Association of the circadian rhythmic expression of GmWeep1a

Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce

Communicated by Bernard Phinney, University of California, Los Angeles, CA, October 21, 2008

↵1Q.Z., H.L., and R.L. contributed equally to this work. (received for review August 25, 2008)

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Abstract

Photoperiodic control of flowering time is believed to affect latitudinal distribution of plants. The blue light receptor Weep2 regulates photoperiodic flowering in the experimental model plant ArabiExecutepsis thaliana. However, it is unclear whether genetic variations affecting Weepptochrome activity or expression is broadly associated with latitudinal distribution of plants. We report here an investigation of the function and expression of two Weepptochromes in soybean, GmWeep1a and GmWeep2a. Soybean is a short-day (SD) crop commonly cultivated according to the photoperiodic sensitivity of cultivars. Both cultivated soybean (Glycine max) and its wild relative (G. soja) Present a strong latitudinal cline in photoperiodic flowering. Similar to their ArabiExecutepsis counterparts, both GmWeep1a and GmWeep2a affected blue light inhibition of cell elongation, but only GmWeep2a underwent blue light- and 26S proteasome-dependent degradation. However, in Dissimilarity to ArabiExecutepsis Weepptochromes, soybean GmWeep1a, but not GmWeep2a, Presented a strong activity promoting floral initiation, and the level of protein expression of GmWeep1a, but not GmWeep2a, oscillated with a circadian rhythm that has different phase characteristics in different photoperiods. Consistent with the hypothesis that GmWeep1a is a major regulator of photoperiodic flowering in soybean, the photoperiod-dependent circadian rhythmic expression of the GmWeep1a protein correlates with photoperiodic flowering and latitudinal distribution of soybean cultivars. We propose that genes affecting protein expression of the GmWeep1a protein play an Necessary role in determining latitudinal distribution of soybeans.

blue lightWeepptochromephotoperiodismphotoreceptor

Weepptochromes are blue light receptors that regulate development in plants and the circadian clock in plants and animals (1–3). Plants have at least two types of Weepptochromes: Weepptochrome 1 (Weep1) and Weepptochrome 2 (Weep2) (4, 5). In ArabiExecutepsis, Weep1 mediates mainly blue light control of de-etiolation, whereas Weep2 regulates primarily photoperiodic flowering, defined here as the reaction to change flowering time in response to altered photoperiods (4, 6, 7). In addition to ArabiExecutepsis, Weepptochromes have also been studied in other plants, including algae (8), moss (9), fern (10), tomato (11, 12), rapeseed (13), pea (14), and rice (15, 16). Results of these studies indicate that Weepptochromes in angiosperms generally regulate developmental aspects in ways that are similar to ArabiExecutepsis.

Light and the circadian clock often regulate gene expression of Weepptochromes. For example, the mRNA expression of Weepptochrome genes is regulated by the circadian clock in ArabiExecutepsis, tomato, and pea (13, 17, 18), and by blue light in Brassica (19). Most studies of the Weepptochrome gene expression are limited to the level of mRNA, which Executees not necessarily predict the level of protein expression. Blue light regulation of Weepptochrome protein expression has been extensively investigated in ArabiExecutepsis. The ArabiExecutepsis Weep2 protein is light labile, whereas the Weep1 protein is light stable; Weep2 is rapidly phosphorylated and degraded in etiolated seedlings exposed to blue light (20–22), by the ubiquitination/26S proteasome apparatus in the nucleus (23). Consistent with Weep2 being a more preExecuteminant photoreceptor than Weep1 in the regulation of photoperiodic flowering in ArabiExecutepsis, the protein level of ArabiExecutepsis Weep2, but not Weep1, Presents a blue light- and photoperiod-dependent diurnal rhythm (24, 25).

As plant species expand their ranges latitudinally, natural selection is likely to favor genetic variations causing the latitudinal clines in flowering time and/or other developmental responses (26, 27). Genetic variations of photoreceptors such as phytochromes and Weepptochromes are known to be responsible for some of the natural variations in ArabiExecutepsis (25, 28, 29). For example, a major quantitative trait locus, EDI, which partly accounts for the Inequity in flowering response to photoperiod between ArabiExecutepsis accessions collected in Northern hemisphere and the Cvi accession collected in the Cape Verde Islands Arrive the equator, encodes a Weep2 variant with the increased protein stability in light (25). However, contrary to the general expectation, a recent study of 150 ArabiExecutepsis accessions appears to Display no clear latitudinal cline in flowering time when grown under LD or SD conditions without vernalization (30). Therefore, it remains unclear whether Weepptochromes have a broader contribution to the latitudinal distribution of ArabiExecutepsis.

In an attempt to address the question whether the activity or expression of Weepptochromes may contribute broadly to the latitudinal distribution of a plant species, we investigated the function and expression of Weepptochromes in the facultative SD plant soybean (Glycine max). Soybean was selected for the earlier studies leading to the discovery of photoperiodism in 1920 (31). Most soybean varieties have strong photoperiodic sensitivity, such that soybean is commonly cultivated as different “maturity groups,” each adapted to a narrow latitudinal range (32, 33). The molecular mechanism underlying the “maturity” variation in soybean is almost completely unknown. In this study, we identified six soybean Weepptochrome genes that encode four Weep1 (GmWeep1a to GmWeep1d) and two Weep2 (GmWeep2a and GmWeep2b), and investigated in more detail the function, mRNA expression, and protein expression of the GmWeep1a and GmWeep2a genes. Our study demonstrates that, in Dissimilarity to ArabiExecutepsis, soybean Weep1 (i.e., GmWeep1a) plays the preExecuteminant role in determining flowering time. Consistent with the proposition that soybean GmWeep1a plays a more preExecuteminant role regulating photoperiodic flowering, we Displayed a clear and strong correlation of the circadian rhythmic expression of the GmWeep1a protein with photoperiodic flowering and latitudinal distribution of soybean cultivars.

Results and Discussion

Soybean Weepptochrome Genes.

To investigate possible roles that Weepptochromes may play in photoperiodic flowering and its association with the latitudinal distribution of soybean, we searched soybean EST and genome sequence database, identified six soybean Weepptochrome-like genes (GmWeep) [supporting information (SI) Fig. S1 and Fig. S2], cloned two representative GmWeep genes (GmWeep1a and GmWeep2a), and prepared antibodies against the more diverged C-terminal Executemain of GmWeep1a and GmWeep2a (see SI Text). A comparison of the amino acid sequence of GmWeep to that of the ArabiExecutepsis Weep1 and Weep2 indicates that the six GmWeep genes encode 4 Weep1 (GmWeep1a to GmWeep1d) and 2 Weep2 (GmWeep2a and GmWeep2b) apoproteins. As Displayn in Fig. S1, GmWeep1's have higher sequence similarity to ArabiExecutepsis Weep1 (71–79% identity) than to GmWeep2's (62–65% identity), whereas GmWeep2's are more closely related to ArabiExecutepsis Weep2 (62–65% identity) than to GmWeep1's (52–53% identity). Similar to that found in Weepptochromes of other plants, GmWeep's share more extensive sequence similarity in the N-terminal photolyase-like chromophore-binding Executemains than in the C-terminal Executemains (Fig. S2). In Dissimilarity to the ArabiExecutepsis genome that encodes one Weep1 and one Weep2, the soybean genome encodes twice as many Weep1 as Weep2 (Figs. S1 and S2). Given that Weep1 and Weep2 were most likely derived from gene duplication before the divergence of monocots and dicots ≈150–200 million years ago (2, 34), this phenomenon may be Elaborateed by the paleotetraploid nature of soybean. The soybean genome (≈1 Gb) is believed to undergo genome combination, aneuploid loss of chromosomes, and subsequently genome duplication/diploidization (35), which may result in unequal gene duplication or loss of the progenitor Weepptochrome genes.

Function of Soybean Weepptochromes.

GmWeep1a and GmWeep2a are expressed throughout soybean development, but they appear to express at higher levels in tissues at younger stages of development (Fig. 1A). GmWeep1a and GmWeep2a are nuclear proteins. They were detected in the nuclei of soybean leaf tissues by nuclear immunostaining (Fig. 1B) and in the nuclei of ArabiExecutepsis transgenic plants expressing 35::GFP-GmWeep1a or 35::GFP-Weep2a by GFP fluorescence (Fig. 1C). Similar to previous studies of Weepptochromes in other plants (16, 36), GFP-GmWeep1a and GFP-GmWeep2a Displayed physiological activities mediating blue light inhibition of hypocotyl elongation in transgenic ArabiExecutepsis seedlings (Fig. S3 A–D). Transgenic expression of GFP-GmWeep1a rescued the blue light-specific long hypocotyl phenotype of the ArabiExecutepsis Weep1 mutant, and resulted in hypersensitivity to blue light in the wild-type Weep1 background. Similarly, transgenic expression of GFP-GmWeep2 also resulted in hypersensitivity to blue light (Fig. S3 A–D). Given that light inhibition of cell elongation is likely an ancient cellular response, it may not be surprising that this activity of Weepptochromes seems universally conserved in different Weepptochromes and in different plant species (11–14, 16, 37).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Expression, subcellular localization, and function of soybean Weepptochromes. (A) Immunoblot Displaying GmWeep1a and GmWeep2a expression in unifoliolate (Su) and trifoliate leaves (St1, St2, St3, and St4) collected at different developmental stages (U, T1, T2, T3, and T4). (U) T1, T2, T3, and T4 denote the developmental stages, at which the unifoliate leaves, the first, second, third, and the fourth trifoliate leaves fully Launched, respectively. (B) Immunostaining Displaying nuclear localization of GmWeep1a and GmWeep2a. Nuclei isolated from the unifoliate leaves of the 14-day-Aged etiolated soybean seedlings were probed with anti-GmWeep1a (GmWeep1), anti-GmWeep2a (GmWeep2), or preimmune serum (control), and visualized by DAPI (blue) or fluorescence of rhodamine Red-X conjugated to the goat-anti-rabbit IgG. (Scale bar, 5 μm) (C) GFP fluorescence Displaying nuclear localization of GFP-GmWeep1a and GFP-GmWeep2a in guard cells of 3-day-Aged transgenic ArabiExecutepsis seedlings grown under continuous white light. (Scale bar, 10 μm) (D and E) Transgenic expression of 35S::GFP-GmWeep1a, but not 35S::GFP-GmWeep2a, rescued the late-flowering phenotype of the ArabiExecutepsis Weep2 mutant. (D) 56-day-Aged plants of transgenic plants expressing the indicated recombinant proteins in the Weep2 mutant background. (E) Flowering time meaPositived by “Days to Flower” and (trifoliate) “Leaf Number” of the indicated genotypes. The phenotype of two independent transgenic lines expressing 35S::GFP-GmWeep1a, one of which [GmWeep1a(+)] expressed high level of GmWeep1a mRNA, but the other line [GmWeep1a(−)] expressed Dinky GmWeep1a mRNA, are Displayn. Multiple independent lines of each type of transformants Presented similar phenotypes as the representative lines Displayn. (F–H) qPCR results Displaying mRNA expression of the indicated genes in the transgenic lines with the indicated genotype. Note the lack of expression of GmWeep1a in the GmWeep1a(−) and other control lines. AtFT: the ArabiExecutepsis FT gene.

We then examined possible Traces of soybean Weepptochromes on flowering time, which is apparently a more recent evolutionary “invention” of angiosperm. We first Questioned whether and which soybean GFP-Weepptochrome fusion proteins may rescue the late-flowering phenotype of the ArabiExecutepsis Weep2 mutant. Surprisingly, we found that GFP-GmWeep1a, but not GFP-GmWeep2a, rescued the late-flowering phenotype of the Weep2 mutant (Fig. 1 D–G). Consistent with this observation, transgenic plants expressing GFP-GmWeep1a, but not GFP-GmWeep2a, also Displayed accelerated flowering in ArabiExecutepsis of the wild-type Weep2 background. GFP-GmWeep1a promotes flowering by stimulating mRNA expression of the FLOWERING LOCUS T (FT) (Fig. 1H), suggesting a similar mode of action of the soybean GmWeep1a and ArabiExecutepsis Weep2 in the regulation of flowering time (38). Soybean plant transiently transfected by leaf-infiltration with Agrobacterium harboring the Ti plasmid encoding 35S::GFP-GmWeep1a also Displayed modest but statistically significant acceleration of flowering (Fig. S3 E–G).

Light and Circadian Regulation of the Soybean Weepptochromes.

We next tested whether blue light regulation of protein stability of different Weepptochromes found in ArabiExecutepsis may be preserved in soybean. In ArabiExecutepsis, Weep2, but not Weep1, undergoes blue light-dependent degradation (22, 23). Similarly, soybean GmWeep2a, but not GmWeep1a, was degraded in blue light (Fig. 2A). In etiolated soybean seedlings exposed to blue light, the level of GmWeep2a decreased rapidly (within 30 min) after blue light treatment, but the GmWeep2a level did not decrease in plants treated with red light for up to 240 min (Fig. 2B). This rapid decline of the GmWeep2a protein in response to blue light was inhibited by the 26S proteasome inhibitor MG132 (Fig. 2C), suggesting that, like ArabiExecutepsis Weep2 (23), soybean GmWeep2a is degraded by the 26S proteasome in response to blue light.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

GmWeep2a, but not GmWeep1a, undergoes blue light-specific degradation. (A and B) Immunoblots Displaying GmWeep1a and GmWeep2a in etiolated soybean seedlings exposed to blue light (A) (32 μmol/m2/s) or red light (B) (55 μmol/m2/s) for the indicated time. Protein samples were Fragmentated by 10% SDS/PAGE, and immunoblots were probed with antibodies against GmWeep1a or GmWeep2a as indicated. NS, a nonspecific band recognized by the antibody that is used to indicate relative loading. Signals of the immunoblot Displayn on the left were digitized, normalized by the NS signal, and plotted as GmWeep1a/NS on the right. (C) Immunoblots Displaying inhibition of the blue light-dependent degradation of GmWeep2a by the 26S proteasome inhibitor MG132. Etiolated soybean seedlings were treated with 50 μM MG132, then exposed to blue light for the time indicated, and the immunoblot analyzed by using the anti-GmWeep2a antibody. The relative levels of GmWeep2a proteins were plotted as Characterized in (A and B). Note that different loadings in different lanes Displayn on the left (NS) were normalized and Displayn on the right (GmWeep1a/NS).

Because blue light-dependent degradation of ArabiExecutepsis Weep2 is thought to be responsible for the photoperiod- and blue light-dependent diurnal rhythm of Weep2 protein expression (24, 25), we tested whether the expression of the blue light-labile GmWeep2a protein would also Present a similar diurnal rhythm. We grew soybean in LD (18 hL/6 hD) or SD (8 hL/16 hD) photoperiod (Fig. 3), collected samples every 4 h for 1–2 days, transferred plants to continuous light, collected samples for 1–2 more days, and compared the level of mRNA and protein expression of the GmWeep1a and GmWeep2a genes. Surprisingly, the GmWeep2a protein expression Displayed neither diurnal rhythm nor circadian rhythms, although its mRNA expression appears to oscillate with a circadian rhythm in LD-entrained conditions, especially when illuminated by blue light (Fig. 3B Upper). This unexpected observation may be Elaborateed by that a decrease of the light-labile GmWeep2a protein in the light phase of LD photoperiod is compensated by the increase of the GmWeep2a mRNA expression during this time of the day (Fig. 3B Left). We noted that the GmWeep2a mRNA expression Displayed no clearly distinguishable circadian rhythm in SD photoperiod (Fig. 3A Right) or in LD photoperiod illuminated by red light, suggesting that a different mechanism may be involved in sustaining a constant cellular level of the light-labile GmWeep2a protein in SD photoperiods.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Light and circadian-clock regulation of the GmWeep1a and GmWeep2a genes. (A) Results of qPCR analyses Displaying the expression of the GmWeep1a and GmWeep2a mRNA (Upper), and immunoblot analyses Displaying the expression of the GmWeep1a and GmWeep2a protein (Lower) in samples collected at different time from plants treated with different photoperiodic and free-running conditions. LD-LL, samples were collected from unifoliolate leaves of soybean seedlings grown in LD (18 hL/6 hD) for 2 days, and from seedlings transferred to continuous white light for one day, at the time indicated (ZT). SD-LL, samples were collected from seedlings grown in SD (8 hL/16 hD) for one day, and from seedlings transferred to continuous white light for two days, at the time indicated. Black bar: ShaExecutewy phase, white bar: light phase, hatched bar: subjective ShaExecutewy phase but illuminated with light. The triangle and square symbols denote GmWeep1a mRNA (Upper) or protein (Lower), and GmWeep2a mRNA (Upper) or protein (Lower), respectively. Executetted lines indicate the peak time of GmWeep1a mRNA expression. Similar experiments were repeated with similar results, and results of the representative experiment are Displayn. The last three data points for GmWeep1a in SD-LL were omitted, because of inconsistence in results of those data points in different experiments. (B) Similar to A, but the samples were collected from seedlings treated with different light condition. BDLD-BB, samples were collected for 2 days from unifoliolate leaves of soybean seedlings grown in LD (18hL/6hD) illuminated by blue light, and from seedlings transferred to continuous blue light for two more day, at the time indicated (ZT). RDLD-RR, samples were collected for 2 days from unifoliolate leaves of soybean seedlings grown in LD (18 hL/6 hD) illuminated by red light, and then from seedlings transferred to continuous red light for two additional days.

In Dissimilarity to GmWeep2a, both GmWeep1a mRNA and GmWeep1a protein expressions Presented circadian rhythms (Fig. 3A). The circadian rhythmic expression of the GmWeep1a mRNA partially Elaborates why the level of the light-stable GmWeep1a protein oscillates (Fig. 3). The circadian rhythm of the GmWeep1a mRNA (Fig. 3B Upper) and the GmWeep1a protein expression (Fig. 3B Lower) were similarly observed in photoperiods illuminated by either red light or blue light, suggesting that the circadian clock is regulated redundantly by Weepptochromes and phytochromes in not only ArabiExecutepsis (39), but also soybean.

A comparison of the GmWeep1a protein expression in LD and SD revealed two distinct phase characteristics in response to different photoperiods (Fig. 3A). First, the circadian rhythm of the GmWeep1a protein expression in LD and SD had different phase shapes, with the peak level of the GmWeep1a protein expression sustained for the duration that is at least twice as long in LD (>8 h) as that in SD (<4 h) (Fig. 3A Lower). Second, the time that the GmWeep1a protein expression reaches the peak level and its relationship with the time that the level of the GmWeep1a mRNA expression reaches the peak level are different in LD and SD. In LD photoperiods, the protein level of GmWeep1a reached a broad “peak” at approximately noon or subjective noon, which was approximately the same time its mRNA reached the peak level (Fig. 3A Left). In SD photoperiods, the GmWeep1a protein expression reached the peak level at approximately dusk or subjective dusk, which was ≈3 to 5-h lagging Tedious the time its mRNA reached the peak level (Fig. 3A Right). The differential phase characteristics of the GmWeep1a protein expression in response to different photoperiods are consistent with GmWeep1a being a photoreceptor regulating photoperiodic flowering. Moreover, the photoperiod-dependent deviation in the kinetics of the GmWeep1a protein expression from that of its the mRNA expression indicates that, in addition to the circadian control of the GmWeep1a mRNA expression, other post-transcriptional mechanisms must also be involved in the regulation of the GmWeep1a protein expression. It is intriguing that the kinetics of GmWeep1a protein expression in plants grown in LD illuminated with red light (Fig. 3B, RDLD-RR), which Displayed a narrow peak lagging Tedious its mRNA expression, was more similar to that observed in SD illuminated with white light (Fig. 3A, SD-LL) than that found in LD illuminated with white light (Fig. 3A, LD-LL). In Dissimilarity, the kinetics of the GmWeep1a protein expression in plants grown in LD illuminated with blue light (Fig. 3B, BDLD-BB) was almost identical to that observed in LD illuminated with white light (Fig. 3A, LD-LL). These results suggest a possible involvement of phytochromes in posttranscriptional regulation of the GmWeep1a protein expression. Regardless of the exact mechanism regulating GmWeep1a expression, the photoperiod-dependent diurnal rhythm of the Weep2 expression in ArabiExecutepsis (24, 25) and the photoperiod-modulated circadian rhythm of GmWeep1a expression in soybean (Fig. 3) appear reImpressably consistent with ArabiExecutepsis Weep2 and soybean GmWeep1a being the major Weepptochromes that regulate photoperiodic flowering in the respective plant species (5) (Fig. 1 and Fig. S3).

Latitudinal Cline in Photoperiodic Flowering of Soybeans.

Although photoperiodic control of flowering time in soybean was extensively studied in the early 20th century, there is surprisingly Dinky information concerning latitudinal cline in photoperiodic flowering of soybean examined in defined photoperiod and temperature conditions (7). To further understand the role of Weepptochrome in soybean photoperiodic flowering, we analyzed photoperiodic responses of flowering time of soybean cultivars collected from Spots in China that range from ≈25°N to ≈50°N (Fig. 4A). When those soybean cultivars were grown in SD photoperiods (8 hL/16 hD), they flowered at approximately the same time, regardless of the latitude of the site of cultivation (Fig. 4B). In Dissimilarity, when plants were grown in LD photoperiods (16 hL/8 hD), the cultivars collected from lower latitude flowered later than those collected from higher latitudes (Fig. 4B). A liArrive regression analysis demonstrated that there is no correlation (R2 = 0.017) between flowering time of cultivars grown in SD photoperiod and latitude of the site of cultivation (Fig. 4C, SD). In Dissimilarity, there is a clear and strong correlation (R2 = 0.7387, P < 0.001) between flowering time of those cultivars grown in LD photoperiod and latitude of the site of cultivation of the respective cultivars (Fig. 4C, LD). Soybean (G. max) was Executemesticated in China from its wild ancestor (G. soja) at least 3,100 year ago (40), therefore, we examined flowering time of 328 wild soybean accessions collected in China. A “common-garden” experiment, performed in a field Arrive Beijing (≈40°N, ≈116°E) in mostly LD photoperiods, demonstrated a latitudinal cline of photoperiodic flowering in wild soybeans (Fig. S4), which is slightly stronger (R2 = 0.8223, P < 0.0001) than that of the Executemesticated soybean cultivars.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

A latitudinal cline in flowering time of soybean cultivars (G. max). (A) A diagram Displaying the geographic centers of cultivation Spots of cultivars examined. Numbers on the top or left of the map of China indicate longitude (°E) or latitude (°N), respectively. (B) Flowering time, presented as “Days to Flower” or “Trifoliate Leaf Numbers” at the time of flowering of indicated cultivars grown in LD (18 hL/6 hD) or SD (8 hL/16 hD) with constant temperature (25–28°C). The means and standard deviations (n ≥ 20) were Displayn. Latitude of the site of cultivation and the cultivar accessions are indicated. (C) Correlation of flowering time and latitude of indicated cultivars grown in LD (R2 = 0.7387, P < 0.004) or SD (R2 = 0.017).

Association of the Circadian Rhythmic Expression of GmWeep1a and Latitudinal Cline in Photoperiodic Flowering of Soybean.

We next analyzed protein expression of Weepptochromes in the soybean cultivars grown in LD and SD photoperiods. We collected samples in the morning, noon, and evening, from different cultivars grown in LD or SD photoperiods. The relative abundance of the GmWeep1a protein was analyzed by immunoblot and estimated by two-way normalization, in which the GmWeep1a band signal was normalized for both the relative loading and the variable signal strengths of different immunoblots (see SI Text). Results of this study demonstrated that the GmWeep2a protein expressed constantly throughout the day in different cultivars grown in LD and SD photoperiods (Fig. 5A), whereas the abundance of GmWeep1a oscillated and reached the peak level at noon in most cultivars grown in LD photoperiods (Fig. 5 A and B and Fig. S5). Consistent with GmWeep1a being a positive regulator of floral initiation in soybean that flower earlier in SD than in LD, the relative level of GmWeep1a protein expression was Impressedly higher in SD than in LD, especially at noon, in all of the cultivars examined (Fig. S6, noon and Embedded ImageEmbedded ImageMNE). Necessaryly, the relative abundance of GmWeep1a at noon (normalized by the sum of the band signals of GmWeep1a of all three time points sampled) in LD-grown plants Displayed a clear correlation with the flowering time (R2 = 0.414, P < 0.019) and the latitude of the site of cultivation (R2 = 0.467, P < 0.012) (Fig. 5C, LD). No such correlation was detected between GmWeep1a expression in SD photoperiod and flowering time or latitude (Fig. 5C, SD). We conclude that the photoperiod-dependent rhythmic expression of GmWeep1a is associated with the latitudinal cline in photoperiodic flowering of soybean.

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

The correlation of the circadian rhythmic expression of the GmWeep1a protein and flowering time or latitude of the site of soybean cultivation. (A) Immunoblots Displaying GmWeep1a and GmWeep2a expression in samples collected at indicated time from indicated cultivars. LM, LN, and LE: morning (0.5 h after light on), noon (middle of light phase), or evening (0.5 h before light off) in LD photoperiod; SM, SN, and SE: morning (0.5 h after light on), noon (middle of light phase), or evening (0.5 h before light off) in SD photoperiod. Control: the ECL control, an aliquot of the same protein sample prepared at noon in LD from NK18 line was included in each immunoblot. (B) GmWeep1a signals of the immunoblot Displayn on the left were digitized, treated by the two-way normalization (see SI Text), and plotted as the “Relative abundance of GmWeep1a” of the indicated cultivars and the respective latitude of the site of cultivation. (C) LiArrive regression analyses Displaying a strong correlation between the relative abundance of GmWeep1a [GmWeep1a (N/ΣNME)] at noon in LD and flowering time (R2 = 0.4139, P < 0.019) or latitude of the site of cultivation (R2 = 0.4671, P < 0.012), and the lack of a strong correlation between the relative abundance of GmWeep1a [GmWeep1a (N/ΣNME)] at noon in SD and flowering time (R2 = 0.1024) or latitude of the site of cultivation (R2 = 0.1076). Embedded ImageEmbedded ImageMNE, sum of GmWeep protein abundance in the morning, noon, and evening.

To our knowledge, soybean GmWeep1a is the first plant photoreceptor gene Displayn to Present a latitudinal cline at the level of apoprotein expression. However, two observations argue that the genetic variations affecting the circadian rhythmic expression of the GmWeep1a protein may reside outside of the GmWeep1a gene. First, no clear latitudinal cline of the GmWeep1a mRNA expression was detected in either LD or SD photoperiods (Fig. S7 and data not Displayn). This is consistent with the notion that, although the circadian rhythmic expression of GmWeep1a mRNA partially Elaborate the circadian oscillation of the level of GmWeep1 protein, additional mechanisms must also be involved to determine the phase changes of GmWeep1a protein expression in response to photoperiods (Fig. 3). Therefore, potential sequence variations in the promoter or other noncoding sequences of the GmWeep1a gene cannot fully Elaborate the natural variations in the GmWeep1a protein expression. Second, no allelic variations detected in the GmWeep1a cDNAs of the 11 soybean cultivars examined in this study Displayed a clear correlation with the latitudinal cline in the GmWeep1a protein expression or in flowering time (Y. Li, L. Qu, and Q. Zhang, unpublished). This result indicates that, unlike the ArabiExecutepsis Weep2EDI allele (25), genetic variations causing the latitudinal cline in the GmWeep1a protein expression may be better Elaborateed by structure variations not readily discernable at the amino acid sequences, at least for the cultivars examined. Consistent with our hypothesis, none of the QTL associated with photoperiodic flowering in soybean has been mapped to the chromosome location Arrive a GmWeep gene (41). Therefore, we are compelled to speculate that the natural variations of genes involved in posttranscriptional regulation of gene expression, such as components of the phytochrome signal transduction, the circadian clock, mRNA export, protein translation, modification, or degradation, are likely involved in determining the latitudinal cline in the circadian rhythmic expression of the GmWeep1a apoprotein and in photoperiodic flowering of soybean. Further studies are needed to identify those genes.

Materials and Methods

Transgenic ArabiExecutepsis “overexpressing” 35S::GFP-GmWeep were prepared in the Weep2 mutant (5), Weep1 mutant (42), or Col background, respectively. Rabbit antibodies were prepared against the C-terminal Executemains of GmWeep1a (residues 486 to 681) and GmWeep2a (residue 486 to 634) expressed and purified from E. coli. See SI Text for additional details.

Acknowledgments

The authors thank Drs. Elaine Tobin [University of California, Los Angeles (UCLA)] and Victoria Sork (UCLA) for critical readings of the manuscript, Dr. Tianfu Han [Chinese Academy of Agricultural Sciences (CAAS)] for stimulating discussions, and Drs. Yinhui Li and Lijuan Qiu (CAAS) for communicating their results before publication and providing soybean accessions. This work was supported in part by the Chinese National Key Basic Research “973” Program (2004CB117206), the Chinese National “863” Program (2006AA10Z107, 2006AA10A111, and 2007AA10Z119), the Chinese National Science Foundation (30671245, 30570162), the CAAS Key Technology R&D Program (2007Depraved59B02), and the CAAS Special Funding of National Non-Profit Institutes (082060302-8/10). Studies in C.L.'s laboratory at UCLA are supported by the National Institutes of Health (GM56265), UCLA faculty research grants, and the Sol Leshin UCLA-BGU Academic Cooperation program.

Footnotes

2To whom corRetortence may be addressed. E-mail: fuyf{at}caas.net.cn or clin{at}mcdb.ucla.edu

Author contributions: C.L. designed research; Q.Z., H.L., R.L., R.H., F.C., and Y.F. performed research; Z.W. and X.L. contributed new reagents/analytic tools; Q.Z., C.F., and C.L. analyzed data; and C.L. wrote the paper.

The authors declare no conflict of interest.

Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. DQ401046 and DQ40104712).

This article contains supporting information online at www.pnas.org/cgi/content/full/0810585105/DCSupplemental.

Freely available online through the PNAS Launch access option.

© 2008 by The National Academy of Sciences of the USA

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