Which pigment appears � rst in the corolla — patterned or background ?


 Flowers display a diversity of pigment patterns on petals – spots, stripes, blotches, and varying combinations of these. Such pigment patterns are accompanied and surrounded by a background that is a contrasting shade or colour sometimes white. We ask the question: Do the pattern and background colours appear simultaneously or successively, and if the latter, is there a bias in which one appears first? We studied the morphological development of flowers of 35 species containing both types of pigmentation, sampled from clades across angiosperms (monocots, Ranunculales, Caryophyllales, rosids and asterids) to address this question of timing of occurrence of the two types of pigmentation. In 28 of the species studied, pigment pattern started appearing in the corolla earlier than the background colour. Pigment pattern appeared later in four cases, and simultaneously with background colour in three cases. Thus, our results reveal, for the first time, variation in developmental sequence of pattern and background colour, with an apparent tendency toward earlier appearance of pigment pattern in the corolla. We hypothesize that the mechanisms involve the imperatives of pigment types, reaction kinetics, differential gene expression, and reaction-diffusion models.


32
In angiosperms, flowers, more than any other organ, display a large variety of pigment 33 patterns. Floral colour patterns are localized in clearly confined and generally well- A pigment pattern on the corolla, both in its presence and localization or design, is 52 generally a stable, heritable trait that appears consistently in succeeding generations, with 53 its appearance governed by genes, its shape being either regular (geometric) or irregular. 54 Pigment patterns other than stable, heritable patterns also existe.g., colour break 55 patterns (Hunter et al. 2011) but are not the subject of this study. There are various types 56 of pigment patterns on corolla (Fig. 1), and the major types are streaks, spots, and 57 blotch/es (Wheldale-Onslow, 1925;pers. obs.). 58 It is known that the expression of pigments (anthocyanins, carotenoids, betalains,  ;Schwinn et al. 2006;Nakatsuka et al. 2008;Chiou and Yeh, 2008;Ma et al. 2009;71 Shang et al. 2010;Yamagishi et al. 2010;Albert et al. 2011;Ohno et al. 2011;Yuan et al.

79
The purpose of this study was to understand one aspect of the development of corolla 80 pigment patterns (CPP) in angiosperms. The main question addressed in this study: Is the 81 emergence of the two types of pigmentation on the developing petal (background and 82 patterned) simultaneous or successive?

84
Morphological developmental study 85 A survey of morphological development of CPP and background pigmentation was done on 86 35 plant taxa sampled across angiosperm (Fig. 2). Care was taken to have as broad sampling 87 as possible across the angiosperms, given limitations of availability. We have included 88 monocots, Ranunculales, and core-eudicots --Malvidae (rosid II) and Fabidae (rosid I), 89 Caryophyllales, and seven families from asteridsasterid I (six families) and asterid II (one). said to be due to the total reflection of light in the absence of pigment (Peach, 1955), these 119 tissues may include UV-reflecting or absorbing areas visible to pollinators, but we did not include flowers with CPP on human-white background in our study as we were interested in 121 pigment development.

123
The sequence of appearance of CPP and background pigmentation was observed and 124 recorded for 35 species (Fig. 3, Supplementary Table S1). Pigment pattern was found to start        Out of the 35 species studied 28 showed CPP development starting earlier than the background pigmentation; four taxa, that are marked with pink coloured branches, showed precedence of background pigmentation over CPP; and three taxa here marked with a blue branch had CPP and background pigmentation emerging simultaneously   Three hypotheses (not mutually exclusive), presented starting from the bottom and connected by black arrows, may in combination suggest the early development of pigment pattern observed in the study. I (bottom), Reaction-diffusion: Interactions between morphogens -an activator (e.g., hormone, TF, or miRNA) and an inhibitor -are indicated by straight arrows. Interaction of the activator with the inhibitor molecule inhibits, whereas accumulation of the activator triggers the localized expression of pigmentation. The nature of the 'trigger" (red star) is unknown, but could be the activator molecule itself, and is likely set off during the cell division phase. II (middle), Differential gene expression: The trigger activates transcription factors R2R3-MYB (sub-group 6) (yellow star, early-acting; orange star, late-acting) that differentially regulate pigment-synthesizing structural genes coding for enzymes of the avonoid biosynthetic pathway, e.g., dihydro avonol reductase (DFR); different copies of DFR are activated at different times --the early-expressing copies act in the CPP region, and the late-expressing copies act in the background. III (top), Reaction kinetics: Early formation of pigment molecules in the CPP region allows time for accumulation of greater amount (quantitative) and variety (qualitative) of pigments as might be required by the reaction rate kinetics of the biochemical pathway, possibly including a ratelimiting step (blue star) The dashed circles indicate CPP regions; blue and purple shapes (triangle, rhombus, pentagon and hexagon) represent different pigment molecules in the CPP and background region. Differential expression of DFR genes and their role in pigmentation, and reaction kinetics DFR gene products, is known; a role for reaction-diffusion is plausible but needs to be tested.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download.