Shoaib Munir

Ph.D. Student
Nationality: Language Known: PAKISTANI English, Urdu,

Punjabi

A Brief Introduction about PAKISTAN

Pakistan is one country many people like to see, and having seen it once, wish to return again and again. Islamic Republic of Pakistan is a sovereign country located in South Asia, bordering the Arabian Sea, India on the east and Iran and Afghanistan on the west and China in the north. PAKISTANI culture is considered to be amongst the world's oldest, richest and most diverse. PAKISTAN is very rich in biodiversity, extensive natural gas reserves, petroleum, coal, iron, copper, salt, gold. PAKISTAN is a federation composed of 4 provinces, 1 territory*, and 1 capital territory.

Beautiful Places in PAKISTAN

The Lahore Fort

Faisal Mosque, in Islamabad

Lahore

Swat, Khyber Pakhtunkhwa

Karachi

K2 (second-highest mountain on Earth)

Synchronization of the flowering transition by the tomato TERMINATING FLOWER gene

Cora A MacAlister, Soon Ju Park, Ke Jiang, Fabien Marcel, Abdelhafid Bendahmane, Yinon Izkovich, Yuval Eshed & Zachary B Lippman. November 2012. Nature Genetics

Background of Research
Variation in plant reproductive success and agricultural productivity is largely determined by differences in shoot architecture, and reproductive shoots known as inflorescences show extensive diversity in both branch and flower number.
(Weberling, F.1989)

Vegetative shoots produce inflorescences when endogenous and environmental signals coincide to induce groups of pluripotent cells called shoot apical meristems (SAMs) to transition to flower-producing inflorescence meristems.(Kobayashi, Y. & Weigel,
D.2007)(Turck, F., Fornara, F. & Coupland, G. 2008)

Background of Research
In monopodial plants such as Arabidopsis thaliana and maize, the shoot apical meristems(SAM) persists after the flowering transition, and the influorescence meristem continuously generates flowers laterally, resulting in a limited range of simple architectures. In sympodial plants such as tomato (Solanum lycopersicum), the primary shoot meristem (PSM) terminates in a flower, and compound shoots arise from specialized sympodial meristems. (Fig.)(Bell,
A.D. 1993)(Pnueli, L. et al. 1998)(Lifschitz, E. & Eshed, Y. 2006)(Lippman, Z.B. et al. 2008)

Background of Research
Solanaceae species like domesticated tomatoes generate zigzag inflorescences range from multiple branches with dozens of flowers to solitary flowers. At the base of the first tomato floral meristem a sympodial inflorescence meristem (SIM) initiates and produces one new SIM, before transitioning to the next floral meristem. Vegetative growth from the axil of the youngest leaf through a sympodial vegetative meristem (SYM), generates three leaves before transitioning into a flower. (Child, A. 1979)(Danert, S. 1958)(Rickett, H.W. 1944)

Background of Research
Mutations in the homeobox transcription factor gene COMPOUND INFLORESCENCE the floral specification complex encoded by the F-box gene AN and Its transcription factor partner FALSIFLORA cause highly branched inflorescences by delaying (s mutants) or blocking flower formation.(Park, S.J., Jiang, K.,
Schatz, M.C. & Lippman, Z.B. 2012)

What & Why tmf

We studied a novel tomato mutant called terminating flower (tmf), whose primary inflorescence is a solitary flower. The tmf mutant flowers early and generates a singleflower primary inflorescence with enlarged leaf-like sepals (Fig.1 d,e). (Lukyanenko, A.N., Ochova, E.P. &
Egeyan, M. 1973)

The termination of the primary meristem in the tmf mutant occurs without SYM or SIM formation, leading to temporary growth arrest of the shoot (Fig. 1b,h). The tmf single-flower phenotype shows 50% penetrance in the original mutant background, but unknown modifiers affect the tmf phenotype.

The roles of TMF in shoot organization.

By examination of leaf complexity, which is an indicator of the juvenile-to-adult transition, showed that tmf mutants do not precociously reach adulthood, suggesting that floral identity is adopted at an earlier vegetative meristem stage. (Poethig, R.S. 2003) The leaf-like sepals of the tmf mutant (Fig.1 d,e) suggest that leaf identity is inappropriately incorporated into the first floral organ whorl as the result of an abrupt floral transition.

The roles of TMF in shoot organization.

(a) Diagram of the compound vegetative and inflorescence shoot systems of wild-type (WT) tomato and tmf mutants. The wild-type PSM terminates in the first flower (red circle) after producing its final leaf (L8), and 56 additional flowers (orange and yellow circles) are produced by the specialized SIMs. Vegetative growth continues from a SYM, which produces three leaves and initiates the next multiflowered inflorescence. tmf mutants flower after the production of four leaves (L4), and the PSM does not generate SIMs or SYMs. Canonical axillary meristems (blue arrows) are eventually released from dormancy and become typical side shoots. (b) Schematic of meristem arrangement at the floral transition in WT tomato and tmf mutants. FM, floral meristem; AxM, canonical axillary meristem.

The roles of TMF in shoot organization.

(c) The wild-type tomato cultivar (cv.) Break o' Day produces 5 7 flowers per inflorescence. (df) In contrast, the primary tmf inflorescence is a single flower with abnormally large, leaf-like sepals (d). The axillary meristems of the tmf mutant (e, white arrow) and their inflorescences are generally normal (f) relative to wild-type tomato (c). (g,h) Scanning electron micrograph at the flowering transition in wild-type tomato (g) and tmf mutants (h). Scale bars, 200 m. Leaf (L) and sepal (S) numbers are as marked. (i) Number of leaves produced before the floral transition in wild-type plants, a population of fully penetrant tmf mutants (tmf 100%) and a tmf line of incomplete penetrance by morphological class.

Loss of tmf does not cause an accelerated transition from juvenile to adult growth (a) Representative final leaf produced by the PSM before floral termination of WT (left) and tmf (right). (b) Quantification of leaf complexity (mean number of primary, secondary and intercalary leaflets). Leaf complexity increases over developmental time, but tmf does not exhibit precociously increased complexity, suggesting it is not undergoing a more rapid transition from juvenile to adult growth (phase change), despite precocious flower formation.

Double mutants between tmf and members of the florigen pathway are additive. Activating and repressing components of the florigenmediated flowering pathway are also known regulators of the transition to flowering.(Shalit, A. et al.
2009)

We generated double mutants of tmf and the florigen mutant single flower truss(sft) and the florigen antagonist self-pruning (sp), and observed additive genetic relationships. (Fig.) its indicating that TMF represses flowering independent of the florigen pathway.

Cloning and expression dynamics of TMF.

We isolated a second allele (tmf-2) with a missense mutation that converts a highly conserved threonine to isoleucine using the targeting induced local lesions in genomes (TILLING) approach.(Menda, N., Semel, Y., Peled, D.,
Eshed, Y. & Zamir, D. 2004)(Henikoff, S., Till, B.J. & Comai, L. .2004)

The tmf-2 mutant did not complement the tmf mutant and recapitulated its morphology, incomplete penetrance (9%) and early flowering phenotype. (Fig.)

The tmf-2 TILLING allele phenocopies tmf and carries a mutation that disrupts a highly conserved amino acid

Cloning and expression dynamics of TMF.

TMF is expressed predominantly in the shoot apex (Fig. 2b) TMF is highly expressed in vegetative stages of the PSM, decreases slightly in the reproductive transition meristem and is weakly expressed in the floral meristem and SIM. Its expression in the vegetative SYM is comparable to that in the primary vegetative meristem (Fig. 2c). We proven these expression dynamics using in situ hybridization, which further showed TMF expression at the periphery of vegetative meristems, extending into initiating vasculature cells (Fig. 2dg).

Cloning and expression dynamics of TMF.

(a) The tmf mapping interval. Red lines indicate marker positions, with the number of recombinants shown below. The red triangle marks the position of the RiderTy1-copia-like retrotransposon insertion. (b) Semi quantitative RT-PCR for TMF transcripts in the indicated tissues. Expression was compared to that of the control UBIQUTIN (UBI) gene. (c) Normalized read counts for TMF (RPKM) across five primary meristem stages: the early, middle and late vegetative meristems (EVM, fifth-leaf initiated; MVM, sixth-leaf initiated; and LVM, seventh leaf initiated), the transition meristem (TM, eighth-leaf initiated) and the floral meristem (FM) and the SIM and SYM12. The vertical dashed line separates PSM stages from sympodial meristems.

Cloning and expression dynamics of TMF.

(df) Spatial distribution of TMF transcripts monitored by in situ hybridization in wild-type tomato. Probe, upper right.

(d) TMF is highly expressed in the EVM stage (8 d after germination (d.a.g.).
(e) Cross-section of the EVM stage showing TMF expression as a spotted ring at the periphery of the meristem and marking lateral organ boundaries. The approximate position of the cross-section is indicated by the white dashed line in d. (f) After the floral transition, TMF is expressed again in the SYM. (g) TMF sense probe control. Scale bars, 100 m.

Overexpression of TMF
The expression dynamics of TMF and loss-offunction phenotypes suggested a role for TMF in maintaining a vegetative meristem state.
How to explore this idea..

Overexpression of TMF
we constitutively expressed green fluorescent protein (GFP) fused to TMF under the cauliflower mosaic virus (CaMV) 35S promoter (35S::GFP-TMF). The GFP-TMF fusion exhibited nuclear localization and rescued all tmf phenotypes, including flowering time and sympodial growth (Fig. ac). We also noted that 35S::GFP-TMF inflorescences produced ectopic leaves, reverted to vegetative shoots and exhibited increased branching at a low but consistent frequency (Fig. 3cf), indicating that forcing TMF activity in reproductive meristems can promote a partial vegetative state.

Overexpression of TMF promotes vegetative characteristics and branching within tomato inflorescences.
(a) Confocal image showing nuclear localization of the GFP-TMF fusion protein (green), with cell walls stained with propidium iodide (red). Scale bar, 20 m. (b) In five of six independent 35S::GFP-TMF transgenic lines, the percentage of each population exhibiting the wild-type phenotype (defined as a multiflowered inflorescence with normal sympodial shoot growth) is significantly higher than in tmf mutants. (c) Multiflowered primary inflorescence of a rescued tmf plant with an ectopic leaf. (df) Expression of GFP-TMF in wild-type plants causes gain-offunction inflorescence defects, including ectopic leaf formation (d, arrowheads; also visible in c), reversions to indeterminate vegetative shoots (e, arrow) and branching (f, white asterisks). Scale bars in cf, 1 cm.

Loss of TMF.
To examine changes in the dynamics of gene expression during the transition in tmf plants, we performed quantitative RT-PCR (qRT-PCR) on three meristem stages (Fig. 4b). In the RNA-seq data, we found that the floral meristem identity genes AN and SEPALLATA3 (SEP3) were already expressed in tmf vegetative meristems. (Fig. 4c) (Park, S.J., Jiang, K., Schatz, M.C. &
Lippman, Z.B. 2012)

But expression of flowering transition genes such as S and a homolog of Arabidopsis FUL was greatly delayed, never reaching normal levels (Fig. 4c).

Loss of TMF drives partial precocious activation of floral termination.

(a) Venn diagram showing that only 10% of floral meristem enriched marker genes are upregulated in tmf vegetative meristems. (b) Sequential meristem maturation stages (EVM, TM, FM) from wild-type and tmf mutant plants. Dashed lines delimit tissue collected for qRT-PCR. (c) qRT-PCR of expression dynamics for the flowering transition genes S and a tomato homolog of Arabidopsis FUL compared to those of floral meristem identity genes AN and SEP3. Values are the averages of two biological and four technical replicates. Expression was normalized to that of the control UBI gene and scaled to the highest expression value for each gene between genotypes.

AN and FA are required for the tmf phenotype.

To investigate the mechanism by which TMF prevents precocious adoption of floral fate in the primary shoot

we focused on AN, which functions in the earliest stages of flower formation and is activated extremely early in the tmf mutant (Figs. 4c and 5a,b) Mutations in AN block flower formation, leading to highly branched inflorescences arrested at the SIM stage. (Allen, K.D. & Sussex, I.M. 1996) Double mutants of tmf and an were indistinguishable from an single mutants (Fig. 5ce), showing that early transcriptional activation of AN is required for the tmf mutant phenotype.

AN and FA are required for the tmf phenotype.

In contrast, S was dispensable for the tmf phenotype, consistent with S transcription remaining unchanged in tmf mutants (Figs. 4c and 5fh). In tomato, FA (homolog of LFY) is up regulated 2-fold during PSM maturation in wild-type plants, (Fig. 5i) but FA was already up regulated by 2.2-fold in the vegetative meristem of tmf mutants. Coincidentally, we identified a dominant mutant from an activation-tagging population with identical phenotype to the tmf mutant, which we named tmf 2-D (Fig. 5j,k).

AN and FA are required for the tmf phenotype.

(a,b) In situ hybridization of wild-type (a) and tmf (b) meristems at different stages probed for AN, showing earlier expression in the tmf mutant. Dashed lines mark the outlines of non-visible leaf primordia. Scale bars in a,b, 100 m. (c,d) tmf primary inflorescences produce a single fruit at later developmental stages (c), whereas an inflorescences are highly branched but have defective, cauliflower-like floral tissue (d). (e) an is completely epistatic to tmf.

AN and FA are required for the tmf phenotype.

(f) s inflorescences are also branched but produce normal flowers. (g) Primary inflorescences in tmf and s double mutants are single flowered as in tmf mutants (arrow). (h) Side-shoot inflorescences are branched as in s mutants, indicating additivity. (i) Normalized read counts for FA, TMF, S and AN during five stages of primary meristem development. (j,k) tmf2-D, an activation-tagging allele of FA resulting from a transposon insertion 1.5 kb upstream of the ATG start site (schematic in j) mimics all tmf mutant phenotypes, including single-flower primary inflorescences (j) and normal side-shoot inflorescences (k). (l,m) fa mutants have highly branched leafy inflorescences (l), which are indistinguishable from those of fa and tmf double mutants (m). Scale bars in c h,jm, 1 cm. (n) Quantification of flowering time on the basis of primary-shoot leaf number. Unlike early flowering in tmf mutants, the fa single mutant and tmf and fa double mutant flower later than wild-type plants.

Altering the timing of AN.

We tested the consequences of the timing of AN activation in promoting early flowering and simplified inflorescence architectures by first expressing AN at an early vegetative meristem stage using the Arabidopsis FILAMENTOUS FLOWER (FIL) promoter. All FIL>>AN plants terminated growth in a single flower after the formation of three simple leaves (Fig. 6a), indicating that the overexpression of either FA or AN can accelerate flowering and cause single-flower inflorescences, possibly as a result of feedback regulation.

Altering the timing of AN .

To express AN just before its normal activation in the floral meristem, we used the S gene promoter (S>>AN) (Fig. 6b). Whereas the most strongly affected plants terminated in a single-flower primary inflorescence (Fig. 6c).

Altering the timing of AN transcriptional activation modulates inflorescence architecture.

(a) Transactivation of AN using the promoter of the Arabidopsis early vegetative meristem and lateral organ gene FIL results in a single abnormal flower after the production of only three simple leaves. (b) The DsRed reporter driven by a 3.9-kb promoter fragment upstream of the S gene shows expression in the transition meristem and early SIM stages. Scale bar, 100 m. (c) Transactivation of AN with the S promoter (S>>AN) mimics the tmf singleflower phenotype in lines with the strongest effects. (df) M82 tomato shoot showing typical three-leaf sympodial cycling and successive multiflowered inflorescences (d). Moderate S>>AN plants showing typical sympodial cycling and conversion of multiflowered inflorescences into single flowers (e) and weak S>>AN plants showing two-flowered inflorescences (f). (g,h) The S>>AN plants in e,f recreate the growth patterns of singleflowered Solanaceae species such as N. benthamiana (g) and two-flowered species like S. cleistogamum (h), respectively. Red arrows mark inflorescences. Scale bars in a,ch, 1 cm.

Conclusion
Notably, through analysis of the tmf mutant, we have found that activating AN expression slightly earlier causes a more abrupt termination that permits normal sympodial cycling yet converts successive multi flowered inflorescences into inflorescences of one or two flowers. By timing AN activation, TMF synchronizes flower formation with the gradual reproductive transition, which, in turn, has a key role in determining simple versus complex inflorescences.

Conclusion
Flowering and inflorescence on plants, vary widely between related species and rely on precise coordination of flowering stages. Classical and recent mathematical modeling suggest that variation in inflorescence architecture might trace back to differences in the rates at which meristems adopt floral identity. Transferring cis-regulatory modules between these closely related species may help to uncover the underlying differences.