Examining gene expression in plants

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Plants need to incorporate a wide range of tissue, developmental, and environmental signals to regulate complex patterns of gene expression (Singh, 1998). All of these are controlled by complex networks of transcription factors. They are fundamental for converting signalling outputs into complex gene regulatory outputs such as changes in daylength. Transcription factors also function in gene expression including chromatin remodelling and recruitment (Singh, 1998). The chromatin must be remodelled in order to allow access for TFs and the recruitment of the RNA polymerase II (Pol II). Hence, chromatin remodelling is essential for gene activation and transcription. All TFs are divided into a number of functional classes, some belonging to more than one. TFs also exist in many shapes and sizes. Majority of the transcription factors are activators and repressors. These proteins bind to specific DNA sequences found only in certain promoters. There has been little work on the general mechanism of transcription in plants compared to animals and yeast but the number of transcription factors found in plants are still increasing. Plant TFs have several structural motifs which allow them to bind onto specific DNA sequences. These were first identified in transcription factors taken from animals and yeast systems.

The CCAAT box is one of the most common elements in eukaryotic promoters. It is located 60-100 bp upstream of the transcription start site and is functional in both direct and inverted orientations (Mantovani, 1998). CCAAT boxes are known to be highly conserved within homologous species because all species have similarities in terms of position, orientation, and flanking nucleotides (Mantovani, 1999). There are many DNA-binding proteins which bind to CCAAT boxes (Li et al., 1992). NF-Y is a heterotrimeric ubiquitous CCAAT-binding factor composed of NF-YA (HAP2/CBF-B), NF-YB (HAP3/CBF-A) and NF-YC (HAP5/CBF-C). It has a high affinity and sequence specificity for CCAAT sequence. Three subunits of NF-Y is required for CCAAT binding. Assembly of the NF-Y follows a strict and stepwise pattern. NF-YB and NF-YC interacts in the cytoplasm to form a heterodimer. This dimer then translocates to the nucleus where the third subunit is added, NF-YA. NF-YA is recruited to construct the mature, heterotrimeric NF-Y transcription factor which has a high affinity for DNA. NF-Y subunits have been under research for a long time now and they have been isolated from various organisms. All the subunits in yeast and vertebrates are encoded by single copy genes, however, multiple genes are found to be encoding for each subunit in plant genomes. This is why various NF-Y proteins follow more complex regulatory roles than in other organisms. In Arabidopsis, there are 10 NF-YA, 13 NF-YB and 13 NF-YC homologs. Each of these subunits have their own distinct and highly conserved region. The conserved core of NF-YA has two roles where N-terminal domain is required for subunit interaction with NF-YB - NF-YC heterodimer and the C-terminal domain recognizes the site where DNA binding would occur. NF-YB and NF-YC conserved core regions contain residues in contact with DNA and NF-YB contributes to DNA-binding specificity (Zemzoumi et al., 1999).

The NF-Y complex has not been completely described yet, but individual subunits are known to be involved in several important processes. LEAFY COTYLEDON 1 (LEC1 or NF-YB9) was the earliest cloned and described plant NF-Y (Siefers et al., 2009). It is expressed strongly in the developing embryo and is vital for controlling the process from embryo to adult status. Even though LEC1 has been very well studied for over a decade, the requirements for NF-YA and NF-YC subunits are still subtle. This can be explained in couple ways because of the complex structure of the embryo. It can either be complexity of genetic and biochemical studies or the lack of complete plant NF-Y complexes.

The most important environmental stimuli to plants is light. Light triggers the reprogramming of nuclear gene expression through complex biological pathways (Stephenson et al, 2010). In plants, there are four distinct families of photoreceptors. Photoreceptors are necessary for sensing the quality, quantity, direction and duration of light. They consist of phytochromes, cryptochromes, phototropins and unidentified ultraviolet B photoreceptor. TFs, kinases, phosphatases and degradation-pathway proteins are triggered by these photoreceptors (Chen et al., 2004). Jiao et al. (2005) estimated that the expression of approximately 20% of the genome in Arabidopsis seedlings is regulated by white light and nearly 26% of the 1,363 TF genes found in Arabidopsis are differentially expressed in developing seedlings in response to light (Jiao et al., 2003). The assembly of the NF-Y complex at CCAAT-box in a spinach promoter is regulated by light (Kusnetsov et al, 1999). In Arabidopsis, NF-YA and NF-YB9 are involved in the regulation of light-harvesting chlorophyll a/b binding protein (Stephenson et al, 2010).

Some plant NF-Y subunits are known to be directly involved with other light regulated TFs. Flowering of Arabidopsis is rapidly induced in long photoperiods, hence it is a long-day (LD) plant. Some plants such as the monocot rice are induced to flower in short periods (SD). In Arabidopsis, zinc-finger type transcriptional activator CONSTANS (CO) is the key regulator of photoperiod dependent flowering. The circadian clock controls CO mRNA levels and it fluctuates on a daily basis. The circadian system is needed for the measurement and interpretation of day length. During the day, expression of CO levels must be at maximum level for CO activity because the protein is easily degraded in the dark. NF-YB and NF-YC subunits have been identified as CO-interacting proteins via yeast two-hybrid assays in both tomato (Solanum lycopersicum) and Arabidopsis (Kumimoto et al, 2010). Recent studies have suggested that CO may be directly controlling the FT (FLOWERING LOCUS T) expression. FT protein is a strong promoter responsible for flowering. However, it is still not known how CO integrates with the FT promoter. This is why several recent studies were about NF-Y transcription factors and their role in photo-period dependent flowering.

Arabidopsis researchers have been benefiting from superficial, single-copy integrative transformation of germinating seeds and intact plants via Agrobacterium T-DNA (transfer DNA) vectors (Haag 2007). Plant transformation is the genetic manipulation when foreign genes are introduced and stably integrated in the plant genomes, and the transformed cells are regenerated to obtain transgenic plants (Zhang et al., 2006). The process includes the preparation of transformation-competent plant cells or tissues and the delivery of foreign genes into plant cells by Agrobacterium tumefaciens. There is another method which can be used called biolistic method but this includes the use of tissue culture and plant regeneration so this would greatly increase the time required to produce transgenic plants. Feldmann and Marks (1987) cocultivated germinating seeds of Arabidopsis thaliana with an Agrobacterium tumefaciens strain including a disarmed Ti plasmid and a binary vector. As a result, they obtained astonishingly stable transgenic lines but the transformation rates were very low. Several years later, the Pelletier group used the Agrobacterium vacuum infiltration method to improve the transformation efficiency. The process involves uprooting of flowering Arabidopsis plants, vacuum infiltration of the plants using an Agrobacterium cell suspension, re-planting, harvesting of seed several weeks later and testing for primary transformants on medium containing a suitable agent such as antibiotics or herbicides (Zhang et al., 2006). This process was later improved and simplified by leaving out the steps of uprooting and replanting. By using this transformation procedure, the precise locations for more than 88,000 T-DNA insertions determined and more than 21,700 of the approximately 29,454 predicted Arabidopsis genes were identified. The good thing is that there is a large number of genes for which no function is known or predicted. This gives a great opportunity to create loss-of-function mutations for all of the genes. By doing this, all the differences between the genes can be predicted and detected phenotypically. Thus, the functions of the genes can be revealed and further analysis can be done.

Latest studies about plant NF-Y complexes have been suggested that they act as fundamental regulatory hubs for many process. The only problem with plant NF-Y complexes is that they have a overlapping functionality, which means that multiples genes are encoding for each subunit. In this study, the overall aim was to grow transgenic plants by using genetically modified Arabidopsis seeds. Grown plants were compared to one another in accordance to phenotypic characteristics of Arabidopsis insertion lines. The insertion lines were found and chosen from the SALK database according to their relevance with light. The seeds were ordered from European Arabidopsis Stock Centre (NASC). The sequence information from the model plant Arabidopsis thaliana was used to find the homologs for each NF-YC subunit. This information was used to align all sequences of NF-YC subunits in conjunction with Oryza sativa, Saccharomyces cerevisiae and Triticum aestivum, one sequence from each, respectively. A phylogenetic tree and alignments for all NF-YC subunits were presented. Alignment of the sequences will reveal each subunit's conserved regions and will give information about how they differ from their ancestral genes or from each other.

Materials

Plant Material: Arabidopsis thaliana

Growth Medium: MS

SALK Insertion Lines: SALK_012588, SALK_131890, SALK_003524, SALK_032163, SALK_069697, SALK_058903 and SALK_058911

Solutions: 70% Ethanol, Bleach solution, sterile water

Equipment: Sterile filter paper, sterile petri dishes

METHODS

MS Medium Preparation

For preparation of 1 Litre MS medium, 4.3 g of MS salts [Sigma] and 30 g/L of sucrose were dissolved in 1 L of double distilled water. The solution was adjusted to pH 5.9 by adding NaOH. Finally, solution was poured into a beaker, 8 g/L agar was added and autoclaved.

Seed Sterilization

40-50 seeds from each SALK insertion line were placed in vial tubes and washed with 70% Ethanol for 2 minutes. The ethanol was removed and seeds were rinsed with sterile H2O. Freshly made bleach solution containing 2ml of bleach, 48 ml of sterile H2o and 1 drop of tween was added to seeds and incubated for 15 minutes with regular shakes. Bleach solution was discarded and seeds were rinsed two to three times with distilled H2O.

Seed Propagation

Autoclaved MS medium was poured into Petri dishes and left for drying. 20-25 of sterilized seeds were placed on solidified medium with a good separation distance. Lids of Petri dishes were closed and sealed with microporous tapes then the plates were moved to the fridge for 24 hours. After incubating in the fridge, the plates were stored in the growth chamber.

Database searches for the NF-YC subunit

13 NF-YC subunit sequences for Arabidopsis were retrieved from TAIR (www.arabidopsis.org). The search was done by using each subunits' AGI (Arabidopsis Genome Initiative number) code, also called the AT designation. Obtained sequences were translated into peptide sequences by using EMBOSS Transeq - http://www.ebi.ac.uk/Tools/emboss/transeq/.

NF-YC subunit protein sequence for rice (Oryza sativa) was retrieved from Rice Transcription Database (RiceTFDB, http://ricetfdb.bio.uni-potsdam.de/v3.0). HAP5 (NF-YC) sequence for yeast (Saccharomyces cerevisiae) was retrieved from the Saccharomyces genome database - http://yeastgenome.org. Lastly, the PlantGDB was used for wheat (Triticum aestivum) NF-YC peptide sequence- http://plantgdb.org/.

Alignment and Phylogenies

Peptide sequences were aligned using Clustal W2 from EBI database - http://www.ebi.ac.uk/Tools/msa/clustalw2/. NF-Y subunits contain a core region that is relatively conserved across species, whereas the flanking regions are much less conserved with great differences in sequence identity and length.

RESULTS

Identification of NF-YC Genes Involved in Light-regulated Gene Expression

Information about the candidate NF-YC genes possibly involved in light-regulated gene expression was obtained from TAIR (www.arabidopsis.org). In Arabidopsis, 20% of all genes are induced by light signalling and some studies have shown that NF-YC transcription factors are involved in light-upregulated gene expression. Three NF-YC transcription factors identified are required for photoperiod-dependent flowering; NF-YC3, NF-YC4 and NF-YC9. These transcription factors are very closely related and conserved. Also, NF-YC3 and NF-YC9 are 100% identical throughout their conserved histone fold motifs. They are consistently and strongly expressed in the leaf vasculature [Kumimoto et al., 2010]. To determine if these NF-YC factors are truly required for photoperiod-dependent flowering, nf-yc mutants was quantified under SD (Short day, 8-h light/16-h dark) conditions, and then extended cold treatments. The results have indicated that nf-yc mutants flower much later than the control plant. This proves the point that NF-YC3, YC-4 and YC-9 are primarily involved in photoperiod-dependent flowering.

Light-grown expression patterns of NF-YC factors are also studied and the results were compared with a publication released previously [Warpeha at al., 2007] which examined blue light perception and ABA signalling. Six day old dark-grown seedlings were used along RT-PCR to identify NF-YC expression [Siefers et al., 2009]. As a result, NF-YC1, NF-YC4 and NF-YC9 were found to be expressed. Additionally, NF-YC3 was also included in this group because it is expressed in a similar way like the others. There were some NF-YC factors that are expressed strongly in the dark, NF-YC10, YC-11 and YC-12. NF-Y expression pattern were also examined and images were taken with GUS staining versus microarray scores. This was done in 4 categories; light versus dark-grown plants, 10-d-old rosettes, stage 15 flowers and stage 1 root tips. According to the images and staining, NF-YC expressions in all these 4 categories were similar. But, the similarity was only about the types of NF-YC transcription factors not the expression levels. The expression levels varied between the categories. For example, in all these categories NF-YC1, YC-3, YC-4, YC-9, YC-10, YC-11 and YC -12 were expressed. The others were expressed weakly in 1 or 2 categories. This is presented in Table 1.

Level of Expression

NF-YC

AGI Code

Light

Dark

Root

Flower

10-D Old

Expression Location

1

At3g48590

++

++

++

+

+

Seedling and pollen

2

At1g56170

-

-

-

-

-

Endosperm

3

At1g54830

++

++

++

+

++

Pollen, root and endosperm

4

At5g63470

++

++

++

+

++

Cotyledon and stamen

5

At5g50490

-

-

-

-

-

No probe set

6

At5g50480

-

-

-

-

-

Endosperm

7

At5g50470

-

-

-

-

-

No probe set

8

At5g27910

-

-

-

-

-

Endosperm

9

At1g08970

++

++

++

++

++

Rosette, and hypocotyl

10

At1g07980

+

+

++

-

-

Endosperm

11

At3g12480

++

++

++

+

++

Endosperm and seed coat

12

At5g38140

+

+

-

+

++

Endosperm

13

At5g43250

-

-

-

-

-

No probe set.

Table 1: Expression levels of NF-YC transcription factors (GUS staining). The table represents all NF-YC factors found in Arabidopsis and their expression levels in different categories.

Sequence Comparison

The sequences of each NF-YC transcription factors were obtained from TAIR and translated into protein sequences by using EBI Transeq (European Bioinformatics Institute) http://www.ebi.ac.uk/Tools/emboss/transeq/. Additionally, these sequences were also compared against Oryza sativa (Indica), Triticum aestivum and Saccharomyces cerevisiae. NF-YC protein sequences were retrieved from the Rice Transcription Database (RiceTFDB) (version 3.0, http://plantfdb.bio.uni-potsdam.de/v3.0/), the Saccharomyces genome database (http://yeastgenome.org/) and the PlantGDB website for Triticum aestivum - http://plantgdb.org/. Different amino acid sequences were compared with each other by using ClustalW2 from EBI server - http://www.ebi.ac.uk/Tools/msa/clustalw2/ (Figure 1).

Figure 1: CLUSTAL W results of 13 Arabidopsis NF-YC sequences plus one sequence from Saccharomyces cerevisiae, Triticum aestivum and Oryza sativa. This was used to compare each of the sequences with each other.

Comparison between the Arabidopsis NF-YCs and homologs from other species showed a high degree of sequence identity (Figure 1). The level of sequence similarity was greatest within the central domain. However, studies have shown that the core sequence of NF-YA family of proteins from each of these four plant species is the most highly conserved among the three subunits. Furthermore, the core sequence of the NF-YC proteins is the most divergent among them (Stephenson et al., 2007). NF-YC is similar to histone fold motifs with its structure and amino acid homology. The subunits are related to H2A histones and mature NF-Y causes localized DNA bending by binding to the minor groove of DNA (Ronchi et al., 1995). The distortions in the nucleosomal level leads to activation of transcription by allowing recruitment of chromatin-remodelling enzymes and RNA polymerase II (Matuoka and Chen, 2002).

Required amino acids in most NF-YC were highly conserved. The changes in required amino acids were seen in phylogenetically distant NF-YCs such as NF-YC10 to NF-YC13 (Figure 1). The changes in the protein structure of NF-YC result in changes in hydropathy (Yang et al., 2005). The reason for this is the change of the required amino acid with a more hydrophobic one. Phylogenetic analyses and Clustal W alignment have showed that there are two distinct clades in NF-YC transcription factors. First clade consists of NF-YC1 to -YC4 and -YC9 and is similar to their homologs in other species. The members of the second clade consist of NF-YC5 to -YC8 and NF-YC10 to -YC13 and are highly divergent from the ancestral NF-YC (Siefers et al., 2009).

Phylogenetic Analyses of NF-YC

A phylogenetic tree for NF-YC was constructed buy neighbour joining method by using the conserved regions presented in Figure 1. The tree was constructed and determined by using Clustal W (Figure 2).

Figure 2: Phylogenetic tree of NF-YC subunit family. The tree was created by using the neighbour joining method on the basis of the amino acid sequences of the conserved domains.

Production and Analysis of Transgenic Arabidopsis Plants

Seeds ordered from the SALK collection database were sowed onto agar plates to grow transgenic Arabidopsis plants. Germination of the seeds were followed and noted by comparing the phenotype of the plants with a WT (Table 1).

SALK

Insertion

Lines

Locus

of Polymorphism

Equivalent

Gene Name

Total Number of Plants

Total Number of Germination

% of Plants Germinated

012588

AT1G54830

NF-YC3

45

39

86.7

131890

AT1G54830

NF-YC3

47

41

87.2

003524

AT1G54830

NF-YC3

26

13

50.0

032163

AT5G63470

NF-YC4

52

50

96.2

069697

AT5G63470

NF-YC4

46

45

97.8

058903

AT1G08970

NF-YC9

50

49

98

058911

AT1G08970

NF-YC9

46

36

78.3

Table 2: Analysis of the germinated Arabidopsis plants. The plants were observed for 14 days in the growing room.

SALK Insertion Lines

Hypocotyl and Cotyledon Emergence (Days)

Standard Deviation

Coefficient of Variation

012588

4.23

0.43

10.2

131890

4.41

0.64

14.5

003524

4.54

0.52

11.5

032163

3.92

0.27

6.9

069697

3.96

0.21

5.3

058903

3.94

0.25

6.3

058911

4.34

0.48

11.1

Table 3: Analysis of the germinated Arabidopsis plants. The plants were observed after 4 days of waiting in the growing room. They were only between the seed germination and leaf development stage.

SALK Insertion Lines

5 Rosette Leaves (Days)

Standard Deviation

Coefficient of Variation

012588

12.33

0.67

5.4

131890

12.24

0.60

4.9

003524

13.31

1.25

9.4

032163

11.94

0.24

2.01

069697

11.91

0.29

2.4

058903

11.98

0.14

1.2

058911

12.06

0.33

2.7

Table 4: Analysis of the Arabidopsis plants during their principal growth stage. The plants were observed on the 12th day. The majority of them had at least 5 rosette leaves larger than 1mm in diameter.

Cumulative Percentage of Germination with the SALK Lines(%)

Days Since start

012588

131890

003524

032163

069697

058903

058911

0

0

0

0

0

0

0

0

4

69.8

63.8

15.4

95.7

94.2

98

54.3

8

76.3

71.7

33.5

96.2

97.8

98

65.3

12

84.8

78.7

50

96.2

97.8

98

78.3

Table 5: Germination Count. Percentage of germination is calculated for germination of each line over a period of 12 days.

Figure 2: Cumulative percentage Germination of selected SALK lines over a period of 12 days. Plants were germinated in MS media and placed in a growth cabinet. Each line on the graph represents SALK lines germinating with different rate of growth.

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