SPB1 Initiates Flowering in Antirrhinum majus through the Activation of Meristem Identity Genes

Why Study Flowering?

The flowering of plants does more than provide beauty to the natural world as well as gifts to females from their male counterparts. This event is also the direct result of the transition from a vegetative state to a reproductive state in eudicots, thus marking the development into adulthood. So why is this development important to the human population? Understanding the process behind flowering (thus the development into a reproductive stage) can help researchers develop techniques to induce the optimal amount of flowers per plant as well as prolong the life of these blooms. In other words, studying the development of flowers at a development level can lead to more abundant, longer lived roses for that special someone. Not only can this lead to more flowers per plant, but also more plants since these blooming components contain the reproductive organs and attract pollinators. Also at a molecular level, finding the developmental pathways in these organisms can aid researchers in uncovering the conservation of these developmental genes and help provide insight on the developmental patterns of similar species.

Antirrhinum majus

Image of Antirrhinum majus

Why Antirrhinum majus (Snapdragons)?

Of all the beautiful flowering plants, why study Antirrhinum majus, more commonly known as snapdragons? Antirrhinum majus are model organisms in biology due to their ease of cultivation and mutation as well as the fact that they are a descendant of the common eudicot model Arabidopsis thaliana making them ideal for comparative developmental studies.

What Can Be Discovered?

In order to discover more about the development of these core eudicots, it is important to first understand what is already known. Researchers used the commonly studied and close relative, Arabidopsis thaliana, as a starting point to find out more about the Antirrhinum majus development. A. thaliana flowering time is regulated by Squamosa-Promoter Binding Protein (SBP) by activating key genes such as fruitful (FUl), apetala (AP1), and leafy (LFY) involved in inflorescence and floral meristem identity (Ferrándiz et al., 2000). Previous studies suggest that paralogs of these SBP box proteins in A. majus, SBP1 and SBP2, regulate the AP1 ortholog, SQUAMOUS (SQUA) (Klein et al., 1996). In the A. thaliana these two SBP genes are closest homologs of SPL3, SPL4, and SPL5 which are negatively regulated by miR156 and miR157 microRNAs (Wang et al., 2009). Based on this data, Preston and Hileman used reverse genetics and silencing to provide insight on this flowering time pathways as well as the conservation of key genes in this pathway in core eudicots. Because the following experiments compared genes of the two flowering plants it is important to remember which genes belong to which species which can be seen below (Preston and Hileman 2010).

snap v thaliana

Image of A. thaliana (left) and A. majus (right)

Regulatory Genes of Interest:
A. majus– SBP1 and 2
A. thaliana– SBP3-5

Target Meristem Identity Genes:
A. majus– “Ful-like genes”: AmFul, SQUA, DEFH28
A. thaliana– “LFY-like genes”: FUL, AP1, and LFY

What Did Preston and Hileman Discover?
1. SBP1 is more closely related to SPL3, SPL4 and SPL5 than to SBP2

Using Maximum parsimony, maximum likelihood, and Bayesian analyses of SBP box genes, it was found that SBP1 is a close homolog of the A. thaliana SPL3-5 regulatory genes of interest, but not SBP2 since this gene was in a different clan. Also these tests showed that the gene duplication that lead to SBP1 and SBP2 occurred before eudicots were even diversified and before the duplication events leading to SPL3-5. Because SBP1 was found to be so closely related to the SBP box genes of A. thaliana, SBP1 became a gene of interest for the rest of the study rather than SBP2.

2. SBP and FLO genes are expressed earlier in development than FUL genes

(Preston and Hileman 2010) Figure 1-Inflorescence and floral meristem identity gene expressions, and their upstream regulators, in wild-type Antirrhinum majus tissues.  (b–c) In situ hybridization of an antisense AmFUL probe showing expression (blue staining) in the inflorescence meristem (im) and floral meristems (fm) of Antirrhinum majus. (d) Sense control AmFUL probe showing minimal staining in Antirrhinum majus inflorescences.

(Preston and Hileman 2010) Figure 2-Inflorescence and floral meristem identity gene expressions and their upstream regulators, in wild-type Antirrhinum majus tissues. (a)Stages of development indicated by leaf node number where SBP1/2 and FLO are upregulated before the FUL-like genes, but all genes transcribed at high levels during inflorescence (inf). Expression in the flowering parts indicated by petals (pe), gynoecium (gy), sepals (se), and stamens (st), while rt is the inflorescence RNA – reverse transcriptase control. (b-c) In situ hybridization of antisense AmFUL probe. (d) Sense control AmFUL probe. Expression indicated in the inflorescence meristem (im) and floral meristems (fm) of Antirrhinum majus.

Through RT-PCR analyses of the genes in A. thaliana leaf tissue it was found that SBP1 and SBP2 were up-regulated similarly, where both increased in expression around the same time and their transcripts became detectable in inflorescence tissue after the transition into flowering. Also, the two genes were expressed in the petals and gynoecia (the carpels), but only slightly expressed in the stamens and sepals (Figure 1a).

During vegetative development, AmFUL, DEFH28 and SQUA were found to be similar in time of expression as well as in location, while FLO was not. FLO was upregulated in the leaf tissue around the same time as SBP1 and SBP2 and its mRNA was not detectable in mid stage flowers. As for the other three genes, AmFUL, DEFH28 and SQUA, they were only slightly if at all expressed in leaf tissue, but were expressed in sepals, petals, and gynoecia, though DEFH28 was only expressed as mRNA in petals and gynoecia (much like the SBP1 and SBP2 genes) (Figure 1a).

To get a more detailed description of the gene expressions, an in situ hybridization analyses was examined where AmFUL was expressed in both the apical inflorescence and lateral floral meristems, similar to DEFH28, SBP1 and SBP2, while SQUAis only expressed in the floral meristems (Figure 1b-d). This indicates the AmFUL is not prevented from the floral meristems by SQUA whereas FUL in A. thaliana is.

3. SBP function in flowering time is broadly conserved

(Preston and Hileman 2010) Figure 3- The silencing of SBP1 causes late or no flowering.

(Preston and Hileman 2010) Figure 3- This figure presents data that SBP1 causes late or no flowering. (a) pTRV2-Empty controls (left) compared to positive pTRV2-SBP1 (right), which failed to flower in normal setting as well as after 6 months in the growth chamber. (b)Primary branch of an uninfected plant showing its features indicated on the figure (arrows point to two flowers). (c) Lateral branch positive pTRV2-SBP1 with more spirally arranged leaves compared to control plants. (d) Comparison diagram of wild-type (left) and non-flowering pTRV2-SBP1 positive plants (right), which develop more lateral branches and spirally arranged leaves per branch than wild-type plants. (e) Cumulative flowering of uninfected, pTRV2-Empty and pTRV2-SBP1 plants over developmental time (measured by leaf node number), where all wild-type and pTRV2-Empty, but no pTRV2-SBP1 plants flowered before the 20 leaf node stage.

In order to perform the next experiment, it was first proven that virus-induced gene silencing (VIGS) is an effective reverse genetic approach in A. majus, which primarily acts to silence a gene of interest. This approach was specific for silencing SBP1 and not SBP2, and was useful in producing a pTRV2-Empty positive plant (a control in which the VIGS process was simulated but silencing did not occur) and a pTRV2-SBP1 positive plant (the SBP1 mutant where SBP1 was silenced) to be compared to the wild type A.majus. Silencing the SBP1 gene resulted in late flowering or no flowering at all, but at the time of flowering these plants did not remain positive for pTRV2-SBP1 (SBP1 did not remain silenced) and had normal levels of SBP1 (Figure 2), indicating that SBP1 is needed for the transition into flowering of A. majus.

4. SBP1 positively regulates inflorescence and floral meristem identity genes

In A. thaliana, SPL3-5 upregulate FUL, AP1, and LFY to regulate the time of flowering, so the next step was to see if SBP1 (the close relative of SPL3-5) upregulates the inflorescence meristem identity genes (SQUA, AmFUL, DEFH28, and FLO) in A. majus. Comparing the pTRV2-SBP1 and wild type plants showed that all four meristem gene levels were significantly lower than in the SBP1 silenced plants than in the wild type plants, suggesting that SBP1 acts to upregulate these genes.

5. The direct regulation of FUL-like genes by SBP proteins is likely to be conserved

SBP1/2 and SPL3 were previously found to bind to the same motif of SQUA, FUL, and AP1 genes (500bp from the start codon). (Klein et al., 1996) The core sequence of this motif is known to be the binding site of all SBP-box proteins, so to test whether these sites exist on other FUL gene homologs analyses was performed on the gene sequences of Arabidopsis thaliana CAULIFLOWER (CAL), and Mimulus guttatus FUL-like genes. Most of the genes sequenced showed this motif 700bp from the start codon, suggesting that SBP-box proteins may regulate FUL-like genes in core eudicots.

So What Does This Mean?

The results of these experiments have helped to outline a genetic pathway for the flowering of A. majus. Through reverse genetics it was found that without SBP1, no flowering or late flowering (where SBP1 expression was arose again after being silenced) occurred indicating that SBP1 is needed for the transition into a flowering state. These results also indicate that SBP box proteins positively regulate meristem identity genes in A. majus. SBP1 was also found to be upregulated before the FUL-like genes and resulted in a decrease or absence of FUL-like levels when silenced providing evidence that SBP1 of FUL-like genes. Lastly, the results when compared to the SPL3-5 proteins that regulate LFY-like genes in A. thaliana, indicate that SBP-box proteins are conserved in core eudicots through their regulation of FUL- and LFY-like genes.

What Can Be Done Next? What Can Be Improved?

Though the experiments performed by Preston and Hileman provide a new understanding of the genetic pathway of flowering in A. majus as well as the conservation of the SBP-box proteins in eudicots, little information was found regarding the role of the SBP2 protein. Future tests could be performed on this protein to discover its role in A. majus. Also, the VIGS testing in which SBP1 was silenced was only performed under long-day conditions, so further analysis could be performed under short-day conditions to see if SBP1 has the same effect on FUL-like genes. However, overall Preston and Hileman seemed to successfully show that SBP1 of snapdragons is homologous to SBP3-5 of A. thaliana, thus providing further insight on the flowering pathway of snapdragons as well as the evolutionary differences between the two model organisms.

References

1.Ferrándiz, C., Gu, Q., Martienssen, R. and Yanofsky, M.F. (2000) Redundant regulation of meristem identity and plant architecture by FRUITFULL, APETALA1 and CAULIFLOWER. Development, 12, 725–734.

2.Klein, J., Saedler, H. and Huijser, P. (1996) A new family of DNA binding proteins includes putative transcriptional regulators of the Antirrhinum majus floral meristem identity gene SQUAMOSA. Mol. Gen. Genet. 250, 7–16.

3.Preston, J. C. and Hileman, L. C. (2010), SQUAMOSA-PROMOTER BINDING PROTEIN 1 initiates flowering in Antirrhinum majus through the activation of meristem identity genes. The Plant Journal, 62: 704–712.

4.Wang, J.-W., Czech, B. and Weigel, D. (2009) miR156-regulated SPL transcription factors define an endogenous flowering pathway in Arabidopsis thaliana. Cell, 138, 738–749.

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