Author: HRW

On the Importance of the World’s Smallest Flowers in Flowering Research

Within the aquatic Lemnaceae family are the smallest angiosperms known to exist (Chase,

2004), with some being as small as 0.3mm (Les et al., 2002), having structures barely visible

with the naked eye. These plants have traditionally been categorized as being in the family

Lemnaceae, but genomic evidence is indicating that they should actually be a subfamily within

the family Araceae (Cabrera et al., 2008; Keating, 2004). Systematic studies have determined

that the evolutionary process has so reduced the plant structures in the Lemnaceae that they no

longer have evident stems and leaves;rather, they have a frond structure which functions in

reproduction as a leaf and stem homologue (Lemon and Posluszny, 2000). Some members have

roots or root-like structures whereas others do not (Lemon and Posluszny, 2000; Bernard and

Bernard, 1990). Even though the Lemnaceae primarily use vegetative reproduction (Lemon and

Posluszny, 2000), they are obviously monocotyledonous angiosperms with distinct, working

reproductive organs of anther and ovary (Bernard and Bernard, 1990; Maheshwari and Kapil,

1963; Saeger, 1929).

There are many factors that make the Lemnaceae good subjects for determining triggers of

the flowering process in angiosperms especially since their primary way of reproducing is

vegetatively, but specific environmental factors still can induce their flowering, a mechanism

which initiates sexual reproduction instead. Throughout the years, many have expressed an

interest in the flowering of Lemnaceae. In 1929, Albert Saeger published records of the

flowering of different Lemnaceae going back to 1729. Flowering in these small plants was

considered a rare event and speculation on what initiated flowering peaked early interest. In

1894, H. B. Guppy speculated that constant, optimum temperature and moisture in the aquatic

environment would deter flower production of Lemna minor (Caldwell, 1899). Others observed

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Author: HRW

that more than one species of Lemnaceae would flower at the same time in a pond, suggesting

that chemicals in the pond water were responsible for triggering the process (Saeger, 1929). A.

Saeger (1929) reported that he had successfully induced all stages of flowering in L. minor using

a dilute mineral solution. In 1932, L. E. Hicks was the first to publish research on flower

induction of the Lemnaceae with his series of experiments on L. trisulca, L. cyclostasa, L. minor,

L. minor var. purpureus L. minima and Wolffia columbiana. Hicks tested the effects of the

following: mineral salt deficiency, several nutrient solutions, photoperiodism, light intensity, 60

different chemicals, and ultra-violet rays on these plants. Hicks notes that sodium hydroxide

produced flowers in three of the five species and exposure to ultra-violet light induced flowering

in all. The variety of investigations Hicks was able to perform on these plants is indicative of

factors which make the Lemnaceae good experimental organisms. W. S. Hillman (1976)

addresses several benefits for using Lemnaceae in the laboratory. Since they are free-floating, it

is possible to prepare axenic cultures that allow control of many variables including contact with

soil organisms. Their small floating bodies also make them easy to care for and places plant cells

in close contact with chemicals in the water medium. Further, the Lemnaceae undergo rapid,

vegetative reproduction which produces clonal growths that can be maintained for years with

limited genetic variability. Finally, the Lemnaceae have some structures and mechanisms like

those of larger angiosperms so studies on them can provide insight on the larger plants.

In 1936, M. K. Chailakhyan proposed a theory on the existence of florigen, a flowering

stimulus hormone in plants which triggers the process of flowering (Aksenova et al., 2005;

Macháčková et al., 2001). In his experiments, Chailakhyan induced flowering in tobacco plants

by manipulating their photoperiodic response so that he exposed their leaves to varying day

lengths (Aksenova et al., 2005). He then concluded that at the optimal day length, the plant was

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Author: HRW

receiving something from its leaves that triggered the flowering so Chailakhyan tested this idea

by grafting leaves from induced plants to non-induced plants where he was able to induce

flowering (Aksenova et al., 2005; Macháčková et al., 2001). Since Chailakhyan, many have

tried to isolate the mechanism which induces flowering in angiosperms.

The Lemnaceae have been ideal experimental organisms to test the photoperiodism effect

on inducing flowers, especially since the Lemna frond is a leaf homolog and different species

exhibit different photoperiodic lengths. In his photoperiodic studies on flowering, Hillman

(1962, 1965) exposed short-day (SD) Lemna perpusilla and long-day (LD) Lemna gibba to

different types of lights under long-day (LD) or short-day conditions, and to high and low doses

of various ions in the medium, including copper. L. perpusilla flowered under SD lighting.

When the low concentrations of copper where introduced to the medium, the plant also flowered

under LD lighting, a condition which would normally promote its vegetative growth, instead.

The reverse happened to LD Lemna gibba where the copper inhibits its flowering under LD

conditions. Because low copper uptake promoted flowering in one species and inhibited in the

other, it was determined that in was not a flowering inducer but rather affected both plants’

photoperiodic sensitivity.

Other researchers have investigated the circadian rhythm in plants that control flowering as

a photoperiodic response. Recent genetic analysis of the dicotyledon Arabiodopsis thaliana have

identified late elongated hypocoty (LHY), circadian clock associated1 (CCA1), gigantea (G1),

and early flowering3(ELF3) as circadian clock-related genes involved in photoperiodic flowering

(Alabadi, 2001; Mizoguchi, 2002; Yanovsky, 2002). Genomic comparisons between A. thaliana

and LD Leman gibba strain G3 and SD Lemna paucicostata strain 6746 revealed that the

Lemnas had sequence matching homologs which were, then, analyzed to determine if they, too,

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Author: HRW

were circadian clock genes (Miwa, 2006: Serikawa, 2008). These SD and LD Lemna were

selected for the study because they are evolutionarily close but have different photoperiodic

systems: this makes them genetically good models for studying circadian-related photoperiodism

(Serikawa, 2008). Expression of the Lemna genes were monitored by using a bioluminescent

reporter system with firefly luciferase as circadian expression markers (Miwa, 2006: Serikawa,

2008). The homologs were also analyzed by being over expressed using a plasmid carrying the

appropriate genes or knocked down through RNA interference (Miwa, 2006; Serikawa, 2008).

The results from the various analyses on the Lemna genes compared to analyses on their

homologs in Arabiodopsis indicate that they are orthologs with similar functions, showing that

gene function has been conserved not only between the Lemna but also between dicotyledons

and monocotyledons (Miwa, 2006; Serikawa, 2008).

In searching for a florigen, Cleland found that the honeydew secreted by aphids feeding on

the SD Xanthium strumarum contained a factor that reportedly induced flowering in X.

strumarum in previous studies (Cleland, 1974; Cleland and Ajami, 1974; Raskin 1992). The

aphid honeydew induced flowering in LD L. gibba G3 (Cleland, 1974). He further isolated

substance from the honeydew and identified it as salicylic acid (SA) (Cleland and Ajami, 1974).

L. gibba was grown in medium with added salicylic acid and the plants were exposed to a

marginal day length of 10 hours light to 14 hours dark and a short day length of 9 hours light to

15 hours dark. SA produced flowering in both long days and short days especially at an

optimum concentration of salicylic acid of 5.6 μm (Cleland and Ajami, 1974). Since the L.

gibba does not flower during short days, the results raised the possibility that salicyclic acid was

controlling the flowering of this plant. Tamot et al. (1987) also tested salicylic acid on Wolffiella

hyalina strain 7378, and it, too, induced flowering in this member of the Lemnaceae. In this

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Author: HRW

study, photoperiodically sensitive W. hyalina responded to SA with significant flowering when it

was grown in a short day schedule of 8 hours light to 16 hours of darkness. As the concentration

of salicylic acid in the medium increased so did the flowering percentage until a maximum

concentration of 10-5 M was reached, at which point the flowering percentage dramatically

decreased. In previous experiments, this Wolffiella species did not flower when exposed to

chelating agents such as EDTA, EDDHA, 8-hydroxyquinoline, or hormones which induced

flowering in other plants (Tamot et al., 1987). The argument that SA was not, itself, a chelating

agent was supported by the results showing that W. hyalina only flowered in response to

salicylic acid s (Tamot et al., 1987). As research on flowering inducers continued, though, more

substances were shown to produce similar effects on flowering as SA. For example, both

salicylic acid and benzoic acid induced flowering in photoperiod-insensitive Lemna paucicostata

LP6 with benzoic acid being marginally more effective (Khurana and Cleland, 1992). This

experiment also tested whether salicylic and benzoic acid taken up by the mother fronds had any

flowering effect on the vegetatively produced daughter fronds. Under determined ideal

conditions, the daughter fronds did eventually flower after being separated for the mother plants

which had grown in medium with either SA or benzoic acid. To measure SA or benzoic acid

uptake, mother fronds were then exposed to medium with either radioactive [14C]salicylic acid

and [14C]benzoic acid. Three days after daughter fronds were produced, they were extracted and

radioactivity was measured in each mother and daughter groups. Mother plants were measured

to have normal uptake of both acids. As for the daughter frond samples, a small amount of

radioactivity was recovered in the benzoic acid group, but no relevant radioactivity was

recovered in the SA group. These results indicated that something else was inducing flowering

in the daughters besides salicylic acid (Kurana and Cleland, 1992).

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Author: HRW

Eighty-three years have passed since Chailakhyan proposed the possibility of florigen as a

substance that triggered flowering in a plant (Aksenova et al., 2005; Macháčková et al., 2001).

Since then, the small Lamnaceae have helped identify substances that induce flowering such as

salicylic acid, benzoic acid, chelating agents, copper, and nicotinic acid (Raskin, 1992). None of

these substances, though, have proven to be the elusive florigen which is activated to produce

flowering as a response to some signal induced by a plant’s circadian clock. Some propose that

florigen may not be one agent but rather several (Colasanti and Sundaresan, 2000). As more

plants undergo genomic sequencing, techniques in isolating and manipulating genes so that their

expression is altered are helping to isolate genetic systems that affect flowering. For example,

several studies have suggested that the protein FLOWERING LOCUST T (FT) encodes florigen

since it has shown to be a major activator of flowering in plants such Arabidopsis thaliana and L.

paucicostata (Kai et al., 2008). Since many angiosperms are of agriculture and, therefore, of

economic importance, the search for finding what triggers the induction of reproduction will

continue with genetic studies aiding in the investigation. The distinctive nature of the

Lamnaceae will likely make it as important is genetic studies as it already has been in

physiological studies.

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Author: HRW

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