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Is (are) there any crucial gene(s) for the formation of flower in flowering plants?

Is (are) there any crucial gene(s) for the formation of flower in flowering plants?


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I am interested in qualitative (flowers of some plants have petal or sepal, but some plants have not) and quantitative (number of flowers of plants) differences between flowers of different plants. Formation of flower in flowering plants is under control of which genes? Which genes are the most important ones in flower development, for example which gene tell the flower that you should have for example 2 stigmas. or which genes tell the plant that you should have 4 flowers or your flowers will not have sepal… Thank you


It's in the genes: Research pinpoints how plants know when to flower

Scientists believe they've pinpointed the last crucial piece of the 80-year-old puzzle of how plants "know" when to flower.

Determining the proper time to flower, important if a plant is to reproduce successfully, involves a sequence of molecular events, a plant's circadian clock and sunlight.

Understanding how flowering works in the simple plant used in this study -- Arabidopsis -- should lead to a better understanding of how the same genes work in more complex plants grown as crops such as rice, wheat and barley, according to Takato Imaizumi, a University of Washington assistant professor of biology and corresponding author of a paper in the May 25 issue of the journal Science.

"If we can regulate the timing of flowering, we might be able to increase crop yield by accelerating or delaying this. Knowing the mechanism gives us the tools to manipulate this," Imaizumi said. Along with food crops, the work might also lead to higher yields of plants grown for biofuels.

At specific times of year, flowering plants produce a protein known as FLOWERING LOCUS T in their leaves that induces flowering. Once this protein is made, it travels from the leaves to the shoot apex, a part of the plant where cells are undifferentiated, meaning they can either become leaves or flowers. At the shoot apex, this protein starts the molecular changes that send cells on the path to becoming flowers.

Changes in day length tell many organisms that the seasons are changing. It has long been known that plants use an internal time-keeping mechanism known as the circadian clock to measure changes in day length. Circadian clocks synchronize biological processes during 24-hour periods in people, animals, insects, plants and other organisms.

Imaizumi and the paper's co-authors investigated what's called the FKF1 protein, which they suspected was a key player in the mechanism by which plants recognize seasonal change and know when to flower. FKF1 protein is a photoreceptor, meaning it is activated by sunlight.

"The FKF1 photoreceptor protein we've been working on is expressed in the late afternoon every day, and is very tightly regulated by the plant's circadian clock," Imaizumi said. "When this protein is expressed during days that are short, this protein cannot be activated, as there is no daylight in the late afternoon. When this protein is expressed during a longer day, this photoreceptor makes use of the light and activates the flowering mechanisms involving FLOWERING LOCUS T. The circadian clock regulates the timing of the specific photoreceptor for flowering. That is how plants sense differences in day length."

This system keeps plants from flowering when it's a poor time to reproduce, such as the dead of winter when days are short and nights are long.

The new findings come from work with the plant Arabidopsis, a small plant in the mustard family that's often used in genetic research. They validate predictions from a mathematical model of the mechanism that causes Arabidopsis to flower that was developed by Andrew Millar, a University of Edinburgh professor of biology and co-author of the paper.

"Our mathematical model helped us to understand the operating principles of the plants' day-length sensor," Millar said. "Those principles will hold true in other plants, like rice, where the crop's day-length response is one of the factors that limits where farmers can obtain good harvests. It's that same day-length response that needs controlled lighting for laying chickens and fish farms, so it's just as important to understand this response in animals.

"The proteins involved in animals are not yet so well understood as they are in plants but we expect the same principles that we've learned from these studies to apply."

First author on the paper is Young Hun Song, a postdoctoral researcher in Imaizumi's UW lab. The other co-authors are Benjamin To, who was a UW undergraduate student when this work was being conducted, and Robert Smith, a University of Edinburgh graduate student. The work was funded by the National Institutes of Health, and the United Kingdom's Biotechnology and Biological Sciences Research Council.


Contents

The transition from the vegetative phase to a reproductive phase involves a dramatic change in the plant's vital cycle, perhaps the most important one, as the process must be carried out correctly in order to guarantee that the plant produces descendants. This transition is characterised by the induction and development of the meristem of the inflorescence, which will produce a collection of flowers or one flower, where only one is produced. This morphogenetic change contains both endogenous and exogenous elements: For example, in order for the change to be initiated the plant must have a certain number of leaves and contain a certain level of total biomass. Certain environmental conditions are also required such as a characteristic photoperiod. Plant hormones play an important part in the process, with the gibberellins having a particularly important role. [4]

There are many signals that regulate the molecular biology of the process. The following three genes in Arabidopsis thaliana possess both common and independent functions in floral transition: FLOWERING LOCUS T (FT), LEAFY (LFY), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1, also called AGAMOUS-LIKE20). [5] SOC1 is a MADS-box-type gene, which integrates responses to photoperiod, vernalization and gibberellins. [4]

The meristem can be defined as the tissue or group of plant tissues that contain undifferentiated stem cells, which are capable of producing any type of cell tissue. Their maintenance and development, both in the vegetative meristem or the meristem of the inflorescence is controlled by genetic cell fate determination mechanisms. This means that a number of genes will directly regulate, for example, the maintenance of the stem cell's characteristics (gene WUSCHEL or WUS), and others will act via negative feedback mechanisms in order to inhibit a characteristic (gene CLAVATA or CLV). In this way both mechanisms give rise to a feedback loop, which along with other elements lend a great deal of robustness to the system. [6] Along with the WUS gene the SHOOTMERISTEMLESS (STM) gene also represses the differentiation of the meristematic dome. This gene acts by inhibiting the possible differentiation of the stem cells but still allows cell division in the daughter cells, which, had they been allowed to differentiate, would have given rise to distinct organs. [7]

A flower's anatomy, as defined by the presence of a series of organs (sepals, petals, stamens and carpels) positioned according to a given pattern, facilitate sexual reproduction in flowering plants. The flower arises from the activity of three classes of genes, which regulate floral development: genes which regulate the identity of the meristem, the identity of the flower organ and finally cadastral genes. [8]

  • Meristem identity genes. Code for the transcription factors required to initiate the induction of the identity genes. They are positive regulators of organ identity during floral development.
  • Organ identity genes. Directly control organ identity and also code for transcription factors that control the expression of other genes, whose products are implicated in the formation or function of the distinct organs of the flower.
  • Cadastral genes. Act as spatial regulators for the organ identity genes by defining boundaries for their expression. In this way they control the extent to which genes interact thereby regulating whether they act in the same place at the same time.

The ABC model Edit

The ABC model of flower development was first formulated by George Haughn and Chris Somerville in 1988. [9] It was first used as a model to describe the collection of genetic mechanisms that establish floral organ identity in the Rosids, as exemplified by Arabidopsis thaliana, and the Asterids, as demonstrated by Antirrhinum majus. Both species have four verticils (sepals, petals, stamens and carpels), which are defined by the differential expression of a number of homeotic genes present in each verticil. This means that the sepals are solely characterized by the expression of A genes, while the petals are characterized by the co-expression of A and B genes. The B and C genes establish the identity of the stamens and the carpels only require C genes to be active. Type A and C genes are reciprocally antagonistic. [10]

The fact that these homeotic genes determine an organ's identity becomes evident when a gene that represents a particular function, for example the A gene, is not expressed. In Arabidopsis this loss results in a flower which is composed of one verticil of carpels, another containing stamens and another of carpels. [10] This method for studying gene function uses reverse genetics techniques to produce transgenic plants that contain a mechanism for gene silencing through RNA interference. In other studies, using forward genetics techniques such as genetic mapping, it is the analysis of the phenotypes of flowers with structural anomalies that leads to the cloning of the gene of interest. The flowers may possess a non-functional or over expressed allele for the gene being studied. [11]

The existence of two supplementary functions, D and E, have also been proposed in addition to the A, B and C functions already discussed. Function D specifies the identity of the ovule, as a separate reproductive function from the development of the carpels, which occurs after their determination. [12] Function E relates to a physiological requirement that is a characteristic of all floral verticils, although, it was initially described as necessary for the development of the three innermost verticils (Function E sensu stricto). [13] However, its broader definition (sensu lato) suggests that it is required in the four verticils. [14] Therefore, when Function D is lost the structure of the ovules becomes similar to that of leaves and when Function E is lost sensu stricto, the floral organs of the three outer most verticils are transformed into sepals, [13] while on losing Function E sensu lato, all the verticils are similar to leaves. [14] The gene products of genes with D and E functions are also MADS-box genes. [15]

Genetic analysis Edit

The methodology for studying flower development involves two steps. Firstly, the identification of the exact genes required for determining the identity of the floral meristem. In A. thaliana these include APETALA1 (AP1) and LEAFY (LFY). Secondly, genetic analysis is carried out on the aberrant phenotypes for the relative characteristics of the flowers, which allows the characterization of the homeotic genes implicated in the process.

Analysis of mutants Edit

There are a great many mutations that affect floral morphology, although the analysis of these mutants is a recent development. Supporting evidence for the existence of these mutations comes from the fact that a large number affect the identity of floral organs. For example, some organs develop in a location where others should develop. This is called homeotic mutation, which is analogous to HOX gene mutations found in Drosophila. In Arabidopsis and Antirrhinum, the two taxa on which models are based, these mutations always affect adjacent verticils. This allows the characterization of three classes of mutation, according to which verticils are affected:

  • Mutations in type A genes, these mutations affect the calyx and corolla, which are the outermost verticils. In these mutants, such as APETALA2 in A. thaliana, carpels develop instead of sepals and stamen in place of petals. This means that, the verticils of the perianth are transformed into reproductive verticils.
  • Mutations in type B genes, these mutations affect the corolla and the stamen, which are the intermediate verticils. Two mutations have been found in A. thaliana, APETALA3 and PISTILLATA, which cause development of sepals instead of petals and carpels in the place of stamen.
  • Mutations in type C genes, these mutations affect the reproductive verticils, namely the stamen and the carpels. The A. thaliana mutant of this type is called AGAMOUS, it possesses a phenotype containing petals instead of stamen and sepals instead of carpels.

Techniques for detecting differential expression Edit

Cloning studies have been carried out on DNA in the genes associated with the affected homeotic functions in the mutants discussed above. These studies used serial analysis of gene expression throughout floral development to show patterns of tissue expression, which, in general, correspond with the predictions of the ABC model.

The nature of these genes corresponds to that of transcription factors, which, as expected, have analogous structures to a group of factors contained in yeasts and animal cells. This group is called MADS, which is an acronym for the different factors contained in the group. These MADS factors have been detected in all the vegetable species studied, although the involvement of other elements involved in the regulation of gene expression cannot be discounted. [8]

Genes exhibiting type-A function Edit

In A. thaliana, function A is mainly represented by two genes APETALA1 (AP1) and APETALA2 (AP2) [16] AP1 is a MADS-box type gene, while AP2 belongs to the family of genes that contains AP2, which it gives its name to and which consists of transcription factors that are only found in plants. [17] AP2 has also been shown to complex with the co-repressor TOPLESS (TPL) in developing floral buds to repress the C-class gene AGAMOUS (AG). [18] However, AP2 is not expressed in the shoot apical meristem (SAM), which contains the latent stem cell population throughout the adult life of Arabidopsis, and so it is speculated that TPL works with some other A-class gene in the SAM to repress AG. [18] AP1 functions as a type A gene, both in controlling the identity of sepals and petals, and it also acts in the floral meristem. AP2 not only functions in the first two verticils, but also in the remaining two, in developing ovules and even in leaves. It is also likely that post-transcriptional regulation exists, which controls its A function, or even that it has other purposes in the determination of organ identity independent of that mentioned here. [17]

In Antirrhinum, the orthologous gene to AP1 is SQUAMOSA (SQUA), which also has a particular impact on the floral meristem. The homologs for AP2 are LIPLESS1 (LIP1) and LIPLESS2 (LIP2), which have a redundant function and are of special interest in the development of sepals, petals and ovules. [19]

A total of three genes have been isolated from Petunia hybrida that are similar to AP2: P. hybrida APETALA2A (PhAP2A), PhAP2B and PhAP2C. PhAP2A is, to a large degree, homologous with the AP2 gene of Arabidopsis, both in its sequence and in its expression pattern, which suggests that the two genes are orthologs. The proteins PhAP2B and PhAP2C, on the other hand, are slightly different, even though they belong to the family of transcription factors that are similar to AP2. In addition they are expressed in different ways, although they are very similar in comparison with PhAP2A. In fact, the mutants for these genes do not show the usual phenotype, that of the null alleles of A genes. [20] A true A-function gene has not been found in Petunia though a part of the A-function (the inhibition of the C in the outer two whorls) has been largely attributed to miRNA169 (colloquially called BLIND) ref .

Genes exhibiting type-B function Edit

In A. thaliana the type-B function mainly arises from two genes, APETALA3 (AP3) and PISTILLATA (PI), both of which are MADS-box genes. A mutation of either of these genes causes the homeotic conversion of petals into sepals and of stamens into carpels. [21] This also occurs in its orthologs in A. majus, which are DEFICIENS (DEF) and GLOBOSA (GLO) respectively. [22] For both species the active form of binding with DNA is that derived from the heterodimer: AP3 and PI, or DEF and GLO, dimerize. This is the form in which they are able to function. [23]

The GLO/PI lines that have been duplicated in Petunia contain P. hybrida GLOBOSA1 (PhGLO1, also called FBP1) and also PhGLO2 (also called PMADS2 or FBP3). For the functional elements equivalent to AP3/DEF in Petunia there is both a gene that possesses a relatively similar sequence, called PhDEF and there is also an atypical B function gene called PhTM6. Phylogenetic studies have placed the first three within the «euAP3» lineage, while PhTM6 belongs to that of «paleoAP3». [24] It is worth pointing out that, in terms of evolutionary history, the appearance of the euAP3 line seems to be related with the emergence of dicotyledons, as representatives of euAP3-type B function genes are present in dicotyledons while paleoAP3 genes are present in monocotyledons and basal angiosperms, among others. [25]

As discussed above, the floral organs of eudicotyledonous angiosperms are arranged in 4 different verticils, containing the sepals, petals, stamen and carpels. The ABC model states that the identity of these organs is determined by the homeotic genes A, A+B, B+C and C, respectively. In contrast with the sepal and petal verticils of the eudicots, the perigone of many plants of the family Liliaceae have two nearly identical external petaloid verticils (the tepals). In order to explain the floral morphology of the Liliaceae, van Tunen et al. proposed a modified ABC model in 1993. This model suggests that class B genes are not only expressed in verticils 2 and 3, but also in 1. It therefore follows that the organs of verticils 1 and 2 express class A and B genes and this is how they have a petaloid structure. This theoretical model has been experimentally proven through the cloning and characterization of homologs of the Antirrhinum genes GLOBOSA and DEFICIENS in a Liliaceae, the tulip Tulipa gesneriana. These genes are expressed in verticils 1,2 and 3. [26] The homologs GLOBOSA and DEFICIENS have also been isolated and characterized in Agapanthus praecox ssp. orientalis (Agapanthaceae), which is phylogenetically distant from the model organisms. In this study the genes were called ApGLO and ApDEF, respectively. Both contain open reading frames that code for proteins with 210 to 214 amino acids. Phylogenetic analysis of these sequences indicated that they belong to B gene family of the monocotyledons. In situ hybridization studies revealed that both sequences are expressed in verticil 1 as well as in 2 and 3. When taken together, these observations show that the floral development mechanism of Agapanthus also follows the modified ABC model. [27]

Genes exhibiting type-C function Edit

In A. thaliana, the C function is derived from one MADS-box type gene called AGAMOUS (AG), which intervenes both in the establishment of stamen and carpel identity as well as in the determination of the floral meristem. [16] Therefore, the AG mutants are devoid of androecium and gynoecium and they have petals and sepals in their place. In addition, the growth in the centre of the flower is undifferentiated, therefore the petals and sepals grow in repetitive verticils.

The PLENA (PLE) gene is present in A. majus, in place of the AG gene, although it is not an ortholog. However, the FARINELLI (FAR) gene is an ortholog, which is specific to the development of the anthers and the maturation of pollen. [28]

In Petunia, Antirrhinum and in maize the C function is controlled by a number of genes that act in the same manner. The genes that are closer homologs of AG in Petunia are pMADS3 and floral-binding protein 6 (FBP6). [28]

Genes exhibiting type-D and E functions Edit

The D function genes were discovered in 1995. These genes are MADS-box proteins and they have a function that is distinct from those previously described, although they have a certain homology with C function genes. These genes are called FLORAL BINDING PROTEIN7 (FBP7) and FLORAL BINDING PROTEIN1L (FBP1l). [12] It was found that, in Petunia, they are involved in the development of the ovule. Equivalent genes were later found in Arabidopsis, [29] where they are also involved in controlling the development of carpels and the ovule and even with structures related to seed dispersal.

The appearance of interesting phenotypes in RNA interference studies in Petunia and tomato led, in 1994, to the definition of a new type of function in the floral development model. The E function was initially thought to be only involved in the development of the three innermost verticils, however, subsequent work found that its expression was required in all the floral verticils. [13]


Results

Identification of COL genes in sesame

To identify the COL genes in sesame, the Hidden Markov Model (HMM) search was performed against the sesame protein database using the Zinc-finger B-box motif (PF00643) and CCT (CONSTANS, CONSTANS-like, TIMING OF CAB EXPRESSION 1) domain (PF06203). In total, 37 B-box Zinc-finger genes and 36 CCT domain-containing genes were identified in the sesame genome, respectively (Additional file 1: Table S1). The B-box Zinc-finger genes and the CCT domain-containing genes were then compared with each other and 13 genes of them were found to be the same. Therefore, the 13 genes which contained both Zinc-finger B-box motif and CCT domain were identified and named as sesame COL genes (Table 1). All of the Arabidopsis COL protein sequences were used as queries for the Basic Local Alignment Search Tool (BLAST) to identify sesame COL proteins. However, we had not identified any additional proteins containing both B-box motifs and CCT domain in the sesame genome. All B-box motif and CCT domain in the SiCOLs were validated by the CDD (http://www.ncbi.nlm.nih.gov/cdd/) and simple modular architecture research tool (SMART) analyses.

The SiCOL genes were not evenly distributed on the linkage groups (LGs) of the sesame genome: one gene on LG3, LG15 and LG16, and two genes on LG1, LG2, LG5, LG6 and LG8. The SiCOL proteins ranged from 332 (SIN_1004896) to 461 (SIN_1018340) amino acids (aa) in length, with an average length of approximately 385 aa. Moreover, no tandem duplicate genes were identified for these SiCOLs, although tandem duplication events had been observed in several other sesame gene families [46,47,48].

Phylogenetic analysis of the SiCOL genes

A phylogenetic tree was constructed using the neighbor-joining (NJ) method basing on multiple alignments of sesame and Arabidopsis COL genes (Fig. 1a). The 13 SiCOLs were classified into three groups (I, II, and III) and each group consisted of 6, 3, and 4 SiCOL proteins, respectively. Two SiCOL genes, SIN_1019889 and SIN_1004896 showed the closest relationship with the Arabidopsis CO gene. The Arabidopsis CO protein sequence was also used as query for the BLAST to identify the homologous genes. It showed that SIN_1019889 and SIN_1004896 were the only homologous genes of Arabidopsis CO gene in sesame. Thus, these two genes were referred as SiCOL1 (SIN_1019889) and SiCOL2 (SIN_1004896), respectively. We therefore concluded that these genes might be involved in the photoperiodic regulation of sesame flowering.

Phylogenetic analysis of the SiCOL genes. a A NJ phylogenetic tree of the COL proteins in sesame and Arabidopsis. The bootstrap values were inferred from 1000 replicates. b Phylogenetic relationship among COL proteins. The phylogram was generated from the multiple alignments of the deduced amino acid sequence from SiCOL1 and SiCOL2 and homologous proteins from other plant species. Bootstrap values from 1000 replicates were used to assess the robustness of the tree and the bootstrap values > 50% are showed

Phylogenetic analysis of SiCOL1, SiCOL2, CO and CO homologous proteins in the other 19 plant species was performed. CO homologous proteins from monocots and dicots were clustered into two groups. Both SiCOL1 and SiCOL2 proteins were divided into the dicots group. SiCOL1 protein (GeneBank ID: XP_011085568) displayed the highest similarity to PnCO protein (the CO protein in Pharbitis nil, 53% identity, AF300700) whereas it showed a 44% identity with CO protein from Arabidopsis (NP_197088). SiCOL2 protein (XP_011099077) displayed the highest identity to SlCO protein (60% identity, NP_001233839) and StCO protein (60% identity, ARU77840), which was higher than that of Arabidopsis CO protein (48% identity). However, SlCO was not involved in the control of flowering time of Solanum lycopersicum [49]. Previous research suggested that sesame was taxonomically close to Utricularia gibba, S. lycopersicum and S. tuberosum [43]. However, in this study, UgCO protein was not close to either SiCOL1 or SiCOL2 protein.

Conserved motifs and structure of the SiCOL genes

Using the SiCOL phylogenetic relationship data, we identified structural features of the sesame COLs, including conserved motifs and the locations of exons and introns (Additional file 1: Figure S1). The SiCOL genes of Group I and Group II had a simple gene structure -- one intron and two exons (Additional file 1: Figure S1b), while all genes in Group III had more exons and presented more complex gene structure than that of Group I and Group II. Multiple Em for Motif Elicitation (MEME) analysis confirmed the presence of the B-box motifs and CCT domains in SiCOL gene sequences. All genes in Group I and Group III had two B-box motifs except SiCOL2, which lacked one of the B-box motifs (Additional file 1: Figure S1c).

The protein sequences of SiCOL1 and SiCOL2 were further analyzed (Additional file 1: Figure S2). The result showed that they shared high similarity in amino acid sequence (61.7%), especially in the regions of B-box 2 motif (83.7%) and CCT domain (97.7%). SiCOL1 and SiCOL2 proteins had large differences in the B-box 1 motif region. Most amino acids of B-box 1 motif in SiCOL2 protein were lost. Even the remaining amino acids in B-box 1 motif of SiCOL2 protein were also quite different from that of SiCOL1. B-box motif plays an important role in the regulation of transcription and in mediating protein–protein interaction [50], and the missing of B-box 1 motif may cause loss of partial function of SiCOL2.

Overexpression of SiCOL1 and SiCOL2 in Arabidopsis

To explore the role of SiCOL1 and SiCOL2 in flowering, we constructed SiCOL1 and SiCOL2 overexpression vectors, and transferred into Arabidopsis Col-0 lines, respectively. Ten independent T0 transgenic lines were obtained for each gene. T1 generation transgenic lines planted in LD condition were about 3 days earlier flowering than the wild type. T2 generation plants were significantly earlier flowering (5 days of 35S::SiCOL1 on average, P < 0.001, and 3 days of 35S::SiCOL2 on average, P < 0.001) than the wild type (Fig. 2 and Additional file 1: Table S2). It is noteworthy that the T2 transgenic lines of 35S::SiCOL1 flowered earlier (2 days in average) than that of 35S::SiCOL2. This result might be caused by the loss of B-box 1 motif in SiCOL2 protein. Therefore, we concluded that SiCOL2 might lose partial function of flowering regulation and SiCOL1 was potential functional homologous gene of CO in sesame.

Days to flowering of transgenic Arabidopsis with overexpressed SiCOL1 and SiCOL2 under LD condition. a Flowering phenotype of T2 transgenic Arabidopsis lines with overexpressed SiCOL1 and SiCOL2. Photo was taken at 7 d after flowering of the SiCOL1 transgenic line. b Days to flowering of T2 transgenic Arabidopsis lines with overexpressed SiCOL1 and SiCOL2 under LD condition. The T2 transgenic Arabidopsis lines containing empty vector were used as control. For each test of 35S::SiCOL1, 35S::SiCOL2 and empty vector, days to flowering of ten lines (each contained 10 plants) were counted (Additional file 1: Table S2). The bar indicates standard deviation

To investigate the mechanism of action of SiCOL1 and SiCOL2 in Arabidopsis, we compared the expression patterns of flowering related genes FT in transgenic lines with wild type under LDs. Under LDs, FT is induced by CO and promotes flowering in Arabidopsis [51]. Comparing with the FT in wild type, FT in the transgenic lines expressed in an extremely high level (Additional file 1: Figure S3). The result suggested that SiCOL1 and SiCOL2 promoted Arabidopsis flowering by inducing the expression of FT. Moreover, expression of FT in T2 transgenic lines with 35S::SiCOL1 was much higher than that in the 35S::SiCOL2 transgenic lines, indicating SiCOL1 had higher induction efficiency of FT expression than SiCOL2.

Expression patterns of SiCOL1 and SiCOL2

Five different tissues of sesame were collected from the widely cultivated sesame variety ‘Zhongzhi13’, including root, stem, leaf, capsule and seed. Quantitative real-time polymerase chain reaction (qRT–qPCR) was used to investigate the expression of SiCOL1 and SiCOL2 in these tissues. The result revealed that the expression of SiCOL1 and SiCOL2 in root, stem, capsule and seed were almost in the same level (Fig. 3a and Additional file 1: Figure S4a). However, both the expression levels of SiCOL1 and SiCOL2 in leaf were significantly higher than that in other tissues (P < 0.001).

Relative expression of SiCOL1 in different tissues and development stages of sesame. a Relative expression of SiCOL1 in five tissues of sesame. b Relative expression of SiCOL1 in leaves of different development stages. The red arrow indicates that tiny flower buds begin to appear in the axil of sesame plants. Transcript abundance was quantified using qRT-PCR and expression levels were normalized using sesame actin7 as a reference gene. The bar indicates standard deviation

Expression of SiCOL1 and SiCOL2 in leaf at the different development stages (from 14 days to 50 days after seed sowing) of ‘Zhongzhi13’ was investigated. All samples were collected in the same time (8:00 am) during a day. Generally, the flower buds of the variety ‘Zhongzhi13’ appear in approximately 30 days and ‘Zhongzhi13’ flowers at about 40 days in the growing season at Wuhan, China. The SiCOL1 and SiCOL2 expression increased quickly from 14 to 28 days and reached the highest level in 28 days, which was the exactly time before the flower buds appeared in the axil of sesame (Fig. 3b and Additional file 1: Figure S4b). After the flower bud appeared, the expression of SiCOL1 moderately decreased (from 30 to 40 days). Although sesame is an indeterminate inflorescence species, the expression of SiCOL1 decreased noticeably after the plant flowered (50 days). However, the expression of SiCOL2 slightly increased after sesame flowering. The result suggested that the expression of SiCOL1 and SiCOL2 dynamic changed during the development of sesame floral organ.

Individuals of ‘Zhongzhi13’ were grown in the LD (14 h light) and SD (9 h light) conditions, respectively. In about 3 days before the flower buds appeared, leaves from three individuals were collected during a 24 h period under LD and SD conditions, respectively. Expressions of SiCOL1 and SiCOL2 in the leaves under LD and SD conditions were detected. Although expression of SiCOL2 was higher than SiCOL1 in both LD and SD conditions, the expression patterns of these two genes were extremely similar. Both in LD and SD conditions, the expression of SiCOL1 and SiCOL2 increased during the darkness whereas decreased under light (Fig. 4 and Additional file 1: Figure S5). The peaks of transcript level of SiCOL1 and SiCOL2 in LD and SD conditions were both in the dawn. Under the SD condition, the lowest expression levels of SiCOL1 and SiCOL2 were both found at 1 h before dusk. Whereas, the valleys of the transcript levels for SiCOL1 and SiCOL2 under LD were different. Under LD condition, SiCOL1 and SiCOL2 had the lowest expression levels in 0 am and 8 pm, respectively. Therefore, as the homolog of CO in sesame, SiCOL1 and SiCOL2 exhibited significantly diurnal rhythmic expression and expressed in a high level before the flowering in leaves.

Relative diurnal expression of SiCOL1 under LD and SD conditions. a Relative expression of SiCOL1 under LD condition. b Relative expression of SiCOL1 under SD condition. White boxes below the graphs indicate light periods and dark boxes indicate darkness. The expression data was normalized by sesame actin7. The bar indicates standard deviation

Haplotype variation of SiCOL1 and SiCOL2

In order to analyze the haplotype variations of SiCOL1 and SiCOL2, SNPs of SiCOL1 and SiCOL2 in 132 landrace genomes were obtained from the SesameHapMap database (http://www.ncgr.ac.cn/SesameHapMap/). These landraces were collected from South Asia, Southeast Asia, East Asia and Central Asia. These regions are the main producing regions of sesame with rich germplasm resources. Among these regions, South Asia is also the geographic origin area of sesame [9, 52]. All samples were planted in the summer of Wuhan, China from 2015 to 2017 and their flowering dates were recorded. Previous study revealed that sesame accessions could be divided into south group and north group by the latitude 32°N [13]. In the present study, samples were also divided into south and north groups according to their geographic origin (Additional file 1: Table S3).

In total, 25, 23 and 2 SNPs were found in the promoter, coding region and intron of SiCOL1, respectively (Fig. 5). Among the 23 SNPs in the coding region, 13 SNPs were the synonymous mutations while the other 10 SNPs were the nonsynonymous mutations, which led to amino acid substitutions and might cause functional polymorphism of the SiCOL1 protein. Only one SNP and three SNPs were detected in the CCT domain and Zinc-finger domain, respectively.

Haplotypes of SiCOL1 among landraces from Asia. Reference base is the base in reference genome ‘Zhongzhi13’. SNP number is the mutation number among the 132 landraces. R, S and N in mutation type indicate replacement, synonymous SNP and nonsynonymous SNP, respectively. Numbers in the right column are numbers of cultivars represented in every haplotypes. Total, South and North indicate total landraces, landraces from south group and landraces from north group, respectively. Variations that different from the reference bases are shown in green

Based on the identified SNPs, 16 haplotypes of SiCOL1 were detected in the tested sesame accessions. All bases in Hap1 (Haplotype 1) were the same as the reference genome ‘Zhongzhi13’ [43]. The bases in Hap1 that were different from other haplotypes ranged from 1 to 35. Six of the haplotypes (Hap2 to Hap7) were similar to Hap1 while the other nine haplotypes (Hap8 to Hap16) were quite different from Hap1. There was only one SNP in Hap 2, Hap3, Hap4 and Hap5. But in Hap 14, Hap 15 and Hap16, the different bases reached 33, 34 and 35, respectively.

The variety ‘Baizhima’ (S054 in Additional file 1: Table S3), which had the SiCOL1 of Hap15 was selected and the expression of SiCOL1 and SiCOL2 was investigated. SiCOL2 showed diurnal rhythmic expression in ‘Baizhima’ under both LD and SD conditions (Additional file 1: Figure S5). However, the expression of SiCOL1 was not detected in ‘Baizhima’ under both LD and SD conditions, suggesting that mutated SiCOL1 did not express and might lose the function of photoperiod response in sesame flowering.

Totally, 15 SNPs were identified in SiCOL2, including seven SNPs in promoter, six SNPs in coding regions and two SNPs in intron (Additional file 1: Figure S6). Four SNPs in the coding regions were the nonsynonymous mutations. However, these SNPs were identified in a few samples, indicating that SiCOL2 was more conserved than SiCOL1. Using the 15 SNPs, SiCOL2 was clustered into 12 haplotypes. The haplotypes contained more than 7 accessions (5.30% of the total samples) were regarded as major haplotypes. Therefore, Hap1, Hap3 and Hap8 were identified to be the three major haplotypes. Among these haplotypes, Hap1 was the biggest haplotype, containing 65.2% of the total samples.

To valid the truth of the SNPs in SiCOL1 and SiCOL2, ten accessions were selected and sequenced. All SNPs identified in SiCOL1 and SiCOL2 of the ten samples were the same as them in SesameHapMap. The result suggested that all SNPs of these genes were true and could be used in the haplotype analysis. However, a 6 bp deletion (from 421 bp to 426 bp) in the coding region, which resulted in an Aspartic acid and a Glutamic acid deletion in protein, was detected in Hap15 of SiCOL1 (Additional file 1: Figure S7). Previous study showed that a 36 bp deletion in the coding region of Hd1 was the crucial mutation that led function divergence of Hd1 in rice [2]. This deletion might have potential influence of gene function in the Hap15 of SiCOL1.

As shown in Fig. 6, a network of all haplotypes was constructed. The haplotype number of landraces from south group (15) was much more than that of north group (5), suggesting that SiCOL1 had highly polymorphisms in the landraces of south group. There were four haplotypes contained landraces from both south and north group: Hap1, Hap6, Hap14 and Hap15. These four haplotypes were also the largest haplotypes in number, containing 90.2% (119 of 132 landraces) of the samples. The landraces belonging to south group were concentrated in Hap 1 and Hap6 (54 of 80 landraces), while most of the landraces from north group were in Hap14 and Hap15 (47 of 52 landraces).

Haplotype network of SiCOL1. Haplotypes are showed by colored solid circles. Circle size is proportional to the quantity of samples within a given haplotype. Hollow circles indicate the assumed haplotypes. Lines between haplotypes represent mutational steps between alleles. The numbers next to the lines indicate the nucleotide difference existed between the linked haplotypes. The red color and green color indicate landraces from south group and north group, respectively

The landraces from India presented in Hap1, Hap5, Hap6, Hap8, Hap9, Hap11, Hap12 and Hap13, indicating a high genetic diversity of SiCOL1 in India sesame landraces. If we take all landraces from South Asia (India, Bangladesh, Pakistan and Nepal) into account, more haplotypes could be found, including Hap4, Hap7, Hap10, Hap 15 and Hap16. Therefore, landraces from South Asia could be found in 13 haplotypes totally. For Southeast Asia, East Asia and Central Asia, the haplotypes of landraces from these regions were Hap7, Hap5 and Hap2, respectively. The haplotypes of landraces from South Asia were much more than haplotypes including landraces from other regions, suggesting that South Asia was the genetic diversity center of SiCOL1. This observation is consistent with previous suggestion that crop cultivars from the geographic origin areas tend to have higher genetic diversity [53, 54].

A network of all SiCOL2 haplotypes was also constructed (Additional file 1: Figure S8). Landraces from south group and north group were detected in twelve and five haplotypes, respectively. In the network of SiCOL1, two major haplotypes, Hap14 and Hap15 were dominated by the landraces from north group. However, landraces from south group were more than that from north group in all major haplotypes of SiCOL2 (Hap1, Hap3 and Hap8).

SiCOL1 haplotypes were related to sesame flowering

The flowering date of the 132 landraces from 2015 to 2017 in Wuhan, China (114°33′ E, 30°34′ N) was recorded and analyzed to further examine the relationship between SiCOL1 haplotypes and sesame flowering (Additional file 1: Table S3). The day light in the summer of Wuhan is a standard LD, sustaining from 13 h to 14.5 h. Under LDs, sesame landraces from north group flowering obviously earlier than that from south group. The box-plot showed the flowering date of landraces in Hap1, Hap 6, Hap14 and Hap15 from 2015 to 2017 (Fig. 7). As we described previously, Hap1 and Hap6 mainly contained sesame accessions from south group, while Hap14 and Hap15 included most sesame accessions from north group. Days to flowering time of the samples in Hap1 and Hap6 were significant more than that in Hap14 and Hap15 (Mann-Whitney test, P < 10 − 9 ). Taking flowering time in 2016 for example, the average flowering date of accessions in Hap1, Hap6, Hap14 and Hap15 was 58.5, 53, 46.2 and 46.3 d, respectively. The Pearson correlation coefficient was used to test the correlation between SiCOL1 haplotypes and flowering date. Significant correlations were identified in all 3 years: 2015 (R 2 = 0.32, R = 0.56, P = 3.10 × 10 − 11 ), 2016 (R 2 = 0.28, R = 0.53, P = 5.38 × 10 − 10 ) and 2017 (R 2 = 0.30, R = 0.55, P = 7.80 × 10 − 11 ). The results suggested that SiCOL1 variations were strongly related to the flowering time of sesame.

Box-plot of the flowering date of sesame landraces in major haplotypes. The major haplotypes, Hap1, Hap6 Hap14 and Hap15 contained 48, 10, 10 and 51 sesame accessions, respectively. All sesame landraces were planted from May to October at Wuhan, China in every year. The detailed information of the flowering date of the sesame landraces was provided in Additional file 1: Table S3

Geographic distribution of SiCOL1 haplotype

Comparing to Hap1 of SiCOL1, Hap15 had one 6 bp deletion in the coding region (Additional file 1: Figure S7) and many SNPs in the promoter as well as coding regions (Fig. 5). In addition, Hap15 did not express under both LD and SD conditions. Therefore, Hap15 of SiCOL1 was regarded as nonfunctional allele. Based on the similarity of haplotypes, we divided the 16 haplotypes of SiCOL1 into two groups, south haplotypes with functional alleles and north haplotypes with nonfunctional alleles. The south haplotypes included Hap1 to Hap7 while the north haplotypes contained Hap 8 to Hap 16. To investigate the relationship between the geographic origin and haplotypes of the sesame landraces, a map of Asia was downloaded from Wikimedia Commons (http://commons.wikimedia.org/wiki/Main_Page) and the distribution information of SiCOL1 haplotypes was showed in the map (Fig. 8). The map clearly showed that south haplotypes mainly existed in the south of 32°N while north haplotypes were concentrated in the north of 32°N. For the 13 countries, the proportion of the north haplotypes ranged from 0 (Nepal and Afghanistan) to 100% (Japan and Uzbekistan).

SiCOL1 protein type distribution among countries in Asia. Red solid circles indicate SiCOL1 protein types from Hap1 to Hap7, while the green solid circles represent SiCOL1 protein types from Hap8 to Hap16. The size of the circles is proportional to the quantity of sesame landraces. The latitude 32°N is indicated by dotted line. The original map was downloaded and adapted from “https://commons.wikimedia.org/wiki/File:BlankMap-Asia.png“(Bytebear at the English language Wikipedia). This original map is licensed under the Creative Commons Attribution-Share Alike 3.0 Unported license, which allows us to share and adapt for free with proper attribution

Since alleles contained in north haplotypes all were nonfunctional and very few landraces in the north haplotypes were from the geographic origin center of sesame, north haplotypes were regarded as the domesticated haplotypes of SiCOL1. The frequency of domesticated alleles is an indicator of artificial selection, so the proportion of the north haplotypes was used to examine the domestication and spread of sesame. North haplotypes were in the minority of Southern Asia, Southeast Asia and South China, but they were the dominant haplotypes in Northern China, Northeast Asia and Central Asia. Therefore, the result suggested that SiCOL1 had been strongly selected and might be the important domesticated gene that contributed to the spread of sesame from low-latitude regions to high-latitude regions.

Expression patterns of SiFT in two varieties with different SiCOL1 haplotypes

The homolog of FT in sesame, SiFT (SIN_1009320), was identified by BLAST [55]. Expression of SiFT was detected in ‘Zhongzhi13’ (with SiCOL1 of Hap1) and ‘Baizhima’ (with SiCOL1 of Hap15) under LD and SD conditions. The diurnal rhythmic expression pattern of SiFT was quite similar to that of SiCOL1 under both LD and SD conditions (Fig. 9), indicating that the expression of SiFT might be induced by SiCOL1. Although the expression pattern of SiFT in ‘Zhongzhi13’ and ‘Baizhima’ was similar, the expression level of SiFT in these two varieties was quite different under both LD and SD conditions. These significantly variant expression levels of SiFT in ‘Zhongzhi13’ and ‘Baizhima’ might result from the non-expression of SiCOL1 in ‘Baizhima’.

Relative diurnal expression of SiFT under LD and SD conditions. a Relative expression of SiFT under LD condition. b Relative expression of SiFT under SD condition. White boxes below the graphs indicate light periods and dark boxes indicate darkness. The expression data was normalized by sesame actin7. The bar indicates standard deviation

The peak of SiFT expression appeared later than that of SiCOL1. This phenomenon was in line with the homologue genes, Hd3a and Hd1, in the SD plant rice. Although Hd1 had the expression peak in dark, Hd3a had the highest expression level after dawn under both LD and SD conditions [56].


Floral organ specification

Once it is committed to a flowering fate, a shoot apical meristem of an annual plant becomes an inflorescence meristem from which floral meristems emerge on the periphery. The floral meristems then undergo differentiation that causes the sequential development of flower organ primordia, starting with sepals, continuing with petals and stamens, and terminating in carpels. In dicotyledon plants, these different floral organs are generally arranged in four concentric rings called whorls: sepals occupy the first whorl, petals the second whorl, stamens the third whorl, and carpels the fourth whorl.

In 1991, Meyerowitz and Coen proposed a model to describe the determination of floral organ identity based on phenotypic and genetic analyses of homeotic mutants of Antirrhinum and Arabidopsis. Their model, termed the ABC model ( Bowman et al. 1991 , Coen and Meyerowitz 1991 ), hypothesizes that the activity of three classes of genes called A, B and C specifies the type of floral organ in each whorl. The activity of A class genes alone is sufficient to cause sepals to form in the first whorl. However, the formation of petals in the second whorl requires the combined activities of A and B class genes. The development of stamens in the third whorl is similarly promoted by the collective activities of B and C class genes, whereas the formation of carpels in the fourth whorl is dependent solely on the C class gene activity.

The majority of the ABC genes are members of a large transcription factor family called the MADSbox gene family. It derives its name from some of the earliest known homeotic genes: MCM1 from yeast ( Passmore et al. 1988 ), AGAMOUS from Arabidopsis ( Yanofsky et al. 1990 ), DEFICIENS from Antirrhinum majus ( Sommer et al. 1990 ) and SRF from Homo sapiens ( Norman et al. 1988 ). In plants, this gene family is involved in a myriad of developmental events, including flower initiation, floral patterning, fruit and seed development, and leaf and root formation.

Two of the A class genes that have been isolated from Arabidopsis include AP1 ( Mandel et al. 1992 ) and APETALA2 (AP2) ( Jofuku et al. 1994 ). The product of the former, a MADSbox gene, has dual functions in promoting floral meristem identity at the initial stage of flower development, and in subsequently specifying floral organ identity. On the other hand, AP2, which is a transcription regulator of the AP2/ERF family ( Magnani et al. 2004 ) and does not contain a MADS domain, has been recently implicated as being the target of a microRNAmediated gene regulatory network ( Chen 2004 ). Strong ap1 mutants have mostly bractlike structures in the first whorl and no organs in the second whorl, while maintaining the normal development of the inner two whorls ( Bowman et al. 1993 ). In addition, flowers produced by these plants are often partially converted to inflorescence shoots, alluding to the early role of AP1 in establishing floral meristem identity. In comparison, mutations in the AP2 gene result in the conversion of sepals to leaves or carpels in the first whorl, and petals to staminoid organs in the second whorl, but do not alter the organ identity of the third and fourth whorls ( Bowman et al. 1991 ).

The B class genes identified so far are APETALA3 (AP3) ( Jack et al. 1992 ) and PISTILLATA (PI) ( Goto and Meyerowitz 1994 ). Both are members of the MADSbox gene family. The AP3 and PI genes are active in petals and stamens, consistent with their roles in determining the identity of these organs ( Jack et al. 1992 , Goto and Meyerowitz 1994 ). Disruption of the normal function of either of these genes causes sepals and carpeloid organs to grow in the second and third whorls respectively ( Hill and Lord 1989 , Jack et al. 1992 ).

The most studied member of the C class gene is AGAMOUS (AG) ( Yanofsky et al. 1990 ). The AG gene encodes a MADSbox transcription factor whose function is required for the proper development of stamens and carpels. Mutant Arabidopsis plants without functional AG produce indeterminate flowers in which stamens are converted to petals and the gynoecium is replaced by a secondary flower that has petals in the third whorl and a tertiary flower in the center. The indeterminate nature of flowers produced in the absence of a functional AG shows that, apart from specifying floral organ identity, AG is also involved in terminating floral meristem activity towards the end of flower development by repressing the activity of the meristempromoting gene, WUSCHEL ( Lenhard et al. 2001 ).

Recent studies indicate that other factors are required for specifying the identity of floral organs in conjunction with the ABC genes. Among such factors are the SEPALLATA genes (SEP) ( Pelaz et al. 2000 ). Like most of the ABC genes, the SEP genes are members of the MADSbox gene family. There are four functionally redundant SEP genes ( Pelaz et al. 2000 , Ditta et al. 2004 )—SEP1, SEP2, SEP3 and SEP4—in Arabidopsis, and together they are essential for the specification of organ identity in all four whorls of a flower. In addition to specifying floral organ identity, one of the SEP genes, SEP4, has been shown to play a pivotal role in maintaining flower meristem identity ( Ditta et al. 2004 ). In sep1 sep2 sep3 triple mutants, the inner three whorls of a flower are converted into sepals ( Pelaz et al. 2000 ). In plants bearing mutations in all four SEP genes, floral organs are replaced by leaflike structures ( Ditta et al. 2004 ). The importance of SEP genes in promoting the development of sepals, petals, stamens and carpels has led to the incorporation of these genes, collectively called the E class genes, into the ABC model ( Jack 2004 ).


The Circadian Rhythm Model

Recent work mostly in the long-day plant, Arabidopsis - supports a different model of photoperiodism. This work suggests that the photoperiodic response is governed by the interaction of daylight with innate circadian rhythms of the plant.

  • Virtually all eukaryotes have innate circadian rhythms.
  • These are rhythms of biological activities that fluctuate over a period of approximately 24 hours (L. circa = about dies = day) even under constant environmental conditions (e.g. continuous darkness). Under constant conditions, the cycles may drift out of phase with the environment.
  • However, when exposed to the environment (e.g., alternating day and night), the rhythms become entrained that is, they now cycle in lockstep with the cycle of day and night with a period of exactly 24 hours.
  • In Arabidopsis, the entrainment of the rhythms requires that light is detected by the
    • phytochromes (absorb red light)
    • cryptochromes (absorb blue light)

    Methods

    Whole-genome identification of MADS-box genes in pineapple

    The protein sequences of pineapple, rice and Arabidopsis were obtained from Phytozome (https://phytozome.jgi.doe.gov/pz/portal.html), RGAP (http://rice.plantbiology.msu.edu/) and TAIR (http://www.arabidopsis.org/) databases, respectively. To identify the MADS-box genes in pineapple, the Hidden Markov Model (HMM) profiles of the SFR (type I) domain (PF00319) and the MEF2 (type II) domain (PF09047), downloaded from Pfam database (http://pfam.xfam.org, Pfam 31.0), were used to search the pineapple genome database [43, 44]. All of the proteins with an E-value lower than 0.01 were selected. Secondly, using all Arabidopsis and rice MADS-box genes as queries, the predicted pineapple MADS genes were checked by BLASTP searches (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Finally, the predicted MADS models detected were examined manually. The retrieved pineapple MADS genes were further verified by the NCBI Conserved Domain Database (https://www.ncbi.nlm.nih.gov/cdd).

    Classification of pineapple MADS-box genes

    MADS-box genes in Arabidopsis and rice were used for classifying the pineapple MADS-box genes. Multiple sequence alignments were performed based on protein sequences of MADS-box genes in pineapple, Arabidopsis and rice using MAFFT (https://www.ebi.ac.uk/Tools/msa/mafft/). A phylogenetic tree was then constructed based on multiple sequence alignments using RAxML with the parameters: pair wise gap deletion and 1000 bootstrap iterations [45]. The phylogenetic tree was further annotated by iTOL program (http://itol.embl.de/).

    Gene structure and conserved motif analysis

    To identify the gene structure of pineapple MADS-box genes, the full-length coding sequence (CDS) and genomic sequence of MADS-box genes were used to perform gene structure analysis by Gene Structure Display Server program (http://gsds.cbi.pku.edu.cn/) [46]. Online software MEME was used to search motifs in pineapple MADS-box genes (http://meme-suite.org/tools/meme) with the parameters: maximum number of motifs – 20 and optimum motif width set at ≥6 and ≤ 200. The motifs of MADS-box genes were annotated by the SMART program (http://smart.embl-heidelberg.de/).

    Location of pineapple MADS-box genes on chromosomes

    The pineapple genome has been mapped to 25 chromosomes [24]. To explore the chromosomal location of MADS-box genes, online software MA2C (MapGene2Chromosome v2) (http://mg2c.iask.in/mg2c_v2.0/) was used to map pineapple MADS-box genes onto chromosomes.

    Expression analysis of pineapple MADS-box genes in four tissues

    Expression patterns of MADS-box genes at different tissues (flower, root, leaf and fruit) were analyzed using RNA-Seq data obtained from Ming et al. [24]. Flower, root and leaf tissues were collected from cultivar F153 and fruit tissue was obtained from cultivar MD-2. The tissues were stored at -80 °C for RNA extraction and transcriptome analysis. The FPKM values were calculated by the Cufflinks/Cuffnorm pipeline (http://cufflinks.cbcb.umd.edu/). Genes with no expression (FPKM values equal “0” in all tissues) were filtered. The expression pattern of pineapple MADS-box genes in different tissues was visualized by a heat map.

    Diurnal expression analysis of MADS-box genes

    Green tip (photosynthesis) and white base (non- photosynthesis) leaf tissues were collected from field pineapple cultivar MD-2 grown in Hawaii over a 24-h period to examine the diurnal expression patterns of pineapple genes. Five individual plants were collected as one replicate, and three biological replicates were collected. The method of analyzing circadian rhythm was adopted from Sharma et al. [27]. Online software Haystack was used to analyze the time series expression data (http://haystack.mocklerlab.org/), with parameters: correlation cut off 0.7, P value cut off 0.05, fold change cutoff 2 and background cutoff 1.

    Plant material, RNA extraction and quantitative RT-PCR analysis

    The flower and leaves of pineapple cultivar MD-2 were obtained from the greenhouse of Fujian Agriculture and forestry University (26°4′54″N, 119°13′47″E) on October 25th, 2019. The average temperature of greenhouse is around 28 °C, and the light cycle is from 4:00–20:00. The ways of collecting pineapple samples and designing biological replicates was the same as the protocols in the paper of Ming et al. [24].

    Total RNA was extracted using Trizol protocol. Reverse transcription was performed from 2μg of RNA using TransScript One-Step Supermix kit. The cDNA was diluted ten-fold for the following qRT-PCR verification. Primers for pineapple MADS-box genes were designed using online website (https://www.idtdna.com/PrimerQuest/Home/Index). Primers information are listed in the Additional file 1: Table S1. The qRT-PCR reaction was performed in the 20 μL volume containing 1 μL of cDNA, 1 μL of each primezr and 10 μL of SYBR Green mix and was under the following program: 95 °C for 3 min 32 cycles at 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 30 s 72 °C for 10 min.

    The expression of MADS-box genes in different tissues (flower and leaves), green tip and white base leaves at different time points (6 am, 12 am, 6 pm, 12 pm) were verified by qRT-PCR. All the reactions were performed in three biological replicates.


    Parts of the flower

    There are considerable differences among the flowers of the 300,000 species of Angiosperms. Botanists rely upon a large vocabulary of specialized terms to describe the parts of these various flowers. The most important morphological features of flowers are considered below.

    Flowers can arise from different places on a plant, depending on the species. Some flowers are terminal, meaning that a single flower blooms at the apex of a stem. Some flowers are axial, in that they are borne on the axes of branches along a stem. Some flowers arise in an inflorescence, a branched cluster of individual flowers.

    There are four whorls of organs in a complete flower. From the outside to the inside, one encounters sepals, petals, stamens, and carpels. The sepals are leaf-like organs, which are often green, but can sometimes be brown or brightly colored, depending on the species. The petals are also leaf-like and are brightly colored in most animal-pollinated species but dull in color or even absent in wind-pollinated plants.

    The stamens and carpels, the reproductive organs, are the most important parts of a flower. The stamens are the male, pollen-producing organs. A stamen typically consists of an anther attached to a filament (stalk). The anther produces many microscopic pollen grains. The male sex cell , a sperm, develops within each pollen grain.

    The carpels are the female ovule-producing organs. A carpel typically consists of an ovary, style, and stigma. The stigma is the tip of the carpel upon which pollen grains land and germinate. The style is a stalk that connects the stigma and ovary. After the pollen grain has germinated, its pollen tube grows down the style into the ovary. The ovary typically contains one or more ovules, structures which develop into seeds upon fertilization by the sperm. As the ovules develop into seeds, the ovary develops into a fruit, whose characteristics depend on the species.

    In some species, one or more of the four whorls of floral organs is missing, and the flower is referred to as an incomplete flower. A bisexual flower is one with both stamens and carpels, whereas a unisexual flower is one which has either stamens or carpels, but not both. All complete flowers are bisexual since they have all four floral whorls. All unisexual flowers are incomplete since they lack either stamens or carpels. Bisexual flowers, with stamens and carpels, can be complete or incomplete, since they may lack sepals and/or petals.


    Contents

    Before 1900 Edit

    The origin of the term "morphology" is generally attributed to Johann Wolfgang von Goethe (1749–1832). He was of the opinion that there is an underlying fundamental organisation (Bauplan) in the diversity of flowering plants. In his book The Metamorphosis of Plants, he proposed that the Bauplan enabled us to predict the forms of plants that had not yet been discovered. [5] Goethe was the first to make the perceptive suggestion that flowers consist of modified leaves. He also entertained different complementary interpretations. [6] [7]

    In the middle centuries, several basic foundations of our current understanding of plant morphology were laid down. Nehemiah Grew, Marcello Malpighi, Robert Hooke, Antonie van Leeuwenhoek, Wilhelm von Nageli were just some of the people who helped build knowledge on plant morphology at various levels of organisation. It was the taxonomical classification of Carl Linnaeus in the eighteenth century though, that generated a firm base for the knowledge to stand on and expand. [8] The introduction of the concept of Darwinism in contemporary scientific discourse also had had an effect on the thinking on plant forms and their evolution.

    Wilhelm Hofmeister, one of the most brilliant botanists of his times, was the one to diverge away from the idealist way of pursuing botany. Over the course of his life, he brought an interdisciplinary outlook into botanical thinking. He came up with biophysical explanations on phenomena like phototaxis and geotaxis, and also discovered the alternation of generations in the plant life cycle. [5]

    1900 to the present Edit

    The past century witnessed a rapid progress in the study of plant anatomy. The focus shifted from the population level to more reductionist levels. While the first half of the century saw expansion in developmental knowledge at the tissue and the organ level, in the latter half, especially since the 1990s, there has also been a strong impetus on gaining molecular information.

    Edward Charles Jeffrey was one of the early evo-devo researchers of the 20th century. He performed a comparative analyses of the vasculatures of living and fossil gymnosperms and came to the conclusion that the storage parenchyma has been derived from tracheids. [9] His research [10] focussed primarily on plant anatomy in the context of phylogeny. This tradition of evolutionary analyses of plant architectures was further advanced by Katherine Esau, best known for her book The Plant Anatomy. Her work focussed on the origin and development of various tissues in different plants. Working with Vernon Cheadle, [11] she also explained the evolutionary specialization of the phloem tissue with respect to its function.

    In 1959 Walter Zimmermann published a revised edition of Die Phylogenie der Planzen. [12] This very comprehensive work, which has not been translated into English, has no equal in the literature. It presents plant evolution as the evolution of plant development (hologeny). In this sense it is plant evolutionary developmental biology (plant evo-devo). According to Zimmermann, diversity in plant evolution occurs though various developmental processes. Three very basic processes are heterochrony (changes in the timing of developmental processes), heterotopy (changes in the relative positioning of processes), and heteromorphy (changes in form processes).

    In the meantime, by the beginning of the latter half of the 1900s, Arabidopsis thaliana had begun to be used in some developmental studies. The first collection of Arabidopsis thaliana mutants were made around 1945. [13] However it formally became established as a model organism only in 1998. [14]

    Wikispecies has information related to Arabidopsis thaliana.

    The recent spurt in information on various plant-related processes has largely been a result of the revolution in molecular biology. Powerful techniques like mutagenesis and complementation were made possible in Arabidopsis thaliana via generation of T-DNA containing mutant lines, recombinant plasmids, techniques like transposon tagging etc. Availability of complete physical and genetic maps, [15] RNAi vectors, and rapid transformation protocols are some of the technologies that have significantly altered the scope of the field. [14] Recently, there has also been a massive increase in the genome and EST sequences [16] of various non-model species, which, coupled with the bioinformatics tools existing today, generate opportunities in the field of plant evo-devo research.

    Gérard Cusset provided a detailed in-depth analysis of the history of plant morphology, including plant development and evolution, from its beginnings to the end of the 20th century. [17] Rolf Sattler discussed fundamental principles of plant morphology. [18] [7]

    The most important model systems in plant development have been arabidopsis and maize. Maize has traditionally been the favorite of plant geneticists, while extensive resources in almost every area of plant physiology and development are available for Arabidopsis thaliana. Apart from these, rice, Antirrhinum majus, Brassica, and tomato are also being used in a variety of studies. The genomes of Arabidopsis thaliana and rice have been completely sequenced, while the others are in process. [19] It must be emphasized here that the information from these "model" organisms form the basis of our developmental knowledge. While Brassica has been used primarily because of its convenient location in the phylogenetic tree in the mustard family, Antirrhinum majus is a convenient system for studying leaf architecture. Rice has been traditionally used for studying responses to hormones like abscissic acid and gibberelin as well as responses to stress. However, recently, not just the domesticated rice strain, but also the wild strains have been studied for their underlying genetic architectures. [20]

    Some people have objected against extending the results of model organisms to the plant world. One argument is that the effect of gene knockouts in lab conditions wouldn't truly reflect even the same plant's response in the natural world. Also, these supposedly crucial genes might not be responsible for the evolutionary origin of that character. For these reasons, a comparative study of plant traits has been proposed as the way to go now. [21]

    Since the past few years, researchers have indeed begun looking at non-model, "non-conventional" organisms using modern genetic tools. One example of this is the Floral Genome Project, which envisages to study the evolution of the current patterns in the genetic architecture of the flower through comparative genetic analyses, with a focus on EST sequences. [22] Like the FGP, there are several such ongoing projects that aim to find out conserved and diverse patterns in evolution of the plant shape. Expressed sequence tag (EST) sequences of quite a few non-model plants like sugarcane, apple, lotus, barley, cycas, coffee, to name a few, are available freely online. [23] The Cycad Genomics Project, [24] for example, aims to understand the differences in structure and function of genes between gymnosperms and angiosperms through sampling in the order Cycadales. In the process, it intends to make available information for the study of evolution of seeds, cones and evolution of life cycle patterns. Presently the most important sequenced genomes from an evo-devo point of view include those of A. thaliana (a flowering plant), poplar (a woody plant), Physcomitrella patens (a bryophyte), Maize (extensive genetic information), and Chlamydomonas reinhardtii (a green alga). The impact of such a vast amount of information on understanding common underlying developmental mechanisms can easily be realised.

    Apart from EST and genome sequences, several other tools like PCR, yeast two-hybrid system, microarrays, RNA Interference, SAGE, QTL mapping etc. permit the rapid study of plant developmental patterns. Recently, cross-species hybridization has begun to be employed on microarray chips, to study the conservation and divergence in mRNA expression patterns between closely related species. [25] Techniques for analyzing this kind of data have also progressed over the past decade. We now have better models for molecular evolution, more refined analysis algorithms and better computing power as a result of advances in computer sciences.

    Overview of plant evolution Edit

    Evidence suggests that an algal scum formed on the land 1,200 million years ago , but it was not until the Ordovician period, around 500 million years ago , that land plants appeared. These began to diversify in the late Silurian period, around 420 million years ago , and the fruits of their diversification are displayed in remarkable detail in an early Devonian fossil assemblage known as the Rhynie chert. This chert preserved early plants in cellular detail, petrified in volcanic springs. By the middle of the Devonian period most of the features recognised in plants today are present, including roots and leaves. By the late Devonian, plants had reached a degree of sophistication that allowed them to form forests of tall trees. Evolutionary innovation continued after the Devonian period. Most plant groups were relatively unscathed by the Permo-Triassic extinction event, although the structures of communities changed. This may have set the scene for the evolution of flowering plants in the Triassic (

    200 million years ago ), which exploded the Cretaceous and Tertiary. The latest major group of plants to evolve were the grasses, which became important in the mid Tertiary, from around 40 million years ago . The grasses, as well as many other groups, evolved new mechanisms of metabolism to survive the low CO
    2 and warm, dry conditions of the tropics over the last 10 million years . Although animals and plants evolved their bodyplan independently, they both express a developmental constraint during mid-embryogenesis that limits their morphological diversification. [26] [27] [28] [29] [30]

    Meristems Edit

    The meristem architectures differ between angiosperms, gymnosperms and pteridophytes. The gymnosperm vegetative meristem lacks organization into distinct tunica and corpus layers. They possess large cells called central mother cells. In angiosperms, the outermost layer of cells divides anticlinally to generate the new cells, while in gymnosperms, the plane of division in the meristem differs for different cells. However, the apical cells do contain organelles like large vacuoles and starch grains, like the angiosperm meristematic cells.

    Pteridophytes, like fern, on the other hand, do not possess a multicellular apical meristem. They possess a tetrahedral apical cell, which goes on to form the plant body. Any somatic mutation in this cell can lead to hereditary transmission of that mutation. [31] The earliest meristem-like organization is seen in an algal organism from group Charales that has a single dividing cell at the tip, much like the pteridophytes, yet simpler. One can thus see a clear pattern in evolution of the meristematic tissue, from pteridophytes to angiosperms: Pteridophytes, with a single meristematic cell gymnosperms with a multicellular, but less defined organization and finally, angiosperms, with the highest degree of organization.

    Evolution of plant transcriptional regulation Edit

    Transcription factors and transcriptional regulatory networks play key roles in plant development and stress responses, as well as their evolution. During plant landing, many novel transcription factor families emerged and are preferentially wired into the networks of multicellular development, reproduction, and organ development, contributing to more complex morphogenesis of land plants. [32]

    Evolution of leaves Edit

    Origins of the leaf Edit

    Leaves are the primary photosynthetic organs of a plant. Based on their structure, they are classified into two types - microphylls, that lack complex venation patterns and megaphylls, that are large and with a complex venation. It has been proposed that these structures arose independently. [33] Megaphylls, according to the telome theory, have evolved from plants that showed a three-dimensional branching architecture, through three transformations: planation, which involved formation of a planar architecture, webbing, or formation of the outgrowths between the planar branches and fusion, where these webbed outgrowths fused to form a proper leaf lamina. Studies have revealed that these three steps happened multiple times in the evolution of today's leaves. [34]

    Contrary to the telome theory, developmental studies of compound leaves have shown that, unlike simple leaves, compound leaves branch in three dimensions. [35] [36] Consequently, they appear partially homologous with shoots as postulated by Agnes Arber in her partial-shoot theory of the leaf. [37] They appear to be part of a continuum between morphological categories, especially those of leaf and shoot. [38] [39] Molecular genetics confirmed these conclusions (see below).

    It has been proposed that the before the evolution of leaves, plants had the photosynthetic apparatus on the stems. Today's megaphyll leaves probably became commonplace some 360 mya , about 40 my after the simple leafless plants had colonized the land in the early Devonian period. This spread has been linked to the fall in the atmospheric carbon dioxide concentrations in the late Paleozoic era associated with a rise in density of stomata on leaf surface. This must have allowed for better transpiration rates and gas exchange. Large leaves with less stomata would have heated up in the sun's rays, but an increased stomatal density allowed for a better-cooled leaf, thus making its spread feasible. [40] [41]

    Factors influencing leaf architectures Edit

    Various physical and physiological forces like light intensity, humidity, temperature, wind speeds etc. are thought to have influenced evolution of leaf shape and size. It is observed that high trees rarely have large leaves, owing to the obstruction they generate for winds. This obstruction can eventually lead to the tearing of leaves, if they are large. Similarly, trees that grow in temperate or taiga regions have pointed leaves, presumably to prevent nucleation of ice onto the leaf surface and reduce water loss due to transpiration. Herbivory, not only by large mammals, but also small insects has been implicated as a driving force in leaf evolution, an example being plants of the genus Aciphylla, that are commonly found in New Zealand. The now-extinct moas (birds) fed upon these plants, and the spines on the leaves probably discouraged the moas from feeding on them. Other members of Aciphylla that did not co-exist with the moas were spineless. [42]

    Genetic evidences for leaf evolution Edit

    At the genetic level, developmental studies have shown that repression of the KNOX genes is required for initiation of the leaf primordium. This is brought about by ARP genes, which encode transcription factors. Genes of this type have been found in many plants studied till now, and the mechanism i.e. repression of KNOX genes in leaf primordia, seems to be quite conserved. Expression of KNOX genes in leaves produces complex leaves. It is speculated that the ARP function arose quite early in vascular plant evolution, because members of the primitive group lycophytes also have a functionally similar gene [43] Other players that have a conserved role in defining leaf primordia are the phytohormone auxin, gibberelin and cytokinin.

    One feature of a plant is its phyllotaxy. The arrangement of leaves on the plant body is such that the plant can maximally harvest light under the given constraints, and hence, one might expect the trait to be genetically robust. However, it may not be so. In maize, a mutation in only one gene called abphyl (abnormal phyllotaxy) was enough to change the phyllotaxy of the leaves. It implies that sometimes, mutational tweaking of a single locus on the genome is enough to generate diversity. The abphyl gene was later on shown to encode a cytokinin response regulator protein. [44]

    Once the leaf primordial cells are established from the SAM cells, the new axes for leaf growth are defined, one important (and more studied) among them being the abaxial-adaxial (lower-upper surface) axes. The genes involved in defining this, and the other axes seem to be more or less conserved among higher plants. Proteins of the HD-ZIPIII family have been implicated in defining the adaxial identity. These proteins deviate some cells in the leaf primordium from the default abaxial state, and make them adaxial. It is believed that in early plants with leaves, the leaves just had one type of surface - the abaxial one. This is the underside of today's leaves. The definition of the adaxial identity occurred some 200 million years after the abaxial identity was established. [21] One can thus imagine the early leaves as an intermediate stage in evolution of today's leaves, having just arisen from spiny stem-like outgrowths of their leafless ancestors, covered with stomata all over, and not optimized as much for light harvesting.

    How the infinite variety of plant leaves is generated is a subject of intense research. Some common themes have emerged. One of the most significant is the involvement of KNOX genes in generating compound leaves, as in tomato (see above). But this again is not universal. For example, pea uses a different mechanism for doing the same thing. [45] [46] Mutations in genes affecting leaf curvature can also change leaf form, by changing the leaf from flat, to a crinkly shape, [47] like the shape of cabbage leaves. There also exist different morphogen gradients in a developing leaf which define the leaf's axis. Changes in these morphogen gradients may also affect the leaf form. Another very important class of regulators of leaf development are the microRNAs, whose role in this process has just begun to be documented. The coming years should see a rapid development in comparative studies on leaf development, with many EST sequences involved in the process coming online.

    Molecular genetics has also shed light on the relation between radial symmetry (characteristic of stems) and dorsiventral symmetry (typical for leaves). James (2009) stated that "it is now widely accepted that. radiality [characteristic of most shoots] and dorsiventrality [characteristic of leaves] are but extremes of a continuous spectrum. In fact, it is simply the timing of the KNOX gene expression!" [48] In fact there is evidence for this continuum already at the beginning of land plant evolution. [49] Furthermore, studies in molecular genetics confirmed that compound leaves are intermediate between simple leaves and shoots, that is, they are partially homologous with simple leaves and shoots, since "it is now generally accepted that compound leaves express both leaf and shoot properties”. [50] This conclusion was reached by several authors on purely morphological grounds. [35] [36]

    Evolution of flowers Edit

    Flower-like structures first appear in the fossil records some

    The flowering plants have long been assumed to have evolved from within the gymnosperms according to the traditional morphological view, they are closely allied to the gnetales. However, recent molecular evidence is at odds to this hypothesis, [52] [53] and further suggests that gnetales are more closely related to some gymnosperm groups than angiosperms, [54] and that gymnosperms form a distinct clade to the angiosperms,. [52] [53] [54] Molecular clock analysis predicts the divergence of flowering plants (anthophytes) and gymnosperms to


    The greatest colonisers on earth? Plants. And this is how they did it

    Representational image | Photo: Flickr

    T he world 500 million years ago looked very different to today. The land was bare, with only bacteria, fungi and algae able to survive on it. Everything else lived in the ocean, but once plants moved onto land, they changed almost everything on Earth’s surface. They helped to create soils, rivers and the oxygen-rich atmosphere, which eventually allowed animals to live a life out of water.

    Our study, recently published in Current Biology, found that bursts of new genes helped plants make the move from water to land. The first land plants, like today’s flora, consisted of many cells with multiple functions that were controlled by thousands of genes made of DNA. We compared the complete gene sets of living plant species ranging from wheat to quinoa and were able to discover the genes which first enabled plants to colonise the land and change life on Earth forever.

    We found that two large groups of genes appeared in plants during the transition onto land. This means that the evolution of land plants was driven by the emergence of new genes, previously not seen in close relatives. We know this because natural selection removes genes that aren’t essential for the organism’s functioning, so if these genes didn’t play an important role, they would have been lost.

    Interestingly, these new genes are found in all land plants in our study, which includes the flowering plants (tomato, rice and orchid), as well as the non-flowering plants (conifer, ginkgo and moss). This suggests that these genes were crucial for allowing plants to survive on land, but how did they help the forerunners of land plants adapt to their new environment?

    When land plants evolved, the number of new genes in the plant kingdom exploded. | Alexander Bowles, Author provided

    The first land plants

    Green algae are among the closest living relatives of the first land plants and are mostly found in aquatic ecosystems, like the ocean and rivers. They’re able to absorb water and nutrients from their surroundings. When plants first colonised land, they needed a new way to access nutrients and water without being immersed in it.

    We found the genes that helped early land plants do this by developing rhizoids – root-like structures that helped them stay anchored in the ground and access water and nutrients. We also identified genes involved in gravitropism, which is what helps roots grow in the right direction. After all, a life out of water would have meant needing to know which way was down. These new genes helped plants coordinate the growth of rhizoids downward and ensure shoots grew up to maximise how much light they could absorb.

    The transition of plants from water to land occurred in a landscape of extreme heat and light, and with little water. The genes we identified allowed early land plants to adapt to the stress of living outside of water, ensuring they could establish themselves and tolerate these harsh conditions.

    A big difference between land plants and their close relatives, green algae, is that land plants develop embryos. In mosses and ferns, this embryo takes the form of a spore while in a lot of other plants, it’s the seed. We found genes that allowed the first land plants to produce and protect these embryos with specialised tissues that limited damage from ultraviolet light and heat.

    By protecting the embryo, a plant increases the chances of its genes being passed on to the next generation, making them more likely to disperse and survive and allowing land plants to colonise the barren landscape.

    The diversity of land plants and close algal relatives. | Alexander Bowles, Author provided

    The movement of plants from water to land is one of the most momentous shifts in the history of life on Earth. The number of new genes that emerged as land plants evolved is far larger than at any other point in the evolutionary history of plants, even more than those that came with flowering plants.

    This burst of new genes marks arguably the most important evolutionary development in the history of plant life. Previously, scientists thought gradual changes at the genetic level underpinned the emergence of plants on land. Now we know that the first land plants were able to produce an embryo, tolerate a range of environmental stresses and anchor themselves to land through an explosion of genetic innovations. These new genes enabled plants to dominate dry land, diversify into over 374,000 species and shape the modern ecosystems we see around the world today.

    This article is republished from The Conversation under a Creative Commons license. Read the original article.

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