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Trichomes are hair-like structures found on the surface of virtually all terrestrial plants (Yang et al., 2015). They are epidermal outgrowths that can occur on all of the aerial parts of a plant, varying markedly in size, shape, distribution, and in their ability to produce secondary metabolites. About 30% of all vascular plants carry the glandular trichomes capable of producing secondary metabolites (Glas et al., 2012). Trichomes are vitally important to plants as a defence mechanism, they are highly significant commercially, and they are of interest to plant biologists in that they serve as an excellent model system to study all aspects of plant differentiation at the single-cell level (Hulskamp, 2004). The simple, non-glandular trichomes found in Arabidopsis have been studied extensively. However the glandular trichomes of the kind found on the surface of Antirrhinum are much less well understood. The primary aim of the research reported here is to identify the gene(s) responsible for variation in epidermal hair (trichome) distribution between Antirrhinum species. Following an introduction which provides essential background on trichomes and on Antirrhinum, the thesis is presented in four parts. The first part describes a RAD-seq experiment used to produce linkage maps for the eight chromosomes making up the Antirrhinum genome and estimates the position of the Hairy gene on linkage group 8. The results are cross-validated against maps produced independently by the Xue group. It also describes novel methods developed to address a number of problems that arose during the course of the analysis, and explores the value of imputation methods in helping to overcome gaps and inconsistencies in the data. The second part presents the findings from a fine-mapping Pool-seq experiment designed to estimate the position of Hairy more precisely. The findings suggest that Hairy lies on one of a small number of scaffolds, with Scaffold 1097 being the most likely candidate. Also covered are the findings of another experiment to estimate the position of the gene that determines whether flowers are pale or dark. In this case the results indicate that the gene lies on one of a small number of scaffolds on linkage group 5. The third part presents the results of an RNA-seq experiment which, when combined with the results of the Pool-seq experiment provides evidence that Hairy might be a glutaredoxin gene on Scaffold 1097. Finally the interim results of three experiments designed to confirm that the gene identified as Hairy controls the distribution of trichomes in Antirrhinum are presented.

The genus Antirrhinum (commonly known as snapdragons) contains more than twentyfive recognised species. The genus has been divided into three morphological subsections: Antirrhinum, Streptosepalum and Kickxiella (Rothmaler, 1956). One of the major characteristics distinguishing the three subsections is flower colour. Most species in subsection Antirrhinum have dark pink or yellow flowers, Kickxiella species are white or pale pink and Streptosepalum species have yellow or pale pink flowers. All Antirrhinum species can be crossed to produce fertile hybrids which allow the genes that underlie their differences to be identified. I used quantitative trait locus (QTL) analysis on hybrids of A. majus (dark magenta flowers) and A. charidemi (pale-pink flowers) to map genomic regions underlying differences in flower colour. This identified two major-effect loci, in Linkage Group 3 (LG3) and LG7, that explained most of the differences between these species. I used near-isogenic lines (NILs) to further test involvement of two candidate genes - Rosea (Ros) in LG3, which encodes a regulator of the anthocyanin biosynthesis pathway (ABP) and Incolorata (Inc) in LG7 which encodes a rate-limiting enzyme of the ABP. In both cases, the A. majus allele increased pigmentation. Sequence differences between Ros alleles of A. majus, A. charidemi and A. molle (a Kickxiella species with white flowers) suggest that A. molle carries a ros loss-of-function mutation and that a transposon insertion in the ROS promoter might contribute to differences in expression between A. majus and A. charidemi. Ros genotypes were found to be strongly correlated with pigmentation in the corolla tube in A. majus x A. charidemi hybrids, and to a lesser extent with corolla lobe pigmentation, although NILs suggested that ROS did not correspond to the major-effect QTL indentified in LG3. I also mapped a minor-effect QTL for tube pigmentation to a region of LG4 containing the ABP structural gene Candica. Analysis of NILs revealed that Inc was not the second major-effect QTL mapped to LG7, although sequence differences were detected between Inc alleles of A. majus and A. charidemi. I was further able to narrow down the region containing the second LG7 major-effect QTL to an interval of 11 cM, between two molecular markers, which could be used to determine the likely QTL genotypes of segregating NILs. Surprisingly, several ABP genes, particularly Nivea, Inc and Pallida, were expressed at higher levels in pale flowers that were homozygous for the A. chardemi QTL allele than in their dark flowered siblings that carried an A. majus allele. This suggests that ABP genes might be up-regulated in pale flowers as part of a negative feedback mechanism. Two potential roles of the LG7 QTL are considered 1) its requirement for anthocyanin modification or transport to the vacuole, so that a build-up of cytosolic anthocyanins or their break-down products in pale flowers increases structural gene expression but cannot compensate for the overall reduction in anthocyanin, or 2) a role in promoting production of flavonols at the expense of anthocyanins.

Parmi les signaux floraux, les odeurs florales sont remarquables pour leur complexité en composés odorants et leur variation entre, et au sein des taxa. Elles interviennent dans de nombreuses interactions que les plantes entretiennent avec les organismes de leur environnement. Cette diversité chimique gouverne de multiples fonctions, telles que l’attraction de pollinisateurs, l’encouragement à la constance florale et la défense contre des antagonistes. Bien que les fonctions écologiques des odeurs florales soient relativement bien étudiées, les facteurs évolutifs qui gouvernent la composition et les variations de ce signal complexe sont très mal connus. C’est dans ce contexte que ma thèse s’inscrit. J’ai étudié les variations de ce trait floral particulier : les odeurs florales. Ma thèse se focalise sur une espèce de plante, la gueule-de-loup, Antirrhinum majus, utilisée comme espèce modèle en biologie depuis des décennies. Cette espèce, native des Pyrénées, elle présente deux sous-espèces, l’une à fleurs magenta, A. m. pseudomajus, et l’autre à fleurs jaunes, A. m. striatum. Alors que ces deux sous-espèces peuvent s’inter-féconder, elles ne coexistent jamais dans la nature et leurs hybrides, reconnaissables par une grande diversité de colorations florales, sont peu fréquents. Le mécanisme de cet isolement reproducteur n’est pas connu, mais le comportement des pollinisateurs a été envisagé dans de précédentes études. Les principaux résultats de ma thèse montrent que les deux sous-espèces d’A. majus se distinguent par leurs odeurs florales. Certains composés volatils, en particulier trois benzénoïdes, ne sont émis que par A. m. pseudomajus, et ceci de manière constante entre les populations et pour différents environnements. Quant aux hybrides, les ratios de composés volatils floraux sont très variables par rapport aux signaux reproductibles parentaux, avec un patron de ségrégation chez les hybrides F2. En utilisant des bourdons commercialisés (Bombus terrestris), donc naïfs de toutes odeurs florales, j’ai montré que ces bourdons sont capables de détecter les principaux composés d’odeurs d’A. majus et qu’ils préfèrent de manière innée un mélange de composés volatils d’A. m. striatum. Finalement, en conditions naturelles, c’est-à-dire avec des odeurs florales naturelles et des pollinisateurs sauvages, ces derniers sont attirés préférentiellement par les odeurs florales de leur sous espèce d’origine. J’ai finalement montré que le patron associatif odeur-nectar qu’apprennent les pollinisateurs fait intervenir uniquement les composés odorants floraux et la quantité de nectar, puisque les différences d’odeurs florales entre les deux sous-espèces sont associées à une plus grande quantité de nectar par fleur chez A. m. pseudomajus mais à une plus faible concentration en sucres. En d’autres termes, les plantes contiennent autant de sucre total dans leurs fleurs dans une sous-espèce ou dans une autre. Ces résultats, pris dans leur ensemble, semblent montrer que les composés volatils floraux sont bien impliqués dans l’isolement reproducteur de ces deux sous-espèces. Même si les odeurs florales ne peuvent pas expliquer à elles seules la distribution spatiale des deux sous-espèces d’A. majus, elles peuvent jouer un rôle supplémentaire de barrière aux flux de gènes. En effet, les pollinisateurs sont susceptibles de montrer un phénomène de constance envers l’un des phénotypes floraux, limitant ainsi les flux de gènes entre les deux sous-espèces. Dans cette thèse, je propose différentes perspectives possibles à mes résultats de thèse / Manquant

The distribution of epidermal hairs (trichomes) is an important taxonomic character in the genus Antirrhinum. Most species in subsection Antirrhinum produce trichomes from lower internodes and leaves, then have bald stems and leaf blades after the third node and resume trichomes production again in the inflorescence (the "bald" phenotype). All species in subsection Kickxiella produce trichomes throughout development (the "hairy" phenotype). Populations of some species are polymorphic for trichome distribution-both bald and hairy individuals were observed in A. australe, A. graniticum, A. latifolium and A. meonanthum. Antirrhinum species also varied in trichome morphology. Five types were recognized according to length and the presence or absence of a secretory gland. Some types were present in all species and had similar distributions-for example short glandular trichomes were found on the adaxial midribs of all leaves in all species, and the lower leaves and internodes of all species shared longer glandular and long eglandular trichomes. However, the trichomes on leaf blades and stems at higher vegetative nodes of hairy species and in the inflorescences differed in morphology between species, suggesting that they are regulated differently from trichomes at more basal positions. Other species in the tribe Antirrhineae showed similar variation in trichome morphology and distribution to Antirrhinum, suggesting that the control of trichome development might be conserved within the tribe. To understand the genetic basis for variation in trichome distribution, a near-isogenic line (NIL) was generated by introducing regions of the genome of A. charidemi (hairy, subsection Kickxiella) into the genetic background of A. majus subsp. majus (bald, subsection Antirrhinum). One NIL segregated bald and hairy progeny, with the same trichome distributions as the parent species, in a ratio that suggested a single locus is responsible for the differences and baldness is dominant. The locus was named as Hairy and assumed to act as a suppressor of trichome formation. Progeny of the NIL were used in genome resequencing of bulked phenotype pools (Pool-seq) to map Hairy. No recombination between Hairy and a candidate gene (GRX1) from the Glutaredoxin gene family, was detected in the mapping population. In addition, RNA-seq revealed that GRX1 was expressed in bald parts of bald progeny, but not in the same parts of hairy progeny, and in situ hybridisation showed GRX1 RNA was restricted to epidermal cells, which form trichomes in the absence of Hairy activity. A virus-induced gene silencing (VIGS) method was also developed to test GRX1 function further. Reducing GRX1 activity allowed ectopic trichome formation in the bald NIL. Together, this evidence strongly supported Hairy being GRX1. To investigate evolution of Hairy and its relationship to variation in trichome distribution, the NIL was crossed to other Antirrhinum species. These allelism tests suggested that Hairy underlies variation in trichome distribution throughout the genus, with the exception of A. siculum, which has a bald phenotype but might lack activity of hairy and a gene needed for trichome formation. Hairy sequences were obtained from representative of 24 Antirrhinum species and two related species in the tribe Antirrhineae. The conserved trichome-suppressing function of the sequence from one of these species (Misopates orontium, bald phenotype) was confirmed by VIGS. Gene phylogenies combined with RNA expression analysis suggested that the ancestral Antirrhinum had a bald phenotype, that a single mutation could have given rise to the hairy alleles in the majority of Kickxiella species, that these alleles were also present in polymorphic populations in the other subsections, consistent with transfer from Kickxiella by hybridisation, and that multiple, independent mutations had been involved in parallel evolution of the hairy phenotype in a minority of Kickxiella species. Phylogenetic analysis of GRX proteins suggested that Hairy gained its trichome-repressing function relatively late in the evolutionary history of eudicots, after the Antirrhineae-Phrymoideae split, but before divergence of the lineages leading to Antirrhinum and Misopates. A yeast two-hybrid screen identified members of the TGA and HD-Zip IV transcription factors as potential substrates of the Hairy GRX.

Citrus spp. are known for the accumulation of flavanone glycosides (e.g., naringin comprises up to 70% of the dry weight of very young grapefruit). In contrast, petunia utilizes relatively more naringenin for production of flavonol glycosides and anthocyanins. This investigation addressed whether or not petunia is capable of glucosylation of naringenin and if so, what are the characteristics of this flavanone glucosylating enzyme. Petunia leaf tissue contains some flavanone-7-O-glucosyltransferase (E.C. 2.4.1.185) activity, although at 90-fold lower levels than grapefruit leaves. This activity was partially purified 89-fold via ammonium sulfate fractionation followed by FPLC on Superose 12 and Mono Q yielding three chromatographically separate peaks of activity. The enzymes in the peak fractions glucosylated flavanone, flavonol, and flavone substrates. Enzymes in Mono Q peaks I and II were relatively more specific toward flavanone substrates and peak I was significantly more active. Enzyme activity was not effected by Ca2+, Mg2+, AMP, ADP, or ATP. The petunia enzyme was over 10,000 times more sensitive to UDP inhibition (Ki 0.89 μM) than the flavanone-specific 7GT in grapefruit. These and other results suggest that different flavonoid accumulation patterns in these two plants may be partially due to the different relative levels and biochemical properties of their flavanone glucosylating (7GT) enzymes.

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.

A key question in biology is how changes in gene function or regulation produce new morphologies during evolution. The nectar spur is an evolutionarily labile structure known to influence speciation in a broad range of angiosperm taxa. Here, the genetic basis of nectar spur development, and the evolution of differences in nectar spur morphology, is investigated in Linaria vulgaris and two closely related species of orchid, the primitively longer-spurred Dactylorhiza fuchsii, and more derived short-spurred D. viridis (Orchidinae, Orchidaceae). Despite considerable morphological and phylogenetic differences, nectar spur ontogeny is fundamentally similar in each of the study species, proceeding from an abaxial bulge formed on the ventral petal relatively late in petal morphogenesis. However, spur development is progenetically curtailed in the short-spurred orchid D. viridis. In each case spur development involves class 1 KNOTTED1-like homeobox (KNOX) proteins. KNOX gene expression is not restricted to the spur-bearing petal, indicating that additional components are required to define nectar spur position, e.g. canonical ABC genes, determinants of floral zygomorphy, and additional (currently unknown) factors. However, constitutive expression of class 1 KNOX proteins in transgenic tobacco produces flowers with ectopic outgrowths on the petals, indicating that KNOX proteins alone are, to some degree, capable of inducing structures similar to nectar spurs in a heterologous host. Interestingly, KNOX gene expression is high in the ovary of all study taxa, suggesting that KNOX proteins may also have been involved in the evolution of this key angiosperm feature. Although principally involved in maintaining indeterminacy in the shoot apical meristem (SAM), members of the KNOX gene family have been co-opted in the evolution and development of compound leaves where they suppress differentiation and extend the morphogenetic potential of the leaf. A similar model is presented here to explain the role of KNOX proteins in nectar spur development. Co-option of KNOX gene expression to the maturing perianth delays cellular differentiation, facilitating the development of the nectar spur but requiring additional, unknown factors, to determine nectar spur fate. As facilitators of nectar spur development, changes in the spatio-temporal patterns of KNOX gene expression may alter the potential for nectar spur development and explain the critical length differences observed between the orchids D. fuchsii and D. viridis (and among other angiosperm taxa). Taken together, the available data indicate that KNOX genes confer a meristematic state upon plant tissues in a variety of morphogenetic contexts, making the gene family a potentially versatile tool to mediate a wide variety of evolutionary transformations.

Much is known about how organs acquire their identity, yet we are only beginning to learn how their shape is regulated. Recent work has elucidated the role of coordinated cell division & expansion in determining plant organ shape. For instance, in Antirrhinum, leaf shape is affected in the cincinnata (cin) mutant because of an alteration in the cell division pattern. CIN codes for a TCP transcription factor and controls cell proliferation. It is unclear how exactly CIN-like genes regulate leaf morphogenesis. We have taken biochemical and genetic approach to understand the TCP function in general and the role of CIN-like genes in leaf morphogenesis in Antirrhinum and Arabidopsis. Targets of CINCINNATA To understand how CIN controls Antirrhinum leaf shape, we first determined the consensus target site of CIN as GTGGTCCC by carrying out RBSS assay. Mutating each of this target sequence, we determined the core binding sequence as TGGNCC. Hence, all potential direct targets of CIN are expected to contain a TGGNCC sequence. Earlier studies suggested that CIN activates certain target genes that in turn repress cell proliferation. To identify these targets, we compared global transcripts of WT and cin leaves by differential display PCR and have identified 18 unique, differentially expressed transcripts. To screen the entire repertoire of differentially expressed transcripts, we have carried out extensive micro-array analysis using 44K Arabidopsis chips as well as 13K custom-made Antirrhinum chips. Combining the RBSS data with the results obtained from the micro-array experiments, we identified several targets of CIN. In short, CIN controls expression of the differentiation-specific genes from tip to base in a gradient manner. In cin, such gradient is delayed, thereby delaying differentiation. We also find that gibberellic acid, cytokinin and auxin play important role in controlling leaf growth. Genetic characterization of CIN-homologues in Arabidopsis Arabidopsis has 24 TCP genes. Our work and reports from other groups have shown that TCP2, 4 and 10 are likely to be involved in leaf morphogenesis. These genes are controlled by a micro RNA miR319. To study the role of TCP4, the likely orthologue of CIN, we generated both stable and inducible RNAi lines. Down-regulation of TCP4 transcript resulted in crinkly leaves, establishing the role of TCP4 in leaf shape. To study the function of TCP2, 4 & 10 in more detail, we isolated insertion mutants in these loci. The strongest allele of TCP4 showed embryonic lethal phenotype, indicating a role for TCP4 in embryo growth. All other mutants showed mild effect on leaf shape, suggesting their redundant role. Therefore, we generated and studied various combinations of double and triple mutants to learn the concerted role of these genes on leaf morphogenesis. To further study the role of TCP4 in leaf development, we generated inducible RNAi and miRNA-resistant TCP4 transgenic lines and carried out studies with transient down-regulation and up-regulation of TCP4 function. Upon induction, leaf size increased in RNAi transgenic plants whereas reduced drastically in miR319 resistant lines, suggesting that both temporal & spatial regulation of TCP4 is required for leaf development. Biochemical characterization of TCP domain To study the DNA-binding properties of TCP4, random binding site selection assay (RBSS) was carried out and it was found that TCP4 binds to a consensus sequence of GTGGTCCC. By patmatch search and RT-PCR analysis, we have shown that one among 74 putative targets, EEL (a gene involved in embryo development), was down regulated in the RNAi lines of TCP4. This suggests that EEL could be the direct target of TCP4. We have tested this possibility in planta by generating transgenic lines in which GUS reporter gene is driven by EEL upstream region with either wild type or mutated TCP4 binding site. GUS analysis of embryos shows that transgenic with mutated upstream region had significantly reduced reporter activity in comparison to wild type, suggesting that EEL is a direct target of TCP4. We have further shown that TCP4 also binds to the upstream region of LOX2, a gene involved in Jasmonic acid (JA) biosynthesis (in collaboration with D. Weigel, MPI, Tubingen, Germany). TCP domain has a stretch of basic residues followed by a predicted helix-loop-helix region (bHLH), although it has little sequence homology with canonical bHLH proteins. This suggests that TCP is a novel and uncharacterized bHLH domain. We have characterized DNA-binding specificities of TCP4 domain. We show that TCP domain binds to the major groove of DNA with binding specificity comparable to that of bHLH proteins. We also show that helical structure is induced in the basic region upon DNA binding. To determine the amino acid residues important for DNA binding, we have generated point mutants of TCP domain that bind to the DNA with varied strength. Our analysis shows that the basic region is important for DNA binding whereas the helix-loop-helix region is involved in dimerization. Based on these results, we have generated a molecular model for TCP domain bound to DNA (in Collaboration with Prof. N. Srinivasan, IISc, Bangalore). This model was validated by further site-directed mutagenesis of key residues and in vitro assay. Functional analysis of TCP4 in budding yeast To assess TCP4 function in regulation of eukaryotic cell division, we have introduced TCP4 in S. cerevisiae under the GAL inducible promoter. TCP4 induction in yeast cells always slowed down its growth, indicative of its detrimental effect on yeast cell division. Flow cytometry analysis of synchronized cells revealed that TCP4 arrests yeast cell division specifically at G1→S boundary. Moreover, induced cells showed distorted cell morphology resembling shmoo phenotype. Shmooing is a developmental process which usually happened when the haploid cells get exposed to the cells of opposite mating type and get arrested at late G1 phase due to the inhibition of cdc28-cln2 complex. This suggested that TCP4-induced yeast cells are arrested at late G1 phase probably by the inhibition of cdc28-cln2 complex. To further investigate how TCP4 induce G1→S arrest, we carried out microarray analysis and found expression of several cell cycle markers significantly altered in TCP4-induced yeast cells. Studies on crinkly1, a novel leaf mutant in Arabidopsis To identify new genes involved in leaf morphogenesis, we have identified crinkly1 (crk1), a mutant where leaf shape and size are altered. We observed that crk1 also makes more number of leaves compared to wild type. Phenotypic analysis showed that crk1 leaf size is ~5 times smaller than that of wild type. Scanning electron microscopy (SEM) showed that both cell size and number are reduced in the mutant leaf, which explains its smaller size. We have mapped CRK1 within 3 cM on IV chromosome.

During photosynthesis Plantaginaceae species can produce glucose derivatives such as iridoid glycosides and alcohol sugars that in addition to sucrose can be exported from leaves. Plantago lanceolata transported sorbitol in addition to sucrose especially at warmer leaf temperatures. However, two iridoids, catalpol and aucubin, found in P. lanceolata were not readily labelled from 14CO2 under any conditions examined. In contrast, in two greenhouse, cut-flower cultivars of Antirrhinum majus the iridoids, antirrhinoside and antirrhide, were readily 14C-labelled along with sucrose but little 14C was recovered in alcohol sugars (e.g., mannitol). The amount of 14C-partitioned into antirrhinoside increased at higher temperatures. Exposing leaves of P. lanceolata and A. majus to reduced-photorespiratory conditions (e.g. short-term CO2 enrichment and/or low O2) increased fixation and export. Under low O2 in P. lanceolata sorbitol 14C-labelling increased relative to sucrose and in A. majus 14C-labelling of sucrose increased relative to antirrhinoside. Also 14C-labelling of antirrhide increased more than antirrhinoside. During both short-term and long-term acclimation to high CO2, whole plant NCER, leaf photosynthesis and export increased in A. majus. Taken together the temperature and CO2 enrichment studies show plasticity in Plantaginaceae species to synthesize and transport sucrose and auxiliary glucose esters and alcohol sugars in a species-specific manner (depending on the rate of carboxylation).