) negatively regulates the trichome differentiation
Analysis of trichome development in the tni mutant showed that the TNI protein negatively regulates trichome differentiation alone, without affecting trichome density. TARANI encodes the deubiquitinase enzyme UBP14 in Arabidopsis (Premananda, K., PhD Thesis, 2014). Because of
the G→A point mutation at the junction of third intron and fourth exon, two different transcripts are formed in the tni mutant; the wild type TNI transcript and an aberrant TNIintron transcript where the 3rd intron is incorporated in frame. This result in a two-fold down-regulation of the
TNI transcript in homozygous tni plants compared to Col-0. To determine whether the tni trichome phenotype is caused by this decrease in TNI level or by the presence of the aberrant transcript TNIintron, we generated a transgenic line where TNI, TNIintron and an artificial micro
RNA that targets TNI specifically in the trichome cells under the control of GL2 promoter.
Analysis of the trichome-branching phenotype in these transgenic lines revealed that, interestingly, both under and over-expression of TNI leads to increased trichome branching, emphasizing the requirement for a balanced amount of TNI protein in the cell for proper trichome development.
Genetic analysis of tni with known trichome branching mutants has shown that BLT is epistatic to TNI. Based on the genetic and molecular data, we hypothesize that in tni mutant, there is an increased amount of BLT protein that leads to increased trichome branching. To test this, we
have raised a 35S::HA-BLT transgenic line, which shows hyper branched trichome phenotype. Analysis of the tni;35S::HA-BLT phenotype, and comparison of BLT protein level between Col-0 and tni using anti-HA antibody, would test our hypothesis that BLT is degraded by TNI to
suppress trichome branching. We are currently in the process of generating the tni;35S::HA-BLT line and hope to obtain the data by the time of thesis defense examination.
Thus, we have added one more transcription factor, TCP4 to the existing trichome developmental pathway (Fig. 6). We have investigated the detailed molecular mechanism for TCP-mediated regulation of trichome differentiation and have laid the foundation for further studies on role of TCP genes in trichome initiation. In the mutants of TCP genes, it is known that the pavement cell differentiation is reduced and the studies reported here have shown that the loss of TCP function has an opposite effect on trichome differentiation; the branching is increased in the TCP mutants. It is possible that the TCP proteins collaborate with different partners in these two cell types to bring about opposite effect on differentiation.
From the second part of this study, we have added a protein degradation factor, TNI/UBP14, to the existing trichome differentiation pathway (Fig. 6). We have shown that, the balanced amount of TNI protein is required for normal trichome development. Unpublished data from our laboratory has shown that TNI is involved in conversion of free polyubiquitin chains into monoubiquitin, which is an essential step in proteasome-mediated degradation of all target proteins (Parinita Majumdar and Utpal Nath). BLT could be one such target protein that is upregulated in the tni mutant leading to hyper-branched trichomes. Increased trichome branching in the 35S::HA-BLT supports this hypothesis. Comparative estimation of the BLT levels between Col-0 and tni plants would enable us to demonstrate this
Role of TCP4 Transcription Factor in the Maturation Program of Arabidopsis Life Cycle
No full textTCP4 as an integrator of key developmental events
A striking aspect of plant life is their sedentary life-style. Though it relieves them of the obligation of forming a complex body organization, it exposes them to environmental challenges. Plants have evolved a flexible pattern of post-embryonic growth. The major phases in their life cycle are photomorphogenesis, vegetative growth with phase transitions, reproductive growth and senescence. The phase transitions are coordinated temporally to ensure proper maturation of organism. Flexibility is built in the re-iterated programs of organogenesis, which provides a plant with an option to adopt an architecture best suited to prevailing environmental conditions. Organogenesis occurs by processes of cell division and maturation (expansion). Cell division determines the growth potential by generating the requisite number of cells and cell maturation fulfils the potential by elaborating the organ form. Organ growth requires spatially- and temporally-controlled cellular maturation.
The TCP class of plant-specific transcription factors, conserved from bryophytes to angiosperms, control diverse developmental and morphological traits, such as plant architecture, floral asymmetry, seed germination, male and female gametophyte development and photomorphogenesis (Martín-Trillo and Cubas, 2010). Class II TCPs, which are targets of miR319, are best known for their role in leaf morphogenesis. They are believed to function by redundantly regulating the onset of cellular maturation ( Efroni et al., 2008; Koyama et al., 2007; Nath et al., 2003; Ori et al., 2007; Palatnik et al., 2003; Schommer et al., 2008). To establish the link between level of TCP activity and organ growth, we undertook the approach of hyper-activating the function of TCP4, a representative class II TCP, by fusing it with a strong transactivation domain.
Enhanced level of TCP4 activity reduced organ growth by causing precocious cellular maturation. It also accelerated the process of organ initiation, maturation and its progression into the final stage of senescence. Hyper-active TCP4-expressing plants underwent faster maturation of shoot apex into reproductive phase. In general, hyper-activation of TCP4 advanced cellular, organ and organism maturation programs in Arabidopsis life cycle (Fig. 1).
Traits such as organ initiation rate, organ size, flowering time and seed yield contribute to the fitness of the plant. Faster rate of organ initiation, bigger organ size, early onset of flowering and higher seed yield are obvious desirable traits. However, they rarely occur simultaneously in a mutant or a natural variant, suggesting that there is a trade-off among different traits. Studies have shown that such traits are linked and are controlled by multiple loci that contribute quantitatively to the phenotype. A change that benefits one trait may adversely affect another (Colautti et al., 2011; Kozlowski, 1992; Mendez-Vigo et al., 2010) . Our study shows that TCP4 activity can potentially coordinate these inter-connected traits. Though hyper-active TCP4-expressing plants have faster rate of organ initiation, the final organ size is reduced and senescence is advanced. These plants reach reproductive phase faster, but produce fewer seeds, hence limiting their propagation and lowering their fitness in comparison to the wild type. Such a genetic constraint on the traits limits the phenotypic variation that can be produced in plants and, hence, their adaptation to the environment. Our study suggests that TCP4 can link organ growth with that of the whole organism. It acts as a heterochronic regulator which possibly affects timing of multiple maturation programs. Any perturbation in the TCP activity may have far-reaching effects on plant growth and thus, optimal level of TCP activity is crucial for plant homeostasis.
One possible explanation for the developmental pleitropy in TCP4 hyper-activation line is an alteration in hormone biosynthesis or sensitivity. A combination of microarray and hormone application studies on hyper-active TCP4-expressing line has indicated a reduction in the levels of GA and auxin and an increase in cytokinin and MeJA levels. There may also be an inhibition of auxin signaling and upregulation of MeJA and ethylene signaling. In addition, TCP4 appeared to regulate both GA biosynthesis and response in opposing manner. The molecular mechanisms involved in TCP4-mediated integration of hormonal pathways are still unclear. Answering these questions would require identification of its direct downstream targets
Biochemical, Genetic and Molecular characterization of TCP3 and TCP4 transcription factors in Arabidopsis thaliana
No full textThe TEOSINTE BRANCHED1, CYCLOIDEA, PROLIFERATING CELL FACTORs (TCP) family of proteins consists of plant-specific, non-canonical basic helix-loop-helix transcription factors that perform diverse developmental processes. Based on sequence similarity within the amino acid residues, TCP proteins are divided into two groups – class I and class II. Arabidopsis genome encodes 24 TCP proteins, 13 belonging to class I and 11 to class II, and a high degree of functional redundancy exists within the members of the same class. Eight class II TCP proteins, known as CINCINNATA-like TCPs (CIN-TCPs) redundantly promote leaf differentiation and maturation. Despite the closest sequence similarity between two CIN-TCPs, TCP3 and TCP4; antagonistic functional differences, primarily with respect to their role in auxin response, has been reported, raising a doubt about whether they have truly redundant function. To test this, we have performed a detailed comparative study of these two proteins using biochemical, genetic, and molecular approaches. We also attempted to identify downstream targets of these proteins by functional genetics approach.
The class I TCP proteins bind to the GGNCCCAC sequence element whereas the class II proteins bind to GTGGNCCC consensus element, indicating that members of both the TCP classes bind to distinct but overlapping DNA sequence. In this study, we carried out detailed DNA-binding analysis of these two CIN-TCP proteins - TCP3 and TCP4 and found difference in their DNA-binding properties; TCP3 binds to both class I and class II DNA elements whereas TCP4 binds only to class II sequence. Further analysis suggested that the N-terminal region of TCP3 is responsible for this dual DNA-binding property. Biochemical analysis suggested a divergence in the binding properties of these two redundant members. To study the functional differences, we used various gain of function lines of TCP3 and TCP4 and showed that activation of TCP3 or TCP4 has similar effects on hypocotyl elongation and leaf maturation; both the proteins promote cell elongation in hypocotyl and suppress cell proliferation in leaf primordia. Detailed analysis also suggests similar activity by both the proteins in their temporal effect on leaf primordia maturation. Further, our results suggest that CIN-TCPs are master regulators of compensation in leaves. The transcriptomic comparisons of the differentially regulated genes by TCP3 and TCP4 showed high degree of redundancy between these two proteins in the regulation of the common downstream target genes. Reporter gene analysis demonstrated that both the proteins promote auxin response in leaf primordia. The genetic and molecular studies between these two redundant proteins show convergence in their function. Through functional genetics approach, we identified putative mutants which showed bigger leaves upon TCP4 induction suggesting the probable targets of TCP4 involved in leaf maturation. Further analyses of these putative mutant lines are required to learn the exact effect of the mutation and the mutated genes in leaf growth.
In summary, here we show a difference in DNA-binding property of TCP3 and TCP4, yet these two proteins act as true functionally redundant pair in regulating leaf morphogenesis and auxin response
Role of VILAMBIT Genes Controlling Flowering Time and Jasmonic Acid Signaling in Arabidopsis
No full textThe transition to flowering is an important decision for plants since seed-setting and the survival of the progeny depend on the environmental conditions prevalent during this transition. Therefore, to ensure maximum reproductive success, plants have evolved several regulatory mechanisms to enable them flower at the most appropriate time. Environmental parameters such as light, temperature and nutrient availability as well as endogenous factors such as age and hormonal status of the plant profoundly affect floral transition (Boss et al., 2004; Srikanth and Schmid, 2011). Studies in Arabidopsis and other model plant species have identified several distinct genetic pathways that integrate the information from the endogenous and environmental cues to regulate flowering (Boss et al., 2004; Srikanth and Schmid, 2011). Many components and gene regulatory networks identified in Arabidopsis are conserved in other commercially important species including rice, maize, sorghum, potato and tomato. Therefore, it is important to understand the basic mechanisms that modulate the flowering response in model plants such as Arabidopsis thaliana, the knowledge from which can be used to develop better adapted and high-yielding varieties of crop plants in the wake of challenges like global warming and increasing food demand.
In the present study, we have studied the function of VLB1 and VLB2, genes that code for plant-specific Zn-finger transcription factors. Previous studies from our laboratory (Pratibha Choudhary, Ph.D thesis, 2011) and by other research groups have reported that VLBs redundantly promote flowering in A. thaliana (Yasui et al., 2012; Celesnik et al., 2013). However, the underlying mechanism of this regulation is not well understood. Our data suggests that VLBs redundantly promote the transition to flowering specifically in the photoperiod pathway, the major floral induction pathway in A. thaliana. CO, which is the 93
key regulatory gene in this pathway, is regulated by various factors at the transcriptional as well as post-transcriptional level (Suarez-Lopez et al., 2001; Yanovsky and Kay, 2002; Srikanth and Schmid, 2011). Using genetics, we show that VLBs and CO function together to promote flowering in the photoperiod pathway. Further, our BiFC results reveal that VLBs and CO interact physically. Nevertheless, the physical interaction between VLBs and CO needs to be further validated by in vitro and in vivo by co-immunoprecipitation experiments. We hypothesize that the interaction between VLBs and CO is important to regulate FT expression and hence, flowering. However, whether VLBs interact with CO and promote the CO-stability, or facilitates its recruitment to the FT promoter region, still needs to be determined.
Apart from its role in flowering, VLBs have been recently shown to regulate biotic and abiotic responses in Arabidopsis (Nakai et al., 2013a; Nakai et al., 2013b). Also, even though it has been demonstrated that VLBs code for transcription factors, no direct targets of VLBs have been reported till date. We performed a whole genome trancriptome-profiling and found that several important classes of genes including WRKY, RLPs, NBS-LRR and JAZs were affected suggesting that, in addition to their role in floral transition, VLBs have important functions in other plant processes as well. In fact, vlb1vlb2 mutant showed an early senescence phenotype and many senescence-associated genes (SAGs) were up-regulated in our microarray experiments, which was further validated by qRT-PCR analysis. By comparing the differentially-regulated genes and PatMatch analysis, we have identified 82 putative direct targets of VLBs in the Arabidopsis genome which need to be validated by chromatin immunoprecipitation (ChIP) assay and functional studies. 94
Results of global transcriptome analysis revealed that the expression of several JA-signaling and response genes was significantly down-regulated. JA is an important phytohormone involved in plant defense and other developmental processes such as stamen development, root growth and senescence (Wasternack, 2007). Results from the JA-induced expression analysis and root inhibition assay confirmed that JA-signaling and response are indeed compromised in the vlb1vlb2 double mutant. Moreover, in vitro DNA-binding assay showed that MYC2, the key transcriptional regulator of JA-responsive gene expression, is a direct transcriptional target of VLB2. A recent study reported that loss-of-function of VLB genes impairs plant defense while their overexpression confers biotic stress tolerance in Arabidopsis (Nakai et al., 2013a; Nakai et al., 2013b). Compromised JA signaling in the vlb1vlb2 double mutant might partly explain this reduced tolerance to pathogens. However, whether VLBs are associated with the MYC2 promoter in planta needs to be tested by performing ChIP and other in vivo assays.
In conclusion, our study shows that VLBs have important regulatory roles in diverse processes including control of flowering time, senescence and JA signaling in Arabidopsis. The validation and functional characterization of the direct targets of VLBs will shed more light on the role of VLBs. Since VLBs are conserved in vascular plants, it will be interesting to see if the function of VLBs is also conserved across species and what might be its ancestral function in evolution
Transcriptional regulation of a microRNA encoding gene MIR319C during leaf development in Arabidopsis thaliana
No full textThe evolutionarily conserved microRNA miR319 and its target transcription factors encoded by five CIN-TCPs (TCP2, 3, 4, 10 & 24) regulate leaf morphogenesis in Arabidopsis by triggering the division to differentiation switch of the leaf cells. In a young leaf, the expression of the miR319 encoding gene MIR319C is restricted at the basal region coinciding with the cell proliferation zone, whereas the CIN-TCP transcripts are detected in the more distal region where differentiation is initiated. How the complementary expression patterns of MIR319C and CIN-TCPs are established in leaf primordia is unknown. Moreover, the factors that activate and maintain MIR319C expression in the leaf primordia are yet to be uncovered.
Here, a detailed spatiotemporal analysis of the predominantly expressed TCP4 and MIR319C genes suggested the possibility of CIN-TCP mediated downregulation of MIR319C promoter activity in the leaf primordia. Loss of multiple CIN-TCPs resulted in the distal extension of the MIR319C expression domain, whereas ectopic TCP4 activity restricted the MIR319C domain more proximally. TCP4 was enriched at the MIR319C promoter, and increased TCP4 activity enhanced the deposition of H3K27me3 repressive marks on the MIR319C. Additionally, transgenic lines carrying mutations in TCP binding sites on MIR319C promoter exhibited miR319 overexpression phenotypes. Together with the previous knowledge that miR319 degrades CIN-TCP transcripts, our study suggests the existence of a double-negative feedback loop involving the miR319-CIN-TCP module in regulating leaf morphogenesis in Arabidopsis.
To uncover the activators of MIR319C in leaf primordia, we screened a leaf-specific Arabidopsis transcription factor (TF) library using a yeast one-hybrid assay to isolate proteins that bind to the 2.7 kb promoter of MIR319C. The screen yielded 57 positives including the six NAM/ATAF1/ATAF2/CUC (NAC) domain-containing TFs with DNA-binding preferences similar to that of the CUC sub-group of NAC TFs, i.e., CUC1, 2 & 3. In addition to the ability of the CUC proteins to bind to the MIR319C promoter region in yeast, the expression domain of CUC2 overlaps with that of MIR319C in early leaf primordia, suggesting a role for CUCs in the activation of MIR319C during leaf development. Loss of CUC2 activity significantly reduced the MIR319C expression domain, whereas increased CUC2 level led to a distal expansion of MIR319C expression. Elevated CUC2 level partly rescued the TCP4-mediated suppression of MIR319C expression suggesting that CUC2 and TCP4 interact to establish the domain of MIR319C expression in leaf primordia. Thus, we have identified CUCs as the activators of MIR319C in the leaf primordia.
In conclusion, we propose a model where the CUC proteins initially activate MIR319C throughout early leaf primordia. As development progresses, the CIN-TCP genes are expressed towards the distal end of the primordia by the action of yet unidentified factors, and the onset of CIN-TCP activity results in the downregulation of MIR319C transcription in the distal primordia, possibly by recruiting chromatin modifiers. Strong CUC activity at the base sustains MIR319C expression in the proximal region, where CIN-TCP transcripts are degraded by mature miR319. Thus, our study provides evidence that a CUC-MIR319C-CIN-TCP module patterns a uniformly growing leaf primordium into the proximal and the distal growth domains, where the cells in the basal region continue to divide and grow, whereas cells in the distal region stop dividing and start differentiating
Developmental Basis and Diversity of Polar Growth Patterns in Leaves
No full textGrowth polarity in leaves – a final discussion
Insights into the growth processes of leaf lamina have come from studies on several species including Arabidopsis, Antirrhinum, tobacco and maize. A feature common to the growth of leaf in these distantly related species is the existence of a pronounced growth gradient in the proximo-distal axis -growth at the tip (distal part) is arrested at an early stage while the basal region (proximal part) continues to grow for the longest duration. This is because the cell division is arrested first at the tip at an early stage of development and the arrest progressively spreads towards the base. Along with the strong proximo-distal growth gradient, a milder growth gradient also exists in the medio-lateral axis, such that the cell division arrest travels slightly faster on the leaf margins imparting an overall convex shape to the arrest front. The temporal and spatial progression of the arrest front has not only been implicated in shaping up of a leaf but is also of paramount importance in the maintenance of a flat surface during leaf growth. Although the patterning mechanisms described above seem to operate during leaf growth in many6 species, the molecular mechanisms governing these processes is still in its infancy. Moreover, patterning of leaf growth has been studied only in a handful of model species and, therefore, the information from the vast body of natural variation remains neglected.
Proximo-distal growth patterning by CINCINNATA
Mutant leaves with altered rates or shapes of the arrest front progression deviate significantly from the normal shape and overall flat structure. Mutation in the CIN gene in Antirrhinum and its orthologues in Arabidopsis cause buckling of the leaf due to excess cell proliferation, which in turn is caused by a delayed progression of the arrest front. CIN-like genes code for TCP transcription factors and are expressed in a broad zone of a growing leaf somewhat distal to the proliferation zone. Even though several direct and indirect targets of CIN-like genes have been identified in various plant species, their role in regulating leaf maturity and surface curvature has remained unclear. The comparison of global transcription profile of wild type and cincinnata mutant of Antirrhinum showed that the expression of genes involved in either signaling or biosynthesis of the major growth hormones were altered in the mutant. By combining DNA-protein interaction, expression analysis, chromatin immuno-precipitation and RNA in situ hybridization, we show that CIN maintains surface flatness by regulating the signaling or level of major plant hormones in developing leaves. CIN promotes cytokinin signaling by directly binding to and thereby promoting the expression of a cytokinin receptor, AmHK4, in a spatio¬temporal manner. Furthermore, it also seems to affect GA level indirectly in young leaves by regulating the spatio-temporal as well as levels of GA-biosynthetic and GA-degrading enzymes. Thus, CIN seems to accelerate maturity in leaf cells along the tip-to-base direction through its effect on the cytokinin and GA signaling pathways. In addition, CIN suppresses auxin signaling more at the margin than in the centre by establishing a margin-to-medial expression gradient of a homologue of the auxin suppressor IAA3, thereby suppressing excess cell proliferation on the margin. Our results uncover an underlying mechanism in a developing leaf that controls curvature of the leaf surface by promotion of timely exit from cell proliferation in the proximo-distal as well as the medio-lateral axes via multiple hormone pathways.
Divergent growth polarities in the proximo-distal axis of leaves
The morphogenetic gradient in the proximo-distal axis of a leaf is brought about by the dynamic expression of several heterochronic regulators which can include TCP and GRF classes of transcription factors. Interestingly, many of these transcription factors are also regulated post-transcriptionally by micro RNAs. In case of the studied model species, these factors seem to be associated with basipetal growth. The early arrest in cell proliferation at the tip and continued cell division at the base has served as a paradigm in studying leaf growth and has been used to conceptualize the growth of leaves with different shapes. However, the possibility of the existence of different patterning mechanisms during leaf growth in the highly diverse plant kingdom remains unexplored. Our survey of leaf growth patterns in 75 dicot species reveals the existence of four distinct proximo-distal polarities in growth patterns. Using the law of simple allometry, we also show that the differentially growing regions of leaves bear a constant relationship between them during growth. A combination of cell-size studies, histochemical staining and expression analysis reveals a strong correlation among growth pattern, cell size and the cell proliferation status. The cell size studies also indicate that there is a wide variation in the final cell sizes of leaves and the relative contribution of cell division and cell expansion to the final leaf size can be highly variable.
Furthermore, we find that the varying growth patterns are linked to changes in the expression pattern of miR396, which controls the expression pattern of cell division regulatory transcription factors, the GRFs. Mis-expressing miR396 at the base of the young Arabidopsis leaf caused an early exit from cell division while reducing the expression of the miR396 at the tip allowed cell division to continue for a longer duration near the tip. Our results demonstrate that leaves with similar shapes can be differently patterned and that this divergent patterning is linked to the expression differences in the regulatory micro RNA, miR396
In conclusion, this study shows that regulators like CIN maintain surface flatness of the Antirrhinum leaf during growth by promoting timely exit from cell division along the proximo-distal and the medio-lateral axes; and it does so by regulating multiple hormone pathways. Although the basic mechanism of patterned cell division and differentiation seems to be conserved among species, the polarities of growth can vary. The variability in the growth polarities could be brought about by changes in the trans-regulation or cis-regulatory changes in the patterning genes
Regulation of Leaf Margin Development by TOOTH/MIR160A in Arabidopsis Thaliana
No full textTOOTH/MIR160A regulates leaf margin outgrowth in Arabidopsis thaliana
Unlike animals, a striking aspect of the plant development is that they have evolved a flexible pattern of post embryonic development. This exposes them to the challenges of many biotic and abiotic signals throughout their life. So, plants have to evolve/regulate various mechanisms to modulate their growth and development for accomplishing a successful life cycle in the prevailing environmental conditions.
Auxin is involved in the initiation of lateral organs at the meristem and serration development along the leaf margin (Bilsborough et al., 2011, Hay et al., 2006). These two developmental mechanisms share common molecular players. For example, CUC2 is required for the boundary formation at the SAM and also is shown to be essential for serration formation at the leaf margin. Similarly, tth shows increased leaf serration phenotype as well as defects in the positioning of flowers at the meristem. This demonstrates the functional significance of TTH-regulated ARFs in controlling auxin mediated developmental pathways.
Leaves originate as small lumps of undifferentiated cells at the flanks of the shoot apical meristem which undergo several rounds division and expansion to generate the mature leaf with characteristic size, shape and leaf margin. Both, endogenous as well as environmental factors modulate the growth and development of a leaf. This is evident from the plasticity in leaf form, observed during the life time of a single plant, as well as from the diversity among closely related species living in different habitats. It is well known that pathways controlling leaf form are subjected to the effects of selection and adaptation. Leaf margin is a key feature of the final leaf shape and it contributes to the abundant diversity in leaf form. Leaf margin architecture varies quite significantly from smooth or entire margin to margins with large outgrowths (lobed margins). The evolution and ecological advantages of this diversity is a subject of intense investigation. It also provides a wonderful system to study the mechanistic details of iterative generation of repeated units, which is a common feature in producing many biological shapes.
Recent advances in molecular technologies and the availability of genomic resources ushered the identification of new factors involved in leaf margin development. Our current knowledge of this developmental programme is that CUC2 establishes auxin maxima at the leaf margin by reorienting an auxin efflux carrier PIN1 which ultimately results in serration outgrowth (Bilsborough et al., 2011, Hay et al., 2006). A few missing links in this pathway are the mechanistic details of CUC2 function in reorienting PIN1 and the molecular details of auxin mediated serration outgrowth. Forward genetic screens have been valuable in characterizing a genetic pathway even in the post genomic era. An EMS mutagenesis screen was performed in this context to identify novel factors that can improve our understanding of this intricate mechanism. tooth was identified in the M2 population based on its increased leaf serration phenotype. Genetic analysis showed that tth phenotype is due to a monogenic recessive mutation. Along with increased leaf serration, tth also shows various developmental defects such as aberrant phyllotaxy, narrower cotyledons and narrower leaves. Positional cloning and sequencing analysis showed a G to A transition at the AT2G39175 locus which codes for MIR160A. The mutation is at the 7th base position of the mature miRNA sequence. Functional characterization of miRNAs by isolating mutations is hampered by their small genomic sizes. Till now, only a few miRNAs have been characterized by mutational analysis in plants (Allen et al., 2007, Baker et al., 2005, Cartolano et al., 2007, Chuck et al., 2007, Knauer et al., 2013, Nag et al., 2009, Nikovics et al., 2006). miR160-ARF10 regulatory module is shown to be required for leaf blade out growth and serration, but not leaf complexity in tomato (Hendelman et al., 2012). miR160 is coded by 3 loci in Arabidopsis, MIR160A, B and C. All three loci encode identical mature miRNA that targets 3 Auxin response factors, ARF10, 16 and 17. ARFs are the effector molecules of auxin mediated developmental programmes. Genetic analysis showed that enhanced serration outgrowth in tth is due to the up-regulation of its target genes. Here, we have identified a miRNA that negatively regulates serration outgrowth by repressing ARF10, 16 and 17 whose functional significance in regulating leaf margin development was not known previously.
Extensive genetic interaction studies have shown that TTH acts in parallel to SAW-BP and MIR164-CUC pathways in regulating leaf margin development. We have also shown that CUC2 and PIN1 are absolutely essential for serration development in tth. CUC2 establishes a pattern required for the expression of ARF10 at the leaf margin. In the absence of CUC2, downstream effector molecules such as ARFs can not perform their function. arf10-2 arf16-2 could reduce, but not suppress serration outgrowth in various mutants suggesting their functional redundancy with other ARF family members.
CUC2 establishes auxin maxima at the leaf margin that triggers the degradation of AUX/IAA repressors thereby relieving ARF proteins which mediate serration outgrowth. Whereas, TTH acts at the post transcriptional level for maintaining normal ARF transcript levels
Role of SPYINDLY in Arabidopsis leaf margin development
SPYNDLY encodes an O-linked N-acetyl glucosamine transferase that acts as a negative regulator of GA response. Consistent with its role in GA response, spy mutants show several GA dependent phenotypes such as early flowering and hyper branched trichomes. spy mutants also show several GA independent phenotypes such as aberrant phyllotaxy and smooth leaf margin. We have studied its role in regulating Arabidopsis leaf serration development. Reporter analysis of ARF10::GUS and CUC2::GUS in spy-3 revealed that SPY is not involved in establishing serration pattern. The spy-3 leaves did not show any defects during the early stages of serration development, but the mature leaves display smooth leaf margin indicating that SPY function is required for serration outgrowth. As shown in the present study, TTH regulated ARFs are also involved in serration outgrowth. Analysis of leaf margin phenotype in tth spy-3 showed that SPY activity is not required for ARF mediated serration outgrowth. Similar genetic interaction studies with SAW-BP pathway mutants showed that leaf margin out growth mediated by meristematic genes is not dependent on SPY function.
Genetic interaction studies with MIR164-CUC pathway genes showed that SPY is required for serration outgrowth in these mutants. Interestingly, the cuc2-3 mutant is defective at both patterning and outgrowth of serration. The spy-3 could suppress serration out growth in cuc2-D suggesting that CUC2 mediated serration out growth is dependent on SPY activity. Protein-protein interaction studies between SPY and CUC2 are in progress to demonstrate whether SPY directly interacts with CUC2 or CUC2 derived signal to regulate serration out outgrowth. It is interesting to examine how mutations at SPY locus can abolish serration out growth mediated by CUC2, but does not affect the serration pattern, even though CUC2 is reported to be essential for both the patterning and outgrowth of serration
Map-based Cloning and Characterization of TARANI, a Global Regulator of Arabidopsis Development
No full textForward genetic screen was performed in Arabidopsis thaliana to isolate novel genes involved in leaf development. The tarani (tni) mutant was selected for further study based on its unique cup-shaped lamina with +ve Gaussian curvature. We show that the larger size of tni leaves is due to rapid growth rate due to excess and prolonged cell division. We monitored the front of the receding cell division zone as a function of time and showed that the shape of the front is more concave compared to wild type, leading to positive curvature. Application of gibberellic acids (GA) synthesis inhibitor rescued the positive curvature of tni suggesting a role for GA in maintaining leaf flatness. Overexpression of cell cycle inhibitor KRP2 also flattened the leaf, confirming a role of cell division. The floral organs and seed are also larger in the tni mutant. Besides growth, tni trichomes are hyper-branched which usually happens when there is more endoreduplication. We found that the nuclei of tni trichomes are larger than wild type nuclei, suggesting increased DNA content. Genetic interaction studies showed that TNI works independent of other trichome branching genes such as with TRYPTICHON and FURCA1.
Map-based cloning showed that tni is positioned on left arm of the 3rd chromosome. Using molecular markers, we narrowed down to interval to a 65 kb region, which codes for 19 genes. Sequencing several of them revealed a G→A transition at the 3rd intron - 4th exon junction of At3g20630 gene. RT-PCR analysis showed the presence of an additional full-length transcript with extra un-spliced 3rd intron. Overexpression of this un-spliced variant in wild type plants produced phenotypes like hyperbranched trichomes and cup-shaped leaves; plus additional phenotypes like organ fusion and organ polarity defects. Complementation and allelic tests confirmed that TNI codes for AtUBP14, an ubiquitin protease.
The tni plants have longer stem and roots which grow at faster rate compared to wild type. Confocal microscopic analysis of mature embryos showed that both shoot (SAM) and root apical meristems (RAM) of tni plants are larger in size. In RAM, the numbers of quiescent center (QC) cells and stem cells have increased in tni plants. The tni inflorescence and flowers are bigger than wild type in size. Also the degree of axillary shoots has increased in the tni plants. Overexpression of the splice variant of TNI produced undifferentiated callus-like structures in the shoot apex and in hypocotyl. All these phenotypes show that TNI is involved in meristem proliferation.
The tni siliques produced many un-fertilized ovules and shrunken and malformed seeds suggesting gametic and/or embryo lethality. We observed that tni embryos were mis-patterned at various stages of development. Following the cell division pattern shows that cells arising from the ‘basal cell’ of the embryo take apical cell fate in tni embryos. The topmost cell of the suspensor, which is also the precursor cell of RAM, is not specified as hypophysial cell in several tni embryos.
In the forward genetic screen, we isolated another mutant called tooth (tth), which has deeper serrations at the leaf margin and narrower leaves compared to wild type. It has been mapped to the longer arm of the 2nd chromosome. Genetic interaction studies show that tth is not allelic to other serration mutants such as serrate and mir164a
Role of the ELONGATED GYNOPHORE/ELONGATA2 Protein in Fruit and Root Development in Arabidopsis Thaliana
No full textIn order to identify new players in fruit development, a forward genetic screen was performed on EMS mutagenized plants. A mutant named elongated gynophore (egy) was identified in the M2 population based on altered fruit morphology. Genetic analysis established that the egy phenotype is due to a monogenic and recessive mutation. The egy plants show additional developmental defects including shorter root, narrower cotyledons and malformed leaf lamina. Molecular mapping and whole genome sequencing analyses showed a G/C deletion at the position 4414980 on the AT5G13680 gene locus which is predicted to encode the ELONGATA2 (ELO2) protein. ELO2 is a constituent member of the elongator complex
which helps in transcriptional elongation in association with the phosphorylated form of RNA polymerase II. This complex has been implicated in controlling development, abiotic stress and biotic stress. Genetic complementation test confirmed that egy is indeed allelic to elo2-3.
Surprisingly, the EGY overexpression line 35S::EGY showed loss-of-function phenotype,
suggesting transgene silencing.
In angiosperms, fruit is derived from the fertilized ovary. The initiation of the female reproductive organ commences with a lump of cells which eventually develops into the gynoecium with a stigma, a style, two fused ovaries and a gynophore, arranged from the apical to basal axis in that order. Genetic networks faithfully shapes up the carpel primordium into predetermined gynoecium shape. Following fertilization, siliques elongate concomitantly with developing embryos. Here we show that the egy mutant has apical basal patterning
defect with longer gynophore at the base. This gynophore phenotype resembles the
phenotype found in the mutants with altered auxin and cytokinin levels/signaling. We show
that egy is hypersensitive to cytokinin treatment; egy fruits treated with cytokinin display phenotype similar to the plants expressing IPT7 under fruit-specific promoter. These results suggest that broadened shoulders at the apical region of egy gynoecium possibly results from
higher cytokinin level/response.
Genetic interaction studies have shown that EGY act independent of AGAMOUS and
PEAPOD to suppress the medio-lateral growth of the apical gynoecium region. Genetic and
expression studies suggest that PINOID and TMO5/T5L1 work downstream to EGY, while
ETTIN acts in parallel to EGY.
We also observed larger seeds in the egy mutant and show that this is controlled maternally.
Thus, the gametic lethality in egy can possibly be accounted for by the defective ovules.
We show that egy primary roots are shorter compared to Col-0, though egy seeds have longer embryonic root to begin with, suggesting a defect in cell division. The root cells are arranged radially in a stereotypic pattern in root meristem from the outer epidermis to the inner stele specific the vascular bundles. The four QC cells are also surrounded by stem cells of various
identities. This stereotypic pattern of cell arrangement is perturbed in the egy root. The stele, composed of pericycle and vascular bundles is reduced in the egy mutant, suggesting a positive role of EGY in vascular cell division. Confocal microscopic studies and real-time PCR data suggest that TMO5/T5L1 work downstream to EGY. Thus, the Arabidopsis
„ELONGATOR‟ complex regulates the transcription of target genes that are necessary for plant growth and development.
A proposed genetic network for the role of EGY in fruit and root development. Based on the genetic interaction studies and expression analysis, we have placed EGY in the existing molecular network that control fruit and root vascular development in Arabidopsi
Role of TCP transcription factors in seedling development, leaf morphogenesis and senescence in Arabidopsis thaliana
No full textThe TCP gene family encodes non-canonical bHLH transcription factors that act as key regulatory molecules in diverse developmental processes in plants including organ morphogenesis, plant architecture, leaf maturation, and flowering transition. In this study, we assign a number of new functions to the CINCINNATA-like TCP (CIN-TCP) proteins throughout the life of Arabidopsis thaliana starting from light-mediated seedling morphogenesis, regulation of simple leaf architecture, and hormone homeostasis during leaf senescence.
PART-I: Role of TCP transcription factors in light-mediated Arabidopsis seedling development
Plant growth and morphogenesis rely heavily on the coordination between external and internal cues to cope with the ever-changing surroundings. In Arabidopsis seedlings grown under low light intensity, the embryonic stem (hypocotyl) elongates more in an attempt to reach the light source, a process called skotomorphogenesis. By contrast, seedlings grown under sufficient light grow shorter (photomorphogenesis). We had earlier shown that the CIN-TCPs promote cell elongation during photomorphogenesis. Here we show that this effect of CIN-TCPs is abolished in darkness, suggesting that CIN-TCP-mediated cell elongation is dependent on the light-signaling pathway. By analyzing hypocotyl elongation under various light qualities, we show that TCP4-mediated hypocotyl cell elongation is dependent on phytochrome B (PhyB) photoreceptor under diverse light conditions. Using various biochemical and genetic assays, we demonstrate that TCP4 activation leads to the stabilization of several phytochrome-interacting factor (PIF) proteins through protein-protein interaction. Enhanced PIF level leads to the destabilization of PhyB and indirectly represses the HFR1 protein to promote hypocotyl elongation. Thus, CIN-TCP functions as a major negative regulator of photomorphogenic seedling growth together with PIF.
PART-II: CIN-TCPs actively suppress leaflet emergence to promote simple leaf form
Though all angiosperm leaves are initiated as simple rod-like primordia at the flank of the shoot apical meristem (SAM), they show extensive shape diversity at maturity, based on which they are broadly divided into two forms; simple leaves with intact lamina and compound leaves with lamina dissected into leaflets. Although genetic intervention has converted compound leaves into simpler or more complex variants, it is not clear whether, or to what extent, simple leaves can initiate leaflets and form compound architecture upon endogenous gene manipulation. Here, we show that simultaneous down-regulation of CIN-TCP and class II KNOTTED1-LIKE (KNOX-II) proteins converts simple Arabidopsis lamina to super-compound form with reiterative and indeterminate leaflet emergence, accompanied with sustained reactivation of the meristem-specific genes including KNOX-I and CUPSHAPED COTYLEDON (CUC). CIN-TCPs activate KNOX-II and a dominant CIN-TCP member restores simple leaf form. These results offer a framework of simple leaf development wherein CIN-TCP-KNOX-II forms a strong differentiation module that suppresses the KNOX-I-CUC network and leaflet initiation in the primordia.
PART-III: CIN-TCPs maintain jasmonic acid homeostasis during leaf senescence through an incoherent feed forward loop (IFFL)
The class I and class II TCP transcription factors, divided based on their sequence diversity, display functional antagonism in regulating multiple cellular and physiological processes including jasmonic acid (JA) biosynthesis during leaf senescence, the final stage of leaf development. Five members of miR319-regulated class II TCPs (TCP2, 3, 4, 10 & 24), also called CIN-TCPs, redundantly promote the JA-biosynthetic enzyme-encoding gene LIPOXYGENASE2 (LOX2). For example, TCP4 binds to the LOX2 promoter and directly activates its transcription that induces leaf senescence. By contrast, the class I TCP members TCP9 and TCP20, together with TCP8 and TCP22, are recruited on the LOX2 promoter to repress its transcription. However, a molecular link between these two TCP groups in regulating LOX2 transcription has not been demonstrated. We here demonstrate a novel type I incoherent feed forward loop (IFFL) formed by the direct transcriptional link between class I (TCP9) and class II TCP proteins (TCP4/ TCP10) to balance the LOX2 expression dynamics. By combined mathematical modelling and genetic manipulation, we show that this IFFL filters out the stochastic noise in TCP and maintains a robust level of LOX2. Thus, the TCP4/TCP10-TCP9-LOX2 module regulates JA homeostasis and leaf senescence in Arabidopsis.
In conclusion, we show that the CIN-TCP proteins, along with their class II members, regulate important developmental processes throughout the life of Arabidopsis starting from the seedling establishment, simple leaf shape, and hormone homeostasis. Studies on the role of CIN-TCP homologs in other species would test whether these functions are conserved in evolution
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