Changes in chlorophyll content, carotenoid content and composition, abscisic acid and phaseic acid levels, hydrolytic enzyme activity and polypeptide pattern were monitored during senescence of the primary attached leaves of Hordeum vulgare L. cv. Dyan. Senescence occurred due to the normal course of leaf development or was induced by incubation of leaves in darkness. Loss of chlorophyll and total leaf protein was retarded in light whereas it continued rapidly in leaves from dark-incubated seedlings. Chlorophyll alb ratio increased with the progression of senescence, suggesting that chlorophyll b was referentially degraded during this process. Loss of total protein coincided with enhanced activity of acid and neutral proteases. In contrast, loss of chlorophyll was not accompanied by an increase in· peroxidase activity, suggesting that this enzyme was not responsible for initiating chlorophyll breakdown. Carotenoid and abscisic acid levels were monitored in the same tissue extracts. The results obtained show that the increase in endogenous levels of abscisic acid, induced by senescence, correlated with enhanced epoxidation of the xanthophyll cycle, ie., increased conversion of zeaxanthin to antheraxanthin and all-trans-violaxanthin. In addition, an increase in abscisic acid levels occurred concomitant with a decrease in all-trans-violaxanthin and 9'-cis-neoxanthin, suggesting an apparent 1:1 relationship on a molar basis. It is therefore proposed that enhanced abscisic acid production, due to foliar senescence, arises from fluctuations in carotenoid turnover. Polypeptide patterns in isolated chloroplasts, purified thylakoid and stromal fractions were very similar for leaves incubated in either light or darkness. A decrease in intensity of bands was observed in isolated chloroplasts and stromal fractions. Intensity of bands in thylakoids remained unchanged with the progression of senescence. Protein standards of peroxidase and lipoxygenase co-migrated with proteins of the isolated chloroplast. Although tentative, some proteins of the chloroplast may be representative of precursors of hydrolytic enzymes which are known to increase during senescence.

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Forward 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.

Growth 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.

TOOTH/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.