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Origins of Multicellularity

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1 Origins of Multicellularity
Chlamydomonas Gonium Pandorina Eudorina Pleodorina Volvox As we move from single-celled organisms to multicelled organisms, we need to consider a big question - WHY? Life emerged about 3.5 billion years ago (BYA), eukaryotic organisms about 2 BYA, and multicellularity about 1 BYA (around the divergence of the plant and animal lineages). In order to form multicellular organisms, cells needed to stay together after mitosis and typically exhibit some functional division of labor, typically a split between the soma (body) and the reproductive germ line (gonads/gonidia). Different levels of cooperation are observed among different green algae, ranging from single cells such as Chlamydomonas (A) to cooperating groups of clonally-derived cells that function together, as in Pandorina (C) and Eudorina (D). Volvox (F) consists of a flagellated colony of several thousand cells that forms daughter colonies as well as gametes internal to the colonial sphere. Division of labor, protection and provisioning of offspring all become possible as the soma grows larger and more elaborate. Diversification is an investment strategy pursued by multicellular life forms offering evolutionary benefits (to draw on an Economics model). This human construct of the “Volvocine Lineage” was originally based upon a speculative anatomical comparisons, but has been supported by phylogenetic analyses of the green algae. The evolution of multicellularity as a life strategy also may have been driven by cheaters (remember Dictyostelium) - in forming an integrated body that promotes the passing along of genetically-related gametes (evolutionary fitness) an organism could defend its investment in reproduction. By forming an integral unit, multicellular organisms also became the focus of evolutionary selection (rather than individual cells) and this led to rapid radiation into new organismal forms. cached Structural Progressions in Colonial Green Algae “The Volvocine Lineage”

2 Alternation of Generations
Evolutionary Emphasis on the Diploid Soma Diploid (2n) Phase Haploid (n) mitosis sori 2n Fertilization Meiosis Ferns As in other sexually-reproducing eukaryotes, plants have an alternation of generations. As multicellular organisms emerged, evolution has favored the diploid generation for the elaboration of the soma. Haploidy opens greater possibility for genetic variation (mutations are immediately expressed in haploid cells) and the immediate culling of deleterious alleles; conversely, diploidy (having two copies of each gene) is more tolerant of somatic mutations and promotes mixing/matching with each generation. In single-celled eukaryotes (such as Chlamydomonas) the haploid phase of the life cycle often predominates. In these organisms the haploid cells divide mitotically before differentiating into sexually competent cells (gametes) that seek out a cell of the opposite mating type, fusing and then entering meiosis to regenerate haploid cells (often without an intervening mitotic diploid stage). For “simple” multicellular plants (like mosses and ferns), both haploid and diploid plant forms exist. In mosses, the visible part of the plant is largely a haploid organism (the gametophyte, the gamete-forming plant); the diploid phase (the sporophyte, or spore-forming plant) forms (often growing directly on the gametophye). The diploid (2n) sporophyte undergoes meiosis to make haploid (n) cells and matures in a reduced spore capsule. In ferns, the haploid organism has been reduced to small, flat, almost microscopic growths that generate eggs and sperm that unite to initiate the larger diploid form that we recognize as ferns In “higher” plants (conifers (gymnosperms) and flowering plants (angiosperms), the haploid generation has been reduced to non-mitotic pollen and ovules, analogous to mammalian sperm and eggs, and (as in animals) the diploid form predominates. cached cached cached mitosis 2n n n Mosses

3 Arabidopsis thaliana The Thale or Mouse-Eared Cress
And that brings us to the focus of this chapter, the model flowering plant Arabidopsis thaliana. Thale is a location in Germany where this species occurs naturally as a weed. We have similar members of the Mustard/Brassica family growing as weeds here in the Eastern US. This family also includes the Mustard Plant (mustard is ground seeds), Canola (also known as Rapeseed, from which Canola Oil is obtained), and Brassica oleracea (a single pleiomorphic (many formed) species from which has been selected cabbage/brussel sprouts/broccoli/cauliflower). cached http:// cached Other on-line resources: Here’s another great on-line course on Plant Biology from Jim Haseloff at the University of Cambridge And a page of Arabidopsis floral mutants

4 The Arabidopsis Life Cycle
Embryo (2n) Ovule (n) Pollen (n) For the majority of its life cycle, Arabidopsis is a diploid organism. The dormancy of the embryo is broken upon germination. The seedling root emerges from the protective seed coat, followed by the cotyledons and hypocotyl. This plant then enters into a period of vegetative growth (making a rosette of leaves) before switching into a floral program that creates flowers. Each flower generates (via meiosis) haploid pollen (borne on anthers) and ovules (protected within the carpels of each flower). Fertilization (pollination) and the restoration of the 2n diploid amount of DNA, occurs as pollen lands upon the receptive surface of the stigma and grows (by tip growth, similar to yeast schmooing) through the the style to the ovary. Embryos complete seed development within the carpels (ovaries) of the mother (which grow to become the seed capsule). The seeds mature as the plant dies (a final Shakespearian act), and as the tissues dry out the seeds become dormant and are released as the capsule splits. The cycle of life turns anew! cached flowering rosette growth

5 14% transposable elements 25,500 genes
Nuclear Genome 5 chromosomes 125Mb (10x yeast) 14% transposable elements 25,500 genes (4.5x yeast) 11,000 gene families A chromosome-wide view of the 125 Mb Arabidopsis genome (10x yeast). Arabidopsis has 5 pairs of chromosomes, containing roughly 25,500 actual/predicted genes (4.5x yeast), that was completely sequenced in Each chromosome is larger than the entire yeast genome (12 Mb). The density of genes, ESTs (expressed sequence tags), transposable elements (TEs), organellar DNA insertions (MT/CP) and RNA encoding sequences are shown, ranging from red (high density) to blue (low density). Note that the centromeric regions of the chromosomes (denoted by the blue zones on the red chromosomal representations in the top line of each display) are relatively gene poor, as it almost universally the case. (The centromeric DNA is largely repeated elements that template the assembly of kinetochores during mitosis/meiosis). The close packing of genes, with limited intervening repetitive DNA, makes this a very compact genome for molecular studies. +79 (chloroplast) +58 (mitochondrion)

6 COGs in the Genomics Wheel
At the time the Arabidopsis genome was sequenced in 2000, almost half of the 25,500 genes were unclassified. While this number has dropped, figuring out the function of the entire genome remains a top priority. Functionally, this genome (as all genomes) too is being annotated with respect to COGs or clusters of orthologous groups. While some metabolic functions are shared by all forms of life, some are characteristics of oxidative phosphorylation, and yet others are the hallmarks of a photosynthetic organism. Still others are involved in the multicellular style of life. cached ca 2000

7 Transformation by Agrobacterium the Crown Gall Bacterium
Lateral gene transfer continues to shape genomes across the tree of life. Agrobacterium, the crown gall bacterium, is naturally able to infect plants to create gall-like tumors; it does so by transferring genes involved in hormone biosynthesis to the host to create a growth that harbors the bacteria. Genetic engineers have modified Agrobacterium to retain the ability to transfer DNA (T-DNA vector) to a host, but have deleted the genes involved in tumor formation. This is the most efficient way to transform dicotyledonous plants and it the primary route for the introduction of novel genes/constructs into plants. The floral dip method simply inverts a flowering plant into a solution of Agrobacterium tumefaciens. We will be examining Agrobacterium-transformed Arabidopsis seedlings in this week’s laboratory. cached cached

8 Germination Shoot apex Root apex
imbibition, vernalization -> radicle outgrowth Germination Shoot apex hypocotyl elongation (note apical hook) Photomorphogenesis (hook opening, cotyledon greening) Seeds of different plants require specific cues to germinate. Temperature, day length, light quality (red vs. far red wavelengths) are all environmental information used by plants to determine when to start growth. Dormancy is imposed during seed maturation; genetic mutants (called viviparous) do not arrest and germinate on the mother plant! After imbibition (water absorption), germination in Arabidopsis is more synchronous if the seeds are chilled for several days (vernalization), mimicking a cold season. The embryonic tissues expand and the radicle (root tip) forces a crack in the seed coat and begins to grow out. As the seedling senses light, it undergoes photomorphogenesis - apical hook opening, greening and a thickening of the stem. cached cached cached Root hairs Root apex

9 Cryptochromes and Phytochromes
Photomorphogenesis Cryptochromes and Phytochromes The etiolated seedling on the left shows pronounced hypocotyl elongation and lack of greening that occurs if the seedling germinates in the dark. Upon illumination, hypocotyl elongation ceases, the apical hook opens, and chloroplast chlorophyll biosynthesis is initiated. This complex coordination is regulated by blue light receptors called cryptochromes and the red/far red receptors called phytochromes. Etiolated growth makes the most of the limited store of energy in the seedling; it is only once light is sensed that the plant invests in energetically expensive chlorophyll biosynthesis and receives a photosynthesic payoff. cached

10 Three Primary Tissues Dermal Ground Vascular Epidermis General Tissue
Xylem Phloem The plant body is constructed of three primary tissue types: dermal, ground and vascular. The (epi-) dermal layer covers the surface of the plant whereas the ground tissues literally fill the plant body. The upper panel shows a series of sections through developing leaf bud (called a primordium) (using a stain that highlights cell wall material); note the vascular tissue differentiates within the ground tissue in a contiguous line suggesting the importance of cell-cell interactions as well as position. The vascular system, outlined in the lower image using an enhancer trap GUS construct (an approach we will be using in lab this week), forms conductive tissue that moves solutes around the plant.. Water and inorganic ions travel up the xylem from the roots, and photosynthate travels down the phloem to feed the roots (remember food/photosynthate flows/phloes down). Division of labor means that all partake of the harvest. cached cached Enhancer trap GUS construct

11 Plant Embryogenesis Topless-1 embryos make an apical root
Following fertilization, then new plant undergoes an asymmetric division separating a smaller, apical terminal cell from a larger basal cell (roughly 40/60, think of Caulobacter!). The apical cell divides to generate the embryo proper (yellow), whereas the basal cell generates the suspensor (sort of a plant placenta, pink) connecting to the mother plant’s ovary tissues (later generating the root meristem). This initial asymmetric division requires a gene called Topless. Topless protein is a negative regulator of root production in the shoot cell; tpl-1 mutants make an apical root! Interestingly, Topless is a transcription factor and shows genetic interactions with histone deacetylases (involved in nucleosomal packing). Literature link: Long et al (2006) TOPLESS Regulates Apical Embryonic Fate in Arabidopsis. Science 312(5779: ) DOI: /science The embryo grows from a globular stage through a torpedo stage to a heart-shaped stage as the two cotyledons (seed leaves) develop. Monocots and Dicots are two basic developmental plans used by flowering plants. Arabidopsis is a dicotyledon (or dicot), whereas corn (our next subject) is a monocotyledon (monocot). Embryogenesis establishes an apical-basal axis (shoot meristem, cotyledons, hypocotyl, root) and the radial arrangement of primary tissues (dermal, ground and vascular). Root and shoot meristems are established as well as storage tissue (the cotyledons or “first leaves”). This axial and radial patterning established the body plan of the plant. It has been estimated that as many as 4,000 genes may be involved in the process of embryogenesis. cached tpl-1 embryo images from cached

12 Shaping the Plant Cell Cellulose Synthase Microtubules Overlay
Because plant cells cannot move, the planes of division and elongation play a key role in determining the form of the plant body. Plants are literally held up by osmotic turgor pressure (think of a wilted plant!) keeping the cells turgid (full of water). The cells are surrounded by a wall of cellulose polymer fibers embedded in a matrix of pectins and lignins. Plants sense gravity in both stems and shoots, orienting the plant body appropriately through directed growth. Plant cells synthesize their walls via cellulose polymer “factories” directed in the plane of the cell membrane by intracellular microtubules. These align the wall filaments in a way that permits growth by controlled wall loosening, driven by osmotic pressure not unlike strategies used by prokaryotes (remember those MreB filaments directing murein synthesis!). This wall loosening is governed by the plant hormone auxin. cached Literature link: Desprez T, et al. Organization of cellulose synthase complexes involved in primary cell wall synthesis in Arabidopsis thaliana. Proc Natl Acad Sci U S A Sep 25;104(39): PMID: Cellulose Synthase Microtubules Overlay Complexes

13 Meristems New plant tissues are generated from meristems, small groups of cells that remain permanently embryonic in character which divide to generate cells that differentiate into the three primary tissue types - truly stem cells in a variety of senses. Shoot meristems are autonomous and capable of regrowing an entire plant (in fact, most single plant cells are totipotent and can be used to regenerate entire plants). Plants have a great capacity to regenerate lost tissues and organs by continued meristematic activity. Anyone who has taken a piece of a plant shoot and rooted it in water quite literally has cloned an organism! As the cells of the shoot meristem divide, some of these are crowded out of the meristem proper and begin to differentiate. An important activity is the secondary organization of groups of cells called leaf promordia that will initiate a lateral outgrowth of the shoot tip to form a new leaf. Leaf primordia initiated in a spiral around the shoulder of the meristem generate the basal rosette of leaves during the vegetative phase of the plant. Vegetative shoot meristems ultimately convert into reproductive structures, forming the flowers (which in turn will generate the pollen and ovules). This is very different from animal development where the germ line is set aside early in development. Root meristems are different, growing away from light and down in response cached cached cached

14 Organization of Shoot Meristems
Tunica L3 Radial Zones Layers Organization of Shoot Meristems The Arabidopsis SAM (shoot apical meristem) consists of about 100 cells that are organized into functional groups and layers. These false-colored sections show a shoot apical meristem and two adjacent floral meristems. Radial Zonation - cells within the meristem divide at different rates to form a dome-like structure from which lateral outgrowths emerge to form leaves or floral structures. Thus cell cycle control is important in shaping the organism, and involves populations of cells in the central zone (CZ), peripheral zone (PZ), and rib zone (Rib). As they transit from the meristem proper (the colored zones), groups of cells organize and proliferate to generate outgrowths such as leaves or floral structures, often in a helical arrangement along the shoot axis. Layering - The meristem is also layered. L1 is a single-cell thick surface layer (epidermis) that always divides in a 2D sheet to cover the plant. L2 is a layer immediately below L1 that also divides perpendicularly to the surface. L1 and L2 together are called the tunica (clothing the plant body). L3 (the corpus or plant body) is the remainder of the meristem; these cells divide in all planes and fill the inside of the meristem and contribute the core of the shoot tissues (including cells that will ultimately differentiate into the vascular tissues). Photosynthetic mutations in the cells of one of the L layers results in a chimera (a monster of Greek mythology derived of tissues from multiple animals); these attractive variegated plants are often propagated bu cuttings (cloning) by horticulturists (they don’t breed true because ovules and pollen are produced from L3 cells). cached cached L1/L2/L3 chimeras

15 Embryo/Seedling Mutants
Apical-basal mutations (b-e) Screens for mutations resulting in misshapen seedlings identified genes involved in the apical-basal axis, radial symmetry and organogenesis. gurke (gk), fackel (fk), monopterous (mp), and gnom (gn) all lack segments along the apical-basal axis knolle (kn) and keule (keu) have disturbed radial symmetry fass (fs), knopf (knf), and mickey (mic) have proper axial and radial arrangement, but misshapen organs (wt) is the wild-type seedling, for comparison cached Radial symmetry mutations (f,g) Organogenesis mutations (h-j)

16 Axial Mutants Axial mutants cluster into apical, central and basal effects (an example of each is shown here). This patterning has been likened by some to anterior-posterior pattern formation in Drosophila, although with far fewer segments it may be that there are 3 distinct programs for the shoot, the hypocotyl and the root. cached

17 Root Structure Cross section of root
Root meristems are similar to but distinct from shoot meristems. They too contain zones and layers. Cell files leading back from the root meristem offer a unique view into the differentiation of the vasculature and other tissues as position correlates with developmental time. Note the distinct zones of division, elongation and differentiation as cells become located farther from the root meristem. Literature Link: Ortega-Martinez et al (2007) Ethylene modulates stem cell division in the Arabidopsis thaliana root. Science Jul 27;317(5837): PMID: Literature Link: Veit B. (2007) Plant biology: plumbing the pattern of roots. Nature Oct 25;449(7165): No abstract available. PMID: Images from cached

18 Root Hairs video microtubules
Root hairs are polarized outgrowths of epithelial cells that increase the surface area of the root to enhance absorption of water and nutrients. They grow by localized wall loosening and delivery of material to the tip of the elongating root hair in a process that involves the microfilament and microtubule cytoskeletons (think about yeast growth!). Root hairs often emerge from the root surface in specific patterns. cached http:// cached cached cached microtubules

19 Epidermal Fates gl1 ttg No Leaf Hairs WT But Lots of Roots!
Mutants in the glabra/glabrous (gl1, gl3) and transparent testa glabra (ttg) genes have a variety of epidermal defects, including the loss of leaf hairs (trichomes). These genes have interesting and sometimes counterintuitive effects on root hair development: in gl2, root hairs, like trichomes, are absent, but in ttg almost every root surface cell is converted into a root hair. These loci encode master switch proteins (transcription factors) that are deployed in the epidermis but trigger different events in root vs shoot development. Trichomes and root hairs are important environmental interfaces. Trichomes protect the plant from high light levels, excessive water loss and herbivory (consumption by insects and other chewing animals). Root hairs anchor the roots to soil and promote water and nutrient uptake. Interestingly, cotton fibers are long trichome outgrowths that occur from the seeds within the seed capsules. Human desire for better material for cloth production has resulted in selection for longer, stronger fibers. GL1 (the wild type gene) encodes a DNA binding domain of the MYB class of transcription factors. TTG does not contain a DNA binding domain, and is thought to affect the activity of other proteins that are transcription factors (including GL1 and Glabrous3 (GL3). cached cached cached Scanning electron micrograph (upper left) from cached Epidermal Fates

20 Root Hair Specification
WT has 8 files of root hairs GL2::GUS expressed in N cells Root Hair Specification Root hair patterning is achieved by cell-cell interactions/signaling. Epidermal cells directly over cortical cells (N) will not become root hairs, whereas those over the cortical cell-cell junctions (H) will become root hairs. There are typically 8 longitudinal files of root hairs along a root each separated by 1-2 files (10-12 in total number around the circumference of the root) of non-root hair epidermal cells. The N cells express a transcription factor called Glabra/Glabrous 2 (GL2), shown upper left via expression of a GL2 promoter::GUS fusion. While GL2 is a suppressor of root hair initiation in the root, in the shoot it helps turn on trichome outgrowth and branching. Thus there is sort of a forked decision point that, when a path is chosen, leads to different identities in different tissues. The fate of the cell is determined by the relative abundance of two antagonistic factors, Werewolf (Wer) and Caprice (Cpc), each of which form a heterotrimeric transcription factor complex with TTG and a basic Helix-Loop_Helix transcription factor (bHLH). When Wer is present, Glabra 2 (Gl2) expression is turned on and the cell is inhibited from forming a root hair (thus is an N cell) wer mutants have excessive numbers of root hairs as none of the cells are inhibited When Cpc is present, Glabra 2 (Gl2) expression is suppressed and the cell forms a root hair (thus is an H cell) cpc mutants do not effectively suppress Gl2 and root hairs don’t form most of the time (they still occur but capriciously!) cached ( )

21 The Flowering Stimulus
Day length and Temperature leaf phloem apex FL-C FL-T The Flowering Stimulus Florigen is the protein encoded by the FL-T locus; it is produced in the leaves and moves through the plant to the meristems to transmits the flowering response through the plant Another process of morphogenesis involves the conversion of a vegetative meristem into a floral meristem, often in response to environmental (seasonal) cues. Constans (CO) is a gene expressed in leaves encoding a protein involved in day length perception. Long days activate expression of Flowering Locus T (FL-T) leading to production of “florigen”, the classic flowering “hormone”, a protein that travels via the vascular system to regulate transcription factor activity in the shoot tip, changing patterns of gene expression in the meristem. FL-T is negatively repressed by the FL-C gene product to suppress flowering before an appropriate winter (cold) stimulus. Cold exposure (vernalization) causes changes in histone modifications (epigenetic silencing) at the FL-C locus (mediated by the VernalizationInsensitive3 and Vernalization2 gene products, turning off FL-C protein synthesis and releasing FL-T expression. Interestingly, this control network has been conserved across the flowering plants to the woody trees, where it is also involved in bud development / dormancy in the Fall. Literature Link: Bohlenius H, Huang T, Charbonnel-Campaa L, Brunner AM, Jansson S, Strauss SH, Nilsson O. CO/FT regulatory module controls timing of flowering and seasonal growth cessation in trees. Science May 19;312(5776): Literature Link: Rubio and Deng (2007) Standing on the shoulders of GIGANTEA. Science Oct 12;318(5848): PMID: Images from cached cached cached

22 Conversion to a Floral Meristem
stigma, carpels Conversion to a Floral Meristem Vegetative meristem Floral meristem identity genes (Lfy, Ap1, Cal; Tfl) Floral meristem Mapping genes (define boundaries) (Su) Floral organ identity (homeotic) genes (whorl identity) Sepals (Ap2) Petals (Ap2, Ap3, Pi) Stamens (Ap3, Pi, Ag) Carpels (Ag) Leafy (Lfy), Apetala1 (AP1) and Cauliflower (Cal) are meristem identity genes that are involved in the conversion from a vegetative meristem to a floral one; Terminal Flower (Tfl) maintains floral identity once it is established. lfy mutants replace flowers with leaves. Overexpression of lfy results in earlier flowering Unusual Floral Organs (UFO) is a mutation that has unusual numbers of structures Superman (SU) is a mapping gene that defines whorl boundaries within the floral meristem Apetala2 (AP2), Agamous (AG), Apetala3 (AP3) and Pistillata (PI) are floral organ identity genes. They are homeotic genes in that they specify organ identity and when mutant result in the substitution of one organ identity for another. Class A genes specify sepal development (AP2) Class A and B genes specify petal development (AP2, AP3, PI) Class B and C genes specify stamen (also called anther) development (AP3, PI, AG) Class C genes alone specify carpel development (AG) Interestingly, at least some of these genes (AP2) are under RNA-mediated (RNAi) control cached Floral diagram from cached

23 The ABC Model of Floral Whorl Identity
B (Ap3, Pi) A (Ap2) C (Ag) Sepals Petals Stamens Carpels ag ap2 pi Flowering is the result of cascades of transcription factor activation/expression that govern the expression of downstream genes that shape the organs and result in different developmental fates for the cells in which they are contained. Sepals, petals, stamens and carpels are all the result of mutually exclusive genetic programs that turn on different suites of genes in specific places at specific times. In the ABC model of floral development, the floral meristem is separated into 4 whorls, specified by three overlapping fields of gene activity. Each field determines the identity of two different whorls. This ensures that the organs occur in the correct order, something not necessarily achieved by a system in which specification occurred independently. A = sepals / AB = petals / BC = stamens (which bear the pollen) / C = carpels (which bear the ovules) A and C are antagonistic, in that mutations that remove A function permit C function to expand across the meristem, and vice-versa. AP3 and PI cooperate to form a heterodimeric transcription factor in B. Loss of function mutations in either gene (ap3 or pi) have identical phenotypes. This model, derived in Arabidopsis, is applicable to floral development across the angiosperms; this floral program emerged early in the evolution of the flowering plants and has been maintained ever since. Mutations in the ABC genes are called homeotic mutations because they result in the transformation of an one organ from one identity into another identity (e.g. in agamous, the stamens and carpels are transformed into extra petals). agamous (ag) mutations were identified in 1943 and cloned in 1990; agamous mutants are often selected and maintained by gardeners who appreciate their showy (but often sterile) flowers! cached cached cached cached

24 A Few Questions for Thought
Explain the evolutionary origins of multicellular organisms. What are the benefits and costs of multicellularity? Compare and contrast plant and animal (drawing upon your own general knowledge) body plans. Describe how a new plant is formed during development (from fertilization through germination). What are meristems, and why are they important in plant biology? What are the three primary tissues of the plant body? What is the difference between a shoot and a root meristem? Between a vegetative and a floral meristem? How are organ identities in determined in the flower? What are the role(s) of homeotic genes in this process? Here are a few review questions for thought that highlight some of the major points from this chapter. You may choose to answer these on your own, as part of a small study group, or in your discussions in GLG and/or BQC.

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