The Role of Lipids in Flowering Development of Arabidopsis Enhanced pah1pah2 Plants. Toshiro Ito 1 & Lee Lishi 2

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1 The Role of Lipids in Flowering Development of Arabidopsis Enhanced pah1pah2 Plants Toshiro Ito 1 & Lee Lishi 2 Department of Biological Sciences, Faculty of Science, National University of Singapore, 10 Kent Ridge Road, Singapore ABSTRACT The function of lipids in floral organ development is not well understood. Genetic analysis of pah1pah2 double mutant Arabidopsis thaliana plants suggests that lipids play a role in the signalling pathways that control plant reproductive development. This mutant overexpresses phosphatidic acid, a precursor in cell membrane and nuclear envelope lipid biosynthesis, and shows irregular floral phenotype. We created an enhanced pah1pah2 line (eph) in order to increase the frequency of such phenotype, to better observe the effects of disrupted lipid biosynthesis. Mutagenization of pah1pah2 seed stock with done with methanesulfonic acid ethyl ester (EMS) and with gamma rays. The purpose was to alter the plant genome and create new lines that expressed close to 100% abnormal flowers in association with pah1pah2 background. From the EMS treated seeds, strong anomolous floral phenotype was successfully obtained from two plants in the 110 EMS line, displaying terminal flowers and fused floral buds respectively. Gene mapping and lipid profile analysis will provide answers as to which genes were affected, their role in plant development and possibly lipid biosynthesis, and also the presence of any novel lipids in floral organogenesis. INTRODUCTION In the life cycle of plants, flowering is an important stage for survival of the species. It is influenced by many different factors, including photoperiod and chemical triggers. One of the most interesting aspects of plant reproductive development however, are the genes that control it and how they function. There are four types are genes: flowering time genes, meristem identity genes, floral organ identity genes, and organ building genes (Jack, 2004). CONSTANS (CO) for example promotes flowering when activated by long-day photoperiod. The known genes that control shoot and floral meristem identity include LEAFY (LFY), APETALA1 (AP1), and CAULIFLOWER (CAL) (Buchanan et al 2000). LFY is found to be a key pathway integrator of floral development, and activates floral organ identity genes of AGAMOUS (AG), APETALA2 (AP2), PISTILLATA (PI), and APETALA3 (AP3) which in turn regulate organ identity. According to the ABC model of floral development, these homeotic genes control adjacent and overlapping whorls: AG controls region A (whorl 1 and 2), PI or AP3 controls region B (whorl 2 and 3), and AP3 controls region C (whorl 3 and 4). The combinatorial effect of different genes on each whorl gives rise to sepals, petals, carpels and stamens. The ABCE model of development takes into account 1 Professor and supervisor 2 Student 1

2 the role of SEPALLAT (SEP) genes that contribute to floral determinancy and the formation of petals, carpels, and stamens (Jack. 2004). Lipids are of key interest in plant development due to its role in cell signalling (Buchanan et al 2000). The Arabidopsis pah1pah2 double mutant presents some evidence that lipids are significant to floral organogenesis. The mutant is phosphatidic acid phosphatase deficient, an enzyme that converts phosphatidic acid into diacylglycerols, a key precursor to membrane lipid synthesis (Buchanan et al 2000). Flowers show homeotic conversion and meristemic indeterminancy. One major problem is the low incidence of such phenotype in pah1pah2. Hence the main part of this project was to increase phenotype occurrence by treating pah1pah2 seeds with EMS and gamma rays to induce further mutation. This would be followed by gene mapping to identify the altered gene and its role in reproductive development. Also, obtaining and analyzing a basic lipid profile during each stage of flower development would enable the isolation of novel lipids that function in association of floral development. MATERIALS AND METHODS Plant material Arabidpsis thaliana pah1pah2 mutant line (Nakamura et al, in preparation). EMS (methanesulfonic acid ethyl ester) mutagenesis Seeds were pretreated at 4 C for 4 days, dried at 25 C for 24 hours, and added to 0.2% aqueous solution of EMS for 12 hours. Mutagenized seeds (M 1 ) were expected to have a point mutation per 50 M 2 lines screened. Gamma Ray mutagenesis 0.65 gray/min (200 grays) for 20 min. Done by Dr. Marie-Helene Montane. Seed planting and growth conditions seeds per square of soil was planted, and kept at 4 C overnight to synchronize germination. Plants were grown under 24 hours light condition (fluorescent lights) at 25 C. The seeds of T1 plants were collected, labeled as separate lines, and incubated in 37 C for faster germination. Another seeds per line were planted to screen for T2 plants. Screening Germinated plants were maintained for two months until fully mature and flowering. Flowers were observed under a Nikon SMZ1500 microscope. RESULTS Fig 1: Terminal flowers on eph 1 Fig 2: Fused buds on eph 2 2

3 For EMS treated T2 plants, 2 plants showed strong homeotic conversion and meristemic indeterminancy phenotypes. Both were found in line 110, which consisted of several plant individuals, and subsequently named eph (enhancers of pah1pah2) 1 and 2. As shown in Fig. 1, terminal flowers were observed. Flowers developed at the tip of each shoot, and the plant was shorter. For normal plants, the shoot tip differentiates into leaves arranged in a rosette pattern, while auxiliary shoots and flowers grow from the side of the main shoot. In Fig. 2, buds were fused together, unable to fully develop or open. No fertilization or seed production was possible, thus the line can only be continued by maintaining the heterozygous parent plant. DISCUSSION The problem outlined in the introduction was how to study the role of lipids in floral organ development when the frequency of pah1pah2 phenotype was so low. The discussion of results will be as to whether pah1pah2 phenotype was successfully enhanced, and the implications of the observations. For eph 1, shoot apical meristem (SAM) tissue was prematurely converted to floral organs. This resulted in terminal flowers, shorter plant appearance, and fewer leaves. Normally SAM tissue maintains indeterminate growth, allowing the shoot to continue elongating until scenescence, while auxiliary meristemic tissue differentiate into leaves, floral organs, and auxillary inflourescence shoots. This phenotype is similar to that seen in studies on TFL1 and TFL2. According to Conti et al (2007), tfl1 mutants have terminal flowers, shortened vegetative phase, and produce fewer leaves, shoots, and flowers. TFL1 gene was found to inhibit floral meristem identity genes in SAM, such as LFY and AP1 (Jack, 2004), thus regulating flowering period and location (Hanzawa et al, 2005). The TFL1 protein is a mobile signal that inhibits mitogen activated protein kinase pathways, preventing plant tissues from differentiating into reproductive organs (Jack, 2004), but does not enter peripheral cells of meristemic tissue (Conti et al, 2007). Tfl2 mutants also have terminal flowers, and early flowering. Compared to tfl1, the flowers were less homogenous, plants had a shorter appearance, and were less responsive to photoperiod induction of flowering. (Larsson et al, 1998). TFL2 inhibits floral meristem identity genes, and also regulates meristem identity response to photoperiods (Larsson et al, 1998). By comparing the phenotypes of eph 1, tfl1 and tfl2, we can infer that EMS mutagenesis might also have disrupted the repression of floral meristem identity genes of SAM tissues. Due to the presence of pah1pah2 background, it is possible that the point mutation affected a gene that functions in relation to PA synthesis, increasing the frequency of abnormal floral phenotype found in pah1pah2. The fused buds in eph 2 is also a significant mutation. Organ fusion occurs when the outer epidermal cell wall and cell cuticle are made permeable. During reproductive development, these tissues are modified to allow 'contact-mediated cell interactions' of organ fusion and pollen hydration (Pruitt et al, 2000). Two known genes that are involved in organ fusion are FIDDLEHEAD (FDH) and HOTHEAD (HTH). From the same study done by Pruitt et al (2000), FDH has been found to inhibit such inter-cellular communication during vegetative development. Interestingly, FDH protein is found to be part of lipid biosynthesis in plants, producing long chain lipids that are located in the cuticle. Cuticle permeability can be altered by changing the lipid composition. 3

4 The FDH gene shares conserved regions with a larger class of genes that code for beta-ketoacyl-coa synthases. These enzymes are the first catalysts of beta-ketoacyl-coa esters, via condensation with malony-coa. Also, FDH is similar to genes that produce chalcone synthases, also part of lipid biosynthesis. Pruitt et al (2000) postulates that FDH encodes an enzyme that catalyzes condensation reactions between malonyl-coa and novel lipids. Both the similarity of FDH to genes involved in lipid biosynthesis, and the fact that change in lipid composition of cuticles can lead to increased permeability and organ fusion, provides strong evidence that lipids play a role in floral meristem and organ development. HOTHEAD (HTH) also inhibits interaction between epidermal cells during flowering, allowing each organ to develop separately. Thus, hth mutants have fused organs, due to increased cuticle permeability, although plants still remain semifertile (Krolikowski et al, 2003). Sequence analysis of the genes show that HTH expresses an enzyme related to a family of FAD containing oxidoreductases. Again, the implications of the appearance of organ fusion in eph 2 is that a gene that also controls lipid composition of cuticle and epidermis tissues during floral development was mutated, possibly in association with the pah1pah2 effect. The identification of at least two successfully enhanced pah1pah2 provides a better foundation to study the link between lipids and flowering in plants. The appearance of fused buds in eph 2 especially suggests that lipids help to regulate formation of flowers, a hypothesis strengthened by comparison to fdh mutants. This hypothesis will only be confirmed and the functional lipid isolated after gene mapping and a thorough biochemical analysis of basic lipid profile. REFERENCES Buchanan B., Gruisseim W., Jones R. (2000), Biochemistry and Molecular Biology of Plants, American Society of Plant Physiologists, Maryland. Conti L., and Bradley D. (2007), 'TERMINAL FLOWER1 is a mobile signal controlling Arabidopsis architecture', The Plant Cells, Vol. 19: Hanzawa Y., Money T., and Bradley D. (2005), 'A single amino acid converts a repressor to an activator of flowering', Proceedings of the National Academy of Sciences of the United States of America (PNAS), Vol. 102(21): Jack, T. (2004), 'Molecular and genetic mechanisms of floral control', The Plant Cell, Vol. 16:S1-S7. Krolikowski K.A., Victor J.L., Wagler T.N., Lolle S.J., and Pruitt R.E. (2003), 'Isolation and characterization of the Arabidopsis organ fusion gene HOTHEAD', The Plant Journal, Vol. 35: Larsson A.S., Landberg K., and Meeks-Wagner D.R. (1998), 'The TERMINAL FLOWER2 (TFL2) gene controls the reproductive transition and meristem identity in Arabidopsis thaliana', Genetics, Vol. 149: Pruitt R.E., Vielle-Calzada J.P, Ploense S.E, Grossniklaus U., Lolle S.J. (2000). 'FIDDLEHEAD, a gene required to suppress epidermal cell interactions in Arabidopsis, encodes a putative lipid biosynthetic enzyme', PNAS, Vol. 97(3):

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